Journal articles on the topic 'Microbiology, Lactic Acid Bacteria, Antibiotic resistance, Genome analysis'
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1
Markusková, Barbora, Aneta Lichvariková, Tomáš Szemes, Janka Koreňová, Tomáš Kuchta, and Hana Drahovská. "Genome analysis of lactic acid bacterial strains selected as potential starters for traditional Slovakian bryndza cheese." FEMS Microbiology Letters 366, Supplement_1 (October 22, 2018): i3—i9. http://dx.doi.org/10.1093/femsle/fny257s.
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ABSTRACT Genomes of 21 strains of lactic acid bacteria isolated from Slovakian traditional cheeses were sequenced on an Illumina MiSeq platform. Subsequently, they were analysed regarding taxonomic classification, presence of genes encoding defence systems, antibiotic resistance and production of biogenic amines. Thirteen strains were found to carry genes encoding at least one bacteriocin, 18 carried genes encoding at least one restriction–modification system, all strains carried 1–6 prophages and 9 strains had CRISPR-Cas systems. CRISPR-Cas type II-A was the most common, containing 0–24 spacers. Only 10% spacers were found to be homological to known bacteriophage or plasmid sequences in databases. Two Enterococcus faecium strains and a Lactococcus lactis strain carried antibiotic resistance genes. Genes encoding for ornithine decarboxylase were detected in four strains and genes encoding for agmatine deiminase were detected in four strains. Lactobacillus paraplantarum 251 L appeared to be the most interesting strain, as it contained genes encoding for two bacteriocins, a restriction–modification system, two CRISPR-Cas systems, four prophages and no genes connected with antibiotic resistance or production of biogenic amines.
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Huang, Zheng, Xingya Zhou, Catherine Stanton, Reynolds Paul Ross, Jianxin Zhao, Hao Zhang, Bo Yang, and Wei Chen. "Comparative Genomics and Specific Functional Characteristics Analysis of Lactobacillus acidophilus." Microorganisms 9, no. 9 (September 20, 2021): 1992. http://dx.doi.org/10.3390/microorganisms9091992.
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Lactobacillus acidophilus is a common kind of lactic acid bacteria usually found in the human gastrointestinal tract, oral cavity, vagina, and various fermented foods. At present, many studies have focused on the probiotic function and industrial application of L. acidophilus. Additionally, dozens of L. acidophilus strains have been genome sequenced, but there has been no research to compare them at the genomic level. In this study, 46 strains of L. acidophilus were performed comparative analyses to explore their genetic diversity. The results showed that all the L. acidophilus strains were divided into two clusters based on ANI values, phylogenetic analysis and whole genome comparison, due to the difference of their predicted gene composition of bacteriocin operon, CRISPR-Cas systems and prophages mainly. Additionally, L. acidophilus was a pan-genome open species with a difference in carbohydrates utilization, antibiotic resistance, EPS operon, surface layer protein operon and other functional gene composition. This work provides a better understanding of L. acidophilus from a genetic perspective, and offers a frame for the biotechnological potentiality of this species.
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Parra-Flores, Julio, Adriana Cabal-Rosel, Beatriz Daza-Prieto, Pamela Chavarria, Eduard Maury-Sintjago, Alejandra Rodriguez-Fernández, Sergio Acuña, and Werner Ruppitsch. "Are Enterobacteriaceae and Enterococcus Isolated from Powdered Infant Formula a Hazard for Infants? A Genomic Analysis." Foods 11, no. 22 (November 8, 2022): 3556. http://dx.doi.org/10.3390/foods11223556.
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Powdered infant formulas (PIF) are the most used dietary substitutes that are used in order to supplement breastfeeding. However, PIF are not sterile and can be contaminated with different microorganisms. The objective of this study was to genomically characterize Enterobacteriaceae (ENT) and Enterococcus strains that were isolated from PIF. Strains were identified by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS). Genomic typing, detection of virulence, and resistance profiles and genes were performed with the Ridom SeqSphere+ software; the comprehensive antibiotic resistance database (CARD) platform; ResFinder and PlasmidFinder tools; and by the disk diffusion method. Nineteen isolates from PIF were analyzed, including ENT such as Kosakonia cowanii, Enterobacter hormaechei, Franconibacter helveticus, Mixta calida, and lactic acid bacteria such as Enterococcus faecium. The strains exhibited resistance to beta-lactams, cephalosporins, and macrolides. Resistance genes such as AcrAB-TolC, marA, msbA, knpEF, oqxAB, fosA, blaACT-7, blaACT-14, qacJ, oqxAB, aac(6’)-Ii, and msr(C); and virulence genes such as astA, cheB, cheR, ompA ompX, terC, ironA, acm, and efaAfm, adem were also detected. All the analyzed strains possessed genes that produced heat-shock proteins, such as IbpA and ClpL. In PIF, the presence of ENT and Enterococcus that are multiresistant to antibiotics—together with resistance and virulence genes—pose a health risk for infants consuming these food products.
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López-García, Elio, Antonio Benítez-Cabello, Javier Ramiro-García, Victor Ladero, and Francisco Noé Arroyo-López. "In Silico Evidence of the Multifunctional Features of Lactiplantibacillus pentosus LPG1, a Natural Fermenting Agent Isolated from Table Olive Biofilms." Foods 12, no. 5 (February 22, 2023): 938. http://dx.doi.org/10.3390/foods12050938.
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In recent years, there has been a growing interest in obtaining probiotic bacteria from plant origins. This is the case of Lactiplantibacillus pentosus LPG1, a lactic acid bacterial strain isolated from table olive biofilms with proven multifunctional features. In this work, we have sequenced and closed the complete genome of L. pentosus LPG1 using both Illumina and PacBio technologies. Our intention is to carry out a comprehensive bioinformatics analysis and whole-genome annotation for a further complete evaluation of the safety and functionality of this microorganism. The chromosomic genome had a size of 3,619,252 bp, with a GC (Guanine-Citosine) content of 46.34%. L. pentosus LPG1 also had two plasmids, designated as pl1LPG1 and pl2LPG1, with lengths of 72,578 and 8713 bp (base pair), respectively. Genome annotation revealed that the sequenced genome consisted of 3345 coding genes and 89 non-coding sequences (73 tRNA and 16 rRNA genes). Taxonomy was confirmed by Average Nucleotide Identity analysis, which grouped L. pentosus LPG1 with other sequenced L. pentosus genomes. Moreover, the pan-genome analysis showed that L. pentosus LPG1 was closely related to the L. pentosus strains IG8, IG9, IG11, and IG12, all of which were isolated from table olive biofilms. Resistome analysis reported the absence of antibiotic resistance genes, whilst PathogenFinder tool classified the strain as a non-human pathogen. Finally, in silico analysis of L. pentosus LPG1 showed that many of its previously reported technological and probiotic phenotypes corresponded with the presence of functional genes. In light of these results, we can conclude that L. pentosus LPG1 is a safe microorganism and a potential human probiotic with a plant origin and application as a starter culture for vegetable fermentations.
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Nicoloff, Hervé, and Françoise Bringel. "ISLpl1 Is a Functional IS30-Related Insertion Element in Lactobacillus plantarum That Is Also Found in Other Lactic Acid Bacteria." Applied and Environmental Microbiology 69, no. 10 (October 2003): 6032–40. http://dx.doi.org/10.1128/aem.69.10.6032-6040.2003.
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ABSTRACT We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum. ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L. plantarum strains as well as in Pediococcus strains. The ISLpl1 pattern changed over many generations of the strain L. plantarum NCIMB 1406. This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L. plantarum. Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.
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Pakroo, Shadi, Armin Tarrah, Rohit Takur, Manyu Wu, Viviana Corich, and Alessio Giacomini. "Limosilactobacillus fermentum ING8, a Potential Multifunctional Non-Starter Strain with Relevant Technological Properties and Antimicrobial Activity." Foods 11, no. 5 (February 26, 2022): 703. http://dx.doi.org/10.3390/foods11050703.
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Lactic acid bacteria (LAB) have gained particular attention among different exopolysaccharide-producing microorganisms due to their safety status and effects on human health and food production. Exopolysaccharide-producing LAB play a crucial role in different ways, such as improving texture, mouthfeel, controlling viscosity, and for low-calorie food production. In this study, we isolated a multifunctional strain with good exopolysaccharide production properties. Limosilactobacillus fermentum ING8 was isolated from an Indian traditional fermented milk (Dahi) and evaluated for its safety, enzymatic activity, NaCl resistance and temperature tolerance, milk coagulation, and storage stability. Finally, the complete genome of this strain was sequenced and subjected to safety in silico evaluation and genomic analysis. The results revealed that L. fermentum ING8 possesses relevant technological properties, such as exopolysaccharide production, antimicrobial activity, and galactose utilization. Besides, this strain showed very high stability to storage conditions at refrigeration temperature. In addition, the genomic analysis did not evidence any possible deleterious elements, such as acquired antibiotic resistance genes, virulence genes, or hemolysis-related genes. However, all structural genes related to the galactose operon and EPS production were detected. Therefore, L. fermentum ING8 can be considered a promising multifunctional bacterium to be proposed as non-starter in different types of dairy productions.
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Mun, So Yeong, Ye Jin Seo, and Hae Choon Chang. "Characterization of the Psychrotrophic Lactic Acid Bacterium Leuconostoc gelidum subsp. aenigmaticum LS4 Isolated from Kimchi Based on Comparative Analyses of Its Genomic and Phenotypic Properties." Foods 10, no. 8 (August 16, 2021): 1899. http://dx.doi.org/10.3390/foods10081899.
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With the aim of developing a new food starter culture, twenty-three psychrotrophic lactic acid bacteria (LAB) were isolated from 16 kimchi samples. One strain, Leuconostoc gelidum subsp. aenigmaticum LS4, which had typical psychrotrophic characteristics, was selected, and its phenotypic and genomic properties as a starter culture were investigated. The complete genome of L. aenigmaticum LS4 consisted of one circular chromosome (1,988,425 bp) and two plasmids (19,308 bp and 11,283 bp), with a guanine–cytosine content of 36.8%. L. aenigmaticum LS4 could grow at 5 °C but not at 37 °C, and maximum cell growth was obtained at 15~25 °C. L. aenigmaticum LS4 did not show any harmful characteristics such as hemolysis, undesirable enzyme activities, biogenic amine production, or antibiotic resistance. L. aenigmaticum LS4 was investigated for its suitability for technological processes (pH, temperature and NaCl treatment). L. aenigmaticum LS4 exhibited strong antimicrobial activity caused by the production of organic acids and bacteriocin, and it produced an exopolysaccharide composed of glucose with a molecular weight of 3.7 × 106 Da. Furthermore, L. aenigmaticum LS4 improved the organoleptic qualities of kimchi juice. Our results indicate that L. aenigmaticum LS4 could be used as a functional starter culture for food (vegetable or fruit) fermentation at low temperatures.
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Wang, Jiali, Chengshun Lu, Qiang Xu, Zhongyuan Li, Yajian Song, Sa Zhou, Le Guo, Tongcun Zhang, and Xuegang Luo. "Comparative Genomics Analysis Provides New Insights into High Ethanol Tolerance of Lactiplantibacillus pentosus LTJ12, a Novel Strain Isolated from Chinese Baijiu." Foods 12, no. 1 (December 22, 2022): 35. http://dx.doi.org/10.3390/foods12010035.
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Lactic acid bacteria have received a significant amount of attention due to their probiotic characteristics. The species Lactiplantibacillus plantarum and Lactiplantibacillus pentosus are genotypically closely related, and their phenotypes are so similar that they are easily confused and mistaken. In the previous study, an ethanol-resistant strain, LTJ12, isolated from the fermented grains of soy sauce aroma type baijiu in North China, was originally identified as L. plantarum through a 16S rRNA sequence analysis. Here, the genome of strain LTJ12 was further sequenced using PacBio and Illumina sequencing technology to obtain a better understanding of the metabolic pathway underlying its resistance to ethanol stress. The results showed that the genome of strain LTJ12 was composed of one circular chromosome and three circular plasmids. The genome size is 3,512,307 bp with a GC content of 46.37%, and the number of predicted coding genes is 3248. Moreover, by comparing the coding genes with the GO (Gene Ontology), COG (Cluster of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases, the functional annotation of the genome and an assessment of the metabolic pathways were performed, with the results showing that strain LTJ12 has multiple genes that may be related to alcohol metabolism and probiotic-related genes. Antibiotic resistance gene analysis showed that there were few potential safety hazards. Further, after conducting the comparative genomics analysis, it was found that strain LTJ12 is L. pentosus but not L. plantarum, but it has more functional genes than other L. pentosus strains that are mainly related to carbohydrate transport and metabolism, transcription, replication, recombination and repair, signal transduction mechanisms, defense mechanisms and cell wall/membrane/envelope biogenesis. These unique functional genes, such as gene 2754 (encodes alcohol dehydrogenase), gene 3093 (encodes gamma-D-glutamyl-meso-diaminopimelate peptidase) and some others may enhance the ethanol tolerance and alcohol metabolism of the strain. Taken together, L. pentosus LTJ12 might be a potentially safe probiotic with a high ethanol tolerance and alcohol metabolism. The findings of this study will also shed light on the accurate identification and rational application of the Lactiplantibacillus species.
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Strøman, Per, Christina C. Müller, and Kim I. Sørensen. "Heat Shock Treatment Increases the Frequency of Loss of an Erythromycin Resistance-Encoding Transposable Element from the Chromosome of Lactobacillus crispatus CHCC3692." Applied and Environmental Microbiology 69, no. 12 (December 2003): 7173–80. http://dx.doi.org/10.1128/aem.69.12.7173-7180.2003.
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ABSTRACT A 3,165-bp chromosomally integrated transposon, designatedTn3692, of the gram-positive strain Lactobacillus crispatus CHCC3692 contains an erm(B) gene conferring resistance to erythromycin at concentrations of up to 250μ g/ml. Loss of this resistance can occur spontaneously, but the rate is substantially increased by heat shock treatment. Heat shock treatment at 60°C resulted in an almost 40-fold increase in the frequency of erythromycin-sensitive cells (erythromycin MIC, 0.047μ g/ml). The phenotypic change was followed by a dramatic increase in transcription of the transposase gene and the concomitant loss of an approximately 2-kb DNA fragment carrying the erm(B) gene from the 3,165-bp erm transposon. In cells that were not subjected to heat shock, transcription of the transposase gene was not detectable. The upstream sequence of the transposase gene did not show any homology to known heat shock promoters in the gene data bank. Significant homology (>99%) was observed between the erythromycin resistance-encoding gene from L. crispatus CHCC3692 and the erm(B) genes from other gram-positive bacteria, such as Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium, and Lactobacillus reuteri, which strongly indicates a common origin of the erm(B) gene for these species. The transposed DNA element was not translocated to other parts of the genome of CHCC3692, as determining by Southern blotting, PCR analysis, and DNA sequencing. No other major aberrations were observed, as judged by colony morphology, growth performance of the strain, and pulsed-field gel electrophoresis. These observations suggest that heat shock treatment could be used as a tool for the removal of unwanted antibiotic resistance genes harbored in transposons flanked by insertion sequence elements or transposases in lactic acid bacteria used for animal and human food production.
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Salvetti, Elisa, Ilenia Campedelli, Ilaria Larini, Giada Conedera, and Sandra Torriani. "Exploring Antibiotic Resistance Diversity in Leuconostoc spp. by a Genome-Based Approach: Focus on the lsaA Gene." Microorganisms 9, no. 3 (February 26, 2021): 491. http://dx.doi.org/10.3390/microorganisms9030491.
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Leuconostoc spp. are environmental microorganisms commonly associated with fermented foods. Absence of antibiotic resistance (AR) in bacteria is a critical issue for global food safety. Herein, we updated the occurrence of AR genes in the Leuconostoc genus through in silico analyses of the genomes of 17 type strains. A total of 131 putative AR traits associated with the main clinically relevant antibiotics were detected. We found, for the first time, the lsaA gene in L. fallax ATCC 700006T and L. pseudomesenteroides NCDO 768T. Their amino acid sequences displayed high similarities (59.07% and 52.21%) with LsaA of Enterococcusfaecalis V583, involved in clindamycin (CLI) and quinupristin-dalfopristin (QUD) resistance. This trait has different distribution patterns in Leuconostoc nontype strains—i.e., L. pseudomesenteroides, L. lactis and L. falkenbergense isolates from fermented vegetables, cheeses, and starters. To better explore the role of lsaA, MIC for CLI and QUD were assessed in ATCC 700006T and NCDO 768T; both strains were resistant towards CLI, potentially linking lsaA to their resistant phenotype. Contrarily, NCDO 768T was sensitive towards QUD; however, expression of lsaA increased in presence of this antibiotic, indicating an active involvement of this trait and thus suggesting a revision of the QUD thresholds for this species.
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Rozman, Vita, Petra Mohar Lorbeg, Primož Treven, Tomaž Accetto, Sandra Janežič, Maja Rupnik, and Bojana Bogovič Matijašić. "Genomic insights into antibiotic resistance and mobilome of lactic acid bacteria and bifidobacteria." Life Science Alliance 6, no. 4 (February 13, 2023): e202201637. http://dx.doi.org/10.26508/lsa.202201637.
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Lactic acid bacteria (LAB) andBifidobacteriumsp. (bifidobacteria) can carry antimicrobial resistance genes (ARGs), yet data on resistance mechanisms in these bacteria are limited. The aim of our study was to identify the underlying genetic mechanisms of phenotypic resistance in 103 LAB and bifidobacteria using whole-genome sequencing. Sequencing data not only confirmed the presence of 36 acquired ARGs in genomes of 18 strains, but also revealed wide dissemination of intrinsic ARGs. The presence of acquired ARGs on known and novel mobile genetic elements raises the possibility of their horizontal spread. In addition, our data suggest that mutations may be a common mechanism of resistance. Several novel candidate resistance mechanisms were uncovered, providing a basis for further in vitro studies. Overall, 1,314 minimum inhibitory concentrations matched with genotypes in 92.4% of the cases; however, prediction of phenotype based on genotypic data was only partially efficient, especially with respect to aminoglycosides and chloramphenicol. Our study sheds light on resistance mechanisms and their transferability potential in LAB and bifidobacteria, which will be useful for risk assessment analysis.
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Sirichoat, Auttawit, Ana Belén Flórez, Lucía Vázquez, Pranom Buppasiri, Marutpong Panya, Viraphong Lulitanond, and Baltasar Mayo. "Antibiotic Susceptibility Profiles of Lactic Acid Bacteria from the Human Vagina and Genetic Basis of Acquired Resistances." International Journal of Molecular Sciences 21, no. 7 (April 8, 2020): 2594. http://dx.doi.org/10.3390/ijms21072594.
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Lactic acid bacteria can act as reservoirs of antibiotic resistance genes that can be ultimately transferred to pathogens. The present work reports on the minimum inhibitory concentration (MIC) of 16 antibiotics to 25 LAB isolates of five Lactobacillus and one Bifidobacterium species from the human vagina. Acquired resistances were detected to kanamycin, streptomycin, chloramphenicol, gentamicin, and ampicillin. A PCR analysis of lactobacilli failed to identify genetic determinants involved in any of these resistances. Surprisingly, a tet(W) gene was detected by PCR in two Bifidobacterium bifidum strains, although they proved to be tetracycline-susceptible. In agreement with the PCR results, no acquired genes were identified in the genome of any of the Lactobacillus spp. strains sequenced. A genome analysis of B. bifidum VA07-1AN showed an insertion of two guanines in the middle of tet(W) interrupting the open reading frame. By growing the strain in the presence of tetracycline, stable tetracycline-resistant variants were obtained. An amino acid substitution in the ribosomal protein S12 (K43R) was further identified as the most likely cause of VA07-1AN being streptomycin resistance. The results of this work expand our knowledge of the resistance profiles of vaginal LAB and provide evidence for the genetic basis of some acquired resistances.
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Qiao, Wanjin, Fulu Liu, Xing Wan, Yu Qiao, Ran Li, Zhenzhou Wu, Per Erik Joakim Saris, Haijin Xu, and Mingqiang Qiao. "Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8." Microorganisms 10, no. 1 (December 27, 2021): 47. http://dx.doi.org/10.3390/microorganisms10010047.
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Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.
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Ma, Qingqing, Zhangming Pei, Zhifeng Fang, Hongchao Wang, Jinlin Zhu, Yuan-kun Lee, Hao Zhang, Jianxin Zhao, Wenwei Lu, and Wei Chen. "Evaluation of Tetracycline Resistance and Determination of the Tentative Microbiological Cutoff Values in Lactic Acid Bacterial Species." Microorganisms 9, no. 10 (October 11, 2021): 2128. http://dx.doi.org/10.3390/microorganisms9102128.
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Lactic acid bacteria (LAB) are widely used as probiotics in the food industry owing to their beneficial effects on human health. However, numerous antibiotic resistance genes have been found in LAB strains, especially tetracycline resistance genes. Notably, the potential transferability of these genes poses safety risks. To comprehensively evaluate tetracycline resistance in LAB, we determined the tetracycline susceptibility patterns of 478 LAB strains belonging to four genera and eight species. By comparing phenotypes with genotypes based on genome-wide annotations, five tetracycline resistance genes, tet(M), tet(W/N/W), tet(L), tet(S), and tet(45), were detected in LAB. Multiple LAB strains without tetracycline resistance genes were found to be resistant to tetracycline at the currently recommended cutoff values. Thus, based on the minimum inhibitory concentrations of tetracycline for these LAB strains, the species-specific microbiological cutoff values for Lactobacillus (para)gasseri, Lactobacillus johnsonii, and Lactobacillus crispatus to tetracycline were first developed using the Turnidge, Kronvall, and eyeball methods. The cutoff values for Lactiplantibacillus plantarum were re-established and could be used to better distinguish susceptible strains from strains with acquired resistance. Finally, we verified that these five genes play a role in tetracycline resistance and found that tet(M) and tet(W/N/W) are the most widely distributed tetracycline resistance genes in LAB.
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WANG, JING, MINGYUE LI, JING WANG, MIAOMIAO LIU, KUN YANG, JIE ZHANG, MINGTAO FAN, and XINYUAN WEI. "Antibiotic Resistance of Coagulase-Negative Staphylococci and Lactic Acid Bacteria Isolated from Naturally Fermented Chinese Cured Beef." Journal of Food Protection 81, no. 12 (November 28, 2018): 2054–63. http://dx.doi.org/10.4315/0362-028x.jfp-18-195.
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ABSTRACT This study provided phenotypic and molecular analysis of the antibiotic resistance within coagulase-negative staphylococci and lactic acid bacteria isolated from naturally fermented Chinese cured beef. A total of 49 strains were isolated by selective medium and identified at the species level by 16S rRNA gene sequencing as follows: Staphylococcus carnosus (37), Lactobacillus plantarum (6), Weissella confusa (4), Lactobacillus sakei (1), and Weissella cibaria (1). All strains were typed by random amplified polymorphic DNA fingerprinting, and their antibiotic resistances profiles to 15 antibiotics were determined as the MIC by using the agar dilution method. All the tested strains were sensitive to ampicillin, and most of them were also sensitive to penicillin, gentamycin, neomycin, norfloxacin, and ciprofloxacin with low MICs. High resistance to streptomycin, vancomycin, erythromycin, roxithromycin, lincomycin, and kanamycin was widely observed, while the resistant levels to tetracycline, oxytetracycline, and chloramphenicol varied. The presence of corresponding resistance genes in resistant isolates was investigated by PCR, with the following genes detected: tet(M) gene in 9 S. carnosus strains and 1 W. confusa strain; erm(F) gene in 10 S. carnosus strains; ere(A) gene in 6 S. carnosus strains; ere(A) gene in 4 S. carnosus strains and 1 L. plantarum strain; and str(A) gene and str(B) gene in 3 S. carnosus strains. The results indicated that multiple antibiotic resistances were common in coagulase-negative staphylococci and lactic acid bacteria strains isolated from naturally fermented Chinese cured beef. Safety analysis and risk assessment should be performed for application in meat products.
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Filipic, Brankica, Natasa Golic, Branko Jovcic, Dejana Cupic-Miladinovic, Svetlana Sokovic, Dusanka Popovic, and Milan Kojic. "Resistance to antibiotics in Lacid acid bacteria - strain Lactococcus." Veterinarski glasnik 69, no. 3-4 (2015): 271–82. http://dx.doi.org/10.2298/vetgl1504271f.
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Lactic acid bacteria (LAB) are widely used in the food industry, especially in the production of fermented dairy products and meat. The most studied species among Lis Lactococcus lactis. L. lactis strains are of great importance in the production of fermented dairy products such as yogurt, butter, fresh cheese and some kind of semi-hard cheese. Although L. lactis acquired the ?Generally Regarded As Safe? (GRAS) status, many investigations indicated that lactococci may act as reservoirs of antibiotic resistance genes, which could be transferred to other bacterial species in human gastrointestinal tract includ?ing pathogens. The genome analysis of L. lactis indicated the presence of at least 40 putative drug transporter genes, and only four multidrug resistance (MDR) transporters are functionally characterized: LmrA, LmrP, LmrCD i CmbT. LmrA is the first described MDR transporter in prokaryotes. LmrCD is responsible for resistance to cholate, which is an integral part of human bile and LmrCD is important for intestinal survival of lactococci that are used as probiotics. Secondary multidrug transporter LmrP confers resistance to lincosamides, macrolides, streptogramins and tetracyclines. CmbT protein has an effect on the host cell resistance to lincomycin, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametox?azole. Since the food chain is an important way of transmitting resistance genes in human and animal population, it is of great importance to study the mechanisms of resistance in lactococci and other LAB, intended for the food industry.
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McGARVEY, JEFFERY A., THAO D. TRAN, ROBERT HNASKO, and LISA GORSKI. "Use of Phyllosphere-associated Lactic Acid Bacteria as Biocontrol Agents to Reduce Salmonella enterica Serovar Poona Growth on Cantaloupe Melons." Journal of Food Protection 82, no. 12 (January 1, 2019): 2148–53. http://dx.doi.org/10.4315/0362-028x.jfp-19-246.
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ABSTRACT Foodborne illness associated with fresh, ready-to-eat produce continues to be a significant challenge to public health. In this study, we created a phyllosphere-associated lactic acid bacteria (PLAB) library and screened it via a high-throughput in vitro fluorescent assay to identify bacteria capable of inhibiting the growth of the pathogenic bacterium Salmonella enterica. One isolate, 14B4, inhibited the growth of S. enterica by >45-fold in vitro; it was able to grow and persist on the surfaces of cantaloupe melons at both ambient (25°C) and refrigerator (5°C) temperatures. Isolate 14B4 inhibited the growth of S. enterica on the surfaces of cantaloupes by >3 log when incubated at 25°C for 24 h and by >4 log when the cantaloupes were stored at 5°C for 3 days and the temperature was shifted to 25°C for 2 days. Genomic DNA sequence analysis of isolate 14B4 revealed that it was Lactococcus lactis and that it did not contain any known antibiotic biosynthesis gene clusters, antibiotic resistance genes, or genes encoding any known virulence factors. Organic acid analysis revealed that L. lactis produces substantial amounts of lactic acid, which is likely the inhibitory substance that reduced the growth of Salmonella on the cantaloupes. HIGHLIGHTS
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Weckx, Stefan, Joke Allemeersch, Roel Van der Meulen, Gino Vrancken, Geert Huys, Peter Vandamme, Paul Van Hummelen, and Luc De Vuyst. "Development and Validation of a Species-Independent Functional Gene Microarray That Targets Lactic Acid Bacteria." Applied and Environmental Microbiology 75, no. 20 (August 14, 2009): 6488–95. http://dx.doi.org/10.1128/aem.01055-09.
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ABSTRACT During the last few years, genome-related information has become available for many microorganisms, including important food-related bacteria. Lactic acid bacteria (LAB) are important industrially in the production of fermented foods such as dairy products, sausages, sourdoughs, and vegetables. Despite their limited metabolic capacity, LAB contribute considerably to important characteristics of fermented foods, such as flavor and texture. In the present study, a species-independent functional gene microarray was developed that targets 406 genes that play key roles in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in the stress response. Also, genes linked to negative traits, such as antibiotic resistance and virulence, are represented. As LAB ecosystems contain a variety of species, there was a more global focus on these specific functional properties. Thus, an algorithm was used to design gene-specific oligonucleotides that preferably hybridize with multiple LAB species, thereby allowing controlled cross-hybridization. For proof of concept, the microarray composed of 2,269 30-mer oligonucleotides focused on LAB species that are prevalent in sourdough ecosystems. Validation hybridizations using DNA and RNA from 18 LAB strains, covering 86% of all the oligonucleotides, showed that there were wide ranges in intensity and high reproducibility between microarrays.
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Gladysheva, Irina V., Sergey V. Cherkasov, Yuriy A. Khlopko, and Andrey O. Plotnikov. "Genome Characterization and Probiotic Potential of Corynebacterium amycolatum Human Vaginal Isolates." Microorganisms 10, no. 2 (January 23, 2022): 249. http://dx.doi.org/10.3390/microorganisms10020249.
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The vaginal microbiome of healthy women contains nondiphtheria corynebacteria. The role and functions of nondiphtheria corynebacteria in the vaginal biotope are still under study. We sequenced and analysed the genomes of three vaginal C. amycolatum strains isolated from healthy women. Previous studies have shown that these strains produced metabolites that significantly increased the antagonistic activity of peroxide-producing lactic acid bacteria against pathogenic and opportunistic microorganisms and had strong antimicrobial activity against opportunistic pathogens. Analysis of the C. amycolatum genomes revealed the genes responsible for adaptation and survival in the vaginal environment, including acid and oxidative stress resistance genes. The genes responsible for the production of H2O2 and the synthesis of secondary metabolites, essential amino acids and vitamins were identified. A cluster of genes encoding the synthesis of bacteriocin was revealed in one of the annotated genomes. The obtained results allow us to consider the studied strains as potential probiotics that are capable of preventing the growth of pathogenic microorganisms and supporting colonisation resistance in the vaginal biotope.
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Li, Yixiao, Yang Song, Zhenzhou Huang, Li Mei, Mengnan Jiang, Duochun Wang, and Qiang Wei. "Screening of Staphylococcus aureus for Disinfection Evaluation and Transcriptome Analysis of High Tolerance to Chlorine-Containing Disinfectants." Microorganisms 11, no. 2 (February 14, 2023): 475. http://dx.doi.org/10.3390/microorganisms11020475.
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The nonstandard use of disinfectants can lead to the disinfectant resistance of bacteria and even increase antibiotic resistance. However, compared with the study of antibiotic resistance, studies of bacterial resistance to disinfectants are relatively few in number. In this study, we explored the standard strain screening procedure for the evaluation of disinfection efficacy. Staphylococcus aureus strains with different sources and substrates were selected from the National Pathogen Resource Center of China and screened the standard strains that could evaluate the long-term bacteriostatic effect of the chlorine-containing disinfectants through the determination of the physical properties, genome-based safety evaluation, and disinfection test evaluation. In this process, one S. aureus strain was more resistant to the long-term bacteriostasis of chlorine-containing disinfectants than the other strains. This strain and the standard strain ATCC 6538 were cultured in the medium containing a low concentration of chlorine-containing disinfectant synchronously. Then, comparative transcriptome analysis was carried out to investigate the potential mechanism of a high tolerance to chlorine-containing disinfectants. The pathway of significant differential expression is related to the oxocarboxylic acid metabolic mechanism, amino acid metabolic mechanism, and pyrimidine mechanism, which may be the molecular mechanism of S. aureus evolution to adapt to chlorine-containing disinfectants. Our study established a technical process for screening and evaluating standard strains for disinfection, which also provided a reference for studying the bacterial evolution mechanism toward chlorine tolerance.
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Pereira, Wellison Amorim, Carlos Miguel N. Mendonça, Alejandro Villasante Urquiza, Viggó Þór Marteinsson, Jean Guy LeBlanc, Paul D. Cotter, Elías Figueroa Villalobos, Jaime Romero, and Ricardo P. S. Oliveira. "Use of Probiotic Bacteria and Bacteriocins as an Alternative to Antibiotics in Aquaculture." Microorganisms 10, no. 9 (August 24, 2022): 1705. http://dx.doi.org/10.3390/microorganisms10091705.
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In addition to their use in human medicine, antimicrobials are also used in food animals and aquaculture, and their use can be categorized as therapeutic against bacterial infections. The use of antimicrobials in aquaculture may involve a broad environmental application that affects a wide variety of bacteria, promoting the spread of bacterial resistance genes. Probiotics and bacteriocins, antimicrobial peptides produced by some types of lactic acid bacteria (LAB), have been successfully tested in aquatic animals as alternatives to control bacterial infections. Supplementation might have beneficial impacts on the intestinal microbiota, immune response, development, and/or weight gain, without the issues associated with antibiotic use. Thus, probiotics and bacteriocins represent feasible alternatives to antibiotics. Here, we provide an update with respect to the relevance of aquaculture in the animal protein production sector, as well as the present and future challenges generated by outbreaks and antimicrobial resistance, while highlighting the potential role of probiotics and bacteriocins to address these challenges. In addition, we conducted data analysis using a simple linear regression model to determine whether a linear relationship exists between probiotic dose added to feed and three variables of interest selected, including specific growth rate, feed conversion ratio, and lysozyme activity.
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Syrokou, Maria K., Spiros Paramithiotis, Eleftherios H. Drosinos, Loulouda Bosnea, and Marios Mataragas. "A Comparative Genomic and Safety Assessment of Six Lactiplantibacillus plantarum subsp. argentoratensis Strains Isolated from Spontaneously Fermented Greek Wheat Sourdoughs for Potential Biotechnological Application." International Journal of Molecular Sciences 23, no. 5 (February 24, 2022): 2487. http://dx.doi.org/10.3390/ijms23052487.
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The comparative genome analysis of six Lactiplantibacillus plantarum subsp. argentoratensis strains previously isolated from spontaneously fermented Greek wheat sourdoughs is presented. Genomic attributes related to food safety have been studied according to the European Food Safety Authority (EFSA) suggestions for the use of lactic acid bacteria (LAB) in the production of foods. Bioinformatic analysis revealed a complete set of genes for maltose, sucrose, glucose, and fructose fermentation; conversion of fructose to mannitol; folate and riboflavin biosynthesis; acetoin production; conversion of citrate to oxaloacetate; and the ability to produce antimicrobial compounds (plantaricins). Pathogenic factors were absent but some antibiotic resistance genes were detected. CRISPR and cas genes were present as well as various mobile genetic elements (MGEs) such as plasmids, prophages, and insertion sequences. The production of biogenic amines by these strains was not possible due to the absence of key genes in their genome except lysine decarboxylase associated with cadaverine; however, potential degradation of these substances was identified due to the presence of a blue copper oxidase precursor and a multicopper oxidase protein family. Finally, comparative genomics and pan-genome analysis showed genetic differences between the strains (e.g., variable pln locus), and it facilitated the identification of various phenotypic and probiotic-related properties.
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Gevers, Dirk, Morten Danielsen, Geert Huys, and Jean Swings. "Molecular Characterization of tet(M) Genes in Lactobacillus Isolates from Different Types of Fermented Dry Sausage." Applied and Environmental Microbiology 69, no. 2 (February 2003): 1270–75. http://dx.doi.org/10.1128/aem.69.2.1270-1275.2003.
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ABSTRACT The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tcr) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)5-PCR DNA fingerprinting technique, the Tcr lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tcr lactic acid bacterial isolates displaying unique (GTG)5-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.
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Vecchione, James J., and Jason K. Sello. "Regulation of an Auxiliary, Antibiotic-Resistant Tryptophanyl-tRNA Synthetase Gene via Ribosome-Mediated Transcriptional Attenuation." Journal of Bacteriology 192, no. 14 (May 7, 2010): 3565–73. http://dx.doi.org/10.1128/jb.00290-10.
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ABSTRACTcis-Acting RNA elements in the leaders of bacterial mRNA often regulate gene transcription, especially in the context of amino acid metabolism. We determined that the transcription of the auxiliary, antibiotic-resistant tryptophanyl-tRNA synthetase gene (trpRS1) inStreptomyces coelicoloris regulated by a ribosome-mediated attenuator in the 5′ leader of its mRNA region. This regulatory element controls gene transcription in response to the physiological effects of indolmycin and chuangxinmycin, two antibiotics that inhibit bacterial tryptophanyl-tRNA synthetases. By mining streptomycete genome sequences, we found several orthologs oftrpRS1that share this regulatory element; we predict that they are regulated in a similar fashion. The validity of this prediction was established through the analysis of atrpRS1ortholog (SAV4725) inStreptomyces avermitilis. We conclude that thetrpRS1locus is a widely distributed and self-regulating antibiotic resistance cassette. This study provides insights into how auxiliary aminoacyl-tRNA synthetase genes are regulated in bacteria.
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Jung, Ji Young, Sang-Soo Han, Z.-Hun Kim, Myung Hoo Kim, Hye Kyeong Kang, Hyun Mi Jin, and Mi Hwa Lee. "In-Vitro Characterization of Growth Inhibition against the Gut Pathogen of Potentially Probiotic Lactic Acid Bacteria Strains Isolated from Fermented Products." Microorganisms 9, no. 10 (October 13, 2021): 2141. http://dx.doi.org/10.3390/microorganisms9102141.
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Lactic acid bacteria (LAB) are probiotic candidates that may restore the balance of microbiota populations in intestinal microbial ecosystems by controlling pathogens and thereby promoting host health. The goal of this study was to isolate potential probiotic LAB strains and characterize their antimicrobial abilities against pathogens in intestinal microbiota. Among 54 LAB strains isolated from fermented products, five LAB strains (NSMJ15, NSMJ16, NSMJ23, NSMJ42, and NFFJ04) were selected as potential probiotic candidates based on in vitro assays of acid and bile salt tolerance, cell surface hydrophobicity, adhesion to the intestinal epithelium, and antagonistic activity. Phylogenetic analysis based on 16S rRNA genes showed that they have high similarities of 99.58–100% to Lacticaseibacillus paracasei strains NSMJ15 and NFFJ04, Lentilactobacillus parabuchneri NSMJ16, Levilactobacillus brevis NSMJ23, and Schleiferilactobacillus harbinensis NSMJ42. To characterize their antimicrobial abilities against pathogens in intestinal microbiota, the impact of cell-free supernatant (CFS) treatment in 10% (v/v) fecal suspensions prepared using pooled cattle feces was investigated using in vitro batch cultures. Bacterial community analysis using rRNA amplicon sequencing for control and CFS-treated fecal samples at 8 and 16 h incubation showed the compositional change after CFS treatment for all five LAB strains. The changed compositions were similar among them, but there were few variable increases or decreases in some bacterial groups. Interestingly, as major genera that could exhibit pathogenicity and antibiotic resistance, the members of Bacillus, Escherichia, Leclercia, Morganella, and Vagococcus were decreased at 16 h in all CFS-treated samples. Species-level classification suggested that the five LAB strains are antagonistic to gut pathogens. This study showed the probiotic potential of the five selected LAB strains; in particular, their antimicrobial properties against pathogens present in the intestinal microbiota. These strains would therefore seem to play an important role in modulating the intestinal microbiome of the host.
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Boguslawska, Joanna, Joanna Zycka-Krzesinska, Andrea Wilcks, and Jacek Bardowski. "Intra- and Interspecies Conjugal Transfer of Tn916-Like Elements from Lactococcus lactis In Vitro and In Vivo." Applied and Environmental Microbiology 75, no. 19 (August 7, 2009): 6352–60. http://dx.doi.org/10.1128/aem.00470-09.
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ABSTRACT Tetracycline-resistant Lactococcus lactis strains originally isolated from Polish raw milk were analyzed for the ability to transfer their antibiotic resistance genes in vitro, using filter mating experiments, and in vivo, using germfree rats. Four of six analyzed L. lactis isolates were able to transfer tetracycline resistance determinants in vitro to L. lactis Bu2-60, at frequencies ranging from 10−5 to 10−7 transconjugants per recipient. Three of these four strains could also transfer resistance in vitro to Enterococcus faecalis JH2-2, whereas no transfer to Bacillus subtilis YBE01, Pseudomonas putida KT2442, Agrobacterium tumefaciens UBAPF2, or Escherichia coli JE2571 was observed. Rats were initially inoculated with the recipient E. faecalis strain JH2-2, and after a week, the L. lactis IBB477 and IBB487 donor strains were introduced. The first transconjugants were detected in fecal samples 3 days after introduction of the donors. A subtherapeutic concentration of tetracycline did not have any significant effect on the number of transconjugants, but transconjugants were observed earlier in animals dosed with this antibiotic. Molecular analysis of in vivo transconjugants containing the tet(M) gene showed that this gene was identical to tet(M) localized on the conjugative transposon Tn916. Primer-specific PCR confirmed that the Tn916 transposon was complete in all analyzed transconjugants and donors. This is the first study showing in vivo transfer of a Tn916-like antibiotic resistance transposon from L. lactis to E. faecalis. These data suggest that in certain cases food lactococci might be involved in the spread of antibiotic resistance genes to other lactic acid bacteria.
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Sutton, Granger, Gary B. Fogel, Bradley Abramson, Lauren Brinkac, Todd Michael, Enoch S. Liu, and Sterling Thomas. "Horizontal transfer and evolution of wall teichoic acid gene cassettes in Bacillus subtilis." F1000Research 10 (May 7, 2021): 354. http://dx.doi.org/10.12688/f1000research.51874.1.
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Background: Wall teichoic acid (WTA) genes are essential for production of cell walls in gram-positive bacteria and necessary for survival and variability in the cassette has led to recent antibiotic resistance acquisition in pathogenic bacteria. Methods: Using a pan-genome approach, we examined the evolutionary history of WTA genes in Bacillus subtilis ssp. subtilis. Results: Our analysis reveals an interesting pattern of evolution from the type-strain WTA gene cassette possibly resulting from horizontal acquisition from organisms with similar gene sequences. The WTA cassettes have a high level of variation which may be due to one or more independent horizontal transfer events during the evolution of Bacillus subtilis ssp. subtilis. This swapping of entire WTA cassettes and smaller regions within the WTA cassettes is an unusual feature in the evolution of the Bacillus subtilis genome and highlights the importance of horizontal transfer of gene cassettes through homologous recombination within B. subtilis or other bacterial species. Conclusions: Reduced sequence conservation of these WTA cassettes may indicate a modified function like the previously documented WTA ribitol/glycerol variation. An improved understanding of high-frequency recombination of gene cassettes has ramifications for synthetic biology and the use of B. subtilis in industry.
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BRASHEARS, M. M., D. JARONI, and J. TRIMBLE. "Isolation, Selection, and Characterization of Lactic Acid Bacteria for a Competitive Exclusion Product To Reduce Shedding of Escherichia coli O157:H7 in Cattle." Journal of Food Protection 66, no. 3 (March 1, 2003): 355–63. http://dx.doi.org/10.4315/0362-028x-66.3.355.
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Lactic acid bacteria (LAB) were selected on the basis of characteristics indicating that they would be good candidates for a competitive exclusion product (CEP) that would inhibit Escherichia coli O157:H7 in the intestinal tract of live cattle. Fecal samples from cattle that were culture negative for E. coli O157:H7 were collected. LAB were isolated from cattle feces by repeated plating on deMan Rogosa Sharpe agar and lactobacillus selection agar. Six hundred eighty-six pure colonies were isolated, and an agar spot test was used to test each isolate for its inhibition of a four-strain mixture of E. coli O157:H7. Three hundred fifty-five isolates (52%) showed significant inhibition. Seventy-five isolates showing maximum inhibition were screened for acid and bile tolerance. Most isolates were tolerant of acid at pH levels of 2, 4, 5, and 7 and at bile levels of 0.05, 0.15, and 0.3% (oxgall) and were subsequently identified with the API system. Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus delbreukii, Lactobacillus salivarius, Lactobacillus brevis, Lactobacillus cellobiosus, Leuconostoc spp., and Pediococcus acidilactici were the most commonly identified LAB. Nineteen strains were further tested for antibiotic resistance and inhibition of E. coli O157:H7 in manure and rumen fluid. Four of these 19 strains showed susceptibility to all of the antibiotics, 13 significantly reduced E. coli counts in manure, and 15 significantly reduced E. coli counts in rumen fluid (P < 0.05) during at least one of the sampling periods. One of the strains, M35, was selected as the best candidate for a CEP. A 16S rRNA sequence analysis of M35 revealed its close homology to Lactobacillus crispatus. The CEP developed will be used in cattle-feeding trials.
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Apostolakos, Ilias, Spiros Paramithiotis, and Marios Mataragas. "Comparative Genomic Analysis Reveals the Functional Traits and Safety Status of Lactic Acid Bacteria Retrieved from Artisanal Cheeses and Raw Sheep Milk." Foods 12, no. 3 (February 1, 2023): 599. http://dx.doi.org/10.3390/foods12030599.
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Lactic acid bacteria (LAB) are valuable for the production of fermented dairy products. We investigated the functional traits of LAB isolated from artisanal cheeses and raw sheep milk, assessed their safety status, and explored the genetic processes underlying the fermentation of carbohydrates. Lactiplantibacillus plantarum had the largest and more functional genome compared to all other LAB, while most of its protein-encoding genes had unknown functions. A key finding of our analysis was the overall absence of acquired resistance genes (RGs), virulence genes (VGs), and prophages, denoting that all LAB isolates fulfill safety criteria and can be used as starter or adjunct cultures. In this regard, the identified mobile genetic elements found in LAB, rather than enabling the integration of RGs or VGs, they likely facilitate the uptake of genes involved in beneficial functions and in the adaptation of LAB in dairy matrices. Another important finding of our study was that bacteriocins and CAZymes were abundant in LAB though each species was associated with specific genes, which in turn had different activity spectrums and identified applications. Additionally, all isolates were able to metabolize glucose, lactose, maltose, and sucrose, but Lactiplantibacillus plantarum was strongly associated with the fermentation of rhamnose, mannose, cellobiose, and trehalose whereas Levilactobacillus brevis with the utilization of arabinose and xylose. Altogether these results suggest that to fully exploit the beneficial properties of LAB, a combination of strains as food additives may be necessary. Interestingly, biological processes involved in the metabolism of carbohydrates that are not of direct interest for the dairy industry may yield valuable metabolites or activate pathways associated with beneficial health effects. Our results provide useful information for the development of new probiotic artisanal cheeses and probiotic starter cultures.
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Escobar-Sánchez, Monserrat, Ulises Carrasco-Navarro, Carmen Juárez-Castelán, Luis Lozano-Aguirre Beltrán, M. Lourdes Pérez-Chabela, and Edith Ponce-Alquicira. "Probiotic Properties and Proteomic Analysis of Pediococcus pentosaceus 1101." Foods 12, no. 1 (December 22, 2022): 46. http://dx.doi.org/10.3390/foods12010046.
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Pediococcus pentosaceus 1101 was identified by using 16S rRNA and MALDI-Biotyper. The strain was exposed to conditions that resemble the gastrointestinal tract (GT) to evaluate its probiotic properties. That included the growth kinetics, proteolytic and inhibitory activities within a pH range, survival at low pH and in the presence of bile salts, antagonistic activity, cell-adhesion properties, and antibiotic resistance. The evaluation was followed by a genomic and proteomic analysis that involved the identification of proteins obtained under control and gastrointestinal conditions. The strain showed antagonistic activity against Gram-negative and Gram-positive bacteria, high resistance to acidity (87% logarithmic survival rate, pH 2) and bile salts (99% logarithmic survival rate, 0.5% w/v), and hydrophobic binding, as well as sensitivity to penicillin, amoxicillin, and chloramphenicol. On the other hand, P. pentosaceus 1101 has a genome size of 1.76 Mbp, with 1754 coding sequences, 55 rRNAs, and 33 tRNAs. The proteomic analysis showed that 120 proteins were involved in mechanisms in which the strain senses the effects of acid and bile salts. Moreover, the strain produces at least one lytic enzyme (N-acetylmuramoyl-L-alanine amidase; 32 kDa) that may be related to the antimicrobial activity. Therefore, proteins identified might be a key factor when it comes to the adaptation of P. pentosaceus 1101 into the GT and associated with its technological and probiotic properties.
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Devirgiliis, Chiara, Doriana Coppola, Simona Barile, Bianca Colonna, and Giuditta Perozzi. "Characterization of the Tn916 Conjugative Transposon in a Food-Borne Strain of Lactobacillus paracasei." Applied and Environmental Microbiology 75, no. 12 (April 24, 2009): 3866–71. http://dx.doi.org/10.1128/aem.00589-09.
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ABSTRACT Food-borne antibiotic-resistant lactic acid bacteria have received growing attention in the past few years. We have recently identified tetracycline-resistant Lactobacillus paracasei in samples of milk and natural whey starter cultures employed in the manufacturing process of a typical Italian fermented dairy product, Mozzarella di Bufala Campana. In the present study, we have characterized at the molecular level the genetic context of tetracycline resistance determinants in these natural strains, which we have identified as tet(M). This gene was present in 21 independent isolates, whose fingerprinting profiles were distributed into eight different repetitive extragenic palindromic groups by cluster analysis. We provide evidence that the gene is associated with the broad-host, conjugative transposon Tn916, which had never before been described to occur in L. paracasei. PCR analysis of four independent isolates by use of specifically designed primer pairs detected the presence of a circular intermediate form of the transposon, carrying a coupling sequence (GGCAAA) located between the two termini of Tn916. This novel coupling sequence conferred low conjugation frequency in mating experiments with the recipient strain JH2-2 of Enterococcus faecalis.
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Baillo, Ayelen, Julio Villena, Leonardo Albarracín, Mikado Tomokiyo, Mariano Elean, Kohtaro Fukuyama, Sandra Quilodrán-Vega, Silvina Fadda, and Haruki Kitazawa. "Lactiplantibacillus plantarum Strains Modulate Intestinal Innate Immune Response and Increase Resistance to Enterotoxigenic Escherichia coli Infection." Microorganisms 11, no. 1 (December 25, 2022): 63. http://dx.doi.org/10.3390/microorganisms11010063.
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Currently, probiotic bacteria with not transferable antibiotic resistance represent a sustainable strategy for the treatment and prevention of enterotoxigenic Escherichia coli (ETEC) in farm animals. Lactiplantibacillus plantarum is among the most versatile species used in the food industry, either as starter cultures or probiotics. In the present work, the immunobiotic potential of L. plantarum CRL681 and CRL1506 was studied to evaluate their capability to improve the resistance to ETEC infection. In vitro studies using porcine intestinal epithelial (PIE) cells and in vivo experiments in mice were undertaken. Expression analysis indicated that both strains were able to trigger IL-6 and IL-8 expression in PIE cells in steady-state conditions. Furthermore, mice orally treated with these strains had significantly improved levels of IFN-γ and TNF-α in the intestine as well as enhanced activity of peritoneal macrophages. The ability of CRL681 and CRL1506 to beneficially modulate intestinal immunity was further evidenced in ETEC-challenge experiments. In vitro, the CRL1506 and CRL681 strains modulated the expression of inflammatory cytokines (IL-6) and chemokines (IL-8, CCL2, CXCL5 and CXCL9) in ETEC-stimulated PIE cells. In vivo experiments demonstrated the ability of both strains to beneficially regulate the immune response against this pathogen. Moreover, the oral treatment of mice with lactic acid bacteria (LAB) strains significantly reduced ETEC counts in jejunum and ileum and prevented the spread of the pathogen to the spleen and liver. Additionally, LAB treated-mice had improved levels of intestinal IL-10 both at steady state and after the challenge with ETEC. The protective effect against ETEC infection was not observed for the non-immunomodulatory TL2677 strain. Furthermore, the study showed that L. plantarum CRL1506 was more efficient than the CRL681 strain to modulate mucosal immunity highlighting the strain specific character of this probiotic activity. Our results suggest that the improved intestinal epithelial defenses and innate immunity induced by L. plantarum CRL1506 and CRL681 would increase the clearance of ETEC and at the same time, protect the host against detrimental inflammation. These constitute valuable features for future probiotic products able to improve the resistance to ETEC infection.
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Wang, Zhiren, Xuanyang Fan, Shuyi Wang, Shuguang Li, Yue Gao, Hui Wang, and Henan Li. "Emergence of Colistin-Resistant Acinetobacter junii in China." Antibiotics 11, no. 12 (November 24, 2022): 1693. http://dx.doi.org/10.3390/antibiotics11121693.
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The increasing number of multidrug-resistant Gram-negative bacteria presents a serious threat to global health. However, colistin-resistant Acinetobacter junii has rarely been reported. We identified a colistin-resistant A. junii clinical isolate, AJ6079, in blood. The colony of AJ6079 presented a dry phenotype, and it was difficult to form a bacterial suspension, whilst transmission electron microscopy revealed that AJ6079 possessed a thick outer membrane. The phenotypic and genomic comparisons were conducted with one colistin-susceptible A. junii, which had the same antibiotic susceptibility profile except for colistin, and had the same KL25 capsule biosynthesis locus. The AJ6079 exhibited a slower growth rate, indicating that colistin-resistant A. junii possesses a higher fitness cost. The genome of AJ6079 had a G+C content of 38.7% and contained one 3,362,966 bp circular chromosome with no plasmid or mobile colistin resistance (mcr) gene. Comparative genomic analysis revealed that the AJ6079 contained several previously unreported point mutations in colistin-resistance-related genes involving amino acid substitutions in PmrB (N5K, G147C), LpxA (I107F, H131Y), and LpxD (F20I, K263R), which might be correlated with colistin resistance in A. junii. Further research is needed for verification as the genetic background was not exactly the same between the two isolates.
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Barbosa, Joana, Helena Albano, Beatriz Silva, Maria Helena Almeida, Teresa Nogueira, and Paula Teixeira. "Characterization of a Lactiplantibacillus plantarum R23 Isolated from Arugula by Whole-Genome Sequencing and Its Bacteriocin Production Ability." International Journal of Environmental Research and Public Health 18, no. 11 (May 21, 2021): 5515. http://dx.doi.org/10.3390/ijerph18115515.
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Lactiplantibacillus plantarum is one of the lactic acid bacteria species most used as probiotics and starter cultures in food production. Bacteriocin-producers Lpb. plantarum are also promising natural food preservatives. This study aimed to characterize Lpb. plantarum R23 and its bacteriocins (R23 bacteriocins). The genome sequence of Lpb. plantarum R23 was obtained by whole-genome sequencing (WGS) in an Illumina NovaSeq platform. The activity of Lpb. plantarum R23-produced bacteriocin against two Listeria monocytogenes strains (L7946 and L7947) was evaluated, and its molecular size was determined by tricine-SDS-PAGE. No virulence or antibiotic resistance genes were detected. Four 100% identical proteins to the class II bacteriocins (Plantaricin E, Plantaricin F, Pediocin PA-1 (Pediocin AcH), and Coagulin A) were found by WGS analysis. The small (<6.5 kDa) R23 bacteriocins were stable at different pH values (ranging from 2 to 8), temperatures (between 4 and 100 °C), detergents (all, except Triton X-100 and Triton X-114 at 0.01 g/mL), and enzymes (catalase and α-amylase), did not adsorb to the producer cells, had a bacteriostatic mode of action and their maximum activity (AU/mL = 12,800) against two L. monocytogenes strains occurred between 15 and 21 h of Lpb. plantarum R23 growth. Lactiplantibacillus plantarum R23 showed to be a promising bio-preservative culture because, besides being safe, it produces a stable bacteriocin or bacteriocins (harbors genes encoding for the production of four) inhibiting pathogens as L. monocytogenes. Further studies in different food matrices are required to confirm this hypothesis and its suitability as a future starter culture.
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Lee, Ju-Hoon, and Daniel J. O’Sullivan. "Sequence Analysis of Two Cryptic Plasmids from Bifidobacterium longum DJO10A and Construction of a Shuttle Cloning Vector." Applied and Environmental Microbiology 72, no. 1 (January 2006): 527–35. http://dx.doi.org/10.1128/aem.72.1.527-535.2006.
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ABSTRACT Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.
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Rodríguez, Javier, Lucía Vázquez, Ana Belén Flórez, and Baltasar Mayo. "Phenotype testing, genome analysis, and metabolic interactions of three lactic acid bacteria strains existing as a consortium in a naturally fermented milk." Frontiers in Microbiology 13 (September 23, 2022). http://dx.doi.org/10.3389/fmicb.2022.1000683.
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This work reports the characterization of three lactic acid bacteria (LAB) strains –Lactococcus lactis LA1, Lactococcus cremoris LA10, and Lactiplantibacillus plantarum LA30– existing as a stable consortium in a backslopping-inoculated, naturally fermented milk (NFM). This study aimed at uncovering the biochemical and genetic basis of the stability of the consortium and the cooperativity among the strains during milk fermentation. All three strains were subjected to phenotyping, covering the utilization of carbohydrates, enzyme activity, and antibiotic resistance. The strains were grown in milk individually, as well as in all possible combinations, and the resulting fermented product was analyzed for sugars, organic acids, and volatile compounds. Finally, the genomes of the three strains were sequenced and analyzed for genes associated with technological and safety properties. As expected, wide phenotypic diversity was seen between the strains. Lactococcus cremoris LA10 was the only strain to reach high cell densities and coagulate milk alone after incubation at 22°C for 24 h; congruently, it possessed a gene coding for a PrtP type II caseinolytic protease. Compared to any other fermentation, acetaldehyde concentrations were greater by a factor of six when all three strains grew together in milk, suggesting that its production might be the result of an interaction between them. Lactococcus lactis LA1, which carried a plasmid-encoded citQRP operon, was able to utilize milk citrate producing diacetyl and acetoin. No genes encoding virulence traits or pathogenicity factors were identified in any of the strains, and none produced biogenic amines from amino acid precursors, suggesting them to be safe. Lactiplantibacillus plantarum LA30 was susceptible to tetracycline, although it harbors a disrupted antibiotic resistance gene belonging to the tetM/tetW/tetO/tetS family. All three strains contained large numbers of pseudogenes, suggesting that they are well adapted (“domesticated”) to the milk environment. The consortium as a whole or its individual strains might have a use as a starter or as starter components for dairy fermentations. The study of simple consortia, such as that existing in this NFM, can help reveal how microorganisms interact with one another, and what influence they may have on the sensorial properties of fermented products.
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Shani, Noam, Simone Oberhaensli, and Emmanuelle Arias-Roth. "Antibiotic susceptibility profiles of Pediococcus pentosaceus from various origins and their implications for the safety assessment of strains with food-technology applications." Journal of Food Protection, December 15, 2020. http://dx.doi.org/10.4315/jfp-20-363.
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In the fight against the spread of antibiotic resistance (ABR), authorities usually require that strains “intentionally added into the food chain” be tested for their antibiotic susceptibility. This applies to strains used in starter or adjunct cultures for the production of fermented foods, such as many strains of Pediococcus pentosaceus . The European Food Safety Authority (EFSA) recommends testing strains for their antibiotic susceptibility based on both genomic and phenotypic approaches. Furthermore, it proposes a set of antibiotics to assess, as well as a list of microbiological cutoffs (MCs) allowing classifying lactic acid bacteria as susceptible or resistant. Accurate MCs are essential, on the one hand, to avoid false negative strains, which may carry ABR genes and remain unnoticed, and on the other, to avoid false positive strains, which may be discarded while screening potential candidates for food-technology applications. Due to relatively scarce data, MCs have been defined for the whole Pediococcus genus, although differences between different species should be expected. In this study, we investigated the antibiotic susceptibility of thirty-five strains of P. pentosaceus isolated from various matrices in the last seventy years. Minimal inhibitory concentrations (MICs) were determined using a standard protocol, and MIC distributions were established. Phenotypic analyses were complemented with genome sequencing and by seeking known antibiotic resistance genes. The genomes of all the strains were free of known antibiotic resistance genes, but most displayed MICs above the currently defined MCs for chloramphenicol, and all showed excessive MICs for tetracycline. Based on the distributions, we calculated and proposed new MCs for chloramphenicol (16 instead of 4 mg/L) and tetracycline (256 instead of 8 mg/L).
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Morovic, Wesley, Paige Roos, Bryan Zabel, Claudio Hidalgo-Cantabrana, Anthony Kiefer, and Rodolphe Barrangou. "Transcriptional and Functional Analysis of Bifidobacterium animalis subsp. lactis Exposure to Tetracycline." Applied and Environmental Microbiology 84, no. 23 (September 28, 2018). http://dx.doi.org/10.1128/aem.01999-18.
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ABSTRACT Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes show various inhibition levels, suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019 and employed RNA sequencing to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to number of viable genome copies from droplet digital PCR and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multidrug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was upregulated only during subinhibitory tetracycline concentrations, while two novel tetracycline resistance genes were upregulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated, while genes for complex carbohydrate utilization, protein metabolism, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems were downregulated. These results provide high-throughput means for assessing antibiotic resistances of two highly related probiotic strains and determine the genetic network that contributes to the global tetracycline response. IMPORTANCE Bifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.
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Yuan, Shenglei, Yundan Wang, Fangqing Zhao, and Le Kang. "Complete Genome Sequence of Weissella confusa LM1 and Comparative Genomic Analysis." Frontiers in Microbiology 12 (September 28, 2021). http://dx.doi.org/10.3389/fmicb.2021.749218.
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The genus Weissella is attracting an increasing amount of attention because of its multiple functions and probiotic potential. In particular, the species Weissella confusa is known to have great potential in industrial applications and exhibits numerous biological functions. However, the knowledge on this bacterium in insects is not investigated. Here, we isolated and identified W. confusa as the dominant lactic acid bacteria in the gut of the migratory locust. We named this strain W. confusa LM1, which is the first genome of an insect-derived W. confusa strain with one complete chromosome and one complete plasmid. Among all W. confusa strains, W. confusa LM1 had the largest genome. Its genome was the closest to that of W. confusa 1001271B_151109_G12, a strain from human feces. Our results provided accurate evolutionary relationships of known Weissella species and W. confusa strains. Based on genomic analysis, the pan-genome of W. confusa is in an open state. Most strains of W. confusa had the unique genes, indicating that these strains can adapt to different ecological niches and organisms. However, the variation of strain-specific genes did represent significant correlations with their hosts and ecological niches. These strains were predicted to have low potential to produce secondary metabolites. Furthermore, no antibiotic resistance genes were identified. At the same time, virulence factors associated with toxin production and secretion system were not found, indicating that W. confusa strains were not sufficient to perform virulence. Our study facilitated the discovery of the functions of W. confusa LM1 in locust biology and their potential application to locust management.
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Surachat, Komwit, Duangporn Kantachote, Monwadee Wonglapsuwan, Arnon Chukamnerd, Panchalika Deachamag, Pimonsri Mittraparp-arthorn, and Kongpop Jeenkeawpiam. "Complete Genome Sequence of Weissella cibaria NH9449 and Comprehensive Comparative-Genomic Analysis: Genomic Diversity and Versatility Trait Revealed." Frontiers in Microbiology 13 (May 19, 2022). http://dx.doi.org/10.3389/fmicb.2022.826683.
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Lactic acid bacteria (LAB) in the genus Weissella spp. contain traits in their genome that confer versatility. In particular, Weissella cibaria encodes several beneficial genes that are useful in biotechnological applications. The complete genome of W. cibaria NH9449 was sequenced and an in silico comparative analysis was performed to gain insight into the genomic diversity among members of the genus Weissella. A total of 219 Weissella genomes were used in a bioinformatics analysis of pan-genomes, phylogenetics, self-defense mechanisms, virulence factors, antimicrobial resistance, and carbohydrate-active enzymes. These investigations showed that the strain NH9449 encodes several restriction-modification-related genes and a CRISPR-Cas region in its genome. The identification of carbohydrate-active enzyme-encoding genes indicated that this strain could be beneficial in biotechnological applications. The comparative genomic analysis reveals the very high genomic diversity in this genus, and some marked differences in genetic variation and genes among Weissella species. The calculated average amino acid identity (AAI) and phylogenetic analysis of core and accessory genes shows the possible existence of three new species in this genus. These new genomic insights into Weissella species and their biological functions could be useful in the food industry and other applications.
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Zhang, Shenwei, Jee-Hwan Oh, Laura M. Alexander, Mustafa Özçam, and Jan-Peter van Pijkeren. "D-Alanyl-D-Alanine Ligase as a Broad-Host-Range Counterselection Marker in Vancomycin-Resistant Lactic Acid Bacteria." Journal of Bacteriology 200, no. 13 (April 23, 2018). http://dx.doi.org/10.1128/jb.00607-17.
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ABSTRACTThe peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending ind-alanyl-d-lactate and binds with high affinity to peptidoglycan ending ind-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a natived-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genusLactobacillus. Finally, our system represents the first “plug and play” counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCEThe genusLactobacilluscontains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selectedLactobacillusspecies. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)—an essential enzyme involved in peptidoglycan synthesis—increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance inLactobacillus; 81% ofLactobacillusspecies are intrinsically resistant to vancomycin, which makes our tool broadly applicable.
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Giddings, Lesley-Ann, Kevin Kunstman, Bouziane Moumen, Laurent Asiama, Stefan Green, Vincent Delafont, Matthew Brockley, and Ascel Samba-Louaka. "Isolation and Genome Analysis of an Amoeba-Associated Bacterium Dyella terrae Strain Ely Copper Mine From Acid Rock Drainage in Vermont, United States." Frontiers in Microbiology 13 (May 23, 2022). http://dx.doi.org/10.3389/fmicb.2022.856908.
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Protozoa play important roles in microbial communities, regulating populations via predation and contributing to nutrient cycling. While amoebae have been identified in acid rock drainage (ARD) systems, our understanding of their symbioses in these extreme environments is limited. Here, we report the first isolation of the amoeba Stemonitis from an ARD environment as well as the genome sequence and annotation of an associated bacterium, Dyella terrae strain Ely Copper Mine, from Ely Brook at the Ely Copper Mine Superfund site in Vershire, Vermont, United States. Fluorescent in situ hybridization analysis showed this bacterium colonizing cells of Stemonitis sp. in addition to being outside of amoebal cells. This amoeba-resistant bacterium is Gram-negative with a genome size of 5.36 Mbp and GC content of 62.5%. The genome of the D. terrae strain Ely Copper Mine encodes de novo biosynthetic pathways for amino acids, carbohydrates, nucleic acids, and lipids. Genes involved in nitrate (1) and sulfate (7) reduction, metal (229) and antibiotic resistance (37), and secondary metabolite production (6) were identified. Notably, 26 hydrolases were identified by RAST as well as other biomass degradation genes, suggesting roles in carbon and energy cycling within the microbial community. The genome also contains type IV secretion system genes involved in amoebae resistance, revealing how this bacterium likely survives predation from Stemonitis sp. This genome analysis and the association of D. terrae strain Ely Copper Mine with Stemonitis sp. provide insight into the functional roles of amoebae and bacteria within ARD environments.
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Jeon, Soomin, Hyaekang Kim, Youngseok Choi, Seoae Cho, Minseok Seo, and Heebal Kim. "Complete Genome Sequence of the Newly Developed Lactobacillus acidophilus Strain With Improved Thermal Adaptability." Frontiers in Microbiology 12 (September 24, 2021). http://dx.doi.org/10.3389/fmicb.2021.697351.
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Lactobacillus acidophilus (L. acidophilus) is a representative probiotic and is widely used in many industrial products for its beneficial effects on human and animal health. This bacterium is exposed to harsh environments such as high temperatures for manufacturing industrial products, but cell yield under high temperatures is relatively low. To resolve this issue, we developed a new L. acidophilus strain with improved heat resistance while retaining the existing beneficial properties through the adaptive laboratory evolution (ALE) method. The newly developed strain, L. acidophilus EG008, has improved the existing limit of thermal resistance from 65°C to 75°C. Furthermore, we performed whole-genome sequencing and comparative genome analysis of wild-type and EG008 strains to unravel the molecular mechanism of improved heat resistance. Interestingly, only two single-nucleotide polymorphisms (SNPs) were different compared to the L. acidophilus wild-type. We identified that one of these SNPs is a non-synonymous SNP capable of altering the structure of MurD protein through the 435th amino acid change from serine to threonine. We believe that these results will directly contribute to any industrial field where L. acidophilus is applied. In addition, these results make a step forward in understanding the molecular mechanisms of lactic acid bacteria evolution under extreme conditions.
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Anumudu, Christian K., Osaze Omoregbe, Abarasi Hart, Taghi Miri, Ukpai A. Eze, and Helen Onyeaka. "Applications of Bacteriocins of Lactic Acid Bacteria in Biotechnology and Food Preservation: A Bibliometric Review." Open Microbiology Journal 16, no. 1 (August 23, 2022). http://dx.doi.org/10.2174/18742858-v16-e2206300.
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Introduction: Due to the growing prevalence of antibiotic resistance in microorganisms and the demand for safe food, there is increasing interest in using natural bioproducts such as the antimicrobial peptides bacteriocins to extend the shelf-life of foods. This is because of their spectrum of activity, ease of synthesis and applicability. This study reports on the global trends in lactic acid bacteria (LAB) bacteriocins based research publications in the Web of Science core collections within the last 20 years (2000-2019), with specific focus to their applications in biotechnology and food science. Methods: Data analysis was undertaken using VOSviewer and HistCite software to evaluate relationships between articles and visualise research linkages amongst authors, institutions and countries. Results: In the 20 years under review, a total of 1741 bacteriocin related articles were published, with the most cited publication examining the anti-infective activity of Lactobacillus salivarius. The highest research output was recorded by the United States, followed by Spain and China. However, Europe as a continent had the highest research output with a higher inter-institution collaboration network and stronger food safety legislations. Discussion: The bibliometric analysis gave insights into the research areas, cooperation network of authors, co-citation maps and co-occurrence of keywords utilized in the research field and indicates that bacteriocin-based research is highly multidisciplinary with a global reach. Conclusion: Key focus is on the control of foodborne disease pathogens, search for new producer organisms and approaches to improve bacteriocin yield and application. This class of antimicrobial peptides has the potential to replace chemical food preservatives in the future.
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Smith, Derek D. N., Ashley N. Williams, Jennifer N. Verrett, Nathanael T. Bergbusch, Viola Manning, Kristin Trippe, and John Stavrinides. "Resistance to Two Vinylglycine Antibiotic Analogs Is Conferred by Inactivation of Two Separate Amino Acid Transporters in Erwinia amylovora." Journal of Bacteriology 201, no. 9 (February 11, 2019). http://dx.doi.org/10.1128/jb.00658-18.
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ABSTRACT Erwinia amylovora is the causal agent of fire blight of apple and pear trees. Several bacteria have been shown to produce antibiotics that antagonize E. amylovora, including pantocins, herbicolins, dapdiamides, and the vinylglycines, 4-formylaminooxyvinylglycine (FVG) and 4-aminoethoxyvinylglycine (AVG). Pantoea ananatis BRT175 was previously shown to exhibit antibiotic activity against E. amylovora via the production of Pantoea natural product 1 (PNP-1), later shown to be FVG; however, exposure of E. amylovora to FVG results in spontaneously resistant mutants. To identify the mechanism of resistance, we used genome variant analysis on spontaneous FVG-resistant mutants of E. amylovora and identified null mutations in the l-asparagine permease gene ansP. Heterologous expression of ansP in normally resistant Escherichia coli was sufficient to impart FVG susceptibility, suggesting that FVG is imported through this permease. Because FVG and AVG are structurally similar, we hypothesized that resistance to AVG would also be conferred through inactivation of ansP; however, ansP mutants were not resistant to AVG. We found that spontaneously resistant Ea321 mutants also arise in the presence of AVG, with whole-genome variant analysis revealing that resistance was due to inactivation of the arginine ABC transporter permease subunit gene artQ. Heterologous expression of the predicted lysE-like transporter encoded within the Pantoea ananatis BRT175 FVG biosynthetic cluster, which is likely responsible for antibiotic export, was sufficient to confer resistance to both FVG and AVG. This work highlights the important roles of amino acid transporters in antibiotic import into bacteria and the potential utility of antimicrobial amino acid analogs as antibiotics. IMPORTANCE The related antibiotics formylaminooxyvinylglycine (FVG) and aminoethoxyvinylglycine (AVG) have been shown to have activity against the fire blight pathogen Erwinia amylovora; however, E. amylovora can develop spontaneous resistance to these antibiotics. By comparing the genomes of mutants to those of the wild type, we found that inactivation of the l-asparagine transporter conferred resistance to FVG, while inactivation of the l-arginine transporter conferred resistance to AVG. We also show that the transporter encoded by the FVG biosynthetic cluster can confer resistance to both FVG and AVG. Our work indicates the important role that amino acid transporters play in the import of antibiotics and highlights the possible utility in designer antibiotics that enter the bacterial cell through amino acid transporters.
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Aziz, Ghazal, Muhammad Tariq, and Arsalan Haseeb Zaidi. "Mining indigenous honeybee gut microbiota for Lactobacillus with probiotic potential." Microbiology 167, no. 3 (March 1, 2021). http://dx.doi.org/10.1099/mic.0.001032.
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The present study was done to explore the diversity of lactic acid bacteria (LAB) associated with the gastrointestinal tract (GIT) of honeybee species endemic to northeastern Pakistan. Healthy worker bees belonging to Apis mellifera, A. dorsata, A. cerana and A. florea were collected from hives and the surroundings of a major apiary in the region. The 16S rRNA amplicon sequencing revealed a microbial community in A. florea that was distinct from the others in having an abundance of Lactobacillus and Bifidobacteria. However, this was not reflected in the culturable bacteria obtained from these species. The isolates were characterized for safety parameters, and 20 LAB strains deemed safe were evaluated for resistance to human GIT stresses like acid and bile, adhesion and adhesiveness, and anti-pathogenicity. The five most robust strains, Enterococcus saigonensis NPL780a, Lactobacillus rapi NPL782a, Lactobacillus kunkeei NPL783a, and NPL784, and Lactobacillus paracasei NPL783b, were identified through normalized Pearson (n) principal components analysis (PCA). These strains were checked for inhibition of human pathogens, antibiotic resistance, osmotic tolerance, metabolic and enzymatic functions, and carbohydrate utilization, along with antioxidative and cholesterol-removing potential. The findings suggest at least three strains (NPL 783a, 784 and 782a) as candidates for further in vitro and in vivo investigations of their potential health benefits and application as novel probiotic adjuncts.
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He, Yang, Xuan Liu, Yuanyang Dong, Jiaqi Lei, Koichi Ito, and Bingkun Zhang. "Enterococcus faecium PNC01 isolated from the intestinal mucosa of chicken as an alternative for antibiotics to reduce feed conversion rate in broiler chickens." Microbial Cell Factories 20, no. 1 (June 28, 2021). http://dx.doi.org/10.1186/s12934-021-01609-z.
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Abstract Background The development and utilization of probiotics had many environmental benefits for replacing antibiotics in animal production. Bacteria in the intestinal mucosa have better adhesion to the host intestinal epithelial cells compared to bacteria in the intestinal contents. In this study, lactic acid bacteria were isolated from the intestinal mucosa of broiler chickens and investigated as the substitution to antibiotic in broiler production. Results In addition to acid resistance, high temperature resistance, antimicrobial sensitivity tests, and intestinal epithelial cell adhesion, Enterococcus faecium PNC01 (E. faecium PNC01) was showed to be non-cytotoxic to epithelial cells. Draft genome sequence of E. faecium PNC01 predicted that it synthesized bacteriocin to perform probiotic functions and bacteriocin activity assay showed it inhibited Salmonella typhimurium from invading intestinal epithelial cells. Diet supplemented with E. faecium PNC01 increased the ileal villus height and crypt depth in broiler chickens, reduced the relative length of the cecum at day 21, and reduced the relative length of jejunum and ileum at day 42. Diet supplemented with E. faecium PNC01 increased the relative abundance of Firmicutes and Lactobacillus, decreased the relative abundance of Bacteroides in the cecal microbiota. Conclusion E. faecium PNC01 replaced antibiotics to reduce the feed conversion rate. Furthermore, E. faecium PNC01 improved intestinal morphology and altered the composition of microbiota in the cecum to reduce feed conversion rate. Thus, it can be used as an alternative for antibiotics in broiler production to avoid the adverse impact of antibiotics by altering the gut microbiota. Graphic Abstract
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Li, Ouyang, Huijian Zhang, Wenjing Wang, Yuxin Liang, Wenbi Chen, Ahmad Ud Din, Li Li, and Yingshun Zhou. "Complete genome sequence and probiotic properties of Lactococcus petauri LZys1 isolated from healthy human gut." Journal of Medical Microbiology 70, no. 8 (August 16, 2021). http://dx.doi.org/10.1099/jmm.0.001397.
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Introduction. Lactococcus petauri LZys1 ( L. petauri LZys1) is a type of lactic acid bacteria (LAB), which was initially isolated from healthy human gut. Hypothesis/Gap Statement. It was previously anticipated that L. petauri LZys1 has potential characteristics of probiotic properties. The genetic structure and the regulation functions of L. petauri LZys1 need to be better revealed. Aim. The aim of this study was to detect the probiotic properties L. petauri LZys1 and to reveal the genome information related to its genetic adaptation and probiotic profiles. Methodology. Multiple in vitro experiments were carried out to evaluate its lactic acid-producing ability, resistance to pathogenic bacterial strains, auto-aggregation and co-aggregation ability, and so on. Additionally, complete genome sequencing, gene annotation, and probiotic associated gene analysis were performed. Results. The complete genome of L. petauri LZys1 comprised of 1 985 765 bp, with a DNA G+C content of 38.07 %, containing 50 tRNA, seven rRNA, and four sRNA. A total of 1931 genes were classified into six functional categories by Kyoto Encyclopaedia of Genes and Genomes (KEGG) database. The neighbour-joining phylogeny tree based on the whole genome of L. petauri LZys1 and other probiotics demonstrated that L. petauri LZys1 has a significant similarity to Lactococcus garvieae . The functional genes were detected to expound the molecular mechanism and biochemical processes of its potential probiotic properties, such as atpB gene. Conclusion. All the results described in this study, together with relevant information previously reported, made L. prtauri LZys1 a very interesting potential strain to be considered as a prominent candidate for probiotic use.
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Sałański, Przemysław, Magdalena Kowalczyk, Jacek K. Bardowski, and Agnieszka K. Szczepankowska. "Health-Promoting Nature of Lactococcus lactis IBB109 and Lactococcus lactis IBB417 Strains Exhibiting Proliferation Inhibition and Stimulation of Interleukin-18 Expression in Colorectal Cancer Cells." Frontiers in Microbiology 13 (May 25, 2022). http://dx.doi.org/10.3389/fmicb.2022.822912.
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Lactic acid bacteria (LAB) are Gram-positive bacteria which are considered for use as adjuvant therapeutics in management of various disease ailments, including obesity, irritable bowel syndrome, lactose intolerance and cancer. To investigate the possible use of Lactococcus lactis strains from our collection in treatment of gastrointestinal cancer, we tested them for the ability to arrest proliferation of human colorectal adenocarcinoma cells (Caco-2). Results of the BrdU assay showed that the anti-proliferative activity of L. lactis cells is strain-specific. We found that particularly, two strains, L. lactis IBB109 and L. lactis IBB417, exhibited the most potent inhibitory effect. Moreover, both strains triggered interleukin 18 gene expression, normally inhibited in Caco-2 (cancer) cells. To examine the probiotic potential of the two strains, we tested them for bile salts and acid tolerance, as well as adhesion properties. Both isolates exhibited probiotic potential—they survived in the presence of 0.3% bile salts and tolerated exposure to low pH and osmotic stress. Notably, we found that L. lactis IBB417 displayed better adherence to mucus and Caco-2 cells than L. lactis IBB109. Additionally, by microdilution tests we confirmed that both strains are sensitive to all nine antibiotics of human and veterinary importance listed by the European Food Safety Authority. Finally, by in silico investigations of whole genome sequencing data, we revealed the genetic features of L. lactis IBB109 and L. lactis IBB417 that can be associated with functional (e.g., adhesion and carbohydrate metabolic genes) and safety (e.g., virulence and antibiotic resistance) aspects of the strains, confirming their health-promoting potential.
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Caballero Gómez, Natacha, Julia Manetsberger, Nabil Benomar, Sonia Castillo Gutiérrez, and Hikmate Abriouel. "Antibacterial and antibiofilm effects of essential oil components, EDTA and HLE disinfectant solution on Enterococcus, Pseudomonas and Staphylococcus sp. multiresistant strains isolated along the meat production chain." Frontiers in Microbiology 13 (October 10, 2022). http://dx.doi.org/10.3389/fmicb.2022.1014169.
Full textAbstract:
The spread of multidrug resistant (MDR) bacteria and resistance genes along the food chain and the environment has become a global, but silent pandemic. To face this challenge, it is of outmost importance to develop efficient strategies to reduce potential contamination by these agents. In the present study, 30 strains of Enterococcus sp., Staphylococcus sp. and Pseudomonas sp. isolated from various surfaces throughout the meat production chain in a goat and lamb slaughterhouse were characterized as MDR bacteria harboring several antibiotic resistance genes (ARGs). The antimicrobial efficacy of natural essential oil components “EOCs” (carvacrol “CA,” cinnamaldehyde “CIN,” eugenol “EU,” geraniol “GE,” limonene “LI” and thymol “TH”), HLE disinfectant solution (3–6% H2O2; 2.2–4.4% lactic acid and 12.5–25 mM EDTA in water) and EDTA was tested against these MDR bacteria. Results showed that Minimum Inhibitory Concentrations (MIC) were compound and strain dependent. In addition, the synergistic effect of these antimicrobials was evaluated at 1/2 MIC. Here our study showed particularly promising results regarding the inhibitory effect at sub-inhibitory concentrations, which were confirmed by the analysis of bacterial growth dynamics over 72 h. Furthermore, the inhibitory effect of EOCs, HLE disinfectant solution and EDTA or their combinations was studied in developing and established biofilms of MDR bacteria obtaining variable results depending on the morphological structure of the tested strain and the phenolic character of the EOCs. Importantly, the combination of EOCs with HLE or EDTA showed particularly positive results given the effective inhibition of biofilm formation. Moreover, the synergistic combinations of EU and HLE/EDTA, TH, CA, GE, LI or CIN + EDTA/HLE caused log reductions in established biofilms of several strains (1–6 log10 CFU) depending on the species and the combination used, with Pseudomonas sp. strains being the most susceptible. Given these results, we propose novel antimicrobial formulations based on the combination of sub-inhibitory concentrations of EOCs and HLE or EDTA as a highly promising alternative to currently used approaches. This novel strategy notably shows great potential to efficiently decrease the emergence and spread of MDR bacteria and ARGs in the food chain and the environment, thus supporting the decrease of resistomes and pathogenesis in clinical and industrial areas while preserving the antibiotic therapeutic action.