Journal articles on the topic 'Bacterial typing techniques'

Until recently, the relatedness of bacterial isolates has been determined solely by testing for one or several phenotypic markers, using methods such as serotyping, phage typing, biotyping, antibiotic susceptibility testing, and bacteriocin typing. However, there are problems in the use of many of these phenotype-based methods. For example, phage and bacteriocin typing systems are not available for all bacterial species and serotyping can be labor-intensive and costly. In addition, phenotypic markers may not be stably expressed under certain environmental or culture conditions. In contrast, some of the newer molecular typing methods involving the analysis of DNA offer many advantages over traditional techniques. One of the more important advantages is that since DNA can always be extracted from bacteria, all bacteria should be typeable. Another is that the discriminatory power of DNA-based methods is greater than that of phenotypic procedures. This review focuses on the basics of molecular typing along with the advantages and disadvantages of several of the newer genotypic typing techniques. This includes methods such as plasmid typing, pulsed-field gel electrophoresis, ribotyping and its variations, and polymerase chain reaction-based methods such as random amplified polymorphic DNA analysis. Molecular typing of microorganisms has made great strides in the last decade, and many food microbiology laboratories have become more knowledgeable and better equipped to carry out these new molecular techniques. Molecular typing procedures can be broadly defined as methods used to differentiate bacteria, based on the composition of biological molecules such as proteins, fatty acids, carbohydrates, etc., or nucleic acids. The latter can also be more specifically defined as genotyping, and is the subject of this review.

Thirty-eight bovine mammary Staphylococcus aureusisolates from diverse clinical, temporal, and geographical origins were genotyped by pulsed-field gel electrophoresis (PFGE) afterSmaI digestion of prokaryotic DNA and by means of binary typing using 15 strain-specific DNA probes. Seven pulsed-field types and four subtypes were identified, as were 16 binary types. Concordant delineation of genetic relatedness was documented by both techniques, yet based on practical and epidemiological considerations, binary typing was the preferable method. Genotypes of bovine isolates were compared to 55 previously characterized human S. aureusisolates through cluster analysis of binary types. Genetic clusters containing strains of both human and bovine origin were found, but bacterial genotypes were predominantly associated with a single host species. Binary typing proved an excellent tool for comparison ofS. aureus strains, including methicillin-resistant S. aureus, derived from different host species and from different databases. For 28 bovine S. aureus isolates, detailed clinical observations in vivo were compared to strain typing results in vitro. Associations were found between distinct genotypes and severity of disease, suggesting strain-specific bacterial virulence. Circumstantial evidence furthermore supports strain-specific routes of bacterial dissemination. We conclude that PFGE and binary typing can be successfully applied for genetic analysis of S. aureusisolates from bovine mammary secretions. Binary typing in particular is a robust and simple method and promises to become a powerful tool for strain characterization, for resolution of clonal relationships of bacteria within and between host species, and for identification of sources and transmission routes of bovine S. aureus.

Abstract An increasing prevalence of hospital acquired infections and foodborne illnesses caused by pathogenic and multidrug-resistant bacteria has stimulated a pressing need for benchtop computational techniques to rapidly and accurately classify bacteria from genomic sequence data, and based on that, to trace the source of infection. BacWGSTdb (http://bacdb.org/BacWGSTdb) is a free publicly accessible database we have developed for bacterial whole-genome sequence typing and source tracking. This database incorporates extensive resources for bacterial genome sequencing data and the corresponding metadata, combined with specialized bioinformatics tools that enable the systematic characterization of the bacterial isolates recovered from infections. Here, we present BacWGSTdb 2.0, which encompasses several major updates, including (i) the integration of the core genome multi-locus sequence typing (cgMLST) approach, which is highly scalable and appropriate for typing isolates belonging to different lineages; (ii) the addition of a multiple genome analysis module that can process dozens of user uploaded sequences in a batch mode; (iii) a new source tracking module for comparing user uploaded plasmid sequences to those deposited in the public databases; (iv) the number of species encompassed in BacWGSTdb 2.0 has increased from 9 to 20, which represents bacterial pathogens of medical importance; (v) a newly designed, user-friendly interface and a set of visualization tools for providing a convenient platform for users are also included. Overall, the updated BacWGSTdb 2.0 bears great utility in continuing to provide users, including epidemiologists, clinicians and bench scientists, with a one-stop solution to bacterial genome sequence analysis.

Background: The identification and molecular characterisation of bacteria associated with neonatal necrotising enterocolitis (NNE) and sepsis is very important in clarifying the role such bacteria play in the development of this disease. Methods: The present multicentre study was aimed at characterising bacteria isolated from haemocultures obtained from 20 neonates suffering NNE by using traditional and molecular methodologies (16 S rRNA subunit sequencing and multilocus sequence typing - MLST). Results: NNE incidence in hospitals in Bogotá was also estimated, finding rates similar to and higher than those reported in the literature (1 to 3 cases of NNE per 1,000 live-births). Staphylococcus epidermidis ST2, ST81, and ST126 sequence types were identified by using these two molecular techniques; the Escherichia coli ST394 sequence type was also identified. Species could not be identified for the Pantoea agglomerans isolate due to the high degree of intra-species identity. Interestingly, the bacterial isolates from the two neonates who died were classified in the same sequence type (i.e. S. epidermidis ST81), even though they came from different hospitals. Conclusion: Such molecular techniques allow characterising bacterial pathogen populations occurring in one or more hospitals in a particular city or determined geographical area and support taking more specific preventative measures directed against such particular clones. Key words: Necrotising enterocolitis (NNE), sepsis, molecular characterisation, multilocus sequence typing (MLST).

The epidemiology of bacterial infection is investigated by the use of identification procedures at a sub-species level by such techniques as serotyping, phage typing, various antigen recognition tests, plasmid profiling and DNA probes. Fungal epidemiology has tended to lag behind in the use of this technology, particularly with the filamentous fungi, though several techniques have been developed for yeasts and especially forCandida albicans. This review will briefly describe the application of these methods to outbreaks ofC. albicansinfection, describe the limited methods available for the investigation of filamentous fungal infection and indicate the necessity for increased research in this area.

Microbiological monitoring of consumer products and the efficiency of early warning systems and outbreak investigations depend on the rapid identification and strain characterisation of pathogens posing risks to the health and safety of consumers. This study evaluates the potential of three rapid analytical techniques for identification and subtyping of bacterial isolates obtained from a liquid hand soap product, which has been recalled and reported through the EU RAPEX system due to its severe bacterial contamination. Ten isolates recovered from two bottles of the product were identified as Klebsiella oxytoca and subtyped using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF MS), near-infrared Fourier transform (NIR FT) Raman spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Comparison of the classification results obtained by these phenotype-based techniques with outcomes of the DNA-based methods pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis of whole-genome sequencing (WGS) data revealed a high level of concordance. In conclusion, a set of analytical techniques might be useful for rapid, reliable and cost-effective microbial typing to ensure safe consumer products and allow source tracking.

Campylobacter is well recognized as the leading cause of bacterial foodborne diarrheal disease worldwide; while, poultry has been identified as a significant cause of campylobacter infection in humans. The C. jejuni has been found to be the predominant species isolated from poultry samples and, yet, responsible for the majority of human campylobacteriosis. Campylobacter spp. are small, oxidase positive, microaerophilic, curved gram-negative rods exhibiting corkscrew motility and colonize the intestinal tract of most mammalian and avian species. From its very first description in late 19th century by Theodor Escherich until nowadays, a lot of research has been carried out providing a wealth of information regarding its microbiological properties. Since novel technologies constantly emerge, increasingly advanced methods for detection, identification and typing of Campylobacter spp. are becoming available. The aim of this article is to review the recent bibliography on Campylobacter focusing, especially, on its survival and growth characteristics, the laboratory methods used for its detection and isolation from clinical, animal, environmental, and food samples, the reported methods applied for its speciation, as well as the typing systems developed for subtyping of Campylobacter.

Background: The identification and molecular characterisation of bacteria associated with neonatal necrotising enterocolitis (NNE) and sepsis is very important in clarifying the role such bacteria play in the development of this disease. Methods: The present multicentre study was aimed at characterising bacteria isolated from haemocultures obtained from 20 neonates suffering NNE by using traditional and molecular methodologies (16 S rRNA subunit sequencing and multilocus sequence typing - MLST). Results: NNE incidence in hospitals in Bogotá was also estimated, finding rates similar to and higher than those reported in the literature (1 to 3 cases of NNE per 1,000 live-births). Staphylococcus epidermidis ST2, ST81, and ST126 sequence types were identified by using these two molecular techniques; the Escherichia coli ST394 sequence type was also identified. Species could not be identified for the Pantoea agglomerans isolate due to the high degree of intra-species identity. Interestingly, the bacterial isolates from the two neonates who died were classified in the same sequence type (i.e. S. epidermidis ST81), even though they came from different hospitals. Conclusion: Such molecular techniques allow characterising bacterial pathogen populations occurring in one or more hospitals in a particular city or determined geographical area and support taking more specific preventative measures directed against such particular clones.

Curtobacterium flaccumfaciens pv. flaccumfaciens is a Gram-positive bacterium and has reemerged as an incitant of bacterial wilt in common (dry, edible) beans in western Nebraska, eastern Colorado, and southeastern Wyoming. Curtobacterium flaccumfaciens pv. flaccumfaciens is diverse phenotypically and genotypically and is represented by several different pathogen color variants. The population structure of 67 strains collected between 1957 and 2009, including some isolated from alternate hosts, was determined with 3 molecular typing techniques: amplified fragment length polymorphism (AFLP), repetitive extragenic palindromic polymerase chain reaction (rep-PCR), and pulsed-field gel electrophoresis (PFGE). All 3 typing techniques showed a great degree of population heterogeneity, but they were not congruent in cluster analysis of the C. flaccumfaciens pv. flaccumfaciens populations. Cluster analysis of a composite data set (AFLP, PFGE, and rep-PCR) using averages from all experiments yielded 2 distinct groups: cluster A included strains with colonies of yellow, orange, and pink pigments, and cluster B had strains of only yellow pigment. Strains producing purple extracellular pigment were assigned to both clusters. Thus, C. flaccumfaciens pv. flaccumfaciens is diverse phenotypically and genotypically.

ABSTRACT The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

Staphylococcus aureus remains a major health problem responsible for many epidemic outbreaks. Therefore, the development of efficient and rapid methods for studying molecular profiles of S. aureus strains for its further typing is in high demand. Among many techniques, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI TOF MS) represents a timely, cost-effective, and reliable strain typing approach, which is still rarely used due to insufficient knowledge about the impact of sample preparation and analysis conditions on the molecular profiles and strain classification efficiency of S. aureus. The aim of this study was to evaluate the effect of the culture conditions and matrix type on the differentiation of molecular profiles of various S. aureus strains via the MALDI TOF MS analysis and different computational methods. The analysis revealed that by changing the culture conditions, matrix type, as well as a statistical method, the differentiation of S. aureus strains can be significantly improved. Therefore, to accelerate the incorporation of the MALDI-based strain typing in routine laboratories, further studies on the standardization and searching of optimal conditions on a larger number of isolates and bacterial species are of great need.

Whole genome sequencing (WGS) is rapidly replacing other molecular techniques for identifying and subtyping bacterial isolates. The resolution or discrimination offered by WGS is significantly higher than that offered by other molecular techniques, and WGS readily allows infrequent differences that occur between 2 closely related strains to be found. In this investigation, WGS was used to identify the changes that occurred in the genomes of 13 strains of bacterial foodborne pathogens after 100 serial subcultures. Pure cultures of Shiga-toxin-producing Escherichia coli, Salmonella enterica, Listeria monocytogenes, and Vibrio parahaemolyticus were subcultured daily for 100 successive days. The 1st and 100th subcultures were whole-genome sequenced using short-read sequencing. Single nucleotide polymorphisms (SNPs) were identified between the 1st and final culture using 2 different approaches, and multilocus sequence typing of the whole genome was also performed to detect any changes at the allelic level. The number of observed genomic changes varied by strain, species, and the SNP caller used. This study provides insight into the genomic variation that can be detected using next-generation sequencing and analysis methods after repeated subculturing of 4 important bacterial pathogens.

Escherichia coli O157:H7, a Shiga toxin–producing E. coli, has been the causative agent of many cases of severe, often life-threatening foodborne illness. Because of the importance of E. coli O157:H7 to public health, many molecular typing methods have been developed to determine its transmission routes and source of infection during epidemiological investigations. Pulsed-field gel electrophoresis (PFGE) is currently used by public health organizations to track infections of E. coli O157:H7 and other foodborne pathogens. In this study, we compared the ability of PFGE, multilocus sequence typing (MLST), and repetitive-element PCR (Rep-PCR) to distinguish among 92 E. coli O157:H7 isolates from cattle, food, and infected humans. Several virulence genes, including the intimin gene (eaeA), the hemolysin gene (hlyA), and the H7 fimbrial gene (fliC), and a housekeeping gene for β-glucuronidase (uidA) were included in MLST. Rep-PCR reactions were performed using a commercially available typing kit (Bacterial Barcodes Inc., Houston, Tex.) with the provided Uprime-RI primer set. Results of the study indicated that PFGE provided the most discrimination among the techniques, identifying 72 distinct PFGE profiles for the isolates; Rep-PCR elucidated 14 different profiles, whereas MLST generated five profiles. Additionally, there did not appear to be any correlation among the typing methods examined in this study. Therefore, to date, PFGE remains the technique of choice for molecular subtyping of E. coli O157:H7.

One of the problems of modern medicine is diagnosis, treatment and prophylaxis of infections caused by multidrug-resistant strains of bacteria. Fast and correct identification of these pathogens is of utmost importance, as it enables early implementation of effective therapy. Therefore, rapid, modern and affordable methods are of outstanding value as they make it possible to conduct a reliable analysis in a very short period of time. One of such techniques is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which in recent years is increasingly used in clinical microbiological laboratories. This method enables cheap, fast and reliable identification of microorganisms based on their protein profiles. At present the possibilities of use of MALDI-TOF MS method become more broad, e.g. for identification of microorganisms directly in positive blood culture samples, typing of bacterial strains in epidemiological investigation of an outbreak as well as detection of multidrug-resistant strains, including producers of carbapenemases.

Comparative study of the genetic characteristics among three Acidithiobacillus caldus strains isolated from different typical environments in China was performed using a combination of molecular methods, namely sequencing analysis of PCR-amplified 16S rRNA genes and 16S-23S rRNA gene intergenic spacers (ITS), repetitive element PCR (rep-PCR), arbitrarily primed PCR (AP-PCR) fingerprinting and random amplified polymorphic DNA (RAPD). Both of the 16S rRNA gene and 16S-23S rRNA gene intergenic spacers sequences of the three strains exhibited small variations, with 99.9-100%, 99.7-100% identity respectively. In contrast, according to the analysis of bacterial diversity based on rep-PCR and AP-PCR fingerprinting, they produced highly discriminatory banding patterns, and the similarity values between them varied from 61.97% to 71.64%. RAPD analysis showed that banding profiles of their genomic DNA exhibited obvious differences from each other with 53.44-75% similarity. These results suggested that in contrast to 16S rRNA genes and 16S-23S rRNA gene intergenic spacers sequencing analysis, rep-PCR, AP-PCR fingerprinting and RAPD analysis possessed higher discriminatory power in identifying these closely related strains. And they could be used as rapid and highly discriminatory typing techniques in studying bacterial diversity, especially in differentiating bacteria within Acidithiobacillus caldus.

ABSTRACT Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with Bactrocera oleae, one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among which Acetobacter tropicalis was predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such as Anopheles stephensi (G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) or Drosophila melanogaster (C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established between A. tropicalis and B. oleae. Using an A. tropicalis-specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that different A. tropicalis strains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.

ABSTRACT There is a paucity of knowledge on microbial community diversity and naturally occurring seasonal variations in agricultural soil. For this purpose the soil microbial community of a wheat field on an experimental farm in The Netherlands was studied by using both cultivation-based and molecule-based methods. Samples were taken in the different seasons over a 1-year period. Fatty acid-based typing of bacterial isolates obtained via plating revealed a diverse community of mainly gram-positive bacteria, and only a few isolates appeared to belong to the Proteobacteria and green sulfur bacteria. Some genera, such as Micrococcus,Arthrobacter, and Corynebacterium were detected throughout the year, while Bacillus was found only in July. Isolate diversity was lowest in July, and the most abundant species, Arthrobacter oxydans, and members of the genus Pseudomonas were found in reduced numbers in July. Analysis by molecular techniques showed that diversity of cloned 16S ribosomal DNA (rDNA) sequences was greater than the diversity among cultured isolates. Moreover, based on analysis of 16S rDNA sequences, there was a more even distribution among five main divisions,Acidobacterium,Proteobacteria, Nitrospira, cyanobacteria, and green sulfur bacteria. No clones were found belonging to the gram-positive bacteria, which dominated the cultured isolates. Seasonal fluctuations were assessed by denaturing gradient gel electrophoresis. Statistical analysis of the banding patterns revealed significant differences between samples taken in different seasons. Cluster analysis of the patterns revealed that the bacterial community in July clearly differed from those in the other months. Although the molecule- and cultivation-based methods allowed the detection of different parts of the bacterial community, results from both methods indicated that the community present in July showed the largest difference from the communities of the other months. Efforts were made to use the sequence data for providing insight into more general ecological relationships. Based on the distribution of 16S rDNA sequences among the bacterial divisions found in this work and in literature, it is suggested that the ratio between the number ofProteobacteria andAcidobacterium organisms might be indicative of the trophic level of the soil.

Abstract Whole genome sequencing (WGS) has revolutionized the genotyping of bacterial pathogens and is expected to become the new gold standard for tracing the transmissions of bacterial infectious diseases for public health purposes. Traditional genomic epidemiology often uses WGS as a verification tool, namely, when a common source or epidemiological link is suspected, the collected isolates are sequenced for the determination of clonal relationships. However, increasingly frequent international travel and food transportation, and the associated potential for the cross-border transmission of bacterial pathogens, often lead to an absence of information on bacterial transmission routes. Here we introduce the concept of ‘reverse genomic epidemiology’, i.e. when isolates are inspected by genome comparisons to be sufficiently similar to one another, they are assumed to be a consequence of infection from a common source. Through BacWGSTdb (http://bacdb.org/BacWGSTdb/), a database we have developed for bacterial genome typing and source tracking, we have found that almost the entire analyzed 20 bacterial species exhibit the phenomenon of cross-border clonal dissemination. Five networks were further identified in which isolates sharing nearly identical genomes were collected from at least five different countries. Three of these have been documented as real infectious disease outbreaks, therefore demonstrating the feasibility and authority of reverse genomic epidemiology. Our survey and proposed strategy would be of potential value in establishing a global surveillance system for tracing bacterial transmissions and outbreaks; the related database and techniques require urgent standardization.

Sepsis is a life-threatening condition arising from a dysregulated host immune response to infection, leading to a substantial global health burden. The accurate identification of bacterial pathogens in sepsis is essential for guiding effective antimicrobial therapy and optimising patient outcomes. Traditional culture-based bacterial typing methods present inherent limitations, necessitating the exploration of alternative diagnostic approaches. This study reports the successful application of Fourier-transform infrared (FT-IR) spectroscopy in combination with chemometrics as a potent tool for the classification and discrimination of microbial species and strains, primarily sourced from individuals with invasive infections. These samples were obtained from various children with suspected sepsis infections with bacteria and fungi originating at different sites. We conducted a comprehensive analysis utilising 212 isolates from 14 distinct genera, comprising 202 bacterial and 10 fungal isolates. With the spectral analysis taking several weeks, we present the incorporation of quality control samples to mitigate potential variations that may arise between different sample plates, especially when dealing with a large sample size. The results demonstrated a remarkable consistency in clustering patterns among 14 genera when subjected to principal component analysis (PCA). Particularly, Candida, a fungal genus, was distinctly recovered away from bacterial samples. Principal component discriminant function analysis (PC-DFA) allowed for distinct discrimination between different bacterial groups, particularly Gram-negative and Gram-positive bacteria. Clear differentiation was also observed between coagulase-negative staphylococci (CNS) and Staphylococcus aureus isolates, while methicillin-resistant S. aureus (MRSA) was also separated from methicillin-susceptible S. aureus (MSSA) isolates. Furthermore, highly accurate discrimination was achieved between Enterococcus and vancomycin-resistant enterococci isolates with 98.4% accuracy using partial least squares-discriminant analysis. The study also demonstrates the specificity of FT-IR, as it effectively discriminates between individual isolates of Streptococcus and Candida at their respective species levels. The findings of this study establish a strong groundwork for the broader implementation of FT-IR and chemometrics in clinical and microbiological applications. The potential of these techniques for enhanced microbial classification holds significant promise in the diagnosis and management of invasive bacterial infections, thereby contributing to improved patient outcomes.

Recently, Pseudomonas aeruginosa infections proportions have increased significantly. Molecular typing and virulence analysis are good techniques, which can lead us to know P. aeruginosa infections. P. aeruginosa isolates were identified by using molecular method (16S rDNA gene) via PCR technique for accurate identification. The highest percent 41.26% of P. aeruginosa bacteria was found in the burn infections followed by 28.57% in wound swabs, 17.46% in ear discharge and lowest percentage were obtained from sputum samples. All isolates classified into six groups (A-F) according to classes of antibiotics. Of the 63 bacterial isolates, 100% were resistant to carbencillin, whereas 31.74% were resistant to ticarcillin and all isolates susceptible to imipenem. In addition all of clinical isolates indicated multidrug resistant (MDR) patterns, the highest rate of MDR was observed with pattern C these isolates were able to resist (9-12) antibiotics. All isolates were typed genotypically by using two methods of amplification, ERIC and RAPD-PCR. The results of the ERIC-PCR typing of P. aeruginosa bacteria that 96.82% showed amplification bands ERIC-PCR also revealed 17 groups of genotypes (A-R) and 4 unique isolates. The results of RAPD-PCR fingerprint revealed 12 groups of genotypes (A-M) of 40–90% similarity according to coefficient values and 4 unique isolates, except 7.93% were untypeble. QS genes (lasI, lasR, rhlI, rhlR), screen showed all isolates 100% were positive for one or more QS genes, in the other hand 82.53% carrying lasI, lasR, rhlI, and rhlR, while the 15.87% carrying lasI, rhlI, and rhlR and 1.58% carrying lasI, lasR, and rhlR genes. ERIC genotyping significantly correlated resistance patterns but not with virulence control QS genes. RAPD genotyping significantly correlated with source of infection, resistance patterns and virulence control QS genes. These results can help initial diagnosis MDR P. aeruginosa outbreaks associated with specific genotyping patterns.

Bacterial cellulose (BC) is receiving a great deal of attention due to its unique properties such as high purity, water retention capacity, high mechanical strength, and biocompatibility. However, the production of BC has been limited because of the associated high costs and low productivity. In light of this, the isolation of new BC producing bacteria and the selection of highly productive strains has become a prominent issue. Kombucha tea is a fermented beverage in which the bacteria fraction of the microbial community is composed mostly of strains belonging to the genus Komagataeibacter. In this study, Kombucha tea production trials were performed starting from a previous batch, and bacterial isolation was conducted along cultivation time. From the whole microbial pool, 46 isolates were tested for their ability to produce BC. The obtained BC yield ranged from 0.59 g/L, for the isolate K2G36, to 23 g/L for K2G30—which used as the reference strain. The genetic intraspecific diversity of the 46 isolates was investigated using two repetitive-sequence-based PCR typing methods: the enterobacterial repetitive intergenic consensus (ERIC) elements and the (GTG)5 sequences, respectively. The results obtained using the two different approaches revealed the suitability of the fingerprint techniques, showing a discrimination power, calculated as the D index, of 0.94 for (GTG)5 rep-PCR and 0.95 for ERIC rep-PCR. In order to improve the sensitivity of the applied method, a combined model for the two genotyping experiments was performed, allowing for the ability to discriminate among strains.

Brucellosis is still a global health issue, and surveillance and control of this zoonotic disease in livestock remains a challenge. Human outbreaks are mainly linked to the consumption of unpasteurized dairy products. The detection of human pathogenic Brucella species in food of animal origin is time-consuming and laborious. Bacteriophages are broadly applied to the typing of Brucella isolates from pure culture. Since phages intracellularly replicate to very high numbers, they can also be used as specific indicator organisms of their host bacteria. We developed a novel real-time PCR (qPCR) assay targeting the highly conserved helicase sequence harbored in all currently known Brucella-specific lytic phages. Quality and performance tests determined a limit of detection of <1 genomic copy/µL. In raw milk artificially contaminated with Brucella microti, Izv phages were reliably detected after 39 h of incubation, indicating the presence of viable bacteria. The qPCR assay showed high stability in the milk matrix and significantly shortened the time to diagnosis when compared to traditional culture-based techniques. Hence, our molecular assay is a reliable and sensitive method to analyze phage titers, may help to reduce the hands-on time needed for the screening of potentially contaminated food, and reveals infection risks without bacterial isolation.

Molecular typing techniques are utilized to determine genetic similarities between bacterial isolates. However, the use of environmental DNA profiling to assess epidemiologic links between patients and their environment has not been fully explored. This work reports the development and validation of two high-throughput short sequence typing (HiSST) schemes targeting the opportunistic pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia, along with a modified SM2I selective medium for the specific isolation of S. maltophilia. These HiSST schemes are based on four discriminative loci for each species and demonstrate high discriminating power, comparable to pairwise whole-genome comparisons. Each scheme includes species-specific PCR primers for precise differentiation from closely related taxa, without the need for upstream culture-dependent methods. For example, the primers targeting the bvgS locus make it possible to distinguish P. aeruginosa from the very closely related Pseudomonas paraeruginosa sp. nov. The selected loci included in the schemes are adapted to massive parallel amplicon sequencing technology. An R-based script implemented in the DADA2 pipeline was assembled to facilitate HiSST analyses for efficient and accurate genotyping of P. aeruginosa and S. maltophilia. We demonstrate the performance of both schemes through in silico validations, assessments against reference culture collections, and a case study involving environmental samples.

ABSTRACT The introduction of multilocus sequence typing (MLST) for the precise characterization of isolates of bacterial pathogens has had a marked impact on both routine epidemiological surveillance and microbial population biology. In both fields, a key prerequisite for exploiting this resource is the ability to discern the relatedness and patterns of evolutionary descent among isolates with similar genotypes. Traditional clustering techniques, such as dendrograms, provide a very poor representation of recent evolutionary events, as they attempt to reconstruct relationships in the absence of a realistic model of the way in which bacterial clones emerge and diversify to form clonal complexes. An increasingly popular approach, called BURST, has been used as an alternative, but present implementations are unable to cope with very large data sets and offer crude graphical outputs. Here we present a new implementation of this algorithm, eBURST, which divides an MLST data set of any size into groups of related isolates and clonal complexes, predicts the founding (ancestral) genotype of each clonal complex, and computes the bootstrap support for the assignment. The most parsimonious patterns of descent of all isolates in each clonal complex from the predicted founder(s) are then displayed. The advantages of eBURST for exploring patterns of evolutionary descent are demonstrated with a number of examples, including the simple Spain23F-1 clonal complex of Streptococcus pneumoniae, “population snapshots” of the entire S. pneumoniae and Staphylococcus aureus MLST databases, and the more complicated clonal complexes observed for Campylobacter jejuni and Neisseria meningitidis.

Coagulase-negative staphylococci, especially Staphylococcus epidermidis, are increasingly important nosocomial pathogens, particularly in critically ill neonates. A 3-year prospective surveillance of nosocomial infections in a neonatal intensive care unit (NICU) was performed by traditional epidemiologic methods as well as molecular typing of microorganisms. The aims of the study were (i) to quantify the impact of S. epidermidis on NICU-acquired infections, (ii) to establish if these infections are caused by endemic clones or by incidentally occurring bacterial strains of this ubiquitous species, (iii) to evaluate the use of different methods for the epidemiologic typing of the isolates, and (iv) to characterize the occurrence and the spread of staphylococci with decreased glycopeptide susceptibility. Results confirmed that S. epidermidis is one of the leading causes of NICU-acquired infections and that the reduced glycopeptide susceptibility, if investigated by appropriate detection methods such as population analysis, is more common than is currently realized. Typing of isolates, which can be performed effectively through molecular techniques such as pulsed-field gel electrophoresis but not through antibiograms, showed that many of these infections are due to clonal dissemination and, thus, are potentially preventable by strict adherence to recommended infection control practices and the implementation of programs aimed toward the reduction of the unnecessary use of antibiotics. These strategies are also likely to have a significant impact on the frequency of the reduced susceptibility of staphylococci to glycopeptides, since this phenomenon appears to be determined either by more resistant clones transmitted from patient to patient or, to a lesser extent, by strains that become more resistant as a result of antibiotic pressure.

Bovine tuberculosis is a serious infectious disease affecting a wide range of domesticated and wild animals, representing a worldwide economic and public health burden. The disease is caused by Mycobacteriumbovis and infrequently by other pathogenic mycobacteria. The problem of bovine tuberculosis is complicated when the infection is associated with multidrug and extensively drug resistant M. bovis. Many techniques are used for early diagnosis of bovine tuberculosis, either being antemortem or postmortem, each with its diagnostic merits as well as limitations. Antemortem techniques depend either on cellular or on humoral immune responses, while postmortem diagnosis depends on adequate visual inspection, palpation, and subsequent diagnostic procedures such as bacterial isolation, characteristic histopathology, and PCR to reach the final diagnosis. Recently, sequencing and bioinformatics tools have gained increasing importance for the diagnosis of bovine tuberculosis, including, but not limited to typing, detection of mutations, phylogenetic analysis, molecular epidemiology, and interactions occurring within the causative mycobacteria. Consequently, the current review includes consideration of bovine tuberculosis as a disease, conventional and recent diagnostic methods, and the emergence of MDR-Mycobacterium species.

Pseudomonas syringae pv. syringae, which causes bacterial canker, is the most polyphagous bacterium in the P. syringae complex because of its broad host range. This pathogen is considered the major bacterial disease in cherry orchards. In this study, several samples were collected from infected sweet cherry (Prunus avium L.) trees in different locations of the Marmara region in Turkey between 2016 and 2018. Sixty-three isolates were identified as P. syringae pv. syringae by pathogenicity, LOPAT, GATTa, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry tests. Total genomic DNA was extracted to confirm identity, followed by PCR amplification of syrB and cfl genes. Out of 63 isolates, 12 were randomly selected for repetitive element sequence-based PCR and multilocus sequence typing analyses to gain insight into the relationships of those isolates. The cluster analysis of enterobacterial repetitive intergenic consensus-, repetitive extragenic palindromic-, and BOX-A1R-based repetitive extragenic-palindromic-PCR techniques could classify the isolates into two distinct clusters. Phylogenetic analysis was carried out to obtain the relation between isolates and the location. The multilocus sequencing typing analysis of gyrB, rpoDp, rpoDs, and gltA genes allowed a clear allocation of the isolates into two separate main clusters. The relationships among the isolates were also evaluated by constructing a genealogical median-joining network (MJN). The isolates from six locations produced 11 haplotypes that were illustrated in the MJN. The results of this study proved that location could not be an indicator for showing the genetic diversity of P. syringae pv. syringae from cherry orchards. As the genetic variability of Pseudomonads has been demonstrated, this study also showed high diversity among different isolates even within the populations. While more research is recommended, the results of this study contributed to a better understanding of the evolutionary progress of P. syringae pv. syringae and the genetic diversity of sweet cherry isolates.

Listeria monocytogenes es un patógeno ubicuo intracelular, causante de la Listeriosis, la cual se considera una enfermedad transmitida por alimentos (ETA). En la actualidad existe una creciente demanda de consumidores de productos alimenticios tratados mínimamente que pueden favorecer la proliferación de este microorganismo. Es necesario contar con programas de vigilancia que incluyan métodos fiables para la detección de este patógeno en casos de brotes epidémicos. Esta revisión bibliográfica compara las ventajas y desventajas de las técnicas fenotípicas y genotípicas utilizadas en la determinación de L. monocytogenes con el fin de definir la más adecuada que permita obtener resultados confiables y en el menor tiempo posible. Se realizó una búsqueda bibliográfica en bases de datos como Pubmed, Science Direct, Proquest y Ovid, en inglés y español, utilizando los siguientes descriptores: L. monocytogenes, molecular typing, diagnosis, PCR y bacterial typing techniques. Estos se combinaron de diferentes maneras para, finalmente, recopilar setenta artículos que cumplieron con los criterios de selección propuestos. Como resultado se presentan las técnicas de diagnóstico fenotípico y genotípico que representan una opción útil para el aislamiento e identificación de este patógeno a partir de diferentes orígenes. Las técnicas revisadas permiten la diferenciación entre especies patógenas y no patógenas, así como de serotipos y genotipos con base en la implementación de procedimientos cuya fundamentación puede diferir, pero que igualmente pueden ser complementarias.

Ruminant mastitis is a welfare and economic problem of livestock and the dairy industry. It is a major animal health problem in countries where livestock are raised for the purpose of milk production. This Review focuses on ruminant mastitis, etiologic agents, diagnostic test and associated risk factors. In this narrative review, a comprehensive literature search of all relevant articles was performed to identify articles published in Science Direct, Web of Science, PubMed, Scopus, MEDLINE, Google Scholar, LISTA and AJOL. English articles published from 2010 to 2023 were reviewed and used. The findings revealed that even though Staphylococcus aureus, Non-aureus Staphylococci species, and Streptococci are the major mastitis pathogens. Others like Escherichia coil, Klebsiella, Citrobacter, Enterobacter, Pseudomonas, Proteus, Serratia, Bacillus, and Corynebacterium etc were isolated from milk samples. Diagnosis was observed to be based on observation of milk and cardinal signs of inflammation of the mammary glands, the use of diagnostic test, like bacterial culture, Strip cup test, White side test (WST), Surf field mastitis test (SFMT), and California Mastitis test (CMT). Molecular typing techniques including PCR, Sequencing, MALDI-TOF MS, MLST, PFGE, spa typing etc have also been observed important role in characterizing agents of mastitis. Major risk factor observed is age, parity and stage of lactations. Clinical management of this condition in ruminants is based on the understanding of the etiology, and treatment options.

Population and evolutionary analyses of pathogenic bacteria are frequently hindered by sampling strategies that concentrate on isolates from patients with invasive disease. This is especially so for the gram-negative diplococcus Neisseria meningitidis, a cause of septicemia and meningitis worldwide. Meningococcal isolate collections almost exclusively comprise organisms originating from patients with invasive meningococcal disease, although this bacterium is a commensal inhabitant of the human nasopharynx and very rarely causes pathological effects. In the present study, molecular biology-based techniques were used to establish the genetic relationships of 156 meningococci isolated from healthy young adults in the Czech Republic during 1993. None of the individuals sampled had known links to patients with invasive disease. Multilocus sequence typing (MLST) showed that the bacterial population was highly diverse, comprising 71 different sequence types (STs) which were assigned to 34 distinct complexes or lineages. Three previously identified hyperinvasive lineages were present: 26 isolates (17%) belonged to the ST-41 complex (lineage 3); 4 (2.6%) belonged to the ST-11 (electrophoretic type [ET-37]) complex, and 1 (0.6%) belonged to the ST-32 (ET-5) complex. The data were consistent with the view that most nucleotide sequence diversity resulted from the reassortment of alleles by horizontal genetic exchange.

Abstract Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.

In accordance with the global action plan on antimicrobial resistance adopted by the World Health Assembly in 2015, there is a need to develop surveillance programs for antimicrobial resistant bacteria. In this context, we have analyzed the clonal diversity of Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) isolated from aquatic environments and human and food samples in Spain, with the aim of determining possible clonal complexes (CCs) that act as markers of the potential risk of transmission of these resistant bacteria. The phylogenetic groups, sequence types (STs) and CCs were determined by different Polymerase Chain Reaction (PCR) and Multilocus Sequence Typing (MLST) techniques. Phylogroup A was prevalent and was mainly present in food and water strains, while human strains were mostly associated with phylogroup B2. According to the observed prevalence in the different niches, CC23 and CC10 are proposed as markers of phylogroups A and C, related with the spread of blaCTX-M1 and blaCTX-M15 genes. Similarly, CC131 and CC38 could be associated to the dissemination of pathogenic strains (phylogroups B2 and D) carrying mainly blaCTX-M14 and blaCTX-M15 genes. Some strains isolated from wastewater treatment plants (WWTPs) showed identical profiles to those isolated from other environments, highlighting the importance that water acquires in the dissemination of bacterial resistance. In conclusion, the detection of these genetic markers in different environments could be considered as an alert in the spread of ESBL.

SUMMARYNinety-six isolates of presumptive or confirmedListeria monocytogeneswere obtained from local clinical (30 isolates) or food laboratories (66 isolates). Minimal biochemical analysis identified only 80% of these isolates asL. monocytogenesthe remaining includedL. seeligeri, 1%, or the non-haemolyticL. innocua, 19%. The 27 clinical and 50 food isolates, mainly from meat products, frozen confectionaries, and cheeses, confirmed asL. monocytogeneswere compared biochemically and serologically. Twenty-one isolates, including some strains ofL. innocuaandL. seeligeri, were examined for pathogenicity in immunocompromized mice and 44 typed using bacterial restriction endonuclease DNA analysis (BRENDA). Only isolates ofL. monocytogeneswere found to be pathogenic. Biovar-typing of the isolates was unreliable and provided poor discrimination. Serogroups 1/2 and 4 predominated among clinical and food isolates and BRENDA provided better discrimination among isolates. Ten stable and reproducible restriction patterns were observed among theListeriasp. isolates studied. Overall, a combination of techniques gave the best discrimination and indicated their potential for use as epidemiological tools.

The first phytopathogenic bacterium with its DNA entirely sequenced is being detected and isolated from different host plants in several geographic regions. Although it causes diseases in cultures of economic importance, such as citrus, coffee, and grapevine little is known about the genetic relationships among different strains. Actually, all strains are grouped as a single species, Xylella fastidiosa, despite colonizing different hosts, developing symptoms, and different physiological and microbiological observed conditions. The existence of genetic diversity among X. fastidiosa strains was detected by different methodological techniques, since cultural to molecular methods. However, little is know about the phylogenetic relationships developed by Brazilian strains obtained from coffee and citrus plants. In order to evaluate it, fAFLP markers were used to verify genetic diversity and phylogenetic relationships developed by Brazilian and strange strains. fAFLP is an efficient technique, with high reproducibility that is currently used for bacterial typing and classification. The obtained results showed that Brazilian strains present genetic diversity and that the strains from this study were grouped distinctly according host and geographical origin like citrus-coffee, temecula-grapevine-mulberry and plum-elm.

Bacterial resistance to antibiotics has proven difficult to control over the past few decades. The large group of multidrug-resistant bacteria includes carbapenemase-producing bacteria (CPB), for which limited therapeutic options and infection control measures are available. Furthermore, carbapenemases associate with high-risk clones that are defined by the sequence type (ST) to which each bacterium belongs. The objectives of this cross-sectional and retrospective study were to describe the CPB population isolated in a third-level hospital in Southern Spain between 2015 and 2020 and to establish the relationship between the ST and the epidemiological situation defined by the hospital. CPB were microbiologically studied in all rectal and pharyngeal swabs and clinical samples received between January 2015 and December 2020, characterizing isolates using MicroScan and mass spectrometry. Carbapenemases were detected by PCR and Sanger sequencing, and STs were assigned by multilocus sequence typing (MLST). Isolates were genetically related by pulsed-field gel electrophoresis using Xbal, Spel, or Apal enzymes. The episodes in which each CPB was isolated were recorded and classified as involved or non-involved in an outbreak. There were 320 episodes with CPB during the study period: 18 with K. pneumoniae, 14 with Klebisella oxytoca, 9 with Citrobacter freundii, 11 with Escherichia coli, 46 with Enterobacter cloacae, 70 with Acinetobacter baumannii, and 52 with Pseudomonas aeruginosa. The carbapenemase groups detected were OXA, VIM, KPC, and NDM with various subgroups. Synchronous relationships were notified between episodes of K. pneumoniae and outbreaks for ST15, ST258, ST307, and ST45, but not for the other CPB. There was a major increase in infections with CPB over the years, most notably during 2020, coinciding with the COVID-19 pandemic. This study highlights the usefulness of gene sequencing techniques to control the spread of these microorganisms, especially in healthcare centers. These techniques offer faster results, and a reduction in their cost may make their real-time application more feasible. The combination of epidemiological data with real-time molecular sequencing techniques can provide a major advance in the transmission control of these CPB and in the management of infected patients. Real-time sequencing is essential to increase precision and thereby control outbreaks and target infection prevention measures in a more effective manner.

This study aimed to assess bovine tuberculosis-like cases in three slaughterhouses in North Central Algeria and to confirm these suspected cases using microscopic, bacteriological examination, and molecular biology technique. We highlighted the factors influencing the prevalence of the disease. Also, the genomic profiles of Mycobacterium tuberculosis complex (MTBC) strains isolated by Oligonucleotide typing technique (Spoligotyping) were determined. At the abattoir level, bovine carcasses were routinely inspected to detect visible abnormalities including suspicious lesions of bovine tuberculosis (BTB). At the laboratory level (Pasteur Institute, Algiers), Ziehl-Neelsen staining, bacterial culture, biochemical study (nitrate reduction test), and spoligotyping were performed to confirm suspected cases. On a total of 1300 bovine carcasses, 100 presented BTB-like cases (7.69%). Animals over 5 years of age were more affected compared to other age groups. Lesions were observed more often in females than in males and cattle of local breed were the most exposed to BTB-like cases (P&lt;0.05). Bacilloscopy data were positive for 44 of the 100 suspected samples (44%) while bacteriology showed that 56 cultures were positive, while 35 were negative and 9 were contaminated. Molecular spoligotyping of 40 Mycobacterium strains samples showed 19 spoligotype profiles of M. bovis, of which 50% of profiles have been previously detected in the Mediterranean area and the three spoligotype patterns not previously reported were named SB2651, SB2652, SB2653 (by http:// www.Mbovis.org). Measures and means to prevent TB transmission among animals and to humans should be recommended, and more intensive investigations are required using both routine and molecular diagnostic techniques to understand and further explore MTBC.

SUMMARY The introduction of in vitro nucleic acid amplification techniques, led by real-time PCR, into the clinical microbiology laboratory has transformed the laboratory detection of viruses and select bacterial pathogens. However, the progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. Microarray analysis has the capability to offer robust multiplex detection but has just started to enter the diagnostic microbiology laboratory. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. One aim of this paper is to review microarray technology, highlighting technical differences between them and each platform's advantages and disadvantages. Although the use of microarrays to generate gene expression data has become routine, applications pertinent to clinical microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and analysis of microbial infections using host genomic expression and polymorphism profiles.

Introduction: There are numerous methods of isolating and detecting organisms that are similar and closely related; one of the most reliable method is molecular typing of 16S rRNA. Apart from being omnipresent as a multigene family, or operons; it is evolutionarily stable; the 16S rRNA gene (1,500 bp) is large enough for informatics purposes. Materials and Method: This study employed molecular sequencing of 16S rRNA by Sanger method to reveal the specific organisms’ nucleotides and blasting (BLASTn) to show the similarities between the resulting organisms and existing organisms. The 16S rRNA remains the best choice of identification process for bacteria because of its distinguishing sizes and evolutionary stability. Results: All isolates were Gram positive rods and were positive in Biochemical tests such as oxidase, catalase, citrate, and protease but were in turn negative in coagulase and indole test tests. On sensitivity test; 80% of all the isolates were resistant to common antibiotics except ciprofloxacin and ceftriaxone. Based on the sequence difference in the variable region (V1) of 16S rRNA as observed from the molecular sequencing results; four isolates out of ten were identified. Six were different strains of B cereus. Others isolates include: wiedmannii, thuringensis, toyonensis and pseudomycoides. Sequence analysis of the primer annealing sites showed that there is no clear‐cut difference in the conserved region of 16S rRNA, and in the gyrB gene, between B. cereus and B. thuringiensis strains. Phylogenetic analysis showed that four isolates showed high similarity to each other; hence the limited number of deletions when subjected to alignments by maximum neighborhood joining parsimony using MEGA X software. B. toyonensis, B. wiedmannii and thuringensis were distantly related. Introduction Authors Pathogens cause illness and death in some countries and it also causes infections and gastrointestinal diseases in other countries thereby causing public health concern. Pathogens are organisms capable of causing diseases. Reliable methods are needed for the detection of pathogens due to pathogen evolution as a result of new human habits or new industrial practices. Microbial classification of organisms ranges from genus to specie level depending upon the technique used either phenotypic or genotypic. Presently, molecular methods now obtain advances to allow utilization in microbiology [1]. There are numerous molecular methods which are of fast and simple application to the detection of pathogen. Among the pathogens involved in human health, Bacillus cereus is interesting due to their ability to survive in various habitats [2]. The genus Bacillus is aerobic or facultative anaerobic bacteria, gram positive spore forming rod shaped bacteria. Which can be characterized by two morphological forms, the vegetative cell which range from 1.02 to 1.2 um in width and from 3.0 to 5.0 in length, it can be straight or slightly curve, motile or non-motile, and the endospore (the non-swelling sporangium). The genus Bacillus is been characterized by the presence of endospore, which is not more than one per cell and they are resistant to many adverse environmental conditions such as heat, radiation, cold and disinfectants. It can also respire either in the presence or absence of oxygen [3]. Cell diameter of Bacillus cereus, sporangium and catalase test do not allow differentiation, where as important in differentiation among B. anthracis, B. cereus, B. thuringiensis can be considered by parasporal crystals and the presence of capsule. [4] Showed a B. thuringiensis strain capable of producing a capsule resembling that of B. anthracis. Most species of the genus display a great kind in physiological characteristics such as degradation of cellulose, starch, pectin, agar, hydrocarbons, production of enzymes and antibiotics and other characteristic such as acidophile, alkalinophile, psychrophile, and thermophile's which allows them to adapt to various environmental conditions [5]. In differentiating between species of the genus Bacillus it was difficult at early attempts when endospore formation and aerobic respiration were the main character used for classification. As reported by many authors that at molecular method level, the differentiation between B. thuringiensis and B. cereus is also very difficult. cereus can survive at the temperature between 4°c and 55°c. The mesophile strains can grow between the temperature of 10°c and 42°c, while psychotropic strains can survive at 4°c, whereas other strains are able to grow at 52 to 55°c. B. cereus vegetative cells grow at pH between 1.0 and 5.2. Heat resistant strain can survive and multiply in wet low acid foods in temperature ranging from 5 to 52°c. The survivability of B. cereus spores at 95°c decreases when the pH level decreases from 6.2 to 4.7 [6]. B. cereus can grow in the presence of salt with concentration up to 7.5% depending on the pH value. thuringiensis possesses a protein crystal that is toxic to insects. This toxin protein was first known as parasporal crystalline inclusion but was later referred to as π - endotoxin or in other ways known as insecticidal crystal protein [7]. Strains of B. thuringiensis bacteria possess a wide range of specificity in various orders of insects such as Lepidoptera, dipteral, coleoptera. These strains of bacteria produce crystalline proteins known as cry protein during sporulation. When B. thuringiensis infects an insects, it will cause the insect to loose appetite, enhances slow movement and over time the insect will die due to crystals of proteins that have been dissolved in the insect's stomach. In the cultivation of vegetable crops, the plant can be attack by many types of pests. Hence, in overcoming pest attacks farmers often use pesticides that contain active synthetic materials. Many negative effects arise from the folly use of chemical pesticides. Among the negative effect is the increase of pest population, resistance, death of natural enemy population and increase in residue level on Agricultural product which makes it unsafe for public consumption [8]. Therefore, it is necessary to find an alternative method in the control of crop pest. The best alternative that can be done is to replace the chemical insecticide with biological control which involves the use of living things in the form of microorganisms. In these profiling microbial communities, the main objective is to identify which bacteria and how much they are present in the environments. Most microbial profiling methods focus on the identification and quantification of bacteria with already sequenced genomes. Further, most methods utilize information obtained from entire genomes. Homology-based methods such as [1–4] classify sequences by detecting homology in reads belonging to either an entire genome or only a small set of marker genes. Composition-based methods generally use conserved compositional features of genomes for classification and as such they utilize less computational resources.Using the 16S rRNA gene instead of whole genome information is not only computational efficient but also economical; Illumina indicated that targeted sequencing of a focused region of interest reduces sequencing costs and enables deep sequencing, compared to whole-genome sequencing. On the other hand, as observed by [8], by focusing exclusively on one gene, one might lose essential information for advanced analyses. We, however, will provide an analysis that demonstrates that at least in the context of oral microbial communities, the 16S rRNA gene retains sufficient information to allow us detect unknown bacteria [9, 10]. This study aimed at employing 16S rRNA as an instrument of identification of seemingly close Bacillus species. Abbreviations BLAST, Basic Local Alignment sequence Tools; PCR, Polymerase Chains reactions; rRNA, ribosomal RNA; Material and methods T Sample collection. Soil samples were collected from three sources from Rice, Sugar Cane, vegetables and abandoned farmland in January 2019. The samples were labeled serially from Sample 1 to Sample 10 (S1 to S10). Bacterial culture: A serial dilution of 10 folds was performed. Bacterial suspension was diluted (10-10) with saline water and 100 μl of bacterial suspension werespread on Nutrient Agar plate and incubated for 24 hours. Bacterial colonies were isolated and grown in Nutrient Broth and nutrient agar. Other microbiological solid agar used include: Chocolate, Blood Agar, EMB, MacConkey, Simon citrate, MRS Agar. Bacteria were characterized by conventional technique by the use of morphological appearance and performance on biochemical analysis [11]. Identification of bacteria:The identification of bacteria was based on morphological characteristics and biochemical tests carried out on the isolates. Morphological characteristics observed for each bacteria colony after 24 h of growth included colony appearance; cell shape, color, optical characteristics, consistency, colonial appearance and pigmentation. Biochemical characterizations were performed according to the method of [12] Catalase test: A small quantity of 24 h old culture was transferred into a drop of 3% Hydrogen peroxide solution on a clean slide with the aid of sterile inoculating loop. Gas seen as white froth indicates the presence of catalase enzyme [13] on the isolates. DNA Extraction Processes The extraction processes was in four phase which are: Collection of cell, lyses of cell, Collection of DNA by phenol, Concentration and purification of DNA. Collection of cell: the pure colonyof the bacteria culture was inoculated into a prepared sterile nutrient broth. After growth is confirmed by the turbidity of the culture, 1.5ml of the culture was taken into a centrifuge tube and was centrifuge at 5000 rpm for 5 minutes; the supernatant layer was discarded leaving the sediment. Lyses of cell: 400 microns of lyses buffer is added to the sediment and was mixed thoroughly and allow to stand for five minutes at room temperature (25°c). 200 microns of Sodium Dodecyl Sulfate (SDS) solution was added for protein lyses and was mixed gently and incubated at 65°c for 10 minutes. Collection of DNA by phenol; 500 microns of phenol chloroform was added to the solution for the separation of DNA, it was mixed completely and centrifuge at 10,000 rpm for 10 minutes. The white pallet seen at the top of the tube after centrifugation is separated into another sterile tube and 1micron of Isopropanol is added and incubated for 1hour at -20°c for precipitation of DNA. The DNA is seen as a colorless liquid in the solution. Concentration and purification of DNA: the solution was centrifuge at 10,000 rpm for 10 minutes. The supernatant layer was discarded and the remaining DNA pellets was washed with 1micron of 17% ethanol, mixed and centrifuge at 10,000 rpm for 10 minutes. The supernatant layer was discarded and air dried. 60 micron TE. Buffer was added for further dissolving of the DNA which was later stored at -40°c until it was required for use [14]. PCR Amplification This requires the use of primers (Forward and Reverse), polymerase enzyme, a template DNA and the d pieces which includedddATP, ddGTP and ddTTP, ddNTP. All this are called the master mix. The PCR reactions consist of three main cycles. The DNA sample was heated at 940c to separate the two template of the DNA strand which was bonded by a hydrogen bond. Once both strand are separated the temperature is reduced to 570c (Annealing temperature). This temperature allows the binding of the forward and reverse primers to the template DNA. After binding the temperature is raised back to 720c which leads to the activation of polymerase enzyme and its start adding d NTPs to the DNA leading to the synthesize of new strands. The cycles were repeated several times in order to obtain millions of the copies of the target DNA [15]. Preparation of Agarose Gel One gram (1 g) of agarose for DNA was measured or 2 g of agarose powdered will be measured for PCR analysis. This done by mixing the agarose powder with 100 ml 1×TAE in a microwaveable flask and microwaved for 1-3 minutes until the agarose is completely dissolved (do not over boil the solution as some of the buffer will evaporate) and thus alter the final percentage of the agarose in the gel. Allow the agarose solution to cool down to about 50°c then after five minutes 10µL was added to EZ vision DNA stain. EZ vision binds to the DNA and allows one to easily visualize the DNA under ultra violet (UV) light. The agarose was poured into the gel tray with the well comb firmly in place and this was placed in newly poured gel at 4°c for 10-15 mins or it sit at room temperature for 20-30 mins, until it has completely solidified[16]. Loading and Running of samples on Agarose gel The agarose gel was placed into the chamber, and the process of electrophoresis commenced with running buffer introduced into the reservoir at the end of the chamber until it the buffer covered at least 2millimeter of the gel. It is advisable to place samples to be loaded in the correct order according to the lanes they are assigned to be running. When loading the samples keep the pipette tip perpendicular to the row of the wells as by supporting your accustomed hand with the second hand; this will reduce the risk of accidentally puncturing the wells with the tip. Lower the tip of the pipette until it breaks the surface of the buffer and is located just above the well. Once all the samples have been loaded it is advised to always avoid any movement of the gel chamber. This might result in the sample spilling into adjacent well. Place the lid on the gel chamber with the terminal correctly positioned to the matching electrodes on the gel chamber black to black and red to red. Remember that DNA is negatively charged hence the movement of the electric current from negatively charged to the positively charged depending on the bandwidth in Kilobytes. Once the electrode is connected to the power supply, switch ON the power supply then set the correct constant voltage (100) and stopwatch for proper time. Press the start button to begin the flow of current that will separate the DNA fragment.After few minutes the samples begins to migrate from the wells into the gel. As the DNA runs, the diaphragm moves from the negative electrode towards the positive electrode [17]. PCR mix Components and Sanger Sequencing This is made up of primers which is both Forward and Reverse, the polymerase enzyme (Taq), a template DNA and the pieces of nucleotides which include: ddNTP, ddATP, ddGTP and ddTTP. Note that the specific Primer’s sequences for bacterial identification is: 785F 5' (GGA TTA GAT ACC CTG GTA) 3', 27F 5' (AGA GTT TGA TCM TGG CTC AG) 3', 907R 5' (CCG TCA ATT CMT TTR AGT TT) 3', 1492R 5' (TAC GGY TAC CTT GTT ACG ACT T) 3' in Sanger Sequencing techniques. BLAST The resulting genomic sequence were assembled and submitted in GenBank at NCBI for assignment of accession numbers. The resultant assertion numbers were subjected to homology search by using Basic Local Alignment Search Tool (BLAST) as NCBI with the assertion number MW362290, MW362291, MW362292, MW362293, MW362294 and MW362295 respectively. Whereas, the other isolates’ accession numbers were retrieved from NCBI GenBank which are:AB 738796.1, JH792136.1, MW 015768.1 and MG745385.1.MEGA 5.2 software was used for the construction of phylogenetic tree and phylogenetic analysis. All the organisms possess 100% identities, 0% gaps and 0.0% E.value which indicated that the organisms are closely related to the existing organisms. The use of 16S rRNA is the best identification process for bacteria because 16S rRNA gene has a distinguishing size of about 500 bases until 1500bp. Rather than using 23S rRNA which is of higher variation, The 16S rRNA is adopted in prokaryotes. 18S rRNA is used for identification in Eukaryotes Results The results of both the conventional morphological and cultural identification was correlated with the molecular sequencing results. Six isolates were confirmed B. cereus species while the other four isolates were. B. wiedmannii, B. thuringiensis, B. toyonensis and B. pseudomycoides.The 16S rRNA sequence of six isolates MW 362290.1- MW362295.1 were assigned accession numbers and deposited in the GenBank while the other four sequences were aligned to those available in the NCBI database. The alignment results showed closely relatedness to LT844650.1with an identity of 100% to 92.2% as above. The six isolates of Bacillus cereus great evolutionary relatedness as shown in the phylogenetic tree constructed using MEGA X software. Results The results of both the conventional morphological and cultural identification was correlated with the molecular sequencing results. Six isolates were confirmed B. cereus species while the other four isolates were. B. wiedmannii, B. thuringiensis, B. toyonensis and B. pseudomycoides.The 16S rRNA sequence of six isolates MW 362290.1- MW362295.1 were assigned accession numbers and deposited in the GenBank while the other four sequences were aligned to those available in the NCBI database. The alignment results showed closely relatedness to LT844650.1with an identity of 100% to 92.2% as above. The six isolates of Bacillus cereus great evolutionary relatedness as shown in the phylogenetic treeconstructed using MEGA X software. Discussion The results obtained in this study is consistent with the previous studies in other countries22,23 The results of the phylogenetic analysis of the 16S rRNA isolate of in this study was similar to the housekeeping genes proposed by [18, 19]. In comparing this study with the earlier study, B. cereus group comprising other species of Bacillus was hypothesized to be considered to form a single species with different ecotypes and pathotype. This study was able to phenotypically differentiated B. thuringiensis, B. pseudomycoides, B. toyonensis, B. wiedmannii and B. cereus sensu strito. Despite differences at the colonial appearance level, the 16S rRNA sequences have homology ranging from 100% to 92% providing insufficient resolution at the species level [6, 7, 18].After analysis through various methods, the strain was identified as Gram-positive bacteria of Bacillus cereus with a homology of 99.4%. Cohan [20] demonstrated that 95–99% of the similarity of 16S rRNA gene sequence between two bacteria hints towards a similar species while >99% indicates the same bacteria.The phylogenetic tree showed that B. toyonensis, B. thuringiensis and B. wiedmanniiare the outgroups of B. cereus group while B. pseudomycoides are most closely related to B. cereus group [19, 21, 22]. Conclusion In the area of molecular epidemiology, genotypic typing method has greatly increased our ability to differentiate between micro-organisms at the intra and interspecies levels and have become an essential and powerful tool. Phenotypic method will still remain important in diagnostic microbiology and genotypic method will become increasingly popular. After analysis through various methods, the strain was identified as Gram-positive bacteria of Bacillus cereus with a homology of between 100% and 92.3%. Acknowledgments Collate acknowledgments in a separate section at the end of the article before the references, not as a footnote to the title. Use the unnumbered Acknowledgements Head style for the Acknowledgments heading. List here those individuals who provided help during the research. Conflicts of interest The Authors declare that there is no conflict of interest. References: Simpkins Meyer F.; Paarmann D.; D’Souza M.; Olson R.; Glass EM.; Kubal M.; Paczian T.; Rodriguez A.; Stevens R. Wilke A The metagenomics rast server–a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics. 2008, 9(1), 386. Segata N.; Waldron L.; Ballarini A.; Narasimhan V.; Jousson O.; Huttenhower C. Metagenomic microbial community profiling using unique clade-specific marker genes. Nature methods. 2012, 9(8), 811–814. Brady A.; Salzberg SL. Phymm and phymmbl: metagenomic phylogenetic classification with interpolated markov models. 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Abstract Background Extended-spectrum b-lactamase (ESBL)-producing bacteria are resistant to several types of antibiotics excluding carbapenems. Consequently, infections caused by ESBL-producing bacteria are difficult to treat. Especially for the immunocompromised including hematologic malignancies ESBL-producing bacteria are emergent pathogens. In addition, there are no evidence-based guidelines specifying the infection control for ESBL-producing bacterial outbreak. Along with the recent trend of wide spreading ESBL genes in the community, carriers ESBL-producing bacteria in the community become common. Methods An ESBL-producing Enterobacteriaceae (ESBL-E) outbreak observed between July 2012 and August 2012 in Kagawa University Hospital was surveyed using various molecular microbiology techniques. In this study, we attempted to identify the source of an outbreak of ESBL-producing bacteria in a medical oncology and immunology care unit. We used pulsed-field gel electrophoresis (PFGE) and PCR-based ESBL gene analysis as molecular typing methods. DNA extracted from ESBL-E was digested overnight with Xba I. Tenover criteria were used for the separation and identification of each band. ESBL-producing bacteria were screened by PCR/DNA for the detection of blaTEM, blaSHV, and blaCTX-M. Subsequently, ESBL genes with specific CTX-M subtypes (groups 1, 2, 8 and 9) were characterized using multiplex PCR amplification. The antibiotic susceptibility of strains isolated from clinical samples was assessed by microdilution methods using an IA20MIC mkII system (Koden Industry Co., Japan). Results In total, 21 isolates (12 K. pneumoniae and 9 E. coli) were obtained from clinical samples, including four control strains (two each of both bacteria), that were physically different from those obtained from other inpatient units in our hospital. PFGE for K. pneumoniae (digested by Xba I) produced similar patterns excluding one control strain. PCR classification of the ESBL gene for K. pneumoniae revealed that all strains other than the control strain carried CTX-M9. On another, ESBL genes derived from E. coli strains were varied in each strain with TEM, SMV, CTX-M-1, CTX-M-2, or CTX-M-9. This result was reconfirmed by direct DNA sequencing. Although the outbreak of K. pneumoniae was considered to be "clonal," PFGE and PCR classification of the ESBL genes for E. coli uncovered at least six different "non-clonal" strains possessing individual ESBL gene patterns. The pattern of antimicrobial susceptibility was more variable for K. pneumoniae than for E. coli. Conclusion Typing by PFGE and ESBL gene PCR analysis is practical for discriminating various organisms. In our cohort, two outbreaks were concomitantly spread with different transmission strategies, namely clonal and non-clonal, in the same unit. This might represent clinical evidence that transmissibility differs according to the type of strain. Our speculation is that the ability to acquire ESBL genes differs among bacteria, and the penetrance of resistance is strain-dependent. ESBL-E outbreaks can occur concurrently, as shown in this study. We observed that patient-to-patient transmission of ESBL-E occurred according to the properties of each individual strain and strain-specific maneuver could be optimal. Disclosures No relevant conflicts of interest to declare.

Urinary tract infections (UTIs) are the leading cause of hospitalization due to bacterial infection, and the frequency of multidrug-resistant Escherichia coli isolates from these infections is increasing worldwide. The current study aims to isolate and characterize antibiotic-resistant Escherichia coli and their antibiogram typing from urine samples of humans. From April to December 2019, a total of 60 human urine samples were collected aseptically and treated to primary isolation by propagation in nutrient broth followed by culture on various agar media. Gram’s staining, string techniques, biochemical characterization, PCR, and Sanger sequencing were performed to confirm E. coli. The Kirby-Bauer disk diffusion technique was used to test the susceptibility of all bacterial strains to thirteen typically prescribed antibiotics. The overall prevalence of E. coli in UTIs was 66.67%. Three variations were noted in E. coli, all of which were single substitutions (A>T, C>T, and T>A). Phylogenetic analysis of the 16S rRNA revealed that the E. coli discovered in this study belonged to the genus Escherichia, but was distinct from those identified in other countries. The antibiograms revealed that all the isolates (100%) were resistant to penicillin, ampicillin, and amoxicillin; 94.87% to doxycycline; 79.16% to gentamycin; 75.48% to ciprofloxacin; 73.07% to erythromycin; 71.66% to levofloxacin; 47.36% to ceftriaxone; and 46.66% to tetracycline. In contrast, all E. coli strains were sensitive to amikacin (95%), vancomycin (92.50%), and azithromycin (92.50%). People with a urinary tract infection (UTI) often have multidrug-resistant E. coli in their urine samples, which calls for a one-health strategy to deal with this rapidly changing condition.

Symptoms resembling papaya bacterial crown rot (1,3) attributed to Erwinia papayae were first observed on ‘Waimanalo’ and ‘Solo Sunrise’ papaya on the island of Tongatapu, Kingdom of Tonga in July 2009. Spreading, dark green, water-soaked lesions formed on juvenile stem tissue and developed into a foul-smelling, wet rot that destroyed large sections of the stem. Coalescing, brown, angular, marginal, and intercostal lesions killed large areas of the lamina. Elongated lesions on petioles resulted in breakage and leaf death. Symptoms on stems typically moved toward the crown with the growing point being killed or the whole crown breaking off at a canker below. Isolations at 28°C on King's medium B (KB) yielded slow-growing, raised, white, mucoid colonies that produced a conspicuous, nondiffusable blue pigment in the medium. Two-day-old suspensions (1 × 108 CFU/ml) of two cultures were injected into juvenile stem tissue, petioles, and laminae of four healthy papaya seedlings of ‘Solo Sunrise’ with a sterile 1-ml insulin syringe. Sterile water was used as a negative control. Typical water-soaked lesions appeared at all bacterial inoculation sites on all plants within 5 days but not on controls. Pigment-producing colonies similar to those used for inoculation were reisolated from four different stem lesions. Bacteria isolated from diseased tissues were gram negative, facultative anaerobes, oxidase negative, nonfluorescent on KB, induced a hypersensitive reaction on tobacco leaves, but could not cause soft rot on potato slices. Those characteristics, combined with blue pigment production, are consistent with the bacterium E. papayae. A partial sequence of the 16S rDNA gene of ~804 bp was amplified from four Tongan isolates (ICMP18248–18251) using primers 27f and 1492r (4). Sequences of these strains were 100% identical to each other (GenBank Nos. HQ286366–HQ286369), 99 and 98% identical to the 16SrDNA sequences of E. mallotivora strains LMG2708 (Z96084.1) and DSM4565 (AJ233414.1) respectively, and 97% identical to the 16SrDNA sequence of E. papayae strain NCPPB 4294 (AY131237.1). E. mallotivora and E. papayae cause different diseases, a leaf spot on Mallotus japonicus (2) and bacterial canker on papaya, respectively. They are closely related and in the laboratory are distinguished by only very few biochemical characteristics (1). E. papayae produces a blue pigment on KB and can utilize arabinose but not mannitol. E. mallotivora does not produce a blue pigment and can utilize mannitol but not arabinose. The four Tongan strains produced a blue pigment and could utilize mannitol and arabinose. Symptoms caused by the strains isolated from Tonga are typical of those caused by E. papayae and the strains identified share most of the characteristics of E. papayae. Because the Tongan strains were able to utilize mannitol as well as arabinose and their 16S rDNA was only 97% similar to E. papayae, these strains are referred to as an E. papayae-like bacterium. The taxonomic position of these isolates will be resolved with techniques such as Multilocus Sequence Typing analysis. To our knowledge, this is the first report of this highly destructive papaya disease in the Kingdom of Tonga and of a pathogen closely related to E. papaya in the country. References: (1) L. Gardan et al. Int. J. Syst. Bacteriol. 54:107, 2004. (2) M. Goto. Int. J. Syst. Bacteriol. 26:467, 1976. (3) N. H. Maktar et al. New Dis. Rep. 17:4, 2008. (4) F. Martin-Laurent et al. Appl. Environ. Microbiol. 67:2354, 2001.

Abstract Background Burkholderia cepacia complex including B. gladioli are opportunistic pathogenic bacteria affecting the immunocompromised population. For prognosis and appropriate treatment, rapid and accurate species identification is particularly important for those diagnosed with cystic fibrosis (CF). Conventional biochemical identification techniques are insensitive and problematic for identifying Burkholderia spp., leading to common misidentification or inconclusive results. Recent studies have successfully employed attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy for rapid, reagent-free and cost-effective microbial identification. In the present study, identification of Burkholderia spp. by this technique is investigated. Methods A total of 59 isolates belonging to 7 species of Burkholderia were included in this study; all these isolates had been well-characterized by VITEK 2, 16S rRNA sequencing, random amplification of polymorphic DNA (recA typing) and/or matrix-assisted laser desorption/ionization time of flight mass spectrometry. ATR-FTIR spectra were acquired directly from colonies on 5% blood agar plates. Results A spectral database containing ATR-FTIR spectra of over 4300 bacterial isolates, encompassing over 70 genera and 190 species, was updated to include spectra of 39 isolates collected in this study and employed in the identification of the other isolates (n = 20). All isolates were correctly identified as Burkholderia by a multitier search approach. For Burkholderia species identification, spectra belonging to 39 isolates representative of all 7 species were used to construct a spectral database employed to identify the other 20 isolates [B. anthina (n = 2), B. gladioli (n = 8), B. multivorans (n = 7), and B. vietnamiensis (n = 3)]. Compared with VITEK 2 (30% correct species identification), ATR-FTIR spectroscopy correctly identified all but one isolate, resulting in overall correct species identification of 95%. Prospectively (10 months), 5 of 1100 isolates collected were identified as Burkholderia spp. by ATR-FTIR spectroscopy in concordance with VITEK 2. Conclusion ATR-FTIR spectroscopy can provide the means of rapid Burkholderia spp. identification for appropriate treatment of those diagnosed with CF. Disclosures All authors: No reported disclosures.

ABSTRACT A set of lactobacilli were investigated by polyphasic analysis. Multilocus sequence analysis, DNA typing, microarray analysis, and in silico whole-genome alignments provided a remarkably consistent pattern of similarity within the Lactobacillus acidophilus complex. On microarray analysis, 17 and 5% of the genes from Lactobacillus johnsonii strain NCC533 represented variable and strain-specific genes, respectively, when tested against four independent isolates of L. johnsonii. When projected on the NCC533 genome map, about 10 large clusters of variable genes were identified, and they were enriched around the terminus of replication. A quarter of the variable genes and two-thirds of the strain-specific genes were associated with mobile DNA. Signatures for horizontal gene transfer and modular evolution were found in prophages and in DNA from the exopolysaccharide biosynthesis cluster. On microarray hybridizations, Lactobacillus gasseri strains showed a shift to significantly lower fluorescence intensities than the L. johnsonii test strains, and only genes encoding very conserved cellular functions from L. acidophilus hybridized to the L. johnsonii array. In-silico comparative genomics showed extensive protein sequence similarity and genome synteny of L. johnsonii with L. gasseri, L. acidophilus, and Lactobacillus delbrueckii; moderate synteny with Lactobacillus casei; and scattered X-type sharing of protein sequence identity with the other sequenced lactobacilli. The observation of a stepwise decrease in similarity between the members of the L. acidophilus group suggests a strong element of vertical evolution in a natural phylogenetic group. Modern whole-genome-based techniques are thus a useful adjunct to the clarification of taxonomical relationships in problematic bacterial groups.

Knowledge of etiology causes of diarrheal illness is essential for development and implementation of public health measures to prevent and control this disease syndrome. There are few published studies examining diarrhea in children aged <5 years in Iraq. This study aims to investigate the occurrences and epidemiology of selected bacterial (Salmonella spp. and Campylobacter spp.), viral (adenovirus, norovirus GI and GII, and astrovirus), and parasitic (Entamoeba spp. and Giardia spp.) agents in stool samples from 155 child diarrheal cases enrolled between March and August 2017, in a hospital-based cross-sectional study in Thi-Qar, southeastern Iraq. Using molecular techniques and sequence-based characterization, adenovirus was the most frequently detected enteropathogen (53/155 (34.2%)), followed by Salmonella spp. (23/155 (14.8%)), Entamoeba spp. (21/155 (13.5%)), and Campylobacter spp. (17/155 (10.9%)). Mixed infection with Salmonella spp. and Campylobacter spp. was evident, and the same was revealed between various enteric viruses, particularly adenovirus and norovirus. The most frequent co-infection pattern was between adenovirus and Campylobacter spp., in seven cases (7/155 (4.5%)). Whole-genome sequencing-derived typing data for Salmonella isolates (n = 23) revealed that sequence type 49 was the most prevalent in this sample set (15/23 (65.2%)). To the best of our knowledge, this study provides the first report on detection and identification of floR, blaCARB-2, and mphA antimicrobial resistance genes in Salmonella isolated from children in the Middle East region. Logistic regression analysis pointed to few enteropathogen-specific correlations between child age, household water source, and breastfeeding patterns in relation to the outcome of detection of individual enteropathogens. This study presents the first published molecular investigation of multiple enteropathogens among children <5 years of age in Iraq. Our data provide supporting evidence for planning of childhood diarrhea management programs. It is important to build on this study and develop future longitudinal case-control research in order to elaborate the epidemiology of enteropathogens in childhood diarrhea in Iraq.

Abstract Background: Multiple-drug resistant Acinetobacter have widely spread in the last decades imposing a serious nosocomial source of infection. Nevertheless, little knowledge was gaimed on tracing the development of antibiotic resistance in Acinetobacter species. Objectives: Explore Acinetobacter spp. via antimicrobial susceptibility, plasmid profiles, and random amplified polymorphism DNA polymerase chain reaction (RAPD-PCR) typing. Methods: One hundred twelve Acinetobacter isolates (including 66 A. baumannii and 46 non-Acinetobacter baumannii strains) were obtained from three university hospitals. The source of infection of these isolates included blood, urine, wound, and respiratory tract. Their susceptibilities to 17 antibiotics were tested and then all Acinetobacter isolates were typed by plasmid analysis and RAPD-PCR method. Results: A. baumannii isolates revealed nine different patterns of antibiotic resistance. Of those, non- A. baumannii, were associated with plasmid and RAPD-PCR typings (p <0.05). A. baumannii was more resistant to multiple antibiotics than non-A. baumannii (p <0.05). Seven different plasmid profiles were observed among 112 Acinetobacter isolates. Plasmids were found in 107 (95.5%) of the 112 isolates. Unlike in RAPD-PCR typing, there was no difference between the type of Acinetobacter, A. or non-A. baumannii strains and plasmid profiles (p >0.05). By RAPD-PCR, six profiles were found for each A. and non-A. baumannii strains. The pattern 6 was the most common pattern among the isolates. Both plasmid and RAPD-PCR typing showed no association between plasmid profiling and site of infection (p >0.05). Conclusion: There is a wide spread of multi-drug resistant Acinetobacter spp., particularly A. baumannii, in the Middle East region that can be traced efficiently by plasmid and genotyping typing of Acinetobacter. More care should be taken for tracing the development of antimicrobial resistance of Acinetobacter using precise molecular typing techniques.

The study aimed to isolate and diagnose bacterial species from clean room air samples for caesarean sections, natural childbirth, sterile preterm infants halls and the sub-corridors leading to these halls and main corridors of the hospital and from the air outside hospital building with isolation and diagnosis of bacterial species from non- living surface and clinical samples that were withdrawn from inside Al-Zahra Hospital for Women and Children in AL-Muqdadiya district in Diyala Governorate – Iraq that extends at latitudes (-3.33 and -36.6) north and longitudes (-44.22 and 45.56) east, during the period from 25 December 2021 to 19 February 2022 in light of the lack of studies that are concerned with this aspect, as 192 air samples were taken distributed to the hospital halls, such as the surgery hall and its sub-corridor leading to it, the delivery hall and its sub-corridor leading to it, the sterile preterm infant unit and its sub-corridor leading to it, the main corridor of these halls, and the air outside the hospital environment, where the air samples distributed into two groups, the first included 96 samples collected in the morning before the start of work at 8 am and the second also included 96 samples collected during Work (noon) at 12 noon ; also, 208 different swabs were collected from patients included 85 swabs after surgeries (caesarean section after suture lift after 7-10 days after delivery), 60 vaginal swabs from patients inside the delivery hall and 63 swabs from newborns lying inside the sterile preterm hall. Also, swabs were collected from non-living surfaces amounting to 100 swabs withdrawn from the natural delivery bed, caesarean section, floors, ventilation outlets, anesthesia device, fluid withdrawal device, incubators for preterm infants, ventilator and dressing cart. The total number of samples withdrawn by the effective method using the SAS air sampling device was 192, with 24 samples for each site, of which 172 samples were given a positive growth rate of 89.5%, the gram-positive bacteria constituted 69%, while the gram-negative bacteria formed an isolation rate of 31%. Sixty vaginal swabs were collected from women in the delivery hall during the examination before the birth began, 33 swabs gave a positive growth rate of 55%, of which 33 were bacterial isolated, the gram negative bacteria formed the isolates by 69.7%. 63 swabs were taken from newborns in the neonatal unit (either normal or caesarean section), those who were admitted to the preterm care hall as a result of complications during childbirth (suffering from shortness, rapid breathing and high bile in the first place, and from inflammation of the respiratory tract and urinary tract infection in the second degree), 38 swabs gave a positive growth rate of 63.3%, of which 38 bacterial isolates were mostly gram-negative bacteria. 85 swabs were taken from caesarean section wounds (7-10 days after caesarean section when the suture is lifted), 29 swabs gave a positive growth rate of 34%, the majority of which were 75.7% gram-negative bacteria. Twenty-six surface swabs gave a positive growth with a rate of 26%, of which 26 different bacterial isolates. The data of the bacterial typing analysis of the three strains of E. coli (multi-resistant bacteria) used in the molecular study by using MLST technique by using housekeeping genes showed that they are globally registered strains on the site of the Institute Pasteur in France, as the results showed that two bacterial isolates, which bear numbers (17,119) of air samples and clinical samples respectively, belong to the same strain (sequence type (ST)=741), while isolates No. (72) of surface samples belong to a different strain (ST=390). Molecular typing (MLST) was performed on three isolates of K. pneumonia (multi-resistant bacteria) where housekeeping genes were used for the purpose of testing. The strains obtained are new strains that have not been isolated before in Iraq, which means that our isolates are not registered in the MLST database at the Institute Pasteur in France.

CRISPR/Cas is a molecular mechanism to prevent predatory viruses from invading bacteria via the insertion of small viral sequences (spacers) in its repetitive locus. The nature of spacer incorporation and the viral origins of spacers provide an overview of the genetic evolution of bacteria, their natural viral predators, and the mechanisms that prokaryotes may use to protect themselves, or to acquire mobile genetic elements such as plasmids. Here, we report on the CRISPR/Cas genetic structure, its spacer content, and strain epidemiology through MLST and CRISPR typing in Acinetobacter baumannii, an opportunistic pathogen intimately related to hospital infections and antimicrobial resistance. Results show distinct genetic characteristics, such as polymorphisms specific to ancestor direct repeats, a well-defined degenerate repeat, and a conserved leader sequence, as well as showing most spacers as targeting bacteriophages, and several self-targeting spacers, directed at prophages. There was a particular relationship between CRISPR/Cas and CC113 in the study of Brazilian isolates, and CRISPR-related typing techniques are interesting for subtyping strains with the same MLST profile. We want to emphasize the significance of descriptive genetic research on CRISPR loci, and we argue that spacer or CRISPR typing are helpful for small-scale investigations, preferably in conjunction with other molecular typing techniques such as MLST.

Background: Study of drug resistance of commensal bacteria in both humans and animals can determine the scale of the drug resistance problem. Usage of antimicrobials to treat infections in humans and animals has generated extensive antimicrobial pressure not only on targeted pathogens but also on commensal bacteria. Commensal Escherichia coli appears to be the major reservoir for resistant genes implicated in the transmission of genetic traits from one bacterium to another. Antimicrobial resistance in Enterobacteriaceae has increased dramatically worldwide in the last decade. An increasing number of community-onset extended-spectrum beta-lactamase (ESBL)-producing bacterial infections, especially those caused by ESBL-producing E. coli, have been reported in many countries, including Thailand. Moreover, ESBL-producing E. coli have been widely detected in food-producing animals and the environment. The increased use of ESBLs in food animals is a serious public health problem. The objective of the study was to determine the prevalence and antimicrobial resistance pattern of ESBL-producing E. coli isolated from pigs, layers, farm workers and stagnant water, in order to increase awareness about antimicrobial usage on farms and to minimize the expansion of the antimicrobial resistance phenomenon in farm settings.Materials, Methods & Results: A total of 588 samples were collected from 107 pig farms and 89 layer farms in Chiang Mai–Lamphun and Chon Buri provinces during May 2015-April 2016. Double-disk diffusion method according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines was used for detection. The results demonstrated that 36.7% (216/588) of samples were ESBL-producing E. coli-positive, including rectal swabs 74.8% (80/107), pig farm worker stool swabs 57.0% (61/107), stagnant water on pig farms 21.5% (23/107), healthy layer rectal swabs 6.7% (6/89) and layer farm worker stool swabs 51.7% (46/89). Most of the isolates were resistant against ampicillin (99.5%), followed by erythromycin (98.6%) and ceftriaxone (96.3%). All of them were classified as multidrug-resistant strains. Moreover, AMP-CRO-E-TE-C-SXT-CN was the most frequent phenotype pattern detected in animals, humans and the environment, followed by AMP-CRO-E-TE-C-SXT-NA-CN.Discussion: The present study offers clear evidence that the prevalence of ESBL-producing E. coli in healthy pigs is higher than in layers. One possible explanation is that a large amount and variety of antimicrobials are used on pig farms, resulting in a common and significant source of drug-resistant ESBL-producing E. coli. The lower incidence of ESBL-producing E. coli in samples from a pig farm environment than in samples of animal origin indicate that pigs are a reservoir of a reservoir for resistant bacteria and a source of environmental contamination. Antimicrobial resistance patterns of ESBLproducing E. coli detected in all sample types and study locations were quite similar. In almost all ESBL-producing E. coli isolates, resistance was shown against ampicillin, erythromycin, ceftriaxone, tetracycline and chloramphenicol. Moreover, multidrug resistance was found in all isolates of ESBL-producing E. coli. The differences in antimicrobial agent resistance patterns can be used to differentiate sources by employing analytical tools such as discriminant function analysis. A molecular typing protocol is recommended for use in a discriminant function analysis for pattern determination of pathogen spreading. However, genetic fingerprinting techniques for microbial source tracking are more expensive, and facilities with appropriate equipment and expertise are required.