Tesi sul tema "Human pathogenic bacterium"

Clostridioides difficile est une cause majeure de diarrhée nosocomiale. La physiopathologie de C. difficile est régie par des réseaux de régulation complexes, incluant des mécanismes basés sur l'ARN tels que les riboswitches. Les riboswitches, situés dans la région 5' non traduite des ARNm, se lient à des ligands spécifiques, induisant des changements de conformation qui modulent l'expression du gène en aval. Chez C. difficile, 16 riboswitches répondent à la molécule de signalisation c-di-GMP. Le c-di-GMP est un régulateur contrôlant la transition d'un mode de vie planctonique libre à un mode de vie sessile associé à la régulation des facteurs de virulence. Plusieurs riboswitches à c-di-GMP régulent des gènes impliqués dans la formation des flagelles, l'assemblage des pili de type IV, le développement du biofilm, l'adhésion et la production de facteurs de virulence tels que les toxines chez C. difficile. De plus, le c-di-GMP inhibe la sporulation chez C. difficile, mais le mécanisme sous-jacent n'est pas connu.Nous avons caractérisé ici des riboswitches à c-di-GMP qui n'ont pas encore été étudiés. Nos analyses bioinformatiques ont révélé que 5 d'entre eux sont situés directement en amont de gènes codant des petites protéines (PPs) de 58 acides aminés (AA). De manière intéressante, un alignement de ces 5 protéines a montré qu'elles sont presque identiques en séquence. De plus, une recherche d'homologie a permis de découvrir 2 protéines supplémentaires de 60 AA, très similaires aux cinq premières, bien que leurs gènes ne soient pas précédés d'un riboswitch. Cette nouvelle famille de protéines est conservée dans toutes les souches de C. difficile, mais n'a pas d'homologues en dehors de l'espèce. Nous avons construit une version étiquetée d'une PP et l'avons détectée par immunoblotting de fractions cellulaires, confirmant sa nature protéique et révélant qu'elle est associée à la membrane.Des données de séquençage de l'ARN (RNA-seq) ont démontré que le c-di-GMP régule négativement l'expression des 5 gènes de PP en aval des riboswitches ainsi que des 2 gènes supplémentaires. Nous avons également observé que le c-di-AMP, un autre dinucléotide cyclique impliqué dans l'osmorégulation, réprime l'expression des 7 gènes. Des expériences de fusions transcriptionnelles entre la région promotrice d'une PP et un gène rapporteur ont confirmé que le c-di-GMP nécessite le riboswitch pour moduler l'expression des gènes en aval. En revanche, le c-di-AMP régule leur expression indépendamment du riboswitch en modulant l'activité du promoteur. Ainsi, le c-di-GMP et le c-di-AMP influencent l'expression des PP par des mécanismes distincts.Pour étudier le rôle des PP chez C. difficile, nous avons surexprimé l'une d'elles et comparé son transcriptome à celui de la souche sauvage. Cela a révélé l'induction de l'expression de plus de 100 gènes impliqués dans la sporulation dans la souche surexprimant la PP. Conformément à ces données, la surexpression de cette PP a conduit à un phénotype d'hypersporulation. En outre, la délétion des 7 gènes de PP (mutant Δ7) a entraîné une réduction de la sporulation, avec des phénotypes intermédiaires pour les souches où seuls certains gènes des PP ont été délétés. Le défaut de sporulation de Δ7 est similaire à celui d'une souche produisant des niveaux élevés de c-di-GMP, suggérant que l'impact du c-di-GMP sur la sporulation pourrait être médié par la régulation des PP. Pour tester cette hypothèse, nous avons créé un mutant Δ7 produisant de fortes concentrations de c-di-GMP. Le défaut de sporulation de cette souche était équivalent à celui du mutant Δ7 non affecté dans sa production de c-di-GMP, indiquant que les effets des délétions des PP et de la surproduction de c-di-GMP ne sont pas cumulatifs.Dans l'ensemble, nos résultats démontrent que cette nouvelle famille de petites protéines est régulée à la fois par le c-di-GMP et le c-di-AMP et joue un rôle clé dans le contrôle de la sporulation chez C. difficile
Clostridioides difficile is the leading cause of nosocomial diarrhea in adults in industrialized countries. The pathophysiology of C. difficile is governed by complex regulatory networks, including RNA-based mechanisms like riboswitches. Riboswitches, located in the 5' untranslated region of mRNAs, bind specific ligands, inducing conformational changes that either promote or inhibit the expression of the downstream gene. In C. difficile, 16 riboswitches respond to the signaling molecule cyclic di-GMP (c-di-GMP). C-di-GMP acts as a second messenger and is recognized as a central regulator controlling the transition from a free planktonic to a sessile lifestyle associated with biofilm formation and virulence factor regulation. Several of the c-di-GMP-responding riboswitches have been well-studied in C. difficile and shown to regulate genes involved in flagella formation, type IV pili assembly, biofilm development, adhesion, and the production of virulence factors such as toxins. Moreover, c-di-GMP inhibits sporulation in C. difficile, but the underlying mechanism remains unclear.In this PhD work, we sought to characterize c-di-GMP-responding riboswitches that have not yet been studied. Our bioinformatics analyses revealed that 5 of them are located directly upstream of predicted genes encoding small proteins (SPs) of 58 amino acids. Interestingly, an alignment of these 5 proteins showed that they are almost identical in sequence. Moreover, a homology search uncovered two additional proteins of 60 amino acids, highly similar to the first five, though their genes are not preceded by a c-di-GMP riboswitch. This novel family of proteins is conserved across C. difficile strains but lacks homologs outside the species. We built a tagged version of one SP and detected it by immunoblotting of cell fractions, confirming its protein nature and revealing that it is primarily localized to the cell membrane.RNA sequencing (RNA-seq) data demonstrated that c-di-GMP negatively regulates not only the expression of the 5 SP genes downstream of the riboswitches but also the 2 additional genes. Unexpectedly, we also observed that c-di-AMP, another cyclic dinucleotide primarily involved in osmoregulation, repressed the expression of all seven genes. We performed reporter assays in different strain backgrounds to explore how these small proteins are regulated by both c-di-GMP and c-di-AMP. These experiments indicated that c-di-GMP required the riboswitch for modulation of downstream gene expression. In contrast, c-di-AMP regulated their expression independently of the riboswitch by modulating the promoter activity. Thus, c-di-GMP and c-di-AMP influence SP expression through distinct mechanisms.To investigate the role of these small proteins in C. difficile physiology, we overexpressed one SP and compared its transcriptome to that of the wild-type strain using RNA-seq. This revealed the upregulation of more than 100 genes involved in sporulation in the overexpressing strain. Consistent with these data, overexpression of this SP led to a hypersporulation phenotype. Furthermore, deletion of all 7 SP genes (Δ7 mutant) resulted in a significant reduction in sporulation, with intermediate phenotypes in strains where only some of the SP genes were deleted. Interestingly, the sporulation defect in the Δ7 mutant was mirrored in a strain producing elevated levels of c-di-GMP, suggesting that the impact of c-di-GMP on sporulation could be mediated by SP regulation. To test this hypothesis, we created a Δ7 mutant producing high concentrations of c-di-GMP. The sporulation defect in this strain was equivalent to that of the Δ7 mutant unaffected in its c-di-GMP production, indicating that the effects of SP gene deletions and c-di-GMP overproduction were not cumulative.Overall, our findings demonstrate that this novel family of small proteins is regulated by both c-di-GMP and c-di-AMP and plays a key role in controlling sporulation in C. difficile

Probiotic bacteria have been investigated in the prevention and treatment of various diseases and allergies. The current study was undertaken to determine the effect of eight probiotic Lactobacillus species against bacterial human skin pathogens using several techniques. Antimicrobial activity of lactobacilli against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Propionibacterium acnes was evaluated using lactobacilli broth cultures (BCB) and cell free supernatant (CFS). Antimicrobial activity was significantly greater with BCB compared with CFS especially for Lactobacillus plantarum and Lactobacillus acidophilus. Lactobacilli and pathogen aggregation, biofilm formation and adhesion to keratin were assessed. L. casei and L. plantarum were selected for further study as they showed the greatest co-aggregation (18.02 ± 1.34% with L. casei and 14.92 ± 1.45% with L. plantarum) with the pathogens (16.63 ± 1.65% with S. aureus 3761 and 14.58 ± 1.68% with P. aeruginosa) and prevention of biofilm formation by the pathogens. The antimicrobial activity of human beta defensin-2 (hBD-2) alone or with L. plantarum against pathogens was assessed. The results with hBD-2 showed that hBD-2 (10 μg / ml for 5 h) and L. plantarum together were significantly more inhibitory against S. aureus than hBD-2 alone. The presence of NaCl reduced the effectiveness of hBD-2 alone and with L. plantarum. In the presence of L. plantarum, inactivation of mprF and dlt genes led to increased binding of hBD-2 by the bacterial cell wall, and then inhibition growth of bacterial cell wall. Studies investigated the effect of exposure of methicillin resistant Staphylococcus aureus (MRSA) to the supernatant of L. plantarum the susceptibility of MRSA to β-lactams. MRSA became sensitive to β-lactams when treated with culture supernatant of L. plantarum. Gene expression studies demonstrated that the mecR1-mecI-mecA-PBP2 signalling pathway was impeded by exposure to culture supernatant of L. plantarum and β-lactams. The studies reported here demonstrate a possible alternative approach to dealing with skin pathogens, which may have clinical implications especially with regard to MRSA infections, and continued research is advised.

Coincident with human activity in recent decades, human-associated microorganisms have arrived to the Antarctic region, possibly linked to increasing presence of scientific bases and ship-borne tourists. In the Arctic, humans have been present for a very long time, and the few parts of the Arctic without human activities is decreasing with time. The studies in this thesis investigate the occurrence of different pathogens in Antarctic and Arctic wildlife, especially in birds. The first study shows the existence of Enteropatogenic Escherichia coli (EPEC) in Antarctic fur seals. The EPEC isolates were so called atypical EPECs, carrying the eae gene but lacking the bfp gene. This is the first record of a diarrheogenic E. coli in wild animals in the Antarctic. The second study displays that spreading of antibiotic resistance mechanisms appears to be much more efficient than previously was known. Enterococcus faecium isolated from Alaskan birds showed high resistance to vancomycin and teicoplanin, but also to ampicillin and ciprofloxacin. These isolates also carried vanA genes and the virulent esp gene, which places the isolates in the clinical clone CC17 and indicates the isolates had a human origin. Bacteria from birds that reside in the Bering Strait region in the third study, demonstrates that only six of 145 E. coli from 532 birds had reduced antibiotic susceptibility. Despite this, selective screen on E. coli showed only four ESBL-producing isolates. The four E. coli isolates carried CTX-M genes. One isolate belonged to the E. coli O25b - ST131 genotype, which is a successful clone with a global spread. In the fourth study, 123 seawater samples and 400 fresh penguin feces were analyzed. From these, 71 E. coli strains were isolated and only one E. coli from penguins was resistant to one antibiotic (cloramfenicol), whereas in E. coli from seawater, resistance against ampicillin, tetracycline, streptomycin and trim-sulfa were detected. E. coli carrying ESBL type CTX -M genes were also detected and Multilocus Sequencing Typing (MLST) showed six different sequence types (ST) previously reported in humans: ST131, ST227, ST401, ST410, ST685 and ST937. In the short time interval between the second study (2005) and the third study (2010) in relation to the fifth study (2012) we found a dramatic increase in antibiotic-resistant genes in the Arctic region. Enterococci, E. coli, and Kl. pneumoniae carried antibiotic resistance genes to an extent and variety not previously reported. E. coli from Arctic birds showed resistant to 1, 2, 3, 4, and 5 different antibiotics. Resistant gene type vanA was confirmed in enterococci and ESBL genes type TEM, SHV and CTX-M in E. coli and Kl. pneumoniae was detected. Multilocus Sequencing typing (MLST), indicating that both E. coli and Kl. pneumoniae carrying ESBL markers that connects them to the humans. In summary, the combined studies strengthen that bacteria that cause infections in humans could spread to relatively pristine environments. We concluded that human and associated antibiotic-resistant bacteria has reached a global level, then we showed that ESBL- carrying bacteria circulating nowadays also in the last ESBL-free continent, Antarctica.

This thesis contributes significantly to the understanding of phase-variable gene expression in Haemophilus influenzae and phasevarions in Streptococcus pneumoniae, and to the broader fields of bacterial genetics and epigenetics. This thesis has: (i) demonstrated that particular forms of lipooligosaccharide (LOS) are selected for during NTHi invasive infection; (ii) described the autotransporter Lav in NTHi, demonstrating that this protein is phase-variable, is an important virulence factor, and is a potential NTHi vaccine candidate; (iii) informed vaccine development by characterising the prevalence of Hgps across Haemophilus influenzae, while identifying expression in an invasive NTHi isolate collection; (iv) effectively doubled publicly available genomes for Haemophilus influenzae biogroup aegyptius isolates; and (v) carried out a detailed analysis of the pathobiology of the SpnIII system of S. pneumoniae. As the body of work that constitutes each Chapter of this thesis is in manuscript format, abstracts can be found in these Chapters.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
Griffith Health
Full Text

Bacterial water pollution is a significant problem because it is associated with reduction in the ‘quality’ of water systems with a potential impact on human health. Faecal indicator bacteria (FIB) are usually used to monitor the quality of water, and to indicate the presence of pathogens in water bodies. However, enumeration alone does not enable identification of the precise origin of these pathogens. This study aimed to monitor the quality of bathing water and associated fresh water in and out of the ‘bathing season’ in the UK, and to evaluate the use of microbial source tracking (MST) such as the host-specific based polymerase chain reaction (PCR) and quantitative PCR (qPCR) to recognize human and other animal sources of faecal pollution. The culture-dependent EU method of estimating FIB in water and sediment samples was performed on beach in the South Sands, Kingsbridge estuary, Devon, UK- a previously ‘problematic’ site. FIB were present at significant levels in the sediments, especially mud, as well as fresh water from the stream and pond flowing onto South Sands beach. However, the quality of bathing water was deemed to be ‘good’ and met with the EU bathing water directive 2006. Using MST it was possible to successfully classify the nature of the source from which the bacteria came. PCR was applied to detect the Bacteroides species 16S rRNA genetic markers from human sewage and animal faeces. All water and sediment samples displayed positive results with a general Bacteroides marker indicating the presence of Bacteroides species. Host-specific PCR showed the human Bacteroides genetic marker only in the sediment of the stream. However, limitations in the ‘types’ of probes available and in the persistence of these markers were identified. Thus, novel dog-specific Bacteroides conventional PCR and qPCR primer sets were developed to amplify a section of the 16S rRNA gene unique to the Bacteroides genetic marker from domestic dog faeces, and these were successfully used to quantify those markers in water samples at a ‘dog permitted’ and ‘dog banned’ beach (Bigbury-on-Sea, Devon, UK). Generic, human and dog Bacteroides PCR primer sets were also used to evaluate the persistence of Bacteroides genetic markers in controlled microcosms of water and sediment at differing salinities (< 0.5 and 34 psu) and temperature (10 and 17 ºC). The rates of decline were found did not differ significantly over 14 and 16 days for the water and sediment microcosms, respectively. Beach sediments which were studied in this project may act as a reservoir for adhesive FIB, and this was confirmed using fluorescence in situ hybridisation (FISH). The similarity in the persistence of these Bacteroides 16S rRNA genetic markers in environmental water and sediment suggests that viable but non-culturable (VBNC) Bacteroides spp. do not persist in the natural environment for long. Therefore, 16S rRNA genetic markers can be of value as additional faecal indicators of bathing water pollution and in source tracking. Thus, in this study MST methods were successfully used and in future applications, dog-specific primer sets can be added to the suite of host-specific Bacteroides genetic markers available to identify the source(s) of problem bacteria found on failing beaches.

The presence of human pathogens in water indicates the sanitary risk associated with different types of water utilization. This study surveyed the sources of human pathogens in urban waters. In order to evaluate the microbiological water quality of urban water, the enumeration of various indicator bacteria (total coliform, fecal coliform, E.coli and enterococci) is usually used.

The abundance of indicator bacteria in urban water indicates the level of fecal contamination and the presence of other human pathogens such as protozoan pathogens (Giardia lamblia & Cryptosporidium parvum).

Fecal pollution of urban waters can be from human and animal origin. Point sources of fecal contamination in an urbanized area are the effluents of urban wastewater treatment plants. While non-point sources are usually originated from diffuse sources such as (runoff from roads, parking lots, pets, leaks, failing septic systems and illegal sewer connections to storm drains). urban stormwater is considered as a major carrier for delivering human pathogens from diffuse sources to receiving waters. Increases in urban stormwater volumes have resulted from increasing urbanization and growth of impervious surfaces.

In order to reduce high amounts of human pathogens in urban waters, different methods are used nowadays to develop urban wastewater treatment plants technologies and urban stormwater management practices.

Oral Biology
M.S.
Objectives: Most bacterial species implicated as pathogens in human periodontitis are anaerobic in their metabolism. Systemic administration of metronidazole, an antibiotic specifically active against anaerobic bacteria, has been shown in multiple clinical trials to be beneficial in enhancing periodontal therapeutic outcomes beyond that attained by conventional mechanically-based forms of periodontal therapy alone, in large part by the drug inducing better reductions of major anaerobic pathogens in periodontal pockets. However, systemic metronidazole regimens in the treatment of periodontitis require multiple patient-administered drug doses per day, which may compromise treatment benefits in patients less compliant with prescribed oral drug consumption schedules. Tinidazole, a second-generation 2-methyl-5-nitroimidazole class antibiotic similar to metronidazole, also possesses marked antibacterial activity against anaerobic bacteria, and exhibits pharmacokinetic properties that enable its bioavailability with only a once-a-day oral drug dose, which may be an advantage for use in periodontitis patients unable to comply with more frequent drug dosing regimens. Little comparative data is available assessing the potential antimicrobial effects of tinidazole, as compared to metronidazole, against anaerobic periodontal pathogens, particularly “wild-type” clinical strains isolated from severely-diseased human periodontal pockets. As a result, this study tested fresh clinical subgingival isolates of selected anaerobic red and orange complex periodontal pathogens for their in vitro susceptibility to tinidazole, metronidazole, and three other antibiotics frequently employed in periodontal therapy. Methods: Paper point subgingival plaque biofilm specimens were removed from 31 adults with severe periodontitis, and transported in VMGA III medium from variousUnited States private periodontal practices to the Oral Microbiology Testing Service Laboratory at Temple University School of Dentistry. Within 24 hours, the samples were serial diluted and plated onto enriched Brucella blood agar plates with either no antimicrobials added, or supplemented with either tinidazole at 16 mg/L, metronidazole at 16 mg/L, doxycycline at 4 mg/L, amoxicillin at 8 mg/L, or clindamycin at 4 mg/L, which represent recognized non-susceptible drug breakpoint concentrations for each of the antibiotics. After incubation at 37°C for 7 days in an 85% N2-10% H2-5% CO2 anaerobic atmosphere, all plates were examined with established phenotypic criteria for selected anaerobic red and orange complex periodontal pathogens, including Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, and Fusobacterium nucleatum group species. In vitro antibiotic resistance was noted when any of the test bacterial species displayed growth on one or more of the antibiotic-supplemented enriched Brucella blood agar plates. A paired t-test compared mean total subgingival proportions of the evaluated anaerobic red and orange complex periodontal pathogens per patient which were resistant in vitro to non- susceptible drug threshold concentrations of tinidazole as compared to metronidazole, as well as to doxycycline, amoxicillin, and clindamycin, with a P-value of < 0.05 required for statistical significance. Results: The study patients yielded an average 25.8% per patient of total subgingival proportions of the selected anaerobic red and orange complex periodontal pathogens. Among these species, P. micra was isolated from all (100%) study patients, and P. intermedia/nigrescens and F. nucleatum from 93.5% and 90.3% patients, respectively, with mean subgingival proportions of these species in positive patientsranging from 1.8% to 9.7%. T. forsythia at mean subgingival levels of 1.8% was recovered from 54.8% of the patients, whereas subgingival P. gingivalis averaged 9.1% in 5 (16.1%) patients. Tinidazole and metronidazole at 16 mg/L threshold concentrations inhibited in vitro growth of all test periodontal pathogens, except for a tinidazole-resistant strain of P. intermedia/nigrescens in one patient that was additionally resistant in vitro to doxycycline, amoxicillin and clindamycin. No statistically significant differences were found between tinidazole and metronidazole in mean total subgingival proportions of anaerobic red and orange complex periodontal pathogens per patient exhibiting in vitro resistance to a 16 mg/L drug concentration (P = 0.327, paired t-test). However, significantly greater total subgingival proportions of anaerobic red and orange complex periodontal pathogens per patient were resistant in vitro to breakpoint concentrations of either doxycycline, amoxicillin, or clindamycin, as compared to tinidazole or metronidazole (all P-values < 0.006, paired t-test). Conclusions: Tinidazole performed in vitro similar to metronidazole, but significantly better than doxycycline, amoxicillin, or clindamycin, in antimicrobial activity against freshly-isolated clinical strains of human subgingival anaerobic red and orange complex periodontal pathogens. As a result of its similar spectrum of antimicrobial inhibition against anaerobic bacteria, and its more convenient once-a-day oral drug dosing properties, tinidazole may be prescribed for clinical systemic use in place of metronidazole in severe human periodontitis treatment regimens where patient compliance with multiple dose per day systemic drug consumption is anticipated to be poor or difficult to attain.
Temple University--Theses

Gram-positive pathogens are responsible for a wide range of global diseases, including nosocomial infections. The increasing incidence of antibiotic-resistant strains warrants the development of novel therapeutic strategies to combat these organisms.

In this study, a simple protocol was developed to test the survival of a human rotavirus (HRV), rhinovirus type 14 (RV-14), and parainfluenzavirus type 3 (HPIV-3) as well as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) on human hands. HPIV-3 lost nearly all of its infectivity within the first 60 minutes on the fingerpads. In contrast to this, nearly 16% of infectious RV-14 could be recovered even after 3 hours. The poor survival of HPIV-3 on human hands prompted us to investigate its survival on stainless steel disks (1 cm in diam.) under ambient conditions. The ability of the two respiratory viruses to survive well on non-porous inanimate surfaces indicates that, once contaminated, these surfaces may act as a potential virus source. To study the patterns of virus transfer, the following three experimental models were developed and tested: finger-to-finger, finger-to-disk and disk-to-finger. The results suggest that human hands and environmental surfaces, singly or in combination, have the potential to spread rotaviral infections, particularly in institutional settings. The comparatively rapid loss of HPIV-3 infectivity on hands may reduce their potential as vehicles for transmission. These results also suggest a role for environmental surfaces in the contamination of hands with respiratory viruses. We compared the efficiency of paper-, cloth- and an electric blow dryer in further reducing the level of infectious rotavirus and E. coli remaining on fingerpads washed with either 70% isopropanol, Savlon in water (1:200), an unmedicated liquid soap, or tap water alone. Irrespective of the hand-washing agent used, warm air drying produced the greatest and the cloth the smallest reduction in the numbers of both test organisms. These findings indicate the importance of the proper drying of washed hands, particularly when less effective hand-washing agents are used. The results of this study show that HRV, RV-14 and S. aureus can survive on human hands long enough to permit their spread through hands. In contrast to this, parainfluenzaviruses and E. coli appeared to be limited in their capacity to survive on hands. The differences found in the efficacy of commonly used hand-washing agents against HRV and the bacteria tested point to the importance of testing such products by proper in vivo protocols using representative viruses. (Abstract shortened by UMI.)

Studying microbial organisms at the level of single genetic variants (strains) is key not only for human pathogens but also for commensal members of the human microbiome. However, several limitations make isolation-based methods only partially effective in surveying the complexity of host-associated microbial communities. Novel biotechnological advances are revolutionizing the study of host-associated microbes, enabling the transition from low-resolution cultivation-based typing to cultivation-free metagenomic characterizations. In my doctoral work, I tested the hypothesis that appropriate analytical tools applied to genomic and metagenomic data can provide information about microbes at a resolution comparable to that of cultivation-based methods. To this end, I employed a set of integrated methods to reconstruct the genome and analyse the functional and transmission patterns of pathogenic and commensal microbes across human and non-human hosts in different contexts. We initially focused on the whole-genome sequencing of a cohort of 184 Staphylococcus aureus infections from patients with a set of diverse diseases at multiple departments of Meyer’s Children Hospital in Florence, Italy. By applying a combination of isolation-based techniques and computational analysis, we surveyed the epidemiology, transmission patterns, and genomic features associated with both highly-studied and under-investigated S. aureus clones. We identified new infective clones and two novel variants of the beta-lactam resistance cassette. We moreover profiled the virulence and resistance factors typically associated with this opportunistic pathogen, observed the dispensability of genes previously considered as putative targets for vaccine development, and tracked the transmission of a newly-emerging epidemic clone. We then focused on the challenging task of extracting strain-level genomic information from cultivation-free metagenomic sequencing of stool samples obtained from mothers and their infants during the first year of life. By applying genetic and pangenomic profiling tools, we showed that the spread of microbiome members can be inferred from metagenomes directly, and we tracked the vertical transmission of microbial strains from mother to her infant and their corresponding transcriptional profiles. This pilot study laid the foundations for larger cohort studies investigating microbiome transmission via metagenomic sequencing. The next step was the application of cultivation-free approaches to identify and survey currently neglected host-associated microbes. In order to explore those species lacking relatively close already sequenced genomes, we performed a large-scale assembly-based analysis to reconstruct high-quality microbial genomes for species and strains in the under-investigated microbiome of non-human primates (NHPs). Overall, less than one-quarter of the recovered genomes were assigned to known species or species previously observed in the human microbiome. The remaining genomes were assigned to over 1,000 new species, which improved the mappable fraction of NHP metagenomes by over 600%. The analysis of this newly-established catalog of NHP-associated species in the context of available human-associated microbial genomes further exposed the loss of biodiversity from wild and captive NHPs to non-Westernized and Westernized human populations, showing that microbiome members shared between NHPs and humans mostly belong to uncharacterized species that are heavily lifestyle-dependent. Through characterization of a cohort of Staphylococcus aureus isolates, tracking of transmission of commensals from mother to infant, and recovering of microbial dark matter associated with non-human primates, we showed that cultivation-free profiling of known and unknown host-associated species can achieve a resolution for comparative genomics that is close to that available for isolate sequencing.

Studying microbial organisms at the level of single genetic variants (strains) is key not only for human pathogens but also for commensal members of the human microbiome. However, several limitations make isolation-based methods only partially effective in surveying the complexity of host-associated microbial communities. Novel biotechnological advances are revolutionizing the study of host-associated microbes, enabling the transition from low-resolution cultivation-based typing to cultivation-free metagenomic characterizations. In my doctoral work, I tested the hypothesis that appropriate analytical tools applied to genomic and metagenomic data can provide information about microbes at a resolution comparable to that of cultivation-based methods. To this end, I employed a set of integrated methods to reconstruct the genome and analyse the functional and transmission patterns of pathogenic and commensal microbes across human and non-human hosts in different contexts. We initially focused on the whole-genome sequencing of a cohort of 184 Staphylococcus aureus infections from patients with a set of diverse diseases at multiple departments of Meyer’s Children Hospital in Florence, Italy. By applying a combination of isolation-based techniques and computational analysis, we surveyed the epidemiology, transmission patterns, and genomic features associated with both highly-studied and under-investigated S. aureus clones. We identified new infective clones and two novel variants of the beta-lactam resistance cassette. We moreover profiled the virulence and resistance factors typically associated with this opportunistic pathogen, observed the dispensability of genes previously considered as putative targets for vaccine development, and tracked the transmission of a newly-emerging epidemic clone. We then focused on the challenging task of extracting strain-level genomic information from cultivation-free metagenomic sequencing of stool samples obtained from mothers and their infants during the first year of life. By applying genetic and pangenomic profiling tools, we showed that the spread of microbiome members can be inferred from metagenomes directly, and we tracked the vertical transmission of microbial strains from mother to her infant and their corresponding transcriptional profiles. This pilot study laid the foundations for larger cohort studies investigating microbiome transmission via metagenomic sequencing. The next step was the application of cultivation-free approaches to identify and survey currently neglected host-associated microbes. In order to explore those species lacking relatively close already sequenced genomes, we performed a large-scale assembly-based analysis to reconstruct high-quality microbial genomes for species and strains in the under-investigated microbiome of non-human primates (NHPs). Overall, less than one-quarter of the recovered genomes were assigned to known species or species previously observed in the human microbiome. The remaining genomes were assigned to over 1,000 new species, which improved the mappable fraction of NHP metagenomes by over 600%. The analysis of this newly-established catalog of NHP-associated species in the context of available human-associated microbial genomes further exposed the loss of biodiversity from wild and captive NHPs to non-Westernized and Westernized human populations, showing that microbiome members shared between NHPs and humans mostly belong to uncharacterized species that are heavily lifestyle-dependent. Through characterization of a cohort of Staphylococcus aureus isolates, tracking of transmission of commensals from mother to infant, and recovering of microbial dark matter associated with non-human primates, we showed that cultivation-free profiling of known and unknown host-associated species can achieve a resolution for comparative genomics that is close to that available for isolate sequencing.

The chickens are colonised by and shed Campylobacter spp. and non-typhoidal Salmonella spp. which can be an important source of infection to humans. The most important source of Campylobacter and Salmonella infections in low- and middle-income countries, is thought to be close contact with live animals, and animal and human faecal contaminated environments. This arises as a result of unsafe water supply, and poor hygiene practices and sanitation. This study examined the risks that may contribute to the acquisition of animal-related foodborne pathogens in infants and young children in two rural districts of central Tanzania. Growth stage was commonly mentioned by questionnaire survey respondents (44.4%) as the main cause of diarrhoea in children under five years of age, among many causes listed. Among others, use of drinking water from open wells (OR = 0.46, 95% CI. 0.39 - 0.54; p < 0.001) and public tap (OR = 0.51, 95% CI. 0.44 - 0.61; p < 0.001) in the dry season was protective against childhood diarrhoea, compared to stream, river, pond or dam water. Sharing water sources with animals in the dry season (OR=1.48, 95% CI. 1.29 - 1.70; p < 0.001) and chickens roosting inside the home overnight (OR = 1.39, 95% CI. 1.20 - 1.60; p < 0.001) were associated with an increase in children diarrhoea incidences. Households headed by members of the Sukuma (p = 0.005) ethnic group and washing hands in running water (p = 0.007) were associated with higher height-for-age z-scores. Campylobacter jejuni, Campylobacter coli and Salmonella spp. were detected in chicken cloacal, latrine and kitchen floor swabs during dry and rainy seasons and several Salmonella serovars of public health importance were isolated from chickens. Prevention of gastrointestinal infections of animal origin should be directed at improving local water supplies, hygiene practices and sanitation, and introducing good animal husbandry skills which consider available resources and existing cultural practices.

Introduction: Pantoea agglomerans is a Gram negative ubiquitous bacteria commonly isolated from plant surfaces, seeds, fruits and animal/human feces usually introduced to human by ingestion of infected fruits/vegetables, thorn pricks and gastrointestinal translocation in lack of stomach acidity. However, the pathogen can also cause opportunistic human infection especially when the immune system is impaired. The aim of this case report is to investigate clinical features in a patient with P. agglomerans bacteremia and bring attention the opportunistic infection by this rare bacteria. Case presentation: We present a case of 57 year old caucasian lady with past medical history of Chronic Obstructive Pulmonary Disease, Atrial fibrillation, Immunoglobulin (IgG) deficiency, recurrent pneumonia, urine infection, oral/vaginal candidiasis, Gastro-esophageal reflux disease who presents with one week history of increased shortness of breath, chest tightness and productive cough without fever/chills. She also had high INR of 4.7 (target 2-3) despite taking normal dose of warfarin. She denies plant exposure. Her vitals were stable, saturation maintained with oxygen supplementation. Chest exam revealed very poor air entry bilaterally suggesting exacerbation of COPD. Oral thrush was present. Recent IgG level within last 6 months was low. Blood culture grew Pantoea agglomerans, pan-sensitive to most of the antibiotics. Chest X ray, CT scan abdomen and urine studies could not localize the source of infection. She was treated with Ceftriaxone, INR normalized to therapeutic range and she improved to baseline after 10 days of treatment. Discussion and conclusion: P. agglomerans is a rare cause of bacteremia which usually presents as fever, chills and general toxicity, however could also present as a cause of exacerbation of chronic diseases. Spontaneous infection can occur in a immunocompromised host, however the pathogen is of low virulence. The link between upper GI symptoms along with antacid receipt and spontaneous P. agglomerans infection could be possible, however needs further study. Hence, P. agglomerans should be considered one of the possible cause of spontaneous bacteremia in a immunocompromised host.

Mycobacterium tuberculosis (Mtb) is the pathogen causing tuberculosis (TB), a disease most often affecting the lung. 1.5 million people die annually due to TB, mainly in low-income countries. Usually considered a disease of the poor, also developed nations recently put TB back on their agenda, fueled by the HIV epidemic and the global emergence of drug-resistant Mtb strains. HIV-coinfection is a predisposing factor for TB, and infection with multi-drug resistant and extremely drug resistant strains significantly impedes and lengthens antibiotic treatment, and increases fatality. Mtb is transmitted from a sick individual via coughing, and resident macrophages are the first cells to encounter the bacterium upon inhalation. These cells phagocytose intruders and subject them to a range of destructive mechanisms, aiming at killing pathogens and protecting the host. Mtb, however, has evolved to cope with host pressures, and has developed mechanisms to submerge macrophage defenses. Among these, inhibition of phagosomal maturation and adaptation to the intracellular environment are important features. Mtb profoundly alters its phenotype inside host cells, characterized by altered metabolism and slower growth. These adaptations contribute to the ability of Mtb to remain dormant inside a host during latent TB infection, a state that can last for decades. According to recent estimates, one third of the world’s population is latently infected with Mtb, which represents a huge reservoir for active TB disease. Mtb is also intrinsically tolerant to many antibiotics, and adaptation to host pressures enhances tolerance to first-line TB drugs. Therefore, TB antibiotic therapy takes 6 to 9 months, and current treatment regimens involve a combination of several antibiotics. Patient noncompliance due to therapeutic side effects as well as insufficient penetration of drugs into TB lesions are reasons for treatment failure and can lead to the rise of drug-resistant populations. In view of the global spread of drug-resistant strains, new antibiotics and treatment strategies are urgently needed. In this thesis, we studied the interplay of the primary host cell of Mtb, human macrophages, and different Mtb phenotypes. A low-burden infection resulted in restriction of Mtb replication via phagolysosomal effectors and the maintenance of an inactive Mtb phenotype reminiscent of dormant bacteria. Macrophages remained viable for up to 14 days, and profiling of secreted cytokines mirrored a silent infection. On the contrary, higher bacterial numbers inside macrophages could not be controlled by phagolysosomal functions, and intracellular Mtb shifted their phenotype towards active replication. Although slowed mycobacterial replication is believed to render Mtb tolerant to antibiotics, we did not observe such an effect. Mtb-induced macrophage cell death is dependent on ESAT6, a small mycobacterial virulence factor involved in host cell necrosis and the spread of the pathogen. Although well-studied, the fate of ESAT6 inside infected macrophages has been enigmatic. Cultivation of Mtb is commonly carried out in broth containing detergent to avoid aggregation of bacilli due to their waxy cell wall. Altering cultivation conditions revealed the presence of a mycobacterial capsule, and ESAT6 situated on the mycobacterial surface. Infection of macrophages with this encapsulated Mtb phenotype resulted in rapid ESAT6-dependent host cell death, and ESAT6 staining was lost as bacilli were ingested by macrophages. These observations could reflect the earlier reported integration of ESAT6 into membranes followed by membrane rupture and host cell death. In conclusion, the work presented in this thesis shows that the phenotype of Mtb has a significant impact on the struggle between the pathogen and human macrophages. Taking the bacterial phenotype into account can lead to the development of drugs active against altered bacterial populations that are not targeted by conventional antibiotics. Furthermore, deeper knowledge on Mtb virulence factors can inform the development of virulence blockers, a new class of antibiotics with great therapeutic potential.

Détection et quantification de bactéries à faible taux par SPRi Résumé: Le protocole standard pour le diagnostic des bactéries dans le domaine médical et agro-alimentaire reste la culture microbienne, qui prend plusieurs jours pour identifier l'agent pathogène. Cependant, ces temps de latence ne sont pas compatibles avec l'urgence d'analyser rapidement la présence de bactéries pathogènes. Il ya un besoin croissant de vouloir développer de nouveaux outils pour identifier les bactéries pathogènes en un temps plus court. Afin atteindre cet objectif, la technologie de l'imagerie par Résonance des Plasmons de Surface (SPRi) a été utilisée avec succès pour la détection spécifique durant leur croissance des populations bactériennes en utilisant des protéines et/ou des anticorps comme molécule de bio reconnaissance des surfaces bactériennes. Ainsi, la détection spécifique de un à plusieurs milliers de pathogènes/ml peut être réalisé en quelques heures seulement. Dans le présent travail, plusieurs souches bactériennes ont été utilisées comme modèle : Streptococcus pneumoniae R6, Escherichia coli K12 ; ainsi que des agents pathogènes réels (Salmonella enteritidis) apportant la preuve que cette méthode peut être élargie à d'autres souches. Les résultats peuvent être quantitatifs par l'établissement d'une courbe d'étalonnage, permettant ainsi de remonter à la concentration initiale d'un échantillon contaminé. La stratégie développée au cours de ce travail se base sur le couplage simultané de la culture bactérienne et de la détection/identification spécifique, en utilisant l'imagerie SPR. Mots clés en français : Imagerie par résonance plasmonique de surface (SPRi), Streptococcus pneumoniae, E.coli K12, Salmonella enteritidis, croissance bactérienne, interactions, détection, capture, diagnostic, agro-alimentaire, pathogènes.

Infectious diseases are the world’s leading cause of premature deaths in humans and animals. The resistance to antibiotics and the emergence of new infectious diseases has increased the need for additional effective antimicrobial products. Despite numerous publications investigating antimicrobial activity of plant extracts it appears that no effective single product antimicrobial has yet been developed from plants. In many cases, however crude plant extracts have excellent activity and may provide useful products. Plants are frequently selected based on traditional use. Traditional healers usually use aqueous extracts of plants which in our experience generally have very low activities and it may be one of the reasons why no new products were developed from plants. Another approach to select plants for research is to use the taxonomic approach based on the premises that: (1) there is a correlation between active chemical compounds and antimicrobial activity; and (2) species in a family or order may have similar activities if the chemical precursors are inherited from a common ancestor. Future screening programmes could then concentrate on close relatives of species within these promising families and orders. The main aim of this study was to randomly screen leaf extracts of several hundred southern African tree species against important microbial pathogens to determine which taxa have the highest activity and may yield useful products to treat infections in human and animal health markets. A wide selection of plant species improved the possibility of finding promising extracts and has the advantage that active compounds may be discovered from plants that are not used traditionally. To ensure sustainable use only leaves of trees were examined. A spin off of this study would also indicate the susceptibility of different organisms, correlate the antimicrobial activities of the different organisms and determine what minimum inhibitory concentration (MIC) represents a good activity based on investigating many extracts against many microbes. The antimicrobial activity was determined by using a sensitive serial dilution microplate method. Acetone extracts were tested against two Gram-positive bacteria, two Gram-negative bacteria and two fungi, i.e. Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and Cryptococcus neoformans. Small and mostly insignificant differences were found between the susceptibility of the microbial pathogens to the extracts. E. faecalis was the most sensitive bacterium and C. neoformans the most sensitive fungal organism. The strongest correlations in activities among the pathogens were between C. albicans and C. neoformans, and among the pathogen classes between Gram-positive and Gram-negative bacteria. The tree extracts analysed in the present study had a wide range of activities against the different pathogens. Twenty six per cent of the extracts inhibited the pathogens at MIC levels of 0.16 mg/ml. This clearly shows that 0.16 mg/ml is not low enough to discriminate between promising species. Some of the extracts inhibited the growth of more than one pathogen while other extracts had selective activities and could be the most promising to follow up. The study identified families and orders with either statistically significantly higher or lower antimicrobial activities. Among the large families, Combretaceae and Fabaceae had high mean activities against all test pathogens. The families Anacardiaceae and Moraceae had high activities against both Gram-positive and Gram-negative bacteria whereas the families Proteaceae and Meliaceae had higher antifungal activities. Among the large orders, Fabales had relatively high activities against all the pathogen classes. Considering that plants in related taxa often contain similar compounds and therefore similar activities, future studies could analyse more representative species in the promising taxa. Many tree species, genera, families and orders, including well-known and lesser known medicinal taxa in southern Africa, were identified with promising activities. To evaluate the potential use of these results, additional cytotoxicity, phytochemical and pharmacological studies should be carried out. The study, although still exploratory, underlined the potential of southern African tree extracts as sources of antimicrobial products. Application of these results within the Phytomedicine Programme has led to patents and products that were as good as commercial products in animal and field trials. We hope that our results will provided a starting point for discovering new products with useful activities.
Thesis (PhD)--University of Pretoria, 2014.
gm2014
Paraclinical Sciences
Unrestricted

Human excreta contain plant nutrients and have the potentialto be used as a fertiliser in agriculture. Urine contributesthe major proportion of the nutrients (N, P and K) in domesticwastewater whereas faeces contribute a smaller amount andinvolves greater health risks if reused due to the possiblepresence of enteric pathogens. Human urine does not generallycontain pathogens that can be transmitted through theenvironment.

Source-separation of urine and faeces is possible by usingurine-separating (or urine-diverting) toilets, available assimple dry toilets or porcelain flush toilets with dividedbowls. The risk for transmission of disease when handling andreusing the urine is largely dependent on thecross-contamination by faeces. In this research, the presenceof human faeces in urine samples was successfully determined byanalysing for faecal sterols. Cross-contamination was evidentin 22% of the samples from urine collection tanks, and in thesequantified to an average (± SD) of 9.1 ± 5.6 mgfaeces per litre urine. Testing for indicator bacteria wasshown to be an unsuitable method for determining faecalcontamination in human urine sinceE. colihad a rapid inactivation in the urine and faecalstreptococci were found to grow within the system.

The fate of any enteric pathogens present in urine iscrucial for the risk for transmission of infectious diseases.Gram-negative bacteria (e.g.SalmonellaandE. coli) were rapidly inactivated (time for 90%reduction, T₉₀<5 days) in source-separated urine at itsnatural pH-value of 9. Gram-positive faecal streptococci weremore persistent with a T₉₀of approximately 30 days. Clostridia sporenumbers were not reduced at all during 80 days. Similarly,rhesusrotavirus andSalmonella typhimuriumphage 28B were not inactivated inurine at low temperature (5°C), whereas at 20°C theirT₉₀-values were 35 and 71 days, respectively.Cryptosporidiumoocysts were less persistent with a T₉₀of 29 days at 4°C. Factors that affect thepersistence of microorganisms in source-separated human urineinclude temperature, pH, dilution and presence of ammonia.

By using Quantitative Microbial Risk Assessment (QMRA), therisks for bacterial and protozoan infections related tohandling and reuse of urine were calculated to be<10^-3for all exposure routes independent of the urinestorage time and temperature evaluated. The risk for viralinfection was higher, calculated at 0.56 for accidentalingestion of 1 ml of unstored urine. If the urine was stored at20°C for 6 months the risk for viral infection was reducedto 5.4 × 10^-4.

By following recommendations for storage and reuse, whichare dependent on the type of crop to be fertilised, it ispossible to significantly decrease the risk for infections. Sofar, the level of risk that is acceptable is unknown. Theacceptable risk will be one of the main factors determining thefuture utilisation of source-separated human urine inagriculture.

Keywords:urine-separation, urine, wastewater systems,wastewater reuse, recycling, enteric pathogens, faecal sterols,indicator bacteria, hygiene risks, microbial persistence,microbial risk assessment, QMRA, fertiliser, crop.

Streptococcus pyogenes, également appelé Streptocoque du Goupe A (SGA), est un pathogène à l'origine d'une grande diversité d'infections, allant d'infections superficielles comme l'angine aux infections invasives, comme la fasciite nécrosante et les endométrites. Au 19ème siècle, une femme sur dix mourait après l'accouchement de fièvre puerpérale, notamment d'endométrite. En France, les infections gynéco-obstétricales correspondent encore de nos jours à 27 % des infections invasives à SGA chez les femmes. Les souches de SGA présentent une forte diversité génétique et de répertoire de facteurs de virulence. Le génotype emm28 est le troisième génotype le plus prévalent en France et il est associé aux endométrites. Nous avons analysé par deux axes complémentaires les facteurs et mécanismes impliqués dans les endométrites à SGA. Par des approches de biochimie et de biologie cellulaire, nous avons caractérisé l'interaction entre les cellules de l'hôte et R28, une protéine de surface spécifique du génotype emm28. Le domaine N-terminal de R28 (R28Nt) et ses deux sous-domaines favorisent la fixation des bactéries à des cellules endométriales, cervicales et déciduales. Ils fixent de manière directe les intégrines α3β1, α6β1 et α6β4. Par ailleurs, R28Nt promeut aussi l'adhésion à des cellules épithéliales de la peau et des poumons. Ces résultats suggèrent que la fixation des intégrines par R28Nt concourt, non seulement, aux endométrites dues au génotype emm28, mais aussi, et de manière plus générale, à la prévalence de ce génotype. Afin de mieux caractériser les étapes précoces essentielles au développement des endométrites à SGA, nous avons développé un modèle original d'infection : nous infectons ex vivo la décidue humaine, qui correspond à la membrane utérine durant la grossesse. Nous avons analysé les effets de l'infection de la décidue par des techniques de microscopie et d'analyse d'image de pointes. SGA adhère au tissu et se multiplie au contact de celui-ci grâce à des éléments sécrétés par le tissu. Sur ce tissu, SGA forme des biofilms composés d'ultrastructures ressemblant, pour certains, à des fils reliant deux coques d'une même chaine et, pour d'autres, à des filaments reliant plusieurs chaînettes ; certains s'organisent en réseau. GAS envahit en profondeur le tissu, ce qui dépend de l'expression de la cystéine protéase SpeB. SGA induit la mort de la moitié des cellules en moins de 4 h à travers la sécrétion de différents facteurs, dont la Streptolysine O (SLO). Enfin, GAS est capable de restreindre la réponse immunitaire du tissu à l'échelle transcriptomique et protéique, le contrôle protéique dépendant de l'expression de SLO et de SpeB
Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is a Gram-positive pathogen responsible for a wide range of diseases. GAS induces superficial infections such as pharyngitis, with 700 million cases/year worldwide, life threatening invasive infections such as necrotizing fasciitis, with 160 000 deaths, and post-infectious sequelae such as rheumatic fever. Altogether, GAS is responsible for 517 000 deaths in the world annually. GAS strains are genetically diverse and their genotyping involves the sequencing of the emm gene 5' end; emm encodes the M protein, a major virulence factor. More than 250 emm-types have been identified and they harbor specific virulence factor repertoires. During the pathogenesis of GAS infections, GAS adheres to the tissue, multiplies, affects the tissue integrity and invades it, resists and controls the immune response. During my PhD, we focused on deciphering the molecular mechanisms involved in GAS early critical steps of human tissue infection, with gyneco-obstetrical sphere infections, including endometritis, as a model. The first to third most prevalent emm-types eliciting invasive infections in Europe, emm28, is associated with these infections. In a first part of my project, we sought receptors for the emm28 specific R28 surface protein and which domains are involved in promoting adhesion to cells. In the second part, we set up an innovative ex vivo model of human decidual infection and we characterized the contribution of virulence factors to the colonization and invasion of this tissue. By cell adhesion experiments, we show that the R28 N-terminal domain (R28Nt) is responsible for an increase of GAS adhesion to human primary decidual cells. We have subdivided R28Nt into two subdomains; each is involved in binding to decidual, cervical and epithelial endometrial cells. We identified several putative R28Nt receptors and focused on R28Nt interaction with integrins. R28Nt and both subdomains directly interact with the integrins alpha3beta1, alpha6beta1 and alpha6beta4. Since R28Nt also increases the binding to the surface of pulmonary and skin epithelium cells, tissues encountered in GAS induced infections, we suggest that the R28Nt-integrin interactions contribute not only to emm28 endometritis, but also to the overall prevalence of the emm28 strains. In the second part of my project, to better characterize the mechanisms involved in GAS infections, we developed an ex vivo model of tissue infection, using human decidua, a tissue encountered during endometritis. We infected the maternal side of feto-maternal membrane, i. e. decidua, from healthy caesarians with an emm28 endometritis clinical isolate and its derived mutants. Using state of the art imaging set-up, image processing and analysis, we followed and quantified in real time different early infection steps. The bacteria multiply until they colonize the entire tissue surface in up to eight hours and this multiplication is triggered by the tissue. The bacteria form a multilayer biofilm of up to 14 µm thick. GAS readily and actively invades the decidua in a time-dependent manner, which depends on the presence of the cysteine protease SpeB. GAS induces dramatic cell cytotoxicity, with up to 50% of cells killed in the first four hours of infection; Streptolysin O (Slo) is involved in this cytotoxicity, confirming the critical importance of this factor in the early steps of infection. Cytokine overexpression and secretion in the tissue after infection indicate that GAS induces a limited immune response and the inflammatory response does not critically depend on the presence of Slo or SpeB. In conclusion, this study demonstrates the importance of several virulence factors, R28, SpeB and Slo, in the GAS emm28 early steps of infection, such as colonization, biofilm formation, cytotoxicity and tissue invasion, indicating their involvement not only in endometritis, but in other emm28 infections

L'objectif principal de ce travail etait d'explorer les bactéries pathogènes que recèle le tube digestif des gorilles. Pour ce faire, un total de 48 échantillons de selles, provenant du Cameroun, appartenant à 21 gorilles ont été analysés. D'abord la culturomique et le pyroséquençage ont été utilisés pour évaluer exhaustivement la diversité bactérienne. En appliquant la culturomique, 86 conditions de culture dont des milieux fabriqués à base de plantes tropicales, sur un échantillon de selles de gorille, 12 800 colonies microbiennes ont été isolées et testées, et 147 espèces bactériennes identifiées. De nombreux pathogènes opportunistes ont été observés, dont 8 qui sont fréquemment associés à des maladies chez l'homme: Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus et Clostridium botulinum. En utilisant la PCR en temps réel pour cribler des pathogènes bactériens dans les 48 échantillons de selles de gorilles, des bactéries fastidieuses telles que Bartonella spp. Borrelia spp., Coxiella burnetii, Tropheryma whipplei ont été observées. Nous avons estimé la prévalence de ces agents pathogènes qui varie entre 4,76% et 85,7%. Ce travail a permis de savoir que l'homme et le gorille ont en commun plusieurs espèces bactériennes dont des pathogènes émergents. Par conséquent, les gorilles sauvages peuvent servir de réservoir et de source pour l'émergence et/ou la réémergence des bactéries pathogènes pour l'homme
The main objective of this work is to explore the gorilla's potential role as a reservoir for pathogenic bacteria. We used both microbial culturomics and pyrosequencing to analyze the gorilla gut bacteria. By applying culturomics to one index gorilla, we tested 12,800 colonies and identified 147 different bacterial species, including 5 new species. Many opportunistic human pathogens were observed, including 8 frequently associated with human disease: Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus and Clostridium botulinum. Using specific real-time PCR on 48 gorilla fecal samples, we also observed the fastidious pathogens Bartonella spp. Borrelia spp., Coxiella burnetii, Tropheryma whipplei. Using microsatellite analysis of the gorilla samples, we estimated that the prevalence of these pathogens was between 4.76% and 85.7%. Therefore, the gorilla shares many bacterial pathogens with humans, which suggests that wild gorillas might be a reservoir for the emergence and/or reemergence of these pathogens, especially in areas where human and gorilla habitats overlap and because of the increasing presence of humans in the African equatorial forests

L'objectif de cette thèse est d'appliquer la méthode d'identification bactérienne par spectrométrie de masse MALDI-TOF pour une utilisation en routine dans un laboratoire de microbiologie clinique. Dans un 1er temps et de manière prospective, nous avons évalué la performance et le coût-efficacité de l'identification bactérienne de routine par MALDI-TOF par rapport aux techniques conventionnelles d'identification phénotypique. Durant la période des 16 semaines d'étude, nous avons comparé la performance de la technique par MALDI-TOF aux techniques conventionnelles d'identification phénotypique comprenant la coloration de Gram, la galerie API ANA et le Vitek 2. En cas de résultats discordants entre ces deux techniques, l'identification était réalisée par biologie moléculaire. Nous avons montré que le MALDI-TOF est un moyen efficace et rentable pour l'identification des bactéries de routine. Le MALDI-TOF peut être utilisée en 1ère intention dans l'identification bactérienne avant l'ensemble de techniques phénotypiques. Dans un 2ème temps, nous avons évalué rétrospectivement la performance et le coût-efficacité de l'utilisation exclusive de MALDI-TOF en diagnostic bactériologique de routine en comparaison avec les techniques conventionnelles. En analysant les données des 11 dernières années, nous avons montré que le MALDI-TOF est efficace et tout à fait adaptée pour l'identification d'espèce bactérienne en routine. Nous avons également prouvé que MALDI-TOF est un outil puissant pour identifier les espèces bactériennes rarement impliquées dans les infections humaines. Cette technique pourrait être une alternative aux méthodes moléculaires dans le laboratoire clinique
The objective of this thesis is to apply the method of bacterial identification by MALDI-TOF MS in daily practice in a routine clinical microbiological laboratory. Firstly, we prospectively evaluated the performance and the cost-effective of bacterial identification by MALDI-TOF in comparison with conventional phenotypic identification methods. During a 16-week study, we compared the performance of MALDI-TOF with conventional techniques of identification including Gram staining, API ANA identification strip and automated identification using the Vitek 2. The unmatched identifications between MALDI-TOF and conventional methods were resolved by molecular identification. In this study, we showed that MALDI-TOF was an effective tool and less expensive for the rapid identification of bacterial species in clinical microbiology laboratory. MALDI-TOF can be used in first intention for identification before Gram staining or other phenotypic identification techniques based on physicochemical properties of bacteria. Secondly, we retrospectively evaluated the performance and the cost-effectiveness of the exclusive use of MALDI-TOF in bacteriological diagnosis in comparison with conventional phenotypic identification. 11-year retrospective analysis of data showed that MALDI-TOF was efficient and completely adapted for the routine identification of bacterial species. We also showed that MALDI-TOF had capacity to identify bacterial species that were rarely involved in human diseases. This technique could be an alternative to molecular methods in the clinical laboratory

Les gastroentérites aiguës affectent chaque année entre un quart et la moitié des personnes dans le Monde. Elles sont causes de morbidité, de mortalité et de coûts de santé importants. Leur transmission directe ou indirecte via l’eau, les aliments, l’air ou les surfaces inertes dépend de leur étiologie (virale, bactérienne ou parasitaire) et du contexte local. Bogotá et sa région présentent plusieurs spécificités : des eaux usées rejetées en rivière souvent sans ou après seulement un traitement primaire, la mise en décharge des papiers toilettes, couches et protections souillés par les excréments, et une consommation de fruits et légumes faible et limitée à des produits bon marché irrigués par des eaux pouvant être contaminées fécalement. Notre thèse visait à évaluer les flux de certains pathogènes entériques de l’Homme dans l’environnement à proximité de Bogotá et à essayer de relier ces flux à la santé de la population.La thèse a associé trois contributions. Premièrement, une méthode de culture du norovirus humain a été mise au point en utilisant des villosités intestinales isolées de souris comme modèle cellulaire présentant toute la diversité des cellules épithéliales intestinales. Plusieurs concentrations en trypsine ont été testées pour activer les norovirus ; la méthode a été appliquée à des échantillons fécaux et environnementaux. Deuxièmement, les contaminations en E. coli et en pathogènes entériques de l’Homme ont été suivies dans des eaux (lixiviat de décharge, eau de ruissellement, rivière, eau d’irrigation, eau potable), des légumes-feuilles mangés crus (blettes) et l'air (au-dessus d’une décharge, en zone rurale, en zone urbaine) dans la région de Bogotá. Troisièmement, l’impact des contextes socioéconomiques et des pratiques individuelles (alimentation, hygiène et santé) sur les cas de gastroentérites aiguës a été testé à partir d’enquêtes réalisées dans un district de Bogotá et analysées par divers outils (analyse en composante principale, modélisation …).Nous avons montré que les villosités intestinales isolées de souris permettent l'infection et la réplication du norovirus humain. Le virus doit être activé avec de la trypsine et a un cycle réplicatif moyen de 10 h. Les villosités sont efficaces pour obtenir un matériel biologique abondant et sont idéales pour étudier l'activité biologique du norovirus ou générer des anticorps. Elles ont permis de voir des norovirus non détectés par méthode moléculaire dans certains excréments ou échantillons environnementaux ; les échantillons positifs par méthode moléculaire ou en immunodot-blot contenaient quasiment tous des norovirus infectieux. Au niveau régional, les rejets d'eaux usées dans les rivières Bogotá et Balsillas et dans le marais Tres Esquinas contaminent le réseau d'irrigation de La Ramada au nord-ouest de Bogotá en E. coli et potentiellement en pathogènes entériques de l’Homme. Les blettes récoltées dans cette zone étaient fortement contaminées, en contraste d’autres zones de culture. Leur contamination évoluait de leur production à leur achat dans les commerces de proximité, les lavages pouvant être contaminants ou décontaminants, les manipulations sur l’étal des marchands étant contaminantes. L’air était souvent contaminé par E. coli et par Shigella spp., sans pouvoir attribuer à la décharge Doña Juana un rôle particulier. La présence de Shigella spp. était observée parallèlement dans plus de la moitié des selles des personnes diarrhéiques. Les enquêtes réalisées ont montré que la fréquence annuelle des gastroentérites aiguës diminuait avec l’accroissement de l’âge des personnes ; elle semblait plus faible dans les foyers avec personnes âgées, peut-être en lien avec des pratiques plus strictes en matière d’hygiène, alimentaire notamment
Acute gastroenteritis affect between a quarter and a half of people in the World each year. They are responsible for significant morbidity, mortality and healthcare costs. Their direct or indirect transmissions via water, food, air or inert surfaces depend on their aetiology (viral, bacterial or parasitic) and the local context. Bogotá and its region have several specificities: wastewater are often discharged into rivers without or after primary treatment only, the deposit in landfill of toilet papers and diapers soiled by excrement, and the low consumption of fruits and vegetables largely restricted to a handful of relatively cheap products that may be irrigated by surface freshwaters heavily contaminated with faeces. Our PhD aimed to assess the fluxes of some human enteric pathogens in the region of Bogotá and to try to relate these fluxes to the population health. The PhD combined three contributions. First, a method for culturing the human norovirus has been developed using isolated mouse intestinal villi as a cell model exhibiting the full diversity of intestinal epithelial cells. Several concentrations of trypsin were tested to activate noroviruses; the method was applied to faecal and environmental samples. Second, contamination with E. coli and some human enteric pathogens was monitored in water (landfill leachate, runoff water, river, irrigation water, drinking water), leafy vegetables eaten raw (chards) and air (above a landfill, in rural areas, in urban areas) in the Bogotá region. Third, the impact of socioeconomic contexts and individual practices (food, hygiene and health) on cases of acute gastroenteritis was assessed from surveys carried out in one district of Bogotá and analysed by various tools (principal component analysis, modelling …). We have shown that mouse isolated intestinal villi allow the infection and replication of human norovirus. The virus has to be activated with trypsin and has an average replicative cycle of 10 h. Villi are efficient in obtaining abundant biological material and are ideal for studying the biological activity of norovirus or for generating antibodies. They made it possible to see infectious noroviruses not detected by molecular method in several faeces and environmental samples; almost all samples positive by molecular method or immunodot-blot contain infectious noroviruses. At the regional level, the discharges of wastewater in the Bogotá and Balsillas rivers and in Tres Esquinas march contaminate the irrigation network of La Ramada area in the northwest of Bogotá with E. coli and potentially human enteric pathogens. Chards harvested in this area were heavily contaminated, in contrast to other growing areas. Their contamination evolved from their production to their purchase in nearby stores, washings increasing or decreasing their contamination, and handling on the merchant's stalls increasing contamination. The air was often contaminated with E. coli and Shigella spp.; it was not possible to detect a particular contribution of the Doña Juana landfill in pathogen aerosolization. The presence of Shigella spp. was observed in parallel in more than half of the stools of people with diarrhoea. Surveys have shown that the annual frequency of acute gastroenteritis decreases with increasing age; it seemed less common in households with elderly people, possibly due to stricter food hygiene practices. A transmission model of acute gastroenteritis distinguishing contamination from outside the households and contaminations between people in the same households did not show significant differences between neighbourhoods. Used to simulate numerical experiments, it suggests working on much higher numbers of surveys
La gastroenteritis aguda afecta entre una cuarta parte y la mitad de las personas en el mundo cada año. Son responsables de importantes costos de morbilidad, mortalidad y asistencia sanitaria. Sus transmisiones directas o indirectas a través del agua, alimentos, aire o superficies inertes dependen de su etiología (viral, bacteriana o parasitaria) y del contexto local. Bogotá y su región aledaña tienen varias especificidades: las aguas residuales a menudo se vierten a los ríos sin o solo después de un tratamiento primario, el depósito de papel higiénico y pañales sucios con excrementos son dispuestos generalmente en un relleno sanitario, y el bajo consumo de frutas y verduras restringido en gran medida a un puñado de productos relativamente baratos pueden ser irrigados por aguas dulces superficiales muy contaminadas con excrementos. Nuestra tesis doctoral tuvo como objetivo evaluar los flujos de algunos patógenos entéricos humanos en la región de Bogotá y tratar de relacionar estos flujos con la salud de la población. El doctorado combinó tres contribuciones. En primer lugar, se desarrolló un método para cultivar el norovirus humano utilizando vellosidades intestinales aisladas de ratón como modelo celular que exhibe la diversidad completa de células epiteliales intestinales. Se probaron varias concentraciones de tripsina para activar norovirus; el método se aplicó a muestras fecales y ambientales. En segundo lugar, se evidenció la contaminación de E. coli y patógenos entéricos humanos en el agua (lixiviados de vertedero, agua de escorrentía, río, agua de riego, agua potable), vegetales de hoja que se comen crudos (acelgas) y aire (sobre un vertedero sanitario, así como en áreas rurales y urbanas) en la región de Bogotá. En tercer lugar, se evaluó el impacto de los contextos socioeconómicos y las prácticas individuales (alimentación, higiene y salud) frente a los casos de gastroenteritis aguda a partir de encuestas realizadas en una localidad de Bogotá y analizadas mediante diversas herramientas (análisis de componentes principales, modelización…). Con este doctorado, hemos demostrado que las vellosidades intestinales aisladas de ratón permiten la infección y la replicación del norovirus humano. El virus debe activarse con tripsina y tiene un ciclo replicativo promedio de 10 h. Las vellosidades son eficaces para obtener abundante material biológico y son ideales para estudiar la actividad biológica de los norovirus o para generar anticuerpos. Ellas permitieron ver norovirus infecciosos no detectados por método molecular en varias heces y muestras ambientales; casi todas las muestras positivas por método molecular o inmunodot-blot contienían norovirus infecciosos. A nivel regional, los vertidos de aguas residuales en los ríos Bogotá y Balsillas y en el humedal Tres Esquinas contaminan la red de riego La Ramada en el noroeste de Bogotá con E. coli y potencialmete con patógenos entéricos humanos. Las acelgas recolectadas en esta área resultaron muy contaminadas, a diferencia de otras áreas de cultivo. Su contaminación evolucionó desde la producción hasta su compra en las tiendas cercanas, los lavados aumentaron o disminuyeron su contaminación y la manipulación en los puestos de comercio aumentaron la contaminación. El aire a menudo estaba contaminado con E. coli y Shigella spp., sin poder atribuir al relleno sanitario Doña Juana un rol particular. A su vez la presencia de Shigella spp. se observó en paralelo en más de la mitad de las deposiciones de personas con diarrea. Las encuestas demostraron que la frecuencia anual de gastroenteritis aguda disminuye respecto al aumento en edad; parecía menos común en hogares con personas mayores, posiblemente debido a prácticas de higiene alimentaria más estrictas. Un modelo de transmisión de gastroenteritis aguda que distinguió la contaminación fuera de los hogares y las contaminaciones entre personas dentro de los mismos hogares no mostró diferencias significativas entre vecindarios

Treponema pallidum ssp. pallidum is the causative agent of syphilis, a multi-stage bacterial infection, transmitted sexually or from mother-to-child, with an unparalleled range of symptoms arising from the ability of treponemes to penetrate any tissue and cross immune privileged endothelial barriers to access the brain, the eye, and the fetus. Further, without treatment T. pallidum evades immune clearance and persists within the host to establish a chronic infection. These characteristics suggest that T. pallidum may have evolved unique mechanisms for immune escape and to mediate host-cell interactions. The findings presented in this dissertation contribute to our knowledge of T. pallidum pathogenesis by investigating a previously unexplored host-cell interaction, between T. pallidum and human platelets. These results validate the hypothesis that, as a pathogen which successfully utilizes vascular dissemination, T. pallidum would not only encounter, but interact with human platelets, complex cells now viewed as vascular sentinels that participate in many host-pathogen interactions. This is the first study to demonstrate that T. pallidum interacts with human platelets and to characterize and quantify these interactions using high resolution microscope imaging techniques (video and frame analysis). These interactions were shown to be complex, reversible and mediated by motile treponemes localizing to stationary, (slide-adhered) activated platelets, versus to free-floating, inactive platelets. In addition, it was found that T. pallidum discriminates between the level of platelet activation and preferentially localized to the most activated platelet. Treponema pallidum was also able to induce platelet activation following an extended lag period. Modified chemotaxis assays quantified by flow cytometry, were used to investigate the migration of T. pallidum in response to the plasma of platelets differentially activated with infection-relevant host components (thrombin, collagen). The results herein reveal that T. pallidum discriminates between different mechanisms of platelet activation, with a significant preference towards the secretions of collagen-activated platelets (under these experimental conditions), compared with that of inactive or thrombin-activated platelets. Previously, T. pallidum chemotaxis had been investigated through genomic characterization and molecular interaction studies with recombinant proteins. This investigation is the first time live T. pallidum was utilized for in vitro chemotaxis assays and is also the first study of pathogen chemotaxis in response to the secretions of differentially activated platelets. The body of work in this dissertation provides a foundation to further investigate the role of T. pallidum-platelet interactions during infection, adding a new host-cell interaction to our understanding of T. pallidum pathogenesis. The evidence that the molecular gradients of host components can affect T. pallidum migration suggests an important role for chemotaxis during T. pallidum infection. Together, the characterization of platelet-interactions and treponeme chemotaxis in response to host components, adds to our knowledge of T. pallidum-host interactions, and eludes to additional pathogenic strategies that may facilitate T. pallidum dissemination and immune evasion.
Graduate
2021-12-14

The major objective of the present research work was to determine the effect of sulphur dioxide (SO2) on probiotic bacterial strains (Lactobacillus acidophilus KI, Lactobacillus rhamnosus R11, Lactobacillus plantarum , Bifidobacterium animalis Bo, and Bifidobacterium animalis Bb12) and pathogenic bacterial strains that could affect gastrointestinal system (GIT) (Listeria monocytogenes, Escherichia coli, Salmonella enteritidis, and Bacillus cereus) using an in vitro simulation model. In a first step, growth curves were performed for both probiotic and pathogenic strains in culture media supplemented with SO2 at 1000 mg/L and 500 mg/L and the optical densities were registered. It was observed that SO2 at 500 mg/L did not cause significant reduction of any of the microorganism, however, the higher concentration of SO2 (1000 mg/L) exhibited an inhibitory effect on B. animalis Bo. As for the pathogenic strains, only L. monocytogenes and in a less extent E. coli were inhibited when treated with SO2 (1000 mg/L). The evaluation of the effect of SO2 at 1000 mg/L on the viability of B. animalis Bo was further performed and a reduction of 1 log was registered. When exposed to GIT conditions, the SO2 showed to somewhat protect pathogenic strains from stomach conditions. At intestinal simulated conditions an inactivation effect of SO2 at 1000 mg/L on B. animalis Bo was observed (1.9 log reduction). The other probiotic strains did not suffer a significant inactivation effect, in fact, it seems that are protected by the presence of SO2. Along the GIT simulation, the concentration of SO2 did not change significantly, but according with the zeta potential the chemical form of the compound changes from sulphite (SO32-) to bisulphites (HSO3-) and then to sulphur dioxide (SO2), with the pH modulation from gastric to intestine conditions. Also, the metabolism of B. animalis Bo in the presence of SO2 (1000 mg/L) is highly affected especially the glucose consumption. But no changes were observed in the production of organic acids such as acetic, and propionic acids, but lactic and citric acids were highly affected, succinic acid was somehow inhibited, but no production of butyric acid was observed. As a conclusion, in general, SO2 is not harmful for gut microbiota if ingested with food and it’s a safe compound to use for food preservation. Nevertheless, if ingested jointly with contaminant bacteria or when finding these bacteria at gut, this compound don´t perform any antimicrobial effect.
O presente trabalho de investigação teve como principal objetivo determinar o efeito do dióxido de enxofre (SO2) em estirpes bacterianas probióticas (Lactobacillus acidophilus KI, Lactobacillus rhamnosus R11, Lactobacillus plantarum, Bifidobacterium animalis Bo e Bifidobacterium animalis Bb12) e estirpes bacterianas patogénicas que podem afetar o sistema gastrointestinal (TGI) (Listeria monocytogenes, Escherichia coli, Salmonella enteritidis e Bacillus cereus) usando um modelo de simulação in vitro. Num primeiro passo, foram realizadas curvas de crescimento de estirpes probióticas e patogénicas em meios de cultura suplementados com SO2 a 1000 mg/L e 500 mg/L com base em medições de densidade óptica. Observou-se que o SO2 a 500 mg/L não causou redução significativa de nenhum dos microrganismos, no entanto, a concentração de 1000 mg/L exibiu um efeito inibitório em B. animalis Bo. Quanto às estirpes patogénicas, apenas L. monocytogenes e, em menor extensão, E. coli foram inibidas quando tratadas com SO2 (1000 mg/L). O efeito do SO2 a 1000 mg/L na viabilidade de B. animalis Bo foi posteriormente avaliado tendo-se observado uma redução de 1 log. No sistema gastrointestinal, o SO2 mostrou um efeito de proteção das estirpes patogénicas em relação às condições estomacais. Nas condições simuladas do intestino, foi observado um efeito de inativação do SO2 a 1000 mg/L em B. animalis Bo (redução de 1,9 log). As demais estirpes probióticas não sofreram efeito significativo de inativação, de facto, parece que são protegidos pela presença de SO2. Ao longo da simulação TGI, a concentração de SO2 não se alterou significativamente, mas de acordo com o potencial zeta, a forma química do composto muda de sulfito (SO32-) para bissulfitos (HSO3-) e depois para dióxido de enxofre (SO2), com a mudança do pH das condições gástricas para intestinais. Além disso, o metabolismo de B. animalis Bo na presença de SO2 (1000 mg/L) foi afetado, especialmente o consumo de glicose. Mas não foram observadas alterações na produção de ácidos orgânicos, como os ácidos acético e propiônico, mas os ácidos lático e cítrico foram altamente afetados, o ácido succínico foi de alguma forma inibido, mas não foi observada produção de ácido butírico. Como conclusão, em geral, o SO2 não é prejudicial para a microbiota intestinal nas doees testadas. Também se observou que se ingerido em conjunto com bactérias contaminantes ou ao encontrar essas bactérias no intestino, não exerce efeito antimicrobiano significativo.

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are exploited by human-specific pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella and Haemophilus species. As opsonin-independent phagocytosis of CEACAM-binding Neisseria depends on Src-family protein tyrosine kinase (PTK) phosphorylation of the CEACAM3 cytoplasmic domain, we hypothesized that an SH2-containing protein might be involved in CEACAM3-initiated, phagocytosis-promoting signals. Accordingly, we screened glutathione-S-transferase (GST) fusion proteins containing SH2 domains derived from a panel of signaling and adapter molecules for their ability to associate with CEACAM3. In vitro pull-down assays demonstrated that the SH2 domain of the adapter molecule Nck (GST-Nck SH2), but not other SH2 domains such as the Grb2 SH2 domain, interact with CEACAM3 in a phosphotyrosine-dependent manner. Either deletion of the cytoplasmic tail of CEACAM3, or point-mutation of a critical arginine residue in the SH2 domain of Nck (GST-NckSH2R308K) that disrupts phosphotyrosine binding, both abolished CEACAM3-Nck-SH2 interaction. Upon infection of human cells with CEACAM-binding Neisseria, full-length Nck comprising an SH2 and three SH3 domains co-localized with tyrosine phosphorylated CEACAM3 and associated bacteria as analyzed by immunofluorescence staining and confocal microscopy. In addition, Nck could be detected in CEACAM3 immunoprecipitates confirming the interaction in vivo. Importantly, overexpression of a GFP-fusion protein of the isolated Nck SH2 domain (GFP-Nck-SH2), but not GFP or GFP-Nck SH2 R308K reduced CEACAM3-mediated phagocytosis of CEACAM-binding Neisseria suggesting that the adaptor molecule Nck plays an important role in CEACAM3-initiated signaling leading to internalization and elimination of human-specific pathogens.

Cross species signal transfer mediated induction of antibiotic production by the marine bacteria against common bacterial pathogens was investigated in the present study. Marine bacteria were isolated and analyzed for their efficacy in antibiotic production against common clinical pathogens viz., Pseudomonas sp., Escherichia coli, Bacillus subtilis and Proteus sp. Out of 36 bacterial isolates analyzed, four isolates exhibited significant antibacterial activity against Pseudomonas, Bacillus, Proteus, Klebsiella and Escherichia coli Isolate designated as CW 602 produced antibiotic when co-cultured with Pseudomonas and CW 401 produced antibiotic when co-cultured with Bacillus cells. Cell free extract of CW 602 and CW 401 cultivated in the presence of heat killed Pseudomonas, Bacillus cells was subjected to solvent extraction by ethyl acetate and antimicrobial activity was tested by disc diffusion method. Present study on induced antibiotic production by Pseudomonas, Bacillus in bacteria CW 602 and CW 401 pave the way for the discovery of pathogen targeted/ specific antibiotic production.

Faculty of Science School of Pathology 9903547r robyn.brakin@gmail.com
Mycobacterium tuberculosis is an important human pathogen, claiming more lives per annum than any other single infectious organism. The host environment of M. tuberculosis contains DNA-damaging agents that pose a constant threat to the M. tuberculosis genome, and as a result, the ability to repair damaged DNA is likely to play an important role in bacterial survival. Y-family polymerases perform translesional synthesis and replicate DNA in an error-prone manner. By characterising the Y-family polymerases in mycobacteria, a better understanding the organism’s adaptive mutagenesis may be established. Through gene expression studies, it was found that UV irradiation of Mycobacterium smegmatis resulted in the up-regulation of dinP3, which was determined to be a Y-family polymerase by sequence analysis. DinP3 expression was found to be under control of the SOS response and is the first example of a Y-family polymerase in mycobacteria forming part of the SOS regulon. However, loss of DinP3 did not change the ability of M. smegmatis to tolerate UV irradiation. Mutagenesis studies revealed a complex interaction between the different Y-family polymerases in M. smegmatis. It was shown that spontaneous mutagenesis was increased in the absence of DinP3, whereas UV-targeted mutagenesis was increased in the absence of DinP, another Y-family polymerase. In conclusion, these results reflect the differences in control and in the mutational profiles of the Y-family polymerases in M. smegmatis. Moreover, these polymerases exhibit distinctive features from other bacterial Y-family polymerases, highlighting the different way in which bacteria have adapted to deal with lesions in their genetic material.

Yes
Buruli ulcer (BU) is an insidious neglected tropical disease. Cases are reported around the world but the rural regions of West and Central Africa are most affected. How BU is transmitted and spreads has remained a mystery, even though the causative agent, Mycobacterium ulcerans, has been known for more than 70 years. Here, using the tools of population genomics, we reconstruct the evolutionaryhistoryofM. ulceransbycomparing165isolatesspanning48yearsandrepresenting11endemiccountriesacrossAfrica. The genetic diversity of African M. ulcerans was found to be restricted due to the bacterium’s slow substitution rate coupled with its relatively recent origin. We identified two specific M. ulcerans lineages within the African continent, and inferred that M. ulcerans lineage Mu_A1 existed in Africa for several hundreds of years, unlike lineage Mu_A2, which was introduced much more recently, approximately during the 19th century. Additionally, we observed that specific M. ulcerans epidemic Mu_A1 clones were introduced during the same time period in the three hydrological basins that were well covered in our panel. The estimated time span of the introduction events coincides with the Neo-imperialism period, during which time the European colonial powers divided the African continent among themselves. Using this temporal association, and in the absence of a known BU reservoir or—vector on the continent, we postulate that the so-called "Scramble for Africa" played a significant role in the spread of the disease across the continent.
K.V. was supported by a PhD-grant of the Flemish Interuniversity Council—University Development Cooperation (Belgium). B.d.J. and C.M. were supported by the European Research Council-INTERRUPTB starting grant (no. 311725). T.P.S. was supported by a fellowship from the National Health and Medical Research Council of Australia (1105525). Funding for this work was provided by the Department of Economy, Science and Innovation of the Flemish Government, the Stop Buruli Consortium supported by the UBS Optimus Foundation, and the Fund for Scientific Research Flanders (Belgium) (FWO grant no. G.0321.07N). The computational resources used in this work were provided by the HPC core facility CalcUA and VSC (Flemish Supercomputer Center), funded by the University of Antwerp, the Hercules Foundation and the Flemish Government—department EWI. Aspects of the research in Cameroon and Benin were funded by the Raoul Follereau Fondation France.

Thesis (M.S.)--University of Tennessee, Knoxville, 2004.
Title from title page screen (viewed Sept. 20, 2004). Thesis advisor: F. Ann Draughon. Document formatted into pages (x, 106 p.). Vita. Includes bibliographical references.