First Molecular Characterization of Siphoviridae-Like Bacteriophages Infecting Staphylococcus hyicus in a Case of Exudative Epidermitis. - Related Documents




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477901.0000First Molecular Characterization of Siphoviridae-Like Bacteriophages Infecting Staphylococcus hyicus in a Case of Exudative Epidermitis. Exudative epidermitis (EE), also known as greasy pig disease, is one of the most frequent skin diseases affecting piglets. Zoonotic infections in human occur. EE is primarily caused by virulent strains of Staphylococcus (S.) hyicus. Generally, antibiotic treatment of this pathogen is prone to decreasing success, due to the incremental development of multiple resistances of bacteria against antibiotics. Once approved, bacteriophages might offer interesting alternatives for environmental sanitation or individualized treatment, subject to the absence of virulence and antimicrobial resistance genes. However, genetic characterization of bacteriophages for S. hyicus has, so far, been missing. Therefore, we investigated a piglet raising farm with a stock problem due to EE. We isolated eleven phages from the environment and wash water of piglets diagnosed with the causative agent of EE, i.e., S. hyicus. The phages were morphologically characterized by electron microscopy, where they appeared Siphoviridae-like. The genomes of two phages were sequenced on a MiSeq instrument (Illumina), resulting in the identification of a new virulent phage, PITT-1 (PMBT8), and a temperate phage, PITT-5 (PMBT9). Sequencing of three host bacteria (S. hyicus) from one single farm revealed the presence of two different strains with genes coding for two different exfoliative toxin genes, i.e., exhA (2 strains) and exhC (1 strain). The exhC-positive S. hyicus strain was only weakly lysed by most lytic phages. The occurrence of different virulent S. hyicus strains in the same outbreak limits the prospects for successful phage treatment and argues for the simultaneous use of multiple and different phages attacking the same host.202134305825
463110.9992Genome Analysis of an Enterococcal Prophage, Entfac.MY. BACKGROUND: Bacteriophages are bacterial parasites. Unlike lytic bacteriophages, lysogenic bacteriophages do not multiply immediately after entering the host cells and may integrate their genomes into the bacterial genomes as prophages. Prophages can include various phenotypic and genotypic effects on the host bacteria. Enterococcus spp. are Gram-positive bacteria that cause infections in humans and animals. In recent decades, these bacteria have become resistant to various antimicrobials, including vancomycin. The aim of this study was to analyze genome of an enterococcal prophage. METHODS: In this study, Enterococcus faecium EntfacYE was isolated from biological samples and its genome was analyzed using next-generation sequencing method. RESULTS: Overall, 254 prophage genes were identified in the bacterial genome. The prophage included 39 housekeeping, 41 replication and regulation, 80 structural and packaging, and 48 lysis genes. Moreover, 46 genes with unknown functions were identified. All genes were annotated in DNA Data Bank of Japan. CONCLUSION: In general, most prophage genes were linked to packaging and structure (31.5%) gene group. However, genes with unknown functions included a high proportion (18.11%), which indicated necessity of further analyses. Genomic analysis of the prophages can be effective in better understanding of their roles in development of bacterial resistance to antibiotics. Moreover, identification and study of prophages can help researchers develop genetic engineering tools and novel infection therapies.202236061127
462920.9992Screening and in silico characterization of prophages in Helicobacter pylori clinical strains. The increase of antibiotic resistance calls for alternatives to control Helicobacter pylori, a Gram-negative bacterium associated with various gastric diseases. Bacteriophages (phages) can be highly effective in the treatment of pathogenic bacteria. Here, we developed a method to identify prophages in H. pylori genomes aiming at their future use in therapy. A polymerase chain reaction (PCR)-based technique tested five primer pairs on 74 clinical H. pylori strains. After the PCR screening, 14 strains most likely to carry prophages were fully sequenced. After that, a more holistic approach was taken by studying the complete genome of the strains. This study allowed us to identify 12 intact prophage sequences, which were then characterized concerning their morphology, virulence, and antibiotic-resistance genes. To understand the variability of prophages, a phylogenetic analysis using the sequences of all H. pylori phages reported to date was performed. Overall, we increased the efficiency of identifying complete prophages to 54.1 %. Genes with homology to potential virulence factors were identified in some new prophages. Phylogenetic analysis revealed a close relationship among H. pylori-phages, although there are phages with different geographical origins. This study provides a deeper understanding of H. pylori-phages, providing valuable insights into their potential use in therapy.202539368610
507630.9992Diagnostic microarray for human and animal bacterial diseases and their virulence and antimicrobial resistance genes. Rapid diagnosis and treatment of disease is often based on the identification and characterization of causative agents derived from phenotypic characteristics. Current methods can be laborious and time-consuming, often requiring many skilled personnel and a large amount of lab space. The objective of our study was to develop a spotted microarray for rapid identification and characterization of bacterial pathogens and their antimicrobial resistance genes. Our spotted microarray consists of 489 70mer probes that detect 40 bacterial pathogens of medical, veterinary and zoonotic importance (including 15 NIAID Category A, B and C pathogens); associated genes that encode resistance for antimicrobial and metal resistance; and DNA elements that are important for horizontal gene transfer among bacteria. High specificity and reliability of the microarray was achieved for bacterial pathogens of animal and human importance by validating MDR pathogenic bacteria as pure cultures or by following their inoculation in complex and highly organic sample matrices, such as soil and manure.201020035807
463040.9992Genome Analysis of the Enterococcus faecium Entfac.YE Prophage. BACKGROUND: Bacteriophages are viruses that infect bacteria. Bacteriophages are widely distributed in various environments. The prevalence of bacteriophages in water sources, especially wastewaters, is naturally high. These viruses affect evolution of most bacterial species. Bacteriophages are able to integrate their genomes into the chromosomes of their hosts as prophages and hence transfer resistance genes to the bacterial genomes. Enterococci are commensal bacteria that show high resistance to common antibiotics. For example, prevalence of vancomycin-resistant enterococci has increased within the last decades. METHODS: Enterococcal isolates were isolated from clinical samples and morphological, phenotypical, biochemical, and molecular methods were used to identify and confirm their identity. Bacteriophages extracted from water sources were then applied to isolated Enterococcus faecium (E. faecium). In the next step, the bacterial genome was completely sequenced and the existing prophage genome in the bacterial genome was analyzed. RESULTS: In this study, E. faecium EntfacYE was isolated from a clinical sample. The EntfacYE genome was analyzed and 88 prophage genes were identified. The prophage content included four housekeeping genes, 29 genes in the group of genes related to replication and regulation, 25 genes in the group of genes related to structure and packaging, and four genes belonging to the group of genes associated with lysis. Moreover, 26 genes were identified with unknown functions. CONCLUSION: In conclusion, genome analysis of prophages can lead to a better understanding of their roles in the rapid evolution of bacteria.202235509366
507550.9992Fast and Economic Microarray-Based Detection of Species-, Resistance-, and Virulence-Associated Genes in Clinical Strains of Vancomycin-Resistant Enterococci (VRE). Today, there is a continuous worldwide battle against antimicrobial resistance (AMR) and that includes vancomycin-resistant enterococci (VRE). Methods that can adequately and quickly detect transmission chains in outbreaks are needed to trace and manage this problem fast and cost-effectively. In this study, DNA-microarray-based technology was developed for this purpose. It commenced with the bioinformatic design of specific oligonucleotide sequences to obtain amplification primers and hybridization probes. Microarrays were manufactured using these synthesized oligonucleotides. A highly parallel and stringent labeling and hybridization protocol was developed and employed using isolated genomic DNA from previously sequenced (referenced) clinical VRE strains for optimal sensitivity and specificity. Microarray results showed the detection of virulence, resistance, and species-specific genes in the VRE strains. Theoretical predictions of the microarray results were also derived from the sequences of the same VRE strain and were compared to array results while optimizing protocols until the microarray result and theoretical predictions were a match. The study concludes that DNA microarray technology can be used to quickly, accurately, and economically detect specifically and massively parallel target genes in enterococci.202439409516
581760.9992Comparative genomics reveals the correlations of stress response genes and bacteriophages in developing antibiotic resistance of Staphylococcus saprophyticus. Staphylococcus saprophyticus is the second most common bacteria associated with urinary tract infections (UTIs) in women. The antimicrobial treatment regimen for uncomplicated UTI is normally nitrofurantoin, trimethoprim-sulfamethoxazole (TMP-SMX), or a fluoroquinolone without routine susceptibility testing of S. saprophyticus recovered from urine specimens. However, TMP-SMX-resistant S. saprophyticus has been detected recently in UTI patients, as well as in our cohort. Herein, we investigated the understudied resistance patterns of this pathogenic species by linking genomic antibiotic resistance gene (ARG) content to susceptibility phenotypes. We describe ARG associations with known and novel SCCmec configurations as well as phage elements in S. saprophyticus, which may serve as intervention or diagnostic targets to limit resistance transmission. Our analyses yielded a comprehensive database of phenotypic data associated with the ARG sequence in clinical S. saprophyticus isolates, which will be crucial for resistance surveillance and prediction to enable precise diagnosis and effective treatment of S. saprophyticus UTIs.202338051037
417870.9992Efficacy and food safety considerations of poultry competitive exclusion products. Competitive exclusion (CE) products are anaerobic cultures of bacteria that are applied to poultry hatchlings to establish a protective enteric microbiota that excludes intestinal colonization by human food-borne pathogens. For safety of the poultry flock and human consumers, the identities of bacteria in CE products need to be known. A CE product is a culture of intestinal contents from adult chickens. It may be microbiologically defined by analysis of bacteria isolated from the culture, but many bacteria are hard to reliably isolate, identify, and characterize with conventional techniques. Sequence analysis of 16S ribosomal RNA (rRNA) genes may be more reliable than conventional techniques to identify CE bacteria. Bacteria in CE products may contain antimicrobial drug resistance and virulence mechanisms that could be transferred to the enteric bacteria of the food animal and to the human consumer. Detection methods for specific antimicrobial drug resistance and virulence genes and the integrase genes of conjugative transposons, mostly utilizing PCR technology, are being developed that can be applied to assess these risks in CE bacteria. With improvements in efficacy, bacterial identification, and detection and control of the possible risks of gene transfer, CE product technology can be made a more effective food safety tool.200617039457
462880.9992Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection. To optimize phage therapy, we need to understand how bacteria evolve against phage attacks. One of the main problems of phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage-resistant strains that can be overcome by the analysis of metadata provided by whole-genome sequencing. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strains belonging to the ST-2 clonal complex during a decade (Ab2000 vs. 2010): 9 from 2000 to 9 from 2010. The presence of genes putatively associated with phage resistance was detected. Genes detected were associated with an abortive infection system, restriction-modification system, genes predicted to be associated with defense systems but with unknown function, and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands in the 2000 strains and 32% in the 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems. A moderately higher presence of these genes in the strains of 2010 in comparison with those of 2000 was found, especially those related to the restriction-modification system and CRISPR-Cas system. The presence of these genes in genomic islands at a higher rate in the strains of 2010 compared with those of 2000 was also detected. Whole-genome sequencing and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in possible phage therapy.202135250902
463990.9992Genomic and Phenotypic Characterization of Mastitis-Causing Staphylococci and Probiotic Lactic Acid Bacteria Isolated from Raw Sheep's Milk. Dairy products play a crucial role in human nutrition as they provide essential nutrients. However, the presence of diverse microorganisms in these products can pose challenges to food safety and quality. Here, we provide a comprehensive molecular characterization of a diverse collection of lactic acid bacteria (LAB) and staphylococci isolated from raw sheep's milk. Whole-genome sequencing, phenotypic characterization, and bioinformatics were employed to gain insight into the genetic composition and functional attributes of these bacteria. Bioinformatics analysis revealed the presence of various genetic elements. Important toxin-related genes in staphylococci that contribute to their pathogenic potential were identified and confirmed using phenotypic assays, while adherence-related genes, which are essential for attachment to host tissues, surfaces in the dairy environment, and the creation of biofilms, were also present. Interestingly, the Staphylococcus aureus isolates belonged to sequence type 5, which largely consists of methicillin-susceptible isolates that have been involved in severe nosocomial infections. Although genes encoding methicillin resistance were not identified, multiple resistance genes (RGs) conferring resistance to aminoglycosides, macrolides, and fluroquinolones were found. In contrast, LAB had few inherently present RGs and no virulence genes, suggesting their likely safe status as food additives in dairy products. LAB were also richer in bacteriocins and carbohydrate-active enzymes, indicating their potential to suppress pathogens and effectively utilize carbohydrate substrates, respectively. Additionally, mobile genetic elements, present in both LAB and staphylococci, may facilitate the acquisition and dissemination of genetic traits, including RGs, virulence genes, and metabolic factors, with implications for food quality and public health. The molecular and phenotypic characterization presented herein contributes to the effort to mitigate risks and infections (e.g., mastitis) and enhance the safety and quality of milk and products thereof.202337762186
4393100.9992Mechanisms of Staphylococcus aureus Antibiotics Resistance Revealed by Adaptive Laboratory Evolution. Infection caused by drug-resistant Staphylococcus aureus is a serious public health and veterinary concern. Lack of a comprehensive understanding of the mechanisms underlying the emergence of drug-resistant strains, it makes S. aureus one of the most intractable pathogenic bacteria. To identify mutations that confer resistance to anti-S. aureus drugs, we established a laboratory-based adaptive evolution system and performed 10 rounds of evolution experiments against 15 clinically used antibiotics. We discovered a panel of known and novel resistance-associated sites after performing whole-genome sequencing. Furthermore, we found that the resistance evolved at distinct rates. For example, streptomycin, rifampicin, fusidic acid and novobiocin all developed significant resistance quickly in the second round of evolution. Intriguingly, the cross-resistance experiment reveals that nearly all drug-resistant strains have varying degrees of increased sensitivity to fusidic acid, pointing to a novel approach to battle AMR. In addition, the in silico docking analysis shows that the evolved mutants affect the interaction of rifampcin-rpoB, as well as the novobiocin-gyrB. Moreover, for the genes we got in the laboratory evolution, mutant genes of clinical isolates of human had significant differences from the environmental isolates and animal isolates. We believe that the strategy and data set in this research will be helpful for battling AMR issue of S. aureus, and adaptable to other pathogenic microbes.202539762552
4601110.9991CRISPR tracking reveals global spreading of antimicrobial resistance genes by Staphylococcus of canine origin. The close contact between pets and their owners is a potential source for microorganisms and genetic material exchange. Staphylococcus species considered as harmless inhabitants of animals' and humans' microbiota can act as reservoirs of antimicrobial resistance genes to more virulent species, thereby increasing their potential to resist drug therapy. This process could be inhibited by the antiplasmid immunity conferred by CRISPR systems. On the other hand, CRISPR spacer sequences can be explored as molecular clocks to track the history of genetic invasion suffered by a bacterial strain. To understand better the role of domestic dogs in human health as an antimicrobial resistance genes source, we analyzed 129 genomes of Staphylococcus strains of canine origin for the presence of CRISPR systems. Only 8% of the strains were positive for CRISPR, which is consistent with Staphylococcus role as gene reservoirs. The plasmidial origin or some spacers confirms the unsuccessful attempt of plasmid exchange in strains carrying CRISPRs. Some of these systems are within a staphylococcal cassette chromosome mec (SCCmec), sharing 98% of identity between their harboring strains. These CRISPRs' spacers reveal that this SCCmec was transferred between canine S. pseudintermedius strains, then to S. schleiferi and to Staphylococcus strains isolated from human beings. Our findings shows genetic evidence for the global spreading of pathogenic bacteria and the antimicrobial resistance genes carried by them and reinforce that, in the age of antimicrobial resistance, it is imperative that drug therapies consider the integrated nature of the relationship between pets and humans.201931030846
4648120.9991Potential of phage cocktails in the inactivation of Enterobacter cloacae--An in vitro study in a buffer solution and in urine samples. The objective of this study was to compare the dynamics of three previously isolated phages for Enterobacter cloacae in order to evaluate their ability to treat urinary tract infections (UTI). The phages genomes, survival, host range, were characterized, and the host-phage dynamics was determined in culture medium and urine samples. The presence of prophages in bacteria, host recovery and development of resistance to phage after treatment was also evaluated. The growth of the E. cloacae was inhibited by the three phages, resulting in a decrease of ≈3 log. The use of cocktails with two or three phages was significantly more effective (decrease of ≈4 log). In urine, the inactivation was still effective (≈2 log). Both phages were considered safe to inactivate the bacteria (no integrase and toxin codifying genes). Some bacteria remained viable in the presence of the phages, but their colonies were smaller than those of the non-treated control and were visible only after 5 days of incubation (visible after 24h in the control). A high bacterial inactivation efficiency with phage cocktails combined with the safety of the phages and their long periods of survival, even in urine samples, paves the way for depth studies, especially in vivo studies, to control urinary tract infection and to overcome the development of resistances by the nosocomial bacterium E. cloacae.201626541317
5841130.9991Isolation and Characterization of a Bacteriophage with Potential for the Control of Multidrug-Resistant Salmonella Strains Encoding Virulence Factors Associated with the Promotion of Precancerous Lesions. BACKGROUND: Antimicrobial-resistant bacteria represent a serious threat to public health. Among these bacteria, Salmonella is of high priority because of its morbidity levels and its ability to induce different types of cancer. AIM: This study aimed to identify Salmonella strains encoding genes linked to the promotion of precancerous lesions and to isolate a bacteriophage to evaluate its preclinical potential against these bacteria. METHODOLOGY: An epidemiological approach based on wastewater analysis was employed to isolate Salmonella strains and detect genes associated with the induction of precancerous lesions. Antimicrobial susceptibility was assessed by the disk diffusion method. A bacteriophage was isolated via the double agar technique, and its morphological characteristics, stability, host range, replication dynamics, and ability to control Salmonella under different conditions were evaluated. The bacteriophage genome was sequenced and analyzed using bioinformatics tools. RESULTS: Thirty-seven Salmonella strains were isolated, seventeen of which contained the five genes associated with precancerous lesions' induction. These strains exhibited resistance to multiple antimicrobials, including fluoroquinolones. A bacteriophage from the Autographiviridae family with lytic activity against 21 bacterial strains was isolated. This phage exhibited a 20 min replication cycle, releasing 52 ± 3 virions per infected cell. It demonstrated stability and efficacy in reducing the Salmonella concentration in simulated gastrointestinal conditions, and its genome lacked genes that represent a biosafety risk. CONCLUSION: This bacteriophage shows promising preclinical potential as a biotherapeutic agent against Salmonella.202439599826
4319140.9991Threat and Control of tet(X)-Mediated Tigecycline-Resistant Acinetobacter sp. Bacteria. Tigecycline is regarded as one of the last-resort antibiotics against multidrug-resistant (MDR) Acinetobacter sp. bacteria. Recently, the tigecycline-resistant Acinetobacter sp. isolates mediated by tet(X) genes have emerged as a class of global pathogens for humans and food-producing animals. However, the genetic diversities and treatment options were not systematically discussed in the era of One Health. In this review, we provide a detailed illustration of the evolution route, distribution characteristics, horizontal transmission, and rapid detection of tet(X) genes in diverse Acinetobacter species. We also detail the application of chemical drugs, plant extracts, phages, antimicrobial peptides (AMPs), and CRISPR-Cas technologies for controlling tet(X)-positive Acinetobacter sp. pathogens. Despite excellent activities, the antibacterial spectrum and application safety need further evaluation and resolution. It is noted that deep learning is a promising approach to identify more potent antimicrobial compounds.202541097540
3928150.9991Organic and conventional fruits and vegetables contain equivalent counts of Gram-negative bacteria expressing resistance to antibacterial agents. Resistance to antibiotics is a major public health problem which might culminate in outbreaks caused by pathogenic bacteria untreatable by known antibiotics. Most of the genes conferring resistance are acquired horizontally from already resistant commensal or environmental bacteria. Food contamination by resistant bacteria might be a significant source of resistance genes for human bacteria but has never been precisely assessed, nor is it known whether organic products differ in this respect from conventionally produced products. We showed here, on a large year-long constructed sample set containing 399 products that, irrespective of their mode of production, raw fruits and vegetables are heavily contaminated by Gram-negative bacteria (GNB) resistant to multiple antibiotics. Most of these bacteria originate in the soil and environment. We focused on non-oxidative GNB resistant to third-generation cephalosporins, because of their potential impact on human health. Among them, species potentially pathogenic for immunocompetent hosts were rare. Of the products tested, 13% carried bacteria producing extended-spectrum beta-lactamases, all identified as Rahnella sp. which grouped into two phylotypes and all carrying the bla(RAHN) gene. Thus, both organic and conventional fruits and vegetables may constitute significant sources of resistant bacteria and of resistance genes.201019919536
4750160.9991A Review of Detection Methods for Vancomycin-Resistant Enterococci (VRE) Genes: From Conventional Approaches to Potentially Electrochemical DNA Biosensors. Vancomycin-resistant Enterococci (VRE) genes are bacteria strains generated from Gram-positive bacteria and resistant to one of the glycopeptides antibiotics, commonly, vancomycin. VRE genes have been identified worldwide and exhibit considerable phenotypic and genotypic variations. There are six identified phenotypes of vancomycin-resistant genes: VanA, VanB, VanC, VanD, VanE, and VanG. The VanA and VanB strains are often found in the clinical laboratory because they are very resistant to vancomycin. VanA bacteria can pose significant issues for hospitalized patients due to their ability to spread to other Gram-positive infections, which changes their genetic material to increase their resistance to the antibiotics used during treatment. This review summarizes the established methods for detecting VRE strains utilizing traditional, immunoassay, and molecular approaches and then focuses on potential electrochemical DNA biosensors to be developed. However, from the literature search, no information was reported on developing electrochemical biosensors for detecting VRE genes; only the electrochemical detection of vancomycin-susceptible bacteria was reported. Thus, strategies to create robust, selective, and miniaturized electrochemical DNA biosensor platforms to detect VRE genes are also discussed.202336832060
6248170.9991Characterization of a stable, metronidazole-resistant Clostridium difficile clinical isolate. BACKGROUND: Clostridium difficile are gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15-35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. CONCLUSIONS/SIGNIFICANCE: This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.201323349739
9919180.9991An In Vitro Chicken Gut Model Demonstrates Transfer of a Multidrug Resistance Plasmid from Salmonella to Commensal Escherichia coli. The chicken gastrointestinal tract is richly populated by commensal bacteria that fulfill various beneficial roles for the host, including helping to resist colonization by pathogens. It can also facilitate the conjugative transfer of multidrug resistance (MDR) plasmids between commensal and pathogenic bacteria which is a significant public and animal health concern as it may affect our ability to treat bacterial infections. We used an in vitro chemostat system to approximate the chicken cecal microbiota, simulate colonization by an MDR Salmonella pathogen, and examine the dynamics of transfer of its MDR plasmid harboring several genes, including the extended-spectrum beta-lactamase bla(CTX-M1) We also evaluated the impact of cefotaxime administration on plasmid transfer and microbial diversity. Bacterial community profiles obtained by culture-independent methods showed that Salmonella inoculation resulted in no significant changes to bacterial community alpha diversity and beta diversity, whereas administration of cefotaxime caused significant alterations to both measures of diversity, which largely recovered. MDR plasmid transfer from Salmonella to commensal Escherichia coli was demonstrated by PCR and whole-genome sequencing of isolates purified from agar plates containing cefotaxime. Transfer occurred to seven E. coli sequence types at high rates, even in the absence of cefotaxime, with resistant strains isolated within 3 days. Our chemostat system provides a good representation of bacterial interactions, including antibiotic resistance transfer in vivo It can be used as an ethical and relatively inexpensive approach to model dissemination of antibiotic resistance within the gut of any animal or human and refine interventions that mitigate its spread before employing in vivo studies.IMPORTANCE The spread of antimicrobial resistance presents a grave threat to public health and animal health and is affecting our ability to respond to bacterial infections. Transfer of antimicrobial resistance via plasmid exchange is of particular concern as it enables unrelated bacteria to acquire resistance. The gastrointestinal tract is replete with bacteria and provides an environment for plasmid transfer between commensals and pathogens. Here we use the chicken gut microbiota as an exemplar to model the effects of bacterial infection, antibiotic administration, and plasmid transfer. We show that transfer of a multidrug-resistant plasmid from the zoonotic pathogen Salmonella to commensal Escherichia coli occurs at a high rate, even in the absence of antibiotic administration. Our work demonstrates that the in vitro gut model provides a powerful screening tool that can be used to assess and refine interventions that mitigate the spread of antibiotic resistance in the gut before undertaking animal studies.201728720731
5077190.9991Development of a new integrated diagnostic test for identification and characterization of pathogens. Animal diseases directly cause multi-million dollar losses world-wide. Therefore a rapid, highly specific, cost-effective diagnostic test for detecting a large set of bacterial virulence and antimicrobial resistance genes simultaneously is necessary. Hence, our group, the BCBG (Bacterial Chips Bacterial Genes) group, proposes developing a powerful molecular tool (DNA microarray) to detect a broad range of infectious agents, their endogenous main virulence factors and antibiotic resistance genes simultaneously. Effectively, a 70-mer oligonucleotide microarray capable of detecting the presence or absence of 169 Escherichia coli virulence genes or virulence marker genes as well as their variants, in addition to 30 principal antimicrobial resistance genes previously characterized in E. coli strains was developed by our group. This microarray was validated with a large collection of well characterized pathogenic and reference E. coli strains. Moreover, we are developing a new powerful clinical diagnostic microarray tool, to identify pathogenic bacteria of veterinary interest. The commercialization of this assay would allow same day diagnosis of infectious agents and their antibiotic resistance resulting in early treatment. In addition, this technology is also applicable to microbial quality control of food and water.200617058497