Functional Metagenome Mining of Soil for a Novel Gentamicin Resistance Gene. - Related Documents




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466901.0000Functional Metagenome Mining of Soil for a Novel Gentamicin Resistance Gene. Extensive use of antibiotics over recent decades has led to bacterial resistance against antibiotics, including gentamicin, one of the most effective aminoglycosides. The emergence of resistance is problematic for hospitals, since gentamicin is an important broad-spectrum antibiotic for the control of bacterial pathogens in the clinic. Previous study to identify gentamicin resistance genes from environmental samples have been conducted using culture-dependent screening methods. To overcome these limitations, we employed a metagenome-based culture-independent protocol to identify gentamicin resistance genes. Through functional screening of metagenome libraries derived from soil samples, a fosmid clone was selected as it conferred strong gentamicin resistance. To identify a specific functioning gene conferring gentamicin resistance from a selected fosmid clone (35-40 kb), a shot-gun library was constructed and four shot-gun clones (2-3 kb) were selected. Further characterization of these clones revealed that they contained sequences similar to that of the RNA ligase, T4 rnlA that is known as a toxin gene. The overexpression of the rnlA-like gene in Escherichia coli increased gentamicin resistance, indicating that this toxin gene modulates this trait. The results of our metagenome library analysis suggest that the rnlA-like gene may represent a new class of gentamicin resistance genes in pathogenic bacteria. In addition, we demonstrate that the soil metagenome can provide an important resource for the identification of antibiotic resistance genes, which are valuable molecular targets in efforts to overcome antibiotic resistance.201626699755
467110.9999Detection by metagenomic functional analysis and improvement by experimental evolution of β-lactams resistance genes present in oil contaminated soils. The spread of antibiotic resistance genes has become a global health concern identified by the World Health Organization as one of the greatest threats to health. Many of antimicrobial resistance determinants found in bacterial pathogens originate from environmental bacteria, so identifying the genes that confer resistance to antibiotics in different habitats is mandatory to better understand resistance mechanisms. Soil is one of the most diverse environments considered reservoir of antimicrobial resistance genes. The aim of this work is to study the presence of genes that provide resistance to antibiotics used in clinical settings in two oil contaminated soils by metagenomic functional analysis. Using fosmid vectors that efficiently transcribe metagenomic DNA, we have selected 12 fosmids coding for two class A β-lactamases, two subclass B1 and two subclass B3 metallo-β-lactamases, one class D β-lactamase and three efflux pumps that confer resistance to cefexime, ceftriaxone, meropenem and/or imipenem. In some of them, detection of the resistance required heterologous expression from the fosmid promoter. Although initially, these environmental genes only provide resistance to low concentrations of antibiotics, we have obtained, by experimental evolution, fosmid derivatives containing β-lactamase ORFs with a single base substitution, which substantially increase their β-lactamase activity and resistance level. None of the mutations affect β-lactamase coding sequences and are all located upstream of them. These results demonstrate the presence of enzymes that confer resistance to relevant β-lactams in these soils and their capacity to rapidly adapt to provide higher resistance levels.202235768448
360020.9998Uncultured soil bacteria are a reservoir of new antibiotic resistance genes. Antibiotic resistance genes are typically isolated by cloning from cultured bacteria or by polymerase chain reaction (PCR) amplification from environmental samples. These methods do not access the potential reservoir of undiscovered antibiotic resistance genes harboured by soil bacteria because most soil bacteria are not cultured readily, and PCR detection of antibiotic resistance genes depends on primers that are based on known genes. To explore this reservoir, we isolated DNA directly from soil samples, cloned the DNA and selected for clones that expressed antibiotic resistance in Escherichia coli. We constructed four libraries that collectively contain 4.1 gigabases of cloned soil DNA. From these and two previously reported libraries, we identified nine clones expressing resistance to aminoglycoside antibiotics and one expressing tetracycline resistance. Based on the predicted amino acid sequences of the resistance genes, the resistance mechanisms include efflux of tetracycline and inactivation of aminoglycoside antibiotics by phosphorylation and acetylation. With one exception, all the sequences are considerably different from previously reported sequences. The results indicate that soil bacteria are a reservoir of antibiotic resistance genes with greater genetic diversity than previously accounted for, and that the diversity can be surveyed by a culture-independent method.200415305923
438030.9998Comparative genome analysis of ciprofloxacin-resistant Pseudomonas aeruginosa reveals genes within newly identified high variability regions associated with drug resistance development. The alarming rise of ciprofloxacin-resistant Pseudomonas aeruginosa has been reported in several clinical studies. Though the mutation of resistance genes and their role in drug resistance has been researched, the process by which the bacterium acquires high-level resistance is still not well understood. How does the genomic evolution of P. aeruginosa affect resistance development? Could the exposure of antibiotics to the bacteria enrich genomic variants that lead to the development of resistance, and if so, how are these variants distributed through the genome? To answer these questions, we performed 454 pyrosequencing and a whole genome analysis both before and after exposure to ciprofloxacin. The comparative sequence data revealed 93 unique resistance strain variation sites, which included a mutation in the DNA gyrase subunit A gene. We generated variation-distribution maps comparing the wild and resistant types, and isolated 19 candidates from three discrete resistance-associated high variability regions that had available transposon mutants, to perform a ciprofloxacin exposure assay. Of these region candidates with transposon disruptions, 79% (15/19) showed a reduction in the ability to gain high-level resistance, suggesting that genes within these high variability regions might enrich for certain functions associated with resistance development.201323808957
597740.9998Methods to determine antibiotic resistance gene silencing. The occurrence of antibiotic-resistant bacteria is an increasingly serious problem world-wide. In addition, to phenotypically resistant bacteria, a threat may also be posed by isolates with silent, but intact, antibiotic resistance genes. Such isolates, which have recently been described, possess wild-type genes that are not expressed, but may convert to resistance by activating expression of the silent genes. They may therefore compromise the efficacy of antimicrobial treatment, particularly if their presence has not been diagnosed. This chapter describes the detection of silent resistance genes by PCR and DNA sequencing. A method to detect five potentially silent acquired resistance genes; aadA, bla (OXA-2), strAB, sul1, and tet(A) is described. First, the susceptibility of the isolates to the relevant antibiotics is determined by an appropriate susceptibility testing method, such as E-test. Then the presence of the genes is investigated by PCR followed by agarose gel electrophoresis of the amplification products. If a resistance gene is detected in a susceptible isolate, the entire open-reading frame and promoter sequence of the gene is amplified by PCR and their DNA sequences obtained. The DNA sequences are then compared to those of known resistant isolates, to detect mutations that may account for susceptibility. If no mutations are detected the expression of the gene is investigated by RT-PCR following RNA extraction. The methods described here can be applied to all acquired resistance genes for which sequence and normal expression data are available.201020401584
574650.9998Identification of a Novel Plasmid-Borne Gentamicin Resistance Gene in Nontyphoidal Salmonella Isolated from Retail Turkey. The spread of antibiotic-resistant bacteria presents a global health challenge. Efficient surveillance of bacteria harboring antibiotic resistance genes (ARGs) is a critical aspect to controlling the spread. Increased access to microbial genomic data from many diverse populations informs this surveillance but only when functional ARGs are identifiable within the data set. Current, homology-based approaches are effective at identifying the majority of ARGs within given clinical and nonclinical data sets for several pathogens, yet there are still some whose identities remain elusive. By coupling phenotypic profiling with genotypic data, these unknown ARGs can be identified to strengthen homology-based searches. To prove the efficacy and feasibility of this approach, a published data set from the U.S. National Antimicrobial Resistance Monitoring System (NARMS), for which the phenotypic and genotypic data of 640 Salmonella isolates are available, was subjected to this analysis. Six isolates recovered from the NARMS retail meat program between 2011 and 2013 were identified previously as phenotypically resistant to gentamicin but contained no known gentamicin resistance gene. Using the phenotypic and genotypic data, a comparative genomics approach was employed to identify the gene responsible for the observed resistance in all six of the isolates. This gene, grdA, is harbored on a 9,016-bp plasmid that is transferrable to Escherichia coli, confers gentamicin resistance to E. coli, and has never before been reported to confer gentamicin resistance. Bioinformatic analysis of the encoded protein suggests an ATP binding motif. This work demonstrates the advantages associated with coupling genomics technologies with phenotypic data for novel ARG identification.202032816720
467060.9998Novel Antibiotic Resistance Genes Identified by Functional Gene Library Screening in Stenotrophomonas maltophilia and Chryseobacterium spp. Bacteria of Soil Origin. As one of the most diverse habitats of microorganisms, soil has been recognised as a reservoir of both antibiotics and the antibiotic resistance genes (ARGs). Bacteria naturally inhabiting soil or water often possess innate ARGs to counteract the chemical compounds produced by competitors living in the same environment. When such bacteria are able to cause infections in immunocompromised patients, their strong innate antibiotic resistance mechanisms make treatment difficult. We generated functional gene libraries using antibiotic-resistant Stenotrophomonas maltophilia and Chryseobacterium spp. bacteria isolated from agricultural soils in Lithuania to select for the genetic determinants responsible for their resistance. We were able to find novel variants of aminoglycoside and β-lactam resistance genes, with β-lactamases isolated from the Chryseobacterium spp. functional gene library, one of which is a variant of IND-like metallo-β-lactamase (MBL) IND-17 and the other of which is a previously uncharacterised MBL we named CHM (Chryseobacterium metallo β-lactamase). Our results indicate that soil microorganisms possess a diversity of ARG variants, which could potentially be transferred to the clinical setting.202337047008
626670.9998Bacterial gene loss as a mechanism for gain of antimicrobial resistance. Acquisition of exogenous DNA by pathogenic bacteria represents the basis for much of the acquired antimicrobial resistance in pathogenic bacteria. A more extreme mechanism to avoid the effect of an antibiotic is to delete the drug target, although this would be predicted to be rare since drug targets are often essential genes. Here, we review and discuss the description of a novel mechanism of resistance to the cephalosporin drug ceftazidime caused by loss of a penicillin-binding protein (PBP) in a Gram-negative bacillus (Burkholderia pseudomallei). This organism causes melioidosis across south-east Asia and northern Australia, and is usually treated with two or more weeks of ceftazidime followed by oral antibiotics for three to six months. Comparison of clinical isolates from six patients with melioidosis found initial ceftazidime-susceptible isolates and subsequent ceftazidime-resistant variants. The latter failed to grow on commonly used culture media, rendering these isolates difficult to detect in the diagnostic laboratory. Genomic analysis using pulsed-field gel electrophoresis and array based genomic hybridisation revealed a large-scale genomic deletion comprising 49 genes in the ceftazidime-resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a PBP 3 present within the region of genomic loss. This provides one explanation for ceftazidime treatment failure, and may be a frequent but undetected event in patients with melioidosis.201223022568
335880.9998Novel class 1 integron harboring antibiotic resistance genes in wastewater-derived bacteria as revealed by functional metagenomics. Combatting antibiotic resistance is critical to our ability to treat infectious diseases. Here, we identified and characterized diverse antimicrobial resistance genes, including potentially mobile elements, from synthetic wastewater treatment microcosms exposed to the antibacterial agent triclosan. After seven weeks of exposure, the microcosms were subjected to functional metagenomic selection across 13 antimicrobials. This was achieved by cloning the combined genetic material from the microcosms, introducing this genetic library into E. coli, and selecting for clones that grew on media supplemented with one of the 13 antimicrobials. We recovered resistant clones capable of growth on media supplemented with a single antimicrobial, yielding 13 clones conferring resistance to at least one antimicrobial agent. Antibiotic susceptibility analysis revealed resistance ranging from 4 to >50 fold more resistant, while one clone showed resistance to multiple antibiotics. Using both Sanger and SMRT sequencing, we identified the predicted active gene(s) on each clone. One clone that conferred resistance to tetracycline contained a gene encoding a novel tetA-type efflux pump that was named TetA(62). Three clones contained predicted active genes on class 1 integrons. One integron had a previously unreported genetic arrangement and was named In1875. This study demonstrated the diversity and potential for spread of resistance genes present in human-impacted environments.202133515651
624890.9998Characterization 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
4664100.9998Comprehensive screening of genomic and metagenomic data reveals a large diversity of tetracycline resistance genes. Tetracyclines are broad-spectrum antibiotics used to prevent or treat a variety of bacterial infections. Resistance is often mediated through mobile resistance genes, which encode one of the three main mechanisms: active efflux, ribosomal target protection or enzymatic degradation. In the last few decades, a large number of new tetracycline-resistance genes have been discovered in clinical settings. These genes are hypothesized to originate from environmental and commensal bacteria, but the diversity of tetracycline-resistance determinants that have not yet been mobilized into pathogens is unknown. In this study, we aimed to characterize the potential tetracycline resistome by screening genomic and metagenomic data for novel resistance genes. By using probabilistic models, we predicted 1254 unique putative tetracycline resistance genes, representing 195 gene families (<70 % amino acid sequence identity), whereof 164 families had not been described previously. Out of 17 predicted genes selected for experimental verification, 7 induced a resistance phenotype in an Escherichia coli host. Several of the predicted genes were located on mobile genetic elements or in regions that indicated mobility, suggesting that they easily can be shared between bacteria. Furthermore, phylogenetic analysis indicated several events of horizontal gene transfer between bacterial phyla. Our results also suggested that acquired efflux pumps originate from proteobacterial species, while ribosomal protection genes have been mobilized from Firmicutes and Actinobacteria. This study significantly expands the knowledge of known and putatively novel tetracycline resistance genes, their mobility and evolutionary history. The study also provides insights into the unknown resistome and genes that may be encountered in clinical settings in the future.202033125315
4640110.9998Genome analysis of probiotic bacteria for antibiotic resistance genes. To date, probiotic bacteria are used in the diet and have various clinical applications. There are reports of antibiotic resistance genes in these bacteria that can transfer to other commensal and pathogenic bacteria. The aim of this study was to use whole-genome sequence analysis to identify antibiotic resistance genes in a group of bacterial with probiotic properties. Also, this study followed existing issues about the importance and presence of antibiotic resistance genes in these bacteria and the dangers that may affect human health in the future. In the current study, a collection of 126 complete probiotic bacterial genomes was analyzed for antibiotic resistance genes. The results of the current study showed that there are various resistance genes in these bacteria that some of them are transferable to other bacteria. The tet(W) tetracycline resistance gene was more than other antibiotic resistance genes in these bacteria and this gene was found in Bifidobacterium and Lactobacillus. In our study, the most numbers of antibiotic resistance genes were transferred with mobile genetic elements. We propose that probiotic companies before the use of a micro-organism as a probiotic, perform an antibiotic susceptibility testing for a large number of antibiotics. Also, they perform analysis of complete genome sequence for prediction of antibiotic resistance genes.202234989942
4629120.9998Screening 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
4657130.9998Discovery of the fourth mobile sulfonamide resistance gene. BACKGROUND: Over the past 75 years, human pathogens have acquired antibiotic resistance genes (ARGs), often from environmental bacteria. Integrons play a major role in the acquisition of antibiotic resistance genes. We therefore hypothesized that focused exploration of integron gene cassettes from microbial communities could be an efficient way to find novel mobile resistance genes. DNA from polluted Indian river sediments were amplified using three sets of primers targeting class 1 integrons and sequenced by long- and short-read technologies to maintain both accuracy and context. RESULTS: Up to 89% of identified open reading frames encode known resistance genes, or variations thereof (> 1000). We identified putative novel ARGs to aminoglycosides, beta-lactams, trimethoprim, rifampicin, and chloramphenicol, including several novel OXA variants, providing reduced susceptibility to carbapenems. One dihydropteroate synthase gene, with less than 34% amino acid identity to the three known mobile sulfonamide resistance genes (sul1-3), provided complete resistance when expressed in Escherichia coli. The mobilized gene, here named sul4, is the first mobile sulfonamide resistance gene discovered since 2003. Analyses of adjacent DNA suggest that sul4 has been decontextualized from a set of chromosomal genes involved in folate synthesis in its original host, likely within the phylum Chloroflexi. The presence of an insertion sequence common region element could provide mobility to the entire integron. Screening of 6489 metagenomic datasets revealed that sul4 is already widespread in seven countries across Asia and Europe. CONCLUSIONS: Our findings show that exploring integrons from environmental communities with a history of antibiotic exposure can provide an efficient way to find novel, mobile resistance genes. The mobilization of a fourth sulfonamide resistance gene is likely to provide expanded opportunities for sulfonamide resistance to spread, with potential impacts on both human and animal health.201729246178
4455140.9998A novel method to discover fluoroquinolone antibiotic resistance (qnr) genes in fragmented nucleotide sequences. BACKGROUND: Broad-spectrum fluoroquinolone antibiotics are central in modern health care and are used to treat and prevent a wide range of bacterial infections. The recently discovered qnr genes provide a mechanism of resistance with the potential to rapidly spread between bacteria using horizontal gene transfer. As for many antibiotic resistance genes present in pathogens today, qnr genes are hypothesized to originate from environmental bacteria. The vast amount of data generated by shotgun metagenomics can therefore be used to explore the diversity of qnr genes in more detail. RESULTS: In this paper we describe a new method to identify qnr genes in nucleotide sequence data. We show, using cross-validation, that the method has a high statistical power of correctly classifying sequences from novel classes of qnr genes, even for fragments as short as 100 nucleotides. Based on sequences from public repositories, the method was able to identify all previously reported plasmid-mediated qnr genes. In addition, several fragments from novel putative qnr genes were identified in metagenomes. The method was also able to annotate 39 chromosomal variants of which 11 have previously not been reported in literature. CONCLUSIONS: The method described in this paper significantly improves the sensitivity and specificity of identification and annotation of qnr genes in nucleotide sequence data. The predicted novel putative qnr genes in the metagenomic data support the hypothesis of a large and uncharacterized diversity within this family of resistance genes in environmental bacterial communities. An implementation of the method is freely available at http://bioinformatics.math.chalmers.se/qnr/.201223231464
6267150.9998Beta-lactamase dependent and independent evolutionary paths to high-level ampicillin resistance. The incidence of beta-lactam resistance among clinical isolates is a major health concern. A key method to study the emergence of antibiotic resistance is adaptive laboratory evolution. However, in the case of the beta-lactam ampicillin, bacteria evolved in laboratory settings do not recapitulate clinical-like resistance levels, hindering efforts to identify major evolutionary paths and their dependency on genetic background. Here, we used the Microbial Evolution and Growth Arena (MEGA) plate to select ampicillin-resistant Escherichia coli mutants with varying degrees of resistance. Whole-genome sequencing of resistant isolates revealed that ampicillin resistance was acquired via a combination of single-point mutations and amplification of the gene encoding beta-lactamase AmpC. However, blocking AmpC-mediated resistance revealed latent adaptive pathways: strains deleted for ampC were able to adapt through combinations of changes in genes involved in multidrug resistance encoding efflux pumps, transcriptional regulators, and porins. Our results reveal that combinations of distinct genetic mutations, accessible at large population sizes, can drive high-level resistance to ampicillin even independently of beta-lactamases.202438918379
4913160.9998Multiple Plasmids Contribute to Antibiotic Resistance and Macrophage Survival In Vitro in CMY2-Bearing Salmonella enterica. Multiple drug resistance (MDR) in bacteria represents a notable problem but if carried on plasmid their spread could become a significant threat to public health. Plasmids in members of the Enterobacteriaceae family and in particular Salmonella and Escherichia coli strains have been implicated in the spread of antibiotic resistance genes. However, the mechanisms involved in the transfer of plasmid-borne resistance genes are not fully understood. Here, we analyzed the ability of Salmonella enterica clinical isolates to transfer plasmid-borne MDR to E. coli. We also determined whether possession of an Inc A/C plasmid by a S. enterica isolate would confer increased fitness compared to an isolate not carrying the plasmid. Sixteen human and animal isolates of S. enterica were screened using a three-panel multiplex PCR assay, and simplex PCR for the blaCMY-2 gene. Using these data we selected a suitable strain as a plasmid donor for the construction of a new Salmonella strain with an Inc A/C plasmid. This allowed us to compare isogenic strains with and without the Inc A/C plasmid in multiple growth, fitness, and invasion assays. The results showed that possession of Inc A/C plasmid confers significant fitness advantage when tested in J774 macrophages as opposed to HEp-2 cells where no significant difference was found. In addition, stress assays performed in vitro showed that the possession of this large plasmid by Salmonella strains tested here does not appear to incur a significant fitness cost. Gaining a better understanding of molecular mechanisms of plasmid transfer between pathogenic bacteria will allow us to characterize the role of MDR in pathogenicity of bacteria and to identify methods to reduce the frequency of dissemination of multiple antibiotic resistance genes.201627070176
4631170.9998Genome 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
4381180.9998Specific Gene Loci of Clinical Pseudomonas putida Isolates. Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host's immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA) systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria.201626820467
9922190.9998De novo acquisition of antibiotic resistance in six species of bacteria. Bacteria can become resistant to antibiotics in two ways: by acquiring resistance genes through horizontal gene transfer and by de novo development of resistance upon exposure to non-lethal concentrations. The importance of the second process, de novo build-up, has not been investigated systematically over a range of species and may be underestimated as a result. To investigate the DNA mutation patterns accompanying the de novo antibiotic resistance acquisition process, six bacterial species encountered in the food chain were exposed to step-wise increasing sublethal concentrations of six antibiotics to develop high levels of resistance. Phenotypic and mutational landscapes were constructed based on whole-genome sequencing at two time points of the evolutionary trajectory. In this study, we found that (1) all of the six strains can develop high levels of resistance against most antibiotics; (2) increased resistance is accompanied by different mutations for each bacterium-antibiotic combination; (3) the number of mutations varies widely, with Y. enterocolitica having by far the most; (4) in the case of fluoroquinolone resistance, a mutational pattern of gyrA combined with parC is conserved in five of six species; and (5) mutations in genes coding for efflux pumps are widely encountered in gram-negative species. The overall conclusion is that very similar phenotypic outcomes are instigated by very different genetic changes. The outcome of this study may assist policymakers when formulating practical strategies to prevent development of antimicrobial resistance in human and veterinary health care.IMPORTANCEMost studies on de novo development of antimicrobial resistance have been performed on Escherichia coli. To examine whether the conclusions of this research can be applied to more bacterial species, six species of veterinary importance were made resistant to six antibiotics, each of a different class. The rapid build-up of resistance observed in all six species upon exposure to non-lethal concentrations of antimicrobials indicates a similar ability to adjust to the presence of antibiotics. The large differences in the number of DNA mutations accompanying de novo resistance suggest that the mechanisms and pathways involved may differ. Hence, very similar phenotypes can be the result of various genotypes. The implications of the outcome are to be considered by policymakers in the area of veterinary and human healthcare.202539907470