Novel class 1 integron harboring antibiotic resistance genes in wastewater-derived bacteria as revealed by functional metagenomics. - Related Documents




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335801.0000Novel 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
335710.9999Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics. To detect plasmid-borne antibiotic-resistance genes in wastewater treatment plant (WWTP) bacteria, 192 resistance-gene-specific PCR primer pairs were designed and synthesized. Subsequent PCR analyses on total plasmid DNA preparations obtained from bacteria of activated sludge or the WWTP's final effluents led to the identification of, respectively, 140 and 123 different resistance-gene-specific amplicons. The genes detected included aminoglycoside, beta-lactam, chloramphenicol, fluoroquinolone, macrolide, rifampicin, tetracycline, trimethoprim and sulfonamide resistance genes as well as multidrug efflux and small multidrug resistance genes. Some of these genes were only recently described from clinical isolates, demonstrating genetic exchange between clinical and WWTP bacteria. Sequencing of selected resistance-gene-specific amplicons confirmed their identity or revealed that the amplicon nucleotide sequence is very similar to a gene closely related to the reference gene used for primer design. These results demonstrate that WWTP bacteria are a reservoir for various resistance genes. Moreover, detection of about 64 % of the 192 reference resistance genes in bacteria obtained from the WWTP's final effluents indicates that these resistance determinants might be further disseminated in habitats downstream of the sewage plant.200919389756
340720.9999The culturable soil antibiotic resistome: a community of multi-drug resistant bacteria. Understanding the soil bacterial resistome is essential to understanding the evolution and development of antibiotic resistance, and its spread between species and biomes. We have identified and characterized multi-drug resistance (MDR) mechanisms in the culturable soil antibiotic resistome and linked the resistance profiles to bacterial species. We isolated 412 antibiotic resistant bacteria from agricultural, urban and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16-23 antibiotics, comprising almost all classes of antibiotic. The mobile resistance genes investigated, (ESBL, bla NDM-1, and plasmid mediated quinolone resistance (PMQR) resistance genes) were not responsible for the respective resistance phenotypes nor were they present in the extracted soil DNA. Efflux was demonstrated to play an important role in MDR and many resistance phenotypes. Clinically relevant Burkholderia species are intrinsically resistant to ciprofloxacin but the soil Burkholderia species were not intrinsically resistant to ciprofloxacin. Using a phenotypic enzyme assay we identified the antibiotic specific inactivation of trimethoprim in 21 bacteria from different soils. The results of this study identified the importance of the efflux mechanism in the soil resistome and variations between the intrinsic resistance profiles of clinical and soil bacteria of the same family.201323776501
340630.9999Environmental and Pathogenic Carbapenem Resistant Bacteria Isolated from a Wastewater Treatment Plant Harbour Distinct Antibiotic Resistance Mechanisms. Wastewater treatment plants are important reservoirs and sources for the dissemination of antibiotic resistance into the environment. Here, two different groups of carbapenem resistant bacteria-the potentially environmental and the potentially pathogenic-were isolated from both the wastewater influent and discharged effluent of a full-scale wastewater treatment plant and characterized by whole genome sequencing and antibiotic susceptibility testing. Among the potentially environmental isolates, there was no detection of any acquired antibiotic resistance genes, which supports the idea that their resistance mechanisms are mainly intrinsic. On the contrary, the potentially pathogenic isolates presented a broad diversity of acquired antibiotic resistance genes towards different antibiotic classes, especially β-lactams, aminoglycosides, and fluoroquinolones. All these bacteria showed multiple β-lactamase-encoding genes, some with carbapenemase activity, such as the bla(KPC)-type genes found in the Enterobacteriaceae isolates. The antibiotic susceptibility testing assays performed on these isolates also revealed that all had a multi-resistance phenotype, which indicates that the acquired resistance is their major antibiotic resistance mechanism. In conclusion, the two bacterial groups have distinct resistance mechanisms, which suggest that the antibiotic resistance in the environment can be a more complex problematic than that generally assumed.202134572700
360040.9999Uncultured 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
339150.9999Phenotypic and genotypic analysis of bacteria isolated from three municipal wastewater treatment plants on tetracycline-amended and ciprofloxacin-amended growth media. AIMS: The goal of this study was to determine the antimicrobial susceptibility of bacteria isolated from three municipal wastewater treatment plants. METHODS AND RESULTS: Numerous bacterial strains were isolated from three municipal wastewater treatment facilities on tetracycline- (n=164) and ciprofloxacin-amended (n=65) growth media. These bacteria were then characterized with respect to their resistance to as many as 10 different antimicrobials, the presence of 14 common genes that encode resistance to tetracycline, the presence of integrons and/or the ability to transfer resistance via conjugation. All of the characterized strains exhibited some degree of multiple antimicrobial resistance, with nearly 50% demonstrating resistance to every antimicrobial that was tested. Genes encoding resistance to tetracycline were commonly detected among these strains, although intriguingly the frequency of detection was slightly higher for the bacteria isolated on ciprofloxacin-amended growth media (62%) compared to the bacteria isolated on tetracycline-amended growth media (53%). Class 1 integrons were also detected in 100% of the queried tetracycline-resistant bacteria and almost half of the ciprofloxacin-resistant strains. Conjugation experiments demonstrated that at least one of the tetracycline-resistant bacteria was capable of lateral gene transfer. CONCLUSIONS: Our results demonstrate that multiple antimicrobial resistance is a common trait among tetracycline-resistant and ciprofloxacin-resistant bacteria in municipal wastewater. SIGNIFICANCE AND IMPACT OF THE STUDY: These organisms are potentially important in the proliferation of antimicrobial resistance because they appear to have acquired multiple genetic determinants that confer resistance and because they have the potential to laterally transfer these genetic determinants to strains of clinical importance.201020629799
340860.9998The role of aquatic ecosystems as reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. The widespread and indiscriminate use of antibiotics has led to the development of antibiotic resistance in pathogenic, as well as commensal, microorganisms. Resistance genes may be horizontally or vertically transferred between bacterial communities in the environment. The recipient bacterial communities may then act as a reservoir of these resistance genes. In this study, we report the incidence of antibiotic resistance in enteric bacteria isolated from the Mhlathuze River and the distribution of genetic elements that may be responsible for the observed antibiotic resistance. The resistance of the enteric bacteria isolated over a period of one year showed that resistance to the older classes of antibiotics was high (94.7% resistance to one antibiotic and 80.8% resistance to two antibiotics). Furthermore, antibiotic resistance data of the environmental isolates showed a strong correlation (r = 0.97) with data obtained from diarrhoea patients. PCR based methods demonstrated that class 1 integrons were present in >50% of the environmental bacterial isolates that were resistant to multiple antibiotics. This class of integrons is capable of transferring genes responsible for resistance to beta-lactam, aminoglycoside, sulfonamide and quaternary ammonium antimicrobial agents. Conjugate plasmids were also isolated, but from a small percentage of isolates. This study showed that the Mhlathuze River (a) is a medium for the spread of bacterial antibiotic resistance genes, (b) acts as a reservoir for these genes and (c) due to socio-economic pressures, may play a role in the development and evolution of these genes along this river system.200415318485
340970.9998Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples. Antibiotic resistance is an increasing global problem resulting from the pressure of antibiotic usage, greater mobility of the population, and industrialization. Many antibiotic resistance genes are believed to have originated in microorganisms in the environment, and to have been transferred to other bacteria through mobile genetic elements. Among others, β-lactam antibiotics show clinical efficacy and low toxicity, and they are thus widely used as antimicrobials. Resistance to β-lactam antibiotics is conferred by β-lactamase genes and penicillin-binding proteins, which are chromosomal- or plasmid-encoded, although there is little information available on the contribution of other mobile genetic elements, such as phages. This study is focused on three genes that confer resistance to β-lactam antibiotics, namely two β-lactamase genes (blaTEM and blaCTX-M9) and one encoding a penicillin-binding protein (mecA) in bacteriophage DNA isolated from environmental water samples. The three genes were quantified in the DNA isolated from bacteriophages collected from 30 urban sewage and river water samples, using quantitative PCR amplification. All three genes were detected in the DNA of phages from all the samples tested, in some cases reaching 104 gene copies (GC) of blaTEM or 102 GC of blaCTX-M and mecA. These values are consistent with the amount of fecal pollution in the sample, except for mecA, which showed a higher number of copies in river water samples than in urban sewage. The bla genes from phage DNA were transferred by electroporation to sensitive host bacteria, which became resistant to ampicillin. blaTEM and blaCTX were detected in the DNA of the resistant clones after transfection. This study indicates that phages are reservoirs of resistance genes in the environment.201121390233
387280.9998Functional metagenomic analysis reveals rivers are a reservoir for diverse antibiotic resistance genes. The environment harbours a significant diversity of uncultured bacteria and a potential source of novel and extant resistance genes which may recombine with clinically important bacteria disseminated into environmental reservoirs. There is evidence that pollution can select for resistance due to the aggregation of adaptive genes on mobile elements. The aim of this study was to establish the impact of waste water treatment plant (WWTP) effluent disposal to a river by using culture independent methods to study diversity of resistance genes downstream of the WWTP in comparison to upstream. Metagenomic libraries were constructed in Escherichia coli and screened for phenotypic resistance to amikacin, gentamicin, neomycin, ampicillin and ciprofloxacin. Resistance genes were identified by using transposon mutagenesis. A significant increase downstream of the WWTP was observed in the number of phenotypic resistant clones recovered in metagenomic libraries. Common β-lactamases such as blaTEM were recovered as well as a diverse range of acetyltransferases and unusual transporter genes, with evidence for newly emerging resistance mechanisms. The similarities of the predicted proteins to known sequences suggested origins of genes from a very diverse range of bacteria. The study suggests that waste water disposal increases the reservoir of resistance mechanisms in the environment either by addition of resistance genes or by input of agents selective for resistant phenotypes.201424636906
339490.9998Antibiotic resistance patterns of Pseudomonas spp. isolated from faecal wastes in the environment and contaminated surface water. The Pseudomonas genus, which includes environmental and pathogenic species, is known to present antibiotic resistances, and can receive resistance genes from multi-resistant enteric bacteria released into the environment via faecal rejects. This study was aimed to investigate the resistome of Pseudomonas populations that have been in contact with these faecal bacteria. Thus, faecal discharges originating from human or cattle were sampled (from 12 points and two sampling campaigns) and 41 Pseudomonas species identified (316 isolates studied). The resistance phenotype to 25 antibiotics was determined in all isolates, and we propose a specific antibiotic resistance pattern for 14 species (from 2 to 9 resistances). None showed resistance to aminoglycosides, tetracycline, or polymyxins. Four species carried a very low number of resistances, with none to β-lactams. Interestingly, we observed the absence of the transcriptional activator soxR gene in these four species. No plasmid transfer was highlighted by conjugation assays, and a few class 1 but no class 2 integrons were detected in strains that may have received resistance genes from Enterobacteria. These results imply that the contribution of the Pseudomonas genus to the resistome of an ecosystem first depends on the structure of the Pseudomonas populations, as they may have very different resistance profiles.202031930390
4607100.9998Genetics of resistance to trimethoprim in cotrimoxazole resistant uropathogenic Escherichia coli: integrons, transposons, and single gene cassettes. Cotrimoxazole, the combined formulation of sulfamethoxazole and trimethoprim, is one of the treatments of choice for several infectious diseases, particularly urinary tract infections. Both components of cotrimoxazole are synthetic antimicrobial drugs, and their combination was introduced into medical therapeutics about half a century ago. In Gram-negative bacteria, resistance to cotrimoxazole is widespread, being based on the acquisition of genes from the auxiliary genome that confer resistance to each of its antibacterial components. Starting from previous knowledge on the genotype of resistance to sulfamethoxazole in a collection of cotrimoxazole resistant uropathogenic Escherichia coli strains, this work focused on the identification of the genetic bases of the trimethoprim resistance of these same strains. Molecular techniques employed included PCR and Sanger sequencing of specific amplicons, conjugation experiments and NGS sequencing of the transferred plasmids. Mobile genetic elements conferring the trimethoprim resistance phenotype were identified and included integrons, transposons and single gene cassettes. Therefore, strains exhibited several ways to jointly resist both antibiotics, implying different levels of genetic linkage between genes conferring resistance to sulfamethoxazole (sul) and trimethoprim (dfrA). Two structures were particularly interesting because they represented a highly cohesive arrangements ensuring cotrimoxazole resistance. They both carried a single gene cassette, dfrA14 or dfrA1, integrated in two different points of a conserved cluster sul2-strA-strB, carried on transferable plasmids. The results suggest that the pressure exerted by cotrimoxazole on bacteria of our environment is still promoting the evolution toward increasingly compact gene arrangements, carried by mobile genetic elements that move them in the genome and also transfer them horizontally among bacteria.202438946902
3395110.9998Presence of multidrug-resistant enteric bacteria in dairy farm topsoil. In addition to human and veterinary medicine, antibiotics are extensively used in agricultural settings, such as for treatment of infections, growth enhancement, and prophylaxis in food animals, leading to selection of drug and multidrug-resistant bacteria. To help circumvent the problem of bacterial antibiotic resistance, it is first necessary to understand the scope of the problem. However, it is not fully understood how widespread antibiotic-resistant bacteria are in agricultural settings. The lack of such surveillance data is especially evident in dairy farm environments, such as soil. It is also unknown to what extent various physiological modulators, such as salicylate, a component of aspirin and known model modulator of multiple antibiotic resistance (mar) genes, influence bacterial multi-drug resistance. We isolated and identified enteric soil bacteria from local dairy farms within Roosevelt County, NM, determined the resistance profiles to antibiotics associated with mar, such as chloramphenicol, nalidixic acid, penicillin G, and tetracycline. We then purified and characterized plasmid DNA and detected mar phenotypic activity. The minimal inhibitory concentrations (MIC) of antibiotics for the isolates ranged from 6 to >50 microg/mL for chloramphenicol, 2 to 8 microg/mL for nalidixic acid, 25 to >300 microg/mL for penicillin G, and 1 to >80 microg/mL for tetracycline. On the other hand, many of the isolates had significantly enhanced MIC for the same antibiotics in the presence of 5 mM salicylate. Plasmid DNA extracted from 12 randomly chosen isolates ranged in size from 6 to 12.5 kb and, in several cases, conferred resistance to chloramphenicol and penicillin G. It is concluded that enteric bacteria from dairy farm topsoil are multidrug resistant and harbor antibiotic-resistance plasmids. A role for dairy topsoil in zoonoses is suggested, implicating this environment as a reservoir for development of bacterial resistance against clinically relevant antibiotics.200515778307
4657120.9998Discovery 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
3392130.9998Coselection for resistance to multiple late-generation human therapeutic antibiotics encoded on tetracycline resistance plasmids captured from uncultivated stream and soil bacteria. AIMS: Transmissible plasmids captured from stream and soil bacteria conferring resistance to tetracycline in Pseudomonas were evaluated for linked resistance to antibiotics used in the treatment of human infections. METHODS AND RESULTS: Cells released from stream sediments and soils were conjugated with a rifampicin-resistant, plasmid-free Pseudomonas putida recipient and selected on tetracycline and rifampicin. Each transconjugant contained a single 50-80 kb plasmid. Resistance to 11 antibiotics, in addition to tetracycline, was determined for the stream transconjugants using a modification of the Stokes disc diffusion antibiotic susceptibility assay. Nearly half of plasmids conferred resistance to six or more antibiotics. Resistance to streptomycin, gentamicin, and/or ticarcillin was conferred by a majority of the plasmids, and resistance to additional human clinical use antibiotics such as piperacillin/tazobactam, ciprofloxacin and aztreonam was observed. MICs of 16 antibiotics for representative sediment and soil transconjugants revealed large increases, relative to the Ps. putida recipient, for 11 of 16 antibiotics tested, including the expanded spectrum antibiotics cefotaxime and ceftazidime, as well as piperacillin/tazobactam, lomefloxacin and levofloxacin. CONCLUSIONS: Resistance to multiple antibiotics-including those typically used in clinical Pseudomonas and enterobacterial infections-can be conferred by transmissible plasmids in streams and soils. SIGNIFICANCE AND IMPACT OF STUDY: Selective pressure exerted by the use of one antibiotic, such as the common agricultural antibiotic tetracycline, may result in the persistence of linked genes conferring resistance to important human clinical antibiotics. This may impact the spread of resistance to human use antibiotics even in the absence of direct selection.201424797476
4931140.9998Delineating the Acquired Genetic Diversity and Multidrug Resistance in Alcaligenes from Poultry Farms and Nearby Soil. Alcaligenes faecalis is one of the most important and clinically significant environmental pathogens, increasing in importance due to its isolation from soil and nosocomial environments. The Gram-negative soil bacterium is associated with skin endocarditis, bacteremia, dysentery, meningitis, endophthalmitis, urinary tract infections, and pneumonia in patients. With emerging antibiotic resistance in A. faecalis, it has become crucial to understand the origin of such resistance genes within this clinically significant environmental and gut bacterium. In this research, we studied the impact of antibiotic overuse in poultry and its effect on developing resistance in A. faecalis. We sampled soil and faecal materials from five poultry farms, performed whole genome sequencing & analysis and identified four strains of A. faecalis. Furthermore, we characterized the genes in the genomic islands of A. faecalis isolates. We found four multidrug-resistant A. faecalis strains that showed resistance against vancomycin (MIC >1000 μg/ml), ceftazidime (50 μg/ml), colistin (50 μg/ml) and ciprofloxacin (50 μg/ml). From whole genome comparative analysis, we found more than 180 resistance genes compared to the reference sequence. Parts of our assembled contigs were found to be similar to different bacteria which included pbp1A and pbp2 imparting resistance to amoxicillin originally a part of Helicobacter and Bordetella pertussis. We also found the Mycobacterial insertion element IS6110 in the genomic islands of all four genomes. This prominent insertion element can be transferred and induce resistance to other bacterial genomes. The results thus are crucial in understanding the transfer of resistance genes in the environment and can help in developing regimes for antibiotic use in the food and poultry industry.202438904697
5746150.9998Identification 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
4910160.9998Excreted Antibiotics May Be Key to Emergence of Increasingly Efficient Antibiotic Resistance in Food Animal Production. At a time when antibiotic resistance is seemingly ubiquitous worldwide, understanding the mechanisms responsible for successful emergence of new resistance genes may provide insights into the persistence and pathways of dissemination for antibiotic-resistant organisms in general. For example, Escherichia coli strains harboring a class A β-lactamase-encoding gene (bla(CTX-M-15)) appear to be displacing strains that harbor a class C β-lactamase gene (bla(CMY-2)) in Washington State dairy cattle. We cloned these genes with native promoters into low-copy-number plasmids that were then transformed into isogenic strains of E. coli, and growth curves were generated for two commonly administered antibiotics (ampicillin and ceftiofur). Both strains met the definition of resistance for ampicillin (≥32 μg/mL) and ceftiofur (≥16 μg/mL). Growth of the CMY-2-producing strain was compromised at 1,000 μg/mL ampicillin, whereas the CTX-M-15-producing strain was not inhibited in the presence of 3,000 μg/mL ampicillin or with most concentrations of ceftiofur, although there were mixed outcomes with ceftiofur metabolites. Consequently, in the absence of competing genes, E. coli harboring either gene would experience a selective advantage if exposed to these antibiotics. Successful emergence of CTX-M-15-producing strains where CMY-2-producing strains are already established, however, requires high concentrations of antibiotics that can only be found in the urine of treated animals (e.g., >2,000 μg/mL for ampicillin, based on literature). This ex vivo selection pressure may be important for the emergence of new and more efficient antibiotic resistance genes and likely for persistence of antibiotic-resistant bacteria in food animal populations. IMPORTANCE We studied the relative fitness benefits of a cephalosporin resistance enzyme (CTX-M-15) that is displacing a similar enzyme (CMY-2), which is extant in E. coli from dairy cattle in Washington State. In vitro experiments demonstrated that CTX-M-15 provides a significant fitness advantage, but only in the presence of very high concentrations of antibiotic that are only found when the antibiotic ampicillin, and to a lesser extent ceftiofur, is excreted in urine from treated animals. As such, the increasing prevalence of bacteria with bla(CTX-M-15) is likely occurring ex vivo. Interventions should focus on controlling waste from treated animals and, when possible, selecting antibiotics that are less likely to impact the proximal environment of treated animals.202235867586
3470170.9998A treatment plant receiving waste water from multiple bulk drug manufacturers is a reservoir for highly multi-drug resistant integron-bearing bacteria. The arenas and detailed mechanisms for transfer of antibiotic resistance genes between environmental bacteria and pathogens are largely unclear. Selection pressures from antibiotics in situations where environmental bacteria and human pathogens meet are expected to increase the risks for such gene transfer events. We hypothesize that waste-water treatment plants (WWTPs) serving antibiotic manufacturing industries may provide such spawning grounds, given the high bacterial densities present there together with exceptionally strong and persistent selection pressures from the antibiotic-contaminated waste. Previous analyses of effluent from an Indian industrial WWTP that processes waste from bulk drug production revealed the presence of a range of drugs, including broad spectrum antibiotics at extremely high concentrations (mg/L range). In this study, we have characterized the antibiotic resistance profiles of 93 bacterial strains sampled at different stages of the treatment process from the WWTP against 39 antibiotics belonging to 12 different classes. A large majority (86%) of the strains were resistant to 20 or more antibiotics. Although there were no classically-recognized human pathogens among the 93 isolated strains, opportunistic pathogens such as Ochrobactrum intermedium, Providencia rettgeri, vancomycin resistant Enterococci (VRE), Aerococcus sp. and Citrobacter freundii were found to be highly resistant. One of the O. intermedium strains (ER1) was resistant to 36 antibiotics, while P. rettgeri (OSR3) was resistant to 35 antibiotics. Class 1 and 2 integrons were detected in 74/93 (80%) strains each, and 88/93 (95%) strains harbored at least one type of integron. The qPCR analysis of community DNA also showed an unprecedented high prevalence of integrons, suggesting that the bacteria living under such high selective pressure have an appreciable potential for genetic exchange of resistance genes via mobile gene cassettes. The present study provides insight into the mechanisms behind and the extent of multi-drug resistance among bacteria living under an extreme antibiotic selection pressure.201324204801
3356180.9998Conjugative multiple-antibiotic resistance plasmids in Escherichia coli isolated from environmental waters contaminated by human faecal wastes. AIMS: To better understand the involvement of faecal contamination in the dissemination of antibiotic resistance genes, we investigated the genetic supports of resistances in nine multi-resistant Escherichia coli strains originating from human faecal contamination, and isolated from three different aquatic environments used for producing drinking water. METHODS AND RESULTS: Seven strains harboured at least one large plasmid that we have characterized (size, antibiotic resistance patterns, incompatibility group, capacity of autotransfer, presence of integron). Most of these plasmids were conjugative and carried numerous resistances. One of the plasmids studied, belonging to the IncP incompatibility group, was able to transfer by conjugation to Pseudomonas fluorescens and Aeromonas sp. Only two of the plasmids we studied carried class 1 and/or 2 integron(s). CONCLUSIONS: Conjugative plasmids isolated from multi-resistant E. coli strains explained most of the resistances of their host strains and probably contribute to the spread of antibiotic resistance genes coming from human faecal contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: These results highlight the key role played by plasmids in the multi-resistance phenotype of faecal bacteria and the diversity of these genetic structures. Contaminated water, especially accidentally contaminated drinking water, could be a path back to humans for these plasmids.201525387599
5977190.9998Methods 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