Distribution of the pco Gene Cluster and Associated Genetic Determinants among Swine Escherichia coli from a Controlled Feeding Trial. - Related Documents




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359901.0000Distribution of the pco Gene Cluster and Associated Genetic Determinants among Swine Escherichia coli from a Controlled Feeding Trial. Copper is used as an alternative to antibiotics for growth promotion and disease prevention. However, bacteria developed tolerance mechanisms for elevated copper concentrations, including those encoded by the pco operon in Gram-negative bacteria. Using cohorts of weaned piglets, this study showed that the supplementation of feed with copper concentrations as used in the field did not result in a significant short-term increase in the proportion of pco-positive fecal Escherichia coli. The pco and sil (silver resistance) operons were found concurrently in all screened isolates, and whole-genome sequencing showed that they were distributed among a diversity of unrelated E. coli strains. The presence of pco/sil in E. coli was not associated with elevated copper minimal inhibitory concentrations (MICs) under a variety of conditions. As found in previous studies, the pco/sil operons were part of a Tn7-like structure found both on the chromosome or on plasmids in the E. coli strains investigated. Transfer of a pco/sil IncHI2 plasmid from E. coli to Salmonella enterica resulted in elevated copper MICs in the latter. Escherichia coli may represent a reservoir of pco/sil genes transferable to other organisms such as S. enterica, for which it may represent an advantage in the presence of copper. This, in turn, has the potential for co-selection of resistance to antibiotics.201830340352
360110.9999R factors mediate resistance to mercury, nickel, and cobalt. Fifty-five clinical isolates and laboratory stocks of Escherichia coli and Salmonella were studied for resistance to each of ten metals. Eleven clinical isolates carrying R factors were resistant to mercury, and, in each case, the resistance was mediated by a previously undefined R-factor gene. The gene was phenotypically expressed within 2 to 4 minutes after entry into sensitive bacteria, but the basis for the resistance remains undefined. Fourteen strains, 12 infected with R factors, were resistant to cobalt and nickel, but these resistances were mediated by R-factor genes in only two strains; separate R-factor genes mediated the resistances to nickel and cobalt. These and other results indicate that the genetic composition of R factors is greater than that originally defined.19675337360
491320.9998Multiple 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
360030.9998Uncultured 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
447340.9998The genetics of bacterial trimethoprim resistance in tropical areas. Resistance to trimethoprim in Gram-negative bacteria is largely manifested by two trimethoprim resistant dihydrofolate reductases (types I and II) encoded by genes originally located on resistance plasmids. Although trimethoprim resistance increased markedly after the clinical introduction of trimethoprim in the West, its spread has slowed and, in Edinburgh at least, has actually been declining. This reduction has been accompanied by the migration of a transposon, encoding the type I plasmid resistance gene, into the bacterial chromosome. In tropical areas, the incidence of trimethoprim resistance is very much higher. In Tanzania, it has spilled over into other bacteria outside the Enterobacteriaceae, but it was in India where the major problem existed. The majority (64%) of the Indian Enterobacteriaceae studied were resistant to the drug and most of the resistance genes were located on very large plasmids which also conferred resistance to many other antibacterial drugs. Some Indian plasmids carried a new trimethoprim resistance gene which is not detectable by conventional sensitivity tests and may be spreading unnoticed elsewhere. The proportion of trimethoprim resistance has been related to the volume of antibacterial drugs used.19873318025
997450.9998Role of Plasmids in Co-Selection of Antimicrobial Resistances Among Escherichia coli Isolated from Pigs. Co-selection is thought to occur when resistance genes are located on the same mobile genetic element. However, this mechanism is currently poorly understood. In this study, complete circular plasmids from swine-derived Escherichia coli were sequenced with short and long reads to confirm that resistance genes involved in co-resistance were co-transferred by the same plasmid. Conjugative transfer tests were performed, and multiple resistance genes were transmitted. The genes possessed by the donor, transconjugant, and plasmid of the donor were highly similar. In addition, the sequences of the plasmid of the donor and the plasmid of the transconjugant were almost identical. Resistance genes associated with statistically significant combinations of antimicrobial use and resistance were co-transmitted by the same plasmid. These results suggest that resistance genes may be involved in co-selection by their transfer between bacteria on the same plasmid.202337540099
370160.9998Genetic Determinants for Metal Tolerance and Antimicrobial Resistance Detected in Bacteria Isolated from Soils of Olive Tree Farms. Copper-derived compounds are often used in olive tree farms. In a previous study, a collection of bacterial strains isolated from olive tree farms were identified and tested for phenotypic antimicrobial resistance and heavy metal tolerance. The aim of this work was to study the genetic determinants of resistance and to evaluate the co-occurrence of metal tolerance and antibiotic resistance genes. Both metal tolerance and antibiotic resistance genes (including beta-lactamase genes) were detected in the bacterial strains from Cu-treated soils. A high percentage of the strains positive for metal tolerance genes also carried antibiotic resistance genes, especially for genes involved in resistances to beta-lactams and tetracycline. Significant associations were detected between genes involved in copper tolerance and genes coding for beta-lactamases or tetracycline resistance mechanisms. A significant association was also detected between zntA (coding for a Zn(II)-translocating P-type ATPase) and tetC genes. In conclusion, bacteria from soils of Cu-treated olive farms may carry both metal tolerance and antibiotic resistance genes. The positive associations detected between metal tolerance genes and antibiotic resistance genes suggests co-selection of such genetic traits by exposure to metals.202032756388
597770.9998Methods 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
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
452390.9998Mosaic structure of a multiple-drug-resistant, conjugative plasmid from Campylobacter jejuni. Partial sequence analysis of a tet(O) plasmid from a multiple-drug-resistant clinical isolate of Campylobacter jejuni revealed 10 genes or pseudogenes encoding different aminoglycoside inactivating enzymes, transposase-like genes, and multiple unknown genes from a variety of pathogenic and commensal bacteria. The plasmid could be mobilized by a P incompatibility group plasmid into Escherichia coli, where it apparently integrated into the chromosome and expressed high-level resistance to multiple aminoglycoside antibiotics. This work provides new information about both the nature of drug resistance in C. jejuni and the ability of C. jejuni to exchange genes with other bacterial species.200515917546
6261100.9998Interplay between Amino Acid Substitution in GyrA and QnrB19: Elevating Fluoroquinolone Resistance in Salmonella Typhimurium. Globally, there have been increasing reports of antimicrobial resistance in nontyphoidal Salmonella (NTS), which can develop into severe and potentially life-threatening diarrhea. This study focuses on the synergistic effects of DNA gyrase mutations and plasmid-mediated quinolone resistance (PMQR) genes, specifically qnrB19, on fluoroquinolone (FQ) resistance in Salmonella Typhimurium. By utilizing recombinant mutants, GyrA(S83F) and GyrA(D87N), and QnrB19's, we discovered a significant increase in fluoroquinolones resistance when QnrB19 is present. Specifically, ciprofloxacin and moxifloxacin's inhibitory concentrations rose 10- and 8-fold, respectively. QnrB19 was found to enhance the resistance capacity of mutant DNA gyrases, leading to high-level FQ resistance. Additionally, we observed that the ratio of QnrB19 to DNA gyrase played a critical role in determining whether QnrB19 could protect DNA gyrase against FQ inhibition. Our findings underscore the critical need to understand these resistance mechanisms, as their coexistence enables bacteria to withstand therapeutic FQ levels, posing a significant challenge to treatment efficacy.202438898378
486110.9998Detection of heavy metal ion resistance genes in gram-positive and gram-negative bacteria isolated from a lead-contaminated site. Resistance to a range of heavy metal ions was determined for lead-resistant and other bacteria which had been isolated from a battery-manufacturing site contaminated with high concentration of lead. Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) and Gram-negative (Alcaligenes species) isolates were resistant to lead, mercury, cadmium, cobalt, zinc and copper, although the levels of resistance to the different metal ions were specific for each isolate. Polymerase chain reaction, DNA-DNA hybridization and DNA sequencing were used to explore the nature of genetic systems responsible for the metal resistance in eight of the isolates. Specific DNA sequences could be amplified from the genomic DNA of all the isolates using primers for sections of the mer (mercury resistance determinant on the transposon Tn501) and pco (copper resistance determinant on the plasmid pRJ1004) genetic systems. Positive hybridizations with mer and pco probes indicated that the amplified segments were highly homologous to these genes. Some of the PCR products were cloned and partially sequenced, and the regions sequenced were highly homologous to the appropriate regions of the mer and pco determinants. These results demonstrate the wide distribution of mercury and copper resistance genes in both Gram-positive and Gram-negative isolates obtained from this lead-contaminated soil. In contrast, the czc (cobalt, zinc and cadmium resistance) and chr (chromate resistance) genes could not be amplified from DNAs of some isolates, indicating the limited contribution, if any, of these genetic systems to the metal ion resistance of these isolates.19979342884
4906120.9998Factors that affect transfer of the IncI1 β-lactam resistance plasmid pESBL-283 between E. coli strains. The spread of antibiotic resistant bacteria worldwide presents a major health threat to human health care that results in therapy failure and increasing costs. The transfer of resistance conferring plasmids by conjugation is a major route by which resistance genes disseminate at the intra- and interspecies level. High similarities between resistance genes identified in foodborne and hospital-acquired pathogens suggest transmission of resistance conferring and transferrable mobile elements through the food chain, either as part of intact strains, or through transfer of plasmids from foodborne to human strains. To study the factors that affect the rate of plasmid transfer, the transmission of an extended-spectrum β-lactamase (ESBL) plasmid from a foodborne Escherichia coli strain to the β-lactam sensitive E. coli MG1655 strain was documented as a function of simulated environmental factors. The foodborne E. coli isolate used as donor carried a CTX-M-1 harboring IncI1 plasmid that confers resistance to β-lactam antibiotics. Cell density, energy availability and growth rate were identified as factors that affect plasmid transfer efficiency. Transfer rates were highest in the absence of the antibiotic, with almost every acceptor cell picking up the plasmid. Raising the antibiotic concentrations above the minimum inhibitory concentration (MIC) resulted in reduced transfer rates, but also selected for the plasmid carrying donor and recombinant strains. Based on the mutational pattern of transconjugant cells, a common mechanism is proposed which compensates for fitness costs due to plasmid carriage by reducing other cell functions. Reducing potential fitness costs due to maintenance and expression of the plasmid could contribute to persistence of resistance genes in the environment even without antibiotic pressure. Taken together, the results identify factors that drive the spread and persistence of resistance conferring plasmids in natural isolates and shows how these can contribute to transmission of resistance genes through the food chain.201525830294
4912130.9998Acquisition of plasmids from Shiga toxin-producing Escherichia coli strains had low or neutral fitness cost on commensal E. coli. Although it has been hypothesized that the acquisition of plasmids-especially those bearing virulence factors and antimicrobial resistance genes-increases the energetic burden and reduces the fitness of a bacterium in general, some results have challenged this view, showing little or no effect on fitness after plasmid acquisition, which may lead to change in the view that there are evolutionary barriers for a wide spread of such plasmids among bacteria. Here, to evaluate the fitness impact of plasmid-encoded antibiotic resistance and virulence genes, plasmids from O26:H11, O111:H8, and O118:H16 Shiga toxin-producing Escherichia coli (STEC) human and bovine isolates were transferred to the non-virulent E. coli HS and K-12 MG1655 strains. Sequencing and PCR were used to characterize plasmids, and to identify the presence of antimicrobial resistance and/or virulence genes. The fitness impact of plasmids encoding virulence and antimicrobial resistance upon bacterial hosts was determined by pairwise growth competition. Plasmid profile analysis showed that STEC strains carried one or more high and low molecular weight plasmids belonging to the B/O, F, I, K, P, Q, and/or X incompatibility groups encoding virulence genes (SPATE-encoding genes) and/or antimicrobial resistance genes (aadA1, strAB, tetA, and/or tetB). Competition experiments demonstrated that the biological cost of carriage of these plasmids by the commensal E. coli strain HS or the laboratory strain E. coli K-12 MG1655 was low or non-existent, ranging from - 4.7 to 5.2% per generation. This suggests that there are few biological barriers-or, alternatively, it suggests that there are biological barriers that we were not able to measure in this competition model-against the spread of plasmid encoding virulence and resistance genes from STEC to other, less pathogenic E. coli strains. Thus, our results, in opposition to a common view, suggest that the acquisition of plasmids does not significantly affect the bacteria fitness and, therefore, the theorized plasmid burden would not be a significant barrier for plasmid spread.202438396221
4471140.9998Update on acquired tetracycline resistance genes. This mini-review summarizes the changes in the field of bacterial acquired tetracycline resistance (tet) and oxytetracycline (otr) genes identified since the last major review in 2001. Thirty-eight acquired tetracycline resistant (Tc(r)) genes are known of which nine are new and include five genes coding for energy-dependent efflux proteins, two genes coding for ribosomal protection proteins, and two genes coding for tetracycline inactivating enzymes. The number of inactivating enzymes has increased from one to three, suggesting that work needs to be done to determine the role these enzymes play in bacterial resistance to tetracycline. In the same time period, 66 new genera have been identified which carry one or more of the previously described 29 Tc(r) genes. Included in the new genera is, for the first time, an obligate intracellular pathogen suggesting that this sheltered group of bacteria is capable of DNA exchange with non-obligate intracellular bacteria. The number of genera carrying ribosomal protection genes increased dramatically with the tet(M) gene now identified in 42 genera as compared with 24 and the tet(W) gene found in 17 new genera as compared to two genera in the last major review. New conjugative transposons, carrying different ribosomal protection tet genes, have been identified and an increase in the number of antibiotic resistance genes linked to tet genes has been found. Whether these new elements may help to spread the tet genes they carry to a wider bacterial host range is discussed.200515837373
4510150.9998Environmental concentrations of antibiotics, biocides, and heavy metals fail to induce phenotypic antimicrobial resistance in Escherichia coli. Most anthropogenically affected environments contain mixtures of pollutants from different sources. The impact of these pollutants is usually the combined effect of the individual polluting constituents. However, how these stressors contribute to the development of antimicrobial resistance in environmental microorganisms is poorly understood. Thus, a 30-day exposure experiment to environmental and sub-inhibitory concentrations of oxytetracycline, amoxicillin, zinc, copper, BAC (benzalkonium chloride) 10 and DADMAC (diallyldimethylammonium chloride) 12, was conducted using fully susceptible E. coli ATCC 25922 to ascertain any development of phenotypic or genotypic resistance. Furthermore, wild-type isolates were collected from the same aquatic environment as the stressors, analysed for phenotypic resistance using the disk diffusion method and genotypically through whole genome sequencing. Exposure to the various concentrations and combinations of the stressors did not trigger phenotypic resistance in the experimental bacteria. Furthermore, genotypic analysis of the WGS on the exposed isolates only found the macrolide resistance mdf(A) gene (also present in the control strain) and the disinfectant resistance gene sitABCD. With further analysis for single nucleotide variants (SNV), mutations were detected for 19 genes that encoded for oxidative stress, DNA repair, membrane proteins efflux systems, growth and persister formations except for the robA, a transcription protein subset of the ArcC/XylS family of proteins, which confer multidrug resistance in E. coli. This indicates that exposure to sub-inhibitory concentrations of antibiotics, heavy metals and biocide residues in the aquatic environmental concentrations of the stressors identified in the current study could not induce phenotypic or genotypic resistance but encoded for genes responsible for the development of persistence and tolerance in bacteria, which could be a precursor to the development of resistance in environmental bacteria.202337482346
4908160.9997Low temperatures do not impair the bacterial plasmid conjugation on poultry meat. Conjugation plays an important role in the dissemination of antimicrobial resistance genes. Besides, this process is influenced by many biotic and abiotic factors, especially temperature. This study aimed to investigate the effect of different conditions of temperature and storage (time and recipient) of poultry meat, intended for the final consumer, affect the plasmid transfer between pathogenic (harboring the IncB/O-plasmid) and non-pathogenic Escherichia coli organisms. The determination of minimal inhibitory concentrations (MIC) of ampicillin, cephalexin, cefotaxime, and ceftazidime was performed before and after the conjugation assay. It was possible to recover transconjugants in the poultry meat at all the treatments, also these bacteria showed a significant increase of the MIC for all antimicrobials tested. Our results show that a non-pathogenic E. coli can acquire an IncB/O-plasmid through a conjugation process in poultry meat, even stored at low temperatures. Once acquired, the resistance genes endanger public health especially when it is about critically and highly important antimicrobials to human medicine.202438191970
4497170.9997Detection and expression analysis of tet(B) in Streptococcus oralis. Tetracycline resistance can be achieved through tet genes, which code for efflux pumps, ribosomal protection proteins and inactivation enzymes. Some of these genes have only been described in either Gram-positive or Gram-negative bacteria. This is the case of tet(B), which codes for an efflux pump and, so far, had only been found in Gram-negative bacteria. In this study, tet(B) was detected in two clinical Streptococcus oralis strains isolated from the gingival sulci of two subjects. In both cases, the gene was completely sequenced, yielding 100% shared identity and coverage with other previously published sequences of tet(B). Moreover, we studied the expression of tet(B) using RT-qPCR in the isolates grown with and without tetracycline, detecting constitutive expression in only one of the isolates, with no signs of expression in the other one. This is the first time that the presence and expression of the tet(B) gene has been confirmed in Gram-positive bacteria, which highlights the potential of the genus Streptococcus to become a reservoir and a disseminator of antibiotic resistance genes in an environment so prone to horizontal gene transfer as is the oral biofilm.201931448060
3598180.9997An investigation of horizontal transfer of feed introduced DNA to the aerobic microbiota of the gastrointestinal tract of rats. BACKGROUND: Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes. RESULTS: Plasmid DNA with two resistance genes (nptI and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, were constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. No transformants were identified among 441 tested isolates. CONCLUSIONS: The analyses showed that extensive ingestion of DNA (100 μg plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit < 1 transformant per 1,1 × 10(8) cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed. This study is consistent with other studies suggesting natural transformation is not detectable in the GIT of mammals.201222463741
4380190.9997Comparative 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