Transfer of a bla CTX-M-1-carrying plasmid between different Escherichia coli strains within the human gut explored by whole genome sequencing analyses.
ABSTRACT: Horizontal transfer of antibiotic resistance determinants contributes to dissemination of antibiotic resistance. Such transfer of resistance genes within the human gut has been documented in some in vivo studies. The present study investigated seven bla CTX-M-1-carrying Escherichia coli isolates from three consecutive faecal samples collected from one cystic fibrosis patient in a nine-months period, by analysing whole genome sequencing data. The analyses showed that the seven E. coli isolates represented three genetically diverse strains. All isolates contained bla CTX-M-1-carrying Incl1 plasmids that shared a common 101?kb backbone differing by only four SNPs. The plasmids harboured by the three different E. coli strains varied within limited regions suggestive of recombination events, according to the phylogenetic topology of the genomes of the isolates harbouring them. The findings strongly suggest that horizontal transfer of a bla CTX-M-1-carrying plasmid had occurred within the patient´s gut. The study illustrates the within-host diversity of faecally carried resistant E. coli isolates and highlights the value of collecting multiple bacterial colonies from longitudinally collected samples to assess faecal carriage of resistant enterobacteria. The clustering of the plasmids with the corresponding E. coli strains carrying them indicates that the plasmids appear to have adapted to their respective E. coli hosts.
Project description:Background:Extended spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) causing urinary tract infections often belong to sequence type 131 (ST131), serotype O25, carrying bla CTX-M-15. Aim:The main aim of this study was to examine the conjugational frequencies of E. coli with plasmids carrying bla CTX-M-15 to E. coli isolates from the fecal flora of healthy humans to determine whether ST131 is more likely to uptake or donate ESBL resistance compared to other E. coli clones. Methods: Donors and recipients were all clinical isolates and did not harbor plasmids with identical incompatibility groups (Inc-groups) based on in silico analyses of Inc-groups and restriction/modification systems (R/M-systems). The in vitro conjugation experiments were performed as filter conjugation with verification of transconjugants by random amplified polymorphic DNA (RAPD) PCR and bla CTX-M-15 PCR. Results:The frequencies of conjugation with bla CTX-M-15-carrying plasmids were found to be very rare with detectable conjugation frequencies in the range of 4x10-9-7x10-7 transconjugants/recipient. Recipients of O25/ST131 type yielded significantly lower conjugation frequencies compared to recipients of other O-types (P=0.004). The applied ST131/O25 donors did not yield detectable levels of transconjugants regardless of the applied recipient. Presence of sub-MIC levels of ampicillin increased plasmid transfer frequencies x100 fold (P=0.07). Conclusion:The results indicate that bla CTX-M-15 is rarely transferred by conjugation to E. coli isolates of the intestinal flora, even when the gene is plasmid-borne.
Project description:<i>Escherichia coli</i> carrying <i>bla</i> <sub>CTX-M-</sub> <sub>1</sub> mediating resistance to extended-spectrum cephalosporins was recently described as a new genotype in Norwegian broiler production. The aim of this study was to characterize these isolates (<i>n</i> = 31) in order to determine whether the emergence of the genotype was caused by clonal expansion or horizontal dissemination of <i>bla</i> <sub>CTX-M-</sub> <sub>1</sub>-carrying plasmids. All included isolates were subjected to whole genome sequencing. Plasmid transferability was determined by conjugation, and plasmid replicons in the transconjugants were described using PCR-based replicon typing. Plasmid sizes were determined using S1 nuclease digestion. Plasmids in a subset of strains were reconstructed and compared to plasmids from broiler production in other European countries. The isolates belonged to nine different sequence types (STs), with the largest group being ST57 (<i>n</i> = 12). The vast majority of <i>bla</i> <sub>CTX-M-</sub> <sub>1</sub>-carrying plasmids were conjugative. All transconjugants were positive for the IncI1-Iγ replicon, and several also harbored the IncFIB replicon. Highly similar plasmids were present in different <i>E. coli</i> STs. Additionally, high similarity to previously published plasmids was detected. A reconstructed plasmid from an ST57 isolate harbored both IncI1-Iγ and IncFIB replicons and was considered to be co-integrated. The presence of one large plasmid was confirmed by S1 nuclease digestion. Our results show that dissemination of <i>bla</i> <sub>CTX-M-</sub> <sub>1</sub> in Norwegian broiler production is due to both clonal expansion and horizontal transfer of plasmids carrying <i>bla</i> <sub>CTX-M-</sub> <sub>1</sub>. The <i>bla</i> <sub>CTX-M-</sub> <sub>1</sub>/IncI1-Iγ plasmids grouped into two main lineages, namely clonal complex (CC)-3 and CC-7. The genetic diversity at both strain and plasmid level indicates multiple introductions to Norway. We also show that the <i>bla</i> <sub>CTX-M-</sub> <sub>1</sub> plasmids circulating in Norwegian broiler production are highly similar to plasmids previously described in other countries.
Project description:To study molecular epidemiology of CTX-M-55-carrying Escherichia coli isolates from urinary tract infections (UTIs) in China. 111 bla<sub>CTX-M-55</sub>-positive E.coli isolates from UTIs patients in China were studied. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to analyze the homologies among the strains. Conjugation experiments, S1nuclease PFGE and PCR analysis were performed to characterize plasmids harboring bla<sub>CTX-M-55</sub> and their genetic environment. 111 isolates were clustered into 86 individual pulsotypes and three clusters by PFGE. Fifty-five (49.5%) of the isolates belonged to 8 STs. Most of the ST1193 isolates belonged to one PFGE cluster. Transconjugants (n?=?45) derived from randomly selected bla<sub>CTX-M-55</sub> donors (n?=?58), were found to contain a single 90-kb conjugative plasmid, which mainly belonged to the IncI1 groups (34, 76%). Among the IncI1 plasmids, the bla<sub>CTX-M-55</sub>/IncI1/ST16 predominated (23/34, 68%). The bla<sub>TEM-1</sub> and aac (3')-II genes were frequently detected on the IncI1 plasmids, and the insertion of ISEcp1 or IS26 was observed at the 48?bp or 45?bp upstream of the start codon of bla<sub>CTX-M-55</sub> gene. The dissemination of bla<sub>CTX-M-55</sub> gene among E. coli UTI isolates, appeared to be due to both the major clonal lineage of ST1193 and the horizontal transfer of epidemic plasmid IncI1/ST16.
Project description:<b>Introduction:</b> Human faecal sludge contains diverse harmful microorganisms, making it hazardous to the environment and public health if it is discharged untreated. Faecal sludge is one of the major sources of <i>E. coli</i> that can produce extended-spectrum β-lactamases (ESBLs). <b>Objective:</b> This study aimed to investigate the prevalence and molecular characterization of ESBL-producing <i>E. coli</i> in faecal sludge samples collected from faecal sludge treatment plants (FSTPs) in Rohingya camps, Bangladesh. <b>Methods:</b> ESBL producing <i>E. coli</i> were screened by cultural as well as molecular methods and further characterized for their major ESBL genes, plasmid profiles, pathotypes, antibiotic resistance patterns, conjugation ability, and genetic similarity. <b>Results:</b> Of 296 isolates, 180 were phenotypically positive for ESBL. All the isolates, except one, contained at least one ESBL gene that was tested (<i>bla</i> <sub><i>CTX</i>-<i>M</i>-1</sub>, <i>bla</i> <sub><i>CTX</i>-<i>M</i>-2</sub>, <i>bla</i> <sub><i>CTX</i>-<i>M</i>-8</sub>, <i>bla</i> <sub><i>CTX</i>-<i>M</i>-9</sub>, <i>bla</i> <sub><i>CTX</i>-<i>M</i>-15</sub>, <i>bla</i> <sub><i>CTX</i>-<i>M</i>-25</sub>, <i>bla</i> <sub><i>TEM</i></sub> , and <i>bla</i> <sub><i>SHV</i></sub> ). From plasmid profiling, it was observed that plasmids of 1-211 MDa were found in 84% (151/180) of the isolates. Besides, 13% (24/180) of the isolates possessed diarrhoeagenic virulence genes. From the remaining isolates, around 51% (79/156) harbored at least one virulence gene that is associated with the extraintestinal pathogenicity of <i>E. coli</i>. Moreover, 4% (3/156) of the isolates were detected to be potential extraintestinal pathogenic <i>E. coli</i> (ExPEC) strains. Additionally, all the diarrhoeagenic and ExPEC strains showed resistance to three or more antibiotic groups which indicate their multidrug-resistant potential. ERIC-PCR differentiated these pathogenic isolates into seven clusters. In addition to this, 16 out of 35 tested isolates transferred plasmids of 32-112 MDa to <i>E. coli</i> J53 recipient strain. <b>Conclusion:</b> The present study implies that the faecal sludge samples examined here could be a potential origin for spreading MDR pathogenic ESBL-producing <i>E. coli</i>. The exposure of Rohingya individuals, living in overcrowded camps, to these organisms poses a severe threat to their health.
Project description:Animals are considered important sources of ESBL/AmpC-producing bacteria in humans. We analyzed indications of transfer of ESBL/AmpC genes between pigs and pig farmers in Vietnam by analyzing whole genome sequences of 114 ESBL/AmpC-producing <i>E. coli</i> isolated from the two hosts, and performed conjugation experiments and plasmid profiling to confirm that such transfer could have happened. ESBL-encoding genes detected in pigs and pig farmers included <i>bla</i> <sub>CTX-M-55</sub>, <i>bla</i> <sub>CTX-M-27</sub>, <i>bla</i> <sub>CTX-M-65</sub>, <i>bla</i> <sub>CTX-M-15</sub>, <i>bla</i> <sub>CTX-M-14</sub>, <i>bla</i> <sub>CTX-M-3</sub>, <i>bla</i> <sub>CTX-M-24</sub>, and <i>bla</i> <sub>CARB-2</sub>, and AmpC β-lactamases included <i>bla</i> <sub>CMY-2</sub>, <i>bla</i> <sub>DHA-1</sub>, and <i>bla</i> <sub>CMY-42</sub>. The most frequent ESBL gene, <i>bla</i> <sub>CTX-M-55</sub>, was carried on plasmid with replicons types IncF, IncX, IncH, IncN, IncR, and IncP. The insertion transposases downstream of the <i>bla</i> <sub>CTX-M-55</sub> gene were different in plasmids carried by different strains. The second most detected gene, <i>bla</i> <sub>CTX-M-27</sub>, is found in a stable genetic arrangement with the same flanking transposons seen across strains, and the gene was located on similar conjugal IncF plasmid types, suggesting a horizontal spread of these plasmids. In three strains, we observed a novel <i>bla</i> <sub>CTX-M-27</sub> harboring IncF type of plasmid which had not been reported before. Its closest reference in NCBI was the non-ESBL <i>Salmonella</i> Typhimurium plasmid pB71 that might have experienced an insertion of <i>bla</i> <sub>CTX-M-27</sub>. Our data also point to an emergence of plasmids co-carrying ESBL genes, <i>mcr</i> genes, quinolones and other antimicrobials resistance determinants, and such plasmids require special attention. Plasmids phylogeny confirmed that the <i>bla</i> <sub>CTX-M-55</sub> encoding plasmids varied considerably, while those encoding <i>bla</i> <sub>CTX-M-27</sub> were closely related. Plasmids harboring both ESBL genes were confirmed to be conjugative and not to differ in transfer efficacy. The isolates carrying the plasmids, even those with plasmids of similar types, showed wide genetic variation with high number of SNPs, suggesting horizontal spread of plasmids into different clonal lines. Their virulence profiles did not confirm to known pathotypes, suggesting that unrelated commensals are a main reservoir for ESBL and AmpC β-lactamases in both humans and pigs. Overall, despite evidence of transferability of plasmids in the analyzed strains, our findings do not support that ESBL-producing <i>E. coli</i> from pigs or their ESBL/AmpC encoding plasmids are commonly spread to workers in close contact with the animals.
Project description:pHN1122-1 carrying bla(CTX-M-55), from an Escherichia coli isolate from a dog, was completely sequenced. pHN1122-1 has an IncI2 replicon and typical IncI2-associated genetic modules, including mok/hok-finO-yafA/B, nikABC, and two transfer regions, tra and pil, as well as a shufflon. bla(CTX-M-55) is found within a 3.084-kb ISEcp1 transposition unit that includes a fragment of IncA/C plasmid backbone. pHN1122-1 and closely related plasmids were identified in other E. coli isolates from animals in China.
Project description:Of 15 extended-spectrum beta-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae collected from the First Municipal People's Hospital of Guangzhou, in the southern part of the People's Republic of China, 9 were found to produce CTX-M ESBLs, 3 produced SHV-12, and 3 produced both CTX-M and SHV-12. Eleven isolates produced either TEM-1B or SHV-11, in addition to an ESBL. Nucleotide sequence analysis of the 12 isolates carrying bla(CTX-M) genes revealed that they harbored three different bla(CTX-M) genes, bla(CTX-M-9) (5 isolates), bla(CTX-M-13) (1 isolate), and bla(CTX-M-14) (6 isolates). These genes have 98% nucleotide homology with bla(Toho-2). The bla(CTX-M) genes were carried on plasmids that ranged in size from 35 to 150 kb. Plasmid fingerprints and pulsed-field gel electrophoresis showed the dissemination of the bla(CTX-M) genes through transfer of different antibiotic resistance plasmids to different bacteria, suggesting that these resistance determinants are highly mobile. Insertion sequence ISEcp1, found on the upstream region of these genes, may be involved in the translocation of the bla(CTX-M) genes. This is the first report of the occurrence of SHV-12 and CTX-M ESBLs in China. The presence of strains with these ESBLs shows both the evolution of bla(CTX-M) genes and their dissemination among at least three species of the family Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, isolated within a single hospital. The predominance of CTX-M type enzymes seen in this area of China appears to be similar to that seen in South America but is different from those seen in Europe and North America, suggesting different evolutionary routes and selective pressures. A more comprehensive survey of the ESBL types from China is urgently needed.
Project description:The purpose of this study was to examine the occurrence of fosfomycin-resistant Escherichia coli from chickens and to characterize the plasmids carrying fosA3. A total of 661 E. coli isolates of chicken origin collected from 2009 to 2011 were screened for plasmid-mediated fosfomycin resistance determinants by PCR. Plasmids were characterized using PCR-based replicon typing, plasmid multilocus sequence typing, and restriction fragment length polymorphisms. Associated addiction systems and resistance genes were identified by PCR. PCR-mapping was used for analysis of the genetic context of fosA3. Fosfomycin resistance was detected in 58 isolates that also carried the fosA3 gene. Fifty-seven, 17, and 52 FosA3-producers also harbored bla CTX-M, rmtB, and floR genes, respectively. Most of the 58 fosA3-carrying isolates were clonally unrelated, and all fosA3 genes were located on plasmids belonged to F33:A-:B- (n = 18), IncN-F33:A-:B- (n = 7), IncHI2/ST3 (n = 10), IncI1/ST71 (n = 3), IncI1/ST108 (n = 3), and others. The genetic structures, IS26-ISEcp1-bla CTX-M-55-orf477-bla TEM-1-IS26-fosA3-1758bp-IS26 and ISEcp1-bla CTX-M-65-IS903-iroN-IS26-fosA3-536bp-IS26 were located on highly similar F33:A-:B- plasmids. In addition, bla CTX-M-14-fosA3-IS26 was frequently present on similar IncHI2/ST3 plasmids. IncFII plasmids had a significantly higher frequency of addiction systems (mean 3.5) than other plasmids. Our results showed a surprisingly high prevalence of fosA3 gene in E. coli isolates recovered from chicken in China. The spread of fosA3 can be attributed to horizontal dissemination of several epidemic plasmids, especially F33:A-:B- plasmids. Since coselection by other antimicrobials is the major driving force for the diffusion of the fosA3 gene, a strict antibiotic use policy is urgently needed in China.
Project description:The acquired CTX-M-type extended-spectrum-?-lactamase (ESBL)-producing Enterobacterales are of great concern in clinical settings because they limit therapeutic options for patients infected by the pathogens. An intriguing clonality of CTX-M ESBL-producing Klebsiella pneumoniae blood isolates was observed from a national cohort study, and comparative genomics were assessed for the 115 K. pneumoniae blood isolates carrying the bla CTX-M gene. The plasmid preference of particular clones of a sequence type (ST) was assessed by liquid mating. A quarter of the bla CTX-M gene-carrying K. pneumoniae blood isolates harbor the gene in their chromosome, and most of those with the built-in bla CTX-M gene belonged either to ST307 or ST48. Notably, all 16 K. pneumoniae ST48 isolates harbored two copies of the bla CTX-M-15 gene in the chromosome. The chromosomal integration of the bla CTX-M-15 gene was mostly derived from the ISEcp1-targeting 5-bp AT-rich locus in the chromosome. The IS26-mediated chromosomal integration occurred when the upstream ISEcp1 from the bla CTX-M gene was truncated, targeting the anchor IS26 copy in the chromosome. Higher transfer efficiency of the bla CTX-M-15 gene-carrying FIA:R plasmid was observed in ST17 than that of the bla CTX-M-14 gene-carrying FIB:FII plasmid. The transfer efficiency of the plasmid differed by isolate among the ST307 members. The K. pneumoniae clones ST307 and ST48 harboring the bla CTX-M-15 gene in the chromosome were able to disseminate stably in clinical settings regardless of the environmental pressure, and the current population of K. pneumoniae blood isolates was constructed. Further follow-up is needed for the epidemiology of this antimicrobial resistance.IMPORTANCE Dominant F-type plasmids harboring the gene have been pointed out to be responsible for the dissemination of the CTX-M extended-spectrum-?-lactamase (ESBL)-producing K. pneumoniae Recently, the emergence of K. pneumoniae isolates with the bla CTX-M gene in their chromosomes has been reported occasionally worldwide. Such a chromosomal location of the resistance gene could be beneficial for stable propagation, as was the Acinetobacter baumannii ST191 harboring chromosomal bla OXA-23 that is endemic to South Korea. Through the present study, particular clones were identified as having built-in resistance genes in their chromosomes, and the chromosomal integration events were tracked by assessing their genomes. The cefotaxime-resistant K. pneumoniae clones of this study were particularized as results of the fastidiousness for plasmids to acquire the bla CTX-M gene for securing the diversity and of the chromosomal addiction of the bla CTX-M gene for ensuring propagation.
Project description:OBJECTIVES: The presence of ESBL/AmpC-producing E. coli in cattle has been reported previously, however information on veal calves is limited. This study describes the prevalence and molecular characteristics of E. coli with non-wild type susceptibility to cefotaxime in veal calves at slaughter. METHODS: Faecal samples from 100 herds, 10 individual animals per herd, were screened for E. coli with non-wild type susceptibility for cefotaxime. Molecular characterization of ESBL/AmpC genes and plasmids was performed on one isolate per herd by microarray, PCR and sequence analysis. RESULTS: 66% of the herds were positive for E. coli with non-wild type susceptibility for cefotaxime. Within-herd prevalence varied from zero to 90%. 83% of E. coli producing ESBL/AmpC carried bla(CTX-M) genes, of which bla(CTX-M-1), bla(CTX-M-14) and bla(CTX-M-15) were most prevalent. The dominant plasmids were IncI1 and IncF-type plasmids. CONCLUSIONS: A relatively high prevalence of various bla(CTX-M) producing E. coli was found in veal calves at slaughter. The genes were mainly located on IncI1 and IncF plasmids.