Molecular analysis of florfenicol-resistant Escherichia coli isolates from pigs

OBJECTIVES: The aim of this study was to analyse florfenicol-resistant Escherichia coli isolates from pigs for the genetic basis of florfenicol resistance, and to compare these data with those previously determined for E. coli isolates from cattle and poultry. METHODS: Fourteen porcine E. coli isolates were included in this study and subjected to serotyping, plasmid profiling and macrorestriction analysis. MICs of florfenicol were determined by broth microdilution. The presence of the gene floR was confirmed by hybridization and PCR analysis. Transformation experiments were conducted to isolate florfenicol resistance plasmids. The floR region of a florfenicol resistance plasmid was cloned and sequenced. RESULTS: All florfenicol-resistant E. coli isolates exhibited MICs of florfenicol >128 mg/L and carried the floR gene. A single isolate had a floR-carrying plasmid of approximately 35 kb, designated pMBSF1. Sequence analysis identified the floR gene flanked by truncated transposase genes. Moreover, a truncated copy of Tn5393 with complete streptomycin resistance genes strA and strB was found upstream of the floR gene of pMBSF1. Chromosomally resistant E. coli isolates, which shared the same BlnI macrorestriction pattern, differed in their floR hybridization patterns. CONCLUSION: The plasmid pMBSF1 is the smallest floR-carrying plasmid reported to date. Its floR region differed from those previously found in E. coli isolates from cattle. Variations in the RFLPs of chromosomal EcoRI fragments carrying floR in isolates that had the same macrorestriction pattern might suggest variable chromosomal integration sites.     

Plasmid-borne florfenicol resistance in Pasteurella multocida

OBJECTIVES: A florfenicol-resistant Pasteurella multocida isolate from a calf was investigated for the genetic basis of florfenicol resistance and the location of the resistance gene. METHODS: The P. multocida isolate 381 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 10.8 kb florfenicol-chloramphenicol resistance plasmid, designated pCCK381, was identified by transformation into Escherichia coli. The plasmid was mapped with restriction endonucleases, cloned and sequenced completely. RESULTS: Of the antimicrobials tested, plasmid pCCK381 conferred resistance only to chloramphenicol and florfenicol. It showed extended similarity to the 5.1 kb plasmid pDN1 from Dichelobacter nodosus in the part carrying the mobilization and replication genes. An adjacent 3.2 kb segment was highly homologous to the florfenicol resistance gene region of plasmid pMBSF1 from E. coli. In pCCK381, combined resistance to chloramphenicol and florfenicol was based on the presence of a floR gene that showed 97.2-99.7% identity to so far known floR genes. CONCLUSIONS: The results of this study showed that a plasmid-borne floR gene was responsible for chloramphenicol and florfenicol resistance in the bovine respiratory tract pathogen P. multocida. This is, to the best of our knowledge, the first report of a florfenicol resistance gene in a target bacterium.     

Molecular analysis of tetracycline resistance in Pasteurella aerogenes

Tetracycline-resistant Pasteurella aerogenes isolates obtained from the intestinal tract of swine were investigated for their tet genes by PCR analysis and hybridization experiments. In contrast to Pasteurella isolates from the respiratory tract, tet(H) genes were detected in the chromosomal DNA of only 2 of the 24 isolates, one of which also carried two copies of a tet(B) gene. All other P. aerogenes isolates carried tet(B) genes, which are the predominant tet genes among Enterobacteriaceae. A single isolate harbored a tet(B) gene as part of a truncated Tn10 element on the 4.8-kb plasmid pPAT2. Comparative analysis of the pPAT2 sequence suggested that the Tn10 relic on plasmid pPAT2 is the result of several illegitimate recombination events. The remaining 21 P. aerogenes isolates carried one or two copies of the tet(B) gene in their chromosomal DNA. In the majority of the cases, these tet(B) genes were associated with copies of Tn10 as confirmed by their SfuI and BamHI hybridization patterns. No correlation between the number of tet gene copies and the MICs of tetracycline, doxycyline and minocycline was observed.     

Efflux-mediated resistance to florfenicol and/or chloramphenicol in Bordetella bronchiseptica: identification of a novel chloramphenicol exporter

OBJECTIVES: Twenty florfenicol- and/or chloramphenicol-resistant Bordetella bronchiseptica isolates of porcine and feline origin were investigated for the presence of floR and cml genes and their location on plasmids. METHODS: The B. bronchiseptica isolates were investigated for their susceptibility to antimicrobial agents by broth micro- or macrodilution and for their plasmid content. Hybridization experiments and PCR assays were conducted to identify resistance genes. Transformation and conjugation studies were performed to show their transferability. Representatives of both types of genes including their flanking regions were sequenced. Moreover, inhibitor studies with the efflux pump inhibitor Phe-Arg-beta-naphthylamide (PAbetaN) were performed. RESULTS: The gene floR was found in the chromosomal DNA of 9 of the 18 florfenicol/chloramphenicol-resistant isolates. Sequence analysis revealed that the deduced FloR protein sequence differed by a single amino acid exchange from FloR of Vibrio cholerae. A chloramphenicol-resistant, but florfenicol-susceptible isolate carried a novel plasmid-borne cml gene, designated cmlB1. The CmlB1 protein revealed only 73.8-76.5% identity to known CmlA proteins. The gene cmlB1 was not part of a gene cassette. The results of inhibitor studies with PAbetaN suggested that a so-far unidentified efflux system might play a role in phenicol resistance of the remaining florfenicol- and/or chloramphenicol-resistant isolates. CONCLUSIONS: This is to the best of our knowledge the first report of a floR gene in B. bronchiseptica isolates. The identification of the first member of a new subclass of cml genes, cmlB1 from B. bronchiseptica, extends our knowledge on specific chloramphenicol exporters.     

Plasmid-mediated florfenicol resistance encoded by the floR gene in Escherichia coli isolated from cattle

A florfenicol resistance gene almost identical to floR of Salmonella enterica serovar Typhimurium DT104 was detected on 110- to 125-kb plasmids in Escherichia coli isolates of animal origin. Analysis of the floR gene flanking regions of one of the plasmids showed that they were different from those encountered in S. enterica serovar Typhimurium DT104.     

Tetracycline resistance genes in isolates of Pasteurella multocida, Mannheimia haemolytica, Mannheimia glucosida and Mannheimia varigena from bovine and swine respiratory disease: intergeneric spread of the tet(H) plasmid pMHT1.

Tetracycline-resistant isolates of Pasteurella multocida and Mannheimia spp. from respiratory diseases in cattle and swine were investigated for the classes of tet gene and their chromosomal or plasmid location. The 34 isolates comprised eight P. multocida, 23 Mannheimia haemolytica, two Mannheimia varigena and a single Mannheimia glucosida isolate. Identification of the tet genes was achieved by PCR analysis and hybridization with specific probes. Transformation and hybridization experiments served to confirm the plasmid location of tet genes. Selected tet genes and their adjacent regions were sequenced. The tet genes tet(B), tet(G) and tet(H) were detected. The gene tet(H) was present in 26 isolates. The 4.4 kb tet(H)-carrying plasmid pMHT1 was detected in six isolates representing all four species. In the remaining 28 isolates, copies of tet(B), tet(G) and tet(H) were identified as chromosomal. No correlation between the tet gene type and the MIC of tetracycline, or between the number of tet gene copies and the MIC of tetracycline was observed. Tetracycline resistance in P. multocida and Mannheimia spp. is mediated by at least three different tet genes. A new type of tet(H)- carrying plasmid, pMHT1, was identified. The detection of pMHT1 in M. glucosida and M. varigena is the first report of resistance plasmids in isolates of these two species. For the first time, tet(G) genes were detected in members of the family Pasteurellaceae.     

Nosocomial spread of cephem-resistant Escherichia coli strains carrying multiple Toho-1-like beta-lactamase genes.

Escherichia coli HKY56, which demonstrated resistance to various beta-lactams except carbapenems, was isolated from the throat swab of an inpatient in 1994. Conjugal transfer of cephem resistance from HKY56 to E. coli CSH2 was not successful. Three cefotaxime-resistant E. coli clones harboring plasmid pMRE001, pMRE002, or pMRE003, each of which carried a 3.4-, 5.8-, or 6.2-kb EcoRI fragment insert, respectively, were obtained from HKY56. Although restriction analysis suggested their different origins, these clones showed similar profiles of resistance to various beta-lactams. The sequence of 10 amino acid residues at the N terminus of beta-lactamase purified from E. coli HB101(pMRE001) was identical to that of Toho-1. This Toho-1-like beta-lactamase-1 (TLB-1) was able to hydrolyze cefoperazone and cefotaxime efficiently, but it failed to hydrolyze cephamycins. A Toho-1-specific DNA probe was hybridized with three distinct EcoRI fragments derived from the chromosomal DNA of strain HKY56, and these fragments corresponded to DNA inserts carried by pMRE001, pMRE002, and pMRE003, respectively. PCR and Southern hybridization analysis suggested that all six cephem-resistant E. coli strains, strains HKY273, HKY285, HKY288, HKY305, HKY316, and HKY335, which were isolated in 1996 at the same hospital where strain HKY56 had been isolated, also possessed multiple Toho-1-like beta-lactamase (TLB) genes, and the hybridization patterns obtained with the Toho-1-specific probe were quite similar among these six isolates. The DNA fingerprinting patterns observed by pulsed-field gel electrophoresis revealed that among the E. coli isolates tested, all isolates except HKY56 possessed a similar genetic background. These findings suggested that E. coli strains that carry chromosomally multiplied TLB genes may have been proliferating and transmitted among patients in the same hospital.     

Evidence for transfer of CMY-2 AmpC beta-lactamase plasmids between Escherichia coli and Salmonella isolates from food animals and humans

Escherichia coli is an important pathogen that shows increasing antimicrobial resistance in isolates from both animals and humans. Our laboratory recently described Salmonella isolates from food animals and humans that expressed an identical plasmid-mediated, AmpC-like beta-lactamase, CMY-2. In the present study, 59 of 377 E. coli isolates from cattle and swine (15.6%) and 6 of 1,017 (0.6%) isolates of human E. coli from the same geographic region were resistant to both cephamycins and extended-spectrum cephalosporins. An ampC gene could be amplified with CMY-2 primers in 94.8% of animal and 33% of human isolates. Molecular epidemiological studies of chromosomal DNA revealed little clonal relatedness among the animal and human E. coli isolates harboring the CMY-2 gene. The ampC genes from 10 animal and human E. coli isolates were sequenced, and all carried an identical CMY-2 gene. Additionally, all were able to transfer a plasmid containing the CMY-2 gene to a laboratory strain of E. coli. CMY-2 plasmids demonstrated two different plasmid patterns that each showed strong similarities to previously described Salmonella CMY-2 plasmids. Additionally, Southern blot analyses using a CMY-2 probe demonstrated conserved fragments among many of the CMY-2 plasmids identified in Salmonella and E. coli isolates from food animals and humans. These data demonstrate that common plasmids have been transferred between animal-associated Salmonella and E. coli, and identical CMY-2 genes carried by similar plasmids have been identified in humans, suggesting that the CMY-2 plasmid has undergone transfer between different bacterial species and may have been transmitted between food animals and humans.     

Molecular characterization of an Escherichia coli clinical isolate that produces both metallo-beta-lactamase VIM-2 and extended-spectrum beta-lactamase GES-7: identification of the In8 integron carrying the blaVIM-2 gene

OBJECTIVES: We have previously reported the isolation of a VIM-2-producing Escherichia coli clinical isolate and the coexistence of blaVIM-2 and blaGES-7 in the same isolate. The aim of this study was to elucidate the genetic environment of these genes. METHODS: PCR and sequence analysis were used to identify and analyse the blaVIM-containing integron. The location of the blaVIM-2 and blaGES-7 genes was identified by hybridization experiments on I-CeuI-digested genomic DNA after PFGE. RESULTS: Only the blaVIM-2 gene has been found on a class I integron, designated In8 (1474 bp). Hybridization with the blaVIM-2, the blaGES-7 and the 16S-23S rRNA probes confirmed the chromosomal location of both genes. The blaVIM-2 probe hybridized with a 710 kb fragment whereas the blaGES-7 probe hybridized with a 144 kb fragment of genomic DNA of the E. coli isolate. CONCLUSIONS: This report provides evidence for the chromosomal location of both blaVIM-2 and blaGES-7 genes carried by an E. coli clinical isolate. In8, containing blaVIM-2, presents an emerging threat of carbapenem resistance among E. coli isolates in Greece.     

Chloramphenicol and kanamycin resistance among porcine Escherichia coli in Ontario

OBJECTIVES: The purpose of this study was to compare the distribution of chloramphenicol and kanamycin resistance genes across three populations of porcine Escherichia coli. METHODS: PCR was used to assess the distribution of the major chloramphenicol and kanamycin resistance genes catA1, cmlA and floR, and aphA1, aphA2 and aadB in enterotoxigenic E. coli (ETEC), non-ETEC isolates from cases of diarrhoea and commensal E. coli from healthy pigs. Associations between these genes and resistance genes for other antimicrobials or virulence genes were assessed. RESULTS: The chloramphenicol and kanamycin resistance genes were distributed differently among the three E. coli populations. While aphA1, aphA2 and aadB were evenly distributed among resistant ETEC, non-ETEC and commensals, the catA1 gene was significantly more frequent in ETEC than in non-ETEC and commensals. Transformation experiments confirmed statistical associations by demonstrating that elt, estB, astA, aadA and sul1 were located with catA1 on a large ETEC plasmid. Plasmids carrying cmlA also carried sul3 and aadA. Other plasmids carrying floR and aadB also carried tet(A), sul2, strA/strB, bla(CMY-2) and occasionally aac(3)IV. CONCLUSIONS: The clustering of genes observed is a likely cause for chloramphenicol resistance persistence. Similar to tetracycline, chloramphenicol resistance genes are physically linked to virulence genes. This is not the case for kanamycin resistance determinants, which were linked to other resistance genes only.     

Characterization of Salmonella enterica serovar Typhimurium isolates harboring a chromosomally encoded CMY-2 beta-lactamase gene located on a multidrug resistance genomic island

Since 2004, extended-spectrum cephalosporin (ESC)-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates have been detected from cattle in the northern major island of Japan, Hokkaido. Resistance to ESCs was found to be mediated by CMY-2 type β-lactamase among 22 epidemiologically unrelated isolates showing indistinguishable pulsed-field gel electrophoresis patterns. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that the CMY-2 β-lactamase gene (bla(CMY-2)) was integrated in a 2.5-Mb chromosomal fragment. Genetic analysis of S. Typhimurium isolate L-3553 indicated that bla(CMY-2) was located on a unique 125-kb genomic island, GI-VII-6, which consists of 140 open reading frames. Pairwise alignment of GI-VII-6 and Escherichia coli plasmid pAR060302 (size, 167 kb) revealed that a large proportion of GI-VII-6 (99%) shows a high sequence similarity (>99%) with pAR060302. GI-VII-6 contains 11 antimicrobial resistance genes including sul1, qacEΔ1, aadA2, and dfrA12 in the aadA2 region; sugE1 and bla(CMY-2) in the bla(CMY-2) region; and sul2, strA, strB, tet(A), and floR in the floR region. Two directly repeated IS26 copies were present at both ends of GI-VII-6. Junction regions of GI-VII-6 were flanked by an 8-bp direct repeat, indicating that GI-VII-6 was acquired by transposition involving IS26 transposase. PCR scanning revealed that the overall structure of GI-VII-6 was almost identical in the 22 isolates. Phylogenetic analysis suggested that S. Typhimurium isolates harboring GI-VII-6 belong to a different genomic lineage than other whole-genome-sequenced S. Typhimurium strains. These data indicate that a particular clone of S. Typhimurium harboring GI-VII-6 has spread among the cattle population in Hokkaido, Japan.     

Beta-lactam resistance in clinical isolates of Escherichia coli caused by elevated production of the ampC-mediated chromosomal beta-lactamase.

Among cephalothin-resistant isolates from patients with urinary tract infections, six Escherichia coli strains were found to produce elevated amounts of a beta-lactamase indistinguishable from that coded by the ampC gene of E. coli K-12. The resistance levels displayed by these isolates toward a number of beta-lactams were, for five of them, considerably higher as compared with E. coli K-12 with the same amount of beta-lactamase, implying the importance of intrinsic resistance in these isolates. Cefuroxime, and to a lesser extent cefamandole, were stable to hydrolysis by E. coli chromosomal beta-lactamase but acted as inhibitors of the enzyme. Nevertheless, increased beta-lactamase production mediated an increased resistance toward these drugs. No plasmids were found in the isolates, suggesting a chromosomal location for the respective ampC locus.     

The AmpC inhibitor, Syn2190, can be used to reveal extended-spectrum beta-lactamases in Escherichia coli

AmpC beta-lactamases are not inhibited by clavulanic acid and could potentially mask detection of extended-spectrum beta-lactamases (ESBLs) using the Clinical and Laboratory Standards Institute confirmatory test. Syn2190 (1,5-dihydroxy-4-pyridone monobactam) selectively inhibits AmpC, but not ESBLs. Fifty-four MicroScan ESBL screen-positive strains of Escherichia coli and an unrelated group of 20 cefoxitin-nonsusceptible E. coli strains were tested with the confirmatory ceftazidime-cefotaxime-clavulanate disk method with or without 4 microg/mL of Syn2190 in the agar. Without Syn2190, 8 (14.8%) of 54 E. coli isolates and 0 of 20 cefoxitin-nonsusceptible E. coli isolates were confirmed. With Syn2190, an additional 9 (16.6%) of 54 of the MicroScan screen-positive E. coli isolates and 6 (30%) of 20 of the cefoxitin-nonsusceptible E. coli isolates were found. Multiplex polymerase chain reaction and sequence analysis confirmed the presence of the plasmid-associated beta-lactamase gene bla(CMY-2) in the 2 available MicroScan-screened E. coli isolates and in 5 of 6 of the cefoxitin-resistant group. These data suggest that in the presence of AmpC, ESBLs in E. coli may not be detected by the currently recommended confirmatory test.     

Characterization of antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea

OBJECTIVES: Antimicrobial resistance and class 1 integrons found in Escherichia coli isolates from humans and animals in Korea were characterized. METHODS: E. coli isolates were examined for susceptibility to antimicrobial agents. Integrase genes were amplified. Gene cassette regions for classes 1 and 2 integrons were amplified and sequenced. Conjugal transfer and Southern hybridization were performed to determine the genetic localization of class 1 integrons. The clonal relationship of E. coli isolates carrying an identical cassette array was analysed by PFGE. RESULTS: Commensal E. coli isolates from animals were highly resistant to commonly used antimicrobial agents such as tetracycline, sulfamethoxazole, streptomycin, ampicillin and carbenicillin. Integrons were most prevalent in commensal E. coli isolates from poultry (44%), followed by clinical isolates from humans (33%), commensal isolates from swine (23%) and humans (13%). dfrA17-aadA5, dfrA12-orfF-aadA2 and aadA1 were found most frequently in E. coli isolates from humans, poultry and swine, respectively. Class 1 integrons were mostly located in conjugative plasmids. E. coli isolates carrying an identical cassette array were phylogenetically unrelated. CONCLUSIONS: The use of antibiotics is strongly associated with antimicrobial resistance. E. coli isolates from different sources may select a specific gene cassette by antibiotic selective pressure, which results in differences in class 1 integrons. The horizontal transfer of class 1 integrons through conjugative plasmids seems to be responsible for wide dissemination of a particular type of class 1 integron.     

Detection of aacA-aphD, qacEδ1, marA, floR, and tetA genes from multidrug-resistant bacteria: Comparative analysis of real-time multiplex PCR assays using EvaGreen(®) and SYBR(®) Green I dyes

We have developed multiplex real-time PCR assays that utilize DNA-intercalating dyes, SYBR Green I (SG) and EvaGreen (EG), with two primer sets (set 1=qacEδ1, tetA and aacA-aphD; set 2=tetA, marA, and floR) to simultaneously amplify the qacEδ1, tetA, aacA-aphD, marA, and floR genes. Validity of the multiplex PCR assays was confirmed by testing 83 bacterial isolates, including Staphylococcus aureus (28 isolates), Enterococcus spp. (17 isolates), Salmonella enterica serovar Typhimurium (8 isolates), Citrobacter spp. (9 isolates), Escherichia coli (14 isolates) and Aeromonas veronii (7 isolates), and performing sequence analysis of representative PCR products. Agarose gel analysis revealed the presence of correct size PCR products, and the differences in their thermal melting (T(m)) curves were used to distinguish various PCR products. Although T(m) peaks of different amplicons after EG-based singleplex and multiplex PCR assays were resolved nicely, only one or two peaks were seen for SG-bound amplicons. EG-based multiplex real-time PCR assays provided better peak resolution. There was a good correlation with a better linear relationship between the C(t) and log input DNA concentration for the set 1 and set 2 genes in EG-based assays (R(EG)(2)=0.9813and0.9803) than in SG-based assays (R(SG)(2)=0.5276and0.6255). The sensitivities of detection were 2.5-25fg and 25-250fg of template DNA in EG and SG-based singleplex and multiplex PCR assays, respectively. The assays, which could be completed in less than 45min, offer sensitive and rapid detection of qacEδ1, aacA-aphD, marA, floR, and tetA genes from a diverse group of multiple antibiotic-resistant bacterial strains.     

Molecular epidemiology of trimethoprim resistance among coagulase-negative staphylococci.

A 42% (70 of 167 isolates) incidence of resistance to 20 micrograms of trimethoprim per ml was found among clinical isolates of coagulase-negative staphylococci from two hospitals. A specific trimethoprim resistance gene probe from a conjugative Staphylococcus aereus plasmid was used to investigate the location of the trimethoprim resistance gene among 29 isolates. In 14 trimethoprim-resistant isolates, the probe hybridized with only chromosomal DNA, in 9 it hybridized with only plasmid DNA, and in 1 isolate both plasmid and chromosomal sequences showed hybridization. In five isolates there was no hybridization of the probe with either chromosomal or plasmid DNA. Four of these five nonhybridizing isolates were Staphylococcus haemolyticus. In contrast, all 22 Staphylococcus epidermidis isolates tested hybridized with the probe. The presence of the trimethoprim resistance gene in a chromosomal location was correlated with a lower MIC (median, 80 micrograms/ml) than when it was plasmid encoded (median, 1,250 micrograms/ml). Restriction endonuclease mapping as well as DNA hybridization of cloned plasmid and chromosomal DNA showed that there were 2.7 kilobases of common DNA in the two loci. This included the 500 base pairs of DNA mediating trimethoprim resistance and a total of 2.2 kilobases of 3'- and 5'-flanking sequences. The presence of the same gene and flanking sequences in chromosomal and plasmid locations suggests that the trimethoprim resistance determinant is translocated among different genetic loci.     

Mosaic structure of p1658/97, a 125-kilobase plasmid harboring an active amplicon with the extended-spectrum beta-lactamase gene blaSHV-5

Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum beta-lactamase (ESBL) of the SHV type. The beta-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the blaSHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.     

Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai,China

Although quinolone resistance usually results from chromosomal mutations, recent studies indicate that quinolone resistance can also be plasmid mediated. The gene responsible, qnr, is distinct from the known quinolone resistance genes and in previous studies seemed to be restricted to Klebsiella pneumoniae and Escherichia coli isolates from the University of Alabama in Birmingham, where this resistance was discovered. In Shanghai, the frequency of ciprofloxacin resistance in E. coli has exceeded 50% since 1993. Seventy-eight unique ciprofloxacin-resistant clinical isolates of E. coli from Shanghai hospitals were screened for the qnr gene by colony blotting and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant E. coli J53 as a recipient with selection for plasmid-encoded antimicrobial resistance (chloramphenicol, gentamicin, or tetracycline) and azide counterselection. qnr genes were sequenced, and the structure of the plasmid DNA adjacent to qnr was analyzed by primer walking with a sequential series of outward-facing sequencing primers with plasmid DNA templates purified from transconjugants. Six (7.7%) of 78 strains gave a reproducible hybridization signal with a qnr gene probe on colony blots and yielded strong signals on plasmid DNA preparations. Quinolone resistance was transferred from all six probe-positive strains. Transconjugants had 16- to 250-fold increases in the MICs of ciprofloxacin relative to that of the recipient. All six strains contained qnr with a nucleotide sequence identical to that originally reported, except for a single nucleotide change (CTA-->CTG at position 537) encoding the same amino acid. qnr was located in complex In4 family class 1 integrons. Two completely sequenced integrons were designated In36 and In37. Transferable plasmid-mediated quinolone resistance associated with qnr is thus prevalent in quinolone-resistant clinical strains of E. coli from Shanghai and may contribute to the rapid increase in bacterial resistance to quinolones in China.     

Emerging plasmid-mediated quinolone resistance associated with the qnr gene in Klebsiella pneumoniae clinical isolates in the United States

Although quinolone resistance commonly results from chromosomal mutation, recent studies indicate that such resistance can also be transferred on plasmids carrying the gene responsible, qnr. One hundred ten ciprofloxacin-resistant clinical isolates of Klebsiella pneumoniae and Escherichia coli from the United States were screened for the qnr gene by PCR and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant E. coli J53 as the recipient and selection with azide and sulfonamide, a resistance frequently linked to qnr. EcoRI and BamHI digests of qnr-hybridizing plasmids were subjected to electrophoresis on agarose gels and probed with qnr by Southern hybridization. qnr was detected in 8 (11.1%) of 72 K. pneumoniae strains. These eight positive strains were from six states in the United States. qnr was not found in any of the 38 E. coli strains tested. Quinolone resistance was transferred from seven of the eight probe-positive strains. Transconjugants with qnr-hybridizing plasmids had 32-fold increases in ciprofloxacin MICs relative to E. coli J53. For all eight strains, the sequence of qnr was identical to that originally reported. By size and restriction digests, four plasmids were related to the first-reported plasmid, pMG252, and three were different. Five new qnr plasmids encoded FOX-5 beta-lactamase, as did pMG252, but two others produced SHV-7 extended-spectrum beta-lactamase. Transferable plasmid-mediated quinolone resistance associated with qnr is now widely distributed in quinolone-resistant clinical strains of K. pneumoniae in the United States. Plasmid-determined quinolone resistance contributes to the increasing quinolone resistance of K. pneumoniae isolates and to the linkage previously observed between resistance to quinolones and the latest beta-lactam antibiotics.     

Plasmid-mediated florfenicol resistance in Pasteurella trehalosi

OBJECTIVES: A florfenicol-resistant Pasteurella trehalosi isolate from a calf was investigated for the presence and the location of the gene floR. METHODS: The P. trehalosi isolate 13698 was investigated for its in vitro susceptibility to antimicrobial agents and its plasmid content. A 14.9 kb plasmid, designated pCCK13698, was identified by transformation into Pasteurella multocida to mediate resistance to florfenicol, chloramphenicol and sulphonamides. The plasmid was sequenced completely and analysed for its structure and organization. RESULTS: Plasmid pCCK13698 exhibited extended similarity to plasmid pHS-Rec from Haemophilus parasuis including the region carrying the parA, repB, rec and int genes. Moreover, it revealed similarities to plasmid RSF1010 in the parts covering the mobC and repA-repC genes and to plasmid pMVSCS1 in the parts covering the sul2-catA3-strA gene cluster. Moreover, the floR gene area corresponded to that of transposon TnfloR. In addition, two complete insertion sequences were detected that were highly similar to IS1593 from Mannheimia haemolytica and IS26 from Enterobacteriaceae. Several potential recombination sites were identified that might explain the development of plasmid pCCK13698 by recombination events. CONCLUSIONS: The results of this study showed that in the bovine pathogen P. trehalosi, floR-mediated resistance to chloramphenicol and florfenicol was associated with a plasmid, which also carried functionally active genes for resistance to sulphonamides (sul2) and chloramphenicol (catA3). This is to the best of our knowledge the first report of resistance genes in P. trehalosi and only the second report of the presence of a florfenicol-resistance gene in target bacteria of the family Pasteurellaceae.