Persistence of an antibiotic resistance plasmid in intestinal Escherichia coli of chickens in the absence of selective pressure.

We studied eight strains of Escherichia coli resistant to high levels of trimethoprim that were isolated over a 6-week period in a commercial breeding flock of broilers. The strains originated from fecal samples and from a carcass immediately after slaughter. Seven of eight strains belonged to the same infrequent biotype. They were also resistant to ampicillin and streptomycin, and some were resistant to tetracycline and potassium tellurite. All the strains transferred trimethoprim and ampicillin resistance to E. coli. Analysis of the donors and of the transconjugants by agarose gel electrophoresis after digestion by restriction endonucleases and by nucleic acid hybridization indicated that resistance to trimethoprim (dfrI) and to ampicillin (bla TEM-1) was mediated by a 65-kilobase plasmid, pIP1531. Persistence of resistance to trimethoprim and ampicillin in this flock was therefore due to two cumulative factors, both occurring in the absence of selective pressure, namely the dissemination of a particular plasmid between strains and the ability of an atypical E. coli strain to stably colonize many animals.     

Molecular characterization of plasmids encoding CTX-M-15 beta-lactamases from Escherichia coli strains in the United Kingdom

OBJECTIVES: The UK, like other countries worldwide, has a growing problem with CTX-M beta-lactamase-producing Escherichia coli. Five major clonally related strains have been identified among CTX-M-15 producers. We characterize here the plasmids from clonal strains A and D. METHODS: Plasmids were extracted and transformed into E. coli DH5alpha; conjugative mating was attempted on agar. MICs were determined by agar dilution. beta-Lactamases were typed by isoelectric focusing; antibiotic resistance genes and integrons were identified by PCR and sequenced. Plasmid incompatibility groups were determined by replicon PCR. RESULTS: bla(CTX-M-15) was carried by a 150 kb plasmid in strain A and a 70 kb plasmid in strain D. Conjugative transfer of cefotaxime resistance was only achieved from strain D; plasmids from both strains were transferred by transformation. The plasmid from strain A additionally carried bla(TEM-1) (variably), bla(OXA-1), aac(6')-Ib-cr and tet(A), as well as a class 1 integron with the gene cassettes aadA5 and dfr(17); the plasmid from strain D carried bla(TEM-1) consistently, also bla(OXA-1), aac(6')-Ib-cr, aac3-IIa and tet(A). Both plasmids belonged to incompatibility group FII. CONCLUSIONS: bla(CTX-M-15) was plasmid-mediated in both strains A and D and was linked to other antibiotic resistance genes including aac(6')-Ib-cr, which encodes an acetyltransferase, not previously found in Europe, acting on both aminoglycosides and some fluoroquinolones. Although the plasmids from the two strains differed in size, both were related and conferred similar multi-drug resistance phenotypes, suggesting that they may share a similar genetic scaffold. Both shared features with plasmids encoding CTX-M-15 beta-lactamases in E. coli from Canada and India.     

Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit

OBJECTIVES: To investigate the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in the neonatal intensive care unit of a university hospital in Italy. METHODS: Antibiotic susceptibility was evaluated by disc diffusion and Etest. ESBLs were identified by isoelectric focusing, PCR and DNA sequencing analysis. Genotyping was performed by PFGE analysis. Conjugation was performed by broth mating. RESULTS: Molecular typing of K. pneumoniae isolates identified three distinct PFGE patterns. Isolates of PFGE profile A were isolated during an epidemic in 1996, while isolates of PFGE profiles B and C were sequentially isolated from September 2002 to December 2004, when 233 colonizations and 19 infections by K. pneumoniae occurred. All K. pneumoniae strains of different PFGE types were identified as ESBL producers. DNA sequencing of amplified beta-lactamase genes identified a novel bla(TEM) ESBL (bla(TEM-136)) along with bla(SHV-1) in chromosomal and plasmid DNA from K. pneumoniae of PFGE type A, respectively, and bla(TEM-1) and bla(SHV-12) in plasmid DNA from K. pneumoniae of PFGE types B and C. Conjugation experiments demonstrated that resistance to third-generation cephalosporins, along with an approximately 80 kb plasmid containing bla(SHV-12) and bla(TEM-1), was transferred from K. pneumoniae epidemic strains of PFGE types B and C to a susceptible Escherichia coli host at a frequency of 4 x 10(-6) and 1 x 10(-6) cfu/recipient cell, respectively. CONCLUSIONS: The selection of ESBL-producing clones and the transfer of the bla(SHV-12) ESBL gene between different clones were responsible for the spread of K. pneumoniae in the neonatal intensive care unit.     

Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada

A major outbreak involving an Escherichia coli strain that was resistant to expanded-spectrum cephalosporins occurred in Toronto and surrounding regions in 2000 to 2002. We report the complete sequence of a plasmid, pC15-1a, that was found associated with the outbreak strain. Plasmid pC15-1a is a circular molecule of 92,353 bp consisting of two distinct regions. The first is a 64-kb region that is essentially homologous to the non-R-determinant region of plasmid R100 except for several point mutations, a few small insertions and deletions, and the absence of Tn10. The second is a 28.4-kb multidrug resistance region (MDR) that has replaced the R-determinant region of the R100 progenitor and consists mostly of transposons or partial transposons and five copies of the insertion element IS26. All drug resistance genes found in pC15-1a, including the beta-lactamase genes bla(CTX-M-15), bla(OXA-1), and bla(TEM-1), the tetracycline resistance gene tetA, and aminoglycoside resistance genes aac(6')-Ib and aac(3)-II, are located in the MDR. The bla(CTX-M-15) gene was found downstream of ISEcp1as part of a transposition unit, as determined from the surrounding sequence. Examination of the plasmids from CTX-M-15-harboring strains isolated from hospitals across Canada showed that pC15-1a was found in several strains isolated from a site in western Canada. Comparison of pC15-1a and pCTX15, found in an E. coli strain isolated in India in 1999, revealed that the plasmids had several features in common, including an R100 backbone and several of the resistance genes, including bla(CTX-M-15), bla(TEM-1), bla(OXA-1), tetA, and aac(6')-Ib.     

Dissemination of CTX-M-type beta-lactamases among clinical isolates of Enterobacteriaceae in Paris, France

We analyzed 19 clinical isolates of the family Enterobacteriaceae (16 Escherichia coli isolates and 3 Klebsiella pneumoniae isolates) collected from four different hospitals in Paris, France, from 2000 to 2002. These strains had a particular extended-spectrum cephalosporin resistance profile characterized by a higher level of resistance to cefotaxime and aztreonam than to ceftazidime. The bla(CTX-M) genes encoding these beta-lactamases were involved in this resistance, with a predominance of bla(CTX-M-15). Ten of the 19 isolates produced both TEM-1- and CTX-M-type enzymes. One strain (E. coli TN13) expressed CMY-2, TEM-1, and CTX-M-14. bla(CTX-M) genes were found on large plasmids. In 15 cases the same insertion sequence, ISEcp1, was located upstream of the 5' end of the bla(CTX-M) gene. In one case we identified an insertion sequence designated IS26. Examination of the other three bla(CTX-M) genes by cloning, sequencing, and PCR analysis revealed the presence of a complex sul1-type integron that includes open reading frame ORF513, which carries the bla gene and the surrounding DNA. Five isolates had the same plasmid DNA fingerprint, suggesting clonal dissemination of CTX-M-15-producing strains in the Paris area.     

A TEM-2beta-lactamase encoded on an active Tn1-like transposon in the genome of a clinical isolate of Stenotrophomonas maltophilia

A constitutively expressed beta-lactamase gene from a clinical isolate of Stenotrophomonas maltophilia, J675Ia, has been cloned. Its DNA sequence is almost identical to that of bla(TEM2) (one nucleotide change) and the expressed enzyme is a Bush type 2a penicillinase with an amino acid sequence identical to that of TEM-2. The bla(TEM) gene was present within a novel Tn1/Tn3-type transposon in the genome of isolate J675Ia and the transposon was able to mobilize bla(TEM) on to the broad host-range conjugative plasmid, R388. When transferred to an Escherichia coli recipient, R388::Tn conferred high-level ampicillin resistance. This represents the first identification of a TEM beta-lactamase in S. maltophilia and the first evidence that this important clinical pathogen is able to act as a reservoir for mobile beta-lactamase genes in the hospital environment.     

Escherichia coli resistant to ampicillin/sulbactam.

Escherichia coli strains resistant to ampicillin/sulbactam from hospitals in 4 different geographic locations were examined with respect to type and amount of beta-lactamase produced. A total of 5 strains was examined from each region. The isoelectric points of all of the involved beta-lactamases were 5.4, corresponding to TEM-1. Km and Vmax values of the beta-lactamases among the clinical isolates resembled those from the control TEM-1 strain. In an 18-hour broth culture the highly resistant isolates produced 3 times more beta-lactamase as compared to the ampicillin/sulbactam-susceptible isolates. However, the highly resistant strains contained approximately the same amount of plasmid DNA (size of > 6,500 bp) as the susceptible isolates. In transformation experiments, both the resistance and the degree of resistance appeared to have been transferable by plasmids. The mechanism for resistance is likely to be a baseline overproduction of TEM-1 beta-lactamase due to either an alteration in the control of gene expression or simply to an increase in the number of copies of the beta-lactamase gene in the plasmids.     

Emergence of multidrug-resistant Salmonella enterica serovar typhi in Korea

A chloramphenicol-resistant strain of Salmonella enterica serovar Typhi was first noted in Korea in 1992, when a resistant isolate was detected in a returned traveler. Continued isolation of multidrug-resistant (MDR) strains thereafter in other settings prompted a retrospective analysis of laboratory records and phenotypic and genotypic analyses of 12 chloramphenicol-resistant isolates. Among these, one isolate was resistant only to chloramphenicol, and the other isolates were also resistant to ampicillin and co-trimoxazole. MDR was transferred by conjugation from 9 of the 11 isolates. PCR showed that all isolates had an incompatible group HI1 plasmid, and oriT was detected in 10 isolates, which included strains with an unsuccessful transfer of resistance. All of the ampicillin-resistant isolates had a beta-lactamase band of pI 5.4 and bla(TEM) alleles. A PCR amplicon from an isolate showed that the sequences were identical to those of bla(TEM-1), suggesting that all isolates had a TEM-1 beta-lactamase. All isolates had class 1 integrons: 10 isolates had integrons of ca. 1.2 kb with dhfr7 gene cassettes, and 1 isolate had an integron of ca. 2.3 kb with aacA4 and bla(OXA-1)-like gene cassettes. The pulsed-field gel electrophoresis patterns of 7 of 11 MDR isolates were identical and indistinguishable from those reported for isolates in India and Indonesia. In conclusion, some of the MDR strains in Korea are related to those in other Asian countries. Susceptibility testing became necessary for selection of antimicrobial agents for the optimal treatment of patients with the emergence of MDR Salmonella serovar Typhi in Korea.     

TEM derivative-producing Enterobacter aerogenes strains: dissemination of a prevalent clone

TEM-24 (CAZ-6) extended-spectrum beta-lactamase (ESBL) was detected in 1988 in Clermont-Ferrand, France, in Klebsiella pneumoniae (bla(TEM-24)) and Enterobacter aerogenes (bla(TEM-24b)), and since 1994, a TEM-24-producing E. aerogenes clonal strain has been observed elsewhere in the country. To determine if the spread of this clonal strain was restricted to TEM-24-producing E. aerogenes strains, 84 E. aerogenes strains (non-TEM/SHV-producing strains, TEM-1- or -2-producing strains, and different ESBL-producing strains), isolated from 1988 to 1999 in Clermont-Ferrand (n = 59) and in 11 other French hospitals in 1998 (n = 25), were studied. A clonal strain was found for TEM-24- but also for TEM-3- and TEM-1- or 2-producing isolates. This study shows that there is a clonal strain dependent on acquisition of the TEM-type enzyme (TEM-24 and other TEM types).     

Fitness trade-offs in blaTEM evolution

bla(TEM-1) expression results in penicillin resistance, whereas expression of many bla(TEM-1) descendants, called extended-spectrum beta-lactamases (ESBLs), results simultaneously in resistance to penicillins and extended-spectrum cephalosporins. Despite the expanded resistance phenotypes conferred by many ESBLs, bla(TEM-1) is still the most abundant bla(TEM) allele in many microbial populations. This study examines the fitness effects of the two amino acid substitutions, R164S and E240K, that have occurred repeatedly among ESBL bla(TEM-1) descendants. Using a single-nucleotide polymorphism-specific real-time quantitative PCR method, we analyzed the fitness of strains expressing bla(TEM-1), bla(TEM-10), and bla(TEM-12). Our results show that bacteria expressing the ancestral bla(TEM-1) allele have a fitness advantage over those expressing either bla(TEM-10) or bla(TEM-12) when exposed to ampicillin. This observation, combined with the fact that penicillins are the most prevalent antimicrobials prescribed worldwide, may explain why bla(TEM-1) has persisted as the most frequently encountered bla(TEM) allele in bacterial populations.     

Characterization of a Salmonella enterica serovar Agona strain harbouring a class 1 integron containing novel OXA-type beta-lactamase (blaOXA-53) and 6'-N-aminoglycoside acetyltransferase genes [aac(6')-I30

OBJECTIVE: To characterize by molecular methods a multidrug-resistant Salmonella enterica serovar Agona (S. enterica Agona) isolated from a hospitalized patient in Rio de Janeiro, Brazil. METHODS: The S. enterica Agona strain was screened by PCR and DNA sequencing for TEM, SHV and CTX-M-type beta-lactamase genes, tet(A), (B), (C) and (D) tetracycline resistance genes, chloramphenicol resistance genes and class 1 integrons. Plasmid characterization was carried out by PCR and Southern hybridization analysis. PCR and PFGE were used to characterize nine other S. enterica Agona strains collected from hospitals in Rio de Janeiro. RESULTS: The study strain was found to harbour a 105 kb plasmid, which contained catA1, bla(TEM-1), a class 1 integron with two novel genes labelled bla(OXA-53) and aac(6')-I30, respectively, and an additional unidentified aminoglycoside resistance gene. A second 53 kb plasmid from the same strain contained tet(D) and bla(SHV-5). OXA-53 was shown to provide reduced susceptibility to ceftazidime, and its activity was inhibited in the presence of clavulanic acid. PFGE analysis of the nine other S. enterica Agona strains revealed two clusters of related strains (78% similarity), and PCR analysis showed that all strains contained the novel integron. CONCLUSION: An S. enterica Agona strain was found to harbour three plasmid-encoded beta-lactamases, one (OXA-53) on a novel class 1 integron that also contains a new aminoglycoside resistance gene, aac(6')-I30. The multidrug resistance plasmids appear to have disseminated to other city hospitals via other S. enterica Agona strains.     

Integration of a transposon Tn1-encoded inhibitor-resistant beta-lactamase gene,bla(TEM-67) from Proteus mirabilis,into the Escherichia coli chromosome

Proteus mirabilis NEL-1 was isolated from a urine sample of a patient hospitalized in a long-term care facility. Strain NEL-1 produced a beta-lactamase with a pI of 5.2 conferring resistance to amoxicillin and amoxicillin-clavulanic acid. Sequencing of a PCR amplicon by using TEM-specific primers revealed a novel bla(TEM) gene, bla(TEM-67). TEM-67 was an IRT-1-like TEM derivative related to TEM-65 (Lys39, Cys244) with an additional Leu21Ile amino acid substitution in the leader peptide. The biochemical properties of TEM-67 were equivalent to those described for TEM-65. Analysis of sequences surrounding bla(TEM-67) revealed that it was located on a transposon, Tn1, which itself was located on a 48-kb non-self-transferable plasmid, pANG-1. Electroporation of plasmid pANG-1 into Escherichia coli DH10B resulted in the integration of bla(TEM-67) into the chromosome, whereas it remained episomal in the P. mirabilis CIP103181 reference strain. Further characterization of pANG-1 revealed the presence of two identical sequences on both sides of Tn1 that contained an IS26 insertion sequence followed by a novel colicin gene, colZ, which had 20% amino acid identity with other colicin genes. The characterization of this novel TEM derivative provides further evidence for the large diversity of plasmid-encoded beta-lactamases produced in P. mirabilis and for their spread to other enterobacterial species through transposable-element-mediated events.     

First description of an Escherichia coli strain producing NDM-1 carbapenemase in Spain

A carbapenem-resistant Escherichia coli strain (DVR22) was recovered from a stool specimen from a patient with traveler's diarrhea who had traveled to India. Molecular screening led to the first identification of NDM-1 in Spain. The bla(NDM-1) gene was located in a conjugative plasmid of ca. 300 kb that also contained the bla(CTX-M-15), bla(TEM-1), Δbla(DHA-1), and armA genes. In addition, bla(NDM-1) was preceded by an ISAba125 insertion element only found in Acinetobacter spp.     

Multidrug-resistant Salmonella enterica serovar Muenchen from pigs and humans and potential interserovar transfer of antimicrobial resistance

Salmonella serovars are important reservoirs of antimicrobial resistance. Recently, we reported on multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium strains among pigs with resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline (resistance [R] type AKSSuT) and resistance to amoxicillin-clavulanic acid, ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (R type AxACSSuT). In the present study, 67 isolates (39 from humans and 28 from pigs) of clinically important Salmonella serovar Muenchen were characterized. Among the porcine isolates, 75% showed resistance to seven antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, amoxicillin-clavulanic acid, and kanamycin (R type ACSSuTAxK). One isolate from humans showed resistance to 10 of the 12 antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, amoxicillin-clavulanic acid, kanamycin, gentamicin, cephalothin, and ceftriaxone (R type ACSSuTAxKGCfCro). Pulsed-field gel electrophoresis revealed no clonality between the porcine and the human strains. The porcine and the human MDR strains carried class 1 integrons of 2.0 and 1.0 kb, respectively. Genes specific to the porcine strain included aadA2, aphA1-Iab, and tetA(B). DNA sequencing revealed that the porcine isolates carried bla(OXA-30) on a class 1 integron. Genes specific to the human strain included bla(TEM), strA, strB, cmlA, tetA(A), and aadA2. No bla(CMY-2) gene was detected. Serovar Muenchen strains of porcine and human origin were able to transfer resistance genes to laboratory strain Escherichia coli MG1655 by conjugation. Plasmid restriction with four restriction enzymes, EcoRI, BamHI, HindIII, and PstI, showed that the conjugative plasmids from porcine Salmonella serovar Muenchen and Typhimurium R-type MDR strains isolated from the same farms at the same time were similar on the basis of the sizes and the numbers of bands and Southern hybridization. The plasmid profiles among the Salmonella serovar Muenchen isolates from the two host species were different. This is the first report to show a high frequency of MDR Salmonella serovar Muenchen strains from pigs and a human strain that is similar to the MDR isolates with the AmpC enzyme previously reported among Salmonella serovars Newport and Typhimurium strains. The MDR strains from the two host species independently represent public health concerns, as Salmonella serovar Muenchen is among the top 10 causes of salmonellosis in humans.     

Propagation of TEM- and PSE-Type β-Lactamases among Amoxicillin-Resistant Salmonella spp. Isolated in France

A survey conducted between 1987 and 1994 at the University Hospital of Besan?on, France, demonstrated a dramatic increase (from 0 to 42. 5%) in the prevalence of amoxicillin resistance among Salmonella spp. Of the 96 resistant isolates collected during this period (including 77 Typhimurium), 54 were found to produce TEM-1 beta-lactamase, 40 produced PSE-1 (equivalent to CARB-2), one produced PSE-1 plus TEM-2, and one produced OXA-1 in isoelectric focusing and DNA hybridization experiments. Plasmids coding for these beta-lactamases were further characterized by (i) profile analysis, (ii) restriction fragmentation pattern analysis, (iii) hybridization with an spvCD-orfE virulence probe, and (iv) replicon typing. In addition, isolates of S. typhimurium were genotypically compared by pulsed-field gel electrophoresis of XbaI-macrorestricted chromosomal DNA. Altogether, these methods showed that 40 of the 41 PSE-1 producers were actually the progeny of a single epidemic S. typhimurium strain lysotype DT104. Isolates of that strain were found to harbor RepFIC virulence plasmids with somewhat different restriction profiles, but which all carried the bla(PSE-1) gene. Of these virulence/resistance plasmids, 15 were transmissible to Escherichia coli. TEM-1-producing S. typhimurium displayed much greater genotypic and plasmidic diversities, suggesting the acquisition of the bla(TEM-1) gene from multiple bacterial sources by individual strains. In agreement with this, 32 of the 35 S. typhimurium plasmids encoding TEM-1 were found to be conjugative. These data show that development of amoxicillin resistance among Salmonella, especially in serovar Typhimurium, results from both gene transfers and strain dissemination.     

Molecular characterization of resistance to extended-spectrum cephalosporins in clinical Escherichia coli isolates from companion animals in the United States

Resistance to extended-spectrum cephalosporins (ESC) among members of the family Enterobacteriaceae occurs worldwide; however, little is known about ESC resistance in Escherichia coli strains from companion animals. Clinical isolates of E. coli were collected from veterinary diagnostic laboratories throughout the United States from 2008 to 2009. E. coli isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime (MIC ≥ 16 μg/ml) and extended-spectrum-β-lactamase (ESBL) phenotypes were analyzed. PCR and sequencing were used to detect mutations in ESBL-encoding genes and the regulatory region of the chromosomal gene ampC. Conjugation experiments and plasmid identification were conducted to examine the transferability of resistance to ESCs. All isolates carried the bla(CTX-M-1)-group β-lactamase genes in addition to one or more of the following β-lactamase genes: bla(TEM), bla(SHV-3), bla(CMY-2), bla(CTX-M-14-like), and bla(OXA-1.) Different bla(TEM) sequence variants were detected in some isolates (n = 40). Three isolates harbored a bla(TEM-181) gene with a novel mutation resulting in an Ala184Val substitution. Approximately 78% of the isolates had mutations in promoter/attenuator regions of the chromosomal gene ampC, one of which was a novel insertion of adenine between bases -28 and -29. Plasmids ranging in size from 11 to 233 kbp were detected in the isolates, with a common plasmid size of 93 kbp identified in 60% of isolates. Plasmid-mediated transfer of β-lactamase genes increased the MICs (≥ 16-fold) of ESCs for transconjugants. Replicon typing among isolates revealed the predominance of IncI and IncFIA plasmids, followed by IncFIB plasmids. This study shows the emergence of conjugative plasmid-borne ESBLs among E. coli strains from companion animals in the United States, which may compromise the effective therapeutic use of ESCs in veterinary medicine.     

Epidemiology of nalidixic acid resistance and TEM-1- and TEM-52-mediated ampicillin resistance of Shigella sonnei isolates obtained in Korea between 1980 and 2000

The resistance to ampicillin and nalidixic acid in Shigella sonnei isolates obtained in Korea during the period 1998 to 2000 was characterized. Recently (J. Y. Oh, H. S. Yu, S. K. Kim, S. Y. Seol, D. T. Cho, and J. C. Lee, J. Clin. Microbiol. 41:421-423, 2003) ampicillin and nalidixic acid resistance was found in 49 and 70%, respectively, of the 67 S. sonnei isolates obtained during this period. We analyzed 138 S. sonnei isolates collected during the same period. Ampicillin and nalidixic acid resistance was found in 30 and 86% of the isolates, respectively. The ampicillin resistance was mediated by a TEM-1 beta-lactamase, and TEM-52 extended-spectrum beta-lactamase was identified in one sporadic S. sonnei isolate from 1999. bla(TEM-1) and bla(TEM-52) were located in conjugative R-plasmids. Tn3 was detected in 41% of the ampicillin-resistant isolates. The R-plasmids from the transconjugants that transferred resistance to ampicillin exhibited different restriction fragment length polymorphism patterns, and a bla(TEM-1) probe was hybridized with the different fragments. The nalidixic acid resistance was exclusively associated with an amino acid substitution, Ser83-->Leu (TCG-->TTG), in gyrA. These findings indicate that the genetically related S. sonnei strains readily acquire resistance to ampicillin, streptomycin, trimethoprim, and sulfamethoxazole but not nalidixic acid through conjugative R-plasmids from difference sources when confronted by antibiotic selective pressures.     

In vivo acquisition of ceftriaxone resistance in Salmonella enterica serotype anatum

The emergence of resistance to antimicrobial agents within the salmonellas is a worldwide and severe problem. A case of treatment failure due to the emergence of resistance to ceftriaxone in Salmonella enterica serotype Anatum was studied. S. enterica serotype Anatum and Escherichia coli, both of which are susceptible to ceftriaxone, were initially isolated from a diabetic patient hospitalized for the treatment of wound and urinary tract infections. Resistant S. enterica serotype Anatum and E. coli strains were isolated concomitantly 2 weeks after the initiation of ceftriaxone therapy. The patient eventually died of a sepsis caused by the ceftriaxone-resistant salmonella. PCR, nucleotide sequence analysis, and DNA-DNA hybridization identified a bla(CTX-M-3) gene located on a 95.1-kb plasmid from the ceftriaxone-resistant isolates of S. enterica serotype Anatum and E. coli. The plasmid was proved to be conjugative. Molecular fingerprinting showed that the susceptible and resistant strains were genetically indistinguishable. The emergence of resistance to ceftriaxone in S. enterica serotype Anatum was due to the in vivo acquisition of a plasmid containing the bla(CTX-M-3) gene and was the cause for treatment failure in this patient.     

Multiple antibiotic resistance plasmids in Enterobacteriaceae isolated from diarrhoeal specimens of hospitalized children in Indonesia

We studied the plasmid and antibiotic resistance characteristics of 35 strains of Enterobacteriaceae recovered from faecal specimens of children with diarrhoea in Central General Hospital, Bandung, Indonesia. Twenty three Escherichia coli, three Providencia, three Proteus, three Klebsiella, two Enterobacter and one Citrobacter were examined. All strains were multiply resistant, many carrying six to nine antibiotic resistances. Most of these resistances were transferable to a laboratory E. coli strain and were carried on large-sized plasmids. All recently-described tetracycline resistance determinants (Classes A----D) were represented; the most common was the Class B, or TN10 type. The TEM-1 beta-lactamase was detected in 17 out of 21 ampicillin-resistant strains examined. The OXA-1, PSE-1, and SHV-1 enzymes were also found. Of 23 plasmids tested, all could be classified into one of eight different incompatibility groups: IncFII, IncN, IncB, IncF1, IncI1, IncI2, IncH2 and IncT. These studies demonstrate the existence of large multiresistant transferable plasmids representing common incompatibility groups and bearing common tetracycline and ampicillin resistance determinants in enteric strains isolated from children hospitalized in Indonesia.     

Plasmid-mediated quinolone resistance conferred by qnrS1 in Salmonella enterica serovar Virchow isolated from Turkish food of avian origin

OBJECTIVES: To study the molecular characteristics of the quinolone and associated ampicillin resistance mechanisms present in Salmonella enterica serovar Virchow isolated from Turkish foods. METHODS: Nine epidemiologically unrelated Salmonella Virchow strains isolated from foods (chicken and minced meat) sold in different markets in Ankara were analysed for their susceptibility to 17 antimicrobials. The strains were typed by PFGE and plasmid profiling and investigated by molecular methods (PCR/sequencing) for the presence of several resistance genes, class 1 integrons and mutations in the quinolone resistance-determining regions. Plasmids conferring quinolone resistance were analysed by restriction fragment length polymorphism (RFLP) analysis, DNA hybridization, sequencing, replicon-typing PCR and mating experiments. RESULTS: All strains showed nalidixic acid resistance (MIC >or= 128 mg/L) together with a decreased susceptibility to ciprofloxacin (three strains with an MIC of 1 mg/L and six with an MIC of 0.25 mg/L), associated with mutations within the gyrA gene (Asp-87 --> Tyr-87). In three strains, qnrS1 genes were detected. Ampicillin resistance encoded by a bla(CTX-M3) gene and/or bla(TEM-1-like) gene was found in four strains. Three of these strains carried an approximately 45 kb conjugative plasmid, designated pRQ2006, harbouring qnrS1 and a Tn3-like transposon. Partial sequencing and RFLP of pRQ2006 indicated its similarity to the qnrS1 plasmid pAH03786 found in a Japanese Shigella flexneri 2b isolate. CONCLUSIONS: This is the first study describing the presence of qnrS1 genes in bacterial isolates from Turkey. The pRQ2006 plasmid seems to be more related to the S. flexneri 2b qnrS1 plasmid pAH0376 than to the Salmonella qnrS1-carrying plasmids pINF5 and TPqnrS-2.