Dissemination of transferable CTX-M-type extended-spectrum beta-lactamase-producing Escherichia coli in Korea

AIMS: Among 365 Escherichia coli isolated in 2003, 31 cefotaxime-resistant isolates were obtained from clinical specimens taken from adults hospitalized in Busan, Korea. Six extended-spectrum beta-lactamase (ESBL)-producing isolates were investigated further to determine the mechanism of resistance. METHODS AND RESULTS: These isolates were analysed by antibiotic susceptibility testing, pI determination, plasmid profiles, transconjugation test, PCR-restriction fragment length polymorphism (RFLP), enterobacterial repetitive consensus (ERIC)-PCR and DNA sequencing. All six of these isolates were found to contain the CTX-M-type ESBL genes. Five clinical isolates and their transconjugants produced CTX-M-3. One clinical isolate (K17391) and its transconjugant (trcK17391) produced CTX-M-15. Five clinical isolates also produced another TEM-1. One clinical isolate (K12776) also contained another TEM-52. CTX-M-3 ESBL gene was responsible for the resistance to piperacillin, cephalothin, cefotaxime, cefepime and aztreonam. CTX-M-15 or TEM-52 was especially responsible for the resistance to ceftazidime. CONCLUSIONS: These results appear to represent the in vivo evolution of CTX-M-type beta-lactamase genes (bla(CTX-M-3) --> bla(CTX-M-15)) under the selective pressure of antimicrobial therapy (especially ceftazidime). PCR-RFLP is a reliable method to discriminate CTX-M-15 gene from CTX-M-3 gene. ERIC-PCR analysis revealed that dissemination of CTX-M-3 was not due to a clonal outbreak of a resistant strain but to the intra-species spread of resistance to piperacillin, cephalothin, cefotaxime, cefepime and aztreonam in Korea. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the occurrence of CTX-M-1 cluster ESBLs in Korea. A more comprehensive survey of these ESBL types from Korea is urgently needed because of the in vivo evolution of CTX-M-15 from CTX-M-3. The emergence of these CTX-M-type ESBLs suggests that diagnostic laboratories should screen for ESBLs with ceftazidime as well as cefotaxime; they should still perform clavulanate synergy tests on resistant isolates.     

Substitution of Thr for Ala-237 in TEM-17, TEM-12 and TEM-26: alterations in β-lactam resistance conferred on Escherichia coli

Non-naturally occurring mutants of TEM-17 (E104K), TEM-12 (R164S) and TEM-26 (E104K:R164S) extended-spectrum (ES) beta-lactamases bearing threonine at position 237 were constructed by site-specific mutagenesis and expressed under isogenic conditions in Escherichia coli. Quantification of beta-lactamase activities and immunoblotting indicated that Ala-237-->Thr did not significantly affect expression levels of these ES enzymes. Minimum inhibitory concentrations of beta-lactam antibiotics showed that the presence of threonine at position 237 exerted a dominant effect increasing the enzymes' preference for various early generation cephalosporins over penicillins. Activity against broad-spectrum oxyimino-beta-lactams was also changed. The effect of Ala-237-->Thr on the activity against ceftazidime, aztreonam, cefepime and cefpirome of all three ES TEM enzymes was detrimental. Introduction of Thr-237 improved activity against cefotaxime and ceftriaxone in TEM-12 and TEM-26, but not in TEM-17.     

Characterization of a novel SHV β-lactamase variant that resembles the SHV-5 enzyme

Abstract An SHV type β-lactamase frequently found in enterobacteria isolated in Greek hospitals was analyzed. The enzyme (SHV-5a) conferred resistance to ceftazidime and aztreonam. The DNA sequence of the structural gene was determined. The deduced amino acid sequence showed that positions 70–73 were occupied by the active site tetrad Ser-Thr-Phe-Lys. As in SHV-5, Ser-238 and Lys-240 were present. However, one deletion (Gly-54) and three substitutions (Arg-140 for Ala, Asn-192 for Lys and Val-193 for Leu) differentiate SHV-5a β-lactamase from SHV-5. Asn-192 and Val-193 have been reported to date only in the R974 plasmid-mediated SHV-1 β-lactamase. Hydrolysis studies with SHV-5a and SHV-5 showed that the enzymes behaved similarly. Additional evidence that they were functionally indistinguishable was provided by the similar MICs of β-lactams when the enzymes were expressed under isogenic conditions. The sequence differences, however, indicate that they are derived from different ancestors.     

RNA polymerase heterogeneity in Streptomyces aureofaciens: characterization by antibody-linked polymerase assay

The ability of Escherichia coli with different receptor specificities to interact with meconium was studied. E. coli strains expressing P-fimbriae, specific for Gal alpha 1-4Gal beta-containing receptors, were agglutinated by meconium at high titres. This reaction was inhibited by globotetraosylceramide. The attachment of P-fimbriated E. coli to human colonic epithelial cells of the HT-29 cell line was inhibited by meconium. Some type 1 fimbriated strains were agglutinated by meconium, but the agglutination was rarely blocked by methyl alpha-D-mannoside. The attachment by type 1 fimbriated strains to HT-29 cells was reduced by meconium only in some cases. These results suggest that meconium interacts with the P-fimbriae of E. coli, in a way that may influence bacterial colonization of the neonatal intestine.     

Two novel transferable extended-spectrum β-lactamases from Klebsiella pneumoniae in Tunisia

Two novel beta-lactamases conferring multiresistance to antibiotics including oxyimino beta-lactams have been identified in two nosocomial K. pneumoniae strains isolated in Tunis in 1986 and 1988. Both enzymes were encoded by ca. 150-kilobase plasmids. Donor and transconjugant strains producing these enzymes exhibited highly similar pattern of resistance (CTX phenotype) to beta-lactams including penicillins and oxyimino beta-lactams e.g. cefotaxime, ceftriaxone, ceftazidime, and aztreonam. High and variable synergy (16 to 1066-fold) was obtained when combined to 0.1 microgram/ml of clavulanate (beta-lactamase inhibitor). The isoelectric points of these two enzymes were 5.4 and 6.4. These beta-lactamases differed from TEM types by hydrolysis for cefotaxime or ceftriaxone but were inhibited by clavulanate and cloxacillin. DNA hybridization studies suggested that that the genes of these enzymes may be derived from genes encoding TEM-type enzymes.     

A preliminary survey of extended-spectrum beta-lactamases (ESBLs) in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Japan

We conducted a survey of extended-spectrum beta-lactamases (ESBLs) among 16805 Escherichia coli and 9794 Klebsiella pneumoniae clinical isolates recovered from 196 separate medical institutions during the period January 1997 to January 1998. Using the criteria for minimal inhibitory concentrations (MICs) of oxyimino-cephalosporins of >/=8 microg ml(-1) and confirmation by double-disk test, we detected 15 E. coli and 34 K. pneumoniae isolates producing ESBLs. Genotypes of ESBLs determined by PCR with type-specific primers included one TEM-derived and 24 SHV-derived ESBLs, in addition to 24 Toho-1-type ESBLs, one of the major types of ESBLs reported in Japan. Nucleotide sequence analysis of SHV-specific PCR products revealed that SHV-12 was the dominant type of SHV-derived ESBL. In addition, we also identified TEM-26 and SHV-2. This is the first report characterizing TEM- and SHV-derived ESBLs in Japan.     

鲍曼不动杆菌超广谱β-内酰胺酶检测及耐药性分析

为了解厦门中山医院鲍曼不动杆菌产超广谱β 内酰胺酶(ESBLs)情况及耐药现状,指导临床用药,采用美国临床实验室标准化委员会(NCCLS)推荐的纸片扩散法,测定 2002 ~2003年临床分离的 211株鲍曼不动杆菌产ESBLs的比例及其对头孢噻肟、阿米卡星等 13种抗菌药物耐药性特征。结果表明 211株鲍曼不动杆菌中ESBLs阳性 27株(占 12. 8% ),产ESBLs菌株对 13种抗菌药物的耐药率明显高于非产ESBLs株。鲍曼不动杆菌的产ESBLs株有较高的交叉耐药性,采用纸片确证实验检出ESBLs对于临床用药有一定的指导意义。     

Incidence of Extended-Spectrum β-Lactamases and Characterization of Integrons in Extended-Spectrum β-Lactamase-producing Klebsiella pneumoniae Isolated in Shantou, China

This study is concerned with the level of antibiotic resistance of extended-spectrum β-lactamase （ESBL）-producing Klebsiella pneumoniae, isolated in Shantou, China, and its mechanism. Seventy- four non-repetitive clinical isolates of K. pneumoniae producing ESBLs were isolated over a period of 2 years. Antibiotic susceptibility, carried out by Epsilometer test, showed that most of the isolates were multiresistant. Polymerase chain reaction showed that, among the several types of β-lactamases, SHV was the most prevalent, TEM was the second most prevalent, and CTX-M was the least prevalent. Sixty-nine isolates were positive for integrase gene IntI1, but no IntI2 or IntI3 genes were found. The variable region of class 1 integrons were amplified and further identified by sequencing. Thirteen different gene cassettes and 11 different cassette combinations were detected. Dfr and aadA cassettes were predominant and cassette combinations dfrA12, orfF and aadA2 were most frequently found. No gene cassettes encoding ESBLs were found. Integrons were prevalent and played an important role in multidrug resistance in ESBL-producing K. pneumoniae.     

Contribution of extended-spectrum AmpC (ESAC) beta-lactamases to carbapenem resistance in Escherichia coli

ESAC beta-lactamases have increased catalytic efficiencies toward extended-spectrum cephalosporins and to a lesser extent toward imipenem as compared with the wild-type cephalosporinases. We show here that ESAC expression associated with the loss of both OmpC and OmpF porins conferred in Escherichia coli a high level of resistance to ertapenem and reduced the susceptibility to imipenem. On the contrary, ESAC expressed in the OmpC- or OmpF-deficient E. coli strains or narrow-spectrum cephalosporinase expressed in the OmpC-and OmpF-deficient strain do not confer reduced susceptibility to any of the carbapenems. The production of ESAC beta-lactamase in favorable E. coli background may represent an additional mechanism of resistance to ertapenem.     

Nucleotide sequences of the genes coding for the TEM-like β-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2)

Abstract Two bla _TEM-like genes were characterized that encoded IRT β-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with β-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 β-lactamase resembled the bla _TEM-1B gene, and that for IRT-2 resembled bla _TEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the bla _IRT-1 gene and C to A transversion in the bla _IRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to β-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic p I (p I =5.2) of these IRT enzymes compared to that of TEM-1 (p I =5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this β-lactamase to inhibition by p -chloromercuribenzoate.     

Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo beta-lactamase

The roles of lysine at position 161 and asparagine at position 167 in IMP-1 metallo beta-lactamase were studied by site-directed mutagenesis. These residues are highly conserved in metallo beta-lactamases and are thought to be present in the active-site cavity. Mutant enzymes with alanine or aspartic acid at position 167 showed almost the same properties as the wild-type enzyme. Kinetic parameters for the mutant enzymes differing at position 161 indicated that the positive charge of lysine 161 is required for electrostatic interaction with the carboxyl moiety of the substrate, i.e. C-3 of penicillins or C-4 of cephalosporins.     

OHIO-1 β-lactamase resistant to mechanism-based inactivators

Abstract Spontaneous mutants of OHIO-1 β-lactamase, an SHV-1 family enzyme, resistant to inactivation by clavulanic acid, sulbactam and tazobactam, have been isolated. The resistant mutant (M4) was inhibited by 100 μg/ml ampicillin plus 32 μg/ml clavulanic acid compared to ≤2 μg/ml clavulanic acid required for the parent strain. The pI of the mutant beta-lactamase was 7.0, identical to the parent enzyme. Kinetic parameters showed that the M4 enzyme had an increased V_max/K_m ratio for all beta-lactam substrates compared to the parent enzyme. The apparent K _i for clavulanic acid, sulbactam and tazobactam was 15.1, 182 and 18 μM, respectively, up to 70-fold higher than the parent enzyme. Partial nucleotide sequencing revealed that the mutant enzyme had a predicted methionine₆₉→ isoleucine₆₉ substitution accounting for the observed changes in substrate specificity.     

Natural antimicrobial susceptibilities and biochemical profiles of Yersinia enterocolitica-like strains: Y. frederiksenii, Y. intermedia, Y. kristensenii and Y. rohdei

The natural susceptibility of 131 Yersinia strains of Y. frederiksenii (n=38), Y. intermedia (n=48), Y. kristensenii (n=26) and Y. rohdei (n=19) to 70 antibiotics was tested. Minimum inhibitory concentration (MIC) values were determined with a microdilution procedure in IsoSensitest broth (all strains) and cation-adjusted Mueller Hinton broth (some strains). All species were naturally sensitive or sensitive and of intermediate susceptibility to tetracyclines, aminoglycosides, acylureidopenicillins, numerous cephalosporins, carbapenems, aztreonam, quinolones, chloramphenicol, folate-pathway inhibitors, nitrofurantoin, and fosfomycin. Uniform natural resistance was found with penicillin G, oxacillin, several macrolides, lincosamides, streptogramins, glycopeptides, rifampicin and fusidic acid. Species-specific differences in susceptibility affecting clinical assessment criteria were seen with aminopenicillins (in the presence and absence of beta-lactamase inhibitors), ticarcillin and some cephalosporins. Major medium-dependent susceptibilities were found with fosfomycin. beta-Lactam MIC susceptibility patterns suggested that most strains of the species tested produce both class A and class C (AmpC) beta-lactamases that are characteristic for the species. The present study describes a database concerning the natural susceptibility of some Y. enterocolitica-like species to a wide range of antibiotics, which can be applied to validate forthcoming antibiotic susceptibility tests of these strains and might contribute to their identification. An evaluation of 30 biochemical tests that secured phenotypic identification to the Yersinia species level is presented.     

Salicylate decreases production of AmpC type beta-lactamases and increases susceptibility to beta-lactams in a Morganella morganii clinical isolate

The effect of salicylate, a marRAB inducer, on the resistance to beta-lactams was characterized in an AmpC beta-lactamase hyperproducer Morganella morganii clinical isolate (the M1 strain). Results were compared with those of the effect of salicylate in a wild-type M. morganii strain. Salicylate induced a decreased susceptibility to nalidixic acid, norfloxacin and tetracycline and simultaneously increased the susceptibility to beta-lactams apparently due to the repression of AmpC beta-lactamase synthesis in the M1 strain. Likewise, salicylate only repressed 46 kDa outer membrane protein expression in the wild-type strain, since the clinical isolate M1 did not express it.     

TEM-E1: a novel β-lactamase conferring resistance to ceftazidime

A novel beta-lactamase, conferring resistance to ceftazidime, has been identified to be encoded by a 31 kb plasmid (pUK720) in a clinical E. coli strain isolated in Belgium. The beta-lactamase, new designated TEM-E1, has a pI of approximately 5.4 and lies in between the iso-electric focused bands of the beta-lactamases TEM-1 and TEM-7. The TEM-E1 beta-lactamase has a similar molecular weight of 22,000 to the TEM-1 and it is also inhibited by clavulanic acid. However, the TEM-E1 enzyme differs from TEM-1 by its low rates and efficiency of hydrolysis for ceftazidime and cefotaxime, TEM-E1 has similar efficiency of hydrolysis values for ceftazidime and cefotaxime, but only confers resistance to ceftazidime.     

Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP)

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta-lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.