Increased Hydrolysis of Oximino-ß-Lactams by CMY-107, a Tyr199Cys Mutant Form of CMY-2 Produced by Escherichia coli.

The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an Escherichia coli clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did.

Emergence and maintenance of multidrug-resistant Escherichia coli of canine origin harbouring a blaCMY-2-IncI1/ST65 plasmid and topoisomerase mutations.

OBJECTIVES: To characterize the mechanisms implicated in fluoroquinolone (FQ) and expanded-spectrum cephalosporin (ESC) resistance in three clinical and seven faecal multidrug-resistant (MDR; resistant to at least three antimicrobial classes) Escherichia coli isolates from a dog with atopic dermatitis, also suffering from recurrent otitis, that had already been exposed to prolonged antimicrobial treatment and colonized for a long period. METHODS: MICs of FQs, ESCs and other antimicrobials were determined by the broth microdilution method. Phenotypic tests (efflux pump inhibition and combination disc tests) and isoelectric focusing were combined with genotypic analyses [PCRs, sequencing, conjugation, S1 nuclease PFGE, PCR-based replicon typing, plasmid multilocus sequence typing (pMLST) and PCR mapping] to characterize the molecular basis of FQ and ESC resistance. Isolates were further characterized by MLST and PFGE. RESULTS: Three otitis and five faecal isolates with enrofloxacin MICs of 32 to >128 mg/L displayed the GyrA:S83L+D87N/ParC:E62K/ParE:G545D pattern harbouring novel ParC and ParE substitutions, whereas the two remaining faecal isolates were susceptible or borderline resistant single-step mutants (GyrA:S83L pattern) and carried qnrS1. Efflux pump overexpression also contributed to FQ resistance and the MDR phenotype. The three otitis and five faecal isolates also exhibited cefoxitin/ceftazidime MICs of 32-64 mg/L and harboured blaCMY-2, adjusted to ISEcp1, on an IncI1/ST65 conjugative plasmid, previously described in Salmonella Heidelberg from poultry. Interestingly, all isolates shared an identical MLST type (ST212), with the otitis isolates showing indistinguishable patterns with the high-level resistant faecal E. coli isolates. CONCLUSIONS: The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a blaCMY-2-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.

KPC-producing, multidrug-resistant Klebsiella pneumoniae sequence type 258 as a typical opportunistic pathogen.

The virulence of a KPC-producing Klebsiella pneumoniae sequence type 258 (ST258) strain representing those circulating in Greece was assessed in a mouse septicemia model. The strain was virtually avirulent (50% lethal dose, >10(8) and 5 × 10(7) CFU for immunocompetent and neutropenic animals, respectively). Also, it was highly susceptible to serum killing, rapidly phagocytosed in vitro, and classified as K41, which is not among the virulent capsular types. The findings indirectly support the notion that high ST258-associated mortality is largely due to inefficient antimicrobial treatment.

Sequence of pR3521, an IncB plasmid from Escherichia coli encoding ACC-4, SCO-1, and TEM-1 beta-lactamases.

The sequence of pR3521, a self-transmissible plasmid from Escherichia coli, was determined. pR3521 (110,416 bp) comprised a contiguous IncB sequence (84,034 bp) sharing extensive similarities with IncI replicons and an acquired region (26,382 bp) carrying sequences of diverse origin, containing bla(ACC-4), bla(SCO-1), bla(TEM-1b) (two copies), strA, strB, sul2, and aacC2.

Characterization of metallo-beta-lactamase VIM-27, an A57S mutant of VIM-1 associated with Klebsiella pneumoniae ST147.

VIM-27 metallo-ß-lactamase, an Ala(57) → Ser variant of VIM-1, was identified in three Klebsiella pneumoniae isolates belonging to sequence type 147. bla(VIM-27) was part of a class 1 integron carried by non-self-transferable plasmids. Kinetic parameters and MIC determinations indicated that VIM-27 hydrolyzed most ß-lactams, especially imipenem and cefoxitin, less effectively than VIM-1.

GES-13, a beta-lactamase variant possessing Lys-104 and Asn-170 in Pseudomonas aeruginosa.

GES-13 beta-lactamase, a novel GES variant possessing Lys-104 and Asn-170, was identified in Pseudomonas aeruginosa. bla(GES-13) was the single gene cassette of a class 1 integron probably located in the chromosome. GES-13 efficiently hydrolyzed broad-spectrum cephalosporins and aztreonam. Imipenem was a potent inhibitor of GES-13 but was not hydrolyzed at measurable rates.

Sequence of pNL194, a 79.3-kilobase IncN plasmid carrying the blaVIM-1 metallo-beta-lactamase gene in Klebsiella pneumoniae.

The nucleotide sequence of pNL194, a VIM-1-encoding plasmid, is described in this study. pNL194 (79,307 bp) comprised an IncN-characteristic segment (38,940 bp) and a mosaic structure (40,367 bp) including bla(VIM-1), aacA7, aadA1, aadA2, dfrA1, dfrA12, aphA1, strA, strB, and sul1. Tn1000 or Tn5501 insertion within fipA probably facilitated recruitment of additional mobile elements carrying resistance genes.

CMY-31 and CMY-36 cephalosporinases encoded by ColE1-like plasmids.

Two CMY-2 derivatives, CMY-31 (Gln(215)-->Arg) from Salmonella enterica serotype Newport and CMY-36 (Ala(77)-->Cys and Gln(193)-->Glu) from Klebsiella pneumoniae, were characterized. Both cephalosporinases functionally resembled CMY-2. bla(CMY) alleles occurred as parts of a putative transposon comprising ISEcp1B and a Citrobacter freundii-derived sequence carried by ColE1-like plasmids similar to CMY-5-encoding pTKH11 from Klebsiella oxytoca.

Activity of imipenem against VIM-1 metallo-beta-lactamase-producing Klebsiella pneumoniae in the murine thigh infection model.

The in-vivo activity of imipenem against VIM-1-producing Klebsiella pneumoniae (VPKP) was assessed in a thigh infection model in neutropenic mice. Animals were infected with three VPKP isolates (imipenem MICs 2, 4 and 32 mg/L, respectively) and a susceptible clinical isolate (MIC 0.125 mg/L) that did not produce any beta-lactamase with broad-spectrum activity. Bacterial density at the site of infection was determined after imipenem treatment (30 and 60 mg/kg every 2 h for 24 h). The log(10) reduction in CFU/thigh was greatest for the wild-type isolate, intermediate for the two imipenem-susceptible VPKP isolates, and lowest for the imipenem-resistant VPKP isolate. Whilst in-vivo imipenem activity appeared reduced against in-vitro susceptible VIM-1 producers compared with a VIM-1-negative control, an increased drug dosage could moderate this reduction.

CTX-m-3 beta-lactamase-producing Escherichia coli from Greece.

An Escherichia coli clinical strain resistant to all beta-lactams except carbapenems was isolated in a Greek hospital. Analysis of beta-lactamase content by isoelectric focusing, PCR assays specific for various bla genes, and DNA sequencing showed that the strain produced TEM-1, a Citrobacter freundii AmpC-related cephalosporinase, and CTX-M-3. The blaCTX.M-3 gene was carried by a 120-kb plasmid that was readily transferable to a susceptible E. coli host.

Substitution of Arg-244 by Cys or Ser in SHV-1 and SHV-5 beta-lactamases confers resistance to mechanism-based inhibitors and reduces catalytic efficiency of the enzymes.

The conserved residue Arg-244 was substituted by the smaller uncharged amino acids Cys and Ser in SHV-1 and SHV-5 beta-lactamases by a PCR-based site-specific mutagenesis procedure. The mutant beta-lactamases displayed decreased susceptibility to clavulanate and, to a lesser extent, to tazobactam and sulbactam. As shown in comparative MIC determinations, R244C and R244S enzymes retained a residual penicillinase activity while their activity towards cephalosporins was drastically diminished. The respective SHV-5 mutants were unable to hydrolyze oxyimino-beta-lactams except aztreonam. The impaired catalytic activity of the mutant beta-lactamases was mainly due to the lowering of affinity for beta-lactam substrates. The above alterations were more pronounced in the R244C mutants. These results provide information on the mode of involvement of Arg-244 in (a) inactivation by beta-lactamase inhibitors and (b) the proper positioning of beta-lactams in the active site of SHV enzymes.

Characterization of In111, a class 1 integron that carries the extended-spectrum beta-lactamase gene blaIBC-1.

A class 1 integron, In111, carried by a self-transferable plasmid from an Escherichia coli clinical strain was characterized. The variable region of In111 constituted an array of gene cassettes encoding the extended-spectrum beta-lactamase IBC-1, the aminoglycoside-modifying enzymes AAC(6')-Ib and ANT(3")-Ia, dihydrofolate reductase I and a putative polypeptide (SMR-2) sharing similarity with the Qac transporters. Transcription of the gene cassettes was driven by a hybrid-type P1 promoter located in a typical 5' conserved segment (CS). The 3'CS included sulI, qacEDelta1, orf5 and orf6. In111 was bounded on the right by an inversely oriented IRt. The 5'CS was preceded by an intact IS26 element followed by an aphA1 gene.

Substitution of Thr for Ala-237 in TEM-17, TEM-12 and TEM-26: alterations in beta-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 beta-lactamase variant that resembles the SHV-5 enzyme.

An SHV type beta-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 beta-lactamase from SHV-5. Asn-192 and Val-193 have been reported to date only in the R974 plasmid-mediate SHV-1 beta-lactamase. Hydrolysis studies with SHV-5a and SHV-5 showed that the enzymes behaved similarly. Additional evidence that they are functionally indistinguishable was provided by the similar MICs of beta-lactams when the enzymes were expressed under isogenic conditions. The sequence differences, however, indicate that they are derived from different ancestors.

Two novel plasmid-mediated cefotaxime-hydrolyzing beta-lactamases (CTX-M-5 and CTX-M-6) from Salmonella typhimurium.

Two novel plasmid-mediated beta-lactamases (CTX-M-5 and CTX-M-6) produced by Salmonella typhimurium clinical strains were characterized. The enzymes exhibited a pI of 8.4, hydrolyzed oxyimino-beta-lactams and were susceptible to mechanism-based beta-lactamase inhibitors. The respective bla genes were cloned and sequenced. The deduced amino acid sequences showed a high degree of homology with those of the previously described plasmid class A CTX-M-type enzymes and appeared related to the chromosomal beta-lactamases of Klebsiella oxytoca.

Imipenem resistance in Enterobacter aerogenes is associated with derepression of chromosomal cephalosporinases and impaired permeability.

Enterobacter aerogenes mutants with high-level resistance to imipenem were studied. They were derived from strains characterized by stable over-production of a class-I beta-lactamase. This enzyme (pI = 8.2) exhibited high affinity toward imipenem and hydrolysed the drug slowly. Imipenem-resistant mutants lacked a major 43-kDa outer membrane protein and displayed decreased permeability to cephaloridine. Introduction of a plasmid coding for the regulatory ampD gene abolished beta-lactamase production and rendered the mutants susceptible to imipenem.

Antibiotic susceptibilities of Yersinia enterocolitica and Y. intermedia isolates from aquatic environments.

Of 37 Yersinia isolates from various aquatic environments, seven were Y. enterocolitica and 30 Y. intermedia. These isolates were biotyped, serotyped and tested for their susceptibility to 20 antibiotics. All Y. enterocolitica isolates were of biovar 1; those of Y. intermedia were distributed amongst four biovars (1, 2, 4 and 6). On the basis of combined biotyping and serotyping results, Y. enterocolitica isolates were distributed in five and Y. intermedia in 17 groups. With the exception of one Y. enterocolitica isolate which was resistant to tetracycline and streptomycin, the isolates were sensitive to the non-beta-lactam antibiotics. In contrast, various patterns of beta-lactam insensitivity were detected, including ampicillin and ticarcillin (35 isolates), cephalothin (33 isolates), carbenicillin (32 isolates), amoxycillin/clavulanate (23 isolates) and cefoxitin (22 isolates). No correlation between biotype or serotype and the susceptibility pattern of the isolates was apparent. Both inducible cephalosporinase activity against third-generation cephalosporins and inhibition of resistance to penicillins were detected in all Y. enterocolitica and Y. intermedia isolates by double-disk tests.