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The spreading of antimicrobial-resistant Enterobacteriaceae, especially those co-harboring plasmid-mediated cephalosporin (bla) and colistin (mcr) resistance genes, is becoming increasingly problematic. As a vector, flies carry antimicrobial-resistant bacteria (ARB) into human and livestock habitats. To investigate ARB in flies, we collected 235 flies from 27 sites (18 urban areas, five pig farms and four chicken farms) in Thailand during 2013-2015. Cefotaxime-resistant Enterobacteriaceae (CtxRE) and bla-positive CtxRE were isolated from 70 (29.8%) and 48 (20.4%) flies, respectively. In 93 bla-positive CtxRE isolates that included Escherichia coli, Enterobacter spp., and Klebsiella pneumoniae from 48 flies, the most frequent bla gene was TEM (n = 62), followed by CTX-M-55 (n = 31), CTX-M-14 (n = 26), CMY-2 (n = 24) and SHV (n = 10), and 58 isolates harbored multiple types of these genes. In addition, we detected the mcr-1 (n = 1) and mcr-3 (n = 19) genes in bla-positive CtxRE isolates from 16 flies. In conjugation experiments, 10 mcr-3- and bla-positive isolates exhibited co-transfer of mcr-3 and blaTEM-1 genes. These results suggest that a relatively high proportion of flies in Thailand carries cephalosporin-resistant Enterobacteriaceae harboring co-transmissible cephalosporin and colistin resistance genes.

Eleven genotypically related Klebsiella pneumoniae isolates were obtained from 11 patients. All isolates were resistant to third-generation cephalosporins due to the production of SHV-2a extended-spectrum beta-lactamase. Comparison of the outer membrane protein profiles revealed one isolate that lacked porins. This porin-deficient isolate was also resistant to cefoxitin (MIC 128 microg ml(-1)) and moxalactam (MIC 64 microg ml(-1)) and had elevated MIC of meropenem (2 microg ml(-1)) when compared to porin-expressing isolates (2-8, 4 and0.06-0.125 microg ml(-1), respectively). Higher MICs, associated with loss of porins in outer membrane, were also observed for cefotaxime (4-8-fold), cefepime (2-16-fold), ciprofloxacin (4-16-fold), imipenem and aztreonam (2-16-fold), but there was no significant difference among MICs of ceftazidime. The porin-deficient mutant was probably selected in vivo during ofloxacin therapy.

Six clinical isolates of Enterobacter cloacae isolated in a Portuguese hospital, between April 1999 and November 2000, demonstrated resistance to almost all broad-spectrum cephalosporins, except to cefepime. These isolates were susceptible to quinolones and to aminoglycosides. lsoelectric focusing demonstrated production of beta-lactamases with pIs 8.0 and by all six isolates, exhibiting a cephalosporinase phenotype. The results of pulsed field gel electrophoresis revealed that these isolates were genetically unrelated. The amino acid sequence of six AmpC beta-lactamases (EclolFF, Eclo6FF, Eclo9FF, Eclo1OFF, Eclo11FF and Eclol5FF) shared 97-99% homology with the chromosomal AmpC beta-lactamase from E. cloacae P99 and 86-87% homology with those of two plasmid-mediated AmpC beta-lactamases, MIR-1 and ACT-1. This is the first report of chromosomal AmpC beta-lactamase production by E cloacae isolates in a Portuguese hospital. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Enterobacter cloacae CHE, a clinical strain with overproduced cephalosporinase was found to be highly resistant to the new cephalosporins, cefepime and cefpirome (MICsor =128 microg ml(-1)). The strain was isolated from a child previously treated with cefepime. The catalytic efficiency of the purified enzyme with the third-generation cephalosporins, cefepime and cefpirome, was 10 times higher than that with the E. cloacae P99 enzyme. This was mostly due to a decrease in K(m) for these beta-lactams. The clinical isolate produced large amounts of the cephalosporinase because introduction of the ampD gene decreased ampC expression and partially restored the wild-type phenotype. Indeed, MICs of cefepime and cefpirome remained 10 times higher than those for a stable derepressed clinical isolate (OUDhyp) transformed with an ampD gene. Sequencing of the ampC gene showed that 18 nucleotides had been deleted, corresponding to the six amino acids SKVALA (residues 289--294). According to the crystal structure of P99 beta-lactamase, this deletion was located in the H-10 helix. The ampR-ampC genes from the clinical isolates CHE and OUDhyp were cloned and expressed in Escherichia coli JM101. The MICs of cefpirome and cefepime of E. coli harboring ampC and ampR genes from CHE were 100--200 times higher than those of E. coli harboring ampC and ampR genes from OUDhyp. This suggests that the deletion, confirmed by sequencing of the ampC gene, is involved in resistance to cefepime and cefpirome. However, the high level of resistance to cefepime and cefpirome observed in the E. cloacae clinical isolate was due to a combination of hyperproduction of the AmpC beta-lactamase and structural modification of the enzyme. This is the first example of an AmpC variant conferring resistance to cefepime and cefpirome, isolated as a clinical strain.

A clinical strain of Escherichia coli (strain Ec 41553) that was resistant to ceftazidime produced a TEM-type beta-lactamase with a pI of 5.4. Clavulanic acid restored the ceftazidime activity, thus suggesting an extended spectrum beta-lactamase (ESBL). The gene encoding ESBL was located in a plasmid of 57 kb. After cloning and sequencing, the ESBL (TEM-29B) showed one amino acid replacement with respect to the TEM-1 sequence, Arg-164 to His. This change increased mainly the rate of hydrolysis of ceftazidime but not of cefotaxime and aztreonam. The relevance of this substitution in the increase of ceftazidime MIC is thus stressed.

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.

Multidrug resistance plasmids carrying the bla(CMY-2) gene have been identified in Salmonella enterica serovars Typhimurium and Newport from the United States. This gene confers decreased susceptibility to ceftriaxone, and is most often found in strains with concomitant resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline. The bla(CMY-2)-carrying plasmids studied here were shown to also carry the florfenicol resistance gene, floR, on a genetic structure previously identified in Escherichia coli plasmids in Europe. These data indicate that the use of different antimicrobial agents, including phenicols, may serve to maintain multidrug resistance plasmids on which extended-spectrum cephalosporin resistance determinants co-exist with other resistance genes in Salmonella.

Third-generation cephalosporin resistance is often mediated by TEM- and SHV-type beta-lactamases in Enterobacteriaceae. TEM-type and OXA-1 enzymes are the major plasmid-borne beta-lactamases implicated in amoxicillin-clavulanic acid resistance in Escherichia coli isolates. We have developed a rapid and simple multiplex polymerase chain reaction (PCR) which discriminates bla(TEM), bla(SHV) and bla(OXA-1) genes by generating fragments of 516, 392 and 619 bp respectively. Multiplex PCR analysis of 51 amoxicillin-clavulanate resistant E. coli isolates detected bla(TEM) and bla(SHV) genes in 45 and two strains, respectively, and only one strain harboured a bla(OXA-1) gene. Twenty-three of the 40 cefotaxime-resistant Enterobacteriaceae isolates produced amplicons with a size compatible with the presence of bla(TEM) (13 strains), bla(SHV) (six strains) genes or the association of both genes (four strains). These results were verified by colony hybridisation. Therefore, multiplex PCR is a suitable tool for initial rapid screening of bla genes in Enterobacteriaceae.

Nine clinical isolates of Enterobacteriaceae (six Escherichia coli and three Proteus mirabilis) isolated in three Parisian hospitals between 1989 and 2000 showed a particular extended-spectrum cephalosporin-resistance profile characterized by resistance to cefotaxime and aztreonam but not to ceftazidime. CTX-M-1, CTX-M-2, CTX-M-9, CTX-M-14 and two novel plasmid-mediated CTX-M beta-lactamases (CTX-M-20, and CTX-M-21) were identified by polymerase chain reaction and isoelectric focusing (pI8) and were associated in eight cases with TEM-1 (pI=5.4) or TEM-2 (pI=5.6) beta-lactamases. We used internal ISEcp1 and IS26 forward primers and the CTX-M consensus reverse primer to characterize the CTX-M beta-lactamase promoter regions and showed their high degree of structure diversity. We found upstream of some bla(CTX-M) genes, a 266-bp sequence 100% identical to the sequence upstream of the Kluyvera ascorbata beta-lactamase gene, suggesting that this chromosomal enzyme is the progenitor of the CTX-M-2/5 cluster.

The Asp179Gly mutant of the OHIO-1 beta-lactamase, an SHV enzyme, was constructed to investigate the effect of disruption of the omega loop on beta-lactamase activity in this class A enzyme. In Escherichia coli DH5 alpha the strain possessing the Asp179Gly mutation of the OHIO-1 beta-lactamase demonstrated increased susceptibility to all beta-lactams except ceftazidime and ceftriaxone. The minimum inhibitory concentrations for ceftazidime and ceftriaxone increased from 0.25 and 0.015 microgram/ml to 4.0 and .25 micrograms/ ml respectively. For ceftazidime, a substrate not hydrolyzed by the wild-type enzyme (K(m) > or = 500 microM), the K(m) of the Asp179Gly mutant beta-lactamase was measured to be 7 microM and the Vmax was 0.13 microM/min. The minimum inhibitory concentrations, K(m), and Vmax for all other beta-lactams decreased. Our analysis of this OHIO-1 beta-lactamase mutant suggests that disruption of the salt bridge in the omega loop by substitution of a glycine at position 179 markedly decreases the catalytic efficiency of the enzyme.