A clinical isolate, Escherichia coli MG-1, isolated from a 4-month-old Vietnamese orphan child, produced a β-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. In a disk diffusion test, a typical synergistic effect between ceftazidime or aztreonam and clavulanic acid was observed along with an unusual synergy between cefoxitin and cefuroxime. The gene for VEB-1 (Vietnamese extended-spectrum β-lactamase) was cloned and expressed in E. coli JM109. The recombinant plasmid pRLT1 produced a β-lactamase with a pI of 5.35 and conferred high-level resistance to extended-spectrum (or oxyimino) cephalosporins and to aztreonam. V max values for extended-spectrum cephalosporins were uncommonly high, while the affinity of the enzyme for ceftazidime and aztreonam was relatively low. bla VEB-1 showed significant homology at the DNA level with only bla PER-1 andbla PER-2. Analysis of the deduced protein sequence showed that VEB-1 is a class A penicillinase having very low levels of homology with any other known β-lactamases. The highest percentage of amino acid identity was 38% with PER-1 or PER-2, two uncommon class A extended-spectrum enzymes. Exploration of the genetic environment of bla VEB-1 revealed the presence of gene cassette features, i.e., (i) a 59-base element associated with bla VEB-1; (ii) a second 59-base element just upstream of bla VEB-1, likely belonging to the aacA1-orfG gene cassette; (iii) two core sites (GTTRRRY) on both sides of bla VEB-1; and (iv) a second antibiotic resistance gene 3′ ofbla VEB-1, aadB. bla VEB-1 may therefore be the first class A extended-spectrum β-lactamase that is part of a gene cassette, which itself is likely to be located on a class 1 integron, as sulfamide resistance may indicate. Furthermore,bla VEB-1 is encoded on a large (>100-kb) transferable plasmid found in a Klebsiella pneumoniae MG-2 isolated at the same time from the same patient, indicating a horizontal gene transfer.

Shotgun cloning experiments with restriction enzyme-digested genomic DNA from Morganella morganii 1, which expresses high levels of cephalosporinase, into the pBKCMV cloning vector gave a recombinant plasmid, pPON-1, which encoded four entire genes:ampC, ampR, an hybF family gene, and orf-1 of unknown function. The deduced AmpC β-lactamase of pI 7.6 shared structural and functional homologies with AmpC from Citrobacter freundii, Escherichia coli, Yersinia enterocolitica, Enterobacter cloacae, and Serratia marcescens. The overlapping promoter organization of ampC and ampR, although much shorter in M. morganii than in the other enterobacterial species, suggested similar AmpR regulatory properties. The MICs of β-lactams for E. coli MC4100 (ampC mutant) harboring recombinant plasmid pACYC184 containing either ampC and ampR (pAC-1) orampC (pAC-2) and induction experiments showed that theampC gene of M. morganii 1 was repressed in the presence of ampR and was activated when a β-lactam inducer was added. Moreover, transformation of M. morganii1 or of E. coli JRG582 (ΔampDE) harboringampC and ampR with a recombinant plasmid containing ampD from E. cloacae resulted in a decrease in the β-lactam MICs and an inducible phenotype for M. morganii 1, thus underlining the role of an AmpD-like protein in the regulation of the M. morganii cephalosporinase. Fifteen other M. morganii clinical isolates with phenotypes of either low-level inducible cephalosporinase expression or high-level constitutive cephalosporinase expression harbored the sameampC-ampR organization, with the hybF andorf-1 genes surrounding them; the organization of these genes thus differed from those of ampC-ampR genes inC. freundii and E. cloacae, which are located downstream from the fumarate operon. Finally, an identical AmpC β-lactamase (DHA-1) was recently identified as being plasmid encoded in Salmonella enteritidis, and this is confirmatory evidence of a chromosomal origin of the plasmid-mediated cephalosporinases.

MRSA remained poorly controlled throughout the hospital and was not just a problem in the ICU. MRSA thus requires more effective measures throughout the hospital. ESBL-E was mainly an ICU pathogen and our approach resulted in a clear decrease in the rate of acquisition in the ICU over time.

We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 g/ml) and teicoplanin (MIC, 64 g/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 g/ml) and to low levels of teicoplanin (MIC, 4 g/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 g/ml) but susceptible to teicoplanin (MIC, 0.5 g/ml). (6,7,10,13,16,31), two E. faecalis strains (13), one Enterococcus gallinarum strain (8), and one Enterococcus raffinosus strain (37). Recently, the vanD operon was detected in a Ruminococcus sp., an anaerobic gram-positive bacterium from human bowel flora, suggesting a potential reservoir for vancomycin resistance genes in commensals of the digestive tract (17).VanD-type strains share various characteristics that distinguish them from VanA-and VanB-type enterococci. In particular, resistance to moderate levels of the two glycopeptides is constitutively expressed and is not transferable by conjugation to other enterococci (10,13,16,31). The organization of the vanD gene cluster, located exclusively in the chromosome, is similar to those of vanA and vanB (13,16