The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.

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Over a 12-year period (1989 to 2000), 159 Klebsiella pneumoniae isolates harboring extended-spectrum ␤-lactamases (ESBLs) (4.8% of the total number of K. pneumoniae isolates obtained) were recovered from 58 patients, who were mainly hospitalized in intensive care and surgery units. For 62 representative isolates from 58 patients, 31 clonal types harboring TEM-4 (n ‫؍‬ 5), SHV-2 (n ‫؍‬ 7), SHV2a (n ‫؍‬ 4), SHV-5 (n ‫؍‬ 1), CTX-M-10 (n ‫؍‬ 13), or CTX-M-9 (n ‫؍‬ 1) ␤-lactamases were identified by pulsed-field gel electrophoresis. This is the first report to document the presence of the CTX-M-10 or the CTX-M-9 ␤-lactamase in K. pneumoniae. These ␤-lactamases were previously identified in Escherichia coli isolates from Spain. Only two of five K. pneumoniae TEM-4 clones caused more than a single case of infection, with one of them spreading for 9 months. A single plasmid was detected among these TEM-4 clones. Only two of seven K. pneumoniae clones containing SHV-2 and three of four strains harboring SHV-2a were detected in more than one case of infection. Plasmids encoding SHV-2 or SHV-2a were unrelated. Four of 13 K. pneumoniae CTX-M-10 clones were found in more than one patient, with two of them recovered 2 and 5 years apart. As in the case of the SHV-2 isolates, we were unable to document a common transmissible genetic element that could explain the polyclonal structure of our isolates. Nevertheless, the spread of a single gene may be suggested by the presence of a conserved set of noncoding polymorphisms in the sequences. Most ESBL-producing K. pneumoniae clones were ephemeral, being poorly selected and maintained in the hospital setting, but the genes encoding ESBL persisted successfully over the years that the strains were recovered, probably as a minority gene population in the hospital metagenome.

To evaluate the role of hemolysin production in the virulence of Listeria monocytogenes, we have undertaken the analysis of the chromosomal region containing hlyA, the gene coding for listeriolysin 0. A recombinant cosmid, conferring a hemolytic phenotype to Escherichia coli, was shown to express listeriolysin 0, by immunoblotting with a specific antiserum against listeriolysin 0. The presence of hlyA on the cosmid was demonstrated by DNA hybridization with a probe previously shown to contain part of hlyA. The complete nucleotide sequence of hlyA has been determined. The deduced protein sequence reveals the presence of a putative 25-amino-acid signal sequence: the secreted form of listeriolysin 0 would have 504 amino acids, in agreement with the molecular weight of purified listeriolysin 0 (58,000). The protein sequence is highly homologous to those of streptolysin 0 and pneumolysin. A peptide of 11 amino acids conserved in the three proteins contains the unique cysteine known to be essential for lytic activity. By DNA-DNA hybridization, the listeriolysin 0 gene was detected in all L. monocytogenes strains tested, even in the nonhemolytic type strain. The gene was absent in other species of the genus Listeria.

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Fifteen Enterobacter clinical isolates (11 Enterobacter cloacae isolates, 3 Enterobacter aerogenes isolates, and 1 Enterobacter gergoviae isolate), representing 0.4% of all Enterobacter isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum ␤-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an Escherichia coli recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of E. aerogenes and six different clones of E. cloacae. Four of these E. cloacae clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the E. gergoviae isolate and in all but one of the E. cloacae isolates. The remaining E. cloacae isolate harbored a TEM-27 ESBL, and the three E. aerogenes isolates harbored a TEM-24 ESBL. PFGE revealed that our E. aerogenes strain was indistinguishable from the French TEM-24-producing E. aerogenes endemic clone. Although a low prevalence of ESBL-producing Enterobacter isolates was found in our institution over a 12-year period, a diversity of nonepidemic E. cloacae clones was detected, as was the persistence of the CTX-M-10 ␤-lactamase. The presence of the TEM-24-producing E. aerogenes French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.Plasmid-mediated extended-spectrum ␤-lactamases (ESBLs) were first reported in the mid-1980s (14, 15). Since then they have been responsible for several outbreaks caused by expanded-spectrum cephalosporin-resistant enterobacteria. Klebsiella pneumoniae and Escherichia coli are the most frequently involved organisms, and TEM-1-and SHV-1-derived ␤-lactamases are the most common ESBLs found in these species (25). These enzymes have also been reported at a much lower frequency among inducible AmpC ␤-lactamase-producing members of the family Enterobacteriaceae, such as Enterobacter spp., Citrobacter spp., Morganella morganii, and Serratia spp. (6,10,17,23,27,28,38). The most common ESBLs found in these species also belong to the TEM-and SHV-derived ␤-lactamases.Enterobacter species have been found to be one of the most important causes of nosocomial infections during the last few years (9, 30). These organisms have been associated with several outbreaks generally involving derepressed mutants overproducing their chromosomal ␤-lactamase or, more infrequently, expressing ESBL (7,10,23,30).The aims of this study were to establish the epidemiological relationship among all ESBL-producing Enterobacter isolates recovered in our institution over a decade, as well as to characterize the ESBLs expressed in these isolates and the plasmids harboring ESBL genes. In addition, we investigated if our ESBL-producing Entero...

A cefotaxime-resistant, ceftazidime-susceptible Escherichia coli isolate was obtained from a patient with sepsis in 1997, from which a ␤-lactamase with a pI of 8.1 was cloned. Cephaloridine and cefotaxime relative hydrolysis rates were 167 and 81, respectively (penicillin G rate ‫؍‬ 100), whereas ceftazidime hydrolysis was not detected. The nucleotide sequence revealed a bla gene related to that coding for CTX-M-3. Despite 21 nucleotide substitutions, only 2 determined amino acid changes (Ala27Val and Arg38Gln). The amino acid sequence identity between this enzyme, designated CTX-M-10, and the chromosomal ␤-lactamase of Kluyvera ascorbata was 81%.

The influence of inoculum size, beta-lactamase hyperproduction (multicopy plasmid) and modifications in the outer membrane protein profile on the susceptibility of Escherichia coli to combinations of amoxycillin/clavulanate, amoxycillin/sulbactam, amoxycillin/tazobactam and piperacillin/tazobactam were studied. For all combinations the bacterial susceptibility was affected by factors determining an increase in beta-lactamase (inoculum size or hyperproduction). Clavulanic acid was the most efficient beta-lactamase inhibitor. The absence of the outer membrane proteins, OmpF and OmpC, did not significantly affect susceptibility to the combinations per se but when combined with the presence of beta-lactamase high MICs were observed. Seven out of eight amoxycillin/clavulanate resistant clinical isolates of E. coli had beta-lactamase hyperproduction and a decrease or absence of OmpF.