Alain Philippon

Dissemination of CTX-M-Type β-Lactamases among Clinical Isolates of Enterobacteriaceae in Paris, France

ABSTRACT We analyzed 19 clinical isolates of the family Enterobacteriaceae (16 Escherichia coli isolates and 3 Klebsiella pneumoniae isolates) collected from four different hospitals in Paris, France, from 2000 to 2002. These strains had a particular extended-spectrum cephalosporin resistance profile characterized by a higher level of resistance to cefotaxime and aztreonam than to ceftazidime. The blaCTX-M genes encoding these β-lactamases were involved in this resistance, with a predominance of blaCTX-M-15. Ten of the 19 isolates produced both TEM-1- and CTX-M-type enzymes. One strain (E. coli TN13) expressed CMY-2, TEM-1, and CTX-M-14. blaCTX-M genes were found on large plasmids. In 15 cases the same insertion sequence, ISEcp1, was located upstream of the 5′ end of the blaCTX-M gene. In one case we identified an insertion sequence designated IS26. Examination of the other three blaCTX-M genes by cloning, sequencing, and PCR analysis revealed the presence of a complex sul1-type integron that includes open reading frame ORF513, which carries the bla gene and the surrounding DNA. Five isolates had the same plasmid DNA fingerprint, suggesting clonal dissemination of CTX-M-15-producing strains in the Paris area.

β-Lactamases of Kluyvera ascorbata, Probable Progenitors of Some Plasmid-Encoded CTX-M Types

ABSTRACT Kluyvera ascorbata produces a β-lactamase that results in an atypical susceptibility pattern, including low-level resistance to penicillins, cephalothin, and cefuroxime, but this resistance is reversed by clavulanate. Ten nucleotide sequences of the corresponding gene, blaKLUA, were obtained and were found to have minor variations (96 to 100%). Otherwise, blaKLUA was found to be similar (95 to 100%) to some plasmid-encoded CTX-M-type β-lactamases. Finally, mobilization of blaKLUA on a plasmid was found to be mediated probably by a genetic mobile element like ISEcp1.

Origin and impact of plasmid-mediated extended-spectrum beta-lactamases

Resistance to oxyimino cephalosporins was originally highlighted by the emergence of plasmid-encoded extended-spectrum β-lactamases deriving by mutation from TEM-1, TEM-2 and SHV type enzymes (class A). The broader spectrum of resistance produced by these enzymes is related to more amino acid substitutions, but susceptibility to seven alpha-methoxyimino cephalosporins and carbapenems was preserved until recently. Clavulanate-sensitive extended-spectrum β-lactamases are distributed worldwide, mainly amongKlebsiella pneumoniae isolates. Novel clavulanate-sensitive extended-spectrum β-lactamases deriving from other class A enzymes (e.g. MEN-1 from βlaOXY, OXA-11 inPseudomonas aeruginosa from PSE-2) have been reported. Recently, clavulanate-resistant extended-spectrum β-lactamases (class C) were encountered amongst single isolates, mostlyKlebsiella pneumoniae. These cephalosporinases or cefamycinases (usually chromosomally mediated) have expanded the spectrum of plasmid-encoded resistance to include seven alpha-methoxyimino cephalosporins. Thus far, only two isolates (1Pseudomonas aeruginosa, 1Bacteroides fragilis), both recovered in Japan, with plasmid-mediated resistance to carbapenems have been found.

Salmonella enteritidis : AmpC plasmid-mediated inducible β-lactamase (DHA-1) with an ampR gene from Morganella morganii

ABSTRACT DHA-1, a plasmid-mediated cephalosporinase from a single clinicalSalmonella enteritidis isolate, conferred resistance to oxyimino-cephalosporins (cefotaxime and ceftazidime) and cephamycins (cefoxitin and moxalactam), and this resistance was transferable toEscherichia coli HB101. An antagonism was observed between cefoxitin and aztreonam by the diffusion method. Transformation of the transconjugant E. coli strain with plasmid pNH5 carrying the ampD gene (whose product decreases the level of expression of ampC) resulted in an eightfold decrease in the MIC of cefoxitin. A clone with the same AmpC susceptibility pattern with antagonism was obtained, clone E. coliJM101(pSAL2-ind), and its nucleotide sequence was determined. It contained an open reading frame with 98.7% DNA sequence identity with the ampC gene of Morganella morganii. DNA sequence analysis also identified a gene upstream of ampCwhose sequence was 97% identical to the partial sequence of the ampR gene (435 bp) from M. morganii. The gene encoded a protein with an amino-terminal DNA-binding domain typical of transcriptional activators of the LysR family. Moreover, the intercistronic region between the ampC andampR genes was 98% identical to the corresponding region from M. morganii DNA. AmpR was shown to be functional by enzyme induction and a gel mobility-shift assay. An ampGgene was also detected in a Southern blot of DNA from the S. enteritidis isolate. These findings suggest that this inducible plasmid-mediated AmpC type β-lactamase, DHA-1, probably originated from M. morganii.

Nosocomial outbreak of acute gastroenteritis in a neonatal intensive care unit in tunisia caused by multiply drug resistantSalmonella wien producing SHV-2 beta-lactamase

In a Tunisian hospital 27 babies, including 12 who were premature, in a single intensive care unit suffered acute gastroenteritis in the period from January to May 1988. The mean age at the onset of gastroenteritis was 8.4 days; nine babies died.Salmonella wien was isolated from stools (all babies) and blood (4 babies). It was also isolated from the stools of one nurse and from a mattress. Twelve of the babies had received cefotaxime, which was successfully replaced by oral colimycin. The outbreak was stopped by the implementation of infection control measures. All isolates ofSalmonella wien were of the same biotype, and had the same antibiotic resistance pattern (third generation cephalosporins, monobactams, aminoglycosides, chloramphenicol, trimethoprim and sulphonamides) and plasmid DNA restriction pattern. The isolates were all susceptible to a combination of cefotaxime and clavulanic acid (a β-lactamase inhibitor), which displayed synergy, suggesting the presence of a β-lactamase (geometric mean MICs 11.24 µg/ml for cefotaxime alone and 0.24 µg/ml in combination with 0.1 µg/ml potassium clavulanate). All isolates produced TEM-1 and SHV-2 β-lactamase which was not transferable toEscherichia coli by conjugation. The presence of the SHV-2 enzyme inSalmonella wien may allow it to adapt to newer β-lactams which is a cause for concern in this hospital.

A novel integron in Salmonella enterica serovar Enteritidis, carrying the bla(DHA-1) gene and its regulator gene ampR, originated from Morganella morganii.

ABSTRACT The genetic organization of the gene coding for DHA-1 and the corresponding ampR gene was determined by PCR mapping. These genes have been mobilized from the Morganella morganii chromosome and inserted into a complexsulI-type integron, similar to In6 and In7. However, they are not themselves mobile cassettes. This integron probably includes a specific site for recombination allowing the mobilization of diverse resistance genes, as observed for blaCMY-1 andblaMOX-1.

Dissemination in five French hospitals ofKlebsiella pneumoniae serotype K25 harbouring a new transferable enzymatic resistance to third generation cephalosporins and aztreonam

A strain ofKlebsiella pneumoniae K25 resistant to newerβ-lactam drugs was isolated in clusters in five hospitals in the Paris area. The MICs of ceftazidime and aztreonam (⩾128 mg/l) were higher than that of cefotaxime (16 mg/l) for the strain but when measured in the presence of clavulanic acid, they were ⩽1 mg/l. The donor strains and derivatives produced aβ-lactamase with a pI of 7.75–7.8 and hydrolysing activity against a wide spectrum ofβ-lactams similar to that of SHV-2 and SHV-3, but with significant hydrolysis of ceftazidime. This new enzyme could be designated SHV-4.

Outbreak of nosocomial infections due to Klebsiella pneumoniae producing SHV-4 beta-lactamase.

One hundred and fifty-four clinical isolates ofKlebsiella pneumoniae resistant to broad-spectrum cephalosporins, aztreonam and amikacin were responsible for an outbreak of nosocomial infections lasting eight months in a university hospital in Paris. This outbreak occurred in the intensive care unit (39 patients), haematology units (8 patients) and surgical and medical units (11 patients). Antibiotic resistant strains were isolated from the urinary tract (48 %), wound and drainage fluids (21 %), respiratory tract (14 %), blood (12 %) and stools (5 %). High resistance to oxyimino-beta-lactams was mediated by a plasmid-encoded beta-lactamase with an isoelectric point of 7.8 (SHV-4). This CAZ-type enzyme conferred a higher level of resistance to ceftazidime and aztreonam (geometric mean MIC 135 mg/l) than to cefotaxime (geometric mean MIC 14 mg/l). All isolates were of the same biotype (weakly urease positive and no sucrose fermentation). EightKlebsiella pneumoniae strains isolated in different units and at different times of the outbreak were of the same serotype, had common plasmid patterns and harboured a large self-transferable plasmid of about 180 kilobases encoding resistance to penicillins, oxyimino-beta-lactams, aminoglycosides, tetracycline and trimethoprim. These eight large plasmids had indistinguishableEcoRI restriction patterns. These results suggest that a single strain ofKlebsiella pneumoniae was responsible for this outbreak.

Molecular characterization of the gene encoding SHV-3 beta-lactamase responsible for transferable cefotaxime resistance in clinical isolates of Klebsiella pneumoniae.

In Klebsiella pneumoniae 86-4, cefotaxime resistance was due to a transferable broad-spectrum beta-lactamase, SHV-3. The plasmid-borne gene encoding SHV-3 has been cloned, and the primary structure of the enzyme was deduced from its nucleotide sequence. SHV-3 differs from SHV-1 in two positions. The extended substrate profile of SHV-3 probably results from the substitution of Ser-213 for Gly, as in SHV-2, whereas replacement of Arg-180 by Leu resulted in a decrease in the pI from 7.6 to 7.0. The blashv-3 gene is highly homologous (92% DNA sequence identity) with the chromosomal gene coding for LEN-1 beta-lactamase of K. pneumoniae, suggesting that the origin of the SHV-encoding genes now present on many plasmids may be chromosomal. Images

Detection of extended broad-spectrum beta-lactamases inEnterobacteriaceae in four French hospitals

In 210 strains ofEnterobacteriaceae which were isolated in four hospitals and which showed reduced susceptibility to cefotaxime, high synergy was demonstrated between amoxicillin (20µg) + clavulanate (10µg) and cefotaxime (30µg) using a simple double-disk test. Isoelectric focusing on gel and specific iodometric detection using ceftriaxone identified four extended broad-spectrumβ-lactamases (isoelectric points 7.6, 6.3, 7.0 and 5.9) produced by the strains.