First description of DHA-1 ampCβ-lactamase in Proteus mirabilis

This report describes the first occurrence of the DHA-1 ampCbeta-lactamase gene in Proteus mirabilis. The organism was isolated from the vaginal flora of a pregnant woman in a French hospital. The DHA-1 beta-lactamase gene was identified on the basis of phenotypic characteristics, PCR amplification and sequencing. Antagonism between cefoxitin and the other cephalosporins suggested inducible production of the DHA-1 enzyme.     

Evaluation of clonal relatedness of extended-spectrum β-lactamase-producing Proteus mirabilis isolates by quantitative antibiogram and RAPD typing

Objective: To delineate, using two different typing systems, the clonal relatedness of 40 isolates of extended-spectrum β-lactamase (ESβla)-producing Proteus mirabilis obtained over a period of 7 years in six hospitals in the Paris area and two in Pas-de-Calais.
Method: Random amplified polymorphic DNA (RAPD) polymerase chain reaction typing was applied by using three random primers on the ESβla-producing P. mirabilis isolates and on isogenic Escherichia coli strains with or without plasmids encoding the representative resistance pattern transferred from P. mirabilis. Quantitative antibiogram typing, which was also applied to the P. mirabilis isolates, was used to define the euclidean distance between these strains.
Results: After having demonstrated that P. mirabilis plasmids did not influence chromosomal DNA amplification, we could classify the ESβla-producing P. mirabilis isolates into 12 groups based on RAPD fingerprints. The same isolates were classified into 19 groups by quantitative antibiogram typing. Despite this difference in group numbers, general concordance between the typing systems was observed. This allowed us to show that the greater number of isolates in some hospitals belonged to a single strain and that single isolates obtained in different hospitals generally represented unique strains.
Conclusions: A small number of ESβla-producing P. mirabilis strains was isolated during 7 years in the eight medical centers studied, and the number of different strains identified suggested that inter-hospital transfer had not occurred.     

Long-term study of the frequency of Escherichia coli and Klebsiella pneumoniae isolates producing extended-spectrum β-lactamases

In total, 438 (1.7%) Escherichia coli and 125 (3.98%) Klebsiella pneumoniae isolates were found to be producers of extended-spectrum beta-lactamase (ESBL) during 1995-2003 in southern Spain. There was a significant increase in the frequency of ESBL-producing E. coli isolates, from < 0.36% before 1999 to 4.8% in 2003, while the frequency of ESBL-producing K. pneumoniae isolates decreased during the same period. The most common ESBLs detected in K. pneumoniae were SHV type, whereas both CTX-M and SHV types were detected in E. coli. In addition, E. coli isolates showed greater clonal diversity (84 distinct REP-PCR patterns, compared with five in K. pneumoniae), fewer enzymes per isolate, and a higher number of isolates recovered from outpatients. These differences may have implications for the control measures that should be used for these two microorganisms.     

Spread of novel expanded-spectrum β-lactamases in Enterobacteriaceae in a university hospital in the Paris area, France

In 2002, 28 non-duplicate enterobacterial isolates producing extended-spectrum beta-lactamases (ESBLs) were collected from infected patients at the Bicêtre Hospital in Paris, France. Escherichia coli was the predominant ESBL-positive enterobacterial species, comprising ten (36%) of the isolates. CTX-M enzymes (CTX-M-3, CTX-M-10, CTX-M-14 and CTX-M-15) were produced by 11 (39%) of the isolates (six E. coli, two Enterobacter cloacae, one Enterobacter aerogenes, one Proteus mirabilis and one Citrobacter freundii). Other ESBLs, such as VEB-1 and PER-1, were also detected, but less frequently.     

Evaluation of the Vitek-2 extended-spectrum β-lactamase test against non-duplicate strains of Enterobacteriaceae producing a broad diversity of well-characterised β-lactamases

The Vitek-2 extended-spectrum β-lactamase (ESBL) test was assessed using a collection of 94 ESBL-positive and 71 ESBL-negative non-duplicate isolates of Enterobacteriaceae. These isolates produced a wide diversity of well-characterised β-lactamases, including 61 different ESBLs, two class A carbapenemases and various species-specific β-lactamases. ESBL detection was performed using (i) the conventional synergy test as recommended by the Comité de l'Antibiogramme de la Société Française de Microbiologie, (ii) the CLSI phenotypic confirmatory test for ESBLs, and (iii) the Vitek-2 ESBL test. For Escherichia coli and klebsiellae, the sensitivity/specificity values were 97.3%/96.9% for the synergy test, 91.8%/100% for the CLSI phenotypic confirmatory test, and 91.8%/100% for the Vitek-2 ESBL test. For other organisms, the sensitivity/specificity values were 100%/97.4% for the synergy test, 90.5%/100% for the CLSI phenotypic confirmatory test, and 90.5%/100% for the Vitek-2 ESBL test. The Vitek-2 ESBL test seemed to be an efficient method for routine detection of ESBL-producing isolates of Enterobacteriaceae, including isolates producing AmpC-type enzymes.     

Infrequent detection of acquired metallo-β-lactamases among carbapenem-resistant Pseudomonas isolates in a Greek hospital

Objective  To study the possible distribution of metallo-β-lactamases among nosocomial Pseudomonas isolates in a Greek hospital with a recent high prevalence of carbapenem-resistant Pseudomonas isolates.
Methods  All carbapenem-resistant (imipenem- and/or meropenem-resistant) (MICs > 8 mg/L) Pseudomonas non-replicate isolates recovered from clinical infections in the Microbiology Laboratory of Saint Demetrios Hospital, Thessaloniki, Greece, from April 1998 to November 2000 were studied for the presence of metallo-β-lactamases. They were tested by a disk diffusion test, PCR analysis, and nucleotide sequencing. DNA fingerprints were obtained by pulsed-field gel electrophoresis (PFGE) of Xba I-digested chromosomal DNA.
Results  In total, 24 carbapenem-resistant isolates (23 P. aeruginosa and one P. putida ) were recovered. The serotypes observed among the P. aeruginosa isolates were, in order of decreasing frequency, O:11 (52%), O:3 and O:12 (17% each), and O:6 (13%). PFGE grouped 17 of the P. aeruginosa isolates into four clusters, each containing from two to seven isolates, while the remaining isolates exhibited unique genotypes. bla _VIM-2 was detected in the P. putida isolate and a P. aeruginosa serotype O:3 isolate. The latter strain was genotypically distinct from other contemporaneous or older carbapenem-resistant P. aeruginosa Greek isolates.
Conclusion  These findings suggest that, although the prevalence of metallo-β-lactamases is low, the integron-associated bla _VIM genes can spread to P. aeruginosa serotypes that have not been previously associated with carbapenem resistance in our region, as well as to other pseudomonal species.     

Bactericidal activity of three β-lactams alone or in combination with a β-lactamase inhibitor and two aminoglycosides against Klebsiella pneumoniae harboring extended-spectrum β-lactamases

Objective: To study the bactericidal activity of β-lactam antibiotics (imipenem, cefepime, cefpirome) alone or in combination with a β-lactamase inhibitor (sulbactam) in the presence or absence of aminoglycoside (amikacin or isepamicin) against Klebsiella pneumoniae strains producing extended-spectrum β-lactamases (ESBLs).
Methods: We characterized 10 strains by means of analytic isoelectric focusing and pulsed-field gel electrophoresis. The ESBLs produced by these strains were derived from either TEM (TEM-1, TEM-2) or SHV-1. The killing-curve method was used for this bacterial investigation. Bacteria (final inoculum 5×10 ⁵ CFU/mL) were incubated with antibiotics at clinical concentrations obtained in vivo.
Results: All the combinations with cefepime or cefpirome + sulbactam were bactericidal, with a 4 log₁₀ decrease being obtained within 6 h without regrowth at 24 h, whereas imipenem alone, and combinations, gave a bactericidal effect within 6 h. The two cephalosporins alone decreased the inoculum of 4 log₁₀ at 6 h but regrowth was observed at 24 h. When the aminoglycoside was added, this bactericidal effect was obtained within 3 h with amikacin and within 1 h with isepamicin.
Conclusions: Cefepime + sulbactam or cefpirome + sulbactarn may be an alternative to imipenem for the treatment of patients with ESBL-producing K. pneumoniae. Aminoglycosides are often associated in nosocomial infections due to ESBL-producing K. pneumoniae: isepamicin acted faster than amikacin, but both worked well. To conclude, it may be prudent to avoid extended-spectrum cephalosporins as single agent when treating serious infections due to ESBL-producing K. pneumoniae. Addition of a β-lactamase inhibitor such as sulbactam ± aminoglycoside is advisable to avoid failure of treatment.     

Prevalence of extended-spectrum β-lactamases in isolates of the Enterobacter cloacae complex from German hospitals

In 2002, 119 isolates of the Enterobacter cloacae complex were collected randomly from 11 German laboratories nationwide. Antibiotic susceptibilities were tested by disk-diffusion tests according to CLSI guidelines, and MICs were determined using Etests. PCRs were performed to amplify all TEM and SHV, and most CTX-M and OXA beta-lactamase genes. PCR products were sequenced to identify the precise extended spectrum beta-lactamase (ESBL) types. Isoelectric focusing (IEF) and PM/PML Etests were used to confirm production of the respective ESBLs. According to susceptibility tests and CLSI criteria, 49 (40%) isolates were resistant to extended-spectrum cephalosporins. Seven (5.8%) isolates were positive in at least one of the PCR assays. Sequencing identified production of TEM-1 beta-lactamase genes by three (2.9%) isolates, and ESBL genes of the CTX-M and SHV beta-lactamase families by five (4.2%) isolates. IEF confirmed the production of beta-lactamases in the expected pI ranges of the respective ESBLs, and four of the five ESBL-producers were detected using the PM/PML Etest. All ESBL-producing isolates showed co-resistance to sulphonamides.     

Development of extended-spectrum activity in TEM β-lactamases in hyper-mutable, mutS Escherichia coli

TEM-1 and TEM(pUC19)beta-lactamases can gain activity against ceftazidime and other expanded-spectrum cephalosporins via point mutation. The frequency of emergent resistance to ceftazidime at 4 x MIC was elevated >or= 250-fold in hyper-mutable, MutS-deficient Escherichia coli harbouring these beta-lactamase genes on high- or low-copy plasmids. Moreover, although ceftazidime-resistant mutants, or those with reduced susceptibility, were selected in both the wild-type and mutS hosts, many more mutants in the mutS host showed ceftazidimase-type extended-spectrum beta-lactamase (ESBL) activity. This correlated with a G-A point mutation at position 484 in the bla(TEM-1) and bla(TEM-pUC19) genes, conferring the Arg164His amino-acid substitution found in the TEM-29 ESBL. Non-ESBL mutants lacked changes in bla(TEM).