Comparison of screening methods for TEM- and SHV-derived extended-spectrum β-lactamase detection

Objective   To compare common extended-spectrum β -lactamase (ESBL) screening methods and β -lactams for their ability to detect TEM- and SHV-related ESBL enzymes.
Methods   This study compared disk diffusion testing by NCCLS methodology, the Jarlier double disk test, a disk-on-disk test, a modified three-dimensional test and the E test method for their sensitivity and specificity in detecting TEM- and SHV-related ESBL producers. Three negative and 22 positive controls were studied. These were two Klebsiella pneumoniae and 23 Escherichia coli transconjugants. Seventeen β -lactam antibiotics were tested: cefamandole, cefotetan, cefoxitin, cefuroxime, cefixime, cefoperazone, cefotaxime, cefpodoxime, cefsulodin, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, moxalactam, cefepime, cefpirome and aztreonam.
Results   NCCLS disk diffusion was 14% sensitive with ceftriaxone, 36% with cefotaxime, 64% with aztreonam, 68% with cefpodoxime, and 73% with ceftazidime. Cefoperazone, cefamandole, cefpodoxime and cefpirome showed 91% sensitivity using the Jarlier test. Using the disk-on-disk test, cefsulodin showed 95% sensitivity, and cefoperazone, cefepime and cefamandole showed 91% sensitivity. With the modified three-dimensional test, cefoperazone, cefpodoxime and cefpirome showed 91% sensitivity.
Conclusions   For practical reasons, we would recommend use of either the Jarlier test or the commercial cephalosporin disks containing clavulanic acid to screen for ESBL producers. Cefoperazone, cefamandole, cefpodoxime and cefpirome showed good sensitivity across the methods tested.     

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.     

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.     

Recommendation for treatment of severe infections caused by Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs)

Extended-spectrum β-lactamase (ESBL)-producing organisms are a global problem. No randomized controlled trials have ever been performed to guide optimal treatment. However, in vitro studies and observational studies strongly suggest that carbapenems (imipenem or meropenem) should be regarded as drugs of choice for serious infections due to ESBL-producing organisms. Other β-lactam antibiotics (cefepime, β-lactam/β-lactamase inhibitor combinations) are not suitable as first-line therapy. The increasing frequency of the association between quinolone resistance and ESBL production have greatly limited the role of this class of antibiotic against ESBL producers.     

The use of antibiotic-containing agars for the isolation of extended-spectrum β-lactamase-producing organisms in intensive care units

MacConkey agar containing either cefotaxime 1.0 mg/L or ceftazidime 1.0 mg/L was evaluated for use in screening for extended-spectrum beta-lactamase (ESBL)-producing organisms. The media were evaluated using known ESBL-positive and -negative strains and 630 clinical specimens over a 6-month period. All Enterobacteriaceae isolated were identified and screened for ESBL production by phenotypic methods. In total, 14 ESBL-producing organisms were detected in the clinical samples. All known ESBL-positive strains were also detected. The use of both screening plates was required to detect all ESBLs.     

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.     

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.     

Clinical variables associated with the isolation of Klebsiella pneumoniae expressing different extended-spectrum β-lactamases

Clinical variables associated with the isolation of Klebsiella pneumoniae expressing different extended-spectrum beta-lactamases (ESBLs) were studied. Clinical records of patients with ESBL-positive K. pneumoniae isolates between 1989 and 2003 (n = 80) were reviewed retrospectively. Patients with SHV- and TEM-type ESBLs were identified more frequently in the intensive care units (67% and 78%, respectively), whereas those with CTX-M ESBLs were found in medical wards (52.2%) or were outpatients (17.4%) (p <0.01). The absence of urinary or central catheters was associated with CTX-M-10 (p 0.013 and p <0.01, respectively). Central catheter-related infections and secondary bacteraemia were associated more frequently with SHV- and TEM-type ESBLs, whereas urinary tract infections were associated with CTX-M-10. Previous aminoglycoside use was associated particularly with SHV-type ESBLs (p <0.01), whereas amoxycillin-clavulanate and oral cephalosporins were associated with CTX-M-10 (p <0.01 and p 0.050, respectively). The frequency of adequate empirical treatment was low (22%), and 61% of patients were treated according to the susceptibility testing results. Mortality (22%) and related mortality (14%) did not differ statistically according to the type of ESBL. Different ESBL types in K. pneumoniae were associated with different clinical variables, and this should be taken into account in current and future epidemiological scenarios.     

Molecular diversity of Proteus mirabilis isolates producing extended-spectrum β-lactamases in a French university hospital

Between February 1997 and December 2002, 3340 hospitalised patients yielded samples positive for Proteus mirabilis, of whom 45 (1.3%) were colonised/infected by P. mirabilis producing extended-spectrum beta-lactamases (ESBLs). The gross incidence of patients colonised/infected by ESBL-producing P. mirabilis was 1.61/10(5) days of hospitalisation, with 20% of isolates being collected from patients in urology wards, most frequently (53.3%) from urine samples. Seventeen (37.7%) of the 43 isolates were obtained from samples collected within 48 h of hospitalisation, indicating that they were community-acquired. Isoelectric focusing assays and sequencing identified the TEM-24, TEM-92 and TEM-52 ESBLs. Pulsed-field gel electrophoresis revealed eight pulsotypes (I-VIII), with the two most common pulsotypes, IV and VI, comprising ten (23.3%) and 12 (26.6%) isolates, respectively. These pulsotypes were considered to represent epidemic strains and spread in various wards of the hospital.