Detection of extended-spectrum beta-lactamase (ESBL)-producing strains by the Etest ESBL screen.

Resistance to contemporary broad-spectrum beta-lactams, mediated by extended-spectrum beta-lactamase (ESBL) enzymes, is an increasing problem worldwide. The Etest (AB Biodisk, Solna, Sweden) ESBL screen uses stable gradient technology to evaluate the MIC of ceftazidime alone compared with the MIC of ceftazidime with clavulanic acid (2 micrograms/ml) to facilitate the recognition of strains expressing inhibitable enzymes. In the present study, ESBL-producing strains (17 Escherichia coli transconjugants) were studied to define "sensitive" interpretive criteria for the Etest ESBL screen. These criteria (reduction of the ceftazidime MIC by > 2 log2 dilution steps in the presence of clavulanic acid) defined a group of 92 probable ESBL-positive organisms among the 225 tested strains of Klebsiella species and E. coli having suspicious antibiogram phenotypes. With a subset of 82 clinical strains, the Etest ESBL screen was more sensitive (100%) than the disk approximation test (87%) and was more convenient. The MICs of ciprofloxacin, gentamicin, and tobramycin at which 50% of isolates are inhibited were 16- to 128-fold higher (coresistance) for the ESBL screen-positive group of strains than for the ESBL screen-negative group of strains. Some strains for which cephalosporin MICs were elevated and which were Etest ESBL screen negative were also cefoxitin resistant, i.e., consistent with a chromosomally mediated AmpC resistance phenotype. The Etest ESBL screen test with the ceftazidime substrate appears to be a useful method for detecting or validating the presence of enteric bacilli potentially producing this type of beta-lactamase.     

Clonal dissemination of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates in a Korean hospital

In this study, we investigated the molecular characteristics of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates that were recovered from an outbreak in a Korean hospital. A new multilocus sequence typing (MLST) scheme for K. pneumoniae based on five housekeeping genes was developed and was evaluated for 43 ESBL-producing isolates from an outbreak as well as 38 surveillance isolates from Korea and also a reference strain. Overall, a total of 37 sequence types (STs) and six clonal complexes (CCs) were identified among the 82 K. pneumoniae isolates. The result of MLST analysis was concordant with that of pulsedfield gel electrophoresis. Most of the outbreak isolates belonged to a certain clone (ST2), and they produced SHV-1 and CTX-M14 enzymes, which was a different feature from that of the K. pneumoniae isolates from other Korean hospitals (ST20 and SHV-12). We also found a different distribution of CCs between ESBL-producing and -nonproducing K. pneumoniae isolates. The MLST method we developed in this study could provide unambiguous and well-resolved data for the epidemiologic study of K. pneumoniae. The outbreak isolates showed different molecular characteristics from the other K. pneumoniae isolates from other Korean hospitals.     

Prevalence and characterisation of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in healthy volunteers in Tunisia

The objective of this investigation was to analyse the carriage rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faecal samples of healthy humans in Tunisia and to characterise the recovered isolates. One hundred and fifty samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 μg/ml) for ESBL-positive E. coli recovery. The characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, multilocus sequence typing (MLST) and phylogroup typing were performed by polymerase chain reaction (PCR) and sequencing. The presence and characterisation of integrons and virulence factors were studied by PCR and sequencing. ESBL-positive E. coli isolates were detected in 11 of 150 faecal samples (7.3%) and one isolate/sample was further characterised. These isolates contained the blaCTX-M-1 (ten isolates) and blaTEM-52c genes (one isolate). The ISEcp1 (truncated by IS10 in four strains) and orf477 sequences were found upstream and downstream, respectively, of all bla (CTX-M-1) genes. Seven different sequence types (STs) and three phylogroups were identified among CTX-M-1-producing isolates [ST/phylogroup (number of isolates)]: ST58/B1 (3), ST57/D (2), ST165/A (1), ST155/B1 (1), ST10/A (1), ST398/A (1) and ST48/B1 (1). The TEM-52-producing isolate was typed as ST219 and phylogroup B2. Six ESBL isolates contained class 1 integrons with the gene cassettes dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one). Healthy humans in the studied country could be a reservoir of CTX-M-1-producing E. coli.     

Deep learning model for prediction of extended-spectrum beta-lactamase (ESBL) production in community-onset Enterobacteriaceae bacteraemia from a high ESBL prevalence multi-centre cohort

European Journal of Clinical Microbiology & Infectious Diseases - Adequate empirical antimicrobial coverage is instrumental in clinical management of community-onset Enterobacteriaceae...     

Effects of inoculum and beta-lactamase activity in AmpC- and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates tested by using NCCLS ESBL methodology

Escherichia coli and Klebsiella pneumoniae isolates with extended-spectrum beta-lactamases (ESBLs) or AmpC cephalosporinases generally respond as predicted to NCCLS tests for ESBL production. However, inoculum size may affect MICs. The effect of inoculum level in clinical isolates expressing beta-lactamases were studied at inocula within 0.5 log unit of the standard inoculum, using broth microdilution methodology with ceftazidime, cefotaxime, cefepime, cefpodoxime, and aztreonam. Strains with TEM-1 or no beta-lactamases gave consistent MIC results with inocula of 10(5) and 10(6) CFU/ml. When the bacteria were screened for ESBL production and the lower inoculum was used, several strains with ESBLs, including CTX-M-10, TEM-3, TEM-10, TEM-12, TEM-6, SHV-18, and K1, gave false-negative results for one or more antimicrobial agents (MICs below the NCCLS screening concentration for detecting suspected ESBLs). When the higher inoculum was used, MICs of at least one antimicrobial agent increased at least fourfold in strains producing TEM-3, TEM-10, TEM-28, TEM-43, SHV-5, SHV-18, and K1. All antimicrobial agents showed an inoculum effect with at least one ESBL producer. Confirmatory clavulanate effects were seen for both inocula for all ESBL-producing strains with all antimicrobial agents tested, except for the CTX-M-10-producing E. coli with ceftazidime and the SHV-18-producing K. pneumoniae with cefotaxime. In kinetic studies, cefpodoxime and cefepime were hydrolyzed by ESBLs in a manner similar to that of cefotaxime. When total beta-lactamase activity and hydrolysis parameters were evaluated, however, no single factor was predictive of inoculum effects. These results indicate that the NCCLS screening and confirmation tests are generally predictive of ESBL production, but false-negative results can arise when a lower inoculum is used in testing.     

Antibiotic resistance in orthopaedic surgery: acute knee prosthetic joint infections due to extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae

The aim of this study was to describe the prevalence and characteristics of knee prosthetic joint infections due to extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. From 2000 to 2007, 132 infections out of 5,076 arthroplasties (2.6%) were registered. Seven out of 132 infections (5.3%) were due to ESBL-producing Enterobacteriaceae, Escherichia coli in six cases and Klebsiella pneumoniae in one. Open debridement and retention of the implant was the first surgical approach and all patients received intravenous carbapenems. Relapse was documented in four cases and remission in three. Therefore, debridement without prosthesis removal was associated with a high failure rate.     

Methicillin-resistant staphylococci (MRS) and extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae in companion animals: nosocomial infections as one reason for the rising prevalence of these potential zoonotic pathogens in clinical samples

The ongoing change in the relationship between humans and companion animals is hallmarked by the increasing intensive care provided to companion animals in veterinary medicine, resulting in growing numbers of high-risk animal patients. The emergence of nosocomial infections in small animal clinics is one of the major drawbacks of this development, especially in terms of multidrug-resistance and potentially zoonotic pathogens. This mini-review therefore addresses recent findings regarding the increasing prevalence of multi-resistant bacterial pathogens like methicillin-resistant staphylococci (MRS), including Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) as well as extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae in companion animals. Along with the steady increase of nosocomial infection rates in veterinary clinics, particular attention has recently been drawn to the genetic background of multi-resistant strains, resulting in the identification of certain genetic lineages which frequently appear in both, human and animal samples. These sequence types (ST), included ST254, ST8 and ST22 in terms of MRSA and ST131, ST405 and ST648 for ESBL-producing E. coli. The interspecies distribution of these STs resulted in the assumption that certain extended-host spectrum genotypes (EHSG) might exist both for MRS and ESBL-producing E. coli. These initial findings underline the necessity to investigate the major molecular or functional driving forces facilitating interspecies transferability of such EHSG strains. Due to the zoonotic potential of these multi-resistant bacteria, another aspect of the changing social role of companion animals needs to be addressed: the close contact of pets with their owners, resulting in presumptive new transmission and infection routes. We therefore envision retaliatory actions like initial surveillance and monitoring programs not only in livestock, but also particularly in companion animals. Interdisciplinary approaches including human and veterinary experts should be implemented to develop reliable investigation procedures with respect to the current reality of animal owners and their pets. Additionally, consequent basic hygienic measures, prudent use of antimicrobials in companion animals and efforts regarding implementation of antibiotic stewardships should be fostered.     

Evaluation of the new VITEK 2 extended-spectrum beta-lactamase (ESBL) test for rapid detection of ESBL production in Enterobacteriaceae isolates

Extended-spectrum beta-lactamases (ESBLs) are a large, rapidly evolving group of enzymes that confer resistance to oxyimino cephalosporins and monobactams and are inhibited by clavulanate. Rapid reliable detection of ESBL production is a prerequisite for successful infection management and for monitoring resistance trends and implementation of intervention strategies. We evaluated the performance of the new VITEK 2 ESBL test system (bioMérieux, Inc, Hazelwood, Mo.) in the identification of ESBL-producing Enterobacteriaceae isolates. We examined a total of 1,129 clinically relevant Enterobacteriaceae isolates (including 218 that had been previously characterized). The ESBL classification furnished by the VITEK 2 ESBL test system was concordant with that of the comparison method (molecular identification of beta-lactamase genes) for 1,121 (99.3%) of the 1,129 isolates evaluated. ESBL production was correctly detected in 306 of the 312 ESBL-producing organisms (sensitivity, 98.1%; positive predictive value, 99.3%). False-positive results emerged for 2 of the 817 ESBL-negative isolates (specificity, 99.7%; negative predictive value, 99.3%). VITEK 2 ESBL testing took 6 to 13 h (median, 7.5 h; mean +/- SD, 8.2 +/- 2.39 h). This automated short-incubation system appears to be a rapid and reliable tool for routine identification of ESBL-producing isolates of Enterobacteriaceae.     

Incidence of extended spectrum beta-lactamase (ESBL)-producing Enterobacteria in the area of Caltagirone (Sicily)

The authors report the incidence of extended spectrum b-lactamases (ESBL)- producing strains in 1,602 enterobacteria consecutively isolated, from April 2002 to December 2003, in Caltagirone (CT) Hospital, and their in vitro susceptibility to several antimicrobial agents. The incidence of ESBL-producing Enterobacteria was equal to 1.5%, mainly among Escherichia coli, Klebsiella spp and Citrobacter koseri, isolated by emergency and medicine wards. In vitro, the enterobacteria were fully susceptible to carbapenems and highly susceptible to third-generation cephalosporin, aztreonam, piperacillin, amikacin, and ciprofloxacin.     

Countrywide spread of community- and hospital-acquired extended-spectrum beta-lactamase (CTX-M-15)-producing Enterobacteriaceae in Lebanon

A prospective study was carried out to assess the extent of carriage of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae at both hospital and community levels in Lebanon. A total of 1,442 fecal samples were collected from hospital-based patients and 58 from health care workers of six Lebanese tertiary care general hospitals located in different areas of Lebanon between January and March 2003. A total of 382 fecal samples were also collected from healthy subjects between April and June 2003. The samples analysis led to the identification of 118 strains as ESBL producers based on the synergistic effects between clavulanate and selected beta-lactams (ceftazidime and cefotaxime). These strains were isolated from 72 subjects: 61 patients, 2 health care workers, and 9 healthy subjects. One representative strain per subject was selected, and a total of 72 nonduplicate ESBL producers, including a high majority of Escherichia coli (n = 56), Klebsiella pneumoniae (n = 9), Enterobacter cloacae (n = 6), and Citrobacter freundii (n = 1), were characterized. The molecular analysis revealed that the majority of the strains (83%) express CTX-M-15 ESBL (pI 8.6). SHV-5a ESBL (pI 8.2) was produced by 18% of the strains. DNA macrorestriction analysis of ESBL-producing E. coli presented 38 different genotypes, revealing the absence of clonal link among these strains. In addition to the fact that the present study highlights the emergence and the countrywide dissemination of CTX-M-15-producing E. coli in Lebanon, it represents the first report of an SHV-5a-producing C. freundii.     

New diagnostic microarray (Check-KPC ESBL) for detection and identification of extended-spectrum beta-lactamases in highly resistant Enterobacteriaceae

The performance of a microarray for the detection of extended-spectrum beta-lactamases was determined on a collection of 638 highly resistant members of the family Enterobacteriaceae collected from patients in 18 hospitals in The Netherlands. The microarray had a significantly higher specificity than the phenotypic assays. It also detects carbapenemases and characterizes the resistance genes, providing epidemiological insight.     

Emergence of extended-spectrum beta-lactamase (CTX-M-15 and CTX-M-14)-producing nontyphoid Salmonella with reduced susceptibility to ciprofloxacin among food animals and humans in Korea

Twenty of 1,279 nontyphoid Salmonella strains isolated from food animals and humans produced CTX-M-type extended-spectrum β-lactamase. All expressed CTX-M-15, except two which coexpressed CTX-M-14 and TEM-1. Insertion sequence ISEcp1 was identified upstream of bla(CTX-M) genes. The bla(CTX-M-15) and bla(CTX-M-14) genes were disseminated by large conjugative IncFIIs and IncI1-Iγ plasmids, respectively.     

Extended-spectrum β-lactamase (ESBL) CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae in Sofia, Bulgaria

During a survey of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Bulgaria in 2001-2002, three isolates from Sofia (two Escherichia coli, one Klebsiella pneumoniae) showed cefotaxime MICs that were decreased in the presence of clavulanate and were 2-8-fold higher than those of ceftazidime. Resistance was transferred to a sensitive recipient strain of E. coli. Both wild-type and transconjugant strains produced a cefotaxime-hydrolysing beta-lactamase of pI 8.8. Sequencing of the PCR product obtained with oligonucleotide primers binding outside the coding region identified this beta-lactamase as CTX-M-15. To our knowledge, this is the first report of CTX-M-15 in Bulgaria.     

Clinical importance of extended-spectrum beta-lactamase (PER-1-type)-producing Acinetobacter spp. and Pseudomonas aeruginosa strains.

Recently, an extended-spectrum beta-lactamase (PER-1) was found to be disseminated among Acinetobacter spp. and Pseudomonasaeruginosa isolates in Turkey. A population-based cohort study was conducted to elucidate predictive mortality factors in patients with nosocomial infections caused by Acinetobacter spp. and P. aeruginosa, with particular reference to PER-1-type extended-spectrum beta-lactamase (ESBL) production. The study group comprised 16 and 21 non-survivors and 82 and 126 survivors in cohorts infected with Acinetobacter and P. aeruginosa, respectively. In the Acinetobacter-infected cohort, nosocomial pneumonia, hypotension and infection with a PER-positive isolate were independent predictors of mortality. In the P. aeruginosa-infected cohort, impaired consciousness, a PER-positive isolate, male sex and (with a negative relative risk) urinary tract infection were independent predictors of death. This study demonstrated the relationship of PER-1-type ESBL-producing Acinetobacter spp. and P. aeruginosa with poor clinical outcome.     

Antibiogram and plasmid profiling of carbapenemase and extended spectrum Beta-lactamase (ESBL) producing Escherichia coli and Klebsiella pneumoniae in Abeokuta,South western,Nigeria

Background

The increased reports of ESBL dissemination from various centres in south western, Nigeria and the recent emergence of carbapenem resistant bacteria prompted the conception of this study.

Objectives

To demonstrate the relationship between high molecular weight plasmids and the expression of antibiotic multi-resistance including ESBL and carbapenemase.

Methods

We investigated 97 isolates of selected organisms consisting of 67 E. coli and 30 Klebseilla spp for the presence of plasmids expressing ESBL including carbapenem-hydrolysing enzymes. Beta-lactamase was determined using acidometric method, while ESBL and carbapenemase activity was determined using the double-disk diffusion test as well as the Modified Hodge test (MHT). Plasmid profiles of ESBL and carbapenemase positive isolates were determined according to standard protocols.

Results

An ESBL prevalence rate of 21.6% and carbapenem- resistance rate of 9.3% was recorded. Antibiotic susceptibility profile of ESBL isolates showed 100.0% resistance against Amoxicillin, Cotrimoxazole and Erythromycin. Moderate susceptibility was recorded against the Quinolone class of antibiotics; Meropenem remained the most active antibiotic against ESBL isolates with 62.5% against E. coli and 60% against K. pneumoniae. The plasmid profiles of our study isolates ranged from 11.8kbp to 35.5kbp.

Conclusion

Due to the relationship between high molecular weight plasmids and multi-drug resistance, we hereby recommend regular molecular surveillance of this form in our study setting.     

Prevalence and significance of a negative extended-spectrum beta-lactamase (ESBL) confirmation test result after a positive ESBL screening test result for isolates of Escherichia coli and Klebsiella pneumoniae: results from the SENTRY Asia-Pacific Surveillance Program

A negative extended-spectrum beta-lactamase (ESBL) confirmation test result obtained after a positive ESBL screening test result using Clinical and Laboratory Standards Institute methods has been a common occurrence among isolates of Escherichia coli and Klebsiella pneumoniae in the SENTRY Antimicrobial Surveillance Program in the Asia-Pacific region. Among isolates collected between 1998 and 2004 this screen-positive, nonconfirmed profile (failed to show clavulanate synergy) was observed in 8.9% of 4,515 E. coli isolates and 20.3% of 2,303 K. pneumoniae isolates. We then selected 52 E. coli isolates and 68 K. pneumoniae isolates with a negative ESBL confirmation test, as well as comparable number of isolates with confirmed ESBL-positive tests, and examined them for the presence of TEM, SHV, plasmid-borne ampC, and CTX-M genes. We found that 62% of nonconfirming E. coli isolates and 75% of nonconfirming K. pneumoniae harbored a plasmid-borne AmpC enzyme of the CIT or DHA type. The majority of nonconfirming E. coli and K. pneumoniae from the Asia-Pacific region harbor important beta-lactamases, and a positive screening test alone should be sufficient grounds to report resistance to extended-spectrum cephalosporins in this region.     

Clonal emergence of extended-spectrum beta-lactamase (CTX-M-2)-producing Salmonella enterica serovar Virchow isolates with reduced susceptibilities to ciprofloxacin among poultry and humans in Belgium and France (2000 to 2003)

Antibiotic treatment is not required in cases of Salmonella enterica gastroenteritis but is essential in cases of enteric fever or invasive salmonellosis or in immunocompromised patients. Although fluoroquinolones and extended-spectrum cephalosporins are the drugs of choice to treat invasive Salmonella, resistance to these antibiotics is increasing worldwide. During the period 2000 to 2003, 90 Salmonella enterica serovar Virchow poultry and poultry product isolates and 11 serovar Virchow human isolates were found to produce an extended-spectrum beta-lactamase, CTX-M-2, concomitantly with a TEM-1 beta-lactamase. The bla(CTX-M-2) gene was located on a large conjugative plasmid (>100 kb). Pulsed-field gel electrophoresis indicated a clonal relationship between the poultry and human isolates. All these isolates displayed additional resistance to trimethoprim-sulfamethoxazole and tetracycline as well as a reduced susceptibility to ciprofloxacin (MICs of between 0.5 and 1 mug/ml). CTX-M-2-producing Salmonella with a reduced susceptibility to fluoroquinolones constitutes a major concern, since such strains could disseminate on a large scale and jeopardize classical antibiotic therapy in immunocompromised patients.     

Characterization of extended-spectrum beta-lactamase (TEM-52)-producing strains of Salmonella enterica serovar typhimurium with diverse resistance phenotypes

Two Salmonella enterica serovar Typhimurium strains from different clonal origins, both producing an extended-spectrum beta-lactamase (TEM-52), were isolated from a patient. This enzyme was encoded on a single plasmid and was found at very low levels in one strain, while being encoded on multiple plasmids and in multiple different EcoRI fragments in the other strain.