Emergence of SHV-12 extended spectrum beta-lactamase among clinical isolates of Enterobacter cloacae in Tunisia

A collection of seven multidrug-resistant clinical isolates of Enterobacter cloacae with reduced susceptibility to ceftazidime and cefepime recovered from 2009 to 2010 at the University Hospital of Mahdia, Tunisia, was analysed. PCR analysis and sequencing demonstrated that all study isolates harbored SHV-12 β-lactamase that was transferred by conjugation. Characterization of the regions surrounding the bla_SHV-12 showed that this gene was ?anked by two IS26 elements. pulsed-field gel electrophoresis (PFGE) revealed differents profiles indicating that the study isolates were not clonally related. Diffusion of E. cloacae producing SHV-12 ESBL in our hospital is the consequence of disseminations of plasmids harboring the SHV-12 gene.     

Bacteriocin typing of clinical isolates of Enterobacter cloacae.

A total of 16 selected bacteriocins of Enterobacter cloacae were characterized presumptively. They proved to be noninfectious, sedimentable (105,000 X g), resistant against chloroform and trypsin, and nonfilterable. The host ranges were essentially species specific. Based on susceptibility to one or more of these 16 bacteriocins, 242 of 308 (78.6%) clinical E. cloacae isolates were typed and assigned to 52 provisional bacteriocin types. Several outbreaks of nosocomial cross-infection were discerned retrospectively. Thus, bacteriocin typing of E. cloacae isolates may prove useful for controlling hospital infection.     

Detection of extended-spectrum beta-lactamases in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes

The aim of the present study was to investigate the frequency of extended-spectrum beta-lactamases (ESBLs) in a consecutive collection of clinical isolates of Enterobacter spp. The abilities of various screening methods to detect ESBLs in enterobacters were simultaneously tested. Among the 68 consecutive isolates (56 Enterobacter cloacae and 12 Enterobacter aerogenes isolates) that were analyzed for beta-lactamase content, 21 (25 and 58%, respectively) possessed transferable ESBLs with pIs of 8.2 and phenotypic characteristics of SHV-type enzymes, 8 (14.3%) of the E. cloacae isolates produced a previously nondescribed, clavulanate-susceptible ESBL that exhibited a pI of 6.9 and that conferred a ceftazidime resistance phenotype on Escherichia coli transconjugants, and 2 E. cloacae isolates produced both of these enzymes. Among the total of 31 isolates that were considered ESBL producers, the Vitek ESBL detection test was positive for 2 (6.5%) strains, and the conventional double-disk synergy test (DDST) with amoxicillin-clavulanate and with expanded-spectrum cephalosporins and aztreonam was positive for 5 (16%) strains. Modifications of the DDST consisting of closer application of the disks (at 20 instead of 30 mm), the use of cefepime, and the use of both modifications increased the sensitivity of this test to 71, 61, and 90%, respectively. Of the 37 isolates for which isoelectric focusing failed to determine ESBLs, the Vitek test was false positive for 1 isolate and the various forms of DDSTs were false-positive for 3 isolates.     

Mechanisms of quinolone resistance in clinical isolates of Enterobacter cloacae

Objective: To study and evaluate changes in the gyrA gene and the outer-membrane protein patterns in relation to evolution of resistance against the quinolones in Enterobacter cloacae.
Methods: Strains expressing gyrA -mediated quinolone resistance become susceptible to quinolones upon insertion of the plasmid pNJR3–2. This plasmid (containing wild-type Escherichia coli quinolone-susceptible DNA gyrase A subunits) and pLA2917 (the vector) were introduced into 10 resistant or moderately susceptible clinical isolates of Enterobacter cloacae by conjugation. The transconjugants, the original isolates, the plasmid and the vector control were screened for susceptibility to ofloxacin, ciprofloxacin and sparfloxacin. Additionally, examinations of the outer-membrane proteins were performed.
Results: A reduction of MICs by a factor of 8–32 was found for the transconjugants of five Enterobacter cloacae isolates in the presence of the gene probe, suggesting that these isolates harbored mutations in gyrA. No discernible difference in the patterns of outer-membrane proteins of sensitive and resistant strains could be detected.
Conclusions: It seems that changes in the target site such as alterations in gyrA are important factors leading to a change in the susceptibility of bacteria to the quinolones, whereas there were no evident changes in the outer-membrane proteins to account for evolution of resistance.     

Chromosomal beta-lactamase expression and antibiotic resistance in Enterobacter cloacae

The activities of beta-lactam antibiotics were compared against Enterobacter cloacae clinical isolates and mutants which had inducible, stably-derepressed, and basal expression of a pI 8.4 subtype of the Ia chromosomal beta-lactamase. These activities were correlated with the results of studies of the beta-lactamase-lability and beta-lactamase-inducer-power of the antibiotics. Cefoxitin and ampicillin were labile, and induced beta-lactamase production strongly at concentrations below their MIC values. Consequently, beta-lactamase-inducible and beta-lactamase-stably-derepressed organisms were highly resistant (MIC greater than 256 mg/L) to these antibiotics, whereas enzyme-basal strains and mutants were much more susceptible (MIC 1-16 mg/L). Imipenem also induced beta-lactamase production strongly at concentrations below its MIC, but was more stable than ampicillin and cefoxitin. It was active against enzyme-inducible and stably-derepressed organisms at 0.25-0.5 mg/L and against beta-lactamase-basal organisms at 0.06-0.25 mg/L. Thus the beta-lactamase afforded only very low-level protection against imipenem; this appeared to be by a non-hydrolytic mechanism, with the enzyme binding to the antibiotic in a relatively stable complex. This complex, which probably was an intermediate in a hydrolytic pathway, was isolated by gelfiltration chromatography and shown to have a breakdown half-life of 47 +/- 2 min. Cefotaxime, ceftriaxone and mezlocillin were labile to the pI 8.4 beta-lactamase but induced beta-lactamase production weakly at concentrations below their MIC values. Consequently, beta-lactamase-inducible and beta-lactamase-basal organisms remained equally susceptible (MIC 0.06-4 mg/L), but stably-derepressed organisms were considerably more resistant (MIC greater than 64 mg/L) to these antibiotics.     

Genomic groups and biochemical profiles of clinical isolates of Enterobacter cloacae

A collection of 123 clinical strains presumptively identified as Enterobacter cloacae and 12 type and reference strains of Enterobacter spp. were genotypically investigated by a quantitative bacterial dot method for DNA-DNA hybridization, giving an estimate of delta Tm (difference in thermal denaturation midpoint between homologous and heterologous duplexes). The API 20E system was used for phenotypic characterization. Using discontinuities in the values of delta Tm as criterion, five genomic groups of E. cloacae could be demonstrated, eleven ungrouped strains representing at least one additional group. Nine API profiles were found, the ideal phenotype of E. cloacae (i.e. the phenotype showing the most common reaction for the species in all tests studied) being the most frequently found. The type strain of E. cloacae and the reference strain CDC 1347-71 represented rather small genomic groups of three and eight strains respectively, most of them inositol positive. A majority of 98 isolates formed one single genomic group, biochemically dominated by the ideal phenotype. The genomic groups could not be differentiated phenotypically. At present there seems to be no reason for an attempt to split E. cloacae into two or more species. The type strain of E. dissolvens showed itself to be closely related to the type strain of E. cloacae (delta Tm 2.3 degrees C), indicating that the two species may be regarded as subjective synonyms.     

Antibiotic susceptibility and beta-lactamase production in clinical isolates of Enterobacter spp

The in vitro susceptibility of 237 clinical isolates of Enterobacter spp. (E. aerogenes, E. agglomerans and E. cloacae; 41, 64 and 132 respectively) to 16 different antibiotics is described. Four quinolones (ciprofloxacin, lomefloxacin, norfloxacin and ofloxacin), two new cephalosporins (cefpirome and cefepime) and imipenem, all showed high activity against the three Enterobacter species tested (MIC50 less than or equal to 0.125 mg/l, MIC90 less than or equal to 0.5 mg/l). Also the aminoglycosides gentamicin and tobramycin were highly active antibiotics (MIC50 less than or equal to 0.5 mg/l, MIC90 less than or equal to 1.0 mg/l). The susceptibility of beta-lactam-antibiotics to beta-lactamase produced by Enterobacter spp. was evaluated, and imipenem and cefepime were found to be most stable. Different methods for detection of inducible beta-lactamases were used, the agar dilution method being more sensitive than the double-disc diffusion test. Elevated beta-lactamase production was detected, via induction, in 83% of E. aerogenes strains and 70% of E. cloacae strains, with cefamandole used as the substrate and cefoxitin as the inducer. Constitutive, high level enzyme production was detected in 7 and 13% respectively of the E. aerogenes and the E. cloacae strains. In all the strains of E. agglomerans, 10% of E. aerogenes and 13% of E. cloacae, no beta-lactamases could be detected with the methods studied.     

Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR

Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC beta-lactamases are limited because these organisms are usually resistant to all the beta-lactam antibiotics, except for cefepime, cefpirome, and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Six families of plasmid-mediated AmpC beta-lactamases have been identified, but no phenotypic test can differentiate among them, a fact which creates problems for surveillance and epidemiology studies. This report describes the development of a multiplex PCR for the purpose of identifying family-specific AmpC beta-lactamase genes within gram-negative pathogens. The PCR uses six sets of ampC-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis. ampC multiplex PCR differentiated the six plasmid-mediated ampC-specific families in organisms such as Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella enterica serovar Typhimurium. Family-specific primers did not amplify genes from the other families of ampC genes. Furthermore, this PCR-based assay differentiated multiple genes within one reaction. In addition, WAVE technology, a high-pressure liquid chromatography-based separation system, was used as a way of decreasing analysis time and increasing the sensitivity of multiple-gene assays. In conclusion, a multiplex PCR technique was developed for identifying family-specific ampC genes responsible for AmpC beta-lactamase expression in organisms with or without a chromosomal AmpC beta-lactamase gene.     

Evaluation of methods for AmpC beta-lactamase in gram negative clinical isolates from tertiary care hospitals

The purpose of this study was to simultaneously screen for Extended-spectrum beta-lactamases (ESBL) and AmpC beta-lactamases in gram negative clinical isolates from four tertiary care hospitals and further to compare two detection methods three-dimensional extraction method and AmpC disk test for AmpC beta-lactamases. A total of 272 isolates were screened for ESBL and AmpC beta-lactamase by modified double disk approximation method (MDDM). Synergy observed between disks of ceftazidime/cefotaxime and clavulanate were considered as ESBL producer. Isolates showing reduced susceptibility to either of the test drugs (ceftazidime or cefotaxime) and cefoxitin were considered as presumptive AmpC producers and further confirmed by three-dimensional extraction method and AmpC disk test. A total of 173 (64%) of the isolates were found to be ESBL positive and 61 (23%) showed resistant to cefoxitin. ESBL was detected in 80 (62%) isolates of E. coli and 71 (73%) of Klebsiella spp. The occurrence of AmpC beta-lactamases was found to be 8% (22) of the total isolates and the two detection methods for AmpC beta-lactamase showed concordant results. Screening for ESBL and AmpC can be simultaneously done by MDDM method and confirmation for AmpC beta-lactamase should be carried out routinely in tertiary care hospitals by AmpC disk test, as it is a simple and rapid procedure.     

Detection of resistance due to inducible beta-lactamase in Enterobacter aerogenes and Enterobacter cloacae.

Thirty-six of 36 strains of Enterobacter cloacae and E. aerogenes with inducible beta-lactamase developed resistance when cefoxitin (inducer) was added to cefuroxime disks. Constitutive beta-lactamase producers (n = 23) were all resistant to cefuroxime. Cefuroxime resistance correlated with the amount of induced or constitutive beta-lactamase. Cefuroxime was a better indicator of induced resistance than cefamandole, cefazolin, cephalothin, ceftriaxone, cefotaxime, ticarcillin with or without clavulanic acid, or cefotetan. Induction by addition of cefoxitin to disks occasionally reduced zone sizes but not enough to change interpretations for ceftazidime, ceftizoxime, aztreonam, cefoperazone with or without sulbactam, and piperacillin with or without tazobactam. Most enterobacters were resistant to cefmetazole. The cefoxitin inducer-cefuroxime indicator method can be used in routine clinical laboratories to detect latent resistance due to chromosomally mediated inducible beta-lactamase in enterobacters.     

产AmpC酶大肠埃希菌和肺炎克雷伯菌中整合子与多重耐药的研究

目的探讨整合子介导的耐药机制在产AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药中的作用.方法5株产AmpC酶大肠埃希菌和肺炎克雷伯菌分离自2002年1月-2004年5月间我院呼吸科住院的患者,采用E-test试验条进行药敏试验、电转化试验,筛选、分离耐药质粒.PCR扩增Ⅰ型整合子基因盒插入序列,分子克隆和序列分析.结果所有产酶菌株通过电转化试验可将头孢西丁耐药性传递给受体菌,5个产AmpC酶耐药质粒中,有4个检出整合酶序列,其中3个携带2种抗药性基因盒,包括氨基糖苷乙酰转移酶基因aacA4;氨基糖苷腺苷转移酶基因aadA5;二氢叶酸还原酶基因dfrA17;氯霉素外排蛋白编码基因cmL44.结论整合子介导的抗药性基因盒参与了产质粒AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药的形成,应引起高度重视.     

Extended-spectrum beta-lactamase enzymes in clinical isolates of Enterobacter species from Lagos,Nigeria

Over a 9-month period, 8 of 40 nonduplicate isolates of Enterobacter spp. producing extended-spectrum beta-lactamase (ESBL) were detected for the first time from two hospitals in Lagos, Nigeria. Microbiologic and molecular analysis confirmed the presence of ESBL. Only four isolates transferred ESBL resistance as determined by the conjugation test, and pulsed-field gel electrophoresis showed genetically unrelated isolates.     

Enhancement of beta-lactamase production by glycine in Enterobacter cloacae ATCC.13047

Beta-lactamase production in Enterobacter cloacae ATCC.13047 was enhanced by glycine, and the degree of enhancement was dependent on the concentration of glycine and the medium used. Maximum enhancement occurred with a concentration of 0.05 moles/l of glycine, and of 4 media examined enhancement was greatest on Isosensitest Agar. Enhanced beta-lactamase production evoked by glycine was compared with that following induction by various concentrations of cefoxitin. The amount of beta-lactamase produced under conditions of maximum glycine enhancement was higher than that produced by cultures fully induced by cefoxitin. Other differences in the characteristics of the enhancement of beta-lactamase production by glycine and beta-lactamase induced by cefoxitin suggested that the mechanisms of the 2 phenomena were different. The significance of this finding in terms of the interpretation of in vitro antimicrobial sensitivity tests with beta-lactam antibiotics is discussed.     

The prevalence of extended-spectrum beta-lactamase in environmental isolates of Enterobacter

The incidence of extended-spectrum beta-lactamase (ESBL)-producing strains and multidrug-resistant strains of Enterobacter spp. isolated from the 1312 km long river Narmada was investigated. Out of the 57 isolates of Enterobacter, 73.68% were found to be ESBL producers including the isolates of E. taylorae and isolates of E. agglomerans, which have been characterized for the first time. All the isolates were found susceptible to the antibiotic imipenem. AmpC gene was found in all the Enterobacter strains tested. AmpC beta-lactamase-producing bacterial pathogens may cause major therapeutic failure if not detected and reported in time. It was seen that these enzymes are mainly chromosomally mediated along with several non-AmpC beta-lactamase.     

SHV-type extended-spectrum beta-lactamase production is associated with Reduced cefepime susceptibility in Enterobacter cloacae

Cefepime is a potentially useful antibiotic for treatment of infections with Enterobacter cloacae. However, in our institution the MIC(90) for E. cloacae bloodstream isolates is 16 microg/ml. PCR amplification of bla genes revealed that one-third (15/45) of E. cloacae bloodstream isolates produced SHV-type extended-spectrum beta-lactamases (ESBLs) in addition to hyperproduction of AmpC-type beta-lactamases. The majority (11/15) of ESBL producers also produced the TEM-1 beta-lactamase. The SHV types included SHV-2, -5, -7, -12, -14, and -30. All but two of the ESBL-producing E. cloacae isolates, but none of the non-ESBL-producing strains, had MICs of cefepime of >or=2 microg/ml. The MIC(90) for cefepime for ESBL-producing strains was 64 mug/ml, while for non-ESBL producers it was 0.5 microg/ml. Using current Clinical and Laboratory Standards Institute breakpoints for cefepime, two thirds (10/15) of ESBL-producing isolates would have been regarded as susceptible to cefepime. Phenotypic ESBL detection methods were generally unreliable with these E. cloacae isolates. Based on these results, pharmacokinetic, pharmacodynamic, and clinical reevaluation of cefepime breakpoints for E. cloacae may be prudent.     

Outbreak of infection caused by Enterobacter cloacae producing the novel VEB-3 beta-lactamase in China

Over a 4-month period from November 2002 to February 2003, 27 ceftazidime-resistant or cefotaxime-resistant nonrepetitive Enterobacter cloacae isolates were collected from 27 patients hospitalized at HuaShan Hospital, Shanghai, People's Republic of China. The Etest did not detect extended-spectrum beta-lactamases (ESBLs) in those 27 isolates; however, screening by the NCCLS ESBL disk test and confirmatory tests detected ESBLs in 4 of 27 isolates and PCR detected ESBLs in 23 of 27 isolates. The majority of ESBL producers exhibited the same repetitive extragenic palindromic PCR pattern but harbored different ESBL genes. CTX-M-3 was the most prevalent ESBL in our study. Interestingly, 12 clonally related E. cloacae isolates possessed a novel bla(VEB)-type beta-lactamase, bla(VEB-3). Bla(VEB-3) was encoded by the chromosome and was located in an integron. Nine of the 12 isolates harbored both the bla(VEB-3) and the bla(CTX-M-3)-like ESBLs. This is the first report of a VEB-1-like ESBL in China and the first report of the simultaneous presence of VEB-1 and CTX-M-3-like ESBLs in an isolate.     

Molecular typing and characterization of extended-spectrum TEM, SHV and CTX-M beta-lactamases in clinical isolates of Enterobacter cloacae

Sixty-one non-repetitive Enterobacter cloacae ESBL producers were collected at the Amiens University Hospital in France. Eight beta-lactam resistance phenotypes (a-h) and three aminoglycoside resistance phenotypes (i-k) were identified among these isolates, and 32 different pulsotypes were observed. Of these 61 isolates, 37 were sequenced and found to harbor beta-lactamases with a pI of 5.9 (TEM-4), 6.5 (TEM-24), 7.8 (SHV-4), 8.2 (SHV-12), 8.4 (CTX-M-1) and 8.0 (CTX-M-9). Four imipenem-resistant ESBL-producing E. cloacae isolates did not express the 38kDa OMP, indicating that this resistance is associated with porin deficiency.     

In vitro selective concentrations of cefepime and ceftazidime for AmpC β-lactamase hyperproducer Enterobacter cloacae variants

This study aimed to compare the selective concentrations of cefepime and ceftazidime on Enterobacter cloacae. A mixed culture of a wild-type ceftazidime/cefepime-susceptible (4±10⁷ CFU/mL) strain and an ampC derepressed Enterobacter cloacae (10⁵ CFU/mL) strain (relative proportions 99.75% and 0.25%) was challenged for 4 h with different antibiotic concentrations of ceftazidime and cefepime (0.03–4096 mg/L), and then transferred to drug-free medium. The proportion of wild-type versus derepressed population was evaluated after 24 h.
Ceftazidime and cefepime selected the derepressed variant at concentrations ranging from 1 to 4096 and from 0.12 to 16 mg/L respectively.
These results suggest that serum concentrations attainable with a 2 g/8 h cefepime dosage may be able to suppress the emergence of derepressed ampC mutants.     

In vitro selective concentrations of cefepime and ceftazidime for AmpC β-lactamase hyperproducer Enterobacter cloacae variants

Methods: A mixed culture of a wild-type ceftazidime/cefepime-susceptible (4×10 ⁷ CFU/mL) strain and an ampC derepressed Enterobacter cloacae (10 ⁵ CFU/mL) strain (relative proportions 99.75% and 0.25%) was challenged for 4 h with different antibiotic concentrations of ceftazidime and cefepime (0.03–4096 mg/L), and then transferred to drug-free medium. The proportion of wild-type versus derepressed population was evaluated after 24 h.
Results: Ceftazidime and cefepime selected the derepressed variant at concentrations ranging from 1 to 4096 and from 0.12 to 16 mg/L respectively.
Conclusions: These results suggest that serum concentrations attainable with a 2 g/8 h cefepime dosage may be able to suppress the emergence of derepressed ampC mutants.