Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in diabetic foot infections

Aims: Diabetic foot lesions are a major medical, social, and economic problem and are the leading cause of hospitalization for patients with diabetes, worldwide. ESBL-producing bacteria may not be detectable by routine disc diffusion susceptibility test, leading to inappropriate use of antibiotics and treatment failure. There is not much information on ESBL-producing organisms causing diabetic foot infection. An attempt was therefore made to study the ESBL-producing Escherichia coli and Klebsiella pneumoniae in diabetic foot patients with type 2 diabetes mellitus. Materials and Methods: A total of 134 isolates of E. coli and K. pneumoniae were obtained from tissue, pus swab, and wound swab samples from diabetic foot ulcers submitted for routine microbiological analysis during the period January to December 2005 from patients with diabetic foot infections who had type 2 diabetes mellitus, attending S. L. Raheja Hospital. The above isolates were tested for antimicrobial susceptibility by disc diffusion technique according to clinical and laboratory standards institute (CLSI) guidelines. The screening for ESBL production was done by phenotypic confirmatory test using ceftazidime disc in the presence and absence of clavulanic acid as recommended by CLSI. Results: Among the 134 isolates, 54 (40.29%) were E. coli and 80 (59.70%) were K. pneumoniae; among which, ESBL production was detected in 31 (23.13%) isolates. Of these 31, 15 (48.38%) were E. coli and 16 (51.61%) were K. pneumoniae. All the ESBL-producing isolates were found to be 100% sensitive to carbapenem (imipenem and meropenem). Mortality was found to be 3.22%, the cause of death being septicemia leading to multiple organ failure. Conclusions: The prevalence of ESBLs among members of Enterobacteriaceae constitutes a serious threat to the current beta-lactam therapy, leading to treatment failure and consequent escalation of costs. There is an urgent need to emphasize rational use of drugs to minimize the misuse of available antimicrobials.     

Cefoxitin resistance mediated by loss of a porin in clinical strains of Klebsiella pneumoniae and Escherichia coli

Purpose: Porins are outer membrane protein (OMP) that form water filled channels that permit the diffusion of small hydrophilic solutes like β-lactam antibiotics across the outer membrane. Two major porins that facilitate diffusion of antimicrobials have been described in Klebsiella spp. and Escherichia coli. The present study was carried out to examine the role of porins among Extended Spectrum β-Lactamase (ESBL) and AmpC β-Lactamase positive strains of Klebsiella spp. and E.coli. Methods: Preparation of OMP from phenotypically characterized clinical isolates K.pneumoniae and E.coli and the separation of the proteins by sodium dodecyl sulfate – polyacrylamide gel electrophoresis were performed as per a previously described procedure. Results: OMP analysis revealed that cefoxitin and ceftazidime resistance was mediated by loss of a porin Omp K35 in the isolates of K.pneumoniae and E.coli. Conclusions: Loss of porin mediated resistance mechanism against cefoxitin was observed among the multidrug resistant K.pneumoniae and E.coli.     

Extended-spectrum beta-lactamases in ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae isolates in Turkish hospitals

PURPOSE: To study the prevalence of TEM-, SHV- and GES-type beta -lactamases among Escherichia coli and Klebsiella pneumoniae strains having ceftazidime MICs higher than 2 mg/L. METHODS: A total of 63 E. coli and 41 K. pneumoniae isolated from five different university hospitals were studied for the existence of TEM-, SHV- and GES-type beta -lactamases. Susceptibility tests were carried out according to the criteria of National Committee for Clinical Laboratory Standards. MICs were obtained by agar dilution method. Existence of extended-spectrum beta -lactamases (ESBLs) were assessed by double-disc synergy test (DDST). Existence of the above-mentioned beta -lactamase genes were studied both by PCR with specific oligonucleotide primers and isoelectric focusing methods. RESULTS: None of the isolates were carbapenem-resistant. DDSTs were positive in 50 (79.3%) and 33 (80.5%) of E. coli and K. pneumoniae , respectively. TEM gene was detected in 41 (65.1%) and 19 (46.3%), whereas SHV gene in 18 (28.6%) and 20 (48.8%) of E. coli and K. pneumoniae strains, respectively. GES genes were not detected. CONCLUSIONS: TEM and SHV genes are highly prevalent among ESBL-producing E. coli and K. pneumoniae , whereas GES-type ESBLs are absent and found not to be responsible of ceftazidime resistance in Turkish hospitals.     

儿童感染肺炎克雷伯菌产AmpC酶和ESBLs的检测及耐药性分析

目的了解广州地区儿童感染肺炎克雷伯菌产质粒介导的AmpC酶和超广谱β-内酰胺酶（ESBLs）的情况及其耐药特征,为临床合理用药提供参考依据。方法采用标准纸片扩散法检测ESBLs,头孢西丁三维试验法检测AmpC酶,K—B纸片法测定肺炎克雷伯菌对抗菌药物的敏感性。结果共检出248株肺炎克雷伯菌,其中46株产AmpC酶,阳性率为18．5％;157株产ESBLs,阳性率为63．3％;同时产AmpC酶和ESBLs菌株阳性率为18．1％。产AmpC酶肺炎克雷伯菌对第三代头孢菌素高度耐药。耐药率达80％～100％;对头孢吡肟、含酶抑制剂复合药的耐药率也在56．5％～93．5％之间：但对环丙沙星、阿米卡星的耐药率则在30％以下,对亚胺培南全部敏感。产ESBLs菌株对头孢菌素、含酶抑制剂复合药的耐药率也较高,在50％-91．7％之间,但对阿米卡星、环丙沙星、亚胺培南仍高度敏感。ESBLs阴性肺炎克雷伯菌对所测抗生素的敏感率均在81．2％以上。产酶菌株耐药率明显比非产酶菌株高。结论广州地区儿童感染肺炎克雷伯菌产ESBLs和AmpC酶的状况已十分突出：产酶菌株对常用抗生素的耐药率较高;碳青霉烯类抗生素可作为治疗产AmpC酶和／或ESBLs肺炎克雷伯菌感染的经验用药。     

Prevalence of plasmid-mediated AmpC beta-lactamases in Escherichia coli and Klebsiella pneumoniae in Korea

Cefoxitin-resistant Escherichia coli and Klebsiella pneumoniae are relatively prevalent in Korea, suggesting dissemination of plasmid-mediated AmpC beta-lactamases. In this study, 238 isolates of cefoxitin-resistant E. coli and K. pneumoniae (not including subspecies ozaenae and rhinoscleromatis) were collected in 2003 from 16 Korean hospitals. The prevalence of plasmid-mediated AmpC beta-lactamases was determined by PCR. The AmpC gene alleles detected in E. coli and K. pneumoniae were bla(DHA-1), 10 (8.6%) and 93 (76.2%); bla(CMY-1)-like, 14 (12.1%) and 2 (1.6%); and bla(CMY-2)-like, 38 (32.7%) and 1 (0.8%) isolates, respectively. The genes identified were bla(DHA-1), bla(CMY10)-like, and bla(CMY-2)-like, and a new variant, bla(CMY-18). Plasmidmediated AmpC gene allele-positive isolates were present both in large city and in small province hospitals, as well as in isolates from outpatients. The proportions of plasmid-mediated AmpC gene-positive isolates were similar in both expanded spectrum beta-lactamase (ESBL)-producing and -nonproducing isolates. In conclusion, DHA-1, CMY-2-like, and CMY-10-like plasmid-mediated AmpC beta-lactamase-producing K. pneumoniae and E. coli isolates are widely disseminated in both large city and small province hospitals. Absence of bla(CMY-1) and detection of a novel variant of bla(CMY-2), bla(CMY-18), indicate continued evolution of the prototype genes. Similar proportions of plasmid-mediated AmpC gene-positive isolates in both ESBL-producing and -nonproducing isolates suggest unhindered future spread of these resistances.     

New method for laboratory detection of AmpC beta-lactamases in Escherichia coli and Klebsiella pneumoniae

A new cefoxitin-agar medium (CAM)-based assay was compared to the previously published modified three-dimensional (M3D) assay for the detection of AmpC production in Escherichia coli and Klebsiella pneumoniae. Clinical isolates of cefoxitin-resistant E. coli (n = 5) and K. pneumoniae (n = 7) and multiple control strains with and without AmpC enzymes were tested by both methods. The CAM method with 4 microg of cefoxitin/ml was equivalent to the M3D method for detecting AmpC production in E. coli and K. pneumoniae. This new method is easier to perform and interpret and allows for testing of multiple isolates on a single plate.     

Detection of Klebsiella pneumoniae and Escherichia coli strains producing extended-spectrum beta-lactamases.

Plasmids encoding extended-spectrum beta-lactamases of the TEM, SHV, and AmpC families were introduced into common Escherichia coli and Klebsiella pneumoniae hosts to create a homogeneous panel for evaluating the abilities of five test systems to detect resistance to eight beta-lactam antibiotics. Although MICs, as determined by agar dilution or E test strips, were increased and disk diffusion zone diameters were diminished, breakpoints for resistance were often not reached, and neither approach was sensitive in detecting resistance to oxyimino-beta-lactams. The MicroScan 18-h microdilution or Vitek rapid automated procedures were similarly insensitive. Ceftazidime was the best single test antibiotic for detecting extended-spectrum beta-lactamase production. beta-Lactamases TEM-7 and TEM-12 were particularly difficult to detect. Because of such difficulties, the prevalence of extended-spectrum beta-lactamases is likely to be greater than is currently appreciated.     

Aminoglycoside resistance in clinical Escherichia coli and Klebsiella pneumoniae isolates from Western Norway

Resistance to gentamicin in Escherichia coli from blood culture has shown an increase over the past decade in Norway. This study was done to investigate aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae in Western Norway. The material included 49 blood culture isolates which had shown aminoglycoside resistance collected during 2000-2009. To investigate co-resistance to alternative antibiotics and dynamics involved in aminoglycoside resistance 67 isolates (mostly from urine) exhibiting resistance to both aminoglycosides and extended spectrum beta-lactam antibiotics were also included. MIC values were obtained for amikacin, gentamicin, kanamycin, netilmicin, streptomycin and tobramycin and all isolates were screened using PCR for aac(3)-II and aac(6')-Ib, encoding aminoglycoside modifying enzymes. Resistance to ≥3 aminoglycosides was found in 92% of the isolates and 60.3% showed resistance to gentamicin, netilmicin, tobramycin and kanamycin. Amikacin resistance was low. Co-resistance to ciprofloxacin was found in 88% of the isolates with gentamicin resistance. aac(3)-IIa/c was found in 79.3% and aac(6')-Ib in 37.9% of the isolates and 28.4% harboured both genes. aac(6')-Ib-cr, possibly contributing to ciprofloxacin resistance was found mostly in extended spectrum beta-lactamase producers. The aminoglycoside resistance patterns indicate co-existence of multiple resistance mechanisms. The use of ciprofloxacin and third generation cephalosporins is likely to have contributed to the increase in aminoglycoside resistance in Norway.     

Identification of extended-spectrum, AmpC, and carbapenem- hydrolyzing beta-lactamases in Escherichia coli and Klebsiella pneumoniae by disk tests

Antibiotic disks with and without clavulanic acid, 3-aminophenylboronic acid, or EDTA were tested with a set of 55 Klebsiella pneumoniae and Escherichia coli strains producing well-characterized extended-spectrum, AmpC, or carbapenem-hydrolyzing beta-lactamases. A relatively simple scheme was devised for distinguishing beta-lactamase types in clinical isolates with or without intact outer membrane porins.     

Emergence and outbreaks of CTX-M beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae strains in a Tunisian hospital

Sixty-two isolates of Enterobacteriaceae (35 Escherichia coli and 27 Klebsiella pneumoniae isolates) producing CTX-M-type beta-lactamases were collected between March 2000 and June 2003 in different wards of Charles Nicolle Hospital in Tunis (Tunisia). Sequencing identified the bla(CTX-M-15) determinant in 55 isolates and bla(CTX-M-16) in 7 isolates. The CTX-M-15-producing strains were isolated in several wards and consisted mainly of two successive clonal groups of E. coli and a major clonal group of K. pneumoniae. The second clonal group of E. coli belonged to phylogenetic group B2 and harbored more virulence factors than the first clonal group. Among the 22 transconjugants or electroporants obtained with selected E. coli and K. pneumoniae CTX-M-15-producing strains, a predominant plasmid restriction pattern was obtained with 17 isolates. The four CTX-M-16-producing strains of E. coli yielded the same pulsed-field gel electrophoresis (PFGE) pattern, while the three CTX-M-16-producing strains of K. pneumoniae yielded two different PFGE patterns. All of the CTX-M-16-producing isolates were recovered in the pediatric ward and had the same plasmid restriction pattern.     

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.     

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.     

Emergence and wide dissemination of CTX-M-type ESBLs, and CMY-2- and DHA-1-type AmpC beta-lactamases in Korean respiratory isolates of Klebsiella pneumoniae

Respiratory isolates of Klebsiella pneumoniae in Korea during 2002-2003 were studied to determine the prevalence and types of extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated AmpC beta-lactamases (PABLs). ESBL-production was tested by double-disk synergy, and genotypes of beta-lactamases were determined by PCR and sequencing. ESBLs were detected in 28.4% of 373 isolates, and the most prevalent types were SHV-12 (63 isolates) and CTX-M-14 (9 isolates). Forty of 75 ESBL-producers (53.5%) also had PABLs: 21 isolates with CMY-2-like, 17 with DHA-1-like. Pulsed-field gel electrophoresis showed 19 types and 25 of 74 isolates had an identical pattern, indicating nosocomial spread. Dissemination of ESBL- and PABL-producing K. pneumoniae strains in Korea is a particular concern, as it limits the choice of antimicrobial agents for treatment of infections.     

Beta-lactamase types and beta-lactam resistance ofEscherichia coli strains with chromosomally mediated ampicillin resistance

On the basis of isoelectric focusing six -lactamase types could be distinguished in ampicillin-resistant and ampicillin-sensitive strains ofEscherichia coli. More than 90% of the ampicillin-resistant strains produced the same -lactamase type. The serotypes found in a group of ampicillin-resistant urinary tract infection strains did not represent the distribution usually found in urinary tract isolates. Chromosomal ampicillin resistance was always associated with high cephalothin MIC values and increased resistance to other -lactam antibiotics of the cephalosporin group.     

KPC-2 producing Klebsiella pneumoniae and Escherichia coli co-infection in a catheter-related infection

We describe the first report of simultaneous blood infection with KPC-2 producing Klebsiella pneumoniae and Escherichia coli in a Brazilian patient. We highlight the importance of implementing efficient infection control measures to limit the spread of these phenotypes in a hospital setting.     

Characterization of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from hospital environments in Algeria

For detecting extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in the hospital environment, sedimentation plates were placed in the rooms of two hospitals. Three environmental isolates, two Klebsiella pneumoniae, and one Escherichia coli resistant to extended-spectrum cephalosporins with a phenotype indicating CTX-M enzymes production (the minimum inhibitory concentration [MIC] of cefotaxime was higher than the MIC of ceftazidime) were recovered. By PCR and sequencing, the three isolates were found to produce CTX-M-15. The bla(CTX-M-15) genes in the three isolates were transferred by conjugation. One K. pneumoniae environmental isolate showed an identical and unique RAPD profile with two other K. pneumoniae clinical isolates recovered from urinary tract infection from patients hospitalized in two different wards of another hospital.     

Carbapenem resistance among Escherichia coli and Klebsiella pneumoniae in a tertiary care hospital in south India

Carbapenem resistance among clinical isolates of Enterobacteriaceae, especially Escherichia coli and Klebsiella pneumoniae, is largely conferred by metallo-β-lactamase (MBL). Fifty-one non repetitive isolates of carbapenem-resistant (Meropenem and Imipenem) E. coli and K. pneumoniae were studied to determine the molecular mechanism for resistance. Presence of blaNDM and blaVIM was determined by polymerase chain reaction (PCR) and DNA sequencing. blaNDM was detected from majority of carbapenem-resistant K. pneumoniae (75%) and E. coli (66.6%). Timely detection and appropriate and aggressive infection control measures are required to control the spread of these bacteria in healthcare settings.     

Cell-wall-inhibiting antibiotic combinations with activity against multidrug-resistant Klebsiella pneumoniae and Escherichia coli

The increasing prevalence of hospital and community-acquired infections caused by multidrug-resistant (MDR) bacterial pathogens is rapidly limiting the options for effective antibiotic therapy. Systematic studies on combinations of already available antibiotics that could provide an effective treatment against MDR bacteria are needed. We tested combinations of antibiotics that target one important physiological function (peptidoglycan synthesis) at several steps, and studied Enterobacteriaceae (Klebsiella pneumoniae and Escherichia coli) for which multidrug resistance associated with ESBL-producing plasmids has become a major problem. To measure the effectiveness of antibiotics alone and in combination, we used checkerboard assays, static antibiotic concentration time-kill assays, and an improved in-vitro kinetic model that simulates human pharmacokinetics of multiple simultaneously administered antibiotics. The target strains included an MDR K. pneumoniae isolate responsible for a recent major hospital outbreak. A double combination (fosfomycin and aztreonam) and a triple combination (fosfomycin, aztreonam and mecillinam) were both highly effective in reducing bacterial populations in all assays, including the in vitro kinetic model. These combinations were effective even though each of the MDR strains was resistant to aztreonam alone. Our results provide an initial validation of the potential usefulness of a combination of antibiotics targeting peptidoglycan synthesis in the treatment of MDR Gram-negative bacteria. We suggest that a combination of fosfomycin with aztreonam could become a useful treatment option for such infections and should be further studied.