BACKGROUND: We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-beta-lactamase (MBL)-producing Pseudomonas spp. METHODS: A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum beta-lactamase [GES]-5, 9 New Delhi metallo-beta-lactamase [NDM]-1, 5 Verona integron-encoded metallo-beta-lactamase [VIM]-2, 3 imipenem-hydrolyzing beta-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain. RESULTS: Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively. CONCLUSIONS: Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.
Bacterial Proteins/antagonists & inhibitors/*metabolism ; Boronic Acids/chemistry/pharmacology ; Disk Diffusion Antimicrobial Tests/*methods ; Edetic Acid/chemistry/pharmacology ; Enterobacteriaceae/drug effects/*enzymology ; Enterobacteriaceae Infections/diagnosis ; Humans ; Pseudomonas/drug effects/*enzymology ; Pseudomonas Infections/diagnosis ; Sensitivity and Specificity ; beta-Lactamases/chemistry/*metabolism

BACKGROUND: Our study was to investigate the prevalence of carbapenemase genes in strains of Enterobacteriaceae species exhibiting decreased susceptibility to carbapenems in our hospital. METHODS: The carbapenemase producing Enterobacteriaceae species were confirmed by modified Hodge test (MHT) and EDTA-disc synergy test which indicating the production of class B carbapenemases. PCR and sequencing analysis were used to identify the drug-resistant genes. DNA fingerprinting based on enterobacterial repetitive intergenic consensus (ERIC)-PCR was applied to investigate the homology of Enterobacteriaceae species. RESULTS: From a collection of 1,472 Enterobacteriaceae species, 18 isolates with decreased susceptibility to carbapenem treatment were identified and 9 of which were positive by MHT, and 6 of which produced class B carbapenemases. PCR and sequencing analysis of the 18 isolates revealed 4 different carbapenemase genes (blaIMP-8, blaoxa-1, blaIMP-26, and blaoxa-47) in 10 isolates, with the blaIMP-8 and blaoxa-1 genes being the most common (60-70% prevalence). ERIC-PCR showed 5, 2, and 2 unique genotypes for Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae, respectively. Three E. coli strains isolated from different patients from the urologic surgery department exhibited the same DNA banding pattern, suggesting a possible clonal dissemination. Majority (17/18) of the carbapenem-unsusceptible Enterobacteriaceae species isolates was obtained from the surgery department of our hospital. CONCLUSIONS: The main carbapenemase genes of Enterobacteriaceae species in our hospital were blaIMP-8 and blaoxa-1. Prevalence of carbapenem resistance may be existed in surgery department and infection control should be taken for preventing further dissemination of drug-resistant strains.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/*genetics ; Carbapenems/*pharmacology ; China ; DNA Fingerprinting ; Drug Resistance, Bacterial/drug effects/genetics ; Enterobacteriaceae/*drug effects/*enzymology/isolation & purification ; Enterobacteriaceae Infections/microbiology ; Genotype ; Hospitals, University ; Humans ; Microbial Sensitivity Tests ; Sequence Analysis, DNA ; beta-Lactamases/*genetics

BACKGROUND: The modified Hodge test (MHT) was designed to detect carbapenemase-producing Enterobacteriaceae (CPE). This study evaluated variables to improve the performance of MHT. METHODS: Carbapenem-resistant Enterobacteriaceae isolated from November 2010 to March 2013 at the Asan Medical Center, were evaluated, including 33 metallo-beta-lactamase (MBL) producers and 103 non-CPEs. MHT was performed by using two carbapenem disks (ertapenem and meropenem; Becton Dickinson, USA), three media (Mueller-Hinton agar (MHA), MacConkey agar (MAC), and zinc-enriched MHA), and two inoculums (0.5-McFarland [McF] suspension and a 10-fold dilution of it.) PCR was performed to detect beta-lactamase genes of the MBL, AmpC, and CTX-M types. RESULTS: The sensitivity of MHT for detecting New Delhi metallo-beta-lactamase (NDM) producers was highest using ertapenem and 0.5-McF, 52.0% on MHA and 68.0% on MAC, respectively. NDM-producing Klebsiella pneumoniae (NDMKP) were detected with higher sensitivity on MAC (78.6%) vs. MHA (28.6%) (P=0.016), but VIM-producing Enterobacter, Citrobacter, and Serratia were detected with higher sensitivity on MHA (78.5%) vs. MAC (14.3%) (P=0.004). MBL producers were consistently identified with lower sensitivity using meropenem vs. ertapenem, 39.4% vs. 60.6% (P=0.0156), respectively. The effects of zinc and inoculum size were insignificant. Enterobacter aerogenes producing unspecified AmpC frequently demonstrated false positives, 66.7% with ertapenem and 22.2% with meropenem. CONCLUSIONS: The MHT should be adjusted for the local distribution of species and the carbapenemase type of MBL producers. MAC and ertapenem are preferable for assessing NDMKP, but MHA is better for VIM. Laboratory physicians should be aware of the limited sensitivity of MHT and its relatively high false-positive rate.
Anti-Bacterial Agents/pharmacology ; Carbapenems/pharmacology ; DNA, Bacterial/genetics/metabolism ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Bacterial ; Enterobacteriaceae/drug effects/*enzymology/isolation & purification ; Enterobacteriaceae Infections/microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Phenotype ; beta-Lactamases/genetics/*metabolism

With the abuse of antimicrobial agents in developing countries, increasing number of carbapenem-resistant Enterobacteriaceae (CRE) attracted considerable public concern. A retrospective study was conducted based on 242 CRE strains from a tertiary hospital in Hangzhou, China to investigate prevalence and drug resistance characteristics of CRE in southeast China. Bacterial species were identified. Antimicrobial susceptibility was examined by broth microdilution method or epsilometer test. Resistant β-lactamase genes were identified by polymerase chain reaction and sequencing. Genotypes were investigated by phylogenetic analysis. Klebsiella pneumoniae and Escherichia coli were the most prevalent types of species, with occurrence in 71.9% and 21.9% of the strains, respectively. All strains exhibited high resistance (> 70%) against β-lactam antibiotics, ciprofloxacin, trimethoprim-sulfamethoxazole, and nitrofurantoin but exhibited low resistance against tigecycline (0.8%) and minocycline (8.3%). A total of 123 strains harbored more than two kinds of β-lactamase genes. bla, bla, bla, and bla were the predominant genotypes, with detection rates of 60.3%, 61.6%, 43.4%, and 16.5%, respectively, and were highly identical with reference sequences in different countries, indicating potential horizontal dissemination. IMP-4 was the most frequent class B metallo-lactamases in this study. In conclusion, continuous surveillance and effective prevention should be emphasized to reduce spread of CRE.
Anti-Bacterial Agents ; therapeutic use ; Carbapenem-Resistant Enterobacteriaceae ; drug effects ; enzymology ; genetics ; China ; epidemiology ; Enterobacteriaceae Infections ; epidemiology ; microbiology ; Genotype ; Humans ; Microbial Sensitivity Tests ; Phylogeny ; Prevalence ; Retrospective Studies ; beta-Lactam Resistance ; beta-Lactamases ; genetics

We investigated the occurrence and genetic basis of AmpC beta-lactamase (AmpC)-mediated antibiotic resistance, by examining Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a university hospital, from 2007 to 2010. The ampC genes were detected by multiplex AmpC PCR, and AmpC-positive strains were subjected to DNA sequencing. Extended-spectrum beta-lactamase (ESBL) production was assessed using the ESBL disk test based on the utilization of boronic acid. Carbapenem-resistant isolates were further investigated by the modified Hodge test, a carbapenemase inhibition test and SDS-PAGE experiments. AmpC expression was detected in 1.6% of E. coli (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, 7.2% of K. pneumoniae (39 DHA-1, 45 CMY-2, and 1 CMY-1) isolates, and 2.5% of P. mirabilis (8 CMY-2 and 1 CMY-1) isolates. Of the 198 acquired AmpC producers, 58 isolates (29.3%) also produced an ESBL enzyme. Among the acquired AmpC-producing K. pneumoniae isolates, the minimum inhibitory concentration (MIC) MIC50/MIC90 values for cefoxitin, cefotaxime, cefepime, imipenem, and meropenem were >32/>32, 16/>32, 1/16, 0.25/0.5, and <0.125/0.125 microg/mL, respectively. The MIC values for carbapenem were > or =2 microg/mL for 2 K. pneumoniae isolates, both of which carried the blaDHA-1 gene with a loss of OmpK36 expression, but were negative for carbapenemase production. The acquisition of AmpC-mediated resistance in K. pneumoniae isolates increased, as did the proportion of AmpC and ESBL co-producers among the hospital isolates. The accurate identification of isolates producing AmpCs and ESBLs may aid in infection control and will assist physicians in selecting an appropriate antibiotic regimen.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics ; DNA, Bacterial/genetics ; Enterobacteriaceae Infections/*epidemiology/*microbiology ; Escherichia coli/drug effects/enzymology/isolation & purification ; Hospitals, University/statistics & numerical data ; Humans ; Klebsiella pneumoniae/drug effects/enzymology/isolation & purification/*physiology ; Microbial Sensitivity Tests ; Multiplex Polymerase Chain Reaction ; Proteus mirabilis/drug effects/enzymology/isolation & purification ; Republic of Korea/epidemiology ; beta-Lactamases/*genetics

OBJECTIVE: To analyze the main pathogens of infection after the liver transplantation and their antibiotic resistant patterns. METHODS: The main pathogens of infection after the liver transplantation were retrospectively analyzed. Using 3-dimensional tests, ESBLs (extended-spectrum beta-lactamase), and AmpC were detected among the Gram negative bacilli. beta-Lactamase and Van gene in Enterococcus were determined by the standard agar dilution susceptibility tests and Nitrocefin respectively. RESULTS: The main infected strains were Enterococcus faecalis (15.0%), Enterobacter cloacae (13.9%), fungus (13.3%), and Escherichia coli (10.7%) after the liver transplantation. Among them, 32.4% of Enterobacter cloacae and 36.8% of Escherichia coli produced ESBLs; 33.8% of Enterobacter cloacae and 10.5% of Escherichia coli. produced AmpC beta-lactamases. The detectable rate of VanA gene in Enterococcusfaecalis and Enterococcus faecium was 7.5% and 11.1%; VanB was 3.8% and 7.4%; VanC was 1.3% and 0, respectively. CONCLUSION The infection mainly occurs in the intestinal tract after the liver transplantation. The production of ESBLs and AmpC beta-lactamases is the main mechanism of antibiotic resistance. The increased detectable rate of vancomycin-resistant Enterococcus should be paid attention to.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Drug Resistance, Bacterial ; genetics ; Enterobacteriaceae ; drug effects ; enzymology ; isolation & purification ; Enterobacteriaceae Infections ; microbiology ; Female ; Humans ; Infant ; Liver Cirrhosis ; surgery ; Liver Neoplasms ; surgery ; Liver Transplantation ; adverse effects ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Postoperative Complications ; microbiology ; Retrospective Studies ; Vancomycin Resistance ; genetics

BACKGROUND: The emergence of carbapenemase-producing Enterobacteriaceae (CPE) represents a major clinical problem because these bacteria are resistant to most antibiotics. CPE remain relatively uncommon in Korea. We report the prevalence, clinical characteristics, and molecular epidemiology of CPE isolates collected from five university hospitals in Korea. METHODS: Between January and December 2015, 393 non-duplicated isolates that were nonsusceptible to ertapenem were analyzed. Production of carbapenemase, extended-spectrum β-lactamase, and AmpC β-lactamase was determined by genotypic tests. Antimicrobial susceptibility profiles were determined by using an Etest. Clonality of Klebsiella pneumoniae carbapenemase (KPC)-2-producing and oxacillinase (OXA)-232-producing Klebsiella pneumoniae isolates was determined by pulsed-field gel electrophoresis (PFGE). RESULTS: Of the 393 isolates tested, 79 (20.1%) were CPE. Of these 79 isolates, 47 (59.5%) harbored the bla(OXA-232) gene while the remaining isolates carried genes bla(KPC-2) (n=27), bla(IMP-1) (n=4), and bla(NDM-1) (n=1). Among the 24 KPC-2 K. pneumoniae isolates from hospital B, 100% were resistant to carbapenems, 8% to colistin, and 0% to tigecycline. Among the 45 OXA-232 K. pneumoniae at hospital C, 95% were resistant to ertapenem, 68% to imipenem, 95% to meropenem, 10% to colistin, and 24% to tigecycline. PFGE analysis revealed a unique pattern for KPC-2 K. pneumoniae and identified 30 isolates belonging to the dominant pulsotypes (PT)1 and PT2 among 41 OXA-232 K. pneumoniae isolates. CONCLUSIONS: CPE strains are present in Korea, with the majority of K. pneumoniae isolates producing OXA-232 and KPC-2. The prevalence and predominant genotypes of CPE show hospital-specific differences.
Aged ; Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics/metabolism ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Enterobacteriaceae/drug effects/*enzymology/isolation & purification ; Enterobacteriaceae Infections/diagnosis/epidemiology/*microbiology ; Female ; Genotype ; Hospitals ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Prevalence ; Republic of Korea/epidemiology ; beta-Lactamases/*genetics/metabolism

Production of extended-spectrum beta-lactamase (ESBL) is one of the most important resistance mechanisms that hamper the antimicrobial treatment of infections caused by Enterobacteriaceae. ESBLs are classified into several groups according to their amino-acid sequence homology. While TEM and SHV enzymes were the most common ESBLs in the 1990s, CTX-M enzymes have spread rapidly among Enterobacteriaceae in the past decade. In addition, some epidemiological studies showed that organisms producing CTX-M enzymes had become increasingly prevalent in the community setting in certain areas in the world. Several novel enzymes with hydrolyzing activity against oxyimino-cephalosporins, albeit with additional enzymatic characteristics different from those of original TEM and SHV ESBLs (e.g., inhibitor-resistance), have been discovered and pose a problem on the definition of ESBLs. Although several methods to detect the production of ESBL are available in clinical laboratories, existence of other factors contributing resistance against beta-lactams, e.g., inducible production of Amp-C beta-lactamase by some species of Enterobacteriaceae, or inhibitor-resistance in some ESBLs may hinder the detection of ESBLs with these methods. Carbapenems are stable against hydrolyzing activity of ESBLs and are regarded as the drug of choice for the treatment of infections caused by ESBL-producing Enterobacteriaceae. Although several other antimicrobial agents, such as fluoroquinolones and cephamycins, may have some role in the treatment of mild infections due to those organisms, clinical data that warrant the use of antimicrobial agents other than carbapenems in the treatment of serious infections due to those organisms are scarce for now.
Anti-Bacterial Agents/*pharmacology/therapeutic use ; Carbapenems/pharmacology/therapeutic use ; Disk Diffusion Antimicrobial Tests ; Enterobacteriaceae/drug effects/*enzymology/genetics ; Enterobacteriaceae Infections/*drug therapy/microbiology ; Fluoroquinolones/pharmacology/therapeutic use ; Humans ; Microbial Sensitivity Tests/methods ; beta-Lactamases/*biosynthesis/metabolism ; beta-Lactams/*pharmacology/therapeutic use

BACKGROUND: We investigated the rates of fecal transmission of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) and carbapenem-resistant Enterobacteriaceae (CRE) among patients admitted to intensive care units (ICUs). METHODS: From June to August 2012, rectal cultures were acquired from all patients at ICU admission. For patients not carrying ESBL-E or CRE at admission, follow-up cultures were performed to detect acquisition. A chromogenic assay was used to screen for ESBL-E and CRE. Bacterial species identification and antibiotic susceptibility tests were performed using the Vitek 2 system (bioMerieux, France). ESBL genotypes were determined by PCR, and clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis. RESULTS: Out of 347 ICU admissions, 98 patients were found to be carriers of ESBL-E (28.2%, 98/347). Follow-up cultures were acquired from 91 of the patients who tested negative for ESBL-E at admission; the acquisition rate in this group was 12.1% (11/91), although none was a nosocomial transmission. For CRE, the prevalence of fecal carriage was 0.3% (1/347), and the acquisition rate was 2.9% (4/140). None of the CRE isolates were carbapenemase-producers. CONCLUSIONS: The high prevalence of ESBL-E carriage on admission (28.2%), coupled with rare nosocomial transmission and the very low carriage rate of CRE (0.3%), challenge the routine use of active surveillance in non-epidemic settings. Nevertheless, passive surveillance measures, such as rapid and accurate screening of clinical specimens, will be critical for controlling the spread of CRE.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*metabolism ; Carbapenems/*pharmacology ; Carrier State/epidemiology ; Cross Infection/epidemiology/*transmission ; DNA, Bacterial/analysis ; Drug Resistance, Bacterial/drug effects ; Electrophoresis, Gel, Pulsed-Field ; Enterobacteriaceae/enzymology/genetics/*physiology ; Enterobacteriaceae Infections/epidemiology/*transmission ; Feces/*microbiology ; Genotype ; Humans ; Intensive Care Units ; Polymerase Chain Reaction ; Prevalence ; Republic of Korea/epidemiology ; beta-Lactamases/*metabolism

OBJECTIVETo analyze the distribution of Enterobacteriaceae isolated from West China Hospital, investigate the antibiotic resistance profile of Enterobacteriaceae with decreased susceptibility to carbapenems and explore the molecular mechanism.

METHODSForty-five Enterobacteriaceae strains resistant or with reduced susceptibility to carbapenems were isolated from patients in West China Hospital. The antimicrobial susceptibility and carbapenemase-producing phenotypes of the bacteria were examined and specific PCR were performed to determine the molecular mechanism.

RESULTSOf the 45 isolates, 17, 21 and 36 were resistant or intermediate strains to imipenem, meropenem and ertapenem, respectively. The majority of these isolates showed resistance to cephalosporins. The modified Hodge test resulted in the highest positivity rate (77.8%), followed by EDTA disc test (57.8%) and PBA disc test (22.2%). BlaTEM, blaSHV and blaCTX-M were detected in 60.0%, 53.3% and 15.6% of these strains with reduced susceptibility. The rate of strains carrying 2 or more genes was 44.4%, and the detection rate of blaIMP was 48.9%. BlaKPC was identified in 4 (8.9%) high-level resistant strains and confirmed to locate on the plasmid.

CONCLUSIONProduction of carbapenemase contributes to reduced susceptibility of carbapenems in Enterobacteriaceae. The presence of blaKPC, MBL and ESBL, and their possible combinations can be the main factor contributing to carbapenem resistance or reduced susceptibility in Enterobacteriaceae. The KPC-2 carbapenemase gene located on the plasmids we found in this study can cause potential horizontal transmission across strains.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Carbapenems ; pharmacology ; Cephalosporins ; pharmacology ; Enterobacteriaceae ; drug effects ; enzymology ; genetics ; Gene Amplification ; Imipenem ; pharmacology ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Thienamycins ; pharmacology ; beta-Lactam Resistance ; beta-Lactamases ; genetics ; metabolism ; beta-Lactams ; pharmacology