In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-beta-lactamase nonproducers.
Bacterial Proteins/*genetics/metabolism ; DNA Primers/metabolism ; Databases, Genetic ; Humans ; Klebsiella Infections/microbiology ; Klebsiella pneumoniae/genetics/isolation & purification/metabolism ; *Multiplex Polymerase Chain Reaction ; beta-Lactamases/*genetics/metabolism

The gldA gene coding glycerol dehydrogenase (GDH) was amplified by PCR with the genomic DNA of Klebsiella pneumoniae as the template. The gldA were inserted in pMD-18T to construct the recombinant cloning vector pMD-gldA. After the DNA sequence was determined, the gldA was subcloned into expression vector pET-32a (+) to construct the recombinant expression vector pET-32gldA. Upon lactose induction, soluble GDH was over-produced by E. coli BL21 (DE3) harboring the expression construct. Recombinant GDH purified by Ni-NTA affinity chromatography showed a single band about 54 kD on SDS-PAGE gel, and the specified activity was about 188 u/mg, the purification fold is 3 times and the activity recovery is 67.5%.
Chromatography, Affinity ; Cloning, Molecular ; DNA, Bacterial ; genetics ; Escherichia coli ; genetics ; isolation & purification ; metabolism ; Klebsiella pneumoniae ; enzymology ; genetics ; Sugar Alcohol Dehydrogenases ; biosynthesis ; genetics ; isolation & purification

Objective The aim of our study is to investigate the prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) and the genetic characteristics of the class 1 integron in CRKP on multi-drug resistance.Methods Clinical Klebsiella pneumoniae strains were collected from multiple departments of a hospital in central China. CRKP strains were identified among the isolates, and antibiotics susceptibility of CRKP strains was analyzed. The polymerase chain reaction (PCR) was adopted to amplify the class 1 integron variable area. The integron genetic structure was analyzed with enzyme digestion and DNA sequencing technology. The relation between class 1 integron and drug resistance was analyzed statistically.Results Totally 955 strains of Klebsiella pneumoniae were isolated from varied sites of the hospital, and 117(12.3%) of them were identified as CRKP, with a separation rate of 8.9% (26/292) in 2013, 11.3% (38/336) in 2014 and 16.2% (53/327) in 2015, which shows an increasing trend by year. 44.4% (52/117) of CRKP strains were separated from specimen of ICU, and 61.5% (72/117) were from sputum. Over 95% CRKP strains were resistant to ampicillin/sulbactam, aztreonam, imipenem, meropenem, ceftazidme, cefotaxime, cefepime,and piperacillin, while relatively low resistant rates were found in tigecycline (12.8%) and colistin (35.9%). The class 1 integron was detected in 77.8% (91/117) of CRKP strains. Class 1 integron of CRKP was significantly correlated with the antibiotic resistance to the tobramycin, gentamicin and amikacin (all P<0.01). The gene cassette analysis of variable area of class 1 integron showed that aadA2 accounts for 64.8% (59/91), aacA4-catB8-aadA1 23.1% (21/91), and aadA2-dfrA25 12.1% (11/91).Conclusions CRKP has an increasing trend in a clinical setting in China, and most of them were resistant to multiple antibiotics. Class 1 integron in CRKP has strong ability to capture the genes resistant to aminoglycosides antibiotics from environment, with the aadA2 gene as the most popular one.
Anti-Bacterial Agents ; pharmacology ; Carbapenems ; pharmacology ; Drug Resistance, Bacterial ; Integrons ; Klebsiella pneumoniae ; drug effects ; genetics ; isolation & purification

OBJECTIVETo investigate and take a case study on a Klebsiella pneumoniae outbreak in a newborn intensive care unit (NICU).

METHODSUsing epidemiological investigation method to cultivate bacilli and detect the homology.

RESULTSKlebsiella pneumonia was detected in 4 NICU patients. Based on environmental sample analyses, four Klebsiella pneumonia strains were identified and confirmed to be highly homologous. The outbreak was effectively controlled after the strict implementation of hand hygiene practice and environment disinfection.

CONCLUSIONKlebsiella pneumonia outbreak in NICU may be caused by the route of hand transmission.

Cross Infection ; epidemiology ; microbiology ; Disease Outbreaks ; Female ; Humans ; Infant, Newborn ; Intensive Care Units, Neonatal ; Klebsiella Infections ; epidemiology ; microbiology ; Klebsiella pneumoniae ; genetics ; isolation & purification ; Male

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.
Electrophoresis, Gel, Pulsed-Field ; Hospitals ; Humans ; Klebsiella pneumoniae/*classification/enzymology/genetics/isolation & purification ; Sequence Analysis, DNA ; beta-Lactamases/*biosynthesis ; Mycobacteria, Atypical/*drug effects/genetics/isolation & purification

BACKGROUND: Multidrug-resistant Enterobacteriaceae is a worldwide problem. Although various resistance mechanisms have been recognized with increasing frequency, only a few cases of triple resistance of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae have been reported. This study was designed to evaluate the coexistence of qnr (qnrA, qnrB, and qnrS) and 16S rRNA methylase (armA, rmtA, rmtB, and rmtC) in ESBL-producing K. pneumoniae. METHODS: We tested 44 isolates of ESBL-producing K. pneumoniae at Chungnam National University Hospital from March to September 2006. Antimicrobial susceptibilities were tested by broth microdilution method, and transconjugation test was performed using E. coli J53 with azide resistance. Search for qnr (qnrA, qnrB, and qnrS) and 16S rRNA methylase (armA, rmtA, rmtB, and rmtC) genes was conducted by PCR amplification, and the genotypes were determined by direct nucleotide sequence analysis of the amplified products. Epidemiologic study was performed by Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). RESULTS: All ESBL-positive strains produced qnrB; however, armA was detected in 68.2%. The coproduction rate of qnrB and armA in ESBL-producing K. pneumoniae was 68.2%. Two types (A and B) were dominant in ERIC-PCR results. CONCLUSIONS: K. pneumoniae producing qnrB, armA, and ESBL are spreading widely.
Bacterial Proteins/biosynthesis/*genetics ; Disk Diffusion Antimicrobial Tests ; Drug Resistance, Bacterial/genetics ; Humans ; Klebsiella Infections/microbiology ; Klebsiella pneumoniae/drug effects/*genetics/isolation & purification ; Methyltransferases/biosynthesis/*genetics ; beta-Lactamases/biosynthesis/drug effects/*genetics

OBJECTIVETo study the resistance phenotype and homology of Klebsiella pneumoniae (KPN) in burn patients with infection.

METHODSFifty-four strains of KPN were isolated from wound excretion, blood, sputum, venous catheter, feces, and oral cavity of patients hospitalized in Institute of Burn Research of Southwest Hospital (briefly called our institute) from January 2007 to June 2011. Drug resistance of the 54 strains of KPN to 18 antibiotics commonly used in clinic, including ampicillin, ticarcillin, etc, was tested by K-B paper disk diffusion method after being identified. Extended-spectrum β-lactamase (ESBL)-producing KPN was screened based on the drug resistance result. The positive rates of drug-resistant genes SHV, TEM, and CTX-M of the ESBL-producing KPN were detected by polymerase chain reaction. The homology of the ESBL-producing KPN was analyzed by pulse field gel electrophoresis and clustering methodology. The homology of ESBL-producing KPN isolated in each year was analyzed too.

RESULTS(1) The sensitive rate of the 54 strains of KPN to imipenem, meropenem, and ertapenem was respectively 96.30%, 92.59%, and 81.48%, that of these strains to cefotetan and cefoxitin was respectively 70.37% and 64.81%, and that of these strains to ceftazidime was 57.41%. The sensitive rates of the 54 strains of KPN to the other antibiotics were all lower than 40.00%. (2) Twenty-six ESBL-producing KPN strains were screened and the positive rate of SHV, TEM, and CTX-M was 96.15% (25/26), 76.92% (20/26), and 57.69% (15/26), respectively. Detection rate of ESBL-producing KPN strains carrying three genes at the same time was 42.31% (11/26), that of these strains carrying both SHV and TEM was 34.62% (9/26), and those of these strains carrying only a single gene were all less than 10.00%. (3) The twenty-six ESBL-producing KPN were classified into 9 gene types, with 30.77% (8/26) in type A, 19.23% (5/26) in type B, 15.38% (4/26) in type C, 11.54% (3/26) in type D, 7.69% (2/26) in type E, and the rest four strains respectively in type F, G, H, I [3.85% (1/26)]. (4) The major gene type of ESBL-producing KPN in the year of 2007 and 2010 was type A, respectively accounting for 2/3 and 1/2, while that in the year of 2009 was type B, accounting for 1/2. The three strains in 2008 was respectively in type C, E, and F. The four strains in 2011 was respectively in type A, D, H, I.

CONCLUSIONSKPN in burn patients with infection in our institute are highly resistant to commonly used antibiotics in clinic, but carbapenems antibiotics can be used for the treatment. Most of the ESBL-producing KPN strains carry two or three drug-resistant genes, and the main gene type of them is type A.

Anti-Bacterial Agents ; pharmacology ; Burns ; microbiology ; Carbapenems ; pharmacology ; DNA, Bacterial ; analysis ; Drug Resistance, Bacterial ; genetics ; Genes, Bacterial ; Humans ; Klebsiella pneumoniae ; genetics ; isolation & purification ; Microbial Sensitivity Tests ; Sequence Homology

BACKGROUND: Plasmid-mediated AmpC beta-lactamases (PABLs) have been detected in the strains of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Salmonella spp. PABLs may be difficult to detect and might interfere in the therapeutic and infection-control processes. Although several PABL-detection methods based on phenotypes have been reported, the Clinical and Laboratory Standards Institute currently does not recommend a routine detection method for PABLs. The aim of this study is to compare the performances of 3 phenotypic PABL detection methods. METHODS: Total 276 non-duplicated clinical isolates of E. coli (N=97), K. pneumoniae (N=136), and P. mirabilis (N=43) were collected from 14 hospitals in Korea between April and June 2007 in a non-consecutive and non-random manner. Multiplex PCR was performed to detect the PABL genes. Further, 3 phenotypic detection methods-cephamycin-Hodge test, Tris-EDTA (TE) disk test, and combination-disk test with 3-aminophenylboronic acid (BA)-were performed using cefoxitin and cefotetan disks. RESULTS: PABL genes were detected by multiplex PCR in 122/276 isolates, including 14/97 E. coli, 105/136 K. pneumoniae, and 3/43 P. mirabilis isolates. The combination-disk test with BA showed higher sensitivity (98.4%), specificity (92.2%), and efficiency (96.3%) than the cephamycin-Hodge (76.2%, 96.1%, and 88.6%, respectively) and the TE-disk (80.3%, 91.6%, and 87.9%, respectively) tests. CONCLUSIONS: The combination-disk test with BA is a simple, efficient, and interpretable test that can be applicable in clinical laboratories involved in the detection of PABLs in clinical isolates of E. coli, K. pneumoniae, and P. mirabilis.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*analysis ; Cefotetan/pharmacology ; Cefoxitin/pharmacology ; Disk Diffusion Antimicrobial Tests/*methods ; Escherichia coli/genetics/*isolation & purification ; Humans ; Klebsiella pneumoniae/genetics/*isolation & purification ; Phenotype ; Plasmids ; Proteus mirabilis/genetics/*isolation & purification ; Sensitivity and Specificity ; beta-Lactamases/*analysis

BACKGROUND: The aim of this study was to determine the yearly prevalence and genotype distribution of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae collected over a 3-yr period in Gwangju, Korea. METHODS: Clinical isolates of E. coli and K. pneumoniae collected at Chosun University Hospital from September 15, 2005 to September 14, 2008 were evaluated. Antimicrobial susceptibility testing was performed using the Vitek II system (bioMerieux, USA) and agar dilution methods. Screening for ESBL and AmpC beta-lactamase genes was performed using PCR amplification of plasmid DNA followed by direct sequencing of the PCR products. RESULTS: The percentage of ESBL-producing isolates was 12.6% (196/1,550) for E. coli and 26.2% (294/1,121) for K. pneumoniae. The ESBL gene sequencing results showed that the most prevalent ESBL types were CTX-M (93.5%) and SHV (12.9%) in E. coli, and SHV (73.2%) and CTX-M (46.3%) in K. pneumoniae. The most common ESBL in E. coli was CTX-M-15-like, followed by CTX-M-14-like, SHV-2a-like, and SHV-12-like. The most prevalent ESBL type in K. pneumoniae was SHV-12, followed by CTX-M-14-like and CTX-M-15-like. Fifty-one percent (21/41) of ESBL-producing K. pneumoniae with ESBL types verified by sequencing also had DHA-1-like AmpC beta-lactamases. However, none of the ESBL-producing E. coli was positive in the AmpC beta-lactamase PCR analysis. CONCLUSIONS: In this study, the most common types of class A ESBLs identified were CTX-M-15-like in E. coli and SHV-12-like in K. pneumoniae.
Bacterial Proteins/*genetics ; Drug Resistance, Bacterial/genetics ; Escherichia coli/*genetics/isolation & purification ; Gene Frequency ; Genotype ; Hospitals, University ; Humans ; Klebsiella pneumoniae/*genetics/isolation & purification ; Microbial Sensitivity Tests ; Republic of Korea ; Sequence Analysis, DNA ; beta-Lactamases/*genetics

PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital. MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE). RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microngram/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microngram/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested. CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.
Adult ; Aged ; Aged, 80 and over ; *Disease Outbreaks ; Disease Susceptibility ; *Drug Resistance, Multiple, Bacterial ; Female ; Genotype ; Hospitals ; Humans ; Klebsiella Infections/*epidemiology/*microbiology ; Klebsiella pneumoniae/classification/*enzymology/genetics/isolation & purification ; Korea ; Male ; Middle Aged ; Phenotype ; beta-Lactamases/*classification/genetics/*metabolism