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

Animals ; Chickens ; microbiology ; Cross-Sectional Studies ; Enterobacteriaceae ; genetics ; isolation & purification ; Escherichia coli ; genetics ; isolation & purification ; Escherichia coli Proteins ; genetics ; Food Microbiology ; Klebsiella pneumoniae ; genetics ; isolation & purification ; Meat ; microbiology ; Multiplex Polymerase Chain Reaction ; Proteus mirabilis ; genetics ; isolation & purification ; Raw Foods ; microbiology ; Singapore ; beta-Lactamases ; genetics

Carbapenemase-producing organisms (CPO) are rapidly disseminating worldwide, and their presence in tertiary care hospitals poses a significant threat to the management of nosocomial infections. There is a need to control CPO, especially in intensive care unit (ICU) patients, because these organisms are resistant to most beta-lactam antibiotics and are easily transmitted. At present, the identification of CPO is time-consuming; hence, this study focused on the use of the Xpert CARBA-R assay (Cepheid, USA) to determine intestinal colonization rates of CPO in patients admitted to the ICU of a tertiary care hospital in Korea. Forty clinical stool samples were collected and inoculated both in a CARBA-R cartridge and in conventional culture plates. The CARBA-R assay required only ~one hour to screen CPO, while the time required for conventional culture was over three days. We also found that the prevalences of intestinal colonization by carbapenem-resistant organisms and Enterobacteriaceae were 17.5% (7 out of 40) and 7.5% (3 out of 40), respectively. Among the colonizing strains, three that contained carbapenemase, including Klebsiella pneumonia carbapenemase (KPC), and imipenem (IMP) and Verona integron-mediated metallo-beta-lactamase (VIM) were found. With its convenience, the Xpert CARBA-R assay can be included in CPO surveillance strategies.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics/metabolism ; DNA, Bacterial/analysis ; Drug Resistance, Multiple, Bacterial/genetics ; Enterobacteriaceae/drug effects/genetics/*isolation & purification ; Feces/microbiology ; Humans ; Imipenem/pharmacology ; Intensive Care Units ; Klebsiella pneumoniae/drug effects/genetics/*isolation & purification ; Reagent Kits, Diagnostic ; Real-Time Polymerase Chain Reaction ; Republic of Korea ; Tertiary Healthcare ; beta-Lactamases/*genetics/metabolism

PURPOSE: Tigecycline is one of the drugs used to treat multi-drug resistant Klebsiella pneumoniae (K. pneumoniae) infections, including complicated skin and soft tissue infections, complicated intra-abdominal infection, and community-acquired pneumonia in the Republic of Korea. However, since its commercial release, K. pneumoniae resistance against tigecycline has been reported, and there is a serious concern about the spread of tigecycline resistant bacteria. MATERIALS AND METHODS: In this study, we collected and analyzed 342 isolates from 23 hospitals in the Republic of Korea to determine the mechanisms of tigecycline susceptibility and their clonal types. The hospitals include several from each province in the Republic of Korea, except Jeju, an island province, and nonsusceptibility among the isolates was tested by the disk diffusion method. In our lab, susceptibility was checked again using the broth dilution method, and clonal types were determined using the multilocus sequence typing protocol. Real-time PCR was performed to measure the ramR mutation in the isolates nonsusceptible to tigecycline, which would suggest an increased expression of the AcrAB multidrug pump. RESULTS: Fifty-six K. pneumoniae isolates were found to be nonsusceptible, 16% of the 342 collected. Twenty-seven and nine isolates of the tigecycline nonsusceptible isolates had mutations in the ramR and rpsJ genes, respectively; while 18 nonsusceptible isolates harbored the tetA gene. Comparison of isolates with and without ramR mutation showed a significant statistical difference (p<0.05) for expression of AcrAB. Moreover, the most common clonal types, as observed in our study, appear to be ST11 and ST789. CONCLUSION: Several dominate clonal types infer tigecycline resistance to K. pneumoniae, including ST11, ST768, ST15, ST23, ST48, and ST307. There does not seem to be a transferrable medium, such as plasmid, for the resistance yet, although mutation of the ramR gene may be a common event, accounting for 48% of the nonsusceptibility in this study.
Anti-Bacterial Agents/*pharmacology/therapeutic use ; Bacterial Proteins ; *Drug Resistance, Bacterial ; Humans ; Klebsiella Infections/*drug therapy ; Klebsiella pneumoniae/*drug effects/genetics/*isolation & purification ; Microbial Sensitivity Tests ; Minocycline/*analogs & derivatives/pharmacology/therapeutic use ; Multilocus Sequence Typing ; Plasmids ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Republic of Korea

BACKGROUND: The emergence of carbapenem-resistant Klebsiella pneumoniae poses a serious problem to antibiotic management. We investigated the beta-lactamases in a group of carbapenem-resistant K. pneumoniae clinical isolates from Turkey. METHODS: Thirty-seven strains of K. pneumoniae isolated from various clinical specimens were analyzed by antimicrobial susceptibility testing, PCR for the detection of beta-lactamase genes, DNA sequencing, and repetitive extragenic palindronic (REP)-PCR analysis. RESULTS: All 37 isolates were resistant to ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftazidime, cefoperazone/sulbactam, cefepime, imipenem, and meropenem. The lowest resistance rates were observed for colistin (2.7%), tigecycline (11%), and amikacin (19%). According to PCR and sequencing results, 98% (36/37) of strains carried at least one carbapenemase gene, with 32 (86%) carrying OXA-48 and 7 (19%) carrying NDM-1. No other carbapenemase genes were identified. All strains carried a CTX-M-2-like beta-lactamase, and some carried SHV- (97%), TEM- (9%), and CTX-M-1-like (62%) beta-lactamases. Sequence analysis of bla(TEM) genes identified a bla(TEM-166) with an amino acid change at position 53 (Arg53Gly) from bla(TEM-1b), the first report of a mutation in this region. REP-PCR analysis revealed that there were seven different clonal groups, and temporo-spatial links were identified within these groups. CONCLUSIONS: Combinations of beta-lactamases were found in all strains, with the most common being OXA-48, SHV, TEM, and CTX-M-type (76% of strains). We have reported, for the first time, a high prevalence of the NDM-1 (19%) carbapenemase in carbapenem-resistant K. pneumoniae from Turkey. These enzymes often co-exist with other beta-lactamases, such as TEM, SHV, and CTX-M beta-lactamases.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/*genetics/metabolism ; Carbapenems/*pharmacology ; DNA, Bacterial/chemistry/genetics/metabolism ; Drug Resistance, Bacterial ; Genotype ; Humans ; Klebsiella Infections/diagnosis/microbiology ; Klebsiella pneumoniae/*drug effects/enzymology/isolation & purification ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Turkey ; beta-Lactamases/*genetics/metabolism

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

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

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

OBJECTIVEThis study aimed to investigate drug resistance and genotypes of the extended spectrum β-lactamase (ESBLs) and AmpC β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolated from Uygur and Han newborns in Urumqi.

METHODSDisk diffusion test (Kirby-Bauer) was used for detecting drug resistance of 299 strains to twenty two kinds of antibiotics. Resistance genes of the ESBLs and AmpC β-lactamase-producing strains were amplified by multiplex PCR and subtypes were confirmed by DNA sequence analysis. Total 148 strains were selected with random number table and sequenced, which included TEM-, SHV-, CTX-M-1-, or CTX-M-9-positive ESBLs-producing strains and DHA-, or CIT-positive AmpC β-lactamase-producing strains. Antibiotic resistant rates were analyzed by Whonet 5.4 and statistic analysis was performed by chi-square (χ(2)) test with PEMS 3.1.

RESULTSThe antibiotic resistant rates between Uygur and Han newborns significantly differ in ESBLs-producing Klebsiella pneumoniae to Sulfamethoxazole-Trimethoprim (80.0% (40/50) and 56.0% (28/50), χ(2) = 6.6176, P = 0.0101), in ESBLs-producing Escherichia coli to Sulbactam and Cefoperazone (54.2% (32/59) and 94.0% (47/50), χ(2) = 21.4512, P = 0.0000), and in AmpC β-lactamase-producing Klebsiella pneumoniae to Sulbactam and Cefoperazone (100.0% (20/20) and 72.2% (26/36), χ(2) = 6.7633, P = 0.0093) and to Amikacin (65.0% (13/20) and 25.0% (9/36), χ(2) = 8.6246, P = 0.0033). Although SHV gene of ESBLs-producing Escherichia coli was detected from Uygur newborns at only 3.4% (2/59) and not detectable from Han newborns, TEM, CTX-M-1, and CTX-M-9 group genes were all detected over 38.0% (19/50). Among the detected strains, the subtypes of TEM and CTX-M-1 were mainly TEM-1 and CTX-M-15, respectively; whereas the subtypes of SHV and CTX-M-9 included SHV-1, 2, 11, 12, 27, 61, 99 and CTX-M-9, 14, 24, 27, 65, respectively. The strains of Escherichia coli and Klebsiella pneumoniae carrying two or more kinds of ESBLs genotypes were 56.7% (42/74) - 90.0% (63/70). Two species carrying the AmpC gene in two kinds of newborns were only grouped in the subtypes of DHA-1 and CMY-44, and other subtypes were not detected at all. Moreover, TEM-positive ESBLs-producing Escherichia coli were detected from Uygur newborns at the higher rate than that from Han newborns (71.2% (42/59) and 50.0% (25/50), χ(2) = 5.1291, P = 0.0235), while there was no difference in other genotypes detected between two kinds of newborns (χ(2) < 3.7780, P > 0.05).

CONCLUSIONThere were significant differences in antibiotic resistance and genotype distribution of Klebsiella pneumoniae and Escherichia coli between two nationality newborns, and these two bacteria detected in this study carried multi-resistance genes and showed high resistant to β-lactamase antibiotics.

Bacterial Proteins ; metabolism ; China ; Escherichia coli ; drug effects ; genetics ; isolation & purification ; Escherichia coli Infections ; microbiology ; Ethnic Groups ; Genotype ; Humans ; Infant, Newborn ; Klebsiella Infections ; microbiology ; Klebsiella pneumoniae ; drug effects ; genetics ; isolation & purification ; Microbial Sensitivity Tests ; beta-Lactam Resistance ; genetics ; beta-Lactamases ; metabolism

Carbapenem-resistant Klebsiella pneumoniae isolates producing K. pneumoniae carbapenemases (KPC) were first reported in the USA in 2001, and since then, this infection has been reported in Europe, Israel, South America, and China. In Korea, the first KPC-2-producing K. pneumoniae sequence type (ST) 11 strain was detected in 2010. We report the case of a patient with a urinary tract infection caused by KPC-2-producing K. pneumoniae. This is the second report of a KPC-2-producing K. pneumoniae infection in Korea, but the multilocus sequence type was ST258. The KPC-2-producing isolate was resistant to all tested beta-lactams (including imipenem and meropenem), amikacin, tobramycin, ciprofloxacin, levofloxacin, and trimethoprim-sulfamethoxazole, but was susceptible to gentamicin, colistin, polymyxin B, and tigecycline. The KPC-2-producing isolate was negative to phenotypic extended-spectrum beta-lactamase (ESBL) and AmpC detection tests and positive to modified Hodge test and carbapenemase inhibition test with aminophenylboronic acid.
Aged ; Bacterial Proteins/antagonists & inhibitors/metabolism ; Carbapenems/pharmacology ; Drug Resistance, Bacterial/genetics ; Female ; Humans ; Klebsiella pneumoniae/drug effects/genetics/*isolation & purification ; Microbial Sensitivity Tests ; Republic of Korea ; Sequence Analysis, DNA ; Urinary Tract Infections/*diagnosis/microbiology ; beta-Lactamases/antagonists & inhibitors/biosynthesis/*genetics/metabolism