OBJECTIVETo characterize carbapenem (CPM)-non-susceptible Klebsiella pneumoniae (K. pneumoniae) and carbape-nemase produced by these strains isolated from Beijing Children's Hospital based on a five-year surveillance.

METHODSThe Minimal Inhibition Concentration values for 15 antibiotics were assessed using the Phonix100 compact system. PCR amplification and DNA sequencing were used to detect genes encoding carbapenemases. WHONET 5.6 was finally used for resistance analysis.

RESULTSIn total, 179 strains of CPM-non-susceptible K. pneumoniae were isolated from January, 2010 to December, 2014. The rates of non-susceptible to imipenem and meropenem were 95.0% and 95.6%, respectively. In the 179 strains, 95 (53.1%) strains carried the blaIMP gene, and IMP-4 and IMP-8 were detected in 92 (96.8%) and 3 (3.2%) IMP-producing isolates, respectively. 65 (36.3%) strains carried the blaNDM-1 gene. 6 (3.4%) strains carried the blaKPC gene, and KPC-2 were detected in 6 KPC-producing isolates. In addition, New Delhi-Metallo-1 (NDM-1) producing isolates increased from 7.1% to 63.0% in five years and IMP-4 producing isolates decreased from 75.0% to 28.3%.

CONCLUSIONHigh frequencies of multiple resistances to antibiotics were observed in the CPM-non-susceptible K. pneumoniae strains isolated from Beijing Children's Hospital. The production of IMP-4 and NDM-1 metallo-β-lactamases appears to be an important mechanism for CPM-non- susceptible in K. pneumoniae.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Child ; China ; epidemiology ; Drug Resistance ; Gene Expression Regulation, Bacterial ; physiology ; Gene Expression Regulation, Enzymologic ; physiology ; Hospitals, Pediatric ; Humans ; Klebsiella Infections ; epidemiology ; microbiology ; Klebsiella pneumoniae ; drug effects ; enzymology ; genetics ; Microbial Sensitivity Tests ; Population Surveillance ; Time Factors ; beta-Lactamases ; genetics ; metabolism

Bacterial Proteins ; metabolism ; Inflammation ; metabolism ; Klebsiella pneumoniae ; metabolism ; MAP Kinase Signaling System ; Proteasome Endopeptidase Complex ; metabolism ; Ubiquitin ; metabolism

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

BACKGROUNDKlebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae bacteria, which cause serious disease outbreaks worldwide, was rarely detected in Xiangya Hospital, prior to an outbreak that occurred from August 4, 2014, to March 17, 2015. The aim of this study was to analyze the epidemiology and molecular characteristics of the K. pneumoniae strains isolated during the outbreak.

METHODSNonduplicate carbapenem-resistant K. pneumoniae isolates were screened for blaKPC-2and multiple other resistance determinants using polymerase chain reaction. Subsequent studies included pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, analysis of plasmids, and genetic organization of blaKPC-2locus.

RESULTSSeventeen blaKPC-2-positive K. pneumoniae were identified. A wide range of resistant determinants was detected. Most isolates (88.2%) coharbored blaKPC-2and rmtB in addition to other resistance genes, including blaSHV-1, blaTEM-1, and aac(3)-IIa. The blaKPC-2and rmtB genes were located on the conjugative IncFIB-type plasmid. Genetic organization of blaKPC-2locusin most strains was consistent with that of the plasmid pKP048. Four types (A1, A2, A3, and B) were detected by PFGE, and Type A1, an ST11, was the predominant PFGE type. A novel K. pneumoniae sequence type (ST1883) related to ST11 was discovered.

CONCLUSIONSThese isolates in our study appeared to be clonal and ST11 K. pneumoniae was the predominant clone attributed to the outbreak. Coharbing of blaKPC-2and rmtB, which were located on a transferable plasmid, in clinical K. pneumoniae isolates may lead to the emergence of a new pattern of drug resistance.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; metabolism ; China ; Electrophoresis, Gel, Pulsed-Field ; Hospitals, Teaching ; Klebsiella Infections ; etiology ; Klebsiella pneumoniae ; drug effects ; metabolism ; Methyltransferases ; metabolism ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; beta-Lactamases ; metabolism

OBJECTIVETo construct the KbvR gene of LuxR family deletion mutant and complementation strains from Klebsiella pneumoniae NTUH-K2044 and analyze the effect of KbvR on bacterial growth, biofilm formation and capsular synthesis.

METHODSA KbvR gene deletion mutant strain was constructed using the suicide vector pKO3-Km, and the gene fragment including KbvR coding region, promoter area and transcription termination area were amplified and cloned into pGEM-T-easy plasmid to construct KbvR complementation strain. The growth curves of the wild-type strain, KbvR gene deletion mutant strain and complementation strain were observed to assess the effect of KbvR on bacterial growth. Crystal violet staining method was used to measure the effect of KbvR on biofilm formation; the effect of KbvR on capsular synthesis was detected using string test, centrifugal test and RT-PCR.

RESULTSThe KbvR deletion mutant and complementation strains were constructed successfully. KbvR gene did not affect the growth of the bacteria, but biofilm formation and capsular synthesis were attenuated in KbvR deletion mutant strain.

CONCLUSIONAs a transcription factor of the LuxR family orphans of the quorum sensing system, KbvR positively regulates bacterial biofilm formation by affecting capsular synthesis.

Bacterial Capsules ; Bacterial Proteins ; metabolism ; Biofilms ; Gene Deletion ; Klebsiella pneumoniae ; cytology ; Plasmids ; Promoter Regions, Genetic

1,3-propanediol is an important chemical used as building block for the synthesis of highly promising polyesters such as polytrimethylene terephthalate. A genetically modified Klebsiella pneumoniae LDH526 can use glycerol as sole carbon source and produce 1,3-propanediol with the titer above 90 g/L. A key factor affecting the production of 1,3-propanediol by the mutant K. pneumoniae is the accurate control of the feeding of glycerol. To generate a robust and reproducible fermentation process of 1,3-propanediol, we designed and optimized an automatically feeding strategy of glycerol based on fermentation kinetics. By coupling the substrate feeding rate with easily observed variables -pH and fermentation time, we have achieved self-starting glycerol feeding and dynamic control of the glycerol concentration during the fermentation process. This automated system allowed us to generate a reproducible, consistent and operator-independent process from lab-scale to production scale. The final concentration of 1,3-propanediol was above 95 g/L after 72 h.
Culture Media ; Fermentation ; Glycerol ; Industrial Microbiology ; methods ; Klebsiella pneumoniae ; growth & development ; Propylene Glycol ; Propylene Glycols ; metabolism

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

No abstract available.
Aged ; Female ; Humans ; Klebsiella Infections/*diagnosis/microbiology ; Klebsiella pneumoniae/*enzymology/isolation & purification ; Pneumonia/*diagnosis/microbiology ; Turkey ; beta-Lactamases/*metabolism

BACKGROUNDExtended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) is one of the most popular pathogens that cause refractory respiratory tract infection. The genetic environment, including insertion sequences and the types of promoter, plays a key role in exploration of the mechanism of prevalence and dismission of the ESBL-producing K. pneumoniae isolates. The aim of the investigation was to target analysis the genetic environment and promoter sequences of blaCTX-M, blaSHV and blaTEM, the most popular β-lactamase genes harbored by ESBL-producing K. pneumoniae isolates.

METHODSFrom February 2010 to July 2011, 158 of 416 K. pneumoniae isolates producing ESBL from patients with lower respiratory tract infection were collected from seven tertiary hospitals from Beijing, Anhui, Fujian, Liaoning, Hebei and Inner Mongolia Autonomous Region in China. The genetic environment including promoters of 10 types of blaCTX-M, 18 types of blaSHV and 2 types of blaTEM were analyzed by amplification and direct sequencing with various sets of PCR primers.

RESULTSISEcp1 was located upstream of the 5' end of the blaCTX-M gene in 130 (97.0%) out of 134 K. pneumoniae isolates harboring blaCTX-M and provided a conserved promoter to blaCTX-M. A non-coding sequence preceded by kdpC and recF was identified in all of the blaSHV genes except blaSHV-12 and blaSHV-2a. IS26 was also found upstream of 1 blaCTX-M-15, 10 blaSHV-1 strains, 4 blaTEM-1 and all of the blaSHV-2, blaSHV-2a, blaSHV-5 and blaSHV-12. Eighty-seven of 91 strains harboring blaTEM-1 carried a copy of Tn2 and IS26-blaTEM-1 fragments were also detected in 4 strains. With respect to K. pneumoniae, the genetic environment of blaCTX-M-38, blaSHV-142 and blaTEM-135 were firstly elaborated, and four kinds of novel genetic environment of blaCTX-M-3, blaCTX-M-15 and blaTEM-1 have been detected as well.

CONCLUSIONSPerfective implementation of the genetic environment information of bgr;-lactamase gene needs to be further explored and supplemented. ISEcp1 and IS26 elements are widespread upstream of the blaCTX-M, blaSHV and blaTEM genes and contribute to horizontal transmission and genetic expression.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; China ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Klebsiella pneumoniae ; drug effects ; enzymology ; genetics ; Microbial Sensitivity Tests ; Promoter Regions, Genetic ; genetics ; beta-Lactamases ; genetics ; metabolism

BACKGROUND/AIMS: We evaluated changes of causative pathogen in acute cholangitis and their antimicrobial susceptibility over six years and differences between community-acquired and hospital-acquired acute cholangitis at our institution. METHODS: Medical records of 1,596 patients with acute cholangitis and biliary drainage between August 2006 and August 2012 were reviewed retrospectively. Cases were divided according to time: period 1 (August 2006-December 2008, n=645, 40.4%), period 2 (January 2009-August 2012, n=951, 59.6%). Cases were divided according to community-acquired cholangitis (n=1,397, 87.5%) and hospital-acquired cholangitis (n=199, 12.5%). Causative pathogens and antimicrobial susceptibility were investigated in each group. RESULTS: Causative pathogen was isolated from bile culture in 1,520 out of 1,596 cases (95.2%). The three most frequently isolated Gram-negative bacteria were extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (n=485, 30.4%), E. coli (n=237, 13.2%), and Citrobacter freundii (n=110, 6.9%). Between periods 1 and 2, prevalence of ESBL-producing E. coli and Klebsiella pneumoniae did not show significant change (36.7% vs. 32.1%, p=0.073; 6.6% vs. 6.2%, p=0.732). C. freundii showed a significant increase from period 1 to period 2 (1.7% vs. 13.2%, p=0.000). In both time periods, imipenem was the antimicrobial agent showing the highest rate of susceptibility (93.3% vs. 93.9%, p=0.783). Higher prevalence of ESBL-producing E. coli and C. freundii was observed in the hospital-acquired cholangitis group (52.1% vs. 31.2%, p=0.000; 15.9% vs. 7.3%, p=0.001). CONCLUSIONS: The most common causative pathogen of acute cholangitis was ESBL-producing E. coli. Prevalence of C. freundii increased over the time period. Imipenem should be reserved as an alternative for resistant pathogens.
Acute Disease ; Aged ; Aged, 80 and over ; Anti-Bacterial Agents/*pharmacology ; Cholangitis/diagnosis/*microbiology ; *Citrobacter freundii/drug effects/isolation & purification ; Community-Acquired Infections/microbiology ; Cross Infection/microbiology ; Drug Resistance, Bacterial ; *Escherichia coli/drug effects/isolation & purification ; Female ; Humans ; Imipenem/pharmacology ; *Klebsiella pneumoniae/drug effects/isolation & purification ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Retrospective Studies ; Time Factors ; beta-Lactamases/metabolism