Detection of drug resistance and gene type_3016

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Detection of drug resistance and gene type_3016

Detection of drug resistance and gene type

     Author: Zhengya Fen Zhou Wei Zi Jing Cheng Xiao-

    Na Wen

     Abstract Objective To understand the neonatal nosocomial infection producing extended spectrum β lactamases (ESBLs)

    producing Klebsiella pneumoniae isolates resistant conditions and β lactamase gene encoding TEM, SHV, OXA, PER, GES, VEB, CTX-type situation there. Method in our hospital in July 2004 infection in neonatal wards in the 14 strains of Klebsiella pneumoniae with automatic microbial identification and antibiotic susceptibility analysis of matching cards for susceptibility testing and ESBLs detection and polymerase chain reaction and sequence analysis method Analysis of strain in ultra-broad-spectrum β lactamase. The results of 14

    ESBLs-producing Klebsiella pneumoniae susceptibility results are consistent, ampicillin, ceftriaxone, cefazolin, ceftazidime were resistant to amoxicillin / clavulanic acid, piperacillin / tazobactam resistance is also , for imipenem.

    Were detected blaCTX M and blaTEM genes were not detected blaSHV, blaOXA, blaPER, blaGES and blaVEB genes. Right blaTEM and blaCTX M gene amplification products were sequenced by BLAST program analysis gene TEM 1 and CTX M 3-type.

    Conclusion hospital infection in the popular ESBLs Klebsiella pneumoniae resistant to third generation cephalosporins and resistance to enzyme inhibitors, gene type TEM 1 and CTX

    M 3. ESBLs belonging to the high risk newborns, neonatal diseases property extended spectrum β lactamase bacterial

    infections to be caused by all sides.

     Key words Newborn ultra-extended-spectrum β-lactamase gene

    types of Klebsiella pneumoniae

     Investigation of the antibiotics resistance and resistant

     genotype of

     ABSTRACT Objective To investigate the antibiotics resistance of ESBLs producing Klebsiella pneumoniae (KPN) which were isolated from newborns and the genotype existence of TEM, SHV, OXA, PER, GES, VEB and CTX. Methods The antimicrobial susceptibility and ESBLs detection of fourteen

    KPN isolates from newborns in the Materity and Children Health Hospital of Changzhou, in July, 2004 were performed. The genotype of ESBLs from KPN were identified by polymerase chain reaction (PCR). Results Total of 14 KPN producing ESBLs were

    resistant to ampicillin, ceftriaxone, cefazolin , ceftazidime, amoxicillin / clavulanic acid and piperacillin / tazobactam, while sensitive to imipenem. All 14 KPN carried blaCTX M

    gene and blaTEM gene, whereas blaSHV, blaOXA, blaPER, blaGES

    and blaVEB genes were not detected. blaCTX M gene and

    blaTEM gene were identified as blaTEM 1 and blaCTX M

    3. Conclusion The KPN isolated from newborns were resistant to the third generation cephalosporins and enzyme inhibitors, and the genotype were TEM 1 and CTX M 3. As newborns are

    a high risk group, it should be pay more attention to keep them from the ESBLs producing KPN epidemic outbreak.

     KEY WORDS Newborns; ESBLs; Klebsiella pneumoniae; Genotype

     Nosocomial infection in intensive care unit producing

    extended spectrum β lactamase-resistant strains of

    bacteria isolated and drug resistance genes have been repeated reports of episodes of domestic [1 ~ 4]. Neonatal nosocomial infection producing extended spectrum β lactamase isolated

    bacteria resistance and resistance gene studies have reported relatively few. In our hospital in 2004 have occurred in a neonatal ward of Klebsiella pneumoniae (Klebsiella pneumoniae, KPN) an outbreak of nosocomial infection. According to the

    results of susceptibility due to timely medical treatment and children fully recovered. Now was isolated from 14 Klebsiella pneumoniae resistance and conduct blaTEM, blaSHV, blaOXA, blaPER, blaGES, blaVEB and blaCTX M genetic test results

    reported as follows.

     1 Materials and methods

     1.1 Clinical data

     July 17, 2004 ~ July 19 wards in 14 cases of newborn children start to appear and body temperature, fever, chills. 6 cases in which men and children, female children in 8 cases, all were premature infants, low birth weight infants born in 1 ~ 11d. Most children with normal body temperature before admission, fever, blood sample for culture when the then 18 ~ 24h after the train were Klebsiella pneumoniae, according to the results of susceptibility with imipenem / cilastatin

    treatment of 3d body temperature normal, 6 ~ 9d children were all cured, blood culture was negative for discharge.

     1.2 Strain identification and susceptibility test

     Biphasic blood culture bottles by bacteria, you use Bio

     Merieux VITEK 32 automatic analysis system for microbial identification were identified. Drug sensitivity test using Bio Merieux's GNS 120 drug Minka, antimicrobial

    susceptibility test results to determine based on CLSI / NCCLS

    (2004 years) standard.

     1.3 producing extended-spectrum β lactamases (ESBLs)


     Microbial identification by the VITEK 32 instrument

    expert system for the direct determination. The principle with the CLSI / NCCLS disk diffusion method recommended by pairs of

    similar, according to four test holes (cefotaxime, cefotaxime clavulanic acid, ceftazidime, ceftazidime and clavulanic acid) in the growth of turbidity changes, when not clavulanic acid, the growth of turbidity turbidity of the growth of more than

    Jiakelawei acid ? 50% shall be positive. Standard strains of Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603.

     1.4 β lactamase gene detection

     TEM, SHV, OXA, PER, GES, VEB, and CTX M-type β

    lactamase genes by PCR assay, protease K digestion and

    extraction of bacterial DNA, primers in Table 1. Reaction primers P1, P2 each 0.5μmol / L, dNTPs each 200μmol / L, KCl

    10mmol / L, (NH4) 2SO4 8mmol / L, MgCl2 2mmol / L, Tris HCl

(pH9.0) 10mmol / L, NP40 0.5%, Taq DNA polymerase 1U. All

    reaction volume of 20μl, which board liquid 5μl. Thermal

    cycling parameters: 93 ? predegeneration 3min, then 93 ?

    1min ? 55 ? 1min ? 72 ? 1min, the last a 72 ? extended to

    5min, a total circulation in Table 1 ESBL enzyme encoding gene

    PCR primers were 35 cycles. Results Watch: 10μl PCR products

    obtained with the electrophoresis indicator 2μl mixing,

    point-like and 1% agarose gel. To voltage 100V regulator electrophoresis 30min, gel imager observed and recorded images. PCR kit and positive DNA cloning genetic technologies by the Wuxi Institute.

     2 Results

     2.1 Sensitivity Results

     14 KPN bacteria susceptibility test results the same (Table 2).

     2.2 β lactamase gene test results

     14 KPN bacteria TEM, CTX M genotype PCR testing were positive. Figure 1,2 for the blaTEM, blaCTX M PCR detected

    by electrophoresis. Table 2 14 of the 15 kinds of antibiotic-

    sensitive bacteria KPN test marker from top to bottom, respectively

    1000,900,800,700,600,500,400,300,250,200,150,100,50 bp; CTX

    M-type gene PCR electrophoresis 4 blaTEM gene amplification products were sequenced by BLAST program analysis ( for the blaTEM 1, with the

    Escherichia coli TEM 1-type gene sequence fragment

    (Registration ID: AB 194682) line rate of 100%. On a CTX M-

    type amplified products were sequenced by BLAST program analysis for the CTX M 3 type, and Escherichia coli CTX

     M 3-type gene sequence fragment (Registration No.: AB185842) line rate of 100% . Reposted elsewhere in the paper

    for free download http://

     In this study, TEM 1, CTX M 3-type gene sequence

    has been successfully registered with GenBank ( / nucleotide, registration number: DQ078258, DQ078259).

     3 Discussion

     Antibacterial drug discovery and application to human life extended for at least 15 years. However, as people, livestock overuse of antibiotics, bacterial resistance rate increased year by year. Bacterial resistance to antibiotics has become a public health problem of global concern. Experts estimate that bacterial resistance to antibiotics in the 21st century the threat to human life and health will be no less than AIDS, cancer and cardiovascular disease. Gram-negative

    bacilli β lactam resistance is mainly producing β

    lactamases. β lactamases initially found in the 20th century, 60 years, the main hydrolysis penicillin antibiotics by blaTEM 1, blaTEM 2 and blaSHV 1 gene encoding. But

    following the β -lactam drugs, excessive use of bacterial

    long-term exposure to drugs, the coding gene mutation, resulting in simultaneous resistance to penicillin, cephalosporins one to three generations (some may be resistant to cephalosporins four-generation) drugs, called extended-

    spectrum β lactamases (ESBLs). Has been found that the

    gene encoding family of ESBLs among blaTEM, blaSHV, there blaCTX M, blaOXA, blaPER, blaVEB, blaGES / IBC and so on. Each gene family genotypes have been found several dozens to hundreds of ranges ESBLs producing bacteria are Klebsiella

    pneumoniae, Escherichia coli, producing Klebsiella oxytoca, Enterobacter aerogenes, Mount Morgan's bacteria, Proteus mirabilis, Enterobacter cloacae, Acinetobacter baumannii, Pseudomonas aeruginosa and other gram-negative bacilli [5 ~


     The group of 14 ampicillin, ceftriaxone, cefazolin, ceftazidime KPN are resistant bacteria blaTEM, blaCTX M

    gene PCR tested positive for 4 strains TEM-type gene

    amplification products were sequenced by BLAST program analysis for the blaTEM 1; right one blaCTX M-type gene

    amplification products were sequenced by BLAST program analysis for the CTX M 3-type. The same phenotype and

    genotype. 14 KPN bacteria to imipenem sensitive to imipenem / cilastatin effective treatment. In addition, 14 KPN bacteria

    on amoxicillin / clavulanic acid, piperacillin / tazobactam resistant, ie resistant to the inhibitors. At the same time producing extended-spectrum β lactamases and extended-

    spectrum β producing ultra-lactamase inhibitor resistance

    can be expressed as [12]. Recently, the Japanese reported the neonatal ICU capacity GES 3-type extended spectrum β

    lactamase KPN outbreak of bacterial infections [13]. ESBLs are at high risk newborns, especially premature infants own body resistance is weak, oxygen, suction and other operations are

    more vulnerable to infections caused by neonatal ward outbreak. A recent interview with the third-generation

    cephalosporins therapy is also to promote the emergence and spread of ESBLs high-risk factors, so particular attention to

    the rational use of high-risk groups, and other third-

    generation cephalosporins extended-spectrum β lactam

    antibiotics, attention to drug-resistant strain monitoring,

    the scientific basis of bacterial culture and sensitivity results to guide the application of antibiotics.

     This research was supported by genetic cloning, Wuxi Institute of Technology MI Zu-huang teacher's guidance, the

    greatest importance.


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