Please wait a minute...
Journal of Integrative Agriculture  2012, Vol. 12 Issue (10): 1714-1720    DOI: 10.1016/S1671-2927(00)8705
ANIMAL SCIENCE · VETERINARY SCIENCE Advanced Online Publication | Current Issue | Archive | Adv Search |
Characterization of blaCTX-M Gene in One Klebsiella pneumoniae Isolate from Sick Chickens in China
 HU Gong-zheng, HU Han, LIU Bao-guang, YUAN Li, LIU Jian-hua, PAN Yu-shan, WU Hua, CHEN Yu-xia
1.College of Animal Husbandry and Veterinary Science, Henan Agricultural University, Zhengzhou 450002, P.R.China
2.Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  Two Klebsiella pneumoniae isolates (Kpc1 and Kpc2) were obtained from liver samples of seven dead chickens and identified with Vitek-32 automated identification system. Antimicrobial susceptibilities were determined by the microdilution broth method. Detection of genes encoding class A b-lactamases was performed by PCR amplification, and cloning of the ESBL gene was by plasmid restriction and fragments ligation. Conjugation assay, transformation experiments and plasmid profile analysis were performed. The incompatibility group of ESBL-carrying plasmid was determined by the PCR-based replicon typing method. Lastly, the genetic environment was analysed by direct sequencing of the DNA surrounding the ESBL gene. The genes associated with tetracycline and gentamicin resistance were also sought by PCR. The results revealed that the ESBL phenotype-negative strain Kpc2 only showed resistance to ampicillin, amoxicillin, tetracycline, and doxycycline and carried blaTEM-1 and tet(A) genes. The ESBL-producing strain Kpc1 exhibited multidrug resistant phenotype and harbored blaTEM-1, blaCTX-M-14, tet(A), tet(B), and rmtB genes. K. pneumoniae Kpc1 contained four plasmids with molecular sizes of approximately 59, 6.9, 2.8, and 1.6 kb, but only a 59-kb plasmid, carried blaTEM-1 and blaCTX-M-14 genes, was observed in its transconjugant. The incompatibility group of plasmid carrying blaCTX-M-14 gene could not be determined. The blaCTX-M-14 gene was flanked upstream by an ISEcp1 insertion sequence and downstream by an IS903 element. This work shows that CTX-M-14 is present in K. pneumoniae isolates from chickens in China. The blaCTX-M-14 gene was associated with an upstream ISEcp1 insertion sequence. Our results underline the need for continuous surveillance of the prevalence and evolution of this CTX-M-type b-lactamase in China.

Abstract  Two Klebsiella pneumoniae isolates (Kpc1 and Kpc2) were obtained from liver samples of seven dead chickens and identified with Vitek-32 automated identification system. Antimicrobial susceptibilities were determined by the microdilution broth method. Detection of genes encoding class A b-lactamases was performed by PCR amplification, and cloning of the ESBL gene was by plasmid restriction and fragments ligation. Conjugation assay, transformation experiments and plasmid profile analysis were performed. The incompatibility group of ESBL-carrying plasmid was determined by the PCR-based replicon typing method. Lastly, the genetic environment was analysed by direct sequencing of the DNA surrounding the ESBL gene. The genes associated with tetracycline and gentamicin resistance were also sought by PCR. The results revealed that the ESBL phenotype-negative strain Kpc2 only showed resistance to ampicillin, amoxicillin, tetracycline, and doxycycline and carried blaTEM-1 and tet(A) genes. The ESBL-producing strain Kpc1 exhibited multidrug resistant phenotype and harbored blaTEM-1, blaCTX-M-14, tet(A), tet(B), and rmtB genes. K. pneumoniae Kpc1 contained four plasmids with molecular sizes of approximately 59, 6.9, 2.8, and 1.6 kb, but only a 59-kb plasmid, carried blaTEM-1 and blaCTX-M-14 genes, was observed in its transconjugant. The incompatibility group of plasmid carrying blaCTX-M-14 gene could not be determined. The blaCTX-M-14 gene was flanked upstream by an ISEcp1 insertion sequence and downstream by an IS903 element. This work shows that CTX-M-14 is present in K. pneumoniae isolates from chickens in China. The blaCTX-M-14 gene was associated with an upstream ISEcp1 insertion sequence. Our results underline the need for continuous surveillance of the prevalence and evolution of this CTX-M-type b-lactamase in China.
Keywords:  Klebsiella pneumonia       CTX-M-14       minimum inhibitory concentrations (MICs)       genetic environment  
Received: 13 June 2011   Accepted:
Fund: 

This study was funded by the National Natural Science Foundation of China (31072170).

Corresponding Authors:  Correspondence HU Gong-zheng, Tel/Fax: +86-371-63555354, Mobile: 13838251716, E-mail: yaolilab@163.com     E-mail:  yaolilab@163.com

Cite this article: 

HU Gong-zheng*, HU Han*, LIU Bao-guang*, YUAN Li, LIU Jian-hua, PAN Yu-shan, WU Hua, CHEN Yu-xia. 2012. Characterization of blaCTX-M Gene in One Klebsiella pneumoniae Isolate from Sick Chickens in China. Journal of Integrative Agriculture, 12(10): 1714-1720.

[1]Bae I K, Lee Y N, Hwang H Y, Jeong S H, Lee S J, Kwak HS, Song W, Kim H J, Youn H. 2006. Emergence of CTXM-12 extended-spectrum ?-lactamase-producingEscherichia coli in Korea. Journal of AntimicrobialChemotherapy, 58, 1257-1259.

[2]Bonnet R. 2004. Growing group of extended-spectrum ?-lactamases: the CTX-M enzymes. Antimicrobial Agentsand Chemotherapy, 48, 1-14.

[3]Carattoli A, Bertini A, Villa L, Falbob V, Hopkins K L,Threlfall E J. 2005. Identification of plasmids by PCRbasedreplicon typing. Journal of MicrobiologicalMethods, 63, 219-228.

[4]Chanawong A, M’Zali F H, Heritage J, Xiong J H, HawkeyP M. 2002. Three cefotaximases, CTX-M-9, CTX-M-13,and CTX-M-14, among Enterobacteriaceae in thePeople’s Republic of China. Antimicrobial Agents andChemotherapy, 46, 630-637.

[5]Cheng J, Gao W, Yin J, Yin J, Sun Z, Ye Y, Gao Y F, Li X, LiJ B. 2010. Phenotypic and molecular characterization oftwo novel CTX-M enzymes carried by K. pneumoniae.Molecular Biology Reports, 37, 1261-1267.

[6]Clinical and Laboratory Standards Institute (CLSI). 2008.Performance Standards for Antimicrobial Disk andDilution Susceptibility Tests for Bacteria Isolated fromAnimals Approved Standard. 3rd ed. (M31-A3). CLSI,Wayne, PA.Clinical and Laboratory Standards Institute (CLSI). 2009.Per formance Standards for Ant i m i c robi a lSusceptibility Testing. 19th Informational Supplement(M100-S16). CLSI. Wayne, PA.Diestra1 K, Juan C, Curiao T, Moya B, Miro E, Oteo J,Coque T M, Perez-Vazquez M, Campos J, Canton R,et al. 2009. Characterization of plasmids encodingblaESBL and surrounding genes in Spanish clinicalisol a tes of Escherichia col i and Klebs iel lapneumoniae. Journal of Antimicrobial Chemotherapy,63, 60-66.

[7]Eckert C, Gautier V, Arlet G. 2006. Sequence analysis of thegenetic environment of various blaCTX-M genes. Journalof Antimicrobial Chemotherapy, 57, 14-23.

[8]He L Y, Lei L C, Jia Y, Lei L C. 2008. Establishment of PCRassay for detection of type 3 funbriae Klebsiellapneumoniae. Chinese Journal of Veterinary Science,28, 261-263. (in Chinese)

[9]Jones C H, Tuckman M, Keeney D, Ruzin A, Bradford P A.2009. Characterization and sequence analysis ofextended-spectrum ?-lactamase-encoding eenes fromE. coli, K. pneumoniae, and Proteus mirabilis isolatescollected during tigecycline phase 3 clinical trials.Antimicrobial Agents and Chemotherapy, 53, 465-475.

[10]JouiniA, Vinue L, Slama K B, Sáenz Y, Klibi N, Hammami S,Boudabous A, Torres C. 2007. Characterization of CTXMand SHV extended-spectrum ?-lactamases andassociated resistance genes in Escherichia coli strainsof food samples in Tunisia. Journal of AntimicrobialChemotherapy, 60, 1137-1141.

[11]Lartigue M, Poirel L, Nordmann P. 2004. Diversity of geneticenvironment of blaCTX-M genes. FEMS MicrobiologyLetters, 234, 201-207.

[12]Lartigue M F, Zinsus C, Wenger A, Bille J, Poirel L, Nordmann P. 2007. Extended spectrum ?-lactamases ofthe CTX-M type now in Switzerland. AntimicrobialAgents and Chemotherapy, 51, 2855-2860.

[13]Ma J Y, Zeng Z L, Chen Z L, Xu X G, Wang X Y, Deng Y T,Lu D H, Huang L Z, Zhang Y Y, Liu J H, et al. 2009. Highprevalence of plasmid-mediated quinolone resistancedeterminants qnr, aac(6)-Ib-cr, and qepA amongceftiofur-resistant Enterobacteriaceae isolates fromcompanion and f ood-produc ing An imal s .Antimicrobial Agents and Chemotherapy, 53, 519-524.

[14]Ma L, Lin C J, Chen J H, Fung C P, Chang F Y, Lai Y K, LinJ C, Siu L K. 2009. Widespread dissemination ofaminoglycoside resistance genes armA and rmtB inKlebsiella pneumoniae isolates in Taiwan producingCTX-M-type extended-spectrum ?-lactamases.Antimicrobial Agents and Chemotherapy, 53, 104-111.

[15]Ng L K, Martin I, Alfa M, Mulvey M. 2001. Multiplex PCRfor the detection of tetracycline resistant genes.Molecular and Cellular Probes, 15, 209-215.

[16]Pai H, Choi E-H, Lee H-J, Hong Y, Jacoby G A. 2001.Identification of CTX-M-14 extended-spectrum ?-lactamase in clinical isolates of Shigella sonnei,Escherichia coli, and Klebsiella pneumoniae in Korea.

[17]Journal of Clinical Microbiology, 39, 3747-3749.

[18]Podschun R, Ullmann U. 1998. Klebsiella spp. asnosocomial pathogens: epidemiology, taxonomy, typingmethods, and pathogenicity factors. ClinicalMicrobiology Reviews, 11, 589-603.

[19]Poirel L, Lartigue M F, Decousser J W, Nordmann P. 2005.ISEcp1B-mediated transposition of blaCTX-M inEscherichia col i . Antimicrobial Agents andChemotherapy, 49, 447-450.

[20]Sáenz Y, Brinas L, Domínguez E, Ruiz J, Zarazaga M, Vila J,Torres C. 2004. Mechanisms of resistance in multipleantibiotic-resistant Escherichia coli strains of human,animal, and food origins. Antimicrobial Agents andChemotherapy, 48, 3996-4001.

[21]Saladin M, Cao V T, Lambert T, Donay J L, Herrmann J L,Ould-Hocine Z, Verdet C, Delisle F, Philippon A, ArletG. 2002. Diversity of CTX-M ?-lactamases and theirpromoter regions from Enterobacteriaceae isolated inthree Parisian hospitals. FEMS Microbiology Letters,209, 161-168.

[22]Smet A, Martel A, Persoons D, Dewulf J, Heyndrickx M,Cloeckaert A, Praud K, Claeys G, Catry B, Herman L,et al. 2009. Comparative analysis of extended-spectrum-?-lactamase-carrying plasmids from different membersof Enterobacteriaceae isolated from poultry, pigs andhumans: evidence for a shared ?-lactam resistance genepool? Journal of Antimicrobial Chemotherapy, 63,1286-1288.

[23]van Vo A T T, Duijkeren E, Fluit A C, Gaastra W. 2007.Characteristics of extended-spectrum cephalosporinresistantEscherichia coli and Klebsiella pneumoniaeisolates from horses. Veterinary Microbiology, 124,248-255.

[24]Xu R, Liu J H, Hu G Z, Wu H, Chen Y X. 2011. MultiplexPCR for detection of three tetracycline resistance genesin E. coli isolated from duck. Acta Agricultural Jiangxi,23, 150-152. (in Chinese)

[25]Yuan L, Liu J H, Hu G Z, Pan Y S, Liu Z M, Mo J, Wei Y J.2009a. Molecular characterization of extended-spectrum?-lactamase-producing Escherichia coli isolates fromchickens in Henan province, China. Journal of MedicalMicrobiology, 58, 1449-1453.

[26]Yuan L, Mo J, Hu G Z, Li Z M, Wang H L. 2009b. Detectionof OXA-type ?-lactamases among 49 strains ofEscherichia coli isolated from chickens. ActaAgricultural Jiangxi, 21, 1-3. (in Chinese)
[1] HUANG Min, LIN Li, WU Yi-xin, Honhing Ho, HE Peng-fei, LI Guo-zhi, HE Peng-bo, XIONG Guo-ru, YUAN Yuan, HE Yue-qiu. Pathogenicity of Klebsiella pneumonia (KpC4) infecting maize and mice[J]. >Journal of Integrative Agriculture, 2016, 15(7): 1510-1520.
No Suggested Reading articles found!