Detection of Plasmid-Borne β-Lactamase Genes in Extended-
Spectrum β-Lactamase (ESBL) and Non-ESBL-Producing
Escherichia coli Clinical Isolates

Merlyn C. Cruz1,2 and Cynthia T. Hedreyda2

1Angeles University Foundation, Mac Arthur Highway, Angeles City, Pampanga
2National Institute of Molecular Biology and Biotechnology,
University of the Philippines Diliman, Quezon City

*Corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.



Increasing frequency of infections caused by antibiotic resistant Escherichia coli strains producing extended-spectrum β-lactamase (ESBL) needs to be addressed by continuous surveillance and accurate detection of specific ESBLs genes for more effective treatment. A total of 71 β-lactam drug resistant isolates (26 phenotypically ESBL-producing and 45 non-ESBL-producing) were observed to carry approximately 23 kb plasmids.  These isolates were subjected to β-lactamase gene-targeted PCR to detect plasmid-encoded blaTEM, blaSHV, blaCTX-M group1 and blaCTX-M group9 genes. BLAST analysis of amplicons revealed that plasmid-encoded blaTEM is most prevalent in both ESBL and non-ESBL-producing E. coli isolates. Plasmid-encoded blaSHV gene was only detected in 8 non-ESBL-producing isolates and explanation of such observation awaits additional studies to detect the possibility that the gene could be in the chromosomal DNA or to test the prevalence of the plasmid-encoded gene with more isolates. Twelve isolates of the ESBL-type blaCTX-M were identified from phenotypically identified ESBLs, comparable with 13 isolates detected with blaTEM. This observation suggests that the relatively newly emerging ESBL-type CTX-M is continuously increasing as one of the new β-lactamase derivatives among ESBL-producing E. coli in the clinical setting. This study reveals that there is discrepancy between the results of the phenotypic observation and genotypic analysis showing that the presence of ESBL-associated      β-lactamase genes may be undetected when using the conventional phenotypic approach. Mutation in these unexpressed genes may result to ESBL antibiotic resistance, suggesting that the unexpressed and undetected genes may serve as reservoir for ESBL genes.


Drug resistance among bacteria is largely attributed to their production of β-lactamase enzyme that can hydrolyze or inactivate the β-lactam drugs that interfere with bacterial cell wall synthesis (Bauman 2004). β-lactam drugs include the antibiotics penicillins, cephalosporins, cephamycins, carbapenems, mono-bactams and β-lactamase inhibitors. Infections caused by members of Enterobacteriaceae such as Escherichia coli, are most often difficult to treat due to their resistance particularly to β-lactam drugs. . . . . read more

AHMED OI, EL-HADY SA, AHMED TM, AHMED IZ. 2013. Detection of bla SHV and bla CTX-M genes in ESBL producing Klebsiella pneumoniae isolated from Egyptian patients with suspected nosocomial infections. Egyptian Journal of Medical Human Genetics 14(3): 277-283.
BAUMAN RW. 2004. Controlling microbial growth in the body: Antimicrobial drugs. In: Microbiology: With diseases by body systems. RW Bauman ed. CA: Pearson Benjamin Cummings. 301-310p.
BOMASANG ES, MENDOZA MT. 2003. Prevalence and risk factors associated with extended-spectrum beta-lactamase (ESBL) production among selected Enterobacteriaceae isolates at the Philippine General Hospital. Phil J Microbiol Infect Dis 32(4): 151-158.
BONNET R. 2004. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother 48:1-14.
BRADFORD PA. 2001. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clinical Microbiology Reviews 14: 933-951.
BUSH K, JACOBY GA. 2010.  Minireview: Updated functional classification of β-lactamases. Antimicrobial Agents and Chemotherapy 54:969-976.
[CLSI] Clinical Laboratory Standards Institute. 2013. Performance standards for antimicrobial disk susceptibility testing; Twenty-third Informational Supplement.  CLSI document M100-S23 33(1): 44-49.
CRUZ M, BACANI C, MENDOZA A, HEDREYDA C. 2014. Evaluation of extended-spectrum beta-lactamase production in Escherichia coli clinical isolates in three hospitals in Luzon. Philippine Science Letters 7(2): 438-444.
DALLENNE C, DA COSATA A, DECRE D, FAVIER C, ARLET G. 2010. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. J Antimicrob Chemother  65(3): 490-495.
DHILLON RHP, CLARK J. 2012. ESBLs: A clear and present danger?. Critical Care Research and Practice. Article ID 625170 2012.
JARLIER V, NICOLAS M, FOURNIER G, PHILIPPON A. 1988. Extended spectrum β-lactamase conferring transferable resistance to new β-lactam agents in Enterobacteriaceae: Hospital prevalence and suspicious patterns. Review of Infectious Disease 10: 867-878.
KOMATSU, M, IKEDA N, AIHARA M, NAKAMICHI Y, KINOSHITA S, YAMASAKI K, SHIMAKAWA K. 2001. Hospital outbreak of MEN-1-derived extended spectrum beta-lactamase-producing Klebsiella pneumoniae. J Infect Chemother 7: 94-101.
LARTIGUE MF, POIREL L, AUBERT D, NORDMANN P. 2006. In vitro analysis of ISEcp1B-mediated mobilization of naturally occurring β-lactamase gene blaCTX-M of Kluyvera ascorbata. Antimicrobial Agents and Chemotherapy 50: 1282-1286
LOTA MM, LATORRE AE. 2014. A retrospective study on extended-spectrum beta-lactamase bacteria in the Philippines from 1999-2013. Acta Medica Philippina 48(1): 28-35.
LUCENA MA, METILLO EB, OCLARIT JM. 2012. Prevalence of CTX-M extended-spectrum beta-lactamase-producing Enterobacteriaceae at a private tertiary hospital in Southern Philippines. Philippine Journal of Science 141(1): 117-121.
MEDEIROS AA. 1984. Βeta-Lactamases. Br Med Bull 40: 18-27.
OGBOLU DO, DAINI OA, OGUNLEDUN A, TERRY ALLI OA, WEBBER MA. 2013. Dissemination of IncF plasmids carrying beta-lactamase genes in Gram-negative bacteria from Nigerian hospitals. J Infect Dev Ctries 7(5): 382-390.
PATERSON DL, BONOMO RA. 2005. Extended-spectrum beta-lactamases: A clinical update. Clin Microbiol Rev 18: 657-686.
SCHMITT J, JACOBS E, SCHMIDTT H. 2007. Molecular characterization of extended-spectrum beta-lactamases in Enterobacteriaceae from patients of two hospitals in Saxony, Germany. J Med Micro 56: 241-249.
TIAN GB, GARCIA J, ADAMS-HADUCH JM, EVANGELISTA JP, DESTURA RV, WANG HN, DOI Y 2010. CTX-M as the predominant extended-spectrum Beta-lactamases Among Enterobacteriaceae in Manila, Philippines. J Antimicrob Chemother 65(3): 584-586.
VILLANUEVA FD, TUPASI TE, ABIAD HG, BAELLO BQ, CARDAñO RC. 2003. Extended-spectrum β-lactamase production among Escherichia coli and Klebsiella spp. at the Makati Medical Center: tentative solutions. Phil J Microbiol Infect Dis 32(3): 103-108.
XU L, ZHAI Y, LYU Y, WANG Q, AN S, CHEN J, CHEN Y, LIU L, LI J, GAO Z. 2014. Identification of Klebsiella pneumoniae strains harboring inactive extended-spectrum beta-lactamase antibiotic-resistance genes. Chinese Medical Journal 127(15) 3051-3057.
YAZDI M, NAZEMI A, MIRINARGASI M, JAFARPOUR M, SHARIFI SH. 2012. Genotypic versus phenotypic methods to detect extended-spectrum beta-lactamases (ESBLs) in uropathogenic Escherichia coli. Annals of Biological Research 3(5): 2454-2458.