Antimicrobial Susceptibility and Genetic Prevalence of Extended-Spectrum β-Lactamases in Gram-Negative Rods Isolated from Clinical Specimens in Pakistan

Abstract

The prevalence of extended-spectrum β-lactamase (ESBL) genes has increased remarkably, resulting in multidrug-resistant gram-negative rods (GNRs) in clinical specimens. This cross-sectional study aimed to determine the antimicrobial susceptibility of ESBL-producing GNRs and its correlation with corresponding genes. Two hundred and seventy-two (n = 272) samples were evaluated for the molecular identification of ESBL genes by polymerase chain reaction after confirmation with the modified double-disc synergy test. E. coli 64.0% (n = 174) was the most prevalent ESBL producer, followed by Klebsiella species 27.2% (n = seventy-four), Acinetobacter species 6.6% (n = eighteen) and others 2.2% (n = six). These ESBL-producing isolates showed resistance to β-lactam antibiotics, i.e., sulbactam/cefoperazone (41.5%), piperacillin/tazobactam (39.3%), meropenem (36.0%), imipenem (34.2%) and non- β-lactam antibiotics, i.e., nalidixic acid (89.0%), co-trimoxazole (84.9%), ciprofloxacin (82.4%), gentamicin (46.3%), nitrofurantoin (24.6%), amikacin (19.9%) and fosfomycin (19.9%). The incidences of the ESBLs-producing genes bla_CTX-M, bla_TEM, bla_OXA and bla_SHV were 91.2%, 61.8%, 39.3% and 17.6%, respectively. Among nine multiple-gene combinations, bla_CTX-M + bla_TEM (30.5%) was the most prevalent combination, followed by bla_CTX-M + bla_OXA + bla_TEM (14.0%), bla_CTX-M + bla_OXA (13.6%), bla_CTX-M + bla_TEM + bla_SHV (7.0%), bla_CTX-M + bla_SHV (2.2%), bla_CTX-M + bla_OXA + bla_SHV (2.2%) and bla_OXA + bla_TEM (1.8%). ESBLs producing GNRs carrying bla_CTX-M, bla_TEM, bla_OXA and bla_SHV showed resistances to β-lactam antibiotics, i.e., ampicillin, amoxillin-clavulanic acid, cefotaxime and ceftazidime but were susceptible to carbapenems (meropenem and imipenem), β-lactam-β-lactamase inhibitor combination (piperacillin/tazobactam) and non-β-lactam antibiotics i.e., aminoglycoside (amikacin and gentamicin), nitrofurantoin and fosfomycin. These antibiotics that demonstrated activity may be used to treat infections in clinical settings.

Keywords: CTX-M; OXA; SHV; TEM; disk diffusion method; extended-spectrum β-lactamases (ESBLs); gram-negative bacteria; polymerase chain reaction (PCR).

Figure 1

Visualization of DNA bands by gel electrophoresis. (a) Lane-NC and PC stand for negative and positive control, respectively. Lane-12 is showing band of 392 bp, which is related to bla_SHV gene, and lane-1, -2, -3, -4, -5, -6, -7, -8, -9, -10 and -11 are negative for bla_SHV gene. (b) Lane-NC and PC stand for negative and positive control, respectively. Lane-1, -2, -3, -4 and -6 are showing bands of 860 bp, which is related to bla_TEM gene. Lane-7 and -10 are showing bands of 619 bp, which is related to bla_OXA gene. Lane-8, -9 and -11 are showing both 860 bp and 619 bp bands, which are related to bla_TEM and bla_OXA, respectively. Lane-5 has no band. (c) Lane-NC and PC stand for negative and positive control, respectively. Lane-1 to -12 are showing bands of 593 bp, which is related to bla_CTX-M gene.