1. MOLECULAR CHARACTERIZATION OF EXTENDED SPECTRUM
BETA-LACTAMASE-PRODUCING GRAM-NEGATIVE
UROPATHOGENS AT SPECIALIST HOSPITAL SOKOTO, NIGERIA
*T. Nuhu1, R.O. Bolaji2, H.Y. Ungo1, A. Olowookere1 and B.O. Olayinka2
1Department of Pharmaceutics and Pharmaceutical Microbiology, Usmanu Danfodiyo University, Sokoto. (nuhu.tanko@yahoo.com)
2Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University, Zaria. (busayoolayinka@yahoo.com)
Background: Extended Spectrum Beta-Lactamase (ESBL)-producing Enterobacteriaceae are fast becoming the leading cause of antibiotic resistant infection.
Objective of the study: The study determined the prevalence and genetic profiles of ESBL-producing uropathogens among members of the family Enterobacteriaceae at
Specialist Hospital Sokoto.
Methodology: A total of 365 mid-stream urine samples collected over a period of 4 months were studied. Isolates were identified using Microgen Identification Kit, GN-
ID. Susceptibility testing was performed using the modified Kirby Bauer method, and results were interpreted according to Clinical Laboratory Standard Institute (CLSI),
2012. ESBL production was detected by Double Disc Synergy Test. Molecular characterization of the ESBL positive isolates were confirmed by PCR method.
Results: A total of 64 Gram negative uropathogens were isolated from 365 urine samples. The isolates were made up of E. coli (29.7%), Salmonella arizonae (23.4%),
Klebsiella oxytoca (10.9%), Klebsiella pnuemoniae (4.7%), Enterobacter gergoviae (9.4%), Enterobacter aerogenes (1.6%), Citrobacter freundii (6.3%), Serratia
marscense (6.3%), Proteus mirabilis (1.6%), Edwardsiella tarda (1.6%), Acinetobacter iwoffii (1.6%), Burkholderia pseudomallei (1.6%), and Pseudomonas aeruginosa
(1.6%). The antibiotic susceptibility profile of the isolates to seven (7) commonly prescribed antibiotics used in the treatment of UTI - associated infections showed that
the isolates were resistant to cotrimoxazole (71.9%), nalidixic acid (67.2%), ciprofloxacin (54.7%), norfloxacin (53.1%), gentamicin (50%), amoxicillin/clavulanic acid
(48.4%) and nitrofurantoin (29.7%). Majority of the isolates were multidrug resistant (64.1%), 15.6% were extended drug resistant (XDR), 4.7% were pandrug resistant
(PDR), while 15.6% do not fall into any of the classification. The PCR genomic DNA of the 15 MDR-ESBL-producing isolates showed that 73.3% were CTX-M, while 26.7%
were OXA.
Conclusion: The prevalence of ESBL in this study was 23.4%. Molecular characterization of the selected MDR-ESBL - producing isolates showed that they harboured the
blaCTX-M gene (73.3%) and blaOXA gene (26.7%).
Keywords: Molecular characterization, ESBL, Uropathogens, Sokoto
INTRODUCTION
Urinary Tract Infections (UTIs) has become the most common hospital-acquired infection, accounting for as many as 35% of nosocomial infections, and it is the second most
common cause of bacteremia in hospitalized patients . It is one of the most common bacterial diseases worldwide that is characterized by a wide range of symptoms from mild
irritative voiding to bacteremia, sepsis, or even death . Hawkey (2008), stated that “since the introduction of antibiotics in the 1950s, there has been a steady and significant
increase in the number of resistant strains of bacteria”. The most important draw-back in the treatment of UTIs is the development of antimicrobial resistance. The improper and
uncontrolled use of many antibiotics resulted in the occurrence of antimicrobial resistance, which became a major health problem worldwide.
Antimicrobial resistance among pathogenic bacteria is increasing worldwide especially against beta-lactam drugs, due to the development of beta-lactamase enzymes (NNIS
System, 2003) which destroy the beta-lactam ring of these antibiotics, thus preventing the action of penicillin binding proteins (PBPs) (Livermore, 1995). In the battle against beta-
lactamase mediated bacterial resistance, development of third generation cephalosporins in the early 1980s was a major breakthrough. But soon, a new plasmid encoded beta-
lactamase capable of hydrolyzing the extended spectrum cephalosporins was reported (Knothe et al., 1983; Jacoby and Medeiros, 1991).
Beta-lactamase producing Enterobacteriaceae has become a global problem . The incidence and type of beta-lactamase enzyme varies in different geographical locations. The
ability of bacteria to produce extended-spectrum beta-lactamases (ESBLs) that hydrolyze penicillins, cephalosporins, and monobactams has resulted in intractable infections and
serious consequences for infection control (Marchandin et al., 1999; Philippon et al., 1989).
A commonly used working definition is that, ESBLs are beta-lactamases capable of conferring bacterial resistance to the penicillins; first, second and third generation
cephalosporins; and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these antibiotics, and which are inhibited by beta-lactamase inhibitors such as
clavulanic acid (Paterson and Bonomo, 2005).
ESBL producing bacteria especially E. coli and K. pneumoniae have emerged as serious pathogens both in hospital and community acquired UTIs (Venkatadri et al., 2014). The
study sought to determine the prevalence of ESBL-producing uropathogens and characterize the ESBL genes.
MATERIALS AND METHODS
Sample Collection:Early morning, mid-stream clean catch urine samples were collected by patients in a sterile disposable containers with screw caps. Prior to urine collection,
patients were counseled on how to collect urine sample by observing all aseptic conditions to avoid contamination.
Isolation and Culturing of Urine Samples:Urine samples were inoculated on CLED Agar using calibrated wire loop. Samples with counts up to and greater than 105cfu/ml were
counted microscopically and considered positive for further analysis.
Antibiotic Susceptibility Testing (AST):The antibiotic susceptibility of the isolates was determined against seven (7) commonly prescribed antibiotics in Specialist Hospital Sokoto
using the modified Kirby Bauer disc agar diffusion (CLSI, 2012).
Screening and Phenotypic Confirmatory Test for ESBL Production: Gram-negative isolates were screened for ESBL production. The double disc synergy test (DDST) was
adopted to confirm for the presence of ESBLs.
Molecular Characterization of Resistant Isolates: Genomic DNA extraction was carried out using Zymo Research Quick-gDNATM MiniPrep. To ascertain that genomic DNA was
actually extracted, the eluent was subjected to agarose gel electrophoresis. Amplification of resistant DNA fragments was carried out using Dream TaqTM DNA polymerase, which is
an enhanced Taq DNA Polymerase optimized for high throughput PCR applications. A 2% agarose gel was used to resolve the PCR genomic DNA fragments with their primers .
CONCLUSIONS
E. coli and Salmonella arizonae were the most prevalent Gram-negative isolates. Gram-negative uropathogens
were generally resistant to Cotrimoxazole (73.3%), Nalidixic acid (66.7%), Norfloxacin (53.5%), and
Ciprofloxacin (50.5%). Multidrug resistant isolates were 64.1%, extended drug resistant (15.6%), pandrug
resistant (4.7%) and 15.6% did not fall into the category of classification. All the ESBL producers were MDR,
and their MARI range from 0.3 to 1.0.ESBL production was 23.4% among the Gram-negative isolates. The
incidence of bla CTX-M and bla OXA gene were reported with 26.7% producing (bla CTX-M and bla OXA), 73.3%
produced bla CTX-M only, and 26.7% produced bla OXA only.
29.7
23.410.9
4.7
9.4
1.6
6.3
6.3
1.6
1.6
1.6 1.6
1.6
E. coli
Sal arizonae
Kleb oxytoca
Kleb pneumoniae
E. gergoviae
E. aerogenes
C.freundii
S. marscence
P. mirabilis
E. tarda
A. iwoffii
B. pseudomallei
Ps aeruginosa
Fig. 2: Percentage distribution of Gram-negative isolates from urine.
0
10
20
30
40
50
60
70
80
PercentageSusceptibility
Antibiotics
Sensitive
Resistant
Fig.2: Percentage susceptibility of Gram-negative uropathogens
Fig. 3:Genomic DNA PCR analysis of CTX-M gene in
ESBL isolates
Fig.4: Genomic DNA PCR analysis of OXA gene in ESBL isolates
14th NATIONAL CONFERENCE AND SCIENTIFIC MEETING, NIGERIA ASSOCIATION OF PHARMACISTS IN ACADEMIA. FACULTY OF PHARMACY, OLABISI ONABANJO UNIVERSITY , SAGAMU, OGUN STATE, NIGERIA. 11TH – 15TH JULY, 2016
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•Paterson D.L, and Bonomo R.A. (2005). Extended-spectrum beta-lactamases: A clinical
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•Clinical Laboratory Standard Institute (CLSI) (2012). M100-S24 - Performance Standards for
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