Detection of Extended Spectrum β-Lactamase Producing Klebsiella pneumoniae and Escherichia coli in Two Hospitals in the Federal Capital Territory, Abuja, Nigeria ()
1. Introduction
Antimicrobial resistance has arisen across the globe in both nosocomial and community settings as a consequence of widespread antibiotics consumption [1]. The Beta-lactam antibiotics are among the most widely used antimicrobial agents worldwide. Destruction of these antibiotics by the bacterial enzyme, beta-lactamase is the most frequently encountered mechanism of resistance among Gram-negative microorganisms [2]. Multidrug resistant gram negative bacilli belonging to the family Enterobacteriaceae have been increasingly responsible for infections in many countries [3]. The emergence of multiresistance in the Enterobacteriaceae family needs attention, because these are important causative agents of hospital infections, typically associated with pneumonias, blood stream infections, urinary tract infections, bacteremia and other intra-abdominal infections [4,5]. By definition, ESBLs are β-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, which are inhibited by β-lactamase inhibitors such as clavulanic acid [6].
The presence of ESBLs has tremendous clinical significance due to the fact that ESBLs are frequently plasmid encoded and also in most cases the plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes therefore limiting antibiotic options in the treatment of ESBL-producing organisms [6]. The selection pressure that drives the emergence of ESBLs has usually been attributed to the intense use of oxyimino-beta lactams, mainly the third generation cephalosporins in addition to extensive use of broad spectrum antibiotics, prolonged hospitalization, indwelling devices and severe underlying diseases [7-9].
Several studies have demonstrated that ESBL-producing bacteria are isolated with increasing frequency in many parts of the world [3,10-12] and a number of reports are also available in other parts of this country [13,14]. The aim of this study was to detect ESBL-producing K. pneumoniae and E. coli from clinical isolates from both inpatients and outpatients to evaluate the risk factors that may be inherent in this location. This is necessary because the prevalence of resistant strains of these organisms varies from one geographical location to another. Two hospitals receiving the highest number of patients from the federal capital territory and its environs namely, the University of Abuja Teaching Hospital and National Hospital were chosen for this study. To our knowledge, no published study exists on ESBL in Klebsiella pnuemoniae and E. coli in this locality, probably reflecting the lack of appreciation of the problem.
2. Materials and Methods
2.1. Isolation and Identification of Klebsiella pneumoniae and Escherichia coli
The isolates were collected from the University of Abuja Teaching Hospital Gwagwalada and National Hospital, Abuja. The organisms were isolated from stool, pus, urine, sputum as well as wound and high vaginal swabs (HVS) samples of both out-patients and In-patients. Isolates presumed to be the etiologic agent responsible for the disease condition were used for the study. Isolates considered to be contaminants were not included in the screening for ESBL. Isolates from pus and wound swabs were however included in the screening even though the organisms are not usually associated with infection at these sites.
Briefly, swabs and clinical specimens were inoculated on eosin methylene blue agar (EMB, Oxoid) and MacConkey agar (Oxoid). After inoculation, the plates were incubated at 37˚C for 24 hr. The mucoids and smooth colonies suggesting K. pneumoniae strains were Gram stained. Routinely, Indian ink was used to detect the presence of capsules and isolates were also inoculated onto the screening media for biochemical identification: TSI (triple sugar iron), SIM (sulphate⁄indole⁄motility) and citrate agar (Oxoid), and incubated at 37˚C for 24 h. Colonies showing green metallic sheen on EMB and non mucoid round pinkish colonies on MacConkey were tested for production of indole, methyl red, Voges Proskaeur and citrate utilization (IMVIC).
2.2. Antimicrobial Susceptibility Tests
The K. pneumoniae and E. coli strains isolated were submitted to antimicrobial susceptibility testing according to the recommendations of the Clinical and Laboratory Standards Institute [15]. The turbidity of the suspensions used for sensitivity testing was adjusted to 0.5 McFarland standard and inoculated onto Mueller-Hinton agar medium followed by incubation at 35˚C ± 1˚C for 18 - 24 hrs. The following antimicrobial discs were used: ceftriaxone (CRO) (30 µg), ceftazidime (CAZ) (30 µg), cefepime (FEP) (30 µg), gentamicin (GEN) (10 µg), amikacin (AK) (30 µg), ciprofloxacin (CIP) (5 µg), chloramphenicol (C) (30 µg) and trimethoprim⁄sulfamethoxazole (SXT) (1.25⁄23.75 µg).
Isolates that exhibited a zone of inhibition of growth for ceftazidime and ceftriaxone ≤22 mm and ≤25 mm, respectively, were submitted to the combined disc test in order to check for ESBL-producing strains. The combined disc methodology used to detect ESBLproducing K. pneumoniae and E. coli was performed as recommended by CLSI [15]. The antimicrobials used were: cefotaxime (30 µg) and cefotaxime (30 µg) plus clavulanic acid (10 µg), and ceftazidime (30 µg) and ceftazidime (30 µg) plus clavulanic acid (10 µg). Results were interpreted according to the criteria established by the CLSI [16]. A 5 mm increase in a zone of inhibition of growth for cefotaxime plus clavulanic acid as compared with the zone around the cefotaxime disc, and a 5 mm increase in the zone diameter for ceftazidime plus clavulanic acid as compared with the zone formed by the ceftazidime disc, were confirmatory for the result of ESBL-producing strains.
Interpretation of results for other antibacterial agents was as per the guidelines of The European Committee on Antimicrobial Susceptibility Testing (EUCAST) for enterobacteriaceae [16].
2.3. Statistical Analysis
Chi square was used to analyze data on gender distribution of the isolates, site distribution of the isolates and frequency of ESBL production by K. pneumoniae and E. coli using the software Smith’s Statistical Package (SSP) version 2.80 copyright© 1995-2005 Gary Smith.
3. Results
A total of 215 isolates were collected from the University of Abuja Teaching Hospital and the National Hospital Abuja to determine ESBL production. Out of 215 isolates 128 were E. coli (60%) and 87 were K. pneumoniae (40%). The age range of these patients was between 2 - 78 years.
Figure 1 shows that more females were infected by these pathogens than men with values of 40.63% (52) for E. coli infections in men which was less than the 59.37% (76) for women. Similarly in K. pneumoniae infections the frequency was higher in females n = 52, (55.170%) compared to males n = 39, (44.83%). The observed differences in male and female proportions of the two pathogens were not significant with a p value of 0.74 χ2, 1 df, (p > 0.5).
The distribution of K. pneumoniae and E. coli in the different specimens examined is shown in Figure 2. The number of isolates obtained from different sites was not uniform. The highest numbers of both E. coli as well as K. pneumoniae were obtained from urine at 60 and 36 respectively. For E. coli diarrheic fecal samples, pus, HVS wound swabs and sputum in decreasing order accounted for the remainder of the isolates at 37, 17, 10 and 0 respectively. K. pneumoniae was isolated in decreasing order from sputum (30), pus (9), HVS (8), wound swabs (3) and stool (0). The distribution of E. coli and K. pneumoniae isolates was significantly different at these sites p value of 0.00000 χ2, 5 df, (p < 0.05).
Table 1 shows the antimicrobial susceptibilities of K. pneumoniae isolates to different classes of antibiotics namely third generation cephalosporins (ceftriaxone and ceftazidime), fourth generation cephalosporins (cefepime), aminoglycosides (gentamicin and amikacin),