Extended-spectrum beta-lactamase production and multi-drug resistance among Enterobacteriaceae isolated in Addis Ababa, Ethiopia

Background The global emergence and spread of extended-spectrum beta-lactamases (ESBLs) producing Enterobacteriaceae have been threatening the ability to treat an infection. Hence, this study aimed to determine the prevalence of ESBL-producing and multi-drug resistance (MDR) Enterobacteriaceae (ESBLs-E) from different clinical specimens in Addis Ababa, Ethiopia. Methods A cross-sectional study was conducted from January 1 to May 30, 2017. A total of 426 Enterobacteriaceae isolates were identified from clinical specimens. The isolates were collected from four laboratories. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method on Muller Hinton agar (MHA). All Enterobacteriaceae were screened for ESBLs production using cefotaxime and ceftazidime as per CLSI guideline. Each ESBL screening positive Enterobacteriaceae were confirmed by a combination disk test (CDT). Data were entered and analyzed by using SPSS version-20. Result The most frequent Enterobacteriaceae were E. coli 228 (53.5%) and K. pneumoniae 103 (24.1%). The magnitude of ESBLs-E was 57.7% (246/426). The highest frequencies of ESBLs-E were observed in blood specimesns (84.4%) and the highest ESBLs production was observed in K. pneumoniae (85.4%). The highest resistance level was seen to sulfamethoxazole-trimethoprim (77.0%), amoxicillin with clavulanic acid (71.6%), cefotaxime (62.2%), cefepime (60.3%) and ceftazidime (60.8%). The overall magnitude of multi-drug resistance (MDR) level was 68.3%. Of ESBLs-E, 96.3% of them were MDR (P < 0.001). Conclusion There was a high prevalence of ESBLs-E and MDR isolate in Addis Ababa. Most of ESBLs-E was isolated primarily in blood and urine. The highest ESBLs production was observed among K. pneumoniae. Hence, strong infection control strategies must be implemented in hospital settings of the country.


Introduction
Enterobacteriaceae are Gram-negative, facultative anaerobes, and non-sporing bacilli. These bacteria have become one of the most important causes of nosocomial and community-acquired infections. They can cause urinary tract, respiratory tract, and bloodstream and wound infections. Increasing rates of antimicrobial resistance have become a worldwide problem predominantly caused by Gram-negative bacteria, the Enterobacteriaceae [1,2].
Beta-lactam drugs such as extended-spectrum penicillins, cephalosporins, monobactams, carbapenems, fluoroquinolones (e.g. ciprofloxacin) and aminoglycosides (e.g. gentamicin) are among the most prescribed antibiotics to treat infections caused by Enterobacteriaceae. The widespread use of beta-lactam antibiotics has caused the expansion of resistant Enterobacteriaceae. The most important mechanism of resistance to beta-lactam antibiotics involves the production of beta-lactamases (especially extended-spectrum beta-lactamases) that inactivate beta-lactam antibiotics and this continue to be the prominent cause of β-lactam antibiotics resistance among Enterobacteriaceae worldwide. ESBL-producing Enterobacteriaceae are important members of antibiotic-resistant bacteria that cause hospital and community-acquired infections [3,4].
ESBL is an enzyme that is produced by bacteria to become resistant to extended-spectrum penicillin, cephalosporins, and monobactams except for cephamycins and carbapenems. It is also inhibited by beta-lactamase inhibitors like clavulanic acid. A worrisome increasing trend has been reported on the development of resistance to extended-spectrum cephalosporins caused by ESBL producing Enterobacteriaceae [2,3,5]. Among Enterobacteriaceae, ESBLs have been found mostly in Klebsiella spp. and E. coli as well as in other Enterobacteriaceae families such as Enterobacter spps., Proteus spps, Citrobacter spps, Morganella spps, Providencia spps., Salmonella spp., and Serratia spps [6][7][8].
Being plasmid mediated, ESBL is easily transmitted among members of Enterobacteriaceae. The dissemination of this resistance applies not only to beta-lactams but also to other commonly used antibiotics such as fluoroquinolones, aminoglycosides, and sulphonamides [9,10]. Consequently, many patients need the 'last resort' antibiotics treatment such as carbapenems [2,11]. Again the use of carbapenems has led to the rapid selection of carbapenem-resistant Enterobacteriaceae [12]. Only a few antibiotics (e.g. carbapenems, colistin, tigecycline) are available to treat infection caused by ESBL-producing bacteria, although the in vivo efficacy and/or toxicity of these drugs is not well well-known [13,14].
Assessing ESBL producing Enterobacteriaceae in the local scenario is necessary to understand the epidemiology and the disease burden as well as to design and implement hospital infection control strategies to prevent the further occurrence and spread of such bacteria. However, little is known about the magnitude of ESBL producing Enterobacteriaceae in Addis Ababa, Ethiopia. Moreover, to the best of our knowledge, almost all clinical bacteriology laboratories in Ethiopia do not perform ESBL tests. Hence, this study aimed to determine the prevalence of ESBL producing and MDR Enterobacteriaceae in different clinical specimens in Addis Ababa, Ethiopia.

Study setting
A laboratory-based cross-sectional study was conducted from January to May 2017 at the Ethiopian Public Health Institute (EPHI) Clinical Bacteriology and Mycology National Reference Laboratory in Addis Ababa. This laboratory is Ethiopia's main referral laboratory and is accredited by the Ethiopian National Accreditation Office (ENAO). The Enterobacteriaceae isolates used for this study were collected from four microbiology laboratories: EPHI clinical bacteriology laboratory, International Clinical Laboratories (ICL), Tikur Anbessa Specialized Hospital (TASH), and Yekatit 12 Medical College Hospital Microbiology Laboratory. The isolates were collected using a convenient sampling technique. All consecutive Enterobacteriaceae isolated from clinical specimens in the selected bacteriology laboratories were included in the study. Demographic characteristics of the patients were recorded using a pre-developed worksheet. The isolates were collected using Tryptose Soy Broth (TSB) (Oxoid Ltd., Basingstoke, United Kingdom) containing 20% glycerol and temporarily stored at − 20°C in the respective laboratory. Within a week the isolates were transported to the EPHI clinical bacteriological laboratory using a cold box with ice.

Culture and identification
The isolates preserved at − 70°C were recovered by re-suspension of the stored isolate in Tryptose Soy Broth (Oxoid Ltd., Basingstoke, United Kingdom). After a few hours, the isolates were inoculated and incubated on MacConkey agar (Oxoid Ltd., Basingstoke, United Kingdom) at 37°C for 18-24 h. After incubation, the colony was characterized by colony appearance, Gram stain, and biochemical tests. The isolates were identified by standard microbiological laboratory methods [15]. Antibiotic susceptibility and ESBLs confirmatory tests were done using the pure isolate sub-cultured on to 5% sheep blood agar (HiMEDIA Laboratories Pvt. Ltd., Mumbai, India).

Preparation of clavulanate stock solution
For the combination disk test CDT method, the combined disks (Ceftazidime-clavulanate (30 μg/10 μg), and cefotaxime-clavulanate (30 μg/10 μg) disks) were prepared from in-house made clavulanate solution according to CLSI guideline [16]. From potassium clavulanate analytical standard powder (Sigma-Aldrich Corp, St. Louis, MO USA) stock solution of clavulanate at 1000 μg/ml was prepared, aliquoted, and stored at − 70°C . When we were ready to perform CDT (each day of testing), 10 μL of clavulanate solution was added to ceftazidime (30 μg) and cefotaxime (30 μg) disks (Abtek Biologicals Ltd., Liverpool, United Kingdom) and we allowed about 30 min for the clavulanate to absorb, and the disks to be dry enough for application. The combined disks were used immediately (within 30 min) after they had dried.

Screening for potential ESBL-producing isolate
The isolates that showed an inhibition zone size of ≤22 mm with ceftazidime (30 μg) and/or ≤ 27 mm with cefotaxime (30 μg) were considered as potential ESBL-producer (screening ESBL positive) and were selected for confirmation for ESBLs production using CDT as recommended by CLSI guideline [16].

Quality control and data quality assurance
Quality control for the new batch was performed using ATCC 25922 E. coli standard strain to check the quality of culture media and antibiotics disks. For the ESBL confirmatory test, K. pneumoniae ATCC® 700603 (ESBLs positive) and E. coli ATCC® 25922 (ESBLs negative) control strains were used to check the quality of the commercially purchased antibiotics disks and in-house prepared combination disks [16]. The data collection form was checked for its completeness and accuracy before recording the data. Culture and antibiotics susceptibility test results were recorded carefully before entry to SPSS software (version 20).

Data entry and analysis
Data were entered and analyzed using SPSS software (version 20). Proportions and the actual number of ESBL-producing Enterobacteriaceae isolates were used to describe frequency outputs for categorical variables. The data were presented in table and graphs. Mean and standard deviation were used to describe continuous variables.

Demographic characteristics of the patients
A total of 426 consecutive non-repetitive Enterobacteriaceae isolates were collected from the four microbiology laboratories from January 1 to May 30, 2017. During the study period, we obtained 150 isolates from International Clinical Laboratories (ICL), 118 isolates from Tikur Anbessa Specialized Hospital (TASH), 89 isolates from Clinical Bacteriology and Mycology National Reference Laboratory in Ethiopian Public Health Institute (EPHI), and 69 isolates from Yekatit 12 Medical College Hospital (YMCH). These isolates were identified from different clinical specimens: 272 from urine; 90 from blood; 40 from pus; 11 from body fluids; 6 from sputum; 3 from ear discharge; 2 from eye discharge; and 2 from cerebrospinal fluid (CSF) ( Table 1).

Multi-drug resistant Enterobacteriaceae
Overall, 68.3% (291/426) of the Enterobacteriaceae isolates were multi-drug resistant (MDR, non-susceptible to at least 3 antibiotics belonging to different antibiotics categories), among which E. coli and K. pneumoniae contributed to 35.0% (150/426) and 20% (85/426) of the Ear & Eye discharge (5)     Abbreviations: R0 stands for resistance for zero antibiotics; R1 stands for resistance to one drug, R2 stands for resistance to two drugs and so on; and ≥ R3 stands for resistance to 3 or more antibiotics from different classes; MDR-E stands for multi-drug resistant Enterobacteriaceae   The distribution of ESBL producers varied among different species of Enterobacteriaceae. The highest intra-species frequency of ESBL production was observed among K. pneumoniae 78.6% (81/103) followed by E. coli and Citrobacter species with 52.2% (119/228) and 51.7% (15/29), respectively (Fig. 2). The lowest intra-species ESBL production was observed in P. mirabilis with 20% (1/5) proportion.

Distribution of ESBL-producing Enterobacteriaceae with their MDR level among different clinical specimens
From all specimens included in this study, the highest magnitude of ESBL-producing Enterobacteriaceae (84.4%; 76/90) and MDR (83.3%; 75/90) was found in blood. In the urine specimen, the extent of ESBL-producing Enterobacteriaceae and MDR were 50.7% (138/272) and 66.5% (181/272), respectively (Table 5). Of all ESBL-producing Enterobacteriaceae, 96.3% (237/246) were MDR, whereas only 30% (54/180) of the non-ESBL producers were MDR. There was a significant correlation (Pearson correlation of 0.759, p-value of 0.01) between ESBL production and MDR Enterobacteriaceae. Binary logistic regression or bivariate analysis also showed that being an ESBL producer has statistically significant association with MDR (P < 0.001). That is, the odds of being MDR were 61.4 times (95% CI   Fig. 3.

Discussions
ESBL-producing Enterobacteriaceae have become a serious worldwide problem. Dissemination of ESBLs compromises the activity of broad-spectrum antibiotics creating major therapeutic difficulties with a significant impact on the outcomes for patients [19].

Prevalence of ESBL-producing Enterobacteriaceae
In the present study, the magnitude of ESBL-producing Enterobacteriaceae was 57.7%, which is higher than magnitudes reported by previous researchers in Ethiopia: 38.4% in Jimma by Siraj and his colleagues [20], 36% in Jimma by Mulualem Y and his colleagues [21], 33.3% in Harar [22] and 25% in Adama [23]. The emergence of ESBL-producing Enterobacteriaceae in higher magnitude in Addis Ababa emphasizes the need to implement strong infection control strategies. The magnitude of ESBL-producing Enterobacteriaceae (57.7%) in our study was comparable with a studies in Bahir-Dar-Ethiopia (57.6%) [24], Burkina Faso (58.0%) [25], Uganda (62.0%) [26], Ghana (49.3%) [27], and Karnataka-India (57.5%) [28]. One of the most important factors in the emergence of ESBLs production is the selective pressure caused by the use of 3rd generation cephalosporins [29,30]. Lack of antibiotic surveillance, antibiotics misuse, and weak infection control measures may also contribute to the high magnitude of ESBL.

Multi-drug resistance among Enterobacteriaceae
In the present study, the overall magnitude of MDR among all Enterobacteriaceae isolate (68.3%) was fairly similar with a study done in Dessie, Ethiopia (74.6%) [45], Gondar, Ethiopia (68%) [50], and Nepal (64.04%) [51]. The higher proportion of MDR limits the treatment option for hospital-acquired infections caused by Enterobacteriaceae. On the other hand, our result was lower than findings from other studies in Gondar, Ethiopia (93.5 and 87.4%) [46,52], Bahir-Dar (93.1%) [53], Nepal (96.84%) [43], and Sierra Leone (85.7%) [44]. The difference in magnitude of MDR isolates might be due to the selection of antibiotic from a different class, the definition for MDR, study period and specimen type, and the difference in the study population.
There was an intra-species difference in MDR level. The present study showed that the level of MDR in K. pneumoniae (82.5%) and E. coli (65.3%) was comparable with studies conducted in Equatorial Guinea (E. coli 74.4%) [49], Sierra Leone (K. pneumoniae 73.3%, E. coli 61.5%) [44]. However, our result is lower than studies conducted in Gondar, Ethiopia (K. pneumoniae 95.6%, E. coli 92.9% [52], Khartoum, Sudan (E. coli 92.2%) [48], and Equatorial Guinea (K. pneumoniae 91.7%) [49]. The MDR level among E. coli (50.2%) in Dessie, Ethiopia is lower than our study [45]. The difference in MDR level among K. pneumoniae and E. coli in our study might be due to most K. pneumoniae being isolated from blood specimens collected from hospital inpatients.
In this study 237 (96.3%) of the ESBL-producers were MDR strains, whereas only 54 (30%) of the non-ESBL-producers were MDR strains. The ESBL-producing isolates had increased resistance compared with non-ESBL-producers indicating that MDR is expected to be more common in ESBL-producing bacteria.

Strength of the study
This is the first study done at multiple health facilities on the magnitude of ESBL-producing Enterobacteriaceae in Addis Ababa, Ethiopia. This multi-centered study can reveal the extent of distribution of ESBLs and MDR among Enterobacteriaceae and the degree of resistance to other non-beta-lactam antibiotics. The magnitude of ESBLs and MDR in the city was done in a relatively larger number of specimens and isolates than in earlier studies.

Limitation of the study
-Although combinations of aminoglycosides and fluoroquinolones were tested, other beta-lactams and beta-lactamase inhibitors, such as tigacycline, colistin, and piperacillin/tazobactam, were not tested, as they were beyond the scope of this study. -We are unable to see possible risk factors, certain clinical features and the outcome of the patients infected with ESBL-producing or MDR bacteria, due to lack of adequate resource. -Although most of the study isolates were collected from inpatients, the exact number of nosocomial versus community-acquired bacteria were not differentiated. -The isolates were collected from four bacteriology laboratories in Addis Ababa, but the results may not be applied to the entire city or country.

Conclusion and recommendation
There was a high prevalence of ESBL-producing Enterobacteriaceae and MDR isolates. The majority of ESBL-producing isolates were found primarily in blood and urine specimens. The most frequent ESBL-producing Enterobacteriaceae were K. pneumoniae and E. coli. A higher level of resistance to multiple classes of antibiotics was observed among ESBL producers compared with non-ESBL producers. The better options for the treatment of ESBL-producing Enterobacteriaceae are meropenem, amikacin, and cefoxitin. ESBL-producing isolates showed a high rate of resistance to ciprofloxacin, cefepime, cotrimoxazole, and gentamicin compared with non-ESBL producers. The rise of MDR and ESBLs necessitates the strengthening of clinical bacteriology research and the diagnostic capacity of laboratory professionals for the detection and surveillance of antibiotic resistance. We recommend routine screening of ESBLs production of Enterobacteriaceae along with strong infection prevention strategies.

Funding
Antibiotics disks and media were supplied by EPHI. However, the other costs were covered by the principal investigator. The supplier body had no influence on study design, data collection, analysis and interpretation of data and writing the manuscript.

Availability of data and materials
The current study data sets used for analysis can be obtained from the corresponding author through email (dejenie21@gmail.com) on reasonable request.
Authors' contributions DS: Conceived, designed, analyzed and interpreted the research; and also wrote the manuscript. TL, AA and HK: Participated in the technical laboratory works and data collection: KD and MH: Supervised the study through their critical review of the research and the manuscript write up. All authors read and approved the final manuscript.

Ethics approval and consent to participate
The study proposal was reviewed and approved by the department of research and ethics review committee of the Medical Laboratory sciences, College of Health Sciences; Addis Ababa University (Ref. No. MLS/223/17). Permission was obtained from the respective laboratories in where the isolate and data were collected.

Consent for publication
Not applicable.