Magnitude and antimicrobial susceptibility patterns of extended-spectrum β-lactamase producing Enterobacteriaceae recovered from environmental samples at Tikur Anbessa Specialized Hospital, Ethiopia

Background The hospital environment especially the intensive care units (ICUs) contributes to the spread of extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, which are contributing to increasing morbidity and mortality rates. We aimed to assess the rate of environmental contamination of ESBL producing Enterobacteriaceae and their antimicrobial susceptibility patterns. A Cross-sectional study was conducted at Tikur Anbessa Specialized Hospital (TASH) from June to July, 2018. A total of 65 environmental swab were taken from inanimate items. All isolates from the inanimate environment were cultured by using ESBL ChromoSelect Agar and identied using conventional bacteriological methods. All ESBL producing Enterobacteriaceae isolates were tested for their antimicrobial susceptibility using the disc diffusion method according to the methods and interpretive criteria of the Clinical and Laboratory Standards Institute (CLSI).


Conclusion
The appearance of ESBL producing Enterobacteriaceae among ICUs hospital environment is posing a serious threat for the control of nosocomial infections. The high resistance found from this study shows the need for policies for devising infection control procedures and screening policies for ESBL on a routine basis.

Background
In Gram-negative bacteria, β-lactamase production remains the most important mechanism of resistance to β-lactam group of antibiotics and has become a global challenge in infection control. One group of these enzymes is called extended-spectrum β-lactamases (ESBLs) [1][2][3]. ESBLs hydrolyze a wide range of cephalosporins including the oxyimino group of cephalosporins such as ceftriaxone, ceftazidime, cefotaxime and the monobactam drugs such as aztreonam, but do not hydrolyze cephamycins and carbapenems, and their activity is inhibited by clavulanic acid [3,4].
ESBL producing bacteria have been detected in inanimate surfaces of hospitals which could contribute to their dissemination [5,6]. The detection of bacterial pathogens including ESBL producing bacteria from inanimate hospital environments is due to an innate ability of bacteria to survive on surfaces for long periods [7]. For instance, Klebsiella spp and Escherichia coli, which are the predominant ESBL producing Enterobacteriaceae, have been reported to survive up to 30 and 16 months, respectively, on inanimate dry surfaces [5].
In Ethiopia the rate of ESBLs on clinical isolates has been increased from time to time [8][9][10]. The increased rate of ESBLproducing bacteria in Ethiopia may be due to the cross transmission of strains from patient to patient or from the inanimate hospital environment to the patients and from health care workers to patients and vice versa [6]. Moreover, the horizontal transmission of resistance genes through genetic mobile elements has also important role in adaption of bacteria for various conditions in the hospital environments [11,12]. The contaminated hospital environment in the transmission of ESBL producing Enterobacteriaceae is widely considered the principal cause of hospital acquired infections (HAIs) [13].
Despite the recent worldwide spread of ESBL in K. pneumoniae and E. coli isolates from HAIs, their dissemination has been little studied especially on inanimate hospital environments in Ethiopia. This study was therefore aimed at determining the rate of environmental contamination of ESBL producing Enterobacteriaceae and their antimicrobial susceptibility patterns will have a valuable input to control the emerging problem in this direction.

Study design and Environmental Sampling
This cross-sectional study included 65 environmental swabs at Tikur Anbessa Specialized Hospital from June to July, 2018. The samples were collected from four sites including the intensive care units of Surgical, Pediatric, Medical and Medical-Surgical wards of the hospital. The samples were collected on a single occasion, using pre-moistened sterile swabs from environmental surfaces and medical equipment including beds, monitors, linens, ventilators, suction machine, Lobby (furniture), work station and sinks in accordance to suggestions from previous studies [14,15].

Culture and Bacterial isolates
The swab samples were enriched overnight at 37 °C in brain heart infusion broth (BHI) (Merck, Germany) [16]. A loop full of bacterial growth was transferred to ESBL ChromoSelect Agar (SIGMA-ALDRICH) and incubated for 24 h at 37°C to assess the ESBL production. Bacterial identi cation was achieved using speci c colony color on ESBL ChromoSelect agar and Gram staining combined with panel of biochemical tests including Triple Sugar Iron agar (TSI), urea, citrate, Sul de Indole Motility (SIM) medium, Lysine Iron Agar (LIA), mannitol, malonate and oxidase [17].

Phenotypic con rmation of ESBL
All the identi ed isolates were phenotypically con rmed for ESBL production using Combination Disk (CD) method on Mueller-Hinton Agar (MHA) (Oxoid, UK). Brie y, a cefotaxime disc (30 μg) was placed 20 mm away from a disc containing cefotaxime-clavulanic acid (30/10 μg). When the inhibition zone between at least one of the combination discs and its corresponding single antibiotic disc differed by ≥5 mm, the strain was identi ed as an ESBL producer according CLSI guideline [18]. Escherichia coli ATCC 25922 and Klebsiella pneumonia ATCC 700603 were used as the control strains.
MDR was de ned as acquired non-susceptibility to at least one agent in three or more antimicrobial categories [19].

Ethics approval and consent to participate
The study protocol was reviewed and approved by Department of Microbiology, Immunology and Parasitology Research Ethics Review Committee (DRERC), College of Health Sciences, Addis Ababa University.
We found a high proportion of ESBL producers among E. coli and K. pneumoniae isolates which has similarly been reported from studies in Gondar, Ethiopia [6], Zimbabwe [22], Gaza in Palestine [23] and Algeria [24]. Given the fact that Klebsiella spp and Escherichia coli have capacity to survive up to 30 and 16 months, respectively, on inanimate dry surfaces [5]. Moreover, isolation of such Enterobacteriaceae is highly indicative of fecal contamination and poor hand washing practices amongst health workers and patients [25].
The Pediatrics and Medical-Surgical ICU exhibited the highest ESBL producing Enterobacteriaceae, each with (37.5%, 6/16). This may lead to cross-infections between patients in the same ICU because patients present in these ICUs are likely to have reduced immune system due to illness, surgical and mechanical manipulation, and/or the use of immunesuppressors and other therapeutic drugs, all of which increasing patients susceptibility to infections [27,28].
Bed linens, ventilators, beds and sink were inanimate surfaces observed to be associated with variable degree of ESBL producing Enterobacteriaceae positivity, with 31.3%, 12.5% and 6.5%, respectively. The identi cation of ESBL producing bacteria on linens, sink, bed and other environmental surface is consistent with reports from the literatures [5,6,12,20]. It is generally assumed that ESBL producing Gram-negative bacteria require moist or damp sites for enhanced longevity [12,24]. Moreover, bed linens are considered to be high patient-contact surfaces and therefore the detected pathogens might have been shed by the infected/colonized patients occupying the particular beds.
In regards to level of antimicrobial resistance among our ESBL producing isolates, signi cantly high resistance level was recorded to penicillin groups such as ampicillin (100%) and cephalosporin groups such as cefuroxime (96%), ceftazidime (92%), cefepime (86%), azetronome (80%) and cefotaxime (72%). These results were mostly comparable with results from other studies conducted in Ethiopia (cefpirome, cefpodoxime, ceftazidime, ceftriaxone and amoxicillin with clavulanic acid each with 100% resistance level) [6] and Algeria where ampicillin resistance level was reported at 98.1% [24]. Probably lack of antibiotic resistance screening and con rmatory testing prior to using these drugs could be responsible for accumulation of such high level of resistance among ESBL producing bacteria in the hospital environments.
Reports from other studies showed that multidrug resistance (MDR) to the commonly prescribed antimicrobial agents are more common among ESBL producers found from inanimate hospital surfaces [29,30]. Hence, unwise use of antibiotics can inadvertently favor emergence of multidrug resistant bacterial strains [11,28]. The nding from the current study showed that (15/16, 93.8%) of the ESBL producing bacterial isolates were resistant to at least 3 antibiotics, showing how rampant MDR Enterobacteriaceae are in the hospital environments. This nding is higher than reports from Northwest Ethiopia (75%) [31], Zimbabwe (75%) [22] and Iran (79.4%) [32].

Conclusions
In this study the appearance of ESBL producing Enterobacteriaceae among ICUs hospital environment in the study site poses a serious threat for the control of nosocomial infections. Occurrence of multidrug resistance (MDR) to the commonly prescribed antimicrobial agents is found to be more common among ESBL producers, showing the need for policies to prevent misuse and overuse of antimicrobial agents. Moreover, it also indicates the need for infection control procedures and screening policies for ESBL on a routine basis in the hospital environment.

Availability of data and materials
The dataset supporting the ndings of this article is available from the corresponding author upon request.