Faecal Carriage Rate of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli and Klebsiella Pneumonia Among Apparently Health Food Handlers in Dilla University Student Cafeteria

Background: The rapid spread of extended-spectrum β- lactamases (ESBL) producing enterobacteriaceae among food handlers, which can cause food borne outbreaks, is a public health concern and become a serious world threat. Klebsiella pneumoniae and Escherichia coli are the predominant ESBL-producing enterobacteriaceae. There is no sucient data related to this in underdeveloped countries. Objective: This study was aimed assess the magnitude of ESBL producing Klebsiella pneumoniae and Escherichia coli isolated from stool of apparently health food handlers in the Dilla University Student Cafeteria. Methods: A cross-sectional study was conducted from November to September 2018/2019. A structured questionnaire was used for collection of data on socio- demographics. A total of 220 stool samples were cultured on MacConkey agar and a series of biochemical tests was done for the identication of bacterial species. Double-disk synergy (DDS) method was used for the detection of ESBL-producing strains. A disc of amoxicillin clavulanic acid was placed in the center of the Mueller–Hinton agar plate, and cefotaxime, aztreonam, and ceftazidime were placed at a distance of 20mm from the disk placed at center. Enhanced inhibition zone of any of the cephalosporin discs on the side facing amoxicillin clavulanic acid was considered as ESBL producer. Results: The rate of faecal carriage of E. coli and K. pneumonia among the food handlers of the current study was found to be 66.4%, of which 81.5% was E. coli, while 18.5% was K. pneumonia. From the total bacterial isolates, 25.3% were conrmed to be positive for ESBL production of which 19.9% was E. coli, while 5.5% was K. pneumonia. ESBL-producing faecal carriage showed high resistance to aztreonam, cefotaxime, ceftriaxone, cefuroxime, and ceftazidime with resistance rate ranged from 88-100%, while the least resistance rate was seen against imipenem accounted <26.3%. In this study,


Background
The Enterobacteriaceae are a substantial, heterogeneous group of gram-negative rods whose natural habitat is the intestinal tract of human beings and animals (1). These microorganisms have emerged as one of the most important reasons for nosocomial and community-obtained infections (2)(3)(4)(5). Enterobacteriaceae are typically associated with a range of infections (6), among which urinary tract infections, bloodstream infections, heath facility associated pneumonia, and some of the intra-abdominal infections are the most crucial (7,8). Studies conducted in different underdeveloped countries indicate that a high case-fatality rate associated with bloodstream infection due to Enterobacteriaceae (9,10).
Antibiotics play a vital role in decreasing the load of communicable diseases all around the world (11).
Microbial resistance to antimicrobial agents is remarkably rising globally (12)(13)(14). This rapid spread of resistance among pathogenic microorganisms is a serious problem globally (2,4), because it limits the drug treatment alternative against infections (15). Antimicrobial resistance has presently been recognized as one of the most important problems facing human health by the World Health Organization (WHO) (15,16). Frequent isolation of multidrug resistant (MDR) pathogens in both hospital and community acquired infections further intensi ed the problem of antimicrobial resistance (5). Now a days extended-spectrum β-lactamases (ESBLs) producing Klebsiella pneumoniae and Escherichia coli is a serious public health issue globally (1). They become resistance to beta-lactam antibiotics via the production of beta-lactamases enzymes which inactivate beta-lactam antibiotics and this continues to be the prominent cause of β-lactam antibiotics resistance among Enterobacteriaceae (17,18). It can rapidly develop resistance against a range of important broad-spectrum antimicrobials (19,20). Inappropriate and irrational use of antimicrobial drugs, poor sanitary and infection control practices in developing countries play a critical role in an increased prevalence of resistant bacteria in a community provides favorable conditions for resistant microorganisms to emerge and spread (21,22). This can lead to a proliferation of organisms with broad-spectrum β-lactamase activity that threatens the future of the β-Lactam class in clinical care (21).
The increasing rate of human infections caused by anti-microbial resistance strains of Enterobacteriaceae makes clinical management more di cult by prolonging the illness and compromise the treatment (5).
This can have a potentially serious impact on human health. The situation is more common in developing countries where there is widespread and uncontrolled use of antibiotics (16). Data on extended-spectrum β-lactamases producing Enterobacteriaceae in Ethiopia are limited and there is no data associated with this. Therefore, this study was aimed assess the magnitude of ESBL producing Klebsiella pneumoniae and Escherichia coli isolated from stool of apparently health food handlers in the Dilla University Student Cafeteria.

Study design and period
An institutional-based cross-sectional study was carried out among food handlers to evaluate the magnitude and antimicrobial resistance pattern of ESBL-producing E. coli and K. pneumoniae from stools of apparently health food handlers in Dilla University Student Cafeteria, Southern Ethiopia from November 2018 to September 2019. Three sample selection sites were identi ed prior to data collection. To select representative participants, the nal sample size was proportionally allocated to each stratum, and food handlers were selected using a systematic random sampling technique. Finally, a total of 220 were included in the study based on the single population proportion formula.

Data collection and analysis
Data related to socio-demographic characteristics and personal hygiene practices were collected via face to face interviewing of the patient or guardian of the patient by using a well-structured questionnaire before laboratory sample collection. One environmental health and four medical laboratory professionals were recruited for data collection, supervision, and microbiological analysis. The data collectors were trained for 2 days by the principal investigator on observational data collection and specimen collection procedures.

Laboratory Data Collection and analysis
After interviewing, all respondents were asked to give a fresh stool specimen in a sterile, clean widemouthed plastic, and leak proof container by a clean wooden applicator stick for microbiological analysis.
The isolation and characterization of E. coli and K. pneumoniae were performed based on the standard procedure. Brie y, a mixture of a stool sample (1 mL) was transferred to the selective media, Hicrome ESBL agar base, to assess the ESBL production in 2 hr (Oxoid, Ltd UK). An inoculum from Hicrome ESBL agar base was cultured onto MacConkey agar (Oxoid, Ltd UK). After overnight incubation at 37 °C the growth of E. coli and K. pneumoniae was differentiated by their colony characteristic, pigment production (pink to colorless at or mucoid colonies), motility and Gram-staining techniques (Gram-negative rods, non-sporing, and non-capsulated).
A list of biochemical tests conducted for further identi cation of isolates were made by conformation of motility and other relevant biochemical tests. An isolate was considered as E. coli when it is indole positive(dark pink ring) and methyl-red positive, citrate negative (no change or remained green) and urea negative, gas and acid producer, and motile and wasconsidered as K. pneumoniae when it is indole and methyl-red negative, citrate positive, urea slow producing, and non-motile. In case of delay, the isolated bacteria were kept at 2-8 °C in the nutrient broth for not more than 24 hours until the antimicrobial sensitivity test was done.
Detection method of ESBL-producing E. coli and K. pneumonia Disk-diffusion method was used to screen ESBL producer using ceftazidime (30 µg), cefotaxime (30 µg), and ceftriaxone (30 µg) (Oxoid, UK) as per CLSI criteria. A suspension of pure colony was wrapped on Mueller-Hinton agar (MHA) (Oxoid, UK) with a sterile cotton swab, and antibiotics discs were placed at a distance of 20 mm from each other and incubated at 35 ± 2 °C for 16-18 hours. The isolates with diminished susceptibility with breakpoint of ≤ 22 mm for ceftazidime, ≤ 25 mm for ceftriaxone, and ≤ 27 mm for cefotaxime were suspicion for ESBL production (15). Phenotypic Con rmations of ESBL producers was performed using double disc synergy between the indicator cephalosporin and clavulanic acid, according to EUCAST guidelines (23). A disc of amoxicillin/clavulanic acid (20/10 µg) was placed in the center of the MHA plate, and then cefotaxime (30 µg), ceftazidime (30 µg) and aztreonam (30 µg) were placed at a distance of 20 mm edge to edge from the amoxicillin/clavulanic acid disk. After 24hours incubation, the elevation of ≥ 5 mm zone inhibition between either of the cephalosporin disks and their respective amoxicillin/clavulanic acid disks was interpreted as ESBL producer (24) (23). MDR was de ned as acquired non-susceptibility to at least one agent in three or more antimicrobial categories. The quality and performance of culture media, biochemical tests, and antimicrobial susceptibility discs were checked using a control strain (E. coli ATCC 25922 and K. pneumoniae ATCC 700603) obtained from Ethiopian Public Health Institute, Addis Ababa.

Data Analysis
The descriptive statistical analysis was performed by using SPSS version 24 software. The prevalence of ESBL producers and non-ESBL-producing isolates were categorized by personal hygiene related factors of food handlers and presented by calculating frequencies and percentages. Chi-square test was used with appropriate correction for the observation. Multivariate logistic regression analysis was used to identify risk factors for colonization by ESBL. P values < 0.05 were considered statistically signi cant.

Ethical consideration
Ethical clearance was obtained from the Ethical Review Committee of Dilla University medical and health Science College. Written informed consent was obtained from each study participant. Strict con dentiality was maintained during the interview process as well as anonymity was kept during data processing and report writing. Food handlers who have found to be positive for enteric pathogens (parasite and bacterial) were referred to their respective staff medical center for appropriate anti-parasitic and antimicrobial treatments.

Results
Socio demographic characteristics of study participant A total of 220 food handlers were included in this study. Of these, 90.9% were females. The majority of the study participants were between the age group of 20 and 40 years (84.5%) with the mean age of 31.2 (standard deviation ± 8.8 years). Of the total respondents, 51.4% reported that they always perform periodic medical checkups, 81.8% always trimmed their nger weekly, and 63.2% always wear a clean apron. Similarly, 69.1% wear hair garment regularly in their working area and 79.5% reported that they always use soap and water for hand washing after visiting toilet. The nding from observation also supported that personal hygiene was practiced by the food handlers. From the total, 97.3% use pipe water for drinking. About 86.8% of the study participants were drunken unpasteurized milk, while 42.3% of the study participants eat raw meat (Table 1).
ESBL production faecal carriage rate of E. coli and K. pneumonia from stool culture In the present study, The rate of fecal carriage of E. coli and K. pneumonia among the food handlers of the current study was found to be 146(66.4%), of which 119 (81.5%) were E. coli, while 27 (18.5%) were K. pneumonia. From the total bacterial isolates, 37 (25.3%) were con rmed to be positive for ESBL production. 29(19.9%) of ESBL producing isolates were E. coli, while K. pneumonia 8(5.5%) (Fig. 2).
In this study, ESBL-producers and non ESBL producers isolates were predominantly reported in females with a prevalence rates of 35 (24.0%) and 97(66.4%). E. coli and K. pneumonia producing ESBL were predominantly reported among age group of 20-40 years with a prevalence of 31(21.2%). Most of ESBL producers were reported in food handlers who did not attend a regular medical checkup with a prevalence of 25(17.1%). Majority of E. coli and K. pneumonia producing ESBL were reported among food handlers who did not wash their hands with soap with the prevalence of 24(16.4%). In the present study, majority of the demographic characteristics study participants have no association with E. coli and K. pneumonia producing ESBL. Only food handlers who used antibiotics in the last 3 months had a strong association with the ESBL carriage with P values of 0.001 (Table 1)  In the current study, ESBL-producing isolates showed signi cantly higher resistance not only towards the third generation cephalosporins but also towards other antimicrobial agents tested (p > 0.001). Both E. coli and K. pneumonia ESBL producers were highly resistant to Aztreonam, cefotaxime, ceftriaxone, cefuroxime, and ceftazidime with resistance rates ranged from 87-100%. Almost all ESBL-producing and non-ESBL-producing isolates were highly resistant to ampicillin and tetracycline with a resistance rate of 89.5%. Higher resistance rate of ESBL producing isolate was also reported among cipro oxacin, trimethoprim-sulfamethoxazole, and nitrofurantoin with resistance rate ranged from 67-75%. In the current study, imipenem was the least resistant antibiotic against ESBL producing isolates with a resistant rate of 13.8% (Table 2). sulfamethoxazole. Others ESBL-producing isolates were co-resistant to β -lactams groups, and 3, 4, and 5 types of non-β lactam antibiotics with a resistant rates of 17(45.9%), 12(32.4%), and 8(21.6%), respectively (Table 3).

Discussion
The emergence and rapid spread of multidrug resistance strains of ESBL producing Enterobacteriaceae is a serious public health issue worldwide. Rapid expansion of ESBLs highly affects the activity of broadspectrum antibiotics, creating major therapeutic di culties with a signi cant impact on the outcomes for patients (25). Although it was suspected to be high, few data are available on the prevalence of faecal carriage of ESBL producing Enterobacteriaceae and little attention was given to it in Africa. In this study, we examined the faecal carriage rate of ESBL producers E. coli or K. pneumoniae in food handlers working at the Dilla University student cafeteria.
The phenotypic information obtained in the current study indicates a signi cant prevalence of ESBL producers. The overall magnitude of faecal carriage of ESBL producing E. coli or K. pneumoniae among apparently health food handlers was 25.3%. This indicates a remarkable high number of ESBL productions among food handlers working in food preparation. Our nding is consistent with studies conducted in India (26), Italy (27), Egypt (28) and in Turkey (29) with prevalence ranged from 15-33%.
However, it is lower than the study conducted in Central African (30), Tanzania (31), Nigeria (32), Burkina Faso (33) and Ghana (34) with prevalence ranged from 40-59%. The variation might be explained by methodological differences, differences in study area, quality of media used, type of specimen collected.
The use of low quality, inappropriate use of antibiotics, and weak infection prevention measures may additionally make a contribution to the high magnitude of ESBL production.
In the present study, E. coli was the leading faecal carriage of ESBL producing Enterobacteriaceae with a prevalence of 19.9%, followed by K. pneumonia (5.5%). Our nding agrees with studies conducted in India (26,27), Central African (30), Central India (35) and Burkina Faso (33), where E. coli was the predominant ESBL producer than K. pneumonia. However, study conducted in Uganda (36), Gambia (37) and elsewhere (38) reported a higher prevalence of K. pneumonia than E. coli. Those bacteria can result in the transfer of the resistance genes to other strains of E. coli and K. pneumonia within the gastrointestinal tract that can result in fatal consequences. In addition, when those carriers admitted to hospitals they can easily transmit the infection to other hospitalized patients. Quick adaptation of those strains to the harsh environment, upregulation of intrinsic resistance mechanisms, and rapid acquisition and transferring of drug resistance genes through mobile genetic elements could be a possible explanation for an elevated overall drug resistance prevalence rate against different categories of drugs.
In the current study, the faecal carriage of ESBLs among food-handlers working in the cafeteria was signi cantly associated with food handlers who used antibiotics repeatedly in the last 3months and those food handlers who did not attend regular medical check studies (39,40). In most parts of developing countries including Ethiopia, antibiotics were used without prescription which can lead to overuse or misuse of antibiotics. This can contribute to the emergence and spread of antimicrobial-resistant strains.
Moreover, in developing countries, poor personal hygiene practices may increase the prevalence of bacterial gastrointestinal infections which may play a role in rising antimicrobial resistance. This increases the risk of treatment failure with potentially serious consequences. This can be overcome by performing bacterial culture and performing an antimicrobial susceptibility test to select the most effective antibiotics for suspected cases of gastrointestinal infected patients.
In the present study, ESBL-Producing E. coli and K. pneumonia were highly resistant to ampicillin, cefotaxime, aztreonam, ceftazidime, and ceftriaxone with resistance rates ranged from 88-100%, while the least resistant was found against imipenem with resistance rate 13.8%. Our nding agrees with studies Sierra Leone (45). This indicates ESBL-producing enterobacteriaceae were rapidly emerging in developing and developed countries. In this study, ESBL-producing E. coli and K. pneumonia were not only resistance to third-generation cephalosporins but also to other non-β lactams groups of antibiotics. The nding of this study showed that Imipenem had a better performance against ESBL-Producing enterobacteriaceae than other antibiotics including cephalosporin group. This is inconsistent with study conducted by Moges and his colleagues (46) who reported that imipenem had higher performance for the treatment of ESBL-Producing Enterobacteriaceae.
Our ndings showed that greater than 90% of the ESBL producing isolates were MDR. The MDR nature of ESBL-producing E. coli and K. pneumonia may be explained by the fact that they are plasmid-mediated enzymes which are carrying multi-resistant genes by plasmid, transposon, and integron and they are readily transferred to other bacteria through conjugation, transduction or transformation. The carrier of those bacteria can easily disseminate to the community and hospitalized patient which can result in remarkable consequences. As a result, now a days bacteria with multiple resistances to antibiotics are widely distributed in hospitals and communities and became a serious problem throughout the world (47). This is the most challenging and alarming condition in the management of infectious diseases associated with ESBL producing Entrobacteriaceae.

Limitations Of The Study
The limitations of this study were the lack of a molecular con rmatory test to establish genotypes of ESBLs. The participants were unable to provide the previous prescribed antibiotics to show association with the faecal carriage of ESBLs (recall bias). Most of the study participants were female, which tends to show the gender bis.

Conclusion
In this study, the magnitude of faecal carriage of ESBL-producing E. coli and K. pneumonia is alarmingly high and threat to human health. ESBL-producing faecal carriage showed high resistance to aztreonam, cefotaxime, ceftriaxone, cefuroxime, and ceftazidime. The better option for the treatment of ESBLproducing E. coli and K. pneumonia in this study is imipenem. The occurrence of multidrug resistance to third-generation cephalosporins and non-β lactam antibiotics is more common among ESBL producers. In the present study, ESBL-producing isolates were co-resistant to β -lactams antibiotics plus at least one of the non β -lactam antibiotics. Only food handlers who used antibiotics repeatedly in the last 3months were signi cantly associated with the ESBL faecal carriage. The rise of ESBL-producing Enterobacteriaceae requires strict infection prevention and control strategies and strengthening of the diagnostic capacity of laboratory professionals for the detection and surveillance of antibiotic resistance that have a signi cant role in clinical decision-making. Hence a coordinated effort should be implemented.