High prevalence of fecal carriage of Extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar, Northwest Ethiopia.

BACKGROUND: Fecal carriage of extended-spectrum beta-lactamase and Carbapenemase-producing Enterobacteriaceae is a potential risk for the transmission of infection with resistant strains. Understanding the burden of these resistant strains in asymptomatic people is essential to reduce the chain of infection transmission. However, data on the fecal carriage of Extended-spectrum Beta-lactamase and Carbapenemase-producing Enterobacteriaceae among food handlers were limited in developing countries especially in Ethiopia. The aim of the present study is, therefore, to assess fecal carriage rate, associated factors, and antimicrobial resistance patterns of Extended-spectrum Beta-lactamase and Carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar Cafeterias, Northwest Ethiopia.
MATERIALS AND METHODS: An institution-based cross-sectional study was conducted from February to June 2021 at the University of Gondar cafeterias. A total of 290 stool samples were collected, transported using Cary Blair transport medium, and processed. All isolates were cultured and identified by using MacConkey agar, and routine biochemical tests. Antimicrobial susceptibility testing was done to each isolate following the Kirby Bauer disk diffusion method. If the zone of inhibition was ≤ 22 mm for ceftazidime, ≤25 mm for ceftriaxone, and ≤27 for cefotaxime they were considered as potential ESBL strain and selected for a further phenotypic confirmatory. Moreover, the double-disc diffusion test and the modified carbapenem inactivation method were used for confirmations of Extended-spectrum β-lactamase and Carbapenemase-producing Enterobacteriaceae respectively. If a ≥5mm difference in zone diameter for either antimicrobial agent in combination with clavulanic acid versus the zone diameter of the agent when tested alone (without B-lactamase inhibitor), was confirmed as ESBL-PE and if the zone of inhibition diameter between 6-15mm and 16- 18mm with a pinpoint colony, it was considered as carbapenem resistance Enterobacteriaceae. Data were entered using Epi-data version 4.6 and then exported to SPSS version 26 for analysis. Potential risk factors were assessed using multivariable logistic regression and a p-value less than 0.05 was considered statistically significant.
RESULTS: Out of 290 stool samples, 63 (21.7%) and 7 (2.4%) were confirmed as Extended-spectrum β-lactamase and Carbapenemase-producing Enterobacteriaceae. The most predominant ESBL-PE was E. coli 43 (14.8%) followed by K. pneumoniae 17 (5.9%). Most of the Extended-spectrum β-lactamase and Carbapenemase-producing isolates were resistant to tetracycline, cefotaxime, ceftazidime, and ceftriaxone (100% each). In contrast, a low resistance level was recorded for Meropenem and cefoxitin. The overall Multi-drug resistant Enterobacteriaceae (MDR) was 147 (42.3%). Antibiotics usage in the last 3 months and drinking unpasteurized milk were associated with the carriage of the Extended-spectrum beta-lactamase-Producing Enterobacteriaceae. CONCLUSIONS AND RECOMMENDATIONS: The high fecal carriage rate of Multi-drug resistance isolate, Extended-spectrum β-lactamase, and Carbapenemase-producing Enterobacteriaceae were recorded among food handlers. Therefore, this study gives signals in the spread of drug-resistant bacteria easily to the community. Hence, the need for adjusting and promotion of infection prevention measures to prevent the spread of drug-resistant bacteria should not be underestimated.

Introduction

Enterobacteriaceae are Gram-negative, facultative anaerobes, and non-spore-forming bacilli. It is mainly found in the gastrointestinal tract of humans and is a significant cause of nosocomial and community-acquired infections. Mostly they cause urinary tract infection, respiratory tract bloodstream, and wound infections. Most Enterobacteriaceae are treated by different antimicrobial agents, but if it is improperly used which leads to multidrug-resistant (MDR) or extensively drug resistance (XDR), The rising trend of antimicrobial resistance has become a global problem which is mainly caused by Enterobacteriaceae [1, 2]. Those Enterobacteriaceae are the most important emerging resistance characteristics which correspond to resistance for β-lactam and carbapenems antibiotics [3, 4].

Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are two prevalent Enterobacteriaceae species with pathogenic potential and ESBL-encoding genes [5]. The Infectious Diseases Society of America has listed them as two out of six pathogens for which new drugs are urgently needed to tackle resistance development [6]. Infections with ESBL-producing bacteria are linked to nearly double the mortality rate as infections with non-ESBL producers, according to a meta-analysis [7]. Colonization with ESBL-producing bacteria is a cause for concern even when there is no infection present. Carriage of resistant commensal Enterobacteriaceae strains in the normal gut flora may act as a reservoir for resistance genes that are then acquired by pathogenic strains [8]. E. coli and K. pneumoniae virulence determinants are adhesins, toxins, iron acquisition factors, lipopolysaccharides, polysaccharide capsules, types 1 and 3 fimbriae, and invasins are typically encoded on pathogenicity islands (PAIs), plasmids, and other mobile genetic components [9, 10].

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of MDR bacterial pathogens from different origins including humans, birds, cattle, and fish that increase the need for routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strain [11-18].

Extended-Spectrum Beta-Lactamase (ESBL) and Carbapenemase are enzymes usually produced by micro-organisms in the gut, such as E. coli and Klebsiella spp. The enzymes produced by resistant bacteria can destroy certain antibiotics and render them useless, making the treatment of infections more difficult. Such bacteria can easily be transmitted from person to person, such as by fecal contamination or by touching contaminated equipment or the air of the ward [19, 20]. ESBL-producing Enterobacteriaceae (ESBL-PE) and Carbapenemase-producing Enterobacteriaceae (CPE) are increasing both in hospital and community settings [21, 22].

As drug resistance widely spreads, a major concern is the coexistence of multiple ESBL and Carbapenemase genes that have led to the emergence of organisms that are resistant to nearly all antibiotics [23]. Inappropriate and irrational use of antimicrobial drugs, poor sanitation, and infection control practices in developing countries play a critical role in an increased prevalence of resistant bacteria in a community providing favorable conditions for resistant microorganisms to emerge and spread [24, 25]. This can lead to a proliferation of organisms with the broad-spectrum β-lactamase activity that threatens the future of the βlactam class in clinical care [24].

The reports showed that an increase in the carriage of antimicrobial-resistant bacteria in the community increases the spread of resistant bacteria via human-to-human transmission. In addition, resistance bacteria can be spread from one person to another in healthcare settings through contaminated hands, surfaces, and also through contaminated food or water [26, 27]. Infections related to extended-spectrum beta-lactamase and Carbapenemase-producing Enterobacteriaceae represent a major global health threat [28].

However, such data are limited in food handlers in Ethiopia; therefore, we designed this study aimed to assess fecal carriage rate, associated factors, and antimicrobial resistance patterns of Extended-spectrum Beta-lactamase and Carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar Cafeteria.

Materials and methods

Study design, period, and area

An institution-based cross-sectional study was conducted from February to June 2021 at the University of Gondar cafeterias. The University of Gondar is located in Gondar town, 737 km from Addis Ababa and 180 km from Bahir Dar in Northwest Ethiopia. The Department of Medical Microbiology has four Laboratory sections which include Bacteriology, Mycology, Virology, and Tuberculosis and Leprosy Laboratory. In the Bacteriology section culture is one of the main activities including bacterial isolation, identification, and antimicrobial susceptibility test. At present, the University of Gondar has approximately 27,000 students in regular and extension programs. In the University there are six cafeterias (including Hospital, College of Medicine and Health Sciences, Atse Tewodros, Maraki, Atse Fasile, and Tseda). Currently, those cafeterias serve meals for 22,000 students and more than 600 patients. According to the information obtained from human resource management of the University of Gondar, approximately 650 food handlers are serving in these cafeterias.

Study population, sample size, and sampling technique

The source population was all food handlers who were working at the University of Gondar cafeteria. The study populations were asymptomatic food handlers who were working at the University of Gondar cafeteria food establishment and available during the data collection period and voluntary to participate in the study. Food handlers who had been on antibiotic treatment during data collection were excluded from this study. Food handlers who had been working in the six campuses cafeteria of the University were included in the study. The sample size was calculated based on a single population proportion formula and the calculated sample size was 290. The value of proportion was taken as 25.3% (0.253) from the previous study conducted by Diriba et al., 2020, on “Fecal carriage rate of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae among apparently health food handlers in Dilla University student cafeteria” Southwest Ethiopia [29]. A stratified random sampling technique was used to recruit 290 study participants from the sampling frame of food handlers. To select representative participants, the final sample size was proportionally allocated to each stratum.

Data collection and analysis

Data related to socio-demographic, clinical, and hygiene-related data were collected by face-to-face interviews of the food handlers using a well-structured questionnaire before laboratory sample collection. All data were collected and analyzed by two trained laboratory technologists.

Sample collection and processing

The study participants were instructed to collect approximately 2 grams of stool into a clean, leak-proof container. Following collection, all stool specimens were labeled with sample number, time, and date of sample collection. Each stool sample was transported to the School of Biomedical and Laboratory Sciences, Medical Microbiology laboratory section using Cary-Blair transport media (HiMEDIA Laboratories Pvt. Ltd., Mumbai, India). A loop full of stool sample was inoculated on MacConkey agar (Oxoid Ltd, Basingstoke, UK) using the Streak Plate Method using an aseptic technique and incubated aerobically at 37°C for 18–24 hours [30].

Bacterial isolation and identification

Cultural observation. Preliminary identification of bacteria was done based on colony characteristics of the organisms. Some colony characteristics like (size, shape, color, pigmentation, texture, elevation, and edge [30].

Microbiological analysis of stool specimen. Enterobacteriaceae were identified by inoculating /streaking of stool samples on MacConkey agar (Oxoid Ltd, Basingstoke, UK) based on their color morphology after an incubation time of 18–24 hours at 37°C. The smear was prepared from each different colony observed on the plates and Gram staining was performed. The Results such as gram reaction (gram-negative), arrangements, and shape of bacteria are seen from the examinations using a microscope [31].

Biochemical examination. Biochemical tests were performed on colonies from pure cultures for the identification of the isolates. Triple sugar iron agar (Oxoid Ltd, Basingstoke (for gas production, lactose fermentation, and hydrogen sulfide production), UK), indole test (for tryptophan utilization, Simon’s citrate agar (Oxoid Ltd, Basingstoke, UK) (citrate utilization test), urease agar (Oxoid Ltd, Basingstoke, UK) (urease production test), lysine iron agar (Oxoid Ltd, Basingstoke, UK) (lysine decarboxylase test), and Motility medium (Oxoid Ltd, Basingstoke, UK) (motility test) were included in the biochemical tests for species identification [32].

Antimicrobial susceptibility testing

Following bacterial identification, the antimicrobial susceptibility testing (AST) of the isolates was performed by a modified Kirby-Bauer disk diffusion technique by following the Clinical and Laboratory Standard Institute (CLSI) guideline, 2020 [33]. To make bacterial suspension the pure colonies of a young culture were picked using a sterile wire loop and emulsified in 0.85% sterile normal saline and compared with 0.5 McFarland turbidity standard. Then the bacterial suspension was inoculated onto Muller-Hinton agar (Oxoid, Basingstoke, and Hampshire, UK) using the lawn culture method.

The following antibiotics disks were used as β-lactam combination group (Amoxicillin/clavulanic acid (AUG 20/10 μg), Beta-lactams (Cefotaxime (CTX 30 μg), Ceftazidime (CAZ 30 μg), Ceftriaxone (CRO 30 μg), and Cefoxitin (CXT 30 μg)), Aminoglycosides (Gentamicin (GEN 10 μg)), Carbapenems (Meropenem (MER 10 μg)), Tetracycline (Tetracycline (TE 30 μg), Fluoroquinolones (Ciprofloxacin (CIP 30 μg)), Phenicols (Chloramphenicol (CHL 30μg)), and Sulfonamide (Trimethoprim/Sulphamethoxazole (23.75 μg /1.25μg)). The antibiotic disks used were from BD, BBLTM Company, USA Product. Then the plates were incubated at 37 ℃ for 18–24 hours. After overnight incubation, the zone of inhibition was measured and interpreted as susceptible, intermediate, and resistant based on the recommendation of CLSI, 2020 [33]. Multi-drug resistance patterns of the isolates were identified using the criteria set by Magiorakos et.al. [34].

Detection and confirmation of extended-spectrum β- lactamase (ESBL)

All strains were tested against ceftriaxone, cefotaxime, and ceftazidime for ESBL screening using the Kirby-Bauer disk diffusion method. If the zone of inhibition was ≤ 22 mm for ceftazidime, ≤25 mm for ceftriaxone, and ≤27 for cefotaxime they were considered as potential ESBL strain and selected for a further phenotypic confirmatory test as described below [33]. Phenotypic confirmation of ESBL production was done using the double-disc diffusion test and interpreted by following the CLSI, 2020 guidelines. Pure culture colonies of ESBL-PE isolates were emulsified in 0.85% saline and compared with 0.5 McFarland turbidity standard then inoculated by lawn culture method using sterile swabs on MHA. The following antibiotic disks such as cefotaxime (30μg), cefotaxime/clavulanic acid (30μg/10μg), ceftazidime (30μg), and ceftazidime/clavulanic acid (30μg/10μg), were used as recommended by CLSI, 2020 to establish the status of the ESBL phenotypes. The plates were then incubated aerobically at 37°C for 18 to 24 hours. K. pneumoniae American Type Culture Collection (ATCC) 700603 (positive control) and E. coli strain ATCC 25922 (negative control) were used for quality control. If a ≥5mm difference in zone diameter for either antimicrobial agent in combination with clavulanic acid versus the zone diameter of the agent when tested alone (without B-lactamase inhibitor), was confirmed as ESBL-PE [33, 35].

Detection and confirmation of Carbapenemase-producer Enterobacteriaceae

Carbapenemase-producing Enterobacteriaceae was screened by using Meropenem disks. The suspension of isolated bacteria was inoculated onto MHA, then Meropenem (10μg) disks were placed and incubated at 37°C for18- 24 hrs. If the zone of inhibition is ≤ 19 mm, it was considered as a presumptive CPE [33]. The suspected CPE was confirmed by the Modified carbapenems inactivation methods (mCIM) and EDTA-modified carbapenem inactivation method (eCIM). The isolates of a bacterial colony that was suspected for CPE were diluted with 2 ml of trypticase soya broth and the Meropenem (10μg) disk was immersed in the suspension and 0.5 M EDTA (only for eCIM) was added; then incubated for 4 hours. A pure colony standard strain of Meropenem susceptible E. coli ATCC 25922 was emulsified in 0.85% normal saline and compared with 0.5 McFarland standard then inoculated the whole plate of MHA. After 4 hours of incubation, the Meropenem disk was removed from the test tube and placed on the MHA plate which was inoculated by E. coli ATCC 25922 Meropenem sensitive strain and incubated at 37°C for 18–24 hours. After incubation, if the zone of inhibition diameter between 6-15mm and 16- 18mm with a pinpoint colony, it was considered as carbapenem resistance Enterobacteriaceae [33].

Quality control

The sterility of newly prepared culture media was checked by incubating 5% of the batch at 35–37°C overnight before it was used and was evaluated for possible growth or contamination. The performance testing was done by inoculating known control strains of E. coli ATCC 25922 to check the quality of the 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. K. pneumoniae ATCC 1705 and ATCC 1706 were used as positive and negative quality control for Carbapenemase production respectively [33].

Data processing and analysis

All data were checked for completeness, coded, and entered using Epi-data version 4.6 and exported to SPSS version 26 for statistical analysis. Then data were analyzed using SPSS version 26 to determine the independent variables and the fecal carriage rate of ESBL and CPE frequency analysis and cross tab were used. The Chi-square test was used with appropriate correction for the observation. To determine the associated factors Multivariable Logistic regression analyses were used. A variable with a p-value ≤ 0.2 in bivariate logistic regression was checked in multivariate analysis for a statistically significant association by controlling the possible confounders. Crude and adjusted odds ratios were used to quantify the strength of association between ESBL-PE and CPE carriage rate and risk factors. A variable with a p-value of less than 0.05 at a 95% confidence interval was considered statistically significant.

Ethical approval

Ethical clearance and support letter were obtained from the Ethical Review Committee of the School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, the University of Gondar with reference number SBLS/02/25/2021. Each study participant was informed about the purpose, methods of collection, anticipated benefit, and risk of the study. Written informed consent was obtained from each study participant. Food handlers who were found to be ESBL and CPE positive for Enterobacteriaceae were referred to their respective staff medical center for appropriate antimicrobial treatments and health education was given about infection prevention.

Results

A total of 290 food handlers were involved in this study. Over three fourth study participants were females 242 (83.4%). The majority of the study participants were between the age group of 20 and 40 years (82.8%) with a mean age of 32.10 (Standard deviation = ±8.691 years). Regarding educational status 98 (33.8%) completed primary school. Respondents with a family size of 1–5 were 262 (90.3%) and of the total respondents, 116 (40%) did not know the principle of food safety (Table 1).

Table 1

Socio-demographic and clinical characteristics, and hygienic practices of food handlers at the University of Gondar Comprehensive Specialized Hospital and Student Cafeteria, Northwest Ethiopia, February to June 2021.

Variables	Category	Frequency	Percentage (%)
Gender	Male	48	16.6
	Female	242	83.4
Age in years	<20	8	2.8
	20–40	240	82.8
	>40	42	14.4
Level of education	Illiterate	57	19.7
	Primary	98	33.8
	Secondary and above	135	39.7
Monthly income	Less than 1000 ETB	7	2.4
	Greater than 1500 ETB	283	97.6
Marital status	Married	159	54.8
	Unmarried	131	45.2
Family size	1–5	262	90.3
	>5	28	9.7
Service years	1–5	152	52.4
	More than 5	138	47.6
Knowledge of food safety practice	Yes	174	60
	No	116	40
Medical checkups	Yes	181	62.4
	No	109	37.6
History of medical instrumentation	Yes	8	2.8
	No	282	97.2
History of hospital admission in the last three months	Yes	6	2.1
	No	284	97.9
Used antibiotics without prescription	Yes	93	32.1
	No	197	69.9
History of diarrhea in the last 3 months	Yes	97	33.4
	No	193	66.6
History of urinary tract infection in the last 3 months	Yes	58	20
	No	232	80
Antibiotics used in the last 3 months	Yes	129	45.5
	No	161	55.5
History of chronic disease	Yes	38	13.1
	No	252	86.9
Hand washing habit	Yes	264	91
	No	26	9
Hands wash with soap	Only with water	63	21.7
	With soap	227	78.3
Fingernail status	trimmed	273	94.1
	not trimmed	17	17
Wear hair garment	Yes	259	89.3
	No	31	10.7
Source of water for drinking	pipe	290	100
	hand dug well	0	0
Pit latrine toilet facilities	Yes	284	97.9
	No	6	2.1
Drinking unpasteurized milk	Yes	71	24.5
	No	219	75.5
Eating raw meat	Yes	59	20.3
	No	231	79.7
Eating raw vegetables	Yes	148	51
	No	142	49

Of the total respondents, 181 (62.4%) had periodic medical checkups, 93 (32.1%) of them used antibiotics without prescription, 97 (33.4%) had a history of diarrhea in the last three months, 58 (20%) had a history of urinary tract infection in the last three month, and 129 (45.5%) of them had antibiotics used in the last 3 months. About 71 (24.5%) of the study participant drink unpasteurized milk, while 59 (20.3%) and 148 (51%) of them had the habit of eating raw meat and raw vegetables respectively (Table 1).

Prevalence of Enterobacteriaceae isolates from the stool sample

All of the study participants had culture-positive results and a total of 347 bacterial isolates were identified. The study revealed that 233 of the study participants contained single organisms 57 of the study participants contained two organisms. Out of 347 bacterial isolates, E. coli were the most prevalent 245 (70.6%) followed by K. Pneumoniae 68 (19.6%) and C. freundii 13 (3.7%) (Fig 1).

Fig 1

Prevalence of Enterobacteriaceae isolates from stool sample at University of Gondar Comprehensive Specialized Hospital and students’ cafeteria, February to June 2021.

Phenotypic characteristics of the recovered isolates

is a bacteria that are gram-negative, rod-shaped, and motile, grows on MacConkey agar (red or colorless colonies), lactose fermenter, indole positive, urease negative, Citrate negative, gas producer, hydrogen sulfide negative, and lysine decarboxylase positive.

is a gram-negative, rod-shaped, grow on MacConkey agar (red large mucoid colonies) lactose-fermenting, indole negative, urease positive, Citrate positive, gas producer, hydrogen sulfide negative, non-motile, and lysine decarboxylase positive.

is a gram-negative, rod-shaped bacteria that grow on MacConkey agar (colorless colony) non-lactose fermenter, indole negative, urease positive, motility positive, citrate positive, hydrogen sulfide positive, lysine decarboxylase positive.

is a gram-negative rod-shaped that grow on MacConkey agar (Smooth, convex, translucent, or opaque grey colored with a shiny surface and entire margin; mucoid or rough colonies occasionally), lactose fermenter, indole negative, urease positive, H2S positive, motility positive, gas producer, lysine decarboxylase negative.

are Gram-negative, rod-shaped, grow on MacConkey agar, lactose fermenter, indole negative, urease negative, H2S negative, gas producer, citrate positive, motile, and lysine decarboxylase negative.

Antimicrobial resistance patterns of Enterobacteriaceae isolates

A total of eleven antibiotics from eight classes (beta-lactam, β-lactam combination group, Aminoglycosides, Carbapenems, Tetracycline, Fluoroquinolones, Phenicols, and Sulfonamides) of antimicrobials were used to assess the susceptibility patterns of the isolate. High resistance rates were observed for tetracycline (61.1%), Sulphamethoxazole-trimethoprim (53.3%), and amoxicillin/clavulanic acid (57.3%). In contrast, Meropenem and cefoxitin were the most effective antibiotics with sensitivity rates of (95%) and (92.2%) respectively (Table 2).

Table 2

Antimicrobial resistance patterns of Enterobacteriaceae among food handlers at the University of Gondar Comprehensive Specialized Hospital and students’ Cafeterias, Northwest Ethiopia, February to June 2021.

Classes	Antibiotics	E. coli (N = 245)		K. pneumoniae (N = 68)		E. cloacae (N = 9)		C.freundii (N = 13)		P.mirabilis (N = 2)		Providencia species (N = 3)		M.morganii (N = 3)		Serratia species (N = 4)		Total R (%)
		S (N (%))	R (N (%)	S (N (%)	R (N (%)	S (N (%)	R (N (%)	S (N (%)	R (N (%)	S (N (%)	R (N (%)	S (N (%)	R (N (%)	S (N (%)	R (N (%)	S N (%)	R (N (%)
Aminoglycosides	GEN	161(65.7)	84 (34.3)	37(54)	31(46)	8(89)	1(11)	9 (69.2)	4 (30.8)	2(100)	0	2(67)	1(33)	3(100)	0	2(50)	2(50)	123(35.5)
Beta-lactam combination group	AUG	106(43)	139(57)	30(44)	38(56)	2(22)	7(78)	4(31)	9(69)	1(50)	1(50)	2(67)	1(33)	2(67)	1(33)	1(25)	3(75)	199(57.3)
Beta-lactams	CAZ	188(77)	57(23)	51(75)	17(25)	9(100)	0	9(69)	4(31)	2(100)	0	2(67)	1(33)	3(100)	0	2(50)	2(50)	81(23.3)
	CRO	192(78)	53(22)	50(73)	18(27)	7(78)	2(22)	10(77)	3(23)	1(50)	1(50)	2(67)	1(33)	2(67)	1(33)	3(75)	1(25)	80(23)
	CXT	121(90)	24(10)	65(96)	3(4)	9(100)	0	13(100)	0	2(100)	0	3(100)	0	3(100)	0	4(100)	0	27(7.8)
	CTX	196(80)	49(20)	51(75)	17(25)	9(100)	0	10(77)	3(23)	2(100)	0	2(67)	1(33)	3(100)	0	3(75)	1(25)	71(20.5)
Carbapenems	MER	233(95)	12(5)	62(91)	6(9)	9(100)	0	13(100)	0	2(100)	0	3(100)	0	3(100)	0	4(100)	0	18(5)
Fluoroquinolones	CIP	187(76.3)	58(23.7)	42(62)	26(38)	6(67)	3(33)	6(46.2)	7(53.8)	2(100)	0	3(100)	0	2(67)	1(33)	2(50)	2(50)	97(28)
Phenicols	CHL	208(84.9)	37(15.1)	62(91)	6(9)	9(100)	0	13(100)	0	2(100)	0	3(100)	0	3(100)	0	3(75)	1(25)	44(12.7)
Sulfonamides	SXT	117(48)	128(52)	28(41)	40(59)	8(89)	1(11)	4(31)	9(69)	1(50)	1(50)	1(33)	2(67)	2(67)	1(33)	1(25)	3(75)	185(53.3)
Tetracycline	TE	89(36)	156(64)	28(41)	40(59)	8(89)	1(11)	3(23)	10(77)	0	2(100)	2(67)	1(33)	2(67)	1(33)	2(50)	2(50)	212(61.1)

Key: S = Sensitive, R = Resistance, AUG = Augmentin/Amoxicillin/clavulanic acid; SXT = Sulphamethoxazole-trimethoprim; TE = Tetracycline; CIP = Ciprofloxacin; CHL = Chloramphenicol; GEN = Gentamycin; CXT = Cefoxitin; CRO = Ceftriaxone; CAZ = Ceftazidime; CTX = cefotaxime, MER = Meropenem.

Multi-drug resistant patterns of Enterobacteriaceae among food handlers

The MDR determination was carried out by considering the classification of antibiotics in the following eight antibiotic classes (beta-lactam, β-lactam combination group, Aminoglycosides, Carbapenems, Tetracycline, Fluoroquinolones, Phenicols, and Sulfonamides). The overall MDR prevalence in this study was 147 (42.3%; 95% CI: 39.7–51). Accordingly, from the isolates of C. freundii were 9 (69.2%) MDR followed by P. mirabilis 1 (50%), E. coli 104 (42.4%), and K. pneumoniae 28 (41.2%) (Table 3). The MDR isolates across the six cafeterias were 42, 32, 35, 26, 6, and 6 from the Hospital cafeteria, College of Medicine and Health Science campus cafeteria, Maraki campus cafeteria, Atse Tewodros campus cafeteria, Atse Fasile campus cafeteria, and Tseda campus cafeteria respectively (Table 4).

Table 3

Multidrug resistance profiles of Enterobacteriaceae isolates among food handlers at the University of Gondar Comprehensive Specialized Hospital and Students ‘Cafeterias, Northwest Ethiopia, February to June 2021.

Isolates N = 347	Level of resistance n, (%)
	R0	R1	R2	R3	R4	R5	R6	R7	R8	R9	MDR≥R3
E. coli N = 245	42	46	53	36	20	10	14	16	5	3	104 (42.4)
K.pneumoniae N = 68	16	14	10	9	4	4	5	4	1	1	28 (41.2)
E. cloacae N = 9	1	1	6	1	0	0	0	0	0	0	1 (11)
C. freundii N = 13	2	1	1	3	0	1	3	1	1	0	9 (69.2)
P. mirabilis N = 2	-	-	1	-	1	0	0	0	0	0	1 (50)
Others N = 10	4	1	1	2	2	0	0	0	0	0	4 (40)
Total (N = 347)	65	63	72	51	27	15	22	21	7	4	147 (42.7)

Note; R0: sensitive for all classes of antibiotics, R1: resistant for one class of antibiotics, R2: resistant for two classes of antibiotics, R3: resistant for three classes of antibiotics, etc., MDR = multidrug resistant.

Table 4

Distribution of MDR-E isolates from the six cafeterias, among food handlers at the University of Gondar Comprehensive Specialized Hospital and Students’ Cafeteria, Northwest Ethiopia, February to June 2021.

Study site	Bacterial Isolates
	MDR Level	E. coli (n = 245)	K. pneumoniae (n = 68)	C. freundii (n = 13)	E. cloacae (n = 9)	P. mirabilis (n = 2)	Others *(n = 10)	Total N (%)
HC (N = 75)	Non-MDR N (%)	23 (9.4)	7 (10.3)	1 (7.7)	1 (11.1)	1 (50)	-	33 (44)
	MDR N (%)	26 (10.6)	10 (14.7)	3 (23.1)	1 (11.1)	1 (50)	1 (10)	42 (56)
CMHSC (N = 55)	Non-MDR N (%)	19 (7.8)	3 (4.4)	0	-	-	1 (10)	23 (41.8)
	MDR N (%)	21 (8.6)	8 (11.8)	2 (0.52)	-	-	1 (10)	32 (58.2)
MCC (N = 105)	Non-MDR N (%)	49 (20)	20 (29.4)	0	-	-	1 (10)	70 (66.7)
	MDR N (%)	28 (11.4)	5 (7.4)	1 (7.7)	-	-	1 (10)	35 (33.3)
ATCC (N = 68)	Non-MDR N (%)	28 (11.4)	7 (10.3)	2 (15.4)	4 (44.4)	-	1 (10)	42 (61.8)
	MDR N (%)	20 (8.2)	2 (3)	3 (23.1)	0	-	1 (10)	26 (39.2)
AFCC (N = 24)	Non-MDR N (%)	10 (4.1)	2 (3)	1 (7.7)	2 (22.2)	-	3 (30)	18 (75)
	MDR N (%)	4 (1.63)	2 (3)	0	0	-	-	6 (25)
TsCC (N = 20)	Non-MDR N (%)	12 (4.9)	1 (1.5)	-	1 (11.1)	-	-	14 (70)
	MDR N (%)	5 (2.1)	1 (1.5)	-	0	-	-	6 (30)
Total (N = 347)	Non-MDR N (%)	141(57.6)	40(58.8)	4 (30.8)	8 (88.9)	1 (50)	6 (60)	200 (57.6)
	MDR N (%)	104 (42.4)	28(41.2)	9 (69.2)	1 (11.1)	1(50)	4 (40)	147 (42.3)

Key; HC = Hospital Cafeteria, CHMSC = College of Medicine and Health Sciences Campus Cafeteria, MCC = Maraki campus cafeteria, ATCC = Atse Tewodros Campus Cafeteria, AFCC = Atse Fasile campus cafeteria, and TsCC = Tseda campus cafeteria. Others* M. morganii, Providencia species, and Serratia species.

The fecal carriage rate of ESBL and Carbapenemase-producing Enterobacteriaceae

From the total of 290 study participants, 63 (21.73%) (95% CI: 17.2–26.6) were colonized by ESBL-PE and 7 (2.4%) (95% CI: 0.7–4.5) with CPE. From the total bacterial isolates, 63 (18.2%) and 7 (2.01%) were confirmed to be positive for ESBL and Carbapenemase production respectively. Of these 63 ESBL isolates, 21, 16, 12, 7, 5, and 2 were found in the Hospital cafeteria, College of Medicine and Health Science campus cafeteria, Maraki campus cafeteria, Atse Tewodros campus cafeteria, Atse Fasile campus cafeteria, and Tseda campus cafeteria respectively. All CPE were 7 (100%) ESBL producers. Similarly, all ESBL producing isolates were MDR-E (Table 5).

Table 5

Distribution of ESBL-PE, Carbapenemase-producing Enterobacteriaceae, and MDR isolate among food handlers at the University of Gondar Comprehensive Specialized Hospital and Students’ Cafeterias, Northwest Ethiopia, February to June 2021.

Study site	Isolates (N = 347)	Proportionate of Enterobacteriaceae (N = 347)
		ESBL (N (%))		Carbapenemase (N (%))		% of ESBL from total sample size	% of both ESBL and CPE from total isolates	% Both ESBL and MDR from total isolates
		Positive	Negative	Positive	Negative
Hospital cafeteria (N = 75)	E. coli	13 (26.5)	36 (73.5)	2 (4.1)	47 (95.9)	13 (4.5)	2 (0.58)	13 (3.7)
	K. pneumoniae	5 (29.4)	12 (70.6)	3 (17.6)	14 (82.4)	5 (1.7)	3 (0.86)	5 (1.44)
	C. freundii	2 (50)	2 50)	0	4 (100)	2 (0.7)	-	2 (0.57)
	P. mirabilis	1 (50)	1 (50)	0	2 (100)	1 (0.35)	-	1 (0.288)
	Others	0	3 (100)	0	3 (100)	-	-	-
	Total	21 (28)	54 (72)	5 (6.7)	70 (93.3)	21 (7.2)	5 (1.44)	21 (6.05)
CHMS campus cafeteria (N = 55)	E. coli	9 (22.5)	31 (77.5)	1 (2.5)	39 (97.5)	9 (3.1)	1 (0.288)	9 (2.6)
	K. pneumoniae	7 (63.6)	4 (36.7)	0	11 (100)	7 (2.4)	-	7 (2)
	C. freundii	0	2 (100)	0	2 (100)	-	-	-
	P. mirabilis	-	-	-	-	-	-	-
	Others*	0	2 (100)	0	2 (100)	-	-	-
	Total	16 (29.1)	39 (70.9)	1 (1.8)	54 (98.2)	16 (5.5)	1(0.288)	16 (4.6)
Maraki campus Cafeteria (N = 105)	E. coli	9 (11.7)	68 (88.3)	1 (1.3)	76 (98.7)	9 (3.1)	1 (0.288)	9 (2.6)
	K. pneumoniae	3 (15)	22 (85)	0	25 (100)	3 (1.03)	-	3 (0.86)
	C. freundii	0	1 (100)	0	1 (100)	-	-	-
	P. mirabilis	-	-	-	-	-	-	-
	Others *	-	2 (100)	-	2 (100)	-	-	-
	Total	12 (11.4)	93 (88.6)	1 (0.95)	104 (99.05)	12 (4.13)	1 (0.288)	12 (3.45)
Atse Tewodros campus cafeteria (N = 68)	E. coli	6 (12.5)	42 (87.5)	0	48 (100)	6 (2.06)	-	6 (1.72)
	K. pneumoniae	1 (9.1)	10 (90.9	0	11 (100)	1 (0.35)	-	1 (0.288)
	C. freundii	0	5 (100)	0	5 (100)	-	-	-
	P. mirabilis	-	-	-	-	-	-	-
	Others*	0	4 (100)	0	4 (100)	-	-	-
	Total	7 (10.3)	61 (89.7)	0	68 (100)	7 (2.4)	-	7 (2)
Atse Fasile campus cafeteria (N = 24)	E. coli	4 (28.6)	10 (71.4)	0	14 (100)	4 (1.4))	-	4 (1.15)
	K. pneumoniae	1 (25)	3 (75)	0	4 (100)	1 (0.35)	-	1 (0.288)
	C. freundii	0	1(100)	0	1 (100)	-	-	-
	P. mirabilis	-	-	-	-	-	-	-
	Others*	0	5 (100)	0	5 (100)	-	-	-
	Total	5 (23.8)	19 (76.2)	0	24 (100)	5 (1.75)	-	5 (1.44)
Tseda campus cafeteria (N = 20)	E. coli	2 (11.8)	15 (88.2)	0	17 (100)	2 (0.7)	-	2 (0.58)
	K. pneumoniae	0	2 (100)	0	2 (100)	-	-	-
	C. freundii	-	-	-	-	-	-	-
	P. mirabilis	-	-	-	-	-	-	-
	Others*	0	1 (100)	0	1 (100)	-	-	-
	Total	2 (10)	18 (90)	0	20 (6)	2 (0.7)	-	2 (0.58)
Total (N = 347)		63 (18.2)	284 (81.8)	7 (2)	340 (98)	63 (21.72)	7 (2)	63 (18.2)

*Others: M. morganii, Providencia species, Serratia species.

From the total of ESBL producing isolates leading ESBL producers were E. coli 43 (68%) followed by K. pneumoniae 17 (27%) similarly the leading Carbapenemase-producing isolates were E. coli 4 (57%) followed by K. pneumoniae 3 (43%) (Fig 2).

Fig 2

Proportion of ESBL and Carbapenemase-producing Enterobacteriaceae from the total of ESBL, and Carbapenemase positive isolates at the University of Gondar Comprehensive Specialized Hospital and Students’ Cafeteria, Northwest Ethiopia, 2021.

Antimicrobial resistance patterns of ESBL and Carbapenemase-producing Enterobacteriaceae

In the current study, cefotaxime, ceftriaxone, ceftazidime, tetracycline, and Sulphamethoxazole-trimethoprim had complete (100% each) resistance found in all of the ESBL-producing isolates. In addition, amoxicillin/clavulanic acid resistance was 55 (87.3%) of these isolates. Resistance to other antibiotics such as Gentamicin, cefoxitin, ciprofloxacin, and chloramphenicol was found to be 29 (46%), 20 (31.7%), 23 (36.5%), and 21 (33.3%) respectively. Only 18 (28.6%) of these ESBL-producing isolates were resistant to Meropenem (Table 6).

Table 6

Antimicrobial Resistance patterns of ESBLs and Carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar Comprehensive Specialized Hospital and Students’ Cafeterias, Northwest Ethiopia, February to June 2021.

Antimicrobials	Patterns	ESBL producing isolates (N = 63)					Carbapenemase-producing isolates (N = 7)
		E. coli (N = 43) N (%)	K. pneumoniae (N = 17) N (%)	C. freundii (N = 2) N (%)	P. mirabilis (N = 1) N (%)	Total N (%)	E. coli (n = 4) N (%)	K.pneumoniae (n = 3) N (%)	Total N (%)
AMC	S	7 (16.3)	0	0	1 (100)	8 (12.7)	0	0	0
	R	36 (83.7)	17 (100)	2 (100)	0	55 (87.3)	4 (100)	3 (100)	7 (100)
CAF	S	26 (60.5)	13 (76.5)	2 (100)	1 (100)	42 (66.7)	0	0	0
	R	17 (39.5)	4 (23.5)	0	0	21 (33.3)	4 (100)	3 (100)	7 (100)
CIP	S	30 (69.8)	9 (52.9)	0	1(100)	40 (63.5)	0	0	0
	R	13 (30.2)	8 (47.1)	2 (100)	0	23 (36.5)	4 (100)	3 (100)	7 (100)
TET	S	0	0	0	0	0	0	0	0
	R	43 (100)	17 (100)	2 (100)	1 (100)	63 (100)	4 (100)	3 (100)	7 (100)
CAZ	S	0	0	0	0	0	0	0	0
	R	43 (100)	17 (100)	2 (100)	1 (100)	63 (100%)	4(100%)	3 (100)	7 (100)
CTX	S	0	0	0	0	0	0	0	0
	R	43 (100)	17 (100)	2 (100)	1 (100)	63 (100)	4 (100)	3 (100)	7 (100)
CRO	S	0	0	0	0	0	0	0	0
	R	43 (100)	17 (100)	2 (100)	1 (100)	63 (100)	4 (100)	3 (100)	7 (100)
GEN	S	21 (48.8)	11 (64.7)	1 (50)	1 (100)	34 (54)	1 (25)	1 (33)	2 (28.5)
	R	22 (51.2)	6 (35.3)	1 (50)	0	29 (46)	3 (75)	2 (67)	5 (71.5)
SXT	S	0	0	0	0	0	0	0	0
	R	43 (100)	17 (100)	2 (100)	1 (100)	63 (100)	4 (100)	3 (100)	7 (100)
MER	S	31 (72.1)	11 (64.7)	2 (100)	1 (100)	45 (71.4)	0	0	0
	R	12 (27.9)	6 (35.3)	0	0	18 (28.6)	4 (100)	3 (100)	7 (100)
CXT	S	29 (67.4)	11 (64.7)	2 (100)	1 (100)	43 (68.3)	0	0	0
	R	14 (32.6)	6 (35.3)	0	0	20 (31.7)	4 (100)	3 (100)	7 (100)

Key: S = Sensitive, R = Resistance, AMC = amoxicillin/clavulanic acid; SXT = Sulphamethoxazole-trimethoprim; TET = tetracycline; CIP = ciprofloxacin; CHL = chloramphenicol; GEN = gentamycin; CXT = cefoxitin; CRO = ceftriaxone; CTX = cefotaxime, CAZ = ceftazidime; MER = Meropenem.

All E. coli and K. pneumoniae ESBL producing isolates were found completely resistant to cefotaxime, ceftriaxone ceftazidime, tetracycline, and Sulphamethoxazole-trimethoprim. This study found that these ESBL producing E. coli isolates were also resistant to amoxicillin/clavulanic acid, gentamycin, chloramphenicol, and ciprofloxacin with the rates of 36 (57.1%), 22 (34.9%), 17 (27%), and 13 (20.6%), respectively. Furthermore only 14 (22.2%), and 12 (19%) of these isolates were resistant to cefoxitin and Meropenem respectively. Additionally, K. pneumoniae ESBLs producing isolates were also resistant to amoxicillin/clavulanic acid, ciprofloxacin, gentamicin, and chloramphenicol with the rates of 17 (27%), 8 (12.7%), 6 (9.5%), and 4 (6.3%), respectively. In this study, all CPE isolates of E. coli and K. pneumoniae were resistant to all antibiotics except gentamicin (75%), and (67%) respectively (Table 6).

Factors associated with fecal carriage rate of ESBL-PE

After bivariate logistic regression analysis, lack of knowledge on food safety practice (p≤0.001), medical checkup (p≤0.075), history of admission in the last 3 months (p≤0.016), history of diarrhea in the last 3 months (p≤0.000), history of urinary tract infection in the last 3 months (p≤0.003), Antibiotics used in the last 3 months (p≤0.001), History of chronic disease (p≤0.072), drinking unpasteurized milk (p≤0.000), ate raw vegetables (p≤0.015) were included in a multivariate logistic regression analysis. Of socio-demographic factors, lack of knowledge on food safety practice had a strong association with the ESBL carriage with P-values 0.003. Of the hygiene-related factors, drinking unpasteurized milk had a strong association with the ESBL carriage with P-values of 0.001. Of the clinical risk factors, the uses of antibiotics in the last 3 months with p-values of 0.016 have demonstrated significant association to the carriage of ESBL-PE (Table 7).

Table 7

Factors associated with fecal carriage rate of ESBL-PE among food handlers at the University of Gondar Specialized Hospital and students’ cafeteria, Northwest Ethiopia, February to June 2021.

Variables	Category	Number tested (N = 290)	ESBL Positive (N = 63) N (%)	ESBL Negative (N = 227) N (%)	Bivariate analysis		Multivariable analysis
					COR(95%CI)	p-value	AOR(95%CI)	p-value
Gender	Male	48	7 (14.6)	41 (85.4)	1		1
	Female	242	56 (23.1)	86 (76.9)	2.46 (0.84–7.23)	0.100	0.30 (0.08–1.2)	0.086
Age in years	<20	8	3 (37.5)	5 (62.5)	1		1
	20–40	240	51 (21.3)	189 (78.7)	0.60 (0.10–3.61)	0.577
	>40	42	9 (21.4)	33 (78.6)	1.03 (0.43–2.48)	0.954
Level of Education	Illiterate	57	29 (50.9)	28 (49.1)	0.16 (0.31–0.97)	0.020	0.7 (0.07–0.16)	0.081
	Primary	98	13 (13.3)	85 (86.7)	1.70 (0.71–1.92)	0.371	0.3 (0.24–0.77)	0.246
	Secondary and above	115	21 (18.3)	94 (81.7)	1		1
Monthly income	Less than 1000 ETB	7	3 (42.9)	4 (57.1)	0.52 (0.08–3.45)	0.501
	Greater than 1500 ETB	283	60 (21.2)	223 (78.8)	1
Marital status	Married	159	38 (23.9)	121 (76.1)	0.84 (0.44–1.60)	0.595
	Unmarried	131	25 (19.1)	106 (80.9)	1
Family size	1–5	262	59 (22.5)	203 (77.5)	0.59 (0.17–2.05)	0.407
	Greater than 5	28	4 (14.3)	24 (85.7)	1
Service years	1–5	152	34 (22.4)	118 (77.6)	0.28 (0.38–0.95)	0.398
	Greater than 5	138	29 (21)	109 (79)	1
Knowledge of food safety practice	Yes	174	38 (21.8)	136 (78.2)	1
	No	116	25 (21.6)	91 (78.4)	3.50 (1.81–6.78)	0.000	1.2 (1.07–1.46)	0.003*
Medical checkups	Yes	181	18 (9.9)	163 (90.1)	1
	No	109	45 (41.3)	64 (58.7)	0.52 (.26–1.0)	0.075
History of admission in the last 3 months	Yes	6	6 (100)	0	0.21 (0.06–0.75)	0.016	2.1 (0.47–9.16)	0.331
	No	284	57 (20)	227 (80)	1		1
History of medical instrumentation	Yes	8	6 (75)	2 (25)	1	0.522	1
	No	282	57 (20.2)	225 (79.8)	0.587 (0.11–3.0)		0.9 (0.18–5.24)	0.970
History of diarrhea in the last 3 months	Yes	93	35 (37.6)	58 (62.4)	0.28 (0.14–0.53)	0.000	1.5 (0.62–3.67)	0.371
	No	197	28 (14.2)	169 (85.8)	1		1
History of UTI	Yes	58	22 (37.9)	36 (62.1)	0.35 (0.18–0.69)	0.003	1.4 (0.56–3.38)	0.510
	No	232	41 (17.7)	191 (82.3)	1		1
Antibiotics used in the last 3 months	Yes	129	45 (34.9)	84 (65.1)	0.19 (0.09–0.39)	0.000	4.14 (1.31–13)	0.016*
	No	161	18 (11.2)	143 (88.8)	1		1
History of chronic disease	Yes	38	13 (34.2)	25 (65.8)	0.48(0.21–1.07)	0.072	0.81 (0.31–2.1)	0.668
	No	252	50 (19.8)	202 (80.2)	1		1
Hand washing habit	Yes	264	60 (22.7)	204 (77.3)	1
	No	26	3 (11.5)	23 (84.5)	0.39 (0.09–1.72)	0.215
Fingernail status	trimmed	273	58 (21.2)	215 (78.8)	1		1	0.566
	Not trimmed	17	5 (29.4)	12 (70.6)	1.59 (0.49–5.13)	0.433	0.69 (0.19–2.49)
Wear hair garment	Yes	259	58 (22.4)	201 (77.6)	1		1	0.795
	No	31	5 (16.1)	26 (83.9)	1.0 (0.36–2.78)	0.993	0.86 (0.27–2.74)
Drinking unpasteurized milk	Yes	71	35 (49.3)	36 (50.7)	8.9 (4.48–17.71)	0.000	10.4 (3.8–28.8)	0.001*
	No	219	27 (12.3)	192 (87.7)	1		1
Eating raw meat	Yes	59	17 (28.8)	42 (71.2)	1.58 (0.77–3.23)	0.211	0.60 (0.24–151	0.282
	No	231	46 (19.9)	185 (80.1)	1		1
Eating raw vegetables	Yes	148	6 (4.1)	142 (95.9)	0.44 (0.22–0.85)	0.015	0.4 (0.15–1.26)	0.125
	No	142	57 (40.1)	85 (59.9)	1		1

Abbreviations, COR = crude odds ratio, AOR = adjusted odds ratio, CI = confidence interval.

Discussion

Among 290 study participants, 347 Enterobacteriaceae isolates were identified. Of these, E. coli accounts for 245 (70.6%) followed by K. pneumoniae 68 (19.6%) and C. freundii (3.7%). Our result was in agreement with reports in Egypt E. coli [36], and Kuwait E. coli [37]. However, this report was lower than a study conducted in Dilla, Ethiopia E. coli. This difference may be due to the technique that we used, geographical difference, and population characteristics.

In the present study fecal carriage rate of ESBL- PE among food handlers was 21. 7% (95% CI: 17.2–26.6). This finding is in line with the studies conducted in Dilla, Ethiopia [29]. However, it is lower than the study conducted in Mexico [38], Italy [39], China [40], India [41], Nepal [42], Chad [43], and Egypt [36], and [44]. In contrast, our result is higher than the study conducted in Japan [45, 46], Switzerland [47], Nigeria [48], and Gambia [49]. The methodological and geographical differences, bacteriological media used may contribute to the high magnitude of ESBL production. These affected food handlers pose a hazard to the spread of MDR gram-negative bacteria in the schools, in their homes, and communities where they prepare and serve food. As demonstrated in a cohort study of food handlers in Japan, if not treated, some of these ESBL carriers could function as long-term reservoirs for the spread of these pathogens [45].

In the current study, E. coli (68%) and K. pneumoniae (27%) were the most common ESBL producing isolates of Enterobacteriaceae. E. coli is a leading cause of ESBL producing isolates. This result is agreed with the report in Nepal [42], China [40], Chad [43], Egypt [36], and Dilla, Ethiopia [29], where E. coli was the predominant ESBL producing Enterobacteriaceae than K. pneumoniae. Additionally, C. freundii isolates were the third ESBL producing isolates with a prevalence of (3%). This finding was in line with the study conducted in Nepal [42]. However, K. pneumoniae as a common ESBL producer was reported in the Gambia E. coli and K. pneumoniae [49] and Bahir Dar, Ethiopia E. coli and K. pneumoniae [50]. These bacteria cause the resistance genes to be transmitted to other strains of E. coli and K. pneumoniae in the gastrointestinal tract, which can be fatal. Furthermore, when those carriers are admitted to hospitals, they can easily spread the infection to other patients. An increased overall drug resistance rate against different categories of drugs could be explained by rapid adaptation of those strains to the harsh environment, up-regulation of intrinsic resistance mechanisms, and rapid acquisition and transmission of drug resistance genes through mobile genetic elements [51].

In this study, the overall prevalence of CPE was 7 (2%) (95%CI = 0.7–4.5), this finding is concordant with other studies such as in the Gambia, [49], Egypt ([44], and China [40]). However, it is lower than studies in Kuwaiti [37, 52]. This discrepancy of isolation may be due to geographical location, population characteristics, poor sanitary practice, incorrect use of antibiotics, cross-border of the population with other high-prevalence countries, sample size, and methodological heterogeneity could all contribute to variations in CPE prevalence.

In this study, the overall prevalence of MDR-E was 147 (42.3%: 95% CI: 39.7–51). This study is consistent with the study done in Egypt [53]. But it was higher than a study conducted in, Kenya [54], Kuwaiti [55], and Qatar [56]. This discrepancy could be due to irrational uses of antibiotics, poor personal and environmental hygiene in the study area, a lack of proper diagnostic tools, consumption of animal products that use antibiotics for growth promotion and treatment, an increase in MDR strains over time, differences in the study population and failure of patient adherence to their medication [57].

Meropenem was found to be the most effective antibiotic for Enterobacteriaceae isolates (sensitivity 95%) which was comparable with the study done in Nigeria [58], and Turkey [59]. In the present study isolates were resistant to tetracycline (61.1%), and amoxicillin/clavulanic acid (57.3%) which was lower than the study conducted in Nigeria amoxicillin/clavulanic acid, and tetracycline [48].

Regarding antimicrobial resistance rate, all ESBL-producing Enterobacteriaceae were resistant to cefotaxime, ceftazidime, and ceftriaxone. This finding agrees with studies conducted in the Gambia [49], Nigeria [48], and Dilla, Ethiopia [29]. This indicates ESBL-producing Enterobacteriaceae were rapidly emerging in developing countries. In this research, ESBL-producing Enterobacteriaceae are found to be not only completely resistant to third-generation cephalosporin but also other non-beta-lactam antibiotics such tetracycline and Sulphamethoxazole-trimethoprim. This finding concords with the study conducted in Nigeria [58], and Dilla, Ethiopia [29]. Additionally, the finding of this study showed that Meropenem had a better performance against ESBL-producing Enterobacteriaceae than other antibiotics including the cephalosporin group. However, our result is different from the study conducted in Nigeria [48].

In this study all ESBL producing isolates were MDR. The MDR nature of ESB-PE may be explained by the fact that they are plasmid-mediated enzymes that carry multi-resistant genes through plasmid, transposon, and integron, and can easily be transferred to other bacteria by conjugation, transduction, or transformation [60].

High fecal colonization of ESBL and Carbapenemase-producing Enterobacteriaceae was attributed to the contribution of different risk factors. The risk factors mainly identified from this study after doing multivariable analysis were lack of knowledge in food safety practices, drinking unpasteurized milk, and history of antibiotics usage in the last 3 months. This result showed that the fecal carriage of ESBLs in food-handlers was significantly associated with, drinking unpasteurized milk, history of antibiotics usage in the last 3 months, and lack of knowledge in food safety practices as found in other studies from China [40], Gambia [49], and Dilla, Ethiopia [29]. However, this finding is inconsistent with the study conducted in Japan reported that history of antibiotic usage had no significant association with the fecal carriages of ESBL-positive Enterobacteriaceae [46].

The extended-spectrum beta-lactamase carriage rate was highest among food handlers with a history of antibiotics usage in the last 3 months compared to those who did not have used antibiotics. The history of antibiotic use in the last 3 months was more than 4 folds higher (AOR, 4.14 (95% CI, 1.3–13)), compared to those who did not have used antibiotics This result is consistent with the reports of the study conducted from China [40], Gambia [49], and Dilla, Ethiopia [29]. This can be explained by the fact that the usage of antibiotics in the past and present can provide a significant competitive advantage for the development of resistant bacteria and the spread of ESBL-producing bacteria in the population. Antibiotics are taken without a prescription in most developing countries, such as Ethiopia, which can lead to antibiotic overuse or misuse, which triggers the emergence and spread of antimicrobial-resistant bacteria [61]. Furthermore, poor personal hygiene habits in developing countries may increase the prevalence of bacterial gastrointestinal infections, which may contribute to the rise in antibiotic resistance. This raises the possibility of treatment failure, which could have profound consequences. This can be avoided by conducting a bacterial culture, and antimicrobial susceptibility test, and adhering to proper antibiotic stewardship whenever treating patients with suspected gastrointestinal infections.

Limitation of the study

The present study has the following limitations:

Antibiotic resistance encoding genes (ARGs) of isolates were not detected as a confirmatory test due to a lack of molecular techniques and primers. Because the study was cross-sectional, there was no follow-up to treat ESBL carriers and identify how they could infection remain.

Conclusions and recommendations

In this study, the prevalence of fecal carriage of MDR Enterobacteriaceae, ESBL-PE, and Carbapenemase-producing Enterobacteriaceae among food-handlers were high and this is a threat to the public. The most frequent ESBL-producing Enterobacteriaceae were E. coli and K. pneumoniae. In this study, Meropenem and cefoxitin were the most effective antimicrobial agents compared to other tested antibiotics. The overall MDR prevalence in this study was 147 (42.3%). Accordingly, from the isolates of C. freundii were 9 (69.2%) MDR followed by P. mirabilis 1 (50%), E. coli 104 (42.4%), and K. pneumoniae 28 (41.2%). Antibiotics usage in the last 3 months and drinking unpasteurized milk were associated with the carriage of the ESBL-PE. The result indicates that ESBL and CPE carriage is widespread calling for mass screening of the community for ESBL and CPE. The emergence of ESBL and Carbapenemase-producing Enterobacteriaceae necessitates the implementation of strict infection prevention and control strategies, surveillance of antibiotic resistance, and following proper antibiotic stewardship, which plays an important role in clinical decision-making. The level of evidence is still low and requires further research by incorporating research on molecular confirmation and profiling of the ESBL and Carbapenemase-producing isolates to identify resistance genes.

(RTF)

Click here for additional data file.

The AST interpretation chart (extracted from CLSI, guideline 2020).

Based on CLSI criteria, interpret the zones sizes of each antimicrobial, reporting the organism as ‘Resistant’, ‘Intermediate/Moderately susceptible’, ‘Susceptible see below the table’.

(RTF)

Click here for additional data file.

Lab protocol.

(RTF)

Click here for additional data file.

10 Jan 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Abdelazeem Mohamed Algammal, Prof, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

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2. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files.

3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

4. Please amend the manuscript submission data (via Edit Submission) to include authors Setegn Eshetie, Desie Kasaw, and Feleke Moges.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments to authors:

- The current study has a significant impact, but it needs a major revision:

- The manuscript should be revised for grammar mistakes.

- Please write the scientific names of bacterial pathogens and genes in the correct form all over the manuscript and in the References section (should be italic).

-The title is broad, please modify the title.

- Add more details about the used methods and most prevalent results in the abstract.

-In the introduction: discuss the public health importance of E. coli, and K. pneumonia and their virulence determinants.

-Improve the aim of work.

Methods:

-Explain the methods of isolation and identification in detail??

-Specific references should be added to all the used methods and techniques.

- Antimicrobial susceptibility testing: Add the manufacturing company, city, and country for the used reagents and antimicrobial discs.

-PCR based detection of virulence genes and antimicrobial resistance genes in the most prevalent retrieved bacterial species should be carried out if applicable (or addresses this point in the study limitations)

--Results:

- Discuss in detail the phenotypic characters of the recovered isolates.

-increase the resolution of different Figures: Please improve.

-PCR based detection of virulence genes and antimicrobial resistance genes in the most prevalent retrieved bacterial species should be carried out if applicable (or addresses this point in the study limitations)

-The correlation between the phenotypic and genotypic MDR should be performed.

-Discussion:

- Please improve.

-Please improve the main conclusion of the manuscript.

Reviewer #2: -The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript:

Title:

I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Abstract:

- The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the aim of the work and the main conclusion of your findings.

Introduction: (it needs to be more informative)

-Give a hint about the virulence factors, different infections caused by of E. coli and K. pneumonia, and the mechanism of disease occurrence.

- The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflect the necessity of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Authors could add the following paragraph:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial pathogens from different origins including humans, birds, cattle, and fish that increase the need for routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains. You should cite the following valuable studies:

1.PMID: 33177849

2.PMID: 32994450

3.PMID: 33061472

4.PMID: 33947875

5.PMID: 34445951

6.PMID: 33188216

7.https://doi.org/10.1016/j.aquaculture.2021.737643

8.PMID: 30150182

-Rephrase the aim of the work to be clear and better sound.

Material and methods: Illustrate your methods with subtitles:

-Add this subtitle: Bacterial Isolation and identification:

•Discuss in detail the methods of isolation and identification of E. coli, K. pneumonia, and other members of Enterobacteriaceae. Besides, specific references should be added.

•Add the company, city, and country of the used bacterial media and reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reactions.

- Antimicrobial susceptibility testing:

•Illustrate the antimicrobial classes of the tested antimicrobial agents within the text.

•The authors are advised to classify the tested isolates to MDR , XDR, and PDR as described by Magiorakos et al.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–81. doi:10.1111/j.1469-0691.2011.03570.x.

- Why did you ignore the detection of antibiotic resistance genes in the recovered isolates??

•Please use PCR to detect antibiotic resistance genes, followed by gene sequencing. Afterward, the correlation between phenotypic and genotypic multidrug resistance should be performed.

-Add more details about the software used in the statistical analyses.

-Results:

-Add this subtitle: Phenotypic characteristics of the recovered isolates.

•Illustrate in detail the phenotypic characteristics of the recovered isolates, especially E. coli and K. pneumonia,

-Antimicrobial susceptibility testing:

•-Illustrate in a new table the occurrence of MDR (Multidrug resistance) among the recovered isolates (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains%Type of resistance

R, MDR, and XDRPhenotypic multidrug resistance

(Antimicrobial classes and different antibiotics).The antibiotic -resistance genes

- Increase the resolution of all figures (it should be 600 dpi).

-Discussion:

- The authors are advised to illustrate the real impact of their findings without repetition of results.

-Illustrate the different mechanisms of antimicrobial resistance in E. coli and K. pneumonia.

-Conclusion

- Should be rephrased to be sounded. A real conclusion should focus on the question or claim you articulated in your study, which resolution has been the main objective of your paper?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

17 Feb 2022

Date: 17/02/2022

Response to Reviewers

To reviewers and journal editors:

Dear reviewers,

We appreciate for spending your precious time and forwarding your valuable comments, which have significantly improved our application. We are also grateful for this positive feedback. Please see below, bold, for a point-by-point response to the reviewers. All page numbers refer to the revised manuscript file with tracked changes. All modifications in the manuscript have been highlighted in red.

We are looking forward to hearing from you in due course,

Sincerely,

Azanaw Amare (Corresponding author)

Azanaw03@gmail.com

Title of the manuscript: “High prevalence of fecal carriage of Extended-spectrum beta-lactamase and Carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar, Northwest Ethiopia.”

Manuscript Number: PONE-D-21-38463

Below we provide the point-by-point responses. All modifications in the manuscript have been highlighted in red.

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: Please revise the manuscript according to the reviewer comments. A ma-jor revision is required.

==============================

Please submit your revised manuscript by Feb 20 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and review-er(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original ver-sion. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to en-hance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Abdelazeem Mohamed Algammal, Prof, Ph.D

Academic Editor

PLOS ONE

Authors’ response: we included the DOI link in the Methods section of the manuscript

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: thank you very much. We believed that we are following PLOS ONE’s style requirements.

2. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files.

Response: thank you very much. We accept and correct it accordingly.

3. In your Data Availability statement, you have not specified where the minimal data set under-lying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any addi-tional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying da-ta set as either Supporting Information files or to a stable, public repository and include the rele-vant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the au-thors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Response: thank you. Please update it

4. Please amend the manuscript submission data (via Edit Submission) to include authors Setegn Eshetie, Desie Kasaw, and Feleke Moges.

[Note: HTML markup is below. Please do not edit.]

Response: I amend it and I notify during the revision

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that sup-ports the conclusions. Experiments must have been conducted rigorously, with appropriate con-trols, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exceptions (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in ad-dition to summary statistics, the data points behind means, medians, and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in Standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also in-clude additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments to authors:

comments: The current study has a significant impact, but it needs a major revision:

Response: thank you very much for your positive feedback

Comments: The manuscript should be revised for grammar mistakes.

Response: We are grateful for the suggestion and thank you. We went throughout the entire manuscript to eliminate grammatical and editing mistakes.

Comments: Please write the scientific names of bacterial pathogens and genes in the correct form all over the manuscript and in the References section (should be italic).

Response: We are also grateful for the suggestion and thank you. We went through the en-tire manuscript to italic bacterial pathogens and genes.

Comments: The title is broad, please modify the title.

Response: Thank you very much. We agree with this comment. Hence, we have amended the title as follows: “High prevalence of fecal carriage of Extended-spectrum beta-lactamase and Carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar, Northwest Ethiopia.”

Comments: Add more details about the used methods and most prevalent results in the abstract.

Responses: We agree with this and have incorporated your comments

Comments: In the introduction: discuss the public health importance of E. coli, and K. pneumo-nia and their virulence determinants.

Response: : Thank you for this suggestion. It would have been interesting to explore this as-pect. We tried to add the following suggestion on page 3 (Line 71&72, 77-87).

Comments: Improve the aim of the work.

Response: Thank you very much for the reminder. We have made revisions accordingly as follows, “Therefore, this study aimed to assess fecal carriage rate, associated factors, and antimicrobial resistance patterns of Extended-spectrum Beta-lactamase and Car-bapenemase-producing Enterobacteriaceae among food handlers at the University of Gon-dar Cafeteria.” Page 4 of the revised manuscript.

Methods:

Comments: Explain the methods of isolation and identification in detail??. Specific references should be added to all the used methods and techniques

Response: We agree with the reviewer’s assessment. Accordingly, we have revised this in the method section and added the suggested comments as follows (Page 6; and line 154-170).

Comments: Antimicrobial susceptibility testing: Add the manufacturing company, city, and country for the used reagents and antimicrobial discs.

Response: Thank you very much for this reminder. We have made revisions on method parts accordingly.

Comments: PCR based detection of virulence genes and antimicrobial resistance genes in the most prevalent retrieved bacterial species should be carried out if applicable (or addresses this point in the study limitations)

Response: We agree that this is a potential limitation of the study. We have added this as a limitation on pages 27 and 28 of the revised manuscript. The revised sentence is as follows (Line 445-449). “Antibiotic resistance encoding genes (ARGs) of isolates were not detected as a confirmatory test due to a lack of molecular techniques and primers”.

--Results:

Comments: Discuss in detail the phenotypic characters of the recovered isolates.

Response: Thank you. The general phenotypic characteristics of the recovered isolates espe-cially for E. coli and K. pneumoniae are described in the result sections on page 11, (lines 264-280).

Comments:-increase the resolution of different Figures: Please improve.

Response: thank you very much. We tried to increase the resolution using the PACE digital diagnostic tool.

Comments:-PCR based detection of virulence genes and antimicrobial resistance genes in the most prevalent retrieved bacterial species should be carried out if applicable (or addresses this point in the study limitations)

Response: We agree that this is a potential limitation of the study. We have added this as a limitation on pages 27 and 28 of the revised manuscript. The revised sentence is as follows (Line 445-449). “Antibiotic resistance encoding genes (ARGs) of isolates were not detected as a confirmatory test due to a lack of molecular techniques and primers.”

Comments:-The correlation between the phenotypic and genotypic MDR should be performed.

Response: We agree that this is a potential limitation of the study. We have added this as a limitation on pages 27 and 28 of the revised manuscript. The revised sentence is as follows (Line 445-449). “Antibiotic resistance encoding genes (ARGs) of isolates were not detected as a confirmatory test due to a lack of molecular techniques and primers”.

-Discussion:

Comments: Please improve.

Response: Thanks for your kind reminders. We revised the discussion section (page 25 and 26, lines 364-417).

Comments: Please improve the main conclusion of the manuscript.

Response: We revised according to the amended title of the manuscript

Reviewer #2:

Comments: The current study is interesting; however, the authors should address the following comments to improve the quality of the manuscript:

Response: Thank you very much for this positive feedback

Title:

Comments: I think the work would benefit from the title that contains the main conclusion of the study (should be derived from the conclusion). Please modify the title.

Response: Thank you very much for pointing out this suggestion. We agree with this com-ment and suggestion. Therefore, we have amended the title as follows: “High prevalence of fecal carriage of Extended-spectrum beta-lactamase and Carbapenemase-producing Enter-obacteriaceae among food handlers at the University of Gondar, Northwest Ethiopia.”

Abstract:

Comments: The abstract must illustrate the used methods and the most prevalent results (give more hints about methods and results). Besides, rephrase the aim of the work and the main con-clusion of your findings.

Response: We agree with this and have incorporated your suggestions and comments (page 2, line 36-38, 40&41; 43-45, 47-52, 59-61, 62-64).

Comments: Introduction: (it needs to be more informative)

Response: thank you very much we tried and revised it to be more informative.

Comments: Give a hint about the virulence factors, different infections caused by E. coli and K. pneumonia, and the mechanism of disease occurrence.

Response: Thank you for this suggestion. It would have been interesting to explore this as-pect. We tried to add the following suggestion on page 3 (Line 71&72, 77-87).

Comments: The authors should illustrate the public health importance concerning the emergence of multidrug-resistant (MDR) bacterial pathogens that reflect the necessity of new potent and safe antimicrobial agents. Several studies proved the widespread MDR- bacterial pathogens;

Responses: Agree. We have made the following changes on page 4 (lines 88-92).

Comments: Authors could add the following paragraph:

Multidrug resistance has been increased all over the world that is considered a public health threat. Several recent investigations reported the emergence of multidrug-resistant bacterial path-ogens from different origins including humans, birds, cattle, and fish that increase the need for routine application of the antimicrobial susceptibility testing to detect the antibiotic of choice as well as the screening of the emerging MDR strains. You should cite the following valuable stud-ies:

1. PMID: 33177849

2. PMID: 32994450

3. PMID: 33061472

4. PMID: 33947875

5. PMID: 34445951

6. PMID: 33188216

7.https://doi.org/10.1016/j.aquaculture.2021.737643

8. PMID: 30150182

Response: thank you very much for your direction, suggestion, and comments. We agree with this and have incorporated your suggestion in the revised manuscript on page 4 (line 88-92).

Comments: Rephrase the aim of the work to be clear and better sound.

Response: Thank you very much for pointing this out. We revised the objective as follows: “Therefore, this study aimed to assess fecal carriage rate, associated factors, and antimicro-bial resistance patterns of Extended-spectrum Beta-lactamase and Carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar Cafete-ria.” Page 4 of the revised manuscript

Comments: Material and methods: Illustrate your methods with subtitles:

Response: thank you very much. We incorporated this as the subtitle

Comments: Add this subtitle: Bacterial Isolation and identification:

Response: thank very much, we incorporate this as the subtitle

Comments: Discuss in detail the methods of isolation and identification of E. coli, K. pneumonia, and other members of Enterobacteriaceae. Besides, specific references should be added.

Response:

Comments: Add the company, city, and country of the used bacterial media and reagents that were used in the biochemical identification of isolates. Also, enumerate all used biochemical reac-tions.

Response: We inserted the suggested comments accordingly

- Antimicrobial susceptibility testing:

Comments: Illustrate the antimicrobial classes of the tested antimicrobial agents within the text.

Response: Thank you for this suggestion. We inserted it with text on page 7, (line 178-183)

Comments: The authors are advised to classify isolated isolates to MDR, XDR, and PDR as de-scribed by Magiorakos et al.

Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pan drug-resistant bacteria: An international expert pro-posal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18:268–81. doi:10.1111/j.1469-0691.2011.03570.x.

Response: Thank you for this suggestion. The antimicrobial classes of the tested antimicro-bial agents are indicated in Table 2 for each type of isolates, while the MDR, is presented in table 3 page 14. But for testing XDR, and PDR, we agree with potential limitations because we did not test all antimicrobial from the antimicrobial class of the drugs.

Comments: Why did you ignore the detection of antibiotic resistance genes in the recovered iso-lates??

Response: We agree that this is a potential limitation of the study. We have added this as a limitation and stated as: “Antibiotic resistance encoding genes (ARGs) of isolates were not detected as a confirmatory test due to a lack of molecular techniques and primers.”

Comments: Please use PCR to detect antibiotic resistance genes, followed by gene sequencing. Afterward, the correlation between phenotypic and genotypic multidrug resistance should be performed.

Response: We agree that this is a potential limitation of the study. We have added this as a limitation on page 27 and 28 of the revised manuscript. The revised sentence is as follows (Line 345-349). “Because of the lack of molecular methods and primes, antibiotic resistance encoding genes (ARGs) of isolates were not detected as a confirmatory test”.

Comments: Add more details about the software used in the statistical analyses.

Response: We think this is an excellent suggestion. However, we believe that these soft wares used for statistical analyses (Epi-Data version 4.6 for data entry and SPSS version 26 for statistical analysis) are open access soft wares everybody can access them. The instructions for use are freely available. Therefore, describing these statistical tools beyond the level of this detail that is presented on the test description is slightly excessive.

-Results:

Comments: Add this subtitle: Phenotypic characteristics of the recovered isolates.

Response: thank you very much for your suggestions, we incorporate this subtitle

Comments: Illustrate in detail the phenotypic characteristics of the recovered isolates, especially E. coli and K. pneumonia,

Response: Thank you. The general phenotypic characteristics of the recovered isolates espe-cially for E. coli and K. pneumoniae are described in the result sections on page 11, lines 263-280.

-Antimicrobial susceptibility testing:

Comments: Illustrate in a new table the occurrence of MDR (Multidrug Resistance) among the recovered isolates (illustrate the names of the antimicrobial classes and different antibiotics):

No. of strains%Type of resistance

R, MDR, and XDR Phenotypic multidrug resistance (Antimicrobial classes and different antibi-otics).

Response: Thank you for this suggestion. The antimicrobial classes of the tested antimicro-bial agents are indicated in Table 2 for each type of isolated, while the MDR, is presented in table 3 on page 14. But for testing XDR, and PDR, we agree with potential limitations be-cause we did not test all antimicrobial from the antimicrobial class of the drugs.

Comments: The antibiotic-resistance genes

Response: We agree that this is a potential limitation of the study. We have added this as a limitation and stated as: “Antibiotic resistance encoding genes (ARGs) of isolates were not detected as a confirmatory test due to a lack of molecular techniques and primers”.

Comments: Increase the resolution of all figures (it should be 600 dpi).

Response: thank you very much. We tried to increase the resolution using the PACE digital diagnostic tool.

-Discussion:

Comments: The authors are advised to illustrate the real impact of their findings without repeti-tion of results.

Response: Thanks for your kind reminders. We revised the discussion as follows (page 25 and 26, lines 365-417)

Comments: Illustrate the different mechanisms of antimicrobial resistance in E. coli and K. pneumonia.

Response: Thanks for your kind reminders. We tried to add some point’s mechanisms of antimicrobial resistance in E. coli and K. pneumonia on pages 25 and 26, lines 387-392, page, 27 line 418-421).

-Conclusion

Comments: This should be rephrased to be sounded. A real conclusion should focus on the ques-tion or claim you articulated in your study, which resolution has been the main objective of your paper?

Response: We Revised according to the amended title of the manuscript

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Reviewer #1: No

Reviewer #2: No

Response: Yes

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Response: Thank you for your reminder about the figure format and after confirmation, we checked and submitted them.

Submitted filename: Response to reviewers file.docx

Click here for additional data file.

18 Feb 2022

High prevalence of fecal carriage of Extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar, Northwest Ethiopia

PONE-D-21-38463R1

Dear Dr. Amare,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Abdelazeem Mohamed Algammal, Prof, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: All comments have been addressed

**********

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #2: The authors have carried out significant changes to the manuscript. They have addressed all the suggested corrections and comments. Really, it's an interesting study that has a significant impact. Now, the manuscript could be accepted.

Congratulations.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: No

8 Mar 2022

PONE-D-21-38463R1

High prevalence of fecal carriage of Extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar, Northwest Ethiopia.

Dear Dr. Amare:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Abdelazeem Mohamed Algammal

Academic Editor

PLOS ONE