Prevalence and risk factors for faecal carriage of multidrug resistant Escherichia coli among slaughterhouse workers

doi:10.21203/rs.3.rs-243806/v1

Download PDF

Research Article

Prevalence and risk factors for faecal carriage of multidrug resistant Escherichia coli among slaughterhouse workers

https://doi.org/10.21203/rs.3.rs-243806/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

The increasing occurrence of antimicrobial-resistant Escherichia coli in human and animal population has become a global public health problem that requires immediate intervention. We aimed to investigate prevalence and risk factors for faecal carriage of drug-resistant E. coli among slaughterhouse workers. We conducted this cross-sectional study among 118 apparently healthy workers in the largest slaughterhouses in Abuja and Lagos from July to December 2020. E. coli was isolated from stool samples of slaughterhouse workers and antimicrobial susceptibility testing performed using the Kirby-Bauer disk diffusion method. Multi-drug resistance (MDR) was defined as resistance to three or more classes of antibiotics. Majority were males: 88.1% (n = 104), aged > 41years: 28.8% (n = 34), married: 70.3% (n = 83), and were butchers: 53.4% (n = 63). Prevalence of MDR E. coli was 50% (n = 59), highest among butchers compared to Slaughterhouse cleaners. Of 75 E. coli isolates identified, 25.3% (n = 19) were ESBL producers; 78.7% (n = 59) were MDR. Keeping animals (p = 0.01); eating at the Slaughterhouse (p = 0.03) and collecting waste (p = 0.02) remained independent risk factors for acquiring MDR E. coli. Prevalence of resistant E. coli was highest among butchers and associated with keeping animals at home, eating at work, and waste-collection. Hand-hygiene and responsible use of antibiotics among slaughterhouse workers should be encouraged.

General Microbiology

Health Economics & Outcomes Research

Escherichia coli

prevalence

slaughterhouse workers

risk factors

Globally, Escherichia coli, which is known as an indicator bacteria, is a common source of foodborne illnesses especially in sub-Saharan Africa including Nigeria ^1,2. The emergence of multi-drug resistant (MDR) foodborne pathogens has further worsened the public health implications of antimicrobial resistance (AMR) resulting in increased length of hospital stay, mortality, and economic burden³. Sub-Saharan Africa, particularly Nigeria is not exempted from the challenges posed by AMR^1,2 as a review conducted in West Africa reported majority of the studies were from Nigeria⁴. The World Health Organization (WHO) has reported that a high level of AMR observed in the human population is due to misuse or abuse of antimicrobials in the food and agriculture sectors⁵.

Food, particularly meat is a major source of MDR-E. coli among humans^6,7. In many developing economies, food-producing animals are slaughtered and processed under very poor hygienic conditions⁸, hence, constituting an occupational risk to slaughterhouse workers (SHWs). MDR-E. coli, often detected in the guts of apparently healthy individuals, may not directly cause disease, but serve as a reservoir of AMR genes^9,10. These resistant genes can further spread to other Gram-negative pathogenic bacteria in the gut and potentially become harmful hence causing disease but can also spread due to open defecation and poor hygiene¹¹.

Slaughterhouses in some settings are hotspots for MDR pathogens, facilitating their spread to the environment¹². Evidence shows that SHWs are at risk of being exposed to certain bacterial infections because of their occupation^13,14. Antimicrobial drugs (AMDs) are also easily accessible to livestock farmers without any prescription from the veterinarian, thus, encouraging antimicrobial use (AMU) indiscriminately in food-producing animals in Nigeria due to easy access¹⁵.

Few studies conducted in Nigeria have examined the presence of MDR-E.coli in human patients and food-producing animals, however, the risk factors for faecal carriage of MDR-E. coli among SHWs is not yet known^16,17. A recent review has reported that human exposure to environmental sources was an important risk factor for the acquisition of MDR-E. coli¹⁰. Most slaughterhouse environments in Nigeria are poorly managed with very poor sanitary conditions, hence promoting the spread of resistant pathogens¹⁸. Nigeria has a growing population of livestock characterized by an extensive system of management making it difficult to track AMU in this food-animal population because of their constant movement in search of food. Besides, there are problems of illegal importation and sale of antimicrobials for use in livestock¹⁹. It is also practically impossible to estimate the public health risks encountered by SHWs when they share the same environment with livestock and their waste products².

There is increased AMU among livestock in Nigeria with resultant increase in the development of AMR among enteric pathogens and indicator bacteria such as E. coli. It is therefore imperative to generate data on the resistance profile of indicator bacteria among apparently healthy individuals exposed to livestock by their occupation for proper diagnosis and treatment of bacterial infections.

We hypothesized that livestock harboring MDR-E. coli facilitates the transmission of the pathogen to workers exposed to livestock and the slaughterhouse environment. This study aimed to investigate the prevalence and risk factors for faecal carriage of MDR-E. coli among SHWs to generate baseline data for the establishment of the AMR Animal-Health Surveillance system in Nigeria. Prevalence was determined by antimicrobial susceptibility testing while risk was accessed based on interviews and multivariate analysis.

Of the 118 SHWs interviewed, majority were males: 88.1% (n = 104), aged > 41years: 28.8% (n = 34), married: 70.3% (n = 83), Muslims: 70.3% (n = 83), had Islamic education: 31.4% (n = 37), lived in urban areas: 96.6% (n = 114), of Hausa ethnicity: 73.7% (n = 87) and were butchers: 53.4% (n = 63) (Table 1).

Table 1

Demographic Characteristics of Slaughterhouse workers in Abuja and Lagos - Nigeria, 2020
Variables	n	%
Age Group (years)
18–25	23	19.5
26–33	29	24.6
34–41	32	27.1
> 41	34	28.8
Marital Status:
Divorced	1	0.9
Married	66	54.6
Single	55	45.5
Widowed	2	1.7
Educational level:
None	6	5.1
Islamic education	37	31.4
Primary	17	14.4
Secondary	34	28.8
Tertiary	24	20.3
Occupation:
Butchers	63	53.4
Slaughterhouse cleaners	21	17.8
Vets/Para-vets	11	9.3
Meat Carriers	10	8.5
Meat Sellers	6	5.1
Livestock Traders	5	4.2
Poultry seller	2	1.7
Duration of work:
0–5 years	35	29.7
≥ 6 years	83	70.3
Location:
Abuja	84	71.2
Lagos	34	28.8
Residence:
Rural	4	3.4
Urban	114	96.6
Religion:
Christianity	35	29.7
Islam	83	70.3
Ethnicity:
Ibo	4	3.4
Hausa	87	73.7
Yoruba	18	15.3
Others	9	7.6

Prevalence of MDR E. coli among Slaughterhouse workers

Fifty-nine SHWs of the 118 included in the study tested positive for MDR-E. coli giving a prevalence of 50%. A majority of these, 69.5% (n = 41) resided in Abuja and the highest prevalence of 50.9% was among butchers (prevalence OR = 1.00, 95% Confidence Interval [CI] = 0.37–2.69; p = 0.00) when compared to slaughterhouse cleaners.

Out of 75 E. coli isolates identified, 25.3% (n = 19) were ESBL-E. coli while 78.7% (n = 59) were MDR. The isolates were resistant to ciprofloxacin: (81.3%, n = 61), tetracycline: (77.3%, n = 58), sulfonamides: (72%, n = 54), trimethoprim: (72%, n = 54), ampicillin: (72%, n = 54), nalidixic acid: (57.3%, n = 43), cefotaxime: (25.3%, n = 19), gentamicin: (20%, n = 15), chloramphenicol: (13.3%, n = 10), ceftazidime: (13.3%, n = 10), and azithromycin: (10.7%, n = 8) (Fig. 1).

All the E. coli isolates were 100% susceptible to cefoxitin, colistin, imipenem, meropenem and nitrofurantoin. The maximum number of ESBL-E. coli was isolated from butchers: 57.9% (n = 11) when compared to other SHWs. The resistance rates of the E. coli isolates to ciprofloxacin, ampicillin, tetracycline, sulfonamides, trimethoprim, nalidixic acid, cefotaxime, and chloramphenicol were higher among butchers when compared to the rest of the SHWs. All the isolates were, however, sensitive to cefoxitin, colistin, imipenem, meropenem, and nitrofurantoin (Table 2).

Table 2

Resistance profile of *E. coli* isolates recovered from slaughterhouse workers at Abuja and Lagos Slaughterhouses to the antibiotics tested
Drug Class	Drug	Resistance break point µg/mL	Overall, n = 75 (%)	Butchers n = 42 (%)	Others n = 33 (%)
Quinolones	Ciprofloxacin (5µg)	≤ 20	61 (81.3)	32 (76.2)	29 (87.9)
Quinolones	Nalidixic acid (30µg)	≤ 13	43 (57.3)	22 (52.4)	21 (63.6)
Tetracyclines	Tetracycline (30µg)	≤ 11	58 (77.3)	30 (71.4)	28 (84.9)
Folate Pathway antagonists	Sulfonamides (300µg)	≤ 12	54 (72.0)	28 (66.7)	26 (78.8)
Folate Pathway antagonists	Trimethoprim (5µg)	≤ 10	54 (72.0)	28 (66.7)	26 (78.8)
Penicillins	Ampicillin (10µg)	≤ 13	54 (72.0)	32 (76.2)	22 (66.7)
Cephalosporins	Cefotaxime (30µg)	≤ 22	19 (25.3)	11 (26.2)	8 (24.2)
Cephalosporins	Ceftazidime (30µg)	≤ 17	10 (13.3)	5 (11.9)	5 (15.2)
Aminoglycosides	Gentamicin (10µg)	≤ 12	15 (20.0)	5 (11.9)	10 (30.3)
Phenicols	Chloramphenicol (30µg)	≤ 12	10 (13.3)	6 (14.3)	4 (12.1)
Macrolides	Azithromycin (15µg)	≤ 12	8 (10.7)	3 (7.2)	5 (15.2)
Resistance to 3 or more classes of antibiotics	MDR	n/a	59 (78.7)	30 (71.4)	29 (87.9)

In this study, 6.6% (n = 5) of the isolates were susceptible to all the AMDs tested, 4% (n = 3) were resistant to only one AMD while 9.3% (n = 7) were resistant to only two AMDs. Surprisingly one isolate was resistant to 11 different AMDs including those of critical importance for human health. In 72% (n = 54) of the isolates, a MAR index greater than 0.2 was observed (Fig. 2).

Among the 19 ESBL-E. coli isolates, high resistance was observed for cefotaxime: 100% (n = 19), ciprofloxacin: 94.7% (n = 18), tetracycline: 94.7% (n = 18), sulfonamides: 94.7% (n = 18), trimethoprim: 94.7% (n = 18), ampicillin: 94.7% (n = 18) and nalidixic acid: 78.9% (n = 15).

Antimicrobial Use patterns of Slaughterhouse workers

Among 59 SHWs colonized with MDR-E. coli, 61% (n = 36) reported that they had taken antibiotics in the last month before stool collection. Less than half 39% (n = 23) reported that the last antibiotics they had taken were Ampiclox (a combination of ampicillin and cloxacillin) especially when they had a knife cut. About a third 35.6% (n = 21) of the workers reported that they self-prescribed antimicrobials more than three times in the last three months before sampling. A majority 93.2% (n = 55) reported that they usually purchased antibiotics from a chemist although 89.8% (n = 53) reported that they usually received direction for use from the pharmacist. More than two-thirds 71.2% (n = 42) reported that they usually completed their dosage while 30.5% (n = 18) reported that they stopped their medication whenever they felt better.

Risk factors for MDR- E. coli among SHWs

Occupational hazards reported among the SHWs were quite a few. Nearly half (49.2%; n = 58) of the workers reported that a major hazard they faced was knife cuts while slaughtering the animals. Over half (56.9%; n = 33) reported that they however had to continue processing slaughtered animals even after sustaining a knife cut, hence their wounds get exposed to infection. Nearly half (48.3%; n = 57) reported that they often handle fetuses recovered from slaughtered pregnant animals.

The factors associated with MDR-E. coli among SHWs were: aged over 41years old (OR = 2.79, 95% CI = 1.21–6.45); keeping animals at home (OR = 2.13, CI = 1.02–4.44); eating at the slaughterhouse while working (OR = 2.13, CI = 1.02–4.44); collecting slaughterhouse waste (OR = 6.53, 95% CI = 1.38–30.92); and washing hands with soap (OR = 0.42, 95% CI = 0.19–0.93) (Table 3).

Table 3

Bivariate Analysis of Factors Associated with Multidrug resistant *E. coli* recovered from Slaughterhouse Workers in Abuja and Lagos-Nigeria, 2020
Work Exposure	MDR (Yes)	MDR (No)	Odds Ratio (95% Confidence level)	P-value
Age
≥ 41 years	23	11	2.79 (1.21–6.45)	0.02*
18–41 years	36	48
Level of Education
Less than Secondary School	29	31	0.87 (0.42–1.80)	0.71
Secondary and above	30	28
Occupation
Butcher	30	33	0.82 (0.40–1.68)	0.58
Others	29	26
Work Exposure
≥ 6 years	41	42	0.92 (0.42–2.03)	0.84
1–5 years	18	17
Slaughtering animal after sustaining an injury
Yes	18	15	1.80 (0.63–5.16)	0.27
No	10	15
Keeping animals at home
Yes	34	23	2.13 (1.02–4.44)	0.04*
No	25	36
Eating while working at Slaughterhouse
Yes	36	25	2.13 (1.02–4.44)	0.04*
No	23	34
Collecting Slaughterhouse waste
Yes	11	2	6.53 (1.38–30.92)	0.01*
No	48	57
Washing hands with soap
Yes	19	34	0.42 (0.19–0.93)	0.03*
No	28	21
Diarrhoea in last 3 months
Yes	23	27	0.76 (0.36–1.57)	0.46
No	36	32
*Values that were significant at bivariate analysis.

Five factors that were statistically significant at bivariate analysis (p ≤ 0.05) were included in the unconditional logistic regression model used in this study. These factors include age over 41years, keeping animals at home, eating at the slaughterhouse while working, collecting slaughterhouse waste, and washing hands with soap. There were fewer female workers hence gender was considered as a potential confounder and included in the final model used for analysis. Using a stepwise elimination approach and controlling for gender, all factors remained statistically significant in the final logistic regression model. We identified four independent risk factors and one protective factor. Being ≥ 41 years (Adjusted OR [AOR] = 4.47, 95% CI = 1.59–12.60); keeping animals at home; (AOR = 4.04, 95%CI = 1.48–11.05); eating at the slaughterhouse (AOR = 3.01, 95%CI = 1.14–7.91) and collecting slaughterhouse waste (AOR = 10.31, 95%CI = 1.54–69.10) remained independent risk factors for faecal carriage of MDR-E. coli among SHWs. Washing hands with soap (AOR = 2.72, 95%CI = 1.06–6.96), however, remained a protective factor against MDR-E. coli among SHWs (Table 4).

Table 4

Factors found to be significantly associated with MDR *E. coli* among Slaughterhouse Workers in Abuja and Lagos, Nigeria 2020 in a Logistic Regression Model
Work Exposure Factors	Adjusted Odds Ratio	95% Confidence interval	P value
Age > 41 years	4.47	1.59–12.60	0.01*
Keeping animals at home	4.04	1.48–11.05	0.01*
Eating at the Slaughterhouse while working	3.01	1.14–7.91	0.03*
Collecting Slaughterhouse waste	10.31	1.54–69.10	0.02*
Washing hands with soap	2.72	1.06–6.96	0.04^#
Independent risk factors for MDR E. coli* that remained significant in the logistic regression model. ^#Only protective factor against MDR E. coli that was significant in the final logistic regression model used for the multivariate analysis. Gender was included in the final model to control for possible confounders.

Unconditional logistic regression analysis showed that SHWs had 4 times the odds of shedding MDR-E. coli in their stool if they were ≥ 41years of age (p = 0.01), kept animals at home (p = 0.01), and 3 times more likely if they ate at the slaughterhouse while processing slaughtered cattle (p = 0.03). Workers were 10 times more likely to shed MDR-E. coli in their stool if they collected slaughterhouse - waste (p = 0.02). They were 3 times more likely to be protected from being colonized by MDR-E. coli if they washed their hands with soap and clean water (p = 0.04).

To the best of our knowledge, this is the first study to document the prevalence and risk factors for faecal carriage of MDR-E. coli among SHWs in Abuja and Lagos, Nigeria. The present study recorded a high prevalence of 50% for MDR-E. coli among SHWs in both locations when compared with 36.6% and 36.8% observed in the general population respectively ^23,24. A similar study conducted among poultry workers in Abuja reported a lower prevalence of 39.7% for MDR-E. coli ⁹. The high prevalence observed in the present study might be due to AMU in animals which have been associated with the development of bacterial resistance in humans exposed to these food-producing animals ^25–27. The present study demonstrates that 72% of MDR-E. coli recovered from the SHWs had a MAR index above 0.2. This implies that the isolates may have been recovered from sources with high frequency of AMU ²⁸ although multiple AMR is usually detected when plasmids encode AMR phenotype ²².

Our findings show a high prevalence of MDR-E. coli but a low prevalence of ESBL-E. coli colonization in the study population possibly because ceftazidime and cefotaxime are not one of the commonly self-prescribed antimicrobials in the present study. This supports the findings from a survey in the Mekong Delta in Vietnam where a prevalence of 81.3% was observed for MDR-E. coli, while a prevalence of 3.2% was recorded for ESBL-E. coli ²⁹. In contrast to our findings, a study conducted in Pakistan reported a much higher ESBL-E. coli prevalence among cattle and healthy individuals ³⁰. A related study conducted in southern Nigeria also reported a much higher ESBL and MDR-E. coli prevalence among pig SHWs ³¹.

This study observed that the MDR-E. coli isolates recovered from SHWs were resistant to ciprofloxacin, tetracycline, ampicillin, sulfonamides, trimethoprim and nalidixic acid. Inappropriate AMU in humans and animals has been documented in literature to promote selective increase in some bacterial populations as well as dissemination of resistant strains ^32,33. A higher ciprofloxacin resistance compared to nalidixic acid resistance was observed among MDR-E. coli isolates supporting recent reports of a similar study in southern Nigeria where a high frequency of ciprofloxacin resistance was observed in isolates recovered from pig slaughterhouse workers ³¹. A possible explanation for this observation is likely due to qnr genes where qnrS1 is the most common in Africa especially since fluoroquinolone resistance in different regions have different characteristics ³⁴. This is not surprising as ciprofloxacin is one of the most commonly prescribed quinolones that is affordable and available over the counter in Nigeria ³⁵. The MDR-E. coli isolates were susceptible to meropenem and imipenem consistent with recent studies conducted among pig slaughterhouse workers in southern Nigeria ³¹. This is not surprising as these antimicrobials are normally given as intravenous injections however, resistance started to emerge in poultry and pigs in EU with VIM and IMP genes detected in E. coli and Salmonella including a few Oxa-48 genes ³⁶. Acquired MDR does not occur only in pathogenic bacteria, but in the microflora of exposed individuals and animals. According to WHO, the important drivers for the colonization of MDR pathogens is overuse and misuse of antimicrobial drugs ⁵ although studies have reported that occupation, diet, health, sanitation, and cultural traditions are also drivers for AMR ³⁷. This could probably explain the high prevalence of MDR-E. coli observed in the present study especially since some SHWs reported self-prescription of antibiotics, incomplete dosages, poor hygiene and sanitation amongst others.

A related study among pig SHWs in Europe reported that the workers were more likely to be colonized by resistant bacteria if they were involved in the early slaughtering processes ³⁸. This supports our study results as butchers who are involved in these early processes were the most affected possibly because of close contact with gut content at the beginning of slaughtering especially on open slabs.

Factors associated with MDR-E. coli in the present study were age, exposure to animals at home, eating at the slaughterhouse while working, collecting waste and hand hygiene. While the former are independent risk factors, the latter is a protective factor. A related study done in Nigeria among poultry workers reported that history of diarrheoa and work exposure of over 10 years were risk factors for feacal carriage of MDR-E. coli ⁹ however the present study did not observe any association with these factors among SHWs. A similar study done among poultry SHWs confirms that some risks do exist from occupational exposure for the acquisition of MDR-E. coli ³⁹.

Washing hands with soap was found to be a protective factor against faecal carriage of MDR-E. coli among the workers. According to WHO, lack of clean water and sanitation facilitates the spread of antimicrobial resistant microbes ⁵. It has been documented that washing hands with soap and clean water is very effective in removing faecal bacteria from the hands ⁴⁰ confirming our study results. The importance of hand-hygiene and sanitation should be encouraged in the fight against AMR.

Mechanism of AMR spread from animals to humans is not clear however, literature shows that animals are reservoirs for E. coli found in humans ⁴¹. Our results show that SHWs were colonized by MDR-E. coli resistant to commonly used antibiotics in humans and livestock. In addition, we identified slaughterhouse waste collection as a risk factor for the acquisition of MDR-E. coli among SHWs. Poor sanitary slaughterhouse environment ^31,42 and occupational exposure to food animals may have been responsible for this observation. There is paucity of studies on risk factors for MDR-E. coli in the target population. Some studies reported travel history and diet as risk factors for faecal carriage of MDR-E. coli however the present study did not consider these factors ^10,43.

Apparently healthy SHWs could harbor and shed MDR E. coli in their stool hence posing as a public health hazard to the general population. Prevalence of resistant E. coli was highest among butchers. Risk factors for faecal carriage of MDR E. coli were age, animal exposure at home, eating at work and waste-collection. Handwashing with soap and clean water was protective against acquiring MDR E. coli among the workers. This study provides additional evidence demonstrating the occupational hazards for people working in slaughterhouses. We recommend that competent authorities organize regular AMR awareness programs targeted at SHWs to mitigate public health risks. Hand-hygiene and responsible use of antibiotics among SHWs should be encouraged.

Study area

This study was conducted in two major cities; Abuja metropolis, North-Central part of Nigeria (9.01145° N, 7.57961° E) with a population of approximately 3 M people and Lagos city, South-Western Nigeria (6.64983° N, 3.32023° E) inhabited by 14 M people, respectively.

Study Design

A cross-sectional study approach was applied among 118 apparently healthy workers who were randomly selected from the two largest slaughterhouses situated in Abuja (84 samples) and Lagos (34 samples) from July to December 2020. All SHWs aged ≥ 18yrs, who volunteered were enrolled as study participants.

An open data kit (ODK) installed on a smartphone was used to administer a structured interviewer-administered questionnaire to SHWs for data collection on demographic characteristics and exposure factors.

Sampling Method

The largest slaughterhouses each in Abuja and Lagos were selected based on the daily volume of livestock slaughter. Using a simple random sampling method, we selected workers from both slaughterhouses for the study. We collected freshly passed stool samples (one sample per worker) using sterile stool containers and transported the samples in cool boxes to the National Reference Laboratory (NRL), Nigeria Centre for Disease Control, Abuja. The samples collected in Abuja were processed within 3 hours of sample arrival in the laboratory for the presence of E. coli. Samples collected in Lagos were stored at -80°C thereafter they were transported to the NRL, Abuja for processing. Positivity was determined as the presence of MDR-E. coli in a stool sample.

Ethics approval

We obtained ethical approval from the Scientific and Ethical committee of the FCT Health Research Ethics Committee (Approval Number: FHREC/2020/01/40/04-05-20) and sought permission from the management of each slaughterhouse before the study commenced.

The study participants were also assured of confidentiality of the information obtained. All procedures were performed in accordance with the ethics committee’s guidelines and requirements.

Bacteriological Analysis

Isolation of E. coli isolates

Briefly, we inoculated one gram of stool sample in 9 ml of buffered peptone water (enrichment broth) and incubated at 37°C for 24 h. Thereafter, a 10 ul loop-full culture from buffered peptone water was streaked onto the selective media, MacConkey agar, and incubated at 37°C for 24 h. Three well-isolated suspected E. coli colonies were streaked, usually pink to red onto the indicative media, Eosin Methylene Blue agar, and incubated at 37°C under aerobic conditions for 24h ²⁰. Elevated, moist colonies with a greenish metallic sheen, suggestive of E. coli, were sub-cultured onto the non-selective media, Trypticase Soy agar plates, and incubated for 24h at 37°C for the isolation of pure cultures. Conventional biochemical tests were performed on all E. coli isolates ²⁰ and confirmed using a commercially available biochemical test strip, Microbact GNB 24E (Oxoid, UK), according to the Manufacturer’s instructions.

Antimicrobial susceptibility testing ofE. coliisolates

The Kirby Bauer disk diffusion method was applied to perform antimicrobial susceptibility testing of the E. coli isolates according to the Clinical and Laboratory Standards Institute (CLSI, 2020) recommendations ²¹. A panel of 16 different antibiotics of 12 classes was applied including ampicillin (10µg), azithromycin (15µg), tetracycline (30µg), gentamicin (10µg), chloramphenicol (30µg), colistin (10µg), sulfonamides (300µg), trimethoprim (5µg), cefoxitin (30µg), ciprofloxacin (5µg), nalidixic acid (30µg), nitrofurantoin (300µg), imipenem (10µg), meropenem (10µg), ceftazidime (30µg), and cefotaxime (30µg). For quality control, we used E. coli ATCC 25922. In this study, we defined MDR as the bacteria isolate being resistant to three or more classes of antibiotics commonly used in medical and veterinary practice in Nigeria.

Multiple Antibiotic Resistance (MAR) indexes

We calculated the MAR index for each E. coli isolate using the formula MAR = a/b, where ‘a’ was the number of antibiotics that the isolate was resistant to while ‘b’ was the total number of antibiotics that the isolates were subjected to ²².

ESBL phenotype detection by disk diffusion

Presumptive extended-spectrum beta-lactamase producing E. coli (ESBL- E. coli) isolates were further confirmed using the disk diffusion method according to CLSI 2020 ²¹ testing for synergy applying the following disks: ceftazidime (30µg) (CAZ), ceftazidime + clavulanic acid (30/10µg) (CAC), cefotaxime (30µg) (CTX), cefotaxime + clavulanic acid (30/10µg) (CEC). Following growth, the zone-diameters were measured and recorded. An increase in the zone-diameter by ≥ 5 mm around the disks containing cephalosporin with the inhibitor over the disks containing solely cephalosporin indicates ESBL production (synergy). Controls: E. coli ATCC 25929 and K. pneumoniae ATCC 700603 were used as negative and positive controls, respectively.

Risk factor survey

We administered electronic questionnaires to SHW using ODK collect app on a smartphone to determine the risk factors for MDR E. coli colonization. We assessed the association between demographics, AMU, exposure factors, and acquiring MDR-E. coli. We obtained permission from slaughterhouse management before administering the questionnaire to the workers. We pretested the questionnaire at the Dei-Dei slaughterhouse, however; we did not include data obtained from the pretesting in the final analysis.

Statistical Analysis

The data collected with ODK collect app was exported as a Microsoft Excel spreadsheet to a computer using the ODK briefcase tool. Data were analyzed using Epi Info version-7 by computing frequencies and proportions. To identify risk factors for MDR-E. coli, we performed bivariate analysis using odds-ratios (OR) as a measure of association. Risk factors identified as statistically significant at bivariate analysis were included in an unconditional logistic regression model used for multivariate analysis at a 5% significance level. All relevant data are within the paper and available as supporting information.

Acknowledgement

The authors would like to appreciate Dr. Pernille Nilsson of Denmark Technical University and Prof. Iruka Okeke of University of Ibadan, Nigeria for their contributions towards the completion of this research. We would like to acknowledge the support of the Chief Veterinary Officer of Nigeria, Dr. Alabi Olaniran and Dr. Peter Umanah at Department of Veterinary & Pest Control Services, Federal Ministry of Agriculture and Rural Development, Abuja. The authors appreciate Dr. Chikwe Ihekweazu, Director General, Nigeria Center for Disease Control, Abuja for providing laboratory support towards this research at the National Reference Laboratory (NRL), Abuja. We acknowledge the intellectual contributions of Miss Eme Ekeng, Mr. Micheal Popoola and Mr. Akinpelu Muftan of NRL, Abuja towards the success of this research. The authors appreciate the support Dr. Stephen Okeme at Karu Slaughterhouse, Abuja and Dr. Ayokunle Omileye at Oko-oba Agege Slaughterhouse, Lagos.

Authors’ contributions

RH and MA made substantial contributions to conception and design. MA was the principal investigator, designed data collections tools, collected data, conducted laboratory work, analyzed and interpreted the data. DA and BH assisted in designing data collection tool. DA supported with data collection. RH and NM supervised the laboratory aspect of the research. MA wrote the first draft of the manuscript. RH, BH, and NM revised article critically for important intellectual content. All authors read and approved the final manuscript.

Competing interests

The authors have declared that there are no competing financial interests.

Data Availability Statement

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Kariuki, S. & Dougan, G. Antibacterial resistance in sub-Saharan Africa: an underestimated emergency. Ann. N. Y. Acad. Sci. 1323, 43–55 (2014).
Katakweba, A. A. S. et al. First Report on a Randomized Investigation of Antimicrobial Resistance in Fecal Indicator Bacteria from Livestock, Poultry, and Humans in Tanzania. Microb. Drug Resist. 24, 260–268 (2018).
Dadgostar, P. Antimicrobial resistance: implications and costs. Infection and Drug Resistance. 12, 3903–3910 (2019).
Bernabé, K. J., Langendorf, C., Ford, N., Ronat, J. B. & Murphy, R. A. Antimicrobial resistance in West Africa: a systematic review and meta-analysis. Int. J. Antimicrob. Agents. 50, 629–639 (2017).
WHO & Antimicrobial resistance. WHO https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance (2020).
Yamaji, R. et al. A Population-Based Surveillance Study of Shared Genotypes of Escherichia coli Isolates from Retail Meat and Suspected Cases of Urinary Tract Infections. mSphere 3(2018).
Saliu, E. M., Vahjen, W. & Zentek, J. Types and prevalence of extended-spectrum beta-lactamase producing Enterobacteriaceae in poultry. Animal Health Research Reviews. 18, 46–57 (2017).
Sebsibe, M. A. & Asfaw, E. T. Occurrence of multi-drug resistant escherichia coli and escherichia coli o157:H7 in meat and swab samples of various contact surfaces at abattoir and butcher shops in Jimma town, southwest district of Ethiopia. Infect. Drug Resist. 13, 3853–3862 (2020).
Aworh, M. K., Kwaga, J., Okolocha, E., Mba, N. & Thakur, S. Prevalence and risk factors for multi-drug resistant Escherichia coli among poultry workers in the Federal Capital Territory, Abuja, Nigeria. PLoS One. 14, e0225379 (2019).
Hu, Y., Matsui, Y. & Riley, W. L. Risk factors for fecal carriage of drug-resistant Escherichia coli: A systematic review and meta-analysis. Antimicrob. Resist. Infect. Control. 9, 31 (2020).
Szmolka, A. & Nagy, B. Multidrug resistant commensal Escherichia coli in animals and its impact for public health. Front. Microbiol. 4, 258 (2013).
Savin, M. et al. Antibiotic-resistant bacteria and antimicrobial residues in wastewater and process water from German pig slaughterhouses and their receiving municipal wastewater treatment plants. Sci. Total Environ. 727, 138788 (2020).
Neyra, R. C., Vegosen, L., Davis, M. F., Price, L. & Silbergeld, E. K. Antimicrobial-resistant bacteria: An unrecognized work-related risk in food animal production. Safety and Health at Work. 3, 85–91 (Elsevier Science B.V. (2012).
Abdullahi, A. et al. Occupational hazards among the abattoir workers associated with noncompliance to the meat processing and waste disposal laws in Malaysia. Risk Manag. Healthc. Policy. 9, 157–163 (2016).
Adesokan, H. K., Akanbi, I. O., Akanbi, I. M. & Obaweda, R. A. Pattern of antimicrobial usage in livestock animals in South-Western Nigeria: The need for alternative plans. Onderstepoort J. Vet. Res. 82, 1–6 (2015).
Ibrahim, A., Junaidu, A. & Garba, M. Multiple antibiotic residues in meat from slaughtered cattle in Nigeria. Internet J. Vet. Med. 8, 6–10 (2012).
Igwe, J., Olayinka, B., Ehnimidu, J. & Onaolapo, J. Virulent Characteristics of Multidrug Resistant E. coli from Zaria, Nigeria.Clin. Microbiol. Open Access5, (2016).
Alhaji, N. B. & Baiwa, M. Factors affecting workers’ delivery of good hygienic and sanitary operations in slaughterhouses in north-central Nigeria. Sokoto J. Vet. Sci. 13, 29–37 (2015).
Nigeria Centre for Disease Control. Antimicrobial use and resistance in Nigeria.1–158(2017).
Bhoomika, Shakya, S., Patyal, A. & Gade, N. E. Occurrence and characteristics of extended-spectrum β-lactamases producing Escherichia coli in foods of animal origin and human clinical samples in Chhattisgarh, India. Vet. World. 9, 996–1000 (2016).
Clinical and Laboratory Standards Institute. CLSI M100 30th Edition. Journal of Services Marketing vol. 30th (2020).
Sandhu, R., Dahiya, S. & Sayal, P. Evaluation of multiple antibiotic resistance (MAR) index and Doxycycline susceptibility of Acinetobacter species among inpatients. Indian J. Microbiol. Res. 3, 299 (2016).
Adesoji, A. T. & Liadi, A. M. Antibiogram studies of escherichia coli and salmonella species isolated from diarrheal patients attending malam mande general hospital dutsin-ma, Katsina State, Nigeria. Pan Afr. Med. J. 37, 1–13 (2020).
*Shitu, S., Gambo, B. A., Musa, M. O., Abubakar, A. A. & Attahiru, M. Prevalence of Multidrug Resistant Escherichia Coli In Suspected Cases of Urinary Tract Infection Among Patients Attending Ahmadu Bello University Medical Center, Zaria. UMYU J. Microbiol. Res. 5, 123–130 (2020).
Zhuge, X. et al. Population Structure and Antimicrobial Resistance Traits of Avian-origin.0–2(2017) doi:10.1111/tbed.13222.
Landers, T. F., Cohen, B., Wittum, T. E. & Larson, E. L. A review of antibiotic use in food animals: Perspective, policy, and potential. Public Health Rep. 127, 4–22 (2012).
Van, T. T. H., Yidana, Z., Smooker, P. M. & Coloe, P. J. Antibiotic use in food animals worldwide, with a focus on Africa: Pluses and minuses. Journal of Global Antimicrobial Resistance. 20, 170–177 (2020).
Thenmozhi*, S., Rajeswari, P., Suresh Kumar, B. T., Saipriyanga, V. & Kalpana, M. Multi-drug resistant patterns of biofilm forming Aeromonas hydrophilia from urine samples. Int. J. Pharm Sci Res. 5(7): 2908–2918 http://ijpsr.com/bft-article/multi-drug-resistant-patterns-of-biofilm-forming-aeromonas-hydrophila-from-urine-samples/?view=fulltext (2014).
Campbell, J. I., Nhung, N. T., Nhung, H. N., Van Minh, P. & Wagenaar, J. A. Prevalence and risk factors for carriage of antimicrobial-resistant Escherichia coli on household and small-scale chicken farms in the Mekong Delta of Vietnam.2144–2152(2015) doi:10.1093/jac/dkv053.
Saleem, R., Ejaz, H. & Zafar, A. S. Y. Phenotypic characterization of extended-spectrum-beta – lactamase producing E. coli from healthy individuals, patients, sewage sludge, cattle, chickens and raw meat. 33(2017).
Egbule, O. S. & Iweriebor, B. C. Beta-Lactamase-Producing Escherichia coli Isolates Recovered from Pig Handlers in Retail Shops and Abattoirs in Selected Localities in Southern Nigeria: Implications for Public Health(2021).
Singh, S., Shukla, S., Tandia, N., Kumar, N. & Paliwal, R. Pharma science monitor. 5, 184–197 (2014).
Daniels, J. B. et al. Role of Ceftiofur in Selection and Dissemination of blaCMY-2-Mediated Cephalosporin Resistance in Salmonella enterica and Commensal Escherichia coli Isolates from Cattle. Appl. Environ. Microbiol. 75, 3648–3655 (2009).
Qin, T. T. et al. SOS response and its regulation on the fluoroquinolone resistance. Ann Transl Med. 3, 358 (2015).
Suleiman, I. A. & Tayo, F. Comparative costs of antibacterial usage in sexually transmitted infections in a Nigerian teaching hospital. Trop. J. Pharm. Res. 9, 549–555 (2010).
Nordmann, P., Naas, T. & Poirel, L. Global Spread of Carbapenemase- producing Enterobacteriaceae. Emerg. Infect. Dis. 17, 1791–1798 (2011).
Hendriksen, R. S. et al. Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage.Nat. Commun.10, (2019).
Dohmen, W. et al. ESBL carriage in pig slaughterhouse workers is associated with occupational exposure. Epidemiol. Infect. 145, 2003–2010 (2017).
van den Bogaard, A. E. Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry slaughterers. J. Antimicrob. Chemother. 47, 763–771 (2001).
Burton, M. et al. The effect of handwashing with water or soap on bacterial contamination of hands. Int. J. Environ. Res. Public Health. 8, 97–104 (2011).
Founou, L. L., Founou, R. C. & Essack, S. Y. Antibiotic Resistance in the Food Chain: A Developing. 7, 1–19 (2016).
De, N., Ayobami, B. O. & Bosede, A. Microbiological Assessment of Agege Abattoir Situated In Lagos State, Nigeria. IOSR J. Environ. Sci. Ver. I. 9, 2319–2399 (2015).
Huijbers, P. M. C. et al. Extended-spectrum and AmpC β-lactamase-producing Escherichia coli in broilers and people living and/or working on broiler farms: prevalence, risk factors and molecular characteristics. J. Antimicrob. Chemother. 69, 2669–2675 (2014).

No competing interests reported.

Download PDF

Editorial decision: Major revision
12 May, 2021
Reviews received at journal
24 Apr, 2021
Reviewers agreed at journal
16 Apr, 2021
Reviewers agreed at journal
08 Apr, 2021
Reviewers invited by journal
08 Apr, 2021
Editor assigned by journal
08 Apr, 2021
Editor invited by journal
04 Mar, 2021
Submission checks completed at journal
04 Mar, 2021
First submitted to journal
15 Feb, 2021

You are reading this latest preprint version

Prevalence and risk factors for faecal carriage of multidrug resistant Escherichia coli among slaughterhouse workers

Status:

Version 1

Abstract

Figures

Introduction

Results

Antimicrobial Use patterns of Slaughterhouse workers

Discussion

Methods

Study area

Study Design

Sampling Method

Ethics approval

Bacteriological Analysis

Isolation of E. coli isolates

Multiple Antibiotic Resistance (MAR) indexes

ESBL phenotype detection by disk diffusion

Risk factor survey

Statistical Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1