Available online on 15.06.2022 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2022 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                                               Research Article 

Incidence and antibiotic profile of gram-positive and gram-negative bacteria isolated from aprons and tables used in abattoir located in Abraka and Obiaruku, Delta State, Nigeria

Anie Clement Oliseloke¹ , Okafo Sinodukoo Eziuzo²* , Anthony Amelia-Jane Oluchi¹ , Egbon Kayode Temitope³ 

¹ Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, Delta State University, Abraka, Nigeria

² Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Delta State University, Abraka, Nigeria

³ Department of Pharmaceutical Microbiology, Federal University, Oye Ekiti, Nigeria

INTRODUCTION

An abattoir is a place where livestock such as cattle, goats and others are slaughtered, processed and distributed for human consumption as well as for other industrial purposes.¹ The abattoir industry is an extremely important component of livestock industry in Nigeria as it provides domestic meat supply to over 150 million people and employment opportunities for the teeming population.² Notwithstanding, the abattoir industries are less developed in developing countries like Nigeria.² Meat serves as a major source of protein and so its daily demand is high. In Nigeria, there has been a continuous increase in the development and growth of livestock production, as it serves as a means of providing steady supply of food animals for slaughter and processing for human consumption.³ The poor state of abattoirs and meat processing plants, lack of meat inspection services with a resultant consumption of unhealthy meat by the public have become a major cause of concern to all stakeholders and public in general and despite the passage of time, there has not been reasonable improvement in abattoir practices in Nigeria.³ There are various means by which contamination of meat occurs in an abattoir. The major sources of contamination of the meat are the animals being slaughtered (from the hides and intestinal tract), the workers, the abattoir environment and to a lower level from the air through aerosols and from the dressing water used for carcass.^4,5 Surfaces which are contaminated with microorganisms have the potential risk of transmitting the pathogens to food during processing; therefore, food borne diseases which occur as a result of contact with hands and surfaces depends largely on the level of contamination.^3,6 Antimicrobial resistance is a major challenge in the management of severe food borne illness, since antimicrobial use in animals selects for resistant food borne pathogens that may be transmitted to humans as food contaminants. This study presents a comprehensive review on gram-positive and gram-negative bacteria, assessing their susceptibility and resistance to antimicrobial agent, pointing out their significance in the health sector as main causes of infections resistant to common antimicrobial agents.

MATERIAL AND METHODS

Materials

Materials used are: Nutrient agar, Peptone water, Nutrient broth, Muller Hinton agar, Simmons Citrate agar, MIU broth media, Crystal violet, safranine stain, Lugol iodine, ethanol, hydrogen peroxide, Kovac’s reagent, glass wares, wire loop, glass rods, lead acetate paper.

Collection of Samples (Apron and Table samples)

A total of hundred (100) samples of apron and table specimen were obtained of which fifty (50) samples were obtained from the aprons of butchers and vendors working in an abattoir located in Abraka and fifty (50) samples from tables used for sectioning the carcasses and displaying the raw meat from an abattoir located in Obiaruku using sterile swab sticks labelled. After collection, the swab sticks were sealed properly and stored at a temperature of 4 °C in the fridge prior to use within 2 hrs of collection.

Test for Microbial Growth and Isolation of Bacteria

The work bench was swabbed using a disinfectant and Nutrient agar previously prepared according to Manufacturers specification and dispensed aseptically on hundred (100) sterile petri- dishes and were inoculated with apron and table specimen with previously preserved swab stick containing collected specimen labelled (1 – 100). Inoculated Petri- dishes were inverted and placed in an incubator at a temperature of 37 ºC for 24 hrs, after which Petri-dishes were examined for microbial growth, observations were recorded.^7,8 Gram test was carried out to classify organisms on their ability to retain the colour of the dye. Catalase, fermentation, oxidase, indole, H²S, MR - VP (Methyl Red and Voges - Proskauer test), urease, citrate utilization test, coagulase and MIU (Motility Indole Urease) test were carried out to identify the organisms isolated. 

Antimicrobial Susceptibility Test

Antimicrobial susceptibility test was carried out using Kirby-bauer disc diffusion method for an already prepared antibiotic multidisc. The antibiotic disc contained the following antibiotic and concentration; Ciprofloxacin 10 µg, Norfloxacin 10 µg, Gentamycin 10 µg, Amoxil 20 µg, Streptomycin 30 µg, Rifampicin 20 µg, Erythromycin 30 µg, Chloramphenicol 30 µg, Ampiclox 20 µg and Levofloxacin 20 µg. 

The table was swabbed using disinfectant and the media used (Mueller-Hinton Agar) was prepared and sterilized according to Manufacturer’s instructions on the label. It was then allowed to cool before aseptically dispensed into sterile Petri - dishes. The agar medium was allowed to solidify at room temperature on the working bench. The isolate was inoculated on the Mueller-Hinton agar using sterile swab stick by dipping it in the broth medium containing the isolate and rubbing it gently on the surface of the Mueller-Hinton agar uniformly. The antibiotic disc was gently and firmly placed on the agar plates using sterile forceps. The impregnated agar plates were then incubated at 37°C for 24hrs and the zone of inhibition was determined.

RESULTS

In this Study, a total of one hundred (100) swab samples comprising 50 samples from aprons of butchers and meat vendors and 50 samples from the tables of butchers and meat vendors were aseptically collected from an abattoir located at Abraka and Obiaruku respectively using sterile swab sticks and these were bacteriologically analyzed for the isolation of gram-positive and gram-negative bacteria. On inoculation, one hundred (100) samples yield growth and they were identified. A total number of 100 bacterial isolates were obtained from both locations. 

Synopsis of Isolated Gram-negative and Gram-positive Bacteria

The synopsis of isolated gram-negative and gram-positive bacteria and their frequency of occurrence is given in Table 1. Proteus spp. was the most dominant gram-negative bacteria species isolated (Table 1) while Mycobacterium spp, Enterococcus spp, Streptococcus spp were the gram-positive isolated (Table 2).

Table 1: Synopsis of Gram-negative Bacteria Isolates

Table 2: Synopsis of gram-positive bacteria isolates and their frequency of occurrence

Key: n- sample size

Zone of Inhibition of Gram-Negative Organisms 

The zones of inhibition for the different organisms revealed that levofloxacin (LEV) and ciprofloxacin (CPX) were the optimized antibiotics for the different gram-negative organisms (Figure 1).

Figure 1: Shows the zone of inhibition of the gram-negative and gram positive bacteria against the various antibiotics

LEV: Levofloxacin; APX: Ampliclox, CH: Chloramphenicol, E: Erythromycin, RD: Rifampicin, S: Streptomycin, AML: Amoxicillin, CN: Gentamicin, NB: Norfloxacin, CPX: Ciprofloxacin

Zones of Inhibition for Gram-positive Organisms

The zones of inhibition for the different organisms revealed that levofloxacin (LEV) and ciprofloxacin (CPX) were the optimized antibiotics for the different gram-positive organisms (Figure 2)

Figure 2: Shows the zone of inhibition of the gram-negative and gram positive bacteria against the various antibiotics

LEV: Levofloxacin; APX: Ampliclox, CH: Chloramphenicol, E: Erythromycin, RD: Rifampicin, S: Streptomycin, AML: Amoxicillin, CN: Gentamicin, NB: Norfloxacin, CPX: Ciprofloxacin

DISCUSSION

It is a fact that contaminated food is the primary source of transmission for many pathogenic bacteria and it is the major cause of enteric diseases in developing countries.³ This study describes the incidence of bacterial pathogens in aprons and tables used in abattoir in Abraka and Obiaruku environs, Delta state. Out of the 50 apron samples collected, majority of the organism sampled were gram-negative. This finding corroborates earlier findings in which majority of the organism identified were gram-negative. 

The microorganisms isolated from aprons are listed and arranged in descending order from the most to the least incident. Proteus spp. and Citrobacter spp. were the most predominant organisms. Contrary result was obtained by Ugwu et al.³ In another related study, Escherichia coli was the predominant organisms.⁶ The presence of these microbes on the aprons goes a long way to show the poor hygienic practices by butchers and meat vendors. Several studies have shown that meat handlers harbor microorganisms especially bacteria asymptomatically.^9,10 Meat handlers with poor personal hygiene and inadequate knowledge working in establishments like abattoir could be possible sources of infections of many enteropathogenic bacteria.¹¹ Similarly, meat handlers who harbor pathogenic bacteria may contaminate foods with their fingers/aprons during food processing and finally lead to infection of consumers.¹² 

The gram-negative bacteria species isolated from the tables are similar to those isolated from aprons with the exception of Pseudomonas. Proteus spp. and Citrobacter spp. were the most prevalent bacteria isolate. However, recent study identified Escherichia coli as the predominant bacteria isolate.⁴ The presence of these microbes on the tables used for sectioning carcasses and displaying meat is an indication that meat placed on such tables could have been contaminated in the process.³ Other factors such as unhygienic practices may also account for the contaminations of the tables after the daily normal sales.³ Proteus spp., Citrobacter spp., and Salmonella spp. were the most prevalent bacteria isolate from both the apron and table samples. Bacteria of the genus Proteus are part of the normal flora of the intestinal tract of humans and animals and are widespread in the environment.^13,14 Proteus species are members of the bacteria family called Enterobacteriaceae and are usually regarded as commensals in the gut and they are mostly recognized clinically as a cause of urinary tract infections.^15,16 Proteus spp. has been recently identified as potential pathogens in Crohn’s disease recurrence after intestinal resection.^17,18 They possess many virulence factors which are potentially vital to gastrointestinal pathogenicity, they include: motility; adherence; the production of urease, hemolysins and IgA proteases; and the ability to acquire antibiotic resistance.¹⁵ Gastrointestinal disease conditions which have been linked to Proteus spp. include: gastroenteritis (both spontaneous and foodborne), nosocomial infections, appendicitis, colonization of devices such as nasogastric tubes and Crohn’s disease (the association of Proteus spp. with Crohn’s disease was very strong in particular).¹⁵

Citrobacter spp. are commensal inhabitants of the intestinal tract of humans and other animals.¹⁹ They have also been recovered from water, sewage and soil.^20,21 Citrobacter spp. are opportunistic pathogens of humans and have been associated with a variety of infections which include: urinary tract infections (UTIs) gastroenteritis, wound infections, pneumonia, brain abscesses, septicemia, meningitis and endocarditis, in particular in neonates and immunocompromised hosts.²² As bacterial contaminants, they have been partly responsible for the cause of foodborne diseases and are often transmitted through food and water.^23,24 Consequently, food-handlers with poor personal hygiene could be potential sources of infections by these microorganisms.²³ 

The Presence of Shigella among the Bacterial isolates points to the fact that the sanitary practices among meat vendors are not satisfactory³, this is because Shigella organisms do not have any natural reservoirs in animals and can only spread from person to person.²⁵ The Centers for Diseases control and Prevention (CDC) estimates that 48million cases of food-borne illness occur in the United States annually and many of these illnesses are caused by Salmonella spp.³ Biological contaminants such as bacteria, viruses, fungi, protozoa and helminths constitute the major cause of food-borne disease with varying degree of severity ranging from mild indisposition to chronic or life-threatening illness or both. In developing countries, such contaminants are responsible for food-borne diseases such as cholera, campylobacteriosis, E. coli, gastroenteritis, salmonellosis, Shigellosis among others.^3,26 The Unhygienic condition of the tables on which the meat is displayed for sale may also contribute to the contamination of meat. Often, the knives and cutlass used in cutting meat are also important contaminants since they are rarely sterilized and the lack of facilities for sterilization of tools at the slaughter houses and meat stalls can be a major cause of this. In addition, the indiscriminate use of these tools in the cutting of offal like the intestines constitute another possible source of meat contamination as the gut contents can easily be spread to the table and the entire meat to be sold. The microbiological safety of food is achieved by as far as possibly ensuring the absence of pathogenic microorganisms and by all means preventing their multiplication. 

The isolates showed varying degree of susceptibility to the different antibiotics used. The antibiotic susceptibility test results showed that all the gram-positive bacteria isolates were sensitive to gentamicin, levofloxacin, chloramphenicol, erythromycin, rifampicin, streptomycin and ciprofloxacin. This may be as a result of the presence of thick peptidoglycan layer which absorbs antibiotics more than gram-negative isolates. Majority of the gram-positive isolates were resistant to norfloxacin except Enterococcus. The gram-positive bacteria isolates were all resistant to amoxicillin and Ampiclox (ampicillin/cloxacillin) with no zone of inhibition. Streptococcus spp. was resistant to amoxicillin and Ampiclox. This result is in tandem with similar results.³ The susceptibility result of bacteria isolated showed that the gram-negative organisms are more resistant than the gram-positive organism; which is expected due to the intrinsic nature of gram-negative cell wall.^3,27

CONCLUSION

This study reveals that the aprons and tables of butchers and meat vendors at the abattoirs are a reservoir of various bacteria, some of which are food borne pathogens and are multidrug resistant.The high prevalence of some of these organisms in this study coupled with their high antibiotic resistance profile is reflective of the poor hygiene practices carried out at the abattoirs in Abraka and Obiaruku and thus pose a serious public health concern to the consumers of meat from such abattoirs.

REFERENCES

1. Onuoha SC, Eluu SC, Okata MO. In-vitro Antimicrobial Resistance of Shigella and Salmonella species Recovered from abattoir effluent in Afikpo, South Eastern Nigeria. International Journal of Current Microbiology and Applied science 2016; 5(4):488-497. https://doi.org/10.20546/ijcmas.2016.504.058

2. Narfarnda WD, Ajayi IE, Shawulu JC, Kawe MS, Omeiza GK, Sani NA, Tenuche OZ, Dantong DD, Tags SZ. Bacteriological Quality of Abattoir Effluents Discharged into Water Bodies in Abuja, Nigeria. International Scholarly Research Network. 2012; Vol.2012, Article ID 515689, 5 pages. https://doi.org/10.5402/2012/515689

3. Ugwu MC, Abuchi U, Gugu T, Ugwu BC, Okezie U, Unachukwu C, Stanley CN. Incidence and antibiotic susceptibility pattern of Gram-negative bacteria isolated from aprons of meat vendors in Awka, Anambra State, Nigeria. World Journal of Pharmacy and Pharmaceutical sciences 2016; 5(4):269-277.

4. Birhanu W, Weldegebriel S, Bassazin G, Mitku F, Birku L, Tadasse M. "Assessment of microbiological quality and meat handling practices in butcher shops and abattoirs found in Gondar town, Ethiopia," International Journal of Microbiological Research 2017; 8(2):59-68.

5. Bell RG, Hathaway SC. "The hygiene efficiency of conventional and inverted lamb dressing systems," Journal of Applied Bacteriology 1996; 81(3):225-234. https://doi.org/10.1111/j.1365-2672.1996.tb04322.x

6. Eruteya OC., Akpan SA. and Ubogu M. Antibiotic sensitivity of bacteria isolated from aprons of beef vendors in Port Harcourt, Nigeria. African Journal of Food sciences 2012; 6(15):401-406. https://doi.org/10.5897/AJFS12.004

7. Okafo SE, Anie CO, Nwanua MC. Formulation and Evaluation of Antimicrobial Topical Creams from Ethanolic Extract of Vernonia ambigua Leaves. Nigeria Journal of Pharmaceutical Research 2019; 15(2):249-255. https://doi.org/10.4314/njpr.v15i2.12

8. Okafo SE, Anie CO, Omoh JO. Evaluation of herbal creams formulated using ethanolic extract of Carica papaya leaves. International Journal of Biology, Pharmacy and Allied Sciences 2022; 11(5):2179-2190. https://doi.org/10.31032/IJBPAS/2022/11.5.5942

9. Senthilkumar B, Prabakaran G. Multidrug resistant Salmonella typhi in asymptomatic typhoid carriers among food handlers in Namakkal district, Tamil Nadu. Indian J. Med. Microbiol. 2005; 23:92-94. https://doi.org/10.1016/S0255-0857(21)02646-3

10. Iyer A, Kumosani T, Yaghmoor S, Barbour E, Azhar E, Harakeh S. Escherichia coli and Salmonella spp. in meat in Jeddah, Saudi Arabia J infect Dev Ctries 2013; 7(11):812-818. https://doi.org/10.3855/jidc.3453

11. Akagha TN, Gugu TH, Enemor EC, Ejikeugwu PC, Ugwu BC, Ugwu MC. Prevalence and Antibiogram of Salmonella Species and Staphylococcus aureus in retail meats sold in Awka metropolis, Southeast Nigeria. International Journal of Biological & Pharmaceutical research 2015; 6(12):924-929.

12. Elhadi N. Prevalence and antimicrobial resistance of Salmonella spp in raw retail frozen imported freshwater fish to eastern province of Saudi Arabia. Asian Pac.J.Trop. Biomed. 2014; 4(3):234-238. https://doi.org/10.1016/S2221-1691(14)60237-9

13. El-Sokkary MA, El-Sokkary MMA, Aabed R, Barwa R. Identification, antibiotic resistance and distribution of different classes of integrons among Proteus species isolated from different sources in Dakahleia and Damietta Egyptian Governorates. African Journal of Microbiology Research 2015; 9(19):1312-1321. https://doi.org/10.5897/AJMR2015.7486

14. Ibezim EC, Kenechukwu FC, Odimegwu DC, Builders PF, Kabele-Toge B, Anie C, Igwilo CO, Otuu FC, Onyechukwu C. Antimicrobial efficacy of a syrup formulation from methanol extract of Garcinia kola seed, Afr J Pharm Res Dev, 2011; 3(1):22-27.

15. Hamilton AL, Kamm MA, Ng SC, Morrison M. Proteus spp. as putative gastrointestinal pathogens. Clinical Microbiology Reviews 2018; 31(3):1-19. https://doi.org/10.1128/CMR.00085-17

16. Anie CO, Arhewoh MI, Okeri HA. Antimicrobial Activity of Crude Extracts of Diospyros monbuttensis (Fam: Ebenaceae) Root and Stem Barks. International Journal of Biomedical Research 2011; 2(1):18‐24. https://doi.org/10.7439/ijbr.v2i1.76

17. Mondot S, Lepage P, Seksik P, Allez M, Treton X, Bouhnik Y, Colombel JF, Leclerc M, Pochart P, Dore J, Marteau P. Structural robustness of the gut mucosal microbiota is associated with Crohn's disease remission after surgery. GUT 2016; 65:954-962. https://doi.org/10.1136/gutjnl-2015-309184

18. Wright EK, Kamm MA, Wagner J, Teo SM, Cruz P, Hamilton AL, Ritchie KJ, Inouye M, Kirkwood CD. Microbial factors associated with postoperative Crohn's disease recurrence. J Crohns Colitis 2017; 11:191-203. https://doi.org/10.1093/ecco-jcc/jjw136

19. Liu L, Lan R, Liu L, Wang Y, Zhang Y, Wang Y, Xu J. Antimicrobial Resistance and cytotoxicity of Citrobacter spp. in MaanshanAnhui Province, china. Front. Microbiol. 2017; 8:1357. https://doi.org/10.3389/fmicb.2017.01357

20. Bae IK, Park I, Lee JJ, Sun HI, Park KS, Lee JE. Novel variants of the qnr B, qnrB22 and qnrB23, in Citrobacter Werkmanii and Citrobacter freundii. Antimicrob. Agents chemother. 2010; 54:3068-3069. https://doi.org/10.1128/AAC.01339-09

21. Nada T, Baba H, Kawamura K, Ohkura T, Torii K, Ohta M. A small outbreak of third generation cephem-resistant Citrobacter Freundii infection on a Surgical ward. Jpn. J.Infect. Dis. 2004; 57:181-182.

22. Doran TI. The role of Citrobacter in Clinical disease of Children: review. Clin. Infect. Dis. 1999; 28:384-394. https://doi.org/10.1086/515106

23. Ifeadike CO, Ironkwe OC, Adogu PO, Nnebue CC, Emelumadu OF, Nwabueze SA. Prevalence and pattern of bacteria and intestinal parasites among food handlers in the Federal Capital Territory of Nigeria. Nigeria. Med.J. 2012; 53:166-171. https://doi.org/10.4103/0300-1652.104389

24. Anie CO, Okafo SE. Microbiological evaluation of some oral antacid suspensions sold in Delta State, Nigeria, Journal of Applied Sciences and Environmental Management 2021; 25(2):283-285. https://doi.org/10.4314/jasem.v25i2.23

25. Dagne M, Tiruneh M, Moges F, Gizachew M. Bacterial Profile and Antimicrobial Susceptibility Pattern among food handlers at Gondar University Cafeteria, Northwest Ethiopia. J. Infect. Dis. Ther. 2013; 1:105.

26. Edema MO, Omemu AM, Bankole MO. Microbiological Safety and quality of ready-to-eat foods in Nigeria. In:The Book of Abstract of the 29 Annual Conference & General Meeting (Abeokuta 2005) on Microbes As agents of sustainable Development, organized by Nigerian Society for Microbiology (NSM), University of Agriculture, Abeokuta, from 6th-10th November 2005, p.26.

27. Enwa F, Anie C, Oghenejobo M, Ilaya S. Evaluation of the comparative activity of alcohol-based hand sanitizers and toilet soaps against some bacterial isolates. Global J Sci Front Res, 2015; 15(3):1-7.