An epifluorescence-based technique accelerates risk assessment of aggregated bacterial communities in carcass and environment

https://doi.org/10.1016/j.envpol.2020.113950Get rights and content

Highlights

  • Foodborne disease reflected great risk to the health of people all over the world. Therefore, there is a need for rapid, precise detection of contamination risk in the field environment.

  • Epifluorescence-based techniques (EBT) combined with automatic image counting software could be efficient for rapid and direct counting of bacterial biofilm in abattoir. Therefore, EBT could be an alternative candidate used to bacterial cell count.

  • The assembly of multiple different extracellular polysaccharide complexes with different maturation stages as voids, microbubbles, channels or mushroom) in carcass, environment and equipment samples, formed by either pathogenic microbes or spoilage microbes (E. coli O157:H7 and Enterobacteriaceae) were identified.

Abstract

The severe and pervasive effects of multispecies foodborne microbial biofilms highlight the importance of rapid detection and diagnosis of contamination risk in the field using epifluorescence-based techniques (EBT) combined with automatic image-counting software. This study screened the hygiene quality of the environment, the carcass and the slaughtering equipment in the El-Kharga abattoir, New Valley Province, Egypt, to assess possible contamination during slaughter process. In addition, biofilm was assessed, and bacteria was enumerated by epifluorescence microscopy. Using both conventional and EBT, the highest bacterial counts were observed for the slaughtering equipment (6.6 and 5.2 cfu/cm2, respectively), followed by different parts of the carcass (4.1 and 4.4 cfu/cm2, respectively) and environmental samples (3.9 and 4.1 cfu/cm2, respectively). A high prevalence of E. coli O157:H7 was observed on the slaughtering equipment (25%), which also led to carcass (1%) contamination. Moreover, Enterobacteriaceae members were detected during examination, such as Klebsiella pneumoniae, Enterobacter aerogenes, and Raoultella ornithinolytica. Despite the relatively good hygiene quality of the abattoir environment, there is also a high risk associated with biofilm formation by pathogenic microorganisms on the slaughtering equipment. Moreover, EBT showed different structures of the biofilm, including those formed at different maturation stages, such as voids, microbubbles, channels and mushroom shapes. (EBT) microscopy combined with image-counting software could be a candidate substitute to estimate efficiently, precisely and rapidly the microbial aggregation and exposure risk in field than the conventional counting techniques.

Introduction

Foodborne disease poses a considerable risk to human health, particularly in Africa, which contains most of the developing countries in the world. In Africa, one-third of the population is subjected to foodborne and waterborne diarrhoeal diseases each year (Decun et al., 2005). Foodborne diseases lead to the death of approximately 2.2 million people annually (FAO & WHO, 2006). In Egypt, food poisoning could be considered a common health problem among the population, with 1748 cases recorded from January to June (Abd-Elhaleem & Abd-Elkarim, 2011). Foodborne diseases are a growing public health problem in both developed and developing countries, and approximately 1000 disease outbreaks have been recorded every year (CDC, 2011). Low quality of food handling and sanitation, improper food safety, the absence of methods to improve equipment safety, lack of regulatory systems, and poor education of food handlers are considered to be major causes of foodborne illness worldwide (Abdullahi et al., 2016).

Abattoirs are among the major sites of food processing responsible for the occurrence of foodborne diseases, and food-associated public health hazards will remain a concern unless basic food hygiene is achieved (Haileselassie et al., 2013). Contaminated hide, blood, carcass remnants, meat and organs may fall on the ground during slaughter and could accumulate in corners or on surfaces such as floors, walls, conveyors, tables, boxes, knives, and swivels. All of these sites function as endless culture systems in which pathogenic and spoilage bacteria can survive in food processing environments and equipment, acting as permanent sources of contamination (Bower et al., 1996; Wirtnanen & Salo, 2003). The abattoir environment and equipment affect the safety and quality of meat (Ntanga, 2013). In such an environment, insufficient cleaning and disinfection of surfaces or equipment can promote bacterial aggregation.

Pathogenic outbreaks are usually linked to biofilm formation (Aarnisalo et al., 2007). Multiplication and survival of microorganisms leads not only to carcass contamination but also to the formation of complex microbial biofilms. Many factors in food processing environments promote biofilm formation, such as the presence of nutrients, moisture and microbial inocula in the crude materials (Myszka & Czaczyk, 2011). Some bacterial strains can construct biofilms within hours (Krysinski et al., 1992). The risk is a result of changes in the microenvironments of the microbial members during biofilm formation, which is followed by changes in gene expression. Biofilms favour paradigmatic niches for the exchange of plasmids or extrachromosomal DNA. The plasmid transfer process occurs at a higher rate among biofilm cells than among free-living cells (planktonic cells) (Ehlers & Bouwer, 1999).

Biofilms are considered to be highly resistant to cleaning and disinfection. This property of biofilms may be a result of the inability of antimicrobial agents to permeate the social organization of the biofilm, the role of gene expression in the stressed response and the apparent growth of the biofilm structure (Myszka & Czaczyk, 2011). Bacterial components of biofilms include spoilage and pathogenic types such as Escherichia coli O157:H7, Campylobacter species, Salmonella species, Klebsiella species, Listeria species and Pseudomonas species, which pose a serious contamination risk in meat and can form mono-species or mixed-species biofilms. Mixed-species biofilms may be stiffer than mono-species biofilms (Zhao et al., 2017; Mosteller & Bishop, 1993). The ability of bacteria to adhere to biotic or abiotic surfaces to construct biofilms is a principal concern in the food industry, especially in meat production and processing (Chmielewski & Frank, 2003). Cross contamination could occur in meat production plants if the equipment and contact surfaces are not sufficiently cleaned and sanitized (Gill et al., 2001).

Several diverse approaches have been used to determine the viability of bacterial cells. The most commonly used method is estimation of the number of CFUs, which reflects the ability of the cells to reproduce (Barer & Harwood, 1999). However, this method is not decisive because a fraction of live bacterial cells cannot divide unless standard growth conditions are applied and because some bacteria are killed by the oxidative stress that occurs upon plating. It is very important to quantify both the dead and alive cells because the dead cells in these structured communities play an important role in the development and spreading of the communities (Webb et al., 2003). In addition, there are several uncertainties associated with biofilm cultivation. Biofilm cells may be destroyed during mixing and agitation, while insufficient mixing may lead to the presence of clumps and inaccurate results (O’Toole et al., 2000). Accordingly, numerous studies have reported the importance of direct microscopy for gaining reliable results regarding the aggregation of bacterial communities on surfaces. Microscopic imaging provides fascinating images that play important roles in the identification of biofilm communities. The great advantage of EBT is the ability to quantitatively analyse biofilms without the need for harvesting and re-suspension, thereby allowing maintenance of the natural structures (Wilson et al., 2017). The use of dyes and fluorescence allows the acquisition of a large amount of data regarding spatiotemporal cellular viability and function without destruction of the biofilm (Heydorn et al., 2000). Therefore, EBT microscopic images can be used directly for bacterial quantification using imaging software.

There is a lack of studies addressing the presence and growth of multispecies bacterial biofilms in meat production facilities at different stages of maturation. The purpose of this study was to use an EBT to as accurate, rapid and reliable counting of various multispecies food borne pathogens and its biofilm at different maturation stages to assess the severity and risk under field conditions. Moreover, identification of foodborne pathogens that play important roles in biofilm communities, even in visibly clean environments and on equipment surfaces in abattoirs, and identification of vital aspects for cleaning and disinfection of abattoirs will help decrease the rate of contamination.

Section snippets

Methods

This study was carried during summer season from E-lkharga abattoir at the northern part of the New Valley governorate, Egypt (between June and August 2016) during which the temperature at sampling, early morning was 39 ± 2C and relative humidity 38  ± 4%.

Results

The overall contamination picture of the El- Kharga abattoir using TBC was represented in Table 1, Table 2. Table (1) recorded a uniform pattern of the slaughtering equipment contamination. A high log TBC (2.2–8.1 cfu cm-2) with no significant difference (P > 0.05) was observed for the examined slaughtering equipment (stamp, swivel and knives) before slaughtering (after disinfection) and after slaughtering comparing with environmental samples (3.2–4.6 cfu cm−2). In addition, no significant

Discussion

Food borne diseases represent a major risk to public health worldwide. Indeed, most of the microbial contamination of food and food products, especially meat, is biofilm relevant (Cappitelli et al., 2014). The transmission of bacteria from the contact surfaces of various environments and equipment leads to microbial contamination of meats, which when exceed the recommended limits; can affect the shelf life of any product, making the product unwholesome and unsuited for human consumption (Akinro

Conclusion

This research highlighted how critical and necessary it is to have a rapid method for the diagnosis of contamination of carcasses and the surrounding environment during the first step of meat production. Moreover, the study indicated the ability of EBTto provide useful information regarding viability of sessile cells, the potential to be used for direct observation of cells in situ and, finally, the high sensitivity. Therefore, direct enumeration by EBT could be an interesting alternative to

Author contributions statement

M.A. modification of the research idea, preparation of all media for bacterial counting and isolation, viteck identification, writing of the paper and submission, R.S. idea, counting and isolation of bacteria, writing of the paper, H.H. epifluorscent microscopic imaging and counting.

CRediT authorship contribution statement

M.A.M. Mahmoud: Conceptualization, Methodology, Validation, Investigation, Resources, Data curation, Formal analysis, Writing - review & editing, Supervision, Project administration. R.S. Zaki: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Writing - original draft. H.H. Abd- Elhafeez: Methodology, Resources, Software.

Declaration of competing interest

(No personal, professional or financial relationships constructed a potential conflict of interest relevant to this article was reported).

Acknowledgements

We thank [Dr. Alaa Khaleel Soliman, the manager of El-kharga abattoir] for facilitating in collecting the samples, and We would also like to show our gratitude to the [Marina Benyamin, Assistant Lecturer, faculty of science, Department of mathematics, New-valley university] for her great work in improving and formating the manuscript.

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