Escherichia coli isolates from meat and abattoirs environment in Egypt: molecular characterization and control by nanosilver particles

ABSTRACT Three hundred samples, including meat from the slaughtered carcass and water, air samples, and swabs from the floor, wall, and employees' hands, were collected from five municipal abattoirs spread across several Egyptian provinces. The Escherichia coli was isolated from floor swabs, meat, air, wall, hand, and water samples. Serotyping of the recovered isolates clarified the presence of various serotypes, including enterohemorrhagic serotypes (O111: H4, O128: H2, and O127: H6) and enterotoxigenic serotypes (O44: H18 and O125: H21). The isolates were resistant to cefotaxime (100%), amoxiclav (80%), then rifampin (66.7%). The stx1 gene, stx2 gene, eaeA gene, blaCMY2 gene and iss gene were detected in 10-80 % of the isolates. Nanosilver (AgNPs) showed that 12.5 ppm was the lowest concentration that prevented bacterial growth. It was observed that 12% of workers wore a clean white coat, only 24% washed their hands between activities during work, only 14% used soap for hand washing, and 42% utilized the same knife for meat and its offal.


Introduction
Any appropriate locations that are formally authorized and recognized by the regulating authorities and where animals are slaughtered and prepared for human consumption are known as abattoirs, slaughterhouses, or meat plants.The primary objective of constructing an abattoir, slaughterhouse, or meat processing facility is to supply safe and hygienic meat under veterinarian supervision and sanitary conditions (Zailani et al. 2016).
Specific mandatory requirements must have been met in abattoir activities to provide the fundamental operational and environmental factors required for the production of food hygiene.
These preliminary programs cover standard operating processes, proper animal management techniques, and basic hygiene strategies (Bolton et al. 2001).
One of the principal factors contributing to foodborne diseases and deaths is the consumption of pathogens like Escherichia coli in infected meat (Bersisa et al. 2019).Pathogenic microorganisms are typically discovered on the skins of living animals exposed to feces, blood, hairs, vomit, or inside the healthy cattle's digestive tract.When processing meat post-slaughter, feces and dirt stuck to the hides can be transported to the surface of previously sterile flesh, especially if the processing is done on the floor without a carcass suspension system (Abdelwahed and Abdelgadir 2019).
Suppose the product needs to be handled correctly or communicated to consumers.In that case, the gastrointestinal materials will diffuse onto the surface of the meat and start to penetrate organs, causing the flesh to degrade rapidly (Abdelwahed and Abdelgadir 2019).There are additional sources of external germs in slaughterhouses, such as inefficient employee management, contaminated tools, and contaminated air (Bintsis 2017).Additionally, using subpar water for machinery and carcasses throughout preparation and slaughtering might contribute to contamination (Parveen et al. 2007).
Numerous employees are unaware of the need to maintain adequate personal hygiene, which makes it easier for employees to grossly pollute items with their fingers and clothing or expose meat to insects, bugs, rats, dust, and other particles of filth (Pradhan et al. 2018).To maintain a high degree of personal hygiene, employees who deal with food should dress appropriately, wear clean shoes, never eat or drink while at work, and refrain from wearing jewellery or watches.Poor food processing and hygiene procedures, insufficient food safety rules and regulations, a shortage of financial resources to participate in secure devices, and an absence of training for food handlers are additional factors contributing to the spread of foodborne diseases, particularly in developing countries (Jeffer et al. 2021).All of the contaminating causes stated previously cause changes in meat's biological and chemical features, which cause it to spoilage or disseminate illnesses across the population (Olaoye and Onilude 2010).
Recently, using silver nanoparticles (AgNPs) as antibacterial and antifungal agents have increased worldwide.Due to their distinct size-dependent optical, electrical, and magnetic properties, silver nanoparticles are particles smaller than 100 nm that have been thoroughly studied and discovered to have been used in catalysis, optics, electronics, and other fields of science and technology.Applications of AgNPs are lovely because of their antibacterial characteristics (Qing et al. 2018).
In the current study, we intended to isolate and recognize E. coli from the abattoir's surroundings and from meat to determine the exact antibiogram profiles of strains and perform molecular detection of some virulence and resistance genes in bacterial isolates.Additionally, use the plate diffusion technique to ascertain the adverse influence of AgNPs on separates.The design of the current study is shown in Figure 1.

Materials
All chemicals were obtained from Merck, UK and media from Oxoid, USA.

Study area
The present work was performed in five municipal slaughterhouses in different provinces of Egypt from June 2022 to December 2022.Abattoirs are manually operated slaughterhouses.They were well-designed with a fence and had a condemnation chamber, an emergency animal slaughter chamber, a quarantined barrier, and eviscerated chambers.The daily cow slaughter capacity is about 200 heads.Depending on how many heads were admitted for slaughter, the slaughter procedures typically began at 6:00 am and continued until 10:00 to 12:00.After each working day, the slaughtering area is regularly cleaned.

Sampling
Three hundred isolated samples were taken from floors, walls, employees' hands, and samples of water, air, and butchered meat (50 samples each).Samples were taken during twice-weekly visits, tagged, and transported in an icebox to the Animal Health Research Institute's laboratory in Cairo, Dokki, Egypt.

Meat samples
Under careful laboratory conditions, 25 g of each isolated sample were aseptically put into a sterile blender flask with 225 mL of sterile peptone water (0.1%) and homogenized at 1400 rpm for 2-5 min (Lopez 2009).

Water samples
Isolated water samples were collected from known operable containers and water taps.Samples were obtained using sterilized plastic screw-capped flasks (500 mL capacity).With little delay, samples were marked and brought in coolers to the lab.Shaking the contents of the sampling vials allowed one millilitre to be transported to a sterilized tube containing sterile peptone water (Lopez 2009;Elekhnawy et al. 2021).

Air samples
Air samples were taken using an impinger filled with 250 mL of peptone water.The impinge's output was joined to the top (inlet) of the trap, while the trap's outlet (side arm) was joined to the pump's inlet.The external record, report, and calibration were joined to the impedance inlet.After carefully combining the contents of the sampling vials, one ml was injected using a sterile pipette into a sterilized tube containing peptone water (Hamet et al. 1991).

Floor and wall swabs
Using sterile cotton swabs, isolated samples were taken from floors and walls in about one cubic centimetre.Then, they were placed in sterilized peptone water and transferred to the lab while being kept cold.

Hand swabs of slaughterhouse workers
Workers at each of the five abattoirs investigated were given 50 hand swabs (10 each).They were collected using sterile swabs placed in a sterile test tube containing buffer peptone water.Each tube was covered with a sterile cotton plug before being transported to the laboratory in an ice box.

Isolation and identification of E. coli
E. coli was isolated and identified according to (Abdel-Rahman et al. 2023).The samples were preenriched in buffered peptone water and incubated aerobically at 37°C for 24 h.Then, a loopful was inoculated onto MacConkey agar and eosin methylene blue agar plates.The inoculated plates were incubated overnight at 37°C.The obtained colonies were subjected to morphological and biochemical identification.Also, antisera against somatic (O) antigens (Denka Seiken Co, Japan) were utilized for serotyping of the E. coli isolates.

Molecular characterization of E. coli
DNA was extracted from the tested bacteria broth using QIAamp DNA Mini Kit (Qiagen, Germany).PCR was used to detect the virulence and antimicrobial resistance genes in a Biometra thermal cycler, T3000 (Germany).The primers are shown in Table S1.The PCR products were separated by electrophoresis on agarose gel.The gel was photographed and analyzed using a gel documentation system (Biometra BDA digital, Germany).The amplification conditions of the primers during PCR are shown in Table S2.

Antibiotic resistance profiles in E. coli strains
The disc diffusion pattern was utilized to test the sensitivity of the E. coli isolates to six antimicrobials (Alotaibi et al. 2022).The Muller-Hinton agar (MHA) medium surface was inoculated with bacteria and then streaked with swab sticks to conduct the test.Agar plates inoculated with discs were then incubated for 24 h at 37°C.With a ruler, the zone diameters were measured in millimetres.Following the criteria previously outlined, isolates were classified as sensitive or resistant, as indicated in Table S3.

Preparation of colloidal AgNPs suspension
The AgNPs solution was synthesized as previously described (Abdelsalam et al. 2019).In a nutshell, two grams of raw rice starch were progressively dissolved by stirring 0.3 g of sodium hydroxide in an alkaline solution with a pH of 11.The mixture was constantly stirred until all of the starch had been dissolved.On the contrary, 100 mL of deionized water was used to dissolve 0.5 g of silver nitrate.The starch solution was stirred at 60°C while the silver nitrate solution was added dropwise.The colour gradually changed from an opaque white to a translucent yellow colour after 15 min with the addition of silver nitrate solution, denoting the creation of AgNPs.

Susceptibility of the chemically synthesized AgNPs against the isolated bacteria by disc diffusion method and minimum inhibitory concentration (MIC) determination
After performing the disc diffusion method as previously described, the MICs of the synthetic chemical AgNPs against the therapeutically isolated antibiotic-resistant bacterial isolates were measured using the broth microdilution technique in the 96-well microtiter plates (Abdelaziz et al. 2019;Abdelkader et al. 2022).Equal volumes of the bacterial cultures and the AgNPs (50 μL each) were applied horizontally and vertically, respectively, to each well.The development control wells on each plate had Muller-Hinton broth (MHB) medium with the tested bacteria to determine the viability of the bacteria.In contrast, the sterility test wells had just sterilized MHB to test the sterility of the medium employed.The plates were incubated at 37°C for 18-20 h and loosely covered with cling film to prevent the bacteria from dehydrating.The MIC is the lowest concentration of each antibacterial agent that prevented bacterial growth (El-Banna et al. 2019;Elekhnawy et al. 2021).

Isolation and identification of E. coli
It was observed that the highest isolation rate of E. coli was from floor swabs (14%), followed by meat samples (12%), air samples (10%), and lastly, wall swabs, hand swabs, and water samples (8% for each) as shown in Figure 2a.
Moreover, serotyping of the recovered isolates was presented in Figure 2b and clarified the presence of various serotypes, including enterohemorrhagic serotypes (O 111 : H 4, O 128 : H 2, and O 127 : H 6 ) and enterotoxigenic serotypes (O 44 : H 18 and O 125 : H 21 ) with different rates.

Antibiogram pattern
The resistance profile of the recovered E. coli isolates (n = 30) obtained from different examined samples was illustrated in Figure 2c and clarified that isolates were resistant to cefotaxime (100%), amoxiclav (80%), then rifampin (66.7%).At the same time, it was found that all isolates were sensitive to gentamicin (100%) and ciprofloxacin (80%).A representative example is shown in Figure S1.

Virulence and antibiotic resistance genes
The molecular detection of some virulence genes in selected E. coli isolates obtained from different examined samples was carried out in Figure 3.It was found that both stx1 and stx2 genes were perceived in 10% of the isolates.The eaeA gene was detected in 50% of the isolates.In addition, molecular detection of specific resistance genes in the E. coli isolates clarified that the blaCMY2 gene was recognized in 40% of the isolates, and the iss gene was identified in 80% of the isolates.

Sociodemographic features of the slaughterhouses workers in the investigated abattoirs
As shown in Table 1, an analysis of the sociodemographic characteristics of the workers revealed that 90% of workers were males.The age group ranged from 15 to 30 years scored the highest level (48%), followed by the age group more than 30 years (38%) and lastly, the age group less than 15 years (14%).

Impact of meat handling practices of the slaughterhouses' workers in investigated abattoirs
As shown in Table 2, it was observed that only 12% of workers were using clean white coats, only 48% washed their hands between activities during work, only 14% used soap for hand washing, and 84% used the same knife for offal and meat.Additionally, cleaning was done with water and soap (no disinfectants were used).

Characters of AgNPs
It was characterized that AgNPs display strong absorption peaks that could be argued to the surface plasmon excitation.The wavelength of the characterized AgNPs was compared with the blank sample of starch.TEM examination is shown in Figure S2.

Inhibitory effect of AgNPs on E. coli isolates
AgNPs exhibited an inhibitory effect on the tested E. coli isolates recovered from the environments of investigated abattoirs.It was found that 12.5 ppm of AgNPs was the lowest concentration that prevented E. coli growth.A representative example of the disc diffusion is revealed in Figure S1.

Discussion
In cases of poor hygiene, the slaughterhouse may be an essential source of meat infection.Meat quality and the danger of ingestion of pathogenic microorganisms to the population are evaluated by studying the microbiological quality of the slaughterhouse and the meat, which reveals the level of sanitation of the slaughterhouses (Adebowale et al. 2010;Almukainzi et al. 2022).
Air pollution in slaughterhouses is one of the important sources of environmental contaminants (Elekhnawy and Negm 2022).Lifting the blood from dead animals left on the ground without cleaning properly causes an offensive odour.It poses health risks owing to the spreading of microorganisms to the surface of the meat and the water in slaughterhouses, especially in hotter climates (Magaji and Chup 2012).Meat contamination and cross-contamination occurred primarily due to inadequate hygienic conditions and poor handling in slaughterhouses (Koo et al. 2013).Since animal hygiene is critical to safeguard the meat and the meat handlers from cross-contamination, the current investigation was employed to study a critical food safety issue related to hygienic measures in local abattoirs.It was observed that the highest isolation rate was obtained from floor swabs (14%), followed by meat samples (12%), air samples (10%), and lastly, wall swabs, hand swabs, and water samples (8% for each).Moreover, serotyping of the recovered isolates revealed the presence of various serotypes, including enterohemorrhagic serotypes (O 111 : H 4, O 128 : H 2, and O 127 : H 6 ) and enterotoxigenic serotypes (O 44 : H 18 and O 125 : H 21 ).Serotyping is still a significant tool for epidemiologic investigations, even though molecular techniques for recognizing specific virulence genes are valuable procedures.
The antibiogram pattern of the recovered E. coli isolates (n = 30) obtained from different examined samples revealed that isolates were resistant to cefotaxime (100%), amoxiclav (80%), then rifampin (66.7%) while it was found that all isolates were sensitive to gentamycin (100%) and ciprofloxacin (80%).The antibiotic biogram of the tested E. coli revealed a marked fluctuation to different antibiotics owing to the misuse of such therapeutic agents while raising the animals.The overuse and misuse of these chemotherapeutics in animals not only affects them but also affects humans.So, its use should be limited only to sick animals with precautions.In Iran, Ayatollahi et al. (2013) found that 78% of the tested E. coli isolates were susceptible to ciprofloxacin.Also, Bouzari et al. (2018) found that 76.9% of the tested E. coli isolates were resistant to ampicillin, 69.2% to cephalothin, and 76.9% were susceptible to gentamicin.Llorente et al. (2014) found that the E. coli isolates from ground beef were sensitive to ciprofloxacin.The differences in the susceptibility profile of E. coli varied among the different studies based on the antibiotics used in animal treatment in the regions of studies.Antimicrobial resistance's rise and dissemination are significant public health concerns, and they are complicated issues caused by a variety of interrelated variables.The selection of the best medical therapy for veterinary infections is greatly aided by in vitro antimicrobial sensitivity testing (Radwan et al. 2016;Negm et al. 2021).
The molecular revealing of some virulence genes in E. coli isolates obtained from different examined samples was employed in the current study.
With its widespread use in science and technology for creating new substances at the nanoscale, AgNPs have recently attracted increasing interest as they possess antimicrobial effects against bacteria (Cho et al. 2005).Here, we found that AgNPs revealed antibacterial activity against the tested isolates with MIC value of 12.5 ppm.AgNPs have superior antibacterial properties than metallic silver and can improve contact with microorganisms due to their large surface area (Toker  (Youssef and Abdel-Aziz 2013).Silver is a potent antimicrobial agent which is used in the form of nanomaterial or as metal salts for antimicrobial applications.Antimicrobial agents have a major role in water treatment, chemical industries, food preservation, aquaculture ponds, agricultural productivity and biomedical applications.Presently, due to emergence of nanoscience and technology metallic AgNPs are used as antimicrobial agent and is synthesized by following various protocols (Youssef and Abdel-Aziz 2013).It has been shown that AgNPs showed activity against E. coli isolates and an increased activity of the antibiotic ciprofloxacin.AgNPs associated with antimicrobial agents can be a therapeutic option for the treatment of bacterial infections.Analysis of sociodemographic characteristics of the workers revealed that 90% of workers were males, and the age group ranged from 15 to 30 years, followed by the age group more than 30 years and lastly, the age group less than 15 years.This finding was supported with that of Kalio and Ali-Uchechukwu (Kalio and Ali-Uchechukwu 2019), who exposed that higher percentages (100%) of butchers in the slaughters were males.In addition, data analysis about the level of education clarified that 24% of workers were informal (had no education), 42% had primary education only, 34% had secondary education, and none had a higher education degree.Also, data analysis about the service duration clarified that 52% of workers had a period of service ranging from 1 to 5 years, and 48% had more than five years of experience.The slaughterhouse operators at the examined sites have relatively low education levels, making it challenging for them to understand the motivations behind particular operations.Stresses the significance of education and knowledge in hygiene during operations, pointing out that meat contamination and a poor understanding throughout meat processing will eventually damage the quality of the meat produced from such processes.
Improving hygiene in slaughterhouses, which will reduce microbial contamination of carcasses and meat, can be achieved by training slaughterhouse workers to acquire knowledge and understanding of the importance of hygiene during slaughtering and meat processing (Wamalwa et al. 2012;El-Banna et al. 2019).Approximately 95% of slaughterhouse employees were not instructed in hygienic meat processing.A sizeable portion of workers in the meat processing industry did not obtain basic training in the hygienic handling of meat (Cook et al. 2017;Elekhnawy et al. 2021).This is against the fundamental standards for employees in the food business.Workers in food facilities like slaughterhouses should receive training on food safety issues (Sun and Ockerman 2005).
Here, all workers at abattoirs under investigation confirmed the absence of periodic medical check-ups.Regular medical check-ups check-ups would help to reduce the spread of germs from unwell or possibly pathogenic workers (Gopinath et al. 2012;Attallah et al. 2022).Regular slaughterhouse disinfection should only be done with efficient and effective disinfectants against a wide range of bacteria and do not compromise the meat's marketability.A slaughterhouse's inadequate sanitation and poor disinfection program increase carcass contamination.All feasible steps should be taken to limit or eradicate such contamination because meat handlers are likely sources of microbial infection (Muinde and Kuria 2005;Hasan Khudhair et al. 2022).
Therefore, use of the AgNPs could be effective strategy to prevents infection and contamination by E. coli.

Conclusion
E. coli was found in the meat and the abattoir environment, which harms meat safety.The primary risk factors for bacterial contamination of carcasses are the ground-based execution of the animals and the subsequent skinning and evisceration in the exact location under unsanitary conditions.As a result, it necessitates careful consideration by all relevant authorities to implement and enforce basic sanitary slaughtering practices.Abattoirs should be built with a high level of sanitation to minimize the initial bacterial count with the application of mechanical techniques in slaughtering to reduce human intervention, and antibiotic treatment of animals should be applied only with proper documentation to ensure maximum safety and lower the carcass contamination with E. coli.

Figure 1 .
Figure 1.Schematic diagram of the current study.

Figure 2 .
Figure 2. a) Percentage of E. coli isolates in the different specimens.b) Percentages of the different E. coli serotypes in the examined samples.c) Percentages of E. coli isolates (n=30) to the tested antibiotics.

Figure 3 .
Figure 3. Molecular detection of virulence and resistance genes in E. coli isolates a) stx1 and stx2 genes, b) eaeA gene, c) blaCMY gene, and d) iss gene.

Table 2 .
Meat handling practices of the slaughterhouses' workers in investigated abattoirs.et al. 2013).They also have little volatility and are stable at high temperatures