Detection of Escherichia coli O157H7 and Campylobacter jejuni in Bovine Carcasses in Two Slaughterhouses in Bio-Bío District,Chile

Abstract

Escherichia coli O157:H7 (E. coli O157:H7) and Campylobacter jejuni (C. jejuni) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2, and eaeA. Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.

Introduction

Meat microbiological quality is a multifactorial issue, depending on the animal’s physiological status at culling, storage temperature, and hygiene during meat processing. Red meats are a good medium for bacteria growth, owing to their elevated water activity (a_w) (closely to 0.99), pH levels (5.1 to 5.6), and reductive capacity with anaerobic bacteria growing at the center and aerobic bacteria at the surface (Jay, 1997). Exsanguination is a critical process during meat microbial contamination owing to the increased likelihood of dragging bacteria from the animal’s surface into the meat while carotids are cut (Mazurier et al., 1992). Enterobacterial infections usually do not manifest clinical signs in animals, passing unadverted during ante and postmortem veterinary inspection, and butchers also must be considered as potential vectors (Arcos-Ávila et al., 2013).

Shigatoxigenic Escherichia coli (STEC) is a foodborne illness seen in developed and developing countries (Baltasar et al., 2014; Botkin et al., 2012). Infection in humans leads to stomachache and hemorrhagic diarrhea, with 61% of patients undergoing hemolytic uremic syndrome (HUS) (Botkin et al., 2012; Baltasar et al., 2014; Sheerin and Glover, 2019; Goldstein and Bentancor, 2022), with clinical signs including anemia and kidney failure and lethality levels of 5% to 10% (Goldstein and Bentancor, 2022). The clinical findings are owing to cytotoxins known as Shiga toxins, which are synthesized by stx1 and stx2 genes of lysogenic bacteriophages that are inserted into their genome in a stable manner. The STEC group presents virulence factors that increase its pathogenicity, such as intimin, an external membrane protein codified by eaeA gen, whose function is adherence to epithelial cells on the colonic epithelium (Baltasar et al., 2014).

Cattle are the main reservoir of Escherichia coli O157:H7 (E. coli O157:H7) for human contamination and are considered as carriers because of no toxic damage (Kalchayanand et al., 2018). One study found that approximately 9% of cattle have a condition known as “Gifted,” leading them to spread up to 10⁴ CFU/g E. coli O157:H7 in feces (Nychas et al., 2008). Although the optimal growing temperature for this pathogen is between 35° and 40°C, some pathogenic strains can grow between 7°C and 46°C and even survive at refrigeration temperatures from 1 to 5 weeks. E. coli O157:H7 has been found in minced meat stored for 9 months at −20°C, and it is also capable of growing at pH 4.5 (Roberts and Hobbs, 1997).

Campylobacter jejuni (C. jejuni) is the main cause of campylobacteriosis, a disease with acute clinical manifestation. This infection is endemic in developing countries, affecting mainly newborns and toddlers (Day et al., 2000). Also, approximately 90% of all Campylobacter infections in humans are due to C. jejuni (Jeantet et al., 2006), through contaminated meat manipulation or ingestion, especially poultry meat (Poppi et al., 2015). There is a lack of information about the role of bovine meat in C. jejuni infections in humans; however, recent studies point to cattle as important contributors to human campylobacteriosis, through, for example, the contamination of carcasses with feces (Ocejo et al., 2019; Roux et al., 2013; Sheppard et al., 2009; Clark et al., 2003. C. jejuni is very susceptible to physical and chemical agents such as desiccation (a_w under 0.97), heat, wind, low pH, gamma radiation, and disinfectants. Nevertheless, it can survive up to 4 weeks in an aqueous medium at 4°C, but inactivation happens in a few days with temperatures over 15°C (Vadillo et al., 2002). Detection of viable cells after storage and refrigeration is important because the infecting dose can be as low as 500 cells (Mainil, 2013).

In Chile, no studies have been carried out on the levels of STEC strains and C. jejuni in slaughterhouses for cattle. Although it is not mandatory to look for E. coli O157:H7 and C. jejuni in slaughterhouses in Chile, it is so in other countries such as the United States, Israel, Canada, and Costa Rica (SAG, 2008). This study is the first, to our knowledge, to show E. coli O157:H7 and C. jejuni levels in cattle carcasses, gathered from two different culling stages, the post-shower and after frigorific maturation, using microbiological and molecular techniques such as PCR and multiplex PCR.

Materials and Methods

Sampling

The samples were obtained from two slaughterhouses in the Bío-Bío district, Chile. The carcass sampling protocol carried out is described by the livestock agricultural service (SAG) in “Muestreo microbiológico de canales y carcasas en plantas faenadoras de exportación” (SAG, 2004). Sterile swabs (BBL Culture Swab^®) were used to collect samples.

E. coli O157:H7 272 detection was carried from 136 bovine carcasses at the time of post-shower after evisceration and 136 carcasses were sampled at the time before leaving the cold chamber. Two samples were obtained for each carcass, one from the neck (on the trapezius muscle cervical portion) and one from the chest (on pectoral muscles), with 544 samples in total being obtained. Sampling for C. jejuni was carried out similarly. From each selected bovine carcass, a sample was taken by swabbing areas of 100 cm² each (10 cm × 10 cm) that were delimited by a template. The surface of the carcass was swabbed in the outlined area, first 10 times vertically and then 10 times horizontally. After the sampling was completed, the samples were labeled with the carcass number, the sampled area, and the slaughterhouse from which the sample was obtained. The samples were transferred while maintaining the cold chain and analyzed in laboratories of the Faculty of Veterinary Medicine, University of Concepcion, Bío-Bío district, Chile.

Escherichia coli detection

Microbiological detection

Enrichment in selective medium: Sample swabs were placed individually in 5 mL of lauryl sulfate tryptose broth (Merck^®) and incubated at 35°C for 48 h in the stove (Wise Cube^®).

Isolation in selective agar: Samples from the lauryl sulfate tryptose broth were plated on MacConkey agar (Merck^®) and incubated at 37°C for 24 h. Confirmation of the presence of E. coli was done by using an IMVIC biochemical test battery, which includes the indole, methyl red, Voges–Proskauer, and citrate tests, and after incubation at 37°C for 24 h.

Multiplex PCR

Samples that were confirmed for the presence of E. coli through the IMVIC biochemical battery were subsequently subjected to multiplex PCR, which was carried out to detect the genes that define the serotype O157:H7 (rfbE and fliC).

Another multiplex PCR was carried out to detect the genes that encode the most important pathogenicity factors, such as stx1, stx2, and eaeA. The primers for PCRs are described in Supplementary Table S1 (Wang et al., 2002). For DNA extraction, 1 mL of bacterial culture was centrifuged at 21,000 g for 5 min, washed in saline solution, and recentrifuged. The supernatant was discarded and the pellet resuspended with 5 μL of sterile distilled water. Cell suspensions were put in Eppendorf vials and incubated in a boiling water bath for 10 min and then centrifuged again at 21,000 g for 5 min. The supernatant was adjusted to an OD260 of 0.15 by dilution in sterile distilled water, and 5 μL was used in the PCR assay (Mazurier et al., 1992). The extracted microbial DNA was quantified with the internal control 1.9 infinite 2000 Pro^® program in the Nanoquam DNA quantifier model Infinite M200 Pro (Tecan^®). The thermal cycler model used was XP cycler (Bioer^®). The Takara sapphireamp^® fast PCR mix was used, and the amplification protocol was used as described by the manufacturer.

Electrophoresis

A 1% agarose gel was prepared using 0.8 g of agarose (Merck^®), 80 mL of 1% TAE buffer (Santa Cruz Biotechnology Inc.^®), and 1.8 μL of Gel Red Nucleic Acid Stain (Biotum^®). A 4 μL of DNA sample and 2 μL of the molecular weight (Maestrogen^®) were seeded independent of the volume of the chamber used. The Voltronix power supply (NYX Technix^®) ran at 80 V for 75 min. The results were visualized in the Labnet^® Enduro GDS program.

Campylobacter jejuni detection

Detection for C. jejuni was carried out as in previous studies (ISP, 2014).

Enrichment in selective medium: Sample swabs were placed in 5 mL of Bolton broth (Oxoid^®) and incubated under microaerophilic conditions at two temperatures; initially at 37°C for 4 h to 6 h and then at 41.5°C for a further 44 h. The microaerophilic environment was achieved using a microaerophilic chamber (Merck^®).

Isolation in selective agar: Samples were placed in selective agar Karmali (Oxoid^®) and incubated (Wise Cube^®) under microaerophilic conditions at 41.5°C for 44 h. The microaerophilic environment was achieved using a microaerophilic chamber.

Microbiological and biochemical confirmation

Colonies with a morphology presumptive of C. jejuni were stained using the Gram (Merck^® REF) and the oxidase test (Merck ^®). PCR was carried out for the detection of the mapA gene, which is specific for C. jejuni and has 589 bp (Singh et al., 2011). The DNA extraction, quantification procedure, and electrophoresis were carried out as previously described for the multiplex PCR.

Statistical analysis

Chi-square test, contingency coefficient V of Cramer and Cohen’s kappa concordance index, and MxN tables were used to describe the results obtained for the detection of E. coli O157:H7 and C. jejuni. The confidence level used was set at 95%. The data were analyzed using Infostat and Epidat 4.1 statistical software.

Results

The results obtained were organized in tables, for each slaughterhouse and phase of the production line, where the percentages of positivity for both E. coli O157:H7 and C. jejuni obtained in the different analyses were observed. For the analysis and comparison of the positive results by the slaughterhouse, phase of the production line, and sample area, a chi-square test and contingency C analysis were carried out. The data have been distributed in MxN tables.

Escherichia coli detection

In total, 156 samples were positive (28.6%) for E. coli using traditional microbiological detection methods and biochemical tests from both phases of the production line (Table 1; Supplementary Table S2). Using traditional methods, 91 carcasses were found to be positive for E. coli (33.4%) (Supplementary Table S3). In some cases, E. coli was isolated in the neck and chest, but after performing the multiplex PCR, only 11 of the 91 positive samples for E. coli were found to be positive for serotype O157:H7 (12%) (Supplementary Table S4), and only 6 of the 11 samples (54.5%) encoded for the stx1, stx2, and eaeA toxins genes (Supplementary Table S4).

Table 1.

E. coli Positive Strains According to Sampling Moment and Area

Sample	Post shower				Sample	Post cold chamber
	IMVIC		PCR			IMVIC		PCR
	Positive	%	Positive	%		Positive	%	Positive	%
Neck (n = 136)	43	31.6	3	2.2	Neck (n = 136)	28	20.5	2	1.4
Chest (n = 136)	54	39.7	4	2.9	Chest (n = 136)	31	22.7	2	1.4
Total (n = 272)	97	35.6	7	2.5	Total (n = 272)	59	21.6	4	1.4

Campylobacter jejuni detection

Twenty-two positive samples were found to be positive using microbiological and biochemical determination (8%) (since in all cases where there was a positive sample, it was one per carcass, from the neck or chest sample, but not both). Using traditional PCR, no samples were found to be amplified for the mapA gene (Supplementary Table S5).

Slaughterhouse A had higher positivity for both pathogens compared with Slaughterhouse B (Tables 2 and 3).

Table 2.

Positive Bovine Carcasses to E. coli According to Slaughterhouse

Bovine carcasses	Slaughterhouse A				Bovine carcasses	Slaughterhouse B
	IMVIC		PCR			IMVIC		PCR
	Positive	%	Positive	%		Positive	%	Positive	%
Post shower (n = 68)	39	57.3	5	7.3	Post shower (n = 68)	17	25.0	2	2.9
Post cold chamber (n = 68)	22	32.3	3	4.4	Post cold chamber (n = 68)	13	19.1	1	1.4
Total (n = 136)	61	44.8	8	5.8	Total (n = 136)	30	22.0	3	2.2

Table 3.

Bovine Carcasses Positive for C. jejuni according to Slaughterhouse

Bovine carcasses	Slaughterhouse A				Bovine carcasses	Slaughterhouse B
	Biochemical tests		PCR			Biochemical tests		PCR
	Positive	%	Positive	%		Positive	%	Positive	%
Post shower (n = 68)	11	16.1	0	0	Post shower (n = 68)	4	5.8	0	0
Post cold chamber (n = 68)	5	7.3	0	0	Post cold chamber (n = 68)	2	2.9	0	0
Total (n = 136)	16	11.7	0	0	Total (n = 136)	6	4.4	0	0

Escherichia coli detection in slaughterhouse and sample moment

For the case of E. coli, statistically significant results could be obtained in only two comparisons.

First was the case of carcasses positive for E. coli detected by traditional microbiological techniques depending on the slaughterhouse, with a small effect ratio and with an insignificant degree of agreement, where greater number of positive carcasses for E. coli were detected in Slaughterhouse A (61 carcasses) compared with Slaughterhouse B (30 carcasses), indicating that it is more likely to detect contaminated carcasses in Slaughterhouse A than in Slaughterhouse B (Table 4).

Table 4.

Carcasses Positive for E. coli by Microbiological Determination in the Different Slaughterhouses

Slaughterhouse	Carcasses result		Total
Slaughterhouse	Positive	Negative	Total
A	61	75	136
B	30	106	136
Total	91	181	272

Pearson’s chi-square value was 0.0001; Cramer’s V contingency coefficient was 0.17. The observed agreement was 0.61. Cohen’s kappa index obtained from this comparison was k = 0.22 [−0.2392, 0.3214].

In second place were the results obtained from positive carcasses according to the moment of sampling with a small degree relationship and an insignificant degree of agreement, in which they indicated that a greater number of carcasses positive for E. coli were obtained in the post-shower moment (56 carcasses) when compared with the post-cold chamber moment (35 carcasses), indicating that it is more likely to detect carcasses contaminated with E. coli at the time of the productive slaughter (Table 5).

Table 5.

Carcasses Positive for E. coli by Microbiological Determination in the Different Phases of the Production Line

Line phase	Carcasses result		Total
Line phase	Positive	Negative	Total
Post shower	56	80	136
Post cold chamber	35	101	136
Total	91	181	272

Pearson’s chi-square value was 0.0070; Cramer’s V coefficient was 0.12. The observed agreement was 0.57. Cohen’s kappa index obtained from this comparison was: k = 0.15 [−0.2681, –0.2826].

Discussion

This study found a higher positivity in samples after the carcass had been cleaned (showered), compared with those obtained at the time of exit from the cold chamber. This could be explained by the cold treatment received at the chamber, as lowering the temperature on the surface of the carcass can prevent microbial growth (Méndez et al., 2013). Also, this study showed that the most prone moment during slaughtering was at the time of skin removal or prior to the post-shower, which is similar to findings of previous studies (Tshabalala et al., 2012; Visvalingam and Holley, 2018). Another factor affecting the growth of superficial bacterial is the use of 2% lactic acid, if it is applied within 2 hours after service (Hardin et al., 1997; Castillo et al., 1998; Quinn and Markey, 2010; Jure et al., 2015). In this study, none/both slaughterhouses used/did not use lactic acid, which could be/not be the cause of our results. Slaughterer A kept the carcasses in the cold chamber for 2 days before dispatch to their final destination, whereas meat at Slaughterhouse B remained for 7 to 14 days, which could have influenced the results obtained, with lower positivity in Slaughterhouse B. These results are in accordance/contrast with other studies that compared different performance times in cold chambers (Maziero et al., 2010; Quinn and Markey, 2010).

Particularly in the case of C. jejuni, only 8.0% of the samples were positive using traditional microbiological methods, and none of the samples was amplified when performing traditional PCR for the detection of the mapA gene. We suggest that cold treatment of the carcass upon entering the chamber (4°C) reduced the growth and detection of the pathogen on the carcasses. This is supported by previous studies that showed that C. jejuni does not grow at low temperatures (Kalchayanand et al., 2018). Another characteristic of C. jejuni is its viable but noncultivable form (VNC), which at low temperatures can be triggered, preventing its growth in selective media (Tamborini et al., 2012), and this may have been the case in our study. However, a potentially higher risk can occur when consuming VCN bacteria exceeding the 10⁴ minimum inoculum; although in some cases, it can be highly infective, requiring only an inoculum of 500 microorganisms (Franz et al., 2017).

Commensal microbiota in meat at a concentration of 10⁵ cells/g could contribute to the control of pathogens, and low prevalence was detected, as similarly described by other authors (ISP, 2014). Tshabalala et al. (2012) inoculated strains of E. coli O157:H7 and Lactobacillus plantarum (a microorganism that produces a bacteriocin called plantaricin) at the same time in bovine carcasses, in similar quantities and in greater quantities. It was found that Lactobacillus plantarum had a direct inhibitor effect on E.coli O157:H7 in meat, with E. coli concentration decreasing in the first week and not being recovered in the third week, when the meat was kept at 4°C. The inhibitory effect was mainly owing to different types of Lactobacillus spp., which acted as controllers of pathogens. This has been studied in the case of meat, where it was proved that Lactobacillus spp. have a direct effect by preventing pathogenic microorganisms from developing and growing exponentially in meat (García, 2004; Tshabalala et al., 2012; Poppi et al., 2015). Similarly, it has been documented that these commensal bacteria have a direct effect on the growth of C. jejuni (Poppi et al., 2015). However, we did not determine meat microbiota, and further studies should be conducted to elucidate if this was the case in our study.

Amplification being poor for genes related to pathogenicity in E. coli (fliC, rfbE, stx1, stx2, and eaeA genes) could be owing to the fact that the genes encoding these factors come from bacteriophages and plasmids, which may or may not be present, acquired or lost (Maziero et al., 2010). Most of the time, the infection of strains by these bacteriophages results in the establishment of lysogeny, triggering lysis in very few cases (García, 2004). Specifically, the stx1 and stx2 genes that encode verotoxins 1 and 2 are found in a bacteriophage integrated into the bacterial chromosome and are associated with the development of HUS, particularly stx2 (Gallegos et al., 2019). Our study did not analyze the presence of bacteriophages, and future studies should be conducted to clarify that our results are owing to this factor.

Results obtained for the detection of E. coli O157:H7 are similarly low as other studies but are of great importance owing to their pathogenic potential. For example, Masana et al. (2010) obtained samples from the surface of bovine carcasses after these were passed through the cold chamber and reported only 2.6% positivity for E. coli O157:H7 and encoding to pathogenicity genes (multiplex PCR with similar genes was carried out in this study). Similarly, Varela et al. (2007) carried out a study in Mexico that detected 2.7% positive samples for E. coli O157:H7. Finally, Gallegos et al. (2019) detected 1.1% of E. coli in two slaughterhouses in Mexico.

In this work, the results obtained by confirmation were low prevalence for both E. coli and C. jejuni; this may be a product of multiple related variables, from the diet, handling, preservice environment, specific characteristics of bacteria, saprophytic bacteria community, slaughter plant protocols, and previous animal stress level, among others (Romero et al., 2010; Amagliani et al., 2018; Paquette et al., 2018). To decrease the risk of possible outbreaks, multiple management is of utmost importance, considering correct animal management, with the application of animal welfare protocols in productive systems, which ensure a good state of health, and application of animal service protocols; adequate handling of derived products for final consumption; and understanding that the doses to achieve an infection are low, whereas the health repercussions are high.

Conclusions

Using traditional microbiological techniques of microbiological isolation and biochemical identification, 28.6% of presumptive E. coli samples were detected from carcasses of the two slaughterhouses. When multiplex PCR was performed to confirm whether samples were positive for E. coli O157:H7, 11 positive carcasses (4.0%) were obtained, of which 6 (2.2%) were able to detect pathogenicity genes.

In the case of C. jejuni, 8.0% of presumptive C. jejuni samples were detected from the carcasses obtained from the two slaughter plants. It was not possible to confirm the presence by molecular PCR technique, as there were no positive samples for the amplification of the mapA gene.

The results obtained for the identification of E. coli through traditional microbiological techniques classified by phase of the production line and by the slaughterhouse and the results obtained for the identification of carcasses positive for C. jejuni using microbiological techniques were the only ones that were statistically significant with a small degree of relationship and negligible agreement.

Footnotes

Acknowledgments

The authors thank Dr. Juana Lopez and Dr. Hernan Cañon for their support and willingness to publish this article.

Author Contributions

F.F. contributed to the data collection, design, and execution of the study. F.F., G.I., and K.F.-C. contributed to the data interpretation and article preparation. All authors have read and agreed to the published version of the article.

Disclosure Statement

The authors declare no conflict of interest.

Funding Information

The present study received funding from the University of the Américas.

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

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