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Introduction
Campylobacter species are responsible for the highest percentage of gastroenteritis worldwide and are found in the intestinal track of domestic animals (Silva et al., 2011; Kaakoush et al., 2015; Pintar et al., 2015; Scallan et al., 2015). The most frequently isolated and reported species in humans with gastroenteritis are Campylobacter jejuni and Campylobacter coli (Silva et al., 2011; Noormohamed & Fakhr, 2013; Kaakoush et al., 2015). The ability of both species to colonize and survive in a wide variety of animal species and habitats makes it challenging to control these foodborne pathogens (Epps et al., 2013). A large portion of Campylobacter infections has been attributed to the consumption of contaminated raw or undercooked poultry, unpasteurized milk or dairy products, untreated water, as well as direct contact with farm animals (Smole Možina & Uzunović-Kamberović, 2005; Domingues et al., 2012; Pintar et al., 2015). Additionally, malpractices during handling, cooking, or post-cooking storage of poultry meat products may contribute to campylobacteriosis. C. jejuni and C. coli can lead to campylobacteriosis, an infection characterized with diarrhea, cramps, abdominal pain and fever. The diarrhea may be bloody and can be accompanied by nausea and vomiting (Domingues et al., 2012; CDC (Centers for Disease Control & Prevention), 2015; Kaakoush et al., 2015; Skarp et al., 2016). The infection usually lasts about one week. In severe cases of C. jejuni infections, some individuals may develop the autoimmune neuropathies, Guillain-Barré or Miller Fischer syndromes, and reactive arthritis in some instances (Epps et al., 2013; Kaakoush et al., 2015; Skarp et al., 2016).
The importance of some virulence mechanisms, implicated in conferring Campylobacter an ability to cause disease in humans, has been documented. In particular, flagella-mediated motility, bacterial adherence to intestinal mucosa, and the ability to produce toxins have been identified as clinically-relevant phenotypes (van Vliet et al., 2001; Dasti et al., 2010; Silva et al., 2011). The genes coding for flagellin and cytolethal distended toxin are considered to be the primary and defining virulence and toxin factors contributing to campylobacteriosis (van Vliet et al., 2001; Poly & Guerry, 2008; Silva et al., 2011).
Chicken meat is a nutritious, healthy food since it’s an excellent source of protein, vitamins, and minerals and is also low in fat and cholesterol when compared to other meats (Skarp et al., 2016). In Mexico, the popularity of chicken meat is due to sensory and dietary reasons as well as economic considerations. Chicken continues to be one of the preferred and most affordable animal proteins in Mexico for the low and middle-income population (Hernández & Parrish, 2017). Among the principal chicken producing states in northern and central Mexico, the poultry sector has significantly increased in the last decades and is expected to continue growing (Salazar et al., 2005; Hernández & Parrish, 2017). This increased consumption of poultry products has led consequently to an increase in human foodborne disease (Corry & Atabay, 2001; Hussain et al., 2007; Zaidi et al., 2012; Miri et al., 2014).
A limited number of published reports examined Campylobacter spp. in raw poultry meat in Mexico. In particular, a study conducted in Guadalajara in central Mexico showed that a total of 33 % of raw chicken thighs were found to be positive for Campylobacter spp., and genotyping assays confirmed that 50 % of the isolates were C. coli (Castillo-Ayala et al., 1993). The most extensive survey examining the presence of Campylobacter spp. in samples from municipal markets and butcheries in multiple cities in Mexico revealed that this foodborne pathogen was found to be present in 58.3 % of raw chicken meat and in 93.6% of chicken intestine samples, indicating a higher percentage when compared to other meats, such as pork or beef samples (Zaidi et al., 2012). More recently, Campylobacter spp. was detected in 74 % and 89 % of fresh and frozen retail chicken, respectively, purchased at food markets in the state of Durango in northern Mexico (Rodríguez Ceniceros et al., 2016).
For the effective isolation and detection of Campylobacter from food or environmental samples, methodologies employ the use of a variety of enrichment media in conjunction with the incorporation of antibiotics to reduce microbial flora and promote the growth and isolation of campylobacters when present in low numbers (Man, 2011; Kaakoush et al., 2015). By taking advantage of the increased motility of campylobacteria, the use of a membrane filtration protocol has proven to effectively recover campylobacteria from clinical, environmental and food samples (Lastovica & Le Roux, 2000; Quiñones et al., 2007; Speegle et al., 2009; Miller et al., 2014; Miller et al., 2017).
Given that the consumption of poultry meat and its processed products are of public health significance, the objective of the present study was to identify the presence of C. jejuni and C. coli from chicken meat sold at the retail markets in the metropolitan Culiacan Municipality in Sinaloa, one of the states with significant chicken production industry in Mexico (Hernández & Parrish, 2017). To achieve this goal, a membrane filtration method was employed to achieve an efficient recovery of campylobacters from food samples. Currently, there is very limited information on the levels of C. jejuni and C. coli in retail chicken meat products sold at retail markets in this region in northwestern Mexico; therefore, the results from this study will aid in the development of surveillance measures to improve food safety and quality among consumers in this agricultural region in Sinaloa, Mexico.
Material and Methods
Sampling
A total of thirty whole chicken carcasses were purchased from eleven different retail markets in the central part of the metropolitan city of Culiacan in the state of Sinaloa, Northwestern Mexico from February to April 2016 (Figure 1). The retail markets sell raw chicken carcasses processed by one of the largest distributors of chicken meat in the state of Sinaloa. After purchase, the raw chicken samples were immediately transported to the laboratories for microbiological research at the Autonomous University of Sinaloa, Mexico and kept at 4 °C until processing within a 24 h time lapse.
Raw chicken carcasses were purchased from eleven food markets (yellow circles with numbers 1-11), representative of the main retailers for raw chicken meat in the northern part of the city of Culiacan. The raw meat products were immediately transported to the laboratories for microbiological research at The Autonomous University of Sinaloa (yellow circle number 12) for further processing as described in the Materials and Methods. The scale bar corresponds to 400 meters.
Figure 1 Location of the retail markets sampled in Culiacan, Sinaloa, Mexico.
Microbiology analysis
Before opening, outside plastic wrapping of the chicken carcasses was disinfected with 70 % ethanol solution. The package liquid from each carcass was collected and kept at 4 °C for 30 min, and 5 mL of the package liquid was further added to 45 mL of Oxoid™ anaerobe basal broth [(Thermo Fisher Scientific, Waltham, MA, USA), amended with Oxoid™ CAT supplement] cefoperazone (8 µg/mL), teicoplanin (4 µg/ mL), amphotericin B (10 µg/mL); Thermo Fisher Scientific] in a sterile cell culture flask with a 0.2 µm vented cap. The flask was incubated at 42 °C for 24 h with gentle shaking (30 rpm) under microaerophilic conditions generated by the GasPak™ EZ Campy Pouch System (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). A membrane filtration method was then employed to improve the recovery of the bacterial isolates from the enrichment broths ( Lastovica & Le Roux, 2000; Quiñones et al., 2007). A 250 µL sample of enrichment broth was added in small droplets onto a sterile Whatman® 0.65 µm mixed cellulose ester membrane filter (Millipore Corporation, Billerica, MA, USA), which was placed on the surface of Oxoid™ anaerobe basal agar (ABA) (Thermo Fisher Scientific) plates amended with Oxoid™ CAT supplement (Thermo Fisher Scientific) and 5% laked horse blood. Samples were passively filtered for 30 min at room temperature under the ambient atmosphere in a laminar flow hood. Subsequently, the filters were carefully removed, and the ABA plates were incubated for 48 h at 42 °C under microaerophilic conditions. The presumptive Campylobacter isolates with a typical colony morphology (pale orange colonies on ABA plates) were collected and stored by using Microbank™ beads (Pro-Lab Diagnostics, Round Rock, TX, USA) and stored at −80 °C. Cells from each colony were further visualized with a LABOMED CXL Binocular Microscope (Labo America Inc., Fremont, CA, USA) to examine the single cell spiral morphology typical of Campylobacter spp., as in previous studies (Quiñones et al., 2007).
Campylobacter species identification
Campylobacter isolates were plated onto Oxoid™ anaerobe basal agar (Thermo Fisher Scientific) at 42 °C for 48 h and were incubated under microaerophilic conditions by using the GasPak™ EZ Campy Pouch System (Becton, Dickinson and Company). Template DNA from each isolate was prepared from crude lysates after resuspending Campylobacter cells from ABA plates in 100 µL of HyPure™ molecular biology-grade water (HyClone Laboratories, Inc., Logan, UT, USA) and further incubation at 95 °C for 20 min (Amézquita-López et al., 2014). The lysates were centrifuged at 2000 × g for 5 min and the supernatants were collected. Crude lysates were stored at −20 °C until further use as a template for PCR amplification.
A PCR assay was conducted for the simultaneous identification of Campylobacter species based on the amplification of the lpxA gene, which codes for the enzyme LpxA, an UDP-N-acetylglucosamine acyltransferase (Klena et al., 2004). All oligonucleotides were purchased from Eurofins Genomics (Huntsville, AL, USA). The multiplex PCR amplifications consisted of a 50 µL reaction mixture, each containing 50 ng of genomic DNA (5 µL of crude lysate), 10 pmol/µL of each forward primer (lpxAC. jejuni) 5’-ACAACTTGGTGACGATGTTGTA-3’, (lpxAC. coli) 5’-AGACAAATAAGAGAGAATCAG-3’, 30 pmol/µL of the reverse primer (lpxARRK2m) 5’-CAATCATGDGCDATATGASAATAHGCCAT-3’ and 22.5 µL of 2× GoTaq® Green Master Mix (Promega Corporation, Madison, WI, USA). The reaction mixtures were placed in an Eppendorf 5331 MasterCycler Gradient Thermal Cycler (Eppendorf Latin America, São Paulo, Brazil) using the following PCR cycling conditions: 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min, followed by a final extension time of 5 min at 72 °C (30 cycles total). Amplified products were analyzed in 2 % agarose gels containing 0.04 μL/mL GelRed Nucleic Acid Stain (Phenix Research, Candler, NC, USA). As positive controls for the PCR assay, C. jejuni strain RM3145 (HB93-13) and C. coli strain RM4661 were used (Nachamkin et al., 2001; Zautner et al., 2015). The positive samples were analyzed based on the expected sizes of the lpxA amplified fragments, which corresponded to 330 bp for C. jejuni and 390 bp for C. coli, as described in previous reports (Klena et al., 2004; Quiñones et al., 2007).
Statistical analysis
Descriptive statistics were performed to determine the prevalence of Campylobacter spp., in the examined whole chicken carcasses from the various sampling locations by using Microsoft Excel with the Analysis Toolpak Add-In (Office 365, Version 1808; Microsoft Corporation, Redmond, WA, USA).
Results and Discussion
The aim of the present study was to determine the presence of Campylobacter spp. recovered from chicken carcasses in Culiacan, Sinaloa, a major agricultural region in the northwestern part of Mexico. Chicken carcasses were sampled from retail markets at eleven sampling sites in the central part of the city of Culiacan (Figure 1). The retail markets selected for the present study sell raw chicken carcasses, which are processed by large distributors of chicken meat in the state of Sinaloa. During the three months of the sampling (February to April 2016), selective enrichment and membrane filtration methodologies were employed to effectively recover Campylobacter spp. from a total of 30 raw chicken carcasses purchased from the 11 retail markets.
The results indicated that presumptive Campylobacter spp. colonies were recovered from 43 % (13/30) of the chicken carcasses after membrane filtration and plating on selective media, and the Campylobacter-positive carcasses were identified in 64 % (7/11) of the retail markets examined (Table 1). Further analysis by phase contrast microscopy of the recovered colonies revealed a spiral-corkscrew single cell morphology (data not shown), which is typical of campylobacters (Quiñones et al., 2007). The presumptive Campylobacter spp. colonies were further genotyped by PCR after amplification of a species-specific region in the lpxA gene, and the analysis revealed that 33.3 % (10/30) of the chicken samples tested positive for C. jejuni (Table 1). Among the presumptive Campylobacter isolates, a total of 28.7 % (27/94) of the colonies were typed as C. jejuni. C. coli was not detected in any of the chicken carcasses examined (Table 1). Our study also revealed that carcasses from retail market site 1 had the highest C. jejuni prevalence with 54.5 % (12/22). The remaining markets had a prevalence ranging from 8.3 % to 25 % with four markets testing negative for presumptive Campylobacter spp. Interestingly, market site 9 had the highest number of presumptive isolates; however, only 20.8 % (5/24) of the isolates were typed as C. jejuni, suggesting the presence of other members of the Campylobacteraceae family in these raw chicken samples.
Table 1 Presence of Campylobacter spp. in raw chicken carcasses purchased at retail markets in Culiacan, Sinaloa, Mexico.
| Retail market (n=11) |
Chicken carcass (n=30) |
No. of presumptive Campylobacter isolates (n=94) |
No. of positive C. jejuni isolates (%) |
No. of positive C. coli isolates (%) |
|---|---|---|---|---|
| 1 | 3 | 22 | 12 (54.5) | ND |
| 2 | 3 | 8 | 2 (25) | ND |
| 3 | 3 | ND† | ND | ND |
| 4 | 3 | 4 | 1 (25) | ND |
| 5 | 2 | ND | ND | ND |
| 6 | 2 | 12 | 3 (25) | ND |
| 7 | 3 | 12 | 3 (25) | ND |
| 8 | 2 | 12 | 1 (8.3) | ND |
| 9 | 4 | 24 | 5 (20.8) | ND |
| 10 | 1 | ND | ND | ND |
| 11 | 4 | ND | ND | ND |
†ND, not detected.
Campylobacter is a leading cause of foodborne illnesses worldwide, accounting for an estimated global burden of 166 million diarrheal illnesses (Kaakoush et al., 2015; Devleesschauwer et al., 2016). Given that poultry are the main reservoir for thermophilic campylobacters in the food supply (Corry & Atabay, 2001; Kaakoush et al., 2015; Skarp et al., 2016), the present study employed a membrane filtration method for examining the presence of Campylobacter spp. in raw chicken carcasses available at local retail markets in the metropolitan city of Culiacan within the state of Sinaloa in Northwestern Mexico. Sinaloa is known as one of the principal chicken producing states in Mexico (Hernández & Parrish, 2017), and the domestic consumption of chicken meat by consumers is expected to continue increasing since it is viewed as a preferred and affordable source of animal protein (Salazar et al., 2005; Hernández & Parrish, 2017).
Due to the difficulty of detecting Campylobacter spp. from food samples due to the high levels of background microbial flora, a cellulose membrane filtration method was employed in conjunction with an enrichment step and selective media to increase the efficiency for isolation of campylobacters. The method is known as the “Cape Town Protocol” and was initially developed for the isolation of campylobacters from human stool samples (Lastovica & Le Roux, 2000). In subsequent studies, this membrane filtration methodology was then adapted for the isolation of multiple members of the Campylobacteraceae family from raw chicken carcasses as well as environmental samples (Quiñones et al., 2007; Speegle et al., 2009; Miller et al., 2014; Miller et al., 2017). In the present study, a membrane pore size of 0.65 µm was selected, as previously shown for its efficacy in the recovery of campylobacters from package liquid from raw chicken carcasses (Quiñones et al., 2007; Speegle et al., 2009; Miller et al., 2014; Berrang et al., 2017). During the slaughtering process, the birds’ gastrointestinal contents can contaminate the skin of the chicken carcass, resulting in the presence of campylobacters in the package liquid (Quiñones et al., 2007; Speegle et al., 2009; Berrang et al., 2017). Improper handling and cross contamination of this carcass package liquid with other fresh produce or fomites can take place during food preparation either at homes or at retailers serving ready-to-eat foods. Thus, it is imperative to develop accurate and reliable methods to assess the presence of campylobacters on raw food products.
By employing this filtration technique, the present study has demonstrated for the first time the recovery of C. jejuni isolates in 33.33 % (10/30) of chicken carcasses samples from 64 % retail markets examined in Culiacan, Sinaloa, Northwestern Mexico. The observed C. jejuni prevalence in raw chicken carcasses from retail markets in Culiacan, Mexico appears to be similar to what has been previously documented in studies from other geographical locations (Hussain et al., 2007; EFSA (European Food Safety Authority), 2010; Williams et al., 2012; Hungaro et al., 2015; Lopes et al., 2018). However, a total of 36 % of the examined retail markets showed no prevalence of Campylobacter spp. One possible explanation for this absence of campylobacters in some carcasses is probably due to the presence of indigenous microbial communities prevalent in raw chicken carcasses that could potentially impede the recovery of campylobacters (Ae Kim et al., 2017) 2017. Interestingly, presumptive Campylobacter isolates that were non-C. jejuni/C. coli were identified in raw chicken meat products for most markets examined in this study. These observations suggest the presence of other members of the Campylobacteraceae family such as Arcobacter butzleri, Campylobacter lari, Campylobacter upsaliensis, or Campylobacter concisus, which have been reported in poultry products and are considered emerging human pathogens (Skovgaard, 2007; Kaakoush & Mitchell, 2012; Ferreira et al., 2016).
Horizontal transmission of Campylobacter in the farm environment represents the most likely route of transmission to broilers. Once colonized, broilers remain colonized until they are slaughtered, and contamination of raw meat and dissemination has been shown to occur in the slaughterhouse (Keener et al., 2004). Given the importance of C. jejuni to public health as only 300 colony-forming units may cause an infection in humans (Hara-Kudo & Takatori, 2011), future work on the development of methodologies and virulence genes for monitoring Campylobacter will provide additional information regarding the prevalence of this foodborne in environmental, clinical, and other food samples.
Conclusions
Campylobacter species are the causative agent of human gastroenteritis worldwide. In Mexico, a limited number of studies have previously documented the presence of Campylobacter spp., in raw chicken meat. This is the first report documenting C. jejuni isolation in raw chicken carcasses from retail markets within Culiacan, Sinaloa. Given the increased consumption of poultry products in Mexico, and the importance as a public health concern, the development of effective methodologies for detecting campylobacters in chicken carcasses purchased from local retail markets will aid in the development of control strategies to ensure a safe food supply for consumers.
