Abstract
A total of 765 samples were collected from beef carcasses, knives, cutting table surfaces, beef, hands, air, and water from four cattle slaughterhouses of Kerala, South India, to determine the occurrence and antibiotic susceptibility of Listeria species and Staphylococcus aureus. Listeria spp. were isolated from beef carcasses (2.0%), knives (3.7%), cutting table surfaces (1.9%), beef (0.7%), and water (1.3%). The identified species were Listeria monocytogenes (0.1%), Listeria innocua (0.9%), and Listeria ivanovii (0.4%). Most of the Listeria spp. were susceptible to majority of the antibiotics tested. The virulence genes were not detected in Listeria spp. However, all the L. innocua isolates were found to harbor the iap gene. The overall occurrence of S. aureus in slaughterhouses was 50.8%. The highest occurrence was observed on hands of abattoir workers (79.6%) and beef carcasses (59.9%). The isolates were commonly resistant to penicillin (38.0%), followed by ceftriaxone (31.9%), ampicillin (29.0%), amoxicillin (28.8%), tetracycline (24.4%), and chloramphenicol (23.9%). Overall, 53.0% of S. aureus isolates were resistant to three or more antibiotics. Vancomycin and methicillin resistance were observed in 8.5% and 5.4% of S. aureus isolates, respectively. Eight methicillin-resistant S. aureus isolates were found to harbor the mecA gene. In conclusion, Listeria spp. was only rarely found in the slaughterhouse environment and on beef. Nevertheless, the recovery of L. monocytogenes from a water reservoir containing sea water that was used to wash carcasses indicates the potential risk of contamination of the carcasses with L. monocytogenes when using sea water. S. aureus was frequently isolated from abattoir workers and beef carcasses, and the occurrence of S. aureus differed significantly between slaughterhouses. The high occurrence of S. aureus, which were often resistant toward different antibiotics, represents a significant public health concern.
Introduction
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Staphylococcus aureus is commonly found on the skin and nasal passages of most humans and animals (Jackson et al., 2013). Many S. aureus strains produce enterotoxins and cause food intoxications or infections such as bacteremia and pneumonia (Jackson et al., 2013). Infected animals and humans are a major source of contamination in the slaughterhouse, from where the bacteria may spread to processing facilities (Alarcon et al., 2006). The organism can grow in a wide range of temperatures, pH, and salt concentrations, and it can adapt, survive, and colonize even on potentially dry and stressful environments, which may favor the growth of the organism in a slaughterhouse environment and in meat (Alarcon et al., 2006; Jackson et al., 2013).
The increase in the incidence of antibiotic-resistant strains isolated from meat is an important public health concern worldwide (Dan et al., 2015). For instance, methicillin-resistant S. aureus (MRSA) has emerged as a health concern and has been found in several meat-producing animal species, including cattle, pigs, and chickens (Bhargava et al., 2011; Waters et al., 2011; Jackson et al., 2013). In developing countries like India, antimicrobial agents are extensively used for prophylaxis and to treat clinical and subclinical infections in animals, which has resulted in the emergence of resistant strains in food-producing animals.
Meat may get contaminated by a variety of sources, including the animal, dirty equipment, and meat handlers (Bhandare et al., 2007). Despite several studies on the occurrence of these pathogens in meat, milk, and the environment (Dhanashree et al., 2003; Kalorey et al., 2005; Thaker et al., 2013), there are no data on the occurrence of these pathogens in Indian cattle slaughterhouses. Since India is the largest exporter of beef, exporting over 2 million metric tons every year (Agricultural and processed food products export development authority [APEDA], 2015), more data regarding the occurrence and antibiotic resistance of Listeria spp. and S. aureus are needed to assess the potential risk to public health. Therefore, this study determined the occurrence and antibiotic susceptibility of Listeria spp. and S. aureus in cattle slaughterhouses of Kerala, South India.
Materials and Methods
Experimental design
This study was performed from August 2013 to January 2014, in three unorganized (I, II, and III) and one organized (IV) cattle slaughterhouse(s) located at different districts of Kerala state, South India. Unorganized slaughterhouses are traditional slaughterhouses with only basic slaughter equipment and without refrigeration units. All procedures (bleeding, dehiding, evisceration, and pluck removal) were carried out manually on the floor. Organized slaughterhouses are mechanized and automated slaughterhouses with proper infrastructure and trained manpower. The capacity of the organized slaughterhouse was 50 animals per day. Bleeding was carried out on the floor. Dehiding, evisceration, pluck removal, splitting, and washing of the carcasses were carried out hanging on the slaughter line. In all the slaughterhouses, all animals were culled cattle (the primary reasons for culling these cattle were reproduction failure, mastitis, and low production). Sampling was carried out on three sampling days per slaughterhouse.
Sample collection and preparation
Beef carcasses, knives, and cutting table surfaces were swabbed (100 cm2 area) using sterile cotton swabs. For beef carcasses, 500 cm2 area was swabbed (neck, brisket, loin, flank, and outer round region; 100 cm2 area each) after evisceration. All swabs were transferred into 100 mL of 0.1% peptone water.
For beef samples, around 250 g of meat was collected from the neck, loin, brisket, outer round, and flank regions of the carcass after evisceration (slaughterhouse I, II, and III) or after deboning (slaughterhouse IV). A representative subsample of 25 g was used for further analysis. Hand samples were collected from randomly selected individuals involved in beef slaughter or deboning operations by collecting the rinse after washing with 100 mL of 0.1% peptone water. For air samples, duplicate Petri dishes containing Polymyxin-Acriflavin-Lithium chloride-Ceftazidime-Aesculin-Mannitol (PALCAM, Himedia, India) and Baird-Parker (BP, Himedia, India) agar were directly exposed in different processing rooms for 15 min (Evancho et al., 2001). Water samples (250 mL) were collected from the main water tank that was used as processing water in the slaughterhouses. In slaughterhouse, I, II, and IV, fresh water was used as processing water, whereas slaughterhouse III used sea water. All the samples were transported to the laboratory under refrigerated conditions and analyzed within 2 h of collection.
Swab and water samples were thoroughly agitated with a cyclomixer (Remi labworld, India) at 8000 rpm for 3 min. Beef samples were homogenized in 225 mL of 0.1% peptone water using a stomacher blender (AES Chemiuex, France) for 2 min.
Isolation and identification of Listeria spp.
Twenty-five milliliter of homogenate was inoculated in 225 mL of University of Vermont broth (UVM I, Himedia, India) and incubated at 37°C for 24 h. One milliliter of the primary enrichment was transferred to 9 mL of UVM II and incubated at 37°C for 48 h (Ryser and Donelly, 2001). A loopful of enriched inoculum from UVM II was streaked on PALCAM agar, which was incubated at 37°C for 24 h. Typical colonies were confirmed by biochemical tests (Barrow and Feltham, 1993).
Isolation and identification of S. aureus
For the isolation of S. aureus, the remaining homogenate (75 mL for hand samples and 200 mL for beef samples) was incubated at 37°C for 24 h. After incubation, a loopful of enriched inoculum was streaked on BP agar and incubated at 37°C for 24–48 h. Typical colonies were identified up to species level using biochemical tests (Lancette and Benett, 2001).
Antibiotic susceptibility
Antimicrobial susceptibility was performed by the Kirby-Bauer disk diffusion method on Mueller-Hinton agar, incubated at 37°C for 24 h. After incubation, results were interpreted according to the guidelines of Clinical and Laboratory Standard Institute (CLSI, 2010).
Polymerase chain reaction assays
Single polymerase chain reaction assays were used to detect the presence of plcA, prfA, actA, hlyA, and iap genes in Listeria spp. and the mecA gene in MRSA strains using specific primers adapted from Rawool et al. (2007) and Frey et al. (2013). Amplification reactions were performed in a 25-μL mixture, containing primers at a concentration of 40 pmol/μL for plcA and prfA, 30 pmol/μL for actA, 10 pmol/μL for hlyA, 20 pmol/μL for iap, and 10 pmol/μL for mecA gene, 1 U of Taq polymerase, 1 × reaction buffer, 0.2 mM dNTP mix, 2 mM MgCl2, and 2.5 μL of cell lysate.
The following conditions were used: initial denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, primer annealing at 60°C for 30 s, and extension at 72°C for 1 min (35 cycles of amplification), and a final extension at 72°C for 10 min. For mecA, primer annealing was done at 55°C. The amplified products were analyzed by gel electrophoresis with 2% agarose gel using ethidium bromide staining.
Statistical analysis
The occurrence of organisms between slaughterhouses was compared using logistic regression analyses, including the sampling visit as random effect when necessary. Bonferroni corrections were applied for multiple testing. To compare differences in resistance, the proportion of resistant isolates was compared between MRSA and methicillin-sensitive S. aureus (MSSA) strains for each of the antibiotics tested using a two-sample test of portions. All analyses were carried out in STATA 11 (StataCorp, 2011).
Results
Occurrence and antimicrobial susceptibilities of Listeria spp.
Overall, 1.4% (11/765) of the samples were positive for Listeria spp. (Table 1). L. monocytogenes was only isolated from one water sample. Listeria innocua was detected in all slaughterhouses, and was isolated from beef carcasses, knives, and cutting tables (Table 1). Listeria ivanovii was isolated from a knife, cutting table, and beef sample. All the L. innocua isolates harbored the iap gene, but other virulence genes were not detected. No virulence genes were detected in L. monocytogenes and L. ivanovii.
n, number of samples; Spp. 1, Listeria monocytogenes; Spp. 2, Listeria innocua; Spp. 3, Listeria ivanovii.
The resistance of Listeria spp. toward commonly used antibiotics is shown in Table 2. The L. monocytogenes isolate was susceptible to majority of the antibiotics, although was resistant to clindamycin and gentamicin. All L. innocua isolates (n = 7) were sensitive to streptomycin and erythromycin. Resistance was observed toward ampicillin, amoxicillin, ceftazidime, and doxycycline. Most of the L. ivanovii isolates were susceptible to the antibiotics tested.
R*, resistant; I*, intermediate; S*, sensitive.
Occurrence and antimicrobial susceptibilities of S. aureus
The overall occurrence of S. aureus in slaughterhouses was 51% (389/765). The percentage of samples that was positive for S. aureus was higher in slaughterhouses I (70%) and III (62%) compared to slaughterhouses II (39%) and IV (38%) (p < 0.001). Among the samples, the highest occurrence was found on hands (80%) and beef carcasses (60%) (Table 3). The proportion of S. aureus-positive beef carcasses was significantly higher in slaughterhouses I and III than in slaughterhouses II and IV (p < 0.05). S. aureus was less frequently isolated from cutting tables in slaughterhouse II (23%) than in slaughterhouses III and IV (62%; p = 0.030). In slaughterhouse I, S. aureus was isolated from 73% of water samples, which was significantly higher than the 13% of water samples in slaughterhouse IV (p = 0.009). S. aureus was isolated from all hand samples in slaughterhouse I, whereas in slaughterhouse IV, only 53.1% of the hands were S. aureus positive.
n, number of samples; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. Aureus.
Among the 389 S. aureus isolates, 81.5% was sensitive to methicillin, followed by vancomycin (77.6%) and oxacillin (76.6%). The isolates exhibited resistance to penicillin (38.0%), ceftriaxone (31.9%), ampicillin (29.0%), amoxicillin (28.8%), tetracycline (24.4%), and chloramphenicol (23.9%) (Table 4). None of the isolates was simultaneously resistant to all the antibiotics. Overall, 53.0% of S. aureus strains were resistant to three or more of the antibiotics tested.
Out of the 389 S. aureus isolates, 21 (5.4%) were resistant to all β-lactam antibiotics, including methicillin and oxacillin (MRSA). The mecA gene was detected in eight of these MRSA isolates (38.1%). Most MRSA were recovered from beef carcasses (6.6%) and hands (4.6%). Out of the 91 and 86 S. aureus isolates that were recovered from beef carcasses and hands, 11.0% and 5.8% were MRSA, respectively. Although S. aureus was frequently detected in water samples, none of the isolates was MRSA. In slaughterhouse IV, only MSSA was recovered. Among the MRSA isolates, 28.6% and 19.0% were resistant to erythromycin and cotrimoxazole compared to 14.1% and 11.7% in MSSA isolates (Table 4; p = 0.071 and p = 0.314, respectively). For chloramphenicol, 9.5% of MRSA compared to 24.7% of MSSA strains showed resistance (p = 0.112). Resistance toward tetracycline, neomycin, and vancomycin was very similar in MRSA and MSSA strains (p > 0.5).
Discussion
The observed occurrence of Listeria spp. in this study was low (1.4%). This is in agreement with AjayKumar (2014) who reported a similar occurrence of L. monocytogenes, L. innocua, and L. ivanovii in cattle (0.6%), soil (0.6%), and water (0.4%) in the same region. In other parts of the world, a higher prevalence of these Listeria species in beef slaughterhouses and meat have been reported, in prevalences up to 78.4% (Barros et al., 2007; Pesavento et al., 2010). Besides the variation in prevalence rates due to the difference in sampling and isolation methods, management practices, and environmental factors (Nightingale et al., 2004; Zhu et al., 2012), the lower occurrence of Listeria spp. in this study and other studies in the same region may be related to the high ambient temperature, which might not have provided an opportunity for L. monocytogenes to compete with other bacteria (Wu et al., 2015). Nevertheless, the isolation of L. monocytogenes from the water tank in slaughterhouse III indicates the potential risk of cross contamination of carcasses when using sea water. L. monocytogenes may contaminate sea water due to sewage and farming effluents and may survive in sea water due to its halophilic nature.
This investigation revealed a high occurrence of S. aureus in cattle slaughterhouses of Kerala, South India. This may be due to poor hygiene and sanitation and excessive handling of the carcasses. In unorganized slaughterhouses, animals were slaughtered on the floor and all the process were carried out on the floor. The position of workers was changing in these slaughterhouses and a single person was carrying out all the procedures. The organized slaughterhouse was automated and mechanized. However, the slaughter line was not automated and carcasses were moved by hand. In slaughterhouse IV, S. aureus was observed on 41.4% of beef carcasses, which indicates that the hygienic conditions maintained in slaughterhouse IV were also unsatisfactory Other studies have shown a much lower occurrence of S. aureus on beef carcasses as Phillips et al. (2001) and Schlegelova et al. (2004) reported only 24.3% and 7.5% S. aureus-positive beef carcasses in Australia and the Czech Republic, respectively. In slaughterhouse I, II, and III, dressing was carried out on the floor and standard hygienic practices were not performed. With over 90% of carcasses being S. aureus positive, the occurrence of S. aureus was significantly higher in slaughterhouse I compared to the other slaughterhouses, which may be related to the high proportion of S. aureus-positive water samples (73%). Slaughterhouses I, II, and IV were supplied with water from a nearby river, which might have been contaminated due to animal or human activities (Lechevallier and Seidler, 1980). This water may thus represent an important source of contamination of meat and the slaughterhouse environment as it is used to wash carcasses, knives, hands, and the slaughterhouse environment.
The lower occurrence of S. aureus on hands of the abattoir workers in the organized slaughterhouse (IV) may be related to better training and resulting hand hygiene practices compared to other slaughterhouses.
In this study, cutting table surfaces and knives represented an important source of (cross) contamination since over 50% of knife and table samples in slaughterhouses I, III, and IV were S. aureus positive. The relatively low occurrence of S. aureus on knives and cutting tables in slaughterhouse II could be related to the better cleaning and disinfection practices after termination of the slaughtering activities compared to other slaughterhouses, and warrant a thorough cleaning of slaughterhouses and equipment after completion of the operations. In slaughterhouse IV, sanitation of knives was carried out by steam sterilization. However, changing of knives after manipulation of each carcass was not strictly followed. This might have affected the contamination process during slaughtering.
Indiscriminate use of antibiotics in cattle results in the development of resistant strains and dissemination of resistance genes within the bacterial population (Lipsitch et al., 2002). In this investigation, L. monocytogenes showed sensitivity to most of the antibiotics. However, intermediate resistance was noticed toward cotrimoxazole, which is of concern for public health because of its second-line use in case of allergy to β-lactams (Caplan et al., 2014). Although Listeria spp. are generally reported as susceptible to antibiotics active against Gram-positive bacteria (Charpentier and Courvalin, 1999), L. innocua isolates showed variable susceptibility patterns, which is in line with a general worldwide pattern of an increasing prevalence of antibiotic resistance.
In this study, none of the antibiotics had shown 100% sensitivity against all the S. aureus isolates and 53% of S. aureus isolates were resistant to three or more antibiotics tested. This is in close agreement with Alian et al. (2012) who reported 82.6% of S. aureus isolates resistant to one or more antimicrobial agents in raw meat in Iran. Contrarily, a low occurrence (4%) of multidrug-resistant S. aureus was reported in beef products in Georgia (Jackson et al., 2013). The World Health Organization (WHO) has recommended the prohibition of chloramphenicol in all food-producing animals, particularly in lactating cows (WHO, 2012). The resistance of S. aureus isolates toward chloramphenicol in this study (23.9%) was much higher than the 0.5% reported from retail ground meats in the United States (Kelman et al., 2011). The proportion of resistance toward erythromycin was also higher in this study, whereas resistance toward penicillin was very similar in both studies. In contrast, the proportion of isolates that were resistant toward tetracycline was much lower in this study (69% compared to 24%) (Kelman et al., 2011). The variation in the resistance pattern may be expected considering the geographical location and differences in restrictions on the use of antibiotics in food animals.
Conclusion
The results of this study indicate that Listeria spp. is not common in cattle slaughterhouses, whereas S. aureus is widespread in cattle slaughterhouses in Kerala, South India. There were some differences in the rate of bacterial contamination between the slaughterhouses, with a lower occurrence of S. aureus on beef carcasses and beef in slaughterhouse II and IV compared to other slaughterhouses. The high occurrence of S. aureus in processing water in slaughterhouses indicates that water may be an important source of contamination of meat and the slaughter environment. Most MRSA were recovered from beef carcasses and hands of the abattoir workers. Transmission of MRSA might have occurred between animals and humans, which addresses the importance of avoiding cross contamination in the slaughterhouse. The isolates displayed varying degrees of resistance to the antibiotics tested and none of the antibiotics showed 100% sensitivity against all the S. aureus isolates. Vancomycin, methicillin, and multiple-drug resistant S. aureus were also noticed and warrant a regular monitoring of animals and abattoir workers for the occurrence of drug-resistant bacteria. These results provide useful information to assess the possible risk associated with the consumption of contaminated beef produced in these slaughterhouses.
Footnotes
Acknowledgments
We would like to thank Kerala Veterinary and Animal Sciences University (KVASU) for providing necessary infrastructure and facilities for conducting the study, also, Kerala State Council for Science and Technology and Environment (KSCSTE) for the financial support.
Disclosure Statement
No competing financial interests exist.