Salmonella is an important and one of the most frequently occurring food-borne pathogens with significant economic and public health significance globally (Abdulhaleem et al., 2019; Salihu et al., 2013; Salihu, 2015; Wattiau, & Bertrand, 2011). It is especially common in foods prepared with contaminated poultry meat or egg. Although infection with non- typhoid Salmonella strains is mostly associated with mild self-limiting gastroenteritis, occasionally, severe invasive infections can result [19, 20]. Moreover, the emergence of multidrug-resistant (MDR) isolates are characterized by reduced susceptibility to the commonly used antimicrobials portend an even severe health hazard. Therefore, surveillance programs aimed at the timely detection of Salmonella contaminations in the entire food chain, including live animals, abattoirs, retail outlets, and food restaurants, is so much desired.
This investigation was undertaken to examine Salmonella isolates from food sold at restaurants, chicken meats sold at supermarkets and wet night market in Malaysia, as well as samples from live chickens from selected poultry farms located within the central region of Peninsular Malaysia in order to assess the antimicrobial susceptibility and the genetic relatedness of the Salmonella pathogen. In total, 47 S. Enteritidis were identified after culture, isolation, biochemical characterization, and serotyping was done. In order to their genetic relatedness, while genome sequencing wgMLST and PFGE were conducted.
The antimicrobial susceptibility analysis of all isolates from the food source, chicken meat, and chicken cloacal swab exhibited susceptibility and varying resistance characteristics to the antimicrobial panel tested. As mentioned above, all of the 47 isolates were confirmed to be S. Enteritidis serotype upon slide agglutination test. The phenotypic antimicrobial resistance result showed that the majority of the isolates were resistant (57.45%) to the antimicrobials tested. Although only two isolates had multiple resistance (resistant to 3 or more), the majority were resistant to Tetracycline. The majority of the Salmonella with a multi-drug resistant profile are resistant to Tetracycline, which has gained popularity as a clinically and agriculturally relevant antibiotic (Brunelle, & Bearson, 2013). The National Pharmaceutical Regulatory Agency (NPRA), which is the drug control authority of Malaysia under the Ministry of Health (MOH), Malaysia and the Department of Veterinary Services (DVS) under the Ministry of Agriculture have granted approvals for the use of Tetracycline in the treatment of disease, as prophylaxis and as growth promoters (Azmi et al., 2018). However, the inappropriate use of these antibiotics in the food-producing animals constitutes a serious public health hazard. More so, the emergence of multiple antibiotic resistance, as observed in 2 isolates in this study, may progressively undermine the viability of many of the routinely used antibiotics.
Sequence analysis of the isolates found that the most common sequence type (ST) observed among all the isolates were ST1925 (30) followed by ST11 (12). While novel STs reported in Malaysia for the first time (ST292, ST365, ST329, and ST2132) were also detected in one isolate each. Comparison of our result against the MLST database indicates that ST1925 is relatively common among Salmonella Enteritidis isolated from human and avian species from the United Kingdom, United States, Australia, and Malaysia (http://enterobase.warwick.ac.uk/species/senterica/search_strains?query=st_search). While ST11 had been reported in humans, poultry, food, and some wild animal species, including reptiles in many countries from Asia, Africa, South America, and European countries (http://enterobase.warwick.ac.uk/species/senterica/search_strains?query=st_search).
Importantly, ST1925 has been reported to be a single cell variant of ST11, and both sequence types are known to be geographically widespread and have previously been reported food, human and animal (Alikhan et al., 2018; Aung et al., 2019). The detection of these STs from different sources may also indicate their ability to adapt to, and persist in, different hosts or types of samples. The detection of ST11 in food may imply possible transmission from neighboring countries like Singapore and China, where the ST type prevails . In this study, the whole-genome sequencing platform was used for the MLST analysis against the traditional PCR based MLST, followed by Sanger sequencing. However, due to the availability of the new generation high-throughput sequencing, whole-genome sequence (WGS) data for typing . This is because of its superior discriminatory power and efficiency in genetic detection variability between isolates, in addition to the fact that the traditional method is both costly and time-consuming .
ST292, ST365, and ST2132 have all been previously reported in Singapore (aquatic), India (human & environmental samples), and United States (poultry) while ST329 was reported among isolates obtained from poultry feed in Peru (Adesiji, & Karunasagar, 2014; Castellanos et al., 2018; Liu et al.,2011; Wiesner et al., 2009). In this study, ST292 was detected in chicken meat, while ST365, ST329 and ST2132 were all detected in the chicken cloacal swab. When analyzed in the MLST database, these strains were not as common as the other STs detected except for ST365, and ST2135 was primarily found among poultry in the US from Salmonella isolates belonging to the Kentucky serotype. Also, while ST365 showed no antimicrobial resistance against the drugs tested, ST292 and ST2132 showed resistance to sulfadimidine/trimethoprim as well as streptomycin and Tetracycline respectively. Therefore, having demonstrated similar antimicrobial susceptibility profiles as well as a common source, this may be suggestive of possible strain relatedness, and potential for transmission between chicken and food.
The pulsed-field gel (PFGE) analysis of the isolates revealed that most of the S. Enteritidis isolate examined exhibited unique genetic relatedness, albeit with some variability. Cluster analysis identified ten (10) pulsotypes with the majority belonging to pulsotype 6 and 8, which further had seventeen subtypes that shared 100% identity pattern between the poultry and food source. From the results of the PFGE analysis, it was evident that PFGE revealed more significant differentiation (10 profiles) compared to the MLST, which produced six (6) sequence types. The earlier observation supports this finding that the diversity indices with PFGE produced the highest rate of variability over MLST and the phenotypic antimicrobial susceptibility testing (Stepan et al., 2011). On the other hand, a one-to-one correlation between PFGE types and ST revealed that some isolates belonging to the same PFGE type had multiple STs and vice versa. The result of this study showed that both MLST and PFGE had a high index of discrimination (D) above the 0.95 recommended. The slight disparity with respect to the Simpson’s index between MLST and PFGE observed in this study has previously been reported where MLST was found to exhibit higher discriminatory power with respect to the typing of ESBL E. coli . The authors argued that such disparity may be attributed to the spectrum of changes detected by PFGE and MLST. In other words, while PFGE detect changes in nucleotide sequence associated with insertions or deletions of DNA, MLST typing detects nucleotide changes within an amplified gene fragment . However, in recent years, the potentials of molecular techniques in discriminating between strains of S. enteritidis have become more pronounced. Methods with the highest discriminatory power are more specific and therefore better recommended during investigations of closely related isolates (Shaaly et al., 2005).
Furthermore, the PFGE analysis was able to delineate the genetic variability between the S. Enteritidis isolates from a different source based on the distinct DNA fingerprints generated. Except for two isolates, all other isolates were typeable and the technique reproducible, which could be very useful as an epidemiological tool for disease outbreak investigation. The present study also showed that various PFGE subtypes identified are present in both fresh chicken meat, live birds, and even cooked food ready for eating. Although the DNA profiles of most of the Salmonella serotype Enteritidis isolates from various sources differed, which may indicate that the isolates belong to different clones as revealed by the MLST analysis.