SURVEILLANCE OF β-LACTAMASE GENES OF SALMONELLA FROM CHICKEN IN WET MARKETS OF METRO MANILA, PHILIPPINES

Article Highlights
- bla_CTX-M gene in 24.7% while bla_TEM gene in 11.2% of Salmonella isolates.
- Coexistence of bla_CTX-M groups II and IV in all bla_CTX-M-positive isolates.
- Dominance of bla_CTX-M corroborated with phenotypic β-lactam resistances.
- bla_CTX-M-positive isolates had extended-spectrum β-lactamase and multidrug resistance.

Abstract
Salmonella sp. is a foodborne pathogenic bacterium causing millions of cases with hundred thousand death incidents. Infection by Salmonella can diversely manifest as gastroenteritis, bacteremia, and enteric fever. Salmonella can be transmitted through direct consumption of contaminated foods especially animal-based foods, such as chicken meat and its derivatives. Over the years, antimicrobial resistance (AMR) and diverse β-lactamase (bla) gene-carrying Salmonella strains have been reported. These facts are alarming given that cephalosporins are a major class of β-lactam antibiotics used in clinical settings. Hence, the main objective of this study was to molecularly detect the occurrence of different bla genes by Polymerase Chain Reaction (PCR) and profile the phenotypic antimicrobial susceptibility of Salmonella collected from various chicken sample types in wet markets of Metro Manila, Philippines. Of the 89 Salmonella isolates, bla_CTX-M had the highest occurrence, detected in 22 isolates (24.7%), while bla_TEM was detected in 10 isolates (11.2%). Genotypic and phenotypic resistance corroboration was observed in nearly all bla_CTX-M-positive Salmonella tested, with all strains showing resistance to ampicillin and nitrofurantoin (100%) and 21 out of 22 (95.5%) exhibiting resistance to both non-extended and extended-spectrum cephalosporins. In addition, bla_CTX-M groups II and IV genes were co-detected and multidrug resistance (MDR) profiles were also observed in all bla_CTX-M-positive isolates. The high AMR patterns of Salmonella isolates suggest potential threats to food safety and public health. Additionally, the corroboration of phenotypic and genotypic resistance and the high occurrence of MDR among Salmonella isolates highlight the importance of continued surveillance of AMR genes and regulation of antimicrobial use to combat AMR.

INTRODUCTION

Salmonella sp. is a Gram-negative, facultative anaerobic, mesophilic, rod-shaped foodborne bacterium that belongs to the family Enterobacteriaceae(Ethelberg et al., 2014). Given that poultry is a significant reservoir for Salmonella, the risk of transmission increases through improper handling, trade, or slaughter of raw chicken(Eng et al., 2015). Infection can occur through the fecal-oral route, involving the ingestion of contaminated food, especially animal-based foods, such as chicken meat, or through water containing Salmonella(Eng et al., 2015). Due to their easy transmission from raw/improperly cooked meat, the high consumption of chicken in the Philippines can pose a risk for Salmonella infection. In the Philippines, the average annual poultry consumption per capita in the period of 2009-2018 was 13.4 kg(D.A.-B.A.I., 2022).

Although Salmonella consists of only two species, enterica and bongori, with S. enterica containing six subspecies, most infections are caused by S. enterica subsp. enterica(Desai et al., 2013). Infections can manifest as enteric fever, gastroenteritis, bacteremia, or a chronic carrier state depending on the Salmonella serovars. Enteric fever is caused by S. Typhi or S. Paratyphi A, B, and C which are typhoidal Salmonella. All other Salmonella strains are designated as nontyphoidal Salmonella (NTS), which causes mild infections, including gastroenteritis. Although typhoidal Salmonella is more likely to follow a human-to-human transmission route, NTS transmission is more likely associated with animal reservoirs, such as contaminated chicken meat. The most common serovars responsible for NTS are S. Enteritidis, S. Typhimurium, and S. Newport. The symptoms of enteric fever include headache, abdominal pain, diarrhea/constipation, fever, rose spots, and in severe cases, bloody diarrhea. Conversely, the symptoms of gastroenteritis are usually self-limiting and include headache, abdominal cramps, vomiting, non-bloody diarrhea, nausea, and muscle ache(Eng et al., 2015).

NTS and typhoidal Salmonella may eventually lead to bacteremia and a chronic carrier state. Bacteremia occurs when Salmonella penetrates the intestinal barrier and invades the bloodstream, while a chronic carrier state is characterized by fecal shedding of Salmonella more than one year after the acute stage of Salmonella infection(Eng et al., 2015). In 2017, there were approximately 14.3 million cases of typhoidal Salmonella and approximately 95.6 million cases of NTS globally(Stanaway et al., 2019). Approximately 136,000 died from typhoidal Salmonella in the same year, and 128,000 died from NTS(Stanaway et al., 2019). In the Philippines,(Santos et al., 2020)found S. enterica in meat sold at wet markets in Metro Manila, highlighting the need to investigate the presence of bla genes among S. enterica isolates.

Due to numerous Salmonella-related deaths annually, antibiotic treatment becomes important for treatments against invasive diseases. Extended-spectrum cephalosporins (ESC) are some of the antibiotics used to treat Salmonella infections(Calayag et al., 2021). The treatments for NTS also include ciprofloxacin and ceftriaxone, and for severe complications, may include cefixime and cefotaxime(Gut et al., 2018). Cephalosporins rely on cell wall synthesis interference through the inhibition of transpeptidases, which causes bacterial cell lysis and death(Cantón, 2007). However, antimicrobial resistance (AMR) threatens the efficacy of these treatments. Some Salmonella strains have acquired resistance to these antibiotics because of their ability to produce β-lactamase enzymes that hydrolyze class A β-lactam antibiotics, including cephalosporins(Cantón et al., 2012).

β-lactamase enzymes, such as temoneira (TEM), cefotaximase (CTX-M), and sulfhydryl variable (SHV) are encoded by bla_TEM, bla_CTX-M,and bla_SHVgenes, respectively, and are among the most common ESBL genes(Ejaz et al., 2021). Additionally, bla_CTX-Mgenes are highly diverse and prevalent among Enterobacteriaceae, due to transmissions, mutations, and recombinations. They include numerous groups that confer resistance to different generations of cephalosporins, primarily against cefotaxime and ceftriaxone, with some enhanced variants capable of acting even against ceftazidime(, 2008). Some extended-spectrum β-lactamases (ESBLs) also have the capability to hydrolyze broad-spectrum third- and fourth-generation cephalosporins(Cantón, 2007), posing a significant threat in clinical treatment settings. Determining the occurrence of these genes and phenotypic resistance of S. enterica is important for understanding its transmission and dynamics. Hence, this study determined the occurrence of bla_TEM, bla_CTX-M, and bla_SHV, in S. enterica isolated from raw chicken from wet markets in Metro Manila, Philippines. Moreover, this study is among the first in the Philippines to detect the simultaneous occurrence of bla_CTX-Mgene groups and determined the phenotypic antimicrobial susceptibility profile of S. enterica possessing those gene groups.

MATERIALS AND METHODS

Salmonella Isolates

Following standard procedures under ISO 6579-1:2017(Standardization, 2017)and Santos et al. (2020), 25 g of raw chicken meat samples from leg, thigh, and breast parts were aseptically minced and placed in sterile Whirl-Pak® bags. Then, 225 mL of buffered peptone water (BPW) (BD Diagnostics System, NJ, USA) was added, followed by homogenization for 30 seconds and incubation at 37 °C for 18-24 hours. Subsequently, 100 μL of the BPW culture was transferred to 9 mL of Rappaport Vassiliadis (RV) (BD Diagnostics System, NJ, USA) and incubated at 42 °C for 18-24 hours. The resulting RV cultures were then streaked on xylose lysine deoxycholate (XLD) agar (BD Diagnostics System, NJ, USA) and incubated at 37 °C for 18-24 hours. Black colonies on red XLD agar were then subcultured on nutrient agar (NA) (BD Diagnostics System, NJ, USA) plates and incubated at 37 °C for 18-24 hours. The resulting colonies were then subjected to DNA extraction, Salmonella confirmation,

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