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_SHV genes, respectively, and are among the most common ESBL genes (Ejaz et al., 2021). Additionally, bla_CTX-M genes 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-M gene 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, and bla gene detection. A total of 89 S. enterica isolates from five cities in Metro Manila, namely, Quezon, Manila, Pasay, Malabon, and Valenzuela, were randomly selected.

DNA Extraction

Three to four colonies from NA were transferred into 100 μL of 1× TE buffer (10 mM Tris, 1 mM EDTA at pH 8.0) for DNA extraction through boil lysis. The suspension was boiled at 100 °C for 10 minutes on a heat block to lyse bacterial cells. After boil lysis, suspensions were subjected to centrifugation at 6,000 rpm for 5 minutes (Calayag et al., 2017). The supernatant containing DNA extract was transferred/decanted into a sterile microcentrifuge tube and stored at −20 °C prior to use in further experiments.

PCR Confirmation of S. enterica

Each PCR reaction consisted of 1 µL of previously extracted DNA as the template, 6.25 μL of GoTaq® G2 Green Master Mix (Promega, WI, USA), 0.5 μL each of 10 μM forward and reverse primers for invA gene, and 4.25 μL of nuclease-free water. DNA extracted from S. enterica subsp. enterica American Type Culture Collection (ATCC) 14028 served as the positive control, and Escherichia coli ATCC 25922 served as the negative control (Ng & Rivera, 2015). A no template control (NTC) was used to check for PCR master mix contamination with nuclease-free water to substitute the DNA template. The details of the primer sequence, amplicon size, PCR protocols, and references for the invA gene for S. enterica detection are shown in Table 1.

Multiplex PCR of bla Genes

invA-positive (confirmed Salmonella) DNA samples were subjected to PCR-based bla_TEM, bla_CTX-M, and bla_SHV detection. The volumes and concentrations of MyTaq^TM HS Red Mix (Bioline, London, UK), forward and reverse primers, and nuclease-free water were the same as those for the invA gene. The details of the primer sequence, amplicon size, PCR protocols, and references for the bla genes are also shown in Table 1. For bla_TEM and bla_CTX-M, the positive controls were S. enterica isolates from the Pathogen-Host-Environment Interactions Research Laboratory (PHEIRL) that tested positive for these genes (Calayag et al., 2021) and the bla_SHV-positive control was Klebsiella pneumoniae ATCC 700603. The negative control for all bla genes was E. coli ATCC 25922 (Pitout et al., 2004). An NTC was also used to check for PCR master mix contamination. PCR products were then subjected to agarose gel electrophoresis (AGE).

PCR detection of blaCTX-M gene groups

Singleplex PCR was performed for bla_CTX-M groups I, II, and IV because of the similarity in their molecular weight. Each PCR reaction consisted of 1 µL of previously extracted DNA as the template, 6.25 μL of GoTaq® G2 Green Master Mix, 0.5 μL each of 10 μM forward and reverse primers, and 4.25 μL of nuclease-free water. The details of the primer sequence, amplicon size, PCR protocols, and references for bla_CTX-M groups are shown in Table 1. Positive controls were isolates that show positive result in PCR amplification and sequenced for confirmation, while E. coli ATCC 25922 served as the negative control. An NTC was also used to check for PCR master mix contamination. PCR products underwent AGE to visualize results. To confirm PCR results, representative amplicons from each bla_CTX-M group underwent Sanger sequencing (Macrogen, Inc., South Korea). Sequence trimming and alignment were performed using BioEdit v 7.2.5 and MEGA v 11.0.13, respectively. The identities of the nucleotide sequences were confirmed using the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST).

Agarose Gel Electrophoresis

PCR products were visualized through AGE in 2% (w/v) agarose (Vivantis, Malaysia) stained with GelRed® Nucleic Acid Gel Stain (Biotium, CA, USA). Amplicons were separated under 280 V for 30-40 minutes. PCR product molecular weight was estimated using HyperLadder™ 100bp (Bioline, Meridian Bioscience, London, UK).

Table 1.Primers, product size, PCR conditions, and references of invA and bla genes

Gene	Primers (5′–3′)	Product size (bp)	PCR condition	Reference
blaCTX-M	F: ATG TGC AGY ACC AGT AAR GTK ATG GC R: TGG GTR AAR TAR GTS ACC AGA AYC AGC GG	593	Initial denaturation: 95 °C (3 minutes) 30 cycles denaturation: 95 °C (30 seconds) Annealing: 55 °C (30 seconds) Extension: 72 °C (1 minute) Final extension: 72 °C (10 minutes)	(Monstein et al., 2007)
blaTEM	F: TCG CCG CAT ACA CTA TTC TCA GAA TGA R: ACG CTC ACC GGC TCC AGA TTT AT	445
blaSHV	F: ATG CGT TAT ATT CGC CTG TG R: TGC TTT GTT ATT CGG GCC AA	747
blaCTX-M group I	F: GAC GAT GTC ACT GGC TGA GC R: AGC CGC CGA CGC TAA TAC A	499	Initial denaturation: 96 °C (3 minutes) 30 cycles denaturation: 96 °C (30 seconds) Annealing: 56 °C (30 seconds) Extension: 72 °C (1 minute) Final extension: 72 °C (10 minutes)	(Pitout et al., 2004)
blaCTX-M group II	F: GCG ACC TGG TTA ACT ACA ATC C R: CGG TAG TAT TGC CCT TAA GCC	351
blaCTX-M group IV	F: GCT GGA GAA AAG CAG CGG AG R: GTA AGC TGA CGC AAC GTC TG	474	Initial denaturation: 96 °C (3 minutes) 30 cycles denaturation: 96 °C (30 seconds) Annealing: 60 °C (30 seconds) Extension: 72 °C (1 minute) Final extension: 72 °C (10 minutes)
invA	F: ACA GTG CTC GTT TAC GAC CTG AAT R: AGA CGA CTG GTA CTG ATC GAT AAT	244	Initial denaturation: 95 °C (2 minutes) 30 cycles denaturation: 95 °C (30 seconds) Annealing: 60 °C (30 seconds) Extension: 72 °C (30 seconds) Final extension: 72 °C (5 minutes)	(Chiu & Ou, 1996)

Antimicrobial Susceptibility Tests

The Vitek® 2 Compact 60 ID/AST System (AST-GN70 card panel, bioMérieux, Marcy-l’Étoile, France) was used to generate antimicrobial susceptibility profiles of 22 isolates to 16 antimicrobial agents including ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, cefazolin, ceftriaxone, cefepime, aztreonam, ertapenem, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, tigecycline, nitrofurantoin, and trimethoprim/sulfamethoxazole and an ESBL test. The ESBL test included the following antimicrobials alone and in combination with clavulanic acid: cefepime, ceftriaxone, and ceftazidime. Preparation of isolates for Vitek® 2 followed standard procedures (Calayag et al., 2021). An isolate was considered multidrug-resistant (MDR) when it displayed nonsusceptibility to at least one antimicrobial agent in three or more antimicrobial categories ((Magiorakos et al., 2012)). Interpretive criteria and breakpoints were based on the Clinical and Laboratory Standards Institute (CLSI) (2022) 32^nd Edition. The negative control used was S. enterica ATCC 25241.

RESULTS AND DISCUSSION

The occurrence of bla genes in S. enterica from wet market chicken samples across the five Metro Manila cities is summarized in Table 2. None of those isolates showed positive results for bla_SHV gene. The occurrence of bla_TEM and bla_CTX-M genes in the tested isolates were 10 (11.24%) and 22 (24.72%) , respectively. The highest occurrence for bla_TEM was found among the isolates sampled in Valenzuela City, while for bla_CTX-M, isolates sampled from Quezon City showed the highest occurrence.

The bla_CTX-M gene showed the highest prevalence among isolates in this study, which contrasted with some studies among Enterobacteriaceae. Most studies reported bla_TEM as the most predominant bla gene over bla_CTX-M and bla_SHV among Salmonella isolated from raw poultry meat and fecal samples from diarrheic children (B-l et al., 2015); (Wu et al., 2015); (Ghazaei, 2018); (Sales et al., 2021). In the Philippines, (Cruz & Hedreyda, 2017) showed the occurrence of bla_TEM, bla_CTX-M, and bla_SHV in β-lactam-resistant clinical E. coli isolates at 56.3%, 18.3%, and 11.3%, respectively. Although the frequency estimates of bla_SHV, bla_TEM, and bla_CTX-M exist for E. coli, the surveillance of the relative frequencies of these genes within the Philippines is limited among Salmonella isolates. (Calayag et al., 2021) estimated the frequency of bla genes in Salmonella among hog tonsils and jejunum isolates from slaughterhouses in Metro Manila, wherein bla_TEM was detected with the highest frequency, followed by bla_CTX-M. However, the only detected bla_CTX-M subtypes were bla_CTX-M-1 from group I and bla_CTX-M-2 from group II (Calayag et al., 2021). This shows that the occurrence of bla genes varies across different geographical areas and sample sources. There are also some studies on other Enterobacteriaceae that were consistent with this study. (Gundran et al., 2019) showed frequencies of 89.9%, 58.0%, and 27.5%, respectively for bla_CTX-M, bla_TEM, and bla_SHV genes, among E. coli from poultry. Similarly, (Li et al., 2016) reported E. coli isolates from chicken fecal samples and showed that 88.8%, 66.3%, and 3.1% tested positive for bla_CTX-M, bla_TEM, and bla_SHV, respectively. These results suggested the high cephalosporin resistance potential of Salmonella isolated from retail poultry in Metro Manila.

Upon detection of bla_CTX-M groups I, II, and IV genes, all 22 bla_CTX-M-positive isolates were simultaneously positive for bla_CTX-M groups II and IV. Interestingly, none of the tested samples were positive for bla_CTX-M group I. The co-carriage of these bla_CTX-M gene groups had been previously reported and was consistent with findings in other studies (He et al., 2013) (Li et al., 2016) (Gundran et al., 2019). To confirm the bla_CTX-M group gene identities, PCR products of DNA templates of a bla_CTX-M group I positive control and representative Salmonella isolates from bla_CTX-M groups II and IV underwent Sanger sequencing. The BLAST results from the aligned DNA sequences of representative bacterial isolates corresponded to the established sequences of bla_CTX-M groups I, II, and IV, confirming the gene identities. The presence of bla_CTX-M groups II and IV suggested higher resistance potential to cephalosporins which was confirmed by checking the antimicrobial susceptibility of these isolates.

For phenotypic resistance, the bla_CTX-M-positive isolates were subjected to VITEK® 2 automated susceptibility testing. Resistance profiles of Salmonella carrying bla_CTX-M to several antimicrobial agents are shown in Table 3. All isolates (100%) displayed resistance to both ampicillin and nitrofurantoin. Meanwhile, 21 isolates (95.5%) were resistant to a non-extended spectrum cephalosporin (NESC) and an ESC. While ESC-resistant isolates expressed nonsusceptibility to a third generation cephalosporin (ceftriaxone), they remained susceptible to a fourth generation cephalosporin (cefepime). The 21 isolates also displayed resistance to a monobactam and two aminoglycoside class antibiotics. Interestingly, one (4.5%) bla_CTX-M-positive isolate did not display resistance to either NESC or ESC antibiotic classes; however, it was resistant to ampicillin with sulbactam, a β-lactamase inhibitor, suggesting other β-lactam resistance mechanisms. Resistance rates of isolates subjected to VITEK® 2 in this study were comparable to the study of Calayag et al. (2017) for ampicillin, nitrofurantoin, cefazolin, gentamicin, and tobramycin. In the study of Calayag et al. (2017), 70.5% of isolates are nonsusceptible to ampicillin, 93.4% to nitrofurantoin, and 100% to cefazolin, gentamicin, and tobramycin. However, the current study showed higher rates for bla_CTX-M gene with variations in bla_CTX-M gene groups detected.

Corroboration of phenotypic and genotypic resistance was observed in 21 bla_CTX-M-positive isolates that displayed resistance to the NESC and ESC antibiotic classes. This was not the case for one isolate that remained susceptible to NESC and ESC. This could suggest that the one isolate susceptible to NESC and ESC is carrying silent copies of bla_CTX-M (Cruz & Hedreyda, 2017); (Calayag et al., 2021). A mutation in the structural gene or regulatory region could prevent the expression of the bla_CTX-M, which may lead to the inactivation of phenotypic resistance (Cruz & Hedreyda, 2017). Another mechanism for gene silencing is the expression of silencing proteins that interfere with the transcription of the AMR genes. However, gene silencing can be conditional wherein they can be activated depending on the culture media and the presence of antimicrobial selection pressure (Deekshit & Srikumar, 2022). In this case, it is possible that the bla_CTX-M genes of one isolate remained silent despite the individual administration of NESC and ESC antimicrobials. Additionally, the ESBL phenotype of this isolate could have been caused by other ESBL genes instead of the detected bla_CTX-M genes.

Shi et al. (2021) claimed that bla_CTX-M groups I and IV play roles in ceftriaxone resistance mechanisms. In their study, 64.2% CTX-M-producing isolates that were resistant to ceftriaxone carried bla_CTX-M group I genes, while 35.8% carried bla_CTX-M group IV genes (Shi et al., 2021). Although excessive antibiotic use is one of the reasons attributed to the dissemination of bla_CTX-M (Cantón, 2007); (Cantón et al., 2012), it cannot be concluded that excessive use of ceftriaxone was the main reason for the dominance of bla_CTX-M genes in this study. Since cephalosporins are not often used in Philippine poultry farms (Barroga et al., 2020), the high occurrence of bla_CTX-M genes warrants further investigation. However, the high resistance rates of the isolates to aminoglycosides and nitrofuran may be associated with excessive antimicrobial usage because these antibiotic classes are among the most utilized antibiotics in Philippine poultry farms alongside fluoroquinolones and tetracyclines (Imperial et al., 2022). The predominance of bla_CTX-M may also be due to the mobilization of bla_CTX-M genes and co-selection through resistance to other antibiotics (Cantón, 2007); (Cantón et al., 2012).

ESC-resistant isolates may acquire AMR through horizontal gene transfer from abiotic surfaces due to the prolonged survival of bacteria harboring AMR genes (Warnes et al., 2012) (Imperial et al., 2022). (Warnes et al., 2012) showed that cefotaxime-sensitive E. coli acquired a bla_CTX-M group I gene via horizontal gene transfer on a stainless-steel surface. (Imperial et al., 2022) documented the acquisition of a gene that encodes resistance for macrolides, lincosamides, streptogramin B, and oxazolidinones (ermB) in bacteria from chicken fecal samples where the chicken host was not subjected to any antibiotic treatments. Hence, the acquisition of the AMR gene via horizontal gene transfer can be attributed to contamination of surfaces during feeding, cage cleaning, and animal handling.

Although bla_CTX-M group I and IV have been considered as the most common groups of bla_CTX-M genes (Cantón et al., 2012), this study reported the absence of bla_CTX-M group I, while bla_CTX-M groups II and IV have the highest occurrences. While the absence of bla_CTX-M group I requires further investigation, it is plausible that plasmids and insertion sequences (IS) played a role in the spread and co-existence of bla_CTX-M groups II and IV. Specifically, ISEcp1 and IS903B are associated with bla_CTX-M group IV, while the ISCR1 element is associated with bla_CTX-M groups II and IV (Cantón, 2007); (Cantón et al., 2012); (Ferreira et al., 2014); (Nguyen et al., 2021); (Shi et al., 2021))=. Moreover, genes under these bla_CTX-M groups II and IV might recombine and produce novel β-lactamases as in the case of bla_CTX-M-123, which is a hybrid of group I bla_CTX-M-15 and group IV bla_CTX-M-14(He et al., 2013). This could eventually lead to more β-lactamases that can counter cephalosporins and render antibiotic treatment ineffective.

Additionally, carrying additional AMR genes that encode the resistance to fluoroquinolones and aminoglycosides of bla_CTX-M-carrying bacteria favors their survival, which may contribute to the spread of bla_CTX-M (Cantón, 2007). Co-carriage of bla_CTX-M, bla_TEM, and a quinolone resistance gene (qnr) were observed in S. enterica isolates of (Calayag et al., 2021). Unfortunately, the coexistence of different AMR genes in a bacterial isolate may lead to MDR. Plasmids that harbor ESBL genes may also carry genes encoding resistance for aminoglycosides, trimethoprim, sulfonamides, tetracycline, and chloramphenicol (Paterson, 2000) (Shi et al., 2021). This is important considering that MDR is observed in all bla_CTX-M-positive S. enterica isolates where each isolate is resistant to at least six antimicrobial classes. However, this study is limited by not detecting other AMR gene classes. The MDR profile of isolates is shown in Table 4. Among the 22 bla_CTX-M positive isolates, 18 (81.8%) were also resistant to six antibiotic classes. Meanwhile, three isolates (13.6%) were resistant to seven antibiotic classes and one (4.5%) was resistant to eight antibiotic classes. Alarmingly, all isolates displayed ESBL phenotypes.

Due to the high occurrences of bla genes and MDR among S. enterica isolates, it is important to promote national surveillance of AMR and antimicrobial use in the agricultural and veterinary sectors to combat AMR. Although regulations regarding the sale, distribution, and prescription of antibiotics for animal use exist in the Philippines, there is a weak implementation of standards for veterinary medicinal products and enforcement of animal antibiotic use policies (Barroga et al., 2020); (Imperial et al., 2022). As a result, there may be a discrepancy between the declared use of antibiotics in animals and the actual use of antimicrobials in farms (Imperial et al., 2022). For this reason, research in phenotypic and genotypic AMR and antimicrobial use in farms is highly critical to assess the current situation of AMR in the Philippines. Doing so will aid in identifying MDR S. enterica in poultry and implementing policies that will curb the spread of AMR and MDR. Continuing surveillance of AMR genes can aid in policymaking that will counter the spread of AMR and, hopefully, prevent cases of invasive Salmonella infections that persist despite antimicrobial treatments.

Table 2.Occurrence of blaTEM, blaCTX-M, and blaSHV among S. enterica isolates obtained from different markets of Metro Manila

City	No. of S. enterica isolates	blaTEM	blaCTX-M	blaSHV
Quezon	16	0	7 (43.8%)	0
Manila	16	2 (12.5%)	3 (18.8%)	0
Pasay	18	0	7 (38.9%)	0
Malabon	20	4 (20.0%)	0	0
Valenzuela	19	4 (21.1%)	5 (26.3%)	0
Total	89	10 (11.2%)	22 (24.7%)	0

Table 3.Nonsusceptibility levels of 22 blaCTX-M-positive S. enterica isolates against different antimicrobial agents

Class	Antimicrobial	% Nonsusceptibility
Penicillin	Ampicillin	100
Penicillin/β-lactamase inhibitor	Ampicillin/sulbactam	9.1
Antipseudomonal penicillin/ β-lactamase inhibitor	Pipercillin/Tazobactam	0
Non-extended spectrum cephalosporin	Cefazolin	95.5
Extended-spectrum cephalosporin	Ceftriaxone	95.5
	Cefepime	0
Monobactam	Aztreonam	95.5
Carbapenem	Ertapenem	0
	Meropenem	0
Aminoglycoside	Amikacin	0
	Gentamicin	95.5
	Tobramycin	95.5
Fluoroquinolone	Ciprofloxacin	13.6
Glycycline	Tigecycline	4.5
Nitrofuran	Nitrofurantoin	100
Folate pathway inhibitor	Trimethoprim/ Sulfamethoxazole	13.6

Table 4.Multidrug resistance patterns of 22 blaCTX-M-positive S. enterica isolates

Multidrug resistance pattern*	Number of isolates
Pen, Pen/BI, NESC, ESC, Mon, Ami, Flu, Nit	1
Pen, NESC, ESC, Mon, Ami, Flu, Nit	1
Pen, NESC, ESC, Mon, Ami, Nit, FPI	2
Pen, Pen/BI, Flu, Gly, Nit, FPI	1
Pen, NESC, ESC, Mon, Ami, Nit	17

CONCLUSION

This study reported the predominance of bla_CTX-M and the coexistence of bla_CTX-M groups II and IV in all bla_CTX-M positive S. enterica isolates. These suggest high cephalosporin resistance potential. Aside from the corroboration of phenotypic and genotypic resistance among bla_CTX-M-positive isolates, MDR was also observed with ESBL phenotypes detected in all isolates. Given the high occurrence of bla genes, the co-existence of two bla_CTX-M gene groups, and high phenotypic resistance among bla_CTX-M positive S. enterica isolates, the continued surveillance of bla and other AMR genes and phenotypic resistances are thus crucial to monitor the extent and dissemination of resistance and combat their emergence and spread through policy recommendations and regulations.

References

1. Barroga T.R.M., Morales R.G., Benigno C.C., Castro S.J.M., Caniban M.M., Cabullo M.F.B., Dorado-Garcia A.. Antimicrobials used in backyard and commercial poultry and swine farms in the Philippines: A qualitative pilot study. Front Vet Sci. 2020; 7(329)
2. Calayag A.M.B., Paclibare P.A.P., Santos P.D.M., Bautista C.A.C., Rivera W.L.. Molecular characterization and antimicrobial resistance of Salmonella enterica from swine slaughtered in two different types of Philippine abattoir. Food Microbiol. 2017; 65:51-6.
3. Calayag A.M.B., Widmer K.W., Rivera W.L.. Antimicrobial susceptibility and frequency of bla and qnr genes in Salmonella enterica isolated from slaughtered pigs. Antibiotics. 2021; 10(12)
4. Cantón R.. Wiley Online Library: Washington DC (US; 2007.
5. Cantón R., González-Alba J.M., Galán J.C.. CTX-M enzymes: Origin and diffusion. Front Microbiol. 2012; 3(110)DOI
6. Chiu C.H., Ou J.T.. Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and spvC, by an enrichment broth culture-multiplex PCR combination assay. J Clin Microbiol. 1996; 34(10):2619-22.
7. C.L.S.I.. Clinical and Laboratory Standards Institute. 2022.
8. B-l Coculescu, A-M Palade, C Delcaru, G Pircalabioru, Ș Berteșteanu. Phenotypic and genotypic characterization of Salmonella Typhimurium strains producing extended-spectrum beta-lactamases (ESBLs) isolated from children (under 4 years of age) with diarrhea in Romania. Rom Biotechnol Lett. 2015; 20(3):10478-85.
9. Cruz M.C., Hedreyda C.T.. Detection of plasmid-borne β-lactamase genes in extended-spectrum β-lactamase (ESBL) and non-ESBL-producing Escherichia coli clinical isolates. Philipp J Sci. 2017; 146(2):167-75.
10. D.A.-B.A.I.. Department of Agriculture-Bureau of Animal Industry. 2022.
11. Deekshit V.K., Srikumar S.. To be, or not to be”-The dilemma of “silent” antimicrobial resistance genes in bacteria. J Appl Microbiol. 2022; 133(5):2902-14.
12. Desai P.T., Porwollik S., Long F., Cheng P., Wollam A., Bhonagiri-Palsikar V., McClelland M.. Evolutionary Genomics of Salmonella enterica Subspecies. mBio. 2013; 4(2)
13. Ejaz H., Younas S., Abosalif K.O.A., Junaid K., Alzahrani B., Alsrhani A., Hamam S.S.M.. Molecular analysis of blaSHV, blaTEM, and blaCTX-M in extended-spectrum β-lactamase producing Enterobacteriaceae recovered from fecal specimens of animals. PLoS ONE. 2021; 16(1)
14. Eng S.K., Pusparajah P., Ab Mutalib N.S., Ser H.L., Chan K.G., Lee L.H.. Salmonella: A review on pathogenesis, epidemiology and antibiotic resistance. Front Life Sci. 2015; 8(3):284-93.
15. Ethelberg S., Mølbak K., Josefsen M.H.. Encyclopedia of Food Safety. Academic Press: Academic Press; 2014:501-14.
16. Ferreira J., Penha Filho R., Andrade L., Berchieri A., Darini A.. Detection of chromosomal blaCTX-M-2 in diverse Escherichia coli isolates from healthy broiler chickens. Clin Microbiol Infect. 2014; 20(10)
17. Ghazaei C.. Phenotypic and molecular detection of β-lactamase genes blaTEM, blaCTX, and blaSHV produced by Salmonella spp. isolated from poultry meat. Gene Cell Tissue. 2018; 5(4)DOI
18. Gundran R.S., Cardenio P.A., Villanueva M.A., Sison F.B., Benigno C.C., Kreausukon K., Punyapornwithaya V.. Prevalence and distribution of blaCTX-M, blaSHV, blaTEM genes in extended-spectrum β-lactamase-producing E. coli isolates from broiler farms in the Philippines. BMC Vet Res. 2019; 15(2019):1-8.
19. Gut A.M., Vasiljevic T., Yeager T., Donkor O.N.. Salmonella infection: Prevention and treatment by antibiotics and probiotic yeasts: A review. Microbiology. 2018; 164(11):1327-44.
20. He D., Partridge Shen, J Zeng, Z Liu, L Rao, L Liu, J.H.. CTX-M-123, a novel hybrid of the CTX-M-1 and CTX-M-9 group β-lactamases recovered from Escherichia coli isolates in China. Antimicrob Agents Chemother. 2013; 57(8):4068-71.
21. Imperial I.C., Pabustan P.M., Valencia K.A., Nicdao M.A., Ibana J.. Emergence of resistance genes in fecal samples of antibiotic-treated Philippine broilers emphasizes the need to review local farming practices. Trop Biomed. 2022; 39(1):150-9.
22. Standardization I.S.O.] International Organization. Microbiology of the food chain-Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1: Detection of Salmonella spp. 2017; 1(2017)Publisher Full Text
23. Li S., Zhao M., Liu J., Zhou Y., Miao Z.. Prevalence and antibiotic resistance profiles of extended-spectrum β-lactamase-producing Escherichia coli isolated from healthy broilers in Shandong Province, China. J Food Prot. 2016; 79(7):1169-73.
24. Magiorakos A.P., Srinivasan A., Carey R.B., Carmeli Y., Falagas M.E., Giske C.G., Monnet. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18(3):268-81.
25. Monstein H.J., Östholm-Balkhed Å., Nilsson M.V., Nilsson M., Dornbusch K., Nilsson L.E.. Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae. APMIS. 2007; 115(12):1400-8.
26. Ng K.C.S., Rivera W.L.. Multiplex PCR–based serogrouping and serotyping of Salmonella enterica from tonsil and jejunum with jejunal lymph nodes of slaughtered swine in Metro Manila, Philippines. J Food Prot. 2015; 78(5):873-80.
27. Nguyen M.N., Hoang H.T.T., Xavier B.B., Lammens C., Le H.T., Hoang N.T.B., Malhotra-Kumar S.. Prospective One Health genetic surveillance in Vietnam identifies distinct blaCTX-M-harbouring Escherichia coli in food-chain and human-derived samples. Clin Microbiol Infect. 2021; 27(10)
28. Paterson D.L.. Recommendation for treatment of severe infections caused by Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs. Clin Microbiol Infect. 2000; 6(9):460-3.
29. Pitout J.D.D., Hossain A., Hanson N.D.. Phenotypic and molecular detection of CTX-M-β-lactamases produced by Escherichia coli and Klebsiella spp. J Clin Microbiol. 2004; 42(12):5715-21.
30. The spread of CTX-M-type extended-spectrum beta-lactamases. Clin Microbiol Infect. 2008; 14:33-41.
31. Sales A.J., Naebi S., Nasiri R., Bannazadeh-Baghi H.. The antibiotic resistance pattern and prevalence of blaTEM, blaSHV, blaCTX-M, blaPSE-1, sipB/C, and cmlA/tetR genes in Salmonella Typhimurium isolated from children with diarrhea in Tabriz, Iran. Int J Health Life Sci. 2021; 7(4)DOI
32. Santos P.D.M., Widmer K.W., Rivera W.L.. PCR-based detection and serovar identification of Salmonella in retail meat collected from wet markets in Metro Manila, Philippines. PLoS One. 2020; 15(9)
33. Shi Q., Ye Y., Lan P., Han X., Quan J., Zhou M., Jiang Y.. Prevalence and characteristics of ceftriaxone-resistant Salmonella in Children’s Hospital in Hangzhou, China. Front Microbiol. 2021; 12:764-87.
34. Stanaway J.D., Parisi A., Sarkar K., Blacker B.F., Reiner R.C., Hay S.I., Crump J.A.. The global burden of non-typhoidal Salmonella invasive disease: A systematic analysis for the global burden of disease study 2017. Lancet Infect Dis. 2019; 19(12):1312-24.
35. Warnes S.L., Highmore C.J., Keevil C.W.. Horizontal transfer of antibiotic resistance genes on abiotic touch surfaces: Implications for public health. 2012; 3(6)
36. Wu H., Wang Y., Wu Y., Qiao J., Li H., Zheng S., Yang B.. Emergence of β-lactamases and extended-spectrum β-lactamases (ESBLs) producing Salmonella in retail raw chicken in China. Foodborne Pathog Dis. 2015; 12(3):228-34.