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Microbiology and Biotechnology Letters

Research Article(보문)

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Food Microbiology  |  Food Borne Pathogens and Food Safety

Microbiol. Biotechnol. Lett. 2023; 51(4): 390-402

https://doi.org/10.48022/mbl.2306.06010

Received: June 26, 2023; Revised: September 28, 2023; Accepted: October 6, 2023

Virulence and Antimicrobial Resistance Gene Profiling of Salmonella Isolated from Swine Meat Samples in Abattoirs and Wet Markets of Metro Manila, Philippines

Rance Derrick N. Pavon and Windell L. Rivera*

Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, Philippines

Correspondence to :
Windell Rivera, Diliman,         wlrivera@science.upd.edu.ph

Abstract

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Salmonella are Gram-negative pathogenic bacteria commonly found in food animals such as poultry and swine and potentially constitute risks and threats to food safety and public health through transmissible virulence and antimicrobial resistance (AMR) genes. Although there are previous studies in the Philippines regarding genotypic and phenotypic AMR in Salmonella, there are very few on virulence and their associations. Hence, this study collected 700 Salmonella isolates from swine samples in abattoirs and wet markets among four districts in Metro Manila and characterized their genotypic virulence and β-lactam AMR profiles. Gene frequency patterns and statistical associations between virulence and bla genes and comparisons based on location types (abattoirs and wet markets) and districts were also determined. High prevalence (>50%) of virulence genes was detected encompassing Salmonella pathogenicity islands (SPIs) 1-5 suggesting their pathogenic potential, but none possessed plasmid-borne virulence genes spvR and spvC. For bla, blaTEM was detected with high prevalence (>45%) and revealed significant associations to four SPI genes, namely, avrA, hilA, mgtC, and spi4R, which suggest high resistance potential particularly to β-lactam antibiotics and relationships with pathogenicity that remain mechanistically unestablished until now. Lastly, comparisons of location types and districts showed variations in gene prevalence suggesting effects from environmental factors throughout the swine production chain. This study provides vital data on the genotypic virulence and AMR of Salmonella from swine in abattoirs and wet markets that suggest their pathogenicity and resistance potential for policymakers to implement enforced surveillance and regulations for the improvement of the Philippine swine industry.

Keywords: Antimicrobial resistance genes, association, prevalence, Salmonella, virulence genes

Graphical Abstract


Introduction

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Salmonella are one of the four major causes of diarrheal diseases worldwide; while they often result in mild cases, they can also induce life-threatening infections [1]. Nonetheless, they have been classified as class 3 according to damage-response frameworks wherein these pathogens cause damage to host regardless of their immune condition [2]. Transmission is primarily by fecal-oral route especially from food animals such as swine and poultry, and eventually leads to complex pathogenicity programs involving diverse virulence gene expressions and effector protein functions for colonization, invasion, and proliferation in host cells [3]. Salmonella virulence genes are currently found in up to 23 known Salmonella pathogenicity islands (SPIs); however, only SPIs 1−5 are well-distributed and documented across different serovars [4]. In the Philippines, studies predominantly report incidences along with serogroups and serovars of Salmonella [5−8], which was observed to be the leading known microbiological cause of food poisoning from 2005 to 2018 [8]. However, there are only few studies on Salmonella virulence genes in the Philippines, with few genes such as spvC [6, 7] and invA, which was used as a confirmatory test for Salmonella identification in these studies [5].

Antimicrobial resistance (AMR) is the ability of microorganisms to survive exposure to antimicrobials that were originally effective in treating infections caused by these microorganisms [9]. While intrinsic resistance to antimicrobials, which involves inherent properties in bacteria, is common, other mechanisms such as selective pressure and horizontal gene transfer from resistant strains can also facilitate the emergence and dissemination of AMR [10]. In 2010, the global antimicrobial consumption in food animal industries, excluding aquaculture, was estimated at 63,151 tons, predicting to increase by 67% by 2030, with Asia potentially increasing by up to 129% [11]. In 2017, food animal antimicrobial consumption was estimated at 93,309 tons, with Asia consuming the largest percentage [12]. In the US alone, almost 90% of antimicrobials are for growth promotion and infection prevention purposes rather than for therapy [13]. In addition, many antimicrobials in this industry and clinical settings are similar or the same [14]. Accordingly, it is predicted that by 2050, AMR would cause 10 million deaths per year, highest in Asia [15]. Salmonella are also reservoirs for AMR, carrying transmissible resistance genes, and labeled as serious threats by the Centers for Disease Control and Prevention (CDC) [16]. High phenotypic resistances including multidrug resistance (MDR) or non-susceptibility to at least one drug from three or more classes of antibiotics, were observed in Salmonella isolated from slaughtered swine in abattoirs [17−19] and raw chicken from wet markets [20] within Metro Manila, Philippines. In genotypic studies, Calayag et al. [19] also reported high prevalence of some β-lactamase (bla) and quinolone resistance (qnr) genes in Salmonella from slaughtered swine. Since the discovery of penicillin, β-lactam antibiotics continue to be developed and used in different settings [13]. These also allowed for diversification of bla genes into several subtypes, which among the most common especially in Enterobacteriaceae are TEM (blaTEM), SHV (blaSHV), and CTX-M (blaCTX-M) [21]. Some subtypes can confer and transmit resistance to third generation cephalosporins and aztreonam, also known as extended-spectrum β-lactamases (ESBLs) [22]. The CDC also listed ESBL-producing Enterobacteriaceae as critical priorities posing a serious threat worldwide [16].

Swine are among common reservoir hosts for Salmonella [23]. Developing countries, including the Philippines, contribute to 60% of the world’s swine production [24]. However, approximately more than 70% of Philippine swine production involves backyard farms [25], which can be defined as ≤10 sows [26]. In the study of Calayag et al. [18], higher incidences of Salmonella enterica were detected in locally registered abattoirs than in accredited abattoirs with high MDR among isolates. Hence, the current study collected Salmonella isolates from various swine samples in abattoirs and wet markets of four districts in Metro Manila and characterized virulence and AMR gene profiles using multiplex PCR. This study aimed to determine the pathogenicity and resistance potential of Salmonella that can help in drafting policies, laws, and guidelines in the improvement of the Philippine swine industry.

Materials and Methods

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Meat sample collection

Sample collection was conducted in abattoirs and wet markets of four districts of Metro Manila, Philippines, namely, Northern, Eastern, Southern, and Capital. For abattoirs, the tonsils and jejunum of freshly slaughtered swine were aseptically collected using sterilized scissors and forceps as described by Ng and Rivera [17] and Calayag et al. [18]. While for wet markets, various swine meat samples such as ground pork, pork chop, pork shoulder (kasim), cured pork (tocino), pork sausage (longganisa), meat ball, ham, menudo cut, and pork riblets (buto-buto) were collected from different market stalls. Meat samples obtained were then placed in clean zip-lock bags and into a cooler with ice before transporting to the laboratory for immediate processing. Ethical review and approval were waived for this study due to informed consent obtained from the Philippine National Meat Inspection Service. Animal slaughter and evisceration were performed according to national regulations. Informed consent was also obtained from veterinarians in charge of the abattoirs for sample collection.

Enrichment and isolation of Salmonella

Following standardized protocols [19], each meat sample bag was aseptically opened, and 25 g of meat sample was sliced with sterilized scissors and forceps and weighed in sterilized aluminum foil. Samples were then transferred in 225 ml of buffered peptone water (BPW) (BD Diagnostics System, USA) contained in Whirl-Pak® bags (Nasco, USA), followed by agitation for 2 min and incubation at 37℃ for 18−24 h. A 100 μl of resulting BPW cultures was then transferred to 10 ml Rappaport Vassiliadis (RV) broth (BD Diagnostics System) and incubated at 42℃ for 18−24 h. Subsequently, 10 μl of resulting RV cultures was streaked into xylose lysine deoxycholate (XLD) agar (BD Diagnostics System) and incubated at 37℃ for 18−24 h. Colonies that grew with typical phenotypic characteristics (black colonies on red media) on XLD were then subcultured to nutrient agar (NA) (BD Diagnostics System) and incubated at 37℃ for 18−24 h before DNA extraction and other downstream processes.

DNA extraction

DNA extraction was performed through a boil-lysis method as previously described [18, 19]. Then, 2−3 colonies of presumptive Salmonella in NA were suspended in 50 μl 1× Tris-EDTA buffer and subjected to boiling temperature at 100℃ for 10 min. The resulting suspension was then subjected to centrifugation at 2,656 ×g for 5 min, and the supernatant containing the DNA was collected for Salmonella confirmation and virulence and AMR genes detection.

Molecular confirmation of Salmonella

Primer sequences, amplification product size, and reference for invA gene PCR can be found in Table 1. DNA extracts were subjected to invA gene detection that is generally considered as a genetic marker for Salmonella confirmation due to their wide distribution across Salmonella serovars [27]. Following previously established protocols [5, 18, 28], each PCR reaction mixture was 12.5 μl in volume and contained 6.25 μl of 2× GoTaq Green Master Mix (Promega, USA), 4.25 μl of nucleasefree water, 0.5 μl of invA forward and reverse primers at 10 μM concentrations, and 1 μl of DNA extract. For the PCR, the protocol was indicated as 95℃ for 2 min (initial denaturation), then 30 cycles of three steps, namely, 95℃ for 30 s (denaturation), 60℃ for 30 s (annealing), and 72℃ for 30 s (extension), and finally, 72℃ for 5 min (final extension). Isolates that were positive for invA gene were considered as confirmed Salmonella and were then subjected to molecular detection for virulence and AMR genes.

Table 1 . Primers, sequences and amplification product size for virulence genes.

GenePrimerSequenceProduct size (bp)Reference
invAinvAF: 5’-ACAGTGCTCGTTTACGACCTGAAT-3’244[27]
invAR: 5’-AGACGACTGGTACTGATCTAT-3’
avrAavrAF: 5’-GTTATGGGACGGAACGACATCGG-3’385[29]
avrAR: 5’-ATTCTGCTTCCCGCCGCC-3’
hilAhilAF: 5’-CTGCCGCAGTGTTAAGGATA-3’497[29]
hilAR: 5’-CTGTCGCCTTAATCGCATCGT-3’
sseCsseCF: 5’-TATGGTAGGTGCAGGGGAAG-3’121[30]
sseCR: 5’-CTCATTCGCCATAGCCATTT-3’
mgtCmgtCF: 5’-TGACTATCCAATGCTCCAGTGAAT-3’655[31]
mgtCR: 5’-ATTTACTGGCCGCTATGCTGTTG-3’
SPI4SPI4F: 5’-GATATTTATCAGTCTATAACAGC-3’1,269[32]
SPI4R: 5’-ATTCTCATCCAGATTTGATGTTG-3’
pipBpipBF: 5’-TAATGTGCCACATACAGGTAACGC-3’789[33]
pipBR: 5’-TTCTGGAGGATGTCAACGGGTG-3’
spvCspvCF: 5’-ACTCCTTGCACAACCAAATGCGGA-3’571[27]
spvCR: 5’-TGTCTTCTGCATTTCGCCACATCA-3’
spvRspvRF: 5’-ATGGATTTCATTAATAAAAAATTA-3’894[34]
spvRR: 5’-TCAGAAGGTGGACTGTTTCAGTTT-3’


Multiplex PCR detection of virulence genes

Primer sequences, amplification product sizes, and references for virulence genes detection can be found in Table 1. Confirmed Salmonella isolates were further subjected to genotypic virulence analysis using multiplex PCR optimized in a previous study [35]. A total of eight virulence genes, excluding invA (previously used for Salmonella confirmation), were detected among isolates. Representing SPIs 1−5 were avrA and hilA for SPI 1, sseC for SPI 2, mgtC for SPI 3, spi4R for SPI 4, and pipB for SPI 5, whereas two genes, namely, spvC and spvR, are plasmid-borne genes. For multiplex PCR of avrA, sseC, mgtC, and pipB genes, the protocol involved 94℃ for 4 min (initial denaturation), then 35 cycles of three steps, namely, 94℃ for 1 min (denaturation), 58℃ for 2 min (annealing), and 72℃ for 2 min (extension), and finally, 72℃ for 5 min (final extension). For multiplex PCR of hilA and spvR genes, as well as singleplex of spvC gene, the protocol involved 95℃ for 3 min (initial denaturation), then 34 cycles of three steps, namely, 95℃ for 30 s (denaturation), 50℃ for 30 s (annealing), and 72℃ for 30 s (extension), and finally, 72℃ for 5 min (final extension). For singleplex PCR of spi4R gene, the protocol involved 94℃ for 4 min (initial denaturation), then 34 cycles of three steps, namely, 94℃ for 1 min (denaturation), 58℃ for 1 min (annealing), and 72℃ for 2 min (extension), and finally, 72℃ for 5 min (final extension). Each multiplex PCR reaction mixture was 12.5 μl in volume and contained 6.25 μl 5× MyTaq HS Red Mix (Bioline, UK), variable amounts of nuclease-free water depending on the volume of primers, 0.25 μl of forward and reverse primers at 10 μM concentrations, and 2 μl of DNA template. Singleplex PCR reactions contained identical mixtures as invA gene PCR except for spi4R that contained 20 μM primer concentrations. For positive controls, S. enterica subsp. enterica ATCC (American Type Culture Collection) serovars from Kwik-Stik (Microbiologics, USA) were used. Namely, Typhimurium (ATCC 14028) and Enteritidis (ATCC 13076) for avrA, sseC, mgtC, pipB, and spi4R, whereas Choleraesuis (ATCC 7001) for hilA, spvC, and spvR.

Multiplex PCR of AMR genes

Primer sequences, amplification product sizes, and references for AMR genes detection can be found in Table 2. Confirmed Salmonella isolates were also subjected to genotypic AMR analysis using multiplex PCR. A total of three AMR genes, all β-lactam resistance encoding genes, were detected among isolates, namely, blaTEM, blaCTX-M, and blaSHV. Each multiplex PCR reaction mixture was 12.5 μl in volume and contained 6.25 μl 5× MyTaq HS Red Mix (Bioline), 2.75 μl of nuclease-free water, 0.25 μl of forward and reverse primers wherein blaTEM primers at 10 μM, blaCTX-M and blaSHV at 30 μM, and 2 μl of DNA template. Following previous studies [19, 36], multiplex PCR protocol for bla genes involved 95℃ for 3 min (initial denaturation), then 30 cycles of three steps, namely, 95℃ for 30 s (denaturation), 60℃ for 30 s (annealing), and 72℃ for 1 min (extension), and finally, 72℃ for 10 min (final extension). For positive controls, Klebsiella pneumoniae (ATCC 700603) was used for blaSHV, whereas in in-house laboratory controls from Calayag et al. [19], positive samples were used for blaTEM and blaCTX-M.

Table 2 . Primers, sequences and amplification product size for antimicrobial resistance genes.

GenePrimerSequenceProduct size (bp)Reference
blaTEMblaTEMF: 5’-TCGCCGCATACACTATTCTCAGAATGA-3’244[36]
blaTEMR: 5’-ACGCTCACCGGCTCCAGATTTAT-3’
blaCTX-MblaCTX-MF: 5’-ATGTGCAGYACCAGTAARGTKATGGC-3’385
blaCTX-MR: 5’-TGGGTRAARTARGTSACCAGAAYCAGCGG-3’
blaSHVblaSHVF: 5’-ATGCGTTATATTCGCCTGTG-3’497
blaSHVR: 5’-TGCTTTGTTATTCGGGCCAA-3’


Data analysis

Descriptive statistics using Fisher’s exact test through SPSS version 26 (IBM) was used to find significant associations between virulence and AMR genes among isolates to determine relationships of pathogenicity and AMR in Salmonella. Virulence and AMR gene patterns were visualized using R version 4.0.5 in R studio (R Foundation) with the UpSetR 1.4.0 package to generate intersecting plots and determine frequencies of gene patterns. Genotypic virulence and AMR profiles were determined in frequencies to reveal the general prevalence of these genes across Salmonella isolates. Prevalence was also compared based on location types whether abattoir or wet market and Metro Manila districts whether Northern, Eastern, Southern, or Capital.

Results

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Prevalence and associations of virulence and AMR genes in Salmonella

A total of 700 Salmonella isolates were collected for DNA extraction and genotypic analysis. High AMR potential for β-lactam antibiotics was also found in this study. For bla genes, blaTEM was detected at a high prevalence of 46.43% among Salmonella isolates, followed by blaSHV at 1.00% and blaCTX-M with only 0.29% (Fig. 1). For SPIs 1−5 virulence genes, prevalence was more than 50%, whereas no isolates harbored plasmidborne spvC and spvR genes, suggesting high pathogenic potential of these Salmonella isolates (Fig. 1). From highest to lowest detected virulence genes, hilA and avrA from SPI 1 were at 68.43% and 65.29%, respectively, followed by mgtC (64.71%) from SPI 3, pipB (57.29%) from SPI 5, sseC (54.29%) from SPI 2, and spi4R (54.00%) from SPI 4 (Fig. 1).

Figure 1.Prevalence of virulence and AMR genes among Salmonella isolates (n = 700).

Fisher's exact test showed some significant associations (p < 0.05) between virulence and AMR genes detected among Salmonella isolates (Table 3). AMR gene blaTEM showed associations with virulence genes avrA, hilA, mgtC, and spi4R, but not sseC and pipB (Table 3). In contrast, blaCTX-M and blaSHV were not subjected to this analysis due to their low prevalence across Salmonella isolates.

Table 3 . Fisher's exact test on the associations between virulence and AMR genes of Salmonella isolates (n = 700).

Virulence and AMR gene associationsFisher's exact test (two-sided p-value)
blaTEM and avrA0.001*
blaTEM and hilA<0.001*
blaTEM and sseC0.404
blaTEM and mgtC<0.001*
blaTEM and spi4R<0.001*
blaTEM and pipB0.146


Patterns of virulence and AMR gene prevalence in Salmonella

Using the UpSetR package in RStudio to determine intersecting plots in virulence and AMR gene prevalence across Salmonella, the highest intersecting pattern (8.86%) with bla and SPI genes involved blaTEM, avrA, hilA, mgtC, spi4R, and pipB, whereas if including sseC, it was 7.43% of the isolates (Fig. 2). Interestingly, the highest pattern (9.57%) involved sseC alone with no other virulence or AMR genes, whereas the third highest pattern (8.29%) possessed all six SPIs 1−5 genes, namely, avrA, hilA, sseC, mgtC, spi4R, and pipB (Fig. 2). Among the seven blaSHV positive isolates, six (85.71%) also possessed blaTEM gene, whereas all two blaCTX-M positive isolates also possessed blaTEM, suggesting association, and potential relationship of bla genes in Salmonella and other Enterobacteriaceae (Fig. 2). In addition, three isolates positive for both blaTEM and blaSHV possessed avrA, hilA, sseC, mgtC, and spi4R (Fig. 2). Similarly, the two blaTEM and blaCTX-M positive isolates also possessed multiple SPIs 1−5 virulence genes (Fig. 2). Collectively, these data suggest not only pathogenic and AMR potential but also interactions or associations between pathogenicity and AMR in Salmonella.

Figure 2.Patterns and intersections of virulence and AMR genes among Salmonella isolates (n = 700) using UpSetR 1.4.0 package R version 4.0.5 in R studio.

Virulence and AMR gene prevalence based on location types

Comparing abattoirs with 436 isolates to wet markets with 264 isolates, some differences were observed for virulence and AMR gene prevalence (Fig. 3). For virulence genes, avrA, hilA, mgtC, spi4R, and pipB generally showed higher prevalence in abattoir isolates than in wet market isolates, whereas sseC was higher (61.36%) in wet market Salmonella isolates than in abattoirs (50.00%) (Fig. 3). hilA showed the highest difference, followed by mgtC between location types, which suggest possible variations in the incidence and potential pathogenicity of Salmonella across these settings. For bla genes, blaTEM prevalence was higher in abattoir (50.69%) than in wet market (39.39%) isolates (Fig. 3). The two blaCTX-M positive isolates all originated from abattoirs, whereas blaSHV prevalence was slightly higher in wet markets (1.52%) than in abattoirs (0.69%) (Fig. 3). These suggest high AMR potential against β-lactam antibiotics in abattoirs and wet markets considering that blaTEM was generally widely distributed among Salmonella across location types.

Figure 3.Comparison of virulence and AMR gene prevalence across abattoirs and wet market Salmonella isolates.

Virulence and AMR gene prevalence based on Metro Manila districts

Comparing the four Metro Manila districts, namely, Northern (n = 197), Eastern (n = 178), Southern (n = 184), and Capital (n = 141), some differences were also observed for virulence and AMR gene prevalence across Salmonella (Fig. 4). For virulence genes, the most prevalent in avrA gene was among Capital isolates (76.60%), hilA among Southern isolates (82.07%), sseC among Northern isolates (63.96%), mgtC among Capital isolates (70.92%), spi4R among Northern isolates (60.91%), and pipB among Capital isolates (61.70%) (Fig. 4). These suggest that isolates from these locations have higher pathogenic potential and thus require further surveillance. For bla genes, the most prevalent in blaTEM and blaSHV was found in Northern isolates (65.48% and 1.52%, respectively), whereas the two blaCTX-M positive isolates were found in Eastern and Northern districts (Fig. 4). These suggest that Salmonella isolated from the Northern district showed the highest AMR potential among other districts.

Figure 4.Comparison of virulence and AMR gene prevalence across four Metro Manila districts (Northern, Eastern, Southern, Capital) isolates.

Discussion

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The high prevalence of virulence and bla genes detected among Salmonella isolated from swine in this study suggests their high pathogenicity and AMR potential that pose a significant threat to the Philippine swine industry and throughout the value chain. hilA was the most commonly detected among virulence genes at 68.43% which is generally at lower rates than most studies [37−39]. hilA, as the highest, has a pivotal role in Salmonella pathogenicity as a master transcriptional regulator for the expression of invasion-associated genes [40]. Another study of Salmonella from chicken feces in South Africa showed very low prevalence (9%) of hilA [41]. However, hilA prevalence may be affected by differences in serovars as can be observed in the study of Thung et al. [42], which showed less prevalence among serovars Agona and Newport. hilA was followed by avrA and mgtC showing lower prevalence but still at > 60%, which was in contrast to a study in Egypt that showed only 30% detection rates in chicken meats [43]. However, many studies report higher prevalence (> 90%) of avrA and mgtC that can also differ based on Salmonella serovars [29, 44−46]. avrA has been associated with several functions from host immune inhibition [47−49], intracellular survival of Salmonella [50], chronic infections and carcinogenesis [51]. Zou et al. [52] and Suez et al. [53] reported the variable presence of avrA among Salmonella strains. The presence of avrA and spvC has also been associated with systemic Salmonella serovars such as Pullorum and Gallinarum [54]. mgtC facilitates intramacrophage proliferation by maintaining ATP homeostasis during phagosome acidification and repression of cellulose production [55]. Mutation experiments involving the mgt locus have also resulted in growth impairment of Salmonella in-host and non-host environments [56]. Among virulence genes, < 60% are pipB, sseC, and spi4R. Thi et al. [57] reported a pipB gene prevalence of only < 50% and mgtC of < 40% among Salmonella from swine. In contrast, Fazl et al. [30] reported 100% prevalence of pipB and sseC among Salmonella Typhimurium from humans and poultry in Iran. Another study of Salmonella from stool samples of children with diarrhea in Iraq showed < 2% sseC gene occurrence present only in one serovar Typhi isolate [58]. pipB encodes for a type III secretion system 2 (TTSS 2) released effector protein with involvement for enteric, but not systemic salmonellosis [33, 59, 60]. Meanwhile, sseC encodes a translocon protein with a role in the intracellular survival [61, 62]. spi4R gene has been reported at higher prevalence (> 90%) by studies of Salmonella from clinical and environmental samples [31, 63], whereas among Salmonella from broiler chickens, spi4R was not present in serovar Infantis, which was suggested to be due to deletion or mutation [64]. Meanwhile, spi4R was predicted to encode for the type I secretion system in Salmonella [65]. The lack of spvC and spvR virulence genes among Salmonella isolates in this study was also reflected by several reports of either very low or no prevalence in other studies [45, 63, 66], which may be due to their plasmid-borne attribute and their presence primarily in systemically disseminated Salmonella serovars [67, 68].

The high prevalence of blaTEM (46.43%) over other bla genes, namely, blaCTX-M and blaSHV in this study was also reflected in other studies such as in India and Nigeria and household pets from Thailand and humans from Egypt [44, 46, 66, 69]. Correlations of blaTEM with resistance to ampicillin, a widely used β-lactam antibiotic worldwide, have been previously established and associated with selective pressure [44, 70]. This suggests the possible anthropological contributions that require proper surveillance and regulation. Although, this study detected lower incidences of blaCTX-M and blaSHV which contrasts to reports of prevalence up to 80% among Salmonella Enteritidis and Typhimurium from chickens and rats [41], these two bla genes are often attributed to ESBL phenotypes [71]. It is plausible that this may be due to higher prices and subsequently less common usage of higher cephalosporins that may potentially correspond [66]. Nonetheless, their presence may be associated with phenotypic β-lactam resistance including ESBLs, thus posing a threat to public health [72−74]. The co-carriage of blaTEM and blaCTX-M has also been reflected in other studies that may indicate ESBL phenotypes [19].

Associations between virulence and AMR have long baffled researchers due to their complex relationships. This phenomenon is supported by the notion of MDR bacteria such as Escherichia coli ST131 several other bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus causing extensive infections [75, 76]. With a multitude of virulence genes [4, 77, 78], additional roles aside from conferring virulence factors and facilitating host-pathogen interactions may exist. This study revealed four significant associations involving blaTEM AMR gene and Salmonella virulence genes avrA, hilA, mgtC, and spi4R. Various Salmonella serovars from clinically diarrheic humans in Egypt showed associations of several virulence genes, namely, invA, avrA, ssaQ, mgtC, siiD, sopB, and bcfC, with chloramphenicol resistance phenotype [79]. Meanwhile, Higgins et al. [80] also revealed strong associations of several virulence genes, including avrA, mgtC, and spvC, to phenotypic MDR status but not to single AMR status among clinical Salmonella isolates. Positive correlations were also detected between several virulence and AMR genes, namely, orgA and blaPSE1, spaN and tolC with cmlA, and tolC with sul1 in Salmonella from dogs and cats [66]. Treatment of MDR Salmonella Typhimurium with chloramphenicol and tetracyclines upregulated hilA, prgH, and invF genes that subsequently increased invasiveness, but other antibiotics such as ampicillin and streptomycin had no effect [81, 82]. Plasmid-borne virulence genes spvC and spvR have also been demonstrated to correlate with various AMR and even MDR phenotypes [45, 83, 84]. An in silico study has also evaluated associations of mobile genetic elements that annotate AMR, virulence genes, and plasmids in Salmonella [85]. Interestingly, hybrid plasmids containing both AMR and virulence gene sequences have been found in Salmonella [86]. However, negative relationships or lack of associations between virulence and AMR in Salmonella have also been found. Some examples include nitrofurantoin resistance and virulence gene bapA [84] and lack of statistical associations between mgtC, avrA, ssaQ, and sopB to all AMR phenotypes in Salmonella from swine in Argentina [45]. Hence, the relationship of virulence and AMR in Salmonella remains complex and requires further elucidation.

External factors can affect Salmonella prevalence and diversity that subsequently contributes to their virulence and AMR potential. Salmonella can contaminate meat products throughout the entire food animal production chain that is affected by environmental aspects whether sanitation, transport, processing, or handling, including areas or sites of breeding, slaughter, and retail [42, 87, 88]. All gene prevalence, except for sseC and blaSHV, were higher in abattoir than in wet market swine isolates, and comparisons of four Metro Manila districts also showed drastic differences in virulence and AMR gene prevalence, which illustrate variations. In comparison, the pork production chain in China showed no significant difference in the number of ARGs possessed by Salmonella from abattoirs and markets; however, there were differences in the types of genes identified [89]. Variations across the broiler production chain in Trinidad and Tobago have also been observed, from the types of Salmonella serovars detected, AMR genes particularly and virulence factors with serovars also contributing to differences [90]. Meanwhile, comparison of intensive and backyard farm swine production in Argentina showed that while no significant difference was observed for virulence genes profile, higher AMR phenotypes among Salmonella were observed in intensive than in backyard farming, suggesting risks in animal production [45].

In conclusion, the virulence and AMR gene profiles of Salmonella from swine samples in abattoirs and wet markets in Metro Manila districts obtained in this study provide valuable insights to support the need for further research and constant surveillance of the entire swine production chain as well as inform policymakers to enforce and improve farming standards, antimicrobial usage, as well as processing and retail monitoring and regulations. High prevalence of virulence genes reported in this study corroborates with other studies indicating pathogenic potential and genetic diversity among Salmonella and supports the wide distribution of SPIs 1−5 virulence genes. High detection rates for blaTEM suggest significant AMR potential for β-lactams, which are among the most utilized antibiotics worldwide, which poses a concern to therapeutics and public health. Some statistical associations and frequency patterns between virulence and AMR genes in Salmonella were also detected that add to the growing repertoire of relationships among pathogenicity and AMR in pathogens that remain complicated and unestablished. Lastly, drastic variations in virulence and AMR gene prevalence were observed comparing abattoirs and markets as well as among Metro Manila districts that suggest contributions of external factors throughout the swine production chain that needs proper surveillance as they remain significant threats to the Philippine swine industry and consumer health.

Acknowledgments

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The authors acknowledge Paolo D.G. Mendoza, Camille Andrea R. Flores, and Alyzza Marie B. Calayag for the technical support. This study was supported financially by the Department of Agriculture-Biotechnology Program Office (DABIOTECH-R1808) of the Philippines.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

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