1907-770XBIOTROPIABIOTROPIA1907-770X0215-6334SEAMEO BIOTROP10.11598/btb.2024.31.3.2207SURVEILLANCE OF β-LACTAMASE GENES OF SALMONELLA FROM CHICKEN IN WET MARKETS OF METRO MANILA, PHILIPPINESMadayagMilesPhilippinesPavonRance DerrickPhilippinesMoraJonah FelizaPhilippinesBalagaKhristinePhilippinesRiveraWindellPhilippineswlrivera@science.upd.edu.phPathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, PhilippinesCahyaHaritzhttps://orcid.org/0000-0001-9202-8382ArdiansyahDr RhomiIndonesiaDepartment of Forest Resources Conservation, Faculty of Forestry, Universitas Gadjah MadaCorresponding author: Windell Rivera, Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, Philippines .Email:wlrivera@science.upd.edu.ph1012202431333934827220242272024Copyright (c) 2024 Miles Madayag, Mr., Ms., Ms., Windell Rivera2024Miles Madayag, Mr., Ms., Ms., Windell Riverahttp://creativecommons.org/licenses/by-nc-nd/4.0This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.Authors who publish with this journal agree with the following terms:
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blaCTX-M gene in 24.7% while blaTEM gene in 11.2% of Salmonella isolates.
Coexistence of blaCTX-M groups II and IV in all blaCTX-M-positive isolates.
Dominance of blaCTX-M corroborated with phenotypic β-lactam resistances.
blaCTX-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, blaCTX-M had the highest occurrence, detected in 22 isolates (24.7%), while blaTEM was detected in 10 isolates (11.2%). Genotypic and phenotypic resistance corroboration was observed in nearly all blaCTX-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, blaCTX-M groups II and IV genes were co-detected and multidrug resistance (MDR) profiles were also observed in all blaCTX-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.
antimicrobial resistanceblaCTX-M groupsbla genesoccurrenceSalmonellaFile created by JATS EditorJATS EditorINTRODUCTION
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 blaTEM, blaCTX-M, and blaSHV genes, respectively, and are among the most common ESBL genes (Ejaz et al., 2021). Additionally, blaCTX-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 blaTEM, blaCTX-M, and blaSHV, 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 blaCTX-M gene groups and determined the phenotypic antimicrobial susceptibility profile of S. enterica possessing those gene groups.
MATERIALS AND METHODSSalmonella 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 blaTEM, blaCTX-M, and blaSHV detection. The volumes and concentrations of MyTaqTM 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 blaTEM and blaCTX-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 blaSHV-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 blaCTX-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 blaCTX-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 blaCTX-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).
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) 32nd 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 blaSHV gene. The occurrence of blaTEM and blaCTX-M genes in the tested isolates were 10 (11.24%) and 22 (24.72%) , respectively. The highest occurrence for blaTEM was found among the isolates sampled in Valenzuela City, while for blaCTX-M, isolates sampled from Quezon City showed the highest occurrence.
The blaCTX-M gene showed the highest prevalence among isolates in this study, which contrasted with some studies among Enterobacteriaceae. Most studies reported blaTEM as the most predominant bla gene over blaCTX-M and blaSHV 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 blaTEM, blaCTX-M, and blaSHV in β-lactam-resistant clinical E. coli isolates at 56.3%, 18.3%, and 11.3%, respectively. Although the frequency estimates of blaSHV, blaTEM, and blaCTX-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 blaTEM was detected with the highest frequency, followed by blaCTX-M. However, the only detected blaCTX-M subtypes were blaCTX-M-1 from group I and blaCTX-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 blaCTX-M, blaTEM, and blaSHV 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 blaCTX-M, blaTEM, and blaSHV, respectively. These results suggested the high cephalosporin resistance potential of Salmonella isolated from retail poultry in Metro Manila.
Upon detection of blaCTX-M groups I, II, and IV genes, all 22 blaCTX-M-positive isolates were simultaneously positive for blaCTX-M groups II and IV. Interestingly, none of the tested samples were positive for blaCTX-M group I. The co-carriage of these blaCTX-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 blaCTX-M group gene identities, PCR products of DNA templates of a blaCTX-M group I positive control and representative Salmonella isolates from blaCTX-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 blaCTX-M groups I, II, and IV, confirming the gene identities. The presence of blaCTX-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 blaCTX-M-positive isolates were subjected to VITEK® 2 automated susceptibility testing. Resistance profiles of Salmonella carrying blaCTX-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%) blaCTX-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 blaCTX-M gene with variations in blaCTX-M gene groups detected.
Corroboration of phenotypic and genotypic resistance was observed in 21 blaCTX-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 blaCTX-M(Cruz & Hedreyda, 2017); (Calayag et al., 2021). A mutation in the structural gene or regulatory region could prevent the expression of the blaCTX-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 blaCTX-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 blaCTX-M genes.
Shi et al. (2021) claimed that blaCTX-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 blaCTX-M group I genes, while 35.8% carried blaCTX-M group IV genes (Shi et al., 2021). Although excessive antibiotic use is one of the reasons attributed to the dissemination of blaCTX-M(Cantón, 2007); (Cantón et al., 2012), it cannot be concluded that excessive use of ceftriaxone was the main reason for the dominance of blaCTX-M genes in this study. Since cephalosporins are not often used in Philippine poultry farms (Barroga et al., 2020), the high occurrence of blaCTX-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 blaCTX-M may also be due to the mobilization of blaCTX-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 blaCTX-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 blaCTX-M group I and IV have been considered as the most common groups of blaCTX-M genes (Cantón et al., 2012), this study reported the absence of blaCTX-M group I, while blaCTX-M groups II and IV have the highest occurrences. While the absence of blaCTX-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 blaCTX-M groups II and IV. Specifically, ISEcp1 and IS903B are associated with blaCTX-M group IV, while the ISCR1 element is associated with blaCTX-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 blaCTX-M groups II and IV might recombine and produce novel β-lactamases as in the case of blaCTX-M-123, which is a hybrid of group I blaCTX-M-15 and group IV blaCTX-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 blaCTX-M-carrying bacteria favors their survival, which may contribute to the spread of blaCTX-M(Cantón, 2007). Co-carriage of blaCTX-M, blaTEM, 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 blaCTX-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 blaCTX-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.
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
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
Multidrug resistance patterns of 22 blaCTX-M-positive S. enterica isolates
This study reported the predominance of blaCTX-M and the coexistence of blaCTX-M groups II and IV in all blaCTX-M positive S. enterica isolates. These suggest high cephalosporin resistance potential. Aside from the corroboration of phenotypic and genotypic resistance among blaCTX-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 blaCTX-M gene groups, and high phenotypic resistance among blaCTX-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.
ACKNOWLEDGMENTS
We thank the Department of Agriculture, Biotechnology Program Office, Philippines (Project No. DABIOTECH-R2302) for the financial support. We also acknowledge Vanessa Yvonne B. Meclat for the technical assistance. We also state that Miles G. Madayag and Rance Derrick N. Pavon contributed equally to this work.
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