Abstract
Background
The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years.
Objective
This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia.
Methods
One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s).
Results
Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [blaTEM ], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3″)-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected.
Conclusion
Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.
Keywords: Salmonella, chickens, drug resistance, bacterial, genes
INTRODUCTION
Salmonella is a foodborne pathogen of significant public and animal health concern worldwide. More than 2,500 serovars have been identified to date, and non-typhoidal Salmonella (NTS)-related invasive infections are progressively being reported in several countries [1]. NTS can cause self-limiting gastroenteritis in humans and animals. In certain individuals, severe complications may be life-threatening when the bacteria invade normally sterile sites, causing bacteremia and meningitis [2]. In recent years, the Salmonella National Surveillance Program conducted by the Department of Veterinary Services (DVS) Malaysia identified an increasing trend in the proportion of Salmonella enterica subsp. enterica serovar Brancaster isolated from chickens in the country. Salmonella Enteritidis was the dominant serovar in poultry throughout the 1990s, and it is listed as a notifiable serovar due to its economic impact and zoonotic potential [3], along with serovar Typhimurium, Pullorum, and Gallinarum. Since 2014, S. Brancaster has been the most commonly isolated serovar from chickens in Malaysia. It was isolated from every aspect of the processing line, including the floor, chopping board, wash water, and chicken cuts [4]. In Europe and West Africa, S. Brancaster was isolated from patients with diarrhea and implicated in fatal cases among infants and elderly patients [5,6]. Few reports indicate that the prevalence of S. Brancaster ranged from 5%–21% in the Asian region, suggesting that S. Brancaster could be an emerging serovar [7,8].
Salmonella infection in humans has been linked to poultry farms as the pathogen can colonize healthy, asymptomatic chickens [9]. The increasing multidrug-resistant (MDR) Salmonella isolated from foods of animal origins signifies a global public health concern [10]. In 2017, an MDR S. Brancaster isolated from chicken meat was reported, with a 5,036,442 bp genome containing various antimicrobial resistance genes towards aminoglycosides, fluoroquinolones, fosfomycin, chloramphenicols, sulphonamides, β-lactams, tetracycline, macrolides, and trimethoprim [11]. In the ongoing evolution of bacteria across various phyla and habitats, frequent gene transfer events and gene acquisition under selective pressures from the host, the environment, and antimicrobials represent the opportunities and challenges for the emergence of MDR pathogens.
The genes associated with survival fitness, antibiotic resistance, and virulence are located on the Salmonella pathogenicity island (SPI), encoding the type III secretion systems (T3SS) responsible for consequent pathological sequences in host phagocytes [12]. The conventional Salmonella serotyping using slide agglutination tests is based on the surface somatic (O) cell wall and flagellar (H) antigen recognition. In the interest of protecting public and animal health, such serotyping data based on the White-Kauffmann-Le Minor scheme was unlikely to identify invasive NTS and is insufficient to provide a deeper analysis in understanding the characteristics and potential pathogenicity of this emerging serovar [13]. Therefore, molecular virulence profiling using polymerase chain reaction (PCR) to detect virulence genes has been proposed to augment the conventional Salmonella serotyping method [14]. This study aims to investigate the virulence genes and antimicrobial resistance profiles of the S. Brancaster isolated from chickens in Malaysia.
MATERIALS AND METHODS
S. Brancaster isolates
This study used 100 S. Brancaster isolates archived at the Veterinary Research Institute (VRI), Ipoh, Malaysia. These isolates originated from chicken cloacal swabs and raw chicken meat from farms and abattoirs sent to the DVS Malaysia regional laboratories as part of the routine national Salmonella surveillance program between 2017 and 2022. Isolates identified as Salmonella spp. through selective media and biochemical tests were submitted to VRI for serotyping. Serotyping was performed using the slide agglutination method with specific O and H antisera (SSI Diagnostica, Denmark), and S. Brancaster isolates were identified based on the antigenic formulae of 1,4,12,27:z29:- [15].
DNA extraction
The bacterial DNA was extracted by heat lysis from an overnight culture at 37°C [16]. A single colony was transferred into 100 µL nuclease-free water and incubated at 95°C in a thermal block for 10 min. After cooling to room temperature and centrifuging for 10 min at 13,000 rpm, the supernatant was used as the DNA template.
Detection of virulence genes by PCR
Two PCR reactions were performed to examine for the presence of eight virulence genes using the primers listed in Table 1 [17]. Reaction one amplified the invasion protein gene (invA), Salmonella iron transporter gene (sitC), Salmonella pathogenicity islands gene (spiA), and cytolethal-distending toxin B gene (cdtB), while reaction two amplified the Salmonella invasion protein gene (sipB), Salmonella plasmid virulence gene (spvB), Salmonella-induced filament gene (sifA), and inositol phosphate phosphatase gene (sopB). The reference strain used was Salmonella Typhimurium ATCC 14028. The amplifications were performed in a 50-µL reaction mixture containing 25 µL of 2× MyTaq Mix (Bioline, UK), 1 µL of each 10 µM forward and reverse primers (1st Base, Malaysia), 5 µL of DNA template, and topped up with 12 µL nuclease-free water. The PCR was performed using a thermal cycler (Eppendorf, Germany) as follows: 95°C for 5 min followed by 25 cycles of denaturation (94°C for 30 sec), annealing (66.5°C for 30 sec), and extension (72°C for 2 min). A final extension step was carried out at 72°C for 10 min. The amplified DNA was electrophoresed in a 1.5% agarose stained with SYBR Safe DNA Gel Stain (Invitrogen, USA) at 90 V for 50 min in 1× Tris-borate-EDTA buffer. The PCR products were visualized using a UV transilluminator (Uvitec, UK) and compared to the 100 bp DNA HyperLadder (Bioline).
Table 1. PCR primers used in this study.
| Gene target | Primer sequence (5′–3′) | Amplicon size (bp) | Reference | |
|---|---|---|---|---|
| Virulence-associated gene (multiplex PCR reaction 1) | ||||
| invA | CTG GCG GTG GGT TTT GTT GTC TTC TCT ATT | 1,070 | [17] | |
| AGT TTC TCC CCC TCT TCA TGC GTT ACC C | ||||
| sitC | CAG TAT ATG CTC AAC GCG ATG TGG GTC TCC | 768 | ||
| CGG GGC GAA AAT AAA GGC TGT GAT GAA C | ||||
| spiA | CCA GGG GTC GTT AGT GTA TTG CGT GAG ATG | 550 | ||
| CGC GTA ACA AAG AAC CCG TAG TGA TGG ATT | ||||
| cdtB | ACA ACT GTC GCA TCT CGC CCC GTC ATT | 268 | ||
| CAA TTT GCG TGG GTT CTG TAG GTG CGA GT | ||||
| Virulence-associated gene (multiplex PCR reaction 2) | ||||
| sipB | GGA CGC CGC CCG GGA AAA ACT CTC | 875 | [17] | |
| ACA CTC CCG TCG CCG CCT TCA CAA | ||||
| spvB | CTA TCA GCC CCG CAC GGA GAG CAG TTT TTA | 717 | ||
| GGA GGA GGC GGT GGC GGT GGC ATC ATA | ||||
| sifA | TTT GCC GAA CGC GCC CCC ACA CG | 449 | ||
| GTT GCC TTT TCT TGC GCT TTC CAC CCA TCT | ||||
| sopB | CGG ACC GGC CAG CAA CAA AAC AAG AAG AAG | 220 | ||
| TAG TGA TGC CCG TTA TGC GTG AGT GTA TT | ||||
| Antimicrobial resistance gene (singleplex PCR) | ||||
| blaPSE-1 | CGC TTC CCG TTA ACA AGT AC | 419 | [19] | |
| CTG GTT CAT TTC AGA TAG CG | ||||
| blaTEM | GCA CGA GTG GGT TAC ATC GA | 310 | [20] | |
| GGT CCT CCG ATC GTT GTC AG | ||||
| floR | CTG AGG GTG TCG TCA TCT AC | 673 | [16] | |
| GCT CCG ACA ATG CTG ACT AT | ||||
| aadA2 | CGG TGA CCA TCG AAA TTT CG | 249 | [20] | |
| CTA TAG CGC GGA GCG TCT CGC | ||||
| strA | CTT GGT GAT AAC GGC AAT TC | 548 | [20] | |
| CCA ATC GCA GAT AGA AGG C | ||||
| ant(3”)-Ia | GTG GAT GGC GGC CTG AAG CC | 526 | [19] | |
| ATT GCC CAG TCG GCA GCG | ||||
| sul-1 | ATC GCA ATA GTT GGC GAA GT | 797 | [19] | |
| GCA AGG CGG AAA CCC GCG CC | ||||
| sul-2 | AGG GGG CAG ATG TGA TCG AC | 249 | [20] | |
| GCA GAT GAT TTC GCC AAT TG | ||||
| sulA | CAC TGC CAC AAG CCG TAA | 360 | [20] | |
| GTC CGC CTC AGC AAT ATC | ||||
| Antimicrobial resistance gene (multiplex PCR) | ||||
| tetA | GCT ACA TCC TGC TTG CCT TC | 210 | [21] | |
| CAT AGA TCG CCG TGA AGA GG | ||||
| tetB | TTG GTT AGG GGC AAG TTT TG | 659 | ||
| GTA ATG GGC CAA TAA CAC CG | ||||
| tetG | CAG CTT TCG GAT TCT TAC GG | 468 | ||
| GAT TGG TGA GGC TCG TTA GC | ||||
PCR, polymerase chain reaction; invA, invasion protein gene; sitC, Salmonella iron transporter gene; spiA, Salmonella pathogenicity islands gene; cdtB, cytolethal-distending toxin B gene; sipB, Salmonella invasion protein gene; spvB, Salmonella plasmid virulence gene; sifA, Salmonella-induced filament gene; sopB, inositol phosphate phosphatase gene; blaPSE-1 , β-lactamase gene; blaTEM , β-lactamase temoneira; floR, florfenicol/chloramphenicol resistance gene; aadA2, aminoglycoside adenyltransferase gene; strA, streptomycin resistance gene; ant(3”)-Ia, aminoglycoside nucleotidyltransferase gene; sul-1, sul-2, sulA, sulfonamides resistance gene; tetA, tetB, tetG, tetracycline resistance gene.
Antimicrobial susceptibility test
The disk diffusion method using Muller-Hinton agar (Oxoid, UK) was used to determine the antimicrobial susceptibility of S. Brancaster towards ampicillin (10 µg), amoxicillin/clavulanic acid (30 µg), chloramphenicol (30 µg), streptomycin (10 µg), gentamicin (10 µg), sulfamethoxazole/trimethoprim (25 µg), tetracycline (30 µg), nalidixic acid (30 µg), and ciprofloxacin (5 µg; Oxoid). Results were obtained after incubating the samples for 16–18 h at 37°C, and the inhibition zones were measured and categorized as susceptible, intermediate, or resistant according to the Clinical and Laboratory Standards Institute guidelines [18]. Escherichia coli ATCC 25922 was used as the quality control reference strain.
Detection of antimicrobial resistance gene in selected S. Brancaster isolates by PCR
Twelve pairs of primers were used to target antimicrobial resistance genes (β-lactamase gene [blaPSE-1 ], β-lactamase temoneira [blaTEM ], florfenicol/chloramphenicol resistance gene [floR], aminoglycoside adenyltransferase gene [aadA2], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3")-Ia], sulfonamides resistance gene [sul-1, sul-2, sulA], tetracycline resistance gene [tetA, tetB, and tetG]) in S. Brancaster isolates that confer phenotypic ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT)-resistance pattern [16,19,20,21]. Amplifications were conducted in a 50-µL reaction mixture containing 25 µL of 2× MyTaq™ Mix (Bioline), 1 µL of each 10 µM forward and reverse primers (1st Base), 5 µL of DNA template, and topped up with the appropriate volume of nuclease-free water. All PCR were optimized and performed as follows: 95°C for 5 min followed by 30 cycles of denaturation (95°C for 1 min), annealing (56°C for 1 min), and extension (72°C for 1 min). A final extension step was carried out at 72°C for 7 min. S. Typhimurium strain DT104 (NCTC 13348) with ACSSuT-resistance pattern was used as the positive control. Amplified PCR products (blaTEM , strA, and sul-2) without the positive control were verified by DNA sequencing and sent to a commercial sequencing laboratory (Apical Scientific Sdn Bhd, Malaysia). The resulting sequences were assembled using SeqMan Pro software (DNAStar Lasergene, USA) and confirmed using the National Centre for Biotechnology Information database and BLAST program. Singleplex and multiplex PCR primer sequences and the expected amplicon sizes of the amplified products are listed in Table 1.
RESULTS
Virulence genes of S. Brancaster
All S. Brancaster isolates carried the invA, sitC, spiA, and sipB. Almost all isolates are positive for sopB (99%), while sifA (59%) and cdtB (26%) are variably present in the tested isolates. The spvB is present in just one isolate (1%). All S. Brancaster tested in this study had at least four virulence genes, as presented in Table 2.
Table 2. Detection of virulence genes by polymerase chain reaction.
| No. of isolates | Virulence gene | |||||||
|---|---|---|---|---|---|---|---|---|
| invA | sitC | spiA | cdtB | sipB | spvB | sifA | sopB | |
| 24 | + | + | + | + | + | − | + | + |
| 38 | + | + | + | − | + | − | − | + |
| 2 | + | + | + | + | + | − | − | + |
| 34 | + | + | + | − | + | − | + | + |
| 1 | + | + | + | − | + | − | − | − |
| 1 | + | + | + | − | + | + | + | + |
| Prevalence (%) | 100 | 100 | 100 | 26 | 100 | 1 | 59 | 99 |
invA, invasion protein gene; sitC, Salmonella iron transporter gene; spiA, Salmonella pathogenicity islands gene; cdtB, cytolethal-distending toxin B gene; sipB, Salmonella invasion protein gene; spvB, Salmonella plasmid virulence gene; sifA, Salmonella-induced filament gene; sopB, inositol phosphate phosphatase gene.
Antimicrobial resistance profiles of S. Brancaster
In the current study, S. Brancaster indicated the highest resistance rates to ampicillin (91%), followed by tetracycline (86%), sulfamethoxazole/trimethoprim (62%), chloramphenicol (61%), gentamicin (45%), nalidixic acid (43%), streptomycin (33%), ciprofloxacin (9%), and amoxicillin/clavulanic acid (4%), as shown in Table 3.
Table 3. Antimicrobial resistance of Salmonella Brancaster isolates detected from cloacal swab and raw chicken meat.
| Group of antimicrobials | Antimicrobial disc | Isolate with resistance | Frequency (%) | |
|---|---|---|---|---|
| Cloacal swab (n = 68) | Raw chicken meat (n = 32) | |||
| β- lactams | Ampicillin | 60 | 31 | 91 |
| Amoxicillin/Clavulanic acid | 3 | 1 | 4 | |
| Phenicols | Chloramphenicol | 42 | 19 | 61 |
| Aminoglycosides | Streptomycin | 22 | 11 | 33 |
| Gentamicin | 34 | 11 | 45 | |
| Sulfonamides | Sulfamethoxazole/Trimethoprim | 45 | 17 | 62 |
| Tetracycline | Tetracycline | 59 | 27 | 86 |
| Quinolones | Nalidixic acid | 30 | 13 | 43 |
| Fluoroquinolones | Ciprofloxacin | 4 | 5 | 9 |
Overall, 85% (85/100) of S. Brancaster isolates were identified as MDR, which confers resistance to at least three classes of antimicrobials tested [22]. The S. Brancaster antimicrobial resistance profiles are presented in Table 4. They displayed 36 antibiogram patterns, with AMP-C-SXT-TE-NAL (12 isolates) being the most frequent resistance type. Three isolates of S. Brancaster were found to be susceptible to all antimicrobials tested, while four isolates were found resistant to all seven antimicrobial classes.
Table 4. Antimicrobial resistance patterns of Salmonella Brancaster isolates.
| No. of antibiotic classes | Resistance phenotype | No. of isolates | ACSSuT-resistance type |
|---|---|---|---|
| 7 | AMP-AMC-C-S-SXT-TE-NAL-CIP | 3 | Yes |
| AMP-C-S-SXT-TE-NAL-CIP | 1 | Yes | |
| 6 | AMP-C-S-CN-SXT-TE-NAL | 5 | Yes |
| AMP-C-S-SXT-TE-NAL | 4 | Yes | |
| AMP-C-CN-SXT-TE-NAL | 7 | Yes | |
| AMP-C-S-SXT-TE-CIP | 1 | Yes | |
| AMP-C-SXT-TE-NAL-CIP | 2 | - | |
| 5 | AMP-C-S-CN-SXT-TE | 4 | Yes |
| AMP-C-S-SXT-TE | 2 | Yes | |
| AMP-C-CN-SXT-TE | 6 | Yes | |
| AMP-C-SXT-TE-NAL | 12 | - | |
| AMP-S-CN-SXT-TE-NAL | 1 | - | |
| 4 | AMP-S-CN-SXT-TE | 1 | - |
| S-CN-SXT-TE-NAL | 1 | - | |
| AMP-C-SXT-TE | 3 | - | |
| AMP-C-CN-TE | 2 | - | |
| AMP-C-TE-NAL | 2 | - | |
| AMP-CN-TE-NAL | 3 | - | |
| AMP-CN-SXT-TE | 2 | - | |
| AMP-S-SXT-CIP | 1 | - | |
| C-SXT-TE-NAL | 1 | - | |
| 3 | AMP-S-CN-TE | 4 | - |
| AMP-AMC-SXT-TE | 1 | - | |
| AMP-C-TE | 4 | - | |
| AMP-CN-TE | 7 | - | |
| AMP-TE-NAL | 1 | - | |
| AMP-S-SXT | 2 | - | |
| C-SXT-TE | 1 | - | |
| 2 | S-CN-TE | 1 | - |
| AMP-TE | 4 | - | |
| AMP-C | 1 | - | |
| AMP-SXT | 1 | - | |
| AMP-CIP | 1 | - | |
| 1 | S-CN | 1 | - |
| AMP | 3 | - | |
| S | 1 | - | |
| 0 | Susceptible | 3 | - |
ACSSuT, ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline; AMP, ampicillin; AMC, amoxicillin/clavulanic acid; C, chloramphenicol; S, streptomycin; CN, gentamicin; SXT, sulfamethoxazole/trimethoprim; TE, tetracycline; NAL, nalidixic acid; CIP, ciprofloxacin.
About 33% (33/100) of the S. Brancaster isolates exhibited the phenotypic ACSSuT-resistance pattern. Among them, seven resistance genes to ampicillin (blaTEM ), chloramphenicol (floR), streptomycin (strA), gentamicin (ant(3″)-Ia), sulphonamides (sul-1 and sul-2), and tetracycline (tetA) were detected at the rates of 93.9%, 69.7%, 6.1%, 57.6%, 3.0%, 12.1%, and 72.7%, respectively (Table 5).
Table 5. Antimicrobial resistance genes identified from Salmonella Brancaster isolates with ACSSuT-resistance pattern.
| ACSSuT-resistance type isolate number | PCR result for | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Ampicillin | Chloramphenicol | Streptomycin | Gentamicin | Sulfonamides | Tetracycline | |||||||
| blaPSE-1 | blaTEM | floR | aadA2 | strA | ant(3”)-Ia | sul-1 | sul-2 | sulA | tetA | tetB | tetG | |
| 2 | − | + | + | − | − | + | − | − | − | + | − | − |
| 6 | − | − | − | − | − | − | − | − | − | − | − | − |
| 8 | − | + | + | − | − | + | − | − | − | + | − | − |
| 10 | − | + | + | − | − | − | − | − | − | + | − | − |
| 18 | − | + | + | − | − | + | − | − | − | + | − | − |
| 23 | − | + | + | − | − | − | − | − | − | + | − | − |
| 24 | − | + | + | − | − | − | − | − | − | + | − | − |
| 34 | − | + | + | − | − | + | − | − | − | + | − | − |
| 37 | − | + | + | − | − | + | − | − | − | + | − | − |
| 38 | − | − | − | − | − | − | − | − | − | − | − | − |
| 39 | − | + | − | − | − | − | − | − | − | − | − | − |
| 42 | − | + | + | − | − | − | − | − | − | + | − | − |
| 52 | − | + | − | − | − | + | − | − | − | − | − | − |
| 54 | − | + | + | − | − | + | − | − | − | + | − | − |
| 55 | − | + | + | − | − | − | − | − | − | + | − | − |
| 56 | − | + | + | − | − | + | − | − | − | + | − | − |
| 59 | − | + | − | − | − | − | − | + | − | − | − | − |
| 61 | − | + | − | − | − | − | − | − | − | − | − | − |
| 62 | − | + | + | − | − | − | − | − | − | + | − | − |
| 63 | − | + | − | − | − | − | − | − | − | − | − | − |
| 65 | − | + | + | − | + | + | − | + | − | + | − | − |
| 69 | − | + | + | − | − | + | − | − | − | + | − | − |
| 70 | − | + | + | − | − | + | − | − | − | + | − | − |
| 72 | − | + | + | − | + | + | − | + | − | + | − | − |
| 74 | − | + | + | − | − | + | − | − | − | + | − | − |
| 79 | − | + | + | − | − | − | − | − | − | + | − | − |
| 80 | − | + | − | − | − | + | + | − | − | + | − | − |
| 82 | − | + | + | − | − | + | − | − | − | + | − | − |
| 91 | − | + | − | − | − | − | − | + | − | − | − | − |
| 94 | − | + | + | − | − | + | − | − | − | + | − | − |
| 96 | − | + | − | − | − | + | − | − | − | − | − | − |
| 97 | − | + | + | − | − | + | − | − | − | + | − | − |
| 98 | − | + | + | − | − | + | − | − | − | + | − | − |
| Rate (%) | 0/33 | 31/33 (93.9%) | 23/33 (69.7%) | 0/33 | 2/33 (6.1%) | 19/33 (57.6%) | 1/33 (3.0%) | 4/33 (12.1%) | 0/33 | 24/33 (72.7%) | 0/33 | 0/33 |
ACSSuT, ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline; PCR, polymerase chain reaction; blaPSE-1 , β-lactamase gene; blaTEM , β-lactamase temoneira; floR, florfenicol/chloramphenicol resistance gene; aadA2, aminoglycoside adenyltransferase gene; strA, streptomycin resistance gene; ant(3”)-Ia, aminoglycoside nucleotidyltransferase gene; sul-1, sul-2, sulA, sulfonamides resistance gene; tetA, tetB, tetG, tetracycline resistance gene.
DISCUSSION
S. Brancaster is an emerging serovar that has been isolated frequently from chicken processing environments, wet markets, and chicken meat in recent years but has been reported infrequently in humans [6]. For several decades, Salmonella Pullorum and S. Enteritidis are typically the most common serovars with economic impact responsible for Salmonella infection in poultry and humans. However, this predominance has decreased in many regions of the world, where other examples of NTS, such as Salmonella Infantis, Salmonella Derby, and Salmonella Anatum, have become the dominant serovars, reflecting the current situation in many countries [8,23]. The S. Brancaster in this study were all isolated from healthy chicken flocks with no reported clinical disease or mortality. The persistence of S. Brancaster in poultry farms has been related to the viability adaptive mechanism of biofilm formation on contact surfaces to ensure persistent colonization [4]. Besides, the widespread dissemination of S. Brancaster in poultry farms has been related to the occurrence of genes at the SPI and plasmid associated with fitness, virulence, and antimicrobial resistance. In this study, S. Brancaster exhibits six combinations of virulence genes tested. This serovar carries multiple virulence genes, including invA, sopB, spvB, and cdtB, that have epidemiological association with other clinically significant and invasive serovars to cause human salmonellosis, such as S. Enteritidis and S. Typhimurium [24].
The presence of the cdtB in S. Brancaster is of major concern as the toxin is associated with severe human bloodstream infection, as it has been discovered in human clinical isolates [25]. Previously, the cdtB was only reported in the Salmonella Typhi typhoidal strain. However, studies indicated that the cdtB is becoming more prevalent in NTS serovars of human and chicken origin that are capable of causing invasive Salmonella infection, including serovars Javiana, Montevideo, Schwarzengrund, Indiana, Enteritidis, and Agona [26]. Previous research has shown that identifying virulence genes that play an important role in the invasive mechanism can predict disease-inducing ability and improve understanding of the potential risks of human infection [22].
Several studies have assessed the distribution of specific virulence-associated genes that can be proposed to distinguish enteric and invasive strains, where the invA, sipB, and spvB have been regarded as key markers for virulotyping studies [24]. One S. Brancaster in the current study tested positive for the spvB linked to a medically important NTS that causes enteric infection and non-typhoid bacteremia [27]. There have been varying reports on virulence gene profiling from different geographical locations. A virulence genotyping study of Salmonella in South Korea reported seven virulence profiles across eight serovars, including Albany, Montevideo, Virchow, Typhimurium, and Senftenberg [22]. Meanwhile, Kuang et al. [28] reported nine virulence profiles of Salmonella Newport in China. All the virulence factors are found with varying frequencies across serovars, making it difficult to set standard essential virulence factors in virulotyping profiling studies. These virulence genes or plasmids, which are discovered to be conjugative and contain genetic information, managed to spread genes throughout bacterial populations, responsible for S. Brancaster’s capacity to induce salmonellosis in humans. However, it is noteworthy that the presence of these virulence genes is not a definitive determinant of their virulence, as the degree of expression also contributed to the pathogen’s invasive properties [29].
The present study indicated that MDR S. Brancaster is widely distributed in chickens at two important points along the food production chain: the farm and poultry meat products. One-third (33%) of them were found to exhibit phenotypic ACSSuT-resistant type similar to the MDR S. Typhimurium DT104 strain. Besides, they also exhibit resistance to other classes of antimicrobials, including quinolones and fluoroquinolones. The resistance to ciprofloxacin, a critically important antimicrobial in treating Salmonella infection or typhoid fever in humans, was at 9%, i.e., much higher than those reported by others ranging from 0%–3.8% [16]. Invasive infections caused by ciprofloxacin-resistant Salmonella have been reported to cause pneumonia, gastroenteritis, fever, septic arthritis, and meningitis in children or immunocompromised adults [8]. MDR Salmonella has been reported to be more virulent and cause a direct threat to human health when treatment is hampered by resistance [30]. Bacteria can share their genetic material and spread resistance through horizontal gene transfer across commensal bacteria and pathogens. If the resistance genes were encoded by a transferable plasmid, they could spread horizontally to other human pathogens, causing an indirect threat, and this phenomenon has been noticed in Salmonella [31]. In this study, the phenotypic and genotypic MDR profiles of the S. Brancaster with ACSSuT-resistant pattern studied only partially matched, and they diverged in some antimicrobial classes more strongly than in other classes. They were mainly positive for the blaTEM (93.9%), floR (69.7%), ant(3″)-Ia (57.6%), and tetA (72.7%) resistance genes. These findings agree with previous studies that reported these resistance genes in all Salmonella samples isolated from poultry and processing facilities, including serovars Brancaster, Albany, Corvallis, and Enteritidis [4,16].
In comparison, phenotypic resistance to streptomycin and sulfonamides corresponding to resistance genes strA (6.1%), sul-1 (3.0%), and sul-2 (12.1%) were found at a much lower frequency. A study reported that the blaPSE-1 and sul-1 resistance genes were primarily found in S. Typhimurium [19]. Phenotypic resistance is the interplay between genotype and environmental conditions. The inconsistency between MDR phenotypes and antimicrobial resistant (AMR) genes may be related to the resistance genes located at mobilizable or conjugative plasmids that may disappear when there is a change in ecological settings, such as time of storage and subculturing that changes the plasmid’s fitness [32,33]. Whole genome sequencing (WGS)-based AMR prediction is currently an alternative to conventional molecular PCR and phenotypic AMR characterization studies. Campioni et al. [34] who employed WGS analysis to predict AMR in S. Enteritidis, also only found partial matches between phenotypic and genotypic AMR among the isolates, with tetracycline being the strongest correlation found. There are limitations to these WGS prediction approaches, including challenges for plasmid detection in short read length, high error rate, and detection relies much on the database. Therefore, the AMR genomic database requires ongoing data input, validation, maintenance, and curation to strengthen the pipeline’s robustness to become a powerful tool for AMR prediction.
The emergence of AMR Salmonella in poultry may be a result of the widespread use of antibiotics commonly used in veterinary medicine. It is a common practice to administer small doses via drinking water in integrated chicken farming to reduce stress, prevent diseases, and enhance poultry growth and production [35]. According to a recent survey conducted on poultry farms in Malaysia, the most commonly used antibiotics are enrofloxacin and amoxicillin, which belong to the same class of antibiotics as ciprofloxacin and ampicillin [36]. Therefore, the Malaysian government is taking proactive measures to phase out and even banned a few medically important antibiotics as antimicrobial growth promoters in livestock animals’ production, including colistin, erythromycin, tilcomycin, tylosin, neomycin, and fosfomycin [37]. Consequently, alternatives such as essential oils, organic acids, and natural olive oil by-products to promote intestinal health and mucosal integrity can be used to reduce salmonellosis in poultry farms [30].
This study provides initial evidence for the occurrence of MDR S. Brancaster in Malaysian chicken farms. Furthermore, the isolates were found to co-harbor various combinations of virulence and resistance genes, indicating a risk of salmonellosis infection among consumers through chicken products and personnel involved in the pre and harvesting phases due to contaminated environments. Salmonellosis control management in farms needs to be improved by including the currently dominant serovar as a possible pathogen of public health concern in Malaysia. Likewise, appropriate husbandry practices and the prudent use of therapeutic antimicrobials are key veterinary practices for enhancing farm biosecurity management to control salmonellosis in chicken production. The limitation of this study is its inability to detect a wide array of resistance genes using conventional PCR. Further study into the plasmid-mediated quinolones resistance genes that impede the treatment of Salmonella infections in humans should be encouraged in surveillance studies so that they can be used to guide public health measures and treatments.
ACKNOWLEDGMENTS
The authors would like to acknowledge the Director-General of Department of Veterinary Services (DVS), the Director of the Veterinary Research Division, and DVS for granting permission to conduct and publish this study.
Footnotes
Funding: This research was supported by the Faculty of Veterinary Medicine, Universiti Putra Malaysia (GP/2018/9617100), and the Veterinary Research Institute, Ipoh, Malaysia.
Conflict of Interest: The authors declare no conflicts of interest.
- Conceptualization: Khoo E, Ahmad NI, Roslee R, Zakaria Z.
- Data curation: Khoo E.
- Formal analysis: Khoo E.
- Funding acquisition: Ahmad NI, Roslee R, Zakaria Z.
- Investigation: Khoo E.
- Methodology: Khoo E.
- Project administration: Khoo E, Ahmad NI.
- Resources: Ahmad NI, Roslee R, Zakaria Z.
- Supervision: Ahmad NI, Roslee R, Zakaria Z.
- Validation: Khoo E, Ahmad NI, Roslee R, Zakaria Z.
- Visualization: Ahmad NI, Roslee R, Zakaria Z.
- Writing - original draft: Khoo E.
- Writing - review & editing: Ahmad NI.
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