Molecular identification and antimicrobial resistance of coagulase-negative Staphylococcus in wild boars from BraziABSTRACT:
The role of wild boars as long-term reservoirs of pathogenic agents and antimicrobial resistance has become a global concern in recent years. This study isolated Staphylococcus spp. from different anatomical sites of 26 apparently healthy, free-range wild boars. A total of 260 samples were collected during necropsies and subjected to bacteriological culture followed by phenotypic analysis. Molecular identification of Staphylococcus spp. was performed through amplification and sequencing of the rpoB gene. Antimicrobial susceptibility testing was conducted using the Kirby-Bauer disk diffusion method against 13 antimicrobials, and the mecA gene was screened by PCR. Twenty-five isolates presumptive of Staphylococcus spp. were selected for further analysis. Fifteen isolates were confirmed as coagulase-negative staphylococci (CoNS), identified as S. agnetis (n = 6), S. chromogenes (n = 7), S. hyicus (n = 1), and S. saprophyticus (n = 1). The remaining isolates were identified as Micrococcus spp. This is the first report, to the authors’ knowledge, describing the isolation of S. agnetis from wild boars. Among the CoNS isolates, moderate to high occurrence of resistance to trimethoprim-sulfamethoxazole (13%), clindamycin (20%), and cefoxitin (40%), were observed, including one multidrug-resistant isolate (S. agnetis). Considering these findings, especially regarding cefoxitin and multidrug resistance, there is a need to monitor wild boar populations to minimize the sanitary risks posed to livestock in high-density farming areas, as well as to humans who may be exposed to these animals.
Key words:
coagulase-negative staphylococci (CoNS); multidrug resistance;
Staphylococcus agnetis
;
Staphylococcus hyicus
; wild boar;
Sus scrofa
RESUMO:
O papel de suídeos asselvajados (javalis) como reservatórios de longo prazo de agentes patogênicos e resistência a antimicrobianos tem se tornado uma preocupação global nos últimos anos. Este estudo teve como objetivo isolar Staphylococcus spp. de diferentes locais anatômicos de 26 javalis, aparentemente saudáveis. Um total de 260 amostras foi coletado durante necropsias e submetido a cultura bacteriológica, seguida de análise fenotípica. A identificação molecular de Staphylococcus spp. foi realizada por meio da amplificação e sequenciamento do gene rpoB. O teste de suscetibilidade a antimicrobianos foi realizado pelo método de difusão em disco de Kirby-Bauer contra 13 antimicrobianos, e o gene mecA foi investigado por PCR. Vinte e cinco isolados presumidos de Staphylococcus spp. foram selecionados para análises posteriores. Quinze isolados foram confirmados como estafilococos coagulase-negativa (CoNS), identificados como S. agnetis (n = 6), S. chromogenes (n = 7), S. hyicus (n = 1) e S. saprophyticus (n = 1). Os demais isolados foram identificados como Micrococcus spp. Este é o primeiro relato, até onde os autores têm conhecimento, descrevendo o isolamento de S. agnetis de javalis. Entre os isolados CoNS, observou-se uma ocorrência moderada a alta de resistência a trimetoprima-sulfametoxazol (13%), clindamicina (20%) e cefoxitina (40%), incluindo um isolado multirresistente (S. agnetis). Considerando os achados descritos, especialmente no que se refere à cefoxitina e multirresistência, há uma necessidade de monitorar as populações de javalis, a fim de minimizar os riscos sanitários para os animais de produção em áreas com alta densidade de criação, bem como para os humanos potencialmente expostos a estes animais.
Palavras-chave:
estafilococos coagulase-negativa (CoNS); multirresistência;
Staphylococcus agnetis
;
Staphylococcus hyicus
; javali;
Sus scrofa
INTRODUCTION
The genus Staphylococcus includes members of the microbiota of both animals and humans (QUINN et al., 2005), colonizing various anatomical sites such as the skin, mouth, mammary glands, gastrointestinal tract, genital tract, and upper respiratory tract of mammals (NAGASE et al., 2001). While coagulase-positive staphylococci (CoPS) are typically considered the most pathogenic, recent studies have highlighted an increasing association of coagulase-negative staphylococci (CoNS) with serious infections and multidrug resistance (BECKER et al., 2014). This has led to a reevaluation of their classification as less or non-pathogenic. There are approximately 50 species of CoNS, capable of causing human and animal disease (BECKER et al., 2014; KOSECKA-STROJEK et al., 2019).
Among nosocomial pathogens, increasing antibiotic resistance rates are of greater concern for CoNS compared to Staphylococcus aureus, further limiting therapeutic options (BECKER et al., 2014). A significant virulence factor across staphylococci, irrespective of their coagulase profile, is the expression of an additional penicillin-binding protein (PBP), known as PBP2a, which confers resistance to β-lactam antibiotics, including penicillins, most cephalosporins, and carbapenems (BECKER et al., 2014). PBP2a is encoded by the mecA gene, a key molecular marker for β-lactam resistance, particularly methicillin resistance (KATAYAMA et al., 2000). Although, further research is required, CoNS of both animal and human origins are considered important reservoirs of genetic elements related to resistance to β-lactam antibiotics and other antimicrobial classes (BECKER et al., 2014).
Antimicrobial resistance extends beyond domestic animals, affecting various wild species such as the wild boar. Given its ecological behavior, the wild boar is particularly prone to acquiring and harboring resistance and/or virulence genes (TORRES et al., 2020). These genes are often encoded on mobile genetic elements, enabling horizontal transfer between microorganisms, regardless of their pathogenicity (PARTRIDGE et al., 2018). In Brazil, studies have been conducted to assess the role of wild boars as carriers of microorganisms that may pose risks to human and livestock health, particularly swine (LESSA et al., 2011; DE SOUZA et al., 2021; KMETIUK et al., 2021; DAL SANTO et al., 2022).
Although, Staphylococcus spp. are not considered environmental pathogens, their ability to survive in hostile conditions due to metabolic adaptations, such as thermotolerance and halotolerance under osmotic stress, enables them to persist in diverse environments (ONYANGO & ALRESHIDI, 2018). Recent studies have identified a high incidence of Staphylococcus in wild boars, which are recognized as significant reservoirs of microorganisms (MEEMKEN et al., 2013; MONECKE et al., 2016; MAMA et al., 2019; PÉREZ et al., 2021). In some regions, wild boar meat is part of the human diet, and its consumption has been linked to public health risks (MAMA et al., 2019). The most recent study reporting wild boar’s microbiota demonstrated that wild boars can serve as a reservoir for extended-spectrum beta-lactamase and multidrug-resistant Escherichia coli (HOLTMANN et al., 2021).
This study isolated Staphylococcus spp. from various anatomical sites of free-ranging wild boars and to investigate the presence of methicillin resistance. Molecular identification was performed through 16S rRNA sequencing, revealing significant species including Staphylococcus agnetis, Staphylococcus chromogenes, and Staphylococcus saprophyticus. Additionally, we assessed the antimicrobial susceptibility profiles of the isolates and evaluated multidrug resistance to 13 different antimicrobial agents. This study provided critical data on the systemic dissemination of CoNS in wild boars, with implications for both veterinary and public health.
MATERIALS AND METHODS
Animals and sampling
Twenty-six apparently healthy wild boars (10 females and 16 males) were slaughtered as part of the official wild boar management program regulated by the Secretaria de Agricultura Pecuária e Irrigação (SEAPI) in Rio Grande do Sul (RS), Brazil. A veterinary pathologist examined the animals and performed the sampling in two farms localized in two municipalities in the west of RS: Alegrete (29°47’02”S, 55°47’28”W) (n = 16) and Quaraí (30°23’16”S, 56°27’03”W) (n = 10). The sampling was conducted in collaboration with wild boar population monitoring agents, in accordance with Normative Instruction No. 03, issued on January 31, 2013 (IBAMA, 2013). Sampling was performed following guidelines from a manual on necropsy sample collection in swine, with particular attention to wild boars (RECH et al., 2014). Swabs from the rectum, nasal cavities (both nostrils), and the vagina or foreskin were aseptically collected and stored in Stuart transport medium. Tissue samples from the heart, kidney, liver, lung, lymph nodes (parotid, mediastinal, mesenteric, and inguinal), spleen, and tonsils were also aseptically excised and stored in sterile plastic bags. All samples were refrigerated and analyzed in the laboratory within 72 hours.
Bacteriological culture and phenotypic identification
All samples (n = 234, seven to nine samples from each animal) were plated onto 5% sheep blood agar (Himedia®) and incubated aerobically at 37 °C for 48 hours, with visual evaluation every 12 hours to avoid contamination by Proteus spp. Isolated colonies with morphological profiles suggestive of Staphylococcus spp. were sub-cultured on another 5% blood agar plate for Gram staining and catalase testing. Strains showing Gram-positive cocci morphology and catalase-positive results were stored at -20 °C in nutrient freezing medium for further analysis. To differentiate Staphylococcus spp. from Micrococcus spp., susceptibility to bacitracin (BAC, 0.04 U) was tested using the disk diffusion method, with Staphylococcus spp. being resistant and Micrococcus spp. sensitive. Additional tests were performed to identify species, including the coagulase test, novobiocin (NOV, 5 µg, Laborclin®) and polymyxin B (PXB, 300 units, Laborclin®) susceptibility, urease testing, and sugar assimilation (mannitol, maltose, trehalose, xylose, and sucrose), following the protocols described by QUINN et al. (2005) and MCFADDEN et al. (2020).
Molecular identification of Staphylococcus spp.
DNA was extracted from all isolates presumptively identified as Staphylococcus spp. and Micrococcus spp. using the boiling method. The presence of the rpoB gene (encoding the beta subunit of RNA polymerase) in Staphylococcus spp. was confirmed using primers rpob1418 (5’-CAATTCATGGACCAAGC-3’) and rpob3554 (5’-CCGTCCAAGTCATGAAAC-3’), which amplify a region of 899 base pairs (bp) as described by MELLMANN et al. (2006). DNA sequencing of the PCR products was performed by ACT Gene Análises Moleculares LTDA (Centro de Biotecnologia, Brazil) using an ABI-PRISM 3100 Genetic Analyzer (Applied Biosystems). Sequences were analyzed for consensus using the Staden Package Gap 4 program (STADEN et al., 2000) and aligned with reference sequences from the National Center for Biotechnology Information (NCBI) database using DNASIS software (v.2.5; Hitachi Software Engineering Co.).
Antimicrobial susceptibility test and screening of mecA gene
Following the Clinical and Laboratory Standards Institute (CLSI, 2024) guidelines, the Kirby-Bauer disk-diffusion method was applied to assess the susceptibility profile of 25 isolates against the following antimicrobials (n = 13) / classes (n = 8): β- Lactams [penicillin G (10 UI), and cefoxitin (30 µg)], Aminoglycoside [gentamicin (10 µg)], Chloramphenicols [chloramphenicol (30 µg), Fluoroquinolones [ciprofloxacin (5 µg), levofloxacin (5 µg) and norfloxacin (10 µg)], Macrolides [azithromycin (15 µg) and erythromycin (15 µg)], Lincosamides [clindamycin (2 mcg)], Sulfonamides [trimethoprim (1.25 µg)-sulfamethoxazole (23.75µg)] and Tetracyclines [doxycycline (30 µg), tetracycline (30 µg)]. The standard strain E. coli ATCC 25922 was used as quality control for the tests. Multidrug resistance was determined as resistance to three or more distinct antimicrobial classes (MAGIORAKOS et al., 2012). The resulting zone diameter breakpoints were translated into clinical categories of susceptible, intermediate, and resistant.
To detect methicillin resistance in staphylococci, typically mediated by the mecA, the Clinical and Laboratory Standards Institute (CLSI, 2024) recommends the use of cefoxitin (30 µg) disk-diffusion as the preferred screening method. Cefoxitin is a potent inducer of the mecA operon and provides a more reliable phenotypic expression of resistance, including detection of heteroresistant subpopulations. To confirm methicillin resistance at the molecular level, the mecA gene was analyzed by PCR, as described by MURAKAMI et al. (1991), with slight modifications. Genomic DNA was extracted from bacterial colonies using the boiling method. After centrifugation, 1 µL of the supernatant was used as the DNA template. PCR was performed using the primer pair mecA-F (5′-AAAATCGATGGTAAAGGTTGGC-3′) and mecA-R (5′-AGTTCTGCAGTACCGGATTTGC-3′), which amplifies a 533-bp fragment of the mecA gene. Amplifications were carried out in 25 µL reaction volumes containing 0.25 µM of each primer under the following thermal cycling conditions: initial denaturation at 94 °C, followed by 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 5 minutes. PCR products were resolved by electrophoresis on a 1.5% agarose gel.
RESULTS AND DISCUSSION
A total of 260 samples (78 swabs and 182 tissue fragments) were collected, and 25 isolates from 17 of the 26 animals sampled were selected for further analysis based on colonies showing Staphylococcus profile. The presence of contaminants and microorganisms exhibiting a swarming phenomenon, such as Proteus spp., led to cultures that were unsuitable for colony isolation, despite checking the plates every 12 hours. Sixty-six samples (25.4%) showed negative or insignificant (less of four colonies sharing the same profile) bacterial growth after 48 hours of incubation.
Because staphylococci are generally resistant to benzalkonium chloride (BAC) (FALK & GUERING, 1983), we initially used BAC susceptibility as a preliminary screening test to distinguish Staphylococcus spp. from Micrococcus spp. Based on this screening, 16 of the 25 isolates were tentatively classified as Staphylococcus spp. and 9 as Micrococcus spp. However, since this phenotypic test was only provisional, we confirmed each isolate’s identity by PCR. PCR analysis revealed that three isolates initially classified as Micrococcus were actually Staphylococcus: they were confirmed as Staphylococcus agnetis, S. chromogenes, and S. saprophyticus (Table 1). These isolates exhibited an atypical BAC susceptibility profile (they were sensitive to BAC), consistent with previous reports (BAKER et al., 1986). The remaining BAC-resistant isolates were PCR-confirmed as S. agnetis (n = 5), S. chromogenes (n = 6), and S. hyicus (n = 1). We note that distinguishing Micrococcus from Staphylococcus was not a primary objective of this study, but rather part of the overall characterization of the isolates. The biochemical profiles of these isolates (Table 1) revealed a high metabolic variability within species, confirming the need for molecular-based methods for the identification of CoNS species. The sequencing results and the GenBank accession numbers for the isolates are shown in table 1.
This investigation, which analyzed samples from multiple anatomical sites, provided firsthand evidence of the presence of CoNS in organs such as the heart, tonsils, and lymph nodes, where the animals carry these agents asymptomatically. These findings offer new insights into the systemic dissemination of CoNS, since many recent studies focused primarily on nasal swabs or serological tests. In Spain, MAMA et al. (2019) recovered a diverse array of CoNS from nasal swabs of 371 wild boars, with S. sciuri, S. xylosus, and S. chromogenes being the most frequently identified species. PÉREZ et al. (2021) also found that all CoNS isolated from nasal swabs of 90 wild boars belonged to the S. sciuri group. Therefore, the absence of CoPS in our assessment aligns with the low prevalence of CoPS observed in wild boars (MAMA et al., 2019; PÉREZ et al., 2021).
To the best of our knowledge, the occurrence of S. agnetis has never been demonstrated in domestic pigs, and this is the first report of its isolation in wild boars (6 of 26 animals; 23.1%). In addition to its presence in the nasal cavity, S. agnetis was also detected in the tonsils and lymph nodes. S. agnetis was first described in 2012 as a pathogen associated with subclinical and mild clinical mastitis in cattle (TAPONEN et al., 2012). This emerging pathogen has also been linked to significant economic losses in the poultry industry, where it causes bacterial chondronecrosis with osteomyelitis, endocarditis, and septicemia in broilers (SZAFRANIEC et al., 2020). Moreover, distinguishing S. agnetis from S. hyicus and S. chromogenes is challenging without genotypic identification methods, making misidentification a common issue (POULSEN et al., 2017; SZAFRANIEC et al., 2020). Without molecular assays, it is likely that S. agnetis would have been misidentified in this evaluation. In agreement with our findings, S. chromogenes is one of the most isolated CoNS from wild boars (MAMA et al., 2019).
Wild boars are significant reservoirs of antimicrobial-resistant pathogens, playing a crucial role in wildlife as sources and spreaders of resistance. However, because the isolates originate from a wild species, no clinical therapeutic breakpoints have been established for many of the specific antibiotic-pathogen combinations. Thus, susceptibility results for CoNS isolates in the present study were analyzed using the CLSI VET01 (2024) guideline, as it provides the most appropriate breakpoints for veterinary contexts, and in parallel, a comparative analysis was performed with interpretive criteria from CLSI M100 (2024) and BrCAST/EUCAST (2024), given that these human-oriented standards are widely recognized internationally. In this context, table 2 reports the results found to provide a comparative interpretive approach - grounded in the detection of probable resistance mechanisms. This strategy offers a broader and more informative assessment of their antimicrobial resistance profile, underscoring the potential risk of disseminating resistance genes across wildlife, human, and livestock interfaces.
Interest in CoNS has increased due to their acquisition of resistance genes to multiple antibiotics (BOAMAH et al., 2017). Although, most of the CoNS isolates analyzed in this study were susceptible to the tested antimicrobials, only one isolate (6.7%) was classified as multidrug-resistant S. agnetis from F7 (nasal) was resistant to four antimicrobial classes: β-lactams - ampicillin, cefoxitin, penicillin; macrolides - azithromycin, erythromycin; tetracyclines - doxycycline, tetracycline, and lincosamides - clindamycin. These findings are consistent with recent studies (MAMA et al., 2019). The complete antimicrobial susceptibility profile of the 25 isolates is presented in table 3.
Among the CoNS isolates, moderate to high occurrence of resistance to trimethoprim-sulfamethoxazole (13%), clindamycin (20%), and cefoxitin (40%), were observed. In Brazil, LESSA et al. (2011) reported higher resistance rates against ampicillin (17%), clindamycin (19%), and erythromycin (44%) in Staphylococcus spp. isolated from wild boars. PÉREZ et al. (2021) found that 7 methicillin-resistant CoNS (MRS) isolates from 90 wild boars were resistant to gentamicin (n = 5), penicillin (n = 5), oxacillin (n = 5), and clindamycin (n = 7). Additionally, MAMA et al. (2019) observed resistance to clindamycin (8.7%) and tetracycline (12.4%) among CoNS. The resistance to cefoxitin (40%) found in this study warrants further attention, once cefoxitin is a surrogate marker for methicillin resistance.
Although, the cefoxitin resistance phenotype was observed in 40% of the CoNS isolates, none were classified as methicillin-resistant based on mecA gene screening. PÉREZ et al. (2021) identified a 5.5% prevalence of MRS in wild boars, all of which belonged to the S. sciuri group. In contrast, no MRS were found by MEEMKEN et al. (2013) in wild boars from Germany (n = 117), nor by MONECKE et al. (2016) in wild boars from Germany (n = 24), Austria (n = 46), or Sweden (n = 90). Most recently, PETERSEN et al. (2020) highlights that wild boars from Denmark constitute a low risk of transmitting MRS. This pattern suggested that wild boars may not be significant reservoirs of methicillin resistance. However, further investigation of additional mec genes is recommended due to the genetic diversity of methicillin resistance, which may be a limitation of this study.
Despite the growth of research on pathogens in wildlife, more studies are needed, particularly at the livestock-wildlife interface. The present study offers novel data on the detection of S. agnetis in wild boars, highlighting the species’ role as a long-term reservoir of pathogenic agents. Considering the antimicrobial resistance data presented here, wild boars represent a potential health risk to other species, including humans. Therefore, biosecurity measures and monitoring of free-ranging wild boars should be maintained, enhanced, and focused on areas with high cattle and swine densities.
ACKNOWLEDGMENTS
The authors thank the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Brazil, for financial support (process n. 7/2551-000090- 0/ARD-2017) and Instituto Federal Farroupilha for providing a scientific initiation scholarship and financial support, as well as the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing access to scientific literature through its Portal de Periódicos.
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BIOETHICS AND BIOSECURITY COMMITTEE APPROVAL
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CR-2024-0556.R2
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This study was approved by the Ethics Committee on Animal Experimentation of the Instituto Federal Farroupilha (CEUA), Rio Grande do Sul, Brazil, under protocol number 3256260617.
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DATA AVAILABILITY STATEMENT
The raw data supporting the findings of this study are available directly from the corresponding author upon reasonable request.
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DECLARATION OF USE OF ARTIFICIAL INTELLIGENCE
No artificial intelligence tools were used in the preparation, writing, review, or improvement of this manuscript, nor in the processing of images or in the study methodology.
Edited by
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ASSOCIATE EDITOR:
Rudi Weiblen (0000-0002-1737-9817)
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SCIENTIFIC EDITOR:
Juliana Felipetto Cargnelutti (0000-0002-3160-3643)
The raw data supporting the findings of this study are available directly from the corresponding author upon reasonable request.
