Fecal Carriage of S. aureus and the mecA Gene in Resident Wild Birds and Its Zoonotic Potential

Resistant Staphylococcus (S.) aureus in general and MRSA, in particular, have received great attention in both veterinary and human health sectors. The importance of fecal carriage of staphylococci is rarely encountered. This study aimed to investigate the role of wild birds in Giza governorate, Egypt in spreading resistant S. aureus from winter 2019 to summer 2021. Cloacal swabs and fecal droppings were collected from different species of wild birds (rock pigeons, laughing doves, cattle egrets, and hooded crows). Isolation and identification of Staphylococcus spp. were performed using Columbia agar base with 5% defibrinated sheep blood and mannitol salt agar. Moreover, molecular detection of the coa, nuc, and mecA genes has been investigated via the polymerase chain reaction (PCR) assay. Out of 166 fecal samples examined, staphylococci had been confirmed in 100 samples (60.2%), with S. aureus representing 70% of the obtained staphylococci; however, non-aureus staphylococci represented the remaining 30% of the isolates. The mecA gene carriage was (57.1%) in S. aureus. This study highlighted the zoonotic potential of staphylococci isolated from resident wild birds in Giza, Egypt. Presences of such pathogenic microorganisms with their resistance traits around and in the human habitat add to the microbial community present around human dwellings in the study area. They may play a role in the spreading of various illnesses. ـــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
aureus is one of the clinically significant pathogens of humans and animals. This bacteria has been linked to various illnesses of various severity and has been found to be life-threatening, with fatality rates higher than those of AIDS, viral hepatitis, and tuberculosis combined (Van Hal et al., 2012). The ability of Livestock-associated S. aureus to infect human populations and the community-associated S. aureus adds a considerable burden on the healthcare system (Dweba et al., 2019).
Although antimicrobial resistance is usually more frequent in poultry, it has also been detected in bacteria isolated from wild birds (Benskin et al., 2009). Free-living birds that are often encountered in the human environment naturally harbor harmful antibiotic-resistant microorganisms (Literak et al., 2010; Wang et al., 2017). This is especially noticed in crows due to their growing numbers and continuous mobility that aids in the spreading of harmful pathogens (Literak et al., 2010).
Nowadays, antimicrobial resistance has been identified by the world health organization (WHO) as one of the most serious threats to public health and food security (WHO, 2020). Moreover, methicillinresistant S. aureus (MRSA) strains are a significant global health issue (Gajdács and Zsoldiné Urbán, 2019). The mecA gene that encodes PBP2a (penicillinbinding protein 2a) is responsible for the majority of MRSA infections, and its detection helps in the identification of methicillin resistance in S. aureus (Shrestha et al., 2002). According to CLSI 2020 guidelines, S. aureus strains that test positive for the mecA should be reported as MRSA (CLSI, 2020).
Furthermore, methicillin-resistant coagulasenegative staphylococci (MRCNS) have long been identified to cause human and animal diseases (Chen et al., 2016). Meanwhile, special attention has been paid to other coagulase-positive staphylococci and nonaureus staphylococci representing a significant threat. Although they are usually found as commensals in humans and animals, however exchange of these bacteria between animals and humans can sometimes cause severe or even lethal infections (González-Martín et al., 2020).
Despite being recognized as major reservoirs or carriers for transmission during the last decade, there has been a rising interest in MRSA incidence in wildlife. Still, little data are available (Silveira et al., 2021). Understanding MRSA's general epidemiology at the national level is essential for healthcare professionals and policymakers to support successful preventive and control initiatives. Pigeons, doves, and hooded crows are among wild birds extensively dispersed in Egypt. Because of their colonial nesting behavior around human dwellings, the vast majority of their excrement concentrate in these areas, contributing to the microbial load that may contain potentially harmful pathogens to human health.
Despite extensive research on MRSA in humans and animals, there is still a scarcity of data about the level of infection, carriage, and the zoonotic importance of this bacterium from wildlife. Here we report the carriage of the mecA gene in S. aureus isolated from droppings and cloacal samples of resident wild birds in Giza, Egypt, and highlight its zoonotic importance to public health.

Ethical statement:
Protocols for sample collection and laboratory examination for this study were reviewed and approved by Faculty of Veterinary Medicine, Cairo University's Institutional Animal Care and Use Committee (No. VetCU10102019087).

Sampling:
A total of 166 fecal samples were collected from resident wild birds (rock pigeons, laughing doves, cattle egrets, and hooded crows) from different regions in Giza governorate, Egypt, during the period from winter 2019 to summer 2021. Traps were used to capture the birds and either cloacal swabs or the top surface of fresh droppings were obtained before the release of the birds from the net. Occasionally, a professional hunter was hired to shoot some crows and doves when the birds identified the traps at the collection sites and avoided them. Samples were transported in an ice box as soon as possible and a microbiological examination was performed within 24 h in Zoonoses Department Research Laboratory, Faculty of Veterinary Medicine, Cairo University.

Isolation and identification of S. aureus:
Isolation and identification of S. aureus were carried out as described earlier (Quinn et al., 2011). Fecal specimens were incubated aerobically into 9 ml of brain heart infusion broth (Oxoid, Hampshire, UK) at 37˚C for 12-24 h. For each sample, two loops from the incubated broth were streaked on Columbia agar base supplemented with 5% defibrinated sheep blood (Oxoid, Hampshire, UK) and mannitol salt agar (Oxoid, Hampshire, UK). Both plates were incubated at 37˚C ± 1˚C and the characteristic growth on each medium was recorded after 24-48 h.
Isolates were presumed to be staphylococci based on colony morphology, catalase response, Gram staining, and oxidative-fermentative tests. Following the identification of the genus Staphylococcus, the enzyme coagulase was identified in all isolates using slide and tube methods (Quinn et al., 2011). Coagulase-negative isolates that showed resistance to methicillin (based on detection of the mecA gene, see below) were submitted to species identification using the API-Staph Kit (BioMerieux, France) as described before (Petzer et al., 2013). A single pure colony from each identified strain was kept on brain heart infusion broth for additional testing and PCR analysis.

Molecular confirmation of S. aureus isolates and detection of the coa and mecA genes:
All staphylococci isolates were refreshed on mannitol salt agar plates at 37°C overnight before DNA extraction. A single bacterial colony was picked from each plate and placed in 200 μl deionized distilled water. The QIAamp Mini DNA Extraction Kit (Qiagen, Hilden, Germany) was used to extract genomic DNA according to the manufacturer's instructions. Primer sequences of the coa gene specific for coagulase production, the nuc gene specific for S. aureus, and the mecA gene specific for methicillin resistance in staphylococci were used in conventional PCR protocols previously described (Table 1).
The reactions were carried out in 25 μl reaction mixtures containing 5 µl of DNA as a template, 1 µl (20 pmol) of each primer, 12.5 μl of 1× PCR master mix (Dream Taq Green PCR Master Mix, Fermentas Life Science) and 5.5 μl molecular grade water. Expected amplification bands were photographed using Gel documentation system (Alpha Innotech) after PCR amplification products had been electrophoresed through 1.5% agarose gel (Sigma, USA) with ethidium bromide (0.5 μg ml-1) (Sigma, USA) in 1x TBE buffer.

RESULTS
Out of 166 fecal samples from wild birds, based on phenotypic (culture characteristics, slide and tube coagulase testing), API kit testing and genotypic identification of the coa gene, staphylococci were detected in 100 fecal samples (60.2%). S. aureus predominated among the recovered Staphylococcus spp. (70 isolates, 70%), while other non-aureus staphylococci were detected at a lower level (30 isolates, 30%) (  Staphylococci have been identified as an appropriate model for "One Health" investigations, as some species and clones have been demonstrated to "jump" through the three ecosystems of interest (human, animals, and the environment) (Abdullahi et al., 2021). In the present study, staphylococci were detected in 60.2% (100/166) of the wild birds' fecal samples, among which S. aureus (70%, 70/100) were phenotypically and genotypically identified. Fecal carriage of staphylococci is not frequently encountered among birds generally and wild birds in particular. However, in a recent study, staphylococci were detected (45.9%) in cloacal samples from wild birds from street markets in Rio de Janeiro, Brazil, with S. aureus representing 11% of the total staphylococci (Matias et al., 2018).
Also, S. aureus has been identified in faeces of corvids, marine and migratory birds in an earlier study (Hubálek, 2004 et al., 2019). Despite the usual presence of some of these agents as commensals, detecting such pathogenic bacterial species from wild birds around human dwellings highlights their significance in spreading illnesses to inhabitants, especially if these bacteria carry virulence or antimicrobial resistance determinants.
Coagulase-negative staphylococci (CoNS); S. haemolyticus, S. chromogenes, S. simulans, S. hyicus, S. hominis, S. saccharolyticus, S. carnosus, and S. lugdunensis were isolated from human and domestic mammalian hosts as reported earlier (Becker et al.,  2014). Although CoNS may be recovered at a lower level, special attention should be paid to these species due to their opportunistic behaviour. In this regard, S. haemolyticus has been linked to septicemia, human endocarditis, and urinary tract infection (Kloos and Bannerman, 1994). Similar to results in the present study, but at a lower level, S. chromogenes, S. haemolyticus, and S. simulans were isolated from cloacal swabs of wild birds in Rio de Janeiro, Brazil (Matias et al., 2018).
The spread of antimicrobial resistance through staphylococci is a significant problem both in veterinary and human medicine, posing a global challenge since some pathogenic species have developed resistance to most antibiotics that limit the therapeutic choices. The appearance of MRSA has become a global public health issue, with these resistant strains identified from wild animal species and birds Most methicillin resistance in S. aureus is controlled by the mecA gene that encodes PBP2a (penicillin-binding protein 2a), which is reported to be the primary cause of the penicillin and methicillin resistance (Pournaras et al., 2015). Due to prolonged COVID-19 lockdown, we can't revive staphylococci isolates for antimicrobial sensitivity testing which was an unintentional limitation to this study. However, alternatively, the presence of the mecA gene responsible for methicillin resistance in S. aureus was assessed.
In the present study, the mecA gene was detected in 57.1% (40/70) S. aureus isolates. Similarly, the mecA gene (22%) had been detected in S. aureus and some coagulase-negative staphylococci recovered from wild bird cloacal samples in Rio de Janeiro, Brazil (Matias et al., 2018). On the other hand, along the shores of Gulf of California, although coagulasepositive staphylococci had been recovered from droppings of migratory seabirds (Heermann's Gulls and Elegant Terns), none of them carried the mecA resistance gene (Contreras-Rodríguez et al., 2019). This may be related to the nature of the surrounding environment and the microbial load where birds from these studies are distributed.
Overall, the dissemination of such resistant bacteria is merely through anthropogenic sources such as industrial and household wastewater effluents, runoff from agriculture, and garbage, between wild animals and the human environments. Once transmitted to wild animals, some bacteria can then be responsible for disseminating various resistance genes, mobile genetic elements, and epidemic clones to several places

CONCLUSION
This study highlighted the zoonotic importance of staphylococci isolated from resident wild birds in Giza, Egypt. The presence of such pathogenic microorganisms with their resistance traits around and in the human habitat has public health implications. It may play a role in spreading various illnesses and resistant bacteria. There is an urgent need to develop control systems to restrict bacterial spread throughout different ecosystems, minimize the emergence of more antimicrobial resistance, and maintain the efficacy of presently available antibiotics. Implementation of regular monitoring policies in different environments is necessary to have a clear image of the role of other wild species in transmitting certain zoonotic agents to humans.