A Novel Methicillin-Resistant Staphylococcus aureus t11469 and a Poultry Endemic Strain t002 (ST5) Are Present in Chicken in Ebonyi State, Nigeria

Background The changing epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) from a hospital-associated pathogen to an organism commonly found in the community and in livestock reflects an organism well-equipped to survive in diverse environments and adjust to different environmental conditions including antimicrobial use. Methods We investigated the molecular epidemiology of S. aureus and MRSA in poultry in Ebonyi State, Nigeria. Samples were collected from 1800 birds on 9 different farms within the state. Positive isolates were tested for antibiotic susceptibility and molecular typing. Results Prevalence in birds was 13.7% (247/1800). MRSA prevalence in poultry was 0.8%. The prevalence of MRSA in broilers and layers was 1.2% and 0.4%, respectively. All tested isolates were susceptible to vancomycin. Molecular analysis of the isolates revealed 3 spa types: t002, t084, and a novel spa type, t11469. The novel spa type t11469 belonged to sequence type ST5. Conclusion The detection of t002 in chicken suggests the presence of livestock-associated MRSA in poultry in Ebonyi State. The detection of the new spa type t11469 in poultry that has not been characterised to ascertain its pathogenic potential remains a cause for concern, especially as some were found to carry PVL genes, a putative virulence factor in staphylococcal infection.

S. aureus has been detected in several species of animals and products from animals [8][9][10][11][12], including poultry. The cloacae and nostrils of poultry remain important sites for the recovery of S. aureus and MRSA [13]. Intensity of contact with live animals is documented a risk factor for MRSA colonization [14]. While colonization alone does not harm the host, livestock-associated MRSA has also been reported to cause 2 BioMed Research International severe infections in humans, including endocarditis [15,16]. However, while studies have been carried out investigating livestock-associated S. aureus in Europe, North America, and Asia, relatively few studies have been carried out in Africa. This study investigated the presence of S. aureus, including potential livestock-associated types, in poultry in Ebonyi State, Nigeria.

Sample Collection and Culture.
Cloacae and nasal samples from 1800 birds (900 broilers and 900 layers) were collected using sterile swabs. Birds were selected at random from 9 poultry farms in the 3 senatorial districts in Ebonyi State, Nigeria (3 farms from each senatorial district). Swabs were processed within 24 hours by inoculation into 6.5% NaCl Staph enrichment broth. Isolation and identification of S. aureus were achieved through the use of mannitol salt plates, CNA plates with 5% sheep blood, and ChromAgar plates (CHROMagar, France). Isolates were confirmed as S. aureus using catalase and coagulase tests and further confirmed by Staph Latex Agglutination test (Pastorex Staphplus, Bio Rad). Animal owners consented to testing of farmowned poultry.

Molecular
Testing. S. aureus isolates were tested for the Panton-Valentine leukocidin (PVL) genes, methicillin resistance gene (mecA), and staphylococcal protein A (spa) gene as previously described [17]. Multilocus sequence typing (MLST) was carried out as described by Enright et al. [18] and the detection of the tetracycline resistance gene tetM as described by Weigel et al. [19], and SCCmec typing was performed according to [20].

Antibiotic Susceptibility
Testing. The resistance of isolates to a panel of 11 antibiotics was determined by the Kirby-Bauer-Clinical and Laboratory Standards Institute (CLSI) modified disc agar diffusion (DAD) method [21]. Isolates were tested for resistance to the following antibiotics: augmentin, cefoxitin, cefuroxime, chloramphenicol, doxycycline, erythromycin, gentamycin, levofloxacin, tetracycline, trimethoprim/sulfamethoxazole, and vancomycin.

Statistical Analysis.
The data obtained were analyzed by ANOVA using the statistical package for social sciences (SPSS) version 20.2, Chicago, Illinois, USA. A value of 0.05 or less was considered statistically significant.

Prevalence of S. aureus.
A total of 1800 birds consisting of 900 broilers and 900 layers from 9 farms in Ebonyi State were swabbed. S. aureus was recovered from the cloacae and nostrils of both broilers and layers. The number of broilers and layers positive for S. aureus was 122 (13.5%) and 125 (13.8%), respectively. The overall prevalence of S. aureus was 247/1800 (13.7%); see Table 1. Of the birds where S. aureus was recovered, 52.2% (129/247) of the colonized birds were positive in both the cloacae and nostrils, while 27.1% (67/247)  were positive only in the cloacae, and 20.6% (51/247) of the colonized birds were positive only in the nostrils. The percentage prevalence of broilers and layers varied between farms but were similar overall. Total prevalence ranged from 11.0% to 16.5%, in broilers, from 10.0% to 19.0%, and in layers, from 8.0% to 19.0% (Table 1).
Statistical analysis using ANOVA of birds with S. aureus in both the cloacae and nostrils, cloacae alone, and nostrils alone showed that there was a significant difference between the groups ( ≤ 0.05). Examining the prevalence of S. aureus recovered from broilers and layers showed a value of 0.85, which was not significant.

Discussion
S. aureus and methicillin-resistant S. aureus were recovered from both broilers and layers in this study. The percentage of animals with S. aureus recovered from both cloacae and nostrils were higher than from cloacae and nostrils alone. This suggests that cloacae and nostrils are important sites for the recovery of S. aureus, a combination which gives better yield. This was consistent with earlier work in Belgium [13], which reported higher recovery of S. aureus from multiple sites than the sites screened singly.
All the isolates exhibited varying degrees of resistance to the panel of 11 antibiotics tested. Almost half (45.7%) of tested samples were resistant to tetracycline. Tetracycline is the most commonly used food supplement and growth promotion factor in poultry farms in this area. Others include trimethoprim/sulfamethoxazole, chloramphenicol, erythromycin, and quinolones. These drugs are also used for disease treatment in these farms. Regulation of antibiotic use in poultry in Ebonyi state and Nigeria more broadly is lacking, which could contribute to the high levels of resistance observed in this environment.
Of 30 isolates examined, only 1 (3.3%) was positive for the tetM gene, one of the genes encoding for tetracycline resistance. The low percentage of tetM recorded might suggest the involvement of other tetracycline resistance genes in tetracycline resistance observed in this study. Fluit et al. [22] have reported that tetracycline resistance is determined by several tetracycline (tet) genes, and the involvement of other tetracycline resistance genes in poultry has been reported elsewhere [23].
The percentage recovery of MRSA from broilers was 1.2% and .4% in layers. Reasons for the differences in the recovery of MRSA from broilers and layers were not clear, as their rearing conditions were similar. Though there was no significant difference between number of S. aureus recovered from broilers and layers, there was a significant difference in MRSA recovered from both, though this was a comparison of small numbers and caution should be taken not to overinterpret results. spa typing showed that 3 spa types of S. aureus, including a livestock-indicator spa type t002, were circulating among chicken in Ebonyi State. Lowder et al. [12] and Köck et al. [16] previously reported this spa type in poultry. A novel spa type t11469 was also detected, which to the best of our knowledge has never been characterised in Ebonyi or elsewhere. Our data showed that 6 isolates were positive for PVL gene, out of which 5 were MSSA while one was MRSA. The finding of PVL-carrying isolates in chicken may portend a serious health hazard to the poultry farm workers and their families due to possibility of farm-to-family transmission and the association of PVL with heightened virulence, though this remains controversial [24]. Furthermore, the isolation of lukS-lukF carrying isolates of S. aureus in this study might constitute a health risk to the general public as chicken are reared in homes and live chicken are sold on the streets and in rural markets where little attention is paid to hygiene. Prior reports have shown that PVL-positive MSSA is common in West Africa [25].

BioMed Research International
The discovery of spa type t002 in ST15 and ST121 was intriguing, as available reports have not demonstrated t002 in these sequence types. Its presence in these sequence types could either suggest adaptation to different ecological condition, as previously reported [27], or local dispersion of spa types in different regions and environment [28], for example, by horizontal transmission of the spa gene into genetic backgrounds of ST15 and ST121 strains. Furthermore, the recovery t084 in poultry suggests an organism highly equipped to cross interspecies borders. It could also be suggestive of the movement of MRSA from its niche in human clinics to livestock as has been variously reported [29]. All MRSA isolates tested carried SCCmec type V, which has been found both in the community [30] and in livestock, including ST398 in pigs [8,15].
There were important limitations to this study. Though birds within the farms were randomly selected, a convenience sample of farms participated, potentially biasing the study. However, it is unlikely that farmers had any knowledge of the S. aureus status of their farms prior to enrolment. Samples from individuals or the environment were not collected, and as budgetary issues allowed only a subset of positive samples to be molecularly typed (30 of 247 positive isolates), we may have missed some of the diversity in our broader sample. Further research studies should examine birds within live markets as well as on farms in order to assess risk to consumers in addition to farm workers.

Conclusion
The detection of a new spa type t11469 carrying the PVL gene could portend a health hazard if it is commonly transmitted to humans. The isolation of this new spa type in poultry especially as it has not been characterized to establish its pathogenic potential or its inherent virulence factors remains a public health threat. Additional sequencing and characterization of t11469 are necessary to establish the pathogenic potential of this novel spa type. Poultry farm workers in this environment should ensure the highest level of hygiene, including constant hand washing to prevent interspecies transfer of this new strain, or other strains of S. aureus that may be present on Nigerian poultry farms.

Conflicts of Interest
The authors declare no conflicts of interest.