spa typing and antimicrobial resistance of Staphylococcus aureus from healthy humans , pigs and dogs in Tanzania

Introduction: Staphylococcus aureus is an opportunistic pathogen causing infections in humans and animals. Here we report for the first time the prevalence of nasal carriage, spa typing and antimicrobial resistance of S. aureus in a Tanzanian livestock community. Methodology: Nasal swabs were taken from 100 humans, 100 pigs and 100 dogs in Morogoro Municipal. Each swab was enriched in Mueller Hinton broth with 6.5% NaCl and subcultured on chromogenic agar for S. aureus detection. Presumptive S. aureus colonies were confirmed to the species level by nuc PCR and analysed by spa typing. Antimicrobial susceptibility patterns were determined by disc diffusion method. Results: S. aureus was isolated from 22 % of humans, 4 % of pigs and 11 % of dogs. A total of 21 spa types were identified: 13, 7 and 1 in human, dogs, and pigs, respectively. Three spa types (t314, t223 and t084) were shared between humans and dogs. A novel spa type (t10779) was identified in an isolate recovered from a colonized human. Antimicrobials tested revealed resistance to ampicillin in all isolates, moderate resistances to other antimicrobials with tetracycline resistance being the most frequent. Conclusion: S. aureus carrier frequencies in dogs and humans were within the expected range and low in pigs. The S. aureus spa types circulating in the community were generally not shared by different hosts and majority of types belonged to known clones. Besides ampicillin resistance, moderate levels of antimicrobial resistance were observed irrespective of the host species from which the strains were isolated.


Introduction
Staphylococcus aureus is an opportunistic pathogen often carried asymptomatically by humans and animals.Approximately 25%-30 % of healthy humans carry S. aureus in their nasal cavity, and nasal carriage is a recognized risk factor for S. aureus infection [1,2].Other hosts of S. aureus include pigs, where a similar prevalence of carriage as in humans has been reported in Switzerland [3]. S. aureus can also be found in dogs and cats; however, in dogs a related coagulase-positive staphylococcal specie, S. pseudintermedius, is more prevalent than S. aureus [4].According to previous studies [5], dogs could be a possible source of S. aureus infection to humans, and it was demonstrated previously [6] that undistinguishable S. aureus strains have been isolated among human and dogs from the same geographical area.
The sequence-based typing method, S. aureus protein A spa typing, offers excellent intra-and inter-laboratory reproducibility and it is commonly applied for population studies of staphylococci [7].A high number of types are recognized in the international database representing typing of more than 300,000 strains (http://www.spaserver.ridom.de).There are reports on spa types in hospital settings in Tanzania [8], but it is unknown which types circulate in the community.In addition to the general lack of information on the S. aureus lineages that are present in human and animal populations in Tanzania, there is also a lack of knowledge of the patterns of antimicrobial susceptibility to commonly used antimicrobials in the country; furthermore S. aureus is an important source of antimicrobial resistance determinants [9,10].Therefore this study was aimed to fill these knowledge gaps by investigating prevalence of S. aureus nasal carriage in healthy humans, pigs and dogs in Tanzania, by characterizing S. aureus using spa gene typing and antimicrobial resistance among human and veterinary isolates.

Ethical clearance and human subjects' consent
All human participants were informed of the objective and methods of the study and voluntarily signed an informed consent form.The National Institute for Medical Research of Tanzania (NIMR) approved the study (ethical clearance certificate No 936 dated 01/4/2010 (NIMR/HQ/R.8a/Vol.IX/936).The Ethical Committee-of Sokoine University of Agriculture (SUA) approved the animal sampling study.

Sample collection
Nasal swabs were collected from 100 epidemiologically unrelated healthy humans, 100 pigs and 100 dogs from urban and peri-urban Morogoro Municipality, Tanzania, from December 2011 through March 2012.Adult pigs were sampled at different pens and houses within eight rearing places representative of the study area.A single sterile cotton swab was inserted 1-2 cm into both nostrils and rotated along the nasal mucosa for 5 -10 s.Swabs were rubbed well by rotating 5 times over the inner wall of the ala and nasal septum.Swabs were stored in sterile tubes containing Cary Blair transport medium and were transported in a cool box to the laboratory.

Isolation and identification of S. aureus
After enrichment in Mueller Hinton Broth (Oxoid Ltd, Basingstoke, UK) containing 6.5% NaCl for 24 hours at 37°C, 10µl of the enrichment culture were streaked on Oxacillin Resistance Screening Agar Base (ORSAB) (Oxoid Ltd, Basingstoke, UK) without selective supplement.Blue colored colonies were harvested and stored at -80°C in Muller-Hinton Broth (Oxoid Ltd) with 50% glycerol.Subsequently, isolates were streaked on blood agar and presumptive staphylococci were tested for coagulase activity in horse plasma.Coagulase-positive isolates were screened by nuc-PCR to confirm S. aureus [11].mecA [12] and mecC PCR were also carried out [13,14].All PCR primers used in nuc, mecA and mecC were obtained from TAG Copenhagen A/S Kong Georgs Vej 12 DK-2000 Frederiksberg in Denmark.Positive controls included S. aureus strain ATCC 25923 from the National Medical research Institute Tanzania [15] used for nuc-PCR, S. aureus 50A247 for mecA PCR [16] and for mecC PCR; S. aureus strains 68529-2 and 11-2 were obtained from University of Copenhagen laboratory.Vials without DNA were likewise included as negative controls.

spa typing
All S. aureus isolates were spa typed and sequenced and cluster analysis based on sequence types were performed using the Ridom StaphType software (Ridom GmbH, Würzburg, Germany) as previously described [17].The spa server database [18] was consulted to extrapolate the spa types to previously reported STs or cloned complexes (CC).Alternatively, presumed STs and CCs were assigned based on associations with spa types reported in the scientific literature.
Results of antimicrobial susceptibility testing are shown in Table 2.All isolates were resistant to ampicillin.Tetracycline resistance was observed in strains isolated from the three host species.No isolates were resistant to oxacillin.

Discussion
The present study provides novel information about carriage frequency and diversity of S. aureus in healthy humans, dogs and pigs outside hospital and clinical settings in Tanzania.None of the isolates were positive in PCR targeting the mecA and mecC genes, indicating that none of the isolates were MRSA positive.
The observed frequency of S. aureus isolation from humans (22%) was within the range shown in other studies, where about 20% (range 12%-30%) of individuals are persistent S aureus nasal carriers, approximately 30% are intermittent carriers (range 16%-70%), and about 50% (range 16%-69%) are noncarriers [20].Since this study was designed as a crosssectional study, we were not able to distinguish between permanent and intermittent carriers.
The prevalence of S. aureus carriage in dogs (11%) was in agreement with previous studies reporting isolation frequencies between 9%-12 % in this animal reservoir [21,5].Other studies [6] reported S. aureus carriers of 5.9%, 17.6% and 17.6% in pharynx, rectum and nasal cavity, respectively.This is valuable information to understand the epidemiology of S. aureus in this animal specie [4] as the majority of previous studies in dogs focused on MRSA [21,22].Among the STs isolated from dogs, ST1, a lineage associated to humans, was the most frequent.The human epidemic clone ST22, also known as E-MRSA 15, has previously been isolated from dogs in other geographical regions [6,22,23], supporting the notion that this S. aureus lineage may have adapted to the canine host.
S. aureus was isolated from 3 out of 8 pig farms and only 4 pigs were carriers.MRSA occurrence in intensive pig farming in Europe and South East Asia is high [3,24], however little is known about occurrence of non MRSA in pigs, not to mention in the semioutdoor production system used by smallholder pig farmers in Tanzania.This system might offer few opportunities for S. aureus to colonize and/or persist on the pigs and in the farm environment as reported in outdoor studies in France/Spain [25].Epidemiology studies on MRSA in intensive pig farming have shown that once this type of S. aureus is present in the farm, it colonizes a large proportion of the pigs [26].Contrary  to MRSA, the low in-farm prevalence (0%-3%) shown in this study suggests low transmission rates of susceptible S. aureus.A longitudinal study showed the highest transmission rates for MRSA in young pigs [26].Our sample was withdrawn from pigs of approximately 4-6 months of age, and this may have contributed to the low prevalence.
spa typing is routinely used as the golden standard in S. aureus epidemiology studies [7].All the porcine isolates in the current study were of the same spa type, t131 and to the best of our knowledge this is the first report of this type in Tanzania; spa type t131 has been associated to ST80, an important community-acquired MRSA lineage in humans [27,28] that has previously been associated to zoonotic transmission between domestic animals and humans [29].The isolation of only one spa type in pigs indicates that this lineage is prevalent among the farms in the study area.It should be noted that farmers bought piglets from independent suppliers and did not share veterinarians, feed supply or other common utensils.Thus the four porcine S. aureus isolates were apparently not epidemiologically related.
Three spa types were common to humans and dogs: t314 (ST121), t084 (ST15 and ST18) and t223 (ST22).Since none of our samples were paired samples from owners and their dogs, it appears that people and dogs share specific S. aureus lineages; whether they are maintained in the two hosts by bidirectional transmission remains to be demonstrated.Apart from one new spa type (t10779), all other spa types detected in the current study have previously been reported elsewhere [18].spa types mentioned above have been reported in other African countries but with isolation from other parts of the body.For example, studies from Nigeria [30] reported these spa types in wounds (t127, t314, t331, t084 and t002), otitis media (t084 and t311), blood stream (t084) and urinary tract infection (t311).
All isolates were resistant to ampicillin, often resistant to tetracyclines, but rarely resistant to aminoglycosides, rifampicin, sulfonamides and phenicols.Resistances to ampicillin and tetracycline have been reported worldwide [5], including referral hospitals in Tanzania [31,32] and community settings in other African countries such as Ghana [33].Resistance to gentamicin was reported in 41.5% of human clinical isolates derived from a hospital in Tanzania [34] but it was low among human and animal isolates investigated by our study.The elevated frequency of tetracycline resistance found in this study (43.2% of the isolates) may be related to the frequent use of this antimicrobial in both animals [35] and humans [8] in Tanzania.The latter study reported a prevalence of 52.4% tetracycline-resistant isolates from wounds, pus, and nasal swabs well corresponding to the 45.5% of resistance observed in nasal swabs from humans in our study.Resistance was generally higher in human isolates than in animal strains.
This study used spa typing as the only tool [36] to type the S. aureus species, which makes it impossible to draw safe conclusion on the population structure of the clones circulating in Tanzania.Other studies have combined spa typing in combination with based upon repeat pattern (BURP) multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) [37].A similar approach would have strengthened the current study.Also the samples size was small, and we cannot rule out that more types would have been detected, had more samples been analyzed.
Further studies are warranted in Tanzania to cover a larger area and to be more specific in identification and typing the isolates in relation to pets and their owners as well as for pig keepers and their pigs.Cats should also be included in further studies.A combination of spa typing and other laboratory tools should be used.

Conclusion
mecA and mecC positive isolates were not detected.Humans and dogs were found to carry S. aureus with frequencies that are within the range of reports from other countries.Prevalence determined in pigs may be considered low considering the availability of data on the occurrence of MRSA in intensive pig farming in Europe and South East Asia; however, generally little is known on carrier frequency among pigs, alongside with MRSA in Tanzania.Antimicrobial resistance was generally low with the exception of ampicillin and tetracycline.spa typing of S. aureus revealed specific spa types shared by humans and canine isolates, suggesting possible transmission between these two host species.The community-associated lineage ST80 spa type t131 was isolated only from pigs.Further studies with a larger collection of strains are needed to determine whether pigs are the sole reservoir or also humans carry this lineage in Tanzania

Acknowledgements
This study was supported by the Danish National Aid organization, DANIDA, trough the project Transforming Smallholder Livestock Farms into Profitable Enterprises (Livestock Enterprises) with grant number 66-08-LIFE.Great thanks are extended to livestock keepers and individuals who voluntarily participated in this study.We would like to thank Mariam Kitojo at Pest Management Centre of Sokoine University of Agriculture Tanzania for Sulphamethoxazole/ trimethoprim (STX), tetracycline (TE), ampicillin (AMP), rifampicin (RD), amoxycillin-clavulanic acid (AMC), oxacillin (OX ), chloramphenicol (C ), gentamicin (CN ).

Table 1 .
spa type associated STs and spa cluster analysis of S. aureus isolated from healthy humans, dogs and pigs in Tanzania.

Table 2 .
Antibiotic resistance in isolates (%) of S. aureus from Tanzania.