Misidentification of coagulase negative Staphylococcus aureus due to gene variant

Aim of study: An accurate, rapid and specific identification of microorganism is crucial for bovine mastitis therapy and prevention. In particular, the identification of Staphylococcus aureus represents the basis of any udder control program. Coagulase tube test with rabbit plasma is the phenotypical method normally used in laboratory routine. A negative reaction usually determines the inclusion of the tested microorganism in the group of coagulase-negative staphylococci (CNS), considered as minor pathogens, without any other distinction. However, in literature several rare cases of staphylococci negative at coagulase test but furtherly identified as S. aureus by other phenotypic or molecular methods are described (Akineden et al., 2011; Sunagar et al., 2013). Those strains could be a bias in the detection of infections with major impact on udder health and lack of control of their spread within the herd. The present study aimed to characterize some of these S. aureus strains by genotyping with RS-PCR and analyzing a set of virulence genes, including coa gene. Materials and Methods During the daily diagnostic activity at the Department of Veterinary Medicine, 31 staphylococci (4 from bulk tank samples, 8 from clinical mastitis, 3 from quarter and 16 from composite samples) from 7 different farms were presumptively identified as S. aureus. Unexpectedly, these isolates resulted negative at coagulase test after both 4 and 24 hours of incubation. The typical morphology and in particular the beta-hemolysis on blood agar made it necessary to test the isolates onto Mannitol Salt Agar (MSA) and Baird Parker with Rabbit Plasma (RPF-BP). Bacterial DNA from S. aureus was extracted by using a protocol previously described (Cremonesi et al., 2013). Then, polymerase chain reactions were performed for each isolate targeting nuc (thermonuclease coding gene) and coa (coagulase coding gene), as previously described (Cremonesi et al., 2013). A subset of strains was analyzed for some virulence genes (clfA, lukE, lukM, tsst, eta, etb, cna, chp, sak, scn, fmtb and ten different enterotoxins) by using protocols described in literature (Cremonesi et al., 2013) and genotyped by RS-PCR (Cremonesi et al., 2013). Finally, the coa gene of six strains, with no correspondence between genotype and phenotype, was sequenced on a paired 2x250 bp run on a MiSeq platform (Illumina, San Diego, CA, USA). Main results and conclusions All the 31 isolates resulted nuc positive (i.e. S. aureus), despite the coagulase negative test. All the isolates yielded positive reaction on MSA and negative on RPF-BP, excepted 5 isolates positive on the latter. Furthermore, he isolates resulted positive to different virulence factors such as lukE, cna, lukM, sak, fmtb, scn, sea, demonstrating their pathogenic potential. RS-PCR genotyping of 13 out of the 31 strains resulted in the following profiles: GTCI (20%) from the isolates of two different farms, GTBQI (16%) from the isolates of one farm and GTRVII from one isolate from one farm. At present, the coa gene sequences are under examination. Epidemiology, virulence, cure rate, and the impact of economic loss for the farmer could be deeply different comparing S. aureus to staphylococci other than S. aureus infections (Graber et al., 2013). The isolation of coagulase-negative S. aureus has a sporadic frequency, yet the identification of the issue is essential to avoid its spreading and persistence that could complicate diagnosis on a larger scale. Hemolysis is an important diagnostic tool in classical bacteriology, so attention should be payed by culture on blood agar beside specific and selective media (Graber et al., 2013). Actually RPF-BP could fail as demonstrated by the negativity of most isolates. Hemolysis, even followed by coagulate tube test, could not be enough to identify S. aureus. Phenotypic and molecular method should be integrated in a diagnostic path able to recognize all the variants of S. aureus.