Cross-sectional study of Staphyloccus lugdunensis prevalence in cats

Staphylococcus lugdunensis is a commensal bacterium in humans and other animals that can cause serious infections. The aim of this research was to estimate the frequency of S. lugdunensis in pet cats and to characterize the S. lugdunensis isolates obtained. The prevalence of S. lugdunensis was 0.77% (4/523) in healthy cats and 1.23% (1/81) in sick cats. The isolates (N = 5), which colonized conjunctival sacs, nares, and the anus, were almost fully phenotypically sensitive to antibiotics, but harbored resistance genes to four chemotherapeutic groups. Their sequence types (STs) included ST2, ST3, ST9, and ST15. There was detected a far lower prevalence of S. lugdunensis in pet cats than is reported in the human population. Nevertheless, the phenotypic and genotypic properties of S. lugdunensis isolates found in the current study were very similar to those described previously in isolates of human origin.

www.nature.com/scientificreports/ were used to collect samples from four anatomical sites (nares, conjunctival sacs, groin, anus) in 523 healthy and 81 sick cats, and additionally from a wound, if present, on sick cats. Additionally, each cat owner filled out the questionnaire considering potential factors connected with staphylococci colonization in cats under investigation, such as animal features (age, sex, breed, medical history) and household's factors (medical occupations or previous hospitalization of household members, other animals kept in the same household and their medical history). Collected swabs were placed into 2 ml of liquid brain-heart infusion broth (BHI) (Oxoid, United Kingdom) and incubated at 37 °C for 24 h. Then the material was subcultured in Mannitol Salt Agar and blood agar plate (Oxoid, United Kingdom) and incubated for 24 h. The preliminary identification of staphylococci was according to the colony morphology, Gram staining, and detection of enzyme production (coagulase tube test; IBSS Biomed, Poland). S. lugdunensis species was identified by polymerase chain reaction (PCR) with species-specific primers performed according to previously detailed reaction conditions 9 . All the isolates of S. lugdunensis were screened for antibiotic susceptibility using both methods: phenotypic [disc diffusion and MIC (Sensititre, Staphylococcus MIC plates, Thermo Fisher Scientific, Waltham, MA, USA)] and genotypic (detections of genetic determinants of resistance). The phenotypic and genotypic antibiotic resistance of each isolate was determined as described previously 10 . S. lugdunensis was tested for slime production by the Congo red agar method, microtiter plate test, and a standard PCR technique for icaA and bap genes detection 10 . Prevalence rates of S. lugdunensis were calculated for the sick cat group and for the healthy cat group by the bootstrap method in the R Statistical Package v. 2.11.1. The statistical analysis of potential risk factors, associated with S. lugdunensis colonization in cats under investigation, was not performed due to a small number of colonized animals in both groups of cats.
Staphylococcus lugdunensis allele sequence types (STs) were determined by multi-locus sequence typing (MLST) with a sequence alignment tool (MEGA X 10.1.) focusing on seven housekeeping loci described by Chassain et al. 11 . MLST was performed with sequence and profile data from Institute Pasteur https ://bigsd b.paste ur.fr/staph lugdu nensi s/staph lugdu nensi s.html). I determined the ST of each S. lugdunensis isolate under investigation by using the search tool with a combination of S. lugdunensis loci in the PasteurMLST database (https ://bigsd b.paste ur.fr/cgi-bin/bigsd b/bigsd b.pl?db=pubml st_staph lugdu nensi s_isola tes&page=profi les).

Results
In total, five distinct S. lugdunensis isolates were identified, including four from healthy cats and one from the conjunctival sack of a cat with conjunctivitis and sneezing symptoms (GenBank accession numbers of 16S RNA sequences of isolated S. lugdunensis, MT1880032-MT1880036). In most cases, S. lugdunensis was isolated alone; in the conjunctival sack of one healthy cat it was isolated with S. epidermidis. Information about the cats colonized with S. lugdunensis is summarized in Table 1.
The prevalence of S. lugdunensis in cats from Wrocław city area was 0.77% [95% confidence interval (CI) 0.02-1.51%] in healthy cats and 1.23% (95% CI 0.0-3.64%) in sick cats. The antibiotic resistance profiles and biofilm-forming properties of the investigated isolates are presented in Table 2. Although isolates exhibited robust biofilm formation on polystyrene plates, none harbored the icaA or bap gene. Four different S. lugdunensis STs were found ( Table 2). The ST of S. lugdunensis isolates were deposed in the Institut Pasteur MLST database (https ://bigsd b.paste ur.fr/staph lugdu nensi s/staph lugdu nensi s.html), identified as isolates 113-117.

Discussion
The present data indicate that S. lugdunensis is likely to be much more rare among pet cats population under investigation (~ 1%) than among humans 30-50% 12 . Notwithstanding, given the potential risk of Staphylococcus interspecies transmission, especially to human surgery patients, the prevalence of S. lungdunensis in pets should be monitored. This study provides some information about S. lugdunesnsis characteristics and carriage sites in cats, but, despite sampling a representative group of cats, the small number of isolates found limits the power of the analysis. There was observed colonization of the perineum, as has also been documented in humans 1,13 . Interestingly, two isolates were found in cats' conjunctival sac samples. To the best of knowledge, there have been no reports of conjunctivitis or keratitis caused by S. lugdunensis in pets. However, there have been a few such cases in human patients 14,15 . Moreover, the identification of S. lugdunensis as a potential causative pathogen of cat bacterial conjunctivitis may be indicative of a wide spectrum of possible infection sites for the bacterium, which is relevant to both veterinary and human medicine.
Contrary to other CoNS, S. lugdunensis remains sensitive to most antibiotics despite its pathogenicity 1,4-6,12 . Among the presently analyzed isolates, only resistances to sulfamethoxazole and ampicillin were identified. Others have identified penicillin-and erythromycin-resistant S. lugdunensis isolates, as well as S. lugdunensis isolates with susceptibility to all antibiotics tested 3,12 . Reports of S. lugdunensis isolates collected from humans harboring antibiotic resistance genes, especially genes that can confer resistance to penicillin (blaZ), macrolides (ermB/C), and tetracyclines (tetK/L/M/O) 16 indicate that S. lugdunensis has the potential to develop phenotypic resistance to antibiotic drugs. Furthermore, a report showing that bacteria exhibit lower antibiotic resistance when they are grown in plankton than when they are grown in a biofilm, indicate that standard in vitro phenotypic antibiotic resistance testing may not fully reflect the in vivo efficiency of chemoterapeutics towards S. lugdunensis 17 . The present observation of strong S. lugdunensis biofilm-forming properties is consistent with prior observations 18,19 There are currently 20 S. lugdunensis STs catalogued in the Institut Pasteur MLST database 11 . The STs identified in the current study, ST2 and ST3, are the most frequent S. lugdunensis STs found in humans, accounting for 30% of deposed isolates thus far. Further research into the occurrence of S. lugdunensis in pet animals is needed to elucidate the pathogenic potential of this ubiquitous species and its interspecies transmission risk.
conclusion The current study characterized the possible carriage sites for S. lugdunensis in cats in Wrocław city area, which could be used in future research design. There was detected a far lower prevalence of S. lugdunensis in pet cats than is reported in the human population. Nevertheless, the phenotypic and genotypic properties of S. lugdunensis isolates found in the current study were very similar to those described previously in isolates of human origin. Further studies are necessary, to better understand the emergence of as a veterinary and zoonotic pathogen, to evaluate the risks of interspecies transmission and potential factors connected with S. lugdunensis colonization, and to determine appropriate household infection control practices.