Detection and Genetic Environment of Pleuromutilin-Lincosamide-Streptogramin A Resistance Genes in Staphylococci Isolated from Pets

Increasing emergence of staphylococci resistant to pleuromutilins, lincosamides, and streptogramin A (PLSA) and isolated from humans and pets is a growing public health concern worldwide. Currently, there was only one published study regarding one of the PLSA genes, vga(A) detected in staphylococci isolated from cat. In this study, eleven pleuromutilin-resistant staphylococci from pets and two from their owners were isolated and further characterized for their antimicrobial susceptibilities, plasmid profiles, genotypes, and genetic context of the PLSA resistance genes. The gene sal(A) identified in 11 staphylococcal isolates was found for the first time in Staphylococcus haemolyticus, Staphylococcus epidermidis, and Staphylococcus xylosus. Moreover, these 11 isolates shared the identical regions flanking the sal(A) gene located in the chromosomal DNA. Two S. haemolyticus isolates from a cat and its owner carried similar vga(A)LC plasmids and displayed indistinguishable PFGE patterns. A novel chromosomal multidrug resistance genomic island (MDRGI) containing 13 resistance genes, including lsa(E), was firstly identified in S. epidermidis. In addition, vga(A)LC, sal(A), and lsa(E) were for the first time identified in staphylococcal isolates originating from pet animals. The plasmids, chromosomal DNA region, and MDRGI associated with the PLSA resistance genes vga(A), vga(A)LC, sal(A), and lsa(E) are present in staphylococci isolated from pets and humans and present significant challenges for the clinical management of infections by limiting therapeutic options.

To date, the antimicrobial resistance mechanisms of staphylococci in pets have received less attention than those in food-producing animals. Given the close relationship between pets and their owners, resistant bacteria and their mobile resistance determinants may be exchanged in either direction between pets and humans (Schwarz et al., 2016). The increasingly frequent isolation of methicillin-and multidrugresistant staphylococci from pets, particularly from dogs with pyoderma and otitis (Cain, 2013), and the occurrence of the same resistance genes in pets and in humans (Butaye et al., 2001;Simjee et al., 2002), may underline the potential role of pets in the transmission of antimicrobial-resistant staphylococci to humans. Currently, there is one published study regarding one of the PLS A genes detected in staphylococci isolated from a cat (Weiß et al., 2013). In the present study, we investigated the distribution of PLS A resistance genes in staphylococci of pet origin and analyzed the locations and genetic environments of these genes.

Ethics Statement
This research was carried out according to the principles of the Declaration of Helsinki. The involved pet animals were treated with the best practice veterinary care and the informed consent was obtained from pets' owners. The study protocol was approved by the Ethics Committee of China Agricultural University.

Bacterial Strains and Antimicrobial Susceptibility Testing
A total of 300 anal swabs or nasal samples were used in this study which were collected from dogs (n = 269), cats (n = 10), and some of their owners (n = 21) in the Veterinary Teaching Hospital of China Agricultural University, Beijing in 2013. The pleuromutilin-resistant isolates were selected by incubation on mannitol salt agar (Luqiao, Beijing, China) supplemented with 2 µg/ml of valnemulin at 37 • C for 16-24 h.
The MIC (Minimal Inhibitory Concentration) determinations were done by a standard broth microdilution test following the recommendations of the Clinical and Laboratory Standards Institute [CLSI] (2015). Staphylococcus aureus ATCC 29213 was served as the quality control strain.

Molecular Methods
The genomic DNAs of the valnemulin-resistant isolates were obtained using a Wizard R Genomic DNA Purification Kit (Promega, Madison, WI, USA), and were screened for the vga, lsa, and sal gene types by PCR assays (Novotna and Janata, 2006;Hot et al., 2014;Li et al., 2014;Wendlandt et al., 2015). Furthermore, the species assignment of the resistant isolates was done by 16S rDNA sequencing and confirmed by MALDI-TOF MS (Bruker Daltonik, Bremen, Germany).
The clonality of the valnemulin-resistant isolates was analyzed by pulsed-field gel electrophoresis (PFGE) as described previously (Wang et al., 2011), and the PFGE results were analyzed using InfoQuestFP software (version 4.5).
The S1 nuclease-PFGE (S1-PFGE) and subsequent Southern blot hybridization were performed to locate the valnemulin resistance genes as described before (Barton et al., 1995). The Low Range PFGE Marker (New England BioLabs, Beverly, MA, USA) served as the size marker.

Characterization and Sequence Analysis of the PLS A Genes
Genomic DNAs of S. sciuri isolate 100N carrying sal(A) and S. epidermidis isolate 138N carrying sal(A) and lsa(E) were submitted to high-throughput whole-genome sequencing (WGS), and preceded by library construction on a HiSeq 2500, which produced 150 bp paired-end reads (Berry Genomics Company, Beijing, China).
Draft assembly of the genomic DNA sequences was analyzed by CLC Genomics Workbench 5 (CLC Bio, Aarhus, Denmark). All contigs with the average coverage of >100-fold were searched for the PLS A genes using BLAST analysis. The regions flanking the PLS A genes were identified using de novo assembly as earlier described (Zerbino and Birney, 2008) and the random primer walking strategy (Zhang et al., 2009). Sequence analysis was conducted using the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and BLAST functions (http://blast.ncbi.nlm.nih.gov/Blast.cgi). To obtain more information about the genetic environments of the PLS A genes in valnemulin-resistant isolates, the random primer walking strategy and inverse PCR were performed.

Characterization of Valnemulin-Resistant Staphylococcal Isolates and Antimicrobial Resistance Profiles
Amongst the 300 anal swabs and nasal samples, 13 (4.3%) valnemulin-resistant isolates with valnemulin MICs of ≥8 µg/ml were detected, which were identified to species level as Staphylococcus sciuri (n = 8), Staphylococcus haemolyticus (n = 2), S. epidermidis (n = 2), and Staphylococcus xylosus (n = 1) ( Table 1). Nine (eight S. sciuri and one S. xylosus) of the 13 isolates carried only sal(A), one S. haemolyticus and one S. epidermidis isolate carried vga(A) LC and vga(A), respectively, one S. haemolyticus isolate carried both vga(A) LC and sal(A), and one S. epidermidis isolate carried both lsa(E) and sal(A) ( Table 1). Notably, the lsa(E) gene was only detected in one S. epidermidis isolate of dog origin, and the vga genes were detected in staphylococcal isolates originating from both humans and cats. In contrast, sal(A) was widespread among the valnemulin-resistant staphylococcal isolates from pets. As the sal(A) gene has been previously found exclusively in S. sciuri isolates (Hot et al., 2014;Wendlandt et al., 2015), this finding describes for the first time the occurrence of sal(A) in S. haemolyticus, S. epidermidis, and S. xylosus.

Genotyping by PFGE
Using a cut-off of 90% similarity, the eight S. sciuri isolates, which carried highly conserved (>99% nucleotide identity) sal(A) genes, clustered into five PFGE types (A-E), with type A accounting for the majority (3/8) of the isolates (Table 1;  Figure 1). Remarkably, the feline S. haemolyticus isolate 131A and the human S. haemolyticus isolate 131R from the corresponding pet owner, which carried identical copies of vga(A) LC , showed the same PFGE type H, indicating that they were closely related. This finding supports the idea that these isolates might have been exchanged between the cat and its owner.
Genetic Environment of PLS A Genes S1-PFGE and Southern blot hybridization indicated that all vgatype genes in the three isolates were located on plasmids, while all lsa(E) and sal(A) genes in the respective staphylococci were located in chromosomal DNA (Table 1; Figure 2).
To determine the regions flanking the vga-type genes in isolates 131R, 131A, and 132R, inverse PCRs were performed using the primers vga-F (5 -CAAGCTGAAAAGCCAACAAGG-3 ) and vga-R (5 -CCTCGTCAATTTCCCATATAGT-3 ), which are located inside the conserved regions of vga(A) and vga(A) LC genes. Three amplicons of 6056-bp (plasmids p131A and p131R) and 7209-bp (p132R) were obtained ( Figure 3A). Plasmids p131A and p131R from the feline and human S. haemolyticus isolates differed in only nine base pairs, and showed 99% nucleotide sequence identity to plasmid pUR2355 from human S. aureus (accession no. JQ312422). Plasmid p132R shared 99% nucleotide identity with the S. epidermidis plasmid pUR3036 of cat origin (accession no. JQ312423). The most notable difference between p132R and p131A/p131R was the presence of 13 amino acid substitutions in the Vga protein. Surprisingly, the lincomycin MICs of isolates 131A, 131R, and 132R were 16, 16, and 64 µg/ml, respectively, which is in contrast to the finding that vga(A) LC confers higher MICs to lincosamides than does vga(A) (Novotna and Janata, 2006).
Frontiers in Microbiology | www.frontiersin.org in the conserved regions of the aspS, sal(A), and alaS genes ( Figure 3B), respectively, were designed, and long-range PCRs were preformed to investigate the genetic environments of sal(A) in the other nine sal(A)-positive staphylococcal isolates ( Table 1). Two amplicons of 7.4 and 7.7 kb were obtained, and identical sequences were found in all nine isolates.
A 20,154-bp lsa(E)-carrying fragment containing 21 ORFs was obtained in the canine S. epidermidis isolate 138N (Figure 3C) after the conjoint analysis of the results of WGS and modified random primer walking strategy. Similar to the corresponding regions in porcine E. faecalis E533 (accession no. KX156278), this lsa(E)-carrying fragment also contained the macrolide-lincosamide-streptogramin B resistance gene erm(B), the aminoglycoside resistance genes aacA-aphD, aadE, and aphA-3, the spectinomycin resistance gene spw, the streptothricin resistance gene sat4, and the lincosamide resistance gene lnu(B). Further downstream of the lnu(B) gene, a second copy of the genes aadE-sat4-aphA-3 was detected ( Figure 3C). This multidrug resistance gene region confers resistance to seven classes of antimicrobial agents and explained why isolate 138N exhibited resistance not only to PLS A antibiotics, but also to spectinomycin, gentamicin and erythromycin ( Table 1). In addition, the hp4 gene encoded a predicted protein related to an acetyltransferase enzyme GNAT (GCN5-related N-acetyltransferase, with no confirmed function) from Enterococcus.

DISCUSSION
Increasing emergence of staphylococci that are resistant to PLS A and are isolated from humans and pets is a growing public health concern worldwide. Each of these three classes of antimicrobial agents contains important drugs for human and veterinary medicine. The pleuromutilin antibiotics valnemulin and tiamulin are frequently used in veterinary medicine. Retapamulin was the first pleuromutilin approved for topical use in skin infections of humans caused by staphylococci (Jacobs, 2007). Lincosamides, such as clindamycin and lincomycin, have generally been indicated for the treatment of osteomyelitis but also skin and soft tissue infections caused by staphylococci in humans and pets (Xue et al., 1996;Zetner et al., 2003). Virginiamycin is a two-component streptogramin with a broad spectrum of activity against Gram-positive bacteria. Some antimicrobial agents approved for use in human medicine are also applied to nonfood-producing animals under specific regulations, such as the Animal Medicinal Drug Use Clarification Act (AMDUCA) in the USA. In this study, all 13 valnemulinresistant isolates displayed resistance or high MICs to pleuromutilins (valnemulin, tiamulin, and retapamulin), lincomycin, virginiamycin M1, indicating that all PLS A genes including vga(A), vga(A) LC , sal(A), and lsa(E) of these isolates were functional. Interestingly, genes sal(A) and vga(A) LC coexisted in one feline S. haemolyticus isolate 131A, and genes sal(A) and lsa(E) were co-located in the chromosomal DNA of the canine S. epidermidis isolate 138N. Isolate 138N exhibited significant differences in the MICs of valnemulin, lincomycin, and virginiamycin M1 compared with other isolates that carried only one type of PLS A genes ( Table 1). There have been no studies regarding to sal, vga, and lsa coexisting in the same isolate, while both vga(A) and lsa(E) were identified in four bovine staphylococcal isolates (Wendlandt et al., 2015). Notably, vga(A) LC , sal(A), and lsa(E) were for the first time identified in staphylococcal isolates originating from pet animals.
The presence of PLS A resistance genes in pet-and humanassociated staphylococci indicates the adaptation of these bacteria to antibiotic pressure, given that PLS A resistance genes confer resistance to antimicrobial agents used in both pets and humans. Additionally, S. epidermidis 138N carried at least 13 resistance genes conferring resistance to seven classes of antimicrobial compounds (pleuromutilins, lincosamides, streptogramins, macrolides, aminoglycosides, aminocyclitols, and streptothricins), indicating that the use of any of the abovementioned antimicrobial agents may lead to co-selection of the MDR genomic island (MDRGI) in isolate 138N. The plasmids, chromosomal DNA region, and MDRGI associated with the multiresistance genes vga(A), vga(A) LC , sal(A), and lsa(E) exist in staphylococci isolated from pets and humans, which enhances the dissemination of PLS A -resistant staphylococci among pets and humans and presents significant challenges for the clinical management of infections by limited therapeutic options.
The three canine sal(A)-positive S. sciuri isolates 139N, 140N, and 145N not only shared the identical regions flanking the sal(A) gene, but also had the uniform PFGE pattern (subtype A), suggesting that these isolates have originated from a single clone. Remarkably, plasmids p131A and p131R, the two almost identical vga(A) LC -carrying plasmids present in isolates with the same PFGE subtype (subtype H), were derived from a cat and its owner. This fact suggests that these two isolates might have been exchanged between the cat and its owner due to their extensive contact. In this regard, it is important to consider the current role of dogs and cats as actual family members in many households and, consequently, the close contact to their owners and other family members (Schwarz et al., 2016).

CONCLUSION
This is the first description of the PLS A genes in staphylococci of pet origin and also the description of a novel lsa(E)-carrying MDRGI in S. epidermidis. Moreover, we identified the sal(A) gene for the first time in S. haemolyticus, S. epidermidis, and S. xylosus. Pets are likely reservoirs of antimicrobial-resistant bacteria, warranting the prudent use of all antimicrobials in pet animal medicine.

AUTHOR CONTRIBUTIONS
YW and SS designed research; FD, HW, and YL performed research; FD, JL, AF, and GM analyzed data; FD, SS, and YW wrote the paper. All authors listed have approved research for publication.