Introduction

Staphylococcus pseudintermedius is an important opportunistic pathogen in canine companions and is commonly associated with skin infections [1]. This bacterium is sporadically associated with human infections because it can be transmitted easily via close contact with animals, and it has the potential to cause severe disease [2]. Methicillin-resistant staphylococci of the intermedius group (SIG) emerged in canines in 1999 [3], and S. pseudintermedius was first described in 2005 [4]. Methicillin-resistant S. pseudintermedius (MRSP) has been spreading worldwide through the expansion and dissemination of dominant clonal lineages with specific genetic characteristics, including the sequence type (ST) 71 in Europe, ST68 in North America and ST45/ST112 in Asia [5, 6]. The first infection of MRSP in humans was reported in 2006 in Belgium [7] and the first MRSP isolated from a human patient in Argentina was reported in 2020 [8]. Furthermore, dominant clones are multi-drug resistant (MDR), suggesting that the spread of horizontally transferrable resistance genes is a contributing factor for the dissemination of certain sequence types [9].

Antimicrobial resistance patterns differ in the three most prevalent MRSP clonal lineages [5]. Clonal complexes (CCs) are groups of sequence types (STs) sharing at least six identical alleles of the seven S. pseudintermedius MLST genes (ack, cpn60, fdh, pta, purA, sar, and tuf), with the primary founder being the ST with the largest number of single locus variants (SLVs) and all other strains diverge from the predicted clonal ancestor [10]. MRSP belonging to clonal complexes CC71 and CC68 often contain several genes that confer resistance to multiple antimicrobials in addition to the mecA gene located within the SCCmec cassette [11, 12]. For CC45, isolates often harbor resistance genes and mutations that make them resistant to almost all antimicrobials used in veterinary medicine [13].

In 2010, the global population structure of MRSP gradually started to change and it became more heterogenous than previously described, with evidence of dissemination through clonal expansion of MRSP dominant lineages over large distances [14]. In Europe, there was an apparent decrease of ST71 [6, 15, 16] with the emergence of two novel MRSP lineages (ST258 and ST496) of European and Australian origin [6, 17]. Likewise, ST71 clones began to spread worldwide over more distant locations and this clone has now been reported in Asia and in North and South America, with high prevalence in many countries in these regions. This change in the global population structure of S. pseudintermedius may be the consequence of importation from other countries due to the mobilization of animals and people across geographical locations [9, 18, 19]. In other parts of the world, the MRSP population appears to be more diverse. In Argentina, the MRSP population consists of genetically distinct STs not closely related to the more prevalent ST71 and ST68 lineages [20].

Staphylococcal chromosome cassette mec (SCCmec) typing is one of the molecular techniques currently used to understand the epidemiology and the clonal relationships of methicillin-resistant S. aureus (MRSA) strains [21]. Consequently, SCCmec typing for S. pseudintermedius has been progressively adapted from the work done for S. aureus. Existing reports of S. pseudintermedius SCCmec type III (previously described as II-III by Descloux et al. [22]) associated it with the European epidemic clone ST71, and ΨSCCmec57395 was significantly associated with ST45 [5, 11, 13]. To date, no knowledge exists regarding S. pseudintermedius belonging to the ST71 and ST45 clones in Argentina. Here we report for the first time in Argentina ST71-SCCmec III and ST45-ΨSCCmec57395.

Main text

Methods

Isolate selection

Thirty S. pseudintermedius isolates from dogs with pyoderma collected during 2016 from the Buenos Aires Metropolitan Area (Ciudad Autónoma de Buenos Aires, Gran Buenos Aires and La Plata, Argentina) were selected randomly from the strain collection of the Laboratory of Bacteriology and Antimicrobials, Department of Microbiology, Faculty of Veterinary Sciences, National University of La Plata, Argentina (Laboratorio de Bacteriología y Antimicrobianos, Departamento de Microbiología, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, Argentina). Identification was confirmed by MALDI-TOF and whole genome sequencing (WGS) at the National Veterinary Services Laboratories (NVSL) in Ames, Iowa, U.S.A. Twenty-three S. pseudintermedius isolates were identified as methicillin-resistant (MRSP) due to the presence of the mecA gene, which encodes methicillin resistance, through WGS analysis (described below).

Whole genome sequencing and genomic analysis

Sequencing was performed with the Illumina MiSeq platform using 2 × 250 paired-end chemistry and the NexteraXT library preparation kit. Multilocus sequence typing (MLST) was determined using ABRicate (https://github.com/tseeman/abricate/) with the S. pseudintermedius PubMLST database, and new alleles and sequence types (STs) were submitted to PubMLST (http://pubmlst.org/spseudintermedius) for curation and number designation by Vincent Perreten (vincent.perreten@vetsuisse.unibe.ch). SCCmec types were determined using SCCmecFinder 1.2 [23] (https://cge.food.dtu.dk/services/SCCmecFinder-1.2/), a database with SCCmec types I through XII, including SCCmec IV and V subtypes (as of the preparation of this manuscript), based on those identified in S. aureus. For the predicted SCCmec types III and V, additional manual alignment/mapping was performed using the available reference sequences for these SCCmec types for S. aureus and S. pseudintermedius (AB03671.1, AM904732.1 for SCCmec type III; HE984157.2 for ΨSCCmec57395; and FJ544922.1, ERR175868, AB512767.1, AB505629.1, AB462393.1, AB121219.1 for SCCmec type V), using Geneious Prime v11.0.9 (Biomatters Ltd., NZ).

Results

For the 23 MRSP isolates analyzed, a total of 14 sequence types (STs) were identified, five previously described: ST339 (n = 7), ST1412 (n = 3), ST71 (n = 2), ST45 (n = 1) and ST313 (n = 1); and nine newly identified STs (Table 1).

Table 1 Multilocus sequence types (MLST) and SCCmec types of methicillin resistant Staphylococcus pseudintermedius isolates obtained from dogs with pyoderma in Argentina
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SCCmecFinder successfully classified twelve isolates as SCCmec type IIIa (n = 2), SCCmec type V (5C2) (n = 3) and SCCmec type V (5C2&5) (n = 7). The remainder of the isolates could not be typed.

The two isolates classified as SCCmec type III belonged to ST71. These were mapped against the S. pseudintermedius KM1381 (AM904732.1) genome reference that harbors a hybrid SCCmec type II-III, described to be a combination of SCCmec II from S. epidermidis and SCCmec III from S. aureus, but lacking the cadmium resistance operon [22]. Both isolates showed high homology (99.9%) to this reference (Fig. 1A).

For one isolate identified as ST45,a SCCmec type could not be determined using SCCmecFinder, but alignment/mapping to HE984157.2 resulted in high homology (98.8%) classifying it as ΨSCCmec57395 (Fig. 1B).

Of the SCCmec type V, three were predicted as SCCmec type V (5C2), with only one ccrC1 recombinase, and seven were predicted as SCCmec type V (5C2&5), with two ccrC1 recombinases. When these 10 isolates were compared against SCCmec V subtype references (Va, Vb and Vc), isolates with SCCmec type V (5C2) (BI-1991, BI-2002, BI-2008) showed 79.4–90.6% homology to the S. aureus type Va (5C2) reference strain. The rest showed 84.8–99.8% homology to S. pseudintermedius 06-3228 (FJ544922.1) and S. pseudintermedius 23,929 (ERR175868), which are both references for S. pseudintermedius SCCmec V (5C2&5) [12, 24]. We classified five of these isolates as SCCmec Vb due to their homology with S. aureus AB462393.1 (Vb). Furhtermore, two of these SCCmec Vb (BI-1980, BI-1990) showed evidence of harboring a truncated mecR1 gene. Finally, we classified two isolates (BI-1991, BI-2003) as SCCmec Vc (5C2&5) because they harbored the czrC gene that is present in the SCCmec Vc but is absent in Vb. (Figure 1C, D and E).

Fig. 1
figure 1

 A, Alignment of S. pseudintermedius BI-1983 and BI-1985 SCCmec elements to S. pseudintermedius KM1381 (AM904732, first described as hybrid II-III) and S. aureus 85/2082 (AB037671.1, SCCmec III). B, Alignment of BI-1989 to S. pseudintermedius 57,395 (HE984157.2, ΨSCCmec57395). C, Alignment of SCCmec V (5C2) predicted elements for BI-1991, BI-2002 and BI-2008 to S. aureus SCCmec Va (5C2) [AB121219.1]. D, Alignment of SCCmec Vb (5C2&5) predicted elements for BI-1979, BI-1980, BI-1984, BI-1990, and BI-2004 to S. aureus Vb (5C2&5) [AB462393.1; AB512767.1] and S. pseudintermedius SCCmec VT (FJ544922.1). E. Alignment of SCCmec Vc (5C2&5) predicted elements BI-1981 and BI-2003 to S. aureus Vc (5C2&5) [KM369884].

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Discussion

This study is the first report of S. pseudintermedius ST71-SCCmec III and ST45-ΨSCCmec57395 in Argentina, obtained from a cohort of isolates recovered from dogs with pyoderma in the Buenos Aires Metropolitan Area in 2016. A previous study in Argentina described a population of MRSP from dogs with clinical disease that consisted of six genetically distinct STs: ST339, ST649, ST919, ST920, ST921, and ST922 [20]. Here, among 23 MRSP, ST339 (n = 7) was also identified, as well as an additional thirteen sequence types, including ST1412 (n = 3), ST71 (n = 2), ST45 (n = 1), ST313 (n = 1) and nine newly identified STs (ST2233-2237, ST2242-2244 and ST2261). These data contribute to the characterization of the population structure of MRSP in Argentina, which now includes two globally prevalent clones (ST71 and ST45). ST71 was initially described as the predominant clone in Europe but is now spread worldwide, whereas ST45 was described as the most prevalent clone in Asia [5]. Gagetti et al. [20] identified two isolates with sequence type ST339 in Argentina. The first MRSP recovered from a human patient in Argentina was ST1412 [8]. Interestingly ST1412 is a double locus variant of ST45, the sequence type that originated in Asia.

The ST71 clone has mainly been associated to SCCmec type III [11]. This SCCmec, first identified in 2005, was initially classified as a hybrid SCCmec II-III [22]. The distribution of this clone was primary found in Europe, but is now disseminated worldwide [23, 25]. The first report of an ST71 MRSP in South America was from a dog in Brazil in 2013 [26] and this study is the first report of this clone in Argentina. As in previous reports, the two isolates identified in this study as ST71 harbored SCCmec type III.

Pseudo (Ψ) SCCmec elements have been identified in S. haemolyticus with no evidence of ccr genes, but with a mec complex [27, 28]. A novel ΨSCCmec57395 was described in MRSP CC45 from companion animals in Thailand and Israel [13]. In Australia, MRSP belonging to ST45 was also associated to this novel ΨSCCmec57395 element [18]. Even though no particular SCCmec type is usually associated to MRSP-ST45 [25, 29], some reports identified ΨSCCmec57395 with this clone [13, 18]. The results from this study show evidence to also classify the MRSP-ST45 isolate from Argentina as an ST45-ΨSCCmec57395, making this the first report of this element in the country.

Lastly, almost half (10/23) of the isolates were predicted as SCCmec V. SCCmec V has been associated to different STs [5], and variation has been observed in SCCmec type V for S. pseudintermedius in comparison to S. aureus. Currently, this element is classified into three subtypes for S. aureus, according to Uehara [30]: Va (5C2), Vb (5C2&5) and Vc (5C2&5). To provide clarity, it’s important to mention how the classification for subtype Vb has evolved. Initially, it was classified as VT (AB462393.1) [31]. Later, Black et al. [12] described a homologous SCCmec type V element in S. pseudintermedius (FJ44922.1), which only differed in a deleted section of a gene in S. pseudintermedius with respect to S. aureus. Then, Takano et al. [32] proposed reclassification of Vb to as SCCmec type VII. Finally, Perreten et al. [11], described an SCCmec V in S. pseudintermedius that was highly homologous to the previously named VT or VII from S. aureus, which was designated as SCCmec V (5C2&5). In this study, three MRSP isolates, belonging to ST339, showed one ccrC1 recombinase only and were most homologous to SCCmec V (5C2). In contrast, the remaining seven MRSP isolates showed two ccrC1 recombinases and were most homologous to SCCmec V (5C2&5). Additionally, there was evidence to suggest that the mecR1 gene was truncated in two of these isolates (BI-1979 and BI-1980). Worthing et al. [18] reported similar results for the SCCmec VT identified in their study. Prior to our study, SCCmec V (5C2&5) was the only SCCmec type reported in MRSP in Argentina [20].

Conclusion

Using whole-genome sequencing we identified two MRSP isolates, one belonging to sequence type 71 and carrying staphylococcal cassette chromosome mec type III (ST71-SCCmec III), and the other belonging to sequence type 45 and carrying the ΨSCCmec57395 (ST45-ΨSCCmec57395), neither of which had been previously reported in Argentina. Even though these sequence types were first identified and distributed in Europe and Asia, respectively, our results support the current worldwide spread observed for these S. pseudintermeius clones. These findings highlight the importance of WGS for understanding the circulating populations of MRSP and the spread of multidrug-resistant S. pseudintermedius in companion animals, which can consequently have a significant impact on public health.

Limitation

  • Complete fragment coverage of the SCCmec elements was limited due to the inevitable gaps present in assemblies from short read technology, therefore fully closed genomes were not available.

  • There are inconsistencies in the literature regarding nomenclature and classification of SCCmec elements, which makes interpretation and comparative analysis more complex.

  • There is an evident need for a formal SCCmec nomenclature that includes SCCmec elements from Staphylococcus pseudintermedius and other Staphylococcus species.