https://orcid.org/0000-0003-1401-2961
Figures
Abstract
This study investigated the carriage of Staphylococcus (S.) intermedius group (SIG) isolates in marine mammals in the Pacific coastal region of Southern California, USA. We screened 452 samples from the oral cavities of California sea lions (Zalophus californianus), northern elephant seals (Mirounga angustirostris), harbor seals (Phoca vitulina), northern fur seals (Callorhinus ursinus), Guadalupe fur seals (Arctocephalus townsendi), long beaked common dolphin (Delphinus delphis bairdii), Pacific white-sided dolphins (Sagmatias obliquidens), and pygmy sperm whales (Kogia breviceps). Forty-two S. pseudintermedius and four S. delphini were obtained. Isolates were characterized by a polyphasic approach, which included antimicrobial susceptibility testing, molecular identification and characterization by rpoB/nuc sequencing, PCRs, DNA-based microarray, genotyping via spa and MLVA, and whole-genome sequencing of selected isolates. Resistance to penicillin, penicillin/tetracycline, and tetracycline alone was observed. Spa typing resulted either in novel types or in isolates that were non‑typeable. Twenty-nine MLVA types were detected. Multi-locus sequence typing revealed four STs: ST453 (previously described) and three novel STs: ST3046−8) among S. pseudintermedius and various virulence-associated genes were detected. Long-term monitoring remains essential to fully map its epidemiology, pathogenicity, and evolution of SIG in marine mammals.
Citation: Loncaric I, Palmer L, Szostak MP, Rosel AC, Ruppitsch W, Rupprecht M, et al. (2026) Diversity among isolates of Staphylococcus intermedius group from Pacific marine mammals of Southern California. PLoS One 21(8): e0356088. https://doi.org/10.1371/journal.pone.0356088
Editor: Kunyan Zhang, University of Calgary, CANADA
Received: March 14, 2026; Accepted: July 29, 2026; Published: August 24, 2026
Copyright: © 2026 Loncaric et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are in the manuscript and/or supporting information files.
Funding: Open Access Fund by the University of Veterinary Medicine Vienna provides support for the APC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The North American Pacific coastline is home to a diverse assemblage of marine mammals, encompassing approximately 50 cetacean and16 pinniped [1]. Common pinnipeds in this region include the California sea lion (Zalophus californianus), northern fur seal (Callorhinus ursinus), harbor seal (Phoca vitulina), and northern elephant seal (Mirounga angustirostris), as well as threatened species such as the Guadalupe fur seal (Arctocephalus townsendi). These animals are recognized sentinels of ocean health, with their population health and disease status reflecting the equilibrium of the marine ecosystems they inhabit [2,3]. In California, primary health threats to marine mammals include human-inflicted trauma, biotoxins from harmful algal blooms, and bacterial infections [4]. Critically, because these species share coastal environments and trophic resources with humans, monitoring their health is central to a One Health approach for assessing ocean health and mitigating potential zoonotic disease risks [5].
The Staphylococcus intermedius group (SIG) comprises closely related coagulase-positive bacteria, including S. intermedius, Staphylococcus pseudintermedius, and Staphylococcus delphini. More recently, novel species have been included in the group, such as Staphylococcus cornubiensis, isolated from a human clinical case [6–9] and Staphylococcus ursi, initially isolated from an American black bear [10,11].
SIG members typically colonize the skin and mucous membranes of a broad range of hosts, including companion animals, livestock, and wildlife such as minks, dolphins, pigeons, dogs, horses, and cats [7,12–17] and are involved in the etiology of bacterial otitis, dermatitis, and other soft-tissue infections in domestic animals and wildlife as primary or secondary pathogens [12,15,18]. Although less common, human infections caused by SIG members have also been reported, and some cases historically attributed to S. aureus were later reclassified as SIG species [16,17,19]. Recent molecular and genomic studies have refined the taxonomy of this group, revealing significant genetic diversity among SIG members. Many isolates previously identified as S. intermedius have been correctly reclassified as S. pseudintermedius [12,18,20–22]. Continued investigation of the SIG taxonomy and host associations is essential to improve diagnostic accuracy and to better understand their epidemiological and zoonotic potential.
The Californian coastline includes many densely populated urban areas capable of generating large volumes of contaminated wastewater (1.58 billion m3 in 2015 [23]), which deposit into the ocean potentially leading to widespread dispersal of the zoonotic SIG, including antibiotic-resistant variants. Consequently, microorganisms traditionally associated with terrestrial mammals may infect marine mammals, posing potential health risks to these marine species. The aim of this study is to characterize the diversity of S. intermedius group isolates circulating in marine mammals along the Pacific coast of Southern California, USA.
Materials and methods
Isolation of Staphylococcus intermedius group strains
Oral samples were collected from stranded marine mammals from October 2019 to September 2022 upon admission to the Marine Mammal Care Center, Los Angeles (MMCCLA) as part of a diagnostic health evaluation, and, therefore, the approval of the local ethics committee was not required. In total, 451 oral samples from 345 California sea lions (Z. californianus), 75 northern elephant seals (M. angustirostris), 11 harbor seals (P. vitulina), nine northern fur seals (Callorhinus ursinus), five Guadalupe fur seals (Arctocephalus townsendi), three long beaked common dolphins (D. delphis bairdii), two Pacific white-sided dolphins (S. obliquidens), and one pygmy sperm whale (K. breviceps) (S1 Table). For the isolation of staphylococci each oral swab was incubated at 37°C overnight in tryptic soy broth (BD, Heidelberg, Germany) with 6.5% (w/v) NaCl and subsequently streaked onto BD™ Columbia CNA Agar with 5% Sheep Blood, Improved II (BD, Heidelberg, Germany). Bacterial colonies showing the typical comorphology for members of SIG were further confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik, Bremen, Germany) and were cryo-preserved at −80°C for subsequent analyses.
We determined the proportions of SIG-positive per species and estimated 95% confidence intervals (CIs) using the Clopper-Pearson exact method via the binom.test function in R [24].
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed using agar disk diffusion according to CLSI [25]. The following antimicrobial agents were used: penicillin (10 units), oxacillin (1 μg), ciprofloxacin (5 μg), gentamicin (10 μg), tetracycline (30 μg), erythromycin (15 μg), clindamycin (2 μg), chloramphenicol (30 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), nitrofurantoin (300 μg), rifampicin (5 μg), and linezolid (30 μg) (all from Becton Dickinson, Heidelberg, Germany). S. aureus ATCC® 25923 served as the quality control strain.
Molecular characterization of isolates
Genomic DNA of staphylococci was extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany). Prior to extraction, initial lysis was performed using the lysis enhancer and buffer provided with the INTER-ARRAY Genotyping Kit S. aureus (Bad Langensalza, Germany). For final species allocation, rpoB and RNA polymerase β-nuc sequencing were used [26,27]. Phylogenetic relationships among isolates were inferred using the unrooted neighbor-joining method. Sequence alignment, analysis, and visualization were conducted with Molecular Evolutionary Genetics Analysis software version 11 (MEGA 11) [28].
All isolates were subjected to staphylococcal protein A (spa) typing following the protocol of Zukancic et al. [29]. PCR products yielding positive bands by gel electrophoresis were Sanger sequenced. spa types have been assigned as described previously [30] and based on the offline database provided by Arshnee Moodley. In addition, isolates were genotyped using Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA), which relies on variable numbers of tandem repeats (VNTRs) [31]. To identify loci suitable for MLVA of S. pseudintermedius, an in silico analysis was performed on reference strain ED99 (GenBank accession CP002478) [32] using Tandem Repeats Finder, (https://tandem.bu.edu/trf/home, last accesses February 2026, [33]. Eight identified loci were selected for this study, with primers and annealing temperatures listed in S2 Table. This selection was based on the following criteria: (i) minimum repeat size of 30 bp, (ii) conservation between the tandem repeats (>90%), and (iii) a copy number ≥2. Primers were designed for the region flanking each VNTR using Primer3 (https://primer3.ut.ee/) [34]. Amplicon sizes were determined by gel electrophoresis. The number of repeats at each locus was calculated using the formula:
and rounded up to an even number [35]. This created an eight-digit code of repeats in each locus and was utilized for generating the MLVA types. Genetic relationships were analyzed using the categorical coefficient (Hamming distance), with clustering performed via the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) integrated in PHYLOViZ 2.0 [36]. All isolates were screened with PCR for six virulence-associated genes, including three exfoliative toxin genes (siet, expA, and expB) [37–40], the leucocidin locus lukI (comprising lukS and lukF) [41], and the canine staphylococcal enterotoxin C gene (secCanine) [40]. In addition, all isolates were analyzed using the Genotyping Kit S. aureus (INTER-ARRAY, fmzb GmbH, Bad Langensalza, Germany) following the manufacturer’s instructions [42,43].
Eight isolates were selected for whole-genome sequencing (WGS), seven S. pseuditermedius isolates were selected based on phenotypic resistance, spa type, and MLVA type. None of the isolates shared all characteristics. Additionally, one S. dephini isolate was selected at random. Isolate DNA extraction, WGS and genome assembly were performed as previously described [44]. Briefly, Genomic DNA was isolated using the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany), and sequencing libraries were prepared with the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA). Paired-end sequencing (2 x 150 bp) was performed on an Illumina NextSeq 2000 platform, targeting a minimum coverage depth of 30-fold. Following quality trimming of the raw reads, de novo genome assembly was carried out using SPAdes v3.11.1. The species allocation was verified by JSpecies workspace using the ANIb (average nucleotide identity via BLAST) analysis tool [45]. Sequence types (ST) were assigned using the database hosted at PubMLST (https://pubmlst.org/organisms/staphylococcus-pseudintermedius) [46]. The clonal relationships of the identified STs were assessed by integrating them with entries from the PubMLST Staphylococcus pseudintermedius database. All database entries available at the time of analysis (February 2026) were clustered using the goeBURST algorithm implemented in the PHYLOViZ 2.0 [36]. To identify acquired resistance genes and/or chromosomal mutations, ResFinder 4.7.2. with default setting (https://genepi.food.dtu.dk/resfinder/, accessed February 2026) was used [47]. ABRicate [48] v1.2.0 was used to identify antimicrobial resistance genes using Comprehensive Antibiotic Resistance Database (CARD) [49], as well as genes associated with biocide resistance with Antibacterial Biocide and Metal Resistance Genes Database (BacMet) [50]. Virulence-associated genes were identified by using Virulence Factor Database (VFDB, https://www.mgc.ac.cn/VFs/main.htm, last accessed February 2026) [51].The Bacterial and Viral Bioinformatics Resource Center (BV-BRC) [52] was used for gene prediction and annotation. All genomes were uploaded to BV-BRC, and genes were annotated and predicted using the Genome Annotation tool. The DNA sequences of the siet and expB genes from the reference genome ED99 (CP002478.1) were BLASTed against all our genomes to confirm their presence. PathogenFinder 2 was used to predict pathogenic capacity on humans from analyzed isolates (https://genepi.food.dtu.dk/pathogenfinder, last accessed February 2026 [53]. A custom ad hoc core‑genome multilocus sequence typing (cgMLST) scheme was generated using Ridom SeqSphere (Client Version 10.5.4, Server Version 10.5.0; Ridom GmbH) to assess the genetic relatedness of the 7 S. pseudintermedius isolates, that were whole- genome sequenced. The S. pseudintermedius strain NCTC5661 (Accession NZ_LR134267.1) was used as the seed genome for target definition. A total of 65 query genomes were included in the target discovery analysis. After automated locus identification and quality filtering, the ad hoc scheme comprised 1,079 cgMLST targets. Additionally, 1,223 loci were classified as accessory genome targets and 66 loci were discarded due to insufficient conservation or failing quality criteria.
The sequences generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) and are available under BioProject accession number PRJNA1484583. Basic information about the genomic sequences is presented in S3 Table.
Results
Isolates
Overall, 46 (10.2%; 95% CI: 7.6–13.4) isolates belonging to SIG were detected in the 451 oral samples: 41 from Z. californianus (11.9; 95% CI: 8.7–15.8), including 38 S. pseudintermedius and three S. delphini; two from M. angustirostris (2.7%; CI: 0.3–9.3), including two S. pseudintermedius; two from Callorhinus ursinus (22.2%; CI: 2.8–60.0), including bothS. pseudintermedius and S. delphini; and a single S. pseudintermedius isolate from Phoca vitulina (9.1%; CI: 0.2–41.3) (Table 1).
Antimicrobial susceptibility testing and detection of resistance genes
Thirty-nine isolates, including all S. delphini, were susceptible to all tested antimicrobial agents. Five isolates were only resistant to penicillin. One isolate displayed combined resistance to penicillin and tetracycline, and one isolate was solely resistant to tetracycline. DNA microarray analysis revealed that in all penicillin-resistant isolates, blaZ genes were detected, whereas resistance to tetracycline was mediated by the tet(M) gene (Table 1). Analyses of genomes via ResFinder and CARD confirmed that blaZ operon (blaZ, blaI, blaR) was present in penicillin resistant isolates, and tet(M) in tetracycline resistant isolate. None of the isolates analyzed were multidrug-resistant [54], and no methicillin-resistant S. pseudintermedius (MRSP) were observed. Among antibacterial biocide and metal-resistance genes, BacMet database, that is a bioinformatics resource that provides comprehensive information on antibacterial biocide and metal resistance genes, search revealed arsB (Arsenic pump membrane protein) and fabI (Enoyl-[acyl-carrier-protein] reductase [NADPH]) in all isolates. In addition, arsC was observed solely in S. delphini.However all had homology slightly higher than 80%.
Genotyping of SIG-species and their virulence characteristic
Forty-two isolates shared the highest rpoB and nuc DNA sequence similarities with the type strain of S. pseudintermedius (S. pseudintermedius LMG22219T), whereas four isolates shared the highest rpoB and nuc DNA sequence similarities with the type strain of S. delphini (S. delphini NCTC12225T) (S1 and S2 Figs). In twenty-six isolates spa region could be amplified and sequenced and all yielded novel unknown spa types (Table 1). Twenty isolates were non-typable. A total of 29 MLVA types were identified (S3 Fig.), with their eight-digit profiles presented in Table 1. MLVA type 9 was the most prevalent (n = 5, 17.2%), followed by type 19 (n = 4, 13.8%) and type 6 (n = 3, 10.3%). Types 2, 10, 11, 14, 17, 20, 22, and 29 were each detected in two isolates (n = 2, 6.9%), while the remaining 18 types occurred as singletons (n = 1, 3.4%) (Table 1, S3 Fig.3).
Among eight selected isolates, wherefrom whole genomes were sequenced, seven were identified by JSpecies as S. pseudintermedius while one was S. delphini. Overall, four STs were observed among S. pseudintermedius, one isolate belonged to ST453 and three to novel STs (ST3046, ST3047, and 3048) (Fig 1). cgMLST also revealed similarities between isolates of the same STs (four allelic differences between ST3046 isolates and 9–11 allelic differences between ST3047 isolates) (Fig 2).
A tree was constructed by connecting nodes at the Single-Locus Variant level to illustrate how STs were related.
Colors correspond to the sequence type.
All isolates tested positive by PCR for the virulence-associated genes siet, lukS, and expB. The leukocidin subunit gene lukF was detected in all but one isolate (Table 1). secCanine or expA were not observed. Various virulence associate genes were detected, ebpS, spsE, hlgA, hlgB, siet, lukS-I, lukF-I, lip, expB were observed in all S. pseudintermedius isolates, whereas icaA, and icaD were detected in four isolates (Table 2). PathogenFinder 2 predicted that all isolates posed pathogenic potential on humans (all with mean prediction >0.8).
Discussion
The present study provides a detailed microbiological analysis of S. pseudintermedius and S. delphini isolated from the oral cavities of pinnipeds, assessing their antimicrobial susceptibility, genetic diversity, and pathogenic potential at the human-wildlife interface. While studies on SIG in animals, especially S. pseudintermedius in companion animals (dogs and cats), have been widely documented, information on infections, carriage, and role of these pathogens in marine mammals is scarce. Recent studies have advanced understanding of the pinniped intestinal microbiome and intestine-associated samples [55–57]. Knowledge of their skin- and mucosa-associated microbiota, particularly within the SIG, remains limited [55–57]. S. pseudintermedius has been previously isolated from the aural discharge in Atlantic harbor seals (Phoca vitulina concolor) from the Atlantic coast in the USA [58] and in the oral cavity of Weddell seals (Leptonychotes weddellii) on James Ross Island and Seymour Island, Antarctica [59]. S. delphini has been previously isolated from purulent skin lesions of captive dolphins in Italy, although the specific host species was not identified in the original manuscript [7]. The presence of blaZ and tet(M) resistance genes is a common observation in S. pseudintermedius [60,61].
In line with previous studies, spa typing of S. pseudintermedius was an effective tool for specific isolates but lacked universal applicability across all lineages [62]. Furthermore, the MLVA scheme developed during the present study demonstrated superior discriminatory power. Its cost-effectiveness and technical simplicity make it a valuable first and/or additional step in the polyphasic molecular characterization of S. pseudintermedius and/or S. delphini consistent with previous studies applying it on other bacteria [63]. Additionally, MLVA serves as an ideal genotyping tool in laboratories with limited genomic infrastructure. Very recently, another MLVA scheme for S. pseudintermedius was developed [64] and demonstrated to be well suited for typing S. pseudintermedius. In contrast to our study, Afema et al. [64] utilized capillary electrophoresis, which enables high throughput in laboratories equipped with this technology. Both MLVA schemes could potentially be merged for use with classical electrophoresis if they meet the necessary criteria, and immediately for capillary electrophoresis in future studies to achieve even greater discriminatory power.
A direct comparison of the isolates from the previously mentioned reports with those obtained in our study presents several methodological challenges, namely, remote geographical origin and different host species. In the S. pseudintermedius PubMLST isolate database, there is a single entry associated with the marine mammals. This strain was isolated from the oral cavity of a healthy Weddell seal (Leptonychotes weddellii) in Antarctica. This particular strain belonged, to ST723 which is only associated with this animal species, and they are only distantly related (Triple Locus Variants (TLV)) STs in PubMLST database (S. pseudintermedius PubMLST, https://pubmlst.org/organisms/staphylococcus-pseudintermedius, last accesses February 2026.). Among four STs obtained during the present study, ST453 has already been established and were associated with canine urinary tract infections from Germany. ST453 had 13 Single Locus Variants (SLV). Three other novel STs ST3046, ST3047, and ST3048 have one SLV (ST2125, originated from canine MRSP in Brazil), no SLV, and one SLV (ST1992, from a healthy dog from Texas, USA), respectively (S. pseudintermedius PubMLST, https://pubmlst.org/organisms/staphylococcus-pseudintermedius, last accesses February 2026). All of these STs including loci combination of S. pseudintermedius P8688 (4-12-1-1-102-1-1 (novel ST still not generated, [59]) are distinct lineages and none are from widely recognized, large-scale lineages often associated with various infections in terrestrial animals like clonal complex (CC) 71, CC45 or CC68 [60].
The nearly ubiquitous presence of exfoliative toxin genes (siet, expB) and the leucocidin locus lukI, is in concordance with previous reports [29,62]. Furthermore, WGS revealed additional virulence-associated genes ebpS (elastin-binding protein), spsE (fibrinogen-binding protein), hlgA and hlgB (gamma-hemolyins), and lip (lipase). In some isolates, biofilm regulation genes icaA and icaB were observed, further aligning with the previous studies [59,60].
Our study, while providing unique insights into the presence of SIG within marine mammal populations, has some limitations. First, the study relied exclusively on samples collected from stranded animals. This cohort may not be representative, potentially leading to sampling bias. The geographical scope of this study is restricted to coastal Los Angeles County, which limits the representativeness of the findings. Based on our data, we cannot conclusively determine whether SIG in marine mammals represents transient colonization or is part of the normal microflora of these animals. However, the identification of very similar isolates belonging to the same ST across different individuals, locations, and temporal scales suggests that SIG may indeed constitute a component of the natural marine mammal microbiome. Finally, the potential for environmental or terrestrial contribution cannot be dismissed, because most of the Los Angeles metropolitan area drains into Santa Monica Bay. The influence of terrestrial runoff and sewage effluent on the microbial profiles observed in these marine hosts remains a critical variable for future investigations.
The detection of zoonotic SIG species (S. pseudintermedius and S. delphini) in stranded marine mammals raises important considerations for public health and wildlife management. While S. pseudintermedius is recognized as an emerging zoonotic pathogen capable of causing opportunistic infections in humans [65,66], the zoonotic potential of S. delphini has only recently been documented [67]. The presence of these bacteria in the oral cavities of marine mammals frequenting beaches and rehabilitation facilities along the highly urbanized Southern California coastline creates potential interfaces for human exposure. However, the absence of multidrug resistance and methicillin resistance in our isolates suggests that, at present, these marine-associated lineages pose a low clinical threat. Nevertheless, the potential for horizontal gene transfer in coastal environments, where terrestrial runoff, wastewater discharge, and marine ecosystems converge, could facilitate the acquisition of resistance determinants by these marine-adapted lineages. The distinct phylogenetic positioning of our novel STs (ST3046, ST3047, ST3048) and their separation from major terrestrial clonal complexes (CC71, CC45, CC68, CC551, CC556, CC1431, CC363 and CC1631 [68] may reflect ecological barriers to gene flow between marine and terrestrial SIG populations. Understanding whether marine mammals serve as transient carriers, environmental accumulators, or true reservoirs for zoonotic SIG lineages is essential for informed risk assessment.
Conclusion
This investigation reveals that marine mammals along the Southern California coast harbor distinct and diverse lineages of S. pseudintermedius and S. delphini (both zoonotic), largely separate from the common terrestrial clones associated with domestic animals and humans. The identification of novel SIG lineages in these marine species underscores the unexplored microbial diversity present in oceanic ecosystems and challenges our current understanding of staphylococcal ecology and host adaptation. By leveraging stranded marine mammals as sentinels of ocean health, this study identifies novel SIG lineages in these marine species, underscoring the unexplored microbial diversity present in oceanic ecosystems, thereby challenging our current understanding of staphylococcal ecology and host adaptation. It also establishes a critical baseline for One Health surveillance at the interface of marine wildlife, environmental, and human health.
Supporting information
S2 Table. Primers and amplification conditions of the VNTR loci.
Locus name was determined by the gene position in reference genome ED99 and “sp” standing for Staphylococcus pseudintermedius.
https://doi.org/10.1371/journal.pone.0356088.s002
(XLSX)
S3 Table. Basic information about the genomic sequences.
https://doi.org/10.1371/journal.pone.0356088.s003
(XLSX)
S1 Fig. Neighbor-Joining method phylogenetic tree generated of rpoB sequences including type strains of SIG.
Bootstrap values (expressed as percentages of 100 replications) greater than 75% are shown at branching points. The evolutionary distances were computed using the Maximum Composite Likelihood method.
https://doi.org/10.1371/journal.pone.0356088.s004
(TIF)
S2 Fig. Neighbor-Joining method phylogenetic tree generated of nuc sequences including type strains of SIG.
Bootstrap values (expressed as percentages of 100 replications) greater than 75% are shown at branching points. The evolutionary distances were computed using the Maximum Composite Likelihood method.
https://doi.org/10.1371/journal.pone.0356088.s005
(TIF)
S3 Fig. Clustering analysis of MLVA of isolates tested using the categorical coefficient (also called Hamming’s distance) and the unweighted pair group method with arithmetic mean clustering method (UPGMA).
Isolates sharing the same MLVA type are separated by a “/”.
https://doi.org/10.1371/journal.pone.0356088.s006
(JPG)
Acknowledgments
We are grateful to all of the people participating in the rescue and rehabilitation of stranded marine animals. We would also like to thank Katrin Berghamer and Michael Steinbrecher for their technical assistance, and the whole team at Marine Mammal Care Center Los Angeles (MMCCLA).
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