Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Survival and adaptation of Streptococcus phocae in host environments

  • Daniela Numberger ,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing

    num0289@gmail.com

    Affiliation Institute for Microbiology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

    ⨯
  • Ursula Siebert,

    Roles Conceptualization, Resources, Supervision, Writing – review & editing

    Affiliation Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation, Buesum, Germany

    ⨯
  • Peter Valentin Weigand

    Roles Conceptualization, Resources, Supervision, Writing – review & editing

    Affiliation Institute for Microbiology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

    ⨯

Abstract

Marine mammals are sentinel species representing the “health” of our oceans on which we are dependent. There are many threats to marine mammals including infectious diseases that increase with climate change and pollution of the marine environment. Streptococcus phocae has frequently been isolated from diseased or dead marine mammals. However, its pathogenicity and contribution to disease in marine mammals is still unknown. As bacteria including (potential) pathogens has to deal with different host environments during colonization or infection, we investigated the survival of S. phocae in fresh porcine and phocid blood, in seawater and in the presence of macrophages and (epithelial) cells from harbor seals and pigs. Furthermore, we tested adherence on and invasion of different (marine) mammalian cells by S. phocae. Our results showed that S. phocae can survive in seawater for at least 11 and 28 days at 16°C and 4°C, respectively. It is able to grow in blood of harbor and grey seals, but not in porcine blood. Furthermore, S. phocae is adherent and invasive to cells from seals and pigs, while the portion of invasive cells was higher in seal derived cells. Macrophages of harbor seals were more efficient in killing S. phocae than porcine macrophages. Our results indicate that S. phocae has strategies enabling it to adapt to the marine environment and seal hosts.

Citation: Numberger D, Siebert U, Valentin Weigand P (2024) Survival and adaptation of Streptococcus phocae in host environments. PLoS ONE 19(1): e0296368. https://doi.org/10.1371/journal.pone.0296368

Editor: Karolina Goździewska-Harłajczuk, Wrocław University of Environmental and Life Sciences: Uniwersytet Przyrodniczy we Wroclawiu, POLAND

Received: June 15, 2023; Accepted: December 12, 2023; Published: January 30, 2024

Copyright: © 2024 Numberger 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 relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by a grant of DN and PVW from the Deutsche Forschungsgemeinschaft (DFG, https://www.dfg.de/en/index.jsp), Bonn, Germany (NU 422/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This Open Access publication was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) - 491094227 "Open Access Publication Funding" and the University of Veterinary Medicine Hannover, Foundation.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The marine ecosystem is challenged by many factors such as anthropogenic pollution, fisheries, shipping, offshore-constructions, harmful algal blooms, acidification and climate change [1–7]. These factors can be stressors for marine mammals, considered as sentinels for the marine ecosystem [8, 9], and might lead to a higher susceptibility for infectious diseases through suppressive effects on their immune system [6, 10].

Beta-hemolytic streptococci are frequently found in marine mammals and are known to have pathogenic potential (for review see Numberger et al., 2021 [11]). One of the predominant species is Streptococcus phocae [11] that was first isolated from dead harbor seals in Norway 1988 suffering mostly from pneumonia [12]. In the following years, S. phocae was still mainly isolated from marine mammals including cape fur seal [13], grey seal [14], ringed seal, harbor porpoise [15], Caspian seal [16], California sea lion [17], short-beaked common dolphin [18], spotted seal [19], bottlenose dolphin [20], white whale [21], stellar sea lion [22], Guadalupe fur seal, elephant seal [23] and southern sea otters [24]. To our knowledge there is no isolation of S. phocae from healthy marine mammals reported. A recent publication describes the first isolation of S. phocae from a terrestrial mammal, the mink (Neovison vison) [25]. It was isolated from three unrelated cases of farmed minks suffered from pneumonia and dermal infection with lesions. Genome sequences revealed a close relation to harbor seal isolates suggesting a possible cross-species transmission, where fish as diet might serve as a vector [25]. The subspecies S. phocae subsp. salmonis is a severe pathogen of Atlantic salmon causing disease and lethality [26], but differs from harbor seal isolates described as S. phocae subsp. phocae. While the contribution of S. phocae in infectious disease of pinnipeds and cetacean is largely unknown, S. phocae infections in sea otters of Alaska were significantly associated with a high morbidity and mortality [24]. In the Kachemak Bay and Resurrection Bay, Alaska, where many northern sea otters died from strep syndrome, S. phocae could be detected via PCR in seawater and mussel samples [27]. The authors also observed a high presence of harbor seals in the Kachemak Bay and suggested a possible association between sea otter and harbor seal infections [27]. These data are based on PCR and do not allow any conclusions on the survival rate of S. phocae in seawater and its tolerance to high salinity and low temperatures.

Despite many reports on the wide distribution/occurrence of S. phocae in marine mammals, particularly pinnipeds, little is known about its persistence in the host and the marine environment or the possible transmission between hosts. In general, pathogenic and commensal bacteria of marine mammals are challenged by different host environments and thus need different adaptation strategies for survival and colonisation in various host niches. Such host environments include (mucosal and epithelial) cells, blood, macrophages and also seawater, which might play an important role in the transmission of bacteria between marine hosts. The first barrier are epithelial cells, to which infecting bacteria need to adhere in order to colonize the host. For dissemination, bacterial pathogens may invade the cells or tissue in order to translocate into the blood stream. The ability of a pathogen to survive in host blood is a prerequisite for a successful systemic infection and is unknown for S. phocae. In the tissue, resident immune cells are crucial for bacterial clearance. For S. pyogenes it has been described that tissue resident macrophages [28] and dendritic cells [29] play a crucial role to limit streptococcal dissemination and initiate protective immune defences. Marine mammals have direct contact to the marine environment including the seawater. Thus, having marine mammals as host seawater is another environment that S. phocae needs to challenge with, e.g. for transmission through seawater.

In this study we investigated the behaviour of three different S. phocae isolates from harbor seal, grey seal and harbor porpoise in the three aforementioned “host environments” of harbor seals, including seawater, seal cells, blood and monocyte-derived macrophages (MDM) as immune-active cells. We incubated three different isolates of S. phocae in seawater from the German North Sea at 4°C and 16°C and inoculated fresh blood of harbor seals. Additionally, we co-incubated S. phocae with monocyte-derived macrophages from harbor seals. The results were compared to experiments in the porcine system as a terrestrial mammal to see adaptations to the marine host.

Material and methods

Streptococcus phocae and Streptococcus suis isolates

Streptococcus phocae isolates were collected during examinations of dead harbor seals (Phoca vitulina), grey seals (Halichoerus grypus) and harbor porpoises (Phocoena phocoena) found during 1996–1999 (Table 1). All isolates were identified as S. phocae by sequencing and MALDI TOF MS Microflex LT/SH (Biotyper) with MBT Compass Library revision L (Nov. 2020; Bruker Corporation, MA, USA). Streptococcus suis, serotype 2 was kindly provided by H. Smith (Lelystad, Netherlands [30]) S. phocae and S. suis were cultured in BD Bacto™ Todd Hewitt Broth (THB, Becton Dickinson, Heidelberg, Germany) or on Columbia agar plates supplemented with 7% (v/v) sheep blood (OxoidTM, Thermo Fisher Scientific, Waltham, MA, USA). All experiments were done with S. phocae stocks take from the log phase.

thumbnail
Download:
Table 1. Information of Streptococcus phocae isolates used in this study.

https://doi.org/10.1371/journal.pone.0296368.t001

Sampling and preparation of seawater and blood from harbor seals

North Sea Water was collected in sterile 2 L glass bottles at the coast of Büsum, Germany and transported to the lab at 4°C. Seawater was filtered through 0.22 μm filters for sterilization without changing the chemical or physical properties and stored at 4°C until use.

Fresh blood samples were taken from healthy harbor and grey seals at the seal rehabilitation station in Friedrichskoog, Germany during medical examination and transported to the lab in Lithium-Heparin S-Monovettes® (Sarstedt, Nümbrecht, Germany) within 3 hours at 4°C. The medical examination was part of the preparation for releasing the animals to the wild after successful rehabilitation. No animals were harmed or sacrificed. Porcine blood was provided by the Clinic for Swine and Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover, Foundation as part of a practical teaching course.

Incubation of S. phocae isolates in seawater and blood samples

For testing the persistence of S. phocae in seawater, 100 ml of sterile-filtered seawater were inoculated with 106 colony forming units (CFU). Positive controls were run in THB and sterile-filtered seawater was tested negatively for the presence of S. phocae and any other microorganisms. To test different temperatures, the inoculated samples and controls were incubated at 4°C and 16°C. Experiments were repeated three times (biological replicates). Every hour or on a daily basis subsamples were taken for plating in triplicates (technical replicates).

For each S. phocae isolate 1 ml of fresh blood from harbor seals (n = 3), grey seals (n = 3) or pigs (n = 3) were inoculated with 106 CFU and incubated at 37°C while rotating. Every hour subsamples were taken for plating in triplicates (technical replicates). Controls in 1 ml THB were incubated and plated in parallel. Blood without bacterial inoculum was plated as negative control.

As all three S. phocae isolates were not able to grow in seawater and decreased over time when incubating in seawater, we were interested if the salinity could be a limiting factor. So, we checked the salt tolerance of one of the S. phocae isolates (Sp55) in comparison to the non-marine Streptococcus suis. To do so, we used THB medium, in which both streptococcal species can grow and added different concentration of sodium chloride. The average salinity of the North Sea is 35 PSU (practical salinity unit and the average salinity of the Baltic Sea is 18 PSU. As we can find harbor seals in both marine environments, we tested these two salinities and added 18 g/l = 18 PSU or 35 g/l = 35 PSU sodium chloride to THB medium. After sterilization the two bacterial species were incubated in triplicates at 37°C in 50 ml tubes and CFU was determined by plating subsamples on blood agar plates after three, six and 24 hours. S. suis was also incubated at 4°C in seawater from the North Sea and checked daily by plating a subsample in comparison to only THB. Statistical analyzes to test for significant differences between the strains were performed by using unpaired t-test (p < 0.05) in Prism 9 by GraphPad Software Version 9.0.0 (121).

Isolation of monocytes

Peripheral blood mononuclear cells (PBMC) from harbor seals and pigs were isolated from fresh heparin blood according to Reichel et al., 2015 [31]. After dilution of heparinized blood with PBS (phosphate buffered saline; Sigma-Aldrich, Taufkirchen, Germany) containing 0.02% EDTA (1:3 ratio; Carl Roth, Karlsruhe, Germany), 20 mL of a Biocoll® separating solution (1.077 g/ml; Bio&SELL GmbH, Nürnberg, Germany) was used to separate 12 mL of diluted blood by centrifugation (800 ×g for 45 min, 4°C). The lymphocyte/monocyte layer was collected and washed three times in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) (400 ×g, 10 min, 4°C). Phocid cells from 12 mL blood were seeded into a 75 cm2 tissue plastic flask (Greiner Bio-One, Frickenhausen, Germany), previously coated with 2% sterile gelatin (Carl Roth, Karlsruhe, Germany) solution followed by autologous harbor seal plasma and allowed to adhere in RPMI 1640 medium supplemented with 10% FCS (fetal calf serum), 1% penicillin/streptomycin (Sigma-Aldrich, Taufkirchen, Germany). For porcine cells uncoated flasks were used. Cells were incubated at 37°C and 5% CO2. Non-adherent cells were removed after 24 h and adherent monocytes were supplied with fresh medium. After one week of differentiation, monocyte-derived macrophages (MDM) were used for subsequent experiments. An unpaired t-test (p < 0.05) was used for statistical analyzes (GraphPad Prism Version 9.0.0 (121)).

Killing assay with monocyte-derived macrophages (MDMs)

Adherent MDM, isolated from porcine or seal blood, were washed twice with PBS and detached using accutase (PAN-Biotech GmbH, Aidenbach, Germany) following the manufacturer’s protocol. The cells were resuspended in RPMI 1640 medium and counted using a Neubauer chamber. MDM were infected with a multiplicity of infection (MOI) of 10:1 by co-incubating 2.5 × 106 bacterial CFU/ml of each S. phocae isolate with 2.5 × 105 MDM cells/ml in RPMI 1640 medium (w/o antibiotics). The experiments were run in triplicates. Subsamples were plated on blood agar plates in triplicates every hour for CFU counting. A control of bacteria was run in RPMI 1640 medium without MDM. Data were tested for statistical significance by using unpaired t-test (p < 0.05) in Prism 9 by GraphPad Software Version 9.0.0 (121).

Immortalized cell lines and primary cells

Cell lines of the kidney (SEK2b) and dermis (SED) of a harbor seal were kindly provided by Dr. Matthias König from the Friedrich Löffler Institute, Germany. The cells were obtained from a dead harbor seal dissected in the seal breeding station Friedrichskoog, Germany and immortalized via transfection of the SV40 T-antigen and the pSV3neo plasmid [32, 33]. New-born pig tracheal epithelial cells (NPTr) were kindly provided by F. Meurens (Nantes, France) [34]. Primary cells were obtained from kidneys of freshly dead harbor seals (pvPRC) or harbor porpoises (ppPRC) necropsied at the Institute for Terrestrial and Aquatic Wildlife research, University of Veterinary Medicine Hannover, Foundation, Büsum, Germany. This was part of a monitoring program that is funded by Schleswig-Holstein, Germany and approved by the responsible ministry. No animals were harmed or sacrificed for this study. Fresh pieces of the kidney were incubated for 48–72 hours at 4°C in Dulbecco’s Modified Eagle Medium (DMEM; high glucose; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.1 mg/ml Penicillin-Streptomycin, 2.5 μg/ml amphotericin B, 1 mg/ml protease and 10 μg/ml DNase (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). The kidney was cut in a sterile petri dish into small pieces (~ 0.5 cm3) and a 50 mL tube was prepared with 50 mL DMEM supplemented with heat-inactivated FCS to which a cell strainer (100 μm) was applied to collect the renal cells. The small pieces of the kidney were disrupted by mincing and washing them through the cell strainer by using the plunger of a syringe. The cells were then centrifuged at 250 ×g for 5 min at 4°C. In addition, the medium in which the kidney had been incubated for 48–72 hours was also centrifuged to collect potential detached cells. The supernatants were discharged, and the cell pellets were washed twice with cold phosphate buffered saline (PBS, Sigma-Aldrich, Taufkirchen, Germany). Following, the cell pellets were resuspended in Renal Epithelial Cell Medium 2 (RECM, PromoCell GmbH, Heidelberg, Germany) supplemented with 10% (v/v) FCS + 0.1 mg/ml Penicillin-Streptomycin, 2.5 μg/ml amphotericin B and 50 μg/ml gentamicin (Carl Roth, Karlsruhe, Germany) and incubated in a cell culture dish (Ø 150 mm; Sarstedt, Nümbrecht, Germany) for 2 hours at 37°C and 5% CO2 to allow fibroblasts to adhere. The supernatants were finally transferred to collagen I (Sigma-Aldrich, Taufkirchen, Germany)-coated T-75 cell culture flasks (Sarstedt, Nümbrecht, Germany) in RECM medium including supplements mentioned above. Medium was changed after 24 hours and then every 2 days. If required, primary cells were detached and split using a cell scraper.

Infection of mammalian cells with S. phocae

Mammalian cells were seeded on 24-well plates and infected with the three different S. phocae isolates (Table 1) at MOI = 5 in duplicates (technical replicates). Each infection was repeated three times for biological replicates. Infected cells were incubated at 37°C and 5% CO2. For double immune fluorescence (DIF) infection was additionally performed on glass cover slips (thickness 1 round, Ø: 12 mm; Carl Roth, Karlsruhe, Germany). Each sample that was taken for CFU counting was diluted serially and several dilutions were plated in triplicates on blood agar plates. For determining the bacterial growth, the supernatant was plated at 0, 3, 6 and 20 hours post infection (p.i.). To get the CFU of adherent plus invasive bacteria, the cells were washed three times with PBS, detached with 1× Trypsin-EDTA (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and lysed with 1% saponin (Carl Roth, Karlsruhe, Germany). The lysed cell suspension was again plated for CFU counting. Invasive bacteria only were quantified by lysis after extracellular antibiotic treatment with 100 μg/mL penicillin and 100 μg/mL gentamicin and plated for CFU counting. Data were statistically analyzed using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, San Diego, CA, USA). Figures show means and standard errors (SEM). Statistical significance was determined by unpaired, nonparametric Mann-Whitney test unpaired t-test using Prism 9 by GraphPad Software Version 9.0.0 (121) (p < 0.05 was considered statistically significant).

Double-Immune-Fluorescence (DIF) microscopy

Coverslips with adhered infected cells were washed with PBS and blocked with 5% FCS for 20 min at room temperature. Then, the coverslips were again washed three times with PBS and incubated with the primary polyclonal rat antibody against S. phocae (1:100, Davids Biotechnology, Regensburg, Germany) for 90 min in a dark humid chamber. After washing the coverslips three times with PBS the Alexa Fluor® 488 goat-anti-rat IgG secondary antibody (1:1000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was added and incubated for 30 min in a dark humid chamber. Another three wash steps in PBS were followed by permeabilization of mammalian cells with 0.2% Triton® X 100 (Carl Roth, Karlsruhe, Germany) for 5 min. Then, intracellular bacteria were stained by repeating the steps above, but using Alexa Fluor® 568 goat-anti-rat IgG secondary antibody (1:1000, Invitrogen, Thermo Fisher Scientific,Waltham, MA, USA) to distinguish between adherent and invasive bacteria. After washing three times in PBS, nuclei were stained by 0.5 μg/ml DAPI (Cell Signaling Technology, Leiden, Netherlands) for 10 min and cover slips were mounted with ProLong® Gold antifade reagent (Cell Signaling Technology, Leiden, Netherlands) at 4°C overnight. Microscopy of infected cells was performed with a Nikon Eclipse Ti-S microscope equipped with a Nikon DS-QiMC-U2 camera controlled by NIS-Elements BR version 4.51.01 (Nikon, Tokyo, Japan). For display purposes, images were identically adjusted for contrast and brightness using Fiji-ImageJ version 1.53c.

Results

Persistence of S. phocae in seawater at 4 and 16°C

As a colonizer/pathogen of marine mammals it is inevitable that S. phocae is exposed to seawater. Thus, we tested the survival rate of S. phocae in seawater at 4°C and 16°C using sterile-filtered Seawater from the North Sea, Germany. At 4°C, S. phocae was still culturable after 28 days (Fig 1A). The first 17 days all S. phocae isolates were stable at ~ 104 CFU/ml and decreased from day 17 to day 28 to an average of 9.9 × 102 CFU/ml (Sp16, Sp55) and 3.3 × 101 CFU/ml (Sp1). S. phocae survived in seawater at 16°C for at least 11 days while a decrease in viability could be observed starting from 24 hours on (Fig 1B). The first 6 hours all three isolates were quite stable at an average of 3.0 × 104 CFU/ml, respectively. From day 1 to day 11 all three S. phocae isolates decreased from an average of 7.1 × 103 CFU/ml to an average of 4.4 × 101 CFU/ml, respectively. Statistical analyzes showed that all three isolates behave similarly in seawater, as there were no significant differences.

thumbnail
Download:
Fig 1.

Persistence of S. phocae in seawater at 4°C (A.) and 16°C (B.). 100 ml of sterile-filtered seawater from the North Sea was inoculated with 106 CFU of three S. phocae isolates Sp1 (white circle), Sp16 (light grey square) and Sp55 (dark grey triangle). Graphs show the mean with standard error of mean (SEM).

https://doi.org/10.1371/journal.pone.0296368.g001

The salt tolerance of S. phocae was tested in Todd Hewitt Broth supplemented with 18 g/l NaCl (18 PSU) and 35 g/l (35 PSU) and compared to a non-marine streptococcal species, the porcine pathogen S. suis. As there were no differences between the S. phocae isolates, we only used Sp55 for the comparison to S. suis. While S. phocae was growing in 18 and 35 PSU, S. suis was not able to grow. Also, S. suis did not survive as long as S. phocae in seawater at 4°C but died within 14 days (S1 Fig).

Survival and growth of S. phocae in porcine and seal blood

Blood is a host environment that many pathogens are exposed to during their dissemination. Therefore, we analyzed the survival of S. phocae in blood from harbor seals, grey seals and compared it with the survival in porcine blood as a terrestrial mammal. All three S. phocae isolates showed growth in both harbor seal and grey seal blood increasing two magnitudes, but not in porcine blood (Fig 2). It was able to increase from average 3.3 × 105 CFU/ml to 1.2 × 107 CFU/ml within 6 hours in harbor seal blood and from average 6.9 × 104 CFU/ml to 3.4 × 106 CFU/ml in grey seal blood. In porcine blood S. phocae showed a drop within the first two hours and was then stable from two to six hours at an average of 5.2 × 104 CFU/ml. No significant differences between the three isolates were observed. Besides, a drop of bacterial concentration within the first hour could be observed as a result of acclimatization of bacteria. The inoculation dose was hence a “target dose” od 106 CFU. As it was not relevant for showing the growth of S. phocae, we decided to remove that data points from Fig 2.

thumbnail
Download:
Fig 2.

Incubation of S. phocae in blood in harbor seals (A.), grey seals (B.) and pigs (C.). 1 ml of heparin blood was inoculated with 106 CFU of three S. phocae isolates Sp1 (white circle), Sp16 (light grey square) and Sp55 (dark grey triangle). Graphs show the mean with standard error of mean (SEM).

https://doi.org/10.1371/journal.pone.0296368.g002

Co-incubation of S. phocae with monocyte-derived macrophages (MDM)

When entering the host, S. phocae might be attacked by the host immune system.

To investigate, if S. phocae is killed by phocid and porcine macrophages, we isolated monocytes from blood of seals and swine. After differentiation to (monocyte-derived) macrophages (MDM), they were incubated with the three S. phocae isolates Sp1, Sp16 and Sp55 in RPMI 1640 medium (10% FCS, w/o antibiotics). The CFU of S. phocae decreased within 120 minutes independent of the host origin (seal or swine) (Fig 3). However, a stronger decrease could be observed with seal MDM in comparison to porcine MDM. While seal MDM cause a decrease of two magnitudes from average 3.3 × 106 CFU/ml to 1.7 × 104 CFU/ml, S. phocae decreased only one magnitude with porcine MDM (average 2.9 × 106 CFU/ml to 3.4 × 105 CFU/ml). Again, no significant differences between the three isolates were observed as revealed by unpaired t-test.

thumbnail
Download:
Fig 3. Killing assay with S. phocae and monocyte-derived macrophages (MDM) from harbor seals and pigs.

MDM were co-incubated with three S. phocae isolates Sp1 (white circle), Sp16 (light grey square) and Sp55 (dark grey triangle) at MOI = 10. Graphs show the mean with standard error of mean (SEM).

https://doi.org/10.1371/journal.pone.0296368.g003

Adherence to and invasion of mammalian cells

Pathogens are able to adhere to and sometimes to invade epithelial (mucosal) cells. To test the adherence and invasion of S. phocae to cells from the dermis and kidney of harbor seals and compared it to the infection of a porcine cell line, the NPTr (newborn pig trachea) cells. The infection experiments demonstrated that the three S. phocae isolates Sp1, Sp16 and Sp55 were both adherent and invasive in all tested cell types (Fig 4). In all experiments, the CFU of bacteria associated with cells (adherent and invasive) increase from three to six hours, while in most cases the number of invasive bacteria alone differ only slightly from three to six hours. Statistical analyzes did not show any significant differences between the three S. phocae isolates. However, the highest number of invasive bacteria was observed in the seal dermis (SED) and seal kidney (SEK2b) cell lines and the lowest number in the NPTr cell line. Statistical analyzes did not reveal significant differences. However, the p-value for invasive bacteria in NPTr versus SEK2b cells was 0.0569, so quite close to significance. The highest number of invasive bacteria in NPTr cells was Sp16 with 3.6 × 103 CFU/ml at 6 hours, while the SEK2b cells had up to 1.5 × 106 CFU/ml and the SED cells up to 4.9 × 105 CFU/ml invasive bacteria at six hours p.i.

thumbnail
Download:
Fig 4. Adherence and invasion of S. phocae to different (marine) mammalian cells.

Cells were infected with three S. phocae isolates Sp1 (white), Sp16 (light grey) and Sp55 (dark grey) at MOI = 5. Bars show the mean with standard error of mean (SEM). NPTr (new-born pig trachea cells); SED (seal dermis cells); SEK2b (seal kidney cells); pvPRC (Phoca vitulina primary renal cells); ppPRC (Phocoena phocoena primary renal cells). The data at 20 h p.i. are not shown, because the cells started to die. No significant differences could be detected using GraphPad Prism version 8.0.1 for Windows (GraphPad Software, San Diego, CA, USA) and unpaired, nonparametric Mann-Whitney test.

https://doi.org/10.1371/journal.pone.0296368.g004

Double-immunofluorescence analysis is based on antibody staining of extracellular bacteria followed by staining of intracellular bacteria after permeabilization. While intracellular bacteria were shown with red fluorescence only, some of the adherent bacteria showed a yellowish staining due to the double-staining and overlapping of red and green fluorescence. It supported the results on adherence and invasion by showing a clear distinction of adherent and invasive bacteria (S2 Fig).

Discussion

In this study we investigated the survival in and adaptation of S. phocae to seal host environments to which the bacteria are exposed during colonization and invasion. For this, we tested the survival of S. phocae in blood and seawater, investigated bacterial adherence to and invasion of host epithelial cells and analyzed phagocytic killing by monocyte-derived macrophages. For comparison to a terrestrial mammal we used porcine blood and host cells, respectively.

In comparison to the swine pathogen S. suis, S. phocae was able to grow in Todd Hewitt Broth supplemented with 35 g/L NaCl (35 PSU) indicating a high salt tolerance and an adaptation to the marine environment. Furthermore, S. phocae was able to survive at least 11 and 28 days in seawater at 16°C and 4°C, respectively. This shows that the survival rate in the marine environment increases with lower water temperatures confirming an adaptation to the marine environment another adaptation, as most pinnipeds and many other marine mammals live in temperate to polar regions [35]. Bacteria are known to reduce their metabolic activity at lower temperatures to protect themselves against cold shock [36, 37]. We assume that growth of S. phocae was limited by nutrients in our experiment, as a tendency for longer survival was observed in the THB control. Thus, S. phocae might be able to persist for even longer times in the marine environment, where the availability of nutrients might be better through natural nutrient cycles or plankton and algal blooms [38, 39]. To conclude, the transmission of S. phocae through seawater is a possible scenario including filtrating organisms (e.g. mussels) that might accumulate S. phocae and are the prey of several marine mammalian species such as sea otters [40, 41]. Further studies are required to identify factors that influence the survival rate of S. phocae such as particle association and high nutrient concentrations. It is known that bacteria including fecal bacteria have higher survival rates in sediment than in water [42] as well as when associated with particles [43, 44] demonstrating the protective features of those environments. In addition, particles and sediment often have higher concentrations of nutrients and organic matter and thus, may increase survival rates [45–47]. This could also be true for S. phocae. A recent study could detect S. phocae in 37% of nasopulmonary mite samples (family Halarachnidae) from southern sea otters and California sea lions indicating those mites as potential vector for bacterial pathogens [48]. Moreover, the association with zooplankton has protective and promotive features for bacteria including pathogens, which again might enhance the bacterial survival rate [49].

The results of the incubation of S. phocae in fresh seal blood showed that it is able to grow within 6 hours, indicating pathogenic potential after entering the seal host bloodstream. In contrast, S. phocae could not grow in porcine blood, but persisted for at least 6 hours. This indicates an adaptation to the marine mammalian host or seals in particular, e.g. by enhanced strategies for immune evasion in the marine host. The streptococcal M-protein, for instance, is known to bind host fibrinogen as immune evasion strategy by inhibiting the complement system [50, 51]. This might be one of many processes that are host specific as it has been shown for canine fibrinogen-binding proteins of Staphylococcus pseudintermedius [52].

Co-incubation with phocid and porcine monocyte-derived macrophages showed a clear killing effect of S. phocae. One explanation could be that S. phocae is able to evade in the blood by binding host proteins such as fibrinogen, which is not present in co-incubations with only MDM. Another possible evasion factor of many streptococcal species is the antiphagocytic capsule of streptococci, which can also depend on complement factors in the host blood [53, 54]. The multiplication of encapsulated S. suis in murine blood is restricted by the host complement system, as shown by a significantly higher multiplication rate in wild-type blood than in blood lacking the complement factor C3 [54]. In the presence of S. suis antiserum, no multiplication could be observed [54]. In another study authors could show that 48% of bovine mammary macrophages ingested encapsulated Streptococcus uberis with a killing rate of 35% and after opsonization of S. uberis with homologous antiserum it was 84% and 52%, respectively [53]. Further studies are necessary including co-cultivation with other blood components such as neutrophils and the complement system. The stronger decrease of S. phocae with seal MDM in comparison to porcine MDM could be an adaptation of the seal host to S. phocae infections and thus, be more efficient in killing S. phocae in contrast to porcine MDM. The MDM of the two different hosts might have different receptors for the recognition of S. phocae, as it has been shown for complement receptors of murine and human MDM and the pathogen Salmonella typhi [55].

Infection experiments with S. phocae investigated the adherence to and invasion in different host epithelial cells. We did not observe any significant differences by statistical tests. However, there was a higher number of invasive bacteria in seal derived cells, particularly SEK2b cells than in the porcine NPTr cells. An unpaired t-test revealed a p-value of 0.0569, which is close to significance. Nonetheless, further studies are necessary to confirm these differences. Although adherence is mainly mediated by specific adhesins and other mechanisms that might be host-dependent [56, 57], passive and host-independent hydrophobic interactions might play a role in unspecific adherence [58]. In contrast, invasion needs active interaction with the host-cell and might therefore be host-dependent and–specific, e.g. receptor binding [59]. Host-specificity of S. phocae was also demonstrated in infection experiments of Atlantic salmon (Salmo salar) by González-Contreras et al. [60], where only the S. phocae isolate from salmon, not from seal caused mortality or pathological signs in fish. Subsequently, two subspecies were described based on more differences between salmon and seal isolates, Streptococcus phocae subsp. salmonis and S. phocae subsp. phocae, respectively [61]. A role as a commensal is also possible as it has been demonstrated for S. suis showing pathogenic and commensal behavior [62]. However, data on the prevalence of S. phocae in healthy individuals are still missing and therefore should be investigated in further studies.

Our data suggest the idea of a co-evolution of S. phocae and the seal host. S. phocae seems to be adapted to the seal by its ability to proliferate in seal blood and enhanced invasion mechanisms of cells from seals than pigs. The seal host showed a more efficient killing of S. phocae by macrophages than the porcine host. Following studies should focus on the mechanisms behind our experiments to support and explain our findings.

Supporting information

S1 Fig.

Incubation of Streptococcus suis and S. phocae Sp55 in Todd Hewitt Broth (THB) supplemented with 35 g/l (= 35 PSU) or 18 g/l (= 18 PSU) sodium chloride at 37°C to test for salt tolerance between the two streptococcal species [A+B]. A control of THB without salt (= 0 PSU) was included [C]. The growth of S. suis in sterile-filtered seawater from the North Sea and THB as a control was additionally investigated [D]. Experiments were run in triplicates. PSU = practical salinity unit (1 PSU = 1g /l).

https://doi.org/10.1371/journal.pone.0296368.s001

(PDF)

S2 Fig. Double-immune fluorescence of different mammalian cells infected with S. phocae.

Only three samples are shown representing the three situations for a qualitative evaluation of the adherence and invasion behaviour of S. phocae: adherent (green/yellowish) and invasive (red) bacteria (first row, NPTr cells, Sp16, 3 h p.i.); only invasive (red) bacteria (second row, ppPRCs, Sp16, 20 h p.i.) and only adherent (green/yellowish) bacteria (third row, SED cells, Sp16, 6 h p.i.).The first column shows the nucleus of eukaryotic cells by the blue fluorescence of DAPI. The second column shows the adherent bacteria by the green or fluorescence of the second antibody Alexa Fluor® 488 goat-anti-rat IgG. The third column shows the invasive bacteria by the red fluorescence of the second antibody Alexa Fluor® 568 goat-anti-rat IgG. The fourth column shows the merged channels including differential interference contrast (DIC) capture to see the cell body. Scale bar 10 μm. All images original magnification 600×.

https://doi.org/10.1371/journal.pone.0296368.s002

(PDF)

S1 Graphical abstract.

https://doi.org/10.1371/journal.pone.0296368.s003

(TIF)

Acknowledgments

We are thankful for sampling and sending the organs from harbor seals as well as North Sea water by the veterinarians Dr. Stephanie Groß, Jana Christina Klink, Simon Rohner, Dr. Luca Aroha Schick, Lotte Caecilia Striewe and Jan Lakemeyer from the Institute for Terrestrial and Aquatic Research in Büsum, Germany. We would like to thank the seal rehabilitation center in Friedrichskoog for providing fresh blood samples from harbor and grey seals. Furthermore, we are very grateful to Dr. Matthias König and Dr. Sylvia Kohler from the Friedrich-Löffler-Institute for providing the cell lines SED and SEK2b. Finally, we want to thank the Clinic for Swine and Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover, Foundation for providing blood from pigs, which was taken as part of a practical course for students. We are also thankful for the support with statistical analyzes by Jörg Merkel.

References

  1. 1. Shuval H. Estimating the global burden of thalassogenic diseases: human infectious diseases caused by wastewater pollution of the marine environment. J Water Health. 2003;1: 53–64. pmid:15382734
  2. 2. Nelms SE, Barnett J, Brownlow A, Davison NJ, Deaville R, Galloway TS, et al. Microplastics in marine mammals stranded around the British coast: ubiquitous but transitory? Scientific Reports. 2019;9: 1075. pmid:30705316
  3. 3. Desforges J-PW, Sonne C, Levin M, Siebert U, De Guise S, Dietz R. Immunotoxic effects of environmental pollutants in marine mammals. Environment International. 2016;86: 126–139. pmid:26590481
  4. 4. Bearzi G, Politi E, Agazzi S, Azzellino A. Prey depletion caused by overfishing and the decline of marine megafauna in eastern Ionian Sea coastal waters (central Mediterranean). Biological Conservation. 2006;127: 373–382.
  5. 5. Fire SE, Wang Z, Byrd M, Whitehead HR, Paternoster J, Morton SL. Co-occurrence of multiple classes of harmful algal toxins in bottlenose dolphins (Tursiops truncatus) stranding during an unusual mortality event in Texas, USA. Harmful Algae. 2011;10: 330–336.
  6. 6. Sanderson CE, Alexander KA. Unchartered waters: Climate change likely to intensify infectious disease outbreaks causing mass mortality events in marine mammals. Global Change Biology. 2020;26: 4284–4301. pmid:32558115
  7. 7. Hofmann GE, Barry JP, Edmunds PJ, Gates RD, Hutchins DA, Klinger T, et al. The effect of ocean acidification on calcifying organisms in marine ecosystems: an organism-to-ecosystem perspective. Annual Review of Ecology, Evolution, and Systematics. 2010;41: 127–147.
  8. 8. Bossart GD. Marine mammals as sentinel species for oceans and human health. Vet Pathol. 2011;48: 676–690. pmid:21160025
  9. 9. Moore SE. Marine mammals as ecosystem sentinels. J Mammal. 2008;89: 534–540.
  10. 10. Ross PS. The role of immunotoxic environmental contaminants in facilitating the emergence of infectious diseases in marine mammals. Human and Ecological Risk Assessment: An International Journal. 2002;8: 277–292.
  11. 11. Numberger D, Siebert U, Fulde M, Valentin-Weigand P. Streptococcal infections in marine mammals. Microorganisms. 2021;9: 350. pmid:33578962
  12. 12. Skaar I, Gaustad P, Tønjum T, Holm B, Stenwig H. Streptococcus phocae sp. nov., a new species isolated from clinical specimens from seals. International Journal of Systematic and Evolutionary Microbiology. 1994;44: 646–650. pmid:7981096
  13. 13. Henton MM, Zapke O, Basson PA. Streptococcus phocae infections associated with starvation in Cape fur seals: case report. Journal of the South African Veterinary Association. 1999;70: 98–99.
  14. 14. Vossen A, Abdulmawjood A, Lämmler C, Weiß R, Siebert U. Identification and molecular characterization of beta-hemolytic streptococci isolated from harbor seals (Phoca vitulina) and grey seals (Halichoerus grypus) of the German North and Baltic Seas. J Clin Microbiol. 2004;42: 469–473. pmid:14715808
  15. 15. Raverty SA, Gaydos JK, Nielsen O, Ross PS. Pathologic and clinical implications of Streptococcus phocae isolated from pinnipeds along coastal Washington State, British Columbia and Arctic Canada. 35th Annual Conference of the International Association of Aquatic Animal Medicine, Galveston, TX. 2004. Available: https://www.vin.com/doc/?id=6696240
  16. 16. Kuiken T, Kennedy S, Barrett T, Van de Bildt MWG, Borgsteede FH, Brew SD, et al. The 2000 canine distemper epidemic in Caspian seals (Phoca caspica): pathology and analysis of contributory factors. Vet Pathol. 2006;43: 321–338. pmid:16672579
  17. 17. Johnson S, Lowenstine L, Gulland F, Jang S, Imai D, Almy F, et al. Aerobic bacterial flora of the vagina and prepuce of California sea lions (Zalophus californianus) and investigation of associations with urogenital carcinoma. Veterinary microbiology. 2006;114: 94–103. pmid:16326045
  18. 18. Díaz-Delgado J, Sierra E, Vela AI, Arbelo M, Zucca D, Groch KR, et al. Coinfection by Streptococcus phocae and cetacean morbillivirus in a short-beaked common dolphin Delphinus delphis. Diseases of Aquatic Organisms. 2017;124: 247–252. pmid:28492181
  19. 19. Hueffer K, Lieske CL, McGilvary LM, Hare RF, Miller DL, O’Hara TM. Streptococcus phocae isolated from a spotted seal (Phoca largha) with pyometra in Alaska. Journal of Zoo and Wildlife Medicine. 2011;42: 108–112. pmid:22946378
  20. 20. Ruiz CL, Jensen ED, Johnson SP, Lutmerding BA, Smith CR, Meegan JM, et al. Strangles-like clinical presentation of Streptococcus phocae in three bottlenose dolphins (Tursiops truncatus). 42nd Annual Conference of the International Association for Aquatic Animal Medicine. 2011. Available: https://www.vin.com/doc/?id=6698522
  21. 21. Lair S, Martineau D, Measures LN. Causes of mortality in St. Lawrence estuary beluga (Delphinapterus Leucas), from 1983 to 2012. Canadian Science Advisory Secretariat Ottawa, Canada; 2014.
  22. 22. Lee K, Kim J-Y, Jung SC, Lee H-S, Her M, Chae C. First isolation of Streptococcus halichoeri and Streptococcus phocae from a steller sea lion (Eumetopias jubatus) in South Korea. Journal of Wildlife Diseases. 2015;52: 183–185. pmid:26555114
  23. 23. Taurisano ND, Butler BP, Stone D, Hariharan H, Fields PJ, Ferguson HW, et al. Streptococcus phocae in marine mammals of northeastern pacific and arctic canada: a retrospective analysis of 85 postmortem investigations. Journal of Wildlife Diseases. 2017;54: 101–111. pmid:28982020
  24. 24. Imai D, Jang S, Miller M, Conrad PA. Characterization of beta-hemolytic streptococci isolated from southern sea otters (Enhydra lutris nereis) stranded along the California coast. Veterinary Microbiology. 2009;136: 378–381. pmid:19117701
  25. 25. Nikolaisen NK, Lindegaard M, Lyhs U, Strube ML, Hansen MS, Struve T, et al. First finding of Streptococcus phocae infections in mink (Neovison vison). Research in Veterinary Science. 2021;139: 145–151. pmid:34311216
  26. 26. Romalde JL, Ravelo C, Valdés I, Magariños B, de la Fuente E, Martín CS, et al. Streptococcus phocae, an emerging pathogen for salmonid culture. Veterinary Microbiology. 2008;130: 198–207. pmid:18280675
  27. 27. Rouse NM. Interactions of Streptococcus infantarius ss coli and Streptococcus phocae in Resurrection and Kachemak Bays, Alaska. PhD Thesis, University of Alaska Anchorage. 2018.
  28. 28. Mishalian I, Ordan M, Peled A, Maly A, Eichenbaum MB, Ravins M, et al. Recruited macrophages control dissemination of group A Streptococcus from infected soft tissues. The Journal of Immunology. 2011;187: 6022–6031. pmid:22025550
  29. 29. Loof TG, Goldmann O, Medina E. Immune recognition of Streptococcus pyogenes by dendritic cells. Infection and Immunity. 2008;76: 2785–2792. pmid:18391010
  30. 30. Smith HE, Damman M, van der Velde J, Wagenaar F, Wisselink HJ, Stockhofe-Zurwieden N, et al. Identification and Characterization of the cps Locus of Streptococcus suis Serotype 2: the capsule protects against phagocytosis and is an important virulence factor. Infection and Immunity. 1999;67: 1750–1756.
  31. 31. Reichel M, Muñoz-Caro T, Sanchez Contreras G, Rubio García A, Magdowski G, Gärtner U, et al. Harbor seal (Phoca vitulina) PMN and monocytes release extracellular traps to capture the apicomplexan parasite Toxoplasma gondii. Developmental & Comparative Immunology. 2015;50: 106–115. pmid:25681075
  32. 32. Stoner GD, Kaighn ME, Reddel RR, Resau JH, Bowman D, Naito Z, et al. Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells. Cancer Research. 1991;51: 365–371. pmid:1703038
  33. 33. Parris CN, Masters JRW, Green MHL. pSV3neo transfection and radiosensitivity of human cancer cell lines. Cancer Letters. 1990;55: 171–175. pmid:2176129
  34. 34. Ferrari M, Scalvini A, Losio MN, Corradi A, Soncini M, Bignotti E, et al. Establishment and characterization of two new pig cell lines for use in virological diagnostic laboratories. Journal of Virological Methods. 2003;107: 205–212. pmid:12505635
  35. 35. Kovacs KM, Aguilar A, Aurioles D, Burkanov V, Campagna C, Gales N, et al. Global threats to pinnipeds. Marine Mammal Science. 2012;28: 414–436.
  36. 36. Weber MH, Marahiel MA. Bacterial cold shock responses. Science progress. 2003;86: 9–75. pmid:12838604
  37. 37. Barria C, Malecki M, Arraiano CM. Bacterial adaptation to cold. Microbiology. 2013;159: 2437–2443. pmid:24068238
  38. 38. Alderkamp A-C, Van Rijssel M, Bolhuis H. Characterization of marine bacteria and the activity of their enzyme systems involved in degradation of the algal storage glucan laminarin. FEMS Microbiology Ecology. 2007;59: 108–117. pmid:17233748
  39. 39. Giroldo D, Ortolano PIC, Vieira A.H. Bacteria–algae association in batch cultures of phytoplankton from a tropical reservoir: the significance of algal carbohydrates. Freshwater Biology. 2007;52: 1281–1289.
  40. 40. Rouse NM, Counihan KL, Goertz CEC, Duddleston KN. Competency of common northern sea otter (Enhydra lutris kenyoni) prey items to harbor Streptococcus lutetiensis and S. phocae. Diseases of Aquatic Organisms. 2021;143: 69–78. pmid:33570041
  41. 41. Rouse NM, Counihan KL, Boege Tobin DD, Goertz CEC, Duddleston KN. Habitat associations between Streptococcus bovis/equinus complex and Streptococcus phocae, the causative agents of strep syndrome in sea otters, and the marine environment. Marine Ecology. 2022;43: e12689.
  42. 42. Howell JM, Coyne MS, Cornelius PL. Effect of sediment particle size and temperature on fecal bacteria mortality rates and the fecal coliform/fecal streptococci ratio. Journal of Environmental Quality. 1996;25: 1216–1220.
  43. 43. Gutiérrez-Cacciabue D, Cid AG, Rajal VB. How long can culturable bacteria and total DNA persist in environmental waters? The role of sunlight and solid particles. Science of The Total Environment. 2016;539: 494–502. pmid:26379262
  44. 44. Myers EM, Juhl AR. Particle association of Enterococcus sp. increases growth rates and simulated persistence in water columns of varying light attenuation and turbulent diffusivity. Water Research. 2020;186: 116140. pmid:33096438
  45. 45. Haller L, Amedegnato E, Poté J, Wildi W. Influence of freshwater sediment characteristics on persistence of fecal indicator bacteria. Water Air Soil Pollut. 2009;203: 217–227.
  46. 46. Pote J, Haller L, Kottelat R, Sastre V, Arpagaus P, Wildi W. Persistence and growth of faecal culturable bacterial indicators in water column and sediments of Vidy Bay, Lake Geneva, Switzerland. Journal of Environmental Sciences. 2009;21: 62–69. pmid:19402401
  47. 47. He Y, Bai M, He Y, Wang S, Zhang J, Jiang S, et al. Suspended particles are hotspots for pathogen-related bacteria and ARGs in coastal beach waters of northern China. Science of The Total Environment. 2022;817: 153004. pmid:35026254
  48. 48. Pesapane R, Chaves A, Foley J, Javeed N, Barnum S, Greenwald K, et al. Nasopulmonary mites (Acari: Halarachnidae) as potential vectors of bacterial pathogens, including Streptococcus phocae, in marine mammals. PLOS ONE. 2022;17: e0270009. pmid:35709209
  49. 49. Perera IU, Fujiyoshi S, Nishiuchi Y, Nakai T, Maruyama F. Zooplankton act as cruise ships promoting the survival and pathogenicity of pathogenic bacteria. Microbiology and Immunology. 2022;66: 564–578. pmid:36128640
  50. 50. Horstmann RD, Sievertsen HJ, Knobloch J, Fischetti VA. Antiphagocytic activity of streptococcal M protein: selective binding of complement control protein factor H. PNAS. 1988;85: 1657–1661. pmid:2964038
  51. 51. Kantor FS. Fibrinogen precipitation by streptococcal m protein: II. renal lesions induced by intravenous injection of M protein into mice and rats. Journal of Experimental Medicine. 1965;121: 861–872. pmid:14278235
  52. 52. Pickering AC, Vitry P, Prystopiuk V, Garcia B, Höök M, Schoenebeck J, et al. Host-specialized fibrinogen-binding by a bacterial surface protein promotes biofilm formation and innate immune evasion. PLOS Pathogens. 2019;15: e1007816. pmid:31216354
  53. 53. Almeida RA, Oliver SP. Antiphagocytic effect of the capsule of Streptococcus uberis. Journal of Veterinary Medicine, Series B. 1993;40: 707–714. pmid:8128805
  54. 54. Seitz M, Beineke A, Singpiel A, Willenborg J, Dutow P, Goethe R, et al. Role of capsule and suilysin in mucosal infection of complement-deficient mice with Streptococcus suis. Infect Immun. 2014; IAI.00080–14. pmid:24686060
  55. 55. Ishibashi Y, Arai T. A possible mechanism for host-specific pathogenesis of Salmonella serovars. Microbial Pathogenesis. 1996;21: 435–446. pmid:8971684
  56. 56. Hasty DL, Ofek I, Courtney HS, Doyle RJ. Multiple adhesins of streptococci. Infection and immunity. 1992;60: 2147–2152. pmid:1587582
  57. 57. Nobbs AH, Lamont RJ, Jenkinson HF. Streptococcus adherence and colonization. Microbiol Mol Biol Rev. 2009;73: 407–450. pmid:19721085
  58. 58. Nesbitt WE, Doyle RJ, Taylor KG. Hydrophobic interactions and the adherence of Streptococcus sanguis to hydroxylapatite. Infection and Immunity. 1982;38: 637–644.
  59. 59. Ó Cróinín T, Backert S. Host epithelial cell invasion by Campylobacter jejuni: trigger or zipper mechanism? Frontiers in Cellular and Infection Microbiology. 2012;2. Available: https://www.frontiersin.org/articles/ pmid:22919617
  60. 60. González‐Contreras A, Magariños B, Godoy M, Irgang R, Toranzo AE, Avendaño‐Herrera R. Surface properties of Streptococcus phocae strains isolated from diseased Atlantic salmon, Salmo salar L. Journal of Fish Diseases. 2018;34: 203–215. pmid:21306587
  61. 61. Avendaño-Herrera R, Balboa S, Castro N, González-Contreras A, Magariños B, Fernández J, et al. Comparative polyphasic characterization of Streptococcus phocae strains with different host origin and description of the subspecies Streptococcus phocae subsp. salmonis subsp. nov. International Journal of Systematic and Evolutionary Microbiology. 2014;64: 1775–1781.
  62. 62. Vötsch D, Willenborg M, Weldearegay YB, Valentin-Weigand P. Streptococcus suis–The “two faces” of a pathobiont in the porcine respiratory tract. Front Microbiol. 2018;9. pmid:29599763