Staphylococcus aureus enhances biofilm formation, aerotolerance, and survival of Campylobacter strains isolated from retail meats

In retail meat products, Campylobacter jejuni, C. coli, and Staphylococcus aureus have been reported in high prevalence. The polymicrobial interaction between Campylobacter and other bacteria could enhance Campylobacter survival during the adverse conditions encountered during retail meat processing and storage. This study was designed to investigate the potential role of S. aureus from retail meats in enhancing the survival of Campylobacter exposed to low temperature, aerobic conditions, and biofilm formation. Results indicated that viable S. aureus cells and filter-sterilized cell-free media obtained from S. aureus prolonged the survival of Campylobacter at low temperature and during aerobic conditions. Biofilm formation of Campylobacter strains was significantly enhanced in the presence of viable S. aureus cells, but the results were inconclusive when extracts from cell-free media were used. In conclusion, the presence of S. aureus cells enhances survivability of Campylobacter strains in adverse conditions such as low temperature and aerobic conditions. Further investigations are warranted to understand the interaction between Campylobacter and S. aureus, and effective intervention strategies are needed to reduce the incidence of both foodborne pathogens in retail meat products.

Staphylococcus aureus can grow aerobically within a wide range of temperatures 31 ; consequently, it might provide a better environment for survival of co-contaminant Campylobacter during food processing and storage. A few reports exist documenting the coexistence of C. jejuni and S. aureus in biofilms 28,29 ; however, the influence of S. aureus strains from retail meats on the survival of C. jejuni and C. coli at low temperatures and aerobic conditions is unclear. In this study, we studied the influence of S. aureus strains from retail meat products on Campylobacter survival at low temperature, in aerobic conditions and within biofilms. Cell-free S. aureus were included to investigate the effect of extracellular metabolites on Campylobacter survival during adverse environmental conditions.

Materials and methods
Bacterial strains and culture conditions. Five Campylobacter strains, including two and three strains of C. jejuni and C. coli, respectively, and two S. aureus strains were sourced from retail meat products and used in this study (Table 1). These strains were previously isolated, characterized and sequenced in our laboratory [1][2][3][4]12,13,22,[32][33][34][35][36][37] . These strains were selected because the genomic sequences were available, and they represent different sources of retail meats. The clinical strain C. jejuni NCTC11168 was used as a reference strain. Campylobacter strains were subcultured from stock culture (stored at − 70 °C) in Mueller Hinton Agar (MHA) supplemented with 5% laked horse blood (at 42 °C) in microaerobic conditions (Oxoid CampyGen 3.5 L sachet, Thermo Scientific or Thermo Forma tri-gas incubator) for 48 h. Subcultures of Campylobacter cultures were maintained on MHA plates or Mueller Hinton Broth (MHB) as needed. S. aureus strains were grown and subcultured in Mannitol Salt Agar (MSA) at 37 °C in aerobic conditions. Preparation of cell-free extracts from S. aureus. Staphylococcus aureus strains B4-59C and B6-55A were grown in MSA for 24 h; cells were then subcultured in freshly-prepared MHB (50 ml) and incubated at 37 °C at 100 rpm for 24 h with aerobic conditions. Cell pellets were obtained after centrifugation (5000 rpm, 5 min), washed in PBS (pH 7.4), suspended in fresh MHB, and adjusted to OD 600 = 1.0. Twenty milliliters of cell suspension was added to 200 ml MHB and incubated at 4 °C for 48 h. Similarly, 10 ml of cell suspension was added to 200 ml MHB in conical flasks (250 ml) and incubated separately at 25 °C and 37 °C with 100 rpm agitation for 24 h. After incubation, inoculated MHB media were filter-sterilized (0.45 µm, Nalgene Rapid-Flow), and sterility was verified on MHA, MHA supplemented with 5% laked horse blood and MSA incubated in aerobic and microaerobic conditions at 37 °C and ambient temperature for 48 h. For each temperature setting (4 °C, 25 °C and 37 °C), experiments were performed in triplicate; filter-sterilized media were mixed and stored at − 20 °C. These sterilized media included: S. aureus grown in MHB at 4 °C (strains B4-49C-4 and B6-55A-4), S. aureus grown in MHB at 25 °C (strains B4-59C-25 and B6-55A-25), S. aureus grown in MHB at 37 °C (strains B4-59C-37 and B6-55A-37), and non-inoculated MHB (control); these media were used in survival, biofilm and aerotolerance assays of Campylobacter strains. The protein concentration of inoculated and non-inoculated MHB was measured by the BCA protein Assay kit (Pierce™).
Fractionation of media used to cultivate S. aureus. Filter-sterilized media from S. aureus cultures were stored at − 20 °C and thawed overnight at 4 °C prior to use. Filter-sterilized media were fractionated using a Ultracel-30 kDa filter (Millipore, Burlington, Massachusetts, USA), and the eluate (≤ 30 kDa) was centrifuged for 30 min at 5000 rpm. For all samples, 20 µl of non-inoculated MHB and both fractions (≤ 30 kDa and ≥ 30 kDa) were separated in 12% polyacrylamide gels by electrophoresis.

Biofilm assays. Biofilm formation using cell free extracts and < 30 kDa media fractions. Campylobacter
strains and cell-free extracts from S. aureus B4-59C-4, B4-59C-25, B4-59C-37, B655A-4, B6-55A-25 and B6-55A-37 were prepared as described above in aerotolerance and survival assays, respectively. Campylobacter monocultures and cells amended with S. aureus cell-free extracts or the ≤ 30 kDa flow-through fraction were analyzed for biofilm formation as described previously 18  One-way ANOVA (Brown-Forsythe and Welch ANOVA tests) and unpaired t-test as per need in GraphPad (Prism 9), and illustrations were created in GraphPad and Microsoft Excel.

Results
Campylobacter survival at low temperature. Prolonged survival of Campylobacter at 4 °C was found when cells were co-cultivated with S. aureus B6-55A or B4-59C as compared to cells consisting of C. jejuni and C. coli monoculture (Fig. 1A, Supplementary Fig. S1). Likewise, cell-free extracts from S. aureus B4-59C-4 and B6-55A-4 increased survival of most Campylobacter strains at 4 °C when added to the media as compared to the non-amended control (MHB) (Fig. 1B-G). Cell-free extracts from S. aureus cultured at 25 °C and 37 °C (B4-59C-25, B4-59C-37, B6-55A-25 and B6-55A-37) also enhanced prolonged survival of C. jejuni and C. coli at 4 °C as compared to MHB (Fig. 1B-G). Among most of the Campylobacter strains (both C. jejuni and C. coli strains), effects of growth medium (MHB vs cell free extracts from S. aureus cultures), time, and time X growth medium interaction were found significant on survival at lower temperature (Supplementary Table S2 Campylobacter survival during aerobic conditions. During aerobic incubation at 25 °C and 42 °C, Campylobacter survival was enhanced throughout the incubation period when cells were co-incubated with S. aureus ( Fig Biofilm formation. Mixed populations of Campylobacter and S. aureus cells produced significantly larger biofilms than Campylobacter monocultures at both 25 °C and 42 °C (Fig. 3A,B, Supplementary Fig. S3). Biofilm formation was also greater with mixed populations of Campylobacter and S. aureus B4-59C than S. aureus monocultures at both temperatures. However, biofilm formation by S. aureus B6-55A monocultures was higher at 42 °C than with mixed cultures (Fig. 3B). Significant effect of growth condition (polymicrobial culture or monoculture) (p < 0.0001) was found at both temperatures (25 °C and 42 °C) on Campylobacter biofilm formation with S. aureus cells in two-way ANOVA analysis (Supplementary Table S2.7). Variable results were obtained when Campylobacter and cell-free extracts from S. aureus media were incubated in microaerobic and aerobic conditions at 42 °C ( Fig. 3C-H). B4-59C-37 was able to enhance biofilm formation of C. coli WA3-33 and C. jejuni NCTC11168 strains in both microaerobic and aerobic condition when compared to control MHB. Higher biofilm formation was also found in B6-55A-37 for C. jejuni NCTC11168 (in aerobic environment) and C. coli HC2-48 (in microaerobic condition). Otherwise, most of the Campylobacter strains produced similar or lower amount of biofilm in cell free extracts from S. aureus media than control MHB in both aerobic and microaerobic environment (Fig. 3C-H). In MHB, all strains except C. coli ZV1-224 produced comparatively higher biofilm in aerobic condition than microaerobic condition. Significantly higher biofilm formation was seen in aerobic condition than microaerobic condition in most tested media except B4-59C-25 for C. coli HC2-48 (Fig. 3G) and likewise result for C. jejuni OD2-67 except in B4-59C-37 (Fig. 3E). Meanwhile, biofilm production was lower in aerobic condition than microaerobic condition for C. coli ZV1-224 in all tested media (Fig. 3H). Although none of the cell-free media significantly enhanced biofilm formation for all Campylobacter strains, growth medium factor (MHB control or cell free extract from S. aureus medium) had significant effect on Campylobacter biofilm formation in both aerobic and microaerobic incubation which was confirmed by statistical analysis with biofilm data from all Campylobacter strains (both C. jejuni and C. coli strains) (Supplementary Table S2. 8). No consistent enhancement of biofilm production was observed when Campylobacter strains were incubated with the ≤ 30 kDa fraction of S. aureus media extracts ( Supplementary Fig. S3C,D). However, biofilm results for C. jejuni OD2-67 in B4-59C-37 (≤ 30 kDa) was found to be significantly higher (p < 0.0001) among all tested media in microaerobic condition. The ≥ 30 kDa fraction could not be analyzed for its impact on biofilm production media due to the insignificant amount of concentrate obtained.

Discussion
In most studies of foodborne pathogens, only a few targeted microorganisms are isolated and characterized 2,4 . However, polymicrobial colonization of retail meat products and meat processing environments is common 16,17,26 , and these polymicrobial conditions could have antagonistic or synergistic effects on foodborne pathogens 39   www.nature.com/scientificreports/ strains in retail meat products originated from the respective animal 3,4,22,34 . Although the virulence of the S. aureus strains used in this study has not been tested, multiple virulence factors were identified in their genomes, including the gene encoding toxic shock syndrome 34 . Prolonged survival of the six Campylobacter strains when cultivated with S. aureus cells or cell-free exudates indicates that S. aureus improves the survival of Campylobacter at lower temperatures. Previous studies regarding the transcriptional landscape of Campylobacter during survival at low temperature indicated that genes involved in quorum sensing and the acquisition of cryoprotectant molecules were differentially expressed [40][41][42] . Furthermore, Campylobacter was shown to utilize exogenous siderophores produced by other microorganisms 43,44 ; these are used for iron acquisition during growth and survival and might also contribute to virulence. Our results showing improved survival of Campylobacter in S. aureus cell-free exudates indicates that metabolites produced by S. aureus improve the survival of Campylobacter. It is also important to note that Campylobacter cells undergo a transition to the VBNC stage during cold stress and remain viable for prolonged periods of time 14 . Hence, the presence of S. aureus cells or cell-free extracts might provide a protective environment for Campylobacter and facilitate viability for prolonged periods at lower temperatures. In this study, we counted culturable Campylobacter cells after incubation at 4 °C; however, this study did not address the impact of S. aureus on the VBNC condition of Campylobacter cells. A previous report documented the detrimental effects of pre-established, organismal biofilms from poultry on the survival of C. jejuni at 10 °C 26 . Organisms identified in biofilms from poultry www.nature.com/scientificreports/ included Pseudomonas spp., Staphylococcus spp., E. coli, Bacillus spp., and Flavobacterium spp. 26 . During cold stress, a percentage of the S. aureus population undergoes a transition to a small colony variant (SCV), which showed alterations in amino acids as compared with normal cells 45 . It is also important to note that extracellular protein excretion by different S. aureus strains differs among strains, growth conditions and growth stage 46 . Various extracellular proteins including phosphoglycerate kinase, succinyl-CoA ligase, peroxiredoxin, superoxide dismutase, transmembrane sulfatase, and chaperonin were differentially expressed in S. aureus during growth at 37 °C at 12, 24 and 48 h 46 . In this study, SDS-PAGE analysis of S. aureus media extracts revealed changes in protein concentration and banding patterns at different pre-incubation temperatures ( Supplementary Fig. S4, Supplementary Table S1). Further work is needed to determine the influence of amino acids and metabolites from S. aureus on Campylobacter survival, especially at lower temperatures. The reduced levels of dissolved oxygen in mixed bacterial populations is another factor that contributes to the survival and growth of Campylobacter 47,48 . It is important to note that S. aureus can survive and multiply at refrigeration temperatures for prolonged periods of time 45,49 ; this would lead to a consumption of available oxygen from media and the creation of an environment favorable for Campylobacter. Furthermore, Campylobacter was shown to tolerate aerobic conditions by metabolic commensalism with Pseudomonas spp. 50 , and a similar relationship might exist between Campylobacter spp. and S. aureus.
Campylobacter is a poor initiator of biofilm production in monoculture but is a well-established secondary colonizer of biofilms produced by other bacterial pathogens 26,51,52 . Variability in biofilm formation among Campylobacter strains in monoculture has been reported 27,29 ; in general, Campylobacter biofilm formation is greater in aerobic than microaerobic conditions 30,38 . However, lower biofilm production in aerobic environment than microaerobic environment for some strains of C. jejuni has also been reported 53 . Nutrient-rich environments like chicken and liver juice were shown to enhance attachment, adhesion and biofilm formation by Campylobacter strains 18,19,29 . Significantly higher levels of biofilm were formed when Campylobacter strains were co-cultivated with S. aureus B4-59C at both 25 °C and 42 °C and suggested a mutually beneficial effect (Fig. 3A,B). In contrast, cumulative biofilm levels by Campylobacter and S. aureus B6-55A were significantly lower than biofilms produced by S. aureus monocultures at 42 °C (Fig. 3A). Similar interactions with mixed biofilm communities have been previously reported between Campylobacter and other organisms and may indicate antimicrobial activities or interspecies competition within the biofilm 27,54 . Meanwhile, previous studies reported better survival of Campylobacter strains in mixed biofilms during aerobic conditions than in monoculture 28,29 . Although flagella and luxS-mediated quorum sensing were suggested to be important for biofilm formation in Campylobacter 55 , the possibility of both flagellum-dependent and flagellum-independent biofilm mechanisms in Campylobacter has been suggested 38 . A previous study found no evidence for the role of interspecies cell signaling via autoinducer-2 among mixed populations, but instead suggested physical contact as the sole mechanism for biofilm formation when Campylobacter was a secondary colonizer 26 . Our results failed to find a specific, consistent influence for S. aureus growth media in Campylobacter biofilm formation, which may suggest that physical contact is needed to stimulate biofilm production in mixed populations. The ratio of the two bacterial pathogens might influence survival, aerotolerance and biofilm formation due to the availability of nutrients and quorum sensing. However, it is important to mention that the cell densities of Campylobacter and S. aureus would be far lower in retail meat environments as compared to the densities used in this study.
In this study, we show that S. aureus frequently occurs as a co-contaminant with Campylobacter in retail meat products. S. aureus enhances the survival of C. jejuni and C. coli strains at low temperatures and during aerobic conditions. Select strains of S. aureus potentially enhance biofilm formation by Campylobacter in aerobic conditions (normal atmospheric environment). Extracellular metabolites and proteins produced by S. aureus at multiple temperatures enhance the survival of Campylobacter strains at low temperature. The extracts produced by S. aureus in media improved the survival of some Campylobacter strains when compared to monocultures in MHB. However, S. aureus media extracts did not foster biofilm formation by Campylobacter strains in both aerobic and microaerobic environments.
In summary, it is well-established that the contamination of retail food products by S. aureus increases the risk of food poisoning. This study shows that contamination of retail meats by S. aureus also enhances the survival of Campylobacter during harsh environmental conditions. Hence, food safety measures are still needed to facilitate improved identification and reduced contamination of foodborne pathogens in mixed populations in retail meat and food products.