Dialogue between Staphylococcus aureus SA15 and Lactococcus garvieae strains experiencing oxidative stress

Clothilde Queiroux, Muriel Bonnet, Taous Saraoui, Pierre Delpech, Philippe Veisseire, Etienne Rifa, Cécile Moussard, Geneviève Gagne, Céline Delbes, Stéphanie Bornes

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Clothilde Queiroux, Muriel Bonnet, Taous Saraoui, Pierre Delpech, Philippe Veisseire, et al. Dialogue between Staphylococcus aureus SA15 and Lactococcus garvieae strains experiencing oxidative stress. BMC Microbiology, 2018, 18, 10.1186/s12866-018-1340-3. hal-02621544

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# Dialogue between *Staphylococcus aureus* SA15 and *Lactococcus garvieae* strains experiencing oxidative stress

Clothilde Queiroux, Muriel Bonnet, Taous Saraoui, Pierre Delpech, Philippe Veisseire, Etienne Rifa, Cécile Moussard, Geneviève Gagne, Céline Delbès*† and Stéphanie Bornes†

**Abstract**

**Background:** *Staphylococcus aureus* is an important foodborne pathogen. *Lactococcus garvieae* is a lactic acid bacterium found in dairy products; some of its strains are able to inhibit *S. aureus* growth by producing H₂O₂. Three strains of *L. garvieae* from different origins were tested for their ability to inhibit *S. aureus* SA15 growth. Two conditions were tested, one in which H₂O₂ was produced (high aeration) and another one in which it was not detected (low aeration). Several *S. aureus* genes related to stress, H₂O₂-response and virulence were examined in order to compare their level of expression depending on the inoculated *L. garvieae* strain. Simultaneous *L. garvieae* H₂O₂ metabolism gene expression was followed.

**Results:** The results showed that under high aeration condition, *L. garvieae* strains producing H₂O₂ (N201 and CL-1183) inhibited *S. aureus* SA15 growth and impaired its ability to deal with hydrogen peroxide by repressing H₂O₂-degrading genes. *L. garvieae* strains induced overexpression of *S. aureus* stress-response genes while cell division genes and virulence genes were repressed. A catalase treatment partially or completely restored the SA15 growth. In addition, the H₂O₂ non-producing *L. garvieae* strain (Lg2) did not cause any growth inhibition. The SA15 stress-response genes were down-regulated and cell division genes expression was not affected. Under low aeration condition, while none of the strains tested exhibited H₂O₂-production, the 3 *L. garvieae* strains inhibited *S. aureus* SA15 growth, but to a lesser extent than under high aeration condition.

**Conclusion:** Taken together, these results suggest a *L. garvieae* strain-specific anti-staphylococcal mechanism and an H₂O₂ involvement in at least two of the tested *L. garvieae* strains.

**Keywords:** *Lactococcus garvieae*, *Staphylococcus aureus*, Antimicrobial, Hydrogen peroxide, Gene expression

**Background**

*Staphylococcus aureus* is an opportunistic human pathogen that can be responsible for food poisoning [1]. Its pathogenic activity is due to the production of various enzymes and toxins. It can be found in different environments including milk and dairy products [2]. In cheese, its level should not exceed 10⁵ CFU.g⁻¹ (European Community Regulation No. 852–853/2004).

*Lactococcus garvieae* is an ubiquitous LAB (Lactic Acid Bacteria) that can be found in various fermented foods including dairy products [3, 4], in fish, ruminant or human

microbiota [5] and can be associated with pathologies such as fish lactococcoses [6, 7]. LABs such as *Lactococcus lactis* or *Lactococcus garvieae* are able to inhibit the proliferation of pathogens in cheese by the production of hydrogen peroxide [8], bacteriocins [9], by competition for nutrients [10, 11] or by acidification of the medium [12–14]. Depending on its concentration, hydrogen peroxide has a bactericide or a bacteriostatic effect on *S. aureus* [15]. *L. garvieae* raises our interest because a specific dairy strain of this LAB, N201 strain, can inhibit *S. aureus* growth by the production of H₂O₂ [2, 16, 17]. Moreover, it has a strong technological potential as a ferment for cheese production [18] and has almost no effect on acidification of the medium, compared to other LABs [2]. The inhibitive

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© The Author(s). 2018 **Open Access** This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

<footnote>* Correspondence: celine.delbes@inra.fr</footnote>

<footnote>†Céline Delbès and Stéphanie Bornes contributed equally to this work.
Université Clermont Auvergne, INRA, UMRF, F-15000 Aurillac, France</footnote>
properties of *L. garvieae* N201 were confirmed on 2 strains of *S. aureus*: a human pathogenic strain, MW2 [19] and a non-pathogenic dairy strain, SA15, isolated from Saint-Nectaire cheese [17]. Delpech et al. [20] showed that *S. aureus* had no effect on *L. garvieae* growth and H₂O₂-related gene expression. On the contrary, *L. garvieae* N201 impaired the capacity of both strains of *S. aureus* to deal with the presence of H₂O₂ which led to growth deficiency [2, 16, 17].

In order to investigate the interaction between *S. aureus* and *L. garvieae* in oxidative stress-inducing culture conditions, genes involved in H₂O₂ metabolism were first choice targets, whose expression has been monitored by Delpech et al. [17, 20] in co-cultures of *L. garvieae* N201 and *S. aureus* SA15 or MW2.

In *L. garvieae*, superoxide dismutase sodA [21, 22] and pyruvate oxidase poxB genes [23, 24] are involved in H₂O₂ synthesis. As they do not have any catalase, LAB generally degrade H₂O₂ using alkyl hydroxyperoxidase (Ahp) [25] or glutathione peroxidase (Gpx) [26]. Thioredoxine reductase (Trx) are involved in response to Reactive Oxygen Species (ROS) [27, 28].

In *S. aureus*, Catalase (KatA) and Alkyl hydroxyperoxidase (Ahp) play a role in H₂O₂ degradation [29, 30]. Ahp leads to a dual function in oxidative-stress resistance, environmental persistence and host-pathogen interaction [29]. Amongst the targeted genes, dnaK is known to be involved in H₂O₂-resistance [31, 32], clpC has an important role in oxidative stress regulation, and ctsR is a transcriptional repressor of stress-genes [33]. Moreover, the 2 latter genes, belonging to the dcw cluster involved in cellular division, might be modulated by H₂O₂-stress, leading to *S. aureus* growth impairment [34]. So, if LAB can modify this genes cluster expression, it could have an inhibiting effect on cellular division.

In addition to *S. aureus* growth modulation, *L. garvieae* may also have an effect on its virulence. Enterotoxins are the main toxins responsible for *S. aureus* food poisoning. Ninety-four per cent of *S. aureus* isolated from cow milk have at least one enterotoxin-encoding gene [35]. Amongst the different enterotoxins, enterotoxin C, encoded by *sec4* gene, is the most frequently involved in food poisoning [36–39]. Cretenet et al. [40] showed that this virulence-related gene expression can be modified by *L. lactis* in cheese. *S. aureus* virulence is under control of the *agr* system which is involved in regulating many stress response and virulence genes [41–43]. *agrA* is a response regulator [44] and is able to induce *hld*, a δ-lysin gene [43], and enterotoxin C encoding gene *sec4* [45]. The *agr* system itself is controlled by several molecular intermediates such as SaeRS and SrrAB. SaeRS is a two-component system involved in response to environmental stress which could inhibit *agr* system [46] and also control virulence genes [47–50]. SrrAB is also a two-component system

activated in an anaerobic environment [51]. It would be involved in virulence gene control [52] in response to H₂O₂. Indeed, in the presence of H₂O₂, srrA is repressed in *S. aureus* [53]. Under anaerobic conditions, SrrA represses *agrA* and *hld* expression [54, 55]. CodY, a regulatory protein involved in repressing virulence gene expression in *S. aureus*, is also involved in controlling *agr* system and virulence genes [56].

The level of aeration can change the level of H₂O₂-production according to the LAB strain. Indeed, it has been demonstrated that the transcriptomes of *L. lactis* and *L. garvieae* are significantly modified by the aeration level [20, 57]. *Lactobacillus crispatus* can produce H₂O₂ in high aeration conditions but not in static conditions [58]. Contrariwise, *Lactobacillus delbrueckii* subsp. *bulgaricus* can also produce H₂O₂ in static conditions, even though the amount produced is lower than in high aeration conditions [59].

The aim of the present study was to compare the transcriptional response involved in the antagonistic interaction between *S. aureus* and different strains of *L. garvieae*. In addition to N201 strain isolated from raw milk Saint-Nectaire cheese and already well described [2, 16], two other *L. garvieae* strains were selected for their different capacities to produce H₂O₂: a H₂O₂-producing strain, CL-1183 (VIVASET, Veterinary Faculty, Complutense University from Madrid) isolated in Brazil from the milk from buffalo cows affected by subclinical mastitis [5] as well as a H₂O₂-non producing strain, Lg2, a fish pathogenic strain isolated in Japan [60]. The expression of *S. aureus* genes related to oxygen metabolism, response to stress, cell division and virulence was measured as well as the expression of *L. garvieae* genes related to oxygen metabolism.

## Results

In order to investigate the interaction between *S. aureus* and *L. garvieae* in oxidative stress-inducing culture conditions, we followed the growth of the strains and measured the amount of H₂O₂ in two culture conditions i.e. under high and low aeration levels. The genes whose expression has been monitored by Delpech et al. [17, 20] were chosen to compare the transcriptional response involved in the antagonistic interaction between *S. aureus* and different strains of *L. garvieae*. The expression of *S. aureus* genes related to oxygen metabolism, response to stress, cell division and virulence was measured as well as the expression of *L. garvieae* genes related to oxygen metabolism (Table 1).

### Ability of *L. garvieae* strains to produce H₂O₂ and to inhibit *S. aureus* growth

The effect of aeration level on microbial growth was tested by determining cellular concentrations of both
<table><caption>**Table 1** Differentially expressed *Staphylococcus aureus* genes in co-culture with 3 *Lactococcus garvieae* strains (N201, CJ-1183 and Lg2), summary table</caption><thead><tr><th rowspan="2">Genes</th><th colspan="3">6 h</th><th colspan="3">9 h</th><th colspan="3">24 h</th></tr><tr><th>N201</th><th>CL-1183</th><th>Lg2</th><th>N201</th><th>CL-1183</th><th>Lg2</th><th>N201</th><th>CL-1183</th><th>Lg2</th></tr></thead><tbody><tr><td colspan="10"><strong>High aeration</strong></td></tr><tr><td>Up-regulated</td><td>-</td><td>dnaK</td><td>-</td><td>clpC<br/>cstR<br/>dnaK<br/>agrA</td><td>agrA</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Down-regulated</td><td>sodA<br/>mraW</td><td>ahpF<br/>katA<br/>mraw<br/>codY</td><td>-</td><td>katA<br/>sodA<br/>mraW<br/>srrA</td><td>katA<br/>mraW<br/>srrA</td><td>-</td><td>katA<br/>sodA<br/>codY<br/>saeS<br/>sec4</td><td>ahpF<br/>sodA<br/>sec4</td><td>ahpF<br/>clpC<br/>dnaK<br/>agrA<br/>codY<br/>hld</td></tr><tr><td colspan="10"><strong>Low aeration</strong></td></tr><tr><td>Up-regulated</td><td>-</td><td>-</td><td>-</td><td>agrA<br/>sec4</td><td>ahpF<br/>agrA<br/>sec4<br/>sel2</td><td>clpC<br/>cstR<br/>dnaK</td><td>-</td><td>codY</td><td>agrA</td></tr><tr><td>Down-regulated</td><td>sec4</td><td>codY</td><td>-</td><td>-</td><td>-</td><td>codY</td><td>-</td><td>-</td><td>sodA<br/>srrA</td></tr></tbody></table>

For detailed values, see Additional files 2 and 3

microorganisms. In pure culture, *S. aureus* growth was almost identical under both high and low aeration conditions. However, the population count reached at 24 h was 1.2 log CFU/ml lower under low aeration than under high aeration (Fig. 1a). The 3 different strains of *L. garvieae* grew as well under high aeration level as

under low aeration level independent of the presence of *S. aureus* (Additional file 1). pH values remained stable (7.0 ± 0.2) in all cultures over the whole experiment (data not shown).

Detectable amounts of H₂O₂ were produced exclusively under high aeration level by N201 and CL-1183

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strains in co-culture with SA15. N201 strain produced from 1.327 mM ± 0.09 to 1.517 mM ± 0.18 of H₂O₂. The strain CL-1183 produced slightly more H₂O₂ than the strain N201, from 1.663 mM ± 0.23 to 2.415 mM ± 0.34. While N201 H₂O₂-production peak was at 9 h, CL-1183 H₂O₂-production stayed stable after 9 h (Table 2).

Under high aeration, when co-cultivated with N201, SA15 growth was 2.1 log [CFU.ml⁻¹] lower than in pure culture as early as 6 h with a maximal growth inhibition of 5.8 log [CFU.ml⁻¹] at 24 h. SA15 growth co-cultivated with CL-1183 was 1.7 log [CFU.ml⁻¹] lower compared to pure culture as early as 6 h. The maximal growth inhibition was observed after 24 h of co-culture (4.5 log [CFU.ml⁻¹] lower). SA15 co-cultivated with Lg2 did not show any significant impairment in growth throughout the experiment compared to pure culture (Fig. 1a).

Under low aeration level and in co-culture with N201, CL-1183 or Lg2, SA15 growth was lower than in pure culture as early as 6 h (0.6 log [CFU.ml⁻¹], 0.9 log [CFU.ml⁻¹] or 0.6 log [CFU.ml⁻¹], with a maximal growth inhibition at 24 h (2.5 log [CFU.ml⁻¹], 3.5 log [CFU.ml⁻¹] or 1.6 log [CFU.ml⁻¹]), respectively) (Fig. 1a).

To determine the involvement of H₂O₂ produced by N201 and CL-1183 in growth inhibition of SA15, co-cultures of SA15 with these 2 strains were performed under high aeration level in presence of catalase (Fig. 1). No detectable amount of H₂O₂ was observed at any time. *S. aureus* cell concentration was not significantly different in presence of catalase compared to control (Fig. 1) during both exponential and stationary phases. Statistical analysis, using ANOVA, showed that in presence of catalase there was no significant difference of SA15 count after 24 h in co-culture with CL-1183 compared to pure culture. In contrary, SA15 cell concentration in co-culture SA15/N201 was still 0.8 log [CFU.ml⁻¹] lower than in pure culture. These results showed that the inhibition of SA15 growth by N201 and CL-1183 was partially or completely suppressed by catalase.

<table><caption>**Table 2** H₂O₂ concentration produced by *Lactococcus garvieae* in pure culture and in co-culture with *Staphylococcus aureus* SA15, under high aeration</caption><thead><tr><th></th><th colspan="4">Hydrogen peroxide concentration (mM)</th></tr><tr><th>Time (h)</th><th>0</th><th>6</th><th>9</th><th>24</th></tr></thead><tbody><tr><td>SA15</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>SA15 + N201</td><td>ND</td><td>1.343 ± 0.141</td><td>1.517 ± 0.180</td><td>1.327 ± 0.090</td></tr><tr><td>SA15 + CL-1183</td><td>ND</td><td>1.663 ± 0.230</td><td>2.415 ± 0.340</td><td>2.409 ± 0.240</td></tr><tr><td>SA15 + Lg2</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr><tr><td>N201</td><td>ND</td><td>1.455 ± 0.027</td><td>1.549 ± 0.040</td><td>1.105 ± 0.030</td></tr><tr><td>CL-1183</td><td>ND</td><td>1.257 ± 0.010</td><td>1.317 ± 0.030</td><td>1.148 ± 0.058</td></tr><tr><td>Lg2</td><td>ND</td><td>ND</td><td>ND</td><td>ND</td></tr></tbody></table>

ND not detected

### Effect of *L. garvieae* strains on *S. aureus* genes expression under high aeration level (Additional file 2)

Whatever the *L. garvieae* strain cultivated with SA15, none of the H₂O₂-response genes tested was up-regulated. When SA15 was cultivated with *L. garvieae* N201 or CL-1183, *katA* was down-regulated, at 9 h and 24 h with N201 (3.7 and 3.0-times, respectively), or at 6 and 9 h with CL-1183 (3.9 and 3.2-times, respectively). When cultivated with N201, *ahpF* expression in SA15 was not modified in comparison with pure culture, whereas in co-cultures SA15/CL-1183 and SA15/Lg2, it was down-regulated (2.1-times at 6 h and 2.9-times at 24 h, 6.9-times at 24 h, respectively). *sodA* was down-regulated at 6, 9 and 24 h in co-cultures with N201 (2.2, 4.4 and 3.9-times, respectively) or at 24 h (2.2-times) with CL-1183. No change in expression of *katA* and *sodA* was observed with Lg2.

*L. garvieae* N201 induced an up-regulation of the three SA15 stress-response genes tested at 9 h; *clpC*, *ctsR* and *dnaK* were 2.0-times, 3.7-times and 2.5-times more expressed than in pure culture, respectively. When cultivated with CL-1183, only *dnaK* was 2.8-times up-regulated at 6 h, while *clpC* and *ctsR* expressions were not affected. Conversely, in co-culture SA15/Lg2, *clpC* and *dnaK* were 3.9-times and 19.8-times down-regulated respectively, while *ctsR* did not show any significant difference in its expression.

In both SA15/N201 and SA15/CL-1183 co-cultures, *mraW* cell division gene was strongly down-regulated at 6 and 9 h (7.0-times and 20.3-times, and 20.0-times and 12.0-times, respectively). In co-culture SA15/Lg2, *mraW* expression was not affected.

In co-culture, the five SA15 virulence-related regulator genes tested displayed contrasted patterns of expression. At 9 h, in both SA15/N201 and SA15/CL-1183 co-cultures, *agrA* was up-regulated (4.0-times with N201 and 3.4-times with CL-1183), while *srrA* was down-regulated (7.1-times or 6.4-times with N201 or CL-1183 respectively). *codY* expression was down-regulated 4.1-times at 24 h by N201 and 3.6-times at 6 h by CL-1183. *saeS* expression was 2.4-times lower at 24 h in co-culture with N201 than in pure culture, whereas it was not affected by CL-1183. *hld* expression remained stable until 24 h when SA15 was in co-culture with N201 or CL-1183. In co-culture SA15/Lg2, at 24 h, *agrA*, *codY* and *hld* expressions were all down-regulated (3.0-times, 2.6-times and 3.9-times, respectively), while *saeS* and *srrA* expressions remained stable.

In co-cultures SA15/N201 and SA15/CL-1183, amongst the 2 enterotoxins-encoding genes tested, only *sec4* expression was modified at 24 h, with a 4.1-times and a 9.0-times down-regulation, respectively. *sel2* expression was not modified whatever the SA15 culture conditions were. In co-culture SA15/Lg2, no enterotoxin gene expression was affected.
### Effect of *L. garvieae* strains on *S. aureus* genes expression under low aeration level (Additional file 3)

Under low aeration level, in presence of CL-1183, the H₂O₂-response gene *ahpF* was 4.2-times up-regulated at 9 h, whereas in presence of Lg2, *sodA* was 6.5-times down-regulated at 24 h. All the stress-response genes tested were up-regulated at 9 h (3.4-times for *clpC*, 4.0-times for *ctsR* and 2.1-times for *dnaK*) with Lg2. No change in stress-response genes expression was observed when SA15 was cultivated with N201 or CL-1183. Concerning the cell division gene *mraW*, its expression was not modified, whatever the strain cultivated with SA15.

Amongst virulence-related regulator genes, *agrA* was up-regulated in all three co-cultures at different stages of growth. It was the only virulence-related regulator gene differentially regulated by N201 (6.0-times up-regulated at 9 h). In the same way, this gene was 4.1-times up-regulated at 9 h by CL-1183, and 2.5-times up-regulated at 24 h by Lg2. *codY* was 2.0-times down-regulated at 6 h but 2.0-times up-regulated at 24 h with CL-1183, while *codY* and *srrA* were 2.1- and 2.7-times down-regulated at 9 and 24 h, respectively with Lg2.

The enterotoxin gene *sec4* was up-regulated in both N201/SA15 and CL-1183/SA15 co-cultures, (3.5- and 2.7-times, at 6 and 9 h respectively, with N201, and 2.5-times at 9 h with CL-1183). *sel2* was 3.5-times up-regulated at 9 h only in the presence of CL-1183.

### Effect of aeration on *L. garvieae* strains (Additional file 4)

To evaluate the strain-specific effect of aeration on *L. garvieae*, expression of 5 H₂O₂-related genes of them (*trxB1*, *ahpC*, *gpx*, *poxB* and *sodA*) was monitored and compared between the 3 *L. garvieae* strains in co-culture with SA15. Overall, most differential expressions concerned H₂O₂-degradation genes. Differences between H₂O₂-producing and non-producing strains were essentially related to *ahpC*: both N201 and CL-1183 overexpressed this gene under low aeration (5.6-times at 24 h in N201, 4.0-times at 6 h and 6.5-times at 9 h in CL-1183). Conversely, Lg2 overexpressed *ahpC* (4.7-times) at 9 h under high level of aeration. Both CL-1183 and Lg2 overexpressed *trxB1* under high level of aeration (2.9-times at 6 h in CL-1183 and 2.1-times 24 h in Lg2). Amongst H₂O₂-synthesis genes, only CL-1183 overexpressed *poxB* (4.1-times at 9 h under low aeration).

### Discussion

The aim of the study was to compare the transcriptional response involved in the antagonistic interaction between *S. aureus* and different strains of *L. garvieae*. Three strains of *L. garvieae* from different origins were used: N201, a dairy-isolated strain known

to be a *S. aureus* inhibiting strain; CL-1183, a strain isolated from milk from buffalo cows suffering from mastitis; and Lg2, a fish pathogenic strain. Under high aeration condition, two of them were shown to be able to produce detectable amount of H₂O₂ (N201 and CL-1183), whereas the third one (Lg2) was not.

### *L. garvieae* strains and aeration level effect on *S. aureus* SA15 growth and on H₂O₂- and stress-responses

Under high aeration level, only *L. garvieae* N201 and CL-1183 inhibited *S. aureus* growth. CL-1183 down-regulated *S. aureus* H₂O₂-degradation genes (*ahpF* and *katA*) and N201 and Lg2 only down-regulated *katA* or *ahpF* respectively. This could mean that SA15 lost its ability to deal with H₂O₂-stress and suggest that the H₂O₂ detoxification occurred predominantly via KatA. These results are in accordance with those obtained by Cosgrove et al. [29], showing that *S. aureus ahpC-katA* mutant was no more sensitive to H₂O₂ than the *katA* mutant. They also found out that, Ahp would have less affinity for H₂O₂ than KatA and that it could be an alternative solution for H₂O₂-degradation when KatA was not functional. In our study, despite N201 only down-regulated *katA* and not *ahpF*, AhpF did not play its compensatory role in H₂O₂ detoxification. This result can be explained by the fact that KatA was responsible for detoxifying high levels of H₂O₂, whereas AhpC was responsible for the removal of low levels of H₂O₂ [29]. This could be correlated with the large amounts of H₂O₂ detected in the co-cultures SA15/N201. The down-regulation of *katA*, at 9 h and 24 h, can be explained by the fact that *srrA* was also down regulated. Mashruwala and Boyd [61] found that *srrAB* mutant strain had decreased transcription of genes encoding for H₂O₂ resistance factors as *katA*. They also reported that SrrAB positively influenced H₂O₂ resistance during periods of high O₂ dependent respiratory activity, but not when cellular respiration was diminished as a result of lower O₂ availability. *S. aureus* SA15 genes involved in stress-response (*clpC*, *ctsR*, *dnaK*) were all up-regulated by N201 and only *dnaK* was up-regulated by CL-1183, whereas *clpC* and *dnaK* were down-regulated by Lg2. These results indicated that in presence N201 and CL-1183, SA15 detected an oxidative stress as a result of H₂O₂ production by these two *L. garvieae* strains. However, the up-regulation of these genes did not allow SA15 to fight against this stress, although precedent studies reported that *S. aureus clpC* or *dnaK* mutants growth was impaired in the presence of H₂O₂ stress [32, 62, 63] and that a basic level of expression of *dnaK* was sufficient in response to this stress [32]. All these data highlighted the complexity of the stress response machinery and the important role of *clpC*, *ctsR* and *dnaK* genes. Moreover, while *mraW* gene was not
differentially expressed in presence of Lg2, it was re-
pressed at 6 and 9 h by N201, confirming results ob-
tained by Delpech et al. [17], and by CL-1183 in our
study, when S. aureus growth was inhibited. The re-
pression of mraW by N201 and CL-1183 was positively
correlated with inhibition of SA15 growth. These re-
sults are supported by Cretenet et al. [40] showing that
L. lactis was able to inhibit ftsH, ftsL and ftsZ genes
also involved in cellular division. Interestingly, several
studies reported that inhibition of cell division protein
is a promising approach for anti-staphylococcal therapy
[64, 65].

Catalase treatment partially reduced SA15 growth inhib-
ition by N201, confirming the result obtained by
Delbes-Paus et al. [2], whereas the inhibition by CL-1183
was completely suppressed. Oogai et al. [66] observed that
in presence of catalase, the S. aureus MW2 growth inhib-
ition by Streptococcus sanguinis was completely sup-
pressed. These observations confirmed the role of H2O2
produced by LAB in growth inhibition of S. aureus. Our
study demonstrated that under high level of aeration, the
inhibition of S. aureus by L. garvieae involved strain spe-
cific mechanisms. Indeed, CL-1183 inhibited SA15 growth
mainly by H2O2 production whereas inhibition due to
N201 may involve the combined action of H2O2 and other
antagonistic mechanism.

Under low aeration condition, S. aureus growth was al-
most identical to that measured under high aeration con-
dition, except after 24 h, when we observed a 1,2 log
reduction in the S. aureus population level that could be
due to the depletion of oxygen. Ledala et al. [67] showed
that the growth rate of S. aureus was independent of oxy-
gen limitation over 12 h although its metabolome was sig-
nificantly affected. S. aureus growth was slightly inhibited
by the three L. garvieae strains, although none of them
could produce detectable amounts of H2O2 (Fig. 1). This
suggests that there was another inhibitory mechanism in-
volved. These results match those obtained with N201 in
the previous studies [2, 17, 20]. Delbes-Paus et al. [2] have
shown that in milk, even if H2O2 was not detected, S. aur-
eus growth was inhibited by L. garvieae N201. However,
no clear hypothesis to explain the mechanism of the
anti-staphylococcal activity under low aeration could be
drawn from the gene expression data. However, the
up-regulation of stress-response genes in the presence of
Lg2 was observed. Indeed, clpC, ctsR and dnaK genes,
were all up-regulated at 6 h in co-culture with Lg2, sug-
gesting that Lg2 triggered a stress on SA15 which led to a
growth inhibition.

L. garvieae strains and aeration level effect on S. aureus SA15 virulence gene expression

Under high level of aeration, *L. garvieae* N201 reduced *S. aureus* virulence-related genes expression confirming

results obtained by Delpech et al. [17]. Cretenet et al.
[40] and Queck et al. [68] have shown that the agr sys-
tem, involved in the regulation of genes linked to S. aur-
eus virulence, was repressed by L. lactis even if L. lactis
does not produce H2O2 in this condition. Molecular inter-
mediaries SaeRS and SrrAB are involved in controlling the
agr system [46]. We showed srrA expression was re-
pressed in co-culture with N201 and CL-1183, when
H2O2 was produced as already observed by Chang et al.
[53]. Moreover, Majerczyk et al. [56] have shown that a
codY mutant could derepressed agr system. In our condi-
tions, codY was repressed by CL-1183 at 6 h explaining
agrA up-regulation at 9 h, whereas repression of codY at
24 h by Lg2 cannot explain agrA down-regulation suggest-
ing that another regulator might be involved. Moreover, at
24 h, despite SA15 growth was not modified, Lg2 caused a
down-regulation of three virulence-related regulator genes
tested in this study (agrA, codY, hld). sec4 and sel2 are two
enteroxin-encoding genes found in S. aureus SA15 [17].
Although enteroxin C-encoding gene sec4 is under the
control of the agr system [45], agrA expression was only
induced by N201 and CL-1183 and sec4 was repressed
under high aeration level as shown by Delpech et al. [17].
Our data showed the repression of virulence associated
genes and of enterotoxin encoding genes as well as simul-
taneous S. aureus growth inhibition in the presence of
N201 or CL-1183, indicating that these 2 strains not only
inhibited SA15 growth but also potentially attenuated its
virulence.

Under low aeration condition, S. aureus over-expressed agrA and enterotoxin-encoding genes sel2 and sec4 in presence of N201 and CL-1183, whereas Lg2 did not induce any modification. In presence of Lg2, agrA was up-regulated at 24 h. This result can be explained by the fact that SA15 srrA gene was down-regulated, at the same time. The same observation was reported by Yarwood et al. [55]. They showed that transcription of RNAIII from the agr locus was inversely dependent on expression of srrAB. Inherently, despite SA15 agrA up-regulation by Lg2, no modification in enterotoxin genes expression was observed. Yarwood et al. [55] also reported that the SA15 srrB mutant growth was significantly slower in anaerobic condition, which is in accordance with SA15 growth inhibition in presence of Lg2.

L. garvieae strain responses to low and high levels of
aeration in co-culture with S. aureus SA15

It seemed that the three strains of *L. garvieae* differed
essentially in their ability to degrade H₂O₂. Presence of
H₂O₂ would not depend only on H₂O₂-synthesis, but
also on H₂O₂-degradation by *L. garvieae* strains, which
is an original mechanism, and in accordance with the re-
sults obtained by Delpech et al. [20].
In high aeration level, Lg2 overexpressed two H₂O₂ degradation genes *ahpC* and *trxB* at 9 and 24 h respectively. The non-detection of H₂O₂ could be explained by the absence of production of H₂O₂ or by its degradation by AhpC and TrxB. The latter mechanism could explain why, under low aeration, despite CL-1183 overexpressed an H₂O₂-synthesis gene, *poxB*, no detectable H₂O₂ was observed. Indeed, at the same time, this strain overexpressed the H₂O₂ degradation gene *ahpC*. Our data showed that the importance of the inhibition varies depending on the presence or absence of H₂O₂. Indeed, inhibition can occur while H₂O₂ is not detected. As regards other potential explanations for the growth inhibition, the only putative bacteriocin identified in the *L. garvieae* N201 genome was homologous to garvieaecin Q (GarQ, data not shown), a class IID bacteriocin [69]. In view of the results of previous experiments on *L. garvieae* N201 [20], the inhibition was probably caused neither by garvieaecin Q nor by a protein, nor by a lipid, and nor by a polysaccharide. In the present study, we evidenced *L. garvieae* strain-specific response in *S. aureus* gene expression which suggests that molecular mechanisms involved in the inhibition could differ between *L. garvieae* strains. Further investigations are needed to elucidate these mechanisms. A detailed analysis of strains physiology is needed to evaluate the potential role of nutritional competition in the inhibition. A global transcriptomic approach of the interaction would shed some light on the metabolic dialogue between these strains, and more particularly the potential involvement of quorum-sensing.

## Conclusions

Our data evidenced an impact of the aeration condition on the interaction between SA15 and *L. garvieae* strains.

Under high level of aeration, only N201 and CL-1183 produced detectable amounts of H₂O₂ and consequently inhibited SA15 growth. Both *L. garvieae* strains activated SA15 stress-response genes and inhibited the expression of several genes involved in H₂O₂-degradation, virulence and cellular division. A catalase treatment partially or completely suppressed this inhibition and no H₂O₂ was detected. Lg2 strain did not produce any detectable amount of H₂O₂ and did not inhibit SA15 growth. However, as N201 and CL-1183, Lg2 inhibited SA15 virulence-related genes and stress-response related genes.

Under low aeration level, none of the *L. garvieae* strains produced any detectable amount of H₂O₂. N201 and CL-1183 were still able to inhibit SA15 growth, as well as, Lg2. While N201 and CL-1183 induced the up-regulation of the SA15 enterotoxin-encoding genes and related regulator *agrA*, Lg2 caused up-regulation of SA15 stress-related genes and down-regulated virulence-related regulators genes.

The antagonistic properties of *L. garvieae* against *S. aureus* were strain- and aeration level-dependent. Under high aeration, they involved H₂O₂ production by two out of the three *L. garvieae* tested strains. This study provides new insights into microbial interactions mechanisms and shows the importance of investigating strain-specific effects.

## Methods

### Strains and culture conditions

Strains *Lactococcus garvieae* N201, CL-1183 and Lg2 and *Staphylococcus aureus* SA15 were cultivated in Brain-Heart Infusion buffered to pH 7 using Potassium Phosphate (BHI, Biokar Diagnostic, Pantin, France) at 30 °C and 37 °C, respectively, under static condition. After 20 h, cells were harvested at 3500 g during 15 min. Cell pellets were resuspended in 2 ml of BHI and syringed 5 times. Cell concentration was then determined using a Petroff-Hausser counting chamber. Fifty milliliters of buffered BHI, containing or not 4000 IU ml⁻¹ of catalase from bovine liver (ref. C100, Sigma), were then co-inoculated at ~ 10⁷ cells per ml for *L. garvieae* and ~ 10⁶ cells per ml for *S. aureus*, or just with *L. garvieae* or *S. aureus* at the same concentration in 250 ml Erlenmeyer flasks or in 50 ml Falcon tubes, as described previously [2, 17].

Then, Erlenmeyer flasks were incubated for 6, 9 or 24 h at 30 °C under shaking condition (150 rpm), corresponding to the high level of aeration condition. The low level of aeration condition corresponded to static fully filled 50 ml Falcon tubes. At 0, 6, 9 and 24 h, 1 ml of the cultures was removed to be syringed and serially diluted in Ringer's solution. After adequate mixing, 100 µl of each dilution were plated onto solid Baird Parker for *S. aureus* and BHI agar for *L. garvieae* for numeration. Colony-forming units were counted after overnight incubation at 37 °C for *S. aureus* and 30 °C for *L. garvieae*. Also, 1 ml was removed from the cultures for H₂O₂ content analysis and 40 ml for RNA extraction. Cell pellets from 6, 9 and 24 h cultures were resuspended in 200 µl of cold Tris-EDTA buffer and then frozen at – 80 °C. The experiment was carried out in triplicate within each of three independent biological replicates, for a total of 9 samples per time point. pH measurements were made with the rest of the cultures.

### Quantitative analysis of H₂O₂ in *L. garvieae* cultures supernatant

Hydrogen peroxide concentration was determined according to Batdorj et al. protocol [70] with slight modifications. Cells were harvested by centrifugation at 11500 g, 15 °C for 10 min. One hundred microliter of the sample supernatant were mixed with 100 µl of 4-aminoantipyrine 4 mg.ml⁻¹ (Sigma-Aldrich, St. Louis, Missouri, USA), 20 µl of water-saturated phenol (Sigma-Aldrich, St. Louis, Missouri, USA), 750 µl of phosphate buffer Na₂HPO₄/
NaH₂PO₄ 0.1 M (pH 7) and 30 µl of horseradish peroxidase type VI-A (500 U·ml⁻¹ in sodium phosphate buffer pH 6 (Sigma-Aldrich, St. Louis, Missouri, USA). Sample was mixed by inverting the tube and OD was measured at 505 nm. Blank was done by replacing sample by sterile medium. H₂O₂ concentration was determined using a standard curve performed with concentrations ranging from 0 to 3 mM. The minimal concentration that could be detected was 0.5 mM.

### RNA extraction and DNase treatments

Cells in Tris-EDTA buffer were thawed out and 25 µl of 20% SDS, 500 µl of phenol (pH 4), 3.5 µl of β-mercaptoethanol and 600 mg of Zirconium beads were added. The cells were broken twice in a tissue homogenizer (Precellys® 24, Bertin Technologies, Montigny-le-Bretonneux, France). Then, 200 µl of chloroform were added and mixed with the solution and sample was centrifuged at 11400 g for 20 min at 4 °C. RNA extraction was performed using NucleoSpin RNA Midi kit (Macherey-Nagel, GmbH & Co. KG, Düren, Germany) following the supplier's instructions. The RNA extracts were treated twice with DNAse I using an Ambion DNA-free kit following the supplier's instructions (Ambion, Inc., Austin, Texas, USA). The RNA extracts were quantified using a Nanodrop™ 2000C (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA).

### Reverse transcription and quantitative PCR

Reverse Transcription (RT) was performed using 0.5 µg of RNA twice treated with DNase, 5 µl of 10X buffer,

2 µl of 25X dNTP, 5 µl of random primers and 2.5 µl of retrotranscriptase (Applied Biosystems®, Life Technologies, Foster City, California, USA). The RT was done in a thermocycler (Techné® Prime, Bibby Scientific, Stone, Staffordshire, UK) with the following parameters: 10 min at 25 °C and 120 min at 37 °C.

### Genes Of Interest (GOI)
Ct were determined in quantitative PCR (qPCR) assays using 2.5 µl of cDNA suspensions 10-fold diluted in RNAse-free water and 10 µl of qPCR mix containing 1.25 µl of each primer (10 mM, Tables 3 and 4), 6.25 µl of qPCR Rotor Gene SybrGreen mix (Qiagen, Hilden, Germany) and 1.25 µl of RNAse-free water. The qPCR was performed according to the protocol on Rotor Gene Q (Qiagen, Hilden, Germany) with the following parameters: 4 min at 94 °C, then for 35 cycles, 30 s at 94 °C, 30 s at 55 °C, 60 s at 72 °C. Each reaction was performed in triplicate within each of three independent biological replicates, for a total of 9 reactions per time point.

Primers efficiencies were determined according to a 10-fold template dilution standard and were all ranging from 1.90 (~ 95%) to 2.293 (~ 115%) according to the following equation: Efficiency = 10^(-1/slope of calibration curve) [71]. The primers used were previously described by others [17]. One reference gene was used for *L. garvieae* data: *tufB* encoding the elongation factor *Tu*. Considering that the lag in growth phase reflects the inhibition of *S. aureus* by *L. garvieae*, we evaluated gene expression at identical time points corresponding to different population levels. Two reference genes whose expression was stable over

<table><caption>**Table 3** Targeted *Staphylococcus aureus* genes</caption><thead><tr><td>Gene</td><td>Description</td><td>Category</td><td>Primers references</td></tr></thead><tbody><tr><td>hu<sup>a</sup></td><td>DNA-binding protein</td><td>Housekeeping gene</td><td>[74]</td></tr><tr><td>recA<sup>a</sup></td><td>Recombinase A</td><td>Housekeeping gene</td><td>[74]</td></tr><tr><td>agrA</td><td>Accessory gene regulator A</td><td>Virulence-related regulator</td><td>[40]</td></tr><tr><td>ahpF</td><td>Alkyl hydroperoxidase F</td><td>H₂O₂-response</td><td>[17]</td></tr><tr><td>clpC</td><td>Clp proteinase C</td><td>Stress-response</td><td>[40]</td></tr><tr><td>ctsR</td><td>Transcriptional repressor of stress-genes</td><td>Stress-response</td><td>[40]</td></tr><tr><td>codY</td><td>Transcriptional repressor</td><td>Virulence-related regulator</td><td>[17]</td></tr><tr><td>dnaK</td><td>Chaperone protein DnaK</td><td>Stress-response</td><td>[40]</td></tr><tr><td>hld</td><td>Deltahemolysin</td><td>Virulence-related regulator</td><td>[40]</td></tr><tr><td>katA</td><td>Catalase</td><td>H₂O₂-response</td><td>[40]</td></tr><tr><td>mraW</td><td>S-adenosyl methyltransferase</td><td>Cell division</td><td>[17]</td></tr><tr><td>saeS</td><td>Histidine protein kinase</td><td>Virulence-related regulator</td><td>[40]</td></tr><tr><td>sec4</td><td>Enterotoxin C</td><td>Enterotoxin</td><td>[40]</td></tr><tr><td>sel2</td><td>Enterotoxin L</td><td>Enterotoxin</td><td>[40]</td></tr><tr><td>sodA</td><td>Superoxide dismutase</td><td>H₂O₂-response</td><td>[40]</td></tr><tr><td>srrA</td><td>Staphylococcal respiratory response</td><td>Virulence-related regulator</td><td>[40]</td></tr></tbody></table>

<footnote><sup>a</sup>Reference genes</footnote>
<table><caption>**Table 4** Targeted *Lactococcus garvieae* genes</caption><thead><tr><th>Gene</th><th>Description</th><th>Category</th><th>Primers references</th></tr></thead><tbody><tr><td>tufb<sup>a</sup></td><td>Elongation factor Tu</td><td>Housekeeping gene</td><td>[20]</td></tr><tr><td>ahpC</td><td>Alkyl hydroxyperoxide reductase</td><td>H<sub>2</sub>O<sub>2</sub>-degradation</td><td>[20]</td></tr><tr><td>gpx</td><td>Glutathione peroxidase</td><td>H<sub>2</sub>O<sub>2</sub>-degradation</td><td>[20]</td></tr><tr><td>poxB</td><td>Pyruvate oxidase</td><td>H<sub>2</sub>O<sub>2</sub>-synthesis</td><td>[20]</td></tr><tr><td>sodA</td><td>Superoxide dismutase</td><td>H<sub>2</sub>O<sub>2</sub>-synthesis</td><td>[20]</td></tr><tr><td>trxB1</td><td>Thioredoxin reductase</td><td>H<sub>2</sub>O<sub>2</sub>-degradation</td><td>[20]</td></tr></tbody></table>

<sup>a</sup>Reference gene

time under the different conditions were used for S. aureus data: housekeeping genes recA, encoding the recombinase A, and *hu*, encoding a DNA-binding protein. The GOI expression was calculated according to the formula introduced by Hellemans et al. [72].

### Comparison of gene expression

Differential gene expression was evaluated by comparing expression between two conditions. First, the influence of aeration on gene expression in pure culture was evaluated for S. aureus. Then, the influence of different strains of L. garvieae (N201, CL-1183 and Lg2) on S. aureus SA15 gene expression was observed under high or low aeration condition. Secondly, L. garvieae gene expression was compared between the different strains in co-culture with S. aureus SA15 under both aeration conditions. A gene was considered as differentially expressed when its expression was changed by at least a factor of 2.

### Statistical analyses

Statistical analyses on microbial counts were performed using R software [73] by one-way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) test. The gene expression statistical analyses were performed using R by ANOVA with Newmann-Keuls post-hoc test. Significance was declared at *P* < 0.05.

### Additional files

**Additional file 1: Figure. S2.** L. garvieae strains growth in pure culture and in coculture with S. aureus SA15, under different levels of aeration. (DOCX 116 kb)

**Additional file 2: Table S5.** Gene expression changes in S. aureus SA15 co culture with 3 L. garvieae strains under high aeration level. (DOCX 24 kb)

**Additional file 3: Table S6.** Gene expression changes in S. aureus SA15 co culture with 3 L. garvieae strains under low aeration level. (DOCX 24 kb)

**Additional file 4: Table S7.** Aeration induced changes in L. garvieae (N201, CL 1183 and Lg2 strains) H2O2-related gene expression in co-culture with S. aureus SA15. (DOCX 17 kb)

### Abbreviations

BHI: Brain heart infusion; GOI: Gene of interest; LAB: Lactic acid bacteria; ROS: Reactive oxygen species

### Acknowledgements

The authors acknowledge Mr. Mar Blanco for the gift of *Lactococcus garvieae* strain CL-1183, as well as Masahira Hattori and Hidetoshi Morita for the gift of *Lactococcus garvieae* strain Lg2 and Maryline Bornes and Kate James for English proofreading.

### Funding

The authors of this work as well as the collection of data were supported by the University Clermont-Auvergne (IUT Clermont-Ferrand) and by the Institut national de la recherche agronomique (INRA). The funding bodies did not have any influence neither in the design of the study, in the analysis and the interpretation of data, nor in the writing of the manuscript.

### Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

### Authors' contributions

CQ performed the RT-qPCR, analyzed genes expression, ran statistical analyses and was a major contributor in writing the manuscript. MB contributed to cultivate the different strains, performed the quantitative analysis of H₂O₂ in L. garvieae cultures, the RNA extraction, DNase treatment and the RT-qPCR, and was also a contributor in writing the manuscript. TS cultivated the different strains, performed the tests with added catalase, and contributed in writing the manuscript. PD helped in RT-qPCR, genes expression analysis and improvement of the manuscript. PV contributed to cultivate the different strains and to extract the RNA. ER contributed to statistical analyses. CM contributed to the adjustment of the method of quantitative analysis of H₂O₂. GG read and corrected the manuscript. SB and CD conceived the study. CD contributed to genes expression analysis and to the structuring and improvement of the manuscript. SB contributed to cultivate the different strains, performed the RNA extraction and was also a contributor in writing the manuscript. All authors read and approved the final manuscript.

### Ethics approval and consent to participate

Not applicable

### Consent for publication

Not applicable

### Competing interests

The authors declare that they have no competing interests.

### Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Received: 21 December 2017 Accepted: 14 November 2018

Published online: 22 November 2018

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