Intraspecific cooperation allows the survival of Staphylococcus aureus staff: a novel strategy for disease relapse

Abstract

Background

The contribution of interspecies interactions between coinfecting pathogens to chronic refractory infection by affecting pathogenicity is well established. However, little is known about the impact of intraspecific interactions on infection relapse, despite the cross-talk of different strains within one species is more common in clinical infection. We reported a case of chronic refractory pulmonary infection relapse, caused by two methicillin-sensitive S. aureus (MSSA) strains (SA01 and SA02) and revealed a novel strategy for relapse via intraspecific cooperation.

Methods

The hemolytic ability, growth curve, biofilm formation, virulence genes and response of G. mellonella larvae to S. aureus infection were analysed to confirm this hypothesis.

Results

SA02 hemolytic activity was inhibited by SA01, along with the expression of hemolysin genes and the virulence factor Hla. Additionally, SA01 significantly enhanced the biofilm formation of SA02. AIP-RNAIII may be a possible pathway for this interaction. Compared with mono-infection, a worse outcome (decreased larval survival and increased microbial burden) of the two MSSA strains coinfected with G. mellonella confirmed that intraspecific interactions indeed enhanced bacterial survival in vivo.

Conclusion

The intraspecific interaction of S. aureus could lead to chronic refractory infection via pathogenicity changes.

Background

Staphylococcus aureus (S. aureus) is an opportunistic human pathogen that is found in over 30% of healthy individuals worldwide. It can lead to various illnesses, including sepsis, pneumonia, osteomyelitis, endocarditis, surgical site infections, and infections of the skin and soft tissues [1]. These infections are often persistent and highly resistant to antibiotic treatment, with methicillin-resistant S. aureus (MRSA) being particularly notorious [2]. In addition to drug resistance, many emerging relapse strategies enable pathogens to survive under intense antibiotic pressure, even in the case of methicillin-sensitive S. aureus (MSSA).

Many strategies are closely associated with chronic refractory infections, such as biofilm formation and persister cells [3]. A common characteristic of these two strategies is that microorganisms exhibit significant antimicrobial resistance without genetic mutations or the presence of antimicrobial resistance genes, making them more difficult to eliminate by the immune system or other disinfectants, which poses a major public health challenge. Research has shown that host conditions, such as low nutrient availability, hypoxia, advanced glycation end products (AGEs), and low pH, can shift bacteria into an antibiotic-tolerant state [4,5,6,7], often manifesting as biofilms. On the other hand, such as quorum sensing (QS), interspecies interactions among co-infecting pathogens may influence virulence, antibiotic resistance, and biofilm formation through mechanisms such as cross-protection and cross-feeding, contributing to chronic refractory infections [8, 9]. QS is a communicating system where autoinducers have been utilized as a signal to coordinate intraspecies behavior. These communicating systems can regulate genetic expressions and enable intraspecies interactions. However, there is limited understanding of the cross-talk between different strains within a single species, which is more common and often overlooked in clinical infections. S. aureus exists as a population of cells within infected tissues, affecting various cell types and strains. The concept of “bet-hedging” from evolutionary biology demonstrates that even under favorable conditions, some members of a population may emerge that would otherwise be deemed non-beneficial; however, if conditions change, these traits may confer a survival advantage [10].

In this study, we report two co-infecting strains of S. aureus (SA01 and SA02), both of which are methicillin-sensitive (MSSA), initially isolated from the bronchoalveolar lavage fluid (BALF) of a pneumonia patient. One of these strains (SA02) served as a reservoir for a relapsing infection, occurring five months after clinical resolution, and contributed to therapeutic failure during vancomycin treatment. To investigate the mechanisms underlying this relapse, we focused on the interactions between the two clinical strains. Our findings revealed that the virulence of the recurrent strain SA02 was significantly inhibited, while biofilm formation was enhanced by SA01 through the AIP-RNAIII pathway, thereby providing protection and increasing the viability of SA02. The key highlight of this study is that intraspecific cooperation between the two S. aureus strains facilitates the survival of these bacteria, making them more susceptible to relapses.

Methods

Bacterial strains and antimicrobial susceptibility testing

Two S. aureus strains (SA01 and SA02) were isolated simultaneously from bronchoalveolar lavage fluid (BALF) specimen of pneumonia patient. The BALF was collected by the pulmonary physician using bronchofibrescopy following the operation standard and delivered to the microbiological laboratory within 30 min. The isolates were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (BioMerieux, Marcy l’Etoile, France) after incubating on Columbia sheep blood agar plate (Jiangmen Caring Trading Ltd., Jiangmen, China) at 37 °C for 48 h in 5% CO₂. Antimicrobial susceptibility was determined using the Vitek2 Compact System (BioMerieux, Marcy l’Etoile, France), and the results were interpreted using the M100-S24 criteria introduced by the Clinical and Laboratory Standards Institute 2023 (CLSI 2023). All strains were stored in sheep blood at -80 °C until use.

Whole-genome sequencing (WGS) of S. aureus strains

Every identified strain of S. aureus from the patient’s BALF specimen was sent to Guangzhou Yike Biotechnology Co., Ltd., for whole-genome sequencing. The genome sequence of the strains was obtained through high-throughput sequencing, and the average nucleotide identity (ANI) value was calculated to estimate the genomic similarities between S. aureus strains isolated using OrthoANIu. The raw sequencing data have been deposited in the NCBI SRA database under BioProject accession number PRJNA1122346. The reviewer link is https://dataview.ncbi.nlm.nih.gov/object/PRJNA1122346?reviewer=e6e437p9848ptr2g4usd48cfh8.

Sequence typing (ST) of S. aureus strains

Sequence typing (ST) was characterized by the multilocus sequence typing (MLST) method. All S. aureus isolates were subjected to MLST analysis in accordance with earlier protocols [11]. Bacterial DNA was extracted using a DNA extraction kit (Tiangen Biotech, Beijing, China) with lysostaphin according to the manufacturer’s instructions. Seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, yqiL) of S. aureus were amplified by polymerase chain reaction (PCR). Gene segments from the product were sequenced and matched to S. aureus allele profiles from a database (http://saureus.mlst.net/).

Preparation of cell-free conditioned medium (CFCM)

CFCMs were prepared by inoculating S. aureus strains (SA01) in Tryptone Soy Broth Medium (TSB) (Oxoid, Hampshire, England). Overnight cultures of S. aureus were inoculated into fresh TSB (1% volume), and the bacteria were allowed to grow for 48 h at 37 °C under shaking conditions (200 rpm). Cultures were centrifuged for 15 min at 12 000 r/min and 4 °C. The supernatants were collected and filtered through a 0.22 μm filter (BIOFIL, Guangzhou, China). The pH of the supernatants was adjusted to 6.8-7.0. The resulting CFCM was then evaporated using a vacuum centrifuge concentrator 600 (CV600) (Beijing JM Technology Co., Ltd., Beijing, China), the dried deposit was resuspended in sterile 0.85% saline, and the final concentration was approximately 20×. The 20% CFCM treated was used in the experiments described herein (at a final concentration of 4× CFCM). Control media(CM)was performed the same treatment above to the TSB but without bacterial growth.

Proximity assay and CAMP test

The proximity assay was performed as described previously [12]. Bacterial suspensions with the same McFarland standard were adjusted by overnight culture of SA01 and SA02. The same volume of bacterial suspension was plated on a blood agar plate in 0.5 cm increments, and the starting distance between SA01 and SA02 was 1 cm. Blood agar plate was incubated at 37 °C for 24 h. The CAMP test was performed according to the standard procedure. SA01 was inoculated as a centre streak on a blood agar plate, and SA02 was inoculated perpendicularly to SA01 one cm apart. Blood agar plate was incubated at 37 °C for 24 h. To analyse the impact of CFCM on the hemolysis of SA02 on blood agar plate, a CFCM interaction hemolysis assay was performed as described elsewhere but with moderate modifications [13]. One milliliter of CFCM from SA01 was equally plated on a blood agar plate. SA02 was inoculated into the plates after drying. The plates were incubated at 37 °C for 48 h. The hemolysis conditions on blood agar plate without CFCM treatment were compared.

Hemolysis assay

The hemolysis assay was performed as described elsewhere with moderate modifications [14]. The prepared CFCM was exposed to 65 °C for one h to disrupt the hemolysin secreted by SA01, and the CM was prepared via the same process. Preprocessed 20% CFCM and CM were cocultured with SA02 strains at 37 °C for 24 h with shaking at 200 rpm. The supernatant was recovered following eight minutes of centrifugation at 10,000 rpm. The ability of the strains to hemolyze was assessed using a 6% human erythrocyte solution. After that, 900 µl of erythrocyte solution and 100 µl of supernatant were combined, incubated for 30 min at 37 °C, and then centrifuged for 10 min at 2500 rpm. The positive control was 0.1% Triton-X 100, and the negative control was TSB. The absorbance (OD405) of the supernatant was determined using a Multiskan FC Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, United States).

Static biofilm formation assay

The biofilm formation ability of the S. aureus isolates was evaluated by the microtiter plate test, as described elsewhere with moderate modifications [15, 16]. After overnight culture in TSB at 37 °C with shaking at 200 rpm, the densities of the isolates were adjusted to 0.5 McFarland standard with sterile distilled water to obtain bacterial suspensions, and the bacterial concentration was approximately 1-1.5 × 10⁸ CFU/ml. To evaluate the biofilm formation ability of SA01 and SA02, 180 µl of TSB and 20 µl of bacterial suspension were added. To analyse the impact of CFCM on the biofilm formation activity of SA02, 140 µl of TSB supplemented with 40 µl of CFCM or CM and 20 µl of bacterial suspension were added. Following incubation at 37 °C for 24 h, the plate was washed with phosphate-buffered saline (PBS) (Boster, Wuhan, China) three times to remove the unattached bacteria. After heat-fixing the biofilms for 120 min at 60 °C, they were dyed for 5 min at room temperature using 0.1% crystal violet (CV). The plate was rinsed under cold running water after the CV was aspirated. After drying the biofilms for 120 min at 35 °C, digital cameras (D-LUX7, Leica, Wetzlar, Germany) were used to capture images. Subsequently, 200 µl of 95% ethanol was used to resolubilize the dyed biofilm, and an optical density (OD) measurement was made using a Multiskan FC Microplate Photometer at 590 nm to assess the biofilm-forming capability. S. aureus ATCC 25,923, S. aureus ATCC 29,213 and TSB broth were used as positive controls, negative controls and blank controls, respectively.

Bacterial growth curve

The bacterial growth curve was calculated as previously described but with moderate modifications [17]. In vitro, the bacterial growth curve was used to evaluate the effect of CFCM on SA02 by measuring the optical absorbance (OD630) of the bacterial culture in TSB. The overnight growth culture was diluted in TSB in the presence of 20% CFCM or CM, and the optical density at OD630 was 0.05. The microplate was then incubated at 37 °C with shaking at 200 rpm. The absorbance of the cultures was measured by a Multiskan FC Microplate Photometer at 630 nm every two hours. The growth curve was plotted with the absorbance data as the vertical coordinate and the time interval as the horizontal coordinate.

Reverse transcription real-time quantitative PCR (qRT‒PCR)

Reverse transcription real-time quantitative PCR was performed as described elsewhere but with moderate modifications [16, 18]. Briefly, overnight cultures of SA02 in the presence or absence of 20% CFCM were centrifuged at 8000 rpm at 4 °C for five minutes to harvest bacterial cells. Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. The purity of the obtained RNA was measured by a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, United States) at 260 nm, and the RNA was then reverse transcribed into cDNA according to the manufacturer’s instructions (All-in-One First-Strand Synthesis MasterMix, Yugong Biolabs, Jiangsu, China). Quantitative reverse transcription PCR (qRT‒PCR) was performed using SYBR Premix Ex Taq (Takara, Tokyo, Japan) and an Applied Biosystems™ 7500 (Thermo Fisher Scientific, Waltham, MA, United States), and the relative expression levels of the target genes were calculated using a formula according to the threshold cycles (Ct values) and were normalized to that of the housekeeping gene 16 S RNA. The primers used are listed in Table S1.

Galleria mellonella larvae survival assay

The Galleria mellonella larvae survival assay was performed as described elsewhere but with moderate modifications [19, 20]. G. mellonella larvae (Huiyude Biotechnology Co., Ltd. Tianjin, China) were stored in the dark at 4 °C and used within seven days of receipt [21]. Stationary phase bacterial cultures were washed three times with sterile normal saline and resuspended. Randomly select ten healthy larva, weighting 200–300 mg to inject bacterial suspensions (measured with a spectrophotometer) through the last left pro-leg using a Hamilton syringe (Hamilton, Shanghai, China). For coinfection, larvae were administered ten µl of bacterial suspensions containing SA01 and SA02 at the indicated inoculum. After injection, the larvae were placed in sterile Petri dishes with filter paper and incubated at 37 °C for three days. Percent survival of the larvae observed every day. Mock-inoculated (sterile normal saline) animals were used as controls.

Determination of the proliferation of SA02 in G. mellonella larvae

To determine the proliferation of the SA02 strain in G. mellonella larvae, an assay was performed as described elsewhere but with moderate modifications [19, 20, 22]. Larvae infected with SA02 strains alone or coinfected with SA01 and SA02 proliferative larvae^− 1 were assessed for more than 72 h. Infected larvae (n = 3) were homogenized using a mortar and pestle with three ml of sterile normal saline, serially diluted and plated onto blood agar plates and incubated at 37 °C for 24 h. Most of the previous workers used selective medium in this assay, but the selective medium was not used here, as it was established that no S. aureus was detected in the sterile normal saline-injected larvae. The bacterial load was calculated as the S. aureus colony-forming units (CFUs) per larva and was based on the number of colonies that grew at specific dilutions.

Preliminary characterization of the active molecule of SA01 CFCM

The characterization of the active molecule was assayed by performing some treatments as described elsewhere but with moderate modifications [23]. CFCM was prepared as described above, either with heat exposure at 65 °C for one h, with 1 mg/ml proteinase K at 37 °C for one h, with 5 mM EGTA at room temperature for one h, or with NaOH to achieve a pH of 11 for one h at room temperature and initial pH return by HCl. Subsequently, the CFCMs treated as described above were equally plated on blood agar plates, and SA02 cells were inoculated. The active molecules were characterized by determining the diameter of the hemolytic halo of SA02.

Statistical analysis

All experiments were performed in triplicate, and the results are expressed as the mean ± standard deviation (SD). The statistical analyses were performed using GraphPad Prism 9.0 software (United States). Survival curves were analysed using the log-rank test, and other assays were analysed using Student´s t test. P < 0.05 was considered to indicate statistical significance.

Results

Case report

A 12-year-old boy with a 15-month history of incomplete eyelid closure and an askew mouth (towards the left) was referred to our clinic and was subsequently diagnosed with ponto-medullary low-grade glioma. Microresection of brain stem lesions was performed, and cefuroxime 0.75 g q/12 h was used to prevent infections during the surgery. On the third postoperative day, the patient developed a fever (body temperature of 37. 5 °C), accompanied by decreased spontaneous breathing. Chest CT revealed pulmonary infection (Fig. 1A), and the antimicrobial regimen was then changed to piperacillin-tazobactam 4.5 g q/8 h. Moreover, two methicillin-susceptible Staphylococcus aureus (MSSA) strains with different phenotypes (SA01 and SA02) were simultaneously isolated from both sputum and BALF specimens, and the susceptibility testing results are listed in Table 1. The two S. aureus strains with different hemolytic phenotypes coexisted on blood agar plate and exhibited irregularly strange hemolytic rings, as shown in Fig. 1B. During the following hospitalization period, these two S. aureus strains were repeatedly isolated from the patient’s BALF, even after vancomycin (1.0 g q/8 h for five days) treatment (previously isolated a strain of MRSA in other hospital before coming to our hospital for treatment). With the improvement of the protopathy, the patient discharged from the hospital after one week, and regular rehabilitation therapy was administered at the community hospital.

Five months later, the patient fell into a sudden coma accompanied by a fever (body temperature of 39.4 °C) and was subsequently diagnosed with septic shock and pneumonia in a community hospital. After five days of anti-infection treatment (piperacillin-tazobactam 4.5 g q/8 h) and supportive care, the patient was transferred to our clinic for further therapy. S. aureus was isolated from BALF again during this hospitalization period. Whole-genome sequencing (WGS) revealed that the most recently isolated S. aureus strain was homologous to SA02 (ANI: 99.70%), indicating that SA02 was not cleared even after challenge with vancomycin and relapsed after five months.

We noted a very special phenomenon in this patient: the patient was infected with two strains of S. aureus at the same time, and the hemolytic ability of one strain (SA02) was affected by that of the other strain (SA01). Even under vancomycin treatment, SA02 still relapsed a few months later. Therefore, we propose the following hypothesis: the intraspecific interaction of S. aureus protects its companions from being killed by host immune cells or antibiotics by reducing virulence, enhancing adhesion, and so on. This study investigated this hypothesis.

Fig. 1

Imaging examination and laboratory examination of the patient: (A) Chest CT image of the patient with pulmonary infection. (B) Two S. aureus strains (SA01 and SA02) with different hemolytic phenotypes coexisted on the blood agar plate: the large beta hemolytic ring of SA02 was inhibited by SA01, which has double smaller hemolytic ring

Table 1 Susceptibility testing results of S. Aureus isolates

MLST analysis

According to MLST analysis, we identified different STs among the S. aureus isolates. The ST type of the SA02 isolate was MSSA ST398, and the SA01 isolate was MSSA ST243.

SA02-mediated hemolytic activity was inhibited by diffusible molecules secreted by SA01

To determine the effects of the soluble diffusible molecules secreted by SA01 on the hemolytic phenotype of SA02, proximity assays and CAMP tests were performed. In the proximity assay, the hemolytic activity of SA02 gradually weakened as the distance between the two isolates decreased (Fig. 2A). In the CAMP test, a triangular arrow of hemolysis could be clearly observed (Fig. 2B). An assay demonstrated that the degree of hemolysis exhibited by SA02 may be affected by soluble diffusible molecules secreted by SA01. Furthermore, a CFCM interaction hemolysis assay showed that the hemolytic activity of SA02 significantly decreased when it was grown on a blood agar plate plated with CFCM from SA01 (Fig. 2C, D), indicating that SA02 hemolysis was inhibited by the diffusible molecules secreted by SA01. Additionally, a hemolysis assay showed that SA01 CFCM decreased the hemolytic ability of SA02 toward human blood cells (P < 0.0001) (Fig. 3A, B).

Fig. 2

Hemolysis of SA02 under the following conditions: (A) Proximity assay with SA01 at 24 h. (B) CAMP test with SA01 at 24 h. (C) SA02 growth in the blood agar plate treated with CFCM. (D) SA02 growth on a blood agar plate treated with CM (as a negative control group). CFCM, cell-free conditioned medium; CM, control medium

Fig. 3

Comparison of the hemolytic ability of SA02 in the presence of SA01 CFCM and CM (as a negative control group): (A) Hemolysis assay in an EP tube. TSB was used as the negative control, and Triton-X was used as the positive control. (B) Comparison of hemolysis by measuring the OD 405. The bar plot shows the OD 405 value, and **** indicates a P value < 0.0001. CFCM, cell-free conditioned medium; CM, control medium

SA01 CFCM promoted SA02 biofilm formation

To evaluate the effect of SA01 CFCM on SA02 biofilm formation, SA02 cells were exposed to 20% CFCM in a microtiter plate test. Crystal violet staining and OD590 values showed that the amount of SA02 biofilm increased by 8.492 ± 0.073 times (P < 0.0001) after treatment with SA01 CFCM (Fig. 4A, B).

Fig. 4

Comparison of the biofilm formation ability of SA02 in the presence of SA01 CFCM and CM (as a negative control group): (A) Cells were stained with crystal violet. (B) Comparison of biofilm formation ability by measuring the OD590. The bar plot shows the OD590 value, and **** indicates a P value < 0.0001. CFCM, cell-free conditioned medium; CM, control medium

SA01 CFCM did not inhibit the growth of SA02

The growth curve of SA02 did not differ in the presence of 20% CFCM or CM (negative control), indicating that SA01 CFCM has no influence on SA02 growth. These findings revealed that the changes in SA02 hemolytic activity and biofilm formation had no relationship with cell growth (P > 0.05) (Fig. 5).

Fig. 5

Comparison of growth curves of SA02 in the presence of SA01 CFCM and CM (as a negative control group) by measuring the OD630. CFCM, cell-free conditioned medium; CM, control medium

SA01 CFCM affected the transcription level of SA02 hemolysin genes and enhanced the transcription of SA02 biofilm genes

After treatment with SA01 CFCM, the expression of hla, hlb and hld in SA02 significantly decreased by 5.2 ± 1.330-, 5.1 ± 1.472-, and 20.6 ± 8.864-fold, respectively (all P < 0.0001), and hlg expression significantly increased by 9.6 ± 4.585-fold (P < 0.05) compared with that in the CM-treated group. In terms of biofilm-related genes, fnbA, fnbB, icaA, and spa expression in the SA02 strain increased 4.4 ± 1.055-fold (P < 0.01), 29.9 ± 16.684-fold (P < 0.05), 15.7 ± 3.242-fold (P < 0.01), and 3.4 ± 0.694-fold (P < 0.01), respectively. The expression of sarA significantly increased by 5.3 ± 2.386-fold (P < 0.05). The expression of RNAIII, the key factor involved in the expression of hemolysin genes and biofilm-related genes, was significantly decreased by 2.8 ± 0.664-fold compared with that in the CM-treated strain (P < 0.001), as shown in Fig. 6.

Fig. 6

Comparison of the gene expression levels in SA02 cells exposed to SA01 CFCM and CM (as a negative control group). The black bar plot shows the relative expression levels (fold change) to the gray bar plot. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001. CFCM, cell-free conditioned medium; CM, control medium

Efficacy of coinfection in G. mellonella

As shown in Fig. 7A and B, larvae that received SA01 (1 × 10⁵ larva^− 1) alone exhibited a survival rate of 96.67 ± 5.77% at 24 h, 90 ± 0% at 48 h and 83.33 ± 5.77% at 72 h post infection, which was not significantly lower than that of the control group. Coinfection of larvae with SA01 (1 × 10⁵ larva^− 1) and SA02 (1 × 10⁶ larva^− 1) resulted in no significant decrease in survival over 48 h [24 h, 83.33 ± 5.77%; 48 h, 56.67 ± 5.77%] but did significantly decrease survival at 72 h (23.33 ± 5.77%) (P < 0.01) compared to that of larvae that received SA02 (1 × 10⁶ larva^− 1) alone at 24 (83.33 ± 5.77%), 48 (63.33 ± 5.77%) and 72 h (53.33 ± 5.77%).

Fig. 7

Percentage survival of G. mellonella larvae. (A) Survival curves of G. mellonella larvae following mono- and coinfection with SA01 (1 × 10⁵ larva^− 1) and SA02 (1 × 10⁶ larva^− 1). The data are from a single representative experiment. (B) The bar plot shows the percentage survival of G. mellonella after injection. Statistical significance was determined by comparing coinfected larvae to the corresponding SA02 monoinfected larvae. ** indicates P < 0.01, and ns represents no significance

The microbial burden of SA02 in Larvae with coinfection increase comparing to that of Larvae infected solely

The microbial burden of SA02 in coinfected [SA01 (1 × 10⁵ larva^− 1) with SA02 (1 × 10⁶ larva^− 1)] larvae was assessed by measuring SA02 c.f.u. larva^− 1 and comparing these results to those of larvae infected by SA02 (1 × 10⁶ larva^− 1) alone. The microbial burden of SA02 in coinfected larvae significantly increased at 24 (2.66 ± 0.52 × 10⁷ c.f.u. larva^− 1, P < 0.01) and 48 (8.20 ± 1.39 × 10⁷ c.f.u. larva^− 1, P < 0.01) h compared to that in larvae infected with SA02 alone at 24 (8.0 ± 1.83 × 10⁶ c.f.u. larva^− 1) and 48 (2.08 ± 0.87 × 10⁷ c.f.u. larva^− 1) h. Coinfection of larvae had no significant effect on the microbial burden at 72 h (1.17 ± 0.04 × 10⁸ c.f.u. larva^− 1, P > 0.05) compared to that in those infected solely with the SA02 strain (7.53 ± 3.02 × 10⁷ c.f.u. larva^− 1) (Fig. 8).

Fig. 8

SA02 c.f.u. larva^− 1 obtained from G. mellonella larvae following monoinfection with SA02 (1 × 10⁶ larva^− 1) or coinfection with SA01 (1 × 10⁵ larva^− 1) over 72 h. The bar plot shows the SA02 c.f.u. larva^− 1. Statistical significance was determined by comparing coinfected larvae to the corresponding SA02 monoinfected larvae. ** indicates P < 0.01, and ns represents no significance

Preliminary characterization of SA01 CFCM suggested that the active molecule is likely AIP

The activity of CFCM was evaluated by comparing the hemolytic halos of SA02 strains on different blood agar plates treated with CFCM via various biochemical tests, and the diameter of the hemolytic halo was measured. The diameter of the hemolytic halo did not significantly differ between the SA02 strains on the plates treated with CFCM exposed to heat, proteinase K or EGTA, while significant differences could be observed after treatment with CFCM exposed to NaOH (Fig. 9). Briefly, apart from NaOH treatment, all the treatments, including exposure to heat, proteinase K and EGTA, retained activity on SA02.

Fig. 9

Characterization of the active molecule present in SA01 CFCM. (A) Hemolysis of SA02 on blood agar plates treated with SA01 CFCM and exposed to heat, proteinase K, EGTA, or NaOH. (B) The bar plot shows the diameter of the SA02 hemolytic ring on blood agar plates treated with SA01 CFCM, as determined by various biochemical tests. **** indicates P < 0.0001, and ns represents no significance. CFCM, cell-free conditioned medium; CM, control medium

Discussion

A common issue in S. aureus infections is disease relapse. MRSA (methicillin-resistant Staphylococcus aureus) has attracted the most attention in S. aureus infections. however, treatment failure is a more complex issue and cannot be explained by resistance development alone, as shown in this case. Here, we report the case of a patient who experienced pulmonary infection relapse caused by two MSSA strains, even after treatment with full-dose and full-range vancomycin, and revealed a novel multiple-strategy approach for relapsing refractory chronic infection via “intraspecific mutualism and cooperation”.

Interactions between different microbial species have been widely reported [24]. Because various individuals within the same species may have diverse activities in the infected host, it is debatable whether species behave as the smallest unit in microbial ecosystems [25]. Although cross-protection and cross-feeding between coinfecting pathogens have been proven by increasing evidence, studies characterizing these interactions have focused only on interspecies interactions, such as those involving Staphylococcus aureus vs. Enterococcus faecalis, Candida albicans vs. Staphylococcus aureus, and coagulase-negative staphylococci vs. Staphylococcus aureus [9, 20, 26]. Currently, several intraspecific variations in the phenotypes and genomes of microbes have been identified [27]. Because they reside in the same habitat for an extended period, different strains of the same species may interact more frequently [28]. Individuals with distinct cells within one species can often be ignored in clinical infections. Similar to how species variation affects community composition and function, intraspecific variation can also have an impact [29]. Although these interactions have received less research attention than interspecific interactions, these interactions are also crucial to the course of the disease [30]. Furthermore, due to the similar colony and microscopic morphology of the same bacterial species, it is difficult to detect different conspecific strains in clinical bacterial infections. Therefore, research on intraspecific interactions has focused mainly on the fields of food microbiology and environmental microbiology, while there is a lack of knowledge on the cross-talk between different cell subgroups within one species in the clinical field. Here, we report the case of a patient infected with different conspecific Staphylococcus aureus strains. Notably, during the patient’s first hospitalization due to pulmonary infection, the bacterial colonies isolated from BALF showed a very strange β-haemolytic phenomenon, manifested as irregular haemolytic rings with sharp edges on the Columbia blood plate. In further experiments, the isolates were identified as two MSSA strains (MSSA ST398 and MSSA ST243) in similar quantities, one of which (MSSA ST243) inhibited the other (MSSA ST398) β-hemolysis, leading to strange hemolytic rings. On the other hand, the poor effectiveness of antibiotic treatment (piperacillin-tazobactam and vancomycin) has plagued clinicians during anti-infection therapy, and MSSA ST398 strain relapse has attracted great attention. In this work, we hypothesized that intraspecific interactions occurred between two different conspecific strains and were attributed to ineffective clinical treatment and infection release. We conducted a series of experiments.

The interaction between coinfecting pathogens may affect growth, virulence, antibiotic resistance and biofilm formation through cross-protection and cross-feeding, leading to chronic refractory infection [9, 20, 26]. We found that the two MSSA strains did not affect each other’s growth rate or antibiotic sensitivity, but strong interactions occurred in terms of virulence and biofilm formation between them, leading to a greater bacterial burden during coinfection. The inhibition of the hemolytic ring suggested that SA01 has a significant inhibitory effect on the virulence of SA02. Further experiments confirmed that the hemolytic activity of SA02 was inhibited by diffusible molecules secreted by SA01, as well as the expression of hemolysin genes and the virulence factor Hla. The retarded virulence helped the SA02 strain survive within infected tissue in a “seemingly harmless” state, against immune responses and as a continual presence despite antibiotic treatment. In addition, SA01 significantly enhanced the biofilm formation of SA02 cells. A complex mixture of bacterial cells, proteins, and DNA encased in polysaccharide intercellular adhesin (PIA) biofilms is crucial for the colonization of bacteria and the development of chronic infections [31]. The development of biofilms by bacteria is a protective mode of growth that decreases the susceptibility of bacteria to antibiotics. Persister cells inside these biofilms are known to be extremely tolerant to antibiotics and have frequently been linked to the recurrence of infection [32]. The G. mellonella infection model showed a worse outcome (decreased larval survival and increased microbial burden) after SA01 and SA02 coinfection than after mono-infection, which confirmed that intraspecific interactions indeed enhance bacterial survival in vivo.

Bacteria collaborate by secreting chemicals that are beneficial to the colony as an entire entity. Cell‒cell signalling pathways frequently control such cooperative actions. Through the identification of autoinducer molecules and the group-wide secretion of these chemicals, bacteria that undergo quorum sensing can collaborate in a density-dependent manner. Numerous bacterial species exhibit phenotypes or substantial intraspecific variation in autoinducer–receptor alleles. Autoinducing oligopeptides (AIPs) are quorum sensing signalling molecules that specifically communicate between bacterial communities [33]. Here, we preliminarily characterized the SA01 CFCM and suggested that the active molecule involved in intraspecific mutualism and cooperation was likely AIP. AIP can inhibit agr-mediated quorum sensing of S. aureus [23], and can drive target gene expression via the posttranscriptional regulator RNAIII [33]. We found that RNAIII of SA02, the key factor in the expression of hemolysin genes and biofilm-related genes, was significantly inhibited by SA01, indicating that AIP-RNAIII may be a possible pathway for intraspecific interactions between SA01 and SA02. Bruno et al. [34] also observed that when co‑culture with S. aureus, non‑aureus staphylococci (NAS) species can generally downregulate the expression of RNAIII, the effector molecule of the QS system. SarA can promote biofilm formation by directly upregulating various extracellular proteins (such as ClfA, SpA, and FnBPA) [35, 36] and by inhibiting the expression of extracellular proteases (such as SspA, SspB, Aur, and ScpA) [37]. This aligns with our observation of a significant upregulation of SarA and a notable enhancement in biofilm formation ability in the treatment group. However, more in-depth research is needed on determining the specific proteins involved.

According to whole-genome sequencing and MLST, the recurrent strain identified in this study was MSSA ST398, which was recently reported to be the most abundant ST type among MSSA strains isolated in Chinese hospitals [38, 39]. In this case, the initial MSSA ST398 strain was isolated repeatedly from the patient’s BALF, even after treatment with vancomycin, and appeared to be in an antibiotic-tolerant state. In contrast to antibiotic-resistant strains, strains that are tolerant to antibiotics can persist in their host for an extended duration, making treatment more challenging, even though they appear to be susceptible to antibiotics in laboratory experiments, resulting in patient suffering and costly hospitalization. As shown in this case, long-term and repeated antibiotic courses due to persistent or recurrent infections negatively impact not only the health of the patient but also the damage of resident microbiota. Even worse, it has been reported that strains with low persistence levels are quickly replaced by strains with high persistence levels, gradually resulting in herd tolerance. Increasing evidence suggests that antibiotic-tolerant pathogens, rather than antibiotic-resistant pathogens, cause many antibacterial drugs to fail to treat bacterial infections and pose major public health problems, such as persistent Streptococcus pulp infection after root canal treatment, persistent Mycobacterium infection in tuberculosis and a variety of recurrent chronic infections (diabetic foot infection, etc.) caused by S. aureus. In addition to antibiotics and other environmental factors (host factors), pathogen‒ interactions play important roles in the pathogenicity and persistence of bacteria. In this case, the interactions between different strains within species could also affect each other’s pathogenicity and biofilm formation ability via intraspecific mutualism and cooperation, leading to a decrease in virulence expression and increased biofilm formation, ultimately resulting in resistance to antibiotics. In this clinical case, further experiments are needed to verify whether it ultimately leads to persister cells, especially tolerance to vancomycin.

There is a limitation that may impact the comprehensiveness of our findings. The inability to identify CFMC using High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) restricts our understanding of its role in this study. The characteristics and potential activity of CFMC could significantly influence the effects of AIP. Therefore, we plan to incorporate HPLC-MS analysis of CFMC in future research to provide more robust evidence supporting our conclusions. We are dedicated to addressing these limitations in our ongoing work to enhance the reliability and depth of our research.

Conclusion

We found that strong interactions occurred in terms of virulence (hemolytic activity and the expression of hemolysin genes) and biofilm formation between them and led to a decreased larval survival rate and increased bacterial burden during coinfection, as confirmed by the G. mellonella infection model in vivo. AIP-RNAIII may be a possible pathway for intraspecific interactions. This study deepens the understanding of mixed infections of different strains within one species and provides new insight into refractory infections caused by S. aureus.