Strain-specific interspecies interactions between co-isolated pairs of Staphylococcus aureus and Helcococcus kunzii from chronic wounds

Abstract

Background: Bacterial infection is major contributor in the delay of wound healing and persistence of non-healing wounds. Bacteria, functioning within complex multicellular communities, facilitate the survival of certain microorganisms in diverse environments. Helcococcus kunzii, a Gram-positive anaerobic commensal coccus, and Staphylococcus aureus, the primary pathogen in chronic wounds, have strong interaction potential, in which H. kunzii decreases the virulence of S. aureus in co-culture in vivo and in vitro.

Results: This paper describes the interaction between H. kunzii and S. aureus clinical strains from chronic wounds by examining their transcriptomic behavior (S. aureus virulence genes) and phenotypic features (hemolysin production, growth and biofilm formation). We used five new clinical pairs of H. kunzii and S. aureus strains co-isolated from diabetic foot ulcers (DFU) and one previously studied clinical pair, not co-isolated from the same DFU. H. kunzii strains were sequenced and compared with two H. kunzii strains previously sequenced with high (H13) or low (H10) impact on S. aureus regulatory virulence effectors. Sequencing showed that all strains were phylogenetically similar to H13, with the exception of NHKT2, allowing to predict a potential "high impact" on S. aureus virulence. After coculture of clinical strain pairs in medium reflective of a chronic wound environment, we observed a downregulation of agrA and hla, two regulatory and virulence genes of S. aureus, supporting previously results obtained with not co-isolated strains. We confirmed a decrease in S. aureus hemolysin production in cocultures. While coculture did not affect the growth of S. aureus, the presence of H. kunzii appeared to influence the dynamics of S. aureus biofilm formation. Specifically, we noted a significant increase in S. aureus biofilm formation in coculture compared to monoculture during the biofilm maturation stage (after 72 h) for 4/5 of the co-isolated pairs.

Conclusions: The effect of reducing the virulence of S. aureus by H. kunzii described previously is not reproducible with co-isolated strains from same DFU. The therapeutic strategy of using H. kunzii as a probiotic should be reconsidered in favor of an approach that targets the underlying mechanisms responsible for this virulence reduction.

Keywords: Helcococcus kunzii; Staphylococcus aureus; Bacterial interaction; Biofilm formation; Coculture experiments; Diabetic foot ulcer; Virulence regulation.

Fig. 1

Genomic analysis of H. kunzii strains. (A) Phylogenetic tree of H. kunzii clinical isolates constructed by whole genome alignment. The FJ, T1, T2, T3, T5 strains corresponded to NHKFJ, NHKT1, NHKT2, NHKT3, NHKT5 respectively. T1 and FJ strains were genetically similar and closely similar to T5, T3, and H13 (reference strain). H10 and T2 were genetically distant. (B) Genome annotation-based comparison of H. kunzii strains with H13 reference isolate. All the H. kunzii genomes harbored a variable number of unique strain related genes, while FJ strain did not harbor unique genes

Fig. 2

Log relative fold-change in mRNA expression of virulence and regulatory genes agrA (A), hla (B) and spa (C) in S. aureus, measured by qRT-PCR. The bacteria were co-cultured with H. kunzii for 48 h in CWM. The results are expressed as the mean fold change under the following conditions:1, Monoculture of S. aureus (serving as the standardized control and reference for all S. aureus strains in monoculture); 2, Coculture of NSAT1-NHKT1; 3, Coculture of NSAT2-NHKT2; 4, Coculture of NSAT3-NHKT3; 5, Coculture of NSAT5-NHKT5; 6, Coculture of NSAFJ-NHKFJ; 7, Coculture of NSA739-H13). Error bars represent the standard deviation. Statistical comparison between the monoculture of each S. aureus strain and its corresponding coculture was carried out using a t-test in GraphPad Prism version 9.2. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 3

Percentage of hemolysin production in S. aureus clinical strains in monoculture compared to coculture with H. kunzii clinical strains. The statistical analysis was conducted using a t-test in GraphPad Prism version 9.2 to compare the monoculture of each S. aureus strain with its corresponding coculture alongside H. kunzii. * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0. 0001

Fig. 4

Enhanced growth of S. aureus in coculture with H. kunzii. A blue horizontal line indicates a fold change of 1, denoting no difference in growth when compared to monoculture at the initial time point. The boxplot visualizes the variation and central tendency of CFU/mL fold changes for clinical S. aureus grown with their co-isolated H. kunzii partner, where each dot represents one of three biological replicates per S. aureus isolate tested. Statistical analysis using Welch’s t test reveals a significant difference between the mean CFU/mL fold change in clinical S. aureus strains cocultured with clinical H. kunzii strains versus those cocultured with the reference strain H13

Fig. 5

Kinetics of early biofilm formation of clinical S. aureus in monoculture and coculture with H. kunzii. The kinetics were determined using the Biofilm Ring Test® (Biofilm Control, France) at 3, 5-and 7 h post-incubation. A: Strains exhibiting rapid biofilm formation kinetics; B: Strains with intermediate biofilm formation kinetics

Fig. 6

Monitoring of CFU/mL over time of S. aureus and H. kunzii embedded within the biofilm matrix in monoculture and coculture. The bars represent the mean viable cell count of S. aureus or H. kunzii after biofilm formation at their respective time point

Fig. 7

Colony Forming Unit per milliliter (CFU/mL) of S. aureus in biofilm at 72 h in coculture with the corresponding H. kunzii clinical strains and the H13 strain. Statistical analysis was performed using a two-way ANOVA