Cross-talk among flesh-eating Aeromonas hydrophila strains in mixed infection leading to necrotizing fasciitis

Abstract

Necrotizing fasciitis (NF) caused by flesh-eating bacteria is associated with high case fatality. In an earlier study, we reported infection of an immunocompetent individual with multiple strains of Aeromonas hydrophila (NF1-NF4), the latter three constituted a clonal group whereas NF1 was phylogenetically distinct. To understand the complex interactions of these strains in NF pathophysiology, a mouse model was used, whereby either single or mixed A. hydrophila strains were injected intramuscularly. NF2, which harbors exotoxin A (exoA) gene, was highly virulent when injected alone, but its virulence was attenuated in the presence of NF1 (exoA-minus). NF1 alone, although not lethal to animals, became highly virulent when combined with NF2, its virulence augmented by cis-exoA expression when injected alone in mice. Based on metagenomics and microbiological analyses, it was found that, in mixed infection, NF1 selectively disseminated to mouse peripheral organs, whereas the other strains (NF2, NF3, and NF4) were confined to the injection site and eventually cleared. In vitro studies showed NF2 to be more effectively phagocytized and killed by macrophages than NF1. NF1 inhibited growth of NF2 on solid media, but ExoA of NF2 augmented virulence of NF1 and the presence of NF1 facilitated clearance of NF2 from animals either by enhanced priming of host immune system or direct killing via a contact-dependent mechanism.

Keywords: Aeromonas hydrophila; intramuscular mouse model; metagenomics; mixed infections; necrotizing fasciitis.

Fig. S1.

Virulence characteristics of A. hydrophila strains NF1 and NF2 in a mouse model of infection. (A) Swiss–Webster mice (n = 5) were infected with the indicated infection doses of the individual NF strains by i.m., and mortality rate was recorded at 7 d. (B) A group of four or five animals was infected with strain NF1 or NF2 at a dose of 2 × 10⁸ cfu per animal i.m. and after 48 h, the entire spleen and liver from each of the surviving animal were excised, and the bacterial counts determined. The horizontal lines represent the arithmetic means of the bacterial counts.

Fig. 1.

Virulence features of mono and mixed infections with A. hydrophila NF strains. (A) A group of five mice was infected with the noted dose of NF1 and NF2-lux or NF1 to NF4 mixture and animal mortality was observed over 7 d. Asterisks denote statistical significance among the indicated groups. (B) Three to five mice were infected with NF1, NF1 and NF2-lux mixture, or NF2-lux at 2 × 10⁸ cfu per animal i.m. Control animals were injected with sterile PBS. After 24 h p.i., severity of inflammatory swelling and necrotic lesions were examined and scored. Based on the severity of necrosis, the scale used was as follows: 0 (no necrosis, e.g., for the PBS group), 1+ (mild necrosis, for the NF1-infected group of mice), 2+ (moderate necrosis, for the NF1 and NF2-infected group of mice), 3+ (severe necrosis, for the NF2-infected group of mice). Representative images are shown.

Fig. 2.

Dissemination characteristics of A. hydrophila NF strains during mixed infection in a mouse model. (A) Mice (n = 5) were injected with a mixture of NF1 to NF4 at an infection dose of 5 × 10⁸ cfu (1.25 × 10⁸ cfu per strain) per animal via i.m. route. After 24 h, whole spleen and liver from each animal were homogenized and an aliquot from each sample was subjected to bacterial colony count. The horizontal lines represent the arithmetic means of the bacterial counts (B). The remaining portion of the homogenates was processed for total DNA isolation. The isolated DNA for some organs representing different animals was combined because of the low yield and was labeled as muscle35, spleen45, and/or spleen123, respectively. The isolated DNA was subjected to deep sequencing and metagenomic analysis to identify the NF strains. Relative distribution of four A. hydrophila strains NF1, NF2, NF3, and NF4 in different metagenomic datasets derived from muscle, spleen, and liver samples is shown.

Fig. S2.

Selective dissemination of A. hydrophila strains in a mouse model. Bacterial dissemination to spleen and liver after 24 h of infection with NF1-lux and NF2, or NF1 and NF2-lux mixed cultures was observed by bioluminescence of colonies. (A) Representative images displayed confirmed that all of the disseminated bacterial colonies are NF1 in liver and spleen (I–IV). Further, DNA isolated from spleen and liver tissue samples from NF1 and NF2 mixed infections were subjected to sequencing and metagenomic analysis to determine identity of the strains (either NF1 or NF2) disseminated to these organs (B). The resulting analysis showed that the bacterial DNA recovered from the peripheral organs was specific to NF1. The quality and the concentration of DNA from liver 2 and spleen 3 samples were not high for sequencing. In addition, mice (n = 3) were infected with a mixture of NF1-lux and NF2, or NF1 and NF2∆exoA at a dose of 2 × 10⁸ cfu (1 × 10⁸ cfu per strain) per animal via i.m. route. After 24 h, entire spleen and liver from each animal were homogenized and bacterial counts were determined. The horizontal lines represent the arithmetic means of the bacterial counts (C). Samples collected from animals infected with NF1 and NF2∆exoA showed no dissemination of bacteria to peripheral organs.

Fig. 3.

Progression of infection from the local site of injection for NF strains in a mouse model. (A) A group of five mice was infected with NF2-lux or NF2-lux and NF1 in mixed culture (1:1 ratio) at 2 × 10⁸ cfu per animal. Immediately after injection and at 24 h p.i., the animals were imaged for bioluminescence signal. (B) Total flux (p/s) was measured for each animal around the bioluminescent spot with the same shape and area across the images. The values between NF2-lux and the NF1 and NF2-lux mixed infection were then compared. (C) After 24 h p.i., the absence of bioluminescence from bacterial colonies recovered from muscle tissue of NF2-lux and NF1 mixed infection indicated elimination of NF2-lux from the site of injection. Bacterial load of the injected muscle tissue is also shown. The horizontal lines represent the arithmetic means of the bacterial counts. (D) Time course for progression of dissemination for NF1-lux strain was monitored for 6 d. Bioluminescence images are for 0 h, 24 h, and 6 d p.i.

Fig. 4.

Role of exoA gene on animal mortality and bacterial dissemination. Mice (n = 5) were infected with NF1 or NF1-exoA (A); WT SSU, SSUΔexoA, or SSUΔexoA::exoA (B); or NF2 or NF2∆exoA (D) at the indicated doses and observed for mortality. Asterisks denote statistical significance among the indicated groups. From mice (n = 5 or 10) infected with NF1 or NF1-exoA, bacterial dissemination was measured in spleen and liver tissues after 24 h p.i. The horizontal lines represent the arithmetic means of the bacterial counts (C).

Fig. 5.

Phagocytic elimination and in vitro growth dynamics of A. hydrophila strains. RAW 264.7 cells were infected with NF1, NF2, or NF1-exoA at a multiple of infection (moi) of 5, and percent bacterial uptake was calculated based on colony counts after gentamicin treatment (1.75 h p.i.) (A). At 2 h after gentamicin treatment, intracellular bacterial counts were determined to estimate percentage of intracellular bacterial survival (B). Furthermore, NF1 and NF2 were grown individually in LB medium at 37 °C for 6 h with 180 rpm shaking in an incubator (New Brunswick Scientific Co., Enfield, CT) and bacterial counts determined (C). At the same time, NF1 and NF2-lux were mixed at a ratio of 5:1 (solid bar) or 10:1 (dotted bar), respectively. The mixed cultures were either incubated at 37 °C on LB agar plates for 6 h (D) or in liquid LB medium with 180 rpm shaking for 6 h (E), and the growth of NF2-lux was measured. Similarly, when NF2 was mixed with NF1-lux at a ratio of 5:1 (solid bar) or 10:1 (dotted bar), respectively, and incubated at 37 °C on LB agar plates for 6 h, the number of NF1-lux was enumerated (F). Results were plotted with arithmetic means ± SD.

Fig. S3.

Bacterial motility assays for A. hydrophila strains. Swimming (A and B) and swarming (C and D) motility of A. hydrophila SSU, SSUΔexoA, SSUΔexoA::exoA, NF1 and NF1-exoA were measured in 60-mm Petri plates. Three independent experiments were performed for each strain, and arithmetic means ± SD were plotted.

Fig. 6.

Schematic showing interaction of NF strains during mixed infections. Left indicates absence of ExoA in strain NF1 resulted in the bacterium being confined to the local site of infection and eventually eliminated by host defense mechanisms. In contrast, Right shows secretion of ExoA by strain NF2 allowed the bacterium to disseminate to peripheral organs, away from the site of infection, by destroying local tissue barriers. Middle reveals pathodynamics of mixed infection with NF1 and NF2. ExoA secreted by NF2 assists dissemination of NF1 to peripheral organs by destroying local tissue barriers. However, NF1 facilitates direct killing and/or host-mediated elimination of NF2 from the site of infection in muscle tissue. Triple arrow indicates overwhelming response compared with that indicated by the single arrow.