The Agr-Like Quorum-Sensing System Is Important for Clostridium perfringens Type A Strain ATCC 3624 To Cause Gas Gangrene in a Mouse Model

Clostridium perfringens type A strains produce toxins that are responsible for clostridial myonecrosis, also known as gas gangrene. Toxin production is regulated by an Agr-like quorum-sensing (QS) system that responds to changes in cell population density. In this study, we investigated the importance of this QS system in a mouse model of gas gangrene. Mice challenged with a C. perfringens strain with a nonfunctional regulatory system developed less severe changes in the injected skeletal muscle compared to animals receiving the wild-type strain. In addition, a synthetic peptide was able to decrease the effects of the QS in this disease model. These studies provide new understanding of the pathogenesis of gas gangrene and identified a potential therapeutic target to prevent the disease.

effects of the 6-R peptide on C. perfringens virulence in this gas gangrene model was tested to further evaluate if the Agr-like QS system is important for gas gangrene.

RESULTS
Construction and genotypic characterization of an ATCC 3624 agrB-null mutant and complementing strain. The present study constructed an agrB-null mutant of type A strain ATCC 3624 in order to begin evaluating whether the Agr-like quorumsensing system regulates the ability of type A strains to produce toxins and cause gas gangrene in a mouse model. For this purpose, the Clostridium-modified TargeTronmediated insertional mutagenesis method (32) was used to construct an ATCC 3624 agrB-null mutant, as demonstrated by PCR using primers that flank the intron insertion site and are specific for internal agrB open reading frame (ORF) sequences (Fig. 1A). With template DNA from wild-type ATCC 3624, these internal PCR primers specifically amplified a PCR product of 536 bp. However, the same primers amplified a PCR product of about 1.5 kb using DNA from the putative agrB mutant, which is consistent with the insertion of a 900-bp intron into the agrB ORF. The intron delivery plasmid was then cured by subculturing for 10 days in the absence of antibiotic selection.
To confirm that only a single intron insertion was present in the putative mutant, DNA was isolated from this strain and subjected to Southern blot analysis using an intron-specific probe (Fig. 1B). The intron-specific probe did not hybridize with wildtype DNA on this Southern blot, as expected. In contrast, this Southern blot experiment revealed that DNA from the agrB-null mutant strain contained a single intron insertion.
A complementing strain was then prepared using a plasmid where the agr operon was cloned into the C. perfringens/Escherichia coli shuttle plasmid pJIR750 (30). After this plasmid was transformed into the ATCC 3624 agrB-null mutant by electroporation, PCR confirmed the presence of the wild-type agrB ORF in the complementing strain. Note that, while Fig. 1A shows the presence of the intron-disrupted agrB gene in the mutant FIG 1 Intron-based mutagenesis to create an agrB-null mutant of C. perfringens type A wild-type ATCC 3624. (A) PCR confirmation of the construction of an agrB-null mutant of wild-type ATCC 3624. Using DNA isolated from wild-type ATCC 3624, a PCR assay amplified a 536-bp product using internal primers, but a larger 1.5-kb product, consistent with the insertion of a 0.9-kb intron, using template DNA isolated from the agrB mutant. A similar PCR analysis confirmed the presence of the 536-bp product in the complementing strain. (B) Southern blot hybridization of an intron-specific probe with DNA from wild-type ATCC 3624. DNA from each strain was isolated and electrophoresed on a 1% agarose gel prior to blotting and hybridization with an intron-specific probe. The size of the DNA fragment, in kilobases (kb), is shown at the right. (C) RT-PCR evaluation of agrB expression. Reverse transcription of extracted RNA from each strain, followed by PCR, was performed. Expression of the agrB gene was demonstrated for the wild-type parent and the complementing strain, but no agrB transcription was detectable for the ATCC 3624 agrB-null mutant. All three ATCC 3624-related strains expressed similar levels of 16S RNA. used to prepare the complementing strain, this larger PCR product was not amplified from the complementing strain. This phenomenon has been observed many times previously with complementation of mutants created by intron-mediated insertional mutagenesis (33)(34)(35)(36). It is due to the primers amplifying products from both the wild-type and intron-disrupted agrB genes in the complementing strain but because of its much smaller size, the PCR product from the wild-type agrB is created more rapidly and greatly increases in relative abundance after each PCR round. An RT-PCR assay was then used (Fig. 1C) to assess agrB expression by 5-h TY broth cultures of wild type, the agrB-null mutant and the complementing strain since an AgrB antibody is not available. This reverse transcription-PCR (RT-PCR) analysis confirmed that wild-type ATCC 3624 expresses agrB transcripts. However, no agrB transcription was detectable for the mutant. The complementing strain showed restored agrB transcription with expression levels similar to those of the wild-type strain. For all three ATCC 3624-related strains, the 16S RNA expression levels were similar.
Comparison of growth rates and toxin production by wild-type ATCC 3624 versus an ATCC 3624 agrB-null mutant or complementing strain in TY medium. When growth curve analyses were performed ( Fig. 2A), wild-type ATCC 3624, its agrB-null mutant, and the complementing strain all grew similarly in TY medium at 37°C.
Since type A strains commonly produce CPA and PFO, Western blot studies were performed to compare CPA and PFO production by ATCC 3624, the agrB-null mutant and the complementing strain after overnight (about 16 h) growth in TY medium at 37°C. The results (Fig. 2B) confirmed CPA and PFO production by wild-type ATCC 3624. However, there was no detectable CPA or PFO production by the agrB-null mutant, even after overnight culture. The agrB complementing strain made similar amounts of CPA and PFO as the wild-type parent. These results indicate that, as reported previously for type A strain 13 (25,30), CPA and PFO production are strongly upregulated by the agr locus in type A strain ATCC 3624.
Virulence of wild-type C. perfringens type A strain ATCC 3624 and its derivatives in a mouse model of gas gangrene. The present study next tested the pathogenicity of C. perfringens type A strain ATCC 3624 and its derivatives in a mouse model of clostridial myonecrosis. For this analysis, ϳ10 6 CFU of wild-type ATCC 3624, the agrB-null mutant and complementing strain were each inoculated intramuscularly into the left hind leg of eight male or female BALB/c mice (weighing 20 to 25 g) per group. A fourth group of eight mice was injected intramuscularly with sterile Dulbecco phosphate-buffered saline (DPBS; control). After 4 h of incubation, the mice were euthanized and examined for gross pathology, and samples were collected for microscopic evaluation and C. perfringens immunohistochemistry (IHC). Grossly, wild-type ATCC 3624 and the complementing strain induced severe changes in challenged mice; these changes were characterized by swelling and darkred discoloration (hemorrhage) of the affected skeletal muscle. In contrast, a similar challenge with the agrB-null mutant induced no gross changes in mouse skeletal muscle (Fig. 3A). The negative control, i.e., an injection of DPBS buffer, also failed to induce any gross changes in skeletal muscle.
Microscopically, wild-type ATCC 3624 induced severe histological changes, characterized by muscle degeneration, necrosis, and inflammation with myriad intralesional rods. Leukostasis was prominent within multiple blood vessels (Fig. 3B). Similar challenge with the agrB mutant produced muscle degeneration, necrosis and inflammation with myriad intralesional rods that were significantly less severe than those observed in mice inoculated with the wild-type strain. The severity of these changes reverted to wild-type levels in animals receiving the complementing strain. Mice receiving DPBS only showed no changes in any of the evaluated parameters ( Fig. 3B and D). Association of C. perfringens with the microscopic lesions in mice inoculated with wild-type ATCC 3624, the agrB mutant ( Fig. 3C), or complementing strain was confirmed by IHC using an indirect immunoperoxidase technique.
Recovery of viable C. perfringens from challenged skeletal muscle. The IHC results indicated that the amounts of total (live and dead) C. perfringens in muscle was similar after challenge with ATCC 3624 or its derivatives (Fig. 3C). To assess whether the numbers of viable C. perfringens present in these tissues were similar, skeletal muscle was aseptically collected from all mice and then plated on C. perfringens selective agar to process for CFU calculations. Similar numbers (ϳ10 6 ) of viable C. perfringens were recovered 4 h after challenge from all mice receiving wild-type ATCC 3624, its agrB-null mutant, or the complementing strain (Fig. 4). Fifteen randomly selected colonies were screened by PCR for the presence of the cpa gene to confirm their identity as C. perfringens. All colonies tested positive (data not shown). No C. perfringens were isolated from mice injected with DPBS buffer alone (controls).
Effects of the 6-R synthetic signaling peptide on in vitro CPA or PFO production by ATCC 3624. The 6-R peptide is a thiolactone ring consisting of the likely natural AIP of C. perfringens plus an additional amino acid that inhibits, by an unidentified mechanism, the Agr-like QS system of several type B and type C wild-type C. perfringens strains, resulting in reduced CPB toxin production by those type B and C strains (31). Therefore, the present study evaluated whether the 6-R peptide can also inhibit CPA or PFO production by type A strain ATCC 3624. For this analysis, three different concentrations of the 6-R peptide were added to TY broth inoculated with wild-type ATCC 3624, followed by CPA or PFO Western blot analysis of CPA or PFO levels in 5-h culture supernatants of those cultures. The results showed ( Fig. 5A) that, at a 100 M concentration, the 6-R peptide caused an ϳ75% reduction in both CPA and PFO production. For PFO production, even a 50 M concentration of the 6-R peptide had significant inhibitory effects (Fig. 5B). This reduction in toxin production was not due to the 6-R peptide affecting bacterial growth (Fig. 5C).
Effects of the 6-R synthetic signaling peptide on C. perfringens type A-induced gas gangrene in vivo. Since the 6-R peptide was able to reduce in vitro production of both CPA and PFO, the present study used this inhibitory peptide to further evaluate involvement of the Agr-like QS system in gas gangrene by testing whether 6-R affects virulence in the mouse model of gas gangrene induced by C. perfringens type A infection. For this experiment, ϳ10 6 CFU of wild-type ATCC 3624 was incubated for 4 h with a 100 M concentration of the 6-R peptide in dimethyl sulfoxide (DMSO) or an equal concentration of DMSO (control) and then injected into the leg muscles of mice. After 4 h, muscle degeneration/necrosis and inflammation were significantly less severe in animals receiving the 6-R peptide (100 M) than in mice receiving wild-type ATCC 3624 alone (Fig. 6A to C). Similar numbers (ϳ10 6 ) of viable C. perfringens were recovered 4 h after challenge from all mice receiving wild-type ATCC 3624 or this strain plus 6-R peptide (100 M) (Fig. 6D). Fifteen randomly selected colonies were screened by PCR for the presence of the cpa gene to confirm their identity as C. perfringens. All colonies tested positive (data not shown). No bacteria were isolated from mice injected with DPBS alone (controls).

DISCUSSION
The present study establishes an important role for the Agr-like QS system in the pathogenesis of gas gangrene induced by C. perfringens type A in a mouse model. Similar QS communication systems are present across many pathogenic firmicute species (37) where they commonly function as regulators for concerted population behaviors, including biofilm formation, sporulation, and virulence (38)(39)(40). It has been hypothesized that the coordinated production of extracellular virulence factors when a  sufficient population density is reached can induce considerable damage to the host, reducing, at the same time, the use of metabolic resources (29).
In appropriate C. perfringens types, the Agr-like QS system regulates in vitro production of most extracellular toxins (25, 28-30, 35, 41). Involvement of the Agr-like QS in C. perfringens infections originating in the intestine was previously demonstrated for type C (28) and type G (29) strains. In the type C study, an agrB-null mutant of type C strain CN3685 showed reduced enteropathogenicity in a rabbit intestinal loop model and less lethality in a mouse enterotoxemia model; both effects were reversible by complementation. Furthermore, attenuation of this agrB mutant was attributed to reduced production of CPB (28). Similarly, the virulence of an agrB-null mutant of type G strain CP1 was attenuated in a chicken necrotic enteritis model in vivo and pathogenicity was restored by complementation (29). Even though NetB production was not quantified in the referred to in vivo study, it is likely that a similar event described for CPB may be responsible for such reduction in pathogenicity in the chicken model since NetB production is controlled by the Agr-like QS and NetB is essential for type G strain-induced chicken necrotic enteritis (29).
Consistent with previous observations (25,30) using type A strain 13, inactivation of the agrB gene in ATCC 3624 also produced a significant in vitro decrease in cpa and pfoA expression at the transcriptional level and production of these protein toxins at the translational level. This reduced toxin expression is likely to be responsible for the significant decrease in the severity of macro-and microscopic lesions induced in the skeletal muscle of mice challenged with the agrB mutant since CPA and PFO play central roles in the development of gas gangrene (7,9). There was a significant reduction in inflammatory cells at the site of injury in mice receiving the ATCC 3624 agrB-null mutant, but many cells of this C. perfringens strain were still visible. This reduced presence of inflammatory cells is consistent with previous observations, in which C. perfringens type A strains deficient in CPA or PFO production induced significantly reduced leukocyte aggregation in mice (42).
To test whether the reduced virulence of the agrB-null mutant in the gas gangrene model was due to a decreased ability of this mutant to survive and reproduce in vivo, skeletal muscle from challenged mice was aseptically collected by the time of euthanasia for C. perfringens counting. The number of viable C. perfringens recovered from muscles 4 h after infection was approximately the same as the challenge dose, indicating that C. perfringens does not significantly grow in this infection model, in agreement with results of another recent study (43). Importantly, no significant differences were observed in the numbers of viable C. perfringens recovered after challenge with the wild-type, mutant, or complemented strains. These results strongly suggest that the reduced virulence of the ATCC 3624 agrB-null mutant in the gas gangrene model was not related to differences in its ability to survive in vivo.
For ethical reasons this study used a 4-h model based on a pilot study in which this time was sufficient to produce gross and microscopic lesions of gas gangrene, reduce animal suffering and death. This is a modification from previous studies that reported significant clinical sign variations using longer periods models (7).
A previous study showed that, via an unidentified mechanism, a C. perfringens AgrD sequence-based synthetic peptide named 6-R was able to interfere with CPB production by several C. perfringens type B and C strains in vitro (31). Why the 6-R peptide blocks or reduces toxin production in only some C. perfringens strains is not understood but it has been speculated that some degree of diversity in C. perfringens AIP receptors may exist (31). The present study determined that the 6-R peptide can also reduce, by ϳ75%, the production of CPA and PFO by ATCC 3624 in vitro. Cultures of wild-type ATCC 3624 incubated in the presence of a 100 M concentration of the 6-R peptide also had reduced ability to produce significant lesions in the skeletal muscle of mice. The reduction of both CPA/PFO production in vitro, as well as the reduced virulence in vivo, was not attributable to the 6-R peptide affecting bacterial growth.
In summary, the present study provides the first evidence that the Agr-like QS system is important for the pathogenesis of gas gangrene in a mouse model of the disease. Given the continuous search for therapies against bacterial pathogens and to avoid the development of antibiotic resistance, interfering with the Agr QS system by using AIP analogues like 6-R may represent a potential candidate as a target to prevent some C. perfringens-mediated diseases, including gas gangrene.

MATERIALS AND METHODS
Bacteria, media, and reagents. C. perfringens type A strain ATCC 3624 was purchased from the American Type Culture Collection (ATCC). The following broth media were used in this study: fluid thioglycolate medium (FTG; Difco Laboratories), TY broth (3% tryptic soy broth [Becton, Dickinson], 1% yeast extract [Becton, Dickinson], and 0.1% sodium thioglycolate [Sigma-Aldrich]), and TGY broth (TY broth supplemented with 2% glucose [Sigma-Aldrich]). The agrB-null mutant and complementing strains were screened using brain heart infusion (BHI) agar (Research Products International) plates containing 15 g ml Ϫ1 chloramphenicol (Sigma-Aldrich). Tryptose-sulfite-cycloserine (TSC) agar plates made of SFP agar base (Becton, Dickinson) with 0.04% D-cycloserine (Sigma-Aldrich) were used for isolation of C. perfringens from challenged skeletal muscle of mice. All other chemical reagents used in this study were purchased from Fisher Scientific, Sigma-Aldrich, or Bio-Rad.
Peptide synthesis. Based upon results of a previous report (31), this study used a synthetic 6-mer (6-R) peptide which consists of the sequence CLWFTH in a thiolactone ring, Synthesis of the 6-R peptide was carried out by the Peptide and Peptoid Synthesis Core Facility Division of the Health Sciences Core Research Facilities (HSCRF) at the University of Pittsburgh. Synthesis was performed using standard FMOC and incubated overnight at 37°C with gentle stirring. The resulting crude reaction mixture was directly loaded onto a Waters Prep 4000 series chromatography system and purified on a Phenomenex Gemini (21.2 ϫ 250 mm) 10-m C 18 column using standard acetonitrile/0.1% TFA gradient conditions. Final analytical determination of peptide purity for the cyclic 6-R-thioester was performed on a Waters Alliance chromatography system using a Phenomenex Gemini (4.6 ϫ 250 mm) 5-m C 18 column along with standard acetonitrile/0.1% TFA gradient conditions. Matrix-assisted laser desorption ionization-time-offlight analysis on an Applied Biosystems Voyager workstation was used for final confirmation of the expected target mass of each peptide. The purity of the final peptide was Ͼ95%. The purified synthetic peptide was resuspended in DMSO (Fisher Scientific) at 50 mM for use and then stored in a -80°C freezer for no longer than 2 weeks. In experiments, the final concentrations of 6-R peptide ranged from 25 to 100 M, as specified.
The agrB complementation vector (CPJVp3) was constructed as described previously (30). This plasmid was electroporated into the ATCC 3624 agrB-null mutant, and transformants were selected on BHI agar containing 15 g ml Ϫ1 of chloramphenicol. The mutant and complementing strains were further confirmed and characterized by PCR, RT-PCR, and Southern blot analyses, as described below.
C. perfringens DNA isolation, PCR, and intron Southern blot analyses. A MasterPure Grampositive DNA purification kit was used for DNA extraction from all C. perfringens strains according to the manufacturer's instructions (Epicenter). PCR for the agrB gene was performed using the NagrBOF and NagrBKOR primers. For the wild-type strain the PCR product amplified using these primers was 536 bp, while for the agrB-null mutant strain the same primer pair amplified a PCR product of about 1,400 bp due to the insertion of a 900-bp intron.
For Southern blotting, aliquots (3 g each) of wild-type or agrB-null mutant DNA samples were first digested overnight with EcoRI at 37°C according to the manufacturer's instructions (New England Biolabs). The overnight-digested DNA samples were then electrophoresed on a 1% agarose gel, followed by transfer onto a positively charged nylon membrane (Roche) for hybridization with an intron-specific probe (46). The intron-specific probe was prepared using the PCR DIG probe synthesis kit (Roche) and the intron primers (IBS and EBS2). After hybridization, Southern blots were developed using reagents from a DIG DNA labeling and detection kit (Roche), according to the manufacturer's instructions.
C. perfringens RNA isolation and RT-PCR analyses. The wild-type parent ATCC 3624 and derivatives (the agrB-null mutant or complementing strain) were grown in TY medium for 5 h at 37°C. Cultures were then pelleted and RNA was extracted using saturated phenol and purified by TRIzol and chloroform (Life Technology and Sigma), as previously described (36). After the absence of DNA was confirmed (36) by subjecting samples to PCR without reverse transcriptase, RNA was quantified by measuring the sample absorbance at 260 nm. An aliquot of 1 l of purified RNA (100 ng) was then used in a one-step RT-PCR containing 10 l of 2ϫ Taq master mix (New England Biolabs), 4 U of avian myeloblastosis virus reverse transcriptase (Promega), and agrB gene primers (described earlier), with ddH 2 O added to reach a 20-l total volume. Similarly, 16S RNA RT-PCR was performed as a loading control (36). Reaction mixtures were incubated for 45 min at 45°C to allow cDNA synthesis, and then regular PCR cycling was performed using the following conditions: (i) 95°C for 2 min; (ii) 30 cycles of 95°C for 15s, 50°C for 30 s, and 68°C for 30 s; and (iii) a final extension of 68°C for 5 min.

Measurement of C. perfringens growth in vitro.
For analysis of C. perfringens in vitro vegetative growth, a 0.2-ml aliquot from overnight FTG cultures of the wild type, agrB-null mutant, or complementing strain was inoculated into 10 ml of TY medium. The cultures were then incubated at 37°C; at various culture times (0, 1, 3, 5, 8, and 24 h) thereafter, a 1-ml aliquot of culture was removed to measure the optical density at 600 nm (OD 600 ) by using a Bio-Rad Smart spectrophotometer.
Western blot analyses of CPA and PFO production. TY cultures of the wild-type, agrB mutant, or complementing strain were adjusted to equal OD 600 s, and 30-l portions of supernatants from those cultures were then mixed with 5ϫ SDS-PAGE loading buffer and boiled for 5 min. Agr locus controls the expression of a number of secreted proteins (27), so loading equal amounts of protein in these experiments is not applicable for comparing the relative production of PFO or CPA by wild-type, the agrB mutant, or complementing strain. Instead, we adjusted the OD 600 of each culture to equivalence (since Agr does not affect growth) and then loaded the same volume of supernatant from that culture. Portions (30 l) of each sample were then electrophoresed on a 10% acrylamide SDS gel, and the separated proteins were transferred onto a nitrocellulose membrane. The membrane was blocked with TBS-Tween 20 (0.05% [vol/vol]) and nonfat dry milk (5% [wt/vol]) for 1 h at room temperature, followed by probing with a 1:1,000 dilution of rabbit polyclonal PFO antibody (36) or a 1:250 dilution of mouse monoclonal CPA antibody (36) overnight at 4°C. Finally, bound antibody was detected with a horseradish peroxidaseconjugated secondary anti-rabbit or anti-mouse antibody (Sigma-Aldrich) and the addition of SuperSignal West Pico chemiluminescence substrate (Fisher Scientific).
Inhibition of toxin production by the 6-R peptide. Wild-type ATCC 3624 was cultured overnight at 37°C in FTG before reculture in fresh TY broth overnight at 37°C. After that second overnight growth, a 15-l aliquot of the TY culture was inoculated into 1 ml of TY with or without the specified concentration of 6-R peptide. The culture without 6-R peptide received 2 l of DMSO as a control since this amount of DMSO was present in cultures receiving the 6-R peptide. After 5 h of culture at 37°C, the supernatants were collected and used for Western blot detection of CPA or PFO production, as described above. ImageJ analysis was performed on three separate Western blots to determine the fold changes of CPA and PFO in the supernatants.
Mouse model of gas gangrene. A 40-l aliquot from overnight FTG cultures of the wild-type, agrB-null mutant, or complementing strain was inoculated into 1 ml of TY medium, followed by incubation at 37°C for 5 h. These cultures were then washed in sterile DPBS. An aliquot of 50 l, equivalent to ϳ10 6 CFU, was injected intramuscularly in the thighs of eight BALB/c mice (20 to 25 g) per group of treatment. Another group of mice was challenged with 50 l of sterile DPBS (control). The animals were euthanized by cervical dislocation 4 h after infection, and samples of skeletal muscle were collected. All experiments involving mice were reviewed and approved by the University of California, Davis, Institutional Animal Care and Use Committee (protocol 20513).
Histopathology. Sections of challenged skeletal muscle were fixed by immersion in 10% buffered formalin (pH 7.2), for 24 to 72 h. Sections (4 m thick) were then prepared routinely and stained with hematoxylin and eosin. The sections were examined microscopically by a pathologist in a blinded fashion. A semiquantitative score of lesion severity was assigned to each section using an ordinal scale from 0 (no lesions observed) to 4 (most severe). The following scoring system was used, based on examination of 5, 200ϫ microscopic fields: 0, no lesions; 1, changes observed in 0 to 25% of the area examined; 2, changes observed in 25 to 50% of the area examined; 3, changes observed in 50 to 75% of the area examined; and 4, changes observed in 75 to 100% of the area examined. The following criteria were considered in this score: inflammation (edema, hemorrhage, and presence of inflammatory cells) and changes in muscle fibers (loss of striations, loss of cytoplasm, vacuolation, swelling, and hypercontraction bands).
C. perfringens immunohistochemistry. Sections of skeletal muscle were processed by an indirect immunoperoxidase technique for C. perfringens as previously described (44) using a Dako EnVision kit (Dako, Carpinteria, CA) according to the instructions of the manufacturer. The primary antibody was rabbit polyclonal C. perfringens antibody (GenWay Bio, San Diego, CA). Samples of skeletal muscle from mice receiving no C. perfringens inoculation were used as negative controls. Additional negative controls consisted of serial tissue sections of the test tissue incubated with normal rabbit serum instead of the specific antibodies. The colon of a goat from which C. perfringens had been isolated was used as a positive control for C. perfringens immunohistochemistry.
Recovery of C. perfringens from challenged tissues. Skeletal muscle was collected aseptically. Tissues were weighed, macerated, and resuspended in DPBS. Serial dilutions (10 Ϫ1 to 10 Ϫ8 ) in DPBS were plated on selective TSC agar plates for C. perfringens and anaerobically incubated overnight at 37°C. After 24 h, black colonies, indicative of C. perfringens (45), were counted to calculate the number of CFU per gram of muscle. A representative number (n ϭ 15) of those colonies was screened by PCR for the cpa gene to confirm that they were C. perfringens. For this, we used the primers CPAF (5=-GCTAATGTTACT GCCGTTGA-3=) and CPAR (5=-CCTCTGATACATCGTGTAAG-3=), which amplify a PCR product of about 324 bp. The PCR program was as follows: initial denaturation at 94°C for 5 min; 30 cycles at 94°C for 45 s, 56°C for 45 s, and 72°C for 60 s; and final extension at 72°C for 7 min.
Inhibition of gas gangrene by the 6-R peptide in a mouse model. Aliquots (40 l) of an overnight FTG cultures of wild-type, agrB-null mutant, or complementing strains were inoculated into 1 ml of TY medium containing 2 l of the 6-R peptide (100 M) in DMSO or an equal concentration of DMSO alone and then incubated at 37°C for 5 h. The cultures were then washed in sterile DPBS, and 1 l of the 6-R peptide (100 M) in DMSO or an equal concentration of DMSO alone was again added. An aliquot of 50 lequivalent to ϳ10 6 CFU-was injected intramuscularly in the left thighs of eight BALB/c mice (20 to 25 g) per treatment group. Another group of mice was injected with 50 l of sterile DPBS (control). The animals were euthanized 4 h after infection, and samples of skeletal muscle were collected.
Statistical analyses. All statistical analyses were performed using R (v3.3.1). Histopathological scores were compared by using a nonparametric Kruskal-Wallis test, followed by the Dunn test as post hoc analysis. For CPA and PFO production in culture supernatants, one-way analysis of variance was applied followed by a post hoc analysis using Tukey's multiple-comparison test. Bacterial counts were compared by negative binomial regression analysis. Differences were considered significant when the P value was Ͻ0.05.