Gram-positive Anaerobic Pathogen Research Articles

Genomic Insights into Virulence Factors and Multi-Drug Resistance in Clostridium perfringens IRMC2505A.

Clostridium perfringens is a spore-forming, Gram-positive anaerobic pathogen that causes several disorders in humans and animals. A multidrug-resistant Clostridium strain was isolated from the fecal sample of a patient who was clinically suspected of gastrointestinal infection and had a recent history of antibiotic exposure and diarrhea. The strain was identified by 16s rRNA sequencing as Clostridium perfringens. The strain's pathogenesis was analyzed through its complete genome, specifically antimicrobial resistance-related genes. The Clostridium perfringens IRMC2505A genome contains 19 (Alr, Ddl, dxr, EF-G, EF-Tu, folA, Dfr, folP, gyrA, gyrB, Iso-tRNA, kasA, MurA, rho, rpoB, rpoC, S10p, and S12p) antibiotic-susceptible genetic species according to the k-mer-based detection of antimicrobial resistance genes. Genome mapping using CARD and VFDB databases revealed significant (p-value = 1 × 10-26) genes with aligned reads against antibiotic-resistant genes or virulence factors, including phospholipase C, perfringolysin O, collagenase, hyaluronidase, alpha-clostripain, exo-alpha-sialidase, and sialidase activity. In conclusion, this is the first report on C. perfringens from Saudi Arabia that conducted whole genome sequencing of IRMC2505A and confirmed the strain as an MDR bacterium with several virulence factors. Developing control strategies requires a detailed understanding of the epidemiology of C. perfringens, its virulence factors, and regional antimicrobial resistance patterns.

Changes in the expression of genes encoding type IV pili-associated proteins are seen when Clostridium perfringens is grown in liquid or on surfaces

BackgroundClostridium perfringens is a Gram-positive anaerobic pathogen that causes multiple diseases in humans and animals. C. perfringens lack flagella but have type IV pili (TFP) and can glide on agar surfaces. When C. perfringens bacteria are placed on surfaces, they become elongated, flexible and have TFP on their surface, traits not seen in liquid-grown cells. In addition, the main pilin in C. perfringens TFP, PilA2, undergoes differential post-translational modification when grown in liquid or on plates. To understand the mechanisms underlying these phenotypes, bacteria were grown in three types of liquid media and on agar plates with the same medium to compare gene expression using RNA-Seq.ResultsHundreds of genes were differentially expressed, including transcriptional regulatory protein-encoding genes and genes associated with TFP functions, which were higher on plates than in liquid. Transcript levels of TFP genes reflected the proportion of each protein predicted to reside in a TFP assembly complex. To measure differences in rates of translation, the Escherichia coli reporter gene gusA gene (encoding β-glucuronidase) was inserted into the chromosome downstream of TFP promoters and in-frame with the first gene of the operon. β-glucuronidase expression was then measured in cells grown in liquid or on plates. β-glucuronidase activity was proportional to mRNA levels in liquid-grown cells, but not plate-grown cells, suggesting significant levels of post-transcriptional regulation of these TFP-associated genes occurs when cells are grown on surfaces.ConclusionsThis study reveals insights into how a non-flagellated pathogenic rod-shaped bacterium senses and responds to growth on surfaces, including inducing transcriptional regulators and activating multiple post-transcriptional regulatory mechanisms associated with TFP functions.

Actinomycosis is not Frequent in the Periapex But is a Persistent Lesion.

Periapical actinomycosis caused by a gram-positive anaerobic pathogen characterizes a typical extra-radicular infection. This study determined the frequency and correlated the content of bacteria colonies with the of periapical actinomycosis size. The study comprised a total of 218 periapical lesions (PL) (cysts, granulomas or abscess). The specimens embedded in paraffin were sliced into 4-µm sections and stained with hematoxylin-eosin, Gram, Periodic Acid-Schiff (PAS) and Grocott's stain. The presence of bacterial colonies composed of filamentous structures labeled with the histochemical stains were described as Actinomyces, and for each case, the bacterial colonies were counted and measured. The correlation between the number and size of bacterial colonies and the size of PL was tested using Pearson's adjusted correlation coefficient. From 218 PL, bacterial colonies were identified in 64 biopsies. Seven cases (0.3%) fulfill the criteria for diagnosis of periapical actinomycosis. All of cases were therapy-resistant and did not showed periapical repair after 12 months of follow-up. Periapical surgery or dental extraction was performed. The correlation test indicated no correlation between the number of bacterial colonies and the lesion size (p=0.752, r=-0.148). However, a larger bacterial colony size generally resulted in a larger periapical lesion (P=0.000, r=0.657). The frequency of periapical actinomycosis was low, and this lesion should be included in the differential diagnosis of PL. The size of the Actinomyces colonies seemed to contribute to increase the size of the periapical lesion.

Immunoactive Clostridial Membrane Vesicle Production Is Regulated by a Sporulation Factor.

Recently, many Gram-positive bacteria as well as Gram-negative bacteria have been reported to produce membrane vesicles (MVs), but little is known regarding the regulators involved in MV formation. We found that a Gram-positive anaerobic pathogen, Clostridium perfringens, produces MVs predominantly containing membrane proteins and cell wall components. These MVs stimulated proinflammatory cytokine production in mouse macrophage-like cells. We suggested that MVs induced interleukin-6 production through the Toll-like receptor 2 (TLR2) signaling pathway. Thus, the MV could have a role in the bacterium-host interaction and bacterial infection pathogenesis. Moreover, we found that the sporulation master regulator gene spo0A was required for vesiculogenesis. A conserved, phosphorylated aspartate residue of Spo0A was indispensable for MV production, suggesting that the phosphorylation of Spo0A triggers MV production. Multiple orphan sensor kinases necessary for sporulation were also required to maximize MV production. These findings imply that C. perfringens actively produces immunoactive MVs in response to the environment changing, as recognized by membrane-spanning sensor kinases and by modulating the phosphorylation level of Spo0A.

Paper #40 - Benzoyl peroxide and clindamycin topical skin preparation decreases Propionibacterium acnes deep colonization in shoulder arthroscopy

Background: Propionibacterium acnes is a gram-positive anaerobic pathogen that can lead to devastating postoperative shoulder infections. Inoculation of the shoulder is thought to occur during skin incision due to colonization of the bacterium in the dermal layer. Dermatology literature demonstrates effective reduction of P. acnes colonization on the skin utilizing a combination benzoyl peroxide 5% and clindamycin phosphate 1% topical gel. The objective of this study was to investigate whether a benzoyl peroxide and clindamycin preoperative surgical skin preparation reduces the incidence of P. acnes inoculation of the shoulder joint during arthroscopy, and to determine the time-related effects of the topical medication on P. acnes colonization. Methods: 49 consecutive shoulder arthroscopy patients from November 2014 to September 2015 were prospectively enrolled. 42 patients completed 21 day cultures and were followed for three months. The first culture was a skin culture was taken at the pre-operative visit from the standard posterior, anterosuperior and anterolateral arthroscopic portal sites. A topical benzoyl peroxide 5% and clindamycin 1% gel was then applied to the shoulder with instructions to apply it every night prior to day of surgery. Routine prophylaxis was used with either Cephalexin 1 gram IV or Vancomycin 1 gram IV or Erythromycin 500 mg IV. The second culture was a skin culture was repeated at the three portal sites in the operating room prior to surgical preparation with 4% chlorhexidine solution, followed by a 2% chlorhexidine gluconate and 70% isopropyl alcohol paint. Shoulder arthroscopy proceeded as indicated with the third culture obtained from within the shoulder at the surgical site via a cannula. Results: Superficial P. acnes colonization of the skin remained similar to prior studies at 57 % (24 of 42 patients.) The topical gel was effective at eliminating 71% (17 of 24 patients) of P. acnes skin colonization by day of surgery. 75 % (3 of 4) of positive intraoperative superficial cultures were found on shoulders that only had 1 preoperative application of benzoyl peroxide and clindamycin. With >1 application (≥2 days prior to surgery), the topical gel was found to be 87.7% (7 of 8 patients) effective in decreasing P. acnes superficial colonization. Surgical site deep colonization was reduced to 4.7% (2 of 42 patients) compared our previous study of 19.6%. Ten control “air swab” cultures were all culture negative at 21 days. Conclusions: Benzoyl peroxide and clindamycin skin preparation is effective in reducing deep shoulder colonization of P. acnes in shoulder arthroscopy. It should be considered for use as a prophylactic topical skin preparation prior to shoulder procedures with a time-related effect of >1 application. Level of Evidence: Level II, prospective cohort study.

Successful management of a cerebral abscess secondary to chronic cholesteatoma caused by Prevotella melaninogenica and Peptococcus anaerobius – A case report and literature review

Abstract Cerebral abscess following cholesteatomatous otomastoiditis is a life-threatening complication and poses diagnostic and therapeutic challenges. We report a case of life-threatening cerebritis and cerebral abscess due to a collection of pus from an aerobic super infections occurring months after the apparent resolution of an otogenic brain abscess in a 67-year-old immunocompetent Italian female. Two gram-positive anaerobic pathogens were isolated during secondary neurosurgical procedures and antibiotic treatment was adopted to resolve the complications. Another objective of this study was to review the literature on gram-positive anaerobic pathogens and brain abscess complications in patients with fistula, and to highlight the importance of short imaging in monitoring treatment during long-term antibiotic therapy for otogenic brain abscess to avoid abscess recurrence. Isolating the strain and monitoring response to treatment with magnetic resonance imaging may improve the prognosis. The study also highlights the need for a close cooperation between infectious disease consultants and neurosurgeons.

Hypermotility in Clostridium perfringens strain SM101 is due to spontaneous mutations in genes linked to cell division.

Clostridium perfringens is a Gram-positive anaerobic pathogen of humans and animals. Although they lack flagella, C. perfringens bacteria can still migrate across surfaces using a type of gliding motility that involves the formation of filaments of bacteria lined up in an end-to-end conformation. In strain SM101, hypermotile variants are often found arising from the edges of colonies on agar plates. Hypermotile cells are longer than wild-type cells, and video microscopy of their gliding motility suggests that they form long, thin filaments that move rapidly away from a colony, analogously to swarmer cells in bacteria with flagella. To identify the cause(s) of the hypermotility phenotype, the genome sequences of normal strains and their direct hypermotile derivatives were determined and compared. Strains SM124 and SM127, hypermotile derivatives of strains SM101 and SM102, respectively, contained 10 and 6 single nucleotide polymorphisms (SNPs) relative to their parent strains. While SNPs were located in different genes in the two sets of strains, one feature in common was mutations in cell division genes, an ftsI homolog in strain SM124 (CPR_1831) and a minE homolog in strain SM127 (CPR_2104). Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model explaining the principles underlying the hypermotility phenotype is presented.

Determination of Clindamycin Phosphate in Different Vaginal Gel Formulations by Reverse Phase High Performance Liquid Chromatography

SUMMARY Clindamycin is an antibiotic effective against Gram-positive aerobes and both Gramnegative and Gram-positive anaerobic pathogens. Clindamycin (its phosphate ester) was used as a model drug for our study in order to check the efficacy of the developed vaginal gel formulations. The aim of this study was to develop an analytical method suitable for the determination of clindamycin phosphate in three different vaginal gel formulations. The main difference between the three formulations was the use of different gelation agents in the formulations (Carbopol® 940, Carbopol® Ultrez 10 and Pemulen® TR 1). A new isocratic high performance liquid chromatography method based on reverse phase separation has been developed for the determination of clindamycin phosphate. The separation was achieved on a Zorbax Eclipse plus C8 column with a mobile phase of acetonitrile and pH 2.5 phosphate buffer and UV detection at 210 nm. A linear response (r>0.99) was observed in the range of 10.0-80.0 μg mL-1. The method shows good recoveries and relative standard deviations were less than 1.0%. The method can be applied to assess the stability of clindamycin phosphate in vaginal gel formulations. It can be used for quality control and stability study samples analysis. Clindamycin phosphate concentration was different in vaginal gel formulations with different gelation agents, so the conclusion is that gelation agents (gel carriers) have a significant influence on the active drug concentration. The obtained results confirm that vaginal gel formulation with Carbopol® 940 shows the maximal stability during the analysis and provides the optimal value of clindamycin phosphate concentration.

Construction and Characterization of a Lactose-Inducible Promoter System for Controlled Gene Expression in Clostridium perfringens

Clostridium perfringens is a Gram-positive anaerobic pathogen which causes many diseases in humans and animals. While some genetic tools exist for working with C. perfringens, a tightly regulated, inducible promoter system is currently lacking. Therefore, we constructed a plasmid-based promoter system that provided regulated expression when lactose was added. This plasmid (pKRAH1) is an Escherichia coli-C. perfringens shuttle vector containing the gene encoding a transcriptional regulator, BgaR, and a divergent promoter upstream of gene bgaL (bgaR-P(bgaL)). To measure transcription at the bgaL promoter in pKRAH1, the E. coli reporter gene gusA, encoding β-glucuronidase, was placed downstream of the P(bgaL) promoter to make plasmid pAH2. When transformed into three strains of C. perfringens, pAH2 exhibited lactose-inducible expression. C. perfringens strain 13, a commonly studied strain, has endogenous β-glucuronidase activity. We mutated gene bglR, encoding a putative β-glucuronidase, and observed an 89% decrease in endogenous activity with no lactose. This combination of a system for regulated gene expression and a mutant of strain 13 with low β-glucuronidase activity are useful tools for studying gene regulation and protein expression in an important pathogenic bacterium. We used this system to express the yfp-pilB gene, comprised of a yellow fluorescent protein (YFP)-encoding gene fused to an assembly ATPase gene involved in type IV pilus-dependent gliding motility in C. perfringens. Expression in the wild-type strain showed that YFP-PilB localized mostly to the poles of cells, but in a pilC mutant it localized throughout the cell, demonstrating that the membrane protein PilC is required for polar localization of PilB.

In Vitro Activity of Ceftaroline against a Broad Spectrum of Recent Clinical Anaerobic Isolates

The in vitro activity of ceftaroline was compared with those of ceftriaxone, clindamycin, imipenem, metronidazole, moxifloxacin, tigecycline, and vancomycin against 514 clinical anaerobic isolates using Clinical and Laboratory Standards Institute (CLSI) standard methodology. Ceftaroline demonstrated good to excellent activity against Gram-positive anaerobic pathogens and limited activity against Gram-negative pathogens, particularly Bacteroides fragilis group isolates.

Transcriptional regulation of hemO encoding heme oxygenase in Clostridium perfringens

A Gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. The ability of infectious bacteria to acquire sufficient iron during infection is essential for the pathogen to cause disease. In the C. perfringens strain 13 genome, a heme oxygenase gene homologue (CPE0214, hemO) was found and its role was examined. The purified recombinant HemO protein showed heme oxygenase activity that can convert heme to biliverdin. hemO transcription was induced in response to extracellular hemin in a dose-dependent manner. The global two-component VirR/VirS regulatory system and its secondary regulator VR-RNA had positive regulatory effects on the transcription of hemO. These data indicate that heme oxygenase may play important roles in iron acquisition and cellular metabolism, and that the VirR/VirS-VR-RNA system is also involved in the regulation of cellular iron homeostasis, which might be important for the survival of C. perfringens in a human host.

Identification of a two-component VirR/VirS regulon in Clostridium perfringens

Clostridium perfringens, a Gram-positive anaerobic pathogen, is a causative agent of human gas gangrene that leads to severe rapid tissue destruction and can cause death within hours unless treated immediately. Production of several toxins is known to be controlled by the two-component VirR/VirS system involving a regulatory RNA (VR-RNA) in C. perfringens. To elucidate the precise regulatory network governed by VirR/VirS and VR-RNA, a series of microarray screening using VirR/VirS and VR-RNA-deficient mutants was performed. Finally, by qRT-PCR analysis, 147 genes (30 single genes and 21 putative operons) were confirmed to be under the control of the VirR/VirS-VR-RNA regulatory cascade. Several virulence-related genes for alpha-toxin, kappa-toxin, hyaluronidases, sialidase, and capsular polysaccharide synthesis were found. Furthermore, some genes for catalytic enzymes, various genes for transporters, and many genes for energy metabolism were also found to be controlled by the cascade. Our data indicate that the VirR/VirS-VR-RNA system is a global gene regulator that might control multiple cellular functions to survive and multiply in the host, which would turn out to be a lethal flesh-eating infection.

Virulence Gene Regulation by the agr System in Clostridium perfringens

A gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissue. The agr system is known to be important for the regulation of virulence genes in a quorum-sensing manner in Staphylococcus aureus. A homologue for S. aureus agrBD (agrBD(Sa)) was identified in the C. perfringens strain 13 genome, and the role of C. perfringens agrBD (agrBD(Cp)) was examined. The agrBD(Cp) knockout mutant did not express the theta-toxin gene, and transcription of the alpha- and kappa-toxin genes was also significantly decreased in the mutant strain. The mutant strain showed a recovery of toxin production after the addition of the culture supernatant of the wild-type strain, indicating that the agrBD(Cp) mutant lacks a signal molecule in the culture supernatant. An agr-virR double-knockout mutant was constructed to examine the role of the VirR/VirS two-component regulatory system, a key virulence regulator, in agrBD(Cp)-mediated regulation of toxin production. The double-mutant strain could not be stimulated for toxin production with the wild-type culture supernatant. These results indicate that the agrBD(Cp) system plays an important role in virulence regulation and also suggest that VirR/VirS is required for sensing of the extracellular signal and activation of toxin gene transcription in C. perfringens.

Characterization of two putative fibronectin-binding proteins of Clostridium perfringens

Clostridium perfringens is a Gram-positive anaerobic pathogen that causes gas gangrene and food poisoning in humans and animals. Genomic analysis of C. perfringens strain 13 revealed that this bacterium contains two genes (CPE0737 and CPE1847) that encode putative fibronectin (Fn)-binding proteins (Fbps). These genes, named fbpA and fbpB, were found to be constitutively expressed in all three strains (13, NCTC8237, CPN50) of C. perfringens, isolated from gas gangrene of human, that were tested. Both fbpA and fbpB were cloned and His-tagged, recombinant FbpA (rFbpA) and recombinant FbpB (rFbpB) were purified by Ni 2+-Sepharose column chromatography from transformed Escherichia coli. These recombinant Fbps were shown to bind to Fn, purified from human serum, in a ligand blotting assay. Additionally, Fn bound to these rFbps in a dose-dependent manner in an enzyme-linked immunosorbent assay. Furthermore, it was found that pre-incubation of Fn with either rFbpA or rFbpB inhibited the binding of Fn to C. perfringens cells.

The luxS gene is involved in cell-cell signalling for toxin production in Clostridium perfringens.

A Gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. C. perfringens possesses a homologue of the luxS gene that is reported to be responsible for the production of autoinducer 2 (AI-2), which participates in quorum sensing in bacteria. The luxS mutant was constructed using C. perfringens strain 13, and the role of the luxS gene in toxin production was examined. The cell-free culture supernatant from wild-type strain 13 greatly stimulated the luminescence of Vibrio harveyi BB170, whereas that from the luxS mutant caused no significant stimulation, indicating that the luxS gene is necessary for AI-2 production in C. perfringens. The luxS mutant showed a reduced level of production of alpha-, kappa- and theta-toxins. In the luxS mutant, the transcription of the theta-toxin gene (pfoA) was lower at mid-exponential growth phase, whereas alpha- and kappa-toxin gene transcription was not significantly affected. The production of toxins in the luxS mutant was stimulated by the addition of the culture supernatant from the wild-type cells, possibly because of the presence of AI-2. Moreover, the expression of the pfoA gene in the luxS mutant was apparently activated when the mutant cells were cultured in the presence of culture supernatants from the wild-type C. perfringens, Escherichia coli DH5alpha carrying the luxS gene of C. perfringens. A deletion analysis of the luxS operon showed that the luxS gene alone is responsible for cell-cell signalling, and that the metB or cysK genes located upstream of luxS are not involved in regulating toxin production. Our results indicate that cell-cell signalling by AI-2 plays an important role in the regulation of toxin production in C. perfringens.