Saeed Banawas

Recent advances in germination of Clostridium spores

Members of Clostridium genus are a diverse group of anaerobic spore-formers that includes several pathogenic species. Their anaerobic requirement enhances the importance of the dormant spore morphotype during infection, persistence and transmission. Bacterial spores are metabolically inactive and may survive for long times in the environment and germinate in presence of nutrients termed germinants. Recent progress with spores of several Clostridium species has identified the germinant receptors (GRs) involved in nutrient germinant recognition and initiation of spore germination. Signal transduction from GRs to the downstream effectors remains poorly understood but involves the release of dipicolinic acid. Two mechanistically different cortex hydrolytic machineries are present in Clostridium spores. Recent studies have also shed light into novel biological events that occur during spore formation (accumulation of transcriptional units) and transcription during early spore outgrowth. In summary, this review will cover all of the recent advances in Clostridium spore germination.

New amino acid germinants for spores of the enterotoxigenic Clostridium perfringens type A isolates

Clostridium perfringens spore germination plays a critical role in the pathogenesis of C. perfringens-associated food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases. Germination is initiated when bacterial spores sense specific nutrient germinants (such as amino acids) through germinant receptors (GRs). In this study, we aimed to identify and characterize amino acid germinants for spores of enterotoxigenic C. perfringens type A. The polar, uncharged amino acids at pH 6.0 efficiently induced germination of C. perfringens spores; L-asparagine, L-cysteine, L-serine, and L-threonine triggered germination of spores of most FP and NFB isolates; whereas, L-glutamine was a unique germinant for FP spores. For cysteine- or glutamine-induced germination, gerKC spores (spores of a gerKC mutant derivative of FP strain SM101) germinated to a significantly lower extent and released less DPA than wild type spores; however, a less defective germination phenotype was observed in gerAA or gerKB spores. The germination defects in gerKC spores were partially restored by complementing the gerKC mutant with a recombinant plasmid carrying wild-type gerKA-KC, indicating that GerKC is an essential GR protein. The gerKA, gerKC, and gerKB spores germinated significantly slower with L-serine and L-threonine than their parental strain, suggesting the requirement for these GR proteins for normal germination of C. perfringens spores. In summary, these results indicate that the polar, uncharged amino acids at pH 6.0 are effective germinants for spores of C. perfringens type A and that GerKC is the main GR protein for germination of spores of FP strain SM101 with L-cysteine, L-glutamine, and L-asparagine.

Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa

Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CDI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.

Bicarbonate and amino acids are co-germinants for spores of Clostridium perfringens type A isolates carrying plasmid-borne enterotoxin gene

Clostridium perfringens type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe) are generally linked to food poisoning, while isolates carrying cpe on a plasmid (P-cpe) are associated with non-food-borne gastrointestinal diseases. Both C-cpe and P-cpe isolates can form metabolically dormant spores, which through germination process return to actively growing cells to cause diseases. In our previous study, we showed that only 3 out of 20 amino acids (aa) in phosphate buffer (pH 7.0) triggered germination of spores of P-cpe isolates (P-cpe spores). We now found that 14 out of 20 individual aa tested induced germination of P-cpe spores in the presence of bicarbonate buffer (pH 7.0). However, no significant spore germination was observed with bicarbonate (pH 7.0) alone, indicating that aa and bicarbonate are co-germinants for P-cpe spores. P-cpe strain F4969 gerKC spores did not germinate, and gerAA spores germinated extremely poorly as compared to wild-type and gerKA spores with aa-bicarbonate (pH 7.0) co-germinants. The germination defects in gerKC and gerAA spores were partially restored by complementing gerKC or gerAA spores with wild-type gerKC or gerAA, respectively. Collectively, this study identified aa-bicarbonate as a novel nutrient germinant for P-cpe spores and provided evidence that GerKC and GerAA play major roles in aa-bicarbonate induced germination.

Clostridium difficile Infections: A Global Overview of Drug Sensitivity and Resistance Mechanisms

Clostridium difficile (C. difficile) is the most prevalent causative pathogen of healthcare-associated diarrhea. Notably, over the past 10 years, the number of Clostridium difficile outbreaks has increased with the rate of morbidity and mortality. The occurrence and spread of C. difficile strains that are resistant to multiple antimicrobial drugs complicate prevention as well as potential treatment options. Most C. difficile isolates are still susceptible to metronidazole and vancomycin. Incidences of C. difficile resistance to other antimicrobial drugs have also been reported. Most of the antibiotics correlated with C. difficile infection (CDI), such as ampicillin, amoxicillin, cephalosporins, clindamycin, and fluoroquinolones, continue to be associated with the highest risk for CDI. Still, the detailed mechanism of resistance to metronidazole or vancomycin is not clear. Alternation in the target sites of the antibiotics is the main mechanism of erythromycin, fluoroquinolone, and rifamycin resistance in C. difficile. In this review, different antimicrobial agents are discussed and C. difficile resistance patterns and their mechanism of survival are summarized.

l-lysine (pH 6.0) induces germination of spores of Clostridium perfringens type F isolates carrying chromosomal or plasmid-borne enterotoxin gene

C. perfringens type F isolates carrying enterotoxin gene (cpe) on the chromosome (C-cpe isolates) are mostly associated with food poisoning, while isolates carrying plasmid-borne cpe (P-cpe isolates) with non-food-borne gastrointestinal diseases. Spore germination is considered the most essential step for initiation of these diseases. Identifying the most effective germinants for spores of C-cpe and P-cpe isolates should help developing novel strategies involving induction of spore germination followed by inactivation of germinated spores with mild treatments. In this study, we showed that (i) l-lysine (pH 6.0) triggered germination of spores of all tested C-cpe and P-cpe isolates; although extremely low concentration of l-lysine (5-10 mM) induced germination of C-cpe spores, 10-fold higher concentration (50 mM) was required for P-cpe spore germination; (ii) P-cpe strain F4969 gerKC spores did not germinate, C-cpe strain SM101 gerKC spores germinated extremely poorly and these gerKC spores released significantly less DPA as compared to wild type spores; and these defects were restored to a nearly wild-type level by complementing gerKC spores with wild-type gerKC; and (iii) F4969 gerAA spores also did not germinate, and released less DPA than wild-type spores in presence of l-lysine (pH 6.0); and these defects were restored partially (germination) and fully (DPA release) by complimenting gerAA spores with wild-type gerAA. Collectively, our current study identified l-lysine as a universal germinant for spores of both C-cpe and P-cpe isolates and provided evidence that GerKC (from SM101 or F4969) and F4969 GerAA play major roles in l-lysine-induced germination.

The prevalence of transfusion-transmitted infections and nucleic acid testing among blood donors in Majmaah, Saudi Arabia

BACKGROUND Few studies discussed the prevalence of TTIs in Saudi donor blood samples. Thus, this study investigated the prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), syphilis and malaria in such samples to determine the efficacy of conducting serological and NATs on blood donors at King Khalid General Hospital in Majmaah, Saudi Arabia. METHODS A total of 3028 donated blood units were collected from August 2015 to March 2017. Serum samples were screened for hepatitis B surface antigens (HBsAgs), HBsAbs, total anti-core antibodies (HBcAbs), HCV antigens and HIV Ab/Ag combinations. Additionally, plasma was screened for syphilis (TPHA) and HTLV. Samples were also tested for malaria with rapid malaria antigen tests. Finally, NATs were performed for the simultaneous direct detection of HBV, HCV and HIV in each sample. RESULTS Out of the 3028 blood samples, 10 (0.33%) reacted to HBsAgs; 12 (0.40%) reacted to HCV antigens; 4 (0.13%) reacted to HIV Ab/Ag combinations; 6 (0.20%) reacted to HTLV antibodies; 297 (9.81%) reacted to HBcAbs and 236 (7.80%) reacted to HBsAbs. Additionally, NATs showed that 14 (0.46%) reacted to NAT-HBV; 20 (0.66%) samples were reacted to NAT-HCV and 2 (0.07%) samples reacted to NAT-HIV. Finally, 16 (0.53%) were positive for syphilis. No samples were positive for malaria. CONCLUSIONS The results indicated that NATs are more effective than serology tests for detecting TTIs. Moreover, correlations between standard serology tests and NATs indicated that using NATs could improve test sensitivities and decrease residual risks of TTIs and ensure safe blood transfusions.