RelQ-mediated alarmone signalling regulates growth, stress-induced biofilm formation and spore accumulation in Clostridioides difficile.

The bacterial stringent response (SR) is a conserved transcriptional reprogramming pathway mediated by the nucleotide signalling alarmones, (pp)pGpp. The SR has been implicated in antibiotic survival in Clostridioides difficile, a biofilm- and spore-forming pathogen that causes resilient, highly recurrent C. difficile infections. The role of the SR in other processes and the effectors by which it regulates C. difficile physiology are unknown. C. difficile RelQ is a clostridial alarmone synthetase. Deletion of relQ dysregulates C. difficile growth in unstressed conditions, affects susceptibility to antibiotic and oxidative stressors and drastically reduces biofilm formation. While wild-type C. difficile displays increased biofilm formation in the presence of sublethal stress, the ΔrelQ strain cannot upregulate biofilm production in response to stress. Deletion of relQ slows spore accumulation in planktonic cultures but accelerates it in biofilms. This work establishes biofilm formation and spore accumulation as alarmone-mediated processes in C. difficile and reveals the importance of RelQ in stress-induced biofilm regulation.

RelQ-mediated alarmone signaling regulates growth, sporulation, and stress-induced biofilm formation in Clostridioides difficile.

The bacterial stringent response (SR) is a conserved transcriptional reprogramming pathway mediated by the nucleotide signaling alarmones, (pp)pGpp. The SR has been implicated in antibiotic survival in Clostridioides difficile, a biofilm- and spore-forming pathogen that causes resilient, highly recurrent C. difficile infections. The role of the SR in other processes and the effectors by which it regulates C. difficile physiology are unknown. C. difficile RelQ is a clostridial alarmone synthetase. Deletion of relQ dysregulates C. difficile growth in unstressed conditions, affects susceptibility to antibiotic and oxidative stressors, and drastically reduces biofilm formation. While wild-type C. difficile displays increased biofilm formation in the presence of sub-lethal stress, the ΔrelQ strain cannot upregulate biofilm production in response to stress. Deletion of relQ slows spore accumulation in planktonic cultures but accelerates it in biofilms. This work establishes biofilm formation and sporulation as alarmone-mediated processes in C. difficile and reveals the importance of RelQ in stress-induced biofilm regulation.

Host-defense piscidin peptides as antibiotic adjuvants against Clostridioides difficile.

The spore-forming intestinal pathogen Clostridioides difficile causes multidrug resistant infection with a high rate of recurrence after treatment. Piscidins 1 (p1) and 3 (p3), cationic host defense peptides with micromolar cytotoxicity against C. difficile, sensitize C. difficile to clinically relevant antibiotics tested at sublethal concentrations. Both peptides bind to Cu2+ using an amino terminal copper and nickel binding motif. Here, we investigate the two peptides in the apo and holo states as antibiotic adjuvants against an epidemic strain of C. difficile. We find that the presence of the peptides leads to lower doses of metronidazole, vancomycin, and fidaxomicin to kill C. difficile. The activity of metronidazole, which targets DNA, is enhanced by a factor of 32 when combined with p3, previously shown to bind and condense DNA. Conversely, the activity of vancomycin, which acts at bacterial cell walls, is enhanced 64-fold when combined with membrane-active p1-Cu2+. As shown through microscopy monitoring the permeabilization of membranes of C. difficile cells and vesicle mimics of their membranes, the adjuvant effect of p1 and p3 in the apo and holo states is consistent with a mechanism of action where the peptides enable greater antibiotic penetration through the cell membrane to increase their bioavailability. The variations in effects obtained with the different forms of the peptides reveal that while all piscidins generally sensitize C. difficile to antibiotics, co-treatments can be optimized in accordance with the underlying mechanism of action of the peptides and antibiotics. Overall, this study highlights the potential of antimicrobial peptides as antibiotic adjuvants to increase the lethality of currently approved antibiotic dosages, reducing the risk of incomplete treatments and ensuing drug resistance.

Unique Features of Alarmone Metabolism in Clostridioides difficile.

The "magic spot" alarmones (pp)pGpp, previously implicated in Clostridioides difficile antibiotic survival, are synthesized by the RelA-SpoT homolog (RSH) of C. difficile (RSHCd) and RelQCd. These enzymes are transcriptionally activated by diverse environmental stresses. RSHCd has previously been reported to synthesize ppGpp, but in this study, we found that both clostridial enzymes exclusively synthesize pGpp. While direct synthesis of pGpp from a GMP substrate, and (p)ppGpp hydrolysis into pGpp by NUDIX hydrolases, have previously been reported, there is no precedent for a bacterium synthesizing pGpp exclusively. Hydrolysis of the 5' phosphate or pyrophosphate from GDP or GTP substrates is necessary for activity by the clostridial enzymes, neither of which can utilize GMP as a substrate. Both enzymes are remarkably insensitive to the size of their metal ion cofactor, tolerating a broad array of metals that do not allow activity in (pp)pGpp synthetases from other organisms. It is clear that while C. difficile utilizes alarmone signaling, its mechanisms of alarmone synthesis are not directly homologous to those in more completely characterized organisms. IMPORTANCE Despite the role of the stringent response in antibiotic survival and recurrent infections, it has been a challenging target for antibacterial therapies because it is so ubiquitous. This is an especially relevant consideration for the treatment of Clostridioides difficile infection (CDI), as exposure to broad-spectrum antibiotics that harm commensal microbes is a major risk factor for CDI. Here, we report that both of the alarmone synthetase enzymes that mediate the stringent response in this organism employ a unique mechanism that requires the hydrolysis of two phosphate bonds and synthesize the triphosphate alarmone pGpp exclusively. Inhibitors targeted against these noncanonical synthetases have the potential to be highly specific and minimize detrimental effects to stringent response pathways in commensal microbes.

Growth in a biofilm sensitizes Cutibacterium acnes to nanosecond pulsed electric fields.

The Gram-positive anaerobic bacterium Cutibacterium acnes (C. acnes) is a commensal of the human skin, but also an opportunistic pathogen that contributes to the pathophysiology of the skin disease acne vulgaris. C. acnes can form biofilms; cells in biofilms are more resilient to antimicrobial stresses. Acne therapeutic options such as topical or systemic antimicrobial treatments often show incomplete responses. In this study we measured the efficacy of nanosecond pulsed electric fields (nsPEF), a new promising cell and tissue ablation technology, to inactivate C. acnes. Our results show that all tested nsPEF doses (250 to 2000 pulses, 280 ns pulses, 28 kV/cm, 5 Hz; 0.5 to 4 kJ/ml) failed to inactivate planktonic C. acnes and that pretreatment with lysozyme, a naturally occurring cell-wall-weakening enzyme, increased C. acnes vulnerability to nsPEF. Surprisingly, growth in a biofilm appears to sensitize C. acnes to nsPEF-induced stress, as C. acnes biofilm-derived cells showed increased cell death after nsPEF treatments that did not affect planktonic cells. Biofilm inactivation by nsPEF was confirmed by treating intact biofilms grown on glass coverslips with an indium oxide conductive layer. Altogether our results show that, contrary to other antimicrobial agents, nsPEF kill more efficiently bacteria in biofilms than planktonic cells.

The (p)ppGpp Synthetase RSH Mediates Stationary-Phase Onset and Antibiotic Stress Survival in Clostridioides difficile.

The human pathogen Clostridioides difficile is increasingly tolerant of multiple antibiotics and causes infections with a high rate of recurrence, creating an urgent need for new preventative and therapeutic strategies. The stringent response, a universal bacterial response to extracellular stress, governs antibiotic survival and pathogenesis in diverse organisms but has not previously been characterized in C. difficile Here, we report that the C. difficile (p)ppGpp synthetase RSH is incapable of utilizing GTP or GMP as a substrate but readily synthesizes ppGpp from GDP. The enzyme also utilizes many structurally diverse metal cofactors for reaction catalysis and remains functionally stable at a wide range of environmental pHs. Transcription of rsh is stimulated by stationary-phase onset and by exposure to the antibiotics clindamycin and metronidazole. Chemical inhibition of RSH by the ppGpp analog relacin increases antibiotic susceptibility in epidemic C. difficile R20291, indicating that RSH inhibitors may be a viable strategy for drug development against C. difficile infection. Finally, transcriptional suppression of rsh also increases bacterial antibiotic susceptibility, suggesting that RSH contributes to C. difficile antibiotic tolerance and survival.IMPORTANCEClostridioides difficile infection (CDI) is an urgent public health threat with a high recurrence rate, in part because the causative bacterium has a high rate of antibiotic survival. The (p)ppGpp-mediated bacterial stringent response plays a role in antibiotic tolerance in diverse pathogens and is a potential target for development of new antimicrobials because the enzymes that metabolize (p)ppGpp have no mammalian homologs. We report that stationary-phase onset and antibiotics induce expression of the clostridial ppGpp synthetase RSH and that both chemical inhibition and translational suppression of RSH increase C. difficile antibiotic susceptibility. This demonstrates that development of RSH inhibitors to serve as adjuvants to antibiotic therapy is a potential approach for the development of new strategies to combat CDI.

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of γ-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

Bacteriological Profile and Antimicrobial Susceptibility Patterns of Bacteria Isolated from Pus/Wound Swab Samples from Children Attending a Tertiary Care Hospital in Kathmandu, Nepal.

In Nepal, little is known about the microbiological profile of wound infections in children and their antimicrobial susceptibility patterns. Total of 450 pus/wound swab samples collected were cultured using standard microbiological techniques and the colonies grown were identified with the help of biochemical tests. The antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique. Methicillin-resistant Staphylococcus aureus isolates were detected by using cefoxitin disc and confirmed by determining minimum inhibitory concentrations (MIC) of oxacillin. 264 (59%) samples were culture positive. The highest incidence of bacterial infections was noted in the age group of less than 1 year (76%). Out of 264 growth positive samples, Gram-positive bacteria were isolated from 162 (61%) samples and Gram-negative bacteria were found in 102 (39%) samples. Staphylococcus aureus (99%) was the predominant Gram-positive bacteria isolated and Pseudomonas aeruginosa (44%) was predominant Gram-negative bacteria. About 19% of S. aureus isolates were found to be methicillin-resistant MIC of oxacillin ranging from 4 µg/mL to 128 µg/mL. Among the children of Nepal, those of age less than 1 year were at higher risk of wound infections by bacteria. Staphylococcus aureus followed by Pseudomonas aeruginosa were the most common bacteria causing wound infections in children.

Assessment of the effectiveness of three different cephalosporin/clavulanate combinations for the phenotypic confirmation of extended-spectrum beta-lactamase producing bacteria isolated from urine samples at National Public Health Laboratory, Kathmandu, Nepal

BACKGROUND: The extended-spectrum ß-lactamase (ESBL) producing bacteria are present as the serious public health problems due to their resistance to large number of antibiotics. The main aims of this study were to determine the prevalence and antibiotic resistance patterns of bacteria producing extended-spectrum ß-lactamases (ESBLs) and to find the suitable cephalosporin/clavulanate combination for phenotypic confirmation of ESBL production. METHODS: During the study period from April 2013 to November 2013, a total of 1003 urine samples from the patients visiting National Public Health Laboratory, Kathmandu, Nepal were collected and processed. The isolates were identified with the help of colony characteristics, gram stain and conventional biochemical tests. Antimicrobial susceptibility testing was performed by Kirby Bauer disc diffusion method. ESBL production screening was done by using ceftriaxone, while ESBL production confirmation was done by using three different 3rd generation cephalosporin/clavulanate combinations. RESULTS: Of the 138 isolates, Escherichia coli was the most predominant with 88 (63.8 %) isolates. Among the antibiotics tested for gram negative bacteria, highest susceptibility was seen toward imipenem followed by amikacin. Of the total isolates, 68 (49.3 %) were suspected as ESBL producers. Of these, 44 (64.7 %) were phenotypically confirmed to be ESBL producers. The majority of ESBL producers were E. coli with 34 (72.3 %) isolates. Of the three different 3rd generation cephalosporin/clavulanate combinations used, ceftazidime/clavulanate combination was found to be most effective for phenotypic confirmation of ESBL producers and was statistically highly significant (P < 0.01). CONCLUSION: Based on the findings of our study, we recommend to use ceftazidime/clavulanate combination for phenotypic confirmation of ESBL producers. Routine ESBL testing for uropathogens along with conventional antibiogram would be useful for proper early management of all the cases of urinary tract infections.

Minimum inhibitory concentration of vancomycin to methicillin resistant Staphylococcus aureus isolated from different clinical samples at a tertiary care hospital in Nepal.

BACKGROUND: Methicillin resistant Staphylococcus aureus (MRSA) has evolved as a serious threat to public health. It has capability to cause infections not only in health care settings but also in community. Due to the multidrug resistance shown by MRSA, there are limited treatment options for the infections caused by this superbug. Vancomycin is used as the drug of choice for the treatment of infections caused by MRSA. Different studies from all around the world have documented the emergence of strains of S. aureus those are intermediate sensitive or resistant to vancomycin. And recently, there have been reports of reduced susceptibility of MRSA to vancomycin, from Nepal also. So the main purpose of this study was to determine the minimum inhibitory concentration (MIC) of vancomycin to methicillin resistant S. aureus isolated from different clinical specimens. METHODS: Total 125 strains of S. aureus isolated from different clinical samples at KIST Medical College and Teaching Hospital, Lalitpur, Nepal from Nov 2012 to June 2013, were subjected to MRSA detection by cefoxitin disc diffusion method. The minimum inhibitory concentrations of vancomycin to confirmed MRSA strains were determined by agar dilution method. Yellow colored colonies in mannitol salt agar, which were gram positive cocci, catalase positive and coagulase positive were confirmed to be S. aureus. RESULTS: Among, total 125 S. aureus strains isolated; 47(37.6%) were MRSA. Minimum inhibitory concentrations of vancomycin to the strains of MRSA ranged from 0.125 µg/ml to 1 µg/ml. CONCLUSION: From our findings we concluded that the rate of isolation of MRSA among all the strains of S. aureus isolated from clinical samples was very high. However, none of the MRSA strains were found to be vancomycin intermediate-sensitive or vancomycin-resistant.