Toxin-Antitoxin Systems Alter Adaptation of Mycobacterium smegmatis to Environmental Stress.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in prokaryotes, but their biological importance is poorly understood. Mycobacterium smegmatis contains eight putative TA systems. Previously, seven TAs have been studied, with five of them being verified as functional. Here, we show that Ms0251-0252 is a novel TA system in that expression of the toxin Ms0251 leads to growth inhibition that can be rescued by the antitoxin Ms0252. To investigate the functional roles of TA systems in M. smegmatis, we deleted the eight putative TA loci and assayed the mutants for resistance to various stresses. Deletion of all eight TA loci resulted in decreased survival under starvation conditions and altered fitness when exposed to environmental stresses. Furthermore, we showed that deletion of the eight TA loci decreased resistance to phage infection in Sauton medium compared with the results using 7H10 medium, suggesting that TA systems might have different contributions depending on the nutrient environment. Furthermore, we found that MazEF specifically played a dominant role in resistance to phage infection. Finally, transcriptome analysis revealed that MazEF overexpression led to differential expression of multiple genes, including those related to iron acquisition. Altogether, we demonstrate that TA systems coordinately function to allow M. smegmatis to adapt to changing environmental conditions. IMPORTANCE Toxin-antitoxin (TA) systems are mechanisms for rapid adaptation of bacteria to environmental changes. Mycobacterium smegmatis, a model bacterium for studying Mycobacterium tuberculosis, encodes eight putative TA systems. Here, we constructed an M. smegmatis mutant with deletions of all eight TA-encoding genes and evaluated the resistance of these mutants to environmental stresses. Our results showed that different TA systems have overlapping and, in some cases, opposing functions in adaptation to various stresses. We suggest that complementary TA modules may function together to regulate the bacterial stress response, enabling adaptation to changing environments. Together, this study provides key insights into the roles of TA systems in resistance to various environmental stresses, drug tolerance, and defense against phage infection.

Cell-Penetrating Antimicrobial Peptides Derived from an Atypical Staphylococcal δ-Toxin.

Revisiting underutilized classes of antibiotics is a pragmatic approach to the identification of alternative therapies for antimicrobial-resistant pathogens. To this end, we designed and screened a set of seven staphylococcal δ-toxin-inspired peptides (STIPs) for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, a pathogen-specific protease was leveraged to generate shorter peptides from these δ-toxin derivatives to expand the screen of putative antimicrobial peptides (AMPs) and to counterscreen against AMP inactivation. Remarkably, a 17-amino acid peptide based on the atypical δ-toxin sequence of Staphylococcus auricularis was discovered to possess an ability to kill MRSA and related pathogens. An alanine scan and series of rational substitutions improved AMP activity, and phenotypic assays characterized the STIPs' ability to rapidly interact with and permeabilize the staphylococcal membrane without causing lysis on a commensurate timescale. Instead of rapid lysis, both l- and d-enantiomers of STIP3-29, an AMP with low micromolar activity, were observed to penetrate and accumulate within cells. Finally, we observed that STIP3-29 was capable of controlling MRSA infection in a three-dimensional skin infection model. Overall, the results suggest that this unconventional source of AMPs can provide promising candidates for further development as therapeutic agents. IMPORTANCE The continued emergence and global distribution of infections caused by antimicrobial-resistant pathogens fuel our perpetual need for new or alternative therapies. Here, we present the discovery and initial characterization of bacterial cell-penetrating AMPs that were based on a family of virulence factors. In contrast to the multitude of AMPs that are sourced from animals, these potential therapeutic molecules have not undergone extensive selection for their antimicrobial properties and have proven to be amenable to activity-optimizing modifications. The staphylococcal toxin-inspired peptides described here represent a source of AMPs that can kill common opportunistic pathogens, such as MRSA, and have the potential to be improved for application in medicine.

Expression of Escherichia coli Heat-Labile Enterotoxin B Subunit in Centella (Centella asiatica (L.) Urban) via Biolistic Transformation.

BACKGROUND: Heat-Labile enterotoxin B subunit (LTB) produced by Escherichia coli, a non-toxic protein subunit with potential biological properties, is a powerful mucosal and parenteral adjuvant which can induce a strong immune response against co-administered antigens. OBJECTIVE: In the present study, LTB protein, encoded by the optimized ltb (also known synthetic ltb, s-ltb) gene in centella plant (Centella asiatica) for use as an antigen, has been discussed. METHODS: The s-ltb gene was cloned into a plant expression vector, pMYO51, adjacent to the CaMV 35S promoter and was then introduced into centella plant by biolistic transformation. PCR amplification was conducted to determine the presence of s-ltb gene in the transgenic centella plant. The expression of s-ltb gene was analyzed by immunoblotting and quantified by ELISA. In vitro activity of LTB protein was determined by GM1-ELISA. RESULTS: PCR amplification has found seven transgenic centella individuals. However, only five of them produced LTB protein. ELISA analysis showed that the highest amount of LTB protein detected in transgenic centella leaves was about 0.8% of the total soluble protein. GM1-ELISA assay indicated that plant LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers. CONCLUSION: The s-ltb gene that was successfully transformed into centella plants by the biolistic method has produced a relatively high amount of plant LTB protein in the pentameric quaternary structure that has GM1-ganglioside binding affinity, a receptor on the intestinal epithelial membrane.

Fatality of Staphylococcus aureus infections in a Greek university hospital: role of inappropriate empiric treatment, methicillin resistance, and toxin genes' presence.

The aim of the present study was to identify predictors of fatality among patients with S. aureus infections requiring hospitalization. Cases hospitalized with S. aureus infections at the University General Hospital of Patras, Greece, during a 4-year period (2013-2016) were studied. mecA, lukS/lukF-PV (Panton-Valentine leukocidin, PVL), tst (toxic shock syndrome toxin), fnbA (fibronectin-binding protein A), eta, and etb (epidermolytic toxins) genes' carriage was detected by PCR in 149 selected patients. Among 464 patients, 346 were included (118 with missing data). Primary bacteremia predominated (44.2%), followed by lower respiratory tract infections (13.6%), deep seated infections (9.8%), osteoarticular (9.5%), and catheter-related bloodstream infections (6.1%). Methicillin-resistant S. aureus (MRSA) represented 33.8% of infections and were less likely to receive appropriate empiric treatment (79.5% versus 97.4%; P < 0.001). Thirty-day fatality was 14.5%. Multivariate analysis revealed that development of septic shock, Charlson Comorbidity Index, lower respiratory tract infection, bacteremia (primary or secondary), MRSA, and CRP was significantly associated with fatality. Appropriate empiric treatment was a predictor of good prognosis. Thirty-two out of 149 S. aureus (21.5%) carried lukS/lukF-PV genes, whereas, 14 (9.4%), 133 (78.7%), four (2.7%), and one (0.7%) carried tst, fnbA, eta, and etb genes, respectively. No difference was found among toxin genes' presence and mortality. PVL was significantly more frequently found among MRSA as compared to MSSA (45.1% versus 9.2%; P < 0.001). MRSA represented one third of the infections requiring hospitalization and were independently associated with fatality, probably since were more likely to receive inappropriate antibiotic treatment as compared to MSSA.

Development of an Efficient Protocol to Obtain Transgenic Coffee, Coffea arabica L., Expressing the Cry10Aa Toxin of Bacillus thuringiensis.

This report presents an efficient protocol of the stable genetic transformation of coffee plants expressing the Cry10Aa protein of Bacillus thuringiensis. Embryogenic cell lines with a high potential of propagation, somatic embryo maturation, and germination were used. Gene expression analysis of cytokinin signaling, homedomains, auxin responsive factor, and the master regulators of somatic embryogenesis genes involved in somatic embryo maturation were evaluated. Plasmid pMDC85 containing the cry10Aa gene was introduced into a Typica cultivar of C. arabica L. by biobalistic transformation. Transformation efficiency of 16.7% was achieved, according to the number of embryogenic aggregates and transgenic lines developed. Stable transformation was proven by hygromycin-resistant embryogenic lines, green fluorescent protein (GFP) expression, quantitative analyses of Cry10Aa by mass spectrometry, Western blot, ELISA, and Southern blot analyses. Cry10Aa showed variable expression levels in somatic embryos and the leaf tissue of transgenic plants, ranging from 76% to 90% of coverage of the protein by mass spectrometry and from 3.25 to 13.88 µg/g fresh tissue, with ELISA. qPCR-based 2-ΔΔCt trials revealed high transcription levels of cry10Aa in somatic embryos and leaf tissue. This is the first report about the stable transformation and expression of the Cry10Aa protein in coffee plants with the potential for controlling the coffee berry borer.

Preparation and immunological characterization of an inactivated canine Clostridium perfringens type A vaccine.

Clostridium perfringens is the main cause of sudden death in dogs and currently there is no vaccine to prevent it. In this study, a canine C. perfringens type A strain was used to prepare a vaccine. C. perfringens was inactivated by formaldehyde and adjuvants were added. The safety and immunological characteristics of the inactivated C. perfringens vaccine were evaluated in mice and dogs. The results showed that the C. perfringens vaccine was safe and had immunoprotective activity. The serum antibody titre of immunized mice reached up to 6·25 × 104 . Both single immunization of 4 ml and dual immunizations of 2 ml each provided good immune protection, with five of five immunized dogs surviving. This study also studied a detoxified crude α-toxin extract vaccine. The results showed that a single immunization with 0·5 ml of the detoxified crude α-toxin extract vaccine provided immune protection, with five of five immunized dogs surviving. The inactivated C. perfringens type A vaccine can be used to prevent canine C. perfringens infections. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium perfringens is the main cause of sudden death in dogs and currently there is no vaccine to prevent it. In this study, an inactivated canine C. perfringens vaccine and a detoxified crude α-toxin vaccine were prepared. The safety and protective effects of these vaccines were evaluated using mouse and dog models. The vaccines were shown to be safe and to provide immune protection effects that can be used to prevent canine C. perfringens infection.

Yunnan Baiyao reduces hospital-acquired pressure ulcers via suppressing virulence gene expression and biofilm formation of Staphylococcus aureus.

Yunnan Baiyao (YB) as a kind of famous Chinese herbal medicine, possessed hemostatic, invigorating the circulation of blood, and anti-inflammatory effects. Identifying strategies to protect patients at risk for hospital-acquired pressure ulcers (HAPU) is essential. Herein, our results showed that YB treatment can effectively reduce the acne wound area and improve efficacy in a comparative study of 60 cases HAPU patients with S. aureus positive of acne wound pathogens. Furthermore, YB inhibited HIa expression and suppressed accessory gene regulator (agr) system controlled by regulatory RNA II and RNA III molecule using pALC1740, pALC1742 and pALC1743 S. aureus strain linked to gfpuvr reporter gene. Moreover, YB downregulated cao mRNA expression and inhibited coagulase activity by RT-PCR, slide and tube coagulase test. Additionally, YB downregulated seb, sec, sed, and tsst-1 mRNA expression to suppress enterotoxin and tsst-1 secretion and adhesion function related genes sarA, icaA, and cidA mRNA expression. Taken together, the data suggest that YB may reduce HAPU via suppressing virulence gene expression and biofilm formation of S. aureus.

Targeted Cancer Cell Killing by Highly Selective miRNA-Triggered Activation of a Prokaryotic Toxin-Antitoxin System.

Although not evolved to function in eukaryotes, prokaryotic toxin Kid induces apoptosis in human cells, and this is avoided by coexpression of its neutralizing antitoxin, Kis. Inspired by the way Kid becomes active in bacterial cells we had previously engineered a synthetic toxin-antitoxin system bearing a Kis protein variant that is selectively degraded in cells expressing viral oncoprotein E6, thus achieving highly selective killing of cancer cells transformed by human papillomavirus. Here we aimed to broaden the type of oncogenic insults, and therefore of cancer cells, that can be targeted using this approach. We show that appropriate linkage of the kis gene to a single, fully complementary, target site for an oncogenic human microRNA enables the construction of a synthetic toxin-antitoxin pair that selectively kills cancer cells overexpressing that particular microRNA. Importantly, the resulting system spares nontargeted cells from collateral damage, even when they overexpress highly homologous, though nontargeted, microRNAs.

Genetically-engineered plants yield an orally immunogenic PirA-like toxin from Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the main etiological agent of human gastroenteritis by seafood consumption and some strains from this species causing the Acute Hepatopancreatic Necrosis Disease in shrimp have been recently reported. The PirA-like toxin from V. parahaemolyticus (ToxA) has been recently reported as an attractive antigen implicated in subunit vaccine development. Since plants are attractive hosts for the production and delivery of vaccines in the present study plants expressing ToxA were developed to account with a low cost platform for the production and oral delivery of ToxA. Tobacco plants were genetically engineered by Agrobacterium-mediated transformation to stably integrate the ToxA-coding gene into the nuclear genome. Transgenic lines were rescued in kanamycin-containing medium and analyzed by ELISA to determine ToxA yields observing levels up to 9 µg g-1 FW leaf tissues. Western blot analysis confirmed the presence of the ToxA protein in plant extracts. Immunogenicity assessment of the plant-made ToxA was performed in mice, comprising a 4-dose oral immunization scheme; revealing the induction of anti-ToxA humoral responses (IgG in serum and IgA in feces). This study opens the path for the development of low cost plant-based vaccines against Vibrio parahaemolyticus.

Virulence mechanisms of plant-pathogenic Streptomyces species: an updated review.

Gram-positive Actinobacteria from the genus Streptomyces are best known for their morphological complexity and for their ability to produce numerous bioactive specialized metabolites with useful applications in human and veterinary medicine and in agriculture. In contrast, the ability to infect living plant tissues and to cause diseases of root and tuber crops such as potato common scab (CS) is a rare attribute among members of this genus. Research on the virulence mechanisms of plant-pathogenic Streptomyces spp. has revealed the importance of the thaxtomin phytotoxins as key pathogenicity determinants produced by several species. In addition, other phytotoxic specialized metabolites may contribute to the development or severity of disease caused by Streptomyces spp., along with the production of phytohormones and secreted proteins. A thorough understanding of the molecular mechanisms of plant pathogenicity will enable the development of better management procedures for controlling CS and other plant diseases caused by the Streptomyces.

Positive and Negative Regulation of the Virulence-Associated Coronafacoyl Phytotoxin in the Potato Common Scab Pathogen Streptomyces scabies.

The potato common scab pathogen Streptomyces scabies produces N-coronafacoyl-l-isoleucine (CFA-Ile), which is a member of the coronafacoyl family of phytotoxins that are synthesized by multiple plant pathogenic bacteria. The CFA-Ile biosynthetic gene cluster contains a regulatory gene, cfaR, which directly controls the expression of the phytotoxin structural genes. In addition, a gene designated orf1 encodes a predicted ThiF family protein and is cotranscribed with cfaR, suggesting that it also plays a role in the regulation of CFA-Ile production. In this study, we demonstrated that CfaR is an essential activator of coronafacoyl phytotoxin production, while ORF1 is dispensable for phytotoxin production and may function as a helper protein for CfaR. We also showed that CFA-Ile inhibits the ability of CfaR to bind to the promoter region driving expression of the phytotoxin biosynthetic genes and that elevated CFA-Ile production by overexpression of both cfaR and orf1 in S. scabies increases the severity of disease symptoms induced by the pathogen during colonization of potato tuber tissue. Overall, our study reveals novel insights into the regulatory mechanisms controlling CFA-Ile production in S. scabies and it provides further evidence that CFA-Ile is an important virulence factor for this organism.

Therapeutic bacteria to combat cancer; current advances, challenges, and opportunities.

Successful treatment of cancer remains a challenge, due to the unique pathophysiology of solid tumors, and the predictable emergence of resistance. Traditional methods for cancer therapy including radiotherapy, chemotherapy, and immunotherapy all have their own limitations. A novel approach is bacteriotherapy, either used alone, or in combination with conventional methods, has shown a positive effect on regression of tumors and inhibition of metastasis. Bacteria-assisted tumor-targeted therapy used as therapeutic/gene/drug delivery vehicles has great promise in the treatment of tumors. The use of bacteria only, or in combination with conventional methods was found to be effective in some experimental models of cancer (tumor regression and increased survival rate). In this article, we reviewed the major advantages, challenges, and prospective directions for combinations of bacteria with conventional methods for tumor therapy.

Application of an Endothelial Cell Culture Assay for the Detection of Neutralizing Anti-Clostridium Perfringens Beta-Toxin Antibodies in a Porcine Vaccination Trial.

BACKGROUND: Beta-toxin (CPB) is the major virulence factor of Clostridium perfringens type C, causing hemorrhagic enteritis in newborn pigs but also other animals and humans. Vaccines containing inactivated CPB are known to induce protective antibody titers in sow colostrum and neutralization of the CPB activity is thought to be essential for protective immunity in newborn piglets. However, no method is available to quantify the neutralizing effect of vaccine-induced antibody titers in pigs. (2) Methods: We developed a novel assay for the quantification of neutralizing anti-CPB antibodies. Sera and colostrum of sows immunized with a commercial C. perfringens type A and C vaccine was used to determine neutralizing effects on CPB induced cytotoxicity in endothelial cells. Antibody titers of sows and their piglets were determined and compared to results obtained by an ELISA. (3) Results: Vaccinated sows developed neutralizing antibodies against CPB in serum and colostrum. Multiparous sows developed higher serum and colostrum antibody titers after booster vaccinations than uniparous sows. The antibody titers of sows and those of their piglets correlated highly. Piglets from vaccinated sows were protected against intraperitoneal challenge with C. perfringens type C supernatant. (4) Conclusions: The test based on primary porcine endothelial cells quantifies neutralizing antibody activity in serum and colostrum of vaccinated sows and could be used to reduce and refine animal experimentation during vaccine development.

Spread of seb-Positive Methicillin-Resistant Staphylococcus aureus SCCmec Type II-ST764 Among Elderly Japanese in Nonacute Care Settings.

We investigated the prevalence and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) among 356 residents of nine long-term care facilities (LTCFs) in Japan during 2015 and 2017. In total, 800 specimens were tested and 39 MRSA isolates were recovered from 31 (8.71%) residents. PCR-based open reading frame typing (POT) and pulsed-field gel electrophoresis typing were performed for the 39 MRSA isolates; five of them showing identical pulsotypes, and POT scores were excluded in further analysis. Staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing, and toxin gene detection were performed for one representative MRSA isolate per resident. Among the 34 unrelated MRSA isolates, 15 (44.1%) and 19 (55.9%) were of SCCmec types II and IV, respectively, and belonged to seven sequence types (STs). Among the 15 SCCmec II isolates, 11 (73.3%), 3, and 1 belonged to ST764 (clonal complex [CC]5), ST5 (CC5), and ST630 (CC8), respectively. Among the 19 SCCmec IV isolates, 13 (68.4%), 3, 2, and 1 belonged to ST1 (CC1), ST474 (CC1), ST8 (CC8), and ST380 (CC8), respectively. Among the 14 CC5 lineage-SCCmec II isolates, one ST5 isolate and 7 of the 11 ST764 isolates (63.6%) carried seb gene, and 14 (87.5%) of 16 CC1 lineage-SCCmec IV isolates had sea gene (p < 0.05). The results indicate that the seb-positive SCCmec type II-ST764 clone has spread in Japanese LTCF environments. As LTCF residents have multiple comorbidities and increased susceptibility to infections, it is necessary to monitor MRSA colonization in LTCFs through periodic screening to prevent dissemination.

Panton-Valentine leukocidin and staphylococcal cassette chromosome mec characterization of community acquired methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Staphylococcus aureus (SA) represents one of the most important microorganism that is part of the normal microflora of humans, but in certain conditions can cause very serious infections. Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for a wide spectrum of nosocomial and community associated infections worldwide. The aim of this study was to determine community acquired MRSA (CA-MRSA), as well as the frequency of Panton-Valentine leukocidin (PVL) genes and staphylococcal cassette chromosome mec (SCCmec) types in isolates obtained from outpatients in the region of 700,000 people (Canton Sarajevo, Bosnia and Herzegovina) Methods: Our investigation included phenotypic and genotypic markers such as antimicrobial resistance, pulsed-field gel electrophoresis (PFGE), SCC typing, and PVL detection. RESULTS: Antimicrobial susceptibility: all MRSA isolates were resistant to the ß-lactam antibiotics tested, and all isolates were susceptible to trimethoprim sulphamethoxazole, rifampicin, fusidic acid, linezolid, and vancomycin. After the PFGE analysis, the isolates were grouped into five similarity groups: A-E. The largest number of isolates belonged to one of two groups: C - 60% and D - 27%. In both groups C and D, SCCmec type IV was predominant (60% and 88.8%, respectively). A total of 24% of the isolates had positive expression of PVL genes, while 76% showed a statistically significantly greater negative expression of PVL genes. CONCLUSIONS: Using combination techniques, we were able to investigate the origin and genetic background of the strains. PFGE analysis revealed two large, genetically related groups of strains consisting of 87 isolates. Our results suggest failure to apply the screening policy, and a lack of knowledge about multiresistant MRSA strains. This study showed the local epidemiological situation which should be the basis of antimicrobial empiric therapy for non-hospitalized patients.

Marjoram extract down-regulates the expression of Pasteurella multocida adhesion, colonization and toxin genes: A potential mechanism for its antimicrobial activity.

Due to the emergence of virulent and antibiotic-resistant microbes, natural antimicrobials from herbal origins have been given more attention as an alternative therapy. This study provides an in vitro research framework to investigate the antibacterial activities of 5 herbal (marjoram, garlic, onion, cinnamon and black seed) oil extracts against 16 multidrug-resistant (MDR) and virulent P. multocida serogroup A isolates recovered from dead and clinically diseased rabbits. Pathogenicity of the screened isolates was further proven experimentally and was verified by PCR analyses of 5 randomly selected virulence genes encoding attachment and colonization proteins (ptfA, pfhA, and omp87), sialidases (nanB) and dermonecrotoxin (toxA). A total of 12 P. multocida isolates were highly pathogenic with the possession of all examined virulence genes, while the other 4 isolates were of lower pathogenicity with expression of the target genes except toxA. In vitro anti-P. multocida activities of the 5 extracts and their synergism rates with 4 antibiotic drugs revealed that marjoram and cinnamon extracts had the highest antibacterial activities and the highest synergism rates against the screened isolates. Pasteurella multocida virulence gene expression profiles were assessed via real-time quantitative reverse transcription PCR (qRT-PCR) in response to marjoram extract. The quantitative analyses showed less than five-fold reduction in the targeted virulence genes expression in presence of marjoram extract compared with the control. The findings from this study document a novel molecular inhibitory activity of marjoram against P. multocida multiple virulence genes and provide a proof of concept for its implementation as an alternative candidate for the treatment of pasteurellosis in farm animals in future.

A Systematic Literature Review for Evidence of Aphanizomenon flos-aquae Toxigenicity in Recreational Waters and Toxicity of Dietary Supplements: 2000⁻2017.

Previous studies of recreational waters and blue-green algae supplements (BGAS) demonstrated co-occurrence of Aphanizomenon flos-aquae (AFA) and cyanotoxins, presenting exposure risk. The authors conducted a systematic literature review using a GRADE PRISMA-p 27-item checklist to assess the evidence for toxigenicity of AFA in both fresh waters and BGAS. Studies have shown AFA can produce significant levels of cylindrospermopsin and saxitoxin in fresh waters. Toxicity studies evaluating AFA-based BGAS found some products carried the mcyE gene and tested positive for microcystins at levels ≤ 1 µg microcystin (MC)-LR equivalents/g dry weight. Further analysis discovered BGAS samples had cyanotoxins levels exceeding tolerable daily intake values. There is evidence that Aphanizomenon spp. are toxin producers and AFA has toxigenic genes such as mcyE that could lead to the production of MC under the right environmental conditions. Regardless of this ability, AFA commonly co-occur with known MC producers, which may contaminate BGAS. Toxin production by cyanobacteria is a health concern for both recreational water users and BGAS consumers. Recommendations include: limit harvesting of AFA to months when toxicity is lowest, include AFA in cell counts during visible blooms, and properly identify cyanobacteria species using 16S rRNA methods when toxicity levels are higher than advisory levels.

Methicillin-resistant Staphylococcus aureus intracranial abscess: An analytical series and review on molecular, surgical and medical aspects.

PURPOSE: Intracranial abscess caused by methicillin-resistant Staphylococcus aureus (MRSA) is rare and unexplored. The aim of the present study is to examine the prevalence, clinical and molecular characteristics, treatment options and outcome of MRSA intracranial abscess over a period of 6 years. PATIENTSAND METHODS: A total of 21 patients were included in this retrospective study. The demographic and clinical details of all the patients were collected. Molecular typing including staphylococcal cassette chromosome mec typing, spa typing and polymerase chain reaction of Panton-Valentine leucocidin toxin (PVL) gene for the latter 6 isolates was performed. RESULTS: The paediatric population was the most affected group (33.3%). The primary route of infection was post-operative/trauma in 7 (33.3%) cases. All the patients were treated surgically either by aspiration or excision. Fifteen (71%) patients received anti-MRSA treatment with vancomycin or linezolid, where linezolid-treated patients showed better prognosis. Of the 11 patients who were on follow-up, unfavourable outcome was observed in 3 (27.3%) cases and 8 (72.7%) cases improved. The molecular typing of six isolates revealed four community-associated (CA) MRSA, one each of livestock-associated (LA) and healthcare-associated MRSA with PVL gene noted in all. CONCLUSION: We propose that timely diagnosis, surgical intervention and appropriate anti-MRSA treatment would contribute to better outcome. The occurrence of CA-MRSA and LA-MRSA infection in the central nervous system signifies the threat from the community and livestock reservoir, thus drawing attention towards surveillance and tracking to understand the epidemiology and implement infection control measures.

CNF1-like deamidase domains: common Lego bricks among cancer-promoting immunomodulatory bacterial virulence factors.

Alterations of the cellular proteome over time due to spontaneous or toxin-mediated enzymatic deamidation of glutamine (Gln) and asparagine (Asn) residues contribute to bacterial infection and might represent a source of aging-related diseases. Here, we put into perspective what is known about the mode of action of the CNF1 toxin from pathogenic Escherichia coli, a paradigm of bacterial deamidases that activate Rho GTPases, to illustrate the importance of determining whether exposure to these factors are risk factors in the etiology age-related diseases, such as cancer. In particular, through in silico analysis of the distribution of the CNF1-like deamidase active site Gly-Cys-(Xaa)n-His sequence motif in bacterial genomes, we unveil the wide distribution of the super-family of CNF-like toxins and CNF-like deamidase domains among members of the Enterobacteriacae and in association with a large variety of toxin delivery systems. We extent our discussion with recent findings concerning cellular systems that control activated Rac1 GTPase stability and provide protection against cancer. These findings point to the urgency for developing holistic approaches toward personalized medicine that include monitoring for asymptomatic carriage of pathogenic toxin-producing bacteria and that ultimately might lead to improved public health and increased lifespans.

Ethanolamine utilization supports Clostridium perfringens growth in infected tissues.

Clostridium perfringens possesses the ethanolamine (EA) utilization (eut) system encoded within the eut operon, which utilizes the EA as a carbon, nitrogen and energy source. To determine the role of the eut system in C. perfringens growth, an in-frame deletion of the eutABC genes was made in strain HN13 to generate the eutABC-deleted mutant strain HY1701. Comparison of HN13 and HY1701 growth in media supplemented with 1.0% glucose and/or 1.0% EA showed that glucose enhanced the growth of both strains, whereas EA enhanced HN13 growth, but not that of HY1701, indicating that the eut system is necessary for C. perfringens to utilize EA. The two-component regulatory system EutVW is needed to induce eut gene expression in response to EA whereas the global virulence regulator VirRS differentially controlled eut gene expression depending on glucose and EA availability. To assess the role of the eut system in vivo, an equal number of HN13 and HY1701 cells were injected into the right thigh muscles of mice. Mice infected with HY1701 showed fewer symptoms than those injected with HN13. The mortality rate of mice infected with HY1701 tended to be lower than for mice infected with HN13. In addition, in infected tissues from mice injected with a mixture of HN13 and HY1701, HN13 outnumbered HY1701. PCR screening demonstrated that C. perfringens isolated from gas gangrene and sporadic diarrhea cases carried both eut genes and the perfringolysin O gene (pfoA) as well as the phospholipase C gene (plc). However, pfoA was not detected in isolates from food poisoning patients and healthy volunteers. Culture supernatants prepared from HN13 grown in media containing 7.5% sheep red blood cells induced significantly higher eutB expression levels compared to those from plc- and/or pfoA-deletion mutants. Together, these results indicate that the eut system plays a nutritional role for C. perfringens during histolytic infection.