Severe Human Infections Research Articles

Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2

Aspergillus fumigatus is an opportunistic fungal pathogen capable of causing severe infection in humans. One of the limitations in our understanding of how A. fumigatus causes infection concerns the initial stages of infection, notably the initial interaction between inhaled spores or conidia and the human airway. Using publicly-available datasets, we identified the Arp2/3 complex and the WAS-Interacting Protein Family Member 2 WIPF2 as being potentially responsible for internalization of conidia by airway epithelial cells. Using a cell culture model, we demonstrate that RNAi-mediated knockdown of WIPF2 significantly reduces internalization of conidia into airway epithelial cells. Furthermore, we demonstrate that inhibition of Arp2/3 by a small molecule inhibitor causes similar effects. Using super-resolution fluorescence microscopy, we demonstrate that WIPF2 is transiently localized to the site of bound conidia. Overall, we demonstrate the active role of the Arp2/3 complex and WIPF2 in mediating the internalization of A. fumigatus conidia into human airway epithelial cells.

Preliminary results on prevalence and persistence of Listeria monocytogenes in different dairy and meat processing plants in Central Italy

Purpose: Listeria monocytogenes is a relevant food - borne pathogen able to cause severe and fatal infections in humans. Understanding correlations among different Listeria monocytogenes strains and evaluating Listeria monocytogenes isolates capacity to persist in processing plant environments is an essential tool for food surveillance. Most outbreaks of human listeriosis seem to be linked to strains able to persist in the environment and to replicate in foods. The aim of this study was to investigate prevalence and persistence of Listeria monocytogenes strains subtypes in different dairy and meat processing plants in the Abruzzo and Molise Region. Methods & Materials: Totally 14 food processing plants have been selected for the study (7 for dairy products – DP - and 7 for meat products - MP). Sampling was performed in 4 session from March to October 2017, from 30 to 35 environmental sampling have been sampled in all the division of the plants, both from food contact surfaces (FCS) and non food contact surfaces (NFCS). Sampling sites were selected as niches for Listeria monocytogenes presence. Samples were tested with FSIS MLG8-10:2017 method. Listeria monocytogenes strains were serotyped using the method described in the U.S. FDA Bacteriological Analytical Manual (Bennett and Weaver, 2001). PFGE subtyping was performed according to the PulseNet protocol (Pulsenet USA, 2013) and macrorestriction patterns were analyzed using the BioNumerics 7.6 (Applied Maths, Kortrijk, Belgium). Results: Results are based on 1772 environmental samples tested, 135 (7.6%) samples were found positive for Listeria monocytogenes (94 NFCS and 41 FCS) in 4 DP and 6 MP. PFGE showed indistinguishable persistent strains in 2 DP and 3 MP in all sampling sessions and in one MP in 3 sampling sessions. Conclusion: The persistence of Listeria monocytogenes underlines the ability to persist in food producing plants, in favourable niches inside the processing environment. To reduce Listeria monocytogenes prevalence, Good Manufacturing Practices and Good Hygiene Practices are needed. Analysis are still ongoing to model the contamination path, NGS on isolated strains will be carried out to confirm the persistence of the strains and to find the presence of gene devoted to resistance and persistence.

PmtA functions as a ferrous iron and cobalt efflux pump in Streptococcus suis

ABSTRACT Transition metals are nutrients essential for life. However, an excess of metals can be toxic to cells, and host-imposed metal toxicity is an important mechanism for controlling bacterial infection. Accordingly, bacteria have evolved metal efflux systems to maintain metal homeostasis. Here, we established that PmtA functions as a ferrous iron [Fe(II)] and cobalt [Co(II)] efflux pump in Streptococcus suis, an emerging zoonotic pathogen responsible for severe infections in both humans and pigs. pmtA expression is induced by Fe(II), Co(II), and nickel [Ni(II)], whereas PmtA protects S. suis against Fe(II) and ferric iron [Fe(III)]-induced bactericidal effect, as well as Co(II) and zinc [Zn(II)]-induced bacteriostatic effect. In the presence of elevated concentrations of Fe(II) and Co(II), ΔpmtA accumulates high levels of intracellular iron and cobalt, respectively. ΔpmtA is also more sensitive to streptonigrin, a Fe(II)-activated antibiotic. Furthermore, growth defects of ΔpmtA under Fe(II) or Co(II) excess conditions can be alleviated by manganese [Mn(II)] supplementation. Finally, PmtA plays a role in tolerance to H2O2-induced oxidative stress, yet is not involved in the virulence of S. suis in mice. Together, these data demonstrate that S. suis PmtA acts as a Fe(II) and Co(II) efflux pump, and contributes to oxidative stress resistance.

Biotechnological Method of Preparation and Characterization of Recombinant Antimicrobial Peptide Avicin A from Enterococcus avium.

Avicin A is a bacteriocin from the gram-positive bacterium Enterococcus avium. It exhibits a high microbicidal activity against bacteria of the genus Listeria, a causative agent of the severe human infection listeriosis. We developed a biotechnological method for obtaining avicin A and characterized its structure and biological activity. We also proposed a possible mechanism of the antimicrobial action of avicin A.

Elizabethkingia miricola infection in Chinese spiny frog (Quasipaa spinosa).

Elizabethkingia miricola is a Gram-negative rod which has been incriminated in severe infections in humans. Recently, a serious infectious disease was identified in Chinese spiny frogs (Quasipaa spinosa), in the Sichuan Province of China; the disease was characterized by corneal opacity, the presence of ascites and neurological symptoms. A Gram-negative bacillus was isolated from the liver, spleen and kidney of the diseased frogs. Experimental infection test revealed that the bacillus could infect the frogs Q.spinosa and the LD50 value was 1.19×106 cfu per frog. The isolated Gram-negative bacillus was identified as E.miricola according to phenotypic characteristics, 16S rRNA and gyrB gene sequence analysis. The isolated strain was only susceptible to florfenicol among all investigated chemotherapeutic agents. Histological examination revealed that E.miricola infection caused pathological lesions to multiple organs and tissues, especially in the liver, brain, kidney. These results confirmed that E.miricola is an emerging pathogen of Chinese spiny frogs.

Dengue Virus Infection of Primary Human Smooth Muscle Cells.

Dengue virus (DENV) infection of humans is presently the most important arthropod-borne viral global threat, for which no suitable or reliable animal model exists. Reports addressing the effect of DENV on vascular components other than endothelial cells are lacking. Dengue virus infection of vascular smooth muscle cells, which play a physiological compensatory response to hypotension in arteries and arterioles, has not been characterized, thus precluding our understanding of the role of these vascular components in dengue pathogenesis. Therefore, we studied the permissiveness of primary human umbilical artery smooth muscle cells (HUASMC) to DENV 1-4 infection and compared with the infection in the previously reported primary human umbilical vein endothelial cells (HUVEC) and the classically used, non-transformed, and highly permissive Lilly Laboratories Cell-Monkey Kidney 2 cells. Our results show that HUASMC are susceptible and productive to infection with the four DENV serotypes, although to a lesser extent when compared with the other cell lines. This is the first report of DENV permissiveness in human smooth muscle cells, which might represent an unexplored pathophysiological contributor to the vascular collapse observed in severe human dengue infection.

Detection of novel astroviruses among rodents of Gabon, Central Africa

Astroviruses (AstVs) are mostly responsible for mild to severe gastroenteritis infections in humans and animals. AstVs infect a wide range of host species, have a large genetic diversity with different circulating variants and are thus a high zoonotic risk for human populations. Among these host species, rodents are known to harbor several AstVs variants. Therefore, it is important to identify in rodent species which AstVs are circulating and evaluate their potential zoonotic risk for humans. In this context, this study aimed to screen the presence of AstVs in 267 rodents trapped in 2012 in Franceville and Makokou, two cities in Gabon. RNA extracted from grinded intestines were used for the screening of AstVs by amplification of a conserved region of the RNA dependent RNA polymerase. Results report the identification of AstVs in 12 individuals (4.6% rate), belonging to three different species including Rattus rattus, Mus musculus and Hybomys univittatus. These findings report the first identification of AstVs in R. rattus and H. univittatus. The phylogenetic analyses indicate host specificity of rodents AstVs. The absence of rodent AstVs within the human AstV clade suggests a low rate of interspecies transmission of these viruses and consequently a low zoonotic risk.

Enhanced Th1/Th2 mixed immune responses elicited by polyethyleneimine adjuvanted influenza A (H7N9) antigen HA1-2 in chickens

Influenza A (H7N9) viruses have caused severe human infections and deaths every year in China since 2013. To reduce the risk of human infection and prevent a new influenza pandemic, there is a pressing need to develop safe and effective anti-H7N9 vaccines for poultry. Polyethyleneimine (PEI) is an organic polycation used extensively as a transfection reagent for decades. Although the adjuvant potential of PEI is well studied in mammals, its applicability and immune characteristics to avian species are still very rare. Here, to investigate the adjuvant activity of PEI, we analyzed the immune responses in chicken peripheral blood mononuclear cells in vitro. PEI significantly upregulated the expression of immune-related cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-18, and IL-1β) and chemokines (CXCLi1, CXCLi2, MIP-1β, and MCP-3), suggesting that PEI promoted immune responses of avian cells. We also assessed the in vivo immune responses to PEI in a chicken model. After the second and third vaccinations, significantly higher IgG titers were observed in the chickens immunized with HA1-2+PEI than that of HA1-2 alone. The HA1-2+PEI group also increased percentages of CD4+ and CD8+ T cells and improved PBMC proliferation. The significantly upregulated IFN-γ and IL-4 levels of splenocytes from HA1-2+PEI vaccinated chickens further indicated that PEI promoted a Th1/Th2 mixed immune responses. This study not only demonstrates the adjuvant potential of PEI when co-administered with influenza H7N9 antigen HA1-2 in chickens, but also supports the use of PEI as a versatile systemic adjuvant platform in poultry.

Fecal Metagenome Sequences from Lactating Dairy Cows Shedding Escherichia coli O157:H7.

Cattle are primary reservoirs of Escherichia coli O157:H7, a causative agent of severe human infections. To facilitate analyses of the communities in which this pathogen is found, we sequenced the fecal metagenomes of 10 dairy cows shedding E. coli O157:H7 and added them to the public domain.

Phosphatase activity of the control of virulence sensor kinase CovS is critical for the pathogenesis of group A streptococcus.

The control of virulence regulator/sensor kinase (CovRS) two-component system is critical to the infectivity of group A streptococcus (GAS), and CovRS inactivating mutations are frequently observed in GAS strains causing severe human infections. CovS modulates the phosphorylation status and with it the regulatory effect of its cognate regulator CovR via its kinase and phosphatase activity. However, the contribution of each aspect of CovS function to GAS pathogenesis is unknown. We created isoallelic GAS strains that differ only by defined mutations which either abrogate CovR phosphorylation, CovS kinase or CovS phosphatase activity in order to test the contribution of CovR phosphorylation levels to GAS virulence, emergence of hypervirulent CovS-inactivated strains during infection, and GAS global gene expression. These sets of strains were created in both serotype M1 and M3 backgrounds, two prevalent GAS disease-causing serotypes, to ascertain whether our observations were serotype-specific. In both serotypes, GAS strains lacking CovS phosphatase activity (CovS-T284A) were profoundly impaired in their ability to cause skin infection or colonize the oropharynx in mice and to survive neutrophil killing in human blood. Further, response to the human cathelicidin LL-37 was abrogated. Hypervirulent GAS isolates harboring inactivating CovRS mutations were not recovered from mice infected with M1 strain M1-CovS-T284A and only sparsely recovered from mice infected with M3 strain M3-CovS-T284A late in the infection course. Consistent with our virulence data, transcriptome analyses revealed increased repression of a broad array of virulence genes in the CovS phosphatase deficient strains, including the genes encoding the key anti-phagocytic M protein and its positive regulator Mga, which are not typically part of the CovRS transcriptome. Taken together, these data establish a key role for CovS phosphatase activity in GAS pathogenesis and suggest that CovS phosphatase activity could be a promising therapeutic target in GAS without promoting emergence of hypervirulent CovS-inactivated strains.

Arboviruses in South Africa, Known and Unknown

The emergence of novel arboviruses of zoonotic potential in South Africa (SA) threatens human health and animal welfare, and affects economic growth and development. These viruses cause severe infections in animals and humans, including neurological diseases, such as encephalitis, resulting in high morbidities, mortalities and economic losses. With increasing reports of Middelburg, Shuni, Sindbis, West Nile and Wesselsbron virus infections in animals and humans in SA, this article reviews and discusses known and currently emerging arboviruses in the country. These reports underscore the need for increased surveillance, vector control management, public health preparedness, focused research, community awareness programs, and the development of rapid and sensitive diagnostic approaches. Furthermore, appropriate medical personnel training and strengthening initiatives for a one-health approach are required to understand and mitigate the emerging arboviral threat to public health.

Molecular characteristics of extended-spectrum \u03b2-lactamase-producing Escherichia coli isolated from the rivers and lakes in Northwest China

BackgroundExtended-spectrum β-lactamases (ESBLs)-producing Escherichia coli (E. coli) isolates in environment water become progressively a potential threat to public health, while the detailed information about the ESBL-producing E. coli isolates in the rivers and lakes in Northwest China is scarce. In the present study, it was aimed to characterize the ESBL-producing E. coli isolated from the surface waters in Northwest China.ResultsA total of 2686 E. coli isolates were obtained from eleven rivers and lakes in Northwest China to screen for ESBL producers. Seventy-six (2.8%) isolates were classified as ESBL producers, and phylogenic groups D and A accounted for 59.2% of the ESBL producers. CTX-Ms were the predominant ESBLs genotype, and they were represented by seven blaCTX-M subtypes. blaCTX-M-14 was the most prevalent specific CTX-M gene, followed by blaCTX-M-9, blaCTX-M-123, blaCTX-M-15, blaCTX-M-27,blaCTX-M-1 and blaCTX-M-65. Moreover, 54 of the 76 ESBL producers carried at least one plasmid-mediated quinolone resistance (PMQR) gene, and aac(6′)-Ib-cr was predominant. The overall occurrence of virulence factors ranged from 1.3% (eae) to 48.7% (traT). Thirty-seven sequence types (STs) were confirmed among the 76 ESBL producers, and the predominant was ST10, which was represented by 10 isolates; importantly, clone B2-ST131, associated with severe infections in humans and animals, was detected three times.ConclusionThe prevalence of ESBL-producing E. coli from the rivers and lakes in Northwest China was low (2.8%), and the extraintestinal pathogenic E. coli (ExPEC) pathotype was the most commonly detected on the basis of the virulence factor profiles. 76.3% of ESBL producers harbored more than one β-lactamase gene, and blaCTX-M-14 was the predominant genotype. Notably, one ST131 isolate from Gaogan Canal simultaneously harbored blaCTX-M-9, blaCTX-M-15, blaCTX-M-123, blaKPC-2, blaNDM-1, blaOXA-2 as well as the PMQR genes qnrA, qnrS and aac(6′)-Ib-cr.

Comparative effectiveness of H7N9 vaccines in healthy individuals

ABSTRACTBackground: Avian H7N9 influenza viruses possess a potential pandemic threat to public health worldwide, and have caused severe infection and high mortality in humans. A series of clinical trials of H7N9 vaccines have been completed. Meta-analyses need to be performed to assess the immunogenicity and safety of H7N9 vaccines.Methods: Database research with defined selection criteria was conducted in PubMed, Cochrane Central Register of Controlled Trials, the World Health Organization’s International Clinical Trials Registry Platform, ClinicalTrials.gov, etc. Data from randomized clinical trials regarding the immunogenicity and safety of H7N9 vaccines were collected and meta-analyzed.Results: For non-adjuvanted H7N9 vaccines, high dose formulations induced limited immunogenicity and increased the risk of local and systemic adverse events, simultaneously. For adjuvanted H7N9 vaccines, on the one hand, ISCOMATRIX, MF59, AS03 and aluminium adjuvants applied in H7N9 vaccines could improve immune responses effectively, and non-aluminium adjuvants had superior performance in saving vaccine dose; on the other hand, aluminium adjuvant had the advantages of safety amongst these adjuvants applied in H7N9 vaccines.Conclusion: H7N9 influenza vaccines with adjuvant might represent the optimal available option in an influenza pandemic, at present.

CapC, a Novel Autotransporter and Virulence Factor of Campylobacter jejuni.

Campylobacter jejuni is recognized as an important causative agent of bacterial gastroenteritis in the developed world. Despite the identification of several factors contributing to infection, characterization of the virulence strategies employed by C. jejuni remains a significant challenge. Bacterial autotransporter proteins are a major class of secretory proteins in Gram-negative bacteria, and notably, many autotransporter proteins contribute to bacterial virulence. The aim of this study was to characterize the C. jejuni 81116 C8J_1278 gene (capC), predicted to encode an autotransporter protein, and examine the contribution of this factor to virulence of C. jejuni The predicted CapC protein has a number of features that are consistent with autotransporters, including the N-terminal signal sequence and the C-terminal β-barrel domain and was determined to localize to the outer membrane. Inactivation of the capC gene in C. jejuni 81116 and C. jejuni M1 resulted in reduced insecticidal activity in Galleria mellonella larvae. Furthermore, C. jejuni capC mutants displayed significantly reduced adherence to and invasion of nonpolarized, partially differentiated Caco-2 and T84 intestinal epithelial cells. Gentamicin treatment showed that the reduced invasion of the capC mutant is primarily caused by reduced adherence to intestinal epithelial cells, not by reduced invasion capability. C. jejuni capC mutants caused reduced interleukin 8 (IL-8) secretion from intestinal epithelial cells and elicited a significantly diminished immune reaction in Galleria larvae, indicating that CapC functions as an immunogen. In conclusion, CapC is a new virulence determinant of C. jejuni that contributes to the integral infection process of adhesion to human intestinal epithelial cells.IMPORTANCECampylobacter jejuni is a major causative agent of human gastroenteritis, making this zoonotic pathogen of significant importance to human and veterinary public health worldwide. The mechanisms by which C. jejuni interacts with intestinal epithelial cells and causes disease are still poorly understood due, in part, to the heterogeneity of C. jejuni infection biology. Given the importance of C. jejuni to public health, the need to characterize novel and existing virulence mechanisms is apparent. The significance of our research is in demonstrating the role of CapC, a novel virulence factor in C. jejuni that contributes to adhesion and invasion of the intestinal epithelium, thereby in part, addressing the dearth of knowledge concerning the factors involved in Campylobacter pathogenesis and the variation observed in the severity of human infection.

Severe human infection with a novel avian-origin influenza A(H7N4) virus

Human infections with influenza H7 subtypes, such as H7N9, have raised concerns worldwide. Here, we report a human infection with a novel influenza A(H7N4) virus. A 68 years-old woman with cardiovascular and cholecystic comorbidities developed rapidly progressed pneumonia with influenza-like-illness as initial symptom, recovered after 23 days-hospitalization including 8 days in ICU. Laboratory indicators for liver and blood coagulation dysfunction were observed. Oseltamivir phosphate, glucocorticoids and antibiotics were jointly implemented, with nasal catheterization of oxygen inhalation for this patient. We obtained the medical records and collected serial respiratory and blood specimens from her. We collected throat, cloacal and/or feces samples of poultry and wild birds from the patient’s backyard, neighborhood, local live poultry markets (LPMs) and the nearest lake. All close contacts of the patient were followed up and sampled with throat swabs and sera. Influenza viruses and other respiratory pathogens were tested by real-time RT-PCR, viral culturing and/or sequencing for human respiratory and bird samples. Micro-neutralizing assay was performed for sera. A novel reassortant wild bird-origin H7N4 virus is identified from the patient and her backyard poultry (chickens and ducks) by sequencing, which is distinct from previously-reported avian H7N4 and H7N9 viruses. At least four folds increase of neutralizing antibodies to H7N4 was detected in her convalescent sera. No samples from close contacts, wild birds or other poultry were tested positive for H7N4 by real-time RT-PCR.

A multifunctional human monoclonal neutralizing antibody that targets a unique conserved epitope on influenza HA

The high rate of antigenic drift in seasonal influenza viruses necessitates frequent changes in vaccine composition. Recent seasonal H3 vaccines do not protect against swine-origin H3N2 variant (H3N2v) strains that recently have caused severe human infections. Here, we report a human VH1-69 gene-encoded monoclonal antibody (mAb) designated H3v-47 that exhibits potent cross-reactive neutralization activity against human and swine H3N2 viruses that circulated since 1989. The crystal structure and electron microscopy reconstruction of H3v-47 Fab with the H3N2v hemagglutinin (HA) identify a unique epitope spanning the vestigial esterase and receptor-binding subdomains that is distinct from that of any known neutralizing antibody for influenza A H3 viruses. MAb H3v-47 functions largely by blocking viral egress from infected cells. Interestingly, H3v-47 also engages Fcγ receptor and mediates antibody dependent cellular cytotoxicity (ADCC). This newly identified conserved epitope can be used in design of novel immunogens for development of broadly protective H3 vaccines.

Dominance of International High-Risk Clones in Carbapenemase-Producing Pseudomonas aeruginosa: Multicentric Molecular Epidemiology Report from India

Background:Pseudomonas aeruginosa is one of the most common opportunistic pathogens that cause severe infections in humans. The burden of carbapenem resistance is particularly high and is on the rise. Very little information is available on the molecular mechanisms and its clonal types of carbapenem-resistant P. aeruginosa seen in Indian hospitals. This study was undertaken to monitor the β-lactamase profile and to investigate the genetic relatedness of the carbapenemase-producing (CP) P. aeruginosa collected across different hospitals from India. Materials and Methods: A total of 507 non-duplicate, carbapenem-resistant P. aeruginosa isolated from various clinical specimens collected during 2014–2017 across seven Indian hospitals were included. Conventional multiplex polymerase chain reaction for the genes encoding beta-lactamases such as extended-spectrum beta-lactamase (ESBL) and carbapenemase were screened. A subset of isolates (n = 133) of CP P. aeruginosa were genotyped by multilocus sequence typing (MLST) scheme. Results: Of the total 507 isolates, 15%, 40% and 20% were positive for genes encoding ESBLs, carbapenemases and ESBLs + carbapenemases, respectively, whilst 25% were negative for the β-lactamases screened. Amongst the ESBL genes, blaVEB is the most predominant, followed by blaPER and blaTEM, whilst blaVIM and blaNDM were the most predominant carbapenemases seen. However, regional differences were noted in the β-lactamases profile across the study sites. Genotyping by MLST revealed 54 different sequence types (STs). The most common are ST357, ST235, ST233 and ST244. Six clonal complexes were found (CC357, CC235, CC244, CC1047, CC664 and CC308). About 24% of total STs are of novel types and these were found to emerge from the high-risk clones. Conclusion: This is the first large study from India to report the baseline data on the molecular resistance mechanisms and its association with genetic relatedness of CP P. aeruginosa circulating in Indian hospitals. blaVIM- and blaNDM-producing P. aeruginosa is the most prevalent carbapenemase seen in India. Majority of the isolates belongs to the high-risk international clones ST235, ST357 and ST664 which is a concern.

Complete Genome Sequence of an Avian Native NDM-1-Producing Salmonella enterica subsp. enterica Serovar Corvallis Strain.

Carbapenems are an important class of β-lactams and one of the last options for treating severe human infections. We present here the complete genome sequence of avian native carbapenemase-producing Salmonella enterica subsp. enterica serovar Corvallis strain 12-01738, harboring a blaNDM-1-carrying IncA/C2 plasmid, isolated in 2012 from a wild bird (Milvus migrans) in Germany.

The Two-Component Signaling System VraSR ss Is Critical for Multidrug Resistance and Full Virulence in Streptococcus suis Serotype 2

Streptococcus suis has received increasing attention for its involvement in severe human infections worldwide as well as in multidrug resistance. Two-component signaling systems (TCSSs) play important roles in bacterial adaptation to various environmental stimuli. In this study, we identified a novel TCSS located in S. suis serotype 2 (SS2), designated VraSRSS, which is involved in bacterial pathogenicity and susceptibility to antimicrobials. Our data demonstrated that the yvqFSS gene, located upstream of vraSRSS , shared the same promoter with the TCSS genes, which was directly regulated by VraSRSS, as shown in electrophoretic mobility shift assays. Notably, YvqFSS and VraSRSS constitute a novel multidrug resistance module of SS2 that participates in resistance to certain groups of antimicrobials. Further analyses showed that VraSRSS inactivation significantly attenuated bacterial virulence in animal models, which, coupled with the significant activation of VraSRSS expression observed in host blood, strongly suggested that VraSRSS is an important regulator of SS2 pathogenicity. Indeed, RNA-sequencing analyses identified 106 genes that were differentially expressed between the wild-type and ΔvraSRSS strains, including genes involved in capsular polysaccharide (CPS) biosynthesis. Subsequent studies confirmed that VraSRSS indirectly regulated the transcription of CPS gene clusters and, thus, controlled the CPS thickness shown by transmission electron microscopy. Decreased CPS biosynthesis caused by vraSRSS deletion subsequently increased bacterial adhesion to epithelial cells and attenuated antiphagocytosis against macrophages, which partially clarified the pathogenic mechanism mediated by VraSRSS Taken together, our data suggest that the novel TCSS, VraSRSS, plays critical roles for multidrug resistance and full virulence in SS2.

A Small Molecule-Screening Pipeline to Evaluate the Therapeutic Potential of 2-Aminoimidazole Molecules Against Clostridium difficile.

Antibiotics are considered to be the first line of treatment for mild to moderately severe Clostridium difficile infection (CDI) in humans. However, antibiotics are also risk factors for CDI as they decrease colonization resistance against C. difficile by altering the gut microbiota and metabolome. Finding compounds that selectively inhibit different stages of the C. difficile life cycle, while sparing the indigenous gut microbiota is important for the development of alternatives to standard antibiotic treatment. 2-aminoimidazole (2-AI) molecules are known to disrupt bacterial protection mechanisms in antibiotic resistant bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus, but are yet to be evaluated against C. difficile. A comprehensive small molecule-screening pipeline was developed to investigate how novel small molecules affect different stages of the C. difficile life cycle (growth, toxin, and sporulation) in vitro, and a library of commensal bacteria that are associated with colonization resistance against C. difficile. The initial screening tested the efficacy of eleven 2-AI molecules (compound 1 through 11) against C. difficile R20291 compared to a vancomycin (2 μg/ml) control. Molecules were selected for their ability to inhibit C. difficile growth, toxin activity, and sporulation. Further testing included growth inhibition of other C. difficile strains (CD196, M68, CF5, 630, BI9, M120) belonging to distinct PCR ribotypes, and a commensal panel (Bacteroides fragilis, B. thetaiotaomicron, C. scindens, C. hylemonae, Lactobacillus acidophilus, L. gasseri, Escherichia coli, B. longum subsp. infantis). Three molecules compound 1 and 2, and 3 were microbicidal, whereas compounds 4, 7, 9, and 11 inhibited toxin activity without affecting the growth of C. difficile strains and the commensal microbiota. The antimicrobial and anti-toxin effects of 2-AI molecules need to be further characterized for mode of action and validated in a mouse model of CDI.