Bile salt hydrolase-mediated inhibitory effect of <Emphasis Type="Italic">Bacteroides ovatus</Emphasis> on growth of <Emphasis Type="Italic">Clostridium difficile</Emphasis>

Clostridium difficile infection (CDI) is one of the most common nosocomial infections. Dysbiosis of the gut microbiota due to consumption of antibiotics is a major contributor to CDI. Recently, fecal microbiota transplantation (FMT) has been applied to treat CDI. However, FMT has important limitations including uncontrolled exposure to pathogens and standardization issues. Therefore, it is necessary to evaluate alternative treatment methods, such as bacteriotherapy, as well as the mechanism through which beneficial bacteria inhibit the growth of C. difficile. Here, we report bile acid-mediated inhibition of C. difficile by Bacteroides strains which can produce bile salt hydrolase (BSH). Bacteroides strains are not commonly used to treat CDI; however, as they comprise a large proportion of the intestinal microbiota, they can contribute to bile acid-mediated inhibition of C. difficile. The inhibitory effect on C. difficile growth increased with increasing bile acid concentration in the presence of Bacteroides ovatus SNUG 40239. Furthermore, this inhibitory effect on C. difficile growth was significantly attenuated when bile acid availability was reduced by cholestyramine, a bile acid sequestrant. The findings of this study are important due to the discovery of a new bacterial strain that in the presence of available bile acids inhibits growth of C. difficile. These results will facilitate development of novel bacteriotherapy strategies to control CDI.     

Molecular typing and antimicrobial susceptibility testing to six antimicrobials of <Emphasis Type="Italic">Clostridium difficile</Emphasis> isolates from three Czech hospitals in Eastern Bohemia in 2011–2012

In 2011–2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.     

Discovery and development of surotomycin for the treatment of <Emphasis Type="Italic">Clostridium difficile</Emphasis>

The primary challenge for treating Clostridium difficile infections (CDI) is maintenance of clinical response after the end of treatment (sustained clinical response). Disease recurrence following a positive clinical response occurs in approximately 6–25 % of patients after the first episode and in up to 65 % for subsequent recurrences. Surotomycin, a novel cyclic lipopeptide antibiotic with a core derived by Streptomyces roseosporus fermentation, disrupts C. difficile cellular membrane activity in both logarithmic and stationary phases and minimally disturbs normal gastrointestinal microbiota because of its lack of activity against Gram-negative anaerobes and facultative anaerobes. Preclinical and clinical evidence indicate that surotomycin has low oral bioavailability, allowing gastrointestinal tract concentrations to greatly exceed its minimum inhibitory concentration for C. difficile. Surotomycin is well tolerated and effective in hamster models of CDI. Phase 2 clinical evidence suggests that surotomycin (250 mg twice daily) is an effective CDI treatment, with statistically lower recurrence rates than vancomycin.     

Porcine and bovine <Emphasis Type="Italic">Clostridium difficile</Emphasis> ribotype 078 isolates demonstrate similar growth and toxigenic properties

Clostridioides (C.) difficile are found in cows, pigs and poultry suggesting that this pathogen is present and more importantly animals could act as a reservoir, via food or environment, of human C. difficile infection. Molecular methods together with phenotypical characterisation bring integrated and important tools to describe diversity and nature of bacteria including C. difficile. Moreover, similar or identical C. difficile types are found in different farm animals. This study aimed to phenotypically characterise C. difficile isolates belonging to ribotype 078 and to identify differences such as growth and toxicity between porcine and bovine isolates. C. difficile isolates were assessed for the growth behaviour (turbidimetry), metabolic potential (Biolog AN) and toxin production (ELISA method) in vitro. The concentration of released either toxin A (TcdA) or toxin B (TcdB) varied greatly between the isolates tested; however, it did not differ between the porcine and bovine ribotypes. Also, the TcdA/TcdB ratio of the isolates did not show a difference either. The most common metabolised substrates were pyruvic acid followed by α-ketobutyric acid. The results show that both porcine and bovine C. difficile RT 078 share similar phenotypical characteristics including growth and production of toxins. The findings may help understand the virulence of C. difficile RT 078 in porcine and bovine species.     

Importance of Molecular Methods to Determine Whether a Probiotic is the Source of <Emphasis Type="Italic">Lactobacillus</Emphasis> Bacteremia

There has been an increasing interest in the use of probiotic products for the prevention of Clostridium difficile infection (CDI). Bio-K+^® is a commercial probiotic product comprising three strains of lactobacilli—Lactobacillus acidophilus CL1285^®, Lact. casei LBC80R^® and Lact. rhamnosus CLR2^®—that have been applied to prevent CDI. Generally considered as safe, lactobacilli have potential to cause bacteremia, endocarditis and other infections. The source of Lactobacillus bacteremia can be normal human flora or lactobacilli-containing probiotic. The aim of this study was to assess whether probiotic lactobacilli caused bacteremia and to show the value of molecular identification and typing techniques to determine probiotic and patient strain relatedness. We report an episode of Lactobacillus bacteremia in a 69-year-old man admitted to a hospital with severe congestive heart failure. During his hospitalization, he required long-term antibiotic therapy. Additionally, the patient received Bio-K+^® probiotic as part of a quality improvement project to prevent CDI. Subsequently, Lactobacillus bacteremia occurred. Two independent blinded laboratory evaluations, using pulse field gel electrophoresis, 16S rRNA gene sequencing and DNA fingerprint analysis (rep-PCR), were performed to determine whether the recovered Lact. acidophilus originated from the probiotic product. Ultimately, the patient strain was identified as Lact. casei and both laboratories found no genetic relation between the patient’s strain and any of the probiotic lactobacilli. This clinical case of lactobacillus bacteremia in the setting of probiotic exposure demonstrates the value of using discriminatory molecular methods to clearly determine whether there were a link between the patient’s isolate and the probiotic strains.     

Clinical and economic impact of the introduction of a nucleic acid amplification assay for <Emphasis Type="Italic">Clostridium difficile</Emphasis>

Background

The clinical outcomes and cost implications of a diagnostic shift from an EIA- to PCR-based assay for Clostridium difficile infection (CDI) have not been completely described in the literature.

Methods

The impact of the PCR-based assay on the incidence and duration of CDI therapy was compared to the EIA assay for patients with a negative CDI diagnostic result. Secondary clinical and economic outcomes were also evaluated. Independent predictors of receipt of antibiotic therapy were assessed via logistic regression.

Results

141 EIA and 140 PCR patients were included. Significantly more patients were started or continued on anti-CDI antibiotic therapy after a known negative assay result in the EIA group (26 patients vs. 8 patients, P = 0.002). Duration of antibiotic therapy after a known negative result was significantly shorter in the PCR group (1 vs. 4 days, P = 0.029) and a 23% reduction in the number of tests obtained per patient was observed (1.41 ± 0.86 vs. 1.82 ± 1.35, P = 0.007). The over fourfold difference in per-test cost of the EIA assay ($8.33 vs. $42.86, P < 0.0001) was offset by the overall medication costs required for the increased treatment in the EIA group ($546.60 vs. $188.96, P = 0.191). Utilization of the EIA-based CDI assay was associated with increased odds of CDI treatment after a negative test (aOR 4.71, 95% CI 1.93–11.46, P = 0.001).

Conclusion

The transition from an EIA to PCR-based assay for diagnosing CDI resulted in a significant decrease in the number of patients treated and the duration of treatment in response to a negative test result. This significant decrease in treatment resulted in decreased costs offsetting the utilization of a more expensive molecular test for patients with a negative CDI diagnostic result.

     

Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> virulence genes in <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis> from Vellore,India

Streptococcus dysgalactiae subsp. equisimilis (SDSE), belonging to the group C and G streptococci, are human pathogens reported to cause clinical manifestations similar to infections caused by Streptococcus pyogenes. To scrutinize the distribution of gene coding for S. pyogenes virulence factors in SDSE, 255 isolates were collected from humans infected with SDSE in Vellore, a region in southern India, with high incidence of SDSE infections. Initial evaluation indicated SDSE isolates comprising of 82.35% group G and 17.64% group C. A multiplex PCR system was used to detect 21 gene encoding virulence-associated factors of S. pyogenes, like superantigens, DNases, proteinases, and other immune modulatory toxins. As validated by DNA sequencing of the PCR products, sequences homologous to speC, speG, speH, speI, speL, ssa and smeZ of the family of superantigen coding genes and for DNases like sdaD and sdc were detected in the SDSE collection. Furthermore, there was high abundance (48.12% in group G and 86.6% in group C SDSE) of scpA, the gene coding for C5a peptidase in these isolates. Higher abundance of S. pyogenes virulence factor genes was observed in SDSE of Lancefield group C as compared to group G, even though the incidence rates in former were lower. This study not only substantiates detection of S. pyogenes virulence factor genes in whole genome sequenced SDSE but also makes significant contribution towards the understanding of SDSE and its increasing virulence potential.     

Innate host defenses against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, can cause life-threatening infections of the central nervous system in immunocompromised and immunocompetent individuals. Cryptococcal meningoencephalitis is the most common disseminated fungal infection in AIDS patients, and remains the third most common invasive fungal infection among organ transplant recipients. The administration of highly active antiretroviral therapy (HAART) has resulted in a decrease in the number of cases of AIDS-related cryptococcosis in developed countries, but in developing countries where HAART is not readily available, Cryptococcus is still a major concern. Therefore, there is an urgent need for the development of novel therapies and/or vaccines to combat cryptococcosis. Understanding the protective immune responses against Cryptococcus is critical for development of vaccines and immunotherapies to combat cryptococcosis. Consequently, this review focuses on our current knowledge of protective immune responses to C. neoformans, with an emphasis on innate immune responses.     

First molecular detection of <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in the hard tick <Emphasis Type="Italic">Rhipicephalus haemaphysaloides</Emphasis> in Taiwan

Anaplasma phagocytophilum is transmitted mainly by hard ticks and can cause potentially fatal granulocytic anaplasmosis in humans, but its occurrence in ticks in Taiwan has never been investigated although this pathogen has been detected in Taiwanese rodents before. Ticks collected from small mammals in Hualien, eastern Taiwan, were assayed for Anaplasma infections; infections of Rickettsia and Apicomplexa protozoans were also studied. Of the 270 individually assayed Rhipicephalus haemaphysaloides ticks, A. phagocytophilum was identified in a nymphal tick. Parasites most similar to Anaplasma bovis, Rickettsia rickettsii, Rickettsia sp. TwKM01, and at least seven apicomplexan species (including genera Cryptosporidium, Hepatozoon, and Theileria) were also identified. This study shows that A. phagocytophilum does occur in the hard tick in Taiwan, although whether R. haemaphysaloides can vector this pathogen remains to be determined. This work also reveals a high diversity of tick-borne bacteria and protozoans circulating in a small region and calls for further research on their potential risks for human health.     

Antibacterial and immunomodulatory activities of bovine lactoferrin against <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 infections in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen that causes food-borne disease in humans ranging from watery diarrhea to bloody diarrhea and severe hemorrhagic colitis, renal failure and hemolytic uremic syndrome. Cattle, the most important source of E. coli O157:H7 transmission to humans, harbor the bacteria in their gastrointestinal tract without showing clinical symptoms. Prevention of E. coli O157:H7 infections in ruminants could diminish the public health risk. However, there is no specific treatment available nor a vaccine or a therapeutic agent which completely prevents E. coli O157:H7 infections in cattle. This paper provides an overview of latest research data on eradicating enterohemorrhagic E. coli O157:H7 in ruminants by use of bovine lactoferrin administration. The article provides insights into the anti-microbial and immunomodulatory activities of bovine lactoferrin against E. coli O157:H7 infections in cattle.     

Methods for the detection and characterization of <Emphasis Type="Italic">Streptococcus suis</Emphasis>: from conventional bacterial culture methods to immunosensors

One of the most important zoonotic pathogens worldwide, Streptococcus suis is a swine pathogen that is responsible for meningitis, toxic shock and even death in humans. S. suis infection develops rapidly with nonspecific clinical symptoms in the early stages and a high fatality rate. Recently, much attention has been paid to the high prevalence of S. suis as well as the increasing incidence and its epidemic characteristics. As laboratory-acquired infections of S. suis can occur and it is dangerous to public health security, timely and early diagnosis has become key to controlling S. suis prevalence. Here, the techniques that have been used for the detection, typing and characterization of S. suis are reviewed and the prospects for future detection methods for this bacterium are also discussed.     

Ultrastructural and cytochemical studies on the infection of wheat spikes by<Emphasis Type="Italic">Fusarium culmorum</Emphasis> as well as on degradation of cell wall components and localization of mycotoxins in the host tissue

The infection process and pathway of spreading ofFusarium culmorum in wheat spikes was examined by means of light, scanning and transmission electron microscopy after spray inoculation and single spikelet inoculation. Macroconidia of the pathogen germinated on the host surfaces, however, hyphal development and penetration of host tissues normally occurred on the inner surfaces of the lemma, glume and palea as well as on the ovary. The pathogen spread downward to the rachilla and rachis node by inter- and intracellular growth from the glume, lemma, palea and ovary. The pathogen extended in the rachis in upward and downward direction by inter- and intracellular growth inside and outside of the vascular bundles of the rachis. The spreading of the hyphae in the host tissues was associated with pronounced alterations including disintegration and digestion of host cell walls, suggesting production of cell wall degrading enzymes during infection and spreading in the host tissues. Immunogold labelling studies revealed that accumulation ofFusarium toxins in infected wheat spike tissue showed a close relationship to pathological changes in the host cells, symptom appearance and pathogen colonisation of the host tissue.Fusarium toxins may play an important role in wheat head blight development.     

Glucosyltransferase-dependent and independent effects of Clostridioides difficile toxins during infection

Clostridioides difficile infection (CDI) is the leading cause of nosocomial diarrhea and pseudomembranous colitis in the USA. In addition to these symptoms, patients with CDI can develop severe inflammation and tissue damage, resulting in life-threatening toxic megacolon. CDI is mediated by two large homologous protein toxins, TcdA and TcdB, that bind and hijack receptors to enter host cells where they use glucosyltransferase (GT) enzymes to inactivate Rho family GTPases. GT-dependent intoxication elicits cytopathic changes, cytokine production, and apoptosis. At higher concentrations TcdB induces GT-independent necrosis in cells and tissue by stimulating production of reactive oxygen species via recruitment of the NADPH oxidase complex. Although GT-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question in the field. In this study we generated novel C. difficile toxin mutants in the hypervirulent BI/NAP1/PCR-ribotype 027 {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 strain to test the hypothesis that GT-independent epithelial damage occurs during CDI. Using the mouse model of CDI, we observed that epithelial damage occurs through a GT-independent process that does not involve immune cell influx. The GT-activity of either toxin was sufficient to cause severe edema and inflammation, yet GT activity of both toxins was necessary to produce severe watery diarrhea. These results demonstrate that both TcdA and TcdB contribute to disease pathogenesis when present. Further, while inactivating GT activity of C. difficile toxins may suppress diarrhea and deleterious GT-dependent immune responses, the potential of severe GT-independent epithelial damage merits consideration when developing toxin-based therapeutics against CDI.     

The first isolation of <Emphasis Type="Italic">Westerdykella dispersa</Emphasis> in a critically burned patient

Patients with critical thermal trauma belong to one of the most high-risk groups for development of infectious complications. Fungal infections are not among frequent complications during therapy of patients with thermal trauma, yet their incidence dramatically aggravates the prognosis for patients with this disorder. In the case report, we present the case of a young man with a critical burn, where Westerdykella dispersa was isolated. Identification of the pathogen was provided with a combination of cultivation and molecular biological confirmation. In this case, the distinction between infection and colonization was very complicated. Histopathological examination for definitive diagnosis of infection was not performed because the material from unburned soft tissue sampling could further compromise the function of the hand. Repeated cultivation and molecular identification W. dispersa before and after the necrectomy is indicative of infection rather than colonization. It is the second documented case of positive cultivation with this pathogen in humans and the first such case in a non-neutropenic host.     

Clinical infection in house rats (<Emphasis Type="Italic">Rattus rattus</Emphasis>) caused by <Emphasis Type="Italic">Streptobacillus notomytis</Emphasis>

Rat bite fever is an under-reported, under-diagnosed emerging zoonosis with worldwide distribution. Besides Spirillum minus, Streptobacillus moniliformis is the major causative microorganism although it usually colonises rats without any clinical signs. A group of house rats (Rattus rattus) kept in a zoo exhibition for educational purposes suffered from neurological signs including disorientation, torticollis, stall walking, ataxia and death. Gross pathological and histo-pathological examinations of the investigated rats revealed high-grade otitis interna et media, from which Streptobacillus notomytis was isolated in pure culture or as the predominant microorganism. This case series underlines a previously expressed hypothesis that R. rattus might be naturally colonised with S. notomytis, whereas the traditional rat bite fever organism, S. moniliformis, might be restricted to the Norway rat (Rattus norvegicus). However, the general paucity of Streptobacillus isolates, especially from their respective animal hosts, precludes definitive proof of these host tropisms. This is the first report of S. notomytis detection outside Asia and Australia and the first evidence for its role as a facultative pathogen in house rats.     

Colon-Targeted Delivery of IgY Against <Emphasis Type="Italic">Clostridium difficile</Emphasis> Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl₂ 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).     

Environmental Factors Associated with the Carriage of Bacterial Pathogens in Norway Rats

Worldwide, Norway rats (Rattus norvegicus) carry a number of zoonotic pathogens. Many studies have identified rat-level risk factors for pathogen carriage. The objective of this study was to examine associations between abundance, microenvironmental and weather features and Clostridium difficile, antimicrobial-resistant (AMR) Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) carriage in urban rats. We assessed city blocks for rat abundance and 48 microenvironmental variables during a trap-removal study, then constructed 32 time-lagged temperature and precipitation variables and fitted multivariable logistic regression models. The odds of C. difficile positivity were significantly lower when mean maximum temperatures were high (≥ 12.89°C) approximately 3 months before rat capture. Alley pavement condition was significantly associated with AMR E. coli. Rats captured when precipitation was low (< 49.40 mm) in the 15 days before capture and those from blocks that contained food gardens and institutions had increased odds of testing positive for MRSA. Different factors were associated with each pathogen, which may reflect varying pathogen ecology including exposure and environmental survival. This study adds to the understanding of how the microenvironment and weather impacts the epidemiology and ecology of zoonotic pathogens in urban ecosystems, which may be useful for surveillance and control activities.     

Antimicrobial Stewardship and Environmental Decontamination for the Control of <Emphasis Type="Italic">Clostridium difficile</Emphasis> Transmission in Healthcare Settings

We implement an agent-based model for Clostridium difficile transmission in hospitals that accounts for several processes and individual factors including environmental and antibiotic heterogeneity in order to evaluate the efficacy of various control measures aimed at reducing environmental contamination and mitigating the effects of antibiotic use on transmission. In particular, we account for local contamination levels that contribute to the probability of colonization and we account for both the number and type of antibiotic treatments given to patients. Simulations illustrate the relative efficacy of several strategies for the reduction of nosocomial colonizations and nosocomial diseases.     

The extracellular proteases produced by <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Vibrio parahaemolyticus, a Gram-negative bacterium, inhabits marine and estuarine environments and it is a major pathogen responsible globally for most cases of seafood-associated gastroenteritis in humans and acute hepatopancreatic necrosis syndrome in shrimps. There has been a dramatic worldwide increase in V. parahaemolyticus infections over the last two decades. The pathogenicity of V. parahaemolyticus has been linked to the expression of different kinds of virulence factors including extracellular proteases, such as metalloproteases and serine proteases. V. parahaemolyticus expresses the metalloproteases; PrtV, VppC, VPM and the serine proteases; VPP1/Protease A, VpSP37, PrtA. Extracellular proteases have been identified as potential virulence factors which directly digest many kinds of host proteins or indirectly are involved in the processing of other toxic protein factors. This review summarizes findings on the metalloproteases and serine proteases produced by V. parahaemolyticus and their roles in infections. Identifying the role of V. parahaemolyticus virulence-associated extracellular proteases deepens our understanding of diseases caused by this bacterium.