Aristaeella hokkaidonensis gen. nov. sp. nov. and Aristaeella lactis sp. nov., two rumen bacterial species of a novel proposed family, Aristaeellaceae fam. nov.

Two strains of Gram-negative, anaerobic, rod-shaped bacteria, from an abundant but uncharacterized rumen bacterial group of the order 'Christensenellales', were phylogenetically and phenotypically characterized. These strains, designated R-7T and WTE2008T, shared 98.6-99.0 % sequence identity between their 16S rRNA gene sequences. R-7T and WTE2008T clustered together on a distinct branch from other Christensenellaceae strains and had <88.1 % sequence identity to the closest type-strain sequence from Luoshenia tenuis NSJ-44T. The genome sequences of R-7T and WTE2008T had 83.6 % average nucleotide identity to each other, and taxonomic assignment using the Genome Taxonomy Database indicates these are separate species within a novel family of the order 'Christensenellales'. Cells of R-7T and WTE2008T lacked any obvious appendages and their cell wall ultra-structures were characteristic of Gram-negative bacteria. The five most abundant cellular fatty acids of both strains were C16 : 0, C16 : 0 iso, C17 : 0 anteiso, C18 : 0 and C15 : 0 anteiso. The strains used a wide range of the 23 soluble carbon sources tested, and grew best on cellobiose, but not on sugar-alcohols. Xylan and pectin were fermented by both strains, but not cellulose. Acetate, hydrogen, ethanol and lactate were the major fermentation end products. R-7T produced considerably more hydrogen than WTE2008T, which produced more lactate. Based on these analyses, Aristaeellaceae fam. nov. and Aristaeella gen. nov., with type species Aristaeella hokkaidonensis sp. nov., are proposed. Strains R-7T (=DSM 112795T=JCM 34733T) and WTE2008T (=DSM 112788T=JCM 34734T) are the proposed type strains for Aristaeella hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively.

Utilization of SARS-CoV-2-Positive donors in pediatric renal transplantation.

BACKGROUND: As COVID-19-positive donors are becoming more common, there is an increasing need for the transplant community to evaluate the safety and efficacy of organ transplant from a SARS-CoV-2-infected donor. METHODS: Here we describe outcomes of two pediatric kidney transplant recipients who were vaccinated against COVID-19 and received their allograft from a SARS-CoV-2-positive donor. RESULTS: Both donors did not die from a COVID-19-related illness; the first donor had 1 week of COVID-19 symptoms 4 weeks prior to donation and the second was asymptomatic. Donor 1 had a Ct of 33.4 at 3 days and Donor 2 with a Ct of 37.2 at 16 days prior to donation. The first recipient was positive for SARS-CoV-2 anti-spike IgG on the day of transplant, but the second patient was negative and both patients received IVIg perioperatively. There was no evidence of SARS-CoV-2 transmission or compromised renal function at 86- and 80-day post-transplant, respectively. CONCLUSIONS: This case series suggests favorable short-term outcomes with accepting SARS-CoV-2-positive donors for pediatric renal transplantation, after thorough evaluation of the donor's risk for transmission, assessing the recipient's serologic status to SARS-CoV-2, and considering pre-emptive measures to mitigate the risk for severe COVID-19 should the recipient acquire donor-derived SARS-CoV-2.

A Near Miss of a Retropharyngeal Abscess with MRSA in a 5-Week-Old Boy Due to an Unusual Presentation.

A retropharyngeal abscess (RPA) in early childhood is not uncommon due to at-risk lymph nodes in this deep neck space and is typified by fever, odynophagia, and a constellation of respiratory manifestations. However, RPA is exceedingly rare in the neonatal subpopulation and not part of the usual differential diagnosis algorithm in this age range. Herein, we present a unique case of a previously healthy 5-week-old male infant with protracted "congestion" and difficulty in oral feeding, whose clinical course is confounded by intermittent, positional bradycardia and subsequent apnea. He was eventually diagnosed with a methicillin-resistant Staphylococcus aureus (MRSA) RPA, leading to concurrent vascular and airways compromise in the form of baroreceptor-mediated bradycardia from mass-effect carotid body compression. This clinical case is an important reminder that any infant with positional vital sign changes should prompt urgent and thorough investigation for extraordinary and otherwise uncommon pathophysiologic states. The case also highlights the power of multidisciplinary collaboration across multiple specialties and parental advocacy in unifying a diagnosis for rare pediatric illnesses.

COVID-19 Pandemic Effects on Caregivers of Youth With Type 1 Diabetes: Stress and Self-Efficacy.

Background: Little is known about the coronavirus disease 2019 (COVID-19) pandemic's psychological effects on caregivers of children with type 1 diabetes. Objective: This study aimed to investigate the experience of caregivers of youth with type 1 diabetes during the COVID-19 pandemic. Methods: A 49-item questionnaire using a 5-point Likert scale and open-response questions was distributed via e-mail and type 1 diabetes-related social media platforms from 4 May to 22 June 2020. Quantitative data were analyzed using SPSS v.25 statistical software. Descriptive statistics were used. Relationships were compared using Pearson correlation. Qualitative data were coded and categorized. Results: A total of 272 caregivers participated (mean ± SD respondent age 42.1 ± 7.8 years; 94.5% females; 81.3% with college degree or higher; 52.6% with annual income >$99,000; 80.1% with private insurance). The mean ± SD age of caregivers' children with type 1 diabetes was 11.0 ± 4.1 years, and their mean ± SD diabetes duration was 4.2 ± 3.5 years. Participants reported being diagnosed with or knowing someone with COVID-19 (24.6%), increased stress (71.9%), job loss (10.3%), and financial difficulty (26.8%) as a result of the pandemic. General self-efficacy scores were high (mean ± SD 16.2 ± 2.6, range 8-20) and significantly correlated with COVID-19-related self-efficacy (mean ± SD 12.6 ± 2.1; R = 0.394, P <0.001) and type 1 diabetes self-efficacy during COVID-19 (mean ± SD 17.1 ± 2.5; R = 0.421, P <0.001). Conclusion: Despite reporting high overall self-efficacy, caregivers of children with type 1 diabetes reported greater overall stress and challenges during the pandemic. Health care providers should be prepared to provide families with specific social and mental health support.

Sustained Virologic Suppression Reduces HIV-1 DNA Proviral Levels and HIV Antibodies in Perinatally HIV-Infected Children Followed from Birth.

The extent to which perinatally HIV-infected children, following cART initiation, develop a low proviral reservoir burden over time, as measured by HIV DNA droplet-digital polymerase chain reaction (ddPCR) and the effect on HIV antibody is not well characterized. We measured proviral HIV DNA and plasma RNA virus load (VL) in 37 perinatally HIV-infected children at 6 months of age who initiated stable cART. At 6-11 years of age, HIV proviral DNA, HIV VL (RNA), and HIV antibody by Western Blot (WB) were assessed. CART was initiated before 6 months of age in 13 children and after 6 months in 24. At school age, the HIV DNA levels did not differ by the timing of cART, and the HIV DNA levels were lower in children with negative/indeterminate WB (p = 0.0256). Children with undetectable HIV RNA VL > 50% of the time since cART initiation had lower median DNA VL than children with undetectable VL < 50% of the time (p = 0.07). Long-term viral suppression in perinatally HIV-infected children is associated with a decrease in HIV antibodies and reduced HIV reservoirs.

Cost of breast cancer diagnosis and treatment in India: a scoping review protocol.

INTRODUCTION: Breast cancer is the foremost cause for mortality among women. The non-communicable disease imposes significant economic expenses to communities. Its economic impact includes both direct and indirect healthcare costs. This scoping review will map key concepts underpinning the current direct and indirect expenses of breast cancer in India. METHODS AND ANALYSIS: This scoping review will follow 'Arksey and O'Malley's' approach and updated methodological guidance from the Joanna Briggs Institute. The Cochrane library, Econ Papers, Embase, ProQuest central, PubMed and SCOPUS will be searched for peer-reviewed scientific journal publications from the year 2000 to 2021. Reference lists of included articles and preprint repositories will be searched for additional and unpublished literature. Independent screening (title, abstract and full text) and data extraction will be carried out against the defined inclusion criteria. The results will be narratively summarised and charted under the conceptual areas of this scoping review. The research gaps and scope for future research on the topic will be identified. Findings will be reported using the Preferred Reporting Items for Systematic Reviews extension for Scoping Reviews. ETHICS AND DISSEMINATION: Ethics clearance will not be obligatory because this scoping review will only involve publicly available data. The review's findings will be disseminated through social media and a presentation in a national or international conference related to economics and healthcare. The findings will be published in a scientific journal that is peer-reviewed.

Fasting C-peptide values among adolescents in the National Health and Nutrition Examination Surveys.

AIM: To report fasting C-peptide values and variations across demographics in healthy non-diabetic adolescents included in the National Health and Nutrition Examination Surveys (NHANES) dataset. RESEARCH DESIGN AND METHODS: In this analysis, we used fasting C-peptide data from the 1999 to 2004 NHANES dataset for participants aged 12 to <18 years (n = 2063). RESULTS: The mean ± SE age of the participants was 14.99 ± 0.06 years. The participants included 992 girls and 1071 boys, and more than 80% of participants had a body mass index (BMI) percentile of <85%. Among boys, the most notable finding was the increase in geometric mean C-peptide level from 0.51 nmol/L at age 12, to 0.65 nmol/L at age 15 years. Among girls, levels fluctuated around a geometric mean of 0.67 nmol/L. Girls had significantly higher mean log-transformed C-peptide concentrations than boys (P < 0.0001) after adjusting for age, race and BMI percentile category. After adjusting for age and BMI percentile category, non-Hispanic Black boys and girls had significantly lower C-peptide levels than non-Hispanic White (P = 0.0026 and P = 0.0093) and Mexican American boys and girls (P < 0.0001 and P < 0.0001), respectively. Despite these findings, both insulin and homeostatic model assessment of insulin resistance were greater in non-Hispanic Black compared to non-Hispanic White participants. CONCLUSIONS: Here we describe fasting C-peptide levels in a non-diabetic adolescent population. These data provide crucial insight into evaluating racial differences in endogenous insulin release and clearance and will provide novel information which can be used in assessing residual ß-cell function and response to intervention therapy.

Canine-to-Human Transmission of Mycoplasma canis in the Central Nervous System.

Dog bites remain a common occurrence in our society, particularly in toddlers and small children under the age of 2. Injuries to the head and face, more common in younger children, can often lead to significant morbidity. Additionally, there continues to be considerable clinical equipoise for standardized post-dog bite injury management. Here, we present the only reported pediatric case in the literature of Mycoplasma canis-associated central nervous system (CNS) infection in an 11-month-old infant who sustained a dog bite to the calvarium. The prevalence of dog bites during the SARS-CoV-2 pandemic had interestingly tripled in number after stay-at-home orders in 1 particular pediatric emergency department in Colorado. This observation paired with advances in microbiological identification like MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight mass spectrometer) may lead to the identification of future cases of uniquely canine pathogens that play a role in human infection.

The systemic inflammatory landscape of COVID-19 in pregnancy: Extensive serum proteomic profiling of mother-infant dyads with in utero SARS-CoV-2.

While pregnancy increases the risk for severe COVID-19, the clinical and immunological implications of COVID-19 on maternal-fetal health remain unknown. Here, we present the clinical and immunological landscapes of 93 COVID-19 mothers and 45 of their SARS-CoV-2-exposed infants through comprehensive serum proteomics profiling for >1,400 cytokines of their peripheral and cord blood specimens. Prenatal SARS-CoV-2 infection triggers NF-κB-dependent proinflammatory immune activation. Pregnant women with severe COVID-19 show increased inflammation and unique IFN-λ antiviral signaling, with elevated levels of IFNL1 and IFNLR1. Furthermore, SARS-CoV-2 infection re-shapes maternal immunity at delivery, altering the expression of pregnancy complication-associated cytokines, inducing MMP7, MDK, and ESM1 and reducing BGN and CD209. Finally, COVID-19-exposed infants exhibit induction of T cell-associated cytokines (IL33, NFATC3, and CCL21), while some undergo IL-1ß/IL-18/CASP1 axis-driven neonatal respiratory distress despite birth at term. Our findings demonstrate COVID-19-induced immune rewiring in both mothers and neonates, warranting long-term clinical follow-up to mitigate potential health risks.

Artificial Biosystem for Modulation of Interactions between Antigen-Presenting Cells and T Cells.

T cell activation is triggered by signal molecules on the surface of antigen-presenting cells (APC) and subsequent exertion of cellular forces. Deciphering the biomechanical and biochemical signals in this complex process is of interest and will contribute to an improvement in immunotherapy strategies. To address underlying questions, coculture and biomimetic models are established. Mature dendritic cells (mDC) are first treated with cytochalasin B (CytoB), a cytoskeletal disruption agent known to lower apparent cellular stiffness and reduction in T cell proliferation is observed. It is attempted to mimic mDC and T cell interactions using polyacrylamide (PA) gels with defined stiffness corresponding to mDC (0.2-25 kPa). Different ratios of anti-CD3 (aCD3) and anti-CD28 (aCD28) antibodies are immobilized onto PA gels. The results show T cell proliferation is triggered by both aCD3 and aCD28 in a stiffness-dependent manner. Cells cultured on aCD3 immobilized on gels has significantly enhanced proliferation and IL-2 secretion, compared to aCD28. Furthermore, ZAP70 phosphorylation is enhanced in stiffer substrate a in a aCD3-dependent manner. The biosystem provides an approach to study the reduction of T cell proliferation observed on CytoB-treated mDC. Overall, the biosystem allows distinguishing the impact of biophysical and biochemical signals of APC and T cell interactions in vitro.

A Comprehensive Update on Kawasaki Disease Vasculitis and Myocarditis.

PURPOSE OF THE REVIEW: Kawasaki disease (KD) is a childhood systemic vasculitis of unknown etiology that causes coronary artery aneurysms (CAA), and if left undiagnosed can result in long-term cardiovascular complications and adult cardiac disease. Up to 20% of KD children fail to respond to IVIG, the mainstay of therapy, highlighting the need for novel therapeutic strategies. Here we review the latest findings in the field regarding specific etiology, genetic associations, and advancements in treatment strategies to prevent coronary aneurysms. RECENT FINDINGS: Recent discoveries using the Lactobacillus casei cell wall extract (LCWE)-induced KD vasculitis mouse model have accelerated the study of KD pathophysiology and have advanced treatment strategies including clinical trials for IL-1R antagonist, Anakinra. KD remains an elusive pediatric vasculitis syndrome and is the leading cause of acquired heart disease among children in the USA and developed countries. Advancements in combination treatment for refractory KD with further understanding of novel genetic risk factors serve as a solid foundation for future research endeavors in the field.

Occurrence and expression of genes encoding methyl-compound production in rumen bacteria.

BACKGROUND: Digestive processes in the rumen lead to the release of methyl-compounds, mainly methanol and methylamines, which are used by methyltrophic methanogens to form methane, an important agricultural greenhouse gas. Methylamines are produced from plant phosphatidylcholine degradation, by choline trimethylamine lyase, while methanol comes from demethoxylation of dietary pectins via pectin methylesterase activity. We have screened rumen metagenomic and metatranscriptomic datasets, metagenome assembled genomes, and the Hungate1000 genomes to identify organisms capable of producing methyl-compounds. We also describe the enrichment of pectin-degrading and methane-forming microbes from sheep rumen contents and the analysis of their genomes via metagenomic assembly. RESULTS: Screens of metagenomic data using the protein domains of choline trimethylamine lyase (CutC), and activator protein (CutD) found good matches only to Olsenella umbonata and to Caecibacter, while the Hungate1000 genomes and metagenome assembled genomes from the cattle rumen found bacteria within the phyla Actinobacteria, Firmicutes and Proteobacteria. The cutC and cutD genes clustered with genes that encode structural components of bacterial microcompartment proteins. Prevotella was the dominant genus encoding pectin methyl esterases, with smaller numbers of sequences identified from other fibre-degrading rumen bacteria. Some large pectin methyl esterases (> 2100 aa) were found to be encoded in Butyrivibrio genomes. The pectin-utilising, methane-producing consortium was composed of (i) a putative pectin-degrading bacterium (phylum Tenericutes, class Mollicutes), (ii) a galacturonate-using Sphaerochaeta sp. predicted to produce acetate, lactate, and ethanol, and (iii) a methylotrophic methanogen, Methanosphaera sp., with the ability to form methane via a primary ethanol-dependent, hydrogen-independent, methanogenesis pathway. CONCLUSIONS: The main bacteria that produce methyl-compounds have been identified in ruminants. Their enzymatic activities can now be targeted with the aim of finding ways to reduce the supply of methyl-compound substrates to methanogens, and thereby limit methylotrophic methanogenesis in the rumen.

Lymphotoxin ß receptor-mediated NFκB signaling promotes glial lineage differentiation and inhibits neuronal lineage differentiation in mouse brain neural stem/progenitor cells.

BACKGROUND: Lymphotoxin (LT) is a lymphokine mainly expressed in lymphocytes. LTα binds one or two membrane-associated LTß to form LTα2ß1 or LTα1ß2 heterotrimers. The predominant LTα1ß2 binds to LTß receptor (LTßR) primarily expressed in epithelial and stromal cells. Most studies on LTßR signaling have focused on the organization, development, and maintenance of lymphoid tissues. However, the roles of LTßR signaling in the nervous system, particularly in neurogenesis, remain unknown. Here, we investigated the role of LTßR-mediated NFκB signaling in regulating neural lineage differentiation. METHODS: The C57BL/6J wild-type and GFAP-dnIκBα transgenic mice were used. Serum-free embryoid bodies were cultured from mouse embryonic stem cells and further induced into neural stem/progenitor cells (NSCs/NPCs). Primary neurospheres were cultured from embryonic and adult mouse brains followed by monolayer culture for amplification/passage. NFκB activation was determined by adenovirus-mediated NFκB-firefly-luciferase reporter assay and p65/RelB/p52 nuclear translocation assay. LTßR mRNA expression was evaluated by quantitative RT-PCR and LTßR protein expression was determined by immunohistochemistry and Western blot analysis. Multilabeled immunocytochemistry or immunohistochemistry followed by fluorescent confocal microscopy and quantitative analysis of neural lineage differentiation were performed. Graphing and statistical analysis were performed with GraphPad Prism software. RESULTS: In cultured NSCs/NPCs, LTα1ß2 stimulation induced an activation of classical and non-classical NFκB signaling. The expression of LTßR-like immunoreactivity in GFAP+/Sox2+ NSCs was identified in well-established neurogenic zones of adult mouse brain. Quantitative RT-PCR and Western blot analysis validated the expression of LTßR in cultured NSCs/NPCs and brain neurogenic regions. LTßR expression was significantly increased during neural induction. LTα1ß2 stimulation in cultured NSCs/NPCs promoted astroglial and oligodendrocytic lineage differentiation, but inhibited neuronal lineage differentiation. Astroglial NFκB inactivation in GFAP-dnIκBα transgenic mice rescued LTßR-mediated abnormal phenotypes of cultured NSCs/NPCs. CONCLUSION: This study provides the first evidence for the expression and function of LTßR signaling in NSCs/NPCs. Activation of LTßR signaling promotes glial lineage differentiation. Our results suggest that neurogenesis is regulated by the adaptive immunity and inflammatory responses.

Gene and transcript abundances of bacterial type III secretion systems from the rumen microbiome are correlated with methane yield in sheep.

BACKGROUND: Ruminants are important contributors to global methane emissions via microbial fermentation in their reticulo-rumens. This study is part of a larger program, characterising the rumen microbiomes of sheep which vary naturally in methane yield (g CH4/kg DM/day) and aims to define differences in microbial communities, and in gene and transcript abundances that can explain the animal methane phenotype. METHODS: Rumen microbiome metagenomic and metatranscriptomic data were analysed by Gene Set Enrichment, sparse partial least squares regression and the Wilcoxon Rank Sum test to estimate correlations between specific KEGG bacterial pathways/genes and high methane yield in sheep. KEGG genes enriched in high methane yield sheep were reassembled from raw reads and existing contigs and analysed by MEGAN to predict their phylogenetic origin. Protein coding sequences from Succinivibrio dextrinosolvens strains were analysed using Effective DB to predict bacterial type III secreted proteins. The effect of S. dextrinosolvens strain H5 growth on methane formation by rumen methanogens was explored using co-cultures. RESULTS: Detailed analysis of the rumen microbiomes of high methane yield sheep shows that gene and transcript abundances of bacterial type III secretion system genes are positively correlated with methane yield in sheep. Most of the bacterial type III secretion system genes could not be assigned to a particular bacterial group, but several genes were affiliated with the genus Succinivibrio, and searches of bacterial genome sequences found that strains of S. dextrinosolvens were part of a small group of rumen bacteria that encode this type of secretion system. In co-culture experiments, S. dextrinosolvens strain H5 showed a growth-enhancing effect on a methanogen belonging to the order Methanomassiliicoccales, and inhibition of a representative of the Methanobrevibacter gottschalkii clade. CONCLUSIONS: This is the first report of bacterial type III secretion system genes being associated with high methane emissions in ruminants, and identifies these secretions systems as potential new targets for methane mitigation research. The effects of S. dextrinosolvens on the growth of rumen methanogens in co-cultures indicate that bacteria-methanogen interactions are important modulators of methane production in ruminant animals.

Unique Case of Disseminated Plague With Multifocal Osteomyelitis.

Plague is a disease caused by Yersinia pestis. Septicemic and pneumonic plague have a high mortality rate if untreated. Here we describe the challenges of accurately diagnosing a nonfatal pediatric case of septicemic plague with involvement of multiple organs; to our knowledge, the first documented case of multifocal plague osteomyelitis.

Rumen metagenome and metatranscriptome analyses of low methane yield sheep reveals a Sharpea-enriched microbiome characterised by lactic acid formation and utilisation.

BACKGROUND: Enteric fermentation by farmed ruminant animals is a major source of methane and constitutes the second largest anthropogenic contributor to global warming. Reducing methane emissions from ruminants is needed to ensure sustainable animal production in the future. Methane yield varies naturally in sheep and is a heritable trait that can be used to select animals that yield less methane per unit of feed eaten. We previously demonstrated elevated expression of hydrogenotrophic methanogenesis pathway genes of methanogenic archaea in the rumens of high methane yield (HMY) sheep compared to their low methane yield (LMY) counterparts. Methane production in the rumen is strongly connected to microbial hydrogen production through fermentation processes. In this study, we investigate the contribution that rumen bacteria make to methane yield phenotypes in sheep. RESULTS: Using deep sequence metagenome and metatranscriptome datasets in combination with 16S rRNA gene amplicon sequencing from HMY and LMY sheep, we show enrichment of lactate-producing Sharpea spp. in LMY sheep bacterial communities. Increased gene and transcript abundances for sugar import and utilisation and production of lactate, propionate and butyrate were also observed in LMY animals. Sharpea azabuensis and Megasphaera spp. act as important drivers of lactate production and utilisation according to phylogenetic analysis and read mappings. CONCLUSIONS: Our findings show that the rumen microbiome in LMY animals supports a rapid heterofermentative growth, leading to lactate production. We postulate that lactate is subsequently metabolised mainly to butyrate in LMY animals, producing 2 mol of hydrogen and 0.5 mol of methane per mol hexose, which represents 24 % less than the 0.66 mol of methane formed from the 2.66 mol of hydrogen produced if hexose fermentation was directly to acetate and butyrate. These findings are consistent with the theory that a smaller rumen size with a higher turnover rate, where rapid heterofermentative growth would be an advantage, results in lower hydrogen production and lower methane formation. Together with previous methanogen gene expression data, this builds a strong concept of how animal traits and microbial communities shape the methane phenotype in sheep.

Methane yield phenotypes linked to differential gene expression in the sheep rumen microbiome.

Ruminant livestock represent the single largest anthropogenic source of the potent greenhouse gas methane, which is generated by methanogenic archaea residing in ruminant digestive tracts. While differences between individual animals of the same breed in the amount of methane produced have been observed, the basis for this variation remains to be elucidated. To explore the mechanistic basis of this methane production, we measured methane yields from 22 sheep, which revealed that methane yields are a reproducible, quantitative trait. Deep metagenomic and metatranscriptomic sequencing demonstrated a similar abundance of methanogens and methanogenesis pathway genes in high and low methane emitters. However, transcription of methanogenesis pathway genes was substantially increased in sheep with high methane yields. These results identify a discrete set of rumen methanogens whose methanogenesis pathway transcription profiles correlate with methane yields and provide new targets for CH4 mitigation at the levels of microbiota composition and transcriptional regulation.

Lack of Outer Membrane Protein A Enhances the Release of Outer Membrane Vesicles and Survival of Vibrio cholerae and Suppresses Viability of Acanthamoeba castellanii.

Vibrio cholerae, the causative agent of the diarrhoeal disease cholera, survives in aquatic environments. The bacterium has developed a survival strategy to grow and survive inside Acanthamoeba castellanii. It has been shown that V. cholerae expresses outer membrane proteins as virulence factors playing a role in the adherence to interacted host cells. This study examined the role of outer membrane protein A (OmpA) and outer membrane vesicles (OMVs) in survival of V. cholerae alone and during its interaction with A. castellanii. The results showed that an OmpA mutant of V. cholerae survived longer than wild-type V. cholerae when cultivated alone. Cocultivation with A. castellanii enhanced the survival of both bacterial strains and OmpA protein exhibited no effect on attachment, engulfment, and survival inside the amoebae. However, cocultivation of the OmpA mutant of V. cholerae decreased the viability of A. castellanii and this bacterial strain released more OMVs than wild-type V. cholerae. Surprisingly, treatment of amoeba cells with OMVs isolated from the OmpA mutant significantly decreased viable counts of the amoeba cells. In conclusion, the results might highlight a regulating rule for OmpA in survival of V. cholerae and OMVs as a potent virulence factor for this bacterium towards eukaryotes in the environment.

Interaction between Vibrio mimicus and Acanthamoeba castellanii.

Vibrio mimicus is a Gram-negative bacterium, which causes gastroenteritis and is closely related to Vibrio cholerae. The environmental reservoir of this bacterium is far from defined. Acanthamoeba as well as Vibrio species are found in diverse aquatic environments. The present study was aimed to investigate the ability of A. castellanii to host V. mimicus, the role of bacterial protease on interaction with A. castellanii and to disclose the ability of cysts to protect intracellular V. mimicus. Co-cultivation, viable counts, gentamicin assay, electron microscopy and statistical analysis showed that co-cultivation of wild type and luxO mutant of V. mimicus strains with A. castellanii did not inhibit growth of the amoeba. On the other hand co-cultivation enhanced growth and survival of V. mimicus strains. Vibrio mimicus showed intracellular behaviour because bacteria were found to be localized in the cytoplasm of amoeba trophozoites and remain viable for 14 days. The cysts protected intracellular V. mimicus from high level of gentamicin. The intracellular growth of V. mimicus in A. castellanii suggests a role of A. castellanii as a host for V. mimicus.

Role of melanin pigment in expression of Vibrio cholerae virulence factors.

We identified the mutated gene locus in a pigment-overproducing Vibrio cholerae mutant of strain A1552. The deduced gene product is suggested to be an oxidoreductase based on partial homology to putative homogentisate 1,2-dioxygenase in Pseudomonas aeruginosa and Mesorhizobium loti, and we propose that the gene VC1345 in the V. cholerae genome be denoted hmgA in accordance with the nomenclature for other species. The hmgA::mini-Tn5 mutant showed a nonpigmented phenotype after complementation with a plasmid clone carrying the WT hmgA(+) locus. Microarray transcription analysis revealed that expression of hmgA and the neighboring genes encoding a postulated two-component sensor system was growth phase dependent. Results from quantitative reverse transcription-PCR analysis showed that hmgA operon expression was reduced in the rpoS mutant, but pigment production by the WT V. cholerae or the hmgA mutant was not detectably influenced by the stationary-phase regulator RpoS. The pigmented mutant showed increased UV resistance in comparison with the WT strain. Interestingly, the pigment-producing mutant expressed more toxin-coregulated pilus and cholera toxin than WT V. cholerae. Moreover, the hmgA mutant showed a fivefold increase in the ability to colonize the intestines of infant mice. A possible mechanism by which pigment production might cause induction of the ToxR regulon due to generation of hydrogen peroxide was supported by results from tests showing that externally supplied H(2)O(2) led to higher TcpA levels. Taken together, our findings suggest that melanin pigment formation may play a role in V. cholerae virulence factor expression.