Emergence of the novel sixth Candida auris Clade VI in Bangladesh.

Candida auris, initially identified in 2009, has rapidly become a critical concern due to its antifungal resistance and significant mortality rates in healthcare-associated outbreaks. To date, whole-genome sequencing (WGS) has identified five unique clades of C. auris, with some strains displaying resistance to all primary antifungal drug classes. In this study, we presented the first WGS analysis of C. auris from Bangladesh, describing its origins, transmission dynamics, and antifungal susceptibility testing (AFST) profile. Ten C. auris isolates collected from hospital settings in Bangladesh were initially identified by CHROMagar Candida Plus, followed by VITEK2 system, and later sequenced using Illumina NextSeq 550 system. Reference-based phylogenetic analysis and variant calling pipelines were used to classify the isolates in different clades. All isolates aligned ~90% with the Clade I C. auris B11205 reference genome. Of the 10 isolates, 8 were clustered with Clade I isolates, highlighting a South Asian lineage prevalent in Bangladesh. Remarkably, the remaining two isolates formed a distinct cluster, exhibiting >42,447 single-nucleotide polymorphism differences compared to their closest Clade IV counterparts. This significant variation corroborates the emergence of a sixth clade (Clade VI) of C. auris in Bangladesh, with potential for international transmission. AFST results showed that 80% of the C. auris isolates were resistant to fluconazole and voriconazole, whereas Clade VI isolates were susceptible to azoles, echinocandins, and pyrimidine analogue. Genomic sequencing revealed ERG11_Y132F mutation conferring azole resistance while FCY1_S70R mutation found inconsequential in describing 5-flucytosine resistance. Our study underscores the pressing need for comprehensive genomic surveillance in Bangladesh to better understand the emergence, transmission dynamics, and resistance profiles of C. auris infections. Unveiling the discovery of a sixth clade (Clade VI) accentuates the indispensable role of advanced sequencing methodologies.IMPORTANCECandida auris is a nosocomial fungal pathogen that is commonly misidentified as other Candida species. Since its emergence in 2009, this multidrug-resistant fungus has become one of the five urgent antimicrobial threats by 2019. Whole-genome sequencing (WGS) has proven to be the most accurate identification technique of C. auris which also played a crucial role in the initial discovery of this pathogen. WGS analysis of C. auris has revealed five distinct clades where isolates of each clade differ among themselves based on pathogenicity, colonization, infection mechanism, as well as other phenotypic characteristics. In Bangladesh, C. auris was first reported in 2019 from clinical samples of a large hospital in Dhaka city. To understand the origin, transmission dynamics, and antifungal-resistance profile of C. auris isolates circulating in Bangladesh, we conducted a WGS-based surveillance study on two of the largest hospital settings in Dhaka, Bangladesh.

Emergence of zoonotic sporotrichosis in Brazil: a genomic epidemiology study.

BACKGROUND: Zoonotic sporotrichosis is a neglected fungal disease, whereby outbreaks are primarily driven by Sporothrix brasiliensis and linked to cat-to-human transmission. To understand the emergence and spread of sporotrichosis in Brazil, the epicentre of the current epidemic in South America, we aimed to conduct whole-genome sequencing (WGS) to describe the genomic epidemiology. METHODS: In this genomic epidemiology study, we included Sporothrix spp isolates from sporotrichosis cases from Brazil, Colombia, and the USA. We conducted WGS using Illumina NovaSeq on isolates collected by three laboratories in Brazil from humans and cats with sporotrichosis between 2013 and 2022. All isolates that were confirmed to be Sporothrix genus by internal transcribed spacer or beta-tubulin PCR sequencing were included in this study. We downloaded eight Sporothrix genome sequences from the National Center for Biotechnology Information (six from Brazil, two from Colombia). Three Sporothrix spp genome sequences from the USA were generated by the US Centers for Disease Control and Prevention as part of this study. We did phylogenetic analyses and correlated geographical and temporal case distribution with genotypic features of Sporothrix spp isolates. FINDINGS: 72 Sporothrix spp isolates from 55 human and 17 animal sporotrichosis cases were included: 67 (93%) were from Brazil, two (3%) from Colombia, and three (4%) from the USA. Cases spanned from 1999 to 2022. Most (61 [85%]) isolates were S brasiliensis, and all were reported from Brazil. Ten (14%) were Sporothrix schenckii and were reported from Brazil, USA, and Colombia. For S schenckii isolates, two distinct clades were observed wherein isolates clustered by geography. For S brasiliensis isolates, five clades separated by more than 100 000 single-nucleotide polymorphisms were observed. Among the five S brasiliensis clades, clades A and C contained isolates from both human and cat cases, and clade A contained isolates from six different states in Brazil. Compared with S brasiliensis isolates, larger genetic diversity was observed among S schenckii isolates from animal and human cases within a clade. INTERPRETATION: Our results suggest that the ongoing epidemic driven by S brasiliensis in Brazil represents several, independent emergence events followed by animal-to-animal and animal-to human transmission within and between Brazilian states. These results describe how S brasiliensis can emerge and spread within a country. FUNDING: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil; the São Paulo Research Foundation; Productivity in Research fellowships by the National Council for Scientific and Technological Development, and Ministry of Science and Technology of Brazil.

Bimodal distribution of azole susceptibility in Sporothrix brasiliensis isolates in Brazil.

Sporothrix brasiliensis is an emerging zoonotic fungal pathogen that can be difficult to treat. Antifungal susceptibility testing was performed on the mold phase of a convenience sample of 61 Sporothrix spp. isolates from human and cat sporotrichosis cases in Brazil using the Clinical and Laboratory Standards Institute standard M38. A bimodal distribution of azole susceptibility was observed with 50% (28/56) of S. brasiliensis isolates showing elevated itraconazole minimum inhibitory concentrations ≥16 µg/mL. Phylogenetic analysis found the in vitro resistant isolates were not clonal and were distributed across three different S. brasiliensis clades. Single nucleotide polymorphism (SNP) analysis was performed to identify potential mechanisms of in vitro resistance. Two of the 28 resistant isolates (MIC ≥16 mg/L) had a polymorphism in the cytochrome P450 gene, cyp51, corresponding to the well-known G448S substitution inducing azole resistance in Aspergillus fumigatus. SNPs corresponding to other known mechanisms of azole resistance were not identified in the remaining 26 in vitro resistant isolates.

Optimization and Validation of Candida auris Short Tandem Repeat Analysis.

Candida auris is an easily transmissible yeast with resistance to different antifungal compounds. Outbreaks of C. auris are mostly observed in intensive care units. To take adequate measures during an outbreak, it is essential to understand the transmission route, which requires isolate genotyping. In 2019, a short tandem repeat (STR) genotyping analysis was developed for C. auris. To determine the discriminatory power of this method, we performed STR analysis of 171 isolates with known whole-genome sequencing (WGS) data using Illumina reads, and we compared their resolutions. We found that STR analysis separated the 171 isolates into four clades (clades I to IV), as was also seen with WGS analysis. Then, to improve the separation of isolates in clade IV, the STR assay was optimized by the addition of 2 STR markers. With this improved STR assay, a total of 32 different genotypes were identified, while all isolates with differences of >50 single-nucleotide polymorphisms (SNPs) were separated by at least 1 STR marker. Altogether, we optimized and validated the C. auris STR panel for clades I to IV and established its discriminatory power, compared to WGS SNP analysis using Illumina reads. IMPORTANCE The emerging fungal pathogen Candida auris poses a threat to public health, mainly causing outbreaks in intensive care units. Genotyping is essential for investigating potential outbreaks and preventing further spread. Previously, we developed a STR genotyping scheme for rapid and high-resolution genotyping, and WGS SNP outcomes for some isolates were compared to STR data. Here, we compared WGS SNP and STR outcomes for a larger sample cohort. Also, we optimized the resolution of this typing scheme with the addition of 2 STR markers. Altogether, we validated and optimized this rapid, reliable, and high-resolution typing scheme for C. auris.

Antibiotic therapy with metronidazole reduces endometriosis disease progression in mice: a potential role for gut microbiota.

STUDY QUESTION: Does altering gut microbiota with antibiotic treatment have any impact on endometriosis progression? SUMMARY ANSWER: Antibiotic therapy reduces endometriosis progression in mice, possibly by reducing specific gut bacteria. WHAT IS KNOWN ALREADY: Endometriosis, a chronic condition causing abdominal pain and infertility, afflicts up to 10% of women between the ages of 25 and 40, ~5 million women in the USA. Current treatment strategies, including hormone therapy and surgery, have significant side effects and do not prevent recurrences. We have little understanding of why some women develop endometriosis and others do not. STUDY DESIGN, SIZE, DURATION: Mice were treated with broad-spectrum antibiotics or metronidazole, subjected to surgically-induced endometriosis and assayed after 21 days. PARTICIPANTS/MATERIALS, SETTING, METHODS: The volumes and weights of endometriotic lesions and histological signatures were analysed. Proliferation and inflammation in lesions were assessed by counting cells that were positive for the proliferation marker Ki-67 and the macrophage marker Iba1, respectively. Differences in faecal bacterial composition were assessed in mice with and without endometriosis, and faecal microbiota transfer studies were performed. MAIN RESULTS AND THE ROLE OF CHANCE: In mice treated with broad-spectrum antibiotics (vancomycin, neomycin, metronidazole and ampicillin), endometriotic lesions were significantly smaller (~ 5-fold; P < 0.01) with fewer proliferating cells (P < 0.001) than those in mice treated with vehicle. Additionally, inflammatory responses, as measured by the macrophage marker Iba1 in lesions and IL-1ß, TNF-α, IL-6 and TGF-ß1 in peritoneal fluid, were significantly reduced in mice treated with broad-spectrum antibiotics (P < 0.05). In mice treated with metronidazole only, but not in those treated with neomycin, ectopic lesions were significantly (P < 0.001) smaller in volume than those from vehicle-treated mice. Finally, oral gavage of faeces from mice with endometriosis restored the endometriotic lesion growth and inflammation (P < 0.05 and P < 0.01, respectively) in metronidazole-treated mice. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: These findings are from a mouse model of surgically-induced endometriosis. Further studies are needed to determine the mechanism by which gut bacteria promote inflammation, identify bacterial genera or species that promote disease progression and assess the translatability of these findings to humans. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that gut bacteria promote endometriosis progression in mice. This finding if translated to humans, could aid in the development of improved diagnostic tools and personalised treatment strategies. STUDY FUNDING AND COMPETING INTEREST(S): This work was funded, in part, by: a National Institutes of Health (NIH)/ National Institute of Child Health and Human Development (NICHD) grant (R00HD080742) to RK; Washington University School of Medicine start-up funds to RK; an Endometriosis Foundation of America Research Award to R.K.; and an NIH/NICHD grant (R01HD091218) to IUM. The authors report no conflict of interest.

Microbial communities in placentas from term normal pregnancy exhibit spatially variable profiles.

The placenta is the principal organ nurturing the fetus during pregnancy and was traditionally considered to be sterile. Recent work has suggested that the placenta harbours microbial communities, however the location and possible function of these microbes remain to be confirmed and elucidated. Here, we employed genomic DNA sequencing of multiple variable (V) regions of the bacterial 16S ribosomal gene, to interrogate microbial profiles in term pregnancies, from the basal plate, which is in direct contact with maternal uterine, endothelial, and immune cells; placental villi, which are bathed in maternal blood, and fetal membranes, which encapsulate the amniotic cavity. QIIME, R package "Phyloseq" analysis was used to assess alpha and beta diversity and absolute abundance of the 16S rRNA gene per location. We demonstrate that (1) microbiota exhibit spatially distinct profiles depending on the location within the placenta and (2) "semi-composite" 16S profiles using multiple V regions validated by quantitative PCR analysis confirmed that distinct bacterial taxa dominate in different placental niches. Finally, profiles are not altered by mode of delivery. Together these findings suggest that there is niche-specificity to the placental microbiota and placental microbiome studies should consider regional differences, which may affect maternal, fetal, and/or neonatal health and physiology.

Maternal microbiomes in preterm birth: Recent progress and analytical pipelines.

Worldwide, 10% of babies are born preterm, defined as birth before 37 weeks' gestation. We have had little success in developing strategies to prevent preterm births, the majority of which are due to infection or are idiopathic. An emerging hypothesis is that the maternal microbiome-the bacteria that inhabit the mother's body and play vital functions in normal health-contributes to the etiology of preterm birth. Here, we highlight the latest data revealing correlations between preterm birth and maternal intestinal, vaginal, cervical, and placental microbiomes. Additionally, we describe the most commonly used comparative microbiome analysis methods and highlight important issues to consider when conducting such studies.

Inhibition of autophagy limits vertical transmission of Zika virus in pregnant mice.

Zika virus (ZIKV) infection during pregnancy leads to devastating fetal outcomes, including intrauterine growth restriction and microcephaly. Greater understanding of mechanisms underlying ZIKV maternal-fetal transmission is needed to develop new therapeutic interventions. Here, we define an important role for the autophagy pathway in ZIKV vertical transmission. ZIKV infection induced autophagic activity in human trophoblasts and pharmacological inhibition limited ZIKV infectivity. Furthermore, deficiency in an essential autophagy gene, Atg16l1, in mice limited ZIKV vertical transmission and placental and fetal damage and overall improved placental and fetal outcomes. This protection was due to a placental trophoblast cell-autonomous effect of autophagic activity, not to alterations in systemic maternal ZIKV infection. Finally, an autophagy inhibitor, hydroxychloroquine, approved for use in pregnant women, attenuated placental and fetal ZIKV infection and ameliorated adverse placental and fetal outcomes. Our study reveals new insights into the mechanism of ZIKV vertical transmission and suggests that an autophagy-based therapeutic warrants possible evaluation in humans to diminish the risks of ZIKV maternal-fetal transmission.

TAM Receptors Are Not Required for Zika Virus Infection in Mice.

Tyro3, Axl, and Mertk (TAM) receptors are candidate entry receptors for infection with the Zika virus (ZIKV), an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we used several routes of viral inoculation and compared viral replication in wild-type versus Axl-/-, Mertk-/-, Axl-/-Mertk-/-, and Axl-/-Tyro3-/- mice in various organs. Pregnant and non-pregnant mice treated with interferon-α-receptor (IFNAR)-blocking (MAR1-5A3) antibody and infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR-blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism were observed. These findings indicate that, in mice, TAM receptors are not required for ZIKV entry and infection.

Autophagy regulation of physiological and pathological processes in the female reproductive tract.

Autophagy is a ubiquitous cell recycling pathway that delivers cytoplasmic constituents to the lysosome and is essential for normal cellular function. Autophagic activity is up-regulated under physiological conditions as well as stressful conditions such as nutrient deprivation, oxidative stress, hypoxia, inflammation, and infection. Thus, it is essential to regard the functional importance of the pathway and its components in a given tissue context. Here we review what is known about the involvement of autophagy process during physiological processes in the female reproductive tract and in pregnancy from preimplantation to oocyte function to placental development, parturition, and postpartum remodeling of the uterus; as well as in pathological and adverse events during these processes.

Hydrogen peroxide induced loss of heterozygosity correlates with replicative lifespan and mitotic asymmetry in Saccharomyces cerevisiae.

Cellular aging in Saccharomyces cerevisiae can lead to genomic instability and impaired mitotic asymmetry. To investigate the role of oxidative stress in cellular aging, we examined the effect of exogenous hydrogen peroxide on genomic instability and mitotic asymmetry in a collection of yeast strains with diverse backgrounds. We treated yeast cells with hydrogen peroxide and monitored the changes of viability and the frequencies of loss of heterozygosity (LOH) in response to hydrogen peroxide doses. The mid-transition points of viability and LOH were quantified using sigmoid mathematical functions. We found that the increase of hydrogen peroxide dependent genomic instability often occurs before a drop in viability. We previously observed that elevation of genomic instability generally lags behind the drop in viability during chronological aging. Hence, onset of genomic instability induced by exogenous hydrogen peroxide treatment is opposite to that induced by endogenous oxidative stress during chronological aging, with regards to the midpoint of viability. This contrast argues that the effect of endogenous oxidative stress on genome integrity is well suppressed up to the dying-off phase during chronological aging. We found that the leadoff of exogenous hydrogen peroxide induced genomic instability to viability significantly correlated with replicative lifespan (RLS), indicating that yeast cells' ability to counter oxidative stress contributes to their replicative longevity. Surprisingly, this leadoff is positively correlated with an inverse measure of endogenous mitotic asymmetry, indicating a trade-off between mitotic asymmetry and cell's ability to fend off hydrogen peroxide induced oxidative stress. Overall, our results demonstrate strong associations of oxidative stress to genomic instability and mitotic asymmetry at the population level of budding yeast.