Flow Cytometry and Automated Microscopy Integration for Phagocytic Analysis of Hypervirulent Klebsiella pneumoniae in Dictyostelium discoideum.

This chapter introduces protocols for culturing and maintaining Dictyostelium discoideum and methods for conducting virulence assays in this organism to study bacterial pathogenicity. It outlines advanced techniques, such as automated microscopy and flow cytometry, for detailed cellular analysis and traditional microbiological approaches. These comprehensive protocols will enable researchers to probe the virulence factors of pathogens like Klebsiella pneumoniae and to elucidate the details of host-pathogen interactions within a cost-effective and adaptable laboratory framework.

Emerging fatal gout disease in Chinese goslings linked to acute kidney injury induced by novel goose astrovirus infection.

A novel goose astrovirus (GAstV) has broken out across China in recent years, causing widespread damage to the poultry industry. In goslings infected with GAstV, the leading cause of death is visceral gout. However, our understanding of the mechanism of gout formation in GAstV infection is largely inadequate. The aim of this study was to examine the pathogenicity of a GAstV strain and explore the molecular mechanisms of visceral gout caused by viral infection in goslings. The virulent GAstV strain HR2105/1 was effectively isolated from the visceral tissue of goslings in gout-affected areas. The whole genome of the HR2105/1 strain was sequenced and analyzed. Subsequently, we established a gosling gout models with experimental GAstV infection. Finally, we conducted a study on the mechanism of GAstV induced acute kidney injury. Phylogenetic analysis of the complete genome sequence showed that it was closely related to the strain circulating in China since 2016, and it was grouped within the GAstV-1 cluster. The clinical signs were reproduced by experimental infection of healthy goslings with the isolated strain and were found to be similar to those reported in clinical cases. Moreover, the virus exhibits strong renal tropism. Infection with the GAstV strain HR2105/1 was found to cause acute kidney injury, as evidenced by increased levels of uric acid and creatinine as well as severe pathological damage. Mechanistic experiments with Masson and Picrosirius Red staining revealed fibrosis in renal tissues after GAstV infection. Furthermore, TUNEL staining revealed that GAstV infection triggered renal cell apoptosis. Additionally, RT-qPCR revealed that GAstV infection caused an excessive inflammatory response by upregulating the expression of IL-1ß, IL-6, IL-10, TGF-ß, and iNOS in renal tissues. Overall, our findings demonstrate that GAstV infection causes renal damage by inducing renal cell apoptosis, fibrosis, and excessive inflammatory response, which subsequently leads to hyperuricemia and lethal visceral gout formation. This is the first systematic study on the etiology of lethal gout in goslings caused by GAstV infection, and we believe that the findings can guide vaccine development and therapeutic targets for GAstV-associated renal diseases.

Differential requirement of formyl peptide receptor 1 in macrophages and neutrophils in the host defense against Mycobacterium tuberculosis Infection.

Formyl peptide receptors (FPR), part of the G-protein coupled receptor superfamily, are pivotal in directing phagocyte migration towards chemotactic signals from bacteria and host tissues. Although their roles in acute bacterial infections are well-documented, their involvement in immunity against tuberculosis (TB) remains unexplored. Here, we investigate the functions of Fpr1 and Fpr2 in defense against Mycobacterium tuberculosis (Mtb), the causative agent of TB. Elevated levels of Fpr1 and Fpr2 were found in the lungs of mice, rabbits and peripheral blood of humans infected with Mtb, suggesting a crucial role in the immune response. The effects of Fpr1 and Fpr2 deletion on bacterial load, lung damage, and cellular inflammation were assessed in a murine TB model utilizing hypervirulent strain of Mtb from the W-Beijing lineage. While Fpr2 deletion had no impact on disease outcome, Fpr1-deficient mice demonstrated improved bacterial control, especially by macrophages. Bone marrow-derived macrophages from these Fpr1-/- mice exhibited an enhanced ability to contain bacterial growth over time. Contrarily, treating genetically susceptible mice with Fpr1-specific inhibitors caused impaired early bacterial control, corresponding with increased Mtb persistence in necrotic neutrophils. Furthermore, ex vivo assays revealed that Fpr1-/- neutrophils were unable to restrain Mtb growth, indicating a differential function of Fpr1 among myeloid cells. These findings highlight the distinct and complex roles of Fpr1 in myeloid cell-mediated immunity against Mtb infection, underscoring the need for further research into these mechanisms for a better understanding of TB immunity.

Occurrence of Extended-spectrum ß-lactamase (ESBL) and Carbapenemase-producing Escherichia coli isolated from Childhood Diarrhoea in Yaoundé, Cameroon.

INTRODUCTION: Extended-spectrum ß-lactamase (ESBL)-producing pathogenic E. coli is a global public health issue, especially in sub-Saharan African countries such as Cameroon. It contributes to increase significantly hospital length of stay, morbidity, mortality and economic costs because of treatment failures. This study aims at determining the resistance background and virulence profiles of ESBL-E. coli isolates among childhood diarrhoea during the cholera outbreak occuring in Yaoundé, Cameroon. MATERIALS AND METHODS: During a four-month periods, from March 1st to June 30th, 2023,  a total of 84 stool samples were collected from 90 under five children presenting clinical signs of gastroenteritis and attending four hospitals in Yaoundé, Cameroon. Bacterial identification was done using API20E and antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method. After extraction, genomic DNA was subjected to conventional and multiplex polymerase chain reaction methods (PCRs) for detection of resistance and virulence genes. Statistical analysis was performed using Epi info™ (7.2.5.0). Statistical significance was considered at a p-value < 0.05. RESULTS: Out of 150 patients contacted, 90 patients were enrolled, 84 samples were collected, 52.38%(44/84) and 3.57%(03/84) were confirmed as extended-spectrum ß-lactamase and carbapenemase-producing E. coli respectively. The risk factors were analyzed, and children who drank natural fruit juice (OR: 0.4, p-value: 0.03) were found to be significantly associated with ESBL-producing E. coli. The ESBL-producing E. coli isolates showed a high level of resistance to amoxicillin-clavulanic acid, cefotaxime, ceftazidime, cefepime, colistin, and tetracycline. The blaCTX-M was more prevalent ß-lactamase resistance gene. The tetracycline resistance genes tet(A) and tet(B) were also detected. The most important virulence genes detected were FimH (81.81%) and papA (79.54%). CONCLUSION: These findings suggest implementing routine surveillance and screening for antimicrobial resistance among children under five. Antimicrobial stewardship strategies (ASP) need to be implemented to curb the emergence and dissemination of ESBL-producing E. coli. In addition, a national surveillance program for antimicrobial resistance needs to be implemented at local and regional levels in order to reduce morbidity in Cameroon.

Transcriptomic changes in the PacC transcription factor deletion mutant of the plant pathogenic fungus Botrytis cinerea under acidic and neutral conditions.

OBJECTIVES: Botrytis cinerea, the causal agent of gray mold, is a necrotrophic fungus that can infect a wide variety of plant species and plant tissues. During infection, this pathogen modulates the pH of its environment by secreting organic acids or ammonia. Deletion of the gene encoding the pH-responsive transcription factor PacC revealed the importance of this regulator in different steps of the infection process and particularly in the secretion of organics acids, reactive oxygen species and plant cell wall degrading enzymes. This study aimed to identify the genes controlled by this fungus-specific transcription factor when the fungus is placed under acidic or neutral conditions. DATA DESCRIPTION: Botrytis cinerea B05.10 and the knock-out BcpacC mutant strains were grown on solid non-buffered medium for 3 days on the surface of cellophane membranes before transfer for 4 h onto the surface of liquid medium buffered at pH 5.0 or 7.0 followed by mycelium collection. After RNA sequencing, differentially expressed genes according to strains or pH conditions were listed. These data will be useful in understanding the adaptation process of B cinerea during plant infection.

Virulence plasmid with IroBCDN deletion promoted cross-regional transmission of ST11-KL64 carbapenem-resistant hypervirulent Klebsiella pneumoniae in central China.

BACKGROUND: Carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) caused infections of high mortality and brought a serious impact on public health. This study aims to evaluate the epidemiology, resistance and virulence characteristics of CR-hvKP and to identify potential drivers of cross-regional transmission in different regions of China, in order to provide a basis for developing targeted prevention measures. METHODS: Clinical K. pneumoniae strains were collected from Jiujiang and Nanchang in Jiangxi province between November 2021 to June 2022. Clinical data of patients (age, sex, source of infection, and diagnosis) were also gathered. We characterized these strains for their genetic relatedness using PFGE, antimicrobial and virulence plasmid structures using whole-genome sequencing, and toxicity using Galleria mellonella infection model. RESULTS: Among 609 strains, 45 (7.4%) CR-hvKP were identified, while the strains. isolated from Nanchang and Jiujiang accounted for 10.05% (36/358) and 3.59% (9/251). We observed that ST11-KL64 CR-hvKP had an overwhelming epidemic dominance in these two regions. Significant genetic diversity was identified among all ST11-KL64 CR-hvKP cross-regional transmission between Nanchang and Jiujiang and this diversity served as the primary driver of the dissemination of clonal groups. Virulence genes profile revealed that ST11-KL64 CR-hvKP might harbour incomplete pLVPK-like plasmids and primarily evolved from CRKP by acquiring the hypervirulence plasmid. We found the predominance of truncated-IncFIB/IncHI1B type virulence plasmids with a 25 kb fragment deletion that encoded iroBCDN clusters. CONCLUSION: ST11-KL64 is the most cross-regional prevalent type CR-hvKPs in Jiangxi province, which mainly evolved from CRKPs by acquiring a truncated-IncHI1B/IncFIB virulence plasmid with the deletion of iroBCDN. Stricter surveillance and control measures are urgently needed to prevent the epidemic transmission of ST11-KL64 CR-hvKP.

Cryptococcal invasion: a comprehensive case of adrenal and systemic infection amid immunosuppression.

We report a case of disseminated cryptococcosis, an uncommon fungal infection predominantly affecting the lungs and central nervous system, with the rare involvement of adrenal cryptococcosis, compounded by meningitis and pneumonia. The patient, previously diagnosed with primary myelofibrosis and undergoing oral Ruxolitinib treatment, exhibited immunosuppression. Imaging via chest and abdominal CT scans revealed inflammation in the right lung's middle lobe, splenomegaly, a splenic lesion, and a left adrenal mass, initially prompting considerations of pheochromocytoma. However, unilateral adrenalectomy and subsequent pathological examination disclosed extensive infiltration by inflammatory and multinucleate giant cells, with Periodic acid-Schiff (PAS) staining confirming the diagnosis. The identification of adrenal cryptococcosis was further supported by positive adrenal pus culture and significantly elevated capsular antigens in both serum and cerebrospinal fluid, at titers of 1:2560. Following a month of oral antifungal treatment, marked reductions in capsular antigen levels were noted, to 1:640 and 1:160 in serum and cerebrospinal fluid, respectively. The patient was discharged on a regimen of oral amphotericin B, flucytosine, and fluconazole, with regular outpatient follow-ups showing no signs of recurrence or dissemination.

The GhEB1C gene mediates resistance of cotton to Verticillium wilt.

MAIN CONCLUSION: The GhEB1C gene of the EB1 protein family functions as microtubule end-binding protein and may be involved in the regulation of microtubule-related pathways to enhance resistance to Verticillium wilt. The expression of GhEB1C is induced by SA, also contributing to Verticillium wilt resistance. Cotton, as a crucial cash and oil crop, faces a significant threat from Verticillium wilt, a soil-borne disease induced by Verticillium dahliae, severely impacting cotton growth and development. Investigating genes associated with resistance to Verticillium wilt is paramount. We identified and performed a phylogenetic analysis on members of the EB1 family associated with Verticillium wilt in this work. GhEB1C was discovered by transcriptome screening and was studied for its function in cotton defense against V. dahliae. The RT-qPCR analysis revealed significant expression of the GhEB1C gene in cotton leaves. Subsequent localization analysis using transient expression demonstrated cytoplasmic localization of GhEB1C. VIGS experiments indicated that silencing of the GhEB1C gene significantly increased susceptibility of cotton to V. dahliae. Comparative RNA-seq analysis showed that GhEB1C silenced plants exhibited altered microtubule-associated protein pathways and flavonogen-associated pathways, suggesting a role for GhEB1C in defense mechanisms. Overexpression of tobacco resulted in enhanced resistance to V. dahliae as compared to wild-type plants. Furthermore, our investigation into the relationship between the GhEB1C gene and plant disease resistance hormones salicylic axid (SA) and jasmonic acid (JA) revealed the involvement of GhEB1C in the regulation of the SA pathway. In conclusion, our findings demonstrate that GhEB1C plays a crucial role in conferring immunity to cotton against Verticillium wilt, providing valuable insights for further research on plant adaptability to pathogen invasion.

[The rs2564978(T) Allele Associated with Severe Influenza A Disrupts the Binding Site for Myeloid Differentiation Factor PU.1 and Reduces CD55/DAF Gene Promoter Activity in Macrophages].

The complement inhibitor CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity in influenza A infection and vascular complications in pathologies that involve excessive activation of the complement system. A luciferase reporter system was used to functionally analyze the single nucleotide polymorphism rs2564978 in the U937 human promonocytic cell line. The polymorphism is in the promoter of the CD55 gene, and its minor allele T is associated with a severe course of influenza A(H1N1)pdm09. A decreased activity of the CD55 promoter carrying the minor rs2564978(T) allele was observed in activated U937 cells, which provide a cell model of human macrophages. Using bioinformatics resources, PU.1 was identified as a potential transcription factor that may bind to the CD55 promoter at the rs2564978 site in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was verified by a PU.1 genetic knockdown with small interfering RNAs under specific monocyte activation conditions.

[The Oral Microbiome in the Development of Oral Cancer].

Oral cancer is an aggressive and rapidly progressive disease. The oral cavity is home to over 700 species of microorganisms that regulate metabolism, immune function, and health. There are three types of mechanisms by which bacteria may participate in carcinogenesis. First, bacteria cause chronic inflammation, which stimulates the production of cytokines, including interleukins, interferons, and tumor necrosis factor. Second, bacteria can interact directly with host cells by secreting toxins or by binding to membrane receptors. Finally, the production of metabolites by bacteria may also contribute to carcinogenesis. The importance of the bacteria level and composition in the transition of oral precancerous lesions to cancer has been demonstrated. The relationships of changes in microbiome composition with smoking, inflammation in healthy individuals, as well as with the development of oral cancer in patients, have been studied.

Harnessing RNA interference for the control of Fusarium species: A critical review.

Fusarium fungi are a pervasive threat to global agricultural productivity. They cause a spectrum of plant diseases that result in significant yield losses and threaten food safety by producing mycotoxins that are harmful to human and animal health. In recent years, the exploitation of the RNA interference (RNAi) mechanism has emerged as a promising avenue for the control of Fusarium-induced diseases, providing both a mechanistic understanding of Fusarium gene function and a potential strategy for environmentally sustainable disease management. However, despite significant progress in elucidating the presence and function of the RNAi pathway in different Fusarium species, a comprehensive understanding of its individual protein components and underlying silencing mechanisms remains elusive. Accordingly, while a considerable number of RNAi-based approaches to Fusarium control have been developed and many reports of RNAi applications in Fusarium control under laboratory conditions have been published, the applicability of this knowledge in agronomic settings remains an open question, and few convincing data on RNAi-based disease control under field conditions have been published. This review aims to consolidate the current knowledge on the role of RNAi in Fusarium disease control by evaluating current research and highlighting important avenues for future investigation.

Functional characterization of extracellular and intracellular catalase-peroxidases involved in virulence of the fungal wheat pathogen Zymoseptoria tritici.

Understanding how pathogens defend themselves against host defence mechanisms, such as hydrogen peroxide (H2O2) production, is crucial for comprehending fungal infections. H2O2 poses a significant threat to invading fungi due to its potent oxidizing properties. Our research focuses on the hemibiotrophic fungal wheat pathogen Zymoseptoria tritici, enabling us to investigate host-pathogen interactions. We examined two catalase-peroxidase (CP) genes, ZtCpx1 and ZtCpx2, to elucidate how Z. tritici deals with host-generated H2O2 during infection. Our analysis revealed that ZtCpx1 was up-regulated during biotrophic growth and asexual spore formation in vitro, while ZtCpx2 showed increased expression during the transition from biotrophic to necrotrophic growth and in-vitro vegetative growth. Deleting ZtCpx1 increased the mutant's sensitivity to exogenously added H2O2 and significantly reduced virulence, as evidenced by decreased Septoria tritici blotch symptom severity and fungal biomass production. Reintroducing the wild-type ZtCpx1 allele with its native promoter into the mutant strain restored the observed phenotypes. While ZtCpx2 was not essential for full virulence, the ZtCpx2 mutants exhibited reduced fungal biomass development during the transition from biotrophic to necrotrophic growth. Moreover, both CP genes act synergistically, as the double knock-out mutant displayed a more pronounced reduced virulence compared to ΔZtCpx1. Microscopic analysis using fluorescent proteins revealed that ZtCpx1 was localized in the peroxisome, indicating its potential role in managing host-generated reactive oxygen species during infection. In conclusion, our research sheds light on the crucial roles of CP genes ZtCpx1 and ZtCpx2 in the defence mechanism of Z. tritici against host-generated hydrogen peroxide.

Physiological and molecular responses of a resistant and susceptible wheat cultivar to the fungal wheat pathogen Zymoseptoria tritici.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), one of the most economically destructive wheat foliar diseases. In this study, we explore the physiological and molecular changes elicited in two wheat cultivars with divergent responses (Taichung 29 = susceptible, and Shafir = resistant) upon infection by Z. tritici. Our aim is to uncover novel insights into the intricate mechanisms that govern wheat defense against Z. tritici infection. Our quantitative histopathological study showed that H2O2 accumulated in the resistant cultivar to a higher degree compared to the susceptible cultivar at the biotrophic and switching phase. Additionally, we combined qPCR with a targeted quantitative HPLC technique to evaluate the expression profiles of 13 defense-related genes and profile the polyphenolic compounds induced differentially in the STB susceptible and resistant cultivar. Our finding indicated that five out of 13 genes were strongly up-regulated in the resistant cultivar compared with that of the susceptible one at eight days post-inoculation (dpi), corresponding to the transition phase present in the infection process of Z. tritici. Finally, our targeted HPLC analysis demonstrated that the traced phenolic compounds were highly elevated in the susceptible cultivar infected by Z. tritici compared with that of the resistant cultivar. In conclusion, our comprehensive analysis unveils a robust defense response in the resistant wheat cultivar Shafir, characterized by heightened H2O2 accumulation, significant up-regulation of key defense-related genes during the transition phase, and a distinct profile of polyphenolic compounds, shedding light on the intricate mechanisms contributing to its resistance against Z. tritici, thereby providing valuable insights for the development of more resilient wheat varieties.

Unveiling the burden of scrub typhus in acute febrile illness cases across India: A systematic review & meta-analysis.

Background & objectives Scrub typhus is an emerging mite-borne zoonotic infection that has been overlooked, despite being one of the most widespread severe vector-borne diseases. With an estimated one billion people at risk worldwide and one million annual cases, it poses a significant public health concern. While various studies have investigated the prevalence of scrub typhus in different regions of India, a comprehensive regional systematic review and meta-analysis on the seropositivity of scrub typhus among acute febrile cases has been lacking. To address this gap, we conducted a systematic review and meta-analysis to compile information on the current seroprevalence of scrub typhus in acute febrile illness cases in India. Methods A literature search of multiple databases on prevalence of scrub typhus in acute febrile illness in India, 60 eligible studies out of 573 studies. The prevalence of individual studies was double arcsine transformed, and the pooled prevalence was calculated using inverse variance method. Results In total, these studies encompassed 34,492 febrile cases. The overall seroprevalence of scrub typhus among acute febrile illness cases in India was found to be 26.41 per cent [95% confidence interval (CI): 22.03-31.03]. Additionally, the pooled case fatality rate (based on data from six studies) among scrub typhus-positive cases yielded a case fatality rate of 7.69 per cent (95% CI: 4.37-11.72). Interpretation & conclusions This meta-analysis shows that scrub typhus is a significant health threat in India. Preventive measures to control scrub typhus need to be given priority.

EpiCandIn: An open online resource for epidemiology of Candida infections in India.

Background & objectives Candida spp. cause candidiasis in humans under conditions disrupting the host defence. While Candida albicans is the most reported cause of candidiasis, there is a surge in the incidence of infections by non-albicans Candida species (NACs), such as C. tropicalis, C. glabrata and C. auris. These species can infect all organs of the human body. To effectively manage these outbreaks, it is important to track the epidemiology of candidiasis. A consolidated resource describing the landscape of candidiasis in India is absent. Methods To address this gap, we have developed an online resource named Epidemiology of Candida Infections in India (EpiCandIn) by manually curating published literature on Candida infections in the Indian population obtained from PubMed and ScienceDirect databases. Results EpiCandIn contains data available since 1972 from 51 sites across 16 States and four Union Territories of India. It provides information on geographical location, Candida species, niche affected, disease characteristics and drug therapy details extracted from the publications. This resource is integrated with visualization tools. Interpretation & conclusions EpiCandIn will be useful for public health researchers and policymakers as it will help them gain insights into the emerging trends and management of Candida infections in India. It can be accessed at epicandin.bicnirrh.res.in.

Phylo-geo haplotype network-based characterization of SARS-CoV-2 strains circulating in India (2020-2022).

Background & objectives Genetic analysis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) strains circulating in India during 2020-2022 was carried out to understand the evolution of potentially expanding and divergent clades. Methods SARS-CoV-2 sequences (n=612) randomly selected from among the sequences of samples collected through a nationwide network of Virus Research Diagnostic Laboratories during 2020 (n=1532) and Indian sequences available in Global Initiative on Sharing All Influenza Data during March 2020-March 2022 (n=53077), were analyzed using the phylo-geo haplotype network approach with reference to the Wuhan prototype sequence. Results On haplotype analysis, 420 haplotypes were revealed from 643 segregating sites among the sequences. Haplotype sharing was noted among the strains from different geographical regions. Nevertheless, the genetic distance among the viral haplotypes from different clades could differentiate the strains into distinct haplo groups regarding variant emergence. Interpretation & conclusions The haplotype analysis revealed that the G and GR clades were co-evolved and an epicentrefor the evolution of the GH, GK and GRA clades. GH was more frequently identified in northern parts of India than in other parts, whereas GK was detected less in north India than in other parts. Thus, the network analysis facilitated a detailed illustration of the pathways of evolution and circulation of SARS-CoV-2 variants.

Incidence, antimicrobial susceptibility & out of pocket expenditure of severe enteric fever in Chandigarh, north India.

Background & objectives Burden estimates of enteric fever are required to make policy decisions on introducing typhoid vaccine in India. Incidence, antimicrobial susceptibility, and out-of-pocket expenditure (OOPE) of enteric fever are estimated in Chandigarh, India. Methods A hybrid (facility and community-based) surveillance system was set up at a secondary care hospital to enrol patients above six months of age, hospitalized with fever, from a defined catchment population from May 2018 to March 2020. Blood samples were collected and cultured using an automated system (BD BACTECTM blood culture system). The Salmonella Typhi and S. Paratyphi isolates were characterized for antimicrobial susceptibility. OOPE was recorded after 14 and 28 days of discharge. Results Blood samples were collected from 97 per cent of the 1650 study participants enrolled. The incidence of enteric fever was 226.8 per 1,00,000 person-years (PY), severe typhoid fever 156.9 per 1,00,000 PY, and severe paratyphoid fever 69.9 per 1,00,000 PY. Salmonella was highly susceptible to ampicillin, azithromycin, and ceftriaxone (99.25%) and least susceptible to ciprofloxacin (11.3%). The OOPE due to hospitalization of individuals infected with S. Paratyphi [INR 8696.6 (USD 116)] was significantly higher than the individuals infected with S. Typhi [INR 7309 (USD 97.5), P=0.01], and among cases who were hospitalized for more than seven days [INR 12,251 (USD 163.3)] as compared with those with a stay of 3-7 days [INR 8038.2 (USD 107.2)] or less than three days [INR 5327.8 (USD 71), P<0.001]. Interpretation & conclusions There was a high incidence of enteric fever, high OOPE, and resistance to ciprofloxacin.

COVID-19 pandemic & neurosciences in India- the CoINstudy: Impact of the pandemic on research related to the neurosciences.

Background & objectives We aimed to assess the impact of COVID-19-related disruptions on ongoing and future projects related to neuroscience research and young researchers in India. Methods We conducted a countrywide online survey using a structured, self-administered questionnaire involving medical trainees, post-doctoral fellows, PhD students, early career faculty members and basic neuroscience researchers. The purpose was to assess the impact of the COVID-19 pandemic on the respondents' ongoing/planned research activities and capture their concerns related to future research. Results Five hundred and four valid responses were analyzed. More than three-fourths of the respondents were in their early careers - 64.1 per cent were resident doctors, and 19.8 per cent were early career consultants. Maximum responses were received from respondents working in neurology (228; 45.2%), followed by psychiatry (192; 38.1%) and neurosurgery (49; 9.7%). More than three-fourths [83.5%, 95% confidence interval (CI): 0.8-0.867] of the respondents reported that the pandemic had affected their research. About one-third of the respondents (171; 33.9%) reported delays in completing research studies. Respondents adapted to the pandemic's circumstances by making methodological changes in their research (155; 30.8%). Most respondents (301; 59.6%) reported being diverted from their traditional work settings to COVID-19-related clinical services. Respondents conducting prospective studies and randomized controlled trials and those diverted to COVID-related services were significantly more likely to report the adverse research impact. Interpretation & conclusions In our survey, an overwhelming majority of the respondents reported that the pandemic adversely impacted their study. This trend was independent of sex, designation, and research output of individual subjects. The serious impact of the COVID-19 pandemic on neurosciences research warrants the attention and concerted efforts of the research supervisors, institutional heads, funding agencies and other stakeholders.

Characterization of blaOXA-232 carrying carbapenem-resistant Klebsiella pneumoniae (CRKP) & their expression profiles under selective carbapenem pressure: An in-depth study from India.

Background & objectives OXA-232 is a five amino acid substitution variant of OXA-48 and is reported in carbapenem-resistant Klebsiella pneumoniae (CRKP), which is associated with nosocomial infections among immunocompromised patients in the intensive care unit. This study aimed to characterise blaOXA-232 in CRKP of clinical origin and investigate its transcriptional response against sub-inhibitory levels of carbapenems. Methods CRKP was isolated from blood (pathogens) and stool cultures (colonisers) of neonates and was characterized for blaOXA-232. Co-existing resistance determinants were investigated in blaOXA-232 positive isolates, followed by horizontal gene transferability assay and PCR-based replicon typing (PBRT). Cloning of blaOXA-232 was performed, and expression of blaOXA-232 in the isolates and their clones under sub-inhibitory concentrations of carbapenems was checked via RT-PCR. Mobile genetic elements associated with blaOXA-232 were investigated, followed by DNA fingerprinting through enterobacterial repetitive intergenic consensus (ERIC) PCR. Results blaOXA-232 with co-carriage of extended-spectrum beta-lactamases (ESBLs), sulphonamides and quinolones were identified in seven CRPK isolates recovered from blood samples of neonates. Transformation and cloning of blaOXA-232 was successful. The sub-inhibitory concentration of carbapenems induces elevated expression of this resistant determinant. ISEcp1 was associated with blaOXA-232 in the upstream region within two haplotypes of CRKP isolates of clinical origin. Interpretation & conclusions Selective carbapenem pressure resulted in higher expression of this gene, which could account for treatment failure. With frequent reports of occurrence among clinical isolates, monitoring and further investigation of this novel variant are necessary to understand its transmission dynamics and to thwart its further dissemination.