National Culture Collection Research Articles

Characterization of a Small Plaque Variant Derived from Genotype V Japanese Encephalitis Virus Clinical Isolate K15P38.

Genotype V (GV) Japanese encephalitis virus (JEV) has been predominantly reported in the Republic of Korea (ROK) since 2010. GV JEV exhibits higher virulence and distinct antigenicity compared to other genotypes, which results in reduced efficacy of existing vaccines. Research on GV JEV is essential to minimize its clinical impact, but the only available clinical strain in the ROK is K15P38, isolated from the cerebrospinal fluid of a patient in 2015. We obtained this virus from National Culture Collection for Pathogens (NCCP) and isolated a variant forming small plaques during our research. We identified that this variant has one amino acid substitution each in the PrM and NS5 proteins compared to the reported K15P38. Additionally, we confirmed that this virus exhibits delayed propagation in vitro and an attenuated phenotype in mice. The isolation of this variant is a critical reference for researchers intending to study K15P38 obtained from NCCP, and the mutations in the small plaque-forming virus are expected to be useful for studying the pathology of GV JEV.

Clinical spectrum, phenotypic and molecular characterization, and antifungal susceptibility of an emerging human pathogen, Acrophialophora, from India.

Acrophialophora is implicated in superficial and invasive infections, especially in immunosuppressed individuals. The present study was undertaken to provide clinical, microbiological, phylogenetic, and antifungal susceptibility testing (AFST) profile of Acrophialophora isolated from India. All the isolates identified as Acrophialophora species at the National Culture Collection for Pathogenic Fungi, Chandigarh, India were revived. Phenotypic and molecular characterization was performed, followed by temperature studies, scanning electron microscopy (SEM), and AFST. We also performed systematic review of all the cases of Acrophialophora species reported till date. A total of nine isolates identified as Acrophialophora species were identified by molecular method as A. fusispora (n=8) and A. levis (n=1), from brain abscess (n=4), respiratory tract (n=3), and corneal scraping (n=2). All patients but two had predisposing factors/co-morbidities. Acrophialophora was identified as mere colonizer in one. Temperature studies and SEM divulged variation between both species. Sequencing of the internal transcribed spacer ribosomal DNA and beta-tubulin loci could distinguish species, while the LSU ribosomal DNA locus could not. AFST showed the lowest minimum inhibitory concentrations (MICs) for triazoles and the highest for echinocandins. Systematic literature review revealed 16 cases (11 studies), with ocular infections, pulmonary and central nervous system infections, and A. fusispora was common species. All the patients except three responded well. High MICs were noted for fluconazole, micafungin, and caspofungin. This is the first study delineating clinical, phenotypic, and genotypic characteristics of Acrophialophora species from India. The study highlights microscopic differences between both species and emphasizes the role of molecular methods in precise identification. Triazoles appear to be the most effective antifungals for managing patients.

An Investigation into the Microbiological Quality of Groundwater in an Industrial Hub in Mid-Hills of Northern India

Frequent water-borne disease outbreaks affecting human health have been reported in district Solan, which is a rapidly industrialing region of Himachal Pradesh, a northern state of India. For ascertaining the cause, microbiological assessment of drinking groundwater sources of the region was undertaken. For this, thermotolerant coliform study and genome typing were undertaken. Multiple tube fermentation technique was employed in selected five sources to detect thermotolerant coliforms during the seasons of monsoon and post-monsoon in the years 2018-19. Thermotolerant coliforms varied in Most Probable Number from 2-34 per 100 ml water indicating contaminated water sources, densities being significantly more in monsoon months (p< 0.05). Four different coliform morphotypes were obtained from all the sources on Eosin Methylene Blue plates. Of these, the only strain, designated as Nalagarh Water Strain (NGW), was recovered from all sources, suggesting its prevalence. Based upon biochemical and morphological attributes, the strain was recognized as Escherichia coli. Conversely, molecular characterization (16S rRNA ribotyping) identified it as Raoultella planticola (GenBank accession No. MK318824). The strain was deposited in a National Culture Collection Centre, National Centre for Microbial Resources, Pune, Maharashtra (India) with deposition accession number MCC 4064 for future utility in public research activities. Microbiological assessment evinced contaminated drinking water sources, probably causing diarrhoeal diseases. 16S rRNA ribotyping assisted in correct identification of the microorganism.

Plant growth-promoting rhizobacteria and biochar as bioeffectors and bioalleviators of drought stress in faba bean (Vicia faba L.).

Plants are subjected to a variety of abiotic stressors, including drought stress, that are fatal to their growth and ability to produce under natural conditions. Therefore, the present study was intended to investigate the drought tolerance potential of faba bean (Vicia faba L.) plants under the co-application of biochar and rhizobacteria, Cellulomonas pakistanensis (National Culture Collection of Pakistan (NCCP)11) and Sphingobacterium pakistanensis (NCCP246). The experiment was initiated by sowing the inoculated seeds with the aforementioned rhizobacterial strains in earthen pots filled with 3kg of sand-mixed soil and 5% biochar. The morphology of biochar was observed with highly porous nature, along with the detection of various essential elements. The biochemical and physiological data showed that phenolic compounds and osmolytes were adversely affected by the induction of drought stress. However, the application of biochar and rhizobacteria boosted the level of flavonoids on average by 52.03%, total phenols by 50.67%, soluble sugar by 82.85%, proline by 76.81%, glycine betaine by 107.25%, and total protein contents by 89.18% in all co-treatments of biochar and rhizobacteria. In addition, stress indicator compounds, including malondialdehyde (MDA) contents and H2O2, were remarkably alleviated by 54.21% and 47.03%, respectively. Similarly, the amplitude of antioxidant enzymes including catalase, peroxidase, superoxide dismutase, ascorbate peroxidase, and guaiacol peroxidase was also enhanced by 63.80%, 80.95%, 37.87%, and 58.20%, respectively, in all co-treatments of rhizobacteria and biochar. Conclusively, biochar and rhizobacteria have a magnificent role in enhancing the drought tolerance potential of crop plants by boosting the physio-biochemical traits and enhancing the level of antioxidant enzymes.

First Report of Plasmid-Mediated Macrolide-Clindamycin-Tetracycline Resistance in a High Virulent Isolate of Cutibacterium acnes ST115.

Cutibacterium acnes, a prevalent skin commensal, has emerged as a significant global challenge due to its widespread antibiotic resistance. To investigate the antibiotic resistance mechanisms and clinical characterization of C. acnes in Korea, we collected 22 clinical isolates from diverse patient specimens obtained from the National Culture Collection for Pathogens across Korea. Among the isolates, KB112 isolate was subjected to whole genome sequencing due to high resistance against clindamycin, erythromycin, tetracycline, doxycycline, and minocycline. The whole genome analysis of KB112 isolate revealed a circular chromosome of 2,534,481 base pair with an average G + C content of 60.2% with sequence type (ST) 115, harboring the potential virulent CAMP factor pore-forming toxin 2 (CAMP2), the multidrug resistance ABC transporter ATP-binding protein YknY, and the multidrug efflux protein YfmO. The genomic sequence also showed the existence of a plasmid (30,947 bp) containing the erm(50) and tet(W) gene, which confer resistance to macrolide-clindamycin and tetracycline, respectively. This study reports plasmid-mediated multi-drug resistance of C. acnes for the first time in Korea.

Founding of the culture collection of antibiotic-resistant strains of zoonotic bacteria in the Russian Federation.

The main purpose of a national bioresource center is to standardize, centralize, preserve, and ensure accessibility of microbial bioresources that accumulate there because of state research programs. The establishment of national bioresource centers for antibiotic-resistant microorganisms allows to solve practical problems in the field of veterinary service, as well as to develop effective chemotherapeutic and disinfectant drugs to overcome the mechanisms of resistance. This study aimed to outline the process of forming a national culture collection of antibiotic-resistant strains of zoonotic bacteria in the Russian Federation using two microbial strains. The object of research was isolates of Salmonella spp., Escherichia coli, Enterococcus spp., Campylobacter spp., Listeria monocytogenes, and Staphylococcus spp., all of which were obtained from biomaterials of farm animals, feed samples, bedding, water from livestock buildings, washouts from environmental objects, and food products. The resistance of bacterial isolates was determined using microbiological and molecular-genetic research methods. During monitoring studies, 1489 bacterial isolates were isolated. In total, 408 bacterial isolates were tested for sensitivity to antimicrobial agents, including E. coli (47.6%), Salmonella spp. (30.4%), Enterococcus spp. (11.3%), and Campylobacter spp. (10.8%). For genetic characterization, 95 isolates of Salmonella enterica, E. coli, Campylobacter spp., L. monocytogenes, Staphylococcus spp., Enterococcus spp. were chosen from the research collection, which was formed as part of the monitoring program for antibiotic resistance. Deposited isolates that underwent whole-genome analysis can be used as positive control samples both in the development and use of methods or test systems for the detection of various resistance genes in zoonotic bacteria. In addition, such isolates can also be used for microbiological studies related to determining the sensitivity of microorganisms to antibacterial drugs, for phenotypic studies in the diagnosis of various bacterial infections in animals and birds, and retrospective analysis of strains from numerous collections.

P425 Approach to identify and isolate C. dubliniensis in laboratory and comparison of various methods for same

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMIntroduction: C. dubliniensis, a newly recognized fungal pathogen, is clinically significant as it develops a stable fluconazole-resistant phenotype on exposure to this antifungal in vitro. C. dubliniensis is overlooked and misidentified as it shares many phenotypic traits with C. albicans. Molecular methods developed to distinguish the two species are not readily applicable. So, an easy-to-perform and an accurate phenotypic test would be a valuable tool.Aim: This study first of its kind from Kashmir Valley (conducted in SKIMS-1200 bedded tertiary care hospital) evaluated seven phenotypic tests with PCR-RFLP as a gold standard. The best methodology for routine identification of this emerging pathogen was developed.Materials: A total of 206 Candida spp. were tested which included 186 in-house C. albicans strains and 20 C. dubliniensis strains. The in-house 186 C. albicans strains were isolated from cancer patients. A total of 500 patients with various malignancies were screened, patients on antifungal drugs and screened once were excluded. All strains were sensitive to both fluconazole and voriconazole. Among 22°C dubliniensis strains, 14 were provided by Dr. Ziauddin Khan (Professor and Chairman, Department of Microbiology, Kuwait University) and 6 by VPCI institute Delhi.Reference strains included in the study: C. albicans 90028; (National culture collection of pathogenic fungi (NCCPF), Department of Medical Microbiology PGIMER Chandigarh) C. dubliniensis (CD36) and (CBS 7987); provided by Dr. Ziauddin KhanMethodsPCR-RFLP using BlnI (AvrII) enzyme was done to confirm the identification of all isolates (Fig. 1). Seven phenotypic tests that were evaluated include Growth on hypertonic Sabouraud Dextrose Agar (SDA), Colony color on Crome Candida Differential Agar, Growth at 45°C, Assimilation of xylose (XYL), Colony color and Chlamydospore formation on Tobacco Agar, Germ tube formation at 39°C and Fluorescence on methyl blue SDA. The variety of tests applied makes our study one of the most detailed studies for the identification of C. dubliniensis till date.ResultsNo C. dubliniensis was found among the isolates recovered from cancer patients by PCR-RFLP.Phenotypic tests that showed the best results wereGrowth on Hypertonic SDAColony color on Crome Candida Differential AgarRelative growth at 45°C.Assimilation of xylose (XAM as well as DISCs)Germ tube formation at 39°C was most rapid. Phenotypic methods proved to be very cost-effective than PCR-RFLP. Based on our results of growth on Hypertonic SDA and XAM and also knowing the fact that 5% of C. dubliniensis assimilate xylose and give false results of xylose assimilation, we developed a novel medium ‘Hypertonic xylose agar medium’ (HXAM). HXAM proved to be 100% accurate, simple, easy to perform, and cost-effective.ConclusionFor routine identification of C. dubliniensis we recommend colony color on Crome Agar and growth on HXAM as they are simple, reliable, inexpensive, reproducible, and readily applicable methods. For retrospective evaluation of stored cultures, we recommend HXAM.Based on our observations, we propose a scheme for the identification of C. dubliniensis (Fig. 2).

P281 Healthy adult with arm swelling and new onset burning pain

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMIntroductionCladosporium cladosporioides is a dematiaceous fungal agent that is known to produce a variety of clinical manifestations such as cutaneous and subcutaneous infection, corneal infection, pulmonary fungal infection, and rarely central nervous system infection. Several cases of subcutaneous mycosis have been reported from Assam, India. We are describing a similar case from Gujarat, India.Case HistoryA 36-year-old healthy, and muscular male farmer residing in central Gujarat presented with painless nodular swelling with normal overlying skin over the posterior aspect of the left arm for the last 3-4 years. He noticed a gradual increase in the size of this swelling and numbness for the last 4 months and burning sensations for the last 10 days before presentation to the surgeon. Local part ultrasound showed a subcutaneous soft tissue lesion. Excision biopsy reported invasive fungal infection and patient was referred for further evaluation to an infectious disease facility (Fig. 1a). He had no other symptoms and comorbidities. Patient didn't recall any trauma in the past. Direct microscopic examination with calcofluor white stain from surgical site scrapping revealed mycelial structure (Fig. 1b) and yielded pure growth of a dematiaceous fungus on Sabouraud dextrose agar medium, brown to blackish colony (Fig. 1c) with olive color on the backside (Fig. 1d) after 6 days. Lactophenol cotton blue stain preparation of culture isolate showed mycelial elements with morphology suggestive of C. cladosporioides (Fig. 1e), which was confirmed at the National Culture Collection for Pathogenic Fungi, PGI Chandigarh. Review of histopathology revealed a nodule with dense acute on chronic inflammation composed of lymphocytes, plasma cells with many neutrophils with abscess containing brightly eosinophilic structures with many PAS positive septate, thin and thick walled, branching, irregularly shaped, bullous hyphae surrounded by neutrophilic boarder (Splendor Hoeppli phenomenon) (Fig. 1f).Patient was treated with capsule itraconazole 200 mg three times a day for three days followed by 200 mg twice a day along with terbinafine and total surgical excision of the subcutaneous nodule. Patient achieved a therapeutic itraconazole level (1.4 mg/l) after 1 week. Patient is currently receiving treatment.DiscussionSubcutaneous mycosis is endemic in Assam. Several cases of subcutaneous chromoblastomycosis caused by C. cladosporidiosis mainly involving the lower limbs were reported from Assam. It mostly affects males engaged actively in outdoor activities during their productive age ranged from 20 to 50 years. It affects a relatively healthy, immunocompetent host and have a long history (in years) of asymptomatic nodule/swelling before diagnosis. Treatment comprises of prolonged antifungal treatment with itraconazole along with surgical excision.

P206 Phenotypic and molecular characterization of emerging Basidiomycete Schizophyllum commune isolated from clinical samples from India

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjective Schizophyllum commune is an environmental basidiomycete capable of causing human infections. Its identification is difficult as often it produces sterile, cottony white colonies without any conidia formation. S. commune is characterized by clamp connections and hyphal spicules. Though it produces basidiocarps with basidiospores it is difficult to induce in vitro. Molecular techniques are essential to confirm its identification. Here, we present the description of Schizophyllum commune isolates collected at our center over the last 5 years.MethodsAll the isolates used in this study was received from various part of India and accessioned at the National culture collection of pathogenic fungi (NCCPF), Chandigarh India. Isolates were grown on Sabouraud dextrose agar and malt-extract agar (MEA) at 25°C for 5-7 days. Lacto phenol cotton blue mounts were prepared and a microscopic examination was done. For observing basidiocarps, MEA plates were incubated for 6-8 weeks. DNA extraction was done using the phenol-chloroform method and ITS region was amplified using pan-fungal primers followed by Sanger sequencing.ResultsAmong total of 22 Schizophyllum commune cultures, 19 were isolated from lower respiratory samples, two from corneal scrapings, and one from the brain. Upon microscopic examination, the major identification feature of this fungi, i.e., clamp connection and spicules were observed only in 8 (36%) isolates. The remaining 14 (64%) were identified up to species level using ITS sequencing. Basidiocarps could be induced only in two isolates.ConclusionAlthough rarely involved in human disease, Schizophyllum commune is being isolated from the clinical specimens. As microscopic identification is difficult and needs expertise, molecular identification is required for early diagnosis and treatment.

P223 First case of Candida auris candidemia in Manipur, Northeast India

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjective Candida auris is known as an emerging ‘superbug’ because of its intrinsic resistance to one or more, sometimes to all available antifungal drugs and spreading globally. It has the ability to cause devastating nosocomial infections. In India, C. auris infection is on the rise with reports from north, south, central and eastern India. Here we present the first case of C. auris fungemia from a tertiary care hospital of Manipur in Northeast India.MethodsA 15-year-old Muslim girl was referred from a private hospital to Regional Institute of Medical Sciences (RIMS) hospital on November 19, 2021 with a history of burning epigastrium, headache, loss of appetite, shortness of breath, dry cough, fever, and generalized weakness for last 3 days. At the time of admission she was cyanotic. Family gave history of congenital heart disease and frequent visits to hospital. Echocardiogram revealed congenital cyanotic heart disease (Tetralogy of Fallot) showing large perimembranous VSD with bidirectional shunt. A complete hemogram showed neutrophilic leukocytosis with shift to left with band form, absolute monocytosis, and increased RBC count with mild anisocytosis. On November 24, 2021, 5 days after admission, her condition deteriorated and she was shifted to ICU. However, the condition of the patient deteriorated and she died on November 29, 2021 due to acute decompensated heart failure. Follow-up of other patients admitted in the same ward revealed no candidemia in next the few weeks.ResultsA single blood culture sent on November 29, 2021 was incubated in an automated blood culture system, BacT Alert and showed growth of budding yeast cells. Growth in SDA revealed it to be Candida sps. and Gram-stained smear examination revealed presence of budding yeast cells but no pseudohyphae. Germ tube test was negative. On CHROM agar, it produces pale yellow colonies at 24 h which progresses to light purple colonies around the rim at 48 h. Further processing in VITEK 2 (Biomerieux) identified it as C. auris. The isolate was sent to National Culture Collection of Pathogenic Fungi, WHO collaborating center, PGIMER and the isolate was confirmed as Candida auris by MALDI-TOF assay.Conclusion Candida auris is spreading irrespective of the level of health care. Blood culture before administration of antibiotics and in febrile sick patients cannot be underestimated. Rapid and accurate identification methods for timely diagnosis and stringent infection control measures with an emphasis on hand hygiene are important to prevent and control C. auris outbreaks.

P248 Fungal meningitis in an immunocompetent individual

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM ObjectiveFungal meningitis is commonly seen in individuals who are immunocompromised. It's a significant cause of morbidity and mortality in people living with HIV worldwide. Among HIV-infected patients of Manipur, it is the most common cause of meningitis and Cryptococcus neoformans is the only species identified so far both in HIV infected population and non-HIV patients in Manipur. The aim of this paper is to highlight the detection of C. gattii meningitis in the high HIV prevalence state of India.MethodA 53-year-old male, mechanic by profession residing in a village in Thoubal district of Manipur attended Medicine OPD with complaints of intractable headache, aggravated by sound and light, generalized weakness, hiccups, vomiting, and decreased appetite for 2 weeks. There was no history of altered sensorium. He is a known case of hypertension and was recently diagnosed with T2DM, not on medication.ResultsAn MRI brain (Fig.1) showed a tiny sub-centric lesion in the bilateral centrum semiovale. Cerebrospinal fluid examination by India Ink preparation revealed capsulated budding yeast cells suggestive of Cryptococcus sps. Cryptococcal antigen testing (CryptoPS, Biosynex) was positive with a high titer. Culture on SDA and BSA showed growth of Cryptococcus sps. For phenotypic identification and antifungal susceptibility testing, the isolate was processed in VITEK 2(Biomerieux) system which identified it as C. gatti (94% probability). The isolate was sensitive to amphotericin B (mic: 0.5 μg/ml) and flucytosine (mic: 2 μg/ml). He tested negative for HIV on two occasions and his CD4 + count was 888 cell/mm3. Blood sugar was 225 mg/dl, with urine glucose leakage in traces. Blood and urine were collected for fungal culture which showed no growth. The patient was put on liposomal amphotericin B 200 mg daily. After 1 week, patient's condition was not deteriorating, although the improvement was minimal. The isolate was sent to the National Culture Collection of Pathogenic Fungi, PGIMER, Chandigarh, and it was confirmed as Cryptococcus gattii by MALDI-TOF assay.ConclusionDetection of C. gattii in an immunocompetent patient in Manipur state, with a high prevalence of HIV is alarming because cryptococcal meningitis is the commonest form of meningitis in this population. In future, whenever Cryptococcus sps is isolated, it will be pertinent to keep in mind the possibility of other species of Cryptococcus to be the causative pathogen. We emphasize the importance and need for surveillance to detect its environmental niche.

P213 Lasiodiplodia theobromae: an emerging human pathogen

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM Objectives Lasiodiplodia theobromae is a dematiaceous fungus, rarely reported to cause infections in humans. This case-series was conducted to identify the potential risk-factors and spectrum of diseases caused by L. theobromae.MethodsWe performed a retrospective analysis of all cases from which the isolates of the L. theobromae were accessioned at the National culture collection of pathogenic fungi (NCCPF), over the last 10 years (January 2012- March 2022). The isolates were retrieved and identified by conventional (colony morphology, color, and microscopic appearances) and molecular (sequencing of ITS1 region of ribosomal DNA) methods. In vitro antifungal susceptibility testing (AFST) was performed by microbroth dilution recommended by the Clinical and Laboratory Standards Institute (CLSI). (M38-A2). The clinical features, demographic details and outcome were recorded.ResultsIn 10 years, a total of 20 patients were identified. The median age of all cases was 39.5 (range: 13-71 years) and 75% were males. The most common clinical presentation was keratomycosis (12,68.6%), followed by soft tissue/sub-cutaneous infection (3,15%), rhinosinusitis (3,15%), onychomycosis (1, 5%), and pneumonia (1, 5%). Most patients were immunocompetent (85%). Among immunocompromised, two patients had acute myeloid leukemia and developed fungal rhinosinusitis while one patient of post-renal transplant on immunosuppressants developed subcutaneous tissue infection in the interscapular region. The fourth patient had decompensated alcoholic hepatitis, encephalopathy, sepsis, and developed pneumonia while he was on mechanical ventilation. Among the various potential risk factors associated with keratomycosis, the most common were, accidental trauma wounds (9, 45%), and mosquito bite in the eye in one patient. The culture on Sabouraud's dextrose agar (SDA) revealed the growth of black mycelial fungus (Fig. 1a). The lactophenol cotton blue (LCB) mount revealed the presence of only dematiaceous septate hyphae which failed to sporulate (Fig. 1b). All the isolates were confirmed by sequencing of ITS (internal transcribed spacer) region of the rDNA using universal primers ITS1 and ITS2. In vitro antifungal susceptibility testing (AFST) was performed using a broth microdilution (BMD) method which revealed variable MIC (μg/ml), i.e., amphotericin (1-2), voriconazole (0.25-2), itraconazole (8-16), posaconazole (1-2). All patients improved on therapy except one patient who succumbed to death due to pneumonia.Conclusion Lasiodiplodia theobromae is an emerging cause of human infections in both immunocompetent and immunosuppressed individuals. It is often difficult to identify due to lack of sporulation making morphological identification challenging. Hence, prompt suspicion and rapid diagnosis with guided therapy are necessary for a better outcome.

P201 Medicopsis romeroi: an emerging cause of subcutaneous infections

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjective Medicopsis romeroi is one of the emerging dematiaceous fungi implicated in subcutaneous human infections. Despite advances in diagnostics, identification of this agent still remains delinquent owing to poor sporulation necessitating molecular modalities. Data on clinical and management profile of M. romeroi are available as case reports. In the present study, we describe an index case of M. romeroi infection and clinical risk factors and management profile of 74 cases of M. romeroi cases from India (n = 32) and those reported in the literature (n = 42) till date.MethodsA detailed history was obtained from the index patient after informed consent. Aspirated fluid was subjected to microbiological investigations. Identification of isolate was done by molecular technique using Sanger's sequencing. All isolates stored at the National culture collection of pathogenic fungi as M. romeroi were retrieved and identity confirmed by ITS sequencing. Demographic and management details were retrieved. We also conducted a systematic literature review of M. romeroi, as per PRISMA guidelines (Fig. 1).ResultsIndex case history: A 59-years-old diabetic female presented with lobulated swelling and sinuses on dorsum of the right hand for 10 months. A provisional diagnosis of mycetoma was made. Calcofluor-potassium hydroxide mount of aspiration fluid revealed dematiaceous septate hyphae and Sabouraud dextrose agar grew non-sporulating greyish black aerial mycelia after 3 days of incubation at 25°C and 37°C (Fig. 2). Molecular identification confirmed isolates as M. romeroi and patient was started on itraconazole with surgical excision. A total of 32 cases of M. romeroi infection from India were included. Mean age of patients was 47.2 years with male:female ratio of 1.3:1. Most common predisposing factors were post-renal transplant (46%) and farming (24%). All the patients presented with nodular or cystic swellings, with frequent involvement of lower limbs (56%). Most of the patients were managed using itraconazole (46%), followed by amphotericin B. All the patients except one responded well to treatment.Literature review: A total of 42 cases have been reported till date, of which 29% are from India. The mean age was 52.3 years, with male:female ratio of 1.4:1. Most common predisposing factors were post-renal transplant (28.5%) and farming/gardening (16.67%). The mean duration to infection in post-transplant cases was 3.26 years and the mean duration to diagnosis in all the cases was 31 months. The noteworthy finding was the absence of predisposing factors in 21.45% cases. A total of 62% presented with skin nodules on the foot, 21.5% on lower limbs, and 11.8% with ocular affliction. Identification was done using molecular modalities in 80% cases. A total of 34% cases were managed using both surgical excision and antifungals, whereas 21% were merely with surgical excision. Another remarkable finding was spontaneous resolution in 5% cases. Antifungals used include itraconazole (25%), followed by voriconazole (21%). MICs of all antifungals showed wide variation (0.25-8 μg/ml for AMB). All the patients except two responded well to treatment and 3 had residual disease.Conclusion Medicopsis romeroi is an emerging cause of subcutaneous infection in India. The present study underlines the significance of molecular tests in the identification of this dematiaceous fungus due to its poor sporulation, hindering the phenotypic characterization.

P407 Standardization of PCR for the diagnosis of invasive aspergillosis and invasive mucormycosis from blood samples

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectives(1) Standardization of PCR-based diagnosis of invasive aspergillosis and invasive mucormycosis in spiked blood samples. (2) Standardization of PCR-based diagnosis of invasive aspergillosis and invasive mucormycosis in proven cases. (3) Validation of the above-standardized protocol in patients with proven/probable invasive aspergillosis or mucormycosis.Materials and Methods: Blood samples were collected from healthy volunteers and patients with proven mucormycosis, proven invasive aspergillosis, and suspected invasive fungal infection. Standard strains of Mucorales and Aspergillus spp. were collected from the National Culture Collection of Pathogenic Fungi, PGIMER and DNA was extracted using a modified protocol of the fungal DNA extraction method by Lee and Taylor (1990). Mucor-specific primer (ZM13), described by Bialek R et al. (2005) was used for mucormycosis samples. Aspergillus specific primer pair was designed from mitochondrial DNA of A. fumigatus using Primer3 software. Genomic DNA was used to determine the optimum annealing temperature and concentration for ZM13 assay. DNA extracted from whole blood samples of healthy volunteers after spiking with a known quantity of A. fumigatus DNA and only genomic DNA was used separately to optimize annealing temperature and concentration of Aspergillus-specific primers. Similarly, whole blood, plasma, and serum samples from healthy volunteers were spiked with various concentrations of Rhizopus arrhizus and A. fumigatus DNA. DNA extracted from spiked samples was used to determine the analytical sensitivity of PCR assays. DNA extracted from blood samples of ten proven mucormycosis and five invasive aspergillosis patients were used to determine the optimum sample dilution factor and template volume for the assay. The final standardized protocols were validated using 28 proven/probable cases of invasive aspergillosis and invasive mucormycosis and thirty cases of suspected invasive fungal infection.ResultsThe optimum annealing temperature and primer concentrations for the assay were 54.3°C and 0.4μm respectively for ZM13 assay and 53°C and 0.45μm respectively for Aspergillus assay. The limit of detection was 0.06fg/μl for ZM13 assay and 0.08fg/μL for Aspergillus specific assay. For mucormycosis assay, the slope of the standard curve was −3.2687 and the percentage efficiency of the assay was 104% with 0.9936 as the coefficient of determination (R2). Likewise, the slope of the standard curve was −3.2695, the percentage efficiency of the assay 102%, and the coefficient of determination (R2) 0.9858 for aspergillosis assay. The best combination of template volume and sample dilution factor was 2.5 μl and 1000x in whole blood samples and, 2.5 μl and 100x in serum samples for mucormycosis assay. It was 1.0 μl and 10x in serum samples for Aspergillus specific assay. The sensitivity, specificity, positive predictive value, and negative predictive value of mucormycosis qPCR using serum samples were 79.3%, 86.4%, 65.7%, and 92.7% respectively. The assay had a positive likelihood ratio of 5.92 and a negative likelihood ratio of 0.24.ConclusionThe real-time PCR protocols standardized for the diagnosis of mucormycosis and invasive aspergillosis from blood samples showed promising results. However, the assay needs further validation in a larger study population.

P393 Clinical spectrum and molecular characterization of Rhytidhysteron spp. (now Gloniopsis) from India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjective Rhytidhysteron spp. are emerging dematiaceous fungi implicated in subcutaneous infections in immunocompromised as well as immunocompetent individuals. Diagnosis of Rhytidhysteron spp is delayed owing to non-sporulation and misidentification. The present study was conducted to characterize Rhytidhysteron spp. isolated from India by molecular techniques.MethodWe retrieved a total of eight isolates of Rhytidhysteron spp from our National Culture Collection for Pathogenic Fungi (NCCPF) that were received from various centers across India. Colony morphology on Sabouraud's dextrose agar (SDA) and potato dextrose agar (PDA) was recorded. Molecular identification was performed by sequencing internal transcribed spacer (ITS) and 28 s region of rDNA. Phylogenetic tree was constructed using the Neighbor-Joining method along with sequences of standard strains retrieved from the NCBI database.Results Rhytidhysteron spp was confirmed by sequencing from cutaneous specimens of eight cases. Six patients were immunocompetent while two were post-renal transplant recipients. Phenotypic features on SDA and PDA revealed growth of greyish-black mycelia on the obverse with black pigmentation on reverse after 7 days of incubation. On microscopic examination of lactophenol cotton blue mount, only dematiaceous septate hyphae without any spores were noted. On phylogenetic analysis of 28 s region, four of our isolates were closely clustered with Gloniopsis calami (Fig.1b), whereas two isolates with G. pneumonia and one each with G. percutanea, and Gloniopsis spp. (Fig.1b). On ITS phylogeny, the same four isolates closely clustered with G. calami (Fig.1a) as in 28 s region, one each isolate clustered with G. pneumonia, and G. percutanea while two isolates formed a separate clade with Rhytidhysteron rufulum (Fig.1a).ConclusionThis fungus is often difficult to identify due to lack of sporulation making morphological identification challenging. Therefore, molecular sequencing is a must for its identification. However, identification using the sequence of ITS and 28 s rDNA does not clearly correlate. Hence to confirm the identity additional genes needs to be sequenced and analyzed.

P441 Molecular identification, genotyping, and antifungal susceptibility of Trichosporon species isolated from clinical samples of patients at various parts of the Indian subcontinent

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectives(1) To study mycological characteristics of strains belonging to Trichosporon and its related genera obtained from clinical samples of patients from India. (2) Molecular identification by intergenic spacer (IGS) region 1 sequencing of the rDNA locus. (3) Genotyping of the major causative agent, T. asahii, and its in vitro drug susceptibility testing.Materials and Methods: A total of 55 clinical isolates of Trichosporon species were collected from NCCPF (National culture collection of pathogenic fungi) PGIMER, Chandigarh along with different health institutions of India. These isolates were recovered from urine, blood, sputum, nail, tissue biopsy, pleural fluid, hair, BAL, and wound discharge over a period of 12 years (2006–2018). The isolates were molecularly characterized and genotyped using IGS-1 region sequencing. In vitro drug susceptibility testing of the isolates was performed against amphotericin-B, fluconazole, itraconazole, voriconazole, and posaconazole according to the CLSI M27-A3 guidelines (CLSI 2008).ResultsPredominant underlying risk factors identified were presence of an indwelling catheter, use of broad-spectrum antibiotics, and presence of comorbid conditions such as diabetes, hypertension, and anemia. A total of 47 (85%) of the 55 isolates were identified as T. asahii, 6 were T. inkin (11%), and 2 were Cutaneo Trichosporon dermatis (3.6%). Trichosporon asahii genotype III (22; 41%) was the most common type, followed by genotype IV (12; 22%), I (8; 15%), and VII (2; 4%). In addition to the 15 known T. asahii genotypes, one novel genotype was identified in this study. Indian T. asahii isolates showed high MIC ranges to amphotericin B (0.06-4 μg/l) and fluconazole (0.25-64 μg/l). Relatively low MIC ranges were found in the case of voriconazole (0.03-1 μg/l), Posaconazole (0.06-1 μg/l), and itraconazole (0.06-1 μg/l). Voriconazole appeared to be the most active drug in maximum T. asahii isolates. The MICs for all the drugs were comparatively lower in the case of non-T. asahii strains.Conclusion Trichosporon asahii remains the most common etiology of Trichosporonosis in India and presents a challenge for both diagnosis and treatment. With increasing drug resistance, therapeutic options are limited, and antifungal regimens with triazoles especially voriconazole appear to be the best. Accurate timely identification, removal of indwelling catheters/central venous lines, and voriconazole-based treatment along with control of underlying conditions were associated with favorable outcomes. Identification of the novel genotype has epidemiological implications and requires further work up.

Characterization and validation of an alternative reference bacterium Korean Pharmacopoeia Staphylococcus aureus strain.

The National Culture Collection of Pathogens (NCCP) is a microbial resource bank in Korea that collects pathogen resources causing infectious disease in human and distributes them for research and education. The NCCP bank attempts to discover strains with various characteristics and specific purposes to provide diverse resources to researchers. Staphylococcus aureus American Type Culture Collection (ATCC) 6538P is used as a reference strain in the microbial assay for antibiotics in the Korean and in the United States Pharmacopoeias. We aimed to analyze domestically isolated microbial resources from the NCCP to replace the S. aureus reference strain. Staphylococcus aureus strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the VITEK-2 system and characterized by multilocus sequence typing, 16S rRNA sequencing, and antibiotic susceptibility testing. Several candidate strains had similar characteristics as the reference strain. Among them, the nucleotide sequence of the 16S rRNA region of NCCP 16830 was 100% identical to that of the reference strain; it was sensitive to six types of antibiotics and showed results most similar to the reference strain. A validity evaluation was conducted using the cylinder-plate method. NCCP 16830 presented valid results and had the same performance as ATCC 6538P; therefore, it was selected as an alternative candidate strain.

Genomic population structure associated with repeated escape of Salmonella enterica ATCC14028s from the laboratory into nature.

Salmonella enterica serovar Typhimurium strain ATCC14028s is commercially available from multiple national type culture collections, and has been widely used since 1960 for quality control of growth media and experiments on fitness ("laboratory evolution"). ATCC14028s has been implicated in multiple cross-contaminations in the laboratory, and has also caused multiple laboratory infections and one known attempt at bioterrorism. According to hierarchical clustering of 3002 core gene sequences, ATCC14028s belongs to HierCC cluster HC20_373 in which most internal branch lengths are only one to three SNPs long. Many natural Typhimurium isolates from humans, domesticated animals and the environment also belong to HC20_373, and their core genomes are almost indistinguishable from those of laboratory strains. These natural isolates have infected humans in Ireland and Taiwan for decades, and are common in the British Isles as well as the Americas. The isolation history of some of the natural isolates confirms the conclusion that they do not represent recent contamination by the laboratory strain, and 10% carry plasmids or bacteriophages which have been acquired in nature by HGT from unrelated bacteria. We propose that ATCC14028s has repeatedly escaped from the laboratory environment into nature via laboratory accidents or infections, but the escaped micro-lineages have only a limited life span. As a result, there is a genetic gap separating HC20_373 from its closest natural relatives due to a divergence between them in the late 19th century followed by repeated extinction events of escaped HC20_373.

Clinical Spectrum, Molecular Characterization, Antifungal Susceptibility Testing of Exophiala spp. From India and Description of a Novel Exophiala Species, E. arunalokei sp. nov.

Introduction Exophiala spp. are important opportunist pathogens causing subcutaneous or even fatal disseminated infections in otherwise both immunosuppressed and healthy individuals but there are no systematic studies on the isolates of Exophiala species from India.MethodsTwenty-four isolates of Exophiala species were retrieved from the National Culture Collection of Pathogenic Fungi (NCCPF) and identified phenotypically and by molecular methods (ITS region sequencing) followed by antifungal susceptibility testing (AFST) as per CLSI-M38A3 guidelines. A review of the literature of cases from India was performed up to 1st January 2021 using the Medline and Cochrane database.Results E. dermatitidis (n = 8), E. jeanselmei (n = 6), E. spinifera (n = 6), E. mesophila (n = 1), E. oligosperma (n = 1), E. xenobiotica (n = 1) were identified and the sequencing of ITS, β-tubulin and β-actin revealed a novel species, E. arunalokei sp. nov. (n = 1). The ITS sequence phylogram of E. jeanselmei revealed that the majority (83%) formed a separate cluster close to type A while majority (75%) of E. dermatitidis were type B. The MIC50 (mg/L) of amphotericin, itraconazole, voriconazole, micafungin, caspofungin, anidulafungin, and posaconazole, was 1, 0.25, 0.125, 0.12, 0.125, 0.062, and 0.062, respectively. Sixteen more cases were identified on the literature review and a significant association of E. dermatitidis with history of surgical procedures (p = 0.013), invasive disease (p = 0.032) and of E. mesophila with tuberculosis (p = 0.026) was seen.ConclusionThis, to the best of our knowledge is the first study from India elucidating the molecular and clinical characteristics of Exophiala species and the first Indian report of human infection due to E. xenobiotica and E. arunalokei.

Diversity and plant growth-promoting potential of actinomycetes associated with the rhizosphere of Arnebia euchroma from Himachal Pradesh (India)

Aim: The present study aimed to explicate the diversity and plant growth promoting (PGP) potential of actinomycetes present in the rhizosphere of an endangered medicinal plant, Arnebia euchroma. Methodology: Rhizospheric soil samples of A. euchroma collected from trans-Himalayan region of Himachal Pradesh were processed for elucidating actinomycetes diversity and load by employing Standard Plate Count Technique. All recovered isolates were screened for their PGP potential using standard protocols. The potential strains were identified through 16SrRNA ribotyping and were deposited in the National Culture Collection Centre, NCMR, Pune, (India). Results: The rhizosphere of A. euchroma harboured a great deal of actinomycetes diversity (33 diverse morphotypes). Starch casein agar was best for isolating the actinomycetes. The same actinomycete isolate showed variations in their morphological features including pigments production on different isolation media. They exhibited multifarious plant growth-promoting activities like P- solubilization, phytase activity, N- fixation, siderophore production etc. The potential isolates were identified as Streptomyces silaceus CA7 (MK836019) and Streptomyces rectiviolaceus NA8 (MK836018). Interpretation: The actinomycetes evinced a considerable plant growth promoting potential that might be helpful in the adaptation and perpetuation of A. euchroma under prevailing harsh environmental conditions of Himachal Pradesh.