HiMedia Laboratories Research Articles

Vancomycin Resistant Enterococci and Trend of Antimicrobial Susceptibility in Urine Cultures

Background: Enterococci are emerging nosocomial pathogen showing resistance to broad spectrum antibiotics. As reported by the center for disease control and prevention, enterococci (13.9%) are the second leading cause of urinary tract infection, after Escherichia coli. After the first report of vancomycin-resistant enterococci in England (1988), India reported its first vancomycin resistant enterococci isolate from New Delhi (1999). The prevalence of vancomycin resistant enterococci ranges from 1-9%, in India. To evaluate the prevalence of vancomycin resistant enterococci among urinary isolates and to check their antimicrobial resistance pattern. Methods: A retrospective observational study conducted at a tertiary care hospital. Data were analysed from November, 2017 to October, 2018. A total of 12,129 urine samples were received and subjected to culture on HiCrome UTI agar (Hi-Media laboratories, Mumbai) and incubated at 37 °C for a period of 18-24 hours. Antimicrobial susceptibility was performed using Kirby–Bauer disc diffusion method. Identification was done on the basis of colony colour and species identification was done by using biochemical test as per standard laboratory protocol. In one case automated siemens microscan walkaway identification system was used for confirmation. Results: Enterococci were isolated in 4.06% from urine cultures out of which 7.52% were vancomycin resistant. Most of the vancomycin resistant enterococci isolate (86.49%) were multi drug resistant. Conclusion: In vancomycin resistant enterococci related urinary tract infection, nitrofurantoin can be used as an appropriate first choice in uncomplicated cases and linezolid should be reserved for serious and complicated urinary tract infections.

Evaluation of the Colistin HiMIC Plate Kit for Colistin Susceptibility Testing of Klebsiella pneumoniae.

Colistin HiMIC Plate Kit (HiMedia Laboratories), a new commercial broth microdilution (BMD) test for colistin susceptibility testing was evaluated. BMD according to ISO standard 20776-1 (2019) with two-fold dilutions from 128 to 0.125 mg/L was used as a reference method. The colistin reference MICs (minimal inhibitory concentration) ranged from 0,25 to 128 mg/L with 15 (20.5%; 15/73) isolates having colistin reference MICs close to the current EUCAST breakpoint (MICs of 2, 4, and 8 mg/L). The study assessed the compliance of a commercial kit with the CLSI criteria, including categorical agreement (CA) and essential agreement (EA ≥90%), very major error (VME rate) <3%, and major error (ME) rate <3%. On 73 carbapenemase-producing Klebsiella pneumoniae isolates Colistin HiMICTM Plate Kit showed CA and EA of 100% (73/73; 95% CI 0.97-1.00) and 82.2% (60/73; 95% CI: 0.72-0.90), respectively. No ME (false-resistant results) and VME (false-susceptible results) were detected. Kit showed acceptable CA, ME, and VME error parameters, whereas the EA did not meet the ≥90% threshold. Laboratories must check for possible limitations of commercial kits before they can be used for colistin susceptibility testing.

Antimicrobial Susceptibility Testing to Erythromycin and Azithromycin of Clinical Isolates of Bordetella pertussis Circulating in Russia

Relevance. Antibacterial drugs are widely used to treat and prevent infections of the upper and lower respiratory tract. The large-scale and unjustified use of antimicrobials to treat these infections has led to the emergence of resistance in most pathogens. The aim. To study antimicrobial susceptibility testing to erythromycin and azithromycin of strains of B. pertussis isolated in Russia. Materials &amp; Methods. The research included 165 strains of B. pertussis isolated in January 2014 to June 2020. Antimicrobial susceptibility to erythromycin and azithromycin was determined by disk diffusion method and MIC test (HiMedia Laboratories Pvt. Limited, India). The A2047G mutation in the 23S rRNA gene was detected by PCR and subsequent sequencing. Results. Disk diffusion zone diameters for erythromycin in the studied strains ranged from 25 to 62 mm (median 44 mm) and disk diffusion zone diameters for azithromycin ranged from 22 to 80 mm (median 50 mm). Isolates with growth inhibition of more than 42 mm in diameter after 7 days of incubation were considered as susceptible. Among the studied strains, 57 (34.5%) were resistant to erythromycin and 23 (13.9%) to azithromycin. Then, MIC of erythromycin and MIC of azithromycin, respectively, were determined for these 57 and 23 strains using the MIC test. The comparison group included 79 isolates that were classified as sensitive to erythromycin (n = 31) and azithromycin (n=48) according to the results of the previous study. A MIC value of 0.12 μg/ml was considered as the cut-off for susceptible strains. All isolates were fully susceptible to erythromycin (MIC ≤ 0.01 μg/ml, median MIC 0.001 μg/ml) and azithromycin (MIC ≤ 0.01 μg/ml, median MIC 0.0001 μg/ml). An A-to-G mutation was not found at position 2047 in the 23S rRNA gene in 80 isolates that had a diameter of growth inhibition zone less than 42 mm. Conclusion. This study demonstrates no significant decrease in the susceptibility to erythromycin and azithromycin among B. pertussis strains isolated in Russia in 2014–2020. The studied B. pertussis strains exhibit a homozygous phenotype for macrolide resistance.

A Comparative Evaluation of the Periodontal Ligament Cell Viability Using Placentrex, Propolis, and Hanks' Balanced Salt Solution: An In-Vitro Study.

Most of the dento-alveolar trauma that occurs frequently in childhood is often linked to avulsion injuries. Two considerable elements influencing the prognosis of tooth avulsion after replantation are extraoral dry duration and the characteristics of the storage media to support the viability of cells. The study aimed to compare and assess the effectiveness of Hanks' balanced salt solution (HBSS) (HiMedia Laboratories, Thane, India) and Placentrex (Albert David Limited, Kolkata, India) against propolis (5% and 10%) as storage media in preserving the vitality of periodontal ligament (PDL) cells. Four groups with 15 samples each were selected for the study. Sixty recently extracted premolars were left to incubate for 30 minutes in 15-ml falcon tubes containing 2.5 ml of collagenase 0.2 mg/ml in phosphate-buffered saline and 2.4 mg/ml of dispase in HBSS, Placentex, propolis 5%, and propolis 10%. After the addition of bovine serum, it was centrifuged for 4 minutes. Trypan blue 0.4% was utilized to recognize the cells, and a hemocytometer was employed for counting the live PDL cells under a light microscope. Propolis 5% and 10%, HBSS, and Placentrex all showed statistically significant differences in PDL cell viability; however, HBSS was significantly outperformed by Placentrex and propolis. Placentrex is a superior substitute storage medium in cases of tooth avulsion as opposed to alternatives such as propolis 5% and 10%.

Polymethylmethacrylate-hydroxyapatite antibacterial and antifungal activity against oral bacteria: An in vitro study

ObjectiveReconstruction of alveolar bone defects resulting from aging, trauma, ablative surgery or pathology, remains a significant clinical challenge. The objective of this study was to investigate the antibacterial and antifungal activities of mixed polymethylmethacrylate-hydroxyapatite (PMMA-HA) against oral microorganisms. Our findings could provide valuable insights into the prospective application of PMMA-HA as a synthetic bone graft material to manage alveolar bone defects via tissue engineering. MethodsHA powder was obtained from the Center for Ceramics in Indonesia and PMMA granules were obtained from HiMedia Laboratories; these were prepared in 20:80, 30:70, and 40:60 ratios. The antibacterial diffusion method was then performed against Staphylococcusaureus, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum, while the antifungal diffusion method was used to test against Candida albicans. Standardized protocols were used for microbial culturing and inhibition zones were measured with digital calipers. Statistical analyses included one-way ANOVA and Kruskal–Wallis tests, supplemented by post-hoc Tukey HSD tests. ResultsA PMMA-HA scaffold with a 20:80 ratio demonstrated the highest antibacterial activity against S. aureus, A. actinomycetemcomitans, P. gingivalis, and F. nucleatum. This was followed by the 30:70 and 40:60 ratios in terms of antibacterial activity. Statistical significance was achieved with p < 0.05 in comparison to controls. However, none of the PMMA-HA ratios showed antifungal activity against C. albicans. ConclusionPMMA-HA scaffolds have significant activity against bacteria, but not against fungi.

Relationship between dental fluorosis, dental caries and salivary levels of Streptococcus mutans.

Worldwide, dental caries is an endemic infection and a significant public health problem. Fluoride reduces caries by helping to prevent demineralisation, by remineralising early carious lesions, and by decreasing the activity and growth of Streptococcus mutans (S. mutans). Fluoride intake above the optimal levels leads to a condition known as dental fluorosis, which causes hypo-mineralisation of the tooth. Some studies have revealed that the severity of fluorosis is directly proportional to caries, but some showed opposite results. Hence, with these contradicting results, this study was undertaken to identify the relationship between different grades of dental fluorosis, dental caries and the most common cariogenic bacteria S. mutans in saliva. A total of 90 subjects within 14- to 16-year age range were selected and categorised depending on the severity of fluorosis into three groups: group I (mild), group II (moderate) and group III (severe) based on modified Dean's fluorosis index criteria (1942). Unstimulated whole saliva samples were collected using the oral rinse technique and processed for quantification of S. mutans using Mitis Salivarius Bacitracin Agar medium. The number of colony-forming units (CFUs) was determined using a colony counter and expressed as ≥102, ≥103, ≥104 and ≥105 CFUs per ml of the sample, in accordance with the scale recommended by the manufacturer (HiMedia Laboratories). The severity of the caries was measured using decayed, missing or filled teeth (DMFT). Observations revealed that the overall DMFT was directly proportional to the level of S. mutans titres. It was observed that 67 to 73% of participants showed fewer colony counts (102 and 103) with minimal DMFT scores and 27 to 33% showed higher counts (104 and 105) with higher DMFT scores in all the groups. 105 CFUs of group III showed the highest mean DMFT scores (2.9) with an increased number of involved subjects than group II (2.3) and group I (1.5). In all, male participants had higher bacterial titres and DMFT scores than females. Fluoride in the drinking water served as anticariogenic agent regardless of the severity of fluorosis. Severe fluorosis with a higher incidence of caries and increased CFUs of S. mutans clearly indicates the importance of preventive measures and early treatment to reduce the severity of fluorosis and prevalence of dental caries.

Изучение биологических свойств и антибиотикорезистентности пробиотических бактерий из Кореи и Японии

The scope of the study is to study the biological properties of dietary supplements line: LactoBalance multiprobiotic, Neobiotic Lactobalance and Neobiotic Lactobalance Baby/Lactobalance Baby, isolation and generic identification of probiotic microorganisms from samples, confirmation of their growth properties and study of their antibiotic resistance. Materials and methods. The study confirmed that the number of probiotic microorganisms in the Lactobalance product line corresponds to those declared by the manufacturer. From biologically active food additives, probiotic microorganisms were isolated, identified and classified according to GOST R 56139-2014, MU 2.3.2.2789-10 [6, 7] and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). Isolation and cultivation of bifidobacteria strains and thermophilic streptococcus were carried out on a nutrient medium for the cultivation and isolation of bifidobacteria (FBSI SSC PMB, Obolensk, Russia), lactobacilli strains – on a nutrient medium MRS (HiMedia Laboratories Pvt. Limited, India). The growth properties of isolated probiotic microorganisms were studied by passaging them on nutrient media and their resistance to the most clinically significant antibiotics (Amoxicillin + clavulanic acid, Cefixime, Ciprofloxacin, Azithromycin, Clarithromycin, Levofloxacin, Oxacillin) and to the antiprotozoal and antimicrobial substance Metronidazole was determined using 2 methods: disco-diffusion method (DDM) and minimum inhibitory concentration (MIC) using strips. The studies used antibiotic discs and MIC strips manufactured by HiMedia Laboratories Pvt. Limited (India), Liofilchem srl. (Italy). Results. From the studied samples of dietary supplements, probiotic bacteria were isolated and identified (11 strains of bifidobacteria, 7 strains of lactobacilli and 1 strain of thermophilic streptococcus, which corresponds to those declared by the manufacturers). The number of living probiotic microorganisms was determined and their growth properties were confirmed. When studying the bacteria included in Lactobalance, almost all isolated strains and, in general, consortia of bacteria had resistance or low natural sensitivity to cefixime and ciprofloxacin, oxacillin. All lactobacilli were naturally resistant to metronidazole. Among bifidobacteria, the B. longum strain was resistant to metronidazole. All bacteria were highly sensitive to levofloxacin, amoxicillin with clavulanic acid, azithromycin and clarithromycin. As part of Neobiotic Lactobalance and Neobiotic Lactobalance Baby/Lactobalance Baby, 84% of the isolated bacteria, as well as the consortium of bacteria, were resistant or had low sensitivity to ciprofloxacin, cefixime, metronidazole and oxacillin. B. bifidum was resistant to azithromycin and clarithromycin. All isolated bacteria showed sensitivity to amoxicillin with clavulanic acid and levofloxacin, but the consortium of bifidobacteria showed high sensitivity only to amoxicillin with clavulanic acid. The decrease in sensitivity to other antibiotics is associated with the synergy of the properties of individual strains. Conclusion. Based on the results of in vitro studies, it can be concluded that the probiotic bacteria included in the Lactobalance dietary supplement line correspond to the declared species and their quantity and retain their growth properties. When taking all dietary supplements in the Lactobalance line while taking antibiotic therapy with cefixime, ciprofloxacin and oxacillin, as well as taking metronidazole, there is a possibility of colonization of the intestinal mucosa with probiotic strains included in their composition. When taking Neobiotic Lactobalance and Neobiotic Lactobalance Baby/Lactobalance Baby, there is a possibility of colonization of the intestinal mucosa with probiotic strains while taking cefixime, metronidazole, ciprofloxacin, oxacillin, azithromycin and clarithromycin. Key words: probiotics, bifidobacteria, lactobacilli, thermophilic streptococcus, natural antibiotic resistance, minimum inhibitory concentration of antimicrobial drugs

P020 Risk factors, speciation ,and antifungal susceptibility in candidemia patients: An observational study

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesTo assess species distribution, antifungal susceptibility pattern, and associated risk factors in cases of candidemia among admitted patients in a tertiary care hospital in New Delhi, India.MethodsAny positive blood culture bottle which revealed budding yeast cells on gram stain was included in the study. The samples were subcultured onto blood agar and HiCrome™ Candida Differential Agar (HiMedia Laboratories Pvt. Limited, Mumbai), after which the colonies were subjected to identification with VITEK MS (bioMerieux, France). The antifungal susceptibility of each isolate was assessed with the help of Vitek 2 AST (bioMerieux, France). Fluconazole, voriconazole, caspofungin, amphotericin B, flucytosine, and micafungin were the antifungals tested for resistance. Antifungal susceptibility by broth microdilution was performed for C. auris and for fluconazole in the case of C. glabrata. Patient demographics, as well as risk factors associated with Candida infections, were collected from case files and by interviewing patients and bystanders.ResultsWe isolated 171 fungal isolates from 160 patients admitted in the study during a period of 1 year from February 1, 2021 to January 31, 2022.Out of the 171 fungal isolates, 162 were Candida spp. Trichosporon spp. Saccharomyces cerevisiae and Fusarium spp. contributed 6, 2, and 1 isolate respectively. Among the Candida isolates, the commonest were C. auris (n = 37) followed by C. tropicalis (n = 34), C. albicans (n = 22), and C. glabrata (n = 22) (Fig. 1). The most common isolate from patients admitted to the ICU/HDU was C. auris (31%). Whereas in wards C. tropicalis (22%) and C. parapsilosis (22%) contributed the maximum number of isolates. Candida pelliculosa (n = 8) and C. tropicalis (n = 7) were the most common isolates among neonates.Antifungal susceptibility results were interpreted as per Clinical Laboratory Standards Institute M27 A2 document. Overall sensitivity was highest for Micafungin followed by amphotericin B. Micafungin was mostly sensitive for C. auris (94.11%), whereas in case of amphotericin B it was 47.22% (Table 1).The most common risk factor observed was the presence of IV line (n = 135), antimicrobial therapy (n = 126), and diabetes (n = 46). In neonates also the most common risk factor was the presence of an IV line (n = 23) followed by outborn status (babies delivered outside in other hospitals and transferred subsequently) (n = 20).A total of 30 days hospital mortality was observed to be 54.05% in patients with C auris isolates.Conclusion Candida BSI etiologies are shifting away from C. albicans and towards species that have a higher propensity for developing resistance, such as the multidrug-resistant C. auris, which is rapidly spreading throughout the world. This highlights the importance of stepping up hospital infection control practices and antimicrobial stewardship initiatives in order to counteract the rapidly increasing antifungal resistance threat.

ANTIBIOTIC RESISTANCE OF CAUSATIVE PATHOGENES OF SURGICAL SITE INFECTION

Microbiological monitoring is remaining a pressing issue in effective control of surgical diseases and prevention of complications. The aim of this study is to determine antibiotic resistance of causative pathogens of surgical site infections isolated in a tertiary medical setting in the Poltava region, Ukraine. This prospective observational study involved 121 middle-aged patients who underwent surgical operations in the surgical department of the Municipal Enterprise “M.V. Sklifosovsky Poltava Regional Clinical Hospital” (Poltava, Ukraine) for 2017-2020. Laboratory investigations of the biological material taken from the patients were conducted at the bacteriological department of the hospital. Pathogens were isolated and identified by an automatised technique, using Vitek2 (Biomérieux, France). 164 isolated and identified strains were determined to have the susceptibility to 30 antibiotics of various groups determined by the disk-diffusion method using standard disks with antibiotics placed on the surface of the Mueller-Hinton culture medium (HiMedia Laboratories Pvt Ltd. India). Statistical analysis was carried out: microorganisms’ susceptibility to antibiotics was calculated in percent. The highest level of resistance to antibacterial drugs was found in Pseudomonas aeruginosa, which, with the exception of 33.3% of strains were susceptible to levofloxacin only, showed resistance to all tested antibiotics. The maximum level of methicillin-resistant strains was found among coagulase-negative staphylococci and amounted to 91.3%. All isolated Klebsiella strains and 18.7% of E. coli strains were producers of extended spectrum beta-lactamases. A significant predominance of multiresistant strains of microorganisms, the resistance of which goes beyond the boundaries of more than two pharmacological groups of antibiotics, has been revealed.

Assessment of antimicrobial activity of bioflavonoid CITROX and chlorhexidine combination against P. gingivalis (in vitro)

The aim of the study - evaluation of the antiseptic properties of the combination of chlorhexidine and bioflavonoid CITROX against P. gingivalis. Clinical isolates of microbial cultures from the collection of the Department of Microbiology, Immunology, Virology of the Moscow State Medical University named after A.I. Evdokimov: P. gingivalis. Primary seeding of the studied material was carried out on nutrient media produced by Himedia Laboratories Pvt. Limited (India): Colombian agar with the addition of 5% defibrinated blood and a selective additive for the isolation of non-spore anaerobes. To conduct the experiment, a thermostatic rotary system - RTS-1 (Biosan, Latvia) was used, which performs a modern type of mixing due to the formation of a vortex-type diffusing effect. The interpretation of the results was carried out by changing the optical density index (the indicator in McFarland units) at a wavelength of λ = 850 nm. The combination of CITROX + chlorhexidine 0.05% more effectively suppresses the growth of periodotopathogenic bacteria than chlorhexidine 0.05%, prolonging the adaptive phase (lag phase) of the growth of P.gingivalis bacteria and can be considered as an alternative to chlorhexidine without additives. Prolongation of the lag phase increases the time until the first clinical symptoms appear, as a result, the body has more time to form an immune response. The combination of the complex of bioflavonoids CITROX and chlorhexidine bigluconate in concentrations of 0.05% and 0.2% is active against P. gingivalis and can be considered as an alternative to chlorhexidine without additives. The inclusion of CITROX in the rinse aid reduces the concentration of chlorhexidine in the rinse aid, which can lead to a decrease in the severity of undesirable effects and be considered as an alternative remedy in the treatment of periodontal diseases.

PSXVI-14 Study of the antagonistic properties of C. utilis in relation to the resident microflora of the animal organism

Abstract C. utilis yeast is widely used in industry for the production of fodder protein, amino acids, vitamins, and enzymes. We have studied the antimicrobial activity of C. utilis CY 302 against such collection strains as E. coli 3912/41, S. flexneri 1a 8516, S. enterica serovar Typhimurium 79, S. epidermidis ATCC 14990, S. aureus ATCC 25923, Proteus vulgaris HX 19 222, L. casei subsp. Rhamnosus ATCC 7469, B. breve ATCC 15701, C. albicans ATCC 10231 by using the agar blocks method and by measuring the inhibition growth zone (IGZ, mm) accompanied by the target cells death zone. The yeast strain was grown on Sabouraud’s medium at 28±1 °C for 3 days, and the test cultures were grown on differential diagnostic media for each bacterial species (HiMedia Laboratories Pvt. Ltd, India) at 35±2 °C overnight. The analyses were done separately for each culture. A weak antimicrobial effect was observed with respect to L. casei and B. breve, the IGZ was 5.0 and 4.0 mm, respectively. The halos of inhibition against pathogenic bacteria were 7 mm for E. coli, 5 mm for S. typhimurium, 5 mm for P. vulgaris, 6 mm for S. fiexneri, 6 mm for S. aureus, and 5 mm for S. epidermidis. After incubation there was noted the absence of DZZR in the wild C. utilis CY 302 strain in relation to the C. albicans ATCC 10231 strain from the collection. As a result of the studies the antagonistic potential of the yeast isolate C. utilis CY 302 was revealed. The work was supported by the Ministry of Science and Higher Education of Russia.

Formation of biofilms by representatives of the oral microflora on the surfaces of basic materials

The aim of the work is to conduct a comparative analysis of the biofilm formation by representatives of the oral microflora on the surfaces of basic materials. Materials and methods. The process of biofilm formation was examined on 7 types of basic plastic samples: Polyan, Breflex, Nylon, Protakryl, Vinakryl, Biocryl, which were used for the manufacture of removable prosthetic basis constructions, and SYNMA, which was used for comparison. Biofilm formation was analyzed by the method Y. Zhang (2017) with minor modifications. The test sample was placed in a test tube with 2.0 ml of nutrient broth Brain Heart Infusion to model the biofilm growth of microorganisms (HiMedia Laboratories Pvt. Ltd., India) supplemented with 1 % glucose, pre-inoculated with test strains at a final concentration of 1 × 104 CFU/ml. The strains were cultivated for 24 hours at a temperature of 37 °C under continuous stirring in a shaker MR-1 (SIA BIOS AN, Latvia) at 20 rpm. Evaluation of the biofilm massiveness was performed after gentian violet staining followed by elution of the stain with ethanol and registration of the eluent optical density (OD). The OD was measured with a Synergy™ HTX S1LFTA microplate multimode photometer (BioTek Instruments, Inc., USA) at 595 nm wavelength using Gen5™ Data Analysis Software. The number of viable bacterial cells in the formed biofilms was determined by the method of ten-fold serial dilutions. The obtained results were converted per unit area of the sample tested. Processing of the results was performed using a two-sample t-test with the software package Statistica 13.0 and Microsoft Office Excel, the differences were considered statistically significant at a P value of &lt; 0.05. Statistical analysis of the obtained data was presented as mean values of measurements ± standard deviation for three independent experiments. Results. According to the microbiological analysis results it was found that α-hemolytic streptococci S. oralis and S. sanguinis showed the ability to form biofilms on the surfaces of basic materials, namely Protacryl and Vinacryl, the total biomass of S. sanguinis biofilms was 47.7 % (P &lt; 0.01) and 14.7 % (P &gt; 0.05) greater, respectively, in comparison to a glass slide. Inhibition of biofilm formation processes was observed on the surfaces of Nylon and Biocryl basic materials. S. oralis and S. gordonii showed the highest ability to survive in biofilms. The intensity of C. albicans biofilms formation on Biocryl basic materials, comparative plastics SINMA and Breflex basic materials was greater than on glass slides by 48.3 %, 43.0 % and 34.9 % (P &lt; 0.01), respectively. The least massive C. albicans biofilms were formed on Breflex surfaces and SINMA comparative plastics in comparison to glass slides by 33.6 % and 24.8 % (P &lt; 0.01), respectively. Both Candida strains had the highest level of fungal viability in biofilms on Breflex, Polyan and Protacryl basic materials (P &lt; 0.01), and C. tropicalis biofilms on Biocryl and Vinacryl basic materials (P &lt; 0.05). Integral coefficients indicated the inhibition of the oral microflora ability to form biofilms on the surfaces of basic materials. Conclusions. Oral α-hemolytic and β-hemolytic streptococci have the ability to intensive biofilm growth on the surfaces of the basic materials Protacryl and Vinacryl. Oral Candida albicans form massive biofilms on the surfaces of Biocryl and Vinacryl basic materials and comparative SYNMA plastics. The basic materials Breflex, Nylon and comparative plastics SYNMA are the most inert to biofilm formation by the oral microflora representatives.

Non-Candida albicans Candida species: virulence factors and species identification in India.

Background and Purpose: The predominant cause of candidiasis was Candida albicans which has recently changed to non-Candida albicans Candida (NCAC) (i.e., Candida spp. other than the C. albicans). The NCAC spp., earlier considered non-pathogenic or minimally virulent, are now considered a primary cause of morbidity and mortality in immunocompromised individuals. Given the NCAC spp.has become more common in clinical cases, this study aimed to determine the prevalence of NCAC spp. in different clinical specimens and assess a few of their virulence factors.Materials and Methods: Routine samples for bacterial culture and sensitivity that showed colony characteristics, like Candida on Blood Agar and microscopic features resembling Candida spp., were processed further. Candida isolates underwent tests for chlamydospore formation and biochemical tests, including sugar fermentation and sugar assimilation tests. These were grown at 42oC, and their colony color was identified using HiCromeTM Candida Differential Agar (HiMedia Laboratories Pvt. Ltd., Mumbai, India), HiCandidaTM Identification Kit (HiMedia Laboratories Pvt. Ltd., Mumbai, India), and VITEK-2® Compact (Biomérieux, France). Virulence factors, such as adherence to buccal epithelial cells (ABEC), biofilm formation, hemolytic activity, and production of coagulase enzyme were also tested.Results: Mean age of the patients was 38.46 years with a male-female ratio of 1.36:1. In total, 137 Candida isolates were recovered; 45.3%, 19.7%, and 13.9% of the isolates were isolated from urine, vaginal swabs, and oropharyngeal swabs, respectively. Moreover, 55 (40.1%) isolates were those of C. albicans and 82 (59.9%) isolates belonged to NCAC spp., with C. tropicalis (23.4%) contributing highest among NCAC species. Furthermore, C. albicans (3; 50%) was the most common spp. in cases of candidemia. Haemolysin production (85.5%) and ABEC (78.2%) were the major virulence factors in C. albicans. C. tropicalis (59.4%) and C. dubliniensis (50%) showed maximum ABEC. Biofilm forming capacity was higher in C. tropicalis (78.1%) than C. albicans (67%).Conclusion: Results of this study suggest varied prevalence and virulence based on geographical locations, even within a subcontinent. It clearly indicates the emergence of the NCAC spp. and their predominance in different body fluids. Identification of Candida to the spp. level should become a routine in all laboratories.

First Report of Fusarium equiseti Causing Seedling Death on Sugar Beet in Minnesota, USA.

In May 2019, sugar beet (Beta vulgaris L.) seedlings with symptoms of wilting and root tip discoloration and necrosis were found in Moorhead (46.5507° N, 96.4208° W), Minnesota, USA. Roots of infected seedlings were surface sterilized with 10% bleach for 15 seconds, rinsed with sterile distilled water and cultured on water agar (MA Mooragar®, Inc, CA) for 3 days at 23 ± 2°C. Isolates were transferred to carnation leaf agar (CLA) and incubated at room temperature (22°C) under fluorescent light for 14 days. Abundant macroconidia were produced in sporodochia. Macroconidia were 5- to 7-septate, slightly curved at the apex, and ranged from 35 to 110 ×1.2 to 3.8 μm. No microconidia were produced. Chlamydospores with thick, roughened walls were observed in chains or in clumps, and were ellipsoidal or subglobose. Single spore was transferred from CLA to potato dextrose agar (HIMEDIA Laboratories, India) produced abundant white mycelium and was pale brown where the colony was in contact with the media. The morphological features of the isolates were consistent with Fusarium equiseti (Corda) Sacc. (Leslie and Summerell 2006, Li et al. 2015). Genomic DNAs (NORGEN BIOTEK CORP, Fungi DNA Isolation Kit #26200) of two representative isolates were used for polymerase chain reaction (PCR). The second largest subunit of RNA polymerase (RPB2) was amplified by PCR with primers 5f2/7cr (O'Donnell et al. 2010). The amplified PCR product was sequenced and deposited in GenBank (accession number MW048778). A BLAST search in Genbank and the Fusarium MLST database showed 100% sequence alignment to F. equiseti with accession MK077037.1 and NRRL 25795, respectively. Pathogenicity testing was done using three sugar beet seedlings (Hilleshög proprietary material, Hilleshög Seed, LLC, Halsey, OR 97348) at cotyledonary stage grown in a pot (4˝×4˝×6˝) with six replicates. Seedlings were inoculated with F. equiseti conidial suspension (104 conidia ml-1 for 8 minutes) by the root dip method (Hanson, 2006). Mock inoculated plants were dipped in sterile water. Inoculated and control plants were placed in the greenhouse at 25 ± 2°C, and 75 to 85% relative humidity. One week later, inoculated seedlings showed root tip tissue discoloration similar to those observed in the field and non-inoculated seedlings were symptomless. This study was repeated. The fungus was re-isolated from diseased roots and confirmed to be F. equiseti based on morphological characters. Fusarium equiseti was reported on freshly harvested and stored beet in Europe but was not found to be pathogenic (Christ et al. 2011). Strausbaugh and Gillen (2009) reported the association of F. equiseti and root rot of sugar beet but did not report pathogenicity. This pathogen is reported in several crops including edible beans that is grown in rotation with sugar beet in several production areas (Jacobs et al. 2018). The most important Fusarium species reported to cause significant economic damage to sugar beet include F. oxysporum and F. secorum (Secor et al. 2014, Webb et. al. 2012). The presence of another pathogenic Fusarium species in sugar beet will require monitoring to determine how widespread it is and whether current commercial cultivars are resistant. To our knowledge, this is the first report of F. equiseti causing disease on sugar beet seedlings in Minnesota, USA.

Cover Image, Volume 138, Issue 15

The image created by Tridib Kumar Bhowmick and colleagues shows Standard Cellulose Powder (Hi-media Laboratories, pvt ltd.) and Purity (>97%, on dry basis). Known as the SEM Analysis Method, the morphology of the standard cellulose was determined using scanning electron microscopy (SEM) (ZEISS, Model: Gemini, Germany). A small amount of cellulose powder was sprinkled on double-sided conductive tape pasted on a metal stub. The sample was coated with gold by a sputter coater and the surface of standard cellulose (STDC) was observed by operating the microscope at 5 kV, with a working distance kept at 6.6 mm. DOI: 10.1002/app.50388

In-vitro hypoglycemic activity of lawsonia inermis Linn. Leaf extracts

in-vitro hypoglycemic activity of lawsonia inermis Linn. Leaf extracts

Coliform contamination on faucet surface of water vending machines in Klang Valley

In Malaysia, water vending machine serves as an alternative to drinking water supply. However, the quality ofdrinking water obtained from water vending machines may vary due to microorganism contamination which caused byinadequate hygienic practices and routine maintenance of the machines. In this study, 100 water vending machines wererandomly selected from 10 districts of Klang Valley. Sterile cotton swabs were used to collect swab samples from all selectedsubjects, with swab samples collected on the outer surface of water faucets. Samples were sent to laboratory for cultureanalyses using Nutrient agars (Oxoid) and HiCromeTM Coliform agars (HiMedia Laboratories). The results showed that noneof the water vending machine was contaminated by faecal coliform, however, with close to 80% of the subjects were foundcontaminated by total coliform (eg. Klebsiella, Enterobacter or Citrobacter species). Although the presence of total coliformmay not be deleterious to the health of end users, our findings highlights the need for authorities and water vending serviceproviders to set an effective sanitation procedure in maintaining the hygienic level of water vending machines.

A reliable fluorimetric method to screen the nitric oxide synthase inhibitors in 96 well plate

In general, 4 amino-5-methylamino-2′,7′-difluorescein diacetate (DAF-FM-DA) dye is used to detect nitric oxide in biological systems through cell imaging. In this study, we have used 96 well plate format to quantify nitric oxide using DAF-FM-DA through a multimode reader (or independently using fluorospectrometer) and could be visualized in a fluorescence microscope. Similar study otherwise will require a high-end instrument. The method has been validated to screen NOS inhibitors in the HEK 293T cell lines over-expressing the NOS isoforms. We observed that the method is very simple to use, adaptive, sensitive and most importantly it saves time. Reagents/toolsEthanol (70% [v/v] in distilled water), Nω-Nitro-l-arginine (l-NAME), 7-Nitro-Indazole (7-NI) (Sigma, St. Louis, MO), HEK 293T cell lines (National Centre for Cell Science (NCCS), Pune, India), DMEM (Himedia laboratories Pvt), Fetal Bovine Serum (FBS) (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a 5% CO2 atmosphere. Hank's Balanced Salt Solution (HBSS) without Phenol Red of pH 7.4 was prepared with the following composition: NaCl, 8.0g, KCl, 0.4g, CaCl2, 0.14g, MgSO4⋅7H2O, 0.1g, MgCl2·6H2O, 0.1g, Na2HPO4·2H2O, 0.06g, KH2PO4, 0.06g, glucose, 1.0g, NaHCO3, 0.35g, H2O, to 1000 ml, Sterilized and refrigerated, Calcium Ionophore A23187 (Sigma Aldrich 52665-69-7) DAF-FM Di Acetate (Molecular Probes Life Technologies), and DAF-FM Di Aceatate was prepared as a stock solution (5 mM) in DMSO, divided into aliquots and stored at −20 °C, followed by dilution to the required concentration in HBSS buffer before use. EquipmentNeubauer chamber, Microtube centrifuges (1.5 mL), Micropipettors,10,100, and 1000 mL with corresponding tips, multimode reader (Tecan, Synergy-HT), inverted fluorescence microscope (Nikon, eclipse Ti-S), black flat bottom Microplates (96-well) (Corning 3603).

Microbiological studies of a new combined drug “Proplantmed” based on propolis phenolic hydrophobic drug

Gastric and duodenal ulcer is one of the most widespread diseases in the world; therefore, creation and research of new effective and safe medicines are of great importance.Aim. To study the microbiological properties of the new combined drug “Proplantmed” based on propolis phenolic hydrophobic drug (PPHD).Materials and methods. The studies of microbiological purity of the drug were performed according to the State Pharmacopoeia of Ukraine (SPhU). The following growth media were used for testing: soybean casein digest medium (“HIMedia Laboratories Pvt. Ltd. India”), soybean casein digest agar (“HIMedia Laboratories Pvt. Ltd. India”), thioglycollate medium for sterility control (“HIMedia Laboratories Pvt. Ltd. India”) and Sabouraud dextrose agar (manufacturing country: India, (“HIMedia Laboratories Pvt. Ltd. India”)).Results. The experimental samples of the drug “Proplantmed” meet the requirements of the SPhU and “Microbiological Purity” test; this fact is an important quality indicator of the finished medicinal product.Conclusions. According to the results of the experiment conducted it has been found that level of microbiological purity of the granules proposed meets the requirements of the SPhU; this indicator is the quality guarantee of this drug.

Anticancer and Cytotoxic Potential of Turmeric (Curcuma longa), Neem (Azadirachta indica), Tulasi (Ocimum sanctum) and Ginger (Zingiber officinale) Extracts on HeLa cell line

Anticancer and Cytotoxic Potential of Turmeric (Curcuma longa), Neem (Azadirachta indica), Tulasi (Ocimum sanctum) and Ginger (Zingiber officinale) Extracts on HeLa cell line