Access Microbiology

Intra-subtype recombination is a common phenomenon among HIV strains which involves transfer of genetic information between two viral strains belonging to the same HIV subtype. However, more research has been done on inter-subtype recombination which is the transfer of genetic information between two strains belonging to different HIV subtypes. In this study HIV 1 subtype C env sequences from Zimbabwean nucleotide sequences (n=2 915) downloaded from Los Alamos HIV databases were used to investigate intra-subtype recombination. Using the GENECUTTER tool, [~]44.4% of whole genomes were selected to have informative env regions (n = 1 295). Clustal was used for Multiple Sequence Alignment and RDP 4 was used for analysis and detection of Intra-subtype recombination. Apparently, GENECONV included in RDP 4 detected 47 recombination events. One of the most informative recombination events was further analysed using UPGMA phylogenetic tree to understand the extent of the intra-subtype recombination.

BackgroundMolecular techniques can enhance the power of epidemiological investigations for tracing HIV transmission networks. This information could be useful for developing strategies for prevention of HIV transmission. Hence, we carried out to a study on the transmission patterns among newly diagnosed HIV cases among High-Risk Groups (HRGs) of North-West India using phylogenomic methods. MethodsPhylogenomic analysis was carried out among 37 randomly selected samples of recently infected HRGs identified through Recent Infections Testing Algorithm (RITA) using Limiting Antigen Avidity Assay. Amplification of the reverse transcriptase region of pol gene (540 base pairs) and sequencing was done. Reference sequences were extracted from HIV Los Alamos database. Sequences aligned by Clustal W and HIV-1 subtype were determined on the basis of phylogenomic analysis of the pol sequence. Phylogenetic trees were constructed using the MEGA (version 11.0). ResultsThe phylogeny clearly depicts that the study isolates RTFSWCHD and RTFSWPB007 cluster with and are related to the Indian reference sequences AY746371 and EU683781 and a Nepalese sequence KX430115.The other study isolates (RTFSWCHD001, RTFSWPB005, RTFSWCHD002, RTFSWPB006, RTFSWHR008, RTFSWHR 009) clustered uniquely among themselves without any interlinking with other references. One study isolate (RTFSWHP004) clustered closely with Zimbabwian isolate AY998351. The phylogeny shows that the study isolate MSMCHD005 clades separately with the Indian references (DQ838761, EU683781and AY746371), but is also very closely related to the references from China (HG421606, JQ658754), Nepal(JN023039) and Myanmar (N223216, JN223183, KC913773). Other study isolates (MSMCHD003, MSMHP007, MSMCHD004, MSMPB001, MSMPB002, and MSMHR006) are highly interrelated among themselves and form a separate unique clade together. The evolutionary tree shows that all the sequences from current study formed a monophyletic lineage, i.e., sequences from India clustered together more than with sequences from any other country. The study sequences showed relatedness only to the Nepal references KX430115 and JN023035. The South African, UK, Norway, China, and Myanmar references are grouped into aseparate clade. ConclusionMolecular epidemiologic methods were able to reveal transmission networks; hence, phylogenomic methods can be used in HIV Sentinel Surveillance to monitor transmission networks.

Staphylococcus aureus has been shown to move across soft agar surfaces by several different mechanisms. S. aureus can move using either spreading (a form of sliding motility producing generally round/frond like colonies) or by forming comets (slime covered aggregates of cells) that result in long thin dendrites branching out from the central colony. Spreading is agreed to be a form of passive motility whilst the comets are a form of gliding motility (i.e. active). Comets occur under similar conditions to spreading round colonies; however it can be difficult to get the comets to form. Here we examine the variables involved in determining whether comets form as well as report further observations of the comets themselves. We found that the conditions that favoured comet formation (and formed the associated dendrites) occurred over a more limited range than those that enabled spreading motility. Comet formation is very sensitive to the solidifying agents used, the amount of media used and the drying time. We further observed that the comets can propel themselves upwards against gravity unlike spreading motility and that S. aureus formed unusual dense aggregates and strand-like structures within comets unlike the normal growing arrangement of S. aureus observed in spreading. These results may aid others in producing motility assays to study spreading and comet formation in S. aureus and provides further insight into how comets behave.

BackgroundAmylase and protease are two of the most demanded and widely used commercial enzymes. Amylases have many industrial uses: starch conversion (food/bakery industry), sizing agent (textile industry and paper industry), degradation of starchy residue from clothes (detergent industry), conversion of starch to fermentable sugar (fuel industry) etc. Their main source is of microbial origin. About two-third of the industrial enzymes (amylase, protease, cellulose, penicillinase, chitinase, nucleases, esterase, lipase etc.) are produced by Bacillus spp. Among bacteria, Bacillus species are specific producers of extracellular enzymes. ObjectiveThe present work comprised the identification of amylase and protease producing Bacillus spp and exposure of the producers to various parameters for the maximum yield of the enzyme. MethodsTo isolate and identify the amylase and protease producing strain, soil samples were collected from different vegetation from the altitude at 4367.35 feet above sea level. The isolates were screened and various biochemical tests and morphological observations were done to identify the isolates. The enzymes were produced by the submerged state fermentation (SmF) from the isolates and purified by dialysis. Effects of temperature, pH, and different carbon and nitrogen sources of the medium using SmF were optimized. ResultsAmong 95 isolates, 36 were identified. Among the identified isolates, Bacillus subtilis and Bacillus thuringiensis were optimized for the amylase and protease production respectively. The maximum amylase production was found at 42C temperature, in fructose as a carbon sugar, peptone as a nitrogen source and at pH 7. Similarly, the maximum protease production was found at 42C temperature, in sucrose as a carbon sugar, ammonium sulphate as a nitrogen source. The producers inhabited the soil of leguminous plant. ConclusionIn the present study, a natural polymer gelatin is used with the nutrient agar medium to help in cell immobilization for maximum production of alkaline protease by strains of Bacillus. More sophisticated process of purification might yield more enzyme compared to the dialysis in our process that yield 42U/ml/min and 52U/ml/min respectively.

The CRISPR-Cas9 system has been used extensively in eukaryotic and prokaryotic systems for various applications. In case of the latter, a couple of previous studies had shown Cas9 protein expression associated toxicity. We studied the same in five microbes, viz Escherichia coli, Salmonella typhimurium, Mycobacterium smegmatis, Xanthomonas campestris and Deinococcus radiodurans. Transformation efficiency of plasmids carrying genes coding for Cas9 or dCas9 was used to gauge toxicity associated with Cas9 protein expression. Results showed differential levels of Cas9 toxicity among the bacteria and lower transformation efficiency for cas9/dcas9 bearing plasmids compared to controls in general. This indicated lethal effect of Cas9/dCas9 expression. While E. coli and S. typhimurium seemed to tolerate Cas9/dCas9 fairly well, in GC rich microbes, M. smegmatis, X. campestris and D. radiodurans, Cas9/dCas9 associated toxicity was acute.

There have been incerases in antibiotic resistant strains of human pathogens and causing treatment of microbial infections difficult. These days more attention is given to seraching new antimicrobial drugs to combat pathogenic microbes. Traditional fermented cottage of Metata Ayib which is naturally enriched with lactic acid bacteria (LAB) and used to preserve cheese for long time in Northern Ethiopia, may have antimicrobial activity against various human pathogens. However, there was no scientific report on the antimicrobial activity of lactic acid bacteria isolated from Metata Ayib. The objective of this study is to evaluate antibacterial activity of lactic acid bacteria from Metata Ayib against clinical and standard human pathogens. The study was laboratory based experiment. Antibiotic production by the LAB was performed by inoculating the LAB isolates into 6.0 ml MRS medium and incubating at 30 {degrees}C. Cell free supernatants (CFS) were collected by centrifugation (10,000 rpm for 15 min at 4 {degrees}C) of the six day fermented broth cultures. The pH of the CFS was adjusted to 6.5 with 4 N NaOH to eliminate the effect of organic acids.The susceptibility of produced antibiotic against test organisms was done by growing on Muller Hinton agar in triplicate using well diffusion method and the inhibition zone was recorded. Preceddingly, MIC and MBC was determined using standard methods. The antibiotic substance exhibited antimicrobial activity towards standard and drug resistant bacteral strains with the inhibition zone ranges upto 25.33{+/-}3.21 mm. The result of MIC against tested ornaisnms showed a considerable antimicrobial activity of the antibiotic substance withn the range values 6.25% to12.5%. In addition, the result of this study showed that LAB obtained from Metata Ayib exhibits antimicrobial activity against standard and pathogenic pathogenic test bacterial species ranged from 12.5-25% of MBC value. This might be due to the production of organic acids, but also other compounds, such as ethanol, hydrogen peroxide, diacetyl, reuterin and bacteriocins. The results of this investigation can also provide baseline for information for future studies about the application of antibacterial substances produced by LAB from fermented Metata ayib.

In microbiology, the estimation of the growth rate of microorganisms is a critical parameter to describe a new strain or characterize optimal growth conditions. Traditionally, this parameter is estimated by selecting subjectively the exponential phase of the growth, and then determining the slope of this curve section, by linear regression. However, for some experiments, the number of points to describe the growth can be very limited, and consequently such linear model will not fit, or the parameters estimation can much lower and strongly variable. In this paper, we propose a tools to estimate growth parameters using a logistic Verhulst model that take into account the entire growth curve for the estimation of the growth rate. The efficiency of such model is compared to the linear model. Finally, the novelty of our work is to propose a "Shiny-web application", online, without any programming or modelling skills, to allow estimating growth parameters including growth rate, maximum population, and beginning of the exponential phase, as well as an estimation of their variability. The final results can be displayed in the form of a scatter plot representing the model, its efficiency and the estimated parameters are downloadable.

Assessment of HIV and Mycobacterium tuberculosis infection is crucial to detect HIV and/or Mycobacterium tuberculosis coinfection and the strategy for infection management and treatment. This study assessed the proportion of students with HIV, Mycobacterium tuberculosis, and HIV/Mycobacterium tuberculosis coinfection. Two hundred and thirty-five university students in Port Harcourt, Nigeria were recruited, ages 16 - 39 years. Samples of blood were collected and processed using standard laboratory procedures. All the students were screened for antibodies to HIV using 2 rapid screening strips and a commercially available enzyme-linked immunosorbent assay (ELISA)-based kit for determination of HIV-1/2/P24/O. The presence of Mycobacterium tuberculosis was done using TB rapid kits and a commercially available ELISA-based kit. The results showed that 3.4% of the students were positive for HIV, 2.1% for Mycobacterium tuberculosis and none for HIV/Mycobacterium tuberculosis coinfection. The age-specific infection rate showed a higher HIV infection rate in the age group 16-24 years (4.7%) than [&ge;]25 years (2.8%). While higher Mycobacterium tuberculosis infection rate occurred in the age group [&ge;]25 years (2.8%) than in <25 years (2.3%). The gender-specific infection rate showed that females had a higher infection rate (HIV, 4.7% and Mycobacterium tuberculosis, 2.3%) than males (HIV, 2.6% and Mycobacterium tuberculosis, 1.7%). Age and sex were the main correlates (P<0.05) of HIV and Mycobacterium tuberculosis. This study further confirmed the presence of HIV and Mycobacterium tuberculosis infections among University students. These findings suggest the need for regular screening of University students for HIV and Mycobacterium tuberculosis.

Being a significant protein L-glutaminases discovers potential applications in various divisions running from nourishment industry to restorative and cure. It is generally disseminated in microbes, actinomycetes, yeast and organisms. Glutaminase is the principal enzyme that changes glutamine to glutamate. The samples were gathered from soil of Taxila, Wah Cantt and Quetta, Pakistan for the isolation of glutaminase producing bacteria. After primary screening, subordinate screening was done which includes multiple testification such as purification, observation of morphological characters and biochemical testing of bacterial strains along with 16S rRNA sequence homology testing. Five bacterial strains were selected showing glutaminase positive test in screening, enzyme production via fermentation and enzymatic and protein assays. Taxonomical characterization of the isolates identified them as Bacillus subtilis U1, Achromobacter xylosoxidans G1, Bacillus subtilis Q2, Stenotrophomonas maltophilia U3 and Alcaligenes faecalis S3. The optimization of different effectors such as incubation time, inducers, carbon source, pH, and nitrogen source were also put under consideration. There was slight difference among incubation of bacterial culture, overall, 36 hours of incubation time was the best for glutaminase production by all the strains. Optimal pH was around 9 in Achromobacter xylosoxidans G1 and Alcaligenes faecalis S3, pH 6 in Bacillus subtilis U1, pH 8 in Stenotrophomonas maltophilia U3, pH 6-8 in Bacillus subtilis Q2. Best glutaminase production was obtained at 37{degrees}C by Bacillus subtilis U1and Bacillus subtilis Q2, 30{degrees}C for Achromobacter xylosoxidans G1, Stenotrophomonas maltophilia U3 and 25{degrees}C by Alcaligenes faecalis S3. The carbon sources put fluctuated effects on activity of enzyme in such a way that glucose was the best carbon source for Bacillus subtilis U1and Bacillus subtilis Q2, Sorbitol for Achromobacter xylosoxidans G1 and Alcaligenes faecalis S3 while xylose was the best for Stenotrophomonas maltophilia U3. Yeast extract and Trypton were among good nitrogen sources for Achromobacter xylosoxidans G1 and of Bacillus subtilis U1 respectively. Glutamine was the best inducer for Bacillus subtilis Q2, Alcaligenes faecalis S3 and Stenotrophomonas maltophilia U3, while lysine for Achromobacter xylosoxidans G1 and glycine act as good inducer in case of Bacillus subtilis U1. After implementation of optimal conditions microbial L-glutaminase production can be achieved and the bacterial isolates have a great potential for production of glutaminase enzyme and their applications.

The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive host for a range of applications in molecular biology and biotechnology. Moreover, the remarkable speed of growth of Vibrio natriegens poses fundamental questions on bacterial physiology and metabolism, energy production, DNA replication and protein synthesis, besides others. In order to address such questions, a solid understanding of the physiological and physical/chemical basis of growth requirements is essential. Here we report a systematic analysis of i) various growth media composition, ii) incubation temperature, iii) pH dependence, and iv) salt concentration requirements for optimal growth of V. natriegens strain DSMZ 759. As a result of the studies, the following optimal conditions were Established: LB medium with 2.5 % NaCl, pH 7.0 - 8.5 and incubation at 37{degrees}C under aerobic conditions. Incubation temperatures above 37 {degrees}C slows growth significantly. Incubation temperatures below 37 {degrees}C slows growth, but at a lower rate. Incubation at or below 28 {degrees}C should be avoided. Under such optimized, standard laboratory conditions, a doubling time of td = 13.6 minutes was observed for V. natriegens measured in mid-log growth phase. The optimized conditions presented here for the growth of V. natriegens can be easily applied in any standardly equipped laboratory. For comparison, identical growth conditions for Escherichia coli were analyzed and are presented as well.\n\nIMPORTANCEGoal of this study was to understand the physiological growths requirements of V. natriegens in routine microbiology and molecular biology laboratory settings. The result is a standardized protocol for the optimized growth of the naturally isolated (wild type) V. natriegens strain DSMZ 759. This protocol can be employed for routine application of V. natriegens for any kind of biochemical, molecular biology and genomic studies and utilization under normal laboratory conditions used by many routinely equipped laboratories.

BackgroundMultidrug resistant (MDR) Pseudomonas aeruginosa isolates harboring genes for virulence and antibiotic resistance, have grown more prevalent lately. These strains pose a major threat to the general population, especially in tertiary care settings. There is a paucity of information on toxigenic and virulence diversity of multidrug resistant P. aeruginosa in Nigeria, hence, the need to characterize and determine the variations of the virulence genes. MethodsSix hundred clinical samples from different anatomical sites were collected aseptically from Lagos University Teaching Hospital (LUTH), University of Medical Sciences, Ondo (UNIMED) and Federal Medical Centre, Abeokuta (FMC). Pseudomonas aeruginosa was isolated using cetrimide agar identified using biochemical tests. Antibiotic sensitivity was done by disc diffusion method. Protease, phospholipase C (lecithinase), caseinase and gelatinase presence were assayed for. Genomic DNA was extracted from P. aeruginosa isolates and screened for the presence of N-Acetylneuraminate synthase (NaN), Elastase B (Las B), Exotoxin A (ExoA), Exoenzyme S (ExoS) and Exoenzyme U (ExoU) virulence genes by PCR. ResultsThree hundred and sixty bacterial isolates identified from clinical samples are as follows: Pseudomonas aeruginosa (11.3%), Escherichia coli (18.0%), Klebsiella pneumoniae (14.3%), Staphylococcus aureus (10.2%), Proteus mirabilis (3.2%), Streptococcus pnuemoniae (2.3%), Enterobacter aerogenes (0.5%) and Acinetobacter baumanni (0.1%). Enzymes detected in the P. aeruginosa isolates were Phospholipase C (77.9%), caseinase (83.9%), gelatinase (98.5%) and protease (88.2%). The P. aeruginosa isolates were all resistant to ampicillin and cloxacillin; 26 (38.2 %) strains exhibited multidrug resistance. Virulence LasB elastase gene was detected in all 14 multi resistant P. aeruginosa, ExoA was detected in 5, ExoS in 4, ExoU in 5 and NaN in 4 isolates: Four (28.6%) ConclusionThe study confirmed presence and variations of toxic genes in Pseudomonas aeruginosa isolated from all the three tertiary hospitals.

ObjectivesThis study compared the resistomes of isolates of Pseudomonas aeruginosa clone ST308 from 2018 and 1997 from India. MethodsTwo ocular clonal type ST308 isolates of Pseudomonas aeruginosa (198 and 219) isolated in 2018 and five historical isolates (31, 32, 33, 35 and 37) isolated in 1997 at the LV Prasad Eye Institute in India were analysed for their susceptibilities to ciprofloxacin, levofloxacin, gentamicin, tobramycin, piperacillin, imipenem, ceftazidime and polymyxin B. DNA was extracted using the DNeasy(R) Blood and Tissue. Paired-end library was prepared using Nextera XT DNA library preparation kit. Libraries were sequenced on Illumina(R) MiSeq bench top sequencer generating 300 bp paired-end reads. Spades v3.12.0 was used for assembly, Resfinder v3.1. for acquired resistance genes and Snippy V2 for variants calling. Integron finder v1.5.1 was used to identify the integrons present in the genomes. ResultsThe recent isolate 219 was resistant to all tested antibiotics except polymyxin while isolate 198 was resistant to ciprofloxacin, levofloxacin, gentamicin and tobramycin. Among historical isolates five were resistant to gentamicin, tobramycin and ciprofloxacin, four were resistant to levofloxacin while two were resistant to polymyxin. Twenty-four acquired resistance genes were present in the 2018 isolates compared to 11 in the historical isolates. All isolates contained the following genes encoding for aminoglycoside aph(6)-Id, aph(3')-lIb, aph(3'')-Ib), beta-lactam (blaPAO), tetracycline (tet(G)), fosfomycin (fosA), chloramphenicol (catB7), sulphonamide (sul1), quaternary ammonium (qacEdelta1) and fluoroquinolone (crpP) resistance. Isolate 198 possessed aph(3')-VI, rmtD2, qnrVC1, blaOXA-488, blaPME-1, while 219 possessed aadA1, rmtB, aac(6')-Ib-cr, blaTEM-1B, blaVIM-2, mph(E), mph(A), msr(E). In the isolate 219 genes blaTEM-1b, blaVIM-2, sul1, qnrvc1, rmtB and aadA1 were carried on class 1 integron. While an incomplete class 1 integron was also found in isolate 198 which was located on the genome where gene rmtB, blaPME-1, qnrVC1 and sul1 genes were positioned. There were no notable differences in the number of single nucleotide polymorphisms, but recent isolates carried more insertions and deletions in their genes. ConclusionP. aeruginosa ocular clonal isolates have changed over time, with strains acquiring genes and having more insertions and deletions in their chromosomal genes that confirm resistance to antibiotics. HighlightsO_LIRecent clonal ocular isolates of Pseudomonas aeruginosa from India have acquired a number of resistance genes compared to historical clones C_LIO_LIConsequently, resistance to antibiotics particularly fluoroquinolones in recent clones of P. aeruginosa appears to have increased. C_LIO_LIThe acquired resistance genes found in the recent P. aeruginosa isolates were related to mobile genetic elements. C_LI

BackgroundScrub typhus a common cause of acute febrile illness in India caused by Orientia tsutsugamushi an obligate intracellular bacterium requiring cell culture for isolation. Cell lines like Vero and L929 are most suitable for isolating and maintaining this organism. This study was undertaken to isolate and characterize of Orientia tsutsugamushi from whole blood samples at a tertiary care centre in Southern India. MethodsThe PBMCs (peripheral blood mononuclear cells) collected from scrub typhus positive (47kDa qPCR positive) patients were inoculated into Vero and L929 cell line at 80% confluence for primary isolation. The inoculated flasks were incubated at 37{degrees}C with 5% CO2 for 30 days and examined for presence of Orientia tsutsugamushi on the day 10, 15, 20 post-inoculation and everyday thereafter for a maximum of 30 days post inoculation. The scrapings were subjected to Giemsa staining, IFA, 47kDa qPCR and transmission electron microscopy (TEM). The isolates were passaged 3-4 times to ensure viability and then stored in DMEM with 10% FBS (-80{degrees}C). Genotyping of the isolates was performed by amplifying a 650 bp segment of the TSA 56 (type specific antigen 56) gene. ResultsAmongst the 50 samples inoculated, three were culture positive as confirmed by 47 kDa qPCR at 24th day of inoculation. This was further confirmed by Giemsa, IFA staining and TEM. The 650bp amplicons showed 99.5 to 100% homology with Orientia tsutsugamushi MW604716, MH003839, MW604718, MW604717, MH922787 and MH003838 strains. Phylogenetic analysis revealed that 2 isolates belong to TA763 genotype and one belongs to Gilliam genotype. ConclusionsWe have successfully isolated and characterised the Orientia tsutsugamushi for the first time at our centre from PBMCs. Based on the partial TSA56 gene sequence our isolates belongs to TA763 and Gilliam genotype. More number of samples are being processed for identifying further isolates followed by genomic analysis.

Four clove clones (BRC1, BRC3 MRC5 and MRC7) were collected from different places of ponmudi hills, Kerala, India. DNA was extracted and RAPD analysis was designed to evaluate clove clonal variation. Upon analysis bacterial sequences were also identified among clove genome sequences. So the clove and microbial sequences were separated and performed an analysis to identify clove associated metagenome. The major microbes present were Mycobacterium canettii Xanthomaonas euvesicateria, Klebsiella and Psuedomonas. A total of 88 genus of microbes were present altogether in four clones of Syzygium aromaticum. There were few fungal species were also identified, Fusarium pseudograminearum was the major fungus associated with all four different clones of clove.

The cyanobacteriochrome GAF domains represent a trove of spectral diversity. These proteins are endemic to cyanobacteria and sense the color and power of light. Multiple mechanisms are used to tune the natural absorbance spectrum of the bound bilin chromophore. In practice, these are difficult to identify from the predicted amino acid sequence. Their individual presence rarely yields a consistent and predictable outcome. The absorbance characteristics of the GAF domain are a complex function of many such tuning mechanisms. This implies that a more combinatoric approach to characterizing the diversity of GAF domains would better to predict spectral tunes. We reviewed the literature and constructed a dataset of predicted/confirmed cyanobacteriochrome GAF domains. This dataset was subjected to multiple sequence alignments and 18 GAF domain families were defined. The amino acid sequence similarity correlated well with known spectral characteristics but there were exceptions. A second approach to predict chromotype involved using Principal Component Analysis to characterize the whole domain architectures of cyanobacteriochrome. This approach identified 7 conserved domain architectures, with some variations. These also offered a correlation to the spectral tune of the GAF domains therein, in addition to the 18 GAF families. The three-dimensional structures of 98 spectrally characterized GAF domains were predicted using Phyre2. Subsequent grouping based on distance maps offered an insight into how the general spectral position of the domain is set. Finer tuning is likely to be achieved by means of six key residues within the binding pocket. Taken together, these insights allowed us to carry out a Multiple Correlation Analysis serving as a mathematical summary of the diversity of cyanobacteriochrome GAF domains. This summary or "cyanobacteriochrome atlas" can be used to make spectral predictions on uncharacterized GAF domains.

Streptomycetaceae is one of the oldest families within phylum Actinobacteria and it is large and diverse in terms of number of described taxa. The members of the family are known for their ability to produce medically important secondary metabolites and antibiotics. In this study, strains showing low 16S rRNA gene similarity (&lt;97.3 %) with other members of Streptomycetaceae were identified and subjected to phylogenomic analysis using 33 orthologous gene clusters (OGC) for accurate taxonomic reassignment resulted in identification of eight distinct and deeply branching clades, further average amino acid identity (AAI) analysis showed lower AAI values or AAI within the range of 60-80 % which was previously observed in related but different genera of bacteria. The whole genome phylogeny based on concatenated core genes and AAI analyses supported the claim that those phylogenetically distinct members may be assigned to 8 novel genera namely Actinoacidiphila, Actinomesophilus, Charcoactinospora, Curviacidiphilus, Kafeoacidiphilus, Mangroviactinospora, Peterkaempfera, and Streptantibioticus. In addition, based on the core genome phylogeny and 16S rRNA tree topology and distinct chemotaxonomic and physiological properties, the sequence belonged to Streptomyces thermoautotrophicus was assigned to a novel genera Charcoactinospora which is placed under novel family Charcoactinosporaceae. Lastly, a clade comprising of strains that showed high 16S rRNA gene similarity (100 %) with similar tree topology in phylogenetic trees was subjected to overall genome related indices analyses such as digital DNA – DNA hybridization, and average nucleotide identity that supported the claim that Streptomyces asterosporus is a later heterotypic synonym of Streptomyces calvus.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsOGCOrthologous gene clusterAAIaverage amino acid identitydDDHdigital DNA-DNA hybridizationANIaverage nucleotide identityView Full Text

Back groundEarlier studies suggested the use of dry swab method for SARS-CoV-2 detection as it does not need VTM and subsequent RNA extraction step making the process cheaper, safer and faster. In this study we explore whether the virus in the dry swab is viable and can be cultured and propagated. MethodSwabs were spiked with SARS-CoV-2 and stored in three different conditions: a) as dry swab (SD, eluted in 1 mL DMEM), b) in 1 mL of Viral Transport Medium (SVTM), and c) in 1 mL of Tris-EDTA buffer (STE). The sample groups were stored either at room temperature (RT, 25{degrees}C{+/-}1{degrees}C) or at 4{degrees}C for 1, 4, 8, 12, 24, 48 and 72 hours before being used as viral inoculums for the propagation studies in Vero cells. ResultsThe RT-qPCR data suggests that SD incubated both at RT and 4{degrees}C harbors viral particles that are viable and culturable at par with SVTM and STE. ConclusionThe dry swab method, in addition to its advantages in detection of the virus, also renders viable viral particles that can be cultured and propagated.

Melanins are ubiquitous black or brown color pigments exhibiting a wide variety of bioactivities. They are stable and insoluble in nature. Melanin are industrially important pigment currently used in cosmetics, medicine and pharmaceuticals, industries. Bacteria mainly produce three types of melanin namely, Eumelanin, pheomelanin and pyomelanin which is usually extracellularly secreted. This makes the downstream processing of bacterial melanin easier. Stress conditions like salt stress, radiation stress etc. triggers the bacteria to produce melanin and several bacterial species were found to produce melanin under different stress induced conditions. In this present study we had isolated melanin producing bacteria from saline sediment sample of Mundra port, Kutch region of Gujarat state of India. The melanin producing bacteria was characterized using staining, biochemical and molecular methods. The melanin produced was extracted and analyzed using physicochemical techniques. The extracted melanin had shown good anti-bacterial and radical scavenging activity. To our knowledge, this is the first report on Bacillus altitudinis producing melanin.

IntroductionThe Mycobacterium. tuberculosis (MTB) detected trace, rifampicin (RIF) resistance indeterminate category of the Xpert MTB/RIF Ultra assay results is usually, non-actionable and requires retesting the samples. We aimed to decipher RIF resistance among tuberculosis patients who have trace and indeterminate results. MethodsFour hundred and three (403) MTB detected trace, RIF resistance indeterminate results, which were obtained in Mycobacteriology (BSL-3) and Molecular Diagnostic laboratories, College of Health Sciences, Makerere University from August 2018 to June 2023, having culture results were identified from the laboratory database. Isolates of those that turned out culture positive were retrieved and sub-cultured in liquid media to perform phenotypic first line Drug Susceptibility tests, first line Line-Probe assays (LPA) and repeat GeneXpert ultra. ResultsA total of 31/403 (7.7%) culture positive isolates were identified from the database of which 77.42% (24/31) were positive for Mycobacterium tuberculosis complex (MTBc). Nineteen (19) out of the identified 24 MTBc were successfully retrieved, cultured and resistance testing performed. Phenotypic Drug susceptibility testing and repeated GeneXpert did not identify any resistance. Only one mutation inhA MUT1 related to isoniazid (INH) resistance was identified using MTBDRplus assay. ConclusionIn this study, we did not identify any missed rifampicin resistance among MTBc culture positive samples that were initially Xpert ultra-trace and rifampicin resistance indeterminate. More studies with bigger sample sizes especially in high MDR-TB settings are required.

Stenotrophomonas maltophilia is a gram-negative opportunistic pathogen that causes respiratory, urinary and bloodstream infections. Due to rising prevalence of difficult to treat S. maltophilia infections, it is considered as one of the important pathogens in many regions, including India. Despite its importance in public health, very few studies provide detailed characterization of the genomes of clinical isolates of S. maltophilia. In this study, we sequence six isolates of S. maltophilia from a tertiary healthcare centre in the Northern India. Along with the culture sensitivity, we report the genomic underpinnings of resistance and virulence in these isolates. In three out of six isolates, we identify a rare beta-lactamase gene kbl-1 that can confer resistance to variety of beta-lactam antibiotics. Though truncated in two isolates, an intact copy of kbl-1 is present in the third isolate. This intact copy of kbl-1 is flanked by insertion elements that are commonly found in several pathogenic species indicating high potential for horizontal gene transfer. To the best of our knowledge, this is a first report of kbl-1 in S. maltophilia from India. ImportanceAntimicrobial resistance (AMR) is one of the biggest global public health challenges. Tackling AMR gets complicated as diverse pathogenic species are involved and resistance is manifested against several different kinds of antibiotics. Genomic surveillance of the resistant pathogens is a key to fight this global threat and needs to extend to important emerging pathogens as well. Here we report genomic sequences and associated characteristics of six Stenotrophomonas maltophilia isolates from Northern India. Along with several resistance and virulence genes we also discover a rare beta-lactamase, blaKBL-1, gene with a high potential of transfer to other species. blaKBL-1 beta-lactamase confers resistance to several beta-lactam antibiotics like ampicillin, amoxicillin, penicillin G, piperacillin, ceftazidime and cefozopran. This is the first report of the presences of this beta-lactamase from India.