Access Microbiology

The effort to discover novel phages infecting Staphylococcus epidermidis contributes to both the development of phage therapy and the expansion of genome-based phage phylogeny. Here, we report the genome of an S. epidermidis-infecting phage SEP1 and compare its genome with five other sequenced phages with high sequence identity. These phages represent a novel siphovirus genus, which was recently reported in the literature. The published member of this group was favorably evaluated as a phage therapeutic agent, but SEP1 is capable of transducing antibiotic resistance. Members of this genus may be maintained within their host as extrachromosomal plasmid prophages, through stable lysogeny or pseudolysogeny. Therefore, we conclude that SEP1 may be temperate and members of this novel genus are not suitable for phage therapy.

BackgroundBacteriophages are ubiquitous, highly diverse, and relatively understudied. Growing interest in phage therapy has underscored the importance of isolating and characterizing novel bacteriophages. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program aims to address this need by involving undergraduates around the world in authentic research. Materials and MethodsNine novel mycobacteriophages - AgentM, Ajay, Aragog, Archetta, ForGetIt, Koko, Ph8s, Phlorence, and Wilkins - were isolated from soil samples in southern California using host Mycobacterium smegmatis mc2155. Each purified phage was characterized using transmission electron microscopy and genome sequencing and annotation. ResultsAll nine bacteriophages were placed into mycobacteriophage Cluster A based on nucleotide similarity with other phages. The average genome length of all nine phages was 50,706 bp. On average, each phage had 87 total coding genes and GC content between 60-63%, consistent with that of other Cluster A phages. Transmission electron microscopy of phage particles revealed they had icosahedral heads and long, flexible tails, consistent with that of the Siphoviridae family. Plaque morphology and genome analysis confirms the nine novel phages are temperate as expected of Cluster A phages. ConclusionsAgentM, Ajay, Aragog, Archetta, ForGetIt, Koko, Ph8s, Phlorence, and Wilkins are all mycobacteriophages that belong to the Siphoviridae family. Comparative genomic analyses revealed genetic mosaicism and diversity among these Cluster A phages. The discovery of these novel phages expands on the existing library of mycobacteriophage genomes.

Several new variants of the SARS-CoV-2 have been isolated in the United States, Mexico, and Canada. Many of the variants contain single variants of functional significance (e.g. S: N501Y increases transmissibility). To study the occurrence and co-circulation of these variants, we have developed an easy-to-use dashboard at janieslab.github.io/sars-cov-2. We created a multiple sequence alignment workflow and processing script to generate a variant dataset, which populates this dashboard. We then use the features of the dashboard, such as visualization of the single and complex nucleotide variants geospatially and in a color-coded matrix format. Users also interact with the dashboard to filter the underlying data to regions of interest and or variants of interest. The user can export reports based on the desired filters, which we intend to be used for regionally specific pandemic response. We find in Genbank, an isolate from Massachusetts containing [(S: Q677H), (ORF3a: Q57H), (M: A85S), (N: D377Y)] collected on September 11, 2020. Moreover, we find that many viral isolates bear a marker of increased transmissibility (S: N501Y) in linkage with at least one variant of concern isolated from Ohio also range across the Untied States and stretch from British Columbia, Canada to Mexico. When we analyze co-circulation of more complex variant constellations with (S: N501Y), we note that the Upper Midwest and Northeast United States contain these isolates. In summary, the viral variants that have raised concern in a few US States in recent reports are widespread. Based on the increase in the proportion of variant viruses being sampled and some empirical evidence in the United Kingdom, South Africa, and Ohio, these variants are likely to lead to increased transmission of SARS-CoV-2 across North America in the coming months.

The concurrent presence of multiple New Delhi metallo-{beta}-lactamase (blaNDM) variants within an isolate often goes undetected without the use of next-generation sequencing. This study detects and characterizes dual blaNDM-variants in Escherichia coli through Sanger and whole-genome sequencing. Additionally, a rapid identification method utilizing restriction digestion was designed for detecting variants carrying M154L mutation. Antibiotic susceptibility, minimal inhibitory concentration for meropenem and ertapenem, PCR and Sanger sequencing of blaNDM along with genome sequencing using Ilumina and Nanopore technology were conducted. Transmissibility and replicon types of blaNDM-harbouring plasmids were evaluated. Restriction digestion using restriction enzyme, BtsCI was developed to distinguish between blaNDM-1 and variants possessing M154L mutations. Two isolates belonging to phylogroups A; ST167 and B1; ST101 and resistant to meropenem and ertapenem ([≥]16mg/L) were recovered from the blood of a neonate and the rectal swab of a pregnant woman respectively. blaNDM was detected by PCR, and Sanger sequences of blaNDM showed two peaks at 262 (G & T) and 460 (A & C) nucleotide positions indicative of more than one blaNDM variant. Hybrid assembly confirmed co-existence of blaNDM-1 and blaNDM-5 in each isolate. blaNDM-1 was located on IncY (ST167) and IncHI1A/HI1B (ST101), while blaNDM-5 was on a IncFIA/FII (ST167) and IncC (ST101) plasmids. Digestion with BtsC1 could discriminate blaNDM-1 and blaNDM-5. Co-existence of multiple blaNDMS, blaNDM-1 and blaNDM-5 in epidemic clones of E. coli is concerning. Restriction digestion method and Sanger sequencing can facilitate quick identification of dual blaNDM variants in single isolate. ImportanceThe global dissemination of antimicrobial resistance genes is a serious concern. One such gene, blaNDM, has spread all across the globe via plasmids. blaNDM confers resistance against all {beta}-lactam antibiotics except monobactams. Most of the earlier literature reported presence of single blaNDM variants. However, this study reports the prevalence of dual blaNDM variants (blaNDM-1 and blaNDM-5) located on two separate plasmids identified in two distinct E. coli epidemic clones ST167 and ST101; isolated from a septicaemic neonate and a pregnant mother respectively. blaNDM-5 differs from blaNDM-1 due to presence of two point mutations i.e., V88L and M154L. This study detected dual blaNDM-variants through Sanger sequences and further validated through hybrid-genome assembly. Detection of multiple blaNDM-variants in a single isolate remains difficult until genome sequencing or southern blotting are carried out. Hence, a simple restriction digestion method was devised for rapid screening of dual blaNDM-variants containing M154L mutation.

Currently the primary method for confirming acute SARS-CoV-2 infection is through the use of molecular assays that target highly conserved regions within the viral genome. Many, if not most of the diagnostic targets currently in use were produced early in the pandemic, using genomes sequenced and shared in early 2020. As viral diversity increases, mutations may arise in diagnostic target sites that have an impact on the performance of diagnostic tests. Here, we report on a local outbreak of SARS-CoV-2 which had gained an additional mutation at position 28890 of the nucleocapsid protein, on a background of pre-existing mutations at positions 28881, 28882, 28883 in one of the main circulating viral lineages in Wales at that time. The impact of this additional mutation had a statistically significant impact on the Ct value reported for the N gene target designed by the Chinese CDC and used in a number of commercial diagnostic products. Further investigation identified that, in viral genomes sequenced from Wales over the summer of 2020, the N gene had a higher rate of mutations in diagnostic target sites than other targets, with 115 issues identified affecting over 10% of all cases sequenced between February and the end of August 2020. In comparison an issue was identified for ORFab, the next most affected target, in less than 1.4% of cases over the same time period. This work emphasises the potential impact that mutations in diagnostic target sites can have on tracking local outbreaks, as well as demonstrating the value of genomics as a routine tool for identifying and explaining potential diagnostic primer issues as part of a laboratory quality management system. This work also indicates that with increasing genomic sequencing data availability, there is a need to re-evaluate the diagnostic targets that are in use for SARS-CoV-2 testing, to better target regions that are now demonstrated to be of lower variability.

A re-usable water bottle was swabbed as part of the citizen science project Swab and Send and a Klebsiella grimontii isolate was recovered on chromogenic agar and designated SS141. Whole genome sequencing of SS141 showed it has the potential to be a human pathogen as it contains the biosynthetic gene cluster for the potent cytotoxin, kleboxymycin, and genes for other virulence factors. The genome also contains blaOXY-6-4 and fosA which is likely to explain the observed resistance to ampicillin, amoxicillin and fosfomycin. We have also shown that SS141 is a potent biofilm former, providing a reasonable explanation for its ability to colonise a re-usable water bottle. With the increasing use of re-usable water bottles as an alternative to disposables, and a strong forecast for growth in this industry over the next decade, this study highlights the need for cleanliness comparable to other re-usable culinary items.

Double stranded RNA (dsRNA) is the genetic material of important viruses and a key component of RNA interference-based immunity in eukaryotes. Previous studies have noted difficulties in determining the sequence of dsRNA molecules that have affected studies of immune function and estimates of viral diversity in nature. Dimethyl sulfoxide (DMSO) has been used to denature dsRNA prior to the reverse transcription stage to improve RT-PCR and Sanger sequencing. We systematically tested the utility of DMSO to improve sequencing yield of a dsRNA virus ({Phi}6) in a short-read next generation sequencing platform. DMSO treatment improved sequencing read recovery by over two orders of magnitude, even when RNA and cDNA concentrations were below the limit of detection. We also tested the effects of DMSO on a mock eukaryotic viral community and found that dsRNA virus reads increased with DMSO treatment. Furthermore, we provide evidence that DMSO treatment does not adversely affect recovery of reads from a single-stranded RNA viral genome (Influenza A/California/07/2009). We suggest that up to 50% DMSO treatment be used prior to cDNA synthesis when samples of interest are composed of or may contain dsRNA.\n\nData SummarySequence data was deposited in the NCBI Short Read Archive (accession numbers: PRJNA527100, PRJNA527101, PRJNA527098). Data and code for analysis is available on GitHub (https://github.com/awilcox83/dsRNA-sequencing/, doi:10.5281/zenodo.1453423). Protocol for dsRNA sequencing is posted on protocols.io (doi:10.17504/protocols.io.ugnetve).

Microorganisms evolve novel mechanisms and pathways to mitigate various stresses. These are developed by the accumulation of beneficial mutations over many generations. Such adaptations are often transferred from mutant microorganisms that develop it intrinsically to wild ones through horizontal gene transfer (HGT). It allows the latter to acquire favourable traits without having to employ resources to natively evolve. We ascertained this in Escherichia coli by first developing tolerance to ethanol, a potent disinfectant, by laboratory evolution and then transmitting it to the wild strain by HGT. Naturally, wild type E. coli cannot survive beyond 35% v/v ethanol in LB media. By serially increasing the concentration of ethanol by 5% v/v and selecting the surviving colonies, we were able to impose an artificial selection pressure. This in vitro microevolution increased the ethanol tolerance in our mutant to 75% v/v. To test if this tolerance could be transferred to the wild strain through HGT, we meticulously exposed the ancestral wild E. coli to the newly tolerant mutants to facilitate the exchange of genetic material. After our exposure, the unadapted wild type E. coli acquired tolerance up to 55% v/v through transformation and 45% v/v through transduction. The baseline tolerance of 35% v/v remained unchanged after conjugation. While our results are still preliminary, they provide interesting insights into the role horizontal gene transfer in developing resistance to bacteriocidal stressors.

The pathogen Streptococcus agalactiae, or Group B Streptococcus (GBS) infection is the leading cause of neonatal sepsis and meningitis in neonates. In this study, we aimed to investigate the occurrence and diversity of the CRISPR-Cas system in S. agalactiae genomes using computational biology approaches. A total of 51 out of 52 complete genomes (98.07%) of S. agalactiae possess CRISPR arrays (75 CRISPR arrays) with 17 strains possessing multiple CRISPR arrays. There were only two CRISPR-Cas systems - type II-A system and type I-C system in S. agalactiae strains. RNA secondary structure analysis through direct repeat analysis showed that the analyzed strains could form stable secondary structures. The 16S rRNA phylogeny exhibited clustering of the strains into three major clades grouped on the type of CRISPR-Cas system. The anti-CRISPRs that contribute to CRISPR-Cas system diversity and prevent genome editing were also detected. These results provide valuable insights into elucidating the evolution, diversity, and function of CRISPR/Cas elements in this pathogen.

Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia, an economically important lung disease of pigs. In draft genomes of two Cypriot clinical A. pleuropneumoniae isolates (MIDG3457 and MIDG3459), we previously identified single genomic regions with homology to Mu-like bacteriophage and presented preliminary evidence of active phage. Here, updated Phastest genomic analysis identified two loci in both MIDG3457 and MIDG3459 that were predicted to encode proteins with high homology to, and whose organization was characteristic of, Mu-like phages. Phylogenetically, the closest matches were with Mannheimia Vb and Glaesserella SuMu phages. Phastest scored the loci as "complete", indicating they produced active phage. PCR amplification of the Mu-like phage c and tail genes from DNase-treated polyethylene glycol 8000 (PEG)-precipitated supernatants of MIDG3457 and MIDG3459 (grown in either Brain Heart Infusion-NAD or Graces Insect Medium-NAD broth) indicated the presence of intact virions. The phages from MIDG3457 and MIDG3459 were named PluMu 3457-1, 3457-2, and PluMu 3459-1 and PluMu 3459-2, respectively. Transmission electron microscopy (TEM) of PEG-precipitated supernatants of broth-grown MIDG3459 identified virions with icosahedral heads and tails, consistent with other Mu-like phages. We conclude that MIDG3459 produces an active Mu- like phage.

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.

The pandemic SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) has created a widespread panic across the globe especially in the developing countries like Pakistan. The lack of resources and technical staff are causing havoc challenges in the detection and prevention of this global outbreak. Therefore, a less expensive and massive screening of suspected individuals for COVID-19 is required. In this study, a user-friendly technique of reverse transcription-loop mediated isothermal amplification (RT-LAMP) was designed and validated to suggest a potential RT-qPCR alternate for rapid testing of COVID-19 suspected individuals. A total of 12 COVID-19 negative and 72 COVID-19 suspected individuals were analyzed. Both RT-qPCR and RT-LAMP assays were performed for all the individuals using open reading frame (ORF 1ab), nucleoprotein (N) and Spike (S) genes. All 12 specimens which were negative using RT-qPCR were also found negative using RT-LAMP assay. Overall 62 out of 72 positive samples (detected using RT-qPCR) were found COVID-19 positive using RT-LAMP assay. Interestingly all samples (45) having Ct values less than 30 showed 100% sensitivity. However, samples with weaker Ct values (i.e., => 35) showed 54% concordance, suggesting potential false negatives or false positives in RT-LAMP or RT-qPCR results, respectively. Overall comparative assessment showed that RT-LAMP assay showed strong sensitivity and specificity and can be used as an alternative strategy for rapid COVID-19 testing. Hence, based on fast processing time, minimal risk of specimens transfer and utilizing available resources, LAMP based detection of COVID-19 is strongly advocated especially for developing countries.

This in vitro study determined the anti-viral efficacy of a unique blend of powder cellulose supplemented with powdered garlic extract (PGE) and a signalling agent. The composition, presented as Nasaleze(R) Cold & Flu Blocker/Nasaleze(R) Travel, was assessed against Human Coronavirus 229E, CoV 229E {ATCC VR-740} in an in vitro experiment. The test substance was used at sub-optimal dosing levels to explore its prevention and treatment capabilities. The virucidal activity of this novel formulation was measured at 48, 72 and 112 hour periods after incubation. Results showed strong reductions in viral titre of Coronavirus 229E compared to a control, while no toxicity to human cells from the test formulation was noted. The extract Nasaleze(R) Cold/Travel showed potential to be used as a therapeutic and preventive agent. The data reconfirms the established anti-viral activity of this formulation acting as a barrier preventing the virus from accessing the nasal mucosa and disrupting its replication.1,2,3

S-gene target failure (SGTF) is neither specific nor accurate for identification of Omicron lineage of SARS-CoV-2. We observed N-gene target failure (NGTF) in 402 out of 412 SARS-CoV2 positive cases from December to mid-January 2022 using a commercially available assay. This phenomenon was not observed with more than 15,000 cases tested previously. We sequenced the genome of five samples with NGTF and compared these results with six cases where NGTF was not seen. We confirm that cases with NGTF were the Omicron lineage while cases with preserved N-gene amplification belonged to Delta lineage. We discovered that the ERS31-33 deletion (nucleotide 28362-28370del) overlaps with N gene probe used, explaining NGTF. As the stealth Omicron variant also harbors ERS31-33 deletion, this approach will work for the detection of stealth Omicron variant as well. We suggest that NGTF can be used as a low cost, rapid screening strategy for detection of Omicron.

The spread and rise of antimicrobial resistance poses a risk to public health due to limited effective treatment options. Alternative antimicrobials that are effective against gram-negative multi-drug resistant pathogens. The increasing rate of carbapenem resistance observed in Klebsiella pneumoniae, indicates the need for alternative antimicrobial options. Bacteriophages that target Klebsiella pneumoniae are promising alternative antimicrobial option, with successful treatments being reported. Here we characterized 30 lytic bacteriophages from various environmental sources and tested their effectiveness against nine clinically relevant carbapenem-resistant K. pneumoniae isolates. These phages were characterized through genomic sequencing, bioinformatic analysis, virulence in liquid medium, and host range on different mediums. Bioinformatic analysis revealed a diverse collection of phages that span 9 ICTV recognized families and 13 genera with genome sizes ranging from 39-349 kbp. The phages were able to inhibit bacterial growth, and no virulence or antibiotic resistance genes were detected within the phage genomes. Host range testing demonstrated phages with broad host range have varying infectivity when plated on different common growth mediums. This study includes candidate phages for further potential development as potential antimicrobial agents against CR-KP, and the complexity in understanding phage-host dynamics of non-capsule phages that target against K. pneumoniae.

We present an optimised method for the recovery of laboratory generated SARS-CoV-2 virus by plaque assay. This method allows easy incorporation into existing standard operating procedures of biological containment level 3 (BCL3) laboratories.

The transmission of SARS-CoV-2 is likely to occur through a number of routes, including contact with contaminated surfaces. Many studies have used RT-PCR analysis to detect SARS-CoV-2 RNA on surfaces but seldom has viable virus been detected. This paper investigates the viability over time of SARS-CoV-2 dried onto a range of materials and compares viability of the virus to RNA copies recovered, and whether virus viability is concentration dependant. Viable virus persisted for the longest time on surgical mask material and stainless steel with a 99.9% reduction in viability by 124 and 113 hours respectively. Viability of SARS-CoV-2 reduced the fastest on a polyester shirt, with a 99.9% reduction within 2.5 hours. Viability on cotton was reduced second fastest, with 99.9% reduction in 72 hours. RNA on all the surfaces exhibited a one log reduction in genome copy recovery over 21 days. The findings show that SARS-CoV-2 is most stable on non-porous hydrophobic surfaces. RNA is highly stable when dried on surfaces with only one log reduction in recovery over three weeks. In comparison, SARS-CoV-2 viability reduced more rapidly, but this loss in viability was found to be independent of starting concentration. Expected levels of SARS-CoV-2 viable environmental surface contamination would lead to undetectable levels within two days. Therefore, when RNA is detected on surfaces it does not directly indicate presence of viable virus even at high CT values. ImportanceThis study shows the impact of material type on the viability of SARS-CoV-2 on surfaces. It demonstrates that the decay rate of viable SARS-CoV-2 is independent of starting concentration. However, RNA shows high stability on surfaces over extended periods. This has implications for interpretation of surface sampling results using RT-PCR to determine the possibility of viable virus from a surface. Unless sampled immediately after contamination it is difficult to align RNA copy numbers to quantity of viable virus on a surface.

A number of bacteria with close resemblance to Vibrio cholerae has been isolated over the years by the Centers for Disease Control and Prevention (CDC), which could not be assigned a proper taxonomic designation based on preliminary identification methods. Nine such isolates have been found to share 16S rRNA gene identity exceeding 99% with V. cholerae, yet DNA-DNA hybridization (60.4-62.1%) and average nucleotide identity values (94.4-95.1%) were below the species cut-off, indicating a potentially novel species. Phylogenetic analysis of core genomes places this group of isolates in a monophyletic clade, within the "Cholerae clade," but distinct from any other species. Extensive phenotypic characterization reveals unique biochemical properties that distinguish this novel species from V. cholerae. Comparative genomic analysis reveals a unique set of siderophore genes, suggesting that iron acquisition strategies could be vital for the divergence of the novel species from a common ancestor with V. cholerae. Based on genetic, phylogenetic, and phenotypic differences observed, we propose these isolates represent a novel species of the genus Vibrio, for which the name Vibrio tarriae sp. nov. is proposed. Strain 2521-89 (= DSM 112461 = CCUG 75318), isolated from lake water, is the type strain. Author NotesThe GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 2521-89 is MW773202.1. The genome sequences (genome assemblies) of strains 2521-89, 2523-88, 2015V-1076, 2016V-1018, 2016V-1062, 2017V-1038, 2017V-1070 and 2017V-1124 are deposited under the accession number NZ_CP022353.1, QKKG01000001.1, QKKH00000000.1, QKKI00000000.1, QKKJ00000000.1, QKKK00000000.1, QKKM00000000.1, and QKKN00000000.1, respectively. All the whole genome sequences are deposited under bioproject ID: PRJNA391152.

Due to the adverse effects of synthetic colours on human health and the environment, there is a rapid shift towards the use of colours from natural sources like plants and microorganisms. Many pigment-producing microorganisms are identified and isolated from extreme environments like glaciers, ice cores, marine surface waters, etc. In this study, we have isolated 4 distinct pigment-producing bacterial strains from an Arctic stone sample collected from the vicinity of the Indian Research Station Himadri (78{degrees}55'N 11{degrees}56'E), located at the International Arctic Research Base, Ny[A]lesund, Svalbard, Norway. Pigment production was optimised by identifying the right growth medium, temperature, pH, and incubation period. The morphological, cultural, and biochemical characteristics were identified using several experiments like Gram Staining, Catalase Test, Oxydative-Fermentative Test, etc. The objective of this study is to identify novel bacterial strains capable of producing distinct pigments for pharmaceutical and industrial applications.

During infection and latency, Mycobacterium tuberculosis (Mtb) primarily relies on lipids as its main energy source. The Mtb mammalian cell entry (Mce) complexes function as lipid transporters with Mkl as the ATPase subunit for substrate translocation. Cholesterol and mycolic acid are essential to the infectiousness and pathogenicity of Mtb. Here, we have investigated the transcriptomic responses of wild type and{Delta} mkl strains of Mtb to cholesterol and mycolic acid through RNAseq analysis. The number of differentially expressed genes was always higher when grown on cholesterol than on mycolic acid. In most cases,{Delta} mkl showed the opposite gene expression pattern compared to the wild type. Among the four mce operons, significant changes in expression patterns were observed mainly in the genes of mce3 and mce4 operons. The upregulation of mce3 operon and the downregulation of mce4 operon in the presence of mycolic acid and cholesterol is intriguing. The transcriptome profile of genes involved in mycolic acid synthesis did not show significant change to either mycolic acid or cholesterol. However, interestingly, both mycolic acid and cholesterol induce upregulation of methyl citrate cycle and dormancy related genes indicating their significance during Mtb persistence and provides insights on Mtbs adaptive strategies under stress through transcriptional remodelling.