- Volume 5, Issue 6, 2023

Enterococcus raffinosus is an understudied member of its genus possessing a characteristic megaplasmid contributing to a large genome size. Although less commonly associated with human infection compared to other enterococci, this species can cause disease and persist in diverse niches such as the gut, urinary tract, blood and environment. Few complete genome assemblies have been published to date for E. raffinosus . In this study, we report the complete assembly of the first clinical urinary E. raffinosus strain, Er676, isolated from a postmenopausal woman with history of recurrent urinary tract infection. We additionally completed the assembly of clinical type strain ATCC49464. Comparative genomic analyses reveal inter-species diversity driven by large accessory genomes. The presence of a conserved megaplasmid indicates it is a ubiquitous and vital genetic feature of E. raffinosus . We find that the E. raffinosus chromosome is enriched for DNA replication and protein biosynthesis genes while the megaplasmid is enriched for transcription and carbohydrate metabolism genes. Prophage analysis suggests that diversity in the chromosome and megaplasmid sequences arises, in part, from horizontal gene transfer. Er676 demonstrated the largest genome size reported to date for E. raffinosus and the highest probability of human pathogenicity. Er676 also possesses multiple antimicrobial resistance genes, of which all but one are encoded on the chromosome, and has the most complete prophage sequences. Complete assembly and comparative analyses of the Er676 and ATCC49464 genomes provide important insight into the inter-species diversity of E. raffinosus that gives it its ability to colonize and persist in the human body. Investigating genetic factors that contribute to the pathogenicity of this species will provide valuable tools to combat diseases caused by this opportunistic pathogen.

Xylanase breaks xylan down to xylose, which is used in industries such as pulp and paper, food and feed, among others. The utilization of wastes for xylanase production is economical, hence this work aimed at producing xylanase through solid-state fermentation and characterizing the enzyme. Xylanase-producing strains of Bacillus megaterium and Aspergillus niger GIO were inoculated separately in a 5 and 10 day solid fermentation study on maize straw, rice straw, sawdust, corn cob, sugarcane bagasse, conifer litters, alkaline-pretreated maize straw (APM) and combined alkaline and biological-pretreated maize straw, respectively. The best substrate was selected for xylanase production. The crude enzyme was extracted from the fermentation medium and xylanase activity was characterized using parameters such as temperature, cations, pH and surfactants. Among different substrates, the highest xylanase activity of 3.18 U ml−1 was recorded when A. niger GIO was grown on APM. The xylanase produced by A. niger GIO and B. megaterium had the highest activities (3.67 U ml−1 and 3.36 U ml−1) at 40 °C after 30 and 45 min of incubation, respectively. Optimal xylanase activities (4.58 and 3.58 U ml−1) of A. niger GIO and B. megaterium , respectively, were observed at pH 5.0 and 6.2. All cations used enhanced xylanase activities except magnesium ion. Sodium dodecyl sulfate supported the highest xylanase activity of 6.13 and 6.90 U ml−1 for A. niger GIO and B. megaterium , respectively. High yields of xylanase were obtained from A. niger GIO and B. megaterium cultivated on APM. The xylanase activities were affected by pH, temperature, surfactants and cations.

Introduction. Culture is the gold-standard diagnosis for urinary tract infections (UTIs). However, most hospitals in low-resource countries lack adequately equipped laboratories and relevant expertise to perform culture and, therefore, rely heavily on dipstick tests for UTI diagnosis.

Research gap. In many Kenyan hospitals, routine evaluations are rarely done to assess the accuracy of popular screening tests such as the dipstick test. As such, there is a substantial risk of misdiagnosis emanating from inaccuracy in proxy screening tests. This may result in misuse, under-use or over-use of antimicrobials.

Aim. The present study aimed to assess the accuracy of the urine dipstick test as a proxy for the diagnosis of UTIs in selected Kenyan hospitals.

Methods. A hospital-based cross-sectional method was used. The utility of dipstick in the diagnosis of UTIs was assessed using midstream urine against culture as the gold standard.

Results. The dipstick test predicted 1416 positive UTIs, but only 1027 were confirmed positive by culture, translating to a prevalence of 54.1 %. The sensitivity of the dipstick test was better when leucocytes and nitrite tests were combined (63.1 %) than when the two tests were separate (62.6 and 50.7 %, respectively). Similarly, the two tests combined had a better positive predictive value (87.0 %) than either test alone. The nitrite test had the best specificity (89.8 %) and negative predictive value (97.4 %) than leucocytes esterase (L.E) or both tests combined. In addition, sensitivity in samples from inpatients (69.2 %) was higher than from outpatients (62.7 %). Furthermore, the dipstick test had a better sensitivity and positive predictive value among female (66.0 and 88.6 %) than male patients (44.3 and 73.9 %). Among the various patient age groups, the dipstick test’s sensitivity and positive predictive value were exceptionally high in patients ≥75 years old (87.5 and 93.3 %).

Conclusion. Discrepancies in prevalence from the urine dipstick test and culture, the gold standard, indicate dipstick test inadequacy for accurate UTI diagnosis. The finding also demonstrates the need for urine culture for accurate UTI diagnosis. However, considering it is not always possible to perform a culture, especially in low-resource settings, future studies are needed to combine specific UTI symptoms and dipstick results to assess possible increases in the test’s sensitivity. There is also a need to develop readily available and affordable algorithms that can detect UTIs where culture is not available.

Streptococcus pneumoniae is one of the pathogenic bacteria causing invasive pneumococcal diseases such as pneumonia, sepsis, and meningitis, which are commonly reported in children and adults. In this study, we investigated the nasopharyngeal carriage rates, serotype distribution, and antimicrobial susceptibility profiles of S. pneumoniae among children with pneumonia and healthy children under 5 years old in Padang, West Sumatra, Indonesia. Nasopharyngeal swabs were collected from 65 hospitalized children with pneumonia in a referral hospital and from 65 healthy children at two day-care centers from 2018 to 2019. S. pneumoniae was identified by conventional and molecular methods. Antibiotic susceptibility was performed with the disc diffusion method. Out of 130 children, S. pneumoniae strains were carried by 53% and 9.2 % in healthy children (35/65) and children with pneumonia (6/65), respectively. Serotype 19F was the most common serotype among the isolated strains (21%) followed by 6C (10%), 14, 34 (7 % each), and 1, 23F, 6A, 6B (5 % each). Moreover, 55 % of the strains (23/42) were covered by the 13-valent pneumococcal conjugate vaccine. Most isolates were susceptible to vancomycin (100%), chloramphenicol (93%), clindamycin (76%), erythromycin (71%), and tetracycline (69%). Serotype 19F was commonly found as a multi-drug resistant strain.

Objective. To review the epidemiology of coagulase-negative staphylococci (CoNS) in England over the recent 12 year period.

Methods. Laboratory-confirmed CoNS reported from sterile sites in patients in England to the UK Health Security Agency (UKHSA) between 2010 and 2021 were extracted from the national laboratory database and analysed.

Results. Overall, 668 857 episodes of CoNS were reported. Unspeciated CoNS accounted for 56 % (374 228) of episodes, followed by Staphylococcus epidermidis (26 %; 174 050), S. hominis (6.5 %; 43 501) and S. capitis (3.9 %; 25 773). Unspeciated CoNS increased by 8.2 % (95 % CI, 7.1–9.3) annually between 2010 and 2016, then decreased annually by 6.4 % (95 % CI: −4.8 to −7.9) until 2021. Speciated CoNS increased by 47.6 % (95 % CI, 44.5–50.9) annually between 2010 and 2016 and increased annually by 8.9 % (95 % CI: 5.1 to 12.8) until 2021. Antimicrobial susceptibility profiles differed by species.

Conclusions. Reports of CoNS from normally sterile body sites in patients in England increased between 2010 and 2016 and remained stable between 2017 and 2021. There has been a striking improvement in species-level identification of CoNS in recent years. Monitoring trends in CoNS epidemiology is crucial for development of observational and clinical intervention studies on individual species.

Brewery spent grain (BSG) has previously been exploited in bioremediation. However, detailed knowledge of the associated bacterial community dynamics and changes in relevant metabolites and genes over time is limited. This study investigated the bioremediation of diesel contaminated soil amended with BSG. We observed complete degradation of three total petroleum hydrocarbon (TPH C10–C28) fractions in amended treatments as compared to one fraction in the unamended, natural attenuation treatments. The biodegradation rate constant (k) was higher in amended treatments (0.1021k) than in unamended (0.059k), and bacterial colony forming units increased significantly in amended treatments. The degradation compounds observed fitted into the elucidated diesel degradation pathways and quantitative PCR results showed that the gene copy numbers of all three associated degradation genes, alkB, catA and xylE, were significantly higher in amended treatments. High-throughput sequencing of 16S rRNA gene amplicons showed that amendment with BSG enriched autochthonous hydrocarbon degraders. Also, community shifts of the genera Acinetobacter and Pseudomonas correlated with the abundance of catabolic genes and degradation compounds observed. This study showed that these two genera are present in BSG and thus may be associated with the enhanced biodegradation observed in amended treatments. The results suggest that the combined evaluation of TPH, microbiological, metabolite and genetic analysis provides a useful holistic approach to assessing bioremediation.

The emergence of multidrug-resistant bacteria has driven the need for novel antibiotics. Our investigations have focussed on lichens as they naturally produce a wide range of unique and very effective defence chemicals. The aim of this study was to evaluate some of the antimicrobial properties of ten common British churchyard lichens. The lichen material was sampled from ten species, namely Caloplaca flavescens, Diploicia canescens, Cladonia fimbriata, Psilolechia lucida, Lecanora campestris subsp. Campestris, Lecanora sulphurea, Pertusaria amara f.amara, Lepraria incana, Porpidia tuberculosa and Xanthoria calcicola. Crude acetone extracts of these lichens were tested against six bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonela typhimurium, Listeria monocytogenes and Lactobacillus acidophilus ) and two fungi (Trichophyton interdigitale and Aspergillus flavus) by the disc-diffusion susceptibility test method. Extracts of Diploicia canescens, Psilolechia lucida, Lecanora sulphurea, Pertusaria amara and Lepraria incana showed clear inhibition of the Gram-positive bacteria tested (S. aureus, L. monocytogenes, L. plantarum). Diploicia canescens, Pertusaria amara and Lepraria incana extracts also inhibited the dermatophyte fungi tested. The Lepraria incana sample tested here was the only extract that showed activity against any of the Gram-negative bacteria tested; it showed inhibition of Pseudomnas aeruginosa. Overall, our results showed that crude extracts of Diploicia canescens and Pertusaria amara had the most potent antimicrobial activity of all the extracts tested. Our results are in general agreement with published findings elsewhere. The activity of the Porpidia tuberculosa margin sample being different from that of the main colony material was an interesting and new finding reported here for the first time.

Introduction. Patients with coronavirus disease-2019 (COVID-19) are prone to develop respiratory bacterial infections irrespective of their need for mechanical ventilatory support.

Hypothesis/Gap Statement. Information about the incidence of concomitant respiratory bacterial infections in COVID- 19 patients from India is limited.

Aim. This study aimed to determine the incidence of concomitant respiratory bacterial pathogens and their drug resistance in these patients.

Methodology. A prospective study was performed by including patients who were admitted to our tertiary care centre from March 2021 to May 2021 to evaluate secondary bacterial respiratory co-infections in patients via real-time PCR (RT-PCR)-confirmed cases of COVID-19 disease caused by SARS CoV-2.

Results. Sixty-nine culture-positive respiratory samples from patients with COVID-19 were incorporated into this study. The most commonly isolated bacterial microorganisms were Klebsiella pneumoniae (23 samples, 33.33 %) and Acinetobacter baumannii (15, 21.73 %), followed by Pseudomonas aeruginosa (13, 18.84 %). Among the microorganisms isolated, 41 (59.4 %) were multidrug-resistant (MDR) and nine (13 %) were extensively drug-resistant (XDR). Among the Gram-negative bacteria isolated, K. pneumoniae showed high drug resistance. Fifty carbapenem-resistant microorganisms were isolated from the patients included in our study. Concerning the hospital stay of the patients enrolled, there was an increased length of intensive care unit stay, which was 22.25±15.42 days among patients needing mechanical ventilation in comparison to 5.39±9.57 days in patients on ambient air or low/high-flow oxygen.

Conclusion. COVID-19 patients need increased length of hospitalization and have a high incidence of secondary respiratory bacterial infections and high antimicrobial drug resistance.

An important public health problem in India is dengue infection, with every year seeing an increase in cases of dengue fever. Dengue affects all individuals irrespective of their gender and age, although the infection rate is higher among males and younger people. Despite low severity in general, dengue virus can cause severe health conditions in some individuals. Genetic characterization of circulating endemic dengue virus (DENV) serotypes plays a significant role in providing epidemiological knowledge and subsequent vaccine development. In the present study, over a 4 year period, we assessed DENV transmission dynamics in major regions of western Uttar Pradesh in North India. ELISA tests were used to diagnose dengue, and PCRs were used to determine the circulating serotype. We found that dengue infection peaks after the rainy season and affects all sexes and ages. A total of 1277 individuals were found positive for dengue; among them, 61.7 % were male and 38.3 % were female. DEN-1 was found in 23.12 %, DEN-2 in 45 %, DEN-3 in 29.06 % and DEN-4 in 1.5 % of the dengue-infected individuals. All four DENV serotypes were circulating in the study area, and DENV serotype-2 (DEN-2) was the most prevalent serotype.

A corrigendum of this article has been published full details can be found at https://doi.org/10.1099/acmi.0.000843

Enterococcus mundtii , a commensal intestinal bacterium, was demonstrated to inhibit the growth of some Mycobacterium tuberculosis complex (MTC) species that cause tuberculosis in humans and mammals. To further explore this preliminary observation, we cross-investigated five E. mundtii strains and seven MTC strains representative of four MTC species using a standardized quantitative agar well diffusion assay. All five E. mundtii strains, calibrated at 10 MacFarland, inhibited the growth of all M. tuberculosis strains with various susceptibility profiles, but no inhibition was observed with lower inoculums. Further, eight E. mundtii freeze-dried cell-free culture supernatants (CFCS) inhibited the growth of M. tuberculosis , Mycobacterium africanum, Mycobacterium bovis and Mycobacterium canettii, the most susceptible MTC species (inhibition diameter 25±1 mm), proportionally to CFCS protein concentrations. The data reported here indicate that the E. mundtii secretome inhibited growth of all MTC species of medical interest, which broadens previously reported data. In the gut, the E. mundtii secretome may modulate the expression of tuberculosis, exhibiting an anti-tuberculosis effect, with some protective roles in human and animal health.

Background. The oesophageal microbiome is thought to contribute to the pathogenesis of oesophageal cancer. However, investigations using culture and molecular barcodes have provided only a low-resolution view of this important microbial community. We therefore explored the potential of culturomics and metagenomic binning to generate a catalogue of reference genomes from the healthy human oesophageal microbiome, alongside a comparison set from saliva.

Results. Twenty-two distinct colonial morphotypes from healthy oesophageal samples were genome-sequenced. These fell into twelve species clusters, eleven of which represented previously defined species. Two isolates belonged to a novel species, which we have named Rothia gullae. We performed metagenomic binning of reads generated from UK samples from this study alongside reads generated from Australian samples in a recent study. Metagenomic binning generated 136 medium or high-quality metagenome-assembled genomes (MAGs). MAGs were assigned to 56 species clusters, eight representing novel Candidatus species, which we have named Ca. Granulicatella gullae, Ca. Streptococcus gullae, Ca. Nanosynbacter quadramensis, Ca. Nanosynbacter gullae, Ca. Nanosynbacter colneyensis, Ca. Nanosynbacter norwichensis, Ca. Nanosynococcus oralis and Ca. Haemophilus gullae. Five of these novel species belong to the recently described phylum Patescibacteria . Although members of the Patescibacteria are known to inhabit the oral cavity, this is the first report of their presence in the oesophagus. Eighteen of the metagenomic species were, until recently, identified only by hard-to-remember alphanumeric placeholder designations. Here we illustrate the utility of a set of recently published arbitrary Latinate species names in providing user-friendly taxonomic labels for microbiome analyses.

Our non-redundant species catalogue contained 63 species derived from cultured isolates or MAGs. Mapping revealed that these species account for around half of the sequences in the oesophageal and saliva metagenomes. Although no species was present in all oesophageal samples, 60 species occurred in at least one oesophageal metagenome from either study, with 50 identified in both cohorts.

Conclusions. Recovery of genomes and discovery of new species represents an important step forward in our understanding of the oesophageal microbiome. The genes and genomes that we have released into the public domain will provide a base line for future comparative, mechanistic and intervention studies.

Clitoria ternatea flowers are rich in anthocyanins and possess various biological activities. Specifically, the antibacterial mechanism of action of C. ternatea anthocyanins remains unknown and was investigated in Escherichia coli . A time–kill assay was used to assess the antibacterial activity and the metabolic perturbations in E. coli were investigated utilizing liquid chromatography–mass spectrometry (LC-MS)-based metabolomics. Pathway analyses were carried out for metabolites showing ≥2-fold changes. The anthocyanin fraction remarkably reduced the growth of E. coli at 4 h by 95.8 and 99.9 % at minimum inhibitory concentration (MIC) and 2× MIC, respectively. The anthocyanin fraction (MIC) had a bacteriostatic effect and was shown to have perturbed glycerophospholipids (1-acyl-sn-glycero-3-phosphoethanolamine, phosphatidylglycerol, diacylglycerol and cardiolipin), amino acids (valine, tyrosine and isoleucine) and energy (ubiquinone and NAD) metabolites at 1 and 4 h. This study demonstrated significant metabolic perturbations of the glycerophospholipid, amino acid and energy metabolism, with these being the key pathways involved in the bacteriostatic activity of anthocyanins from C. ternatea, which may have promise as bacteriostatic agents for E. coli -related infections.

Prophages have important roles in virulence, antibiotic resistance, and genome evolution in Staphylococcus aureus . Rapid growth in the number of sequenced S. aureus genomes allows for an investigation of prophage sequences at an unprecedented scale. We developed a novel computational pipeline for phage discovery and annotation. We combined PhiSpy, a phage discovery tool, with VGAS and PROKKA, genome annotation tools to detect and analyse prophage sequences in nearly 10 011  S . aureus genomes, discovering thousands of putative prophage sequences with genes encoding virulence factors and antibiotic resistance. To our knowledge, this is the first large-scale application of PhiSpy on a large-scale set of genomes (10 011  S . aureus ). Determining the presence of virulence and resistance encoding genes in prophage has implications for the potential transfer of these genes/functions to other bacteria via transduction and thus can provide insight into the evolution and spread of these genes/functions between bacterial strains. While the phage we have identified may be known, these phages were not necessarily known or characterized in S. aureus and the clustering and comparison we did for phage based on their gene content is novel. Moreover, the reporting of these genes with the S. aureus genomes is novel.

Objective. The present study was carried out to establish the chemical profile of the methanolic extract of Polyalthia longifolia stem bark and investigate its antibacterial property against some human pathogenic bacteria.

Methods. The extract was analysed using liquid and gas chromatography coupled to mass spectrometry. Antibacterial activity of P. longifolia extract against some human pathogenic bacteria was screened using the AlamarBlue method, and MIC and MBC were determined.

Results and Conclusion. Liquid chromatography-mass spectrometry (LC-MS) revealed the presence of 21 compounds among which 12 were identified. Gas chromatography-mass spectrometry (GC-MS) allowed identification of 26 compounds, the three major ones being the following: cis vaccenic acid (17.79 %), 3-ethyl-3-hydroxyandrostan-17-one (13.80 %) and copaiferic acid B (12.82 %). P. longifolia extract was active against Gram-positive bacteria with MIC ranging from 1 to 2 mg ml−1 and MBC from 2 to 6 mg ml−1. This study demonstrated the bactericidal effect of the methanolic extract of Polyalthia longifolia stem bark against some human pathogenic bacteria, including methicillin-resistant S. aureus . This effect could be related to the presence in the extract of a broad diversity of well-known compounds with established pharmacological properties. These results support the ethnomedicinal use of P. longifolia stem bark in Cameroon for the management of methicillin-resistant S. aureus (MRSA)-related infections.

Francisella tularensis , the causative agent for tularaemia, is a Tier 1 select agent, and a pan-species pathogen of global significance due to its zoonotic potential. Consistent genome characterization of the pathogen is essential to identify novel genes, virulence factors, antimicrobial resistance genes, for studying phylogenetics and other features of interest. This study was conducted to understand the genetic variations among genomes of F. tularensis isolated from two felines and one human source. Pan-genome analysis revealed that 97.7 % of genes were part of the core genome. All three F. tularensis isolates were assigned to sequence type A based on single nucleotide polymorphisms (SNPs) in sdhA. Most of the virulence genes were part of the core genome. An antibiotic resistance gene coding for class A beta-lactamase was detected in all three isolates. Phylogenetic analysis showed that these isolates clustered with other isolates reported from Central and South-Central USA. Assessment of large sets of the F. tularensis genome sequences is essential in understanding pathogen dynamics, geographical distribution and potential zoonotic implications.

The entomopathogenic fungus Akanthomyces muscarius is commonly used in agriculture to manage insect pests. Besides its use as a commercially important biological control agent, it also presents a potential model for studying host–pathogen interactions and the evolution of virulence in a laboratory setting. Here, we describe the first high-quality genome sequence for A. muscarius. We used long- and short-read sequencing to assemble a sequence of 36.1 Mb with an N50 of 4.9 Mb. Genome annotation predicted 12347 genes, with 96.6 % completeness based on the core Hypocrealen gene set. The high-quality assembly and annotation of A. muscarius presented in this study provides an essential tool for future research on this commercially important species.

Skin flora bacteria, such as Cutibacterium acnes , are the predominant contaminants of blood products used for transfusion. Platelet concentrates (PCs), a therapeutic product used to treat patients with platelet deficiencies, are stored at ambient temperature under agitation, providing ideal conditions for bacterial proliferation. At Canadian Blood Services, PCs are screened for microbial contamination using the automated BACT/ALERT culture system. Positive cultures are processed and contaminating organisms are identified using the VITEK 2 system. Over a period of approximately 2 years, several PC isolates were identified as Atopobium vaginae to a high level of confidence. However, since A. vaginae is associated with bacterial vaginosis and is not a common PC contaminant, a retrospective investigation revealed that in all cases C. acnes was misidentified as A. vaginae . Our investigation demonstrated that the media type used to grow PC bacterial isolates can have a significant impact on the results obtained on the VITEK 2 system. Furthermore, other identification methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALD-TOF MS) and PCR amplification of the 16S RNA gene were only partially successful in the identification of C. acnes . Therefore, our findings support a multiphasic approach when PC isolates are identified as A. vaginae by the VITEK 2 system for proper identification of C. acnes using macroscopic, microscopic and other biochemical analyses.