Bioinformatic analysis of Streptococcus uberis genes and genomes

Streptococcus uberis is a Gram-positive, catalase-negative member of the family Streptococcaceae and is an important environmental pathogen primarily responsible for a significant amount of bovine intramammary infections. This thesis describes the sequencing and comparison of multiple strains from clinical and sub-clinical infections. Following de novo assembly, these are compared to the single reference strain (0140J). The assemblies of strains sequenced with two technologies (Illumina and SOLiD) were compared. From these assemblies, annotation allowed the comparison of gene content, the pan and core genomes and gene gain/loss of coding sequences associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Identification of sequence variants allowed identification of highly conserved and highly variant genes. Inferred intraspecies and interspecies (host-S. uberis) protein-protein interaction networks revealed pathways of bovine proteins enriched with potentially interacting pathogen proteins. These identified known and predicted pathways and also novel interaction partners. This was the first “whole-genome” comparison of multiple S. uberis strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains. These data allowed the first insight into potential evolutionary forces behind virulence differences

Prevalence of Entamoeba species in captive primates in zoological gardens in the UK

The aim of this study was to determine the prevalence of amoebic infection in non-human primates (NHPs) from six Zoological gardens in the United Kingdom. Initially, 126 faecal samples were collected from 37 individually identified NHPs at Twycross Zoo, UK, and were subjected to microscopic examination. A subsequent, nationwide experiment included 350 faecal samples from 89 individually identified NHPs and 73 unidentified NHPs from a number of UK captive wildlife facilities: Twycross Zoo (n = 60), Colchester Zoo (n = 3), Edinburgh Zoo (n = 6), Port Lympne Wild Animal Park (n = 58), Howletts Wild Animal Park (n = 31), and Cotswold Wildlife Park (n = 4). Samples were examined by PCR and sequencing using four specific primer sets designed to differentiate between the pathogenic E. histolytica, the non-pathogenic E. dispar, and non-pathogenic uninucleate cyst-producing Entamoeba species. In the first experiment, Entamoeba was detected in 30 primates (81.1%). Six (16.2%) primates were infected with E. histolytica species complex. The highest carriage of Entamoeba species was found in Old World Colobinae primates. In the nationwide experiment, molecular analysis of faecal samples revealed notable rates of Entamoeba infection (101 samples, 28.9%), including one sample infected with E. histolytica, 14 samples with E. dispar, and 86 samples with uninucleated-cyst producing Entamoeba species. Sequences of positive uninucleated-cyst producing Entamoeba samples from Twycross Zoo clustered with the E. polecki reference sequences ST4 reported in Homo sapiens, and are widely separated from other Entamoeba species. These findings suggest a low prevalence of the pathogenic Entamoeba infection, but notable prevalence of non-pathogenic E. polecki infection in NHPs in the UK

Motion Artifacts Correction from Single-Channel EEG and fNIRS Signals using Novel Wavelet Packet Decomposition in Combination with Canonical Correlation Analysis

The electroencephalogram (EEG) and functional near-infrared spectroscopy (fNIRS) signals, highly non-stationary in nature, greatly suffers from motion artifacts while recorded using wearable sensors. This paper proposes two robust methods: i) Wavelet packet decomposition (WPD), and ii) WPD in combination with canonical correlation analysis (WPD-CCA), for motion artifact correction from single-channel EEG and fNIRS signals. The efficacy of these proposed techniques is tested using a benchmark dataset and the performance of the proposed methods is measured using two well-established performance matrices: i) Difference in the signal to noise ratio ({\Delta}SNR) and ii) Percentage reduction in motion artifacts ({\eta}). The proposed WPD-based single-stage motion artifacts correction technique produces the highest average {\Delta}SNR (29.44 dB) when db2 wavelet packet is incorporated whereas the greatest average {\eta} (53.48%) is obtained using db1 wavelet packet for all the available 23 EEG recordings. Our proposed two-stage motion artifacts correction technique i.e. the WPD-CCA method utilizing db1 wavelet packet has shown the best denoising performance producing an average {\Delta}SNR and {\eta} values of 30.76 dB and 59.51%, respectively for all the EEG recordings. On the other hand, the two-stage motion artifacts removal technique i.e. WPD-CCA has produced the best average {\Delta}SNR (16.55 dB, utilizing db1 wavelet packet) and largest average {\eta} (41.40%, using fk8 wavelet packet). The highest average {\Delta}SNR and {\eta} using single-stage artifacts removal techniques (WPD) are found as 16.11 dB and 26.40%, respectively for all the fNIRS signals using fk4 wavelet packet. In both EEG and fNIRS modalities, the percentage reduction in motion artifacts increases by 11.28% and 56.82%, respectively when two-stage WPD-CCA techniques are employed.Comment: 25 pages, 10 figures and 2 table

Genomic landscape of prominent XDR Acinetobacter clonal complexes from Dhaka, Bangladesh

Background: Acinetobacter calcoaceticus-A. baumannii (ACB) complex pathogens are known for their prevalence in nosocomial infections and extensive antimicrobial resistance (AMR) capabilities. While genomic studies worldwide have elucidated the genetic context of antibiotic resistance in major international clones (ICs) of clinical Acinetobacter spp., not much information is available from Bangladesh. In this study, we analysed the AMR profiles of 63 ACB complex strains collected from Dhaka, Bangladesh. Following this, we generated draft genomes of 15 of these strains to understand the prevalence and genomic environments of AMR, virulence and mobilization associated genes in different Acinetobacter clones. Results: Around 84% (n = 53) of the strains were extensively drug resistant (XDR) with two showing pan-drug resistance. Draft genomes generated for 15 strains confirmed 14 to be A. baumannii while one was A. nosocomialis. Most A. baumannii genomes fell under three clonal complexes (CCs): the globally dominant CC1 and CC2, and CC10; one strain had a novel sequence type (ST). AMR phenotype-genotype agreement was observed and the genomes contained various beta-lactamase genes including blaOXA-23 (n = 12), blaOXA-66 (n = 6), and blaNDM-1 (n = 3). All genomes displayed roughly similar virulomes, however some virulence genes such as the Acinetobactin bauA and the type IV pilus gene pilA displayed high genetic variability. CC2 strains carried highest levels of plasmidic gene content and possessed conjugative elements carrying AMR genes, virulence factors and insertion sequences. Conclusion: This study presents the first comparative genomic analysis of XDR clinical Acinetobacter spp. from Bangladesh. It highlights the prevalence of different classes of beta-lactamases, mobilome-derived heterogeneity in genetic architecture and virulence gene variability in prominent Acinetobacter clonal complexes in the country. The findings of this study would be valuable in understanding the genomic epidemiology of A. baumannii clones and their association with closely related pathogenic species like A. nosocomialis in Bangladesh. 2022, The Author(s).This work was funded by North South University Conference Travel and Research Grants (NSU CTRG) Committee under the grant number: NSU-RP-18-042.Scopu

Normalizing Community Mask-Wearing: A Cluster Randomized Trial in Bangladesh

Background: A growing body of scientific evidence suggests that face masks can slow the spread of COVID-19 and save lives, but mask usage remains low across many parts of the world, and strategies to increase mask usage remain untested and unclear. Methods: We conducted a cluster-randomized trial of community-level mask promotion in rural Bangladesh involving 341,830 adults in 600 villages. We employed a series of strategies to promote mask usage, including free household distribution of surgical or cloth masks, distribution and promotion at markets and mosques, mask advocacy by Imams during Friday prayers, role modeling by local leaders, promoters periodically monitoring passers-by and reminding people to put on masks, village police accompanying those mask promoters, providing monetary rewards or certificates to villages if mask-wearing rate improves, public signaling of mask-wearing via signage, text message reminders, messaging emphasizing either altruistic or self-protection motives for mask-wearing, and extracting verbal commitments from households. The primary objective was to assess which of these interventions would increase proper (covering nose and mouth) wearing of face masks, and secondarily, whether mask promotion unintentionally creates moral hazard and decreases social distancing. This analysis is part of larger study evaluating the effect of mask-wearing on transmission of SARS-CoV-2. Results: There were 64,937 households in the intervention group and 64,183 households in the control group; study recruitment has ended. In the control group, proper mask-wearing was practiced by 13% of those observed across the study period. Free distribution of masks along with role modeling by community leaders produced only small increases in mask usage during pilot interventions. Adding periodic monitoring by mask promoters to remind people in streets and public places to put on the masks we provided increased proper mask-wearing by 29.0 percentage points (95% CI: 26.7% - 31.3%). This tripling of mask usage was sustained over all 10 weeks of surveillance, which includes a period after intervention activities ended. Physical distancing, measured as the fraction of individuals at least one arm’s length apart, also increased by 5.2 percentage points (95% CI: 4.2%-6.3%). Beyond the core intervention package comprised of free distribution and promotion at households/mosques/markets, leader endorsements plus periodic monitoring and reminders, several elements had no additional effect on mask wearing, including: text reminders, public signage commitments, monetary or non-monetary incentives, altruistic messaging or verbal commitments, or village police accompanying the mask promoters (the last not cross-randomized, but assessed in panel data). No adverse events were reported during the study period. Conclusions: Our intervention demonstrates a scalable and cost-effective method to promote mask adoption and save lives, and identifies a precise combination of intervention activities that were necessary. Comparisons between pilots shows that free mask distribution alone is not sufficient to increase mask-wearing, but adding periodic monitoring in public places to remind people to wear the distributed masks had large effects on behavior. The absence of any further effect of the village police suggests that the operative mechanism is not any threat of formal legal sanctions, but shame and people’s aversion to a light informal social sanction. The persistence of effects for 10 weeks and after the end of the active intervention period, as well as increases in physical distancing, all point to changes in social norms as a key driver of behavior change. Our cross-randomizations suggest that improved mask-wearing norms can be achieved without incentives that require costly monitoring, that aesthetic design choices and colors can influence mask-wearing, and that surgical masks with a substantially higher filtration efficiency can be a cost-effective alternative to cloth masks (1/3 the cost) and are equally or more likely to be worn. Implementing these interventions – including distribution of free masks, and the information campaign, reminders, encouragement – cost $2.30-$3.75 per villager, or between $8 and$13 per person adopting a mask. Combined with existing estimates of the efficacy of masks in preventing COVID-19 deaths, this implies that the intervention cost $28,000-$66,000 per life saved. Beyond reducing the transmission of COVID-19, mask distribution is likely to be a cost-effective strategy to prevent future respiratory disease outbreaks

Monitoring antimicrobial susceptibility of Neisseria gonorrhoeae isolated from Bangladesh during 1997- 2006: Emergence and pattern of drug-resistant isolates

Gonorrhoea is one of the most common sexually transmitted infections (STIs) in developing countries, and the emergence of resistance to antimicrobial agents in Neisseria gonorrhoeae is a major obstacle in the control of gonorrhoea. Periodical monitoring of antimicrobial susceptibility of N. gonorrhoeae is essential for the early detection of emergence of drug resistance. In total, 1, 767 gonococcal strains isolated from males and females (general population and those with high-risk behaviour) from different parts of Bangla-desh were studied during 1997- 2006 . Minimum inhibitory concentrations of penicillin, tetracycline, cipro-floxacin, ceftriaxone, spectinomycin, and azithromycin for the isolates were determined by the agar dilu-tion method. Isolates resistant to three or more antimicrobial agents are considered multidrug-resistant. The prevalence of plasmid-mediated penicillinase-producing N. gonorrhoeae (PPNG) and plasmid-mediated tetracycline-resistant N. gonorrhoeae (TRNG) was determined. Nine percent of the isolates were resistant to ciprofloxacin in 1997 compared to 87% in 2006. Multidrug-resistant N. gonorrhoeae have emerged in 1997 , and 44% of the strains (n= 66) isolated during 2006 were multidrug-resistant. Forty-two percent of the isolates in 2006 were both PPNG- and TRNG-positive compared to none in 1997 . The rapidly-changing pattern of gonococcal antimicrobial susceptibility warrants the need for an antimicrobial susceptibility-monitoring programme, and periodical analysis and dissemination of susceptibility data are essential to guide clinicians and for successful STI/HIV intervention programmes

The Impact of Community Masking on COVID-19: A Cluster Randomized Trial in Bangladesh

Background: Mask usage remains low across many parts of the world during the COVID- 19 pandemic, and strategies to increase mask-wearing remain untested. Our objectives were to identify strategies that can persistently increase mask-wearing and assess the impact of increasing mask-wearing on symptomatic SARS-CoV-2 infections. Methods: We conducted a cluster-randomized trial of community-level mask promotion in rural Bangladesh from November 2020 to April 2021 (N=600 villages, N=342,126 adults). We cross-randomized mask promotion strategies at the village and household level, including cloth vs. surgical masks. All intervention arms received free masks, information on the importance of masking, role modeling by community leaders, and in-person reminders for 8 weeks. The control group did not receive any interventions. Neither participants nor field staff were blinded to intervention assignment. Outcomes included symptomatic SARS-CoV-2 seroprevalence (primary) and prevalence of proper mask-wearing, physical distancing, and symptoms consistent with COVID-19 (secondary). Mask-wearing and physical distancing were assessed through direct observation at least weekly at mosques, markets, the main entrance roads to villages, and tea stalls. At 5 and 9 weeks follow-up, we surveyed all reachable participants about COVID-related symptoms. Blood samples collected at 10-12 weeks of follow-up for symptomatic individuals were analyzed for SARS-CoV-2 IgG antibodies. Results: There were 178,288 individuals in the intervention group and 163,838 individuals in the control group. The intervention increased proper mask-wearing from 13.3% in control villages (N=806,547 observations) to 42.3% in treatment villages (N=797,715 observations) (adjusted percentage point difference = 0.29 [0.27, 0.31]). This tripling of mask usage was sustained during the intervention period and two weeks after. Physical distancing increased from 24.1% in control villages to 29.2% in treatment villages (adjusted percentage point difference = 0.05 [0.04, 0.06]). After 5 months, the impact of the intervention faded, but mask-wearing remained 10 percentage points higher in the intervention group. The proportion of individuals with COVID-like symptoms was 7.62% (N=13,273) in the intervention arm and 8.62% (N=13,893) in the control arm. Blood samples were collected from N=10,952 consenting, symptomatic individuals. Adjusting for baseline covariates, the intervention reduced symptomatic seroprevalence by 9.3% (adjusted prevalence ratio (aPR) = 0.91 [0.82, 1.00]; control prevalence 0.76%; treatment prevalence 0.68%). In villages randomized to surgical masks (n = 200), the relative reduction was 11.2% overall (aPR = 0.89 [0.78, 1.00]) and 34.7% among individuals 60+ (aPR = 0.65 [0.46, 0.85]). No adverse events were reported. Conclusions: Our intervention demonstrates a scalable and effective method to promote mask adoption and reduce symptomatic SARS-CoV-2 infections. Trial registration: ClinicalTrials.gov Identifier: NCT04630054 Funding: GiveWell.or

Wastewater-based epidemiological surveillance to monitor the prevalence of SARS-CoV-2 in developing countries with onsite sanitation facilities

Wastewater-based epidemiology (WBE) has emerged as a valuable approach for forecasting disease outbreaks in developed countries with a centralized sewage infrastructure. On the other hand, due to the absence of well-defined and systematic sewage networks, WBE is challenging to implement in developing countries like Bangladesh where most people live in rural areas. Identification of appropriate locations for rural Hotspot Based Sampling (HBS) and urban Drain Based Sampling (DBS) are critical to enable WBE based monitoring system. We investigated the best sampling locations from both urban and rural areas in Bangladesh after evaluating the sanitation infrastructure for forecasting COVID-19 prevalence. A total of 168 wastewater samples were collected from 14 districts of Bangladesh during each of the two peak pandemic seasons. RT-qPCR commercial kits were used to target ORF1ab and N genes. The presence of SARS-CoV-2 genetic materials was found in 98% (165/168) and 95% (160/168) wastewater samples in the first and second round sampling, respectively. Although wastewater effluents from both the marketplace and isolation center drains were found with the highest amount of genetic materials according to the mixed model, quantifiable SARS-CoV-2 RNAs were also identified in the other four sampling sites. Hence, wastewater samples of the marketplace in rural areas and isolation centers in urban areas can be considered the appropriate sampling sites to detect contagion hotspots. This is the first complete study to detect SARS-CoV-2 genetic components in wastewater samples collected from rural and urban areas for monitoring the COVID-19 pandemic. The results based on the study revealed a correlation between viral copy numbers in wastewater samples and SARS-CoV-2 positive cases reported by the Directorate General of Health Services (DGHS) as part of the national surveillance program for COVID-19 prevention. The findings of this study will help in setting strategies and guidelines for the selection of appropriate sampling sites, which will facilitate in development of comprehensive wastewater-based epidemiological systems for surveillance of rural and urban areas of low-income countries with inadequate sewage infrastructure.This research was supported by Water Aid Bangladesh, North South University, Dhaka, COVID-19 Diagnostic Lab, Department of Microbiology, Noakhali Science and Technology University (NSTU), Noakhali, Bangladesh, the International Training Network of Bangladesh University of Engineering and Technology (ITN-BUET) - Centre for Water Supply and Waste Management, and KTH Royal Institute of Technology, Sweden. We acknowledge the sincere help and support of the staff and volunteers of NSTU-COVID-19 Diagnostic Lab, Noakhali Science and Technology University, Bangladesh during the different phases of the study. PB and MTI acknowledge the Life Science Technology Platform, Science for Life Laboratory for the seed funding to initiate the wastewater-based epidemiological studies for SARS-CoV-2 in Bangladesh. We would also like to acknowledge the two anonymous reviewers for their critical comments as well as their thoughtful insights, which has significantly improved the manuscript.Peer reviewe

Virulence related sequences: insights provided by comparative genomics of Streptococcus uberis of differing virulence

Background: Streptococcus uberis, a Gram-positive, catalase-negative member of the family Streptococcaceae is an important environmental pathogen responsible for a significant proportion of subclinical and clinical bovine intramammary infections. Currently, the genome of only a single reference strain (0140J) has been described. Here we present a comparative analysis of complete draft genome sequences of an additional twelve S. uberis strains. Results: Pan and core genome analysis revealed the core genome common to all strains to be 1,550 genes in 1,509 orthologous clusters, complemented by 115-246 accessory genes present in one or more S. uberis strains but absent in the reference strain 0140J. Most of the previously predicted virulent genes were present in the core genome of all 13 strains but gene gain/loss was observed between the isolates in CDS associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Experimental challenge experiments confirmed strain EF20 as non-virulent; only able to infect in a transient manner that did not result in clinical mastitis. Comparison of the genome sequence of EF20 with the validated virulent strain 0140J identified genes associated with virulence, however these did not relate clearly with clinical/non-clinical status of infection. Conclusion: The gain/loss of mobile genetic elements such as CRISPRs and prophage are a potential driving force for evolutionary change. This first “whole-genome” comparison of strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains did not identify simple gene gain/loss rules that readily explain, or be confidently associated with, differences in virulence. This suggests that a more complex dynamic determines infection potential and clinical outcome not simply gene content

Bioinformatic analysis of Streptococcus uberis genes and genomes

Streptococcus uberis is a Gram-positive, catalase-negative member of the family Streptococcaceae and is an important environmental pathogen primarily responsible for a significant amount of bovine intramammary infections. This thesis describes the sequencing and comparison of multiple strains from clinical and sub-clinical infections. Following de novo assembly, these are compared to the single reference strain (0140J). The assemblies of strains sequenced with two technologies (Illumina and SOLiD) were compared. From these assemblies, annotation allowed the comparison of gene content, the pan and core genomes and gene gain/loss of coding sequences associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Identification of sequence variants allowed identification of highly conserved and highly variant genes. Inferred intraspecies and interspecies (host-S. uberis) protein-protein interaction networks revealed pathways of bovine proteins enriched with potentially interacting pathogen proteins. These identified known and predicted pathways and also novel interaction partners. This was the first “whole-genome” comparison of multiple S. uberis strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains. These data allowed the first insight into potential evolutionary forces behind virulence differences