Molecular characterization and distribution of cephalosporin resistance determinants in Escherichia coli and Klebsiella pneumoniae isolated from patients attending Kampala International University Teaching Hospital in Bushenyi, Western Uganda

Cephalosporins are the first-line therapy antibiotics used in the treatment of gram-negative bacterial infections. However, high prevalence of cephalosporins resistance in Klebsiella pneumoniae and Escherichia coli has been reported worldwide. Studies conducted in Uganda reported high incidences of cephalosporin resistance (CR). Successive studies at Mulago National Referral Hospital indicated a decline in the resistance levels pointing to the need for regular antibiotic resistance surveillance. Therefore, this study carried out molecular characterization of CR determinants in E. coli and K. pneumoniae isolated from patients attending Kampala International University Teaching Hospital (KIU-TH). A retrospective study using E. coli and K. pnuemoniae samples previously obtained from surgical wounds and urinary tract infections among patients treated at KIU-TH between September 2016 and August 2018 was conducted. Biochemical assays were used to confirm the identity of the samples. Combined disc and boronic acid assays were used to determine the cephalosporine resistance profile of the isolates. Multiplex PCR amplification was used to characterize the extended spectrum betalactmase (ESBL) encoding genes. The study revealed that E. coli (130/81.2%) isolates were more predominant than K. pneumoniae (30/18.8%) among the archived samples. K. pneumoniae showed the highest phenotypic resistance with a mean prevalence of 90.6% but comparable to that of E. coli (89.3%). Of the 160 isolates screened, 105 (65.6%) were ESBL producers. Multiplex PCR revealed that the most predominant ESBL encoding gene was blaSHV at a prevalence of 42.0%, followed by blaTEM at 27.3%, blaCTX-M at 22.4% and blaCTX-M-15 at 8.4%. The incidence of phenotypic resistance and distribution of ESBL genes were significantly higher in patients of Ishaka division. Our study reports a high prevalence of cephalosporin-resistant E. coli and K. pnuemoniae isolated from patients attending KIU-TH and highlights the need forr routine screening of antimicrobial resistance in health-care facilities so as to guide clinicians on the rational prescription of antibiotics.List of abbreviations: ATCC: American Type Culture Collection; ESBL: extended spectrum betalactmases; AmpC: aminopenicillin cephalosporinase; CTX-M: cephotaxime (M-first detected in Munich) hydrolyzing capabilities; CTX-M-U: cephotaxime hydrolyzing capabilities gene Universal primer; SHV: sulfhydryl variables (variant-2); CMY-2: cephamycins (variant-2); TEM: temoneira; ACT-1: AmpC type (variant-1); Bla: Beta lactam; DNA: deoxyribonucleic acid; PCR: Polymerase chain reaction; MNRH: Mulago National Referral Hospital; MRRH: Mbarara Regional Referral Hospital; KRRH: Kabale Regional Referral Hospital (KRRH); KIU-TH: Kampala International University Teaching Hospital; CDL: Central Diagnostic Laboratory; CoVAB: College of Veterinary Medicine, Animal Resources and Biosecurity; CR: cephalosporin resistanc

A review of phage mediated antibacterial applications

Background: For over a decade, resistance to newly synthesized antibiotics has been observed worldwide. The challenge of antibiotic resistance has led to several pharmaceutical companies to abandon the synthesis of new drugs in fear of bacteria developing resistance in a short period hence limiting initial investment return. To this effect, alternative approaches such as the use of bacteriophages to treat bacterial infections are being explored. This review explores the recent advances in phage-mediated antibacterial applications and their limitations.Methods: We conducted a comprehensive literature search of PubMed, Lib Hub and Google Scholar databases from January 2019 to November 2019. The search key words used were the application of bacteriophages to inhibit bacterial growth and human phage therapy to extract full-text research articles and proceedings from International Conferences published only in English.Results: The search generated 709 articles of which 95 full-text research articles fulfilled the inclusion guidelines. Transmission Electron Microscopy morphological characterization conducted in 23 studies registered Myoviruses, Siphoviruses, Podoviruses, and Cytoviruses phage families while molecular characterization revealed that some phages were not safe to use as they harbored undesirable genes. All in vivo phage therapy studies in humans and model animals against multidrug-resistant (MDR) bacterial infection provided 100% protection. Ex vivo and in vitro phage therapy experiments exhibited overwhelming results as they registered high efficacies of up to 100% against MDR clinical isolates. Phage-mediated bio-preservation of foods and beverages and bio-sanitization of surfaces were highly successful with bacterial growth suppression of up to 100%. Phage endolysins revealed efficacies statistically comparable to those of phages and restored normal ethanol production by completely eradicating lactic acid bacteria in ethanol fermenters. Furthermore, the average multiplicity of infection was highest in ex vivo phage therapy (557,291.8) followed by in vivo (155,612.4) and in vitro (434.5)

Phylogenetic Characterization of Crimean-Congo Hemorrhagic Fever Virus Detected in African Blue Ticks Feeding on Cattle in a Ugandan Abattoir.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the most geographically widespread of the tick-borne viruses. However, African strains of CCHFV are poorly represented in sequence databases. In addition, almost all sequence data collected to date have been obtained from cases of human disease, while information regarding the circulation of the virus in tick and animal reservoirs is severely lacking. Here, we characterize the complete coding region of a novel CCHFV strain, detected in African blue ticks (Rhipicephalus (Boophilus) decoloratus) feeding on cattle in an abattoir in Kampala, Uganda. These cattle originated from a farm in Mbarara, a major cattle-trading hub for much of Uganda. Phylogenetic analysis indicates that the newly sequenced strain belongs to the African genotype II clade, which predominantly contains the sequences of strains isolated from West Africa in the 1950s, and South Africa in the 1980s. Whilst the viral S (nucleoprotein) and L (RNA polymerase) genome segments shared >90% nucleotide similarity with previously reported genotype II strains, the glycoprotein-coding M segment shared only 80% nucleotide similarity with the next most closely related strains, which were derived from ticks in Western India and Northern China. This genome segment also displayed a large number of non-synonymous mutations previously unreported in the genotype II strains. Characterization of this novel strain adds to our limited understanding of the natural diversity of CCHFV circulating in both ticks and in Africa. Such data can be used to inform the design of vaccines and diagnostics, as well as studies exploring the epidemiology and evolution of the virus for the establishment of future CCHFV control strategies

Phylogenetic Characterization of Crimean-Congo Hemorrhagic Fever Virus Detected in African Blue Ticks Feeding on Cattle in a Ugandan Abattoir.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the most geographically widespread of the tick-borne viruses. However, African strains of CCHFV are poorly represented in sequence databases. In addition, almost all sequence data collected to date have been obtained from cases of human disease, while information regarding the circulation of the virus in tick and animal reservoirs is severely lacking. Here, we characterize the complete coding region of a novel CCHFV strain, detected in African blue ticks (Rhipicephalus (Boophilus) decoloratus) feeding on cattle in an abattoir in Kampala, Uganda. These cattle originated from a farm in Mbarara, a major cattle-trading hub for much of Uganda. Phylogenetic analysis indicates that the newly sequenced strain belongs to the African genotype II clade, which predominantly contains the sequences of strains isolated from West Africa in the 1950s, and South Africa in the 1980s. Whilst the viral S (nucleoprotein) and L (RNA polymerase) genome segments shared >90% nucleotide similarity with previously reported genotype II strains, the glycoprotein-coding M segment shared only 80% nucleotide similarity with the next most closely related strains, which were derived from ticks in Western India and Northern China. This genome segment also displayed a large number of non-synonymous mutations previously unreported in the genotype II strains. Characterization of this novel strain adds to our limited understanding of the natural diversity of CCHFV circulating in both ticks and in Africa. Such data can be used to inform the design of vaccines and diagnostics, as well as studies exploring the epidemiology and evolution of the virus for the establishment of future CCHFV control strategies

A survey of biosecurity practices of pig farmers in selected districts affected by African swine fever in Uganda

IntroductionIn Uganda, pig production is an important source of livelihood for many people and contributes to food security. African swine fever (ASF) is a major constraint to pig production in Uganda, threatening the food supply and sustainable livelihoods. Prevention of ASF primarily relies on good biosecurity practices along the pig value chain. Previous studies showed that biosecurity along the pig value chain and on farms in Uganda is poor. However, the biosecurity practices of pig farmers in ASF affected areas of Uganda and their opinions on on-farm ASF morbidity and mortality were previously not comprehensively characterized. The objectives of this study were to document pig farmers’ experiences with ASF in their farms and to describe the pig biosecurity practices in districts of Uganda that were highly affected by ASF.MethodsA total of 99 farmers were interviewed in five districts. Data were collected by way of triangulation through farmer interviews, field observations during the farmer interviews, and a survey of key informants. However, farmer interviews were considered the primary source of data for this study. Farmers’ biosecurity practices were scored using a biosecurity scoring algorithm.ResultsForty-one out of 96 (42.7%) farmers reported having pigs with ASF in the past 12 months. The level of pig farming experience (p = 0.0083) and herd size (p < 0.0001) were significantly associated with the reported occurrence of ASF. Overall, the biosecurity scores for the respondents were considered poor with 99% (98/99) scoring <70% and just one farmer obtaining a fair score of 72.2%. District (p = 0.0481), type of husbandry system (p = 0.014), and type of pig breed raised (p = 0.004) were significantly associated with farmer’s biosecurity score.ConclusionContinued farmer education on ASF and the importance of good biosecurity practices is necessary. More in-depth scientific inquiry into the factors influencing the biosecurity practices among pig farmers in Uganda is necessary

Genetic variability and consequence of Mycobacterium tuberculosis lineage 3 in Kampala-Uganda

Limited data existed exclusively describing Mycobacterium tuberculosis lineage 3 (MTB-L3), sub-lineages, and clinical manifestations in Kampala, Uganda. This study sought to elucidate the circulating MTB-L3 sub-lineages and their corresponding clinical phenotypes.; A total of 141 M. tuberculosis isolates were identified as M. tuberculosis lineage 3 using Single nucleotide polymorphism (SNP) marker analysis method. To ascertain the sub-lineages/sub-strains within the M. tuberculosis lineage 3, the direct repeat (DR) loci for all the isolates was examined for sub-lineage specific signatures as described in the SITVIT2 database. The infecting sub-strains were matched with patients' clinical and demographic characteristics to identify any possible association.; The data showed 3 sub-lineages circulating with CAS 1 Delhi accounting for 55% (77/141), followed by CAS 1-Kili 16% (22/141) and CAS 2/CAS 8% (12/141). Remaining isolates 21% (30/141) were unclassifiable. To explore whether the sub-lineages differ in their ability to cause increased severe disease, we used extent of lung involvement as a proxy for severe disease. Multivariable analysis showed no association between M. tuberculosis lineage 3 sub-lineages with severe disease. The risk factors associated with severe disease include having a positive smear (OR = 9.384; CI 95% = 2.603-33.835), HIV (OR = 0.316; CI 95% = 0.114-0.876), lymphadenitis (OR = 0. 171; CI 95% = 0.034-0.856) and a BCG scar (OR = 0.295; CI 95% = 0.102-0.854).; In Kampala, Uganda, there are three sub-lineages of M. tuberculosis lineage 3 that cause disease of comparable severity with CAS-Dehli as the most prevalent. Having HIV, lymphadenitis, a BCG scar and a smear negative status is associated with reduced severe disease

A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala

Background: Accurate and high-throughput genotyping of Mycobacterium tuberculosis complex (MTBC) may be important for understanding the epidemiology and pathogenesis of tuberculosis (TB). In this study, we report the development of a LightCycler® real-time PCR single-nucleotide-polymorphism (LRPS) assay for the rapid determination of MTBC lineages/sublineages in minimally processed sputum samples from TB patients. Method Genotyping analysis of 70 MTBC strains was performed using the Long Sequence Polymorphism-PCR (LSP-PCR) technique and the LRPS assay in parallel. For targeted sequencing, 9 MTBC isolates (three isolates per MTBC lineage) were analyzed for lineage-specific single nucleotide polymorphisms (SNPs) in the following three genes to verify LRPS results: Rv004c for MTB Uganda family, Rv2962 for MTB lineage 4, and Rv0129c for MTB lineage 3. The MTBC lineages present in 300 smear-positive sputum samples were then determined by the validated LRPS method without prior culturing. Results: The LSP-PCR and LRPS assays produced consistent genotyping data for all 70 MTBC strains; however, the LSP-PCR assay was 10-fold less sensitive than the LRPS method and required higher DNA concentrations to successfully characterize the MTBC lineage of certain samples. Targeted sequencing of genes containing lineage-specific SNPs was 100 % concordant with the genotyping results and provided further validation of the LRPS assay. Of the 300 sputum samples analyzed, 58 % contained MTBC from the MTBC-Uganda family, 27 % from the MTBC lineage 4 (excluding MTBC Uganda family), 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage. Conclusion: The LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients. Electronic supplementary material The online version of this article (doi:10.1186/s12879-015-1121-7) contains supplementary material, which is available to authorized users

Harnessing technology and portability to conduct molecular epidemiology of endemic pathogens in resource-limited settings

Improvements in genetic and genomic technology have enabled field-deployable molecular laboratories and these have been deployed in a variety of epidemics that capture headlines. In this editorial, we highlight the importance of building physical and personnel capacity in low and middle income countries to deploy these technologies to improve diagnostics, understand transmission dynamics and provide feedback to endemic communities on actionable timelines. We describe our experiences with molecular field research on schistosomiasis, trypanosomiasis and rabies and urge the wider tropical medicine community to embrace these methods and help build capacity to benefit communities affected by endemic infectious diseases

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.We thank S. Lecher, S. Li and J. Zallet for technical support. Calculations were performed at the sciCORE scientific computing core facility at the University of Basel. This work was supported by the Swiss National Science Foundation (grants 310030_166687 (S.G.) and 320030_153442 (M.E.) and Swiss HIV Cohort Study grant 740 to L.F.), the European Research Council (309540-EVODRTB to S.G.), TB-PAN-NET (FP7-223681 to S.N.), PathoNgenTrace projects (FP7-278864-2 to S.N.), SystemsX.ch (S.G.), the German Center for Infection Research (DZIF; S.N.), the Novartis Foundation (S.G.), the Natural Science Foundation of China (91631301 to Q.G.), and the National Institute of Allergy and Infectious Diseases (5U01-AI069924-05) of the US National Institutes of Health (M.E.)