Molecular Characterization of Staphylococcus aureus Isolated from Raw Milk and Humans in Eastern Tanzania: Genetic Diversity and Inter-Host Transmission.

Staphylococcus aureus is a common cause of infection in humans and animals, including bovine mastitis, globally. The objective of this study was to genetically characterize a collection of S. aureus isolates recovered from milk and nasal swabs from humans with and without animal contact (bovine = 43, human = 12). Using whole genome sequencing (NextSeq550), isolates were sequence typed, screened for antimicrobial resistance and virulence genes and examined for possible inter-species host transmission. Multi locus sequence typing (MLST) and single nucleotide polymorphism (SNP)-based phylogeny revealed 14 different sequence types, including the following six novel sequence types: ST7840, 7841, 7845, 7846, 7847, and 7848. The SNP tree confirmed that MLST clustering occurred most commonly within CC97, CC5477, and CC152. ResFinder analysis revealed five common antibiotic resistance genes, namely tet(K), blaZ, dfrG, erm©, and str, encoding for different antibiotics. mecA was discovered in one human isolate only. Multidrug resistance was observed in 25% of the isolates, predominantly in CC152 (7/8) and CC121 (3/4). Known bovine S. aureus (CC97) were collected in humans and known human S. aureus lineages (CC152) were collected in cattle; additionally, when these were compared to bovine-isolated CC97 and human-isolated CC152, respectively, no genetic distinction could be observed. This is suggestive of inter-host transmission and supports the need for surveillance of the human-animal interface.

Microbiological Characterisation of Community-Acquired Urinary Tract Infections in Bagamoyo, Tanzania: A Prospective Study.

Urinary tract infections (UTIs) are among the most common infections in sub-Saharan Africa, but microbiological data to guide treatment decisions are limited. Hence, we investigated the bacterial aetiology and corresponding antimicrobial susceptibility patterns in outpatients with UTIs in Bagamoyo, Tanzania. Urine samples from symptomatic individuals were subjected to microbiological examinations for bacterial species identification using conventional methods and disc diffusion-based resistance testing. Subsequently, urine samples were transferred to Germany for confirmatory diagnostics using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and automated resistance testing. Overall, 104 out of 270 (38.5%) individuals had a positive urine culture and 119 putative pathogens were identified. The most frequently detected bacteria were Escherichia coli (23%), Klebsiella spp. (7%), Enterobacter cloacae complex (3%) and Staphylococcus aureus (2%). E. coli isolates showed high resistance against cotrimoxazole (76%), ampicillin (74%), piperacillin (74%) and fluoroquinolones (37%), but widespread susceptibility to meropenem (100%), fosfomycin (98%), piperacillin/tazobactam (97%) and amoxicillin/clavulanic acid (82%). The agreement between E. coli susceptibility testing results in Tanzania and Germany was ≥95%, except for piperacillin/tazobactam (89%) and ciprofloxacin (84%). Given the considerable resistance to frequently prescribed antibiotics, such as cotrimoxazole and fluoroquinolones, future research should explore the potential of oral alternatives (e.g., fosfomycin) for the treatment of UTIs in Tanzania.

Causative agents and antimicrobial resistance patterns of human skin and soft tissue infections in Bagamoyo, Tanzania.

Few epidemiological studies have been carried out to assess the aetiology and antimicrobial susceptibility patterns of pathogens giving rise to skin and soft tissue infections (SSTIs) in sub-Saharan Africa. In the present study from six healthcare facilities in Bagamoyo, Tanzania, wound swabs from outpatients with SSTIs were analysed by a suite of methods, including microbiological culture techniques, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and resistance testing. Among 185 patients with SSTIs, 179 (96.8%) swabs showed microbiological growth. In total, 327 organisms were found, of which 285 were of potential aetiological relevance. Staphylococcus aureus was the predominant pathogen (prevalence: 71.4%), followed by the Gram-negative bacteria Enterobacter cloacae complex (14.6%), Klebsiella pneumoniae (12.4%) and Pseudomonas aeruginosa (11.8%). While one out of three isolates of S. aureus showed resistance to macrolides, tetracyclines, cotrimoxazole and clindamycin, only a single methicillin-resistant S. aureus (MRSA) strain was found. In Gram-negative bacteria, resistance to ampicillin and cotrimoxazole was common, while extended-spectrum beta-lactamases were rarely detected (<1%). We conclude that S. aureus was the most frequently detected pathogen in community-acquired SSTIs in Bagamoyo, Tanzania. Resistance to commonly prescribed oral antibiotics was considerable, but multi-resistant strains were rarely encountered. Monitoring of antibiotic susceptibility patterns in SSTIs is important to provide specific data for tailoring treatment recommendations.

Management of superficial and deep-seated Staphylococcus aureus skin and soft tissue infections in sub-Saharan Africa: a post hoc analysis of the StaphNet cohort.

PURPOSE: The incidence of Staphylococcus aureus skin and soft tissue infection (SSTI) is high in sub-Saharan Africa. This is fueled by a high prevalence of Panton-Valentine leukocidin (PVL), which can be associated with necrotizing disease. The aim was to describe the clinical presentation and the treatment of SSTI in the African setting and to identify challenges in the management. METHODS: Patients (n = 319) were recruited in DR Congo (n = 56, 17.6%), Gabon (n = 89, 27.9%), Mozambique (n = 79, 24.8%) and Tanzania (n = 95, 29.8%) during the prospective observational StaphNet cohort study (2010-2015). A physician recorded the clinical management in standardized questionnaires and stratified the entity of SSTI into superficial (sSSTI) or deep-seated (dSSTI). Selected virulence factors (PVL, ß hemolysin) and multilocus sequence types (MLST) were extracted from whole genome sequencing data. RESULTS: There were 220/319 (69%) sSSTI and 99/319 (31%) dSSTI. Compared to sSSTI, patients with dSSTI were more often hospitalized (13.2 vs. 23.5%, p = 0.03), HIV-positive (7.6 vs. 15.9%, p = 0.11), and required more often incision and drainage (I&D, 45.5 vs. 76.5%, p = 0.04). The proportion of an adequate antimicrobial therapy increased marginally from day 1 (empirical therapy) to day 3 (definite therapy), for sSSTI (70.7 to 72.4%) and dSSTI (55.4 to 58.9%). PVL was a risk factor for I&D (OR = 1.7, p = 0.02) and associated with MLST clonal complex CC121 (OR = 2.7, p < 0.001). CONCLUSION: Appropriate antimicrobial agents and surgical services to perform I&D were available for the majority of patients. Results from susceptibility testing should be considered more efficiently in the selection of antimicrobial therapy.

Community-Associated Staphylococcus aureus from Sub-Saharan Africa and Germany: A Cross-Sectional Geographic Correlation Study.

Clonal clusters and gene repertoires of Staphylococcus aureus are essential to understand disease and are well characterized in industrialized countries but poorly analysed in developing regions. The objective of this study was to compare the molecular-epidemiologic profiles of S. aureus isolates from Sub-Saharan Africa and Germany. S. aureus isolates from 600 staphylococcal carriers and 600 patients with community-associated staphylococcal disease were characterized by DNA hybridization, clonal complex (CC) attribution, and principal component (PCA)-based gene repertoire analysis. 73% of all CCs identified representing 77% of the isolates contained in these CCs were predominant in either African or German region. Significant differences between African versus German isolates were found for alleles encoding the accessory gene regulator type, enterotoxins, the Panton-Valentine leukocidin, immune evasion gene cluster, and adhesins. PCA in conjunction with silhouette analysis distinguished nine separable PCA clusters, with five clusters primarily comprising of African and two clusters of German isolates. Significant differences between S. aureus lineages in Africa and Germany may be a clue to explain the apparent difference in disease between tropical/(so-called) developing and temperate/industrialized regions. In low-resource countries further clinical-epidemiologic research is warranted not only for neglected tropical diseases but also for major bacterial infections.

Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology.

Staphylococcus aureusis a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed anin silicotyping scheme for the software SeqSphere(+)(Ridom GmbH, Münster, Germany). The implemented target genes (n= 182) correspond to those queried by the IdentibacS. aureusGenotyping DNA microarray (Alere Technologies, Jena, Germany). Thein silicoscheme was evaluated by comparing the typing results of microarray and of WGS for 154 humanS. aureusisolates. A total of 96.8% (n= 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosomemecelement types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences.