Molecular profile of gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana.

Doctoral Degree. University of KwaZulu-Natal, Durban.Gram-negative ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp) pathogens are a major healthcare concern globally due to their increasing multidrug resistance and ability to cause debilitating infections. Phenotypic and genotypic characteristics of multidrug resistant Gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana were investigated. Two hundred (200) clinical, non-duplicate Gram-negative bacterial pathogens were randomly selected from various human specimens routinely processed by the diagnostic microbiological laboratory in the hospital. Multidrug resistant (isolates resistant to at least one agent in three or more antibiotic class) isolates selected from each group of Gram-negative ESKAPE pathogens constituted the final sample. Identification and antibiotic susceptibility profiles were carried out using Vitek-2. Identity of isolates for whole genome sequencing was further confirmed by MALDI-TOF MS. Four P. aeruginosa and 10 K. pneumoniae were subjected to whole genome sequencing based on their extensively drug resistant profiles and resistance to third-generation cephalosporins respectively using Illumina MiSeq, after genomic DNA extraction using the NucliSens easyMAG®. Antibiotic resistance genes and plasmids were identified by mapping the sequence data to an online database using ResFinder and plasmidFinder respectively. MLST was also determined from the WGS data. The raw read sequences and assembled whole genome contigs have been deposited in GenBank under project number PRJNA411997. An average multidrug resistance of 89.5% was observed, ranging from 53.8% in Enterobacter spp to 100.0% in Acinetobacter spp and P. aeruginosa. Gram-negative ESKAPE bacteria constituted 48.5% (97) of the 200 isolates. P. aeruginosa (n=4) belonging to ST234 harboured blaDIM-1, blaIMP-34, blaOXA-10, blaOXA-129, blaOXA-50, blaPAO aadA1, aac4 aph(3’)-IIb, fosA, sul1, dfrB5, catB7, arr-2 conferring resistance to β-lactams, aminoglycosides, fosfomycin, sulphonamides, trimethoprim phenicols and rifampin respectively. qnrVC was detected in two of the four isolates . Both blaDIM-1 and blaIMP-34-like positive contigs showed identical DNA sequences and were linked to type 1 integron structures. BlaDIM-1 was 100% identical to the blaDIM-1 prototype gene, while blaIMP-34-like had two base pair (bp) differences T190C and C314G respectively compared to blaIMP-34, leading to one amino acid substitution in IMP-34-like indicating that, the gene may have independently evolved, perhaps due to selection pressure. Blast analysis did not reveal identical genetic structures deposited in NCBI, neither among the nucleotide collection, completed genomes nor among the completed plasmids. β-lactamases (blaCTX-M-15, blaSHV-11, blaTEM-1B) and resistance genes for aminoglycosides (aac(3)-IIa-like,aph(3')-Ia) quinolones/fluoroquinolones (oqxA-like,oqxB-like,qnrB10-like,qnrB2) and others including fosfomycin (fosA), trimethoprim (dfrA14), and sulphonamide (sul2) were found in the K. pneumoniae (n=10). Multiple and diverse mutations of the quinolone resistance-determining regions gyrA, gyrB and parC genes were detected in the K. pneumoniae (n=4), which were clonally distinct. The diversity of resistance genes expressed by Gram-negative ESKAPE pathogens conferring resistance to multiple antibiotics is problematic in a resource-constrained country like Ghana, necessitating urgent antibiotic stewardship and infection prevention and control interventions

Carbapenem resistance determinants acquired through novel chromosomal integrations in extensively drug-resistant pseudomonas aeruginosa

Two novel blaDIM-1- or blaIMP-1-containing genomic islands (GIs) were discovered by whole-genome sequence analyses in four extensively drug-resistant (XDR) Pseudomonas aeruginosa isolates from inpatients at a tertiary hospital in Ghana. The strains were of sequence type 234 (ST234) and formed a phylogenetic clade together with ST111, which is recognized as a global high-risk clone. Their carbapenem resistance was encoded by two Tn402-type integrons, In1592 (blaDIM-1) and In1595 (blaIMP-1), both carrying complete tni mobilization modules. In1595 was bound by conserved 25-bp inverted repeats (IRs) flanked by 5-bp direct repeats (DRs) associated with target site duplication. The integrons were embedded in two GIs that contained cognate integrases and were distinguished by a lower GC content than the chromosomal average. PAGI-97A (52.659 bp; In1592), which encoded a P4-type site-specific integrase of the tyrosine recombinase family in its 3′ border, was integrated into tRNA-Pro(ggg) and bracketed by a 49-bp perfect DR created by 3′-end target duplication. GIs with the same structural features, but diverse genetic content, were identified in 41/226 completed P. aeruginosa genomes. PAGI-97B (22,636 bp; In1595), which encoded an XerC/D superfamily integrase in its 5′ border, was inserted into the small RNA (sRNA) PrrF1/PrrF2 locus. Specific insertions into this highly conserved locus involved in iron-dependent regulation, all leaving PrrF1 intact, were identified in an additional six phylogenetically unrelated P. aeruginosa genomes. Our molecular analyses unveiled a hospital-associated clonal dissemination of carbapenem-resistant ST234 P. aeruginosa in which the XDR phenotype resulted from novel insertions of two GIs into specific chromosomal sites

PHYTOCHEMICAL INVESTIGATION AND ANTI-MICROBIAL ACTIVITY OF CLAUSENA ANISATA (WILLD), HOOK.

Background: Clausena anisata belongs to the family Rutaceae, a shrub widely used in West Africa for the treatment of bacterial and fungal infections of the skin including boils, ringworm and eczema. The study was designed to evaluate the antimicrobial activity and phytochemical screening of ethanol leaf extract of C. anisata (CLE). Method: Antimicrobial activity of CLE was investigated using agar well diffusion and micro-dilution methods against four Gram-positive bacteria (Bacillus substilis NCTC 10073, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Bacillus thuringiensis ATCC 13838) and two Gram-negative bacteria (Pseudomonas aeruginosa ATCC 4853, Proteus vulgaris ATCC 4175) and a clinical isolate of Candida albicans. Results: CLE was active against all test organisms with minimum inhibitory concentration (MIC), range of 0.5 to 7.0 mg/mL against Gram-positive bacteria, 2.5 to 1.0 mg/mL against Gram-negative bacteria and 5.5mg/mL against C. albicans. The MICs of the methanol fraction of CLE were 0.6 mg to 5.0/mL and 1.0 to 3.0 mg/mL for Gram-positive and Gram-negative bacteria respectively. Chloroform fraction had MIC of 3.0 to 7.5 mg/mL and 2.0 to 6.5 mg/mL for Gram-positive and Gram-negative bacteria, respectively and petroleum ether fraction had 4.5 to 8.0 mg/mL for Gram-positive and Gram-negative bacteria. The CLE exhibited static action against all test organisms within a range of 0.5 to 22.0 mg/mL. Phytochemical screening of C. anisata revealed the presence of tannins, flavonoids, steroids, saponins, glycosides and alkaloids. HPLC finger-printing of the CLE and its fractions were determined. Conclusion: These results may justify the medicinal uses of C. anisata for the treatment of microbial infection

Multidrug-resistant gram-negative bacterial infections in a teaching hospital in Ghana

Abstract Background Multidrug-resistant Gram-negative bacteria have emerged as major clinical and therapeutic dilemma in hospitals in Ghana. To describe the prevalence and profile of infections attributable to multidrug-resistant Gram-negative bacteria among patients at the Komfo Anokye Teaching Hospital in the Ashanti region of Ghana. Methods Bacterial cultures were randomly selected from the microbiology laboratory from February to August, 2015. Bacterial identification and minimum inhibitory concentrations were conducted using standard microbiological techniques and the Vitek-2 automated system. Patient information was retrieved from the hospital data. Results Of the 200 isolates, consisting of K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp., E. coli, Yersinia spp., Proteus mirabilis, Pasteurella spp., Chromobacterium violaceum, Salmomella enterica, Vibrio spp., Citrobacter koseri, Pantoea spp., Serratia spp., Providencia rettgeri Burkholderia cepacia, Aeromonas spp., Cadecea lapagei and Sphingomonas paucimobilis, 101 (50.5%) and 99 (49.5%) recovered from male and female patients respectively The largest proportion of patients were from age-group ≥60 years (24.5%) followed by < 10 years (24.0%) and least 10–19 years (9.5%) with a mean patient age of 35.95 ± 27.11 (0.2–91) years. The decreasing order of specimen source was urine 97 (48.5%), wound swabs 47 (23.5%), sputum 22 (11.0%) bronchial lavage, nasal and pleural swabs 1 (0.50%). Urinary tract infection was diagnosed in 34.5% of patients, sepsis in 14.5%, wound infections (surgical and chronic wounds) in 11.0%, pulmonary tuberculosis in 9.0% and appendicitis, bacteremia and cystitis in 0.50%. The isolates showed high resistance to ampicillin (94.4%), trimethoprim/sulfamethoxazole (84.5%), cefuroxime (79.0%) and cefotaxime (71.3%) but low resistance to ertapenem (1.5%), meropenem (3%) and amikacin (11%). The average multi-drug resistance was 89.5%, and ranged from 53.8% in Enterobacter spp. to 100.0% in Acinetobacter spp. and P. aeruginosa. Conclusion Bacterial infections caused by multi-drug resistant (isolates resistant to at least one agent in three or more antibiotic classes) Gram-negative pathogens among patients at Komfo Anokye Teaching Hospital in Kumasi, Ghana are rife and interventions are necessary for their containment

Antimicrobial, Antioxidant, and Wound Healing Properties of Kigelia africana (Lam.) Beneth. and Strophanthus hispidus DC.

Microbial infections of various types of wounds are a challenge to the treatment of wounds and wound healing. The study was to investigate antimicrobial and antioxidant properties of methanol leaf and stem bark extracts of Kigelia africana and methanol leaf and root extracts of Strophanthus hispidus and also to determine wound healing properties of the extracts. The antimicrobial activities of the methanol extracts were determined against two Gram-positive and two Gram-negative bacteria and a fungus using agar diffusion and micro-dilution methods. The antioxidant activity was determined using 1,1-diphenyl-2-picryl–hydrazyl (DPPH) method. The influence of the extracts on rate of wound closure was investigated using the excision wound model and histopathological investigation of treated and untreated wound tissues performed. The MICs of leaf extract of K. africana against test organisms were 2.5–7.5 mg/mL and stem bark extract were 2.25–7.5 mg/mL. The leaf extract of S. hispidus had MIC range of 2.5–7.5 mg/mL and 2.5–10 mg/mL for root extract. The IC50 of leaf and stem bark extracts of K. africana were 56.9 and 13.7 μg/mL, respectively and leaf and root of S. hispidus were 49.8 and 45.1 μg/mL, respectively. K. africana extracts (7.5% w/w) showed significant () wound contraction at day 7 with 72% of wound closure whiles significant () wound contractions were observed on day 11 for stem bark of K. africana, leaf and root extracts of S. hispidus. Wound tissues treated with the extracts showed improved collagenation, re-epitheliazition and rapid granulation formation compared with untreated wound tissues. The extracts were found to contain alkaloids, saponins, tannins, flavonoids, carbohydrates, and sapogenetic glycosides. The HPLC finger-printing of the extracts were developed. The leaf, stem bark and root extracts of K. africana and S. hispidus exhibited antimicrobial, antioxidant, and enhanced wound healing properties and these may justify the medicinal uses of the plants for treatment of microbial infections and wounds

Genomic characterization of multidrug-resistant ESBL-producing Klebsiella pneumoniae isolated from a Ghanaian teaching hospital

Objectives - This study delineated the clonal lineages, antibiotic resistome and plasmid replicon types in multidrug-resistant K. pneumoniae isolates from a teaching hospital in Ghana. Methods - Identification and antibiotic susceptibility testing were done using the MALDI-TOF MS and Vitek-2 automated system. Genomic DNA extraction was carried out using the NucliSens easyMAG® (BioMérieux) kits and the DNA was subjected to whole genome sequencing (WGS) using the Illumina MiSeq platform. Results - Of the 200 isolates obtained, 37 were identified as K. pneumoniae of which 9 were resistant to all second and third-generation cephalosporins. These 9 isolates selected for further genomic analysis were characterized by the presence of 8 diverse sequence types (STs), capsular polysaccharide serotypes (K types and wzi allelic types) and multiple genes encoding resistance to β-lactams (blaCTX-M-15, blaSHV-11, blaTEM-1B, blaOXA-1), aminoglycosides (aac(3)-IIa, strB, strA, aadA16), fluoroquinolones/quinolones (qnrB66, oqxA, oqxB) and other antibiotic classes. Resistance genes were associated with plasmids, predominantly IncFIB(K) and ColRNAI. Multiple and diverse mutations in quinolone resistance-determining regions of gyrA (S83Y, D87A) and parC (S80I, N304S) in isolates resistant to ciprofloxacin (MIC ≥ 4 mg/mL) were found. Global phylogenomic analysis affirmed the diverse clonal clustering and origin of these isolates. Conclusions - The varied clonal clusters and resistome identified in the multidrug-resistant K. pneumoniae isolates is a major threat to the management of infections in Ghana. The molecular characterization of antibiotic resistance is thus imperative to inform strategies for containment

The path to longer and healthier lives for all Africans by 2030: the Lancet Commission on the future of health in sub-Saharan Africa

Sub-Saharan Africa’s health challenges are numerous and wide-ranging. Most sub-Saharan African countries face a double burden of traditional, persisting health challenges, such as infectious diseases, malnutrition, and child and maternal mortality, and emerging challenges from an increasing prevalence of chronic conditions, mental health disorders, injuries, and health problems related to climate change and environmental degradation. Although there has been real progress on many health indicators, life expectancy and most population health indicators remain behind most low-income and middle-income countries in other parts of the world. Our Commission was prompted by sub-Saharan Africa’s potential to improve health on its own terms, and largely with its own resources. The spirit of this Commission is one of evidence-based optimism, with caution. We recognise that major health inequities exist and that health outcomes are worst in fragile countries, rural areas, urban slums, and conflict zones, and among the poor, disabled, and marginalised. Moreover, sub- Saharan Africa is facing the challenges and opportunities of the largest cohort of young people in history, with the youth population aged under 25 years predicted to almost double from 230 million to 450 million by 2050. The future of health in Africa is bright, but only if no one is left behind