John Osei

Antibiotic-resistant Klebsiella pneumoniae is increasingly being implicated in invasive infections worldwide with high mortalities. Forty-two multidrug resistant (MDR) K. pneumoniae isolates were collected over a 4-month period. Antimicrobial susceptibility was determined using Microscan. The evolutionary epidemiology, resistome, virulome and mobilome of the isolates were characterised using whole-genome sequencing and bioinformatics analysis. All isolates contained the blaCTX-M gene, whilst 41/42(97%) contained blaTEM, 36/42(86%) contained blaOXA and 35/42(83%) harboured blaSHV genes. Other resistance genes found included blaLEN, aac(6′)-lb-cr, qnrA, qnrB, qnrS, oqxAB, aad, aph, dfr, sul1, sul2, fosA, and cat genes. Fluoroquinolone and colistin resistance-conferring mutations in parC, gyrAB, pmrAB, phoPQ and kpnEF were identified. The blaLEN gene, rarely described worldwide, was identified in four isolates. The isolates comprised diverse sequence types, the most common being ST152 in 7/42(17%) isolates; clone-specific O and K capsule types were identified. Diverse virulence genes that were not clone-specific were identified in all but one isolate. IncF, IncH and IncI plasmid replicons and two novel integrons were present. The blaCTX-M-15 and blaTEM-1 genes were bracketed by Tn3 transposons, ISEc9, a resolvase and IS91 insertion sequence. There were 20 gene cassettes in 14 different cassette arrays, with the dfrA and aadA gene cassettes being the most frequent. Phylogenetic analysis demonstrated that the isolates were evolutionarily associated with strains from both South Africa and abroad. These findings depict the rich resistome, mobilome and virulome repertoire in clinical K. pneumoniae strains, which are mainly transmitted by clonal, multiclonal and horizontal means in South Africa.

Studies evaluating the new GeneXpert Ultra with other rapid diagnostic assays are limited, particularly in different geographical settings. The performance of the GeneXpert Ultra, the GeneXpert G4, the Line probe assays (LPA) and auramine smear microscopy in detecting TB in pulmonary and extra-pulmonary samples were thus evaluated.
Remnants (n=205 samples) of pulmonary (n=125 samples) and extra-pulmonary (n=80 samples) specimens from TB suspects were prospectively collected. Each sample was divided for diagnosis using microscopy, GeneXpert MTB/RIF assays, and LPA; these were all comparatively evaluated, using the MGIT 960 culture as a gold standard.
The sensitivity and specificity of microscopy, Xpert Ultra, Xpert G4 and MTBDRplus (ver 2) in pulmonary samples were respectively: 82.00% and 90.28%; 88.00% and 58.57%; 79.59% and 90.28%; 80.00% and 11.11%. For extra-pulmonary specimen, the sensitivity and specificity were respectively: 53.85% and 98.51%; 69.23% and 49.25%; 50.00% and 97.01%; 69.23% and 25.37%.
The new and improved GeneXpert Ultra assay was more sensitive than GeneXpert G4 and LPA in both pulmonary and extra pulmonary samples, albeit with lower specificity than the GeneXpert G4. The auramine and LPA tests were also highly sensitive, although the LPA was less specific.

Antibiotic-resistant Escherichia coli is a common occurrence in food, clinical, community and environmental settings worldwide. The resistome, mobilome, virulome and phylogenomics of 20 multidrug resistant (MDR) clinical E. coli isolates collected in 2013 from Pretoria, South Africa, were characterised. The isolates were all extended-spectrum β-lactamase producers, harbouring CTX-M (n=16; 80%), TEM-1B (n=10; 50%) and OXA (n=12, 60%) β-lactamases alongside genes mediating resistance to fluoroquinolones, aminoglycosides, tetracyclines etc. Most resistance determinants were found on contigs containing IncF plasmid replicons and bracketed by composite transposons (Tn3), diverse ISs and class 1 integrons (In13, In54, In369, and In467). Gene cassettes such as blaOXA, dfrA5-psp-aadA2-cmlA1a-aadA1-qac and estX3-psp-aadA2-cmlA1a-aadA1a-qac were encompassed by Tn3 and ISs; several isolates had same or highly similar genomic antibiotic resistance islands. ST131 (n=10), ST617 (n=2) and singletons of ST10, ST73, ST95, ST410, ST648, ST665, ST744 and ST998 clones were phylogenetically related to clinical (human and animal) strains from Egypt, Kenya, Niger, Nigeria, Tanzania, and UK.  A rich repertoire of virulence genes, including iss, gad and iha were identified. MDR E. coli harbouring chromosomal and plasmid-borne resistance genes in same and multiple clones exist in South Africa, which is very worrying for clinical epidemiology and infectious diseases management.

Significance and impact of study
We report on the first clinical Morganella morganii draft genomes from Africa. The isolates were found in the urine of patients presenting with urinary tract infections (UTIs). Notably, they were resistant to important clinical antibiotics, including those used to treat UTIs. Due to the common occurrence of UTIs, particularly among pregnant women for whom drug options are limited, the presence of antibiotic-resistant uropathogens such as M. morganii is a serious public health concern. We therefore characterised the resistance mechanisms and epidemiology of these isolates to provide further insights into their dissemination and background data for future studies.
Abstract
Morganella morganii is an opportunistic bacterial pathogen of the Enterobacteriaceae family that is occasionally isolated from clinical (animal and human) specimens with varying resistance profiles. Detailed genomic analyses of drug-resistant M. morganii strains are relatively limited, particularly in Africa, which is also due to their relatively low isolation rates from clinical settings. Here, we report on two multidrug-resistant clinical M. morganii isolates from urine specimens of two hospitalised patients in South Africa who presented with urinary tract infections in 2013. The isolates, M006 and E042, were only susceptible to carbapenems, amikacin and tigecycline. One strain, M006, had a novel class 1 integron, ln1484, associated with aadA7, sul1and gcuD gene cassettes and a Col3M plasmid replicase gene. The ln1484 intI1:aadA7:sul1 genes were bracketed by a TnAs3 composite transposon while a tet(B) gene was found on an IS4 family transposon. The rare blaDHA-4 and blaDHA-1 AmpC β-lactamase genes were identified on the isolates’ chromosome. The isolates were phylogenetically distant and closely related to other international strains, suggesting that they were not obtained from a single epidemiological source. Further molecular surveillance is necessary to establish the prevalence of these MDR strains in the tertiary hospital. Moreover, antibiotic stewardship and antibiotic sensitivity testing of all clinical isolates should be undertaken after empirical treatment to inform tailored therapy as well as reduce escalation of resistance and associated morbidities and mortalities.

Background

Tuberculosis (TB) remains a main global public health problem. However, a systematic review of TB resistance epidemiology in Africa is wanting.
Methods

A comprehensive systematic search of PubMed, Web of Science and ScienceDirect for English research articles reporting on the molecular epidemiology of Mycobacterium tuberculosis complex resistance in Africa from January 2007 to December 2018 was undertaken.
Results and conclusion

Qualitative and quantitative synthesis were, respectively, undertaken with 232 and 186 included articles, representing 32 countries. TB monoresistance rate was highest for isoniazid (59%) and rifampicin (27%), particularly in Zimbabwe (100%), Swaziland (100%), and Sudan (67.9%) whilst multidrug resistance (MDR) rate was substantial in Zimbabwe (100%), Sudan (34.6%), Ivory Coast (24.5%) and Ethiopia (23.9%). Resistance-conferring mutations were commonly found in katG (n = 3694), rpoB (n = 3591), rrs (n = 1272), inhA (n = 1065), pncA (n = 1063) and embB (n = 705) in almost all included countries: S315G/I/N/R/T, V473D/F/G/I, Q471H/Q/R/Y, S303C/L etc. in katG; S531A/F/S/G, H526A/C/D/G, D516A/E/G etc. in rpoB; A1401G, A513C etc. in rrs; -15C→T, -17G→A/T, -16A→G etc. in inhA; Ins456C, Ins 172 G, L172P, C14R, Ins515G etc. in pncA. Commonest lineages and families such as T (n = 8139), LAM (n = 5243), Beijing (n = 5471), Cameroon (n = 3315), CAS (n = 2021), H (n = 1773) etc., with the exception of T, were not fairly distributed; Beijing, Cameroon and CAS were prevalent in South Africa (n = 4964), Ghana (n = 2306), and Ethiopia/Tanzania (n = 799/635), respectively. Resistance mutations were not lineage-specific and sputum (96.2%) were mainly used for diagnosing TB resistance using the LPA (38.5%), GeneXpert (17.2%), whole-genome sequencing (12.3%) and PCR/amplicon sequencing (9%/23%). Intercountry spread of strains was limited while intra-country dissemination was common. TB resistance and its diagnosis remain a major threat in Africa, necessitating urgent action to contain this global menace.

Objectives: The underlying resistance mechanism and phylogenetic relationship of a colistinresistant Salmonella Enteritidis strain EC20120916 that resulted in fatal meningitis in an immunecompromised patient was investigated by analysing the genome sequence.

Methods: Whole-genome sequencing was performed on strain EC20120916 with the Illumina MiSeq platform. Annotation of the sequence was performed using the prokaryotic genome annotation pipeline (PGAP). Antibiotic resistance gene, plasmid replicons and pathogenicity islands were identified. A phylogenetic tree was constructed using Parsnp and edited with Figtree.

Results: The genome size was 4, 699, 318 bp with a GC content of 55.2% and 4471 proteinencoding genes. The aac(6’)-laa gene, encoding resistance to aminoglycosides, was identified, although this was not expressed phenotypically in the isolate. No colistin resistance-conferring mutations or plasmid-mediated mechanisms were identified to explain the colistin resistance. The strain was phylogenetically related to three international strains, although it was not close enough to suggest importation from outside South Africa.

Conclusion: This is the first report of a colistin-resistant Salmonella Enteritidis isolate causing meningitis in an immune-compromised patient in South Africa. The absence of colistin resistance-conferring mutations or plasmid-mediated resistance mechanisms suggest that a novel mechanism is responsible for the colistin resistance in this isolate. The isolate was acquired locally.

Background: Whole-genome sequence analysis was performed on a multidrug-resistant Providentia rettgeri PR002 clinical strain isolated from the urine of a hospitalised patient in Pretoria, South Africa, in 2013.
Methods: The resistome, mobilome, pathogenicity island(s), as well as virulence and heavy-metal resistance genes of the isolate were characterised using whole-genome sequencing and bioinformatic analysis.
Results: PR002 had a genome assembly size of 4 832 624 bp with a GC content of 40.7%, an A/C2 plasmid replicase gene, four integrons/gene cassettes, 17 resistance genes, and several virulence and heavy metal resistance genes, confirming PR002 as a human pathogen. A novel integron, In1483, harbouring the blaOXA-2 gene was identified, with other uncharacterised class 1 integrons harbouring aacA4cr and dfrA1. Aac(3’)-IIa and blaSCO-1, as well as blaPER-7, sul2 and tet(B) were found bracketed by composite Tn3 transposons, and IS91, IS91 and IS4 family insertion sequences respectively. PR002 was resistant to all antibiotics tested except amikacin, carbapenems, cefotaxime-clavulanate, ceftazidime-clavulanate, cefoxitin, and fosfomycin. PR002 was closely related to PR1 (USA), PRET_2032 (SPAIN), DSM_1131 and NCTC7477 clinical P. rettgeri strains, but not close enough to suggest it was imported into South Africa from other countries.
Conclusion: MDR in P. rettgeri is rare, particularly in clinical settings, making this case a very important incident requiring urgent attention. This is also the first report of an A/C plasmid in P. rettgeri. The array, multiplicity and diversity of resistance and virulence genes in this strain are concerning, necessitating stringent infection control, antibiotic stewardship and periodic resistance surveillance/monitoring policies to pre-empt further horizontal and vertical spread of these resistance genes.

Background: Mycobacterium bovis BCG is a live, attenuated tuberculosis vaccine. While the vaccine protects infants from tuberculosis, complications including, disseminated infections have been reported following vaccination. Genetically diverse BCG sub-strains now exist following continuous passaging of the original Pasteur strain for vaccine manufacture. This genetic diversity reportedly influences the severity of disseminated BCG infections and the efficacy of BCG immunization.
Methods: Mycobacterium bovis BCG were isolated from infants suspected of being infected with tuberculosis. The whole genome of the clinical isolates and BCG Moscow were sequenced using Illumina Miseq and the sequences were analysed using CLC Genomics Workbench 7.0, PhyResSE v1.0 and Parsnp.
Results and conclusion: Genetic variations between the clinical strains and the reference BCG Copenhagen were identified. The clinical strains shared only one mutation in a secretion protein. We identified mutations in various antibiotic resistance genes in the BCG isolates, which suggest their potential as MDR phenotypes. Phylogenetic analysis showed that the two isolates were distantly related, and the M1_S48 clinical isolate was closely related to M. bovis BCG Moscow. Our phylogenomics results imply that two different BCG strains may be circulating in South Africa. However, it is difficult to associate the administered BCG vaccine strain and the supplied BCG strain with specific adverse events as BCGiosis is under-reported. This study presents background genomic information for future surveillance and tracking of the distribution of BCGiosis-associated mycobacteria. It is also the first to report on the genomes of clinical BCG strains in Africa.

Modern advances in genomics provide an opportunity to re-interpret historical bacterial
culture collections. In this study, genotypic antibiotic resistance profiles of Mycobacterium tuberculosis isolates from an historical 20-year-old multidrug-resistant tuberculosis (MDR-TB) culture collection in South Africa are described. DNA samples extracted from the phenotypically MDR-TB isolates (n=240) were assayed by Hain line probe assay (LPA) for the confirmation of MDR-TB and by Illumina Miseq whole genome sequencing (WGS) for the characterization of mutations in eight genes (rpoB, katG, inhA, rpsL, pncA, embB, gyrA and rrs) that are known to code for resistance to commonly used anti-TB agents.
LPA identified 71.3% of the TB isolates as MDR-TB, 18.3% as rifampicin (RIF) mono- resistant, 2% as isoniazid (INH) mono-resistant, and 8.3% as susceptible to both RIF and INH. In a subset of 42 randomly selected isolates designated RIF+INH-resistant by Löwenstein-Jensen (LJ) culture in 1993, LPA and WGS results confirmed MDR-TB. In all five INH mono-resistant isolates by LPA, and in all but one (wild-type) of the 34 successfully sequenced RIF mono- resistant isolates, WGS revealed matching mutations. Only 26% of isolates designated as susceptible by LPA, however, were found to be wild-type by WGS. Novel mutations were found in the rpoB (Thr480Ala, Gln253Arg, Val249Met, Val251Tyr; Val251Phe), katG (Trp477STOP; Gln88STOP; Trp198STOP; and Trp412STOP), embB (Thr11Xaa, Gln59Pro) and pncA (Thr100Ile, Thr159Ala, Ala134Arg; Val163Ala, Thr153Ile, DelGpos7, Phe106Ser) genes. Three MDR-TB isolates showed mutations in both the gyrA and rrs genes, suggesting that extensively drug-resistant tuberculosis existed in South Africa well-before its formal recognition in 2006.

Bacteria and fungi continue to develop new ways to adapt and survive the lethal or biostatic effects of antimicrobials through myriad mechanisms. Novel antibiotic resistance genes such as lsa(C), erm(44), VCC-1, mcr-1, mcr-2, mcr-3, mcr-4, bla KLUC-3 and bla KLUC-4 were discovered through comparative genomics and further functional studies. As well, mutations in genes that hitherto were unknown to confer resistance to antimicrobials, such as trm, PP2C, rpsJ, HSC82, FKS2 and Rv2887, were shown by genomics and transcomplementation assays to mediate antimicrobial resistance in Acinetobacter baumannii,

Background: Carbapenems (CAR), colistin (CST), and tigecycline (TGC) alone or in combination therapy has become the last-resort antibiotics for treating infections caused by multidrug resistant (MDR) bacteria. However, resistance to these reserve antibiotics are increasingly being reported worldwide. Hence, the quest to find other agents that will synergistically restore the efficacy of these antibiotics have increased.

Abstract
A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase-producing Gram-negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans-complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT-PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC-FGH-IJK, mexAB-XY-oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb ) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr-1 gene conferring resistance to colistin was identified via WGS, trans-complementation and a murine thigh infection model studies.Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI-TOF MS, micro-array and real time multiplex PCR hold much promise for the future as new detection tools

The move towards intensive commercial pig production in Ghana is increasing at an unprecedented pace albeit financial, feeding and swine flu challenges. Coupled with religious, traditional and social marginalization, the industry has suffered from poor growth for many years. In order to describe the current state and challenges facing the pig industry and the major factors influencing its commercialization in Ghana, 110 pig farms within five Districts in the Ashanti region were studied. Increasing pressure on land (77.27%), poor markets (87.27%) and disease threats (91.82%) were the major factors influencing the adoption of intensive pig farming which increased farmers’ financial responsibilities. Major problems faced by intensive farmers included scarcity and high cost of feed and frequent disease outbreaks which destroyed their markets that forced many farms to close down annually. The size of the farms, number of staff, source of water, location of the farm, feeding, infrastructure, farm hygiene, marketing and security were all influenced by the financial capital of the farmers. Poor waste management practices and farm locations were potential hazards to public health. The farms were hardly threatened by thieves or wild animals. There is much promise in the pig industry currently. Provision of feed subsidies and credit to farms and stabilizing market prices are interventions government can adopt to woo local and foreign investors, grow the industry, and reduce unemployment and meat import. Improved hygiene and better waste management on farms could reduce disease outbreaks and zoonoses transmission.

The study aimed to describe the prevalence and distribution of protozoal infections and their predisposing risk factors among pupils in the Kwabre East District, Ghana. Intestinal protozoal infections are a common occurrence among school children, especially in tropical climates and underdeveloped societies owing to poor hygiene and sanitation, overpopulation, illiteracy and low living standards. Fresh stool samples from 884 pupils—one sample per person—representing 48 schools were screened for intestinal protozoa using iodine and saline mounts and the formol-ether concentration technique after questionnaire administration. An isolation rate of 55.7% (492/884) of which 59.6%, 24.8% and 15.7% were Giardia lamblia, Entamoeba coli and Entamoeba hystolytica/dispar respectively, was obtained. G. lamblia infection intensity was highest throughout all the circuits within the district. Parents with low education, buying food from vendors, age, poor hand washing practices after visiting the lavatory, drinking borehole/well water, using pit latrines and not taking anti-protozoal medications were factors that predisposed pupils to intestinal protozoal infections. There was a substantial prevalence of G. lamblia, E coli and E. hystolytica infections among the Kwabre East District pupils owing to their ages, toilets used, hand washing practices, feeding practices, parents’ educational status and sources of drinking water.

Research articles describing carbapenemases and their genetic environments in Gram-negative bacteria were reviewed to determine the molecular epidemiology of carbapenemases in Africa. The emergence of resistance to the carbapenems, the last resort antibiotic for difficult to treat bacterial infections, affords clinicians few therapeutic options, with a resulting increase in morbidities, mortalities, and healthcare costs. However, the molecular epidemiology of carbapenemases throughout Africa is less described. Research articles and conference proceedings describing the carbapenemase genetic environment and molecular epidemiology in Africa were retrieved from Google Scholar, Scifinder, Pubmed, Web of Science, and Science Direct databases. Predominant carbapenemase genes so far described in Africa include the blaOXA-48 type, blaIMP, blaVIM, and blaNDM in Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter spp., and Escherichia coli carried on various plasmid types and sizes, transposons, and integrons. Consequently, the true molecular epidemiology of carbapenemases and their genetic environment in Africa is still unknown. Class D and class B carbapenemases, mainly prevalent in A. baumannii, K. pneumoniae, E. cloacae, Citrobacter spp., and E. coli were the commonest carbapenemases. Carbapenemases are mainly reported in North and South Africa as under-resourced laboratories, lack of awareness and funding preclude the detection and reporting of carbapenemase-mediated resistance.

Introduction.Salmonellatyphimuriumisassociatedwithoutbreaksoffood-bornenontyphoidalsalmonellosis(NTS)worldwide withmarkedmultidrugresistance.Objectives.ThisstudyaimedtodeterminetheprevalenceofantibioticresistantSalmonella typhimuriuminpigsinAshantiRegion,Ghana.Methods.Faecesfrom10pigsperpigfarmwerecollectedandmixedtoobtain 108multiply-compositefaecalsamples.Standardmicrobiologyandbiochemicalprocedureswereusedtoisolateandidentifyan S.typhimuriumisolatefromthecompositefaecalsampleofeachfarm.Antibioticsensitivitytestwascarriedouttodetermine thesensitivityoftheisolates.Discussion.Fromthe108multiply-compositefaecalsamples,72S.typhimuriumisolateswere obtainedfrom72separatecompositesamplesrepresenting72differentpigfarms.Ofthe72faecalisolates,32(52.8%)were resistanttoatleastoneantibiotic.Twenty-sevenisolates(71.1%)wereresistanttoamoxicillinandstreptomycin.Resistanceto tetracycline,doxycycline,andciprofloxacinwasfoundin17(44.7%),15(39.5%),and8(21.1%)isolates,respectively.Resistance tonorfloxacin,sulphamethoxazole-trimethoprim,andgentamicinwereexpressedin3(7.9%),3(7.9%),and0(0.0%)isolates, respectively.Conclusion.MultipledrugresistanceiscommoninS.typhimuriumisolates,manyofwhichcouldbelongtothesame clone,frompigsinAshantiRegion,Ghana.