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Increasingly, gut microbiota distortions are being implicated in the pathogenesis of several infectious and noninfectious diseases. Specifically, in the absence of an eubiotic microbiota, mice are more prone to colonization and infection by Mycobacterium tuberculosis (Mtb). In this qualitative analysis, the following were observed: (1) antimicrobials cause long-term gut microbiota perturbations; (2) Mtb causes limited and transient disturbances to the lung-gut microbiota; (3) pathogens (e.g., Helicobacter hepaticus) affect microbiota integrity and reduce resistance to Mtb; (4) dysbiosis depletes bacterial species regulating proper immune functioning, reducing resistance to Mtb; (5) dysregulated immune cells fail to express important pathogen-recognition receptors (e.g., macrophage-inducible C-type lectin; MINCLE) and Mtb-killing cytokines (e.g., IFN-γ, TNF-α, and IL-17), with hampered phagocytic capability; (6) autophagy is central to the immune system's clearance of Mtb, control of inflammation, and immunity-microbiome balance; (7) microbiota-produced short-chain fatty acids, which are reduced by dysbiosis, affect immune cells and increase Mtb proliferation; (8) commensal species (e.g., Lactobacillus plantarum) and microbiota metabolites (e.g., indole propionic acid) reduce tuberculosis progression; and (9) fecal transplants mostly restored eubiosis, increased immune resistance to Mtb, restricted dissemination of Mtb, and reduced tuberculosis-associated organ pathologies. Overuse of antimicrobials, as shown in mice, is a risk factor for reactivating latent or treated tuberculosis.

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 hospitalized 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. SIGNIFICANCE AND IMPACT OF THE 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 characterized the resistance mechanisms and epidemiology of these isolates to provide further insights into their dissemination and background data for future studies.

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.

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.

BackgroundDiagnostics for tuberculosis (TB) and treatment monitoring remains a challenge, particularly in less-resourced laboratories. Further, the comprehensive sputum microbiota of TB patients during treatment are less described, particularly using long-read sequencers.MethodsDNA from sputum samples collected from newly-diagnosed TB patients were sequenced with Oxford Nanopore’s MinION. MG-RAST and R packages (Phyloseq, α/β diversities, functional components, OTUs networks and ordination plots. Statistical significance of the generated data was determined using GraphPad.Results & conclusionAntibiotics reduced the abundance and functional subsystems of each samples’ microbiota from baseline until day 7, when persistent, tolerant, and resistant microbiota, including fungi, grew back again. Variations in microbiota abundance and diversity were patient-specific. Closer microbiome network relationships observed in baseline samples reduced until day 7, when it became closer again. Bacterial microbiota networks and spatial ordination relationships were closer than that of other kingdoms. Actinobacteria phylum and Mycobacterium were more affected by antibiotics than other phyla and genera. Parasites, viruses, and fungi were less affected by antibiotics than bacteria in a descending order. Resistance genes/mechanisms to important antibiotics, plasmids, transposons, insertion sequences, integrative conjugative elements were identified in few samples.MinION can be adopted clinically to monitor treatment and consequent dysbiosis, and identify both known and unknown pathogens and resistance genes to inform tailored treatment choices, specifically in TB.Author summaryTuberculosis (TB), one of the major killers of mankind, continually remains elusive as challenges with early diagnosis and treatment monitoring remain. Herein, we use a single portable sequencer from Oxford Nanopore, the minION, to diagnose TB and monitor its treatment with antibiotics using routine sputum samples. In addition, the presence of other pathogens, important commensals, antibiotic resistance genes, mobile genetic elements, and the effect of the antibiotic treatment on the sputum microbiota were determined from the same data. This makes the minION an important tool that can be used in clinical laboratories to obtain data that can inform live-saving decisions.

The spread of carbapenem- and polymyxin-resistant Enterobacteriaceae poses a significant threat to public health, challenging clinicians worldwide with limited therapeutic options. This review describes the current coding and noncoding genetic and transcriptional mechanisms mediating carbapenem and polymyxin resistance, respectively., The spread of carbapenem- and polymyxin-resistant Enterobacteriaceae poses a significant threat to public health, challenging clinicians worldwide with limited therapeutic options. This review describes the current coding and noncoding genetic and transcriptional mechanisms mediating carbapenem and polymyxin resistance, respectively. A systematic review of all studies published in PubMed database between 2015 to October 2020 was performed. Journal articles evaluating carbapenem and polymyxin resistance mechanisms, respectively, were included. The search identified 171 journal articles for inclusion. Different New Delhi metallo-β-lactamase (NDM) carbapenemase variants had different transcriptional and affinity responses to different carbapenems. Mutations within the Klebsiella pneumoniae carbapenemase (KPC) mobile transposon, Tn4401, affect its promoter activity and expression levels, increasing carbapenem resistance. Insertion of IS26 in ardK increased imipenemase expression 53-fold. ompCF porin downregulation (mediated by envZ and ompR mutations), micCF small RNA hyperexpression, efflux upregulation (mediated by acrA, acrR, araC, marA, soxS, ramA, etc.), and mutations in acrAB-tolC mediated clinical carbapenem resistance when coupled with β-lactamase activity in a species-specific manner but not when acting without β-lactamases. Mutations in pmrAB, phoPQ, crrAB, and mgrB affect phosphorylation of lipid A of the lipopolysaccharide through the pmrHFIJKLM (arnBCDATEF or pbgP) cluster, leading to polymyxin resistance; mgrB inactivation also affected capsule structure. Mobile and induced mcr, efflux hyperexpression and porin downregulation, and Ecr transmembrane protein also conferred polymyxin resistance and heteroresistance. Carbapenem and polymyxin resistance is thus mediated by a diverse range of genetic and transcriptional mechanisms that are easily activated in an inducing environment. The molecular understanding of these emerging mechanisms can aid in developing new therapeutics for multidrug-resistant Enterobacteriaceae isolates.

Background There is a general dearth of information on extrapulmonary tuberculosis (EPTB). Here, we investigated Mycobacterium tuberculosis (Mtb) drug resistance and transmission patterns in EPTB patients treated in the Tshwane metropolitan area, in South Africa. Methods Consecutive Mtb culture-positive non-pulmonary samples from unique EPTB patients underwent mycobacterial genotyping and were assigned to phylogenetic lineages and transmission clusters based on spoligotypes. MTBDRplus assay was used to search mutations for isoniazid and rifampin resistance. Machine learning algorithms were used to identify clinically meaningful patterns in data. We computed odds ratio (OR), attributable risk (AR) and corresponding 95% confidence intervals (CI). Results Of the 70 isolates examined, the largest cluster comprised 25 (36%) Mtb strains that belonged to the East Asian lineage. East Asian lineage was significantly more likely to occur within chains of transmission when compared to the Euro-American and East-African Indian lineages: OR = 10.11 (95% CI: 1.56–116). Lymphadenitis, meningitis and cutaneous TB, were significantly more likely to be associated with drug resistance: OR = 12.69 (95% CI: 1.82–141.60) and AR = 0.25 (95% CI: 0.06–0.43) when compared with other EPTB sites, which suggests that poor rifampin penetration might be a contributing factor. Conclusions The majority of Mtb strains circulating in the Tshwane metropolis belongs to East Asian, Euro-American and East-African Indian lineages. Each of these are likely to be clustered, suggesting on-going EPTB transmission. Since 25% of the drug resistance was attributable to sanctuary EPTB sites notorious for poor rifampin penetration, we hypothesize that poor anti-tuberculosis drug dosing might have a role in the development of resistance.

Background: There is a general dearth of information on extrapulmonary tuberculosis (EPTB). Here, we investigated Mycobacterium tuberculosis (Mtb) drug resistance and transmission patterns in EPTB patients treated in the Tshwane metropolitan area, in South Africa.Methods: Consecutive Mtb culture-positive non-pulmonary samples from unique EPTB patients underwent mycobacterial genotyping and were assigned to phylogenetic lineages and transmission clusters based on spoligotypes. MTBDRplus assay was used to search mutations for isoniazid and rifampin resistance. Machine learning algorithms were used to identify clinically meaningful patterns in data. We computed odds ratio (OR), attributable risk (AR) and corresponding 95% confidence intervals (CI). Results: Of the 70 isolates examined, the largest cluster comprised 25 (36%) Mtb strains that belonged to the East Asian lineage. East Asian lineage was significantly more likely to occur within chains of transmission when compared to the Euro-American and East-African Indian lineages: OR= 10.11 (95% CI: 1.56-116). Lymphadenitis, meningitis and cutaneous TB, were significantly more likely to be associated with drug resistance: OR=12.69 (95% CI: 1.82-141.60) and AR = 0.25 (95% CI: 0.06-0.43) when compared with other EPTB sites, which suggests that poor rifampin penetration might be a contributing factor.Conclusions: The majority of Mtb strains circulating in the Tshwane metropolis belongs to East Asian, Euro-American and East-African Indian lineages. Each of these are likely to be clustered, suggesting on-going EPTB transmission. Since 25% of the drug resistance was attributable to sanctuary EPTB sites notorious for poor rifampin penetration, we hypothesize that poor anti-tuberculosis drug dosing might have a role in the development of resistance.

Background There is a general dearth of information on extrapulmonary tuberculosis (EPTB). We investigated Mycobacterium tuberculosis (Mtb) drug resistance and transmission patterns in EPTB patients treated in the Tshwane metropolitan, South Africa. Methods Consecutive Mtb culture-positive non-pulmonary samples from unique EPTB patients underwent further mycobacterial genotyping and were assigned to phylogenetic lineages and transmission clusters based on spoligotypes. MTBDR plus assay was used to test for isoniazid and rifampin susceptibility. ML algorithms were used to identify clinically meaningful patterns in data. We computed odds ratio (OR), attributable risk (AR) and corresponding 95% confidence intervals (CI). Results Of the 70 isolates examined, the largest cluster comprised of 25 (36%) Mtb strains that belonged to the East Asian lineage. East Asian lineage was significantly more likely to occur within chains of transmission when compared to the Euro-American and East-African Indian lineages: OR= 10.11 (95% CI: 1.56-116). Lymphadenitis, meningitis and skin TB, were significantly more likely to be associated with drug resistance: OR=12.69 (95% CI: 1.82-141.60) and AR = 0.25 (95% CI: 0.06-0.43) when compared with other EPTB sites, which suggests that poor rifampin penetration might be a contributing factor. Conclusions Majority of Mtb strains circulating in the Tshwane metropolis belongs to East Asian, Euro-American and East-African Indian lineages. Each of these are likely to be clustered, suggesting on-going EPTB transmission. Since 25% of the drug resistance was attributable to sanctuary EPTB sites notorious for poor rifampin penetration, we hypothesize that poor anti-tuberculosis drug dosing might have a role in the resistance.

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.

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BackgroundTuberculosis (TB) remains a main global public health problem. However, a systematic review of TB resistance epidemiology in Africa is wanting.MethodsA 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 conclusionQualitative 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; C15T, G17A/T, -A16G etc. in inhA; Ins456C, Ins 172G, 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 were 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.

Background Genotypic characterization of Mycobacteria tuberculosis ( Mtb ) isolates in pulmonary tuberculosis is well documented; however, there is dearth of information on extrapulmonary tuberculosis (EPTB). Here, we investigate Mtb drug resistance and transmission patterns in EPTB patients diagnosed and treated in the Tshwane metropolitan, South Africa. Methods Consecutive Mtb culture-positive non-pulmonary isolates underwent mycobacterial genotyping and were assigned to phylogenetic lineages and transmission clusters based on spoligotypes. The Genotype MTBDR plus assay was used for isoniazid and rifampin susceptibility testing. Ensembles of machine learning (ML) algorithms were used to identify clinically meaningful patterns in data. We computed odds ratio (OR), attributable risk (AR) and their corresponding 95% confidence intervals (CI). Based on ML outputs, we examined factors associated with EPTB sites, drug resistance and clustering for the period between July 1 st , 2014 and January 31 st , 2015. Results A total of 75 isolates were identified during study period: we excluded 5 (7%) Mycobacterium bovis isolates. The overall proven EPTB incidence of 4.43 (95% CI: 3.72-5.23) per 100,000 population per year is lower than prior reports. However, the frequency distribution across most frequently encountered EPTB disease sites, including pleura, peritoneum, meninges and skin, were consistent with those reports. The largest cluster comprised of 25 (36%) Mtb strains belonged to East Asian lineage. East Asian lineage was significantly more likely to occur within a chain of EPTB transmission when compared to Euro-American and East-African Indian lineages: OR= 10.11 (95% CI: 1.56-116). Lymphadenitis, meningitis and skin TB, were significantly more likely to be associated with drug resistance: OR=12.69 (95% CI: 1.82-141.60) and AR = 0.25 (95% CI: 0.06-0.43) when compared with other EPTB sites, which suggests that poor rifampin penetration might be a contributing factor. Conclusions Majority of Mtb strains circulating in the city of Tshwane metropolis belongs to East Asian, Euro-American and East-African Indian lineages, and each of these are likely to be clustered, suggesting on-going EPTB disease transmission. On the other hand, 25% of the drug resistance is attributable to sanctuary EPTB sites notorious for poor rifampin penetration, suggesting role for poor drug dosing.

Whole-genome sequence analysis was performed on a multidrug-resistant Providencia rettgeri PR002 clinical strain isolated from the urine of a hospitalized patient in Pretoria, South Africa, in 2013. The resistome, mobilome, pathogenicity island(s), as well as virulence and heavy-metal resistance genes of the isolate, were characterized using whole-genome sequencing and bioinformatic analysis. PR002 had a genome assembly size of 4,832,624 bp with a GC content of 40.7%, an A/C

The technical limitations of common tests used for detecting pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates pose challenges for comprehensive and accurate descriptions of drug resistance in patients with multidrug-resistant tuberculosis (MDR-TB). In this study, a 606-bp fragment (comprising the pncA coding region plus the promoter) was sequenced using Ion Torrent next-generation sequencing (NGS) to detect associated PZA resistance mutations in 88 recultured MDR-TB isolates from an archived series collected in 2001. These 88 isolates were previously Sanger sequenced, with 55 (61%) designated as carrying the wild-type pncA gene and 33 (37%) showing mutations. PZA susceptibility of the isolates was also determined using the Bactec 460 TB system and the Wayne test. In this study, isolates were recultured and susceptibility testing was performed in Bactec 960 MGIT. Concordance between NGS and MGIT results was 93% ( n = 88), and concordance values between the Bactec 460, the Wayne test, or pncA gene Sanger sequencing and NGS results were 82% ( n = 88), 83% ( n = 88), and 89% ( n = 88), respectively. NGS confirmed the majority of pncA mutations detected by Sanger sequencing but revealed several new and mixed-strain mutations that resolved discordancy in other phenotypic results. Importantly, in 53% (18/34) of these isolates, pncA mutations were located in the 151 to 360 region and warrant further exploration. In these isolates, with their known resistance to rifampin, NGS of pncA improved PZA resistance detection sensitivity to 97% and specificity to 94% using NGS as the gold standard and helped to resolve discordant results from conventional methodologies.

Enterococcus faecalis is a lactic acid-producing Gram-positive bacterium commonly found in the intestinal tract of humans and animals; it is implicated in multidrug-resistant nosocomial infections. The draft genome of this E. faecalis sequence type 6 (ST6) strain consists of 3,215,228 bp, with 37.20% GC content, 3,048 predicted coding sequences, and 61 RNA genes.

Enterococcus faecalis is a lactic acid-producing Gram-positive bacterium commonly found in the intestinal tract of humans and animals; it is implicated in multidrug-resistant nosocomial infections. The draft genome of this E. faecalis sequence type 6 (ST6) strain consists of 3,215,228 bp, with 37.20% GC content, 3,048 predicted coding sequences, and 61 RNA genes.

Background The incidence of multidrug-resistant tuberculosis (MDR-TB) is increasing and the emergence of extensively drug-resistant tuberculosis (XDR-TB) is a major challenge. Controlling resistance, reducing transmission and improving treatment outcomes in MDR/XDR-TB patients is reliant on susceptibility testing. Susceptibility testing using phenotypic methods is labour intensive and time-consuming. Alternative methods, such as molecular assays are easier to perform and have a rapid turn-around time. The World Health Organization (WHO) has endorsed the use of line probe assays (LPAs) for first and second line diagnostic screening of MDR/XDR-TB. Methods We compared the performance of LPAs to BACTEC MGIT 960 system for susceptibility testing of bacterial resistance to first-line drugs: rifampicin (RIF), isoniazid (INH), ethambutol (EMB), and second-line drugs ofloxacin (OFL) and kanamycin (KAN). One hundred (100) consecutive non-repeat Mycobacterium tuberculosis cultures, resistant to either INH or RIF or both, as identified by BACTEC MGIT 960 were tested. All isoniazid resistant cultures (n = 97) and RIF resistant cultures (n = 90) were processed with Genotype®MTBDRplus and Genotype®MTBDRsl line probe assays (LPAs). The agar proportion method was employed to further analyze discordant LPAs and the MGIT 960 isolates. Results The Genotype ®MTBDRplus (version 2) sensitivity, specificity, PPV and NPV from culture isolates were as follows: RIF, 100%, 87.9, 58.3% and 100%; INH, 100%, 94.4%, 93.5% and 100%. The sensitivity, specificity PPV and NPV for Genotype ® MTBDRsl (version 1 and 2) from culture isolates were as follows: EMB, 60.0%, 89.2%, 68.2% and 85.3%; OFL, 100%, 91.4%, 56.2% and 100%; KAN, 100%, 97.7%, 60.0% and 100%. Line probe assay showed an excellent agreement (k = 0.93) for INH susceptibility testing when compared to MGIT 960 system while there was good agreement (k = 0.6–0.7) between both methods for RIF, OFL, KAN testing and moderate agreement for EMB (k = 0.5). A high RIF mono-resistance (MGIT 960 33/97 and LPA 43/97) was observed. Conclusion LPAs are an efficient and reliable rapid molecular DST assay for rapid susceptibility screening of MDR and XDR-TB. Using LPAs in high MDR/XDR burden countries allows for appropriate and timely treatment, which will reduce transmission rates, morbidity and improve treatment outcomes in patients.

We report the first draft genome sequence of an Enterococcus faecium sequence type 18 (ST18) strain isolated from a tuberculosis patient in Africa. The genome is comprised of 3,202,539 bp, 501 contigs, 37.70% GC content, 3,202 protein-encoding genes, and 61 RNA genes. The resistome and virulome of this important pathogen are presented herein.

Modern advances in genomics provide an opportunity to reinterpret historical bacterial culture collections. In this study, genotypic antibiotic resistance profiles of Mycobacterium tuberculosis isolates from a 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 rifampin (RIF) monoresistant, 2% as isoniazid (INH) monoresistant, and 8.3% as susceptible to both RIF and INH (RIF+INH). In a subset of 42 randomly selected isolates designated as RIF+INH resistant by Löwenstein-Jensen (LJ) culture in 1993, LPA and WGS results confirmed MDR-TB. In all five INH-monoresistant isolates by LPA and in all but one (the wild type) of the 34 successfully sequenced RIF-monoresistant 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, 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.

Background The genus Ureaplasma colonises human mucosal surfaces such as urogenital tract of men and women. In men it has been implicated in the aetiology of non-gonococcal urethritis and infertility. Although its pathogenesis is not yet fully understood, it has been suggested that certain serotypes are associated with disease. This study undertook to detect genital Ureaplasma spp. and to characterise Ureaplasma parvum in men with and without urogenital symptoms. Methods Two hundred first void urine specimens were collected from symptomatic (100) and asymptomatic (100) men attending a private clinic. All specimens were cultured in U9 broth and subcultured on A2 agar medium for confirmation. All isolates were tested for susceptibility using the Mycofast Evolution 3 kit. DNA was extracted from all specimens and amplified using a multiplex TaqMan polymerase chain reaction assay targeting the multiple-banded antigen gene for the detection and serotyping of U parvum . Ureaplasma urealyticum was detected by a commercial real-time PCR kit. Results Cultures were positive in 16/100 symptomatic and 12/100 asymptomatic men (p=NS). All isolates were susceptible to doxycycline, pristimycin, roxycycline and azithromycin. One Ureaplasma spp. from an asymptomatic male was resistant to ciprofloxacin and josamycin and intermediately resistant to ofloxacin and another was resistant to ofloxacin. An isolate from a symptomatic man was resistant to ciprofloxacin. There was no significant difference (p=0.16) between the U parvum isolated from symptomatic (11/100) and asymptomatic (18/100) men as well as for U urealyticum from symptomatic 16/100 and asymptomatic 15/100 men (p=0.86). Four men (two from each group) were colonised by both Ureaplasma spp. . The predominant serotype was six, followed by types 1, 14 and 3 with no significant difference between symptomatic and asymptomatic men (p=0.309). Conclusions There is no data of circulating U parvum serotypes from South Africa. The prevalence rate was low and no significant differences were found between symptomatic and asymptomatic men for both Ureaplasma spp. . Serotype 6 was the most common type compared to reports from developed countries which suggests type 3 as being the most common. Macrolides and tetracyclines remain effective drugs for treatment of the Ureaplasma infections. Molecular techniques are valuable identification and characterisation of this fastidious group of bacteria.