Phylogenomic and epidemiological insights into two clinical Mycobacterium bovis BCG strains circulating in South Africa.

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: M. bovis BCG was 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 CONCLUSIONS: Genetic variations between the clinical strains and the reference BCG Copenhagen were identified. The clinical strains shared only one mutation in a secretion protein. Mutations were identified in various antibiotic resistance genes in the BCG isolates, which suggests their potential as multidrug-resistant (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. The phylogenomics results imply that two different BCG strains may be circulating in South Africa. However, it is difficult to associate the BCG vaccine strain administered and the BCG strain supplied 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.

Occurrence of enterococci harbouring clinically important antibiotic resistance genes in the aquatic environment in Gauteng, South Africa.

The development of antibiotic resistance and dissemination of its determinants is an emerging public health problem as it compromises treatment options of infections that were, until recently, treatable. Investigation of outbreaks of vancomycin resistant enterococci (VRE) suggests that the environment serves as a significant reservoir for antibiotic resistance genes (ARGs). However, there is a paucity of data regarding the presence of ARGs in the water sources in South Africa. In this study, water samples collected from wastewater treatment plants (WWTPs), surface water and hospital sewage were screened for enterococci harbouring genes conferring resistance to four classes of antibiotics. Enterococci isolates harbouring ARGs were detected in raw influent and treated wastewater discharge from WWTPs and hospital sewage water. Plasmid and transposon encoded ermB (macrolide), tetM and tetL (tetracycline) as well as aph(3')-IIIa (aminoglycosides) genes were frequently detected among the isolates, especially in E. faecalis. The presence of enterococci harbouring ARGs in the treated wastewater suggest that ARGs are discharged into the environment where their proliferation could be perpetuated. Among the enterococci clonal complexes (CCs) recovered from wastewater were E. faecium CC17 (ST18), which is frequently associated with hospital outbreaks and a novel E. faecalis sequence type (ST), ST780.

Minocycline Immunomodulates via Sonic Hedgehog Signaling and Apoptosis and Has Direct Potency Against Drug-Resistant Tuberculosis.

Drug-resistant tuberculosis represents a global emergency, requiring new drugs. We found that minocycline was highly potent in laboratory strains of Mycobacterium tuberculosis and that 30 drug-susceptible and multidrug/extensively drug-resistant clinical strains were susceptible to clinically achievable concentrations. In the hollow fiber system model, lung concentration-time profiles of 7 mg/kg/day human-equivalent minocycline dose achieved bacterial kill rates equivalent to those of first-line antituberculosis agents. Minocycline killed extracellular bacilli directly. Minocycline also killed intracellular bacilli indirectly, via concentration-dependent granzyme A-driven apoptosis. Moreover, minocycline demonstrated dose-dependent antiinflammatory activity and downregulation of extracellular matrix-based remodeling pathways and, thus, could protect patients from tuberculosis immunopathology. In RNA sequencing of repetitive samples from the hollow fiber system and in independent protein abundance experiments, minocycline demonstrated dose-dependent inhibition of sonic hedgehog-patched-gli signaling. These findings have implications for improved lung remodeling and for dual immunomodulation and direct microbial kill-based treatment shortening regimens for drug-susceptible and drug-resistant latent and active M. tuberculosis infection.

Comparison of line probe assay to BACTEC MGIT 960 system for susceptibility testing of first and second-line anti-tuberculosis drugs in a referral laboratory in South Africa.

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.

Diversity of Multidrug Efflux Genes and Phenotypic Evaluation of the In vitro Resistance Dynamics of Clinical Staphylococcus Aureus Isolates Using Methicillin; a Model ß-lactam.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) across the world often leave clinicians with little or no choice of treatment options. The multi-drug efflux (MDE) genes are bacterial survival mechanisms responsible for the pumping out of antibiotics and other biocides from the cytoplasm. Whilst effort is being made in the development of antibiotic adjuvants such as efflux pumps inhibitors, information is needed on the diversity of these MDEs in the circulating S. aureus and on the growth dynamics of the clinical isolates in response to antibiotics is not regularly examined. METHODS: Here, we evaluated the diversity of MDEs in cinical S. aureus recovered in a tertiary academic hospital, Pretoria, South African hospital using PCR and also employed visual minimum inhibitory concentration and quantitative analysis of spectrophometric measurements of bacterial growth in the presence of a model ß lactam antibiotic (methicillin), to phenotypically elucidate the resistance pattern of these isolates in response to methicillin. RESULTS: Three major distribution patterns of MDEs were observed in the clinical isolates evaluated. Moreover, norA, nor B and tet38 were present in 98.9% of the isolates while other MDE were present in different proportions ranging from 40 to 98.6% of the isolates. In addition, S. aureus isolates, be it of MRSA or MSSA genotype did not habour the same set of MDEs despite being recovered from the same hospital setting. Finally, we showed that MSSA displayed phenotypic resistance to methicilllin despite the non-detection of the mecA resistance gene. CONCLUSIONS: Our data suggest that the growth of S. aureus may be enhanced by ß lactams (methicillin) and that MSSA may also display resistance to methicillin and perhaps other ß lactam antibiotics. The high prevalence of MDEs suggestive of resistance to a broad spectrum of biocides and fluoroquinolones are particularly disturbing.

Trends in the Genetic Background of Methicillin-Resistant Staphylococcus Aureus Clinical Isolates in a South African Hospital: An Institutional-Based Observational Study.

BACKGROUND: This study sought to understand the epidemio-ecological dynamics of MRSA isolates associated with a South African hospital over a period spanning year 2007-8 (a previous study reported in 2009) and year 2010-11 (this study). METHODS: One hundred and ninety three isolates were characterised by molecular fingerprinting methods including pulsed field gel electrophoresis (PFGE), spa typing, agr-typing, SCCmec-typing, and multilocus sequence typing (MLST). The Vitek-2 automated antibiogram of representative isolates was also performed. RESULTS: Our data shows that the distribution of MRSA strains among the different clinical conditions was rarely dependent on the genetic backbone or genotype. Compared to the previous survey in 2009, CA-MRSA isolates increased by 31% while HA-MRSA isolates decreased by 17%. An increase in genetic diversity was also revealed including the detection of three pandemic clonal complexes (spa type t012-ST36/CC30, spa type t037-ST239/CC8, spa type t891-ST22/CC22 and spa type t1257-ST612/CC8). Majority of the genotypes were classified as Spa Cluster B-SCCmec I-agr I 19.2%; (37/193) Spa Cluster A-SCCmercury-agr I 14.5%; (28/193). CONCLUSION: This study reveals that increased diversity in MRSA genetic background was associated with resistance to frontline antibiotics. Also, an increase was recorded in the CA-MRSA/HA-MRSA ratio within a 5-year period despite the continuous dominance of the HA-MRSA genotype.

Semi-quantitative digital analysis of polymerase chain reaction-electrophoresis gel: Potential applications in low-income veterinary laboratories.

AIM: The interpretation of conventional polymerase chain reaction (PCR) assay results is often limited to either positive or negative (non-detectable). The more robust quantitative PCR (qPCR) method is mostly reserved for quantitation studies and not a readily accessible technology in laboratories across developing nations. The aim of this study was to evaluate a semi-quantitative method for conventional PCR amplicons using digital image analysis of electrophoretic gel. The potential applications are also discussed. MATERIALS AND METHODS: This study describes standard conditions for the digital image analysis of PCR amplicons using the freely available ImageJ software and confirmed using the qPCR assay. RESULTS AND CONCLUSION: Comparison of ImageJ analysis of PCR-electrophoresis gel and qPCR methods showed similar trends in the Fusobacterium necrophorum DNA concentration associated with healthy and periodontal disease infected wallabies (p≤0.03). Based on these empirical data, this study adds descriptive attributes ("more" or "less") to the interpretation of conventional PCR results. The potential applications in low-income veterinary laboratories are suggested, and guidelines for the adoption of the method are also highlighted.

The effects of iron limitation and cell density on prokaryotic metabolism and gene expression: Excerpts from Fusobacterium necrophorum strain 774 (sheep isolate).

Fusobacterium necrophorum is a Gram-negative obligate anaerobe associated with several diseases in humans and animals. Despite its increasing clinical significance, there is little or no data on the relationship between its metabolism and virulence. Previous studies have shown that bacteria grown under iron-limitation express immunogenic antigens similar to those generated in vivo. Thus, this paper describes the relationship between F. necrophorum subsp. necrophorum (Fnn) metabolism and the expression of the encoded putative virulence factors under iron-restricted conditions. At the midlog phase, iron limitation reduced Fnn growth but the cell density was dependent on the size of the inoculum. Preferential utilization of glucose-1-phosphate, d-mannitol and l-phenylalanine; production of 2-hydroxycaproic acid and termination of dimethyl sulphide production were major Fnn response-factors to iron limitation. Ultimately, iron restriction resulted in an increased ability of Fnn to metabolize diverse carbon sources and in the expression of stress-specific virulence factors. Iron starvation in low Fnn cell density was associated with the up-regulation of haemagglutinin (HA) and leukotoxin (lktA) genes (2.49 and 3.72 fold change respectively). However, Fnn encoded Haemolysin (Hly), yebN homologue (febN) and tonB homologue, were down-regulated (0.15, 0.79 and 0.33, fold changes respectively). Interestingly, cell density appeared to play a regulatory role in the final bacteria cell biomass, induction of a metabolic gene expression and the expression pattern virulence factors in Fnn suggesting the role of a cell density-associated regulatory factor. This report suggest that future studies on differential expression of bacterial genes under altered environmental condition(s) should consider testing the effect of cell concentrations as this is often neglected in such studies. In conclusion, iron restriction induces preferential utilization of carbon sources and altered metabolism in Fnn with associated changes in the expression pattern of the virulence factors.

Does anaerobic bacterial antibiosis decrease fungal diversity in oral necrobacillosis disease?

Oral necrobacillosis (ON) is a model polymicrobial disease that affects macropods in captivity and livestock. Several studies in humans and animals have focused mainly on the bacterial etiology of this disease with little or no information on the role/association of fungi with ON. Using a Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay and statistical analysis of the fungal community structure in healthy and disease groups, a reduction in the species diversity and drastic reduction (>1000 fold) in the fungal population in wallabies with ON was observed. Furthermore, an in vitro assay revealed a potential anaerobic-bacteria antibiosis mechanism in the observed decrease in fungal population in ON and a synergistic bacterial-fungal interaction in wallabies with healthy oral status. This study contributes to our knowledge of the fungal community structure associated with ON and forms the basis for an investigation at an epidemiological scale in order to exploit the clinical potentials of these findings.

The oral microbial community of gingivitis and lumpy jaw in captive macropods.

Gingivitis and lumpy jaw are diseases of polymicrobial aetiology. Although Fusobacterium necrophorum has been associated with these diseases in macropods, little is known about other organisms associated with these diseases in this animal species. PCR-DGGE analysis revealed the potential pathogens associated with gingivitis and lumpy jaw in macropods. PCR-DGGE profile comparison between the healthy and disease groups indicated a shift in the oral bacterial community structures with similarity coefficients of 48% and 35% for gingivitis and lumpy jaw respectively. Moreover, gingivitis was associated with increase in bacterial diversity (Shannon index = 2.87; PL curve = 45%) while lumpy jaw resulted in a decline in bacterial diversity (Shannon index = 2.47; PL curve = 74%). This study suggest that the establishment of gingivitis and lumpy jaw diseases follows the ecological plaque hypothesis. This forms the basis for an expanded investigation in an epidemiological scale and suggests the need for the appropriate choice of antimicrobial agent(s) and for the effective management and control of polymicrobial diseases.

A molecular ecological approach to the detection and designation of the etiological agents of a model polymicrobial disease.

The application of the original Koch postulates and the molecular Koch postulates in the definition of the etiological agents of polymicrobial diseases has received little or no attention. In the present study, denaturing gradient gel electrophoresis (DGGE) of oral samples (n = 3) from each of 3 categories of animals (healthy, diseased [gingivitis], and then oxytetracycline-treated) was used and revealed different bacterial community structures in a model polymicrobial disease (gingivitis) and after clinical cure. Potential microbes associated with the disease and belonging to the following families were identified: Fusobacteriaceae, Porphyromonadaceae, Flavobacteriaceae, Alcanivoracaceae, Bacteroidaceae, Xanthomonadaceae, and Neisseriaceae. Liquid chromatography-mass spectrophotometric analysis of culturable anaerobic bacteria culture supernatant revealed 3 major compounds (2-hydroxycaproic acid, phenyllactic acid, and indole acetic acid) that differentiated the healthy and disease groups. Results indicate that different microbial community structures were associated with the healthy and disease oral states. The results demonstrate the potential of DGGE as a tool in the detection and designation of etiological agents of polymicrobial diseases.

A molecular survey of a captive wallaby population for periodontopathogens and the co-incidence of Fusobacterium necrophorum subspecies necrophorum with periodontal diseases.

Periodontal diseases (PD) are diseases of polymicrobial aetiology and constitute major health problems in captive macropods. Increasing knowledge of the causal pathogens is therefore crucial for effective management and prevention of these diseases. PCR survey and sequence analyses of potential periodontopathogens in captive wallaby populations revealed a co-incidence of the diseases with the detection of Fusobacterium necrophorum subsp. necrophorum (Fnn) and its encoded leukotoxin (lktA) gene. Sequence analyses showed that the outer membrane protein of Fnn in the GenBank database shared significant homology (99%) with the Fnn encoded haemagglutinin-related-protein gene fragment identified in this study. In addition, this report suggests the existence of a variant of Fnn with no detectable lktA gene and thus warrants further studies. In contrast to reports associating Porphyromonas gingivalis and F. nucleatum with PD, this study revealed that PD in macropods are associated with Porphyromonas gulae and Fnn and raises the question: is there a possible host pathogen co-evolution in the pathogenesis of PD in animals and humans? These findings contribute to the understanding of the aetiology of periodontal disease in macropods as well as opening up a new direction of research into the microbial interactions involved in the pathogenesis of PD in macropods.

"Cycliplex PCR" confirmation of Fusobacterium necrophorum isolates from captive wallabies: a rapid and accurate approach.

Isolation and identification of obligate anaerobic bacteria is labour intensive and time consuming. This has led to the increased application of molecular tools to circumvent part of this problem. We report here the development of a rapid, accurate and cost-effective method to isolate and identify Fusobacterium necrophorum species from South Australian wallaby populations using a supplemented medium (BHIRS) in conjunction with a "Cycliplex PCR" method which involves a stepwise-selective amplification of target PCR products. This report demonstrates the complementation of phenotypic characterization by PCR for accurate and fast identification of F. necrophorum isolates from wildlife origin.

Sustainable remediation--the application of bioremediated soil for use in the degradation of TNT chips.

Environmental contamination by TNT (2,4,6 trinitrotoluene), historically used in civilian industries and the military as an explosive is of great concern due to its toxicity. Scientific studies have however shown that TNT is susceptible to microbial transformation. The aim of this study was to assess the potential of a previously bioremediated hydrocarbon contaminated soil (PBR) to increase TNT degradation rates. This was investigated by adding TNT chips to PBR and uncontaminated soils (PNC) in laboratory based studies (up to 16 weeks). Residual TNT chip analysis showed greater TNT degradation in PBR soils (70%) and significantly higher metabolic rates (4.5 fold increase in cumulative CO(2) levels) than in PNC soils (30%). Molecular analysis (PCR-DGGE-cluster analysis) showed substantial shifts in soil microbial communities associated with TNT contamination between day 0 and week 4 especially in PBR soils. Bacterial communities appeared to be more sensitive to TNT contamination than fungal communities in both soils. Quantitative PCR analysis showed ~3 fold increase in the abundance of nitroreductase genes (pnrA) in PBR soils with a gradual reduction in community evenness (Pareto-Lorenz curves) in contrast to PNC soils. These results suggest that microbial response to TNT contamination was dependent on the history of soil use. The results also confirm that the microbial potential of waste soils such as PBR soil (usually disposed of via landfill) can be successfully used for accelerated TNT chip degradation. This promotes sustainable re-use of waste soils extending the life span of landfill sites.

Molecular characterisation of African swine fever viruses from Nigeria (2003-2006) recovers multiple virus variants and reaffirms CVR epidemiological utility.

Samples collected from wild and domestic suids in Nigeria, over a 3-year period (2003-2006), were evaluated for African swine fever (ASF) virus genome presence by targeting three discrete genome regions, namely the 478-bp C-terminal p72 gene region advocated for genotype assignment, a 780-bp region spanning the 5'-ends of the pB125R and pB646L (p72) genes and the hypervariable central variable region (CVR) encoded within the 9RL ORF (pB602L). ASF virus (ASFV) presence was confirmed in 23 of the 26 wild and domestic pigs evaluated. No evidence of ASF infection was found in two warthogs from Adamawa State; however, one bushpig from Plateau State was positive. Nucleotide sequences of the 478-bp and 780-bp amplicons were identical across all ASFV-positive samples sequenced. However, five discrete CVR variants were recovered, bringing the total number identified to date, from Nigeria, to six. The largest of the CVR variants, termed 'Tet-36' was identical to a virus causing outbreaks in neighbouring Benin in 1997, indicating a prolonged persistence of this virus type in Nigeria. Co-circulation of three tetramer types (Tet-36, Tet-27 and Tet-20) was found in Plateau State in July 2004, whilst in Benue State, two tetramer types (Tet-20 and Tet-21) were present in August 2005. Despite simultaneous field presence, individual co-infection was not observed. This study has reaffirmed the epidemiological utility of the CVR genome region for distinguishing between geographically and temporally constrained genotype I viruses, and has revealed the presence of multiple ASFV variants in Nigeria.