Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock.

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.

Genomic epidemiology and molecular characteristics of blaNDM-1-positive carbapenem-resistant Pseudomonas aeruginosa belonging to international high-risk clone ST773 in the Gauteng region, South Africa.

PURPOSE: The emergence of carbapenem-resistant P. aeruginosa (CRPA) harbouring acquired carbapenemase genes (blaVIM, blaIMP and blaNDM) has become a global public health threat. Three CRPA isolates included in the study had an extensively drug-resistant phenotype with susceptibility to colistin only and were positive for the blaNDM-1 gene. The current study aimed to investigate the genomic epidemiology and molecular characteristics of the blaNDM-1-positive CRPA isolates collected from the Gauteng region, South Africa. METHODS: Short read whole genome sequencing (WGS) was performed to determine sequence types (STs), genetic relatedness, resistome, virulome and the genetic environment of the blaNDM-1 gene. RESULTS: The WGS and phylogenetic analyses revealed that the study isolates belonged to an international high-risk clone ST773 and belonged to the same clade with eight blaNDM-1-positive ST773 isolates from Hungary, India, Nigeria, South Korea and USA. The study isolates harboured a wide repertoire of intrinsic and acquired antibiotic resistance genes (ARGs) related with mobile genetic elements, porins and efflux pumps, as well as virulence factor genes. The clade-specific ARGs (blaNDM-1, floR2/cmlA9, rmtB4, tetG) were found in a putative integrative and conjugative element (ICE) region similar to ICE6660-like. CONCLUSION: As ICE carrying the blaNDM-1 gene can easily spread to other P. aeruginosa isolates and other Gram-negative bacteria, the findings in this study highlight the need for appropriate management strategies and active surveillance of CRPA isolates in the Gauteng region, South Africa.

Salmonella enterica serovar Typhi H58 clone has been endemic in Zimbabwe from 2012 to 2019.

BACKGROUND: Typhoid fever, caused by S. enterica ser. Typhi, continues to be a substantial health burden in developing countries. Little is known of the genotypic diversity of S. enterica ser. Typhi in Zimbabwe, but this is key for understanding the emergence and spread of this pathogen and devising interventions for its control. OBJECTIVES: To report the molecular epidemiology of S. enterica ser. Typhi outbreak strains circulating from 2012 to 2019 in Zimbabwe, using comparative genomics. METHODS: A review of typhoid cases records from 2012 to 2019 in Zimbabwe was performed. The phylogenetic relationship of outbreak isolates from 2012 to 2019 and emergence of antibiotic resistance was investigated by whole-genome sequence analysis. RESULTS: A total 22 479 suspected typhoid cases, 760 confirmed cases were reported from 2012 to 2019 and 29 isolates were sequenced. The majority of the sequenced isolates were predicted to confer resistance to aminoglycosides, ß-lactams, phenicols, sulphonamides, tetracycline and fluoroquinolones (including qnrS detection). The qnrS1 gene was associated with an IncN (subtype PST3) plasmid in 79% of the isolates. Whole-genome SNP analysis, SNP-based haplotyping and resistance determinant analysis showed that 93% of the isolates belonged to a single clade represented by multidrug-resistant H58 lineage I (4.3.1.1), with a maximum pair-wise distance of 22 SNPs. CONCLUSIONS: This study has provided detailed genotypic characterization of the outbreak strain, identified as S. Typhi 4.3.1.1 (H58). The strain has reduced susceptibility to ciprofloxacin due to qnrS carried by an IncN (subtype PST3) plasmid resulting from ongoing evolution to full resistance.

Molecular epidemiology of extended-spectrum beta-lactamase-producing extra-intestinal pathogenic Escherichia coli strains over a 2-year period (2017-2019) from Zimbabwe.

This study was designed to characterize extended-spectrum beta-lactamase (ESBL)-producing extra-intestinal pathogenic Escherichia coli (E.coli) (ExPEC) associated with urinary tract infections in nine different geographic regions of Zimbabwe over a 2-year period (2017-2019). A total of 48 ESBL-positive isolates from urine specimen were selected for whole-genome sequencing from 1246 Escherichia coli isolates biobanked at the National Microbiology Reference laboratory using phenotypic susceptibility testing results from the National Escherichia coli Surveillance Programme to provide representation of different geographical regions and year of isolation. The majority of ESBL E. coli isolates produced cefotaximase-Munich (CTX-M)-15, CTX-M-27, and CTX-M-14. In this study, sequence types (ST) 131 and ST410 were the most predominant antimicrobial-resistant clones and responsible for the increase in ESBL-producing E. coli strains since 2017. Novel ST131 complex strains were recorded during the period 2017 to 2018, thus showing the establishment and evolution of this antimicrobial-resistant ESBL clone in Zimbabwe posing an important public health threat. Incompatibility group F plasmids were predominant among ST131 and ST410 isolates with the following replicons recorded most frequently: F1:A2:B20 (9/19, 47%), F2:A1: B (5/19, 26%), and F1:A1:B49 (8/13, 62%). The results indicate the need for continuous tracking of different ESBL ExPEC clones on a global scale, while targeting specific STs (e.g. ST131 and ST410) through control programs will substantially decrease the spread of ESBLs among ExPEC.

Klebsiella pneumoniae ST307 with blaOXA-181, South Africa, 2014-2016.

Klebsiella pneumoniae sequence type (ST) 307 is an emerging global antimicrobial drug-resistant clone. We used whole-genome sequencing and PCR to characterize K. pneumoniae ST307 with oxacillinase (OXA) 181 carbapenemase across several private hospitals in South Africa during 2014-2016. The South Africa ST307 belonged to a different clade (clade VI) with unique genomic characteristics when compared with global ST307 (clades I-V). Bayesian evolution analysis showed that clade VI emerged around March 2013 in Gauteng Province, South Africa, and then evolved during 2014 into 2 distinct lineages. K. pneumoniae ST307 clade VI with OXA-181 disseminated over a 15-month period within 42 hospitals in 23 cities across 6 northeastern provinces, affecting 350 patients. The rapid expansion of ST307 was most likely due to intrahospital, interhospital, intercity, and interprovince movements of patients. This study highlights the importance of molecular surveillance for tracking emerging antimicrobial clones.

Identification and characterization of Staphylococcus devriesei isolates from bovine intramammary infections in KwaZulu-Natal, South Africa.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are among the leading bacterial causes of bovine mastitis in many dairy-producing countries. Among the challenges associated with the specific diagnosis of CoNS infections is the biochemical heterogeneity of the species in the genus and the unavailability of accurate, cost-effective and up-to-date diagnostic tests. A previous study investigating the diversity of CoNS associated with cases of bovine mastitis in South Africa, resulted in six CoNS isolates which could not be identified despite the use of a combination of different molecular assays. The identification and characterisation of the isolates was pursued further in this study. RESULTS: The six CoNS isolates in question were identified by sequencing multiple housekeeping genes (dnaJ, hsp60, rpoB, 16S rRNA) and characterized through the use of matrix-assisted laser/desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and the Biolog GEN III Microplate™ bacterial identification system. Sequencing of housekeeping genes identified the isolates as S. devriesei. This Staphylococcus species was only described in 2010 and this is the first report documenting the isolation of S. devriesei from cases of bovine IMIs in South Africa. Analysis of mass spectra generated by the six isolates showed intra-species variation which was also observed when evaluating the metabolic profiles of the isolates using the Biolog GEN III system. Neither the MALDI-TOF MS nor the Biolog database are currently populated with data relating to S. devriesei, resulting in the isolates not being identified, in the case of MALDI-TOF MS analysis, or mis-identified as was observed with the Biolog GEN III system. CONCLUSIONS: The phenotyping data collected during this investigation provides useful information concerning Staphylococcus devriesei which could be used to populate user system databases thereby ensuring the accurate identification of isolates in future. The availability of improved diagnostics will in turn facilitate studies to elucidate the epidemiology, pathogenicity and true prevalence of this species in dairy herds.

Etiology of bacterial vaginosis and polymicrobial biofilm formation.

Microorganisms in nature rarely exist in a planktonic form, but in the form of biofilms. Biofilms have been identified as the cause of many chronic and persistent infections and have been implicated in the etiology of bacterial vaginosis (BV). Bacterial vaginosis is the most common form of vaginal infection in women of reproductive age. Similar to other biofilm infections, BV biofilms protect the BV-related bacteria against antibiotics and cause recurrent BV. In this review, an overview of BV-related bacteria, conceptual models and the stages involved in the polymicrobial BV biofilm formation will be discussed.

Surveillance of catheter-related infections: the supplementary role of the microbiology laboratory.

BACKGROUND: The burden of catheter-related infections (CRIs) in developing countries is severe. In South Africa, a standardised surveillance definition does not exist and the collection of catheter days is challenging. The aim of the study was to provide baseline data on the prevalence of CRIs and to describe the epidemiology of CRI events within a tertiary academic hospital. METHODS: Surveillance was laboratory-based and conducted for a six month period. A microbiologically confirmed CRBSI (MC-CRBSI) event was defined as the isolation of the same microorganism from the catheter and concomitant blood cultures (BCs), within 48 h of catheter removal, which were not related to an infection at another site. RESULTS: A total of 508 catheters, removed from 332 patients, were processed by the laboratory, of which only 50% (253/508 removed from 143/332 patients) of the catheters were accompanied by BCs within 48 h. Sixty-five episodes of MC-CRBSI in 57 patients were detected, involving 71 catheters and 195 microbial isolates. The institutional prevalence rate was 3.7 episodes per 1 000 admissions and 5.8 episodes per 10 000 in-patient days. Catheter day data was collected in only six wards of the hospital. The pooled laboratory incidence was 10.1 MC-CRBSI episodes per 1 000 catheter days, whereas the hospital-based central line-associated bloodstream infection (CLABSI) rate was pooled at 5.7 episodes per 1 000 catheter days. The majority of patients had an underlying gastro-intestinal condition (33%; 19/56) with a non-tunnelled, triple-lumen central venous catheter, placed in the subclavian vein (38%; 27/71). The most predominant pathogen was methicillin-resistant Staphylococcus epidermidis (28%; 55/195), followed by extensively-drug resistant Acinetobacter baumannii (18%; 35/195). CONCLUSIONS: Catheter-related infection prevention and control efforts require urgent attention, not only to keep patients safe from preventable harm, but to prevent the spread of multidrug resistant microorganisms.

Antimicrobial susceptibility patterns of Ureaplasma species and Mycoplasma hominis in pregnant women.

BACKGROUND: Genital mycoplasmas colonise up to 80% of sexually mature women and may invade the amniotic cavity during pregnancy and cause complications. Tetracyclines and fluoroquinolones are contraindicated in pregnancy and erythromycin is often used to treat patients. However, increasing resistance to common antimicrobial agents is widely reported. The purpose of this study was to investigate antimicrobial susceptibility patterns of genital mycoplasmas in pregnant women. METHODS: Self-collected vaginal swabs were obtained from 96 pregnant women attending an antenatal clinic in Gauteng, South Africa. Specimens were screened with the Mycofast Revolution assay for the presence of Ureaplasma species and Mycoplasma hominis. The antimicrobial susceptibility to levofloxacin, moxifloxacin, erythromycin, clindamycin and tetracycline were determined at various breakpoints. A multiplex polymerase chain reaction assay was used to speciate Ureaplasma positive specimens as either U. parvum or U. urealyticum. RESULTS: Seventy-six percent (73/96) of specimens contained Ureaplasma spp., while 39.7% (29/73) of Ureaplasma positive specimens were also positive for M. hominis. Susceptibilities of Ureaplasma spp. to levofloxacin and moxifloxacin were 59% (26/44) and 98% (43/44) respectively. Mixed isolates (Ureaplasma species and M. hominis) were highly resistant to erythromycin and tetracycline (both 97% resistance). Resistance of Ureaplasma spp. to erythromycin was 80% (35/44) and tetracycline resistance was detected in 73% (32/44) of Ureaplasma spp. Speciation indicated that U. parvum was the predominant Ureaplasma spp. conferring antimicrobial resistance. CONCLUSIONS: Treatment options for genital mycoplasma infections are becoming limited. More elaborative studies are needed to elucidate the diverse antimicrobial susceptibility patterns found in this study when compared to similar studies. To prevent complications in pregnant women, the foetus and the neonate, routine screening for the presence of genital mycoplasmas is recommended. In addition, it is recommended that antimicrobial susceptibility patterns are determined.

Comparison of the new Mycofast Revolution assay with a molecular assay for the detection of genital mycoplasmas from clinical specimens.

BACKGROUND: Genital mycoplasmas are opportunistic bacteria that are associated with undesirable gynaecologic and reproductive events. Mycoplasmas are fastidious bacteria with increasing resistance to routine antimicrobials and often fail to grow on conventional culture methods. The commercial Mycofast Revolution assay permits the phenotypic detection and identification of genital mycoplasmas. Antimicrobial susceptibility testing against five antimicrobial agents with MICs corresponding to the CLSI guidelines can also be performed. This study aimed to compare the new commercially available Mycofast Revolution assay with a multiplex PCR assay. METHODS: Self-collected swabs were obtained from pregnant women attending the antenatal clinic of a tertiary academic hospital in Pretoria, South Africa from October 2012 to November 2012. These swabs were used to seed UMMt and modified Amies transport media. The seeded UMMt transported medium was used to inoculate the Mycofast Revolution assay for the identification, enumeration and antimicrobial susceptibility testing of genital mycoplasmas. Following DNA extraction from the modified Amies transport medium, specimens were subjected to a multiplex PCR assay for the detection of genital mycoplasmas. RESULTS: The Mycofast Revolution kit had a sensitivity and specificity of 77.3% (95% CI: 62.15% to 88.51%) and 80% (95% CI: 28.81% to 96.70%), respectively, against the PCR assay. The positive and negative predictive values were 97.1% (95% CI: 85.03% to 99.52%) and 28.6% (95% CI: 8.57% to 58.08%). Genital mycoplasmas were detected in 71.4% (35/49) of samples with the Mycofast Revolution assay with 49% (24/49) being Ureaplasma spp. and 22.4% (11/49) mixed strains. The multiplex PCR assay had a positivity rate of 89.8% (44/49) for genital mycoplasmas; mixed strains were present in 51% (25/49) of samples, Ureaplasma spp. in 16.3% (8/49) and M. hominis in 22.4% (11/49) of samples. CONCLUSIONS: There was a fair agreement (κ = 0.319) between the Mycofast Revolution assay and the mPCR assay. With the high prevalence rates of genital mycoplasmas, fast and efficient diagnostic methods are imperative to treat infections and minimise complications. The Mycofast Revolution assay is simple to use, has a short turn-around time and interpretation of results are straightforward. This assay circumvents common problems experienced with conventional culture and molecular methods in diagnostic laboratories where skilled personnel are limited and can be used as an alternative diagnostic assay.

Population structure of Salmonella enterica Typhi in Harare, Zimbabwe (2012-19) before typhoid conjugate vaccine roll-out: a genomic epidemiology study.

BACKGROUND: The continued emergence of Salmonella enterica serovar Typhi, with ever increasing antimicrobial resistance, necessitates the use of vaccines in endemic countries. A typhoid fever outbreak in Harare, Zimbabwe, in 2018 from a multidrug resistant S Typhi with additional resistance to ciprofloxacin was the catalyst for the introduction of a typhoid conjugate vaccine programme. We aimed to investigate the emergence and evolution of antimicrobial resistance of endemic S Typhi in Zimbabwe and to determine the population structure, gene flux, and sequence polymorphisms of strains isolated before a typhoid conjugate vaccine programme to provide a baseline for future evaluation of the effect of the vaccination programme. METHODS: In this genomic epidemiology study, we used short-read whole-genome sequencing of S Typhi isolated from clinical cases of typhoid fever in Harare, Zimbabwe, between Jan 1, 2012, and Feb 9, 2019, to determine the S Typhi population structure, gene flux, and sequence polymorphisms and reconstructed the evolution of antimicrobial resistance. Maximum likelihood time-scaled phylogenetic trees of Zimbabwe isolates in the context of global isolates obtained from the National Center for Biotechnology Information were constructed to infer spread and emergence of antimicrobial resistance. FINDINGS: The population structure of S Typhi in Harare, Zimbabwe, from 2012 to 2019 was dominated by multidrug resistant genotype 4.3.1.1.EA1 (H58) that spread to Zimbabwe from neighbouring countries in around 2009 (95% credible interval 2008·5-2010·0). Acquisition of an IncN plasmid carrying antimicrobial resistance genes including a qnrS gene and a mutation in the quinolone resistance determining region of gyrA gene contributed to non-susceptibility and resistance to quinolone antibiotics. A minority population of antimicrobial susceptible S Typhi genotype 3.3.1 strains were present throughout. INTERPRETATION: The currently dominant S Typhi population is genotype 4.3.1.1 that spread to Zimbabwe and acquired additional antimicrobial resistance though acquisition of a plasmid and mutation in the gyrA gene. This study provides a baseline population structure for future evaluation of the effect of the typhoid conjugate vaccine programme in Harare. FUNDING: Bill & Melinda Gates Foundation and the Biotechnology and Biological Sciences Research Council Institute Strategic Programme.

Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Salmonella Typhi genomes.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions: The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Salmonella Typhi (Typhi) is a type of bacteria that causes typhoid fever. More than 110,000 people die from this disease each year, predominantly in areas of sub-Saharan Africa and South Asia with limited access to safe water and sanitation. Clinicians use antibiotics to treat typhoid fever, but scientists worry that the spread of antimicrobial-resistant Typhi could render the drugs ineffective, leading to increased typhoid fever mortality. The World Health Organization has prequalified two vaccines that are highly effective in preventing typhoid fever and may also help limit the emergence and spread of resistant Typhi. In low resource settings, public health officials must make difficult trade-off decisions about which new vaccines to introduce into already crowded immunization schedules. Understanding the local burden of antimicrobial-resistant Typhi and how it is spreading could help inform their actions. The Global Typhoid Genomics Consortium analyzed 13,000 Typhi genomes from 110 countries to provide a global overview of genetic diversity and antimicrobial-resistant patterns. The analysis showed great genetic diversity of the different strains between countries and regions. For example, the H58 Typhi variant, which is often drug-resistant, has spread rapidly through Asia and Eastern and Southern Africa, but is less common in other regions. However, distinct strains of other drug-resistant Typhi have emerged in other parts of the world. Resistance to the antibiotic ciprofloxacin was widespread and accounted for over 85% of cases in South Africa. Around 70% of Typhi from Pakistan were extensively drug-resistant in 2020, but these hard-to-treat variants have not yet become established elsewhere. Variants that are resistant to both ciprofloxacin and ceftriaxone have been identified, and azithromycin resistance has also appeared in several different variants across South Asia. The Consortium's analyses provide valuable insights into the global distribution and transmission patterns of drug-resistant Typhi. Limited genetic data were available fromseveral regions, but data from travel-associated cases helped fill some regional gaps. These findings may help serve as a starting point for collective sharing and analyses of genetic data to inform local public health action. Funders need to provide ongoing supportto help fill global surveillance data gaps.

Molecular characterization and second-line antituberculosis drug resistance patterns of multidrug-resistant Mycobacterium tuberculosis isolates from the northern region of South Africa.

Despite South Africa being one of the high-burden multidrug-resistant tuberculosis (MDR-TB) countries, information regarding the population structure of drug-resistant Mycobacterium tuberculosis strains is limited from many regions of South Africa. This study investigated the population structure and transmission patterns of drug-resistant M. tuberculosis isolates in a high-burden setting of South Africa as well as the possible association of genotypes with drug resistance and demographic characteristics. A total of 336 consecutive MDR-TB isolates from four provinces of South Africa were genotyped using spoligotyping and mycobacterial interspersed repetitive-unit-variable number tandem repeat (MIRU-VNTR) typing. Drug susceptibility testing for ofloxacin, kanamycin, and capreomycin was performed using the agar proportion method. The results showed that 4.8% of MDR-TB isolates were resistant to ofloxacin, 2.7% were resistant to kanamycin, and 4.5% were resistant to capreomycin, while 7.1% were extensively drug resistant (XDR), and the remaining 83.6% were susceptible to all of the second-line drugs tested. Spoligotyping grouped 90.8% of the isolates into 25 clusters, while 9.2% isolates were unclustered. Ninety-one percent of the 336 isolates were assigned to 21 previously described shared types, with the Beijing family being the predominant genotype in the North-West and Limpopo Provinces, while the EAI1_SOM family was the predominant genotype in the Gauteng and Mpumalanga Provinces. No association was found between genotypes and specific drug resistance patterns or demographic information. The high level of diversity and the geographical distribution of the drug-resistant M. tuberculosis isolates in this study suggest that the transmission of TB in the study settings is not caused by the clonal spread of a specific M. tuberculosis strain.

Comparison between the BACTEC MGIT 960 system and the agar proportion method for susceptibility testing of multidrug resistant tuberculosis strains in a high burden setting of South Africa.

BACKGROUND: The increasing problem of multi-drug-resistant (MDR) tuberculosis (TB) [ie resistant to at least isoniazid (INH) and rifampicin (RIF)] is becoming a global problem. Successful treatment outcome for MDR-TB depends on reliable and accurate drug susceptibility testing of first-line and second-line anti-TB drugs. METHOD: Consecutive M. tuberculosis isolates identified as MDR-TB during August 2007 to January 2008 using the BACTEC MGIT 960 systems and the agar proportion method were included in this study. Susceptibility testing of MDR-TB isolates against ethambutol (EMB) and streptomycin (STR) as well as two second-line anti-TB drugs, kanamycin (KAN) and ofloxacin (OFX) was performed using the BACTEC MGIT 960 systems at a routine diagnostic laboratory. The results were compared to those obtained by the agar proportion method. RESULT: The agreement between the BACTEC MGIT 960 system and the agar proportion method was 44% for EMB, 61% for STR and 89% for both KAN and OFX. The sensitivity and specificity of the BACTEC MGIT 960 system using the agar proportion method as a gold standard was 92% and 37% for EMB, 95% and 37% for STR, 27% and 97% for KAN and 84% and 90% for OFX, respectively. CONCLUSIONS: The BACTEC MGIT 960 system showed acceptable sensitivity for EMB, STR, and OFX; however, the BACTEC MGIT 960 system was less specific for EMB and STR and demonstrated a low sensitivity for KAN. The lower agreement found between the two methods suggests the unreliability of the BACTEC MGIT 960 system for the drugs tested. The reasons for the lower agreement between the two methods need to be investigated and further studies are needed in this setting to confirm the study finding.

Diversity of the gut, vaginal and oral microbiome among pregnant women in South Africa with and without pre-eclampsia.

Background: Changes in microbial communities are a known characteristic of various inflammatory diseases and have been linked to adverse pregnancy outcomes, such as preterm birth. However, there is a paucity of information regarding the taxonomic composition and/or diversity of microbial communities in pre-eclampsia. The aim of this study was to determine the diversity of the gut, vaginal and oral microbiome in a cohort of South African pregnant women with and without pre-eclampsia. The diversity of the gut, vaginal and oral microbiome was determined by targeted next generation sequencing (NGS) of the V3 and V4 region of the 16S rRNA gene on the Illumina MiSeq platform. Results: In this study population, pre-eclampsia was associated with a significantly higher alpha diversity (P = 0.0472; indicated by the Shannon index) in the vaginal microbiome accompanied with a significant reduction in Lactobacillus spp. (P = 0.0275), compared to normotensive pregnant women. Lactobacillus iners was identified as the predominant species of the vaginal microbiome in both cohorts. High inter-individual variation in alpha diversity was observed in the gut and oral microbiome in both cohorts. Although differences in the relative abundance of bacteria at all phylogenetic levels were observed, overall microbial composition of the gut, oral and vaginal microbiome was not significantly different in the pre-eclampsia cohort compared to the normotensive cohort. Conclusion: Collectively, a reduction of Lactobacillus spp., and predominance of L. iners in pregnant women with pre-eclampsia could suggest an unstable vaginal microbiome that might predispose pregnant women to develop pre-eclampsia. The lack of significant structural changes in the gut, oral and vaginal microbiome does not suggest that the characterized communities play a role in pre-eclampsia, but could indicate a characteristic unique to the study population. The current study provided novel information on the diversity of the gut, oral and vaginal microbiome among pregnant women in South Africa with and without pre-eclampsia. The current study provides a baseline for further investigations on the potential role of microbial communities in pre-eclampsia.

Faecal carriage of ESBL producing and colistin resistant Escherichia coli in avian species over a 2-year period (2017-2019) in Zimbabwe.

Introduction: Extended spectrum beta-lactamase (ESBL) producing Escherichia coli have become widespread among food producing animals. These strains serve as a reservoir of antibiotic resistance genes (ARGs) and act as a possible source of infection to humans as transmission can occur by direct or indirect contact. Methods: This study investigated the faecal carriage of ESBL producing and colistin resistant E. coli in poultry over a 2-year period (2017-2019) from Zimbabwe. A total of 21 ESBL positive isolates from poultry cloacal specimens were selected for whole genome sequencing from animal E. coli isolates bio-banked at the National Microbiology Reference laboratory using phenotypic susceptibility testing results from the National Escherichia coli Surveillance Program to provide representation of different geographical regions and year of isolation. Cloacal swabs were collected from 3000 broiler live birds from farm 1 and from farm 2, 40 backyard chickens and 10 ducks were sampled. Antimicrobial susceptibility and ESBL testing were performed as per Clinical Laboratory Standards Institute guidelines. Whole genome sequencing of ESBL producing isolates was used to determine sequence types (STs), ARGs, and phylogroups. Results: Twenty-one of the included E. coli isolates were confirmed as ESBL producers. Three defined sequence type clonal complexes (CCs) were identified (ST10CC, ST155CC and ST23CC), with ST10CC associated with the most antibiotic resistant profile. The ESBL phenotype was linked to the presence of either cefotaximase-Munich-14 (CTX-M-14) or CTX-M-79. Plasmid mediated quinolone resistant determinants identified were qnrB19 and qnrS1 and one ST10CC isolate from farm 1 broiler chickens harbored a mobile colistin resistance gene (mcr-1). Phylogenetic groups most identified were B1, A and unknown. Discussions: The avian ESBL producing E. coli belonged to a diverse group of strains. The detection of several ARGs highlights the importance of implementing enhanced control measures to limit the spread in animals, environment, and humans. This is the first report of mcr-1 in Zimbabwe, which further underscores the importance of the One Health approach to control the spread and development of AMR.

In vitro antibacterial activity of Loxostylis alata extracts and isolated compounds against Salmonella species.

BACKGROUND: Owing to antibiotic resistance, alternative antimicrobials from medicinal plants are receiving attention as leads for anti-infective agents. This study aimed to investigate selected tree species and their constituents for activity against bacterial foodborne pathogens, particularly Salmonella serovars. METHODS: Antibacterial activity of ten plant species was determined by serial microdilution against bacteria implicated in causing gastrointestinal ailments. Active compounds were isolated from Loxostylis alata using bioassay-guided fractionation. Antioxidant activity was determined using free-radical scavenging assays. Cytotoxicity and genotoxicity of the extracts was ascertained on Vero cells, and using the Ames assay respectively. RESULTS: Extracts had low to moderate MIC values from 0.04 to 2.5 mg/mL. Protorhus longifolia and Loxostylis alata were most active and L. alata had the highest selectivity index value (2.51) against Salmonella Typhimurium, as well as high antioxidant activity. Cytotoxicity values ranged from 0.02 to 0.47 mg/mL, while tested extracts were not genotoxic. Bioactive compounds isolated from L. alata included delicaflavone and a polymethoxyflavone. CONCLUSIONS: The Loxostylis alata leaf extract had strong activity against Salmonella serovars but isolated compounds were less active, indicating likely synergistic effects. Extracts of L. alata are promising candidates for development of antimicrobial preparations or food additives against microbial contamination.

Evaluation of the rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test for rapid colistin resistance detection in lactose non-fermenting Gram-negative bacteria.

Introduction. Colistin is one of the last-resort antibiotics for treating multidrug-resistant (MDR) or extensively drug-resistant (XDR) lactose non-fermenting Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii.Gap Statement. As the rate of colistin resistance is steadily rising, there is a need for rapid and accurate antimicrobial susceptibility testing methods for colistin. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test has recently been developed for rapid detection of colistin resistance in P. aeruginosa and A. baumannii.Aim. The present study aimed to evaluate the performance of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test in comparison with the reference broth microdilution (BMD) method.Methodology. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test was performed using a total of 135 P. aeruginosa (17 colistin-resistant and 118 colistin-susceptible) and 66 A. baumannii isolates (32 colistin-resistant and 34 colistin-susceptible), in comparison with the reference BMD method.Results. The categorical agreement of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test with the reference BMD method was 97.5 % with a major error rate of 0 % (0/152) and a very major error (VME) rate of 10.2 %. The VME rate was higher (23.5 %) when calculated separately for P. aeruginosa isolates. The overall sensitivity and specificity were 89.8 and 100 %, respectively.Conclusion. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test performed better for A. baumannii than for P. aeruginosa.

Growth Forms of Gardnerella spp. and Lactobacillus spp. on Vaginal Cells.

Bacterial vaginosis (BV) is a common vaginal condition in women of reproductive age. During BV development, BV-associated bacteria may form a polymicrobial biofilm, which predispose women to recurrent BV. The aim of the study was to investigate the growth forms of Gardnerella spp. and Lactobacillus spp. and to determine the association between the bacterial growth forms and clinical characteristics [urinary tract infection (UTI) symptoms, human immunodeficiency virus (HIV) infection and abnormal vaginal discharge] in women attending a tertiary hospital in Pretoria, South Africa. A first-void urine specimen was collected from 196 women and BV was diagnosed using the Nugent scoring and the Ison-Hay criteria (vaginal smear microscopy). Fluorescence in situ hybridisation (FISH) was performed to classify the growth forms ["dispersed" or "biofilm"]. Bacterial cells were categorized as "dispersed" if cells were scattered separately and as "biofilm" if bacterial aggregates on the vaginal epithelial cells were observed. BV was detected in 52 women (52/196; 27%) and in these women, Gardnerella spp. were predominantly present in biofilms (46/52; 88% for Nugent scoring; and 45/50; 90% for Ison-Hay criteria), whereas Lactobacillus spp. were predominantly present in a dispersed form (38/52; 73% for Nugent scoring; and 37/50; 74% for Ison-Hay criteria). The odds of having BV increased when Gardnerella biofilms were present (p < 0.001), whereas the opposite was observed for Lactobacillus biofilms (p = 0.001). Neither Gardnerella spp. or Lactobacillus spp. (both dispersed or biofilms) had an association with the presence of UTI symptoms, HIV coinfection or abnormal vaginal discharge. In conclusion, this study demonstrated and confirmed that Gardnerella biofilms are associated with BV and that Lactobacillus spp. may form biofilms to protect against BV.