Antibacterial, Resistance Modulation, Anti-Biofilm Formation, and Efflux Pump Inhibition Properties of Loeseneriella africana (Willd.) N. Halle (Celastraceae) Stem Extract and Its Constituents.

This study investigated the antibacterial, resistance modulation, biofilm inhibition, and efflux pump inhibition potentials of Loeseneriella africana stem extract and its constituents. The antimicrobial activity was investigated by the high-throughput spot culture growth inhibition (HT-SPOTi) and broth microdilution assays. The resistance modulation activity was investigated using the anti-biofilm formation and efflux pump inhibition assays. Purification of the extract was carried out by chromatographic methods, and the isolated compounds were characterized based on nuclear magnetic resonance, Fourier transform infrared and mass spectrometry spectral data and comparison with published literature. The whole extract, methanol, ethyl acetate, and pet-ether fractions of L. africana all showed antibacterial activity against the test bacteria with MICs ranging from 62.5 to 500.0 µg/mL The whole extract demonstrated resistance modulation effect through strong biofilm inhibition and efflux pump inhibition activities against S. aureus ATCC 25923, E. coli ATCC 25922 and P. aeruginosa ATCC 27853. Chromatographic fractionation of the ethyl acetate fraction resulted in the isolation of a triterpenoid (4S,4αS,6αR,6ßS,8αS,12αS,12ßR,14αS,14ßR)-4,4α,6ß,8α,11,11,12ß,14α-Octamethyloctadecahydropicene-1,3(2H,4H)-dione) and a phytosterol (ß-sitosterol). These compounds showed antibacterial activity against susceptible bacteria at a MIC range of 31-125 µg/mL and potentiated the antibacterial activity of amoxicillin (at » MIC of compounds) against E. coli and P. aeruginosa with modulation factors of 32 and 10, respectively. These compounds also demonstrated good anti-biofilm formation effect at a concentration range of 3-100 µg/mL, and bacterial efflux pump inhibition activity at ½ MIC and » MIC against E. coli and P. aeruginosa. Loeseneriella africana stem bark extracts and constituents elicit considerable antibacterial, resistance modulation, and biofilm and efflux pump inhibition activities. The results justify the indigenous uses of L. africana for managing microbial infections.

Phenotypic and Genomic Insights into Biofilm Formation in Antibiotic-Resistant Clinical Coagulase-Negative Staphylococcus Species from South Africa.

The work aims to investigate biofilm formation and biofilm/adhesion-encoding genes in coagulase-negative staphylococci (CoNS) species recovered from blood culture isolates. Eighty-nine clinical CoNS were confirmed using the VITEK 2 system, and antibiotic susceptibility testing of isolates was conducted using the Kirby-Bauer disk diffusion method against a panel of 20 antibiotics. Isolates were qualitatively screened using the Congo red agar medium. Quantitative assays were performed on microtiter plates, where the absorbances of the solubilised biofilms were recorded as optical densities and quantified. In all, 12.4% of the isolates were strong biofilm formers, 68.5% had moderate biofilm capacity, and 17.9% showed weak capacity. A subset of 18 isolates, mainly methicillin-resistant S. epidermidis, were investigated for adherence-related genes using whole-genome sequencing and bioinformatics analysis. The highest antibiotic resistance rates for strongly adherent isolates were observed against penicillin (100%) and cefoxitin (81.8%), but the isolates showed no resistance to linezolid (0.0%) and tigecycline (0.0%). The icaABC genes involved in biofilm formation were detected in 50% of the screened isolates. Other adherence-related genes, including autolysin gene atl (88.8%), elastin binding protein gene ebp (94.4%), cell wall-associated fibronectin-binding protein gene ebh (66.7%), clumping factor A gene clfA (5.5%), and pili gene ebpC (22.2%) were also found. The insertion sequence IS256, involved in biofilm formation, was found in 10/18 (55.5%) screened isolates. We demonstrate a high prevalence of biofilm-forming coagulase-negative staphylococci associated with various resistance phenotypes and a substantial agreement between the possession of biofilm-associated genes and the biofilm phenotype.

Genomic Analysis of Antibiotic-Resistant Staphylococcus epidermidis Isolates From Clinical Sources in the Kwazulu-Natal Province, South Africa.

Staphylococcus epidermidis has become an important nosocomial pathogen. Multidrug resistance makes S. epidermidis infections difficult to treat. The study aims to describe the genomic characteristics of methicillin-resistant S. epidermidis (MRSE) isolated from clinical sources, to comprehend the genetic basis of antibiotic resistance, virulence, and potential pathogenicity. Sixteen MRSE underwent whole-genome sequencing, and bioinformatics analyses were carried out to ascertain their resistome, virulome, mobilome, clonality, and phylogenomic relationships. In all, 75% of isolates displayed multidrug resistance and were associated with the carriage of multiple resistance genes including mecA, blaZ, tet(K), erm(A), erm(B), erm(C), dfrG, aac(6')-aph(2''), and cat(pC221) conferring resistance to ß-lactams, tetracyclines, macrolide-lincosamide-streptogramin B, aminoglycosides, and phenicols, which were located on both plasmids and chromosomes. Their virulence profiles were evidenced by the presence of genes involved in adherence/biofilm formation (icaA, icaB, icaC, atl, ebh, and ebp), immune evasion (adsA, capC, and manA), and antiphagocytosis (rmlC, cdsA, and A). The community-acquired SCCmec type IV was the most common SCCmec type. The CoNS belonged to seven multilocus sequence types (MLSTs) and carried a diversity of mobile genetic elements such as phages, insertion sequences, and plasmids. The bacterial anti-phage defense systems clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) immunity phage system and restriction-modification system (R-M system) and the arginine catabolic mobile element (ACME) involved in immune evasion and transport of virulence genes were also found. The insertion sequence, IS256, linked with virulence, was found in 56.3% of isolates. Generally, the isolates clustered according to STs, with some similarity but also considerable variability within isolates. Whole-genome sequencing and bioinformatics analysis provide insights into the likely pathogenicity and antibiotic resistance of S. epidermidis, necessitating surveillance of this emerging pathogen.

Staphylococcus aureus in Intensive Pig Production in South Africa: Antibiotic Resistance, Virulence Determinants, and Clonality.

Although Staphylococcus aureus is a major threat to the veterinary, agricultural, and public health sectors because of its zoonotic potential, studies on its molecular characterisation in intensive animal production are rare. We phenotypically and genotypically characterised antibiotic-resistant S. aureus in intensive pig production in South Africa, using the farm-to-fork approach. Samples (n = 461) were collected from the farm, transport vehicles, and the abattoir using the World Health Organisation on Integrated Surveillance of Antimicrobial Resistance (WHO-AGISAR) sampling protocol. Bacteria were isolated using selective media and identified using biochemical tests and polymerase chain reaction (PCR). Phenotypic resistance was determined using the disk diffusion method. Selected resistance and virulence genes were investigated using PCR. Clonality among the isolates was determined using the repetitive element sequence-PCR. In all, 333 presumptive staphylococcal isolates were obtained, with 141/333 (42.3%) identified as staphylococci biochemically. Ninety-seven (97; 68.8%) were confirmed as S. aureus using PCR, 52.6% of which were identified as methicillin-resistant S. aureus (MRSA) through the mecA gene. All the 97 S. aureus isolates (100%) were resistant to at least one of the antibiotics tested, with the highest resistance observed against erythromycin and clindamycin (84.50% each), and the lowest observed against amikacin (2.10%); 82.47% (80/97) were multidrug-resistant with an average multiple antibiotic resistance index of 0.50. Most of the phenotypically resistant isolates carried at least one of the corresponding resistance genes tested, ermC being the most detected. hla was the most detected virulence gene (38.14%) and etb was the least (1.03%). Genetic fingerprinting revealed diverse MRSA isolates along the farm-to-fork continuum, the major REP types consisting of isolates from different sources suggesting a potential transmission along the continuum. Resistance to antibiotics used as growth promoters was evidenced by the high prevalence of MDR isolates with elevated multiple antibiotic resistance indices >0.2, specifically at the farm, indicating exposure to high antibiotic use environments, necessitating antibiotic stewardship and proper infection control measures in pig husbandry and intensive pig production.

Multidrug-Resistant Coagulase-Negative Staphylococci Isolated from Bloodstream in the uMgungundlovu District of KwaZulu-Natal Province in South Africa: Emerging Pathogens.

Coagulase-negative staphylococci (CoNS) are increasingly associated with nosocomial infections, especially among the immunocompromised and those with invasive medical devices, posing a significant concern. We report on clinical multidrug-resistant CoNS from the uMgungundlovu District, KwaZulu-Natal Province, South Africa, as emerging pathogens. One hundred and thirty presumptive CoNS were obtained from blood cultures. Culture, biochemical tests, and the Staphaurex™ Latex Agglutination Test were used for the initial identification of CoNS isolates; confirmation and speciation were undertaken by the VITEK 2 system. Susceptibilities of isolates against a panel of 20 antibiotics were determined using the Kirby-Bauer disk diffusion method, and the multiple antibiotic resistance (MAR) indices of the isolates were determined. The polymerase chain reaction (PCR) was used to amplify the mecA gene to confirm methicillin resistance. Overall, 89/130 presumptive CoNS isolates were confirmed as CoNS by the VITEK 2 system. Of these, 68 (76.4%) isolates were putatively methicillin-resistant by the phenotypic cefoxitin screen test and 63 (92.6%) were mecA positive. Staphylococcus epidermidis (19.1%), S. hominis ssp. hominis (15.7%), and S. haemolyticus (16.9%) were the most common CoNS species. Isolates showed high percentage resistance against penicillin (100.0%), erythromycin (74.2%), and azithromycin (74.2%) while displaying high susceptibilities to linezolid (95.5%), gentamicin (95.5%), and tigecycline (94.4%). Multidrug resistance (MDR) was observed in 76.4% of isolates. MAR index calculation revealed 71.9% of isolates with MAR index >0.2 and 20.2% >0.5. Isolates with the highest MAR indices (0.7 and 0.8) were recovered from the neonatal intensive care unit. Fifty-one MDR antibiograms were observed. The high prevalence of methicillin resistance and multidrug resistance in several species of CoNS necessitates surveillance of this emerging pathogen, currently considered a contaminant of microbial cultures.

Genomic Investigation into the Virulome, Pathogenicity, Stress Response Factors, Clonal Lineages, and Phylogenetic Relationship of Escherichia coli Strains Isolated from Meat Sources in Ghana.

Escherichia coli are among the most common foodborne pathogens associated with infections reported from meat sources. This study investigated the virulome, pathogenicity, stress response factors, clonal lineages, and the phylogenomic relationship of E. coli isolated from different meat sources in Ghana using whole-genome sequencing. Isolates were screened from five meat sources (beef, chevon, guinea fowl, local chicken, and mutton) and five areas (Aboabo, Central market, Nyorni, Victory cinema, and Tishegu) based in the Tamale Metropolis, Ghana. Following microbial identification, the E. coli strains were subjected to whole-genome sequencing. Comparative visualisation analyses showed different DNA synteny of the strains. The isolates consisted of diverse sequence types (STs) with the most common being ST155 (n = 3/14). Based Upon Related Sequence Types (eBURST) analyses of the study sequence types identified four similar clones, five single-locus variants, and two satellite clones (more distantly) with global curated E. coli STs. All the isolates possessed at least one restriction-modification (R-M) and CRISPR defence system. Further analysis revealed conserved stress response mechanisms (detoxification, osmotic, oxidative, and periplasmic stress) in the strains. Estimation of pathogenicity predicted a higher average probability score (Pscore ≈ 0.937), supporting their pathogenic potential to humans. Diverse virulence genes that were clonal-specific were identified. Phylogenomic tree analyses coupled with metadata insights depicted the high genetic diversity of the E. coli isolates with no correlation with their meat sources and areas. The findings of this bioinformatic analyses further our understanding of E. coli in meat sources and are broadly relevant to the design of contamination control strategies in meat retail settings in Ghana.

Review of Clinically and Epidemiologically Relevant Coagulase-Negative Staphylococci in Africa.

Coagulase-negative staphylococci (CoNS) have engendered substantial interest in recent years as pathogenic causes of infections in both human and veterinary medicine, especially in the immunocompromised, critically ill, long-term hospitalized and in those harboring invasive medical devices such as catheters. They have been implicated in infections such as urinary tract infections, bloodstream infections, and invasive device-related infections, and are responsible for substantial economic losses in livestock production. The advancement of diagnostic techniques has increased our understanding of their molecular mechanisms of pathogenicity, even though distinguishing between innocuousness and pathogenicity is still challenging. The incidence of CoNS varied across the continent in humans and animals (mainly cattle), ranging from 6% to 68% in suspected human infections and from 3% to 61.7% in suspected animal infections, distributed across different geographic locations. Furthermore, there were varying antibiotic resistance patterns observed in CoNS isolates, with high methicillin resistance in some cases, leading to crossresistance against many antibiotics. Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus xylosus were most commonly reported in studies herein reviewed, while the enterotoxin C gene, atl E gene, ica gene, and hemolysin virulence factors were linked with enhanced pathogenicity. Advancement in identification and typing methods, including whole genome sequencing, virulence screening, and the assessment of the immune status of subjects in studies will help to thoroughly assess the true pathogenic potential of isolated CoNS species in developing countries. Careful antibiotic stewardship guidelines should be followed due to the ability of CoNS to develop multidrug resistance.

Systematic Review of Important Bacterial Zoonoses in Africa in the Last Decade in Light of the 'One Health' Concept.

Zoonoses present a major public health threat and are estimated to account for a substantial part of the infectious disease burden in low-income countries. The severity of zoonotic diseases is compounded by factors such as poverty, living in close contact with livestock and wildlife, immunosuppression as well as coinfection with other diseases. The interconnections between humans, animals and the environment are essential to understand the spread and subsequent containment of zoonoses. We searched three scientific databases for articles relevant to the epidemiology of bacterial zoonoses/zoonotic bacterial pathogens, including disease prevalence and control measures in humans and multiple animal species, in various African countries within the period from 2008 to 2018. The review identified 1966 articles, of which 58 studies in 29 countries met the quality criteria for data extraction. The prevalence of brucellosis, leptospirosis, Q fever ranged from 0-40%, 1.1-24% and 0.9-28.2%, respectively, depending on geographical location and even higher in suspected outbreak cases. Risk factors for human zoonotic infection included exposure to livestock and animal slaughters. Dietary factors linked with seropositivity were found to include consumption of raw milk and locally fermented milk products. It was found that zoonoses such as leptospirosis, brucellosis, Q fever and rickettsiosis among others are frequently under/misdiagnosed in febrile patients seeking treatment at healthcare centres, leading to overdiagnoses of more familiar febrile conditions such as malaria and typhoid fever. The interactions at the human-animal interface contribute substantially to zoonotic infections. Seroprevalence of the various zoonoses varies by geographic location and species. There is a need to build laboratory capacity and effective surveillance processes for timely and effective detection and control of zoonoses in Africa. A multifaceted 'One Health' approach to tackle zoonoses is critical in the fight against zoonotic diseases. The impacts of zoonoses include.

Understanding antimicrobial discovery and resistance from a metagenomic and metatranscriptomic perspective: advances and applications.

Our inability to cultivate most microorganisms, specifically bacteria, in the laboratory has for many years restricted our view and understanding of the bacterial meta-resistome in all living and nonliving environments. As a result, reservoirs, sources and distribution of antibiotic resistance genes (ARGS) and antibiotic-producers, as well as the effects of human activity and antibiotics on the selection and dissemination of ARGs were not well comprehended. With the advances made in the fields of metagenomics and metatranscriptomics, many of the hitherto little-understood concepts are becoming clearer. Further, the discovery of antibiotics such as lugdinin and lactocillin from the human microbiota, buttressed the importance of these new fields. Metagenomics and metatranscriptomics are becoming important clinical diagnostic tools for screening and detecting pathogens and ARGs, assessing the effects of antibiotics, other xenobiotics and human activity on the environment, characterizing the microbiome and the environmental resistome with lesser turnaround time and decreasing cost, as well as discovering antibiotic-producers. However, challenges with accurate binning, skewed ARGs databases, detection of less abundant and allelic variants of ARGs and efficient mobilome characterization remain. Ongoing efforts in long-read, phased- and single-cell sequencing, strain-resolved binning, chromosomal-conformation capture, DNA-methylation binning and deep-learning bioinformatic approaches offer promising prospects in reconstructing complete strain-level genomes and mobilomes from metagenomes.

Emerging mechanisms of antimicrobial resistance in bacteria and fungi: advances in the era of genomics.

Bacteria and fungi continue to develop new ways to adapt and survive the lethal or biostatic effects of antimicrobials through myriad mechanisms. Novel antibiotic resistance genes such as lsa(C), erm(44), VCC-1, mcr-1, mcr-2, mcr-3, mcr-4, bla KLUC-3 and bla KLUC-4 were discovered through comparative genomics and further functional studies. As well, mutations in genes that hitherto were unknown to confer resistance to antimicrobials, such as trm, PP2C, rpsJ, HSC82, FKS2 and Rv2887, were shown by genomics and transcomplementation assays to mediate antimicrobial resistance in Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, Saccharomyces cerevisae, Candida glabrata and Mycobacterium tuberculosis, respectively. Thus, genomics, transcriptomics and metagenomics, coupled with functional studies are the future of antimicrobial resistance research and novel drug discovery or design.