Virulence Signatures, Integrons, and Antibiotic Resistance Genes in Bacterial Strains Recovered from Selected Commercial Dairy Products and Fresh Raw Meat.

Bacterial species responsible for food infections and intoxication are sometimes carried through the food production and processing. Very few published literatures exist on integrons among antibiotic-resistant staphylococcal strains from foods of animal origin in Gauteng Province, South Africa, hence this study. A total of 720 samples (360 meat and 360 dairies) from a community abattoir of a research farm in South Africa, using conventional bacteriological and molecular methods. Nine (9) bacterial strains, including Bacillus subtilis AYO-123, Acinetobacter baumannii AYO-241, Staphylococcus lentus AYO-352, among others were identified and submitted to GenBank. More bacterial strains were recovered from raw meat (90.5%) than dairy products (9.5%). Resistance was shown (0-100%) to Imipenem, Meropenem, Norfloxacin, Clindamycin, and 22 other antibiotics, without any carbapenem-resistant Acinetobacter baumannii and methicillin/vancomycin-resistant Staphylococcus species (MRSS/VRSS). Virulence genes for fibronectin-binding protein A (FnbA) were predominant (56.24%) followed by the circulating nucleic acids (cna) gene (43.75%). Others were staphylococcal enterotoxin A (sea, 41%), staphylococcal enterotoxin B (seb, 23.5%). Co-presence of sea and seb genes occurred in 11.76% of the isolates, but no coa genes was amplified. Antibiotic resistance genes (ARGs), tetK (70.58%), linA (29.4%), and ermA (11.76%) were detected, but none of the mecA and vat genes was amplified. Class 2 integron (50%) was more predominantly detected than integron 1 (25%), but no Class 3 integron was detected. Bacteria with both the detected virulence and antibiotic resistance genes are of potential risks to human health.

In vitro profiling and functional assessments of the anti-diabetic capacity of phenolic-rich extracts of Bulbine natalensis and Bulbine frutescens.

AIMS: Bulbine natalensis (BN) and Bulbine frutescens (BF) are recommended in South African traditional medicine to treat diabetes, but their modes of action are unknown. This study assessed the phenolic acid profiles, mineral composition and in vitro functional effects of BN and BF to better understand their glucose-lowering capabilities. METHODS: Phenolic acid and mineral composition of BN and BF methanolic extracts were determined by HPLC and inductively coupled plasma optical emission spectroscopy respectively. Antioxidant capacity was assessed by potassium ferricyanide reducing power and 2,2-diphenyl-2-picrylhydrazyl radical scavenging assays, and inhibition of alpha-amylase, alpha-glucosidase, pancreatic lipase and DPP4 was evaluated by standard enzyme assays. The effects of BN and BF extracts on insulin secretion were investigated using static incubations of isolated mouse islets and molecular docking analysis was used to identify interactions of BN and BF with partners that could mediate stimulatory effects on insulin secretion. RESULTS: Methanolic extracts of BN and BF contained high concentrations of protocatechuic and gallic acids, and high levels of Zn, Mn and Cr. The extracts inhibited alpha-glucosidase, alpha-amylase, pancreatic lipase and DPP4 activities, and they also inhibited free radical generation. Both extracts significantly potentiated glucose-stimulated insulin secretion without significantly affecting basal insulin secretion or islet cell viability. Protocatechuic acid, the most abundant phenolic acid in the extracts, showed high affinity for PKA, PKC, DPP4 and CaMK II in the docking analysis. CONCLUSIONS: BN and BF have multiple beneficial effects on glucoregulatory pathways and they, or their derivatives, could be developed to treat type 2 diabetes.

Draft Genome Sequence of Sweet Pepper Fruit Epiphyte-Associated Bacillus cereus HRT7.7.

This study reports the whole-genome sequence of Bacillus cereus HRT7.7, an epiphyte isolated from red sweet pepper fruits that is capable of stimulating plant growth and development. The genome assembly is 5,109,010 bp in length, with a G+C content of 35.2%.

Whole-Genome Sequence of Paenibacillus polymyxa Strain SRT9.1, a Promising Plant Growth-Promoting Bacterium.

Paenibacillus polymyxa SRT9.1 is an epiphytic bacterium capable of inhibiting plant-pathogenic bacteria. The strain has potential for development as a biocontrol agent for use in agriculture. We report the whole-genome sequence of Paenibacillus polymyxa SRT9.1, consisting of 6,754,470 bp and 7,878 coding sequences, with an average G+C content of 45%.

Computational, chemical profiling and biochemical evaluation of antidiabetic potential of Parkia biglobosa stem bark extract in type 2 model of rats.

The present study investigated the antidiabetic and antioxidant capacity of hydromethanol extract from Parkia biglobosa stem bark (PBSBHM) in fructose-streptozotocin induced type 2 diabetic rats after 28 days of oral administration. Simultaneously, evaluated the phenolic profiles and mineral compositions of crude extract. Molecular docking analysis of protocatechuic acid, the most abundant phenolic acid with potential downstream partners protein kinase A (PKA), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMK II), was investigated. The preliminary results showed that PBSBHM crude extract contained 225.2 ± 18.25 mg GAE/g of total phenolic and 99.28 ± 12.3 mg QE/g of total flavonoid. Both protocatechuic and gallic acids were identified as a prominent phenolic compound through HPLC analysis, while vanillic acid was not detected. High mineral composition of K, Mg, P, Ca while Mn and Cr as trace elements were found in PBSBHM by plasma optical emission spectroscopy. PBSBHM extracts showed a significant radical scavenging activity from a therapeutic point of view, a moderate antioxidant potential and improved glucose tolerance after 30 min of glucose loading. PBSBHM extracts significantly attenuated serum glucose level and glycosylated haemoglobin at the tested dosage. However, it elevated the hepatic hexokinase activity and glycogen level compared with the diabetic untreated rats. PBSBHM ameliorates the decreased activity of pancreatic superoxide dismutase, catalase and reduced glutathione but decreased the MDA level. Docking analysis of protocatechuic acid showed a moderate affinity for the target enzymes compared to the standard drugs. Our data showed that the stem bark extract of this botanical has antidiabetic potential and at least in part substantiates its traditional use in the management of diabetes, possibly due to the synergistic interactions of protocatechuic acid with other biologically active components.

Multi-Strain Probiotics: Synergy among Isolates Enhances Biological Activities.

The use of probiotics for health benefits is becoming popular because of the quest for safer products with protective and therapeutic effects against diseases and infectious agents. The emergence and spread of antimicrobial resistance among pathogens had prompted restrictions over the non-therapeutic use of antibiotics for prophylaxis and growth promotion, especially in animal husbandry. While single-strain probiotics are beneficial to health, multi-strain probiotics might be more helpful because of synergy and additive effects among the individual isolates. This article documents the mechanisms by which multi-strain probiotics exert their effects in managing infectious and non-infectious diseases, inhibiting antibiotic-resistant pathogens and health improvement. The administration of multi-strain probiotics was revealed to effectively alleviate bowel tract conditions, such as irritable bowel syndrome, inhibition of pathogens and modulation of the immune system and gut microbiota. Finally, while most of the current research focuses on comparing the effects of multi-strain and single-strain probiotics, there is a dearth of information on the molecular mechanisms of synergy among multi-strain probiotics isolates. This forms a basis for future research in the development of multi-strain probiotics for enhanced health benefits.

Characterization of Rumen Microbiota of Two Sheep Breeds Supplemented With Direct-Fed Lactic Acid Bacteria.

Supplementation of direct-fed microbials into ruminants' nutrition has shown great potential in manipulating rumen fermentation and enhancing productive animal performance. However, little is known about rumen microbial composition and diversity of Damara and Meatmaster sheep, breeds indigenous to South Africa. The study aimed at exploring and comparing the rumen microbiomes of two breeds with different feeding treatments as follows: no antibiotic, no probiotics (T1), only potential probiotic (T2), only potential probiotic (T3), the combination of potential probiotics (T4), antibiotic (T5); using a metagenomic approach. The results showed that based on the Shannon index, the microbial diversity of Damara was higher (p < 0.05) than Meatmaster, while treatment T4 was higher than treatment T1 (p < 0.05). The principal coordinate analysis showed no significant difference among treatments, while there were significant dissimilarities between sheep breeds and sample-day (p < 0.05). Canonical correspondence analysis (CCA) displayed the dispersion of microbial communities among treatments, where negative control (T1) was distinct from other treatments. Bacteroidetes and Firmicutes were the most abundant microbial phyla across treatments for both breeds. Negative control and the combination of potential probiotics showed lower proportions of Proteobacteria compared to other treatments. At the genus level, Prevotella and Clostridium were abundant across all treatments, while Pseudomonas was abundant only in T2, T3, and T5. In all treatments, Fibrobacter was detected after the feeding trials, while it was not detected in most treatments before trials. The results revealed that the rumen microbiome's structure and abundance were slightly altered by administering lactic acid as a putative probiotic.

Quercetin modulates hyperglycemia by improving the pancreatic antioxidant status and enzymes activities linked with glucose metabolism in type 2 diabetes model of rats: In silico studies of molecular interaction of quercetin with hexokinase and catalase.

Quercetin was assessed for its antihyperglycemic effect in fructose-streptozotocin (STZ) induced diabetic rats. The oral administration of quercetin at the dosage of 25 and 50 mg/kg for 28 days remarkably reduced the level of blood glucose, glycosylated hemoglobin (Hb), and hepatic glycogen but enhanced plasma Hb concentration. The altered activities of glucose-6-phosphatase and hexokinase in diabetic rats were significantly improved upon quercetin treatment. Furthermore, the antioxidant activity of pancreatic superoxide dismutase, catalase (CAT), and reduced glutathione was effectively increased while the value for thiobarbituric acid reactive species was decreased. A significant reduction of glycemia was observed in the glucose tolerance test, 120 min after the glucose pulse. Also, the damage caused by fructose-STZ in the liver and pancreas of diabetic animals were restored to near normal. Molecular docking of quercetin showed a high affinity for hexokinase and CAT with a binding energy of -7.82 and -9.83 kcal/mol, respectively, more elevated than the standard drugs. PRACTICAL APPLICATIONS: Functional foods and nutraceuticals have increasingly interested the consumers in terms of health benefits and have started focussing on the prevention or cure of disease by the foods and their health-enhancing phytochemicals. Quercetin is one of the most potent naturally occurring antioxidants within the flavonoid subclasses, mostly distributed as a secondary metabolite in fruits, vegetables, and black tea. Based on the results exhibited in the present study, we proposed that the consumption of foods rich in quercetin could be a cheap and affordable nutraceutical that can be developed for the treatment of T2DM and its complications. Further studies on the safety aspects of quercetin in long-term usage are strongly recommended before implementing for the treatment of human diseases.

Whole-Genome Sequence of Lactobacillus acidophilus PNW3, Isolated from Weaned Piglets of the Indigenous South African Windsnyer Pig Breed.

A draft genome sequence of Lactobacillus acidophilus PNW3 is reported. The genome assembly is 1,857,655 bp long in 25 contigs with an N 50 value of 230,557 bp and a G+C content of 34.6%. The total number of predicted protein-coding genes is 1,776, with 58 predicted RNAs and 42 predicted pseudogenes.

Complete Genome Sequence of Lactobacillus reuteri PNW1, a Promising Probiotic Candidate.

This study reports the whole-genome sequence of Lactobacillus reuteri PNW1 isolated from gastrointestinal tracts of weaned piglets of the indigenous South African Windsnyer pig breed. A total of 5.2 GB data comprising 8,209,104 paired-end reads were generated. The assembled genome is 2,430,215 bp long in 420 contigs with 39% G+C content.

Safety Evaluation and Colonisation Abilities of Four Lactic Acid Bacteria as Future Probiotics.

The study evaluated the safety and colonisation properties of four lactic acid bacteria (LAB), by determining their cell hydrophobicity and aggregation abilities. In addition, the presence of virulence and resistance genes was assayed in these probiotic candidates. Lactobacillus reuteri ZJ625, Lactobacillus reuteri VB4, Lactobacillus salivarius ZJ614 and Streptococcus salivarius NBRC13956 were tested for cell surface hydrophobicity abilities against xylene, chloroform and ethyl acetate. The isolates were also tested for auto-aggregation and co-aggregation abilities; the optical densities of cell growth were measured after 1, 2, 3 and 4 h of experimental set-up. DNA was extracted from all the four isolates and amplified using PCR with specific primers to detect virulence genes of adhesion collagen protein (ace) and aggregation substances (agg and asa); also, resistance genes of Vancomycin vanA, Vancomycin vanC1 and Vancomycin vanC2/3 were assayed in the four isolates. The isolates showed high hydrophobicity to all solvents: xylene (78-84%), chloroform (68-75%) and ethyl acetate (52-60%). High auto- and co-aggregations ranging from 60 to 70% and from 45 to 56% respectively were observed in the isolates after 4 h of incubation at 37 °C. Some of the tested isolates showed the presence of virulence and resistance genes; however, this does not indicate that these genes are unsafe because their transmission and expression abilities are unknown. Therefore, in this study, the isolates studied are considered safe for use as future probiotics, as revealed from results presented, which generally represents the scanned safety evaluations of the isolates as promising probiotics.

Metagenomic Sequencing of Wastewater from a South African Research Farm.

We sequenced wastewater effluent from the Agricultural Research Council-Animal Production in South Africa that conducts studies on livestock health and farm ecology. Thauera, Oscillibacter, and Pseudomonas were the most abundant genera within the community. Thirty-one different antibiotic resistance genes were identified, 10 of which are associated with tetracycline resistance.

Alpha-Amylase Inhibition and Antioxidative Capacity of Some Antidiabetic Plants Used by the Traditional Healers in Southeastern Nigeria.

Oxidative stress plays a significant role in the pathogenesis of metabolic syndrome including diabetes mellitus (DM). The inhibition of alpha-amylase is an important therapeutic target in the regulation of postprandial increase of blood glucose in diabetic patients. The present study investigated the alpha-amylase inhibitory and antioxidant potential of selected herbal drugs used in the treatment of DM by the traditional healers in Isiala Mbano and Ikwuano regions of southeastern Nigeria. Antioxidant activity was evaluated in terms of free radical scavenging, reducing power, and total phenolic (TPC) and flavonoid content (TFC) in consonance with the TLC profiling. The results showed that methanol crude extracts from Anacardium occidentale (AO) and Ceiba pentandra (CP) recorded higher TPC and TFC, potent free radical scavenging, and efficient reducing power (RP) as compared with other plant samples. All the plant extracts exhibited a relative alpha-amylase inhibition apart from Strophanthus hispidus (SH) extract with a negative effect. We discovered a mild to weak correlation between alpha-amylase inhibition or antioxidative capacity and the total phenol or flavonoid content. At least in part, the results obtained in this work support the traditional use of certain plant species in the treatment of patients with DM.

Isolation, Identification and Screening of Potential Probiotic Bacteria in Milk from South African Saanen Goats.

This study aimed to evaluate lactic acid bacteria isolates from Saanen goats' milk for probiotic attributes, thereby determining their potential as direct-fed microbials for goats. Isolates were identified using API 50CH system, 16S rDNA sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry. All 17 isolates obtained were identified as Lactobacillus plantarum except one identified as Pediococcus acidilactici. Four isolates identified as L. plantarum (Accession numbers KJ026587.1, KM207826.1, KC83663.1 and KJ958428.1) by at least two of the techniques used and isolate 17 differently identified by all the methods used were selected as representatives and then screened for probiotic properties. These isolates displayed phenotypic probiotic attributes including tolerance to acid and bile salts, ability to adhere to intestines and possession of antagonistic activities against Proteus vulgaris, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa and Escherichia coli. The lactic acid bacteria isolated from Saanen goats' milk showed potential to be used as sustainable probiotics in goats' industry. Successful use of probiotics in animals depends upon availability of appropriate isolates originating from the specific host animal. This study is a positive contribution towards identification of isolates with potential for formulation as direct-fed microbials for South African Saanen goats.

Biocidal effects of stem bark extract of Chrysophyllum albidium G. Don on vancomycin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus causes variety of infections in humans and animals worldwide and predominates in surgical wound infections. This study assessed the antimicrobial potential of the stem bark extract of Chrysophyllum albidum against an array of vancomycin resistant Staphylococcus aureus (VRSA) isolated from clinical samples. METHODS: The methanolic crude extract of the plant was preliminary screened for the presence of phytochemicals; after then, the extract was partitioned into n-hexane, chloroform, ethyl acetate and butanol fractions. A range of concentrations of the plant extract fractions was prepared to assess its antimicrobial potency; the minimum inhibitory concentrations (MICs); the minimum bactericidal concentrations (MBCs); the rate of killing; the potassium ion leakage potential and nucleotides leakage ability against the VRSAs. RESULTS: The phytochemical screening revealed the presence of tannins, alkaloids, flavonoids, saponins, steroids, reducing sugars and terpenoids as major phytoconstituents resident in the crude plant extract. The two active fractions (n-hexane and butanol) at a concentration of 10 mg/ml exhibited antibacterial activities with the MIC and MBC values for the fractions ranged between 0.63-10 mg/ml and 1.25-10 mg/ml respectively. The time kill assay revealed that the antibacterial action of the two fractions are time and concentration dependent; the n-hexane and butanol fractions achieved 100 % kill on the test isolates at a concentration of 3 × MIC and 2 × MIC respectively after 120 min of reaction time. Varying amount of potassium ions as well as nucleotides were leaked from the test cells by n-hexane and butanol fractions. CONCLUSIONS: This study has established the possibility of developing antimicrobial agents of natural origin to manage possible infection from vancomycin resistant Staphylococcus aureus that are now developing multi-resistance against many antibiotics.

Probable mechanisms of biocidal action of Cocos nucifera Husk extract and fractions on bacteria isolates.

BACKGROUND: The incidence of resistance to the existing antibiotics by microorganisms demand increased effort in the development of new antibiotics for the treatment of microbial infections and diseases. Infections due to multidrug resistant pathogens are difficult to manage due to relatively limited choices of antimicrobial agents. This study investigated antimicrobial activities of the husk extract of Cocos nucifera on some bacteria that are associated with human diseases. METHODS: Powdered husk of Cocos nucifera was cold extracted using mixture of methanol and distilled water in ration 3:2 (v/v). Extract was partitioned into n-hexane. Chloroform, ethylacetate and n-butanol fractions and thereafter, the minimum inhibitory concentrations (MICs) of the extract and those of the fractions were determined. The ethylacetate fraction was found to be more active and was partially purified by a combination of thin-layer and column chromatography. Finally, the rate of killing, leakages of proteins, potassium ions and nucleotides from the tests bacterial cells were determined. RESULTS: The minimum Inhibitory concentrations (MICs) of the extract ranged between 0.39 and 12.50 mg/ml and those of the fractions ranged between 0.16 and 5.00 mg/ml. The time-kill assay revealed a minimum of 27.8% killed at 1 × MIC after 15 min contact time with the fractions and a minimum of 95% killed after 120 min. Varying amount of proteins, potassium ions as well as nucleotides were leaked from selected bacterial isolates by the four active fractions. The amount of proteins leaked from the cells after 15 min contact time ranged between 3.56 and 19.08 µg/ml at 1 × MIC and between 10.97 and 19.54 µg/ml at 2 × MIC. The amount of potassium ions leaked from the cells after 15 min contact time ranged between 0.182 and 0.379 mg/ml at 1 × MIC and between 0.227 and 0.561 mg/ml at 2 × MIC. The nucleotides leaked from the cells after 15 min contact time ranged between 0.609 and 2.446 µg/ml at 1 × MIC and between 0.897 and 2.841 µg/ml at 2 × MIC. CONCLUSIONS: This study established the possibility of developing antimicrobial agents of natural origin to combat resistance to antimicrobial compounds by some pathogens currently being experienced in agricultural and health care environments.

Evaluation of Antibacterial and Antifungal Properties of Alchornea laxiflora (Benth.) Pax. & Hoffman.

Alchornea laxiflora leaf extract was tested against a range of microorganisms using standard microbiological methods for antimicrobial activities. The extract inhibited the growth of all the bacterial and 15 fungal isolates tested. The zones of inhibition exhibited against the test bacteria ranged between 12 mm and 24 mm and between 11 mm and 24 mm for the extract and the antibiotic streptomycin, respectively. The zones of inhibition observed against the fungal isolates by the extract ranged between 12 mm and 23 mm. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) exhibited by the extract against test bacteria ranged between 0.78 mg/mL-25 mg/mL and 1.56 mg/mL-25 mg/mL, respectively, while the MICs and minimum fungicidal concentrations (MFCs) values for the test fungi ranged between 8.75 mg/mL-35.00 mg/mL and 8.75 mg/mL-35.00 mg/L, respectively. The preliminary phytochemical screening of the extract revealed the presence of alkaloids, tannins, flavonoids, saponins, and reducing sugars as major phytoconstituents in the extract. A. laxiflora leaf extract is a potent source of antibacterial and antifungal compounds; further studies on the extract are ongoing in our laboratories to elucidate the probable mechanism(s) of action on bacteria and fungi found to be susceptible to the extract.

Anti-staphylococcal, anti-HIV and cytotoxicity studies of four South African medicinal plants and isolation of bioactive compounds from Cassine transvaalensis (Burtt. Davy) codd.

BACKGROUND: Medicinal plants represent an important opportunity to rural communities in Africa, as a source of affordable medicine and as a source of income. Increased patient awareness about safe usage is important as well as more training with regards to traditional medicine. The aim of this study was to evaluate the ethnomedicinal prowess of some indigenous South African plants commonly used in Eastern Cape Province of South Africa for the treatment of skin and respiratory tract infections, HIV and their toxicity potential. METHODS: Cassine transvaalensis, Vangueria infausta, Croton gratissimus and Vitex ferruginea were tested for antibacterial activities against Staphylococcus aureus and Staphylococcus epidermidis using Kirby-Bauer disk diffusion and minimum inhibition concentration (MIC). Cytotoxic and anti-HIV-1 activities of plants were tested using MTT Assay (3- (Dimethylthiozole-2-yl-2,5-diphenyltetrazolium bromide)) and anti- HIV-1iib assay. In search of bioactive lead compounds, Cassine transvaalensis which was found to be the most active plant extract against the two Staphylocoous bacteria was subjected to various chromatographic. Thin layer chromatography, Column chromatography and Nuclear Magnetic Resonance (NMR), (1H-1H, 13C-13C, in DMSO_d6, Bruker 600 MHz) were used to isolate and characterize 3-Oxo-28-hydroxylbetuli-20(29)-ene and 3,28-dihydroxylbetuli-20(29)-ene bioactive compounds from C. transvaalensis. RESULTS: The four plants studied exhibited bioactive properties against the test isolates. The zones of inhibition ranged between 16 mm to 31 mm for multi-drug resistant staphylococci species. MIC values varied between 0.6 and 0.02 µg/ml. C. gratissimus and C. transvaalensis exhibited the abilities to inhibit HIV-1iib. Two bioactive compounds were isolated from C. transvaalensis. CONCLUSION: Data from this study reveals the use of these plant by traditional healers in the Eastern Cape. Furthermore, C. transvaalensis and C. gratissimus were found to be more active as against HIV-1iib. While C. transvaalensis was most active against the two Staphylococcus bacteria.

Stem bark extract and fraction of Persea americana (Mill.) exhibits bactericidal activities against strains of bacillus cereus associated with food poisoning.

The study investigates the in vitro antibacterial potentials of stem bark extracts of Persea americana on strains of Bacillus cereus implicated in food poisoning. The crude stem bark extracts and butanolic fraction at a concentration of 25 mg/mL and 10 mg/mL, respectively, exhibited antibacterial activities against test isolates. The zones of inhibition exhibited by the crude extract and the fraction ranged between 10 mm and 26 mm, while the minimum inhibitory concentration values ranged between 0.78 and 5.00 mg/mL. The minimum bactericidal concentrations ranged between 3.12 mg/mL-12.5 mg/mL and 1.25-10 mg/mL for the extract and the fraction, respectively. The butanolic fraction killed 91.49% of the test isolates at a concentration of 2× MIC after 60 min of contact time, while a 100% killing was achieved after the test bacterial cells were exposed to the butanolic fraction at a concentration of 3× MIC after 90 min contact time. Intracellular protein and potassium ion leaked out of the test bacterial cells when exposed to certain concentrations of the fraction; this is an indication of bacterial cell wall disruptions by the extract's butanolic fraction and, thus, caused a biocidal effect on the cells, as evident in the killing rate test results.