Diversity and predicted functional roles of cultivable bacteria in vermicompost: bioprospecting for potential inoculum.

Vermicompost (VC) harbours diverse microbes, including plant growth-promoting microorganisms (PGPM) that are beneficial for sustainable crop production. Hence, this study aimed to analyse bacterial diversity of VC samples as a first high-throughput screening step towards subsequent targeted isolation of potential bacterial inoculum candidates. To achieve this, bacterial communities in VC collected from five production farms were enriched in nutrient-rich media before high-throughput sequence (HTS) analysis of the partial 16S rRNA gene. HTS analysis revealed 572 amplicon sequence variants (ASVs) in all enriched VC samples. Firmicutes, Proteobacteria, Planctomycetes and Bacteroidetes were the most dominant phyla, while Lysinibacillus, Escherichia-Shigella, Bacillus, Pseudomonas, Clostridium sensu stricto 1, Morganella, Vibrio and Aeromonas were the predominant genera across the enriched VC. The presence of Clostridium sensu stricto 1, Escherichia-Shigella and Vibrio genera, which are potentially pathogenic species, suggests the need to improve vermicomposting efficiency and safety. Predicted functional profiling of the bacterial communities using PICRUSt2 showed abundance profiles of nitrogenases, phosphatases and sulfatases. In addition, the potential to produce siderophore, indole acetic acids (IAA) and phytohormone regulator 1-aminocyclopropane-1-carboxylate (ACC) were predicted. Lysinibacillus, Bacillus, Paenibacillus and Pseudomonas were major bacterial communities with potential plant growth-promoting traits and could serve as resources in bacterial inoculum production. The findings in this study provide insight into the community composition, abundance and the potential functional capability of cultivable bacterial species of enriched VC. This study also points to VC as a suitable source of potentially beneficial bacterial candidates for inoculum production.

Insights into the Microbiological Safety of Vermicompost and Vermicompost Tea Produced by South African Smallholder Farmers.

Feedstock used in the production of vermicompost (VC) and vermicompost tea (VCT) may harbour various pathogenic bacteria responsible for a number of animal and human diseases worldwide. The identification and characterisation of such pathogenic organisms is necessary for assessing the safety of these products. In the present study, our goal was to determine the presence of possible pathogens in VC and VCT and, if present, to determine the antimicrobial susceptibility profiles of the organisms. VC and VCT samples were collected from five different farms in the Winterveldt, South Africa. Only one out of 60 VC and VCT samples was found to contain a potentially pathogenic organism. The use of phenotypic procedures aided the final identification of the isolate, which was confirmed to be Salmonella enterica serovar Typhimurium. This isolate tested positive for species specific invA genes. Antibiotic testing using the agar diffusion technique showed that the Salmonella isolate was resistant to only kanamycin. The Salmonella counts that were observed in this study were lower than the generally accepted infective doses of these bacteria. In the light of these findings, it was concluded that VC and VCT produced by the farmers involved presented a low risk in terms of the safety of the products.

Application of European standards for health and quality control of game meat on game ranches in South Africa.

The health and quality compliance of game carcasses (n = 295) intended for the South African export market and aspiring to comply with the strict hygiene requirements of the European Union were compared with game carcasses (n = 330) available for the local market and currently not subjected to meat safety legislation. Samples were collected in similar seasons and geographical areas in South Africa from 2006 to 2009. Aerobic plate counts (APC) of the heart blood verified that both groups possessed similar ante mortem bacterial status. For health compliance APC, tests for Escherichia coli, Salmonella spp. and Staphylococcus aureus were performed on the carcasses. Surfaces of the local carcasses were swabbed using the European Enviro-biotrace sponge technique at 3 and 72 h post mortem. Unskinned but eviscerated export carcasses in the abattoir were skinned and sampled by incision using a corkborer 72 h post mortem. Temperature and pH readings were recorded at 3 and 72 h post mortem from the longissimus dorsi muscle and the readings at 3 h differed (P = 0.035). Temperatures at 72 h were lower for export than local carcasses (P < 0.001) because of earlier introduction and maintenance of the cold chain. The pH readings also differed between groups at 3 and 72 h (P < 0.001). APC results for the local group exceeded the maximum permissible count (< 10(5)). S. aureus results showed differences (P < 0.001), with readings from the local group being higher. The same tendency was exhibited for E. coli (P = 0.008). Imposition of hygiene guidelines for game ranchers producing meat for the local market is therefore recommended.

Bacterial populations associated with bulk packaged beef supplemented with dietary vitamin E.

Five Simmentaler type calves were fed diets supplemented with 500 mg vitamin E per day and five fed control diets, Rump steaks from each carcass were PVC-overwrapped and bulk packaged in 100% CO2 or 20% CO2:80% O2. Bulk packs were stored up to 42 days at 4 degrees C and PVC-overwrapped samples subsequently displayed up to 7 days at 4 degrees C. After display the Aerobic Plate Count (APC) of steaks was determined and four colonies were randomly selected from the highest dilution APC plates showing growth. A total of 627 colonies were obtained. Gram-reaction, catalase, oxidase, morphology and motility of the isolates were determined. The gram-negative and gram-positive isolates were then identified using a dichotomous identification key. Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. predominated on rump steaks from both feeding treatments and in packaging treatments. After 42 days bulk storage Enterobacteriaceae, Pseudomonas spp., lactic acid bacteria and Acinetobacter spp. predominated in 20% CO2:80% O2 and 100% CO2 bulk packaging. Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. predominated on rump steaks, from both feeding and packaging treatments, during the aerobic display period of 7 days.

Combined effect of modified atmosphere bulk packaging, dietary vitamin E supplementation and microbiological contamination on colour stability of Musculus gluteus medius.

Ten Simmentaler type calves were fed diets supplemented with 500 mg vitamin E per day and ten fed control diets. After completion of a 100 day feeding period the cattle were slaughtered and rump steaks (M. gluteus medius) from each carcass PVC-overwrapped and subsequently bulk packaged in 100% CO(2) or 20% CO(2): 80% O(2). Steaks with low levels of bacteriological contamination were also prepared and packaged. Bulk packs were stored at 4°C for 0, 14, 28 and 42 days and the PVC-overwrapped samples subsequently displayed for 0, 4 and 7 days at 4°C. After display saturation, surface metmyoglobin and oxymyoglobin accumulation of the steaks were determined and acceptability of the steaks assessed by sensory evaluation using a trained panel. The dietary vitamin E supplemented steaks were more acceptable than the steaks from cattle not supplemented with vitamin E. Steaks prepared with low levels of bacteriological contamination, supplemented with dietary vitamin E, were more acceptable and discoloured less than all the other treatments. Beef rump steaks bulk packaged in 20% CO(2): 80% O(2) or 100% CO(2) were acceptable and colour stable for up to 14 days bulk storage at 4°C and a subsequent 2 days retail display at 4°C.

Microbiological shelf life of bulk-packaged Musculus gluteus medius supplemented with dietary vitamin E.

Five Simmentaler type calves were fed diets supplemented with 500 mg vitamin E per day and five fed control diets. Rump steaks from each carcass were PVC-overwrapped and bulk packaged in 100% CO, or 20% CO(2):80% O(2). Bulk packs were stored up to 42 days at 4°C and steaks displayed up to 7 days at 4°C. Bacterial counts of rump steaks from either packaging treatment were not significantly influenced during bulk storage or retail display by supplementation with dietary vitamin E. Both packaging treatments delayed bacterial growth during bulk storage. Aerobic plate counts of rump steaks stored in 100% CO(2) were lower than those of rump steaks stored in 20% CO(2): 80%: O(2). This study showed that rump steaks supplemented with dietary vitamin E can be bulk packaged in 20% CO(2): 80% O(2) or 100% CO(2) and stored for up to 42 days with shelf life of 4-7 days.

The taxonomy, ecology and cultivation of bacterial genera belonging to the family Flavobacteriaceae.

The group known as the 'flavobacteria' has previously been regarded as synonymous with the genus Flavobacterium. Today, however, flavobacteria refers to the family Flavobacteriaceae comprising 10 genera. This review deals with the rapid changes in the taxonomy of these bacteria, especially over the last decade. It also briefly reviews the ecology of the genera in this family and describes the media that have been utilized in the general and selective cultivation of these organisms.

A polyphasic taxonomic study of Chryseobacterium strains isolated from dairy sources.

A polyphasic taxonomic study, employing protein electrophoresis (SDS-PAGE), gas chromatographic analysis of cellular fatty acids (FAME), mol% G+C determination and DNA-DNA hybridizations, was undertaken on 103 dairy isolates shown to belong to Chryseobacterium. Reference strains of the Chryseobacterium species, CDC group IIb and Embedobacter brevis were included. SDS-PAGE analysis yielded good differentiation between the investigated species. About half of the strains could be clustered into nine major groups while the other half occupied a separate position. With FAME analysis no clear differentiation of the Chryseobacterium species (except C. meningosepticum) and SDS-PAGE groups could be achieved. FAME analysis, however, gave good differentiation between the Chryseobacterium and Empedobacter strains. The mol% G+C of the isolates tested, ranged between 36.4 and 39.0. The combination of SDS-PAGE and DNA-DNA hybridization identified a large group of dairy isolates as C. indologenes, one isolate as C. gleum and two new genotypic groups, comprising five and 15 dairy isolates respectively, emerged from the polyphasic study. Another large part of strains have a separate or uncertain position in Chryseobacterium and remained classified as Chryseobacterium species CDC group IIb.

The incidence of Weeksella- and Bergeyella-like bacteria in the food environment.

Eighty-five catalase- and oxidase-positive Gram-negative rods and cocci susceptible to penicillin G were isolated from a variety of food sources. The phenotypic relationships of these isolates with reference cultures of Bergeyella-like, Chryseobacterium, Empedobacter, Myroides, Moraxella, Sphingobacterium and Weeksella-like strains were examined by numerical taxonomy. Seventy-three isolates were recovered in five groups; 80% of the isolates clustered in groups 1, 2 and 3 and produced indole, bearing a strong resemblance to Weeksella and Bergeyella. They could not, however, be regarded as belonging to the known species of W. virosa and B. zoohelcum. It is suggested that three species may be necessary to accommodate the environmental Weeksella- or Bergeyella-like bacteria. The isolates in groups 4 and 5 had white colonies and were unable to produce indole, in this way resembling the Moraxella genus.

Technologically important characteristics of Enterococcus isolates from milk and dairy products.

Enterococcus faecium (54 strains), E. faecalis (40 strains), and E. durans (14) were isolated from various dairy products (raw milk, cream, butter and fermented milk products) during a previous study (Wessels et al., 1988). In this article various characteristics of these isolates, which may have a bearing on their significance in dairy products, have been studied. A large percentage of the identified strains of all three species were able to grow at 7 degrees C. Seventy-six percent of the E. faecium strains, 62% E. faecalis and 50% E. durans strains also showed proteolytic activity at psychrotrophic temperatures. The fact that proteolytic activity could be detected within 2 days at 7 degrees C is significant, since bulk cooled milk is normally held for 3 to 4 days at temperatures between 4 and 7 degrees C at farms or factories prior to processing. This examination confirmed that enterococci are proteolytic rather than lipolytic.

The taxonomic relationship of certain environmental flavobacteria to the genus Weeksella.

Forty environmental strains and reference cultures of Flavobacterium, Cytophaga and Weeksella spp. were examined by numerical taxonomy. Twenty-seven strains were recovered in four phena. Phena 1A and 1B comprised 48% of the strains and were sufficiently similar to the genus Weeksella as to suggest possible inclusion in this genus. They could not be accommodated in the existing species W. virosa and W. zoohelcum. Strains from phenon 2 appear to belong neither in the Flavobacterium or the Weeksella genus. Although no reference strains were included in phena 3 and 4 they appear phenotypically to be most similar to F. breve and F. odoratum respectively.

Psychrotrophic, proteolytic and lipolytic properties of Enterobacteriaceae isolated from milk and dairy products.

Most of the Enterobacteriaceae strains (73 out of 75) isolated in a previous study (Wessels et al., 1988) were psychrotrophic on agar plates, with the exception of Enterobacter cloacae strains. The Enterobacteriaceae strains were largely non-proteolytic on milk agar medium although limited numbers of E. cloacae, Serratia rubidaea and Klebsiella oxytoca strains were capable of proteolytic activity at 25 degrees C. The E. cloacae and K. oxytoca strains positive at 25 degrees C were also proteolytic at 7 degrees C. Most of the species tested were non-lipolytic on Victoria blue butterfat agar. The majority of Serratia marcescens and Klebsiella pneumoniae strains and a minority of E. cloacae and K. oxytoca strains, however, were lipolytic on this medium.

Determination of Bacterial ATP in Milk - The Influence of Adenosine Triphosphate-Hydrolyzing Enzymes from Somatic Cells and Pseudomonas fluorescens.

Presence and origin of adenosine triphosphate (ATP) hydrolyzing enzymes in milk and their effect on the ATP assay were determined. Somatic cells, when present in large numbers, produced sufficient enzyme to hydrolyze extracted ATP. This was illustrated by the relationship which existed between ATPase activity and somatic cell counts (polynominal correlation coefficient (R) = 0,82; n = 39). A highly significant relationship [linear correlation coefficient (r) = 0,91; number of observations (n) = 81] was found between the count of a Pseudomonas fluorescens strain and the resultant ATPase activity. This activity, however, did not appear to influence the ATP assay, the enzymes being produced in the late exponential phase and early stationary phase of growth. At this stage the psychrotrophic counts has reached a level of 1 × 108 CFU/ml which could result in off flavors in the milk. Researchers encountering high somatic cell counts in milk are advised to interpret the results of the ATP assay with care.

A numerical taxonomic study of Flavobacterium-Cytophaga strains from dairy sources.

Phenotypic data on 203 Gram-negative non-fermentative bacteria of the Flavobacterium-Cytophaga group isolated from milk and butter were analyzed by numerical taxonomic techniques. Twenty reference strains including species of Flavobacterium, Cytophaga and strains of Pseudomonas paucimobilis were included in the study. Using the matching coefficient of Sokal & Michener with antibiotic susceptibility data included, 189 isolates were recovered in nine clusters. Six of these clusters were linked at or above the 85% S level while three were linked at or above the 79% S level. The largest cluster, representing 46.3% of the isolates, could be equated with Flavobacterium sp. Group IIb. Other clusters could be equated with Flavobacterium sp. L 16/1 (22.7% of isolates), F. balustinum (10.8% of isolates), F. breve (4.4%), F. multivorum (3.5%) and Cytophaga johnsonae (1.5%). The cluster resembling Flavobacterium sp. L 16/1 and a smaller unclassified cluster, were exceptional in being susceptible to the antibiotics cephalothin and penicillin G.

Bacteriophage typing of Staphylococcus aureus strains isolated from Bloemfontein dairy herds.

Bacteriophage typing was performed on 88 coagulase positive Staphylococcus aureus strains isolated during a survey of subclinical mastitis in Bloemfontein dairy herds. Phage typing was performed using two basic international phage typing sets, i.e. the human isolate phage set (HPS) and the bovine isolate phage set (BPS). The results clearly indicated that the BPS could be successfully applied for the phage typing of bovine mastitis S. aureus strains. The majority of the strains was typed as BPS phage group IV (78,4%) and HPS group III (47,7%). The high prevalence of BPS group IV strains is in agreement with other studies. The prevalence of non-typable strains was 3,4% for BPS and 28,4% for HPS. Phages 102, 117, 107, 81, 47, and 6 had high lytic activity. BPS group IV patterns (102/107/117 and 102/117) dominated. The incidence of unique phage patterns was 12,5% for BPS and 26,0% for HPS. A relatively high proportion (71,3%) of the strains was typable with the HPS. As these strains were of possible human origin it indicated the possibility of mutual human-animal transfer of the pathogens. No relationship could be found between phage groups on the one hand and multiple antibiotic resistance on the other, and no phage groups dominated within herds.

Antibiotic susceptibility patterns of mastitis pathogens isolated from Bloemfontein dairy herds.

The antibiotic susceptibility patterns of bacteria associated with subclinical mastitis in Bloemfontein fresh milk dairy herds were determined. A total of 141 bacterial strains tested, consisted of Staphylococcus aureus (93 strains), coagulase negative staphylococci (17), streptococci (12), Corynebacterium bovis (8), Pseudomonas aeruginosa (7) and enterobacteria (4). Antibiotic susceptibility was determined qualitatively using the Kirby-Bauer disc diffusion method and quantitatively by determining the minimal inhibitory concentrations using the agar dilution method. The utilization of commercial intramammary antibiotic preparations on the dairy farms is also discussed. The Gram-positive bacteria were generally not exceptionally resistant to the antibiotics tested. S. aureus susceptibility figures for penicillin G were 66% and for methicillin (or cloxacillin) 100%. The coagulase negative staphylocci in contrast were relatively more resistant than the coagulase positive staphylococci. The enterobacteria and particularly the P. aeruginosa strains, were extremely resistant to all antibiotics tested. In the latter case even carbenicillin and gentamicin susceptibility figures were low. A general mastitis control programme is discussed.

Prevalence and types of bacteria associated with subclinical mastitis in Bloemfontein dairy herds.

Bacteria associated with subclinical mastitis were isolated from machine-milked dairy herds in the Bloemfontein area supplying fresh milk during the period July to December 1980. The 151 quarter milk samples examined, were also subjected to somatic cell counts. Identification of the isolated bacterial strains showed that Staphylococcus aureus was the dominant mastitis-associated organism, constituting 66,4% of all bacteria isolated. Compared with other recent mastitis surveys a low prevalence of classical mastitis streptococci (0,7%) and of Gram-negative bacterial infections (6,3%) was encountered. The Gram-negative bacteria were almost invariably isolated from neglected herds in which the cows were generally in poor condition and the hygienic measures employed were totally inadequate. Other bacterial strains isolated included Corynebacterium bovis (6,3%) and the coagulase negative staphylococci (11,0%). The high somatic cell counts of the quarter milk samples yielding S. aureus, the mastitis streptococci and the Gram-negative bacteria suggested a major pathogenic role for these isolates. The frequent occurrence of C. bovis strains and coagulase negative staphylococci in samples with high somatic cell counts similarly suggested that these organisms were more pathogenic than is generally assumed.

A multicentre study of the susceptibility of a variety of bacteria to cephalothin, cefamandole, tobramycin and gentamicin.

A multicentre study of antibiotic susceptibility was performed in South Africa. Sensitivity to cephalothin, cefamandole, tobramycin and gentamicin was tested on a variety of aerobic and anaerobic bacteria. Two disc susceptibility techniques were used, i.e. the Kirby-Bauer technique (aerobes) and the broth-disc method (anaerobes); minimum inhibitory concentrations (MICs) were determined according to the International Collaborative Study techniques, and regression lines for individual centres were constructed. Satisfactory lines were obtained for cephalosporins, but, in some centres, problems were experienced with the aminoglycosides. Variations in MICs for Haemophilus influenzae were probably due to an inoculum effect. Accumulative percentage tables of the number of strains inhibited were compiled, and the comparative performance of the antibiotics was assessed.