Development and evaluation of a reporter system for prokaryotic cells based on a secreted acid phosphatase from Staphylococcus aureus strain 154

Reporter gene technology has facilitated greatly the analysis of gene expression and the study of individual promoters and their regulation. Although various reporter gene systems are available, none of them are universally applicable and consequently, studies aimed at screening of new reporters are continuing. Toward this end, an acid phosphatase, designated SapS, was identified and characterized from the culture supernatant of a Staphylococcus aureus strain isolated from vegetables. Biochemical characterization of the 30-kDa monomeric enzyme indicated that it displayed optimum activity at 40°C and pH 5, using p-nitrophenyl phosphate (pNPP) as substrate. The enzymatic activity was enhanced by Mg2+, but was inhibited by EDTA and molybdate. Based on its properties and amino acid sequence analyses, SapS was classified as a new member of the bacterial class C family of non-specific acid phosphatases. The S. aureus SapS enzyme was subsequently evaluated as a reporter for host strain evaluation and cell surface display. Bacillus halodurans of which the major cell wall protease gene (wprA) was inactivated was used as expression host, and the cell wall-binding domain of the cwlC gene from B. halodurans was used as an anchoring motif for cell surface display. The results from in vitro enzyme activity assays indicated that extracellular production of the SapS reporter enzyme was improved 3.5-fold in the mutant compared to wild-type B. halodurans strain. Zymographic detection of SapS activity showed that the SapS-CwlC fusion protein was localized in the B. halodurans cell wall fraction, thus demonstrating the potential of SapS as a reporter for cell surface display of heterologous proteins. The versatility of the SapS enzyme as a reporter for gene expression and protein secretion in both Gram-positive and Gram-negative bacteria was also investigated. Transcriptional and translational fusions of the sapS gene with selected heterologous promoters and signal sequences were constructed, and expressed in Escherichia coli, B. subtilis and B. halodurans. The strongest promoter for heterologous protein production in each of the host strains was identified, i.e. the E. coli lacZ promoter in E. coli, the B. halodurans alkaline protease promoter in B. subtilis, and the B. halodurans σD promoter in B. halodurans.Thesis (PhD)--University of Pretoria, 2010.Microbiology and Plant Pathologyunrestricte

Microbiological safety of spinach throughout commercial supply chains in Gauteng Province, South Africa and characterization of isolated multidrug-resistant Escherichia coli

Please read abstract in the article.Water Research Commission; South African Agency for Science and Technology Advancement; Partnerships for Enhanced Engagement in Research United States Agency for International Development; Department of Science and Innovation–National Research Foundation (NRF).http://www.wileyonlinelibrary.com/journal/jamhj2023Plant Production and Soil Scienc

Occurrence, phenotypic and molecular characterization of extended-spectrum- and AmpC-beta-lactamase producing enterobacteriaceae isolated from selected commercial spinach supply chains in South Africa

The increasing occurrence of multidrug-resistant (MDR) extended-spectrum b-lactamase- (ESBL) and/or AmpC b-lactamase-producing Enterobacteriaceae in health care systems, the environment and fresh produce is a serious concern globally. Production practices, processing and subsequent consumption of contaminated raw fruit and vegetables represent a possible human transmission route. The purpose of this study was to determine the presence of ESBL/AmpC-producing Enterobacteriaceae in complete spinach supply chains and to characterize the isolated strains phenotypically (antimicrobial resistance profiles) and genotypically (ESBL/AmpC genetic determinants, detection of class 1, 2, and 3 integrons). Water, soil, fresh produce, and contact surface samples (n = 288) from two commercial spinach production systems were screened for ESBL/AmpC-producing Enterobacteriaceae. In total, 14.58% (42/288) of the samples were found to be contaminated after selective enrichment, plating onto chromogenic media and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identity confirmation of presumptive ESBL/AmpC isolates. This included 15.28% (11/72) water and 12.12% (16/132) harvested- and processed spinach, while 25% (15/60) retail spinach samples were found to be contaminated with an increase in isolate abundance and diversity in both scenarios. Dominant species identified included Serratia fonticola (45.86%), Escherichia coli (20.83%), and Klebsiella pneumoniae (18.75%). In total, 48 (81.36%) isolates were phenotypically confirmed as ESBL/AmpC-producing Enterobacteriaceae of which 98% showed a MDR phenotype. Genotypic characterization (PCR of ESBL/AmpC resistance genes and integrons) further revealed the domination of the CTX-M Group 1 ESBL type, followed by TEM and SHV; whilst the CIT-type was the only plasmid-mediated AmpC genetic determinant detected. Integrons were detected in 79.17% (n = 38) of the confirmed ESBL/AmpC-producing isolates, of which we highlight the high prevalence of class 3 integrons, detected in 72.92% (n = 35) of the isolates, mostly in S. fonticola. Class 2 integrons were not detected in this study. This is the first report on the prevalence of ESBL/AmpC-producing Enterobacteriaceae isolated throughout commercial spinach production systems harboring class 1 and/or class 3 integrons in Gauteng Province, South Africa. The results add to the global knowledge base regarding the prevalence and characteristics of ESBL/AmpC-producing Enterobacteriaceae in fresh vegetables and the agricultural environment required for future risk analysis.The Department of Science and Technology–National Research, Foundation (NRF), Centre of Excellence in Food Security, the Water Research Commission (WRC) funded project “Measurement of water pollution determining the sources and changes of microbial contamination and impact on food safety from farming to retail level for fresh vegetables” (WRC Project No K5/2706/4, Water Research Commission Knowledge Review 2017/18) and the Partnerships for Enhanced Engagement in Research (PEER) a USAID/DST funded project Characterizing and tracking of antimicrobial resistance in the water-plant-food public health interface (Grant no. 48).http://www.frontiersin.org/Microbiologyam2020Plant Production and Soil Scienc

Characterization of multidrug-resistant Escherichia coli isolated from two commercial lettuce and spinach supply chains

Leafy green vegetables have increasingly been reported as a reservoir of multidrug-resistant pathogenic Enterobacteriaceae, with Shiga toxin–producing Escherichia coli frequently implicated in disease outbreaks worldwide. This study examined the presence and characteristics of antibiotic resistance, diarrheagenic virulence genes, and phylogenetic groupings of E. coli isolates (n = 51) from commercially produced lettuce and spinach from farms, through processing, and at the point of sale. Multidrug resistance was observed in 33 (64.7%) of the 51 E. coli isolates, with 35.7% (10 of 28) being generic and 100% (23 of 23) being extended-spectrum β-lactamase/AmpC producing. Resistance of E. coli isolates was observed against neomycin (51 of 51, 100%), ampicillin (36 of 51, 70.6%), amoxicillin (35 of 51, 68.6%), tetracycline (23 of 51, 45%), trimethoprim-sulfamethoxazole (22 of 51, 43%), chloramphenicol (13 of 51, 25.5%), Augmentin (6 of 51, 11.8%), and gentamicin (4 of 51, 7.8%), with 100% (51 of 51) susceptibility to imipenem. Virulence gene eae was detected in two E. coli isolates from irrigation water sources only, whereas none of the other virulence genes for which we tested were detected. Most of the E. coli strains belonged to phylogenetic group B2 (25.5%; n = 13), B1 (19.6%; n = 10), and A (17.6%; n = 9), with D (5.9%; n = 3) less distributed. Although diarrheagenic E. coli was not detected, antibiotic resistance in E. coli prevalent in the supply chain was evident. In addition, a clear link between E. coli isolates from irrigation water sources and leafy green vegetables through DNA fingerprinting was established, indicating the potential transfer of E. coli from irrigation water to minimally processed leafy green vegetables.The U.S. Agency for International Development (USAID) Partnerships for Enhanced Engagement in Research; the Water Research Commission (WRC) and the Department of Science and Innovation (DSI)–National Research Foundation (NRF) Centre of Excellence in Food Security under the Food Safety Programme.https://www.sciencedirect.com/journal/journal-of-food-protectionhj2023Plant Production and Soil Scienc

Profiling bacterial communities of irrigation water and leafy green vegetables produced by small-scale farms and sold in informal settlements in South Africa

AVAILABILITY OF DATA AND MATERIALS : Sequence data are available at NCBI-SRA under submission numbers SUB12270895 and SUB12272756 for BioProject number PRJNA900001.ADDITIONAL FILE 1: TABLE S1. Samples analysed for bacterial community characterisation. TABLE S2. Taxonomic breakdown of core bacterial taxa present in flooding irrigation water. TABLE S3. Bacterial families that are associated with isolation and outbreaks in South Africa.Morogo is an African indigenous term used for leafy green vegetables harvested in the wild or cultivated in small-scale farms and consumed by the local populations of the region. Small-scale farmers have gained recognition as important suppliers of morogo to informal settlements. In commercial production systems, leafy green vegetables have increasingly been reported as associated with foodborne pathogens and disease outbreaks. Little is known of the presence of these organisms on leafy green vegetables in the informal unregulated food systems. This study aimed to profile bacterial communities in irrigation water (flooding and overhead irrigation water) and leafy green vegetables (Brassica rapa L. chinensis and Brassica rapa varieties of morogo) to establish the natural bacterial flora at the water-fresh produce interface from five small-scale farms in two provinces in South Africa. Illumina MiSeq high-throughput sequencing showed that each farm exhibited a unique bacterial community composition, with an overall high relative abundance of Proteobacteria, Firmicutes and Actinobacteria, including prominent families such as Burkholderiaceae (48%), Enterobacteriaceae (34%), Bacillales Family XII (8%), Rhodobacteraceae (3%), Micrococcaceae (1.98%) and Pseudomonadaceae (1.79%). Specific Enterobacteriaceae Serratia, Enterobacter, Salmonella, Shigella, Escherichia coli, Buchnera, Citrobacter, Klebsiella and Proteus were identified, in addition to unique communities associated with plant or irrigation water source. These findings suggest that the edible plant microbiome can play an important role as transient contributor to the human gut and has the potential to affect overall health.The Water Research Commission (WRC) for the funded project “Measurement of water pollution determining the sources and changes of microbial contamination and impact on food safety from farming to retail level for fresh vegetables”, the Department of Science and Innovation (DSI)—National Research Foundation (NRF) Centre of Excellence in Food Security, the Partnerships for Enhanced Engagement in Research (PEER) USAID/DST funded project “Characterizing and tracking of antimicrobial resistance in the water-plant-food public health interface” and in part by the NRF of South Africa.https://cabiagbio.biomedcentral.comhj2023Plant Production and Soil Scienc

Antibiogram signatures of some enterobacteria recovered from irrigation water and agricultural soil in two district municipalities of South Africa

This study was undertaken to evaluate the antibiogram fingerprints of some Enterobacteria recovered from irrigation water and agricultural soil in two District Municipalities of the Eastern Cape Province, South Africa using standard culture-based and molecular methods. The prevalent resistance patterns in the isolates follow the order: Salmonella enterica serovar Typhimurium [tetracycline (92.3%), ampicillin (69.2%)]; Enterobacter cloacae [amoxicillin/clavulanic acid (77.6%), ampicillin (84.5%), cefuroxime (81.0%), nitrofurantoin (81%), and tetracycline (80.3%)]; Klebsiella pneumoniae [amoxicillin/clavulanic acid (80.6%), ampicillin (88.9%), and cefuroxime (61.1%)]; and Klebsiella oxytoca [chloramphenicol (52.4%), amoxicillin/clavulanic acid (61.9%), ampicillin (61.9%), and nitrofurantoin (61.9%)]. Antibiotic resistance genes detected include tetC (86%), sulII (86%), and blaAmpC (29%) in Salmonella enterica serovar Typhimurium., tetA (23%), tetB (23%), tetC (12%), sulI (54%), sulII (54%), catII (71%), blaAmpC (86%), blaTEM (43%), and blaPER (17%) in Enterobacter cloacae., tetA(20%), tetC (20%), tetD(10%), sulI (9%), sulII (18%), FOX (11%) and CIT (11%)-type plasmid-mediated AmpC, blaTEM (11%), and blaSHV (5%) in Klebsiella pneumoniae and blaAmpC (18%) in Klebsiella oxytoca. Our findings document the occurrence of some antibiotic-resistant Enterobacteria in irrigation water and agricultural soil in Amathole and Chris Hani District Municipalities, Eastern Cape Province of South Africa, thus serving as a potential threat to food safety.Table S1: Description of sampling points, Table S2: Primer sequence and PCR cycling conditions used for the molecular detection of members of Enterobacteriales, Table S3: The primer sequence and expected amplicon size used for the screening of resistance genes in members of Enterobacteriales, Table S4: The primer sequence and expected amplicon size used for the screening of AmpC -lactamase and ESBLs in members of Enterobacteriales [44].The South African Medical Research Council (SAMRC), United States Agency for International Development (USAID) and the National Research Foundation (NRF).http://www.mdpi.com/journal/microorganismsam2021Plant Production and Soil Scienc

The pandemic strain of Austropuccinia psidii causes myrtle rust in New Zealand and Singapore

The myrtle rust pathogen, Austropuccinia psidii, was recently detected in New Zealand and Singapore. We used microsatellite markers to identify the strain of A. psidii that caused these incursions. Our results show that the pandemic strain of the pathogen caused outbreaks in both New Zealand and Singapore.The Tree Protection Co-operative Programme (TPCP) and the National Research Foundation of South Africa (Grant specific unique reference numbers UID 78566 and UID 83924) and the DST-NRF Centre of Excellence in Tree Health Biotechnology (CTHB). ARM acknowledges the University of Queensland Development Fellowships (UQFEL1718905) and support from the Department of the Environment and Energy under the Australian Biological Resources Study (grant number RG18-43). WHH and BJRA acknowledge the support from the Ministry for Primary Industries (MPI) Plant Health and Environment Laboratory and the MPI Myrtle Rust Response Team.http://link.springer.com/journal/133132020-05-01hj2020BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant PathologyPlant Production and Soil Scienc

Exploratory study into the microbiological quality of spinach and cabbage purchased from street vendors and retailers in Johannesburg, South Africa

Knowledge of the microbiological quality, prevalence of antibiotic resistance and virulence genes in bacterial isolates from leafy green vegetables supplied by formal (retailers) and informal (street vendors) suppliers in South Africa is limited. Since leafy vegetables have been implicated in food borne disease outbreaks world-wide, a total of 180 cabbage and spinach samples were collected from 3 major retailers and 9 street vendors in Johannesburg, SA. Escherichia coli and coliforms were enumerated using 3M Petrifilm count plates. The prevalence of Listeria monocytogenes, Salmonella spp. and Shigella spp. were determined using real-time PCR analysis. Identities of presumptive E. coli isolates from the fresh produce were confirmed using MALDI-TOF MS. Isolates were characterized using phenotypic (antibiotic resistance) and genotypic (phylogenetic, virulence gene) analysis. Hygiene indicator bacteria levels on spinach from formal and informal retailers exceeded the maximum level specified by the Department of Health guidelines for fresh fruit and vegetables. Street vendor spinach E. coli counts were higher (p= < 0.0789) than retailer spinach counts. E. coli was present in 2 cabbage samples only at 0.0035 CFU/g. L. monocytogenes and Salmonella spp. were detected in 7.2% and 5% of the 180 samples respectively using real-time PCR analysis, while Shigella was not detected. Of the 29 spinach E. coli isolates 37.9% were multi-drug resistant. Virulence genes eae and stx1 were present in 14% and 3% of the spinach E. coli isolates respectively, while the stx2 gene was not detected. Eighty-six percent (86%) of these isolates belonged to phylogroup A, 3% to group C, 7% to group E and 3% to clade 1. The results from the current exploratory study on the microbiological quality of spinach bought from selected retailers highlighted the need for continued surveillance on a larger scale, especially in the informal sector, in order to characterize the potential risk to the consumer.The Department of Science and Technology/National Research Foundation Centre of Excellence in Food Security.http://www.foodprotection.orgpublications/jfp.asp2018-10-30hj2017Plant Production and Soil Scienc

Effect of postharvest practices on the culturable filamentous fungi and yeast microbiota associated wit the pear carpoplane

Information regarding the filamentous fungi and yeast microbiota on pear surfaces is limited when compared to other fruits such as grapes and apples. The effect of commercial postharvest practices on pear fruit surface microbiota and species composition is not known, particularly in terms of the presence of postharvest pathogens and potential biocontrol microorganisms. Pear fruit were collected at harvest in the orchards of four commercial farms, after harvest at a communal pack house following chlorine drenching and after modified atmosphere storage. Microbiological analysis showed that during season one the fungal populations on pears from the four farms were significantly lower after CA storage when compared to populations of orchard pears, however during season two, the opposite trend was observed. The yeast populations were either significantly higher or similar after CA storage compared to the orchard pear counts during both seasons. Commercial drenching led to either an increase or reduction in the filamentous fungi and yeast populations, however a definite trend could not be observed. The postharvest practices decreased the number of viable morphologically different yeast and filamentous fungal species. A total of 16 yeast and 24 filamentous fungal species were isolated. A 76% dominance of Ascomycetes was observed. Known postharvest pathogens Penicilium commune and Penicillium crysogenum were present after CA storage. Potential known biocontrol organisms included Aureobasidium pullulans, Cryptococcus sp. and Sporobolomyces roseus. Knowledge generated could contribute to development of commodity-specific supply-chain management systems and biocontrol strategies based on scientific data to reduce pear fruit losses and for quality control purposes.The National Research Foundation, the Department of Science and Technology Postharvest Innovation program and the University of Pretoria.http://www.elsevier.com/locate/postharvbio2017-08-30Plant Scienc

Prevalence of E. coli O157:H7 strains in irrigation water and agricultural soil in two district municipalities in South Africa

We assessed the prevalence of E. coli O157:H7 in irrigation water and agricultural soil samples in two District Municipalities in South Africa using standard culture-based and molecular techniques. Presumptive E. coli O157:H7 counts in irrigation water and agricultural soil samples ranged from 1.00 Log10 CFU/100 ml to 4.11 Log10 CFU/100 ml and 2.20 Log10 CFU/g to 4.21 Log10 CFU/g respectively (P ≤ 0.05). Thirteen (28%) of the confirmed isolates (n = 46) were Shiga toxigenic and 33 (72%) were Shiga toxin negative. The presence of Shiga toxigenic E. coli O157:H7 strain in irrigation water and agricultural soil samples reported by this study suggests potential risk to food safety and population health.The South African Medical Research Council, United States Agency for International Development and the Department of Science and Technology of South Africa.https://www.tandfonline.com/loi/genv20pm2021Plant Production and Soil Scienc