Extended-spectrum b-lactamase (ESBL) and AmpC b-lactamase-producing Enterobacteriaceae are no longer restricted to the health care system, but represent increased risks related to environmental integrity and food safety. Fresh produce has been increasingly reported to constitute a reservoir of multidrug-resistant (MDR) potential human pathogenic Enterobacteriaceae. This study aimed to detect, identify, and characterize the antimicrobial resistance of ESBL/AmpC-producing Enterobacteriaceae isolates from fresh vegetables at point of sale. Vegetable samples (spinach, tomatoes, lettuce, cucumber, and green beans; n=545) were purchased from retailers in Gauteng, the most densely populated province in South Africa. These included street vendors, trolley vendors, farmers' market stalls, and supermarket chain stores. Selective enrichment, plating onto chromogenic media, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) confirmation of isolate identities showed that 17.4% (95/545) vegetable samples analyzed were contaminated with presumptive ESBL/AmpC-producing Enterobacteriaceae. Dominant species identified included Escherichia coli, Enterobacter cloacae, Enterobacter asburiae, and Klebsiella pneumoniae. Phenotypic antibiotic resistance analysis showed that 96.1% of 77 selected isolates were MDR, while resistance to aminoglycoside (94.8%), chloramphenicol (85.7%), and tetracycline (53.2%) antibiotic classes was most prevalent. Positive phenotypic analysis for ESBL production was shown in 61 (79.2%) of the 77 isolates, and AmpC production in 41.6% of the isolates. PCR and sequencing confirmed the presence of b-lactamase genes in 75.3% isolates from all vegetable types analyzed, mainly in E. coli, Enterobacter spp., and Serratia spp. isolates. CTXM group 9 (32.8%) was the dominant ESBL type, while EBC (24.1%) was the most prevalent plasmidic type AmpC b-lactamase. Our findings document for the first time the presence of MDR ESBL/AmpC-producing Enterobacteriaceae in raw vegetables sold at selected retailers in Gauteng Province, South Africa.

The increasing occurrence of multidrug-resistant (MDR) extended-spectrum β-lactamase-(ESBL) and/or AmpC β-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

The internalisation potential of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7 and Salmonella enterica subsp. enterica serovar Typhimurium in lettuce was evaluated using seedlings grown in vermiculite in seedling trays as well as hydroponically grown lettuce. Sterile distilled water was spiked with one of the four human pathogenic bacteria (10(5) CFU/mL) and used to irrigate the plants. The potential for pathogen internalisation was investigated over time using light microscopy, transmission electron microscopy and viable plate counts. Additionally, the identities of the pathogens isolated from internal lettuce plant tissues were confirmed using polymerase chain reaction with pathogen-specific oligonucleotides. Internalisation of each of the human pathogens was evident in both lettuce seedlings and hydroponically grown mature lettuce plants. To our knowledge, this is the first report of S. aureus internalisation in lettuce plants. In addition, the levels of background microflora in the lettuce plants were determined by plate counting and the isolates identified using matrix-assisted laser ionisation-time of flight (MALDI-TOF). Background microflora assessments confirmed the absence of the four pathogens evaluated in this study. A low titre of previously described endophytes and soil inhabitants, i.e., Enterobacter cloacae, Enterococcus faecalis, Lysinibacillus fusiformis, Rhodococcus rhodochrous, Staphylococcus epidermidis and Staphylococcus hominis were identified.

Rain-, ground-and municipal potable water were stored in low density polyethylene storage tanks for a period of 90 days to determine the effects of long term storage on the deterioration in the microbial quality of the water. Total viable bacteria present in the stored water and the resultant biofilms, were enumerated using heterotrophic plate counts. PCR and Colilert-18® tests were performed to determine if the faecal indicator -2 -bacteria, Escherichia coli, might be present in the water and in the biofilm samples collected throughout the study. The municipal potable water at the start of the study was the only water source that conformed to the South African water quality guidelines for domestic use. After 15 days of storage, this water source had microbiologically deteriorated to levels considered unfit for human consumption. E. coli was detected in the ground-and potable-water and ground-and potable biofilms periodically; whereas, it was detected in the rain water and associated biofilms at every sampling point.Imperfections in the UV resistant inner lining of the tanks revealed to be ecological niches for microbial colonisation and biofilm development. The results from the current study confirmed that long term storage can influence water quality and increase the number of microbial cells associated with biofilms on the interior surfaces of water storage tanks.