Conveyor belts are recognized as common niches for persistent Listeria monocytogenes contamination in food processing environments. This study evaluated the efficacy of 24 commercially available cleaning and disinfection (C&D) agents, UV-C irradiation, and heat treatments against L. monocytogenes, using a laboratory biofilm model on woven conveyor belt material. Selected treatments were further tested on a used conveyor belt from food industry harboring a persistent outbreak strain. Cleaning followed by disinfection, using concentrations recommended by manufacturers, achieved limited reductions (1.2-2.5 log) of L. monocytogenes after 10 min exposure in the laboratory biofilm model. Extreme concentrations of cleaning agents improved efficacy in the laboratory model but failed to eliminate L. monocytogenes from the used belt. All C&D agents applied at recommended concentrations led to >3.8 log reduction against a dried suspension of L. monocytogenes on stainless steel. UV-C treatment resulted in dose-dependent but modest reductions (<= 2.4 log) and the combination of C&D and UV-C improved efficacy in the laboratory model, but failed to eliminate L. monocytogenes from the used belt from food industry. Heat treatment in a water bath at 60 degrees C for 1 h completely eliminated L. monocytogenes (>4 log reduction, negative after enrichment) from both types of conveyor belts, whereas hot dry air was less effective. Extended enrichment times revealed prolonged lag phases of stressed cells, highlighting the risk of false-negative results when standard detection protocols are applied. Overall, the findings highlight the potential of hot water treatment as an effective strategy for eliminating L. monocytogenes from hard-to reach sites where chemical C&D and UV-C treatments are insufficient.

Listeria monocytogenes is pervasive in agricultural environments and difficult to eradicate from food-processing facilities. Consequently, various foods become contaminated, posing health risks to immunocompromised individuals. This surveillance study aimed to enhance the understanding of the genetic diversity, virulence, plasmid content, sanitizer tolerance, and antibiotic resistance of L. monocytogenes from ready-to-eat (RTE) hummus, fresh produce and food-processing environments in the Western Cape, South Africa (2018-2021). Sixty L. monocytogenes isolates were classified as lineage I or lineage II using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Lineage I was notably prevalent overall (57%; n = 34) and significantly associated with fresh produce (88%; n = 7) (p = 0.04). Whole-genome sequencing (WGS) and bioinformatic analysis characterized a subset of 20 L. monocytogenes isolates into seven sequence types (STs) (ST1, ST2, ST3, ST5, ST101, ST121, ST204), and three serotypes (1/2a, 1/2b, 4b). ST204 (38%; n = 3) was most prevalent in the food-processing environment, ST5 (50%; n = 3) in fresh produce, and ST5 (33%; n = 2), ST101 (33%; n = 2) and ST121 (33%; n = 2), in RTE hummus. However, no single serotype or ST was significantly overrepresented in any category (p > 0.05). All isolates carried both Listeria pathogenicity island (LIPI)-1 and LIPI-2 gene clusters. Two isolates (ST1 and ST3) contained all genes comprising LIPI-3. Two ST5 isolates from RTE hummus contained the tetM gene. Isolates from different origins contained the emrC, bcrABC or qacH genes conferring tolerance to benzalkonium chloride, a quaternary ammonium compound class of sanitizer. RTE hummus, fresh produce and the food-processing environment are susceptible to contamination by diverse and virulent L. monocytogenes strains.

Listeria monocytogenes is ubiquitous in nature and persistent in food-processing facilities, farms, retail stores, and home and restaurant kitchens. Current research suggests ready-to-eat (RTE) products (including RTE hummus and fresh produce) to be of increasing interest and concern. These foods are typically stored at refrigeration temperatures suited to the survival of L. monocytogenes and are consumed without further processing. Since L. monocytogenes is ubiquitous in agricultural environments, the cultivation of fresh produce predisposes it to contamination. The contamination of RTE foods originates either from raw ingredients or, more commonly, from cross-contamination within food-processing facilities. Research on the food-processing environment has been recommended to reduce the incidence of L. monocytogenes in foods. The consumption of contaminated foods by immunocompromised individuals causes invasive listeriosis, with a 20% to 30% fatality rate despite treatment. The emergence of antibiotic-resistant strains has reduced the effectiveness of modern medicine and may increase morbidity and mortality. Without epidemiological surveillance and identifying trends in disease determinants, no action can be taken to improve food safety and mitigate the risk of such outbreaks.

Food safety is always of paramount importance globally due to the devasting social and economic effects of foodborne disease outbreaks. There is a high consumption rate of meat worldwide, making it an essential protein source in the human diet, hence its microbial safety is of great importance. The food industry stakeholders are always in search of methods that ensure safe food whilst maintaining food quality and excellent sensory attributes. Currently, there are several methods used in microbial food analysis, however, these methods are often time-consuming and do not allow real-time analysis. Considering the recent technological breakthroughs in artificial intelligence and machine learning, it raises the question of whether these advancements could be leveraged within the meat industry to improve turnaround time for microbial assessments. Hyperspectral imaging (HSI) is a highly prospective technology worth exploring for microbial analysis. The rapid, non-destructive method has the potential to be integrated into food production systems and allows foodborne pathogen detection in food samples, thus saving time. Although there has been a substantial increase in research on the utilisation of HSI in food applications over the past years, its use in the microbial assessment of meat is not yet optimal. This review aims to provide a basic understanding of the visible-near infrared HSI system, recent applications in the microbial assessment of meat products, challenges, and possible future applications.

Listeria monocytogenes is a concern in seafood and its food processing environment (FPE). Several outbreaks globally have been linked to various types of seafood. Genetic profiling of L. monocytogenes is valuable to track bacterial contamination throughout the FPE and in understanding persistence mechanisms, with limited studies from South Africa. Forty-six L. monocytogenes isolates from origins: Fish/seafood products (n = 32) (salmon, smoked trout, fresh hake, oysters), the FPE (n = 6), and clinical (n = 8) were included in this study. Lineage typing, antibiotic susceptibility testing, and screening for two genes (bcrABC and emrC) conferring sanitizer tolerance was conducted. The seafood and FPE isolates originated from seven different factories processing various seafood products with undetermined origin. All clinical isolates were categorized as lineage I, and seafood and FPE isolates were mostly categorized into lineage II (p < 0.01). Seafood and FPE isolates (53%) carried the bcrABC gene cassette and one fish isolate, the emrC gene. A subset, n = 24, was grouped into serotypes, sequence types (STs), and clonal complexes (CCs) with whole genome sequencing (WGS). Eight CCs and ten STs were identified. All clinical isolates belonged to serogroup 4b, hypervirulent CC1. CC121 was the most prevalent in isolates from food and the FPE. All isolates carried Listeria pathogenicity islands (LIPI) 1 and 2. LIPI-3 and LIPI-4 were found in certain isolates. We identified genetic determinants linked to enhanced survival in the FPE, including stress survival islets (SSI) and genes conferring tolerance to sanitizers. SSI-1 was found in 44% isolates from seafood and the FPE. SSI-2 was found in all the ST121 seafood isolates. Isolates (42%) harbored transposon Tn1688_qac (ermC), conferring tolerance to quaternary ammonium compounds. Five plasmids were identified in 13 isolates from seafood and the FPE. This is the first One Health study reporting on L. monocytogenes genetic diversity, virulence and resistance profiles from various types of seafood and its FPE in South Africa.

Fish and seafood are rich sources of protein, vitamins, and minerals, significantly contributing to individual health. A global increase in consumption has been observed. Listeria monocytogenes is a known problem in food processing environments and is found in various seafood forms, including raw, smoked, salted, and ready-to-eat. Without heat treatment and given L. monocytogenes ' ability to multiply under refrigerated conditions, consuming seafood poses a substantial health hazard, particularly to immunocompromised individuals. Numerous global outbreaks of listeriosis have been linked to various fish products, underscoring the importance of studying L. monocytogenes . Different strains exhibit varying disease-causing abilities, making it crucial to understand and monitor the organism's virulence and resistance aspects for food safety. This paper aims to highlight the genetic diversity of L. monocytogenes found in fish products globally and to enhance understanding of contamination routes from raw fish to the final product.

Among the severe foodborne illnesses, listeriosis resulting from the pathogen Listeria monocytogenes exhibits one of the highest fatality rates. This study investigated the application of near infrared hyperspectral imaging (NIR-HSI) for the classification of three L. monocytogenes serotypes namely serotype 4b, 1/2a and 1/2c. The bacteria were cultured on Brain Heart Infusion agar, and NIR hyperspectral images were captured in the spectral range 900-2500 nm. Different pre-processing methods were applied to the raw spectra and principal component analysis was used for data exploration. Classification was achieved with partial least squares discriminant analysis (PLS-DA). The PLS-DA results revealed classification accuracies exceeding 80 % for all the bacterial serotypes for both training and test set data. Based on validation data, sensitivity values for L. monocytogenes serotype 4b, 1/2a and 1/2c were 0.69, 0.80 and 0.98, respectively when using full wavelength data. The reduced wavelength model had sensitivity values of 0.65, 0.85 and 0.98 for serotype 4b, 1/2a and 1/2c, respectively. The most relevant bands for serotype discrimination were identified to be around 1490 nm and 1580-1690 nm based on both principal component loadings and variable importance in projection scores. The outcomes of this study demonstrate the feasibility of utilizing NIR-HSI for detecting and classifying L. monocytogenes serotypes on growth media.

Carbapenem agents are regarded as last-resort antibiotics, however, bacterial resistance towards carbapenems has been reported in both clinical and agricultural settings worldwide. Carbapenem resistance, defined as the resistance of a bacteria towards one or more carbapenem drugs, can be mediated in either of, or a combination of, three mechanisms–although, the mechanism mediated through the production of carbapenemases (β-lactamases that are able to enzymatically degrade carbapenems) is of most significance. Of particular concern is the occurrence of carbapenemase producing Enterobacterales (CPE), with literature describing a dramatic increase in resistance globally. In South Africa, increases of carbapenemase activity occurring in Enterobacter species, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa have recently been reported. CPE can also be found in agricultural environments, as global studies have documented numerous instances of CPE presence in various animals such as pigs, cattle, seafood, horses and dogs. However, most reports of CPE occurrence in agricultural settings come from Northern America, Europe and some parts of Asia, where more extensive research has been conducted to understand the CPE phenomenon. In comparison to clinical data, there are limited studies investigating the spread of CPE in agricultural settings in Africa, highlighting the importance of monitoring CPE in livestock environments and the food chain. Further research is necessary to uncover the true extent of CPE dissemination in South Africa. This review will discuss the phenomenon of bacterial antibiotic resistance (ABR), the applications of the carbapenem drug and the occurrence of carbapenem resistance globally.

Near infrared (NIR) hyperspectral imaging and multivariate data analysis was evaluated for its potential to detect and classify Listeria species. Three Listeria species, namely L. monocytogenes (ATCC 23074), L. innocua (ATCC 33090) and L. ivanovii (ATCC 19119) were grown for single colonies on Brain Heart Infusion agar and imaged in the NIR range of 950–2500 nm. Principal component analysis (PCA) was used for data exploration and to establish pattern recognition. Images were pre-processed with standard normal variate correction and the Savitzky-Golay smoothing technique (third order polynomial with 15 points). Two approaches to data analysis, that is object-wise and pixel-wise analysis, were investigated for discriminant analysis. The PCA score plot showed slight separation between the three groups with L. monocytogenes and L. ivanovii grouping close together. It was possible to visualise separation along PC3 (5.64% sum of squares (SS)) and PC4 (3.44% SS). Based on the loadings, differences in bacteria were attributed to teichoic acids, protein, and carbohydrate composition in the bacterial cell wall within the wavelength range 1000–1900 nm. Using extracted spectral data from the hypercubes, partial least squares discriminant analysis was employed for further classification. Classification accuracies above 90% were achieved for L. monocytogenes, L. innocua and L. ivanovii . This was true for data analysed using both pixel-wise analysis and object-wise analysis. The results demonstrated that hyperspectral imaging has notable potential to classify bacteria within the Listeria genus. Nonetheless, in order to improve model efficiency, model optimisation and incorporation of more bacterial strains need to be investigated in further research. Keywords Agar , food safety , species , multivariate data analysis , near infrared hyperspectral imaging , partial least discriminant analysis , single colonies

Near infrared (NIR) hyperspectral imaging and multivariate data analysis was evaluated for its potential to detect and classify Listeria species. Three Listeria species, namely L. monocytogenes (ATCC 23074), L. innocua (ATCC 33090) and L. ivanovii (ATCC 19119) were grown for single colonies on Brain Heart Infusion agar and imaged in the NIR range of 950–2500 nm. Principal component analysis (PCA) was used for data exploration and to establish pattern recognition. Images were pre-processed with standard normal variate correction and the Savitzky-Golay smoothing technique (third order polynomial with 15 points). Two approaches to data analysis, that is object-wise and pixel-wise analysis, were investigated for discriminant analysis. The PCA score plot showed slight separation between the three groups with L. monocytogenes and L. ivanovii grouping close together. It was possible to visualise separation along PC3 (5.64% sum of squares (SS)) and PC4 (3.44% SS). Based on the loadings, differences in bacteria were attributed to teichoic acids, protein, and carbohydrate composition in the bacterial cell wall within the wavelength range 1000–1900 nm. Using extracted spectral data from the hypercubes, partial least squares discriminant analysis was employed for further classification. Classification accuracies above 90% were achieved for L. monocytogenes, L. innocua and L. ivanovii. This was true for data analysed using both pixel-wise analysis and object-wise analysis. The results demonstrated that hyperspectral imaging has notable potential to classify bacteria within the Listeria genus. Nonetheless, in order to improve model efficiency, model optimisation and incorporation of more bacterial strains need to be investigated in further research.

There have been numerous studies highlighting the efficacy of various bacteriophages (phages) and phage cocktails in the reduction of pathogens in food. Despite approval from legislative bodies permitting phage use in food processing environments, applied via spray or dip, there is still no information on which spray parameters should be used for successful implementation. The study here investigates phage survival diluted to 1% in distilled water (dH2O) and prepared bottled water (PBW), followed by a subsequent spray application through a fixed nozzle (530 μm) and strainer size (74 × 74 μm), with pressures of 3, 5, and 6 Bar. The survival of the phage was determined through sampling the outputs of the spray system and performing double agar overlay plaque assays. PBW decreased the phage concentration (p = 0.18) more than the dH2O (p = 0.73) prior to spray application. It was found that the PBW phage solution was less affected by the various spray parameters (p = 0.045) than the dH2O (p = 0.011). The study showed that unchlorinated water (dH2O), as well as a pressure of 3 Bar, had the highest output phage concentration through the nozzle and strainer, providing valuable information for industrial implementation.

The control of Salmonella in chicken processing plants is an ongoing challenge for many factories around the globe, especially with the increasing demand for poultry escalating processing throughputs. Foodborne outbreaks due to Salmonella still pose a prominent risk to public health. As chicken meat is a good reservoir for Salmonella, it is important for chicken processing plants to continuously optimize methods to reduce the incidence of Salmonella on their products. Current methods include the use of chemical antimicrobials such as chlorine-containing compounds and organic acids. However, these current methods are decreasing in popularity due to the rising rate of Salmonella resistance, coupled with the challenge of preserving the sensory properties of the meat, along with the increasing stringency of antimicrobial use. Bacteriophages are becoming more appealing to integrate into the large-scale hurdle concept. A few factors need to be considered for successful implementation, such as legislation, and application volumes and concentrations. Overall, bacteriophages show great potential because of their host specificity, guaranteeing an alternative outcome to the selective pressure for resistant traits placed by chemicals on whole microbial communities.

Food-borne disease outbreaks are common and offer valuable insights into the causes, impacts, and mechanisms underlying food pathogens. This also serves as a good foundation to validate the performance of current best practice control methods, for example antibiotics, that are used in the fight against food pathogens. Listeriosis outbreaks, caused by Listeria monocytogenes, is no exception. In 2018, South Africa experienced the largest global listeriosis outbreak recorded to date. However, despite the scale of this outbreak, information on the bacterium and its resistance towards antibiotics is still severely lacking. Furthermore, until now it remained to be determined whether L. monocytogenes antibiotic resistance patterns in South Africa mirror resistance patterns elsewhere in the world. The aim of this study was therefore to evaluate the efficacy of antibiotics that are currently used against L. monocytogenes. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disc diffusion method, L. monocytogenes isolates (n = 177) from diverse origins in the Western Cape, South Africa (clinical, food, and environment) were tested for susceptibility against five different antibiotics, namely ampicillin, erythromycin, chloramphenicol, gentamicin, and tetracycline. Isolates were collected over a period of two years (2017–2019). All isolates were susceptible to ampicillin, the currently recommended antibiotic, while a large number of isolates were resistant to chloramphenicol, erythromycin, and tetracycline. Also, patterns of resistance observed here are different to patterns observed elsewhere. The findings of this study demonstrate that it is imperative to continuously monitor the efficacy of currently recommended antibiotics, since resistance patterns can quickly develop when such antibiotics are overutilized, and secondly, that it is crucial to assess local antibiotic resistance patterns in conjunction with global patterns, since the latter is not necessarily generalizable to local scales.

ABSTRACT Listeria monocytogenes is a ubiquitous, intracellular foodborne pathogen that is responsible for invasive listeriosis. The ability of L. monocytogenes to cause disease has some correlation with the serotypes of a specific lineage group, making the identification of lineage groups important for epidemiological analysis. The development of typing methods to link the strains of L. monocytogenes to an outbreak of listeriosis would help minimize the spread of the disease. The aim of this study was to design a PCR-restriction fragment length polymorphism (RFLP) method to differentiate between the lineage groups of L. monocytogenes. PCR-amplified fragments of the hly gene for 12 serotypes of L. monocytogenes were sequenced, aligned, and analyzed with the BioEdit program, and single nucleotide polymorphisms (SNPs) within regions of this gene were identified. Because of the difficulty in acquiring a serotype 4ab reference strain, this serotype was not included in this study. We tested the specificity and accuracy of the PCR-RFLP method on these L. monocytogenes reference strains and validated the method with 172 L. monocytogenes strains recovered from humans, food, and the food processing environment in 2000 to 2002 and 2008 to 2010 from regions within South Africa. PCR-RFLP analysis applied in this study placed L. monocytogenes serotypes into one of three lineage groups based on the sequence differences and SNPs within each lineage group. The SNPs were conserved in a region where RFLP analysis could be applied for a distinction between L. monocytogenes lineage groups. HIGHLIGHTS

BACKGROUND There are no paediatric reports of invasive infection caused by carbapenem-resistant Enterobacteriaceae (CRE) from Africa. OBJECTIVES To document a series of cases of CRE infections at a tertiary children's hospital in Cape Town, South Africa, describing the clinical and microbiological findings in these children. METHODS A retrospective, descriptive study was completed using data from a series of children with invasive CRE infection between 2010 and 2015, sourced from their clinical notes and microbiology results. RESULTS The first of 10 invasive CRE infections during the study period occurred in November 2012. Nine CRE infections were caused by Klebsiella pneumoniae, and one by both K. pneumoniae and Escherichia coli. The median age was 25 months (interquartile range (IQR) 5 - 60). All 10 CRE infections were hospital acquired. The median length of hospitalisation before CRE infection was 28.5 days (IQR 20 - 44). Eight of the children were exposed to carbapenems during the 12-month period prior to invasive CRE infection. Six were treated with colistin and carbapenem combination therapy, of whom 2 died, including 1 of a non-CRE event. The other 4 children received colistin monotherapy. All these children died, including 2 from non-CRE events. CONCLUSIONS Children with invasive CRE infection and severe underlying disease must be treated with combination antibiotic therapy. Strict infection control practice and antibiotic stewardship are necessary to contain the spread of CRE and limit the number of new infections.

Background. During 2013, the haematology/oncology unit at a tertiary level paediatric hospital in South Africa experienced the emergence of infection with vancomycin-resistant Enterococcus (VRE). Objective. To describe the clinical and molecular aspects of the cases identified. Methods. VRE isolates identified from blood culture specimens processed at the National Health Laboratory Service were screened for the presence of the vancomycin resistance genes van A , B and C1, 2 and 3. Further characterisation of these isolates was carried out using pulsed-field gel electrophoresis (PGFE) and multilocus sequence typing (MLST). Clinical records of infected patients were reviewed to identify possible risk factors, while surveillance with rectal swabs was performed to identify VRE-colonised patients. Results. Four patients with haematological malignancies were identified with VRE bloodstream infections. Patients were immunocompromised at the time of the bloodstream infection (BSI), with receipt of vancomycin prior to VRE-BSI, and infections were treated with linezolid. Colonisation with VRE was found in 8 of 55 patients screened. Infected and colonised patients were isolated in the unit during their admission and strict contact precaution infection control practices were instituted. The van A gene was identified in all of the isolates but one. PFGE and MLST results showed a degree of genetic relatedness between certain isolates obtained from rectal swab and blood culture samples, suggesting possible patient-to-patient transmission or persistence of the isolates in the unit. Conclusion. Strict infection control practices are necessary to prevent infection and transmission of resistant organisms among vulnerable patients.

The bacterial pathogen Listeria monocytogenes is responsible for listeriosis, a food-borne disease, which may result in severe illness and possible death. Large outbreaks of listeriosis have been associated with food products including soft cheeses and ready to eat food products. Polymerase chain reaction (PCR) is a molecular identification method for food-borne pathogens; however, a drawback of this method is that false-positive or false-negative results may occur. To validate the accuracy of the PCR as a powerful molecular tool for pathogen detection, it is important that false-negative results be distinguishable from true-negative PCR results. The aim of this study was to design and include an internal amplification control (IAC) within the PCR to coamplify with L. monocytogenes in order to identify false-negative results of L. monocytogenes from ostrich meat and camembert cheese samples. The IAC had to be incorporated into the PCR without loss of specificity and sensitivity on the detection limit of L. monocytogenes and was developed and tested for use in a multiplex PCR detection system. A region of the pUC19 plasmid was selected as the IAC for this study. The optimal concentration at which pUC19 would coamplify with L. monocytogenes was determined to be 0.001 pg/µL. Following an enrichment procedure, the minimum number of organisms detected in a spiked food sample by the PCR was 8 CFU/mL L. monocytogenes; the same detection limit was attained when the pUC19 IAC was included in the PCR. An optimal pUC19 IAC concentration increased the reliability of the PCR for food diagnostic purposes.