Molecular detection and characterization of foodborne bacteria: Recent progresses and remaining challenges.

The global food demand is expected to increase in the coming years, along with challenges around climate change and food security. Concomitantly, food safety risks, particularly those related to bacterial pathogens, may also increase. Thus, the food sector needs to innovate to rise to this challenge. Here, we discuss recent advancements in molecular techniques that can be deployed within various foodborne bacteria surveillance systems across food settings. To start with, we provide updates on nucleic acid-based detection, with a focus on polymerase chain reaction (PCR)-based technologies and loop-mediated isothermal amplification (LAMP). These include descriptions of novel genetic markers for several foodborne bacteria and progresses in multiplex PCR and droplet digital PCR. The next section provides an overview of the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins systems, such as CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13a, as tools for enhanced sensitive and specific detection of foodborne pathogens. The final section describes utilizations of whole genome sequencing for accurate characterization of foodborne bacteria, ranging from epidemiological surveillance to model-based predictions of bacterial phenotypic traits through genome-wide association studies or machine learning.

Manuka Oil vs. Rosemary Oil: Antimicrobial Efficacies in Wagyu and Commercial Beef against Selected Pathogenic Microbes.

Essential oils possessing antimicrobial characteristics have acquired considerable interest as an alternative to chemical preservatives in food products. This research hypothesizes that manuka (MO) and kanuka (KO) oils may possess antimicrobial characteristics and have the potential to be used as natural preservatives for food applications. Initial experimentation was conducted to characterize MOs (with 5, 25, and 40% triketone contents), rosemary oil (RO) along with kanuka oil (KO) for their antibacterial efficacy against selected Gram-negative (Salmonella spp. and Escherichia coli), and Gram-positive (Listeria monocytogenes and Staphylococcus aureus) bacteria through disc diffusion and broth dilution assays. All MOs showed a higher antimicrobial effect against L. monocytogenes and S. aureus with a minimum inhibitory concentration below 0.04%, compared with KO (0.63%) and RO (2.5%). In chemical composition, α-pinene in KO, 1, 8 cineole in RO, calamenene, and leptospermone in MO were the major compounds, confirmed through Gas-chromatography-mass spectrometry analysis. Further, the antimicrobial effect of MO and RO in vacuum-packed beef pastes prepared from New Zealand commercial breed (3% fat) and wagyu (12% fat) beef tenderloins during 16 days of refrigerated storage was compared with sodium nitrate (SN) and control (without added oil). In both meat types, compared with the SN-treated and control samples, lower growth of L. monocytogenes and S. aureus in MO- and RO- treated samples was observed. However, for Salmonella and E. coli, RO treatment inhibited microbial growth most effectively. The results suggest the potential use of MO as a partial replacement for synthetic preservatives like sodium nitrate in meats, especially against L. monocytogenes and S. aureus.

Non-Targeted Metabolomic Profiling Identifies Metabolites with Potential Antimicrobial Activity from an Anaerobic Bacterium Closely Related to Terrisporobacter Species.

This work focused on the metabolomic profiling of the conditioned medium (FS03CM) produced by an anaerobic bacterium closely related to Terrisporobacter spp. to identify potential antimicrobial metabolites. The metabolome of the conditioned medium was profiled by two-channel Chemical Isotope Labelling (CIL) LC-MS. The detected metabolites were identified or matched by conducting a library search using different confidence levels. Forty-eight significantly changed metabolites were identified with high confidence after the growth of isolate FS03 in cooked meat glucose starch (CMGS) medium. Some of the secondary metabolites identified with known antimicrobial activities were 4-hydroxyphenyllactate, 3-hydroxyphenylacetic acid, acetic acid, isobutyric acid, valeric acid, and tryptamine. Our findings revealed the presence of different secondary metabolites with previously reported antimicrobial activities and suggested the capability of producing antimicrobial metabolites by the anaerobic bacterium FS03.

Multitarget preservation technologies for chemical-free sustainable meat processing.

Due to the growing consumer demand for safe and naturally processed meats, the meat industry is seeking novel methods to produce safe-to-consume meat products without affecting their sensory appeal. The green technologies can maintain the sensory and nutritive characteristics and ensure the microbial safety of processed meats and, therefore, can help to reduce the use of chemical preservatives in meat products. The use of chemical additives, especially nitrites in processed meat products, has become controversial because they may form carcinogenic N-nitrosamines, a few of which are suspected as cancer precursors. Thus, the objective of reducing or eliminating nitrite is of great interest to meat researchers and industries. This review, for the first time, discusses the influence of processing technologies such as microwave, irradiation, high-pressure thermal processing (HPTP) and multitarget preservation technology on the quality characteristics of processed meats, with a focus on their sensory quality. These emerging technologies can help in the alleviation of ingoing nitrite or formed nitrosamine contents in meat products. The multitarget preservation technology is an innovative way to enhance the shelf life of meat products through the combined use of different technologies/natural additives. The challenges and opportunities associated with the use of these technologies for processing meat are also reviewed.

Antibacterial efficacy and possible mechanism of action of 2-hydroxyisocaproic acid (HICA).

The exploitation of natural antimicrobial compounds that can be used in food preservation has been fast tracked by the development of antimicrobial resistance to existing antimicrobials and the increasing consumer demand for natural food preservatives. 2-hydroxyisocaproic acid (HICA) is a natural compound produced through the leucine degradation pathway and is produced in humans and by certain microorganisms such as lactic acid bacteria and Clostridium species. The present study investigated the antibacterial efficacy of HICA against some important bacteria associated with food quality and safety and provided some insights into its possible antimicrobial mechanisms against bacteria. The results revealed that HICA was effective in inhibiting the growth of tested Gram-positive and Gram-negative bacteria including a multi-drug resistant P. aeruginosa strain in this study. The underlying mechanism was investigated by measuring the cell membrane integrity, membrane permeability, membrane depolarisation, and morphological and ultrastructural changes after HICA treatment in bacterial cells. The evidence supports that HICA exerts its activity via penetration of the bacterial cell membranes, thereby causing depolarisation, rupture of membranes, subsequent leakage of cellular contents and cell death. The current study suggests that HICA has potential to be used as an antibacterial agent against food spoilage and food-borne pathogenic bacteria, targeting the bacterial cell envelope.

Draft Genome Sequence of Thermoactinomyces vulgaris Strain AGRTWHS02, Isolated from Pasture Soil of a Sheep Dairy Farm in New Zealand.

Thermoactinomyces species are heat-resistant spore-forming bacteria that are capable of producing proteases. Here, we report the draft genome sequence of a new Thermoactinomyces vulgaris strain, AGRTWHS02, with a strong proteolytic activity, which was isolated from a sheep dairy farm environment in New Zealand. The genome is 2.56 Mbp, with a GC content of 47.9%.

An investigation into the anaerobic spoilage microbiota of beef carcass and rump steak cuts using high- throughput sequencing.

The presence of anaerobic microflora on fresh beef carcass and rump steaks, which may contribute to meat spoilage, was explored in this study. A total of 120 carcass and 120 rump steak swabs were collected immediately after slaughtering and boning, respectively from five meat plants, anaerobically incubated and enriched at 4°C for 3 weeks. This was followed by DNA extraction and 16S rRNA amplicon sequencing using the Illumina MiSeq, with subsequent bioinformatics analysis. The enriched microbiota of the samples was classified and grouped into 149 operational taxonomic units (OTUs). The microbiota recovered from both sample types consisted mainly of Carnobacterium, with an average relative abundance of 28.4% and 32.8% in beef carcasses and beef rump steaks, respectively. This was followed by Streptococcus, Serratia, Lactococcus, Enterococcus, Escherichia-Shigella, Raoultella and Aeromonas ranging from 1.5 to 20% and 0.1 to 29.8% in enriched carcasses and rump steak swabs, respectively. Trichococcus, Bacteroides, Dysgomonas, Providencia, Paraclostridium and Proteus were also present ranging from 0 to 0.8% on carcass and 0 to 1.8% on rump steak swabs, respectively. Alpha and beta diversity measurements showed limited diversity between the two sample types, but some differences between samples from the beef plants investigated were evident. This study highlights the presence of potential spoilage bacteria, mainly anaerobic genera on and between carcass and rump steaks, as an indication of contamination on and between these samples.

Applications of novel processing technologies to enhance the safety and bioactivity of milk.

Bioactive compounds in food can have high impacts on human health, such as antioxidant, antithrombotic, antitumor, and anti-inflammatory activities. However, many of them are sensitive to thermal treatments incurred during processing, which can reduce their availability and activity. Milk, including ovine, caprine, bovine, and human is a rich source of bioactive compounds, including immunoglobulins, vitamins, and amino acids. However, processing by various novel thermal and non-thermal technologies has different levels of impacts on these compounds, according to the studies reported in the literature, predominantly in the last 10 years. The reported effect of these technologies either covers microbial inactivation or the bioactive composition; however, there is a lack of comprehensive compilation of studies that compare the effect of these technologies on bioactive compounds in milk (especially, caprine and ovine) to microbial inactivation at similar settings. This research gap makes it challenging to conclude on the specific processing parameters that could be optimized to achieve targets of microbial safety and nutritional quality at the same time. This review covers the effect of a wide range of thermal and non-thermal processing technologies including high-pressure processing, pressure-assisted thermal sterilization, pulsed-electric field treatment, cold plasma, microwave-assisted thermal sterilization, ultra-high-pressure homogenization, ultrasonication, irradiation on the bioactive compounds as well as on microbial inactivation in milk. Although a combination of more than one technology could improve the reduction of bacterial contaminants to meet the required food safety standards and retain bioactive compounds, there is still scope for research on these hurdle approaches to simultaneously achieve food safety and bioactivity targets.

An investigation of the environmental niches of blown pack spoilage causing Clostridium estertheticum and Clostridium gasigenes on New Zealand beef and sheep farms.

The transfer of blown pack spoilage causing Clostridium spores from the farm to the meat plant is of growing concern to the meat industry. This study investigated the environmental niches of these Clostridium spp., specifically Clostridium estertheticum and Clostridium gasigenes in the beef and sheep farm environments in New Zealand. Faecal, soil, grass, drinking water, puddle water and feed (fodder beet, hay, bailage and silage, where available) samples were collected on five beef and sheep farms during Winter and Spring in 2018, in North and South Island, respectively. Beef and sheep farm samples were tested for C. estertheticum and C. gasigenes using enrichment plus PCR, qPCR and direct plating. C. estertheticum was detected in bovine faecal (4%), soil (2-18%) and grass (0-12%) samples at concentration of up to 2.0 log10 cfu/g. C. gasigenes were found in 18-46% of faecal, 16-82% of soil, 12-44% of grass, 0-44.4% of drinking water and 0-58.3% of puddle water samples tested and the direct counts ranged from 2.4 log10 cfu/ml in puddle water to 3.4 log10 cfu/g in soil. C. estertheticum were detected by qPCR in sheep farms in ovine feces (2.3%), soil (2.3%) and fodder beet (10%). All other sample types (grass, drinking water, puddle water, baleage, hay, silage and fodder beet) were negative using direct and enrichment plus PCR methods. In contrast C. gasigenes was detected in of faecal (22.7-38.6%), soil (22.7-84.1%), grass (17.5-34.1%) drinking water (35.7-78.6%), puddle water (33.3-40%), hay baleage (57%), silage (2%) and fodder beet (10%) at concentrations of up to 3.7 log10 cfu/g/ml. It was concluded that C. estertheticum and C. gasigenes were common on beef and sheep farms with the latter having higher incidence and mean concentration.

Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections.

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

Draft Genome Sequence of Pseudomonas saudiphocaensis Strain AGROB56, Isolated from a Dairy Farm in New Zealand.

Here, we report the draft genome sequence of a new Pseudomonas saudiphocaensis strain, AGROB56, with lipolytic potential, isolated from a sheep dairy farm in New Zealand. The genome is 3.61 Mbp, with a GC content of 61.1%, and the genome sequence was found closely related to Pseudomonas saudiphocaensis 20 BNT.

The survival of blown pack spoilage associated Clostridium estertheticum and Clostridium gasigenes spores during the ensiling of grass.

Blown pack spoilage (BPS) of vacuum packaged primals, caused by Clostridium estertheticum and Clostridium gasigenes, is a serious issue for the beef industry. There are multiple sources of these bacteria on beef farms, including grass and associated feed preparations. The aim of this study was to investigate the survival of C. estertheticum and C. gasigenes spores during the ensiling of grass and the subsequent opening of the silos. Grass, harvested from fields, with and without cattle slurry amendment, was inoculated with approximately 100 spores/g and ensiled using a laboratory (silo) model system at 20°C in the dark. Adding formic acid or sucrose resulted in six treatment combination as follows: no slurry (NS), no slurry plus formic acid (NSFA), no slurry plus sucrose (NSS), slurry (S), slurry plus formic acid (SFA) and slurry plus sucrose (SS). During the silage fermentation, samples were removed periodically and tested for C. estertheticum, C. gasigenes, total viable, Escherichia coli, Enterobacteriaceae and lactic acid bacteria (LAB) counts. The pH, ethanol, volatile fatty acids (VFA), lactic acid and ammonia concentrations were also monitored throughout the experiment. C. estertheticum did not survive the ensiling process, regardless of treatment. In contrast, C. gasigenes grew in the early stages and was detected during the entirety of the fermentation for all treatments. Based on these observations, it was concluded that the silage fermentation process described would not remove C. gasigenes and contaminated grass may result in contaminated feed for animals.

Draft Genome Sequence of Clostridium cadaveris Strain AGRFS2.2, Isolated from a Bovine Dairy Farm in New Zealand.

We report the draft genome sequence of a new Clostridium cadaveris strain, AGRFS2.2, isolated from soil in a bovine dairy farm environment in New Zealand. The genome is 3.6 Mbp long with a GC content of 31.3%. The genome sequence was found to be closely related to that of Clostridium cadaveris JCM 1392T.

Draft Genome Sequence of Pseudomonas nitrititolerans Strain AGROB37, Isolated from a Sheep Dairy Farm in New Zealand.

We report the draft genome sequence of a new Pseudomonas nitrititolerans strain, AGROB37, isolated from a sheep dairy farm environment in New Zealand. The genome is 4.19 Mbp long, with a GC content of 63.2%. The genome sequence was found to be closely related to that of the type strain Pseudomonas nitrititolerans GL14.

Draft Genome Sequence of Clostridium cochlearium Strain AGROS13, Isolated from a Sheep Dairy Farm in New Zealand.

We report the draft genome sequence of a new Clostridium cochlearium strain, AGROS13, which was isolated from a sheep dairy farm environment in New Zealand. The genome is 2.7 Mbp, with a GC content of 28.2%. The genome sequence was found to be closely related to that of Clostridium cochlearium ATCC 17787. The new strain harbors a biosynthetic gene cluster coding for an unknown sactipeptide.

Draft Genome Sequence of Clostridium senegalense Strain AGRFS4, Isolated from a Dairy Farm in New Zealand.

We report the draft genome sequence of a new Clostridium senegalense strain, AGRFS4, which was isolated from a dairy farm environment in the Manawatu region in New Zealand. The genome is 3.98 Mb, with a GC content of 27%. The genome sequence was found to be 86.6% similar to that of the type strain Clostridium senegalense JC122. Until now, no Clostridium senegalense strain from New Zealand has been reported.

Farm level survey of spore-forming bacteria on four dairy farms in the Waikato region of New Zealand.

The aim of our study was to determine the occurrence and diversity of economically important spore-forming bacteria in New Zealand dairy farm systems. Farm dairy effluent (FDE) collected from Waikato dairy farms were tested for the presence of spore-forming bacteria, using a new culture-based methodology followed by genomic analysis. An enrichment step in which samples were inoculated in cooked meat glucose starch broth under anaerobic conditions, aided in the differential isolation of Bacillus and Clostridium species. Furthermore, the use of molecular methods such as ERIC genotyping, 16S rRNA gene sequence analysis identified different spore-forming bacteria present in FDE. C. sporogenes signature PCR gave further information on the phylogenetic relationship of the different Clostridium spp. isolated in this study. In total 19 Bacillus spp., 5 Paenibacillus spp. and 17 Clostridium spp. were isolated from farm dairy effluent. Sequence types similar to economically important food spoilage bacteria viz: C. butyricum, C. sporogenes and members of the Paenibacillus Genus were isolated from all four farms, whereas, sequence types similar to potential toxigenic, B. cereus, C. perfringens, C. butyricum, and C. botulinum were found on at least three of the farms. Sampling of farm dairy effluent provides a good indicator of farm level prevalence of bacterial load as it is used to irrigate dairy pasture in New Zealand. This study highlights the presence of various spore-forming bacteria in dairy waste water and indicates the implementation of good hygienic farm practices and dairy waste effluent management.