Hermetia illucens L. larvae–associated intestinal microbes reduce the transmission risk of zoonotic pathogens in pig manure

Black soldier fly (BSF) larvae are considered a promising biological reactor to convert organic waste and reduce the impact of zoonotic pathogens on the environment. We analysed the effects of BSF larvae on Staphylococcus aureus and Salmonella spp. populations in pig manure (PM), which showed that BSF larvae can significantly reduce the counts of the associated S. aureus and Salmonella spp. Then, using a sterile BSF larval system, we validated the function of BSF larval intestinal microbiota in vivo to suppress pathogens, and lastly, we isolated eight bacterial strains from the BSF larval gut that inhibit S. aureus. Results indicated that functional microbes are essential for BSF larvae to antagonise S. aureus. Moreover, the analysis results of the relationship between the intestinal microbiota and S. aureus and Salmonella spp. showed that Myroides, Tissierella, Oblitimonas, Paenalcalignes, Terrisporobacter, Clostridium, Fastidiosipila, Pseudomonas, Ignatzschineria, Savagea, Moheibacter and Sphingobacterium were negatively correlated with S. aureus and Salmonella. Overall, these results suggested that the potential ability of BSF larvae to inhibit S. aureus and Salmonella spp. present in PM is accomplished primarily by gut-associated microorganisms.     

The Intestinal Bacterial Community in the Food Waste-Reducing Larvae of <Emphasis Type="Italic">Hermetia illucens</Emphasis>

As it is known that food waste can be reduced by the larvae of Hermetia illucens (Black soldier fly, BSF), the scientific and commercial value of BSF larvae has increased recently. We hypothesised that the ability of catabolic degradation by BSF larvae might be due to intestinal microorganisms. Herein, we analysed the bacterial communities in the gut of BSF larvae by pyrosequencing of extracting intestinal metagenomic DNA from larvae that had been fed three different diets. The 16S rRNA sequencing results produced 9737, 9723 and 5985 PCR products from larval samples fed food waste, cooked rice and calf forage, respectively. A BLAST search using the EzTaxon program showed that the bacterial community in the gut of larvae fed three different diets was mainly composed of the four phyla with dissimilar proportions. Although the composition of the bacterial communities depended on the different nutrient sources, the identified bacterial strains in the gut of BSF larvae represented unique bacterial species that were unlike the intestinal microflora of other insects. Thus, our study analysed the structure of the bacterial communities in the gut of BSF larvae after three different feedings and assessed the application of particular bacteria for the efficient degradation of organic compounds.     

Development of black soldier fly (Diptera: Stratiomyidae) larvae fed dairy manure

Black soldier flies, Hermetia illucens L., are a common colonizer of animal wastes. However, all published development data for this species are from studies using artificial diets. This study represents the first examining black soldier fly development on animal wastes. Additionally, this study examined the ability of black soldier fly larvae to reduce dry matter and associated nutrients in manure. Black soldier fly larvae were fed four rates of dairy manure to determine their effects on larval and adult life history traits. Feed rate affected larval and adult development. Those fed less ration daily weighed less than those fed a greater ration. Additionally, larvae provided the least amount of dairy manure took longer to develop to the prepupal stage; however, they needed less time to reach the adult stage. Adults resulting from larvae provided 27 g dairy manure/d lived 3-4 d less than those fed 70 g dairy manure. Percentage survivorship to the prepupal or adult stages did not differ across treatments. Larvae fed 27 g dairy manure daily reduced manure dry matter mass by 58%, whereas those fed 70 g daily reduced dry matter 33%. Black soldier fly larvae were able to reduce available P by 61-70% and N by 30-50% across treatments. Based on results from this study, the black soldier fly could be used to reduce wastes and associated nutrients in confined bovine facilities.     

Variation in Resistance to Hydrostatic Pressure among Strains of Food-Borne Pathogens

Among food-borne pathogens, some strains could be resistant to hydrostatic pressure treatment. This information is necessary to establish processing parameters to ensure safety of pressure-pasteurized foods (N. Kalchayanand, A. Sikes, C. P. Dunne, and B. Ray, J. Food Prot. 61:425–431, 1998). We studied variation in pressure resistance among strains of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella species at two temperatures of pressurization. Early-stationary-phase cells in 1% peptone solution were pressurized at 345 MPa either for 5 min at 25°C or for 5, 10, or 15 min at 50°C. The viability loss (in log cycles) following pressurization at 25°C ranged from 0.9 to 3.5 among nine L. monocytogenes strains, 0.7 to 7.8 among seven S. aureus strains, 2.8 to 5.6 among six E. coli O157:H7 strains, and 5.5 to 8.3 among six Salmonella strains. The results show that at 25°C some strains of each species are more resistant to pressure than the others. However, when one resistant and one sensitive strain from each species were pressurized at 345 MPa and 50°C, the population of all except the resistant S. aureus strain was reduced by more than 8 log cycles within 5 min. Viability loss of the resistant S. aureus strain was 6.3 log cycles even after 15 min of pressurization. This shows that strains of food-borne pathogens differ in resistance to hydrostatic pressure (345 MPa) at 25°C, but this difference is greatly reduced at 50°C. Pressurization at 50°C, in place of 25°C, will ensure greater safety of foods.     

Persistence of Mycobacterium avium subsp. paratuberculosis and Other Zoonotic Pathogens during Simulated Composting, Manure Packing, and Liquid Storage of Dairy Manure

Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis (Mycobacterium paratuberculosis). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated M. paratuberculosis, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55°C, manure packing at 25°C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with M. paratuberculosis at 10⁶ CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of M. paratuberculosis was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO₂, while no significant change in carbon content was observed in the liquid slurry. Escherichia coli, Salmonella, and Listeria were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55°C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days. M. paratuberculosis was detected by standard culture only on day 0 in all the treatments, but was undetectable in any treatment at 3 and 7 days. On days 14, 28, and 56, M. paratuberculosis was detected in the liquid storage treatment but remained undetectable in the compost and pack treatments. However, M. paratuberculosis DNA was detectable through day 56 in all treatments and up to day 175 in liquid storage treatments. Taken together, the results indicate that high-temperature composting is more effective than pack storage or liquid storage of manure in reducing these pathogens in dairy manure. Therefore, thermophilic composting is recommended for treatment of manures destined for pathogen-sensitive environments such as those for vegetable production, residential gardening, or application to rapidly draining fields.     

Dynamics of the intestinal bacterial community in black soldier fly larval guts and its influence on insect growth and development

Black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is a prominent insect for the bioconversion of various organic wastes. As a saprotrophic insect, the BSF inhabits microbe-rich environments. However, the influences of the intestinal microorganisms on BSF growth and development are not very clear. In this study, the dynamics of the intestinal bacterial community of BSF larvae (BSFL) were analyzed using pyrosequencing. Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were the most prevalent bacterial phyla in the intestines of all larval instars. The dynamic changes in bacterial community compositions among different larval instars were striking at the genus level. Klebsiella, Clostridium, Providencia, and Dysgonomonas were the relatively most abundant bacteria in the 1st- to 4th-instar BSFL, respectively. Dysgonomonas and Providencia also dominated the 5th- and 6th-instar larvae, at ratios of 31.1% and 47.2%, respectively. In total, 148 bacterial strains affiliated with 20 genera were isolated on different media under aerobic and anaerobic conditions. Among them, 6 bacteria, BSF1–BSF6, were selected for further study. The inoculation of the 6 isolates independently into germ-free BSFL feeding on an artificial diet showed that all the bacteria, except BSF4, significantly promoted BSF growth and development compared with the germ-free control. Citrobacter, Dysgonomonas, Klebsiella, Ochrobactrum, and Providencia promoted BSF development significantly by increasing the weight gains of larvae and pupae, as well as increasing the prepupae and eclosion rates. In addition, Citrobacter, Klebsiella and Providencia shortened the BSF life cycle significantly. The results illustrate the promotive effects of intestinal bacteria on BSF growth and development.     

Inoculating poultry manure with companion bacteria influences growth and development of black soldier fly (Diptera: Stratiomyidae) larvae

The growth and development of black soldier fly, Hermetia illucens (L.), larvae fed chicken manure inoculated with bacteria isolated from black soldier fly larvae and associated larval feed was evaluated. Four strains of Bacillus subtilis were evaluated. B. subtilis strains S15, S16, S19, were isolated from the gut of black soldier fly larvae. B. natto strain D1 was isolated from the diet fed to black soldier fly larvae. These bacteria were added individually into nonsterile 200 g fresh hen manure at 10(6) cfu/g and homogenized. Treated manure was then inoculated with 4-d old black soldier fly larvae. Prepupal weight ranged from 0.0606 g in the control to 0.0946 g in manure treated with the S15 strain. Larval survivorship to the prepupal stage in all treatments ranged from 98.00 ± 2.65% to 99.33 ± 1.15%. Prepupal survivorship to the pupal stage ranged from 91.92 ± 1.87% to 97.95 ± 1.03%. Adult emergence from the pupal stage did not significantly (P < 0.05) differ across treatments and ranged from 98.95 ± 1.82% to 100.00 ± 0.00%. Adult body length resulting from the larvae in each of the treatments was significantly greater than those from the control. Longevity of adults did not differ significantly between treatments. Time from hatching to the development of the first pupa did not differ significantly across treatments; however, development time from hatching to 90% reaching the prepupual stage was significantly different between treatments and ranged from 29.00 ± 1.00 d to 34.33 ± 3.51 d. Development time from hatching to 90% reaching the adult stages was significantly different between treatments. Our results demonstrate that inoculating poultry manure with bacteria from black soldier fly larvae influences the growth and development of conspecific larvae feeding on the manure.     

肠道共生细菌预发酵鸡粪对黑水虻生长发育的影响

以利用黑水虻Hermetia illucens L.肠道共生细菌不同菌株培养液预发酵的鸡粪为培养料,研究了添加不同菌株的发酵鸡粪对黑水虻幼虫生长发育的影响。结果表明,添加不同菌株的预发酵鸡粪能显著增加预蛹和蛹的重量,显著提高化蛹率并增加雌、雄成虫体长,缩短预蛹所需时间,但对幼虫存活率、成虫羽化率、成虫寿命没有显著影响。同时,不同菌株的作用效果有所不同。     

武汉亮斑扁角水虻幼虫肠道微生物对其成虫产卵行为的影响

研究武汉亮斑扁角水虻幼虫肠道微生物对武汉亮斑水虻成虫产卵行为的影响。首先使用LB(Luria-Bertani)培养基对武汉亮斑水虻幼虫肠道微生物进行分离纯培养,筛选得到23株肠道微生物。以灭菌的LB液体培养基为阴性对照,使用已证实引诱产卵效果好的来自武汉亮斑水虻卵表的属于戈登氏菌属的细菌(Gordonia sihwensis)FE06为阳性对照,利用筛选到的武汉亮斑水虻幼虫肠道微生物摇瓶发酵液对武汉亮斑水虻成虫进行产卵行为实验。发现有5株武汉亮斑水虻幼虫肠道微生物(BSFLG01、BSFLG03、BSFLG04、BSFLG05、BSFLG06)对武汉亮斑水虻产卵有显著的积极促进作用,其中3株(BSFLG03、BSFLG05、BSFLG06)对水虻成虫产卵的引诱作用明显强于所设立的阳性对照FE06;菌株BSFLG07为一种毕赤酵母菌(Pichia kudriavzevii),对武汉亮斑水虻产卵有消极影响。经菌落培养特征、菌体形态比较、16SrDNA或18SrDNA序列测定和发育树比对分析,鉴定出这5种具有显著引诱武汉亮斑水虻产卵作用的微生物分别为:枯草芽胞杆菌(Bacillus subtilis)BSFLG01、一种沙雷氏菌(Serratiasp.)BSFLG03、库德毕赤酵母(Pichia kudriavzevii)BSFLG04、皱落假丝酵母(Candida rugosas)BSFLG05、粘质沙雷氏菌(Serratia marcescens)BSFLG06。     

Detection of Salmonella spp. Using a Generic and Differential FRET-PCR

To facilitate the detection of Salmonella and to be able to rapidly and conveniently determine the species/subspecies present, we developed and tested a generic and differential FRET-PCR targeting their tetrathionate reductase response regulator gene. The differential pan-Salmonella FRET-PCR we developed successfully detected seven plasmids that contained partial sequences of S. bongori and the six S. enterica subspecies. The detection limit varied from ∼5 copies of target gene/per PCR reaction for S. enterica enterica to ∼200 for S. bongori. Melting curve analysis demonstrated a T _m of ∼68°C for S. enterica enterica, ∼62.5°C for S. enterica houtenae and S. enterica diarizonae, ∼57°C for S. enterica indica, and ∼54°C for S. bongori, S. enterica salamae and S. enterica arizonae. The differential pan-Salmonella FRET-PCR also detected and determined the subspecies of 4 reference strains and 47 Salmonella isolated from clinically ill birds or pigs. Finally, we found it could directly detect and differentiate Salmonella in feline (5/50 positive; 10%; one S. enterica salamae and 4 S. enterica enterica) and canine feces (15/114 positive; 13.2%; all S. enterica enterica). The differential pan-Salmonella FRET-PCR failed to react with 96 non-Salmonella bacterial strains. Our experiments show the differential pan-Salmonella FRET-PCR we developed is a rapid, sensitive and specific method to detect and differentiate Salmonella.     

Effects of food substrates and moxidectin on development of black soldier fly,Hermetia illucens

Black soldier fly is a common and widely distributed saprophagous species that has an excellent potential for being used for biological conversion of organic wastes on an industrial scale. The main goal of the reported study was expanding the list of wastes suitable for utilization by this species. We compared larval growth on cull potatoes, horse manure and cafeteria food waste in 100‐L bins in a greenhouse. We also conducted laboratory experiments to investigate whether black soldier fly larvae are affected by the presence of moxidectin, a common endectocide used to treat an array of domestic animals and readily excreted in faeces, in their food substrates. Feeding on potatoes resulted in slower growth, and the final size of potato‐fed larvae was smaller compared to the larvae fed on cafeteria waste. Nevertheless, potatoes supported substantial biomass accumulation, and could be a valuable option for rearing fly larvae for commercial feed production. Larvae feeding on horse manure gained very little weight and eventually failed to pupate. Moxidectin had a strong negative effect on larval survivorship; however, ca. 30% of larvae reared in the substrate containing a realistic field concentration of moxidectin still survived to adulthood. Our findings confirm that using black soldier fly larvae is a promising technology for recycling organic wastes, including those of plant origin.     

Soft cheese supplemented with black cumin oil: Impact on food borne pathogens and quality during storage

Black cumin seed oil (BCSO) was tested for its inhibitory effect against some pathogenic bacteria (Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Listeria monocytogenes Scott A and Salmonella enteritidis PT4) in Domiati cheese during cold storage. Physical, chemical and sensorial changes in cheese during storage were recorded. Pasteurized milk was inoculated before renneting with a mixed culture of bacteria at ca. 4 log CFU mL⁻¹. In vitro and in situ supplementation with BCSO showed antimicrobial impact on the growth of S. aureus, E. coli, L. monocytogenes and S. enteritidis inoculated into media and cheese samples. Supplementing of cheese with BCSO (0.1% or 0.2%, w/w) significantly reduced the counts of the inoculated pathogens by ca. 1.3 log and 1.5 log CFU g⁻¹ after 21 days of storage. In addition, BCSO controlled the development of titratable acidity, limited the changes in ripening indices, flavor components and kept considerable physicochemical and sensorial properties of cheese.     

Multiplex PCR and DNA array for the detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. targeting virulence-related genes

A multiplex PCR and DNA array for quick detection of Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella spp. was developed using specific genetic markers derived from virulence-related genes. The genetic markers of cytK, sei, prfA, rfB, and hilA gene specifically amplified DNA fragments of 320 bp, 500 bp, 700 bp, 1.0 kb and 1.2 kb from B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., respectively. These markers are specific for the detection of the corresponding target pathogens. The sensitivity of the genetic markers was down to ～0.5 fg genomic DNA and ～10¹ CFU/ml (one bacterial cell per reaction) of bacterial culture. The combination of mPCR and DNA macroarray hybridization sensitively and specifically detected B. cereus, S. aureus, L. monocytogenes, E. coli O157:H7, and Salmonella spp., in complex mixed cultures and food matrices. Thus, this mPCR and macroarray-based approach serves as rapid and reliable diagnostic tool for the detection of these five pathogens.     

Health Risk from the Use of Roof-Harvested Rainwater in Southeast Queensland,Australia, as Potable or Nonpotable Water,Determined Using Quantitative Microbial Risk Assessment

A total of 214 rainwater samples from 82 tanks were collected in urban Southeast Queensland (SEQ) in Australia and analyzed for the presence and numbers of zoonotic bacterial and protozoal pathogens using binary PCR and quantitative PCR (qPCR). Quantitative microbial risk assessment (QMRA) analysis was used to quantify the risk of infection associated with the exposure to potential pathogens from roof-harvested rainwater used as potable or nonpotable water. Of the 214 samples tested, 10.7%, 9.8%, 5.6%, and 0.4% were positive for the Salmonella invA, Giardia lamblia β-giardin, Legionella pneumophila mip, and Campylobacter jejuni mapA genes, respectively. Cryptosporidium parvum oocyst wall protein (COWP) could not be detected. The estimated numbers of Salmonella, G. lamblia, and L. pneumophila organisms ranged from 6.5 × 10¹ to 3.8 × 10² cells, 0.6 × 10° to 3.6 × 10° cysts, and 6.0 × 10¹ to 1.7 × 10² cells per 1,000 ml of water, respectively. Six risk scenarios were considered for exposure to Salmonella spp., G. lamblia, and L. pneumophila. For Salmonella spp. and G. lamblia, these scenarios were (i) liquid ingestion due to drinking of rainwater on a daily basis, (ii) accidental liquid ingestion due to hosing twice a week, (iii) aerosol ingestion due to showering on a daily basis, and (iv) aerosol ingestion due to hosing twice a week. For L. pneumophila, these scenarios were (i) aerosol inhalation due to showering on a daily basis and (ii) aerosol inhalation due to hosing twice a week. The risk of infection from Salmonella spp., G. lamblia, and L. pneumophila associated with the use of rainwater for showering and garden hosing was calculated to be well below the threshold value of one extra infection per 10,000 persons per year in urban SEQ. However, the risk of infection from ingesting Salmonella spp. and G. lamblia via drinking exceeded this threshold value and indicated that if undisinfected rainwater is ingested by drinking, then the incidences of the gastrointestinal diseases salmonellosis and giardiasis are expected to range from 9.8 × 10° to 5.4 × 10¹ (with a mean of 1.2 × 10¹ from Monte Carlo analysis) and from 1.0 × 10¹ to 6.5 × 10¹ cases (with a mean of 1.6 × 10¹ from Monte Carlo analysis) per 10,000 persons per year, respectively, in urban SEQ. Since this health risk seems higher than that expected from the reported incidences of gastroenteritis, the assumptions used to estimate these infection risks are critically examined. Nonetheless, it would seem prudent to disinfect rainwater for use as potable water.Roof-harvested rainwater has received significant attention as a potential alternative source of potable and nonpotable water in regions where water is scarce (37). To encourage the use of roof-harvested rainwater, governmental bodies of many countries, such as Australia, Denmark, Germany, India, and New Zealand, are providing subsidies to residents to encourage the use of rainwater for domestic purposes. The use of rainwater is quite common in Australia, particularly in rural and remote areas, where reticulated mains or town water is not available. Recent water scarcity in several capital cities prompted the use of rainwater as an alternative source. For instance, the Queensland State Government initiated the “Home Water Wise Rebate Scheme,” which provides subsidies to Southeast Queensland (SEQ) residents who use rainwater as nonpotable water for domestic purposes (49). Over 260,000 householders were granted subsidies up to December 2008, when the scheme was concluded.There is a general community feeling that roof-harvested rainwater is safe to drink, and this is partially supported by limited epidemiological evidence (26). Some studies have reported that roof-harvested rainwater quality is generally acceptable for use as potable water (13, 29). In contrast, the presence of potential pathogens, such as Aeromonas spp. Campylobacter spp., Campylobacter jejuni, Salmonella spp., Legionella pneumophila, Giardia spp., Giardia lamblia, and Cryptosporidium spp., in roof-harvested rainwater samples has been reported (2, 9, 34, 45, 47, 48). Such pathogens can cause gastrointestinal illness in humans, with nausea, vomiting, and/or diarrhea occurring within 12 to 72 h (Salmonella enterica serovar Typhimurium) to 9 to 15 days (Giardia lamblia) after ingestion of contaminated water. L. pneumophila can cause the respiratory infection pneumonia, and the fatality rate can be 50% in immunocompromised patients (57).Direct routine monitoring of the microbiological quality of source water for all possible pathogens is not economically, technologically, or practically feasible. Consequently, traditional fecal indicators, such as fecal coliforms, Escherichia coli, and enterococci, have long been used to determine the presence of pathogens. Most studies assess the quality of roof-harvested rainwater based on the numbers of these fecal indicators (13, 30). However, the major limitation in using fecal bacteria as indicators is their poor correlation with the presence of pathogenic microorganisms in water (2, 30). An alternative is the measurement of pathogens using traditional culture-based methods. However, there are several limitations of such methods, including the underestimation of the bacterial number due to the presence of injured or stressed cells (10) and the fact that certain microorganisms in environmental waters can be viable but not culturable (39). Culture-based methods are also generally laborious and costly. Recent advances in molecular techniques such as PCR technology enable rapid, specific, and sensitive detection of many pathogens. Advances in PCR methodology also enable the quantification of potential pathogens in source water that are otherwise difficult and/or laborious to culture using traditional microbiological methods. In view of this, we used binary PCR (presence/absence)- and quantitative PCR (qPCR)-based assays to first detect and then quantify zoonotic pathogens in samples from roof-harvested rainwater in SEQ residential houses.The aims of the research study were 2-fold: (i) to quantify the number and frequency of occurrence of Salmonella, G. lamblia, and L. pneumophila organisms in a range of domestic water tanks in SEQ by using qPCR-based methods and (ii) to apply quantitative microbial risk assessment (QMRA) analysis in order to estimate the risk of infection from exposure to these pathogens found in roof-harvested rainwater. The uniqueness of this study stems from the fact that instead of measuring fecal indicators, the pathogens that are capable of causing illness were quantified and this information was combined with QMRA to assess the human health risk of using roof-harvested rainwater as potable or nonpotable water.     

Production of Italian Dry Salami: Effect of Starter Culture and Chemical Acidulation on Staphylococcal Growth in Salami Under Commercial Manufacturing Conditions

The effect of starter culture and chemical acidulation on the growth and enterotoxigenesis of Staphylococcus aureus strain S-6 in Italian dry salami under commercial manufacturing conditions was studied. The experimental design included two levels of S. aureus (10⁴ and 10⁵/g), three levels of starter culture (0, 10⁵, and 10⁶/g), three levels of initial pH (pH₀) (6.1, 5.5, and 4.8), two manufacturing plants, and three replications. S. aureus growth in the salami was affected significantly (P < 0.005) by pH₀, initial levels of S. aureus (staph₀) and lactic acid bacteria (LAB₀), day of fermentation, and by the interactions of pH₀ × day, pH₀ × LAB₀, LAB₀ × staph₀, pH₀ × staph₀, and pH₀ × location of fermentation. In general, the lower the pH₀ and the higher the LAB₀, the greater the inhibition of S. aureus. The LAB levels during the fermentation were affected significantly (P < 0.005) by pH₀, LAB₀, day of fermentation, location, LAB₀ × pH₀, and LAB₀ × day. Derived regression equations related level of S. aureus and LAB at any day of fermentation to a number of microbiological and chemical variables. Close similarity of observed and predicted levels of S. aureus and LAB growth demonstrated the usefulness of the experimental approach in evaluating the safety of a process. No detectable enterotoxin or thermonuclease was found at any stage of processing even when S. aureus reached levels of 10⁷/g of salami.     

Nutritional composition of black soldier fly larvae feeding on agro-industrial by-products

Black soldier fly (BSF) larvae, Hermetia illucens L. (Diptera: Stratiomyidae), bio-convert organic side streams into high-quality biomass, the composition of which largely depends on the side stream used. In the present study, BSF larvae were reared on feed substrates composed of dried brewers’ spent grains, each supplemented with either water, waste brewer’s yeast, or a mixture of waste brewer’s yeast and cane molasses to obtain 12 different substrates: barley/water, barley/yeast, barley/yeast/molasses, malted barley/water, malted barley/yeast, malted barley/yeast/molasses, malted corn/water, malted corn/yeast, malted corn/yeast/molasses, sorghum-barley/water, sorghum-barley/yeast, and sorghum-barley/yeast/molasses. The crude protein, fat, ash, and mineral contents of the BSF larvae fed each feed substrate were quantified by chemical analyses. The effect of substrate, supplementation, and their interaction on crude protein, fat, and ash contents of BSF larval body composition was significant. Calcium, phosphorus, and potassium were the most abundant macrominerals in the larvae and their concentrations differed significantly among substrates. These findings provide important information to support the use of BSF larval meal as potential new source of nutrient-rich and sustainable animal feed ingredients to substitute expensive and scarce protein sources such as fishmeal and soya bean meal.     

In Vivo Microbial Coevolution Favors Host Protection and Plastic Downregulation of Immunity

Microbiota can protect their hosts from infection. The short timescales in which microbes can evolve presents the possibility that “protective microbes” can take-over from the immune system of longer-lived hosts in the coevolutionary race against pathogens. Here, we found that coevolution between a protective bacterium (Enterococcus faecalis) and a virulent pathogen (Staphylococcus aureus) within an animal population (Caenorhabditis elegans) resulted in more disease suppression than when the protective bacterium adapted to uninfected hosts. At the same time, more protective E. faecalis populations became costlier to harbor and altered the expression of 134 host genes. Many of these genes appear to be related to the mechanism of protection, reactive oxygen species production. Crucially, more protective E. faecalis populations downregulated a key immune gene, , known to be effective against S. aureus infection. These results suggest that a microbial line of defense is favored by microbial coevolution and may cause hosts to plastically divest of their own immunity.     

Effects of aeration rate and feed on growth,productivity and nutrient composition of black soldier fly (Hermetia illucens L.) larvae

Black soldier fly larvae (Hermetia illucens L.) are widely cultivated as bioconversion agents. These larvae are known to be highly sensitive to changes in the external environment, such as temperature and relative humidity. Therefore, a rearing hive equipped with an instrumentation system known as the Modular Fly Hive was designed to cultivate black soldier larvae. This study aimed to determine the effects of aeration rate (0 – 0.84 m³/s.) and feed type (coconut endosperm waste and soybean curd) on growth and productivity of black soldier fly cultivated in Modular Fly Hives as well as well as feed digestibility and nutrient composition of the larval biomass. Increasing aeration rate decreased the average temperature and relative humidity within the hives. The results showed that the larvae demonstrated a sigmoidal growth curve for all types of feed and the type of feed significantly affects the average wet weight and length of the larvae. The greatest dry biomass productivity (52.85 ± 3.85 g/m²/day) was observed when the larvae were cultivated using soybean curd residue with an aeration rate of 0.84 m³/s, while the lowest dry biomass productivity (35.27 ± 9.72 g/m²/day) was observed when the larvae were cultivated using coconut endosperm waste without aeration. Proximate analysis revealed that the larval biomass had total protein, lipid, ash and carbohydrate content in the range of 37.20 – 48.60%, 9.61 – 20.02%, 4.80 – 6.40%, 33.86 – 38.70%, respectively. Amino acid in the biomass were dominated by glutamic acid (11.11–12.30%), aspartic acid (8.25 – 10.35%), leucine (8.09 – 8.57%), and lysine (6.74 – 8.14%). Lipid isolated from the larval biomass were mainly composed of lauric acid (28.35–61.68%), linoleic acid (6.27 – 30.29%), palmitic acid (7.62 – 15.23%), and myristic acid (5.05 – 14.34%).     

Biofilm Production Potential of Salmonella Serovars Isolated from Chickens in North West Province,South Africa

Bacterial biofilms have recently gained considerable interest in the food production and medical industries due to their ability to resist destruction by disinfectants and other antimicrobials. Biofilms are extracellular polymer matrices that may enhance the survival of pathogens even when exposed to environmental stress. The effect of incubation temperatures (25°C, 37°C, and 40°C) and Salmonella serotype on biofilm-forming potentials was evaluated. Previously typed Salmonella serotypes (55) isolated from the gut of chickens were accessed for biofilms formation using a standard assay. Salmonella Typhimurium ATCC 14028^TM and Salmonella Enteritidis ATCC 13076^TM (positive controls), Escherichia coli (internal control) and un-inoculated Luria Bertani (LB) broth (negative control) were used. The isolates formed no biofilm (11.86–13.56%), weak (11.86–45.76%), moderate (18.64–20.34%), strong biofilms (23.73–54.24%) across the various temperatures investigated. Serotypes, Salmonella Heidelberg and Salmonella Weltevreden were the strongest biofilm formers at temperatures (25°C, 37°C, and 40°C, respectively). The potential of a large proportion (80%) of Salmonella serotypes to form biofilms increased with increasing incubation temperatures but decreased at 40°C. Findings indicate that average temperature favours biofilm formation by Salmonella serotypes. However, the influence of incubation temperature on biofilm formation was greater when compared to serotype. A positive correlation exists between Salmonella biofilm formed at 25°C, 37°C and 40°C (p ≥ 0.01). The ability of Salmonella species to form biofilms at 25°C and 37°C suggests that these serotypes may present severe challenges to food-processing and hospital facilities.Key words: Salmonella, biofilm, biofilm production potential, crystal violet microtitre     

LPS Structure and PhoQ Activity Are Important for Salmonella Typhimurium Virulence in the Gallleria mellonella Infection Model

The larvae of the wax moth, Galleria  mellonella , have been used experimentally to host a range of bacterial and fungal pathogens. In this study we evaluated the suitability of G . mellonella as an alternative animal model of Salmonella infection. Using a range of inoculum doses we established that the LD₅₀ of Salmonella Typhimurium strain NCTC 12023 was 3.6 × 10³ bacteria per larva. Further, a set of isogenic mutant strains depleted of known virulence factors was tested to identify determinants essential for S . Typhimurium pathogenesis. Mutants depleted of one or both of the type III secretion systems encoded by Salmonella Pathogenicity Islands 1 and 2 showed no virulence defect. In contrast, we observed reduced pathogenic potential of a phoQ mutant indicating an important role for the PhoPQ two-component signal transduction system. Lipopolysaccharide (LPS) structure was also shown to influence Salmonella virulence in G . mellonella . A waaL (rfaL) mutant, which lacks the entire O-antigen (OAg), was virtually avirulent, while a wzz _ST/wzz _fepE double mutant expressing only a very short OAg was highly attenuated for virulence. Furthermore, shortly after infection both LPS mutant strains showed decreased replication when compared to the wild type in a flow cytometry-based competitive index assay. In this study we successfully established a G . mellonella model of S . Typhimurium infection. By identifying PhoQ and LPS OAg length as key determinants of virulence in the wax moth larvae we proved that there is an overlap between this and other animal model systems, thus confirming that the G . mellonella infection model is suitable for assessing aspects of Salmonella virulence function.