Category Archives: Research

Research – Bacillus amyloliquefaciens ALB65 Inhibits the Growth of Listeria monocytogenes on Cantaloupe Melons

ABSTRACT

Listeria monocytogenes is a foodborne pathogen that causes high rates of hospitalization and mortality in people infected. Contamination of fresh, ready to eat produce by this pathogen is especially troubling because of the ability of this bacterium to grow on produce under refrigeration temperatures. In this study, we created a library of over 8,000 plant phyllosphere-associated bacteria and screened them for the ability to inhibit the growth of L. monocytogenes in an in vitro fluorescence-based assay. One isolate, later identified as Bacillus amyloliquefaciens ALB65, was able to inhibit the fluorescence of L. monocytogenes by >30-fold in vitro. B. amyloliquefaciens ALB65 was also able to grow, persist, and reduce the growth of L. monocytogenes by >1.5 log CFU on cantaloupe melon rinds inoculated with 5 × 103 CFU at 30°C and was able to completely inhibit its growth at temperatures below 8°C. DNA sequence analysis of the B. amyloliquefaciens ALB65 genome revealed six gene clusters that are predicted to encode genes for antibiotic production; however, no plant or human virulence factors were identified. These data suggest that B. amyloliquefaciens ALB65 is an effective and safe biological control agent for the reduction of L. monocytogenes growth on intact cantaloupe melons and possibly other types of produce.
IMPORTANCE Listeria monocytogenes is estimated by the Centers for Disease Control and Prevention and the U.S. Food and Drug Administration to cause disease in approximately 1,600 to 2,500 people in the United States every year. The largest known outbreak of listeriosis in the United States was associated with intact cantaloupe melons in 2011, resulting in 147 hospitalizations and 33 deaths. In this study, we demonstrated that Bacillus amyloliquefaciens ALB65 is an effective biological control agent for the reduction of L. monocytogenes growth on intact cantaloupe melons under both pre- and postharvest conditions. Furthermore, we demonstrated that B. amyloliquefaciens ALB65 can completely inhibit the growth of L. monocytogenes during cold storage (<8°C).

Research – Bacterial Distribution and Community Structure in Beef Cattle Liver and Bile at Slaughter

Journal of Food Protection

In this study, the distribution of hygienic indicator bacteria in cattle livers and bile was examined at slaughterhouses. First, 127 cattle livers with gallbladders were carefully eviscerated from the carcasses at 10 slaughterhouses. Microbiological examination showed that 9 bile (7.1%) and 19 liver parenchyma (15.0%) samples were positive for the family Enterobacteriaceae (EB) with means ± SD of 3.68 ± 4.63 log CFU/mL and 1.59 ± 2.47 log CFU/g, respectively; thus, bacterial contamination was apparent even at the postevisceration stage. Subsequently, 70 cattle livers were obtained at the postprocessing/storage stage from 7 of the ten slaughterhouses; microbiological analysis revealed greater means of EB in the liver parenchyma (means ± SD of 3.00 ± 3.89 log CFU/g, P =0.011) than those at postevisceration stage, suggesting that bacterial dissemination and/or replication occurred in the liver parenchyma during processing and storage. According to 16S rRNA ion semiconductor sequencing analysis of representative samples from 12 cattle, Proteobacteria , Firmicutes , and Actinobacteria were dominant in both the parenchyma and bile, in which EB/ Escherichia coli were predominate among EB-rich livers. These results suggest that bile plays a role as a vehicle for bacterial transmission to the liver parenchyma. This is the first study to demonstrate bacterial distribution and community structure in the liver and biliary microecosystem of cattle at slaughter. Our data provide possible implication of EB testing in bile to screen cattle livers contaminated with high levels of fecal indicator bacteria.

Research – Prevalence of Salmonella in Chinese Food Commodities: A meta-analysis

Journal of Food Protection

kswfoodworld Salmonella

The objective of the present study was to analyze the prevalence of Salmonella in multiple food commodities in China by performing a meta-analysis. Accordingly, we screened studies that examined the prevalence of Salmonella in PubMed, Embase, and Web of Science databases. Methodological quality assessment and heterogeneity analyses were performed for included studies. The prevalence rate with the 95% confidence interval (95% CI) was selected as the effect size. Subgroup analyses for each food type were conducted and then stratified by regions, food-chain processing points, and seasons. In total, 49 studies were included in the meta-analysis, among them, 8 (16.3%) studies were deemed “High risk”, 13 (26.5%) studies were “Unclear risk”, and 28 (57.2%) studies were “Low risk”. The overall prevalence rate of Salmonella was 20.0 (95%CI: 15.9-24.4)%. The prevalence rate of Salmonella in raw meat products was 23.6 (95%CI: 19.8-27.6)%, which was higher than that in aquatic products (13.7 [95%CI: 3.1-29.9]%), milk products (0.9 [95%CI: 0.0-3.9]%), frozen convenience foods (6.5 [95%CI: 4.4-8.9]%), ready-to-eat foods (2.0 [95%CI: 1.1-3.2]%), vegetables and fruits (0.9 [95%CI: 0.0-5.2]%), and shell eggs (4.2 [95%CI: 3.0-5.7]%). Subgroup analyses revealed that prevalence rates of Salmonella in raw meat products from abattoirs (26.3 [95%CI: 17.4-36.3]%) and retail stores (30.0 [95%CI: 24.6-35.8]%) were higher than those determined from farms (10.2 [95%CI: 7.0-13.9)%; P < 0.05); however, no significant difference was observed in the prevalence of Salmonella stratified by different geographical regions or seasons (P > 0.05). Based on these findings, high levels of Salmonella contamination could be detected in raw meat products in China, and the prevalence rate of Salmonella in raw meat products from abattoirs and retail stores was high.

Research – High-Pressure-Induced Sublethal Injuries of Food Pathogens—Microscopic Assessment

MDPI

High Hydrostatic Pressure (HHP) technology is considered an alternative method of food preservation. Nevertheless, the current dogma is that HHP might be insufficient to preserve food lastingly against some pathogens. Incompletely damaged cells can resuscitate under favorable conditions, and they may proliferate in food during storage. This study was undertaken to characterize the extent of sublethal injuries induced by HHP (300–500 MPa) on Escherichia coli and Listeria innocua strains. The morphological changes were evaluated using microscopy methods such as Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Epifluorescence Microscopy (EFM). The overall assessment of the physiological state of tested bacteria through TEM and SEM showed that the action of pressure on the structure of the bacterial membrane was almost minor or unnoticeable, beyond the L. innocua wild-type strain. However, alterations were observed in subcellular structures such as the cytoplasm and nucleoid for both L. innocua and E. coli strains. More significant changes after the HHP of internal structures were reported in the case of wild-type strains isolated from raw juice. Extreme condensation of the cytoplasm was observed, while the outline of cells was intact. The percentage ratio between alive and injured cells in the population was assessed by fluorescent microscopy. The results of HHP-treated samples showed a heterogeneous population, and red cell aggregates were observed. The percentage ratio of live and dead cells (L/D) in the L. innocua collection strain population was higher than in the case of the wild-type strain (69%/31% and 55%/45%, respectively). In turn, E. coli populations were characterized with a similar L/D ratio. Half of the cells in the populations were distinguished as visibly fluorescing red. The results obtained in this study confirmed sublethal HHP reaction on pathogens cells. View Full-Text

Research – Antimicrobial activity and mechanism of oregano essential oil against Shewanella putrefaciens

Wiley Online

The aim of the present study was to investigate the antimicrobial mechanism of oregano essential oil (OEO) against Shewanella putrefaciens. Antimicrobial activity of OEO against Shewanella putrefaciens was investigated by the agar disc diffusion method. The change of growth curve, electric conductivity, the integrity of cell membrane, alkaline phosphatase (AKP), and lactate dehydrogenase (LDH) activity were measured to evaluate its antibacterial mechanism. The morphology of bacterial cells was observed by scanning electron microscopy (SEM). The interaction between OEO and Shewanella putrefaciens genomic DNA was measured by ultraviolet–visible (UV–Vis) spectroscopy, and DNA ethidium bromide adduct was analyzed by fluorescence. The results showed that the minimum inhibitory concentration of OEO against Shewanella putrefaciens was 0.09% (v/v), and OEO could inhibit the growth of Shewanella putrefacien with a dose-dependent manner. The cell membrane and cell wall of Shewanella putrefaciens were destroyed by OEO, which led to the leakage of nucleic acid, protein, and the release of AKP and LDH. The results of SEM confirmed the damaging effect of OEO on the bacterial morphology. The results of UV–Vis and fluorescence titration indicated that binding of the complexes to DNA was an intercalative mode.

Research – New details behind how the Shigella pathogen delivers bacterial proteins into our cells

Science Daily

Shigella - kswfoodworld

Image CDC

Shigella, a bacterial pathogen that causes dysentery and is the leading cause of childhood diarrheal diseases, inserts a pore called a translocon into an infected person’s intestinal cells and then injects bacterial proteins into the cells. There, the proteins hijack the cells’ machinery to help Shigella multiply. In a study published in mBio, a team at Massachusetts General Hospital (MGH) has uncovered important details about Shigella‘s translocon, which may help researchers develop an effective strategy to block this critical component of infection.

Shigella infects our gut by manipulating our intestinal cells and tricking them into letting the Shigella inside. In fact, there are many bacterial pathogens that use this same, or similar, mechanism to infect us,” says lead author Poyin Chen, PhD, a postdoctoral fellow at MGH. “This translocon pore is essentially the gateway through which bacterial proteins get pumped into our cells. We know that this structure is made of two proteins — IpaB and IpaC — but what we don’t know is how these proteins fit together to make this pore.”

When the investigators used protein mapping techniques to look closely at translocons when they were embedded in cell membranes, they were able to see which of the two proteins — specifically IpaB — makes up the inner ring of the pore. “If you think of the translocon pore as a donut, this would be the walls of the donut hole. This finding is important because this is the part of the translocon pore that directly interacts with bacterial proteins as they are injected into our cells,” explains Chen. “With the findings from this study, we can begin to understand if this pore acts as a slippery tube that bacterial proteins travel through or if the translocon pore can control the flow of bacterial proteins into our cells.”

Such details may help investigators target the translocon and block the entry of Shigella proteins into cells. “For something that is so essential to establishing infection, we know terribly little of how it’s made and how it works,” says Chen. “As we gain a better understanding of its parts, we will be able to approach the structure as a whole and maybe even find ways to neutralize the function of this structure to prevent infection before it can begin.”

Co-authors include Brian C. Russo, Jeffrey K. Duncan-Lowey, Natasha Bitar, Keith Egger and Marcia B. Goldberg.

This work has been supported by the National Institutes of Health, the Massachusetts General Hospital Executive Committee on Research Tosteson Award, and the Charles A. King Trust Postdoctoral Research Fellowship Program.

Research – Editorial: Vibrio Species in the Food Processing Chain

Frontiers in Microbiology

Food Illness

Editorial on the Research Topic
Vibrio Species in the Food Processing Chain

Rising concern about the foodborne illnesses caused by pathogenic Vibrio species (mainly V. parahaemolyticus, V. cholera, and V. vulnificus) has led to a strengthening of research on the characterization of the presence of the genus in food matrices, virulence genes, pandemic markers, and the correlation between clinical and environmental isolates from different ecosystems. The emergence of antimicrobial resistance strains (AMR) in Vibrio spp. may produce a decrease in the effectiveness of commonly used antibiotics, thus posing a threat to public health. Progress in genomic studies has identified motile elements implied in gene transfer that may give birth to developing surveillance strategies for risk mitigation. The development of new infection models that can predict the pathogenesis of Vibrio spp. and the use of high-throughput sequencing techniques for serogroup genes may be useful tools for understanding molecular pathways and the infectivity of Vibrio spp. food isolates. In this Research Topic, different approaches, aiming at characterizing Vibrio spp. from aquaculture, marine, and vegetable ecosystems, together with the evaluation of microbial behavior and the development of new infection and serogroup models, are shown.

A mini-review by Dutta et al. discusses the role and antimicrobial resistance of pathogenic Vibrio spp. They present potential sources of antibiotic resistance genes for Vibrio spp., including the horizontal gene transmission from other pathogens as the main route. This has shown the genetic basis of the emergence of multidrug and extensively multidrug resistant Vibrio spp. through different types of highly mobile elements that can be extensively propagated among bacteria. The use of phage or probiotic therapies as alternative treatments for the inactivation of antibiotic resistant species of Vibrio may be helped by the maintenance of good hygiene practices and processing technologies to protect public health.

Antibiotic resistance genes can also originate from the environment, such as wastewater effluents or sediments in marine or aquaculture habitats. In this regard, Siddique et al. studied the characterization of pathogenic V. parahaemolyticus in a fish farm ecosystem (tilapia, rui, and shrimp). Among the 216 samples, 60.2% were positive for the pathogen, including 323 isolates of which 17 harboured the trh virulence gene gene. They confirm the presence of resistant strains to amoxicillin, ampicillin, and penicillin. Pathogenicity was further confirmed by the fluid accumulation in the ileal loop of rabbits being O8: KUT, the most predominant pathogenic serotype.

The presence and characterization of V. parahaemolyticus and V. vulnificus in marine and estuarine environments was studied by da Silva et al. They found 150 isolates of V. parahaemolyticus, including 52 positives for trh gene, and 129 of V. vulnificus from water and blue crab samples. PFGE and agglutination tests were used for molecular subtyping and determination of antibiotic resistance. The study showed the high presence of the O5 pathogenic serotype, together with the multidrug resistant isolates (41%) and the high genetic diversity of both Vibrio species, as no correlations were found among the sampling sites, antimicrobial resistance profiles, and pathogenicity.

The associated presence of Vibrio spp. in water ecosystems may underestimate their origin from other environmental and food sources. Ready-To-Eat vegetables can harbor pathogenic Vibrio spp. if poor manufacturing, hygiene, and storage practices are followed. Igbinosa et al. evaluated the presence of V. parahaemolyticus in minimally processed vegetables. Among the 63 isolates, they found microbial counts from 1.5 to 1,000 MPN/g and drug resistant isolates to ampicillin and cefotaxime mainly (>60%). They studied the biofilm formation finding that 23.8% of the isolates were strong biofilm producers. Regarding the presence of virulence genes, 100, 14.3, and 31.8% of the isolates harbored the toxR gene, trh, and tdh determinants, respectively.

The microbial behavior of Vibrio spp. can be quantified with predictive models. Posada-Izquierdo et al. investigated the fate of a Vibrio spp. cocktail inoculated in lye-treated table olives for 22 days. A predictive growth model was developed as a function of salt concentration (2–12%) and pH (4–9) using a synthetic medium and table olive brines. They found a higher effect of salt concentration than of pH for the growth inhibition of Vibrio spp. However, they were not able to proliferate in the table olives during fermentation, highlighting that phenolics compounds could exert a clear antimicrobial effect.

The disposal of reliable models to predict the pathogenesis of Vibrio spp. are increasingly needed since the use of virulence markers could not fully elucidate the presence of long-standing virulence indicators. This was demonstrated by Santos et al. using clinical and environmental V. parahaemolyticus isolates in two systemic infection models, namely mice and Galleria mellonella larvae. Interestingly, non-pathogenic environmental isolates produced lethal infections regardless of their source, serotype, and genotype (tdh, orf8, toxRSnew, and vpadF). A high correlation was found in the assayed models, supporting that G. mellonella larvae can be used as an alternative model to study the pathogenesis of V. parahaemolyticus.

Recently, the use of high-throughput sequencing technologies has aided researchers in deciphering the genome of different species. This was essential to provide complete knowledge of the molecular and metabolic pathways of microorganisms and the identification of virulence gene clusters. Bian et al. have developed VPsero, a rapid serotyping tool for V. parahaemolyticus using serogroup specific genes obtained from whole-genome sequencing data. The algorithm, based on the comparison of lipopolysaccharide and capsular polysaccharide gene clusters covered 43 K and 12 O serogroups. The authors showed the high sensitivity and specificity of the tool (>0.91), though limitations could be faced in future studies, such as the addition of new serogroups, the verification of the quality of assembled genomes and the availability of short reads.

This Research Topic presents a collection of manuscripts highlighting relevant findings in the pathogenesis of Vibrio spp. in the food chain and suggests future directions for research, enabling progress in the development of novel analytical methods and surveillance actions to mitigate the emerging risk posed by these human pathogens.

Research – Inactivation of Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes in Tahini by Microwave Heating

MDPI

Tahini (sesame paste) is a traditional food. Numerous foodborne outbreaks have been associated with it. This study aimed to (i) explore the efficiency of 2450 MHz microwave heating at 220, 330, 440, 550, and 660 W on the inactivation of Salmonella spp, Escherichia coli O157:H7, and Listeria monocytogenes in tahini; (ii) determine the impact of desiccation and starvation stresses on pathogen survival; (iii) assess the impact of microwave heating on the physicochemical characteristics of tahini. The inoculated microorganisms in tahini were reduced with higher microwave power levels (p < 0.05) and longer exposure times. The D-values of unstressed Salmonella spp., Escherichia coli O157:H7, and L. monocytogenes ranged from 6.18 to 0.50 min, 6.08 to 0.50 min, and 4.69 to 0.48 min, respectively, at power levels of 220 to 660 W, with z-values of 410, 440, and 460 W, respectively. Generally, desiccation and starvation stress levels prior to heating increased microbial resistance to heat treatment. Microwave heating did not affect acid, peroxide, p-anisidine, or color values of tahini up to 90 °C. These findings reveal microwave heating as a potential method for lowering the risk of Salmonella spp., E. coli O157:H7 and L. monocytogenes in tahini with no compromise on quality. View Full-Text

Research – Uses of Bacteriophages as Bacterial Control Tools and Environmental Safety Indicators

Frontiers in Microbiology

Bacteriophages are bacterial-specific viruses and the most abundant biological form on Earth. Each bacterial species possesses one or multiple bacteriophages and the specificity of infection makes them a promising alternative for bacterial control and environmental safety, as a biotechnological tool against pathogenic bacteria, including those resistant to antibiotics. This application can be either directly into foods and food-related environments as biocontrol agents of biofilm formation. In addition, bacteriophages are used for microbial source-tracking and as fecal indicators. The present review will focus on the uses of bacteriophages like bacterial control tools, environmental safety indicators as well as on their contribution to bacterial control in human, animal, and environmental health.

Introduction

Bacteriophages, also known as phages, are prokaryotes viruses, being the most abundant life form, present in all environments and the predominant entities in the sea (Boehme, 1993Suttle, 2005). Several studies have demonstrated a 1:5 relative abundance between bacteria and bacteriophage (Fuhrman, 1999Balter, 2000Rohwer, 2003). They were discovered independently by Twort (1915), who isolated them from Staphylococcus spp., and from patients with dysentery. D’Herelle (1926) described bacteriophage as a virus that has the capability to parasitize bacteria (Twort, 1915Delbruck, 1942). Bacteriophages vary greatly in morphology and replicative characteristics, containing either RNA or DNA, being these parameters currently used by the International Committee on Taxonomy of Viruses (ICTV) for bacteriophage classification (King et al., 2012Table 1). However, the identification of bacteriophages is difficult since there are no universally conserved markers, unlike e.g., the bacterial 16S rRNA gene (Paul et al., 2002), with only minor parts of bacteriophage genomes being used to determine family specific makers, such as the viral capsid g20 of T4 (Fuller et al., 1998Marston and Sallee, 2003Sullivan et al., 2008).

Research -A Severe Gastroenteritis Outbreak of Salmonella enterica Serovar Enteritidis Linked to Contaminated Egg Fried Rice, China, 2021

Frontiers in Microbiology

Salmonella contamination of eggs and egg shells has been identified as a public health problem worldwide. Here, we reported an outbreak of severe gastrointestinal symptoms caused by Salmonella enterica serovar Enteritidis (S. enteritidis) in China. We evaluated the outbreak by using epidemiological surveys, routine laboratory testing methods, and whole genome sequencing (WGS). This outbreak occurred in a canteen in Beijing, during March 9–11, 2021, 225 of the 324 diners who have eaten at the canteen showed gastrointestinal symptoms. The outbreak had characteristical epidemiological and clinical features. It caused a very high attack rate (69.4%) in a short incubation time. All patients developed diarrhea and high fever, accompanied by abdominal pain (62.3%), nausea (50.4%), and vomiting (62.7%). The average frequency of diarrhea was 12.4 times/day, and the highest frequency of diarrhea was as high as 50 times/day. The average fever temperature was 39.4°C, and the highest fever temperature was 42°C. Twenty strains of S. enteritidis were recovered, including 19 from the patients samples, and one from remained egg fried rice. Antibiotic susceptibility test showed that the 20 outbreak strains all had the same resistance pattern. PFGE results demonstrated that all 20 strains bore completely identical bands. Phylogenetic analysis based on WGS revealed that all 20 outbreak strains were tightly clustered together. So the pathogenic source of this food poisoning incident may was contaminated egg fried rice. Resistance gene analysis showed that the outbreak strains are all multi-drug resistant strains. Virulence gene analysis indicated that these outbreak strains carried a large number of virulence genes, including 2 types of Salmonella pathogenicity islands (SPI-1 and SPI-2). Other important virulence genes were also carried by the outbreak strains, such as pefABCD, rck and shdA. And the shdA gene was not in other strains located in the same evolutionary branch as the outbreak strain. We speculated that this is a significant reason for the serious symptoms of gastroenteritis in this outbreak. This outbreak caused by S. enteritidis suggested government should strengthen monitoring of the prevalence of outbreak clone strains, and take measures to mitigate the public health threat posed by contaminated eggs.

Introduction

World Health Organization (WHO) estimated the global burden of foodborne diseases, the results showed that almost 1 in 10 people fall ill every year from eating contaminated food and 420,000 die as a result (Dewey-Mattia et al., 2018). Salmonellosis is one of the most frequently reported foodborne diseases worldwide. In particular, disease caused by non-typhoid Salmonella is a global public health problem, whether in a high-income country or a low-income country (Feasey et al., 2016). Each year, approximately 40,000 Salmonella infections are reported to the United States Centers for Disease Control and Prevention (CDCs) (Vaughn et al., 2020). Salmonella enterica serovar Enteritidis (S. enteritidis) is the predominant Salmonella serotype accounting for between 40 and 60% of laboratory-confirmed illnesses of salmonellosis in recent years (Quick et al., 2015). Salmonella enteritidis typically cause a self-limiting gastroenteritis with the symptoms of diarrhea, fever, abdominal cramps, and dehydration (Jiang et al., 2020). Salmonellosis is mainly caused by eating eggs and egg products contaminated with S. enteritidis (90%) and has become a serious health problem. It has been attributed to this serovar’s unusual ability to colonize ovarian tissue of hens and to be able to present within the contents of intact shell eggs (Chousalkar et al., 2018).

Here we reported a severe gastroenteritis outbreak of S. enteritidis linked to contaminated egg fried rice. There were 225 cases of diarrhea and fever in a short period of time in a canteen in Beijing within 3 days. Epidemiological investigations and laboratory tests confirmed that the outbreak was caused by S. enteritidis and was related to the undercooked egg fried rice. At present, such a large-scale outbreak with severe clinical symptoms of S. enteritidis caused by undercooked eggs is rarely reported (Li et al., 2020). Therefore, we reported the outbreak and examined its molecular characteristics using whole genome sequencing (WGS).