Intraspecies competition among Salmonella enterica isolates in the lettuce leaf apoplast.

Multiple Salmonella enterica serovars and strains have been reported to be able to persist inside the foliar tissue of lettuce (Lactuca sativa L.), potentially resisting washing steps and reaching the consumer. Intraspecies variation of the bacterial pathogen and of the plant host can both significantly affect the outcome of foliar colonization. However, current understanding of the mechanisms underlying this phenomenon is still very limited. In this study, we evaluated the foliar fitness of 14 genetically barcoded S. enterica isolates from 10 different serovars, collected from plant and animal sources. The S. enterica isolates were vacuum-infiltrated individually or in pools into the leaves of three- to four-week-old lettuce plants. To estimate the survival capacity of individual isolates, we enumerated the bacterial populations at 0- and 10- days post-inoculation (DPI) and calculated their net growth. The competition of isolates in the lettuce apoplast was assessed through the determination of the relative abundance change of barcode counts of each isolate within pools during the 10 DPI experimental period. Isolates exhibiting varying apoplast fitness phenotypes were used to evaluate their capacity to grow in metabolites extracted from the lettuce apoplast and to elicit the reactive oxygen species burst immune response. Our study revealed that strains of S. enterica can substantially differ in their ability to survive and compete in a co-inhabited lettuce leaf apoplast. The differential foliar fitness observed among these S. enterica isolates might be explained, in part, by their ability to utilize nutrients available in the apoplast and to evade plant immune responses in this niche.

Characterization of the Pathogenic Potential of the Beach Sand Microbiome and Assessment of Quicklime as a Remediation Tool.

Beach sand may act as a reservoir for potential human pathogens, posing a public health risk. Despite this, the microbiological monitoring of sand microbiome is rarely performed to determine beach quality. In this study, the sand microbial population of a Northern Adriatic Sea beach sand was profiled by microbiological (CFU counts) and molecular methods (WGS, microarray), showing significant presence of potential human pathogens including drug-resistant strains. Consistent with these results, the potential of quicklime as a restoring method was tested in vitro and on-field. Collected data showed that adding 1-3% quicklime (w/w) to sand provided an up to -99% of bacteria, fungi, and viruses, in a dose- and time-dependent manner, till 45 days post-treatment. In conclusion, data suggest that accurate monitoring of sand microbiome may be essential, besides water, to assess beach quality and safety. Moreover, first evidences of quicklime potential for sand decontamination are provided, suggesting its usage as a possible way to restore the microbiological quality of sand in highly contaminated areas.

Possible Sources of Listeria monocytogenes Contamination of Fresh-cut Apples and Antimicrobial Interventions During Antibrowning Treatments: A Review.

Fresh-cut apples, being rich in antioxidants and other nutrients, have emerged as popular snacks in restaurants, at home, and in school lunch programs, partially due to freshness, convenience, and portion size availability. Two major challenges in processing fresh-cut apples are the browning of cut surfaces and contamination with human pathogens. Regarding human pathogens, contamination by Listeria monocytogenes is a major concern, as evidenced by two outbreaks of whole apples and numerous recalls of fresh-cut apples. Antibrowning agents currently used by the industry have little to no antimicrobial properties. The present review discusses the possible origins of L. monocytogenes in fresh-cut apples, including contaminated whole apples, and contamination via the processing environment and the equipment in fresh-cut facilities. Treatment with antibrowning solutions could possibly be an opportunity for Listeria contamination and represents the last chance to inactivate pathogens. The discussion is focused on the antibrowning treatments where formulations and coatings with antibrowning and antimicrobial properties have been developed and evaluated against Listeria and other microorganisms. In addition, several research needs and considerations are discussed to further reduce the chance of pathogen contamination on fresh-cut apples.

Pathogen flows from on-site sanitation systems in low-income urban neighborhoods, Dhaka: A quantitative environmental assessment.

BACKGROUND: Despite wide usage of on-site sanitation, there is limited field-based evidence on the removal or release of pathogens from septic tanks and other primary treatment systems, such as anaerobic baffled reactors (ABR). In two low-income areas in Dhaka, we conducted a cross-sectional study to explore pathogen loads discharged from commonly used on-site sanitation-systems and their transport in nearby drains and waterways. METHODS: We collected samples of drain water, drain sediment, canal water, and floodwater from April-October 2019. Sludge, supernatant, and effluent samples were also collected from septic tanks and ABRs. We investigated the presence and concentration of selected enteric pathogens (Shigella, Vibrio cholerae (V. cholerae), Salmonella Typhi (S. Typhi), Norovirus Genogroup-II (NoV-GII), and Giardia) and presence of Cryptosporidium in these samples using quantitative polymerase chain reaction (qPCR).The equivalent genome copies (EGC) of individual pathogens were estimated in each sample by interpolation of the mean Ct value to the corresponding standard curve and the dilution factor for each sample type. Absolute quantification was expressed as log10 EGC per 100 mL for the water samples and log10 EGC per gram for the sediment samples. RESULTS: Among all samples tested (N = 151), 89% were contaminated with Shigella, 68% with V. cholerae and NoV-GII, 32% with Giardia, 17% with S. Typhi and 6% with Cryptosporidium. A wide range of concentration of pathogens [range: mean log10 concentration of Giardia = 0.74 EGC/100 mL in drain ultrafiltration samples to mean log10 concentration of NoV-GII and Giardia = 7.11 EGC/100 mL in ABR sludge] was found in all environmental samples. The highest pathogen concentrations were detected in open drains [range: mean log10 concentration = 2.50-4.94 EGC/100 mL], septic tank effluent [range: mean log10 concentration = 3.32-4.65 EGC/100 mL], and ABR effluent [range: mean log10 concentration = 2.72-5.13 EGC/100 mL]. CONCLUSIONS: High concentrations of pathogens (particularly NoV-GII, V.cholerae and Shigella) were frequently detected in environmental samples from two low-income urban neighbourhoods of Dhaka city. The numerous environmental exposure pathways for children and adults make these findings of public health concern. These results should prompt rethinking of how to achieve safe sanitation solutions that protect public health in dense low-income areas. In particular, improved management and maintenance regimes, further treatment of liquid effluent from primary treatment processes, and appropriate application of onsite, decentralised and offsite sanitation systems given the local context.

Virological Quality of Irrigation Water Sources and Pepper Mild Mottle Virus and Tobacco Mosaic Virus as Index of Pathogenic Virus Contamination Level.

Irrigation water is a doorway for the pathogen contamination of fresh produce. We quantified pathogenic viruses [human adenoviruses, noroviruses of genogroups I and II, group A rotaviruses, Aichi virus 1 (AiV-1), enteroviruses (EnVs), and salivirus (SaliV)] and examined potential index viruses [JC and BK polyomaviruses (JCPyVs and BKPyVs), pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV)] in irrigation water sources in the Kathmandu Valley, Nepal. River, sewage, wastewater treatment plant (WWTP) effluent, pond, canal, and groundwater samples were collected in September 2014, and in April and August 2015. Viruses were concentrated using an electronegative membrane-vortex method and quantified using TaqMan (MGB)-based quantitative PCR (qPCR) assays with murine norovirus as a molecular process control to determine extraction-reverse transcription-qPCR efficiency. Tested pathogenic viruses were prevalent with maximum concentrations of 5.5-8.8 log10 copies/L, and there was a greater abundance of EnVs, SaliV, and AiV-1. Virus concentrations in river water were equivalent to those in sewage. Canal, pond, and groundwater samples were found to be less contaminated than river, sewage, and WWTP effluent. Seasonal dependency was clearly evident for most of the viruses, with peak concentrations in the dry season. JCPyVs and BKPyVs had a poor detection ratio and correspondence with pathogenic viruses. Instead, the frequently proposed PMMoV and the newly proposed TMV were strongly predictive of the pathogen contamination level, particularly in the dry season. We recommend utilizing canal, pond, and groundwater for irrigation to minimize deleterious health effects and propose PMMoV and TMV as indexes to elucidate pathogenic virus levels in environmental samples.

Evaluation of exposure scenarios on intentional microbiological contamination in a drinking water distribution network.

Drinking water distribution networks are vulnerable to accidental or intentional contamination events. The objective of this study was to investigate the effects of seeding duration and concentration, exposure pathway (ingestion via drinking of water and tooth brushing and inhalation by taking a shower) and pathogen infectivity on exposure and infection risk in the case of an intentional pathogenic contamination in a drinking water distribution network. Seeding of a pathogen for 10 min and 120 min, and subsequent spreading through a drinking water distribution network were simulated. For exposure via drinking, actual data on drinking events and volumes were used. Ingestion of a small volume of water by tooth brushing twice a day by every person in the network was assumed. Inhalation of contaminated aerosol droplets took place when taking a shower. Infection risks were estimated for pathogens with low (r = 0.0001) and high (r = 0.1) infectivity. In the served population (48 000 persons) and within 24 h, about 1400 persons were exposed to the pathogen by ingestion of water in the 10-min seeding scenario and about 3400 persons in the 120-min scenario. The numbers of exposed persons via tooth brushing were about the same as via drinking of water. Showering caused (inhalation) exposure in about 450 persons in the 10-min scenario and about 1500 in the 120-min scenario. Regardless of pathogen infectivity, if the seeding concentration is 10(6) pathogens per litre or more, infection risks are close to one. Exposure by taking a shower is of relevance if the pathogen is highly infectious via inhalation. A longer duration of the seeding of a pathogen increases the probability of exposure.

Movement of Salmonella serovar Typhimurium and E. coli O157:H7 to Ripe Tomato Fruit Following Various Routes of Contamination.

Salmonella serovars have been associated with the majority of foodborne illness outbreaks involving tomatoes, and E. coli O157:H7 has caused outbreaks involving other fresh produce. Contamination by both pathogens has been thought to originate from all points of the growing and distribution process. To determine if Salmonella serovar Typhimurium and E. coli O157:H7 could move to the mature tomato fruit of different tomato cultivars following contamination, three different contamination scenarios (seed, leaf, and soil) were examined. Following contamination, each cultivar appeared to respond differently to the presence of the pathogens, with most producing few fruit and having overall poor health. The Micro-Tom cultivar, however, produced relatively more fruit and E. coli O157:H7 was detected in the ripe tomatoes for both the seed- and leaf- contaminated plants, but not following soil contamination. The Roma cultivar produced fewer fruit, but was the only cultivar in which E. coli O157:H7 was detected via all three routes of contamination. Only two of the five cultivars produced tomatoes following seed-, leaf-, and soil- contamination with Salmonella Typhimurium, and no Salmonella was found in any of the tomatoes. Together these results show that different tomato cultivars respond differently to the presence of a human pathogen, and for E. coli O157:H7, in particular, tomato plants that are either contaminated as seeds or have a natural opening or a wound, that allows bacteria to enter the leaves can result in plants that have the potential to produce tomatoes that harbor internalized pathogenic bacteria.