Sanitization of hydroponic farming facilities in Singapore: what, why, and how.

This study performed microbial analysis of nutrient film technique (NFT) hydroponic systems on three indoor farms in Singapore (the "what"). To justify the necessity of sanitizing hydroponic systems, strong biofilm-forming bacteria were isolated from the facility and investigated for their influence on Salmonella colonization on polyvinyl chloride (PVC) coupons in hydroponic nutrient solutions (the "why"). Finally, sanitization solutions were evaluated with both laboratory-scale and field-scale tests (the "how"). As a result, the microbiome composition in NFT systems was found to be highly farm specific. The strong biofilm formers Corynebacterium tuberculostearicum C2 and Pseudoxanthomonas mexicana C3 were found to facilitate the attachment and colonization of Salmonella on PVC coupons. When forming dual-species biofilms, the presence of C2 and C3 also significantly promoted the growth of Salmonella (P < 0.05). Compared with hydrogen peroxide (H2O2) and sodium percarbonate (SPC), sodium hypochlorite (NaOCl) exhibited superior efficacy in biofilm removal. At 50 ppm, NaOCl reduced the Salmonella Typhimurium, C2, and C3 counts to <1 log CFU/cm2 within 12 h, whereas neither 3% H2O2 nor 1% SPC achieved this effect. In operational hydroponic systems, the concentration of NaOCl needed to achieve biofilm elimination increased to 500 ppm, likely due to the presence of organic matter accumulated during crop cultivation and the greater persistence of naturally formed multispecies biofilms. Sanitization using 500 ppm NaOCl for 12 h did not impede subsequent plant growth, but chlorination byproduct chlorate was detected at high levels in the hydroponic solution and in plants in the sanitized systems without rinsing. IMPORTANCE: This study's significance lies first in its elucidation of the necessity of sanitizing hydroponic farming systems. The microbiome in hydroponic systems, although mostly nonpathogenic, might serve as a hotbed for pathogen colonization and thus pose a risk for food safety. We thus explored sanitization solutions with both laboratory-scale and field-scale tests. Of the three tested sanitizers, NaOCl was the most effective and economical option, whereas one must note the vital importance of rinsing the hydroponic systems after sanitization with NaOCl.

Pathogenic and transmissional potentials of a Chromobacterium haemolyticum isolate from a hydroponic farm.

AIMS: This study aims to investigate the in vitro pathogenicity of Chromobacterium haemolyticum strain WI5 toward the intestinal tract, its resistance to water treatments, and its potential for foodborne transmission through leafy greens produced in hydroponic systems. METHODS AND RESULTS: C. haemolyticum WI5 caused cytopathic effects in human colon cells HCT116 and exhibited an 8.2-fold higher cell attachment compared to Salmonella serotype Typhimurium. It showed comparable resistance to sodium hypochlorite (NaOCl) and ultraviolet (UV) treatments as Escherichia coli O157: H7 and Pseudomonas aeruginosa but was more susceptible to desiccation. On lettuce, C. haemolyticum WI5 failed to persist, with counts decreasing below the detection limit (≥4 log reductions) after 3 and 2 days at 4 and 25°C, respectively. CONCLUSIONS: C. haemolyticum WI5 demonstrated considerable virulence features and high in vitro pathogenicity toward the intestinal tract. NaOCl and UV treatments were effective in disinfecting C. haemolyticum in water. Due to its high susceptibility to desiccation and poor survivability on lettuce, the foodborne transmission potential of C. haemolyticum is considered limited.

Fate and mitigation of Salmonella contaminated in lettuce (Lactuca sativa) seeds grown in a hydroponic system.

AIMS: We investigated the fate of Salmonella in lettuce seeds grown in a hydroponic system and the potentials of applying photodynamic inactivation (PDI) to enhance microbial safety of hydroponic farming systems. METHODS AND RESULTS: Lettuce was grown from Salmonella-contaminated seeds, and rose bengal-mediated PDI was applied. Without intervention, Salmonella could persist in plants and hydroponic farming environment throughout 6 weeks of lettuce growth. Cross-contamination from Salmonella-inoculated to noninoculated seedlings was observed. PDI significantly decreased Salmonella from 3.90 ± 0.31 log colony-forming unit (CFU) per plant to 2.77 ± 0.49 log CFU per plant without extra illumination needed (p < 0.01) by week six. CONCLUSIONS: Salmonella from contaminated seeds could survive for an extended period in lettuce and hydroponic farming environment and posed serious cross-contamination risks. Rose bengal-mediated PDI showed promise in controlling Salmonella contamination in lettuce in a hydroponic farming setting. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shed light on the serious food safety implications that Salmonella-contaminated lettuce seeds might entail in a hydroponic farming environment and demonstrated rose bengal-mediated PDI as a potential mitigation strategy. These findings contribute to the increasingly relevant field of urban farming systems and their associated food safety concerns.

Lactiplantibacillus plantarum 299v as starter culture suppresses Enterobacteriaceae more efficiently than spontaneous fermentation of carrots.

Fermentation, especially spontaneous fermentation, has become from an ancient food preservation method to a stylish cooking trend in very recent years. Accordingly, the associated food safety should be revisited, particularly since inexperienced people increasingly performing spontaneous fermentation on an ad hoc basis. In this study, three lactic acid bacteria (LAB) strains were tested. Lactiplantibacillus plantarum 299v at high initial inoculation levels (>6 log CFU/mL brine water) effectively eliminated Salmonella spiked in a carrot fermentation system from 4.6 ± 0.2 log CFU/mL to < 1 log CFU/mL after 7 days fermentation at 20 °C. Next, the bacterial enumeration and 16s rRNA gene sequencing results between spontaneous fermentation and fermentation samples using L. plantarum 299v as the starter culture were compared. It was found that the inhibiting effect of L. plantarum 299v could be extended beyond Salmonella spp. to the entire Enterobacteriaceae family during the carrot fermentation. Therefore, our study suggests that in comparison with spontaneous fermentation, selected starter culture such as L. plantarum 299v can consistently improve the safety and quality of fermented foods.

Whole genome sequencing (WGS) fails to detect antimicrobial resistance (AMR) from heteroresistant subpopulation of Salmonella enterica.

Due to rapidly falling costs, whole genome sequencing (WGS) is becoming an essential tool in the surveillance of antimicrobial resistance (AMR) in Salmonella enterica. Although there have been many recent works evaluating the accuracy of WGS in predicting AMR from a large number of Salmonella isolates, little attention has been devoted to deciphering the underlying causes of disagreement between the WGS genotype and experimentally determined AMR phenotype. This study analyzed the genomes of six S. enterica isolates previously obtained from raw chicken which exhibited disagreements between WGS genotype and AMR phenotype. A total of five WGS false negative predictions toward ampicillin, amoxicillin/clavulanate, colistin, and fosfomycin resistance were presented in conjunction with their corresponding empirical phenotypic and/or genetic evidence of heteroresistance. A further case study highlighting the inherent limitations of WGS to detect the underlying genetic mechanisms of colistin heteroresistance was presented. These findings implicate heteroresistance as an underlying cause for false negative WGS-based AMR predictions in S. enterica and suggest that widespread use of WGS in the surveillance of AMR in food isolates might severely underestimate true resistance rates.

Glycogen plays a key role in survival of Salmonella Typhimurium on dry surfaces and in low-moisture foods.

Salmonella enterica is known to survive in desiccate environments and is often associated with low-moisture foods (LMFs). In this work, S. Typhimurium ATCC 14028 was found to survive better by achieving the least reductions (3.17 ± 0.20 Log CFU reduction) compared to S. Tennessee ATCC 10722 (3.82 ± 0.13 Log CFU reduction) and S. Newport ATCC 6962 (6.03 ± 0.36 Log CFU reduction) after 30 days on surfaces with a relative humidity of 49% at ambient temperature. A metabolomic analysis revealed that S. Typhimurium was still active in energy metabolism after 24 h in the desiccate environment and glycogen, an energy reserve, was drastically reduced. We followed up on the glycogen levels over 30 days and found indeed a sharp decline on the first day. However, the glycogens detected on day 7 were significantly higher (P < 0.05) and thereafter remained stable above the original levels until day 30. The expression levels of both glycogen anabolism- and catabolism-related genes (csrA, glgA, glgC, glgX) were significantly up-regulated at all tested points (P < 0.05). The glgA and glgC insertion mutants displayed weaker survivability on both dry surfaces and in representative LMFs (flour and milk powder) compared to the wild-type strain. This work highlights the role of glycogen during different periods of desiccation, which may bring novel insight into mitigating Salmonella by disrupting glycogen metabolism.

Sequential infection of human norovirus and Salmonella enterica resulted in higher mortality and ACOD1/IRG1 upregulation in zebrafish larvae.

Human norovirus (HNoVs) and Salmonella are both very important foodborne pathogens with mixed infection of HNoV and Salmonella reported clinically. With the use of model organism zebrafish (Danio rerio), it was observed that the sequential infection of HNoVs and Salmonella caused lower survival rates (12.5 ± 4.2%) than the single-pathogen infection by Salmonella (31.6 ± 7.3%, P < 0.05) or HNoVs (no mortality observed). Gene expression study with the use of RT-PCR and global transcriptomic analysis revealed that the mortality of zebrafish larvae was very likely due to the harmful inflammatory responses. Specifically, it was noted that the genes encoding aconitate decarboxylase 1 (ACOD1), also known as immunoresponsive gene 1 (IRG1), were significantly upregulated in the sequentially infected zebrafish larvae. The expression of acod1 could lead to mitochondrial reactive oxygen species (ROS) production. The ROS levels were indeed higher in sequentially infected zebrafish larvae than the single-pathogen infected ones (P < 0.05). An immersion treatment of glutathione or citraconate did not affect the microbial loads of HNoVs and Salmonella but significantly reduced the ROS levels and protected the zebrafish larvae by inducing higher survival rates in the sequentially infected zebrafish larvae (P < 0.05). Taken together, this study accumulated new knowledge over the function of ACOD1/IRG1 pathway in infectious diseases, and proposed possible treatment strategies accordingly.

Bacterial antagonism of Chromobacterium haemolyticum and characterization of its putative type VI secretion system.

This study reports the isolation of a new Chromobacterium haemolyticum strain named WI5 from a hydroponic farming facility. WI5 exhibited remarkable bacterial antagonistic properties, eliminating Salmonella, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus (initial inoculum load ∼105 CFU/ml) in dual-species co-culture biofilms. Antagonism was strictly contact-dependent and highly influenced by nutrient availability. Next, we identified a complete suite of putative Type VI secretion system (T6SS) genes in the WI5 genome, annotated the gene locus architecture, and determined the crystal structure of hallmark T6SS tube protein Hcp1, which revealed a hexameric ring structure with an outer and inner diameter of 77 and 45 Å, respectively. Structural comparison with homologs showed differences in the key loops connecting the ß-strands in which the conserved residues are located, suggesting a role of these residues in the protein function. The T6SS is well-known to facilitate interbacterial competition, and the putative T6SS characterized herein might be responsible for the remarkable antagonism by C. haemolyticum WI5. Collectively, these findings shed light on the nature of bacterial antagonism and a putative key virulence determinant of C. haemolyticum, which might aid in further understanding its potential ecological role in natural habitats.

Differential Survivability of Two Genetically Similar Salmonella Thompson Strains on Pre-harvest Sweet Basil (Ocimum basilicum) Leaves.

Although conventionally considered an animal pathogen, recent evidence increasingly suggests that fresh produce may act as significant transmission vehicles and alternative hosts to Salmonella. This study reports the differential survivability of two genetically similar Salmonella Thompson strains (ST 889B and ST 688C) on the adaxial surface of pre-harvest basil (Ocimum basilicum) leaves. Upon inoculation, two distinct phenomena, a dried water-print or a macroscopic lesion, were observed within 24 h. ST 889B survived better than ST 688C on healthy-looking leaves without lesions, possibly due to its higher biofilm-forming ability. Both strains survived better on the leaves with lesions than on the healthy-looking leaves (ST 688C: 4.39 ± 0.68 vs. 2.18 ± 0.29; ST 889B: 4.78 ± 0.12 vs. 2.83 ± 0.18 log CFU per sample at 6 days post-inoculation). ST 889B caused the formation of lesions at a higher frequency [70/117 leaves (59.8%)] than ST 688C [35/96 leaves (36.5%)]. Thus, we highlighted two distinct Salmonella survival strategies in the basil pathosystem and demonstrated gene expression polymorphism (variations in the expression of the same set of genes) as an indispensable strategy in the colonization of plants as hosts by the human pathogens.

Survival of an emerging foodborne pathogen: Group B Streptococcus (GBS) serotype III sequence type (ST) 283-under simulated partial cooking and gastric fluid conditions.

Group B Streptococcus (GBS) was previously not known to be transmitted through food, but an outbreak investigation in Singapore in 2015 documented for the first time an association between GBS Type III Sequence Type 283 infection and consumption of raw fish dishes. As very little is known about the survival of GBS during heat treatment and the stomach transit, its survival under simulated conditions was studied, in comparison with that of Escherichia coli O157:H7 and Listeria monocytogenes. The mean D-values of four GBS strains ranging from 0.72 to 0.88 min in neutral pH tryptone soy broth at 56.4 °C and 0.44-1.43 min at pH 2.35 at 37 °C in simulated gastric fluid, were significantly lower (p < 0.05) than those of E. coli O157:H7 and L. monocytogenes. This study suggests possible factors other than acid or heat resistance of GBS to be instrumental to its pathogenicity.

Characterization of a novel multidrug resistance plasmid pSGB23 isolated from Salmonella enterica subspecies enterica serovar Saintpaul.

BACKGROUND: Salmonella enterica subspecies enterica serovar Saintpaul (S. Saintpaul) is an important gut pathogen which causes salmonellosis worldwide. Although intestinal salmonellosis is usually self-limiting, it can be life-threatening in children, the elderlies and immunocompromised patients. Appropriate antibiotic treatment is therefore required for these patients. However, the efficacy of many antibiotics on S. enterica infections has been greatly compromised due to spreading of multidrug resistance (MDR) plasmids, which poses serious threats on public health and needs to be closely monitored. In this study, we sequenced and fully characterized an S. enterica MDR plasmid pSGB23 isolated from chicken. RESULTS: Complete genome sequence analysis revealed that S. Saintpaul strain SGB23 harbored a 254 kb megaplasmid pSGB23, which carries 11 antibiotic resistance genes responsible for resistance to 9 classes of antibiotics and quaternary ammonium compounds that are commonly used to disinfect food processing facilities. Furthermore, we found that pSGB23 carries multiple conjugative systems, which allow it to spread into other Enterobacteriaceae spp. by self-conjugation. It also harbors multiple types of replicons and plasmid maintenance and addictive systems, which explains its broad host range and stable inheritance. CONCLUSIONS: We report here a novel MDR plasmid pSGB23 harboured by S. enterica. To our knowledge, it carried the greatest number of antibiotic resistance genes with the broadest range of resistance spectrum among S. enterica MDR plasmids identified so far. The isolation of pSGB23 from food sources is worrisome, while surveillance on its further spreading will be carried out based on the findings reported in this study.