Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants

Sublethally injured and VBNC cells may evade detection, resulting in underestimation of a food product’s microbial load. Under favorable conditions, cells may regain their growth capacity and acquire new resistant characteristics, posing a major threat for public health.

In vitro virulence potential, surface attachment and transcriptional response of sublethally injured Listeria monocytogenes following exposure to peracetic acid

The disinfectant Peracetic acid (PAA) can cause high levels of sublethal injury to L. monocytogenes . This study aims to evaluate phenotypic and transcriptional characteristics concerning surface attachment and virulence potential of sublethally injured L. monocytogenes ScottA and EGDe after exposure to 0.75 ppm PAA for 90 min at 4°C and subsequent incubation in TSBY at 4°C. Results showed that injured L. monocytogenes cells (99% of total population) were able to attach (after 2 and 24h) on stainless steel coupons at 4°C and 20°C. In vitro virulence assays using human intestinal epithelial Caco-2 cells showed that injured L. monocytogenes could invade host cells but could not proliferate intracellularly. In vitro virulence response was strain-dependent; injured ScottA was more invasive than EGDe. Assessment of PAA-injury at the transcriptional level showed upregulation of genes ( motB, flaA ) involved in flagellum motility and surface attachment. The transcriptional response of L. monocytogenes EGDe and ScottA was different; only injured ScottA demonstrated upregulation of the virulence genes inlA and plcA . Downregulation of the stress-related genes fri and kat, and upregulation of lmo0669 was observed in injured ScottA. The obtained results indicate that sublethally-injured L. monocytogenes cells may retain part of their virulence properties as well as their ability to adhere on food processing surfaces. Transmission to food products and introduction of these cells in the food chain is therefore a plausible scenario that is worth taking into consideration in terms of risk assessment. Importance L. monocytogenes is the causative agent of listeriosis a serious food-borne illness. Antimicrobial practices, such as disinfectants used for the elimination of this pathogen in food industry can produce a sublethally injured population fraction. Injured cells of this pathogen, that may survive an antimicrobial treatment, may pose a food safety-risk. Nevertheless, knowledge regarding how sublethal injury may impact important cellular traits and phenotypic responses of this pathogen is limited. This work suggests that sublethally injured L. monocytogenes cells maintain the virulence and surface attachment potential and highlights the importance of the occurrence of sublethally injured cells regarding food safety.

Viability, Sublethal Injury, and Release of Cellular Components From Alicyclobacillus acidoterrestris Spores and Cells After the Application of Physical Treatments, Natural Extracts, or Their Components

Alicyclobacillus acidoterrestris is a spoiling microorganism regarded as one of the most important causes of spoilage of fruit juices and acidic products. In this paper, four strains of A. acidoterrestris (type strain-DSM 3922; two wild strains isolated from soil-C8 and C24; wild strain isolated from a spoiled pear juice CB1) were treated through natural extracts/active compounds from essential oils (EOs), and physical treatments were used to assess their susceptibility and the presence of sublethal injury. The characterization of damage was also performed. The results suggest that it is possible to control A. acidoterrestris through alternative approaches, although the effect relied upon the age of spores. In addition to the mere antimicrobial effect, some treatments could cause a sublethal injury on spores. Lemon extract was the most effective treatment for both the antimicrobial effect and the sublethal injury, as evidenced by the release of proteins, and calcium dipicolinate [dipicolinic acid (DPA)] by fresh spores and only DPA (with an exception for C8) by old spores. A sublethal injury with protein release was also found for physical treatments [US (ultrasound) or heating]. For the first time, this paper reports on the existence of a sublethal injury for A. acidoterrestris, and this evidence could also be a challenge, because injured microorganisms could restore their metabolism, or an opportunity to design new preserving treatments.

Research progress on the injury mechanism and detection method of disinfectant-injured Escherichia coli in the drinking water system

Sublethally injured bacteria can still develop into normal bacteria under favorable growth conditions, and their pathogenicity poses a great threat to human health. In the drinking water system, some bacteria cause sublethal injury under the action of disinfectants, that is, disinfectant-injured bacteria. Hence, the detection of disinfectant-injured bacteria and the elucidation of injury mechanisms are of great significance for ensuring the microbial safety of drinking water systems. This article takes the indicator bacteria Escherichia coli as the research object, reviews and summarizes the sublethal injury conditions, damage mechanism, and detection methods of disinfectant-injured bacteria in drinking water, and puts forward a prospect for the future research directions of drinking water disinfection and disinfectant-injured bacteria.

Inactivation and Subsequent Growth Kinetics of Listeria monocytogenes After Various Mild Bactericidal Treatments

This study was carried out to investigate the effects of mild heat, lactic acid, benzalkonium chloride and nisin treatments on the inactivation, sublethal injury, and subsequent growth of Listeria monocytogenes. Results showed that the Bigelow model successfully described the thermal inactivation kinetics, while the Log-linear model with tail consistently offered the most accurate fit to LA, BC, and nisin inactivation curves of cells. Differential plating indicated that percentage of sublethal injury for nisin treated cells was significantly higher than that for the other three treatments. Compared to non-treated cells, significant extension of lag time was observed for all treated cells. The longer exposures to heat treatment contributed to the extended lag time of the survivors. While for LA, BC and nisin treated cells, the longest lag time was not observed at the most severe treatment conditions. The correlation analysis of sublethal injury percentage on the duration of lag time revealed that only heat treatment showed the significant correlation. Overall, the lag time analysis could evaluate a wide range of bacterial injury. Lag time of treated cells was significantly influenced by stress treatments and temperatures of recovery, however, there were not any significant changes in the maximum specific growth rate between treated and non-treated cells under isothermal recovery conditions. The information generated from this study is valuable for utilizing intervention strategies in the elimination or growth inhibition of L. monocytogenes.

Quantifying sublethal Florida manatee-watercraft interactions by examining scars on manatee carcasses

A leading human-related threat to the Florida manatee Trichechus manatus latirostris is collisions with watercraft, which account for 20-25% of reported mortalities. Quantitative threat assessments do not include information on all known manatee-watercraft interactions. These interactions often result in sublethal wounding, usually leaving multiple fresh external wounds in a variety of patterns. These wounds then resolve into well-healed scars. We characterized and quantified watercraft-related scar patterns (1 pattern = 1 strike event) on 2935 nonperinatal carcasses (>150 cm total length) that were recovered from 2007 through 2016 to compare the number of patterns by life stage, sex, and population region and across years. We used generalized linear mixed models to examine the effects of several factors on the probability carcasses having scars and on the number of scar patterns per carcass. The models indicated that approximately 96% of adults, approximately 70% of subadults, and approximately 34% of calves had watercraft-related scars. The raw data showed that 1 in 4 adults had been hit 10 or more times; 5 adult carcasses bore evidence of 40 or more strikes. On average, adult females had more scar patterns than did adult males. Manatees on Florida’s west coast had more scar patterns than did those on the east coast, while carcasses from the less populated Everglades had significantly fewer scar patterns than did those from the rest of the state. These results improve our understanding of the extent of sublethal injury of the Florida manatee caused by boat strikes.