Viable Pathogenic Bacteria Research Articles

Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

Precise, Sensitive Detection of Viable Foodborne Pathogenic Bacteria with a 6-Order Dynamic Range via Digital Rolling Circle Amplification.

The presence of viable pathogenic bacteria in food can lead to serious foodborne diseases, thus posing a risk to human health. Here, we develop a digital rolling circle amplification (dRCA) assay that enables the precise and sensitive quantification of viable foodborne pathogenic bacteria. Directly targeting pathogenic RNAs via a ligation-based padlock probe allows for precisely discriminating viable bacteria from dead one. The one-target-one-amplicon characteristic of dRCA enables high sensitivity and a broad quantitative detection range, conferring a detection limit of 10 CFU/mL and a dynamic range of 6 orders. dRCA can detect rare viable bacteria, even at a proportion as low as 0.1%, which is 50 times more sensitive than the live/dead staining method. The high sensitivity for detecting viable bacteria accommodates dRCA for assessing sterilization efficiency. Based on the assay, we found that, for pasteurization, slightly elevating the temperature to 68 °C can reduce the heating time to 10 min, which may minimize nutrient degradation caused by high-temperature exposure. The assay can serve as a precise tool for estimating the contamination by viable pathogenic bacteria and assessing sterilization, which facilitates food safety control.

Occurrence of viable but non-culturable (VBNC) pathogenic bacteria in tap water of public places

Occurrence of viable but non-culturable (VBNC) pathogenic bacteria in tap water of public places

Bioluminogenic Probe for Rapid, Ultrasensitive Detection of β-Lactam-Resistant Bacteria.

Increasingly difficult-to-treat infections by antibiotic-resistant bacteria have become a major public health challenge. Rapid detection of common resistance mechanisms before empiric antibiotic usage is essential for optimizing therapeutic outcomes and containing further spread of resistance to antibiotics among other bacteria. Herein, we present a bioluminogenic probe, D-Bluco, for rapid detection of β-lactamase activity in viable pathogenic bacteria. D-Bluco is a pro-luciferin caged by a β-lactamase-responsive cephalosporin structure and further conjugated with a dabcyl quencher. The caging and quenching significantly decreased the initial background emission and increased the signal-to-background ratio by more than 1200-fold. D-Bluco was shown to detect a broad range of β-lactamases at the femtomolar level. An ultrasensitive RAPID bioluminescence assay using D-Bluco can detect 102 to 103 colony forming unit per milliliter (cfu/mL) of β-lactamase-producing Enterobacterales in urine samples within 30 min. The high sensitivity and rapid detection make the assay attractive for the use of point-of-care diagnostics for lactam-resistant pathogens.

Current Perspectives on Viable but Non-Culturable Foodborne Pathogenic Bacteria: A Review.

Foodborne diseases caused by foodborne pathogens pose risks to food safety. Effective detection and efficient inactivation of pathogenic bacteria has always been a research hotspot in the field of food safety. Complicating these goals, bacteria can be induced to adopt a viable but non-culturable (VBNC) state under adverse external environmental stresses. When in the VBNC state, pathogens cannot form visible colonies during traditional culture but remain metabolically active and toxic. The resulting false negative results in growth-related assays can jeopardize food safety. This review summarizes the latest research on VBNC foodborne pathogens, including induction conditions, detection methods, mechanism of VBNC formation, and possible control strategies. It is hoped that this review can provide ideas and methods for future research on VBNC foodborne pathogenic bacteria.

Isothermal RNA Amplification for the Detection of Viable Pathogenic Bacteria to Estimate the Salmonella Virulence for Causing Enteritis.

Viable foodborne pathogens can cause intestinal infection and food poisoning. Herein, we reported an RNA assay allowing for sensitive (close to 1 CFU and 1% viable bacteria detectable) and rapid (within 2.5 h) detection of viable pathogenic bacteria by coupling isothermal RNA amplification (nucleic acid sequence-based amplification, NASBA) with a CRISPR/Cas13a system. NASBA allowed direct amplification of 16S rRNA extracted from viable S. enterica (RNAs degrade rapidly in dead bacteria), and the specificity of amplification was ensured using Cas13a/crRNA to recognize the amplicons. We used the CRISPR/Cas13-based NASBA assay (termed cNASBA assay) to investigate the in vivo colonization and intestinal infection of S. enterica in mice. We found that S. enterica was mainly colonized at the cecum, colon, and rectum, and the severity of enteritis caused by S. enterica was determined by the number of viable S. enterica rather than the total count of S. enterica. The cNASBA assay can quantify viable S. enterica and thus can improve the accuracy of virulence estimation compared to qPCR. It shows promise as a reliable tool for monitoring pathogen contamination and biosafety control.

Light-up RNA aptamer signaling-CRISPR-Cas13a-based mix-and-read assays for profiling viable pathogenic bacteria

Viable pathogenic bacteria cause serious human diseases via systemic infections and food poisoning. Herein, we constructed a light-up RNA aptamer signaling-CRISPR-Cas13a assay enabling mix-and-read detection of viable pathogenic bacteria. Directly targeting pathogen RNAs via CRISPR-Cas13a allows precisely discriminating viable bacteria from dead bacteria. We introduced a light-up RNA aptamer, Broccoli, serving as the substate of activated CRISPR-Cas13a to monitor the presence of pathogen RNAs, eliminating the need to use chemically labeled RNA substrate. Sequentially, the assay allows a reverse transcription-free, nucleic acid amplification-free, and label-free quantification of RNA targets and viable pathogenic bacteria. It could detect as low as 10 CFU of Bacillus cereus and precisely quantify viable bacteria with a content ranging from 0% to 100% in 105 CFU total bacteria. The quantification of viable bacteria allows more accurately estimating the ability of B. cereus to spoil food. The RNA assay promises its use in point-of-use detection of viable pathogens and biosafety control.

Direct Metatranscriptome RNA-seq and Multiplex RT-PCR Amplicon Sequencing on Nanopore MinION - Promising Strategies for Multiplex Identification of Viable Pathogens in Food.

Viable pathogenic bacteria are major biohazards that pose a significant threat to food safety. Despite the recent developments in detection platforms, multiplex identification of viable pathogens in food remains a major challenge. A novel strategy is developed through direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing on Nanopore MinION to achieve real-time multiplex identification of viable pathogens in food. Specifically, this study reports an optimized universal Nanopore sample extraction and library preparation protocol applicable to both Gram-positive and Gram-negative pathogenic bacteria, demonstrated using a cocktail culture of E. coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes, which were selected based on their impact on economic loss or prevalence in recent outbreaks. Further evaluation and validation confirmed the accuracy of direct metatranscriptome RNA-seq and multiplex RT-PCR amplicon sequencing using Sanger sequencing and selective media. The study also included a comparison of different bioinformatic pipelines for metatranscriptomic and amplicon genomic analysis. MEGAN without rRNA mapping showed the highest accuracy of multiplex identification using the metatranscriptomic data. EPI2ME also demonstrated high accuracy using multiplex RT-PCR amplicon sequencing. In addition, a systemic comparison was drawn between Nanopore sequencing of the direct metatranscriptome RNA-seq and RT-PCR amplicons. Both methods are comparable in accuracy and time. Nanopore sequencing of RT-PCR amplicons has higher sensitivity, but Nanopore metatranscriptome sequencing excels in read length and dealing with complex microbiome and non-bacterial transcriptome backgrounds.

The synergy of chemical immobilization and electrical orientation of T4 bacteriophage on a micro electrochemical sensor for low-level viable bacteria detection via Differential Pulse Voltammetry

In this work, a wild-type T4 bacteriophage based micro electrochemical sensor (T4B-MES) was developed for specific and sensitive detection of viable pathogenic bacteria. Recently, bacteriophage has been widely applied as recognition elements for bacteria detection due to its low cost, high stability and specificity. Firstly, a systematic study was proposed in this paper to investigate the synergy of externally applied electric field and chemical functionalization on phage immobilization, involving several key factors such as Debye length. According to our experiments, the capture efficiency of the deposited phages had reached the maximum when the Debye length was comparable to the phage size. With the optimized immobilization protocol, the sensitivity of the T4B-MES was then determined with Differential Pulse Voltammetry (DPV), providing a quite low detection limit of 14 ± 5 cfu/mL and a wide dynamic range of 1.9 × 101–1.9 × 108 cfu/mL. In addition, the T4B-MES demonstrated the ability to distinguish viable and dead bacteria cells with high specificity, making it a promising solution in a variety of applications, e.g., water quality monitoring.

Alcohol or Gut Microbiota: Who Is the Guilty?

Alcoholic liver disease (ALD), a disorder caused by excessive alcohol intake represents a global health care burden. ALD encompasses a broad spectrum of hepatic injuries including asymptomatic steatosis, alcoholic steatohepatitis (ASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The susceptibility of alcoholic patients to develop ALD is highly variable and its progression to more advanced stages is strongly influenced by several hits (i.e., amount and duration of alcohol abuse). Among them, the intestinal microbiota and its metabolites have been recently identified as paramount in ALD pathophysiology. Ethanol abuse triggers qualitative and quantitative modifications in intestinal flora taxonomic composition, mucosal inflammation, and intestinal barrier derangement. Intestinal hypermeability results in the translocation of viable pathogenic bacteria, Gram-negative microbial products, and pro-inflammatory luminal metabolites into the bloodstream, further corroborating the alcohol-induced liver damage. Thus, the premise of this review is to discuss the beneficial effect of gut microbiota modulation as a novel therapeutic approach in ALD management.

National Safety Survey of Animal-use Commercial Probiotics and Their Spillover Effects From Farm to Humans: An Emerging Threat to Public Health.

Human-use probiotics have recently been associated with clinical infections and antibiotic resistance transfer, raising public concern over their safety. However, despite their extensive application in aquaculture and animal husbandry, the safety of animal-use probiotics remains poorly described. We evaluated the safety of 92 animal-use probiotics from China. The pattern of spread of pathogens from probiotics and the consequent public health implications were also examined by conducting in-field genomic surveillance at 2 farms. A total of 123 probiotic Bacillus species isolates were obtained from 92 brands of probiotics, of which 45 isolates were resistant to antibiotics. Notably, 33.7% of probiotic products were contaminated with life-threatening pathogens such as Klebsiella pneumoniae. Genomic surveillance at a chicken farm identified an anthrax toxin-positive Bacillus cereus strain in a probiotic product used as a feed supplement, which was transferred into the groundwater and to a nearby fish farm. Following up retrospective analysis of the surveillance data during 2015-2018 in 3 provinces retrieved 2 B. cereus strains from human with intestinal anthrax symptoms and confirmed the transmission of B. cereus from farm to human. Surveillance of anthrax toxin revealed that cya was detected in 8 of 31 farms. This study provides the first national safety survey of animal-use probiotics in China and confirms the spillover effects of probiotics from the farms to human. These results suggest that the large-scale application of pathogen-containing probiotics leads to the transfer of pathogens, with worrisome implications for public health. Good Manufacturing Practice should be implemented during the production of all probiotics.Animal-use probiotic products are frequently contaminated with viable pathogenic bacteria. This study revealed that virulent probiotic organisms and contaminating pathogens were colonized with farm animals and shed into the environment, which facilitated the transfer of pathogens to humans.

Ultrasensitive Detection of Salmonella and Listeria monocytogenes by Small-Molecule Chemiluminescence Probes.

Detection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2-6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy-dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600-fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point-of-care diagnostics.

Ultrasensitive Detection of Salmonella and Listeria monocytogenes by Small‐Molecule Chemiluminescence Probes

AbstractDetection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2–6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy‐dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600‐fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point‐of‐care diagnostics.

The role of urbanization in soil and groundwater contamination by heavy metals and pathogenic bacteria: A case study from Oman

This study assessed the perception of urban residents of A'Seeb city, Oman, about the impact of their activities on environment. A sociological survey using questionnaire was used to know the residents' perceptions about urban gardening, municipal-waste disposal, and soil and water contamination. Viable pathogenic bacteria, water soluble metals, basic cations, salinity, and texture were quantified and identified in soil and groundwater in proximity of urban gardens and municipal-waste disposal sites. The majority of surveyed residents are not paying attention to the negative consequences of their activities on soil and environment. Although the measured heavy metals concentrations in some of the contaminated sites were significant but still below the international standards. Fecal contaminants reported in in some samples from gardens, garbage-disposal sites and groundwater. Human pathogens belonging to risk group-2 including Klebsiella pneumonia, Shigella spp and E. Coil were identified. More socio-environmental studies required to correlate the behavior of urban residents and pollution and to delineate the sources of the detected pathogenic bacteria. Our results set a foundation for future studies on urban soils and associated residence behaviors and practices in Oman and the neighboring Gulf countries.

Evaluation of denaturing gradient gel electrophoresis (DGGE) and next generation sequencing (NGS) in combination with enrichment culture techniques to identify bacteria in commercial microbial-based products

Identification of bacteria in new or existing commercial microbial-based products (MBPs) is important for compliance with government regulations and for human and environmental risk assessment. Research was performed to develop effective methods to identify bacteria present in a MBP using a combined approach of conventional enrichment culture technique and denaturing gradient gel electrophoresis (DGGE) followed by clonal sequencing or next generation sequencing (NGS). Genomic DNA obtained from un-enriched or enriched MBP in MacConkey broth, Azide Dextrose broth, Peptone Water mixed with Polymixine B and Gram Negative (GN) media under three different temperatures (22 °C, 28 °C and 37 °C) were sequenced in two methods for the V3 and V6 hypersensitive regions of 16S ribosomal DNA (rDNA) and compared.Enrichment followed by DGGE and clonal sequence analysis identified 20 bacterial genera in all enriched and un-enriched media. In contrast, NGS was able to identify 114 bacterial families and 134 genera both in V3 and V6 regions. In clonal sequence analysis, in comparison to the un-enriched MBP, the MacConkey broth enriched for Escherichia or Shigella and Morganella species, GN medium enriched for Proteus and Morganella species and Azide Dextrose broth enriched for Vagococcus and Enterococcus species at both 28 °C and 37 °C. Moreover, the enrichment facilitated NGS to record higher numbers of families and genera in all enrichment cultures, comparatively higher variations in V3 region than in V6. More prominently, NGS identified 14 genera and 9 species in the family Enterobacteriaceae compared to only 5 genera identified in the un-enriched control using V6 region variance in MacConkey broth at 28 °C. Increasing the temperature without enrichment identified specific families by V3 and V6 regions.This study indicates that the polyphasic approach with appropriate enrichment and incubation at different temperatures followed by NGS analysis is a promising method for the identification of viable, non-pathogenic or potential pathogenic bacteria in complex MBPs.

Killing mechanism of bacteria within multi-species biofilm by blue light

ABSTRACTObjectives: The aim of the study was to characterize the immediate and delayed effects of non-coherent blue-light treatment on the composition and viability of an in vitro biofilm composed of anaerobic multispecies, as well as the mechanisms involved.Methods: A multispecies biofilm was constructed of Streptococcus sanguinis, Actinomyces naeslundii, Porphyromonas gingivalis and Fusobacterium nucleatum, test groups were exposed to blue light. The multispecies biofilm was explored with a newly developed method based on flow cytometry and confocal microscopy. The involvement of the paracrine pathway in the phototoxic mechanism was investigated by a crossover of the supernatants between mono-species P. gingivalis and F. nucleatum biofilms.Results: Blue light led to a reduction of about 50% in the viable pathogenic bacteria P. gingivalis and F. nucleatum, vs that in the non-exposed biofilm. Biofilm thickness was also reduced by 50%. The phototoxic effect of blue light on mono-species biofilm was observed in P. gingivalis, whereas F. nucleatum biofilm was unaffected. A lethal effect was obtained when the supernatant of P. gingivalis biofilm previously exposed to blue light was added to the F. nucleatum biofilm. The effect was circumvented by the addition of reactive oxygen species (ROS) scavengers to the supernatant.Conclusion: Blue-light has an impact on the bacterial composition and viability of the multispecies biofilm. The phototoxic effect of blue light on P. gingivalis in biofilm was induced directly and on F. nucleatum via ROS mediators of the paracrine pathway. This phenomenon may lead to a novel approach for ‘replacement therapy,’ resulting in a less periodonto-pathogenic biofilm.

The impact of municipal sewage sludge stabilization processes on the abundance, field persistence, and transmission of antibiotic resistant bacteria and antibiotic resistance genes to vegetables at harvest

Biosolids were obtained from four Ontario municipalities that vary in how the sewage sludge is treated. These included a Class B biosolids that was anaerobically digested, a Class A biosolids that were heat treated and pelletized (Propell), and two Class A biosolids that were stabilized using either the N-Viro (N-Rich) or Lystek (LysteGro) processes. Viable enteric indicator or pathogenic bacteria in the biosolids were enumerated by plate count, gene targets associated with antibiotic resistance or horizontal gene transfer were detected by PCR, and a subset of these gene targets were quantified by qPCR. Following application at commercial rates to field plots, the persistence of enteric bacteria and gene targets in soil was followed during the growing season. Carrots, radishes and lettuce were sown into the amended and unamended control plots, and the diversity and abundance of gene targets they carried at harvest determined. All three Class A biosolids carried fewer and less abundant antibiotic resistance genes than did the Class B biosolids, in particular the very alkaline N-Viro product (N-Rich). Following application, some gene targets (e.g. int1, sul1, strA/B, aadA) that are typically associated with mobile gene cassettes remained detectable throughout the growing season, whereas others (e.g. ermB, ermF, blaOXA20) that are not associated with cassettes became undetectable within three weeks or less. At harvest a larger number of gene targets were detected on the carrots and radishes than in the lettuce. Overall, land application of Class A biosolids will entrain fewer viable bacteria and genes associated with antibiotic resistance into crop ground than will amendment with Class B biosolids.

EmPis-1L, an Effective Antimicrobial Peptide Against the Antibiotic-Resistant VBNC State Cells of Pathogenic Bacteria.

The antibiotic-resistant viable but non-culturable (VBNC) pathogenic bacteria are considered as a new threat to public health. Antimicrobial peptides (AMPs), possessing bactericidal effects in a rapid membrane attacking mode, are supposed to be effective against bacteria entering the VBNC state. In the current study, the activity of grouper AMP piscidin killing the VBNC state cells of pathogenic bacteria Escherichia coli O157, Staphylococcus aureus, and Vibrio parahaemolyticus OS4 was studied. After entering the VBNC state, cells of E. coli O157, S. aureus, and V. parahaemolyticus OS4 developed resistance to the antibiotics Ampicillin and Kanamycin. Rather than truncated form of Malabar grouper piscidin 1 (EmPis-1S), full-length Malabar grouper piscidin 1 (EmPis-1L) showed strong activity to kill the above VBNC bacteria. The VBNC state cells (1 × 105CFU/mL) of the three species of bacteria could be totally lysed by 10μmol/L of EmPis-1L in 1h. The VBNC state cells of S. aureus were most susceptible to EmPis-1L, which killed the cells by 100% in 30min at the low concentration of 2.0μmol/L. In addition, EmPis-1L at the concentration of no more than 10μmol/L showed no observed toxicity to human lung carcinoma epithelial cells (A549) and mouse neuroblastoma cells (N2a). Accordingly, EmPis-1L could be a promisingly safe and efficient agent for eliminating the traditional antibiotic-resistant VBNC state cells of pathogenic bacteria, E. coli, S. aureus, and V. parahaemolyticus.

Fates of Salmonella Enteritidis and Cronobacter sakazakii in various multiple‐strain yogurts and kefir during cold storage

AbstractMany multiple‐strain fermented milks such as yogurt and kefir have been developed and consumed to improve their nutritional and functional benefits. Since these fermented milks can be a vehicle of foodborne illness, we investigated the fates of Salmonella Enteritidis and Cronobacter sakazakii in four fermented milks: multiple‐lactic acid bacteria (multi‐LAB) yogurt, multiple‐LAB‐Bifidobacterium (multi‐LAB‐BIF) yogurt, pH 4.5 kefir (mild kefir), and pH 3.6 kefir (strong kefir). Each was inoculated with 5.6 and 5.8 log cfu/ml of S. Enteritidis and C. sakazakii, respectively, and stored at 4°C for 5 days. Strong and mild kefirs exhibited more potent antimicrobial activities than multi‐LAB and multi‐LAB‐BIF yogurts, inactivating all viable pathogenic bacteria within 1 and 5 days, respectively. Despite having lower pH values than mild kefir (pH 4.49), multi‐LAB (pH 4.25) and multi‐LAB‐BIF (pH 4.38) yogurts failed to clear viable S. Enteritidis cells in 5 days (> 5 log cfu/ml cells survived).Practical applicationsYogurt is one of the most popular fermented milk, and has been implicated in several human foodborne outbreaks. Kefir is unique fermented milk containing multiple strains of lactic acid bacteria and yeast. In this study, we investigated the fates of Salmonella Enteritidis and Cronobacter sakazakii in these fermented milks. Two types of commercial yogurt failed to clear viable S. Enteritidis cells in 5 days, whereas kefir successfully killed all viable S. Enteritidis and C. sakazakii cells in 5 and 4 days, respectively, even with significantly higher pH values suggesting that pH might not be a suitable indicator to ensure the microbiological safety of yogurts and kefir, and that pathogens face other antimicrobial hurdles than low pH and high acidity. Considering the potent self‐clearance effects of kefir against foodborne pathogens, novel yogurt products containing strains with potent antimicrobial activity such as kefir microorganisms could be newly developed in the future.

Effect of Simulated Sanitizer Carryover on Recovery of Salmonella from Broiler Carcass Rinsates

Numerous antimicrobial chemicals are currently utilized as processing aids with the aim of reducing pathogenic bacteria on processed poultry carcasses. Carryover of active sanitizer to a carcass rinse solution intended for recovery of viable pathogenic bacteria by regulatory agencies may cause false-negative results. This study was conducted to document the potential carryover effect of five sanitizing chemicals commonly used as poultry processing aids for broilers in a postchill dip. The effect of postdip drip time on the volume of sanitizer solution carryover was first determined by regression of data obtained from 10 carcasses. The five sanitizer solutions were diluted with buffered peptone water at 0-, 1-, and 5-min drip time equivalent volumes as determined by the regression analysis. These solutions were then spiked to 10(5) CFU/ml with a mixture of five nalidixic acid-resistant Salmonella enterica serovars, stored at 4°C for 24 h, and finally enumerated by plate count on brilliant green sulfa agar containing nalidixic acid. At the 0- and 1-min drip time equivalents, no Salmonella recovery was observed in three of the five sanitizers studied. At the 5-min drip time equivalent, one of these sanitizers still exhibited significant (P ≤ 0.05) bactericidal activity. These findings potentially indicate that the currently utilized protocol for the recovery of Salmonella bacteria from postchill sanitizer interventions may lead to false-negative results due to sanitizer carryover into the carcass rinsate.