Direct Viable Count Method Research Articles

Can antimicrobial photodynamic therapy serve as an effective adjunct protocol for disinfecting the necrotic root canal system? A randomized controlled study

BackgroundBacterial infection plays an important role in persistent periapical lesions and inadequate disinfection of root canals is considered the biggest factor responsible for endodontic treatment failure. Antimicrobial Photodynamic Therapy (aPDT) has become the latest choice to eradicate microorganisms in root canals.ObjectiveThis study aims to evaluate the effectiveness of Antimicrobial Photodynamic Therapy (aPDT) in bacterial count reduction compared to Passive Ultrasonic Activation (PUI) and Ca(OH)2 dressings.Materials and methodsForty-five anterior single canal teeth with medium-sized periapical lesions (2–5 mm) were divided into three groups according to the disinfecting technique (each group consists of 15 canals with 1:1:1 allocation ratio): Group A: Ca(OH)2 dressing. Group B: Passive Ultrasonic Activation (PUI). Group C: Antimicrobial Photodynamic Therapy (aPDT). Direct bacterial viable count method was used to count the colonies forming units (CFU) before and after the disinfecting and the bacterial count reduction was estimated, the statistical analysis was performed at a 95% confidence level using the Chi-square and Mann–Whitney U test.ResultsaPDT showed no statistically significant difference when compared to passive ultrasonic irrigation (P > 0.05) but showed higher and more promising results when compared to Ca(OH)2 dressings (P < 0.05).ConclusionsaPDT has the ability and effectiveness as a disinfecting technique in necrotic and infected root canals.Clinical significance of the studyThe results of this clinical trial provide that aPDT can be considered an adjunct method for root canal disinfection with the same effectiveness as passive ultrasonic irrigation.

Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrioparahaemolyticus in Oysters and Mussels.

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

Cronobacter sakazakii local isolates response to acid stress and their resuscitability

Cronobacter spp. has been reported to cause meningitis, necrotizing enterocolitis, and septicemia in a group of infants through the consumption of powder infant formula. These bacteria are reported to withstand various stress conditions such as heating, drying, low water activity, low pH, etc. A local isolate of Cronobacter sakazakii YRt2a was reportedly survived and entered Viable But Non-Culturable (VBNC) conditions during desiccation stress. This study aims to study the behavior of local isolates of Cronobacter spp. in response to acid stress and its resuscitability. C. sakazakii E2 and YRt2a were grown in TSB at pH 3.0±0.2 or 3.5±0.2. The number of culturable cells and viable cells were enumerated by the Total Plate Count and Direct Viable Count methods, respectively. Resuscitation was done by growing the stress or VBNC cells in TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer. The results showed that C. sakazakii E2 and YRt2a entered VBNC state after 60 mins of exposure to pH 3.0±0.2, while remained culturable after 120 minutes exposure to pH 3.5±0.2. TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer could resuscitate the stress or VBNC cells of C. sakazakii. Stress or VBNC state experienced by C. sakazakii in response to acid tends to be transient and can be resuscitated. C. sakazakii experiencing stress or VBNC may pose a risk for food safety.

Determination of the main bioaerosol components using chemical markers by liquid chromatography–tandem mass spectrometry

This work is part of an extensive research project aimed at the determination and characterization of bioaerosol with a multidisciplinary approach.In this context, one of the main objectives of the project has been the development of a comprehensive analytical method for the determination of different chemical biomarkers of the bioaerosol, by liquid chromatography coupled with tandem mass spectrometry.The following biomarkers have been considered, and correlated to specific components of bioaerosol as unambiguous indicators:•ergosterol → fungal components•chlorophylls, phytosterols (stigmasterol and b-sitosterol), α-tocoferol → vegetable cells and algae•cholesterol → animal cells, vegetable cells and algae.•dipicolinic acid → bacterial spores•muramic and meso-2,6-diaminopimelic acid → bacterial cellsTo verify the method, to find diagnostic ratios and to calculate the appropriate conversion factors, fungal spores, bacterial cells and spores, and algae of known species, commonly airborne, were analysed.The material was subjected to freezing and de-freezing cycles, followed by extraction, hydrolysis and purification of the biomarkers. The chromatographic separation of the bacterial biomarkers was achieved by using a polymeric column, based on Hydrophilic Liquid Interaction with the electrospray ionization mass spectrometric detection, whereas sterols and chlorophylls were separated by a reversed phase column, coupled to atmospheric pressure chemical ionization – tandem mass spectrometer. The optimized method was applied to environmental particulate matter sampled in an outdoor site. Bacterial and fungal content was compared to the results obtained from the classical direct viable counting method in the sampled particulate matter.

Sublethal Injury and Viable but Non-culturable (VBNC) State in Microorganisms During Preservation of Food and Biological Materials by Non-thermal Processes.

The viable but non-culturable (VBNC) state, as well as sublethal injury of microorganisms pose a distinct threat to food safety, as the use of traditional, culture-based microbiological analyses might lead to an underestimation or a misinterpretation of the product’s microbial status and recovery phenomena of microorganisms may occur. For thermal treatments, a large amount of data and experience is available and processes are designed accordingly. In case of innovative inactivation treatments, however, there are still several open points with relevance for the investigation of inactivation mechanisms as well as for the application and validation of the preservation processes. Thus, this paper presents a comprehensive compilation of non-thermal preservation technologies, i.e., high hydrostatic pressure (HHP), pulsed electric fields (PEFs), pulsed light (PL), and ultraviolet (UV) radiation, as well as cold plasma (CP) treatments. The basic technological principles and the cellular and molecular mechanisms of action are described. Based on this, appropriate analytical methods are outlined, i.e., direct viable count, staining, and molecular biological methods, in order to enable the differentiation between viable and dead cells, as well as the possible occurrence of an intermediate state. Finally, further research needs are outlined.

Environmental surveillance for toxigenic Vibrio cholerae in surface waters of Haiti.

Epidemic cholera was reported in Haiti in 2010, with no information available on the occurrence or geographic distribution of toxigenic Vibrio cholerae in Haitian waters. In a series of field visits conducted in Haiti between 2011 and 2013, water and plankton samples were collected at 19 sites. Vibrio cholerae was detected using culture, polymerase chain reaction, and direct viable count methods (DFA-DVC). Cholera toxin genes were detected by polymerase chain reaction in broth enrichments of samples collected in all visits except March 2012. Toxigenic V. cholerae was isolated from river water in 2011 and 2013. Whole genome sequencing revealed that these isolates were a match to the outbreak strain. The DFA-DVC tests were positive for V. cholerae O1 in plankton samples collected from multiple sites. Results of this survey show that toxigenic V. cholerae could be recovered from surface waters in Haiti more than 2 years after the onset of the epidemic.

Listeria monocytogenes Can Form Biofilms in Tap Water and Enter Into the Viable but Non-Cultivable State

Listeria monocytogenes is a foodborne pathogen that can be transmitted through contaminated raw food or by ready-to-eat products that have been in contact with contaminated surfaces. Tap water (TW) is used to wash produce, as a processed food constituent and to wash processing surfaces and floors. The main aim of this work was to investigate the formation and survival of L. monocytogenes biofilms on stainless steel (SS) coupons in TW at 4, 22, 30 and 37 °C. For that, coupons with biofilm were visualised in situ while other coupons were scraped to quantify total cells by SYTO 9, cultivable numbers by plating onto brain heart infusion agar and viable numbers by the direct viable count method. Results showed that L. monocytogenes can form biofilms on SS surfaces in TW at any temperature, including at 4 °C. The number of total cells was similar for all the conditions tested while cultivable numbers varied between the level of detection (<8.3 CFU cm(-2)) and 3.5 × 10(5) CFU cm(-2), meaning between 7.0 × 10(4) and 1.1 × 10(7) cells cm(-2) have entered the viable but non-cultivable (VBNC) state. This work clearly demonstrates that L. monocytogenes can form biofilms in TW and that sessile cells can remain viable and cultivable in some conditions for at least the 48 h investigated. On the other hand, VBNC adaptation suggests that the pathogen can remain undetectable using traditional culture recovery techniques, which may give a false indication of processing surface hygiene status, leading to potential cross-contamination of food products.

Comparing electrochemical disinfection with chlorination for inactivation of bacterial spores in drinking water

Drinking water disinfection techniques without the dosage of chemicals are regarded as more advantageous in terms of costs and practical use. Here we investigated the efficacy of electrochemical disinfection for inactivation of Bacillus subtilis spores – a model microorganism of highly resistant pathogens. The effect of electrochemical disinfection with TinO2n−1 ceramic electrodes which generate active chlorine from chloride in situ, was compared to the traditional chlorination in which active chlorine was produced from addition of sodium hypochlorite. Research was performed on a batch scale with a synthetic buffered drinking water containing 35.5 mg/l of chloride ions. Spore viability was analysed with both cultivation and cell potential for dividing (direct viable count method). The results showed that at similar residual disinfectant concentrations x contact time (CT value), electrochemical disinfection was over three times more effective in neutralizing both cultivable B. subtilis spores and those with cell potential for dividing than traditional chlorination. As in chlorination, electrochemical disinfection was shown to be water-pH dependent and the lowest CT value of 112 mg/l min−1 (2-log removal) was obtained at pH 6. The lowest efficiency for both techniques was observed at pH 8. In conclusion, electrochemical disinfection is a viable in situ method even at low levels of chlorides in drinking water and appears to be more effective than simple chlorination with the addition of the active chlorine species when highly resistant microbial forms are analysed, however, to apply the technology on a large scale additional studies on potential formation of disinfection by-products must be performed.

In Vivo Study of the Survival of Lactobacillus delbruecki subsp. bulgaricus CECT 4005T and Streptococcus thermophilus CECT 801 by DVC‐FISH after Consumption of Fermented Milk

Direct Viable Count (DVC) method has been recently combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of Lactobacillus delbrueckii subsp. bulgaricus CECT 4005T and Streptococcus thermophilus CECT 801. This method has been used to determine their in vitro viability to gastrointestinal juices, being the resistance of L. delbrueckii subsp. bulgaricus and S. thermophilus 26.2% and 9.2%, respectively. On the other hand, an in vivo study has been carried out with the application of this technique for their detection in human feces, after consuming fermented milk. Cells of L. delbrueckii subsp. bulgaricus CECT 4005T were not detected, whereas viable cells of S. thermophilus CECT 801 were detected in a number higher than 10(3) cells per gram in a 30% of the samples after 4 wk of consumption. DVC-FISH is a quick and culture-independent useful method, which has been applied for the 1st time in an in vivo survival study of LAB.

Viability kinetics, induction, resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio cholerae O1 in freshwater microcosm

To study the induction of a viable but nonculturable (VBNC) state in Vibrio cholerae O1 in freshwater, in response to cold temperatures (4°C) and starvation. Vibrio cholerae O1 cells were inoculated in freshwater microcosm and incubated at 4°C. The cells became coccoid, rugose and subsequently nonculturable by day 16 on tryptic soy agar (TSA) and by day 23 on TSA-SP, while 87 and 65% of the cells retained their membrane integrity, respectively. Viable cells were observed until day 30 using direct fluorescent antibody-direct viable count method. In vitro resuscitation was demonstrated by temperature upshift. Utilizing 16S rRNA as an endogenous control, the DNA pol II (27·43-fold), fliG (12·44-fold), ABC transporter (27·11-fold), relA (60·76-fold) and flaC (15·29-fold) were significantly up-regulated in VBNC cells, while the expression of fadL-3 was comparable. The expression of DNA pol II, fliG, ABC transporter, relA and flaC was 3·3, 1·1, 5·9, 5·8 and 1·2-fold, respectively, for resuscitated cells. VBNC cells were found to be virulent, as ctxA and tcpA were expressed. Vibrio cholerae undergoes both phenotypic alteration and genotypic modulation to protect itself from stress in freshwater. Induction and resuscitation of the VBNC state in freshwater is important for an understanding of the epidemiology of cholera in the freshwater environment.

A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus

We have developed a direct viable count (DVC)-FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA-gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC-FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. This technique was successfully applied to detect viable cells in inoculated faeces. Results showed that this DVC-FISH procedure is a quick and culture-independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria.

Specific detection of viable Listeria monocytogenes in Spanish wastewater treatment plants by Fluorescent In Situ Hybridization and PCR

Listeria monocytogenes detection in wastewater can be difficult because of the large amount of background microbiota and the presence of viable but non-culturable forms in this environment. The aim of this study was to evaluate a Fluorescent In Situ Hybridization (FISH) assay combined with Direct Viable Count (DVC) method for detecting viable L. monocytogenes in wastewater samples, as an alternative to conventional culture methods. 16S rRNA sequence data were used to design a specific oligonucleotide probe. In order to assess the suitability of the method, the assays were performed on naturally (n = 87) and artificially (n = 14) contaminated samples and results were compared to those obtained with the isolation of cells on selective media and with a PCR method. The detection limit of FISH and PCR assays was 104 cells/mL without enrichment and 10 cells/mL after enrichment. A total of 47 samples, including 3 samples from effluent sites, yielded FISH positive results for L. monocytogenes. Using DVC-FISH technique, the presence of viable L. monocytogenes cells was detected in 23 out of these 47 FISH positive wastewater samples. PCR and culture methods yielded 27 and 23 positive results, respectively. According to these results, FISH technique has the potential to be used as a sensitive method for the detection and enumeration of L. monocytogenes in environmental wastewater samples.

Inactivation of the gene katA or sodA affects the transient entry into the viable but non-culturable response of Staphylococcus aureus in natural seawater at low temperature

We have investigated the fate of Staphylococcus aureus by starving the cells and maintaining them in natural seawater at 22 and 4°C. At 22°C, cells developed a long-term survival state where about 0.037% of the initial population remained culturable over more than 7months, whereas at 4°C, bacteria lost culturability and transiently entered into the viable but non-culturable state (VBNC). However, after 22days of entry into the VBNC state, the number of viable cells detected via the direct viable count method decreased significantly. We show here that mutational inactivation of catalase (KatA) or superoxide dismutase (SodA) rendered strains hypersensitive to seawater stress at 4°C and consequently, part of the seawater lethality on S. aureus at low temperature is mediated by reactive oxygen species (ROS) during microcosm-survival process. Shifting the temperature from 4 to 22°C of totally non-culturable wild-type cells induced a partial recovery of the population. However, deficiencies in catalase or superoxide dismutase prevent resuscitation ability.

A simple technique for water disinfection with hydrodynamic cavitation: Effect on survival of Escherichia coli

Effect of hydrodynamic cavitation on disinfection of Escherichia coli was investigated in laboratory scale device. The cavitation was generated using a rotor, driven by a simple milling cutter, in the thin layer of water which was circulated from and to a reservoir. Disinfection efficacy was analyzed by measuring respiratory activity using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) method and the ability to multiply with direct viable count (DVC) method. Experiments showed that hydrodynamic cavitation was very effective in reducing bacterial ability to divide. Exposure of 3 min using energy input of 490 W/L stopped the division of 75%of E. coli cells. However, analyses with CTC showed that most of the cells sustained respiratory ability, indicating that bacteria enter active but not culturable (ABNC) state. Thus, the potential of bacteria to resuscitate from ABNC state should be further investigated. The study showed that hydrodynamic cavitation is a simple technique and could potentially be used for drinking water disinfection in rural communities.

Nucleic Acids as Viability Markers for Bacteria Detection Using Molecular Tools

A large set of nucleic acid detection methods with good sensitivity and specificity are now available for the detection of pathogens in clinical, food and environmental samples. Given increasing demand, many efforts have been made to combine these methods to assess viability. Genomic DNA PCR amplification has been shown to be inappropriate for distinguishing viable from dead bacteria owing to DNA stability. Many authors have tried to bypass this difficulty by switching to RNA amplification methods such as reverse transcription-PCR and nucleic acid sequence-based amplification. More recently, researchers have developed methods combining specific sample pretreatment with nucleic acid detection methods, notably ethidium or propidium monoazide pretreatment coupled with PCR DNA detection or direct viable count methods and subsequent fluorescent in situ hybridization of 16S rRNA. This review evaluates the performance of these different methods for viability assessment.

Performance of microbiological control by a point-of-use filter system for drinking water purification

Purification capacity of a faucet mounted type water filter for home use was evaluated, particularly with regard to microbiological performance under different running conditions. Biofilms were formed inside the filter, affecting the bacterial quality of the effluent water. Low flow rate, long stagnation period and high filter temperature were found favorable for bacterial growth inside. By commercial analytical profile index (API) kits, ten different bacterial species were identified in drinking water, four of which were probably contributed to the biofilm formation since they were also present in the biofilm. Fluorescence in situ hybridization (FISH) was used to confirm the API identification results, and direct viable count (DVC) method was employed to improve the sensitivity of FISH for the isolated Acinetobacter spp. and Pseudomonas putida as models. Relationship between the filter operating condition and the bacterial community alteration was partly revealed, which could provide the basic knowledge for the filter design and its practical use.

In-situ enumeration and probing of pyrene-degrading soil bacteria

Inferences about which microorganisms degrade polycyclic aromatic hydrocarbons in contaminated soils have largely been obtained using culture-based techniques, despite the low percentage of microorganisms in soil that are believed to be culturable. We used a substrate-responsive direct viable count method to identify and quantify potential polycyclic aromatic hydrocarbon-degrading bacteria in a soil containing petroleum wastes. Bacteria were extracted and their response to substrates determined in the presence of DNA gyrase inhibitors, which cause viable and active cells to elongate. When yeast extract, a widely used carbon source, was added as a growth substrate, together with nalidixic acid, piromidic acid and ciprofloxacin, a significant increase in elongated cells to 47%, 37% and 22%, respectively, was observed within 24 h. With pyrene as the main substrate, 10 mg L(-1) of nalidixic acid or piromidic acid caused 18-22% and 8-12%, respectively, of the cells to elongate within 24 h; whereas the effect of 0.5 mg L(-1) ciprofloxacin was not significant until 53 h later. Enlarged cells were identified and enumerated by fluorescent in situ hybridization, using Alpha-, Beta- and Gammaproteobacteria, and domain Bacteria-specific probes. The Bacteria-specific probe detected 35-71% of the total microorganisms detected by the DNA-binding dye 4,6-diamidino-2-phenylindole. Initially, 44%, 13% and 5% of the total bacteria in the soil extract were Alpha-, Beta- and Gammaproteobacteria, respectively. Without pyrene or a gyrase inhibitor, these subgroups decreased to 30% of the total population but were predominant with piromidic acid or unchanged with ciprofloxacin when pyrene was the main substrate. The proportion of elongated Alpha- and Betaproteobacteria (potential pyrene degraders) increased significantly (P<0.05). This approach links phylogenetic information with physiological function in situ without the conventional cultivation of bacteria and can be used to probe and enumerate degradative groups at even a finer level of discrimination.

A combination of direct viable count and fluorescent in situ hybridization for estimating Helicobacter pylori cell viability

The viable but non-culturable (VBNC) stage of Helicobacter pylori may represent a problem of public health concern, since these cells cannot be detected by traditional culture methods. In this study, the direct viable count method (DVC) was modified and adapted to H. pylori analysis by testing different times of incubation and concentrations of DNA-gyrase inhibitors. The DVC procedure was combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of H. pylori (DVC–FISH). Incubation with 0.5 μg/ml of novobiocin for 24 h provided the optimal conditions for obtaining 3–5 times the original size of Helicobacter viable cells. Field work performed with various types of water (freshwater and seawater) using the DVC–FISH approach enabled us to confirm the presence of VBNC H. pylori cells in 16 of the 45 analyzed samples. The combination of the modified DVC procedure with FISH can provide a rapid and specific method to detect and identify viable cells of H. pylori in environmental samples.

Avirulence of Viable But Non-Culturable Listeria monocytogenes cells demonstrated by in vitro and in vivo models

The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 degrees C and Scott A strain at 4 degrees C. No culturable bacteria were detected in the VBNC state, although 104 active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state.

Effect of alternative on-site wastewater treatment on the viability and culturability of Salmonella choleraesuis

The objective of this study was to determine how alternative on-site wastewater treatment systems (i.e. subsurface flow constructed wetlands, intermittent sand filters and intermittent peat filters) affect the viability and culturability of Salmonella choleraesuis (serotype typhimurium, ATCC 23567). Influent was a high strength septic tank effluent (BOD5 240-344 mgL(-1), TN approximately 100 mgL(-1), TP approximately 15 mgL(-1)) at the Natural Resources Research Institute's (NRRI) alternative treatment system test facility in northern Minnesota. Treatment systems were inoculated with cultures of S. choleraesuis for 5-7 consecutive days in summer and winter during 1998-99. After the seeding, outflow samples were taken until Salmonella counts were sustained at background levels. In addition to culture-based enumeration, S. choleraesuis abundances were also measured using fluorescent in situ hybridization (FISH) alone and in combination with the direct viable count method (DVC) to determine if plate counts underestimated total and viable Salmonella abundances and if the Salmonella cell viability changed after passing through the treatment systems. In most cases, total and viable cell abundances in treatment system effluents were several orders of magnitude higher than cultured cell abundances. Our results indicate that the culture-based method underestimated viable concentrations of the model pathogen, S. choleraesuis. Salmonella cell viability decreased in effluents during the summer but increased during the winter. Using a culture-based enumeration method alone to determine removal efficiencies of bacterial indicators and pathogens for wastewater treatment systems may result in artificially high estimates of effective treatment.