Microbiome of Free-Living Amoebae (FLA) Isolated from Fresh Organic Produce: Potential Risk to Consumers?

In response to growing global interest in organic agriculture, this study delves into the microbial landscape of organically grown raw produce with a focus on food safety. Vegetables that are consumed raw are potential vehicles for the transmission of any type of microorganism capable of causing human disease. Free-living amoebae (FLA) are ubiquitous protozoa found in many ecosystems and can serve as hosts to pathogenic bacteria. So far, data regarding the FLA bacterial microbiome in fresh produce remain scarce and are non-existent for those of organic origin. Thus, the aim of this preliminary work is to characterize the microbiome of FLA in commonly consumed raw vegetables to know their possible implications for consumers. A total of 40 organic cabbage, lettuce, spinach, and strawberry samples were analyzed. FLA were found in all samples, and their bacterial microbiome was obtained via amplicon sequencing using the Illumina MiSeq platform and pair-end protocol. Acanthamoeba spp. and Vermamoeba vermiformis were identified via qPCR in 65.0% and 25.0% of the samples, respectively. Regarding the bacterial microbiome of FLA, the most abundant genera were Pseudomonas (1.8-17.8%) and Flavobacterium (1.7-12.6%). Bacteria not previously related to FLA, such as Prosthecobacter or Cellvibrio, are described in this work. Importantly, several bacterial genera found within the FLA microbiome were identified as potential human pathogens, including Pseudomonas, Flavobacterium, Arcobacter, Klebsiella, Mycobacterium, Salmonella and Legionella. This is the first work in which FLA microbiome isolated from organic products has been characterized, underscoring the significance of understanding FLA's role as carriers of pathogenic bacteria in the context of organic food safety.

Prevalence and Characterization of Beta-Lactam and Carbapenem-Resistant Bacteria Isolated from Organic Fresh Produce Retailed in Eastern Spain.

Fresh fruits and vegetables are potential reservoirs for antimicrobial resistance determinants, but few studies have focused specifically on organic vegetables. The present study aimed to determine the presence of third-generation cephalosporin (3GC)- and carbapenem-resistant Gram-negative bacteria on fresh organic vegetables produced in the city of Valencia (Spain). Main expanded spectrum beta-lactamase (ESBL)- and carbapenemase-encoding genes were also detected in the isolates. One hundred and fifteen samples were analyzed using selective media supplemented with cefotaxime and meropenem. Resistance assays for twelve relevant antibiotics in medical use were performed using a disc diffusion test. A total of 161 isolates were tested. Overall, 33.5% presented multidrug resistance and 16.8% were resistant to all ß-lactam antibiotics tested. Imipenem resistance was observed in 18% of isolates, and low resistance levels were found to ceftazidime and meropenem. Opportunistic pathogens such as Acinetobacter baumannii, Enterobacter spp., Raoultella sp., and Stenotrophomonas maltophilia were detected, all presenting high rates of resistance. PCR assays revealed blaVIM to be the most frequently isolated ESBL-encoding gene, followed by blaTEM and blaOXA-48. These results confirm the potential of fresh vegetables to act as reservoirs for 3GC- and carbapenem-producing ARB. Further studies must be carried out to determine the impact of raw organic food on the spread of AMRs into the community.

Simultaneous Detection of Four Main Foodborne Pathogens in Ready-to-Eat Food by Using a Simple and Rapid Multiplex PCR (mPCR) Assay.

The increasing consumption of organic or ready-to-eat food may cause serious foodborne disease outbreaks. Developing microbiological culture for detection of food-borne pathogens is time-consuming, expensive, and laborious. Thus, alternative methods such as polymerase chain reaction (PCR) are usually employed for outbreaks investigation. In this work, we aimed to develop a rapid and simple protocol for the simultaneous detection of Escherichia coli (E coli), Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus) and Salmonella enterica (S. enterica), by the combination of an enrichment step in a single culture broth and a multiplex PCR (mPCR) assay. The effectiveness of several enrichment media was assessed by culture and PCR. Buffered peptone water (BPW) was selected as the optimum one. Then, mPCR conditions were optimized and applied both to pure co-cultures and artificially inoculated food samples (organic lettuce and minced meat). In the culture medium inoculated at 100 CFU/mL, mPCR was able to detect the four microorganisms. When performed on artificially food samples, the mPCR assy was able to detect E. coli, S. enterica, and L. monocytogenes. In conclusion, BPW broth can effectively support the simultaneous growth of E. coli, S. aureus, L. monocytogenes, and S. enterica and could be, thus, used prior to a mPCR detection assay in ready-to-eat food, thereby considerably reducing the time, efforts and costs of analyzes.

Optimization of pre- treatments with Propidium Monoazide and PEMAX™ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells.

Accurate detection of H. pylori in different environmental and clinical samples is essential for public health strtdudies. Now, a big effort is being made to design PCR methodologies that allow for the detection of viable and viable but non-culturable (VBNC) H. pylori cells, by achieving complete exclusion of dead cells amplification signals. The use of DNA intercalating dyes has been proposed. However, its efficacy is still not well determined. In this study, we aimed to test the suitability of PMA and PEMAX™ dyes used prior to qPCR for only detecting viable cells of H. pylori. Their efficiency was evaluated with cells submitted to different disinfection treatments and confirmed by the absence of growth on culture media and by LIVE/DEAD counts. Our results indicated that an incubation period of 5 min for both, PMA and PEMAX™, did not affect viable cells. Our study also demonstrated that results obtained by using intercalating dyes may vary depending on the cell stress conditions. In all dead cell's samples, both PMA and PEMAX™ pre-qPCR treatments decreased the amplification signal (>103 Genomic Units (GU)), although none of them allowed for its disappearance confirming that intercalating dyes, although useful for screening purposes, cannot be considered as universal viability markers. To investigate the applicability of the method specifically to detect H. pylori cells in environmental samples, PMA-qPCR was performed on samples containing the different morphological and viability states that H. pylori can acquire in environment. The optimized PMA-qPCR methodology showed to be useful to detect mostly (but not only) viable forms, regardless the morphological state of the cell.

Determination of the bacterial microbiome of free-living amoebae isolated from wastewater by 16S rRNA amplicon-based sequencing.

Free-living amoebae (FLA) are ubiquitous protozoa commonly found in water. FLA are well-established hosts for amoeba-resistant bacteria, most of which are pathogenic, and offer them shelter from adverse environmental conditions or water treatments. Since there is very little knowledge about the complete bacterial microbiome of FLA, in this work the bacterial microbiome of FLA isolated from wastewater both after secondary and tertiary treatments was studied by amplicon-based sequencing. FLA were detected in 87.5% and 50.0% of wastewater samples taken after secondary and tertiary disinfection treatments, respectively. The most abundant bacterial phyla were Proteobacteria, Planctomycetes, Bacteroidetes and Firmicutes, which represented 83.77% of the total bacterial FLA microbiome. The most abundant class of bacteria was Gammaproteobacteria, which contains an important number of relevant pathogenic bacteria. The bacteria of public health concern Aeromonas, Arcobacter, Campylobacter, Helicobacter, Klebsiella, Legionella, Mycobacterium, Pseudomonas and Salmonella were detected as part of the FLA microbiome. Although different microbial communities were identified in each sample, there is no correlation between the microbiome of FLA and the extent of wastewater treatment. To our knowledge, this is the first work in which the bacterial microbiome of FLA isolated from wastewater is studied. Obtained results indicate that FLA are hosts of potentially pathogenic bacteria in treated wastewater used for irrigation, which may pose a public health threat.

Evaluation of different culture media for detection and quantification of H. pylori in environmental and clinical samples.

The objective of the present study was to establish the most suitable culture medium for the isolation of H. pylori from environmental and clinical samples. Ten different culture media were compared and evaluated. Four of them had been previously described and were modified in this study. The rest of the media were designed de novo. Three different matrices, tap water, wastewater, and feces, were inoculated with serial dilutions of H. pylori NCTC 11637 strain at a final concentration of 104 and 103 CFU/ml and the recovery rates were calculated. From inoculated tap water and wastewater samples, H. pylori colonies were recovered from four out of the analyzed culture media. When fecal samples were analyzed, the isolation of the pathogen under study was only possible from two culture media. Different optimal media were observed for each type of sample, even for wastewater and stool samples. Nevertheless, our results indicated that the combination of Dent Agar with polymyxin B sulfate did not inhibit the growth of H. pylori and was highly selective for its recovery, regardless of the sample origin. Thus, we propose the use of this medium as a diagnostic tool for the isolation of H. pylori from environmental and clinical samples, as well as for epidemiological studies.

Caenorhabditis elegans as an in vivo model to assess fucoidan bioactivity preventing Helicobacter pylori infection.

Currently, Helicobacter pylori is the unique biological carcinogenic agent. The search for antimicrobial alternatives to antibiotics against this pathogen has been categorized as a priority due to the drastic failure associated with current applied antibiotic therapy. The present study assessed the bioactive antimicrobial capability of fucoidan ("Generally Recognized as Safe" approval - European Commission December 2017) from different species of Phaeophyceae algae (Fucus vesiculosus, Undaria pinnatifida, Macrocystis pyrifera) against H. pylori. All the studied fucoidans showed bacteriostatic and bactericidal effects at the studied concentrations [5-100] µg ml-1 and exposure times [0-7 days]. The most effective anti-H. pylori fucoidan was validated in Caenorhabditis elegans as an in vivo model. C. elegans feed was supplemented with Undaria pinnatifida [0-100] µg ml-1 fucoidan, resulting in a significant improvement in lifespan, lowered H. pylori concentration in the digestive tract, and increased egg-laying pattern. New research lines proposing this compound as an active agent in nutraceutical and preventive novel therapies should be opened.

Deep-amplicon sequencing (DAS) analysis to determine the presence of pathogenic Helicobacter species in wastewater reused for irrigation.

Wastewater has become one of the most important and least expensive water for the agriculture sector, as well as an alternative to the overexploitation of water resources. However, inappropriate treatment before its reuse can result in a negative impact on the environment, such as the presence of pathogens. This poses an increased risk for environmental safety, which can subsequently lead to an increased risk for human health. Among all the emerging wastewater pathogens, bacteria of the genus Helicobacter are some of the most disturbing ones, since they are directly related to gastric illness and hepatobiliary and gastric cancer. Therefore, the aim of this study was to determine the presence of potentially pathogenic Helicobacter spp. in treated wastewater intended for irrigation. We used a next generation sequencing approach, based on Illumina sequencing in combination with culture and other molecular techniques (qPCR, FISH and DVC-FISH), to analyze 16 wastewater samples, with and without an enrichment step. By culture, one of the direct samples was positive for H. pylori. FISH and DVC-FISH techniques allowed for detecting viable Helicobacter spp., including H. pylori, in seven out of eight samples of wastewater from the tertiary effluents, while qPCR analysis yielded only three positive results. When wastewater microbiome was analyzed, Helicobacter genus was detected in 7 samples. The different molecular techniques used in the present study provided evidence, for the first time, of the presence of species belonging to the genus Helicobacter such as H. pylori, H. hepaticus, H. pullorum and H. suis in wastewater samples, even after disinfection treatment.

Correlation among fecal indicator bacteria and physicochemical parameters with the presence of Helicobacter pylori DNA in raw and drinking water from Bogotá, Colombia.

BACKGROUND: The quality of raw and drinking water is a matter of considerable concern due to the possibility of fecal contamination. To assess the quality and public health risk of different types of water, the fecal indicator bacteria (FIB) are used. However, some pathogens, such as Helicobacter pylori, may be present in water when FIB cannot be found. H pylori is recognized as the causative agent of chronic gastritis, peptic and duodenal ulcers, and gastric cancer. The aim of this study was to evaluate the relationships among physicochemical parameters, FIB concentrations, and the presence of H pylori DNA in raw and drinking water from Bogotá, Colombia. MATERIALS AND METHODS: A total of 310 water samples were collected 1 day per week from July 2015 to August 2016, and physicochemical parameters (pH, turbidity, conductivity, and residual free chlorine) were measured. Presence of H pylori DNA was determined and quantified by quantitative polymerase chain reaction (qPCR). Fecal indicator bacteria (total coliforms, Escherichia coli, and spores of sulfite-reducing Clostridia) were enumerated by using standard culture techniques. RESULTS: Thirty of 155 (31%) raw water samples and forty-eight of 155 (38.7%) drinking water samples were positive for the presence of H pylori. No statistically significant relationships were found between physicochemical parameters or FIB with the presence or absence of H pylori in any sample (P < 0.05). CONCLUSIONS: This study provides evidence of the presence of H pylori DNA in raw and drinking water in Bogotá, and shows that the detection and enumeration of FIB and physicochemical parameters in water do not correlate with the risk of contamination with H pylori.

Risk Characterization of Antibiotic Resistance in Bacteria Isolated from Backyard, Organic, and Regular Commercial Eggs.

This study was conducted to assess the risk due to antimicrobial-resistant strains of Salmonella spp., Listeria monocytogenes, and Escherichia coli isolated from the eggshell and the contents of eggs bought in markets in Valencia (Spain). Thirty-four samples from three different production styles were analyzed: standard ( n = 34), organic ( n = 16), and backyard ( n = 10) eggs. L. monocytogenes was not isolated in any style of production. Only one strain of Salmonella was isolated from standard production, which was resistant to ciprofloxacin and amoxicillin. E. coli strains were resistant in 22% of the isolates from organic production, 12.25% from standard production, and 11.23% from backyard production. In all cases, the highest resistance was observed for amoxicillin-clavulanate. None of the isolates from standard and backyard eggs were resistant to chloramphenicol, ciprofloxacin, gentamycin, and streptomycin, while only ceftriaxone was found to be effective against all E. coli isolates from organic eggs. ß-Lactamase genes blaTEM , blaSHV, and blaCMY-2 and the resistance genes for tetracycline tetA, tetB, and tetC were tested. The most commonly detected antimicrobial resistance genes among the E. coli isolates were tetA (49.30%), blaTEM (47.89%), and tetB (36.62%). Overall, a maximum public health risk is associated with ß-lactam antibiotics.

Helicobacter Pylori Detection in Shellfish: A Real-Time Quantitative Polymerase Chain Reaction Approach.

Shellfish is a highly valuated natural food product that is usually consumed minimally processed. Some foodborne pathogens have been associated to marine products and isolated from aquatic environments. Helicobacter pylori emerges as one of the most concerning human pathogens associated to water and, thereby, it could be present in raw and slightly treated marine food products. The present research work aimed to detect the presence of H. pylori in Spanish commercial samples of shellfish (mussels, clams, and cockles) by means of a quantitative real-time polymerase chain reaction (qPCR) approach based on the vacuolating cytotoxin A (vacA) gene specificity. Putative H. pylori amplicons were confirmed by sequencing. qPCR was positive for 12 out of the 100 samples, being 67% (8/12) from mussels, 25% (3/12) from clams, and only 8% (1/12) from cockles. After sequencing, three of the amplicons showed 97-99% homology with the H. pylori vacA gene. Quantitative results indicate that the levels of contamination remained below 102 log10 colony forming units per mL (CFU/mL). The present research shows for the first time the effectiveness of the optimized qPCR in the identification of potentially H. pylori contaminated shellfish products. Our results confirm the presence of H. pylori in shellfish from the Spanish western seacoast and verify the possible relationship between the presence of H. pylori in seawater and the role of contaminated seafoods as vehicles of H. pylori entrance into the food chain.

Detection of Helicobacter pylori in drinking water treatment plants in Bogotá, Colombia, using cultural and molecular techniques.

Helicobacter pylori is one of the most common causes of chronic bacterial infection in humans, and a predisposing factor for peptic ulcer and gastric cancer. The infection has been consistently associated with lack of access to clean water and proper sanitation. H. pylori has been detected in surface water, wastewater and drinking water. However, its ability to survive in an infectious state in the environment is hindered because it rapidly loses its cultivability. The aim of this study was to determine the presence of cultivable and therefore viable H. pylori in influent and effluent water from drinking water treatment plants (DWTP). A total of 310 influent and effluent water samples were collected from three drinking water treatment plants located at Bogotá city, Colombia. Specific detection of H. pylori was achieved by culture, qPCR and FISH techniques. Fifty-six positive H. pylori cultures were obtained from the water samples. Characteristic colonies were covered by the growth of a large number of other bacteria present in the water samples, making isolation difficult to perform. Thus, the mixed cultures were submitted to Fluorescent in situ Hybridization (FISH) and qPCR analysis, followed by sequencing of the amplicons for confirmation. By qPCR, 77 water samples, both from the influent and the effluent, were positive for the presence of H. pylori. The results of our study demonstrate that viable H. pylori cells were present in both, influent and effluent water samples obtained from drinking water treatment plants in Bogotá and provide further evidence that contaminated water may act as a transmission vehicle for H. pylori. Moreover, FISH and qPCR methods result rapid and specific techniques to identify H. pylori from complex environmental samples such as influent water.

High prevalence of Salmonella spp. in wastewater reused for irrigation assessed by molecular methods.

Salmonella spp. is one of the most important causal agents of food-borne illness in developed countries and its presence in irrigation water poses a risk to public health. Its detection in environmental samples is not easy when culture methods are used, and molecular techniques such as PCR or ribosomal rRNA probe hybridization (Fluorescent in situ Hybridization, FISH) are outstanding alternatives. The aim of this work was to determine the environmental risk due to the presence of Salmonella spp. in wastewater by culture, PCR and FISH. A new specific rDNA probe for Salmonella was designed and its efficiency was compared with the rest of methods Serotype and antibiotic resistance of isolated strains were determined. Forty-five wastewater samples (collected from two secondary wastewater treatment plants) were analysed. Salmonella strains were isolated in 24 wastewater samples (53%), two of them after disinfection treatment. Twenty-three Salmonella strains exhibited resistance to one or more antimicrobial agent. Analysis of wastewater samples yielded PCR positive results for Salmonella in 28 out of the 45 wastewater samples (62%). FISH analysis allowed for the detection of Salmonella in 27 (60%) samples. By using molecular methods, Salmonella was detected in four samples after disinfection treatment. These results show the prevalence of Salmonella in reclaimed wastewater even after U.V. disinfection, what is a matter of public health concern, the high rates of resistance to antibiotics and the adequacy of molecular methods for its rapid detection. FISH method, with SA23 probe developed and assayed in this work provides a tool for detecting Salmonella in water within few hours, with a high rate of effectiveness.

Isolation, molecular identification and quinolone-susceptibility testing of Arcobacter spp. isolated from fresh vegetables in Spain.

Some species of the Arcobacter genus are considered emerging foodborne and waterborne enteropathogens. However, the presence of Arcobacter spp. in vegetables very little is known, because most studies have focused on foods of animal origin. On the other hand, quinolones are considered as first-line drugs for the treatment of infection by campylobacteria in human patients, but few data are currently available about the resistance levels to these antibiotics among Arcobacter species. Therefore, the aim of this study was to investigate the presence and diversity of arcobacters isolated from fresh vegetables such as lettuces, spinaches, chards and cabbages. Resistance to quinolones of the isolates was also investigated. One hundred fresh vegetables samples purchased from seven local retail markets in Valencia (Spain) during eight months were analysed. The study included 41 lettuces, 21 spinaches, 34 chards and 4 cabbages. Samples were analysed by culture and by molecular methods before and after enrichment. By culture, 17 out of 100 analysed samples were Arcobacter positive and twenty-five isolates were obtained from them. Direct detection by PCR was low, with only 4% Arcobacter spp. positive samples. This percentage increased considerably, up 20%, after 48 h enrichment. By polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), 17 out of the 25 isolates were identified as A. butzleri and 8 as A. cryaerophilus. Only two A. butzleri isolates showed resistance to levofloxacin and ciprofloxacin. The sequencing of a fragment of the QRDR region of the gyrA gene from the quinolones-resistant isolates revealed the presence of a mutation in position 254 of this gene (C-T transition). This study is the first report about the presence of pathogenic species of Arcobacter spp. in chards and cabbages and confirms that fresh vegetables can act as transmission vehicle to humans. Moreover, the presence of A. butzleri quinolone resistant in vegetables could pose a potential public health risk.

Detection, Identification, and Antimicrobial Susceptibility of Arcobacter spp. Isolated from Shellfish in Spain.

This work aimed to determine the presence of Arcobacter spp. in shellfish and to determine its susceptibility to quinolones. One hundred samples (41 mussels, 37 clams, and 22 cockles) were purchased from different local retail shops in Valencia, Spain, from September 2013 to June 2015. All samples were analyzed simultaneously by culture, after an enrichment step, and by polymerase chain reaction (PCR), directly and after enrichment. The susceptibility to levofloxacin and ciprofloxacin of the isolates was tested using the disk-diffusion test and E-test strips method. To clarify the mechanism of quinolone resistance, a fragment of the quinolone resistance-determining region of the gyrA gene was sequenced. Thirty-seven samples were positive and 49 isolates were obtained by culture, and Arcobacter spp. DNA was detected in 32% of the samples by PCR. However, after 48-h enrichment, the number of positive samples increased, and 68 of the 100 samples yielded the specific Arcobacter spp. PCR product. In addition, 49 isolates were identified by PCR-restriction fragment length polymorphism. The most commonly found species was Arcobacter butzleri (25 isolates, 51.03%) followed by Arcobacter cryaerophilus (19 isolates, 38.77%) and Arcobacter defluvii (5 isolates, 10.20%). Only three isolates of A. butzleri were resistant to both antibiotics. A mutation C to T transition in the position 254 of the gyrA gene was present in the three resistant isolates. This study confirms that pathogenic arcobacters are frequently found in edible shellfish samples. Moreover, this is the first time that A. butzleri and A. cryaerophilus have been isolated from cockles.

Antimicrobial resistance of Escherichia coli isolated in newly-hatched chickens and effect of amoxicillin treatment during their growth.

The use of antimicrobials in food animals is the major determinant for the propagation of resistant bacteria in the animal reservoir. However, other factors may also play a part, and in particular vertical spread between the generations has been suggested to be an important transmission pathway. The objective of this paper was to determine the resistance patterns of Escherichia coli isolated from newly-hatched chickens as well as to study the antibiotic pressure effect when amoxicillin was administered during their growing period. With this aim, meconium from 22 one-day-old Ross chickens was analysed. In addition, during their growth period, amoxicillin treatments at days 7, 21 and 35 were carried out. Results showed a high number of E. coli-resistant strains were isolated from the treated one-day-old chickens, and were the highest for ß-lactams group, followed by quinolone and tetracyclines. After treatment with amoxicillin, the highest percentage of resistances were detected for this antibiotic compared to the others analysed, with significant differences in resistance percentages between control and treated broilers detected in relation to ampicillin, cephalothin, streptomycin, kanamycin, gentamicin, chloramphenicol and tetracycline. Differences in resistances to ciprofloxacin and nalidixic acid between control and treated animals were not observed and there was lack of resistance for amikacin and ceftriaxone. These results suggest the possibility of vertical transmission of resistant strains to newly-hatched chicks from parent flocks, and seem to indicate that the treatment with amoxicillin increased the resistance of E. coli to other antibiotics.

Study of Arcobacter spp. contamination in fresh lettuces detected by different cultural and molecular methods.

Arcobacters are considered potential emerging food and waterborne pathogens. However, there is no data on the presence of Arcobacter spp. in fresh vegetables. Therefore the objective of this research was to study the presence of Arcobacter spp. in fresh lettuces. Fifty fresh lettuces purchased from different local shops in Valencia (Spain) were analyzed. The assay was performed simultaneously by cultural and molecular methods. Isolates were identified by real-time, multiplex PCR and restriction fragment length polymorphism analysis of PCR-amplified DNA fragment (PCR-RFLP). Finally, all the isolates were genotyped using the randomly amplified polymorphic DNA (RAPD-PCR) method. Arcobacter sp. was detected in 10 of the 50 samples (20%) by real-time PCR, being A. butzleri the unique detected species by mPCR. The detection levels obtained by conventional PCR (7 samples/50, 14%) were slightly lower. These seven samples were found to be positive also by culture isolation. All 19 obtained isolates were identified as A. butzleri by multiplex PCR and PCR-RFLP. Great genetic heterogeneity among the isolates was observed by RAPD-PCR profiling. To our knowledge, this is the first study in which Arcobacter spp. is detected in fresh vegetables such as lettuces. Although these foods are generally considered safe, given the large quantities consumed and the fact that further cooking is absent, lettuce could be a source of Arcobacters of public health concern.

Molecular detection of pathogens in water--the pros and cons of molecular techniques.

Pollution of water by sewage and run-off from farms produces a serious public health problem in many countries. Viruses, along with bacteria and protozoa in the intestine or in urine are shed and transported through the sewer system. Even in highly industrialized countries, pathogens, including viruses, are prevalent throughout the environment. Molecular methods are used to monitor viral, bacterial, and protozoan pathogens, and to track pathogen- and source-specific markers in the environment. Molecular techniques, specifically polymerase chain reaction-based methods, provide sensitive, rapid, and quantitative analytical tools with which to study such pathogens, including new or emerging strains. These techniques are used to evaluate the microbiological quality of food and water, and to assess the efficiency of virus removal in drinking and wastewater treatment plants. The range of methods available for the application of molecular techniques has increased, and the costs involved have fallen. These developments have allowed the potential standardization and automation of certain techniques. In some cases they facilitate the identification, genotyping, enumeration, viability assessment, and source-tracking of human and animal contamination. Additionally, recent improvements in detection technologies have allowed the simultaneous detection of multiple targets in a single assay. However, the molecular techniques available today and those under development require further refinement in order to be standardized and applicable to a diversity of matrices. Water disinfection treatments may have an effect on the viability of pathogens and the numbers obtained by molecular techniques may overestimate the quantification of infectious microorganisms. The pros and cons of molecular techniques for the detection and quantification of pathogens in water are discussed.