Modelling the effects of charge on antibiotic diffusion and adsorption in liquid crystalline virus suspensions.

We develop a microscopic model of antibiotic diffusion in virus suspensions in a liquid crystalline state. We then approximate this with an effective homogenised model that is more amenable to analytical investigation, to understand the effect of charge on the antibiotic tolerance. We show that liquid crystalline virus suspensions slow down antibiotics significantly, and that electric charge strongly contributes to this by influencing the effective diameter and adsorptive capacity of the liquid crystalline viruses so that charged antibiotics diffuse much slower than neutral ones; this can be directly and efficiently derived from the homogenised model and is in good agreement with experiments in microbiology. Charge is also found to affect the relationship between antibiotic diffusion and viral packing density in a nontrivial way. The results elucidate the effect of charge on antibiotic tolerance in liquid crystalline biofilms in a manner that is straightforwardly extendable to other soft matter systems.

Efficiency and novelty of using environmental swabs for dry-surface biofilm recovery.

Studies on the epidemiology of dry-surface biofilms (DSBs) within healthcare settings have shown an almost universal distribution across frequently touched items. Despite a growing body of evidence for DSBs in hospitals, little attention has been paid to the recovery capacity of techniques used to detect these microbial communities. Biofilms are inherently difficult to remove from surfaces due to adhesive substances within their matrix and may act as sources of infection, but to what extent is largely unknown. In this study, we evaluate the recovery efficiencies of commonly used environmental swabs against DSBs containing 7.24 log10 Acinetobacter baumannii cm-2, using a drip flow reactor and desiccation cycle. Biofilm presence was visually confirmed using episcopic differential interference contrast microscopy combined with epifluorescence and quantified using sonicated viable plate counts. The swab materials used comprised foam, viscose and cotton, all of which were pre-moistened using a buffer solution. The surfaces were vigorously swabbed by each material type and the resultant microbe populations for both swabs and remaining DSBs were quantified. Our results found foam-tipped swabs to be superior, detecting on average 30 % of the original DSB contamination; followed by viscose (6 %) and cotton (3 %). However, no distinct difference was revealed in the concentration of microbes remaining on the surface after swabbing for each swab type, suggesting there is variation in the capacity for each swab to release biofilm-associated micro-organisms. We conclude whilst environmental swabs do possess the ability to detect biofilms on dry surfaces, the reduced efficiencies are likely to cause an underestimation of the microbes present and should be considered during clinical application.

A light-guiding urinary catheter for the inhibition of Proteus mirabilis biofilm formation.

Catheter-associated urinary tract infection (CAUTI) is a leading cause of hospital-acquired infections worldwide causing debilitating illness for patients as well as a significant financial and treatment burden on health services. CAUTI is linked with the build-up of biofilms on catheter surfaces which act as a reservoir for infection. Additionally, urease-producing bacteria such as Gram-negative Proteus mirabilis (PM), can form crystalline biofilms which encrust catheter surfaces ultimately leading to blockages which require immediate removal of the catheter. Currently there are limited treatments available to prevent the formation of biofilms by PM as well as other urinary tract infection causing bacteria. A novel concept for a light-guiding urinary catheter is presented where a silicone elastomer waveguide incorporated along the length of the catheter is used to irradiate the catheter surfaces with antimicrobial blue light (405 nm) to prevent biofilm formation in situ. The prototype device is mass producible while also easy to fabricate in a lab setting for research studies. The inhibitory effect of blue light on PM biofilm formation over a range of irradiances is described for the first time showing an LD90 at 192-345 J/cm2 and total inhibition at 1,700 J/cm2 In vitro studies show that the light-guiding catheter (LGC) prototypes exhibit a 98% inhibition in PM biofilm formation inside the catheter lumen at an average estimated irradiance of 30-50 mW/cm2 (324-540 J/cm2 fluence) showing that the concept is highly effective, promising to be a powerful and economical antimicrobial approach to prevent catheter associated biofilm development and blockage.

Bactericidal and anti-biofilm effects of uncharged and cationic ultrasound-responsive nitric oxide microbubbles on Pseudomonas aeruginosa biofilms.

Bacterial biofilms are a major and ongoing concern for public health, featuring both inherited genetic resistance traits and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing need for novel methods of drug delivery, to increase the efficacy of antimicrobial agents. This research evaluated the anti-biofilm and bactericidal effects of ultrasound responsive gas-microbubbles (MBs) of either air or nitric oxide, using an in vitro Pseudomonas aeruginosa biofilm model grown in artificial wound medium. The four lipid-based MB formulations evaluated were room-air MBs (RAMBs) and nitric oxide MBs (NOMBs) with no electrical charge, as well as cationic (+) RAMBs+ and NOMBs+. Two principal treatment conditions were used: i) ultrasound stimulated MBs only, and ii) ultrasound stimulated MBs with a sub-inhibitory concentration (4 µg/mL) of the antibiotic gentamicin. The total treatment time was divided into a 60 second passive MB interaction period prior to 40 second ultrasound exposure; each MB formulation was tested in triplicate. Ultrasound stimulated RAMBs and NOMBs without antibiotic achieved reductions in biofilm biomass of 93.3% and 94.0%, respectively. Their bactericidal efficacy however was limited, with a reduction in culturable cells of 26.9% and 65.3%, respectively. NOMBs with sub-inhibitory antibiotic produced the most significant reduction in biofilm biomass, corresponding to a 99.9% (SD ± 5.21%); and a 99.9% (SD ± 0.07%) (3-log) reduction in culturable bacterial cells. Cationic MBs were initially manufactured to promote binding of MBs to negatively charged biofilms, but these formulations also demonstrated intrinsic bactericidal properties. In the absence of antibiotic, the bactericidal efficacy of RAMB+ and NOMB+ was greater that of uncharged counterparts, reducing culturable cells by 84.7% and 86.1% respectively; increasing to 99.8% when combined with antibiotic. This study thus demonstrates the anti-biofilm and bactericidal utility of ultrasound stimulated MBs, and specifically is the first to demonstrate the efficacy of a NOMB for the dispersal and potentiation of antibiotics against bacterial biofilms in vitro. Importantly the biofilm system and complex growth-medium were selected to recapitulate key morphological features of in vivo biofilms. The results us offer new insight for the development of new clinical treatments, for example, in chronic wounds.

Modelling of filamentous phage-induced antibiotic tolerance of P. aeruginosa.

Filamentous molecules tend to spontaneously assemble into liquid crystalline droplets with a tactoid morphology in environments with high concentration on non-adsorbing molecules. Tactoids of filamentous Pf bacteriophage, such as those produced by Pseudomonas aeruginosa, have been linked to increased antibiotic tolerance. We modelled this system and show that tactoids composed of filamentous Pf virions can lead to antibiotic tolerance by acting as an adsorptive diffusion barrier. The continuum model, reminiscent of descriptions of reactive diffusion in porous media, has been solved numerically and good agreement was found with the analytical results, obtained using a homogenisation approach. We find that the formation of tactoids significantly increases antibiotic diffusion times which may lead to stronger antibiotic resistance.

Artificial Human Sweat as a Novel Growth Condition for Clinically Relevant Pathogens on Hospital Surfaces.

The emergence of biofilms on dry hospital surfaces has led to the development of numerous models designed to challenge the efficacious properties of common antimicrobial agents used in cleaning. This is in spite of limited research defining how dry surfaces are able to facilitate biofilm growth and formation in such desiccating and nutrient-deprived environments. While it is well established that the phenotypical response of biofilms is dependent on the conditions in which they are formed, most models incorporate a nutrient-enriched, hydrated environment dissimilar to the clinical setting. In this study, we piloted a novel culture medium, artificial human sweat (AHS), which is perceived to be more indicative of the nutrient sources available on hospital surfaces, particularly those in close proximity to patients. AHS was capable of sustaining the proliferation of four clinically relevant multidrug-resistant pathogens (Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa) and achieved biofilm formation at concentration levels equivalent to those found in situ (average, 6.00 log10 CFU/cm2) with similar visual characteristics upon microscopy. The AHS model presented here could be used for downstream applications, including efficacy testing of hospital cleaning products, due to its resemblance to clinical biofilms on dry surfaces. This may contribute to a better understanding of the true impact these products have on surface hygiene. IMPORTANCE Precise modeling of dry surface biofilms in hospitals is critical for understanding their role in hospital-acquired infection transmission and surface contamination. Using a representative culture condition which includes a nutrient source is key to developing a phenotypically accurate biofilm community. This will enable accurate laboratory testing of cleaning products and their efficacy against dry surface biofilms.

Synergism versus Additivity: Defining the Interactions between Common Disinfectants.

Many of the most common disinfectant and sanitizer products are formulations of multiple antimicrobial compounds. Products claiming to contain synergistic formulations are common, although there is often little supporting evidence. The antimicrobial interactions of all pairwise combinations of common disinfectants (benzalkonium chloride, didecyldimethylammonium chloride, polyhexamethylene biguanide, chlorocresol, and bronopol) were classified via checkerboard assay and validated by time-kill analyses. Combinations were tested against Acinetobacter baumannii NCTC 12156, Enterococcus faecalis NCTC 13379, Klebsiella pneumoniae NCTC 13443, and Staphylococcus aureus NCTC 13143. Synergistic interactions were identified only for the combinations of chlorocresol with benzalkonium chloride and chlorocresol with polyhexamethylene biguanide. Synergism was not ubiquitously demonstrated against all species tested and was on the borderline of the synergism threshold. These data demonstrate that synergism between disinfectants is uncommon and circumstantial. Most of the antimicrobial interactions tested were characterized as additive. We suggest that this is due to the broad, nonspecific mechanisms associated with disinfectants not providing an opportunity for the combined activities of these compounds to exceed the sum of their parts. IMPORTANCE The scarcity of observed synergistic interactions suggests that in the case of many disinfectant-based products, combined mechanisms of interaction may be being misinterpreted. We emphasize the need to correctly differentiate between additivity and synergism in antimicrobial formulations, as inappropriate classification may lead to unnecessary issues in the event of regulatory changes. Furthermore, we question the need to focus on synergism and disregard additivity when considering combinations of disinfectants, as the benefits that synergistic interactions provide are not necessarily relevant to the application of the final product.

Biofilm Development on Urinary Catheters Promotes the Appearance of Viable but Nonculturable Bacteria.

Catheter-associated urinary tract infections have serious consequences, for both patients and health care resources. Much work has been carried out to develop an antimicrobial catheter. Although such developments have shown promise under laboratory conditions, none have demonstrated a clear advantage in clinical trials. Using a range of microbiological and advanced microscopy techniques, a detailed laboratory study comparing biofilm development on silicone, hydrogel latex, and silver alloy-coated hydrogel latex catheters was carried out. Biofilm development by Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis on three commercially available catheters was tracked over time. Samples were examined with episcopic differential interference contrast (EDIC) microscopy, culture analysis, and staining techniques to quantify viable but nonculturable (VBNC) bacteria. Both qualitative and quantitative assessments found biofilms to develop rapidly on all three materials. EDIC microscopy revealed the rough surface topography of the materials. Differences between culture counts and quantification of total and dead cells demonstrated the presence of VBNC populations, where bacteria retain viability but are not metabolically active. The use of nonculture-based techniques showed the development of widespread VBNC populations. These VBNC populations were more evident on silver alloy-coated hydrogel latex catheters, indicating a bacteriostatic effect at best. The laboratory tests reported here, which detect VBNC bacteria, allow more rigorous assessment of antimicrobial catheters, explaining why there is often minimal benefit to patients.IMPORTANCE Several antimicrobial urinary catheter materials have been developed, but, although laboratory studies may show a benefit, none have significantly improved clinical outcomes. The use of poorly designed laboratory testing and lack of consideration of the impact of VBNC populations may be responsible. While the presence of VBNC populations is becoming more widely reported, there remains a lack of understanding of the clinical impact or influence of exposure to antimicrobial products. This is the first study to investigate the impact of antimicrobial surface materials and the appearance of VBNC populations. This demonstrates how improved testing is needed before clinical trials are initiated.

An effective evidence-based cleaning method for the safe reuse of intermittent urinary catheters: In vitro testing.

AIMS: To determine a safe bactericidal cleaning method that does not damage urethral catheters used for intermittent catheterization. In some countries, single-use catheters are the norm; in others, the reuse of catheters is common depending on health insurance, personal preference, or individual concerns about the environment. However, no recent study of cleaning methods has been published to provide evidence for the safe reuse of catheters. METHODS: Using advanced microbiological methods, a laboratory study of eight cleaning methods was conducted. Sections of uncoated polyvinylchloride (PVC) catheters were exposed to bacterial uropathogens in physiologically correct artificial urine media then tested with a range of heat, chemical, and mechanical cleaning methods. Analysis of culturable and viable but nonculturable (VBNC) bacteria was done and direct microscopy was used. Descriptive statistics were used to compare values. RESULTS: Heat treatments, although effective, resulted in catheter surface breakdown and damage. Ultrasonic cleaning and vinegar showed evidence of VBNC populations indicating the methods were bacteriostatic. Detergent and water wash followed by immersion in a commercially available 0.6% sodium hypochlorite solution and 16.5% sodium chloride (diluted Milton) gave consistent bactericidal results and no visible catheter damage. CONCLUSIONS: Combined mechanical and chemical treatment of a detergent and water wash followed by immersion in diluted Milton (the "Milton Method") provided consistent and effective cleaning of uncoated PVC catheters, showing bactericidal action for all uropathogens tested after repeated exposure. If found safe in clinical testing, this method could increase the reuse of catheters, reduce plastic waste in the environment, reduce cost, and increase patient choice.

Bacteria and nanosilver: the quest for optimal production.

Silver nanoparticles (AgNPs) have potential uses in many applications, but current chemical production methods are challenged by scalability, limited particle stability, and the use of hazardous chemicals. The biological processes present in bacteria to mitigate metallic contaminants in their environment present a potential solution to these challenges. Before commercial exploitation of this technology can be achieved, the quality of bacteriogenic AgNPs needs to be improved for certain applications. While the colloidal and morphological stabilities of biogenic AgNPs are widely regarded as superior to chemogenic particles, little control over the synthesis of particle morphologies has been achieved in biological systems. This article reviews a range of biosynthetic reaction conditions and how they affect AgNP formation in bacteria to understand which are most influential. While there remains uncertainty, some general trends are emerging: higher Ag+ concentrations result in higher AgNP production, up to a point at which the toxic effects begin to dominate; the optimal temperature appears to be heavily species-dependent and linked to the optimal growth temperature of the organism. However, hotter conditions generally favor higher production rates, while colder environments typically give greater shape diversity. Little attention has been paid to other potentially important growth conditions including halide concentrations, oxygen exposure, and irradiation with light. To fully exploit biosynthetic production routes as alternatives to chemical methods, hurdles remain with controlling particle morphologies and require further work to elucidate and harness them. By better understanding the factors influencing AgNP production, a foundation can be laid from which shape-controlled production can be achieved.

Viable-but-Nonculturable Listeria monocytogenes and Salmonella enterica Serovar Thompson Induced by Chlorine Stress Remain Infectious.

The microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-tagged Listeria monocytogenes and Salmonella enterica serovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing following incubation on spinach leaves. Culture data revealed that total viable L. monocytogenes and Salmonella Thompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNC L. monocytogenes and Salmonella Thompson was assessed by using Caenorhabditis elegans Ingestion of VBNC pathogens by C. elegans resulted in a significant life span reduction (P = 0.0064 and P < 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturable L. monocytogenes treatments was observed. L. monocytogenes was visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected.IMPORTANCE Many bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogens Listeria monocytogenes and Salmonella enterica It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed in Caenorhabditis elegans that ingested these VBNC pathogens, with VBNC L. monocytogenes as infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.

Novel Insights into the Proteus mirabilis Crystalline Biofilm Using Real-Time Imaging.

The long-term use of indwelling catheters results in a high risk from urinary tract infections (UTI) and blockage. Blockages often occur from crystalline deposits, formed as the pH rises due to the action of urease-producing bacteria; the most commonly found species being Proteus mirabilis. These crystalline biofilms have been found to develop on all catheter materials with P. mirabilis attaching to all surfaces and forming encrustations. Previous studies have mainly relied on electron microscopy to describe this process but there remains a lack of understanding into the stages of biofilm formation. Using an advanced light microscopy technique, episcopic differential interference contrast (EDIC) microscopy combined with epifluorescence (EF), we describe a non-destructive, non-contact, real-time imaging method used to track all stages of biofilm development from initial single cell attachment to complex crystalline biofilm formation. Using a simple six-well plate system, attachment of P. mirabilis (in artificial urine) to sections of silicone and hydrogel latex catheters was tracked over time (up to 24 days). Using EDIC and EF we show how initial attachment occurred in less than 1 h following exposure to P. mirabilis. This was rapidly followed by an accumulation of an additional material (indicated to be carbohydrate based using lectin staining) and the presence of highly elongated, motile cells. After 24 h exposure, a layer developed above this conditioning film and within 4 days the entire surface (of both catheter materials) was covered with diffuse crystalline deposits with defined crystals embedded. Using three-dimensional image reconstruction software, cells of P. mirabilis were seen covering the crystal surfaces. EDIC microscopy could resolve these four components of the complex crystalline biofilm and the close relationship between P. mirabilis and the crystals. This real-time imaging technique permits study of this complex biofilm development with no risk of artefacts due to sample manipulation. A full understanding of the stages and components involved in crystalline encrustation formation will aid in the development of new protocols to manage and ultimately prevent catheter blockage.

Interaction of Legionella pneumophila and Helicobacter pylori with bacterial species isolated from drinking water biofilms.

BACKGROUND: It is well established that Legionella pneumophila is a waterborne pathogen; by contrast, the mode of Helicobacter pylori transmission remains unknown but water seems to play an important role. This work aims to study the influence of five microorganisms isolated from drinking water biofilms on the survival and integration of both of these pathogens into biofilms. RESULTS: Firstly, both pathogens were studied for auto- and co-aggregation with the species isolated from drinking water; subsequently the formation of mono and dual-species biofilms by L. pneumophila or H. pylori with the same microorganisms was investigated. Neither auto- nor co-aggregation was observed between the microorganisms tested. For biofilm studies, sessile cells were quantified in terms of total cells by SYTO 9 staining, viable L. pneumophila or H. pylori cells were quantified using 16 S rRNA-specific peptide nucleic acid (PNA) probes and cultivable cells by standard culture techniques. Acidovorax sp. and Sphingomonas sp. appeared to have an antagonistic effect on L. pneumophila cultivability but not on the viability (as assessed by rRNA content using the PNA probe), possibly leading to the formation of viable but noncultivable (VBNC) cells, whereas Mycobacterium chelonae increased the cultivability of this pathogen. The results obtained for H. pylori showed that M. chelonae and Sphingomonas sp. help this pathogen to maintain cultivability for at least 24 hours. CONCLUSIONS: It appears that M. chelonae may have an important role in the survival of both pathogens in drinking water. This work also suggests that the presence of some microorganisms can decrease the cultivability of L. pneumophila but not the viability which indicates that the presence of autochthonous microorganisms can lead to misleading results when the safety of water is assessed by cultivable methods alone.

Survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and caliciviruses in drinking water-associated biofilms grown under high-shear turbulent flow.

Most of the bacteria in drinking water distribution systems are associated with biofilms. In biofilms, their nutrient supply is better than in water, and biofilms can provide shelter against disinfection. We used a Propella biofilm reactor for studying the survival of Mycobacterium avium, Legionella pneumophila, Escherichia coli, and canine calicivirus (CaCV) (as a surrogate for human norovirus) in drinking water biofilms grown under high-shear turbulent-flow conditions. The numbers of M. avium and L. pneumophila were analyzed with both culture methods and with peptide nucleic acid fluorescence in situ hybridization (FISH) methods. Even though the numbers of pathogens in biofilms decreased during the experiments, M. avium and L. pneumophila survived in biofilms for more than 2 to 4 weeks in culturable forms. CaCV was detectable with a reverse transcription-PCR method in biofilms for more than 3 weeks. E. coli was detectable by culture for only 4 days in biofilms and 8 days in water, suggesting that it is a poor indicator of the presence of certain waterborne pathogens. With L. pneumophila and M. avium, culture methods underestimated the numbers of bacteria present compared to the FISH results. This study clearly proved that pathogenic bacteria entering water distribution systems can survive in biofilms for at least several weeks, even under conditions of high-shear turbulent flow, and may be a risk to water consumers. Also, considering the low number of virus particles needed to result in an infection, their extended survival in biofilms must be taken into account as a risk for the consumer.

Survival of Listeria monocytogenes Scott A on metal surfaces: implications for cross-contamination.

Listeria monocytogenes is an important re-emerging pathogen which is commonly found in the environment. Many outbreaks have been associated with the contamination of food produce, often linked to cross-contamination from surfaces or equipment to prepared foodstuffs. In the present study a number of copper-base metal alloys have been used to assess the survival times of L. monocytogenes on different materials, in comparison with stainless steel. High concentrations (10(7)) of bacteria were placed on metal coupons cut from each alloy. After defined incubation times, coupons were placed in tubes containing phosphate buffered saline and vortexed to remove the cells. Aliquots were then plated onto tryptone blood agar plates and the number of colony forming units counted. The high concentration of bacteria was used to represent a "worst-case" scenario. The results indicate that survival is greatly reduced on a copper-base alloy compared to stainless steel. Viable cells could be detected on stainless steel after 24 h incubation at room temperature. On copper, brass, aluminium bronze and silicon bronze, no viable bacteria could be detected after 60 min incubation, indicating a 5 log reduction (the detection limit of the procedure was 100 bacteria). No cells could be detected from copper nickel and copper nickel zinc alloys, after 90 min incubation. The viability stain, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), confirmed these results, with actively respiring bacteria being clearly labelled on stainless steel after 24 h. The results suggest that careful choice of surface material could reduce the potential risk of cross-contamination in industrial, commercial and domestic environments.

Targeting species-specific low-affinity 16S rRNA binding sites by using peptide nucleic acids for detection of Legionellae in biofilms.

Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.