Critical analysis of methods to determine growth, control and analysis of biofilms for potential non-submerged antibiofilm surfaces and coatings.

The potential uses for antibiofilm surfaces reach across different sectors with significant resultant economic, societal and health impact. For those interested in using antibiofilm surfaces in the built environment, it is important that efficacy testing methods are relevant, reproducible and standardised where possible, to ensure data outputs are applicable to end-use, and comparable across the literature. Using pre-defined keywords, a review of literature reporting on antimicrobial surfaces (78 articles), within which a potential application was described as non-submerged/non-medical surface or coating with antibiofilm action, was undertaken. The most used methods utilized the growth of biofilm in submerged and static systems. Quantification varied (from most to least commonly used) across colony forming unit counts, non-microscopy fluorescence or spectroscopy, microscopy analysis, direct agar-contact, sequencing, and ELISA. Selection of growth media, microbial species, and incubation temperature also varied. In many cases, definitions of biofilm and attempts to quantify antibiofilm activity were absent or vague. Assessing a surface after biofilm recovery or assessing potential regrowth of a biofilm after initial analysis was almost entirely absent. It is clear the field would benefit from widely agreed and adopted approaches or guidance on how to select and incorporate end-use specific conditions, alongside minimum reporting guidelines may benefit the literature.

Eco-friendly non-biocide-release coatings for marine biofouling prevention.

Environmental concerns have been changing the way of looking for solutions to problems. The hydrosphere, together with its biosphere, has been feeling the impact of many pollutants, used for instance in the marine industry for economic reasons or lack of knowledge of their effects. In particular biocides, applied as coatings in paints, are released into the waters becoming toxic and persistent extending their action to an area far beyond the initial coated surface they should protect. In order to minimize these side effects, two biocides, Irgarol (I) and Econea (E), were covalently attached to polyurethane (PU) and foul-release silicone based (PDMS) marine paints through an isocyanate linker. Their antifouling bioactivity was better in PDMS coatings, both for single (Econea) and combined biocides (E/I ratio = 1.5) with contents lower than 0.6 wt%. The treated samples remained almost clean after more than one year immersion in the Portuguese shore of the Atlantic Ocean, and after about 24 weeks under the tropical conditions of Singapore (Fouling rate < 1%). Complementary biofilm adhesion susceptibility tests against Pseudoalteromonas tunicata D2 showed adhesion reduction higher than 90% for PU formulations containing single biocides and close to 100% for PDMS with combined biocides. The eco-toxicity assessment evidenced a low environmental impact, in accordance with the European standards. In addition, shipping field trial tests showed the best antifouling performance for the Econea-based PDMS formulations (E = 0.6 wt%), which remained clean for about nine months in open seawaters, proving the efficacy of this non-release strategy, when applied under dynamic conditions.

Surface modifications for antimicrobial effects in the healthcare setting: a critical overview.

The spread of infections in healthcare environments is a persistent and growing problem in most countries, aggravated by the development of microbial resistance to antibiotics and disinfectants. In addition to indwelling medical devices (e.g. implants, catheters), such infections may also result from adhesion of microbes either to external solid-water interfaces such as shower caps, taps, drains, etc., or to external solid-gas interfaces such as door handles, clothes, curtains, computer keyboards, etc. The latter are the main focus of the present work, where an overview of antimicrobial coatings for such applications is presented. This review addresses well-established and novel methodologies, including chemical and physical functional modification of surfaces to reduce microbial contamination, as well as the potential risks associated with the implementation of such anticontamination measures. Different chemistry-based approaches are discussed, for instance anti-adhesive surfaces (e.g. superhydrophobic, zwitterions), contact-killing surfaces (e.g. polymer brushes, phages), and biocide-releasing surfaces (e.g. triggered release, quorum sensing-based systems). The review also assesses the impact of topographical modifications at distinct dimensions (micrometre and nanometre orders of magnitude) and the importance of applying safe-by-design criteria (e.g. toxicity, contribution for unwanted acquisition of antimicrobial resistance, long-term stability) when developing and implementing antimicrobial surfaces.

Detection of Escherichia coli O157 by peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) and comparison to a standard culture method.

Despite the emergence of non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, E. coli serotype O157 is still the most commonly identified STEC in the world. It causes high morbidity and mortality and has been responsible for a number of outbreaks in many parts of the world. Various methods have been developed to detect this particular serotype, but standard bacteriological methods remain the gold standard. Here, we propose a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method for the rapid detection of E. coli O157. Testing on 54 representative strains showed that the PNA probe is highly sensitive and specific to E. coli O157. The method then was optimized for detection in food samples. Ground beef and unpasteurized milk samples were artificially contaminated with E. coli O157 concentrations ranging from 1 × 10(-2) to 1 × 10(2) CFU per 25 g or ml of food. Samples were then preenriched and analyzed by both the traditional bacteriological method (ISO 16654:2001) and PNA-FISH. The PNA-FISH method performed well in both types of food matrices with a detection limit of 1 CFU/25 g or ml of food samples. Tests on 60 food samples have shown a specificity value of 100% (95% confidence interval [CI], 82.83 to 100), a sensitivity of 97.22% (95% CI, 83.79 to 99.85%), and an accuracy of 98.33% (CI 95%, 83.41 to 99.91%). Results indicate that PNA-FISH performed as well as the traditional culture methods and can reduce the diagnosis time to 1 day.

Rapid detection of urinary tract infections caused by Proteus spp. using PNA-FISH.

We developed a fluorescence in situ hybridization (FISH) method for the rapid detection of Proteus spp. in urine, using a novel peptide nucleic acid (PNA) probe. Testing on 137 urine samples from patients with urinary tract infections has shown specificity and sensitivity values of 98 % (95 % CI, 93.2-99.7) and 100 % (95 % CI, 80,8-100), respectively, when compared with CHROMagar Orientation medium. Results indicate that PNA-FISH is a reliable alternative to traditional culture methods and can reduce the diagnosis time to approximately 2 h.

Detection of Salmonella enterica serovar Enteritidis using real time PCR, immunocapture assay, PNA FISH and standard culture methods in different types of food samples.

Several methods for the rapid and specific detection of Salmonella in food samples have been described. Here, we compare 4 of those methods in terms of assay time, procedure complexity, detection limit, sensitivity, specificity and accuracy. Milk, eggs and mayonnaise samples were artificially contaminated with Salmonella enterica serovar Enteritidis cell concentrations ranging from 1×10(-2) to 1×10(2) CFU per 25 g or ml of food. Samples were then pre-enriched and analyzed by either: i) real-time PCR, using the iQ-Check Salmonella kit; ii) immunocapture, using the RapidChek SELECT Salmonella; iii) a peptide nucleic acid fluorescence in situ hybridization (PNA FISH) method and iv) the traditional bacteriological method ISO 6579:2002. All methods were able to detect Salmonella in the different types of food matrixes and presented a similar detection level of 1CFU per 25 g or ml of food sample. The immunocapture and the PNA FISH methods proved to be very reliable, as their results were 100% in agreement with the ISO method. However, real-time PCR presented a significant number of false positives, which resulted in a specificity of 55.6% (CI 95%, 31.3-77.6) and an accuracy of 82.2% (CI 95%, 63.2-91.4) for this method. Sensitivity was 100% since no false negative results were observed. In conclusion, the implementation of these molecular techniques, mainly the immunocapture and PNA-FISH methods, provides a reliable and less time-consuming alternative for the detection of Salmonella spp. in food samples.

Optimization of culture conditions to improve Helicobacter pylori growth in Ham's F-12 medium by response surface methodology.

Helicobacter pylori is a gastroduodenal pathogen that colonizes the human stomach and is the causal agent of gastric diseases. From the clinical and epidemiological point of view, enhancing and improving the growth of this bacterium in liquid media is an important goal to achieve in order to allow the performance of accurate physiological studies. The aim of this work was to optimize three culture conditions that influence the growth of H. pylori in the defined medium Ham s F-12 supplemented with 5 percent fetal bovine serum by using response surface methodology as a statistical technique to obtain the optimal conditions. The factors studied in this experimental design (Box-Behnken design) were the pH of the medium, the shaking speed (rpm) and the percentage of atmospheric oxygen, in a total of 17 experiments. The biomass specific growth rate was the response measured. The model was validated for pH and shaking speed. The percentage of atmospheric oxygen did not influence the growth for the range of values studied. At the optimal values found for pH and shaking speed, 8 and 130 rpm, respectively, a specific growth rate value of 0.164 h-1, corresponding to a maximal concentration of approximately 1.5x108 CFU/ml, was reached after 8 h. The experimental design strategy allowed, for the first time, the optimization of H. pylori growth in a semi-synthetic medium, which may be important to improve physiological and metabolic studies of this fastidious bacterium.

Application of flow cytometry for the identification of Staphylococcus epidermidis by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) in blood samples.

Staphylococcus epidermidis is considered to be one of the most common causes of nosocomial bloodstream infections, particularly in immune-compromised individuals. Here, we report the development and application of a novel peptide nucleic acid probe for the specific detection of S. epidermidis by fluorescence in situ hybridization. The theoretical estimates of probe matching specificity and sensitivity were 89 and 87%, respectively. More importantly, the probe was shown not to hybridize with closely related species such as Staphylococcus aureus. The method was subsequently successfully adapted for the detection of S. epidermidis in mixed-species blood cultures both by microscopy and flow cytometry.

Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples.

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 x 10(9) +/- 5 x 10(8) CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 x 10(7) +/- 5 x 10(6) CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4',6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.

Effect of chlorine on incorporation of Helicobacter pylori into drinking water biofilms.

The use of a specific peptide nucleic acid (PNA) probe demonstrated that Helicobacter pylori persisted inside biofilms exposed to low concentrations of chlorine (0.2 and 1.2 mg liter(-1)) for at least 26 days, although no culturable cells were recovered. Coupled with data obtained using viability stains in pure culture, this result suggests that H. pylori can survive chlorination but remain undetectable by culture methods, which can be effectively replaced by PNA hybridization.

Development and application of a novel peptide nucleic acid probe for the specific detection of Cronobacter genomospecies (Enterobacter sakazakii) in powdered infant formula.

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of Cronobacter strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several Enterobacteriaceae species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of Cronobacter in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.

Incorporation of natural uncultivable Legionella pneumophila into potable water biofilms provides a protective niche against chlorination stress.

Legionella pneumophila is a waterborne pathogen that has been isolated sporadically from drinking water distribution systems (DWDS). Resistance to disinfectants is mainly attributed to the association of cells with amoebae, but biofilms are also thought to provide some degree of protection. In the present work, a two-stage chemostat was used to form heterotrophic biofilms from drinking water to study the influence of chlorine on the presence of naturally occurring L. pneumophila. The pathogen was tracked in planktonic and sessile biofilm phases using standard culture recovery techniques for cultivable cells and a peptide nucleic acid fluorescence in situ hybridisation assay for total cells. The results showed that the total number of L. pneumophila cells in biofilms was not affected by the concentrations of chlorine tested, and the presence of L. pneumophila could not be detected by culturing. To restrict the outbreaks of disease caused by this bacterium, efforts need to be concentrated on preventing L. pneumophila from re-entering an infectious state by maintaining residual disinfectant levels through the entire DWDS network so that the resuscitation of cells via contact with amoebae is prevented.

Comparison between standard culture and peptide nucleic acid 16S rRNA hybridization quantification to study the influence of physico-chemical parameters on Legionella pneumophila survival in drinking water biofilms.

Legionella pneumophila is a waterborne pathogen that is mainly transmitted by the inhalation of contaminated aerosols. In this article, the influence of several physico-chemical parameters relating to the supply of potable water was studied using a L. pneumophila peptide nucleic acid (PNA) specific probe to quantify total L. pneumophila in addition to standard culture methods. A two-stage chemostat was used to form the heterotrophic biofilms, with biofilm generating vessels fed with naturally occurring L. pneumophila. The substratum was the commonly used potable water pipe material, uPVC. It proved impossible to recover cultivable L. pneumophila due to overgrowth by other microorganisms and/or the loss of cultivability of this pathogen. Nevertheless, results obtained for total L. pneumophila cells in biofilms using a specific PNA probe showed that for the two temperatures studied (15 and 20 degrees C), there were no significant differences when shear stress was increased. However, when a source of carbon was added there was a significant increase in numbers at 20 degrees C. A comparison of the two temperatures showed that at 15 degrees C, the total cell numbers for L. pneumophila were generally higher compared with the total microbial flora, suggesting that lower temperatures support the inclusion of L. pneumophila in drinking water biofilms. The work reported in this article suggests that standard culture methods are not accurate for the evaluation of water quality in terms of L. pneumophila. This raises public health concerns since culture methods are still considered to be the gold standard for assessing the presence of this opportunistic pathogen in water.

Validation of SYTO 9/propidium iodide uptake for rapid detection of viable but noncultivable Legionella pneumophila.

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires' disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l(-1)), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD BacLight). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l(-1) of free chlorine and in 10 min when the concentration is increased to 1.2 mg l(-1). However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.

Incorporation of natural uncultivable Legionella pneumophila into potable water biofilms provides a protective niche against chlorination stress.

Legionella pneumophila is a waterborne pathogen that has been isolated sporadically from drinking water distribution systems (DWDS). Resistance to disinfectants is mainly attributed to the association of cells with amoebae, but biofilms are also thought to provide some degree of protection. In the present work, a two-stage chemostat was used to form heterotrophic biofilms from drinking water to study the influence of chlorine on the presence of naturally occurring L. pneumophila. The pathogen was tracked in planktonic and sessile biofilm phases using standard culture recovery techniques for cultivable cells and a peptide nucleic acid fluorescence in situ hybridisation assay for total cells. The results showed that the total number of L. pneumophila cells in biofilms was not affected by the concentrations of chlorine tested, and the presence of L. pneumophila could not be detected by culturing. To restrict the outbreaks of disease caused by this bacterium, efforts need to be concentrated on preventing L. pneumophila from re-entering an infectious state by maintaining residual disinfectant levels through the entire DWDS network so that the resuscitation of cells via contact with amoebae is prevented.

Persistence of Helicobacter pylori in heterotrophic drinking-water biofilms.

Although the route of transmission of Helicobacter pylori remains unknown, drinking water has been considered a possible transmission vector. It has been shown previously that, in water, biofilms are a protective niche for several pathogens, protecting them from stressful conditions, such as low carbon concentration, shear stress, and less-than-optimal temperatures. In this work, the influence of these three parameters on the persistence and cultivability of H. pylori in drinking-water biofilms was studied. Autochthonous biofilm consortia were formed in a two-stage chemostat system and then inoculated with the pathogen. Total numbers of H. pylori cells were determined by microscopy using a specific H. pylori 16S rRNA peptide nucleic acid probe, whereas cultivable cells were assessed by standard plating onto selective H. pylori medium. Cultivable H. pylori could not be detected at any time point, but the ability of H. pylori cells to incorporate, undergo morphological transformations, persist, and even agglomerate in biofilms for at least 31 days without a noticeable decrease in the total cell number (on average, the concentration was between 1.54 x 10(6) and 2.25 x 10(6) cells cm(-2)) or in the intracellular rRNA content may indicate that the loss of cultivability was due to entry into a viable but noncultivable state. Unlike previous results obtained for pure-culture H. pylori biofilms, shear stress did not negatively influence the numbers of H. pylori cells attached, suggesting that the autochthonous aquatic bacteria have an important role in retaining this pathogen in the sessile state, possibly by providing suitable microaerophilic environments or linking biomolecules to which the pathogen adheres. Therefore, biofilms appear to provide not only a safe haven for H. pylori but also a concentration mechanism so that subsequent sloughing releases a concentrated bolus of cells that might be infectious and that could escape routine grab sample microbiological analyses and be a cause of concern for public health.

Survival of gastric and enterohepatic Helicobacter spp. in water: implications for transmission.

Part of the reason for rejecting aquatic environments as possible vectors for the transmission of Helicobacter pylori has been the preference of this microorganism to inhabit the human stomach and hence use a direct oral-oral route for transmission. On the other hand, most enteric bacterial pathogens are well known for being able to use water as an environmental reservoir. In this work, we have exposed 13 strains of seven different Helicobacter spp. (both gastric and enterohepatic) to water and tracked their survival by standard plating methods and membrane integrity assessment. The influence of different plating media and temperatures and the presence of light on recovery was also assessed. There was good correlation between cultivability and membrane integrity results (Pearson's correlation coefficient = 0.916), confirming that the culture method could reliably estimate differences in survival among different Helicobacter spp. The species that survived the longest in water was H. pylori (>96 h in the dark at 25 degrees C), whereas H. felis appeared to be the most sensitive to water (<6 h). A hierarchical cluster analysis demonstrated that there was no relationship between the enterohepatic nature of Helicobacter spp. and an increased time of survival in water. This work assesses for the first time the survival of multiple Helicobacter spp., such has H. mustelae, H. muridarum, H. felis, H. canadensis, H. pullorum, and H. canis, in water under several conditions and concludes that the roles of water in transmission between hosts are likely to be similar for all these species, whether enterohepatic or not.

Detection of Escherichia coli in biofilms from pipe samples and coupons in drinking water distribution networks.

Fluorescence in situ hybridization (FISH) was used for direct detection of Escherichia coli on pipe surfaces and coupons in drinking water distribution networks. Old cast iron main pipes were removed from water distribution networks in France, England, Portugal, and Latvia, and E. coli was analyzed in the biofilm. In addition, 44 flat coupons made of cast iron, polyvinyl chloride, or stainless steel were placed into and continuously exposed to water on 15 locations of 6 distribution networks in France and Latvia and examined after 1 to 6 months exposure to the drinking water. In order to increase the signal intensity, a peptide nucleic acid (PNA) 15-mer probe was used in the FISH screening for the presence or absence of E. coli on the surface of pipes and coupons, thus reducing occasional problems of autofluorescence and low fluorescence of the labeled bacteria. For comparison, cells were removed from the surfaces and examined with culture-based or enzymatic (detection of beta-d-glucuronidase) methods. An additional verification was made by using PCR. Culture method indicated presence of E. coli in one of five pipes, whereas all pipes were positive with the FISH methods. E. coli was detected in 56% of the coupons using PNA FISH, but no E. coli was detected using culture or enzymatic methods. PCR analyses confirmed the presence of E. coli in samples that were negative according to culture-based and enzymatic methods. The viability of E. coli cells in the samples was demonstrated by the cell elongation after resuscitation in low-nutrient medium supplemented with pipemidic acid, suggesting that the cells were present in an active but nonculturable state, unable to grow on agar media. E. coli contributed to ca. 0.001 to 0.1% of the total bacterial number in the samples. The presence and number of E. coli did not correlate with any of physical and/or chemical characteristic of the drinking water (e.g., temperature, chlorine, or biodegradable organic matter concentration). We show here that E. coli is present in the biofilms of drinking water networks in Europe. Some of the cells are metabolically active but are often not detected due to limitations of traditionally used culture-based methods, indicating that biofilm should be considered as a reservoir that must be investigated further in order to evaluate the risk for human health.

A new model for the transmission of Helicobacter pylori: role of environmental reservoirs as gene pools to increase strain diversity.

Twenty-five years after the first successful cultivation and isolation of Helicobacter pylori, the scientific community is still struggling to understand the way(s) this bacterium is transmitted among the human population. Here, both epidemiologic and microbiologic evidence addressing this matter is reviewed and explored to conclude that most H. pylori successful colonizations are derived from direct person-to-person contact and that even though exposure of humans to H. pylori from environmental sources is a very common event, in most occasions the host is able to fight off infection. In addition, under a new model developed here, we propose that the near elimination of environmental reservoirs is the main responsible for the lower prevalence observed in the more industrialized countries by acting on two levels: by decreasing the number of direct infections and by diminishing the number of intraspecies recombination events for producing strain variation within H. pylori.