Development of an optimized technique for the recovery of H. pylori from water and drinking water biofilms

At present, the route of transmission is perhaps one of the most controversialareas of Helicobacter pylori research. Drinking water and associated biofilms have been suggested as possible environmental reservoirs for the bacterium, however, successful and consistent detection of the bacterium in real systems has not yet been accomplished. A new protocol that offers improved recovery ratings of H. pylori from water and associated-biofilms has been developed and evaluated. This protocol includes optimized incubation atmospheres and a new recovery culture medium, as well as an assessmentof existing techniques for the detachment of H. pylori from surfaces. Even though obvious advantages in terms of cell counts are obtained using this method, certain problems, such as contamination by faster-growing species of the culture medium in heavily contaminated waters may still hinder a successful recovery of the pathogen.Future research will involve further improvement of the protocol and monitoring of real drinking water distribution systems.Unit of Helicobacter/Campylobacter of the Center of Bacteriology of INSA para a Ciência e a Tecnologia (FCT) ; European Commission Research Project SAFER

Tracking persistence of Helicobacter pylori in potable water biofilms using novel 16S rRNA peptide nucleic acid probes

A two stage chemostat system was used to study the pathogenic bacteria H. pylori association to drinking water biofilms. After allowing one week old biofilms to be grown on stainless steel coupons the system was inoculated with the pathogen that was detected using the recently established technique of 16S rRNA peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH). Results show that H. pylori can successfully incorporate within biofilms and its presence was detected for up to five days, either in the basal layer or inside typical biofilm structures such as stacks or fronds. The PNA FISH protocol appears to be a promising new technique for the in situ visualization of microorganisms in biofilms, especially because the hydrophobic nature of the PNA molecule allows a better diffusion through the constituents of the biofilm matrix allowing an improved discrimination of microorganisms inside these naturally occurring structures. A setback in the application of this methodology was the presence of autofluorescent microorganisms. This problem can be minimized by comparing the morphologic characteristics of these suspected false positives with typical H. pylori morphology. If questions subsist, the visualization of the biofilm under different filter blocks can also improve the degree of certainty in the identification, since the reporter probe has usually a very distinctive pattern of fluorescence when compared to the autofluorescent microorganisms

A fluorescence in situ hybridization method using a peptide nucleic acid probe for the detection of Salmonella spp. in biofilms

A novel peptide nucleic acid (PNA) probe for the detection of Salmonella spp. has been developed. The probe was synthesized and the Alexa Fluor dye 594 was attached to the N-terminus in order to allow detection by fluorescence in situ hybridization (FISH). Specificity and sensitivity probe matching theoretical estimates were both of 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 ans sensitivity. Afterwards, the method was successfully adapted to cell detection in suspensions and biofilms. Counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) allowed Salmonella spp. discrimination from heterotrophic consortia of bacteria. However, the direct detection in biofilms presented some limitations for particular types of adhesion materials. These limitations were mainly related with the autofluorescence of the support material at the same wavelength emission as the probe. Nevertheless, this limitation has been overcome by disrupting the biofilm (sonication step) and performing the hybridization on glass slides or in suspension. We hence conclude that PNA FISH represents a reliable tool for biofilm study, allowing specific and direct detection for most support materials, and hence provides spatial organization information for specific groups of microorganisms within mixed/natural biofilms for substrata without a strong autofluorescence signal

Use of fluorescent in situ hybridisation for the visualisation of Helicobacter pylori in real drinking water biofilms

A fluorescently labelled peptide nucleic acid (PNA) probe has been applied for the in situ detection of Helicobacter pylori in drinking water biofilms. The method was originally applied to real pipe samples removed from a drinking water distribution system (DWDS) but the curvature and the heavy fouling of the pipes prevented an accurate detection of the bacterium by epifluorescence microscopy. Therefore, two semi-circular flow cells were placed in a bypass of the DWDS, and coupons with up to 72 days of exposure were regularly sampled and analysed for the presence of H. pylori. In the flat surfaces of the coupons, it was possible to sparsely detect cells exhibiting similar morphology to H. pylori that were emitting the PNA probe fluorescent signal. Coupons were also visualised under the microscope before the hybridisation procedure to serve as negative controls and ensure the validity of the method. This work corroborates the findings already published elsewhere that this bacterium might be present in DWDS biofilms. The method requires, however, highly trained personnel for an accurate detection of the pathogen and will need simplification before being routinely used in standard water analysis laboratories.Fundação para a Ciência e a Tecnologia (FCT) European Commission Research Project SAFE

Detection of H. pylori in biofilms formed in a real drinking water distribution system using petide nucleic acid fluorescence in situ hybridization

Helicobacter pylori has previously been detected in real drinking water distribution systems (DWDS) using PCR based- techniques, but this approach will not discriminate between live and dead cells and their spatial relationship within the biofilm. On the other hand, in situ detection using fluorescence in situ hybridization (FISH) has been used successfully for spatial resolution but only in lab-grown experiments. In the present work, therefore, two flow cells were placed in a by-pass of the DWDS, and coupons were regularly removed for the detection of H. pylori by peptide nucleic acid (PNA) FISH, as well as for the assessment of CFU and total bacteria and visualization under scanning electron and epifluorescence microscopy. Chemical and physical parameters of the water feeding the flow cells were also monitored.Cells exhibiting similar morphology to H. pylori that were PNA FISH positive could be sparsely detected in the coupons. The technique showed promise despite the large morphologicalheterogeneity of microorganismspresent in biofilms and associated autofluorescence.Fundação para a Ciência e a Tecnologia (FCT)European Commission Research Project (SAFER

Development and application of a novel Peptide Nucleic Acid probe for the specific detection of Cronobacter (Enterobacter sakazakii) in powdered infant formula

Cronobacter spp. are causative agents of meningitis, septicemia and necrotizing enterocolitis in neonates and immunocompromised infants. Recently, contaminated powdered infant formula (PIF) has been reported as a source of these infections. In order to minimize the risk of infection, the development of a rapid, sensitive and specific method for the early detection of this bacterium in infant formula is of the utmost importance. Fluorescence in situ hybridization (FISH), a technique that allows direct visualization of whole cells, has been combined with specific peptide nucleic acid (PNA) probes, a new synthetic molecule with a better hybridization performance than DNA probes. In this work, a new FISH method for the detection of Cronobacter spp. using a novel PNA probe is reported. This PNA-FISH method was then adapted for the detection of this bacterium in PIF. The PNA-FISH procedure using the Cronobacter probe proved to be a reliable method for the detection of this pathogen in PIF samples and an alternative to existing molecular methods. It presented high specificity and sensitivity, detected less than 1 CFU per 10g of Cronobacter in infant formula and provided detection in less than 12 hours. Direct visualization of bacterial cells was possible and the method was simple and easy to use, without any special equipment apart from an epifluorescence microscope. The samples can be also analysed by flow cytometry

Use of fluorescent in situ hybridization for the visualization of Helicobacter pylori in real drinking water biofilms

A fluorescently labelled peptide nucleic acid (PNA) probe has been applied for the in situ detection of Helicobacter pylori in drinking water biofilms. The method was originally applied to real pipe samples removed from a drinking water distribution system (DWDS) but the curvature and the heavy fouling of the pipes prevented an accurate detection of the bacterium by epifluorescence microscopy. Therefore, two semi-circular flow cells were placed in a by-pass of the DWDS, and coupons with up to 72 days of exposure were regularly sampled and analysed for the presence of H. pylori. In the flat surfaces of the coupons, it was possible to sparsely detect cells exhibiting similar morphology to H. pylori that were emitting the PNA probe fluorescent signal. Coupons were also visualized under the microscope before the hybridization procedure to serve as negative controls and ensure the validity of the method. This work corroborates the findings already published elsewhere that this bacterium might be present in DWDS biofilms. The method requires however highly trained personnel for an accurate detection of the pathogen and will need simplification before being routinely used in standard water analysis laboratories.Fundação para a Ciência e Tecnologia (FCT) ; European Commission Research Project SAFER

Characterization of multispecies biofilms by peptide nucleic acid fluorescence in situ hybridization (PNA-FISH)

Our current understanding of biofilms in the environment and in health indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the quantification, visualization and discrimination of each population has meant that studies assessing multi-species interactions between sessile microorganisms are scarce and low-throughput. Employing novel peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) methods, we present here a characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven support materials. Ex-situ, we were able to relate quantitatively the populations of ~56 mixed species biofilms up to 48h, regardless of the support material.In situ a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible for most support materials. Regarding biological behavior, composition of mixed-culture biofilm seems to be the final result of competition between microorganisms, both for available nutrients and for free surface to colonize. It is also suggested that the ability to form biofilm is mostly a species-dependent phenomenon rather than surfacedependent, as six of the materials maintained both the species profile and had similar total cell numbers. The exception was copper, that inhibited the biofilm formation for the species tested. Our findings concluded that, using a single method, such as PNA-FISH, to confidently discriminate multispecies early-stage biofilms, researchers can infer about spatial organization, intra- or inter-specie interaction and also assess viable but not cultivable states