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°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°C. A comparison of the two temperatures showed that at 15°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.     

Temporal Variations in the Abundance and Composition of Biofilm Communities Colonizing Drinking Water Distribution Pipes

Pipes that transport drinking water through municipal drinking water distribution systems (DWDS) are challenging habitats for microorganisms. Distribution networks are dark, oligotrophic and contain disinfectants; yet microbes frequently form biofilms attached to interior surfaces of DWDS pipes. Relatively little is known about the species composition and ecology of these biofilms due to challenges associated with sample acquisition from actual DWDS. We report the analysis of biofilms from five pipe samples collected from the same region of a DWDS in Florida, USA, over an 18 month period between February 2011 and August 2012. The bacterial abundance and composition of biofilm communities within the pipes were analyzed by heterotrophic plate counts and tag pyrosequencing of 16S rRNA genes, respectively. Bacterial numbers varied significantly based on sampling date and were positively correlated with water temperature and the concentration of nitrate. However, there was no significant relationship between the concentration of disinfectant in the drinking water (monochloramine) and the abundance of bacteria within the biofilms. Pyrosequencing analysis identified a total of 677 operational taxonomic units (OTUs) (3% distance) within the biofilms but indicated that community diversity was low and varied between sampling dates. Biofilms were dominated by a few taxa, specifically Methylomonas, Acinetobacter, Mycobacterium, and Xanthomonadaceae, and the dominant taxa within the biofilms varied dramatically between sampling times. The drinking water characteristics most strongly correlated with bacterial community composition were concentrations of nitrate, ammonium, total chlorine and monochloramine, as well as alkalinity and hardness. Biofilms from the sampling date with the highest nitrate concentration were the most abundant and diverse and were dominated by Acinetobacter.     

Interaction of <Emphasis Type="Italic">legionella pneumophila</Emphasis> and <Emphasis Type="Italic">helicobacter pylori</Emphasis> 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.     

Impact of Non-Legionella Bacteria on the Uptake and Intracellular Replication of Legionella pneumophila in Acanthamoeba castellanii and Naegleria lovaniensis

In aquatic environments, Legionella pneumophila survives, in association with other bacteria, within biofilms by multiplying in free-living amoebae. The precise mechanisms underlying several aspects of the uptake and intracellular replication of L. pneumophila in amoebae, especially in the presence of other bacteria, remain unknown. In the present study, we examined the competitive effect of selected non-Legionella bacteria (Escherichia coli, Aeromonas hydrophila, Flavobacterium breve, and Pseudomonas aeruginosa) on the uptake of L. pneumophila serogroup 1 by the amoebae Acanthamoeba castellanii and Naegleria lovaniensis. We also investigated their possible influence on the intracellular replication of L. pneumophila in both amoeba species. Our results showed that the non-Legionella bacteria did not compete with L. pneumophila for uptake, suggesting that the amoeba hosts took in L. pneumophila through a specific and presumably highly efficient uptake mechanism. Living and heat-inactivated P. aeruginosa best supported the replication of L. pneumophila in N. lovaniensis and A. castellanii, respectively, whereas for both amoeba species, E. coli yielded the lowest number of replicated L. pneumophila. Furthermore, microscopic examination showed that 100% of the A. castellanii and only 2% of the N. lovaniensis population were infected with L. pneumophila at the end of the experiment. This study clearly shows the influence of some non-Legionella bacteria on the intracellular replication of L. pneumophila in A. castellanii and N. lovaniensis. It also demonstrates the different abilities of the two tested amoeba species to serve as a proper host for the replication and distribution of the human pathogen in man-made aquatic environments such as cooling towers, shower heads, and air conditioning systems with potential serious consequences for human health.     

Phylogenetic diversity of microbial communities in real drinking water distribution systems

Microbial regrowth in drinking water distribution systems (DWDS) is a major concern in the water supply industry. Detailed knowledge of the microbial community in DWDS will be of great importance for assessing the microbiological risks of drinking water. The spatial heterogeneity of microbial community structures in the bulk waters of a large real DWDS was investigated using 16S rRNA clone library analysis. The results indicate that high residual chlorine in drinking water could not control microbial regrowth in DWDS. The bacterial communities in the bulk waters were spatially heterogenic, mainly composed of Alphaproteobacteria and Betaproteobacteria (or Cyanobacteria). Microorganisms from the genera Acinetobacter, Sphingomonas and Gemella were detected, implying there is microbiological risk from drinking water. This work provides new insight into microbial ecology in DWDS.     

Biofilms: the environmental playground of Legionella pneumophila

Legionella pneumophila, the aetiological agent of 90% of legionellosis cases, is a common inhabitant of natural and anthropogenic freshwater environments, where it resides in biofilms. Biofilms are defined as complex, natural assemblages of microorganisms that involve a multitude of trophic interactions. A thorough knowledge and understanding of Legionella ecology in relation to biofilm communities is of primary importance in the search for innovative and effective control strategies to prevent the occurrence of disease cases. This review provides a critical update on the state‐of‐the‐art progress in understanding the mechanisms and factors affecting the biofilm life cycle of L. pneumophila. Particular emphasis is given to discussing the different strategies this human pathogen uses to grow and retain itself in biofilm communities. Biofilms develop not only at solid‐water interfaces (substrate‐associated biofilms), but also at the water‐air interface (floating biofilms). Disturbance of the water surface can lead to liberation of aerosols derived from the floating biofilm into the atmosphere that allow transmission of biofilm‐associated pathogens over considerable distances. Recent data concerning the occurrence and replication of L. pneumophila in floating biofilms are also elaborated and discussed.     

Validation of SYTO 9/Propidium Iodide Uptake for Rapid Detection of Viable but Noncultivable <Emphasis Type="Italic">Legionella pneumophila</Emphasis>

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.     

Replication of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> in Floating Biofilms

Biofilms are a major source of human pathogenic Legionella pneumophila in aquatic systems. In this study, we investigated the capacity of L. pneumophila to colonize floating biofilms and the impact of Acanthamoeba castellanii on the replication of biofilm-associated Legionella. Biofilms were grown in Petri dishes and consisted of Aeromonas hydrophila, Escherichia coli, Flavobacterium breve, and Pseudomonas aeruginosa. Six hours following inoculation, Legionella were detected in floating biofilms in mean concentrations of 1.4 × 10⁴ cells/cm² (real-time polymerase chain reaction) and 8.3 × 10² CFU/cm² (culture). Two-way analysis of variance tests and fluorescent in situ hybridization clearly proved that increased biofilm-associated L. pneumophila concentrations were the result of intracellular replication in A. castellanii. Forty-eight hours after the introduction of A. castellanii in the Petri dishes, 90 ± 0.8% of the amoebae (infection rate) were completely filled with highly metabolic active L. pneumophila (mean infection intensity).     

Characterisation of the Physical Composition and Microbial Community Structure of Biofilms within a Model Full-Scale Drinking Water Distribution System

Within drinking water distribution systems (DWDS), microorganisms form multi-species biofilms on internal pipe surfaces. A matrix of extracellular polymeric substances (EPS) is produced by the attached community and provides structure and stability for the biofilm. If the EPS adhesive strength deteriorates or is overcome by external shear forces, biofilm is mobilised into the water potentially leading to degradation of water quality. However, little is known about the EPS within DWDS biofilms or how this is influenced by community composition or environmental parameters, because of the complications in obtaining biofilm samples and the difficulties in analysing EPS. Additionally, although biofilms may contain various microbial groups, research commonly focuses solely upon bacteria. This research applies an EPS analysis method based upon fluorescent confocal laser scanning microscopy (CLSM) in combination with digital image analysis (DIA), to concurrently characterize cells and EPS (carbohydrates and proteins) within drinking water biofilms from a full-scale DWDS experimental pipe loop facility with representative hydraulic conditions. Application of the EPS analysis method, alongside DNA fingerprinting of bacterial, archaeal and fungal communities, was demonstrated for biofilms sampled from different positions around the pipeline, after 28 days growth within the DWDS experimental facility. The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component. Additionally, the greatest proportion of EPS was located above that of the cells. Fungi and archaea were established as important components of the biofilm community, although bacteria were more diverse. Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.     

The effects of metabolite molecules produced by drinking water-isolated bacteria on their single and multispecies biofilms

The elucidation of the mechanisms by which diverse species survive and interact in drinking water (DW) biofilm communities may allow the identification of new biofilm control strategies. The purpose of the present study was to investigate the effects of metabolite molecules produced by bacteria isolated from DW on biofilm formation. Six opportunistic bacteria, viz. Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp. isolated from a drinking water distribution systems (DWDS) were used to form single and multispecies biofilms in the presence and absence of crude cell-free supernatants produced by the partner bacteria. Biofilms were characterized in terms of mass and metabolic activity. Additionally, several physiological aspects regulating interspecies interactions (sessile growth rates, antimicrobial activity of cell-free supernatants, and production of iron chelators) were studied to identify bacterial species with biocontrol potential in DWDS. Biofilms of Methylobacterium sp. had the highest growth rate and M. mucogenicum biofilms the lowest. Only B. cepacia was able to produce extracellular iron-chelating molecules. A. calcoaceticus, B. cepacia, Methylobacterium sp. and M. mucogenicum biofilms were strongly inhibited by crude cell-free supernatants from the other bacteria. The crude cell-free supernatants of M. mucogenicum and S. capsulata demonstrated a high potential for inhibiting the growth of counterpart biofilms. Multispecies biofilm formation was strongly inhibited in the absence of A. calcoaceticus. Only crude cell-free supernatants produced by B. cepacia and A. calcoaceticus had no inhibitory effects on multispecies biofilm formation, while metabolite molecules of M. mucogenicum showed the most significant biocontrol potential.     

Mycobacterium avium Bacilli Grow Saprozoically in Coculture with Acanthamoeba polyphaga and Survive within Cyst Walls

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-μm-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.     

Evasion of phagotrophic predation by protist hosts and innate immunity of metazoan hosts by Legionella pneumophila

Legionella pneumophila is a ubiquitous environmental bacterium that has evolved to infect and proliferate within amoebae and other protists. It is thought that accidental inhalation of contaminated water particles by humans is what has enabled this pathogen to proliferate within alveolar macrophages and cause pneumonia. However, the highly evolved macrophages are equipped with more sophisticated innate defence mechanisms than are protists, such as the evolution of phagotrophic feeding into phagocytosis with more evolved innate defence processes. Not surprisingly, the majority of proteins involved in phagosome biogenesis (~80%) have origins in the phagotrophy stage of evolution. There are a plethora of highly evolved cellular and innate metazoan processes, not represented in protist biology, that are modulated by L. pneumophila, including TLR2 signalling, NF‐κB, apoptotic and inflammatory processes, histone modification, caspases, and the NLRC–Naip5 inflammasomes. Importantly, L. pneumophila infects haemocytes of the invertebrate Galleria mellonella, kill G. mellonella larvae, and proliferate in and kill Drosophila adult flies and Caenorhabditis elegans. Although coevolution with protist hosts has provided a substantial blueprint for L. pneumophila to infect macrophages, we discuss the further evolutionary aspects of coevolution of L. pneumophila and its adaptation to modulate various highly evolved innate metazoan processes prior to becoming a human pathogen.     

Legionella pneumophila in the Environment: The Occurrence of a Fastidious Bacterium in Oligotrophic Conditions

Legionella pneumophila, a micro-organism encountered in aquatic environments, can cause serious intracellular infections among humans. Since the bacterium is ubiquitous in aquatic habitats, it appears to be impossible to prevent L. pneumophila from entering man-made water systems. However, many questions concerning the survival and/or growth in the environment, the partners and opponents of L. pneumophila remain unanswered. This review focuses on the factors governing the ecology of L. pneumophila, since there is considerable divergence and even contradiction in literature on its environmental requirements. A key question to be resolved is the discrepancy between the fastidious nature of L. pneumophila in axenic cultures (e.g. 400 mg l⁻¹ L-cysteine and 250 mg l^-1 ferric iron) and the nutritionally poor environments in which it is commonly detected. It is assumed that dense microbial communities, as occurring in sediments and biofilms – but not likely in surface and drinking water, – can provide the necessary growth requirements for L. pneumophila. However, most of the studies concerning L. pneumophila have led to the general opinion that the organism can only multiply in the aquatic environment as a parasite in certain protozoa. The discovery of the non-classical siderophore legiobactin also indicates that the iron requirement for survival and autonomous growth is not as high as has been assumed. It thus appears that in order to control Legionella in the environment, focus should be on the eradication of microbial hotspots in which L. pneumophila resides.     

An ignored and potential source of taste and odor (T&O) issues—biofilms in drinking water distribution system (DWDS)

It is important for water utilities to provide esthetically acceptable drinking water to the public, because our consumers always initially judge the quality of the tap water by its color, taste, and odor (T&O). Microorganisms in drinking water contribute largely to T&O production and drinking water distribution systems (DWDS) are known to harbor biofilms and microorganisms in bulk water, even in the presence of a disinfectant. These microbes include T&O-causing bacteria, fungi, and algae, which may lead to unwanted effects on the organoleptic quality of distributed water. Importantly, the understanding of types of these microbes and their T&O compound-producing mechanisms is needed to prevent T&O formation during drinking water distribution. Additionally, new disinfection strategies and operation methods of DWDS are also needed for better control of T&O problems in drinking water. This review covers: (1) the microbial species which can produce T&O compounds in DWDS; (2) typical T&O compounds in DWDS and their formation mechanisms by microorganisms; (3) several common factors in DWDS which can influence the growth and T&O generation of microbes; and (4) several strategies to control biofilm and T&O compound formation in DWDS. At the end of this review, recommendations were given based on the conclusion of this review.

     

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.Helicobacter pylori is a Gram-negative microorganism that colonizes the human stomach and can cause gastric ulcers that can degenerate into gastric carcinoma (5). The route of transmission for this pathogen is not well known, and even though culturable H. pylori has never been isolated from drinking water distribution systems (DWDS), molecular techniques such as PCR have detected the presence of H. pylori DNA in potable water (6, 15, 17), indicating that this environment could act as a reservoir for this bacterium.Chlorine is the most commonly used disinfectant worldwide to ensure the safe distribution of water to the consumer (19). Although studies conducted by Johnson et al. (14) and Baker et al. (4) have shown that H. pylori is inactivated by chlorine, their conclusions were based on the lack of recovery using standard culture plating methods which fail to consider cells that have entered a viable but nonculturable (VBNC) state. Recently, Moreno et al. (16) applied molecular techniques to demonstrate that H. pylori can survive in low concentrations of chlorine in a VBNC state. However, all these studies were performed with pure cultures using suspended cells, and until now, there have been no studies reporting on the effect of chlorination on H. pylori when associated with heterotrophic biofilms where, as is well known, microorganisms become more resistant to the biocide effect of chlorine (10).In a recent study, we demonstrated that H. pylori can be incorporated into drinking water biofilms and remain viable in the lower layers of these structures (11). It is therefore important to understand the ability of this pathogen to be incorporated and survive in heterotrophic biofilms formed in chlorinated waters. If this pathogen can remain viable under these conditions, it might therefore represent a risk to public health when released into the bulk fluid.The aim of this work was to study the effect of low concentrations of chlorine on H. pylori cells both when associated with heterotrophic biofilms and when suspended in pure culture, to assess whether the biofilm can provide protection against disinfection.     

Intracellular multiplication ofLegionella pneumophila in amoebae isolated from hospital hot water tanks

We studied the ability ofLegionella to multiply in potable water samples obtained from investigations of nosocomial legionellosis. AutochthonousLegionella multiplied in three of 14 hospital water samples after incubation at 35°C and 42°C. All three samples were from hot water tanks. Multiplication did not occur when a selected sample was filtered through a 0.45-m membrane and reinoculated with indigenousLegionella. We isolated bothLegionella pneumophila and one or more species of free-living amoebae, primarity members of theHartmannellidae, from each of these hot water tank samples. Amoebae from a total of six hot water tank samples were used for cocultivation studies withL. pneumophila. All amoebae supported multiplication ofLegionella in coculture at 35°C. Four of six isolates of amoebae supported multiplication oflegionella at 42°C, while none supported multiplication at 45°C. Gimenez staining and electron microscopy showed thatLegionella multiplied intracellularly in amoebae. Control of these amoebae in potable water may prevent colonization and multiplication ofLegionella in domestic hot water systems.     

Legionella pneumophila Persists within Biofilms Formed by Klebsiella pneumoniae,Flavobacterium sp., and Pseudomonas fluorescens under Dynamic Flow Conditions

Legionella pneumophila, the agent of Legionnaires'' disease pneumonia, is transmitted to humans following the inhalation of contaminated water droplets. In aquatic systems, L. pneumophila survives much of time within multi-organismal biofilms. Therefore, we examined the ability of L. pneumophila (clinical isolate 130b) to persist within biofilms formed by various types of aquatic bacteria, using a bioreactor with flow, steel surfaces, and low-nutrient conditions. L. pneumophila was able to intercalate into and persist within a biofilm formed by Klebsiella pneumoniae, Flavobacterium sp. or Pseudomonas fluorescens. The levels of L. pneumophila within these biofilms were as much as 4×10⁴ CFU per cm² of steel coupon and lasted for at least 12 days. These data document that K. pneumoniae, Flavobacterium sp., and P. fluorescens can promote the presence of L. pneumophila in dynamic biofilms. In contrast to these results, L. pneumophila 130b did not persist within a biofilm formed by Pseudomonas aeruginosa, confirming that some bacteria are permissive for Legionella colonization whereas others are antagonistic. In addition to colonizing certain mono-species biofilms, L. pneumophila 130b persisted within a two-species biofilm formed by K. pneumoniae and Flavobacterium sp. Interestingly, the legionellae were also able to colonize a two-species biofilm formed by K. pneumoniae and P. aeruginosa, demonstrating that a species that is permissive for L. pneumophila can override the inhibitory effect(s) of a non-permissive species.     

Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems

A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing Legionella pneumophila along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions. The materials varied in their abilities to support biofilm development and the growth of L. pneumophila. Elastomeric surfaces had the most abundant biofilms supporting the highest numbers of L. pneumophila CFU; this was attributed to the leaching of nutrients for bacterial growth from the materials. No direct relationship existed between total biofouling and the numbers of L. pneumophila CFU.     

Experimental Evolution of Legionella pneumophila in Mouse Macrophages Leads to Strains with Altered Determinants of Environmental Survival

The Gram-negative bacterium, Legionella pneumophila, is a protozoan parasite and accidental intracellular pathogen of humans. We propose a model in which cycling through multiple protozoan hosts in the environment holds L. pneumophila in a state of evolutionary stasis as a broad host-range pathogen. Using an experimental evolution approach, we tested this hypothesis by restricting L. pneumophila to growth within mouse macrophages for hundreds of generations. Whole-genome resequencing and high-throughput genotyping identified several parallel adaptive mutations and population dynamics that led to improved replication within macrophages. Based on these results, we provide a detailed view of the population dynamics of an experimentally evolving bacterial population, punctuated by frequent instances of transient clonal interference and selective sweeps. Non-synonymous point mutations in the flagellar regulator, fleN, resulted in increased uptake and broadly increased replication in both macrophages and amoebae. Mutations in multiple steps of the lysine biosynthesis pathway were also independently isolated, resulting in lysine auxotrophy and reduced replication in amoebae. These results demonstrate that under laboratory conditions, host restriction is sufficient to rapidly modify L. pneumophila fitness and host range. We hypothesize that, in the environment, host cycling prevents L. pneumophila host-specialization by maintaining pathways that are deleterious for growth in macrophages and other hosts.