Isolation of pathogenic Legionella species and legionella-laden amoebae in dental unit waterlines

doi:10.1016/j.jhin.2005.05.001

Journal of Hospital Infection

Volume 61, Issue 3, November 2005, Pages 257-262

https://doi.org/10.1016/j.jhin.2005.05.001 Get rights and content

Summary

Legionella released into the air during treatment are a potential source of infection. Water stagnation in dental unit waterlines (DUWLs) creates biofilms and promotes the proliferation of these micro-organisms. This study investigated the presence of amoeba infected with legionella, L. pneumophila and other pathogenic Legionella species in a dental teaching hospital. Water samples were collected in the morning and afternoon from 99 dental units and 16 taps connected to the municipal water supply. Samples were plated on selective media and tested for legionella using the direct immunofluorescent antibody technique and the latex agglutination test. Legionella were found in 33% of the DUWLs and in 47% of the mains taps supplying these units. Legionella-laden amoebae occurred in one mains tap sample and in 20% of DUWLs in a clinic of the teaching hospital. L. micdadei was the predominant species isolated from this clinic. L. pneumophila serogroups 2–14 predominated in the mains water, whereas L. pneumophila serogroup 1 was found in approximately half of the contaminated DUWLs and mains taps irrespective of the time of sampling. Pathogenic Legionella species seeded by municipal water into DUWLs is a potential source of legionella infection for both dental personnel and patients during prolonged dental treatment. This problem is compounded by the presence of legionella-laden amoebae which may contain levels of organism well within the infective dose. The interaction of legionella with amoebae is an important ecological factor that may significantly increase the risk of legionellosis, and thus should be given further consideration in the refinement of risk assessment models.

Introduction

Bio-aerosols generated during dental procedures are the main source of legionella, the agents that cause both Legionnaires' disease and Pontiac fever.1, 2, 3, 4, 5 Raised levels of antibodies to legionella confirm that dental surgeons, nurses, hygienists and patients are exposed to contaminated aerosols. This may reflect continuous exposure to small numbers of these micro-organisms that may result in mild Pontiac fever or subclinical infections.2, 6, 7, 8

Biofilms formed within the small bore tubing of dental units create an environment that favours the attachment and growth of Legionella species. Once colonized, other micro-organisms as well as protozoa may attach to this biofilm.1, 9, 10 Under adverse conditions, amoebae will increase their uptake of bacteria and provide a continuous supply of bacteria that are released into the environment.¹¹ This may be problematic because legionella-laden amoebae may contain infective numbers of organisms.¹² The recognition of amoebae as reservoirs and vehicles for bacterial spread leads to public health concerns.¹³

Many cases of legionella infection are not identified as such.¹⁴ L. pneumophila serogroup 1 is responsible for 80% of reported human cases of legionellosis and is usually the only species isolated during routine testing.15, 16 Specialized laboratory methods for identifying other species are not readily available and may be a reason for them not being routinely identified.³

Studies have reported the presence of legionella in dental unit waterlines (DUWLs) and water supplying these units, but the presence of legionella in amoebae has been rarely reported.¹⁷ This study investigated the presence of legionella-laden amoebae and other pathogenic Legionella species in DUWLs and municipal water supplying the units.

Section snippets

Water sampling

Water samples were obtained from the six clinics housed in the three buildings of the University of the Witwatersrand Oral and Dental Hospital (Table I). The Maxillo-Facial and Oral Surgery Clinic, Restorative Dental Clinic and Periodontology and Oral Medicine Clinic were located in the Oral Dental Hospital that was opened in 1953. The Prosthetic and Orthodontics Clinics were transferred to University Corner in 1980. The Oral Health Clinic was established in Dental House in 1989 and equipped

Results

Almost 40% of all sites tested harboured legionella. They included 31 dental units and six mains taps in the morning, and 35 dental units and nine mains taps in the afternoon (Table I). L. pneumophila serogroups 2–14 and other pathogenic Legionella species were isolated more often than L. pneumophila serogroup 1. They were found in 17 dental units and five mains taps in the morning, and in 22 dental units and six mains taps in the afternoon. Low levels of L. pneumophila serogroups 2–14 were

Discussion

L. pneumophila serogroups 2–14 and other Legionella species including L. micdadei and a blue fluorescent species were isolated more often than L. pneumophila serogroup 1 and occurred at levels from 1 to 73 cfu/mL. This suggests that conditions in the DUWLs did not favour this species. However, low levels should not be ignored because they can fluctuate and become potentially infectious if favourable conditions prevail. Even though legionella may not be present at high concentrations in dental

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Prevalence and investigations of bacterial contamination in dental healthcare associated environment
2022, Journal of King Saud University - Science
Citation Excerpt :
According to some studies, the environment, such as water, air, and surfaces, can play an essential role in pathogen transmission. Biofilm formation, water stagnation and a lack of disinfection in dental unit water systems encourage the growth of microorganisms (Barbeau and Nadeau, 1997; Checchi et al., 1998; Singh and Coogan, 2005; Zanetti et al., 2000). Moreover, generators and air–water infusion pumps can sometimes nebulize the saliva and microorganisms contained in the patient's mouth, contaminating the surrounding air and surfaces (King, 1997).
Microbial contamination of medical substrate surface of dental parts touched or exposed to blood and saliva of patients can act as a reservoir for pathogenic bacteria, resulting in cross-contamination. For this reason, presence of oral microorganisms in dental clinics is a major source of dental contamination and infection worldwide. The work described here aimed to determine the presence of bacteria on variety of dental clinic surfaces including a dental chair arm rest, a sink/faucet, the floor beneath a dental chair; a towel dispenser, handles connected to a light and to an instrument table; dental record paper; an X-ray viewer and finally, a bench and head-rest in five separate departments (pediatric dentistry, implant, dental prosthetics, oral medicine and restorative dentistry) of the King Saud University Dental Hospital, both before and after patient treatment. The isolation and identification of bacterial colonies was achieved using a range of methods such as using a Vitek2 automated system. The results showed that all surfaces in different departments were contaminated with bacteria, most notably in the Implant Department, while much less bacterial contamination was reported in the Department of Oral Medicine. The most commonly isolated bacteria were species of, Staphylococcus, Streptococcus, Pseudomonas, Bacillus and Micrococcus.
Fungal diversity in drinking water distribution systems
2022, Freshwater Mycology: Perspectives of Fungal Dynamics in Freshwater Ecosystems
Ecologically, fungi are saprophytes; however, some species also act as parasites or symbionts. Due to their complex life cycle, extensive hyphal networks, and high nutrient absorption rates, they thrive in various environments. The presence of fungi in biofilms and their relationship with other microorganisms have not yet been thoroughly studied, even though the use of metagenomic methods has provided more accurate information in recent years. Fungi produce secondary metabolites, including toxins, with some of their metabolites recognized as human pathogens or allergens. Not only these secondary metabolites but the fungal cell wall components and fungal loads may also be involved in the development of allergies and other opportunistic and systemic infections, particularly in immunocompromised patients. Fungal infections are a growing concern due to the increasing number of immunocompromised patients and other risk factors.
Viability qPCR, a new tool for Legionella risk management
2017, International Journal of Hygiene and Environmental Health
Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management.
The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test.
In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p < 0.001). Otherwise, no significant differences between methods were found in the rest of the groups.
The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy.
Free living amoebae in water sources of critical units in a tertiary care hospital in India
2015, Indian Journal of Medical Microbiology
Background: Isolation of free-living amoebae (FLA) is reported sparsely from water taps, ventilators, air conditioners, haemodialysis units and dental irrigation systems of hospitals worldwide. Their prevalence in hospital environment especially in wards having immunocompromised patients may pose a risk to this group of susceptible population as they may cause disease themselves or may carry pathogens inside them. No study from India has performed such surveillance. Objective: To evaluate extent of FLA contamination in water sources of bone marrow transplant (BMT) intensive care unit (ICU), transplant ICU, haemodialysis unit and high dependency unit in a tertiary care hospital in India. Materials and Methods: A total of hundred samples including fifty each of tap water samples and swabs from mouth of taps used for drinking, bathing and hand washing purposes in these units were collected according to standard procedure. Samples were inoculated onto non-nutrient agar plates at room temperature followed by morphological confirmation. Molecular identification including polymerase chain reaction (PCR) and sequencing was performed in culture positive samples. Results: Four tap water samples and ten swab samples showed growth of trophozoites and cyst formation. Morphologically, four amoebae resembled Acanthamoeba spp. which was further confirmed by PCR and sequencing showed them to be of T3 and T4 genotypes. Conclusion: The presence of these FLA in hospital water sources emphasises the urgent need of implementing effective preventive measures. Further studies are required to estimate the true prevalence of FLA in Indian hospitals by taking larger number of samples.
Vermamoeba vermiformis does not propagate Legionella pneumophila subsp. pascullei in a simulated laboratory dental-unit waterline system
2014, International Biodeterioration and Biodegradation
Citation Excerpt :
Numerous reports suggest that the DUWLs develop heterogeneous biofilms (Barbeau et al., 1998; Barbeau and Buhler, 2001; Pankhurst, 2003; Szymanska, 2003; Spratt et al., 2004; Hassen et al., 2012), in which consortia of microorganisms survive as interdependent communities (Donlan and Costerton, 2002). Amongst the different types of organisms that reside in DUWL biofilm, the opportunistic, gram negative, human bacterial pathogen Legionella pneumophila has been commonly reported (Borneff, 1986; Oppenheim et al., 1987; Pankhurst and Philpott-Howard, 1993; Williams et al., 1993, 1996; Atlas et al., 1995; Pankhurst et al., 1998; Singh and Coogan, 2005; Rice et al., 2006). Although free living amoebae are also found in fresh-water aquatic environments, their concentration increases considerably, within the DUWL niche (Barbeau and Buhler, 2001).
This study investigated whether Vermamoeba vermiformis and Legionella spp., co-habited within an established, mature biofilm of a simulated laboratory dental-unit waterline (sDUWL) and if this amoebic vector supported the life cycle of Legionella pneumophila. Trophozoites of V. vermiformis, isolated in a previous study, were cultured on a natural mixed biofilm bacterial culture and a pure culture of an avirulent, L. pneumophila (non-Sgp1strain ST707) as food sources. Vermamoeba did not show a preference for L. pneumophila. Legionella species were isolated from the sDUWL discharge water and identified by DNA profiling using 16s bacterial rDNA primers as L. pneumophila subsp. pascullei strain U8W. Electron microscopy was performed to establish whether V. vermiformis was acting as a carrier, whilst within the biofilm, for Legionella species isolated from the sDUWL water. Ultrastructure failed to demonstrate vast numbers of any bacteria within the cytoplasm of V. vermiformis that emerged from the same heterogeneous biofilm organisms. These results were taken to indicate that Vermamoeba was unable to propagate either of the Legionella strains studied.
Biocide sensitivity of Vermamoeba vermiformis isolated from dental-unit-waterline systems
2014, International Biodeterioration and Biodegradation
This study isolated amoebae from two different dental-unit-waterline (DUWL) sources, a simulated laboratory system (sDUWL) and a decommissioned system (dDUWL), within 24 h of it being dismantled from a working clinical practice. Molecular profiles of the isolates were determined and morphological characteristics of the test organisms were examined using microscopy. DNA barcoding identified the amoebae from both sources as being Vermamoeba vermiformis (previously described as Hartmannella vermiformis). These amoebae have been deposited at the Culture Collection of Algae and Protozoa and as CCAP 1534/16, GenBank accession number KC161965 (in-vitro simulated system) and CCAP 1534/17, GenBank accession number KC188996 (decommissioned system). The organisms from both sources exhibited two main forms: motile trophozoites and non-motile cysts. Mature cysts displayed natural fluorescence with excitation and emission wavelength of 488 nm, attributed to the presence of natural flavins and nicotinamide compounds. Both the encysted and the trophozoite forms of V. vermiformis remained unaffected when exposed to the proprietary biocides tested at the concentrations recommended for use in dental surgeries and cysts successfully excysted, to release trophozoites upon subsequent culture. However, two out of three proprietary dental biocides tested on isolated heterotrophic bacteria were effective at controlling bacterial contamination.

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Journal of Hospital Infection

Summary

Introduction

Section snippets

Water sampling

Results

Discussion

References (22)

Biofilm and the dental office

J Am Dent Assoc

Detecting Legionella pneumophila in water systems: a comparison of various dental units

J Am Dent Assoc

A review of the science regarding dental unit waterlines

J Am Dent Assoc

Legionnaires' disease: the infective dose paradox

Commentary

Bacterial infections of free-living amoebae

Res Microbiol

The efficacy of chlorination and filtration in the control and eradication of Legionella from dental chair water systems

J Hosp Infect

Biofilms augment the number of free-living amoebae in dental unit waterlines

Res Microbiol

Legionella contamination of dental-unit waters

Appl Environ Microbiol

Infections caused by Legionella micdadei and Legionella pneumophila among renal transplant recipients

J Infect Dis

Legionnaires disease: update

BMJ

Cited by (63)

Prevalence and investigations of bacterial contamination in dental healthcare associated environment

Fungal diversity in drinking water distribution systems

Viability qPCR, a new tool for Legionella risk management

Free living amoebae in water sources of critical units in a tertiary care hospital in India

Vermamoeba vermiformis does not propagate Legionella pneumophila subsp. pascullei in a simulated laboratory dental-unit waterline system

Biocide sensitivity of Vermamoeba vermiformis isolated from dental-unit-waterline systems