The impact of material surface roughness and temperature on the adhesion of Legionella pneumophila to contact surfaces

The adhesion of bacterial cells to various surfaces is based on physical and chemical interactions between the micro-organisms and the surfaces. The main purpose of this research is to determine the effect of material roughness and incubation temperature on the adhesion of bacteria. To determine the adhesion of the bacterial strain of Legionella pneumophila ATCC 33153 to the glass coupons, a spectrophotometric method of measuring the optical density of crystal violet dye that is released from pre-stained bacterial cells attached to the test surface was used. The intensity of adhesion is in positive correlation to the increase in surface roughness (p < 0.05). The adhesion is the greatest at an optimal temperature of 36 °C, whereas the temperature of 15 °C has a bacteriostatic effect and the temperature of 55 °C a bactericidal effect.     

Isolation and identification of Legionella pneumophila from material reclamation facilities

Sampling points at a material reclamation facility (MRF) were monitored over three months for the presence of Legionella spp. A number of different Legionellae were isolated and typed to identify L. pneumophila serogroup 1, the serotype which is the most common human pathogen. Phenotypic methods resulted in a total of 61 presumptive isolates of Legionella spp. Using latex agglutination, 26 out of the 61 were identified as L. pneumophila serogroup 1, 23 as L. pneumophila serogroups 2–14, and the remaining 12 were Legionella spp. However, on typing using pulse field gel electrophoresis, the 26 L. pneumophila serotype 1 isolates were a diverse group of 25 PFGE types with none persisting in the environment over time. This diversity suggests that there are a number of contamination sources for this important human pathogen in the MRF environment which constitute a risk to health for operatives in these facilities.     

Effects of Japan Sea Proper Water on the growth ofLegionella pneumophila, Escherichia coli, andStaphylococcus aureus

Objective To assess whetherLegionella pneumophila serogroup 1 and serogroup 6,Escherichia coli, andStaphylococcus aureus can survive in Japan Sea Proper Water (JSPW). Methods The inhibitory effects of JSPW, surface seawater (SSW), phosphate buffer solution with 3.5% NaCl of pH 7.0 (3.5% NaCIPBS), and the 10²- and 10⁴-fold dilute solutions with purified water or phosphate buffer solution of pH 7.0, and purified water were investigated. Survival cells were counted immediately after the water and the bacteria were mixed, and at 1,3,5, and 7 days after incubation at 37°C. If the number of surviving cells was decreased more than 2 log units compared with the starting value, we judged the medium to have had an inhibitory effect on the growth of the bacteria. Results The survival cells of the bacteria in JSPW had decreased more than 2 log units compared with the starting value at 1 day after incubation. After 1 day of incubation, the cells ofLegionella pneumophila serogroup 6 andStaphylococcus aureus were found to have decreased more than 2 log units in purified water (PW) used as a control. Furthermore,Legionella pneumophila serogroup 1 in the 10²-fold dilute solution of JSPW was only 1.04 log units lower than the starting value at 7 days after incubation. In the 10²- and 10⁴-fold dilute solutions of JSPW,Escherichia coli survived for 7 days after incubation. These results were almost similar to the results in SSW and 3.5% NaCIPBS. Conclusions The present findings demonstrate thatLegionella pneumophila serogroup 1 andEscherichia coli cannot survive in undiluted JSPW for over a day at 37°C, suggesting the inhibitory effects may be due to the sodium chloride contained in JSPW.     

COVID-19 Co-infection with Legionella pneumophila in 2 Tertiary-Care Hospitals,Germany

We describe screening results for detection of co-infections with Legionella pneumophila in patients infected with severe acute respiratory syndrome coronavirus 2. In total, 93 patients were tested; 1 was positive (1.1%) for L. pneumophila serogroup 1. Co-infections with L. pneumophila occur in coronavirus disease patients and should not be missed.     

Integration of Pseudomonas aeruginosa and Legionella pneumophila in drinking water biofilms grown on domestic plumbing materials

Drinking water biofilms were grown on coupons of plumbing materials, including ethylene-propylene-diene-monomer (EPDM) rubber, silane cross-linked polyethylene (PE-X b), electron-ray cross-linked PE (PE-X c) and copper under constant flow-through of cold tap water. After 14 days, the biofilms were spiked with Pseudomonas aeruginosa, Legionella pneumophila and Enterobacter nimipressuralis (10⁶ cells/mL each). The test bacteria were environmental isolates from contamination events in drinking water systems. After static incubation for 24 h, water flow was resumed and continued for 4 weeks. Total cell count and heterotrophic plate count (HPC) of biofilms were monitored, and P. aeruginosa, L. pneumophila and E. nimipressuralis were quantified, using standard culture-based methods or culture-independent fluorescence in situ hybridization (FISH). After 14 days total cell counts and HPC values were highest on EPDM followed by the plastic materials and copper. P. aeruginosa and L. pneumophila became incorporated into drinking water biofilms and were capable to persist in biofilms on EPDM and PE-X materials for several weeks, while copper biofilms were colonized only by L. pneumophila in low culturable numbers.E. nimipressuralis was not detected in any of the biofilms. Application of the FISH method often yielded orders of magnitude higher levels of P. aeruginosa and L. pneumophila than culture methods. These observations indicate that drinking water biofilms grown under cold water conditions on domestic plumbing materials, especially EPDM and PE-X in the present study, can be a reservoir for P. aeruginosa and L. pneumophila that persist in these habitats mostly in a viable but non-culturable state.     

Plasmid profiles of Legionella spp. isolates,Italy

Plasmid analysis and restriction-endonuclease digestion were used to study 54 clinical and environmental Legionella strains. Plasmids with approximate molecular masses of 40, 50, 70, and 90 megadaltons (Mdal) have been isolated from L. pneumophila serogroup 1 strains. One L. jordanis strain contained two plasmids of 25 and 70 Mdal. Restriction analysis of clinical and related hospital-environmental isolates resulted in identical patterns. Geographic diversity is shown for strains of different origin.     

Inactivation of Bacteriophage MS‐2 and poliovirus in copper,galvanized and plastic domestic water pipes

Survival of bacteriophage MS‐2 and poliovirus was evaluated in new and aged copper, galvanized and plastic polymer (polybutylene, cross‐linked polyethylene, polyvinylchloride) pipes. Inactivation rates (k = — [log₁₀C_t — log₁₀C₀]/t) were calculated as a log₁₀ reduction h^‐1. Levels of copper leached from copper pipes, over a period of 24 h, ranged from 400–800 μg 1^‐1. Numbers of viable MS‐2 were reduced significantly in copper pipes (k = 0.32) compared to plastic polymer pipes (k: = 0.03). New galvanized pipes showed greater inactivation of MS‐2 (k = 2.57) when compared to new copper pipes, and similar rates were also observed in the aged pipes. Poliovirus showed more resistance (k = 0.015 and 0.009 for copper and galvanized pipes, respectively) to the action of copper and galvanized pipes than MS‐2. Addition of 0.20 (μg 1^‐1 free chlorine to water containing 400 (μg 1^‐1 leached copper significantly enhanced the inactivation of both viruses (k = 4.74 for MS‐2 and 0.04 for poliovirus, respectively) when compared to either copper or chlorine alone. Less copper was released from aged copper pipes compared to the new ones, resulting in a significantly lower inactivation of MS‐2 (new, k = 0.32; aged, k = 0.13). The use of copper or galvanized pipes in water distribution systems may serve as an additional protection against viral contamination.     

Bacterial adhesion capacity on food service contact surfaces

The aim of this study was to analyse the adhesion of E. coli, P. aeruginosa and S. aureus on food contact materials, such as polyethylene terephthalate, silicone, aluminium, Teflon and glass. Surface roughness, streaming potential and contact angle were measured. Bacterial properties by contact angle and specific charge density were characterised. The bacterial adhesion analysis using staining method and scanning electron microscopy showed the lowest adhesion on smooth aluminium and hydrophobic Teflon for most of the bacteria. However, our study indicates that hydrophobic bacteria with high specific charge density attach to those surfaces more intensively. In food services, safety could be increased by selecting material with low adhesion to prevent cross contamination.     

Rapid genotyping of Legionella pneumophila serogroup 1 strains by a novel DNA microarray-based assay during the outbreak investigation in Warstein,Germany 2013

Between 1 August and 6 September 2013, an outbreak of Legionnaires’ disease (LD) with 78 cases confirmed by positive urinary antigen tests occurred in Warstein, North Rhine-Westphalia, Germany. Legionella (L.) pneumophila, serogroup (Sg) 1, monoclonal antibody (mAb) subgroup Knoxville, sequence type (ST) 345, was identified as the epidemic strain. This strain was isolated from seven patients. To detect the source of the infection, epidemiological typing of clinical and environmental strains was performed in two consecutive steps. First, strains were typed by monoclonal antibodies. Indistinguishable strains were further subtyped by sequence-based typing (SBT) which is the internationally recognized standard method for epidemiological genotyping of L. pneumophila. In an early stage of the outbreak investigation, many environmental isolates were found to belong to the mAb subgroup Knoxville, but to two different STs, namely to ST 345, the epidemic strain, and to ST 600. A majority of environmental isolates belonged to ST 600 whereas the epidemic ST 345 strain was less common in environmental samples. To rapidly distinguish both Knoxville strains, we applied a novel typing method based on DNA-hybridization on glass chips. The new assay can easily and rapidly discriminate L. pneumophila Sg 1 strains. Thus, we were able to quickly identify the sources harboring the epidemic strain, i.e., two cooling towers of different companies, the waste water treatment plants (WWTP) of the city and one company as well as water samples of the river Wester and its branches.     

Microbial adhesion capacity. Influence of shear and temperature stress

Environmental parameters dictate the conditions for both biofilm formation and deconstruction. The aim of this study is to analyse the impact of hydrodynamic and thermodynamic effects on bacterial detachment. Escherichia coli grown on two stainless steel metal surfaces with different roughness (brushed with roughness of 0.05 μm and electropolished with roughness of 0.29 μm) are exposed to laminar and turbulent (shower) flows of phosphate buffered saline media at temperatures of 8, 20 and 37 °C. Results show that the turbulent flow removes significantly more bacterial cells than laminar flow (p <0.05) on both materials. This indicates that the shear force determines the rate of detached bacteria. It is also observed that detachment of cells is more efficient on brushed than on electropolished contact surfaces because on the latter surface, fewer cells were attached before exposure. Moreover, we demonstrate that the temperature of the washing agent has an impact on bacterial detachment. At the same flow conditions, the exposure to higher temperature results in greater detachment rate.     

Preferential colonization and release of Legionella pneumophila from mature drinking water biofilms grown on copper versus unplasticized polyvinylchloride coupons

Legionella occurrence in premise drinking water (DW) systems contributes to legionellosis outbreaks, especially in the presence of suitable protozoan hosts. This study examined L. pneumophila behavior within DW biofilms grown on copper (Cu) and unplasticized polyvinylchloride (uPVC) surfaces in the presence of Acanthamoeba polyphaga. One year-old DW biofilms were established within six CDC biofilm reactors: three each containing Cu or uPVC coupons. Biofilms were then inoculated with L. pneumophila (uPVC-Lp and Cu-Lp), or L. pneumophila and A. polyphaga (uPVC-Lp/Ap and Cu-Lp/Ap) and compared to sterile water inoculated controls (uPVC- and Cu-Control) over a 4 month period. L. pneumophila appeared more persistent by qPCR within Cu biofilms in the presence of A. polyphaga compared to uPVC biofilms with or without A. polyphaga, but maintained their cultivability in uPVC biofilms compared to Cu biofilms. Also, persistent shedding of L. pneumophila cells (assayed by qPCR) in the effluent water implied colonization of L. pneumophila within Cu-coupon reactors compared to no detection from uPVC-coupon reactor effluent 14 days after inoculation. Hence, L. pneumophila appeared to colonize Cu surfaces more effectively and may be shed from the biofilms at a greater frequency and duration compared to L. pneumophila colonized uPVC surfaces with host amoebae playing a role in L. pneumophila persistence within Cu biofilms.     

Environmental investigation of a legionellosis outbreak in western Sydney: the role of molecular profiling

This investigation used DNA profiling in an attempt to identify the environmental source of a community outbreak of 11 cases of Legionnaires? disease. Nine of these cases were culture positive and a single strain (DNA profile) of Legionella pneumophila serogroup 1 was isolated from eight cases. Spot water samples were collected from 51 cooling towers implicated by case exposure histories; this same strain was isolated from four towers at three separate locations up to 6 km apart. None of these locations had been frequently implicated by case histories. Because we did not perform an analytic epidemiological investigation, we were unable to identify a single environmental source for the outbreak. It is also possible that this outbreak was multifocal. The use of molecular profiling should not overshadow the importance of epidemiological methods in these environmental investigations. More data is needed regarding the prevalence, distribution, and clinical significance (virulence) of environmental L. pneumophila strains. This would aid interpretation of molecular profiling used in investigations of community legionellosis outbreaks.     

Assessment of a low-cost, point-of-use, ultraviolet water disinfection technology

We describe a point-of-use (POU) ultraviolet (UV) disinfection technology, the UV Tube, which can be made with locally available resources around the world for under $50 US. Laboratory and field studies were conducted to characterize the UV Tube's performance when treating a flowrate of 5 L/min. Based on biological assays with MS2 coliphage, the UV Tube delivered an average fluence of 900±80 J/m² (95% CI) in water with an absorption coefficient of 0.01 cm^-1. The residence time distribution in the UV Tube was characterized as plug flow with dispersion (Peclet Number = 19.7) and a mean hydraulic residence time of 36 s. Undesirable compounds were leached or produced from UV Tubes constructed with unlined ABS, PVC, or a galvanized steel liner. Lining the PVC pipe with stainless steel, however, prevented production of regulated halogenated organics. A small field study in two rural communities in Baja California Sur demonstrated that the UV Tube reduced E. coli concentrations to less than 1/100 ml in 65 out of 70 samples. Based on these results, we conclude that the UV Tube is a promising technology for treating household drinking water at the point of use.     

嗜肺军团菌致病性与效应蛋白的研究进展

嗜肺军团菌是一种可以引起军团菌肺炎和庞蒂亚克热的兼性胞内病原菌,主要侵染阿米巴原虫和人类巨噬细胞。该菌在宿主胞内能依靠Dot/Icm IVB型分泌系统产生的效应蛋白成功逃避溶酶体的降解。本文主要对嗜肺军团菌的致病物质、胞内存活与增殖机制及其效应蛋白的生物学功能进行综述,详细介绍嗜肺军团菌的毒力因子与致病机制,为军团病防治的研究提供新思路,也为其他胞内病原菌所致感染性疾病的研究提供重要的借鉴意义。     

Comparison of clinical and environmental samples of Legionella pneumophila at the nucleotide sequence level

Legionella pneumophila serogroup 1 is the most common etiological agent of legionellosis. We have used clinical and environmental isolates from different sources to compare their genetic variability. We have obtained the nucleotide sequence for six protein-coding loci, included in the SBT scheme for L. pneumophila, and three intergenic regions from 127 samples, 47 of environmental origin and 80 from clinical samples. Levels of genetic variability were found to be higher in the environmental than in the clinical samples, but these did not represent a mere subset of the former. Not a single case of full identity between clinical and environmental isolates was found, which raises the possibility that only a specific subset of environmental isolates is actually capable of producing infection in humans. A phylogenetic analysis of the concatenate alignment of the nine loci sequences showed four main groups, each including clinical and environmental isolates, although their distribution was not uniform among them. The comparison of each individual gene tree with the others revealed several cases of incongruence involving samples from both origins, thus suggesting the presence of recombination in the two groups.     

Legionella pneumophila: Population genetics, phylogeny and genomics

Legionella pneumophila is a human pathogen that was recognized only about 30 years ago. It is the causative agent of Legionnaires’ disease, a severe pneumonia that is transmitted through inhalation of aerosols of contaminated water. Shortly after its discovery, the ability of Legionella to multiply intracellularly in fresh water protozoa was discovered. This long lasting co-evolution between the eukaryotic host and Legionella has led to the selection of a panoply of virulence factors, which allow to exploit important cellular processes during infection. Compelling evidence for the importance of protozoa in the evolution of this bacterium comes from analysis of complete genome sequences. A key feature of the L. pneumophila genomes is the presence of a high number and wide variety of eukaryotic like proteins and protein domains probably acquired through horizontal gene transfer and/or convergent evolution.In the last years several different typing methods aiming in investigating the molecular epidemiology of L. pneumophila have been developed. Furthermore, the access to whole genome sequences of several L. pneumophila strains allowed to apply large scale comparative genomic studies using DNA arrays. A higher genetic diversity among environmental isolates with respect to clinical isolates and the presence of specific clones of L. pneumophila overrepresented in human disease or causing legionellosis world wide, were identified. Further studies analyzing the natural populations of Legionella more in detail will allow to gain a better understanding of the population structure and the ecological diversity of this species. This review describes the latest observations about the structure of L. pneumophila populations, the techniques used to study the molecular epidemiology and evolution of L. pneumophila, the knowledge gained from genome analysis, and discusses future perspectives.     

Evaluation of the efficacy of ultraviolet irradiation for disinfection of hospital water contaminated by Legionella

We evaluated the efficacy of the ultraviolet irradiation on hospital water colonized by Legionella pneumophila serogroup 3, by inserting a lamp system on a hot water pipe supplying a small area. Cultures were performed for four months from 5 L samples of water, collected before and after the ultraviolet treatment at the lamp unit and from two distal points. Irradiation was effective immediately after disinfection (<10 cfu/L), even when the incoming water was highly contaminated. One distal point showed little or no contamination (<10–20 cfu/L), while the other showed little to moderate contamination (<10³ cfu/L). We conclude that ultraviolet irradiation is useful to protect the water system in small area; however, because of the lack of residual activity, it should be combined with other methods of disinfection. Maintenance of the water system is also necessary in order to reduce biofilm formation and Legionella recolonization.     

Plasmid profiles of legionella pneumophila strains isolated in Spain

An assay for plasmid DNA content was carried out in 100 strains of Legionella pneumophila of distinct serogroups and isolated from various sources (clinical, environment). The strains were isolated from different geographic regions in our country. The presence of plasmids was proved in one of the 11 clinical isolates and in 68 of the 89 isolated of environmental origin studied. In the strains belonging to serogroup 1 and isolated in our region (Cantabria), three plasmid profiles were observed, whereas in strains of the same serogroup from other geographic regions, two profiles were shown which exhibited differences compared to the former ones. Analysis by means of restriction endonucleases suggested that plasmids of similar size in serogroup 1 strains of different source, were related. The results obtained do not appear to reveal any correlation between plasmid profile and source of isolation or serogroup.Corresponding author.     

Isolation of Legionella pneumophila from hotels of Greece

Twenty water samples collected from 6 hotels situated in various areas of Greece were examined for the presence of Legionella pneumophila and Legionella-like organisms. Five of the six hotels included in this investigation were associated with cases of legionellosis. Legionella pneumophila serogroups 1 and 8 were isolated from four of six hotels, mainly from the hot water supply system. This is the first isolation and identification of L. pneumophila in Greece.Corresponding author.     

Effect of Stainless Steel on Corrosion Behavior of Copper in a Copper-bearing Intrauterine Device

Some copper-bearing intrauterine devices (Cu-IUDs) consist of pure copper and stainless steel. Corrosion of copper in the Cu-IUD was anticipated to be affected by galvanic action due to electrical contact between these two metals. Electrochemical measurements were carried out in physiological saline with or without indomethacin, which was introduced for bleeding control. In the copper/stainless steel couple, the open-circuit potential of stainless steel was found to play a decisive role. In most cases, when stainless steel was in the passive state and acted as the cathode, the contact accelerated copper corrosion. In addition, the area ratio of stainless steel to copper altered copper corrosion behavior. The larger the area of stainless steel, the greater the acceleration of copper corrosion. It was noted that the stainless steel surface might be activated due to improper handling of the IUD. In this case, copper became the cathode of the couple and its corrosion was suppressed.