Detection of infectious rotavirus in naturally contaminated source waters for drinking water production

Aims:  To assess chlorine susceptibility of Legionella pneumophila grown from two amoebic hosts, Acanthamoeba castellanii and Hartmannella vermiformis .
Methods and Results:  After being released from amoebae, Leg. pneumophila were chlorinated at 2 and 5 mg l⁻¹ for 5 min–24 h. Bacterial culturability and cytoplasmic membrane deterioration were quantified by culture assay on BCYEα agar and BacLight stains coupled with a fluorescent microscope, respectively. Chlorination reduced the culturability of Leg. pneumophila by 2·93–4·59 log CFU ml⁻¹ and damaged cellular membrane by 53·8–99·2%. Moreover, cells released from H. vermiformis exhibited significantly lower degrees in culturability reduction ( P  = 0·0008) and membrane deterioration ( P  < 0·0001) when compared with those from A. castellanii . The amoebic genus is the most significant parameter affecting cytoplasmic membrane integrity of chlorinated Legionella ( P  < 0·0001), followed by free chlorine concentration ( P  = 0·042).
Conclusions:  Legionella pneumophila replicated from H. vermiformis possess greater chlorine resistance than the cells from A. castellanii .
Significance and Impact of the Study:  This study shows the heterogeneity of amoebae-grown Leg. pneumophila in chlorine susceptibility, which should be considered in the control of legionellae proliferation, particularly in the systems where H. vermiformis is dominant, e.g. hot water plumbing.     

Non-culturable Legionella pneumophila associated with Acanthamoeba castellanii: detection of the bacterium using DNA amplification and hybridization

J. HAY. D.V. SEAL, B.BILLCLIFFE AND J.H. FREER. 1995. The intracellular localization of Legionella pneumophila serogroup 1 within Acanthamoeba castellanii rendered the bacteria non-culturable on supplemented BCYE agar. DNA amplification, using two 19-mer primers, and hybridization using a 25-mer oligonucleotide probe, permitted detection of Leg. pneumophila in approximately 81% (29/36) of samples where the bacteria could not be detected using culture. A combination of co-cultivation of samples with Leg. pneumophila -naive A. polyphaga or Hartmannella vermiformis , incubation in a defined liquid medium or use of catalase indicated that approximately 31% (9/29) of the samples contained Leg. pneumophila which were viable although not culturable.     

A colony based confirmation assay for Legionella and Legionella pneumophila employing the EnviroAmp™Legionella system and seroagglutination

Modifications to the EnviroAmp^™ Legionella detection system are described which permit the rapid analysis of bacterial colonies taken from Legionella selective media. Capillary PCR permitted twice the number of samples to be analysed with a single kit. When PCR was positive for Leg. pneumophila , this result was confirmed by seroagglutination. The reverse dot blot hybridization assay was only used where PCR indicated a Legionella sp. other than Leg. pneumophila , permitting further savings on detection system components. This technique and standard confirmation procedures were applied to 133 isolates arising from 63 water samples plated to Legionella isolation media. Results agreed except for two isolates which gave a positive result for Legionella spp. by PCR and hybridization but were negative using standard procedures. Raising the annealing/extension temperature of the PCR by 2 °C eliminated the false positive result with these two isolates but did not adversely effect the sensitivity of the assay, as determined by re-testing of 68 environmental isolates and testing of 69 new environmental isolates and 12 Legionella reference species. The modified technique provides a convenient and cost effective alternative to standard confirmation procedures.     

Colonization of components of a model hot water system by Legionella pneumophila

A model hot water distribution network was seeded with a virulent strain of Legionella pneumophila serotype 1. Ten weeks after inoculation, components of the system, which include aluminium discs, copper, stainless steel, silicone tubing, rubber and glass beads, were examined for colonization by L. pneumophila. The samples were stained with fluorescein-labelled antibodies to the strain and were examined with scanning electron microscopy. Colonization, which was accompanied by copious quantities of a slime-like debris, was heaviest on the rubber and least on the copper. Adherence to silicone tubing and stainless steel was observed.     

Colonization of components of a model hot water system by Legionella pneumophila

A model hot water distribution network was seeded with a virulent strain of Legionella pneumophila serotype 1. Ten weeks after inoculation, components of the system, which include aluminium discs, copper, stainless steel, silicone tubing, rubber and glass beads, were examined for colonization by L. pneumophila. The samples were stained with fluorescein-labelled antibodies to the strain and were examined with scanning electron microscopy. Colonization, which was accompanied by copious quantities of a slime-like debris, was heaviest on the rubber and least on the copper. Adherence to silicone tubing and stainless steel was observed.     

Effect of salt concentration and temperature on survival of Legionella pneumophila

The effects of various concentrations of sodium chloride solutions (0·1%–3%) and different temperatures (4, 10, 20, 30 and 37 °C) on survival of Legionella pneumophila were investigated. It was found that at temperatures between 4 °C and 20 °C, Legionella organisms survived in salt solutions up to 3% NaCl. Only the combination of high temperatures, i. e. 30 °C and 37 °C, with NaCl concentrations over 1·5%, reduced cell numbers significantly. It was interesting to note that the addition of small amounts of NaCl (0·1%–0·5%) enhanced survival of Leg. pneumophila , suggesting a protective effect of NaCl. In order to obtain information about conditions encountered in the environment, the survival experiments were repeated in sterile sea water from the Baltic Sea and the North Sea. The marked bacterial die-off, especially at higher temperatures, was not observed in natural sea water. All these results indicate that Leg. pneumophila can survive in the marine environment.     

Genetic control of macrophage susceptibility to infection by Legionella pneumophila

Abstract Legionella pneumophila readily grows in cultures of thioglycollate (TGC)-induced macrophages (MPs) from A/J mice, but not in MPs from BALB/c mice or other mouse strains. In the present study, the growth of Legionella pneumophila in MPs from A/J and BALB/c mice, as well as hybrids of the two strains and back-crossed mice, was investigated to determine whether the permissiveness of growth of these bacteria was due to an inherited trait of the MPs. The MPs from all A/J mice supported the growth of Legionella , regardless of whether they were obtained from TGC or casein injected donors, but the cells from the mice given TGC supported growth of L. pneumophila much better than cells from mice injected with casein. Furthermore, MPs obtained from all BALB/c mice treated with either TGC or casein were nonpermissive for the growth of L. pneumophila . MPs from approximately 46% of the back-crossed ACF1 to A/J mice were permissive for L. pneumophila growth, while MPs from all ACF1 to back-crossed BALB/c mice were found to be nonpermissive. MPs from approximately 19% of ACF2 mice were permissive for L. pneumophila . Killing activities of MPs using temperature sensitive mutants of Salmonella typhimurium were variable and did not correlate with permissiveness or nonpermissiveness for growth of L. pneumophila . In addition, the number of inflammatory cells in the peritoneal cavity induced in response to TGC did not correlate with the permissiveness or nonpermissiveness of the MPs from various mouse strains to Legionella , indicating the permissive nature of the cells is controlled by genetic mechanisms involving a recessive phenotype but differs from resistance genes such as Ity important for replication of S. typhimurium .     

Dendritic cells pulsed with live and dead Legionella pneumophila elicit distinct immune responses

Legionella pneumophila is the causative pathogen of Legionnaires' disease, which is characterized by severe pneumonia. In regard to the pathophysiology of Legionella infection, the role of inflammatory phagocytes such as macrophages has been well documented, but the involvement of dendritic cells (DCs) has not been clarified. In this study, we have investigated the immune responses that DCs generate in vitro and in vivo after contact with L. pneumophila. Heat- and formalin-killed L. pneumophila, but not live L. pneumophila, induced immature DCs to undergo similar phenotypic maturation, but the secreted proinflammatory cytokines showed different patterns. The mechanisms of the DC maturation by heat- or formalin-killed L. pneumophila depended, at least in part, on Toll-like receptor 4 signaling or on Legionella LPS, respectively. After transfer to naive mice, DCs pulsed with dead Legionella produced serum Ig isotype responses specific for Legionella, leading to protective immunity against an otherwise lethal respiratory challenge with L. pneumophila. The in vivo immune responses required the Ag presentation of DCs, especially that on MHC class II molecules, and the immunity yielded cross-protection between clinical and environmental strains of L. pneumophila. Although the DC maturation was impaired by live Legionella, macrophages were activated by live as well as dead L. pneumophila, as evidenced by the up-regulation of MHC class II. Finally, DCs, but not macrophages, exhibited a proliferative response to live L. pneumophila that was consistent with their cell cycle progression. These findings provide a better understanding of the role of DCs in adaptive immunity to Legionella infection.     

Legionella waterline colonization: detection of Legionella species in domestic, hotel and hospital hot water systems

AIMS: An evaluation was made of the prevalence of Legionella species in hot water distribution systems in the city of Bologna (Italy) and their possible association with bacterial contamination (total counts and Pseudomonadaceae) and the chemical characteristics of the water (pH, Ca, Mg, Fe, Mn, Cu, Zn and Total Organic Carbon, TOC). METHODS AND RESULTS: A total of 137 hot water samples were analysed: 59 from the same number of private apartments, 46 from 11 hotels and 32 from five hospitals, all using the same water supply. Legionella species were detected in 40.0% of the distribution systems, L. pneumophila in 33.3%. The highest colonization was found in the hot water systems of hospitals (93.7% of samples positive for L. pneumophila, geometric mean: 2.4 x 10(3) CFU l(-1)), followed by the hotels (60.9%, geometric mean: 127.3 CFU l(-1)) and the apartments with centralized heating (41.9%, geometric mean: 30.5 CFU l(-1)). The apartments with independent heating systems showed a lower level of colonization (3.6% for Legionella species), with no evidence of L. pneumophila. Correlation analysis suggests that copper exerts an inhibiting action, while the TOC tends to favour the development of L. pneumophila. No statistically significant association was seen with Pseudomonadaceae, which were found at lower water temperatures than legionellae and in individual distribution points rather than in the whole network. CONCLUSIONS: The water recirculation system used by centralized boilers enhances the spreading of legionellae throughout the whole network, both in terms of the number of colonized sites and in terms of CFU count. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in Legionella colonization between types of buildings are not due to a variation in water supply but to other factors. Besides the importance of water recirculation, the study demonstrates the inhibiting action of copper and the favourable action of TOC on the development of L. pneumophila.     

Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

Antimicrobial efficacy of a silver-zeolite matrix coating on stainless steel

A silver- and zinc-containing zeolite matrix (AgION) used as a coating for stainless steel was tested for antimicrobial efficacy against Escherichia coli 25922, Staphylococcus aureus 25923, Pseudomonas aeruginosa 27853, and Listeria monocytogenes 7644. Assays were performed on flat coupon surfaces and in formed steel cups. AgION reduced microbial colony-forming units when compared to uncoated steel surfaces under all conditions tested. Percent reductions ranged from 84.536 to 99.999 after 4 h exposure, and from 99.992 to 100 after 24 h in all cases. The durability of the coatings declined most markedly when the coating had been applied with a wet process and scrubbed between uses with a test tube brush. Powder-coated surfaces cleaned with a towel retained a high degree of activity after five cycles of use. Electronic Publication     

负载金属离子的5A 沸石的体外抗菌实验

目的:探讨负载金属离子的5A沸石的体外抗菌作用。方法:选用金黄色葡萄球菌、铜绿假单孢菌、白色念珠菌,利用倍比稀释法对5A沸石组、磺胺嘧啶银组及载不同浓度Ag+、Zn2+5A沸石共15组进行了体外抗菌试验研究,确定最佳抗菌效果离子负载方案。结果:三种细菌的MIC,双金属离子负载在抗菌上具有协同载Ag+5A沸石分别达到了125μg/ml~500μg/ml、31.25μg/ml~500μg/ml、250μg/ml~500μg/ml;附载Zn2+5A沸石分别达到了12.5mg/ml~25mg/ml、6.25mg/ml~50mg/ml、25mg/ml;负载双离子5A沸石分别为250μg/ml~500μg/ml、62.5μg/ml~500μg/ml、500μg/ml。结论:5A沸石负载金属离子后均具有抗菌作用,且抗菌作用与附载金属离子的量正相关;负载相同质量Ag+的5A沸石较附载Zn2+者具有更强的抗菌作用;双金属离子负载在抗菌上具有协同作用;2%Ag++8%Zn2+与2%Ag++10%Zn2+及4%载银组与阳性对照组无显著性差异。     

Adhesion of food-borne bacteria to stainless steel is reduced by food conditioning films

Aims:  Preconditioning of stainless steel with aqueous cod muscle extract significantly impedes subsequent bacterial adhesion most likely due to repelling effects of fish tropomyosin. The purpose of this study was to determine if other food conditioning films decrease or enhance bacterial adhesion to stainless steel.
Methods and Results:  Attachment of Pseudomonas fluorescens AH2 to stainless steel coated with water-soluble coatings of animal origin was significantly reduced as compared with noncoated stainless steel or stainless steel coated with laboratory substrate or extracts of plant origin. Coating with animal extracts also decreases adhesion of other food-relevant bacteria. The manipulation of adhesion was not attributable to growth inhibitory effects. Chemical analysis revealed that the stainless steels were covered by homogenous layers of adsorbed proteins. The presence of tropomyocin was indicated by appearance of proteins with similar molecular weight based in sodium dodecyl sulfate–polyacrylamide gel electrophoresis, in several extracts that reduced adhesion but also extracts not containing this protein reduced bacterial adhesion, indicating that several molecular species may be involved in the phenomenon.
Conclusions:  It is a common perception that food materials facilitate bacterial adhesion to surfaces; however, this study demonstrates that aqueous coatings of food origin may actually reduce bacterial adhesion.
Significance and Impact of the Study:  Compounds from food extracts may potentially be used as nontoxic coatings to reduce bacterial attachment to inert surfaces.     

Detection of Legionella pneumophila at thermal spas

Water samples were collected at three therapeutic thermal spas in the area of Brescia, between February and October 2000: 34.8% of the samples contained Legionella pneumophila; the predominant isolates (30%) belonged to Legionella pneumophila serogroup 1. The microorganism was present in the spa water at high concentrations, generally higher than 10000 cfu/l. The large number of positive Legionella pneumophila samples indicates a potential risk of infection to patients, especially those undergoing inhalation treatment with thermal water, or those using a whirlpool or taking a shower even if, during the study, no clinical cases of Legionnaires' disease were observed. In some inhalators in use we detected Legionella pneumophila: after a treatment to eradicate the microorganism, no sanitary fittings currently show contamination. Thus, in our opinion, they are not sources of infection when they are mantained and serviced properly. Thermal disinfection and service checks at regular intervals are suggested for contaminated systems.     

Role of the 25 kDa major outer membrane protein of Legionella pneumophila in attachment to U-937 cells and its potential as a virulence factor for chick embryos

The gene encoding the 25 kDa major outer membrane protein (MOMP) of Legionella pneumophila was transformed into Escherichia coli JM 83 and the resultant E. coli LP 116 clone expressed the Legionella-MOMP. Compared with the parent E. coli strain, the clone showed a fivefold increase in opsonin-independent binding to U-937 cells. Furthermore, this gene was incorporated by electroporation into a low virulence derivative of Leg. pneumophila which showed reduced expression of the MOMP but enhanced expression of a 31 kDa protein in the OMP profile. After electroporation, the attenuated strain showed an increased expression of the MOMP while the 31 kDa protein was eliminated and virulence for the chick embryo was re-established. The use of a monoclonal antibody specific for the MOMP abolished virulence and adherence. These studies suggest that the 25 kDa MOMP of Leg. pneumophila serves as an adhesive molecule for host cells and that this protein plays a major role in the virulence of the organism for the chick embryo.     

Changes in the strength of attachment of micro-organisms to surfaces following treatment with disinfectants and cleansing agents

Suspensions of Pseudomonas aeruginosa and Staphylococcus epidermidis , and biofilms established (16 h) on submerged glass and stainless steel (216 2B) coupons, were exposed to sodium hypochlorite (0·02% or 0·015% w/v), Dodigen (0·0015% w/v or 0·0006% w/v), sodium dodecylsulphate (6% w/v or 0·1% w/v) and Tween-80 (6% w/v) for 5 min at 20 °C. Survival was assessed by viable counts and blot succession. Biofilm bacteria were significantly less susceptible to these biocides than were planktonic cells, but their attachment to the surfaces was loosened by such treatments. Treatment with the non-ionic surfactant, Tween-80, however, strengthened the attachment of Staph. epidermidis to stainless steel. Such effects on attachment strength, which are species and surface dependent, have profound implications on post-treatment cleansing and possible re-contamination of product in clean-in-place (CIP) systems.     

Vaccination with the major secretory protein of Legionella induces humoral and cell-mediated immune responses and protective immunity across different serogroups of Legionella pneumophila and different species of Legionella.

In a previous study, we demonstrated that immunization of guinea pigs with the major secretory protein (MSP) of Legionella pneumophila, serogroup 1 induced humoral and cell-mediated immune responses to MSP and protective immunity against lethal aerosol challenge with this serogroup of L. pneumophila. Although serogroup 1 L. pneumophila cause most cases of Legionnaires' disease, other serogroups of L. pneumophila and species of Legionella cause many cases. In this study, we have examined if immunization with MSP induces humoral and cell-mediated immune responses and protective immunity across different serogroups of L. pneumophila and species of Legionella. By immunoblot analysis, MSP from L. pneumophila serogroup 1 (Lp1 MSP), L. pneumophila serogroup 6 (Lp6 MSP), and Legionella bozemanii (Lb MSP) shared common epitopes recognized by guinea pig anti-Lp1 MSP antiserum. These MSP molecules, however, were not identical as they had different apparent m.w. Immunization of guinea pigs with MSP induced strong cell-mediated immune responses across the different serogroups and species, as indicated by splenic lymphocyte proliferation and cutaneous delayed-type hypersensitivity in response to both homologous and heterologous MSP. Immunization with MSP induced strong protective immunity across two serogroups of L. pneumophila; overall, 9 survived aerosol challenge with L. pneumophila serogroup 1 compared to 0 of 12 (0%) sham-immunized control animals (p = 3 x 10(-4), Cochran-Mantel-Haenzel chi 2 statistic for pooled data). Immunization with MSP also induced protective immunity across species of Legionella but protection was species-specific. Whereas immunization with Lb MSP induced protective immunity against L. pneumophila, neither immunization with Lp1 MSP nor immunization with Lb MSP induced protective immunity against L. bozemanii, which produces MSP. Not surprisingly, immunization with MSP did not induce protective immunity against MSP-negative Legionella micdadei. In the case of both L. bozemanii and L. micdadei, immunization with a sublethal dose did confer protective immunity to aerosol challenge indicating that these species do contain immunoprotective components. This study demonstrates that immunization with MSP induces humoral and cell-mediated immune responses across different serogroups of L. pneumophila and species of Legionella, but that the capacity of MSP immunization to induce protective immunity is species-specific. Nevertheless, an MSP vaccine has the potential to induce protective immunity against the great majority of cases of Legionnaires' disease.     

Legionella pollution in cooling tower water of air-conditioning systems in Shanghai, China

Aims:  To determine Legionella pollution prevalence, describe the amount of Legionellae with respect to temperature in Shanghai cooling tower water (CTWs) in various types of public sites.
Methods and Results:  Six urban districts were selected as the study fields, adopting multiple-phase sampling methods. Routine culture was used to identify Legionellae. Of the samples, 58·9% (189/321) were observed to be positive, 19·9% were isolated over 100 CFU ml⁻¹. Legionella pneumophila serogroup 1 was the most frequently isolated species (155/189, 82·0%), followed by Leg. micdadei that was at the second place (44/189, 23·3%). The mean CFU ml⁻¹ of Legionellae in CTWs reached its peak from July to September. Over all 15·4% of the samples exceeding 100 CFU ml⁻¹ were observed in a hospital setting.
Conclusions:  The prevalence of Legionella pollution in CTWs, especially in CTWs of subway stations and hospitals, is worrying, and the positive rate and CFU ml⁻¹ of Legionellae in CTWs have a close relationship with air temperature.
Significance and Impact of the Study:  The study demonstrates pollution prevalence rates in different types of sites and various seasons, and provides a proportion of different serogroups of Legionellae . It illuminates an urgent need for dealing with the potential risk of legionellosis in Shanghai, through improved control and prevention strategies.     

Occurrence and genetic diversity of uncultured Legionella spp. in drinking water treated at temperatures below 15 degrees C

Representatives of the genus Legionella were detected by use of a real-time PCR method in all water samples collected directly after treatment from 16 surface water (SW) supplies prior to postdisinfection and from 81 groundwater (GW) supplies. Legionella concentrations ranged from 1.1 x 10(3) to 7.8 x 10(5) cells liter(-1) and were significantly higher in SW treated with multiple barriers at 4 degrees C than in GW treated at 9 to 12 degrees C with aeration and filtration but without chemical disinfection. No Legionellae (<50 CFU liter(-1)) were detected in treated water by the culture method. Legionella was also observed in untreated SW and in untreated aerobic and anaerobic GW. Filtration processes in SW and GW treatment had little effect or increased the Legionella concentration, but ozonation in SW treatment caused about 1-log-unit reduction. A phylogenetic analysis of 16S rRNA gene sequences of 202 clones, obtained from a selection of samples, showed a high similarity (>91%) with Legionella sequences in the GenBank database. A total of 40 (33%) of the 16S rRNA gene sequences obtained from treated water were identified as described Legionella species and types, including L. bozemanii, L. worsleiensis, Legionella-like amoebal pathogen types, L. quateirensis, L. waltersii, and L. pneumophila. 16S rRNA gene sequences with a similarity of below 97% from described species were positioned all over the phylogenetic tree of Legionella. Hence, a large diversity of yet-uncultured Legionellae are common members of the microbial communities in SW and GW treated at water temperatures of below 15 degrees C.     

The effect of Aeromonas strains on the growth of Legionella

Thirty-one environmental and two type strains of Aeromonas were tested for their ability to inhibit the growth of strains of several Legionella species. Of 10 Legionella spp. tested, only Legionella pneumophila and Leg. longbeachae were able to resist the inhibitory effects of some of the Aeromonas strains. Selected Aeromonas strains were also tested for their ability to inhibit the growth of several non-legionella strains. None of the non-legionella strains were inhibited by any of the selected Aeromonas strains. Attempts were made to isolate Aeromonas strains from cooling towers and Aeromonas hydrophila was isolated from one of the cooling towers tested. The results suggest that many strains of Aeromonas can inhibit the growth of Legionella strains on solid media and could affect the isolation of legionellas from water sources.