Evaluation of detection methods for Legionella species using seeded water samples

South African laboratories are currently using various methods in a non-standardised approach to detect Legionella species in environmental samples. In an attempt to provide guidelines for the development of a standard method, a number of currently available detection methods were evaluated, using seeded samples of sterile and non-sterile tap water, cooling water and make-up water. The samples were seeded with a type strain of L. pneumophila serogroup 1 (American Type Culture Collection 33152). The effect of sample concentration by centrifugation and membrane filtration followed by either vortex or sonication for resuspension of organisms was studied. Three currently available culture methods were evaluated: the International standard method (ISO/DIS 11731), the Australian standard method (AS 3896 - 1991) and a locally-developed adaptation of the most probable number method (MPN). In addition, the direct immunofluorescence test and a commercially available latex agglutination test kit were included in the evaluations. The usefulness of treatment with acid or heat prior to culture was also compared. Our results indicated that concentration by membrane filtration using nitro-cellulose filters with a pore size of 0.45 ?m, followed by sonication for 10 min, would be the most appropriate concentration and resuspension method for the samples. In the absence of sample pretreatment with acid or heat, organism recovery from sterile seeded samples on BCYE ranged from 85.9 - 98.7%. However, in the non-sterile samples, these figures dropped to 8.1 - 38.5%. Sample pretreatment resulted in a further loss of at least 50% of organisms in all the samples, regardless of the pretreatment method or culture medium used. In general, the ISO and AS methods were more appropriate than the MPN method for organism recovery from sterile seeded samples. However, for the nonsterile samples, the MPN method yielded better recovery. WaterSA Vol.27(4) 2001: 523-52

Construction and evaluation of a gfp-tagged Salmonella Typhimurium strain for environmental applications

Salmonella enterica ser. Typhimurium was isolated from freshwater sediments and chromosomally labelled with a stable variant of the green fluorescent protein (GFP). The pUT mini-Tn5 Km transposon was used to introduce the gfp gene onto the chromosome of the S. Typhimurium strain by tri-parental mating. Southern Blot hybridisation confirmed that the gene had integrated into the chromosome. The gfp gene was stably maintained and the labelled strain was not growth-rate impaired. The incorporation of the gfp gene did not convey any significant loss of phenotype which would affect the survival and behaviour of the tagged strains. The tagged S. Typhimurium strain was used to spike an established drinking water biofilm and was able to colonise and persist within the biofilm. The tagged strain was also successfully used to study the survival of S. Typhimurium in natural sediments under different temperatures. These tagged strains can therefore be used to study the fate and survival of different Salmonella strains in water environments.Keywords: biofilm, green fluorescent protein, Salmonella Typhimurium, surviva

Evaluation of oxidising disinfectants to control Vibrio biofilms in treated seawater used for fish processing

Marine fish-processing plants often use seawater during their operations. Chlorination and UV are commonly used for disinfection of this water but may not be effective in preventing biofilm formation within the water distribution network. These biofilms negatively impact water quality and could lead to contamination of fish products. During a recent study, Vibrio alginolyticus strains were detected on processed hake. The presence of most Vibrio spp. on fish products is of consumer safety concern and needs to be minimised. Water treatment strategies effective for seawater disinfection but with minimal negative effect on fish quality are required. In this study the effectiveness of chlorine, ozone and hydrogen peroxide (H2O2) in the inhibition of mature biofilms or biofilm formation in natural seawater was investigated. Two V. alginolyticus strains (V590 and V595) isolated from hake fish as well as the type strains of V. alginolyticus LMG 4409 and V. parahaemolyticus LMG 2850 were used. Chlorine was ineffective as experiments showed that strains V590, V595 and V. parahaemolyticus LMG 2850 could form biofilms even in the presence of 4 mg/ℓ of chlorine. When ozone was used, biofilm initiation and formation were completely inhibited for only 2 strains of V. alginoluticus, i.e. LMG 4409 and V590, at 1.6 mg/ℓ or 0.8 mg/ℓ ozone, respectively. Hydrogen peroxide performed the best of all the disinfectants evaluated in this study. Inhibition of biofilm formation was observed for all strains at 0.05% H2O2. The mature biofilms were more resistant to H2O2 but were all eliminated at 0.2% concentrations. This study indicated that H2O2 is the most effective biocide to prevent biofilm formation in seawater distribution networks and could potentially be used as an alternative or supplementary disinfectant of seawater in marine fish-processing plants. Keywords: V. alginolyticus, V. parahaemolyticus, biofilms, H2O2, disinfection, seawate

Optimisation of the PCR-invA primers for the detection of Salmonella in drinking and surface waters following a pre-cultivation step

A polymerase chain reaction (PCR)-based method for the detection of Salmonella species in water samples was optimised and evaluated for speed, specificity and sensitivity. Optimisation of Mg2+ and primer concentrations and cycling parameters increased the sensitivity and limit of detection of PCR to 2.6 x 104 cfu/m.. A 6h non-selective pre-enrichment step further increased the limit of detection to 26 cfu/m.. Out of 14 different Salmonella strains tested, only two, Salmonella arizonae and Salmonella pullorum, did not give positive amplification results with primers homologous to a conserved region of the invA gene. When environmental and drinking waters were assessed, a non-selective pre-enrichment step was included to increasethe detection efficiency of PCR. The PCR method demonstrated specificity in the presence of other competing micro-organismsas confirmed by the conventional culture method. No false positives or negatives were observed when household and environmental water samples were tested by invA-PCR analysis parallel to the culture method

The genetic relatedness of E. coli associated with post- collection drinking water contamination in rural households

Rural households are often dependent on rivers, springs, boreholes or standpipes some distance from their homes for their daily water requirements. Water for drinking and domestic use is consequently stored in containers in-house which are prone to post-collection contamination. The objective of the study was to determine the most likely origin or place of introduction of E. coli associated with post-collection contamination in rural households, by assessing the degree of genetic relatedness of E. coli present in the stored water and other environmental samples. E. coli isolates were obtained using either mFC agar with confirmation of indole production (44 isolates) or Colilert®-18 (52 isolates). Amplified fragment length polymorphism (AFLP) fingerprinting was applied to determine the genetic relatedness of E. coli isolated from in-house storage containers, drinking cups, hand-swab samples, cattle dung and from the source water (spring water). DNA fingerprints of E. coli produced a number of clusters (>85% similarity scores calculated with the cosine coefficient). Identical E. coli genetic patterns were observed at closely linked points within the domestic pathway of water handling, such as between hand-swab and drinking-cup samples, between storage container and source isolates, and between drinking cups, source water and storage containers. The results indicated that AFLP fingerprinting could be applied to determine the genetic relatedness of E. coli isolated from closely linked points within the domestic pathway of water use within a household. However, the high genetic diversity observed for E. coli bacteria isolated from the difffferent water and environmental samples tested in this study, hampered the identification of post collection points of contamination.Keywords: typing, fingerprinting, amplified fragment length polymorphism, E. coli, water quality, genetic relatedness, AFL

Prevalence of free-living acanthamoeba and its associated bacteria in energy-efficient hot water systems in South Africa

As part of the Eskom rebate programme, energy-efficient hot water systems such as solar water heaters (low pressure), heat pumps and energy-efficient showerheads were rolled out to the public as a measure to conserve and save energy. There has been a concern that these systems may not reach the required high temperatures, especially during winter, and, as a result of this, Acanthamoeba and its associated bacteria such as Legionella pneumophila, Pseudomonas aeruginosa and nontuberculous mycobacteria could flourish within these systems causing a potential health risk to consumers. This study examined the relationship between Acanthamoeba and its associated bacteria at different temperature ranges. A total of 156 water (69) and biofilm samples (87) were collected from a solar water heater, heat pump, geyser and showerheads and examined for these organisms using amoebal enrichment and molecular techniques. Amoeba could be cultivated from 45 (65.2%) of the water samples and 56 (64.4%) of the biofilm samples. The study confirmed the presence of Legionella pneumophila, Pseudomonas aeruginosa and nontuberculous mycobacteria in the hot water systems at both of the simulated winter (20°C to 30°C) and higher summer (40°C to 55°C) temperatures as well as the control system. There was a significant positive correlation between the presence of Acanthamoeba and the presence of Pseudomonas. Based on this association it is suggested that Pseudomonas aeruginosa could be investigated as an indicator organism for the presence of Acanthamoeba and opportunistic pathogens

Allergy education and training for physicians.

The increasing prevalence of allergic diseases has placed a significant burden on global healthcare and society as whole. This has necessitated a rapid development of "allergy" as a specialist area. However, as allergy is so common and, for most, relatively easy to diagnose and control, all clinicians need to have basic knowledge and competence  to manage  mild disease and recognize when referral is required. The allergology specialty has not yet been recognized in many countries and even where allergy is fully recognized as a specialty, the approach to training in allergy differs significantly. In the light of recent developments in allergy diagnosis and management, there is an urgent need to harmonize core competences for physicians, as well as the standardization of core principles for medical education and post-graduate training in allergy. All physicians and allied health professionals must appreciate the multidisciplinary team (MDT) approach to allergy, which is key to achieving the highest standards in holistic care. Due to worldwide variation in resources and personnel, some MDT roles will need to be absorbed by the treating physician or other healthcare professionals. We draw particular attention to the role of psychological input for all allergy patients, dietetic input in the case of food allergy and patient education to support all patients in the supported self-management of their condition on a daily basis. A strong appreciation of these multidisciplinary aspects will help physicians provide quality patient-centered care. We consider that harmonization of allergy components within undergraduate curricula is crucial to ensure all physicians develop the appropriate allergy-related knowledge and skills, particularly in light of inconsistencies seen in the primary care management of allergy. This review from the World Allergy Organization (WAO) Education and Training Committee also outlines allergy-related competences required of physicians working with allergic patients and provides recommendations to promote harmonization of allergy training and practice worldwide

Resource-oriented music therapy for psychiatric patients with low therapy motivation: Protocol for a randomised controlled trial [NCT00137189]

BACKGROUND: Previous research has shown positive effects of music therapy for people with schizophrenia and other mental disorders. In clinical practice, music therapy is often offered to psychiatric patients with low therapy motivation, but little research exists about this population. The aim of this study is to examine whether resource-oriented music therapy helps psychiatric patients with low therapy motivation to improve negative symptoms and other health-related outcomes. An additional aim of the study is to examine the mechanisms of change through music therapy. METHODS: 144 adults with a non-organic mental disorder (ICD-10: F1 to F6) who have low therapy motivation and a willingness to work with music will be randomly assigned to an experimental or a control condition. All participants will receive standard care, and the experimental group will in addition be offered biweekly sessions of music therapy over a period of three months. Outcomes will be measured by a blind assessor before and 1, 3, and 9 months after randomisation. DISCUSSION: The findings to be expected from this study will fill an important gap in the knowledge of treatment effects for a patient group that does not easily benefit from treatment. The study's close link to clinical practice, as well as its size and comprehensiveness, will make its results well generalisable to clinical practice

Carbon-focused conservation may fail to protect the most biodiverse tropical forests

As one of Earth’s most carbon-dense regions, tropical forests are central to climate change mitigation efforts. Their unparalleled species richness also makes them vital for safeguarding biodiversity. However, because research has not been conducted at management-relevant scales and has often not accounted for forest disturbance, the biodiversity implications of carbon conservation strategies remain poorly understood. We investigated tropical carbon–biodiversity relationships and trade-offs along a forest-disturbance gradient, using detailed and extensive carbon and biodiversity datasets. Biodiversity was positively associated with carbon in secondary and highly disturbed primary forests. Positive carbon–biodiversity relationships dissipated at around 100 MgC ha–1, meaning that in less disturbed forests more carbon did not equal more biodiversity. Simulated carbon conservation schemes therefore failed to protect many species in the most species-rich forests. These biodiversity shortfalls were sensitive to opportunity costs and could be decreased for small carbon penalties. To ensure that the most ecologically valuable forests are protected, biodiversity needs to be incorporated into carbon conservation planning