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South African Journal of Science
On-line version ISSN 1996-7489
Print version ISSN 0038-2353
S. Afr. j. sci. vol.110 n.11-12 Pretoria Nov./Dec. 2014
Biofilm formation in surface and drinking water distribution systems in Mafikeng, South Africa
Suma George MulamattathilI,II; Carlos BezuidenhoutI; Moses MbeweII
ISchool of Environmental Science and Development, North-West University,Potchefstroom, South Africa
IIDepartment of Water and Sanitation, University of Limpopo, Polokwane, South Africa
Poor quality source water and poorly treated reused wastewater may result in poor quality drinking water that has a higher potential to form biofilms. A biofilm is a group of microorganisms which adhere to a surface. We investigated biofilm growth in the drinking water distribution systems in the Mafikeng area, in the NorthWest Province of South Africa. Analysis was conducted to determine the presence of faecal coliforms, total coliforms, Pseudomonas spp. and Aeromonas spp. in the biofilms. Biofilms were grown on a device that contained copper and galvanised steel coupons. A mini tap filter - a point-of-use treatment device which can be used at a single faucet - was also used to collect samples. Scanning electron microscopy demonstrated that multi-species biofilms developed on all the coupons as well as on the point-of-use filters. Galvanised steel and carbon filters had the highest density of biofilm. Total coliforms, faecal coliforms and Pseudomonas spp. were isolated from raw water biofilm coupons only. Aeromonas spp. and Pseudomonas spp. were isolated from filters. The susceptibility of selected isolates was tested against 11 antibiotics of clinical interest. The most prevalent antibiotic resistance phenotype observed was KF-AP-C-E-OT-K-TM-A. The presence of virulence genes was determined using the polymerase chain reaction. These results indicate that bacteria present in the water have the ability to colonise as biofilms and drinking water biofilms may be a reservoir for opportunistic bacteria including Pseudomonas and Aeromonas species.
Keywords: Aeromonas; biofilm; drinking water distribution system; Pseudomonas; total coliforms
Water is a vital resource for life and access to safe drinking water is a basic right of every individual.1 South Africa is a semi-arid country with very little rainfall, resulting in high water stress; as such, individuals in many communities struggle to access potable water.2 Water scarcity problems can be addressed through the recycling of municipal wastewater for reuse in households - a practice which is increasing worldwide.3 However, reclaimed water may be a major source of pathogenic and opportunistic microorganisms, as well as pharmaceutical waste products.4 The presence of pathogenic microorganisms in treated water sources usually is because they are able to survive the treatment process. Moreover, in most developing countries, water-treatment plants are usually faced with maintenance problems and a lack of qualified personnel.
In aquatic environments, microorganisms have the ability to adhere to solid surfaces and form biofilms.5 Biofilms are bacterial communities embedded in a polysaccharide matrix, which gives them the opportunity to resist destruction by antibiotics, environmental stress, biocides and detergents.6 Bacterial regrowth in the distribution system may result from the detachment of biofilm bacteria, which increases the risk of infection in humans when the water is consumed.6 Generally, most water distribution systems are characterised by the presence of biofilms, regardless of purity, the type of pipe material used for distribution or the presence of a disinfectant.7 Bacteria in drinking water systems can therefore grow in bulk water and as biofilms attached to the walls of pipes.8 Moreover, the development of biofilms inside water distribution pipes facilitates the propagation of mixed microbial populations and is considered the main source of planktonic bacteria in water supply systems.9 This problem is further aggravated by the presence of opportunistic pathogens such as Pseudomonas, Aeromonas, Klebsiella, Mycobacter, Escherichia coli, Helicobacter, Salmonella and Legionella spp. that may increase the health risks associated with the consumption of water from these sources.10
Different materials - such as cast iron galvanised steel, stainless steel, copper and polyethylene - have been used to manufacture water distribution pipes and these materials favour biofilm formation in the water distribution systems.7,11,12 Differences in the pipe materials greatly favour the survival of different bacterial species.13 The presence of biofilms in drinking water distribution pipes usually leads to a number of undesirable effects on the quality of water that is supplied to consumers.14 The development of biofilms in copper pipes facilitates cuprosolvency which increases the release of copper into the distribution system.15 Furthermore, increased carbon influences the growth of heterotrophic plate count bacteria which are also involved in the corrosion of copper.15 The corrosion of lead-containing plumbing materials increases the chances of lead contamination in tap water, which can cause adverse health effects in humans, especially children.16 Detachment of bacteria from the biofilms may affect the quality of the water.17 Therefore, the deterioration of the quality of drinking water as a result of biofilm formation is a major concern for most municipal supply agencies and communities. Biofilms are present in spite of different treatment processes; the occurrence of biofilms in drinking water is attributed to bacterial resistance to disinfectants, ability of the bacterial species to resist chemical compounds released from pipe materials and species association which increases the proportion of viable cells.18,19 Moreover, the use of different disinfectant methods may have long-term effects on the biofilm community.20 Biofilms consisting of Pseudomonas aeruginosa and different faecal bacterial species have been detected in water distribution systems, even in countries that have more advanced water-treatment facilities.21,22
Mafikeng is the capital of the North West Province of South Africa. This city uses both groundwater as well as dam water for drinking water production. Some areas receive a mixture of the two water types and others receive only one or the other. The purification plant for the surface water is at the Modimola Dam and receives treated wastewater. The water purification plant is downstream from the sewage treatment plant and is thus a semi-closed water conservation system.
The study was designed to investigate the biofilm forming ability, antibiotic resistance and virulence gene determinants of biofilm bacteria, especially Pseudomonas and Aeromonas species, in the water distribution systems in Mafikeng.
Materials and methods
Sampling area
Biofilm forming devices were installed at different sites within the water distribution system in the Mafikeng-Mmabatho area: (1) raw untreated water from the Modimola Dam, (2) treated water from household taps, received from the Modimola Dam treatment plant, (3) treated water from Molopo Eye, a natural spring and (4) water from the Modimola Dam treatment plant mixed with chlorinated water from Molopo Eye (mixed water).
Biofilm formation devices
To study biofilm growth, a flow system technique that utilises a biofilm developing device was used (Figure 1). The biofilm developing device pipe system was made from clear plastic pipe with a diameter of 16 mm. The device was installed with copper and galvanised coupons to serve as solid surfaces onto which bacteria could adhere and form biofilms. Coupons were held in place by screws. The device was mounted horizontally to the main pipe of a building of the North-West University in Mafikeng (Figure 1). This building receives mixed water. The coupons were installed at the different sampling points for 6 months. Mini tap filters (Figure 2) - which are point-of-use (POU) treatment devices which can be used at a single faucet under constant flow - were also used to form biofilms during the second collection. The filters were placed on cold water taps in participating locations that received treated groundwater (Molopo Eye water), only Modimola Dam water or mixed water (North-West University, Mafikeng campus). The filters remained at these sampling points for 4 months.
Sampling of biofilm
Biofilm samples were analysed twice during the study period. To collect samples, the pipes containing biofilm developing devices were closed with valves before they were disconnected and the coupons were removed using sterile forceps. The filters in the mini tap filter devices were aseptically removed from the cartridge. Coupons were placed immediately into sterile 100-mL Schott bottles that contained water from the particular sampling site. Samples were transported on ice to the laboratory for analysis. Upon arrival in the laboratory, the coupons were removed from the bottles. Those coupons intended for scanning electron microscopy were stored in 100% alcohol and the remaining coupons were analysed for bacterial growth.
Scanning electron microscopy
The biofilm structure was investigated using scanning electron microscopy (SEM). Biofilm samples were fixed by 2.5% glutaraldehyde and 2% osmium tetroxide, dehydrated sequentially in increasing concentrations of ethanol (70%, 90% and 100%) for 15 min and critically dried in liquid carbon dioxide. The samples were mounted on SEM stubs using double-sided carbon tape. These stubs were then carbon coated. Finally, the samples were super coated with gold/palladium and viewed using a Philips XL30 scanning electron microscope (Philips, Aachen, Germany). Enlargements ranged from 63X to 20 000X.
Isolation of bacteria from the biofilm
Bacteria were isolated using standard methods. Biofilm bacteria on the coupons were removed by swabbing the surfaces with a sterile cotton swab dipped in sterile nutrient broth and immediately streaking it onto selective media for the isolation of targeted organisms. The media used were: mFC agar for the isolation of faecal coliforms, mEndo for total coliforms and Aeromonas selective agar for Aeromonas and Pseudomonas species. All the media used were Biolab agars from Merck (Johannesburg, South Africa). The plates were incubated aerobically at 35 °C for 24 h, except for mFC agar plates which were incubated at 45 °C for 24 h. Blue colonies from mFC agar and metallic-sheen colonies from mEndo agar were considered as presumptive faecal coliforms and total coliforms, respectively. Moreover, yellow and green colonies on Aeromonas selective agar represented Aeromonas and Pseudomonas species, respectively. These isolates were sub-cultured on the respective selective media and plates were incubated aerobically at 35 °C and 45 °C, respectively, for 24 h. Presumptive pure colonies were subjected to specific preliminary and confirmatory biochemical tests. All pure isolates were Gram stained using standard methods.
Preliminary biochemical tests
Triple sugar iron agar test
The triple sugar iron (TSI) test can also be used as a confirmation test for E. coli. The TSI agar obtained from Biolab (Merck, Johannesburg, South Africa) was used to determine the ability of isolated organisms to utilise the substrates glucose, sucrose and lactose at sample concentrations of 0.1%, 1.0% and 1.0%, respectively. The test was performed as per the instructions of the manufacturer and evaluated based on the formation of gas and hydrogen sulphide and fermentation of carbohydrates to produce acids. TSI agar slants were prepared in sterile 15-mL McCartney bottles. The slants were streak and stab inoculated with a sterile inoculation needle containing the selected colony. Following inoculation, the slants were incubated at 37 °C for 24 h. The colour of the slant and butt was recorded (red or yellow) as well as the production of hydrogen sulphide (when the agar blackened) and formation of gas (when the agar split).
Oxidase test
This test was performed using the Test Oxidase™ reagent (PL.390) from Mast Diagnostics (Nesto, Wirral, UK) in accordance with the manufacturer's published protocol. A well-isolated pure colony was placed on filter paper using a sterile wire loop. A drop of Test OxidaseTM reagent was added to the filter paper and mixed. After 30 s the filter was observed for a colour change. Isolates that produced a purple colour were presumptively considered to be E. coli. Oxidase positive colonies were taken as presumptive Aeromonas isolates.
Analytical profile index test
The analytical profile index (API) 20E test was performed in accordance with the manufacturer's protocol (BioMeriéux, Marcy I'Etoile, France). An API 20E strip was used to identify Enterobacteriaceae and other non-fastidious, Gram-negative rods. The strips were inoculated and incubated at 37 °C for 24 h. The indices obtained after reading the results were interpreted using the API web software (BioMeriéux®). The organisms were identified to species level.
Antimicrobial susceptibility test
An antibiotic susceptibility test was performed using the Kirby-Bauer disc diffusion method. The following antibiotic discs (Mast Diagnostics, UK) were used at the final concentrations that are indicated: ampicillin (AP) 10 µg, cephalothin (KF) 5 µg, streptomycin (S) 10 µg, erythromycin (E) 15 µg, chloramphenicol (C) 30 µg, neomycin (NE) 30 µg, amoxycillin (A) 10 µg, ciprofloxacin (CIP) 5 µg, trimethoprim (TM) 25 µg, kanamycin (K) 30 µg, oxytetracycline (OT) 30 µg. These antibiotics were chosen because either they are used in both humans and other animals or they have been reported to be resistant in previous studies.
Three colonies were picked from each sample and each colony was transferred into 3 mL of sterile distilled water to prepare a bacterial suspension. Aliquots of 1 mL from each suspension were spread plated on Mueller-Hinton agar plates. Antibiotic discs were applied to the plates using sterile needles and the plates were incubated at 37 °C for 24 h. After incubation, the antibiotic inhibition zone diameters were measured. Results obtained were used to classify isolates as being resistant, intermediate resistant or susceptible to a particular antibiotic using standard reference values according to the US National Committee for Clinical Laboratory Standards (now called the Clinical and Laboratory Standards Institute). Multiple antibiotic resistance phenotypes were generated for isolates that showed resistance to three or more antibiotics.
Confirmatory DNA test
Genomic DNA extraction
Genomic DNA was extracted from all the presumptive Pseudomonas and Aeromonas isolates using the alkaline lysis method. The concentration and quality of the extracted DNA in solution were determined using a spectrophotometer (NanoDrop ND 1000, Thermo Scientific, USA) and 1% (w/v) agarose gel electrophoresis. The latter was also used for determining the integrity of the genomic DNA.
Polymerase chain reaction for identifying species
The identities of the presumptive Pseudomonas species were confirmed through amplification of the toxA24 and ecfX25 gene determinants and Aeromonas species were confirmed through gyrB23 amplification. Polymerase chain reactions (PCRs) were performed using oligonucleotide primer combinations under the cycling conditions given in Table 1. Standard 25-µL reactions that consisted of 1 µg/uL of the template DNA, 50 pmol of each oligonucleotide primer set, 1X PCR master mix and RNase free water were prepared. Amplifications were performed using a Peltier Thermal Cycler (model-PTC-220DYAD™ DNA ENGINE, MJ Research Inc., Waltham, MA, USA). All PCR reagents used were Fermentas (USA) products supplied by Inqaba Biotec Pty Ltd (Pretoria, South Africa). PCR products were subjected to 1% (w/v) agarose gel electrophoresis.
Polymerase chain reaction for detection of virulence gene markers
Pseudomonas species were screened for the presence of the exoA, exoS and exoT virulence gene determinants26 while a specific PCR for the detection of aerA and hylH genes was performed on all positively identified Aeromonas species27. PCRs were performed using oligonucleotide primer combinations under the cycling conditions given in Table 2. Amplifications were performed using a Peltier Thermal Cycler (model-PTC-220DYAD™ DNA ENGINE). The reactions were prepared in 25-µL volumes that constituted 1 µg/µL of the template DNA, 50 pmol of each oligonucleotide primer set, 1X PCR master mix and RNase free water. All PCR reagents used were Fermentas (USA) products supplied by Inqaba Biotech Pty Ltd (Pretoria, South Africa). pCr products were subjected to 2% (w/v) agarose gel electrophoresis.
Electrophoresis of polymerase chain reaction products
Products of the PCRs were separated by electrophoresis on 2% (w/v) agarose gel. Electrophoresis was conducted in a horizontal Pharmacia Biotech equipment system (model Hoefer HE 99X; Amersham Pharmacia Biotech, Uppsala, Sweden) for 2 h at 60 V using 1X TAE buffer (40 mM Tris, 1 mM EDTA and 20 mM glacial acetic acid, pH 8.0). Each gel contained a 100-bp DNA molecular weight marker (Fermentas, Hanover,MD, USA). The gels were stained in ethidium bromide (0.1 ^g/ml) for 15 min and amplicons were visualised under UV light. A Gene Genius Bio imaging system (Syngene, Synoptics, Cambridge, UK) was used to capture the image using GeneSnap (version 3.07.01) software (Syngene, Synoptics) to determine the relative size of amplicons.
Occurrence and diversity of microorganisms in the bio films
Table 3 indicates the different types of organisms isolated from the biofilm. The results indicate only the numbers of isolates that were positive for the various categories when subjected to preliminary tests (TSI, oxidase and, in the case of Pseudomonas spp. and Aeromonas spp., API 20E). It is evident from Table 3 that total coliforms and faecal coliforms were present in the biofilms from the raw water from Modimola Dam but were not detected in the biofilms of the treated water from Modimola Dam. Furthermore, Aeromonas and Pseudomonas spp. were detected in the biofilms of the raw dam water as well as the drinking water from the POU devices. Only Pseudomonas spp. (27) were isolated from the biofilm of the Modimola Dam raw water (Table 3), whereas both Aeromonas spp. and Pseudomonas spp. were found in the biofilms of the treated Modimola Dam drinking water and the mixed water.
Figures 3 to 5 depict the surfaces of coupons and filters exposed to raw water (Figure 3) and treated water distribution systems (Figures 4 and 5). Different organisms were isolated from the metal surfaces. The SEM revealed the existence of bacterial cells within the biofilm matrix, with a greater amount of bacterial cells found on the galvanised coupons than on the copper coupons (Figures 3 to 5). From these micrographs, it was also evident that high bacterial densities were found in biofilms from the raw water of the Modimola Dam as well as from the drinking water distribution system in Mafikeng. The dam water is treated and supplied to homes for consumption.
When the coupons were removed from the biofilm development device, a green colour was noticed on the copper coupons; this colour is attributed to the corrosion products that are visible (like crystals) in the SEM (Figure 4b). Attached cells in association with exopolysaccharide were visible in galvanised, copper and POU filter surfaces in the SEMs (Figures 3 to 5). It is therefore suggested that the suspended cells have the potential to attach to the surface and participate during biofilm formation. An extensive sponge-like exopolysaccharide layer can be seen on the galvanised coupons in association with bacterial cells (Figure 3a). Figure 5 (POU filters) shows evidence of biofilm formation on the surface of filters from mixed water, dam water and water from the Molopo Eye. Rod-shaped bacteria are the dominating organisms in the biofilm and the aggregation of rod-shaped bacteria entangled in the exopolysaccharide, as seen in the micrographs, usually reflect a mature biofilm. It was found that galvanised coupons from raw and treated water contained thicker biofilms than did the copper coupons. Of the treated water from the three sites, coupons from the mixed water were colonised by a variety of bacteria.
If biofilms contain any pathogenic bacteria, the detachment of biofilms could release these bacteria into drinking water and affect risk levels of consumers.7 In another study, coliform bacteria - originating from biofilms observed on rubber-coated valves - were isolated from drinking water distribution systems.21 Coliforms were not detected in the present study, but that does not mean that they were absent.
Antimicrobial susceptibility test
All the organisms were subjected to an antibiotic sensitivity test using 11 antibiotics of clinical importance. Multiple antibiotic resistance phenotypes were generated for isolates resistant to three or more drugs; the results are shown in Table 4.
All the isolates tested were resistant to ampicillin, amoxicillin, cephalothin, erythromycin, chloramphenicol and trimethoprim. All the organisms tested were susceptible to ciprofloxacin. Four different multiple antibiotic resistance patterns were observed and all the isolates were resistant to three or more classes of antibiotics. The highest level of resistance -with the phenotype KF-AP-C-E-OT-K-TM-A, indicating resistance to eight drugs - was observed for isolates from biofilms. From these results, it is evident that ciprofloxacin and streptomycin were the most effective, because all or a large proportion of the isolates were susceptible to both. These results indicate that biofilm grown organisms may serve as a reservoir for antibiotic-resistant organisms, and therefore may have the potential to cause infections. This finding is a cause for concern, particularly for infants, the elderly and immunocompromised individuals in the Mafikeng community.
Identification and detection of virulence gene isolates were done using gyrB, toxA and ecfX gene fragments through PCR. The gyrB and ecfX gene fragments were amplified. Specific PCR assays for the detection of virulence genes (aerA and hylH in Aeromonas and exoA, exoS and exoT in Pseudomonas) produced DNA fragments of the expected size of some of the markers. Of the 39 isolates that were screened, the combination of virulence genes detected in the isolates from the different areas are shown in Table 5. Aeromonas spp. that were isolated from biofilms from Modimola Dam raw water (8 of 12) and mixed water (1 of 10) harboured the hylH gene (Figure 6). These genes were more prevalent in isolates from raw dam water. However, aerA genes were not detected in the isolates from either site. The exoA gene (Figure 7) was detected in Pseudomonas spp. from the raw water biofilm and biofilm isolates from the treated dam water. Isolates from the biofilms from all sites harboured exoT genes (Figure 8). However, none of the Pseudomonas spp. isolates possessed the exoS gene.
Water is a vital component of life but can serve as an important vehicle for the dissemination of potential pathogens to humans.21 Source water that receives sewage effluent may be polluted with opportunistic pathogenic microorganisms and pharmaceuticals.28 Modimola Dam receives treated sewage effluent. Organisms that survived the treatment process may be able to grow in the aquatic environment. A further concern is that, at times, the treatment processes fail and the potential exists for the presence of opportunistic pathogens such as Aeromonas hydrophila and Pseudomonas aeruginosa. Both of these species have a tendency to form biofilms. This study was thus aimed at determining whether Aeromonas spp. and Pseudomonas spp. occur in biofilms in the drinking water of Mafikeng.
Organisms isolated in this study include faecal coliforms, total coliforms, Aeromonas and Pseudomonas. In a similar study, heterotrophic bacteria were isolated from biofilms in copper plumbing, which included a wide variety of organisms.10Aeromonas species are implicated in gastroenteritis and are generally considered to be waterborne pathogens29 while Pseudomonas species are opportunistic pathogens that cause nosocomial infections in susceptible patients30. Moreover, it is very difficult to eradicate Pseudomonas species because of their high intrinsic resistance to a variety of antibiotics, including ß-lactams, aminoglycosides and fluroquinolones.31Aeromonas has the potential to grow in water distribution systems, especially in biofilms, where it is resistant to chlorination and produces many different putative virulence factors.32 Isolates that are resistant to chlorine may be present in tap water that is intended for human consumption, and which therefore causes disease in humans.
The regrowth and formation of biofilms in drinking water distribution pipes has been detected even in countries with advanced water-treatment and health-care facilities.21 The presence of biofilms has been found to cause significant corrosion of pipe materials and, subsequently, the addition of inorganic and organic matter, which results in a poor aesthetic quality of water.12 Moreover, free chlorine reacts with compounds present in biofilms inside water pipes, producing an unpleasant taste and odour.11 Adherent bacteria are more resistant to antimicrobial agents and could contribute to the planktonic cells present in the bulk water. The prevalence of planktonic bacteria in drinking water may be a result of sloughing of the biofilm - an assumption which is supported by the observation of planktonic bacterial episodes in treated drinking water.5
Biofilm formation is facilitated by many factors including available nutrients, characteristics of the pipe material, disinfectants used, physico-chemical parameters and the ability of the microorganisms to resist destruction by antimicrobial agents.33 The significant release of nutrients from the surface material to the water promotes the growth of bacteria.18
Cementitious, metallic and plastic materials are the three most commonly used types of plumbing materials. The plumbing materials chosen for this study were copper and galvanised steel, which are commonly used in domestic plumbing systems in South Africa. Our results demonstrate biofilm formation on both of the plumbing materials that were used. In another study, a higher density of bacteria was observed on polyethylene and polyvinylchloride surfaces than on galvanised steel.34 In the present study, thicker biofilms were observed on galvanised steel than on copper. However, biofilms on galvanised steel coupons were not compared with those on polyethylene and polyvinylchloride surfaces. It has been observed that biofilms on copper had low concentrations of culturable bacteria.35 It is thus not uncommon to find low levels of culturable bacteria in biofilms on copper coupons. Moreover, it has been reported that the formation of biofilms was slower on copper pipes than on polyethylene pipes. However, after 200 days there was no difference in microbial numbers in biofilms between the two pipe materials.18 This finding implies that in the case of long-term use, biofilm levels on metal surfaces will be similar to those on polyethylene and polyvinylchloride surfaces. However, as a consequence of their nature, biofilms on metal surfaces will contribute to microbial-induced corrosion, which can increase the metal concentration in water distributed by copper pipes,15 which has the potential to cause health problems.
In the present study, we did not focus on the corrosion potential of the biofilms but rather on whether or not Pseudomonas and Aeromonas spp. colonise the biofilms. It has been previously demonstrated that Pseudomonas spp. are opportunistic pathogens that can integrate into drinking water biofilms on materials which are relevant for domestic plumbing systems.35 Biofilms have been implicated in human infections and are particularly recalcitrant to antibiotic compounds.30,36 Final water produced by the water-treatment plant must comply with the South African National Standard (SANS) 241:2011 for drinking water.37
No total coliforms or faecal coliforms were detected in biofilms from the treated drinking water, which thus complied with the SANS 241 standard of 0 CFU/100 mL. This finding is an indication that the treatment process was effective in removing total coliforms and faecal coliforms from the raw water. However, Pseudomonas and Aeromonas spp. were isolated from the biofilms of treated and filtered water. This may be an indication of deteriorating water quality. High levels of Pseudomonas spp. in water may cause taste, odour and turbidity problems.38 There is no available SANS 241 standard for Pseudomonas and Aeromonas spp. in drinking water.
Identities of the organisms were confirmed through PCR using gyrB, toxA, ecfX, aerA and hylH gene fragments. The ecfX gene encodes an extra cytoplasmic function sigma factor, which may be involved in haem uptake or virulence39, whereas the gyrB gene encodes the DNA gyrase subunit B, a protein which plays a crucial role in the DNA replication process40 and the toxA gene encodes the exotoxin A precursor25. PCR assays targeting the ecfX and gyrB genes are highly suitable for the identification of P. aeruginosa.25,39 Application of the PCR technique to target gyrB, aerA and hylH genes is an excellent molecular chronometer for screening potentially virulent Aeromonas species in food and the environment.4041
One rational approach to determine whether Pseudomonas and Aeromonas spp. have the potential to be virulent is the assessment of virulence phenotypes and screening for specific virulence genes. Pseudomonas and Aeromonas spp. isolated in the present study carried some gene sequences encoding toxic proteins, indicating the potential of these organisms to cause diseases in humans. The ability of Pseudomonas spp. to express these virulence determinants also enhances their capabilities to produce biofilms.36Pseudomonas aeruginosa is able to synthesise a large number of virulence proteins that greatly influence pathogenesis.26Pseudomonas species produce extracellular compounds which promote adhesion and the ability of the isolates to attach to surfaces, thereby also increasing the virulence properties. The pathogenicity of Aeromonas is complex and multifactorial and has been linked to exotoxins such as cytolytic enterotoxin, haemolysin/aerolysin, (aerA, hylH, hylA, alt and ast), lipase and protease and various other cell-associated factors.2732 Screening for specific cytotoxin and haemolysin genes appeared to be the most effective way of detecting and characterising Aeromonas virulence factors.41
The desired gene fragments were successfully amplified, which indicates the presence of virulent Pseudomonas and Aeromonas spp. in biofilms from drinking water. From the molecular data it was demonstrated that the exoA, exoT and hylH genes were successfully used for the detection of virulent Pseudomonas and Aeromonas spp. in raw and drinking water biofilm samples. It is thus important to perform molecular confirmation of isolates to ensure accurate results.
Detection of these genes amongst the Pseudomonas and Aeromonas spp. isolated from the drinking water sources of Mafikeng is cause for concern and should be further investigated. PCR assays could provide a powerful supplement to the conventional methods for a more accurate risk assessment and monitoring of potentially virulent Pseudomonas and Aeromonas spp. in the environment.
Bacterial biofilms were detected in all water sources that were sampled and opportunistic pathogens such as Pseudomonas and Aeromonas species were isolated from biofilms in raw water from the Modimola Dam, drinking water and mixed water. These isolates were found to harbour virulence gene determinants indicating that they have the potential to cause diseases in humans. Therefore, it is important to constantly determine the occurrence of these species in water bodies and drinking water distribution systems, in particular, and to determine whether conditions prevail that may allow these opportunistic species to survive water purification processes. Such a strategy will be of particular importance in scenarios in which treated wastewater is reused for drinking water. A clear understanding of the different mechanisms by which biofilm bacteria harbour and distribute virulence factors, as well as protect themselves from the action of disinfectants and antibiotics, is vital to formulate control and management strategies.
We thank the National Research Foundation of South Africa (FA20040101000030 and FA2006040700029) for financial support of this study. We also thank the staff of the microbiology laboratory at Animal Health, NWU (Mafikeng) for their assistance. We acknowledge the assistance received from Mrs Rika Huyser and the employees of Mmabatho water works.
Authors' contributions
S.G.M. is the main author of the article and performed all the research work. C.B. supervised the work and guided the main author and M.M. co-supervised the work.
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16. Goudier M, Bouzid J, Sayadi S, Montiel A. Impact of orthophosphate addition on biofilm development in drinking water distribution systems. J Hazard Mater. 2009;167:1198-1202. http://dx.doi.org/10.1016/j.jhazmat.2009.01.128 [ Links ]
17. Elenter D, Milferstedt K, Zhang W, Hausner M, Morgenroth E. Influence of detachment on substrate removal and microbial ecology in a heterotrophic/ autotrophic biofilm. Water Res. 2007;41:4657-4671. http://dx.doi.org/10.1016/j.watres.2007.06.050 [ Links ]
18. Lehtola MJ, Miettinena IT, Keinanen MM, Kekkia TK, Laineb O, Hirvonen A. Microbiology, chemistry and biofilm development in a pilot drinking water distribution system with copper and plastic pipes. Water Res. 2004;38:p.3769-3779. http://dx.doi.org/10.1016/j.watres.2004.06.024 [ Links ]
19. Simöes M, Simöes L, Vieira M. Species association increases biofilm resistance to chemical and mechanical treatments. Water Res. 2009;43:229-237. http://dx.doi.org/10.1016/j.watres.2008.10.010 [ Links ]
20. Roeder R, Lenz J, Tarne P, Gebel J, Exner M, Szewzyk U. Long-term effects of disinfectants on the community composition of drinking water biofilms. Int J Hyg Environ Health. 2010;213:183-189. http://dx.doi.org/10.1016/j.ijheh.2010.04.007 [ Links ]
21. Kilb B, Lange B, Schaule G, Flemming H, Wingender J. Contamination of drinking water by coliforms from biofilms grown on rubber-coated valves. Int J Hyg Environ Health. 2003;206:563-573. [ Links ]
22. Werner E, Roe F, Bugnicourt A, Franklin M, Haydorn A, Molin S. Stratified gowth in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol. 2004;70(10):6188-6196. http://dx.doi.org/10.1128/AEM.70.10.6188-6196.2004 [ Links ]
23. Qin X, Emerson J, Stapp J, Stapp L, Abe P Burns J. Use of real-time PCR with multiple targets to identify Pseudomonas aeruginosa and other nonfermenting Gram-negative bacilli from patients with cystic fibrosis. J Clin Microbiol. 2003;41:4312-4317. http://dx.doi.org/10.1128/JCM.41.9.4312-4317.2003 [ Links ]
24. Khan A, Cerniglia C. Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR. Appl Environ Microbiol. 1994;60:3739-3745. [ Links ]
25. Lavenir R, Jocktane D, Laurent F, Nazaret S, Cournoyer B. Improved reliability of Pseudomonas aeruginosa PCR detection by the use of the species specific ecfX gene target. J Microbiol Methods. 2007;70:20-29. http://dx.doi.org/10.1016/j.mimet.2007.03.008 [ Links ]
26. Kaszab E, Szoboszlay S, Dobolyi C, Háhn J, Kriszt B. Antibiotic resistance profiles and virulence markers of Pseudomonas aeruginosa strains isolated from compost. Bioresour Technol. 2011;102:1543-1548. http://dx.doi.org/10.1016/j.biortech.2010.08.027 [ Links ]
27. Yogananth N, Bhakyaraj R, Chanthuru A, Anbalagan T, MullaiNila K. Detection of virulence gene in Aeromons hydrophila isolated from fish samples using PCR technique. Global J Biotech Biochem. 2009;4:51-53. [ Links ]
28. Köck-Schulmeyer M, Ginebreda A, Postigo C, López-Serna R, Pérez S, Brix R. Wastewater reuse in Mediterranean semi-arid areas: The impact of discharges of tertiary treated sewage on the load of polar micro pollutants in the Llobregat River (NE Spain). Chemosphere. 2011;82:670-678. http://dx.doi.org/10.1016/j.chemosphere.2010.11.005 [ Links ]
29. Pablos M, Huys G, Cnockaert M, Rodriguez-Calleja J, Otero A, Santos J, et al. Identification and epidemiology relationships of Aeromonas isolates from patients with diarrhoea, drinking water and foods. Int J Food Microbiol. 2011;147:203-210. http://dx.doi.org/10.1016/jjjfoodmicro.2011.04.006 [ Links ]
30. Fricks-Lima J, Hendrickson C, Allegaier M, Zhuo H, Wiener-Kronish J, Lynch S, et al. Differences in biofilm formation and antimicrobial resistance of Pseudomonas aeruginosa isolated from airways of mechanically ventilated patients and cystic fibrosis patients. Int J Antimicrob Agents. 2011;37:309-315. http://dx.doi.org/10.1016/jjjantimicag.2010.12.017 [ Links ]
31. Bredenstein E, De la Fuente-Núnez C, Hancock R. Pseudomonas aeruginosa: All roads lead to resistance. Trends Microbiol. 2011;19:419-426. http://dx.doi.org/10.1016/j.tim.2011.04.005 [ Links ]
32. Pablos M, Rodriguez-Celleja J, Santos J, Garcia-Lopez M. Occurrence of motile Aeromonas in municipal drinking water and distribution of genes encoding virulence factors. Int J Food Microbiol. 2009;135:158-164. http://dx.doi.org/10.1016/j.ijfoodmicro.2009.08.020 [ Links ]
33. Manuel CM, Nunes O, Melo L. Dynamics of drinking water biofilm in flow conditions. Water Res. 2007;47:551-562. http://dx.doi.org/10.1016/j.watres.2006.11.007 [ Links ]
34. Cloete TE. Resistance mechanism of bacteria to antimicrobial compounds. Int Biodeterior Biodegrad. 2003;51:277-282. http://dx.doi.org/10.1016/S0964-8305(03)00042-8 [ Links ]
35. Moritz M, Flemming H, Wingender J. Integration of Pseudomonas aeruginosa and Legionella pneumophila in drinking water biofilms grown on domestic plumbing materials. Int J Hyg Environ Health. 2013;213:190-197. http://dx.doi.org/10.1016/j.ijheh.2010.05.003 [ Links ]
36. Pimenta A, Martino P, Bouder E, Hulen C, Blight M. In vitro identification of two adherence factors required for in vivo virulence of Pseudomonas fluorescens. Microb Infect. 2003;5:1177-1187. http://dx.doi.org/10.1016/j.micinf.2003.09.002 [ Links ]
37. Department of Water Affairs (DWA). Blue drop handbook version 1 [document on the Internet]. [ Links ] c2012 [cited 2014 Mar 23]. Available from: http://www.dwa.gov.za/dir_ws/DWQR/Subscr/ViewNewsDoc.asp?FileID=262
38. World Health Organization (WHO). Guidelines for drinking water. 4th ed. Geneva: WHO; 2011. p. 541. [ Links ]
39. Anuj S, Whiley D, Kidd T, Bell S, Wainwright C, Nissen M, et al. Identification of Pseudomonas aeruginosa by a duplex polymerase chain reaction assay targeting the ecfX and gyrB genes. Diagn Microbiol Infect Dis. 2009;63:127-131. http://dx.doi.org/10.1016/j.diagmicrobio.2008.09.018 [ Links ]
40. Yánez M, Valor C, Catalán V. A simple and cost-effective method for the quantification of total coliforms and Escherichia coli in potable water. J Microbiol Methods. 2006;65:608-611. http://dx.doi.org/10.1016/j.mimet.2005.09.005 [ Links ]
41. Yousr A, Napis S, Rusul G, Son R. Detection of aerolysin and hemolysin genes in Aeromonas spp. isolated from environmental and shellfish sources by polymerase chain reaction. ASEAN Food J. 2007;14(2):115-122. [ Links ]
Suma George Mulamattathil
Department of Water and Sanitation
University of Limpopo
Polokwane campus
Private Bag X1106
Sovenga 0727
South Africa
EMAIL: firstname.lastname@example.com
Received: 27 Sep. 2013
Revised: 09 Jan. 2014
Accepted: 13 Apr. 2014


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5585-01 Skil 7-1/4" Circular Saw Brand new
The Skil 7-1/4 in. Circular Saw features a tough, 13 Amp, ball-bearing motor for fast, powerful cuts along with extra-safety features for the do-it-yourselfer. The Accu-Sight system helps keep your cut on line for straight, accurate cuts. Settings for depth of cut are easy to read and can be changed quickly. It also offers rear-view depth adjustment for clear visibility and quick cut-depth changes. The aluminum, anti-snag lower guard reduces snags when making narrow cut-offs.

subset: commoncrawl


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How We Celebrate Spring and Still Have a Ski RaceApril 9, 2009 at 7:00 am | Posted in children, Families, Holidays, moms, parents, Self Esteem, spirituality, Thoughtful Thursday | Leave a comment
It is Thoughtful Thursday and
I am with my children at the last
of the ski racing events of the year!
It is a CanAm and only
- 30 US girls and 30 Canadian girls
- 30 US boys and 30 Canadian boys
- aged 11 and 12
Kind of a special and elite group of skiers! I am one proud mama for sure!
Yes, in April here we are while
many of you are celebrating
spring and Easter, we are
still in the last throws of
ski racing.
SO what does that do for our celebrating spring?
Celebrating Easter?
Its really easy,
we celebrate spring by
breathing the spring air,
feeling the sun on our faces
with different energy.
WE watch the snow go with sadness
that our wonderful snow sports life MUST
come to an end.
And with excitement that we will
be on our bikes, hiking
and swimming outside soon.
And Easter, that is easy for us as well.
Since the early Christians had
no real date and they borrowed some other
holidays to fill the day,
We have begun to celebrate on the
Greek Orthodox Easter.
It is 6 weeks later- allowing us
to be fully with the holiday.
These ways of alternating our
lifestyles to fit the needs and
desires of our family,
yet staying with our
morals and values
gives my family AND me
the ability to live and breath
with our spirit, soul, self esteem
My children
are able to be confident,
yet relaxed.
capable yet not pushy
confident yet not arrogant,
respectful of themselves and others.
My kids are not special. Yours can be
the same way.
As you go through your days,
weeks, months,
find what fits with your family.
believe your values and morals
are the best for your family.
And let others have their
values and morals.
Have a great day,
and of course
parent with passion, purpose and integrity,
PS I really have enjoyed that
some of you are putting comments on the blog.
It really energizes me and I know
it energize others as well.
Leave a Comment »
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Dysprosium: Achilles Heel Of Hybrid, EV, & Wind Turbine Designs
Periodic table entry for dysprosium. Image credit:Wikimedia.
China produces 95% of the worlds supply of the rare earth metal, dysprosium, a key metal in magnets used in the drive motors for hybrid electric vehicles - up to 100 grams of dysprosium per hybrid car produced, according to a Wikipedia reference. Dysprosium's magnetic properties also make it an important metal for wind turbines and electric vehicles. Could be vital for MagLev trains, too.
Scarcity of the metal is a sustainability and a political issue, according to a recent article in the TimesOnline, Crunch looms for green technology as China tightens grip on rare-earth metals. This does not look good.
Don Burbar, the chief executive of Avalon Rare Metals, said: "The crux of the matter is that there are now a lot of technologies that can't work without rare earths, and China is currently in effective control of the global supply. China has positioned itself to retain control, and meanwhile politicians around the world do not appreciate how the supply side of green technology works."A company called Great Western Minerals Group is exploring for a rare earth minerals, west of Salt Lake City Utah; but, other than that, China controls a vital element for a renewable energy driven future.
In Japan, the world's biggest importer of rare-earth metals, more than 10,000 tonnes per year — about a fifth of the country's total annual consumption — are thought to enter the country through a thriving black import network without which Japan would already be in a severe supply crisis, a senior government official said.
More on magnets that incorporate rare earth metals.
Scientists a Step Closer to Super Magnets for Energy-Efficient ...
The World's First "Magnetic Levitation" Wind Turbines Unveiled in ...
"Magnetic Fridge" Could Be 40% More Efficient


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Your Guide to Commercial Real Estate
At Guiderock Commercial Realty LLC, we understand that you have a myriad of responsibilities in your business. Commercial real estate is an ever-evolving industry, and we strive to help you get the most out of your transactions. Whether you need brokerage services or other assistance, Guiderock Commercial Realty LLC is your guide to commercial real estate, keeping you focused on everything else you need to do.


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A Florida rape victim is suing her county jail and its medical contractor after one employee allegedly refused to give her emergency contraception, citing religious reasons.
After the woman — identified as R.W. — was raped, she went to the jail to identify her assailant. While there, the victim was placed under arrest for an outstanding warrant. It was during this arrest that the incident occurred.
According to the court filing, R.W. had taken one of a two-pill emergency contraception set. But when she needed to take the second pill, the difficulty began:
The next morning, January 28, 2007, Plaintiff requested the second anti-conception pill from Spinelli, explaining that Plaintiff had recently been raped, visited the Rape Crisis Center, and had a prescription from a medical doctor instructing her to take the second pill after twelve hours to prevent the rape from resulting in pregnancy. Spinelli refused to administer the pill to Plaintiff, allegedly stating that it was against her religious beliefs.
This is yet another example of the disturbing trend of conscience-clause excuses for not providing emergency assistance to rape victims. Florida has such a clause, which allows health care providers to deny care if it goes against their religion. Another incident, where a doctor in Oklahoma denied emergency contraceptives to a rape victim, relied on the same "religious beliefs" excuse.
R.W's rape did not result in pregnancy, and she was permitted to take the second pill — though late — under another person's supervision.


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For my Capstone project my main inquiry question was," How can teens and tweens maximize their social media experience?" I did a lot research using my sub questions, which eventually helped me answer my main inquiry question. Once I finished my research, I had to write an essay that would answer my question. Here is my essay:
Is social media a big part of your life? If yes, you probably know social media can be a good place for you. It keeps you connected with your friends and is a positive place for kids. What you might not know is that social media can can be a very negative place if you don't use it well. In fact, according to ABC News 50% of teens say they are victims of cyberbullying. After research I thought of three of the main ways kids can maximize their social media experience. One, try to lift up the social media community with your social media posts. Two, think before you post. Three, remember why you are using social media.
When you are using social media have you ever tried to make yourself look cooler or have tried to make your life seem better than it really is? If you have, you are not the only one. A lot people want to impress, or try to get likes on their posts. For example they are posting the things that are the best parts of their life. It is like they are trying to make a highlight reel of their life. It is not the best way to post because it does not lift anyone up. It just makes the people going through the hard parts of their lives feel worse. According to Psycom.net depression levels for teens went up 3.6% percent since 2005. That is only one year after Facebook came out. Since then all the famous social media platforms were made. Another example is on the Scarsdale Moms Facebook chat. A person was telling everyone her son's grades. They were really good and one person got mad and was making mean comments. In the end she was the one that looked bad.
My second reason to show how kids can maximize their social media experience is to think before you post. Too many people are hurting their lives and their family's lives because they do not take the time to think "Is what I am posting okay? Would I want everyone to see this post?" Recently there have been some top draft prospects in the NFL, such as Josh Allen. Josh Allen was a quarterback from the University of Wyoming. A lot of people thought he would be the number one pick in 2018 draft. Instead he fell to the seventh pick. This happened because in high school he made some racist comments on social media. A lot of people started to doubt his decision making off the football fields. Another reason why you should think before you post is that what you post does not only affect you. It affects the people around you. So if I made a post that said that I made the select soccer team and our team is amazing,
how do you think it would make your friend that didn't make it feel? Probably not so good. These are the reasons why you need to think before you hit the post button.
My third and final way kids can maximize their social media experience is to remember why you are using it. For example if every time you are using social media and it is not benefiting you in any way, just stop using it. What I mean is why use social media if all it is doing is wasting your time and making you feel worse about yourself. Tali Horowitz, an expert on the social media field, works at Common Sense. She said," If it doesn't make you feel good, take a break." Also social media was not made so you could hurt the digital community with negativity. Use social media the right way.
In conclusion, if you can, incorporate these tips into the way you use social media. Social media will be a great tool for you. Remember, social media was not made specifically for you, so you need to use it the right way for you. How will you use social media?


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UN Development Goals Miss the Mark
When world leaders assembled this week in New York to discuss development policy, there was a lot of noise about the failure of rich counties to attain the Millennium Development Goals (MDGs), which were adopted in 2001 by the United Nations member states.
Chief among the goals is halving the proportion of people who are extremely poor – that is, individuals living on less than a dollar a day – by 2015. The other goals target universal primary education; gender equality; child mortality; maternal health; HIV/AIDS and malaria; and environmental sustainability.
As a student and teacher of development policy, I believe it is high time for policymakers to stop wasting their time with the pie-in-the-sky MDGs. In short, while the intention that led UN member states to adopt them is noble, the MDGs themselves are all just talk.
Economist Jeffrey Sachs, an adviser to the UN on development policy, claims the world's wealthiest countries lack the will to meet the goals. As an analogy, think of MDGS as New Year's resolutions: "I will lose weight, quit smoking, exercise more, read one nonfiction book a week, and learn to play the piano this year!" But just as it is better to commit to piecewise lifestyle improvements than to be overly ambitious and try to improve everything all at once, it is best for policymakers to focus on humble, incremental policy objectives.
Over the last 10 years, a revolution has taken place in development policy research which has led to new understanding about whether specific policy interventions actually work and, when they do work, to what extent. The charge has been led by the Massachusetts Institute of Technology's Esther Duflo and her collaborators.
Among the findings:
– Drugs that kill intestinal parasites have been shown to improve the health of treated children and the school attendance of both treated and untreated children as well as, surprisingly, the health of the latter.
– Information campaigns to inform young women about the relative risks of contracting HIV from potential sexual partners have been shown to decrease HIV infection rates among these young women.
– Conditional cash transfer programs, which provide education grants to poor mothers provided they satisfy certain criteria, have been shown to increase school enrollment, among other things.
All three of these findings are the result of asking simple questions and answering them using the best possible data. In my own ongoing research on agricultural value chains in Madagascar and on micro-insurance in Mali, I strive to ask similarly simple questions and answer them using careful research designs.
Instead of making vague promises about delivering overly ambitious results by a specific date, we should promise the implementation of policies that have been shown to work. Not only will this actually help alleviate poverty, it will be a better use of donor funds, which ultimately come from taxpayers in rich countries.
Marc Bellemare is an assistant professor of economics and public policy in the Sanford School of Public Policy at Duke University. This op-ed first ran in the Herald-Sun on Sept. 23, 2010. It also ran in The Philadelphia Inquirer Sept. 28, 2010.


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In 99 incidents the culprits were not found. The incidents are difficult to solve, since the "culprits don't leave a signature," says Van der Valk. Violence against mosques is relatively rare in the big cities, though relatively many Muslims live there, says the researcher.
The Discrimination Internet hotline also gets regular reports of racist statements against Muslims. A spokesperson said that by Friday there were 317 reports of "hate against Muslims", which made up 20% of the total number of reports this year. [Islam in Europe] Read more


text: Volume 2.00 | Jan 8 Immune Regulation News 2.00, January 8, 2010. In this issue: Science News | Current Publications | Industry News | Policy News | Events Subscribe | Unsubscribe TOP STORYDampening CD4+ Immune Cell Function Via the Protein TLR4 University of California scientists have determined that triggering TLR4 on CD4+ T cells dampens their inflammatory function, as TLR4 deficiency in two mouse models of colitis accelerated the development of disease and/or induced more severe disease. [University of California, La Jolla] Isolate Untouched Cells forAntigen Processing and Presentationwith EasySep® REQUEST A POSTER SPECIAL FEATUREVideo: The EasySep® Pour-off – A Simple and Effective Way to Isolate Cells SCIENCE NEWS Enzyme's Key Role in Regulating Immune SystemMice lacking an enzyme known as deoxycytidine kinase have defects in their adaptive immune system, producing very low levels of both T and B lymphocytes, the major players involved in immune response. [University of California, Los Angeles]Inflammatory Mediator Regulates Diarrhea in Inflammatory Bowel Disease The activation of NF-kappaB, an inflammatory mediator, results in diarrhea in inflammatory bowel disease. [Northwestern University, Chicago]What Is The Function Of The Protein CD20?A team of researchers from The Netherlands, has determined that CD20 has a nonredundant role in generating optimal B cell immune responses by analyzing a patient lacking the protein. [Academic Medical Center, Amsterdam] CURRENT PUBLICATIONS CTLA-4 Suppresses the Pathogenicity of Self Antigen-Specific T Cells by Cell-Intrinsic and Cell-Extrinsic MechanismsResults demonstrate that both cell-intrinsic and non-cell-autonomous actions of CTLA-4 operate to maintain T cell tolerance to a self antigen. [Nat Immunol] TLR4 Signaling in Effector CD4+ T Cells Regulates TCR Activation and Experimental Colitis in MiceResearchers show that TLR4 triggering on CD4+ T cells affects their phenotype and their ability to provoke intestinal inflammation. [J Clin Invest] Expansion of FOXP3+ CD8 T cells with Suppressive Potential in Colorectal Mucosa Following a Pathogenic Simian Immunodeficiency Virus Infection Correlates with Diminished Antiviral T Cell Response and Viral Control This study suggests an important but previously unappreciated role for CD8 regulatory T cells in suppressing antiviral immunity during immunodeficiency virus infections. [J Immunol] CD137 Promotes Proliferation and Survival of Human B CellsCD137 (4-1BB)-mediated costimulation plays an important role in directing the fate of antigen-stimulated T cells and natural killer cells, yet the role of CD137 in mediating B cell function is unknown. This study suggests that CD137 costimulation may play a role in defining the fate of antigen-stimulated human B cells. [J Immunol] CTLA4Ig Alters the Course of Autoimmune Disease Development in Lyn-/- MiceResults suggest that when self-reactive B cell clones are unimpeded in acquiring T cell help, they secrete pathogenic IgG autoantibodies that trigger the fulminant autoimmunity normally observed in Lyn-/- mice. [J Immunol] Ins2 Deficiency Augments Spontaneous HLA-A*0201-Restricted T Cell Responses to Insulin In humans, the non-MHC locus conferring the strongest Type 1 diabetes disease susceptibility is the insulin gene, and alleles yielding lower thymic insulin expression are predisposing. Researchers sought to incorporate this characteristic into an HLA-transgenic model of the disease and to determine the influence of reduced thymic insulin expression on CD8+ T cell responses to preproinsulin. [J Immunol] INDUSTRY NEWSTeva Sees Revenue More than Doubling by 2015 Teva Pharmaceutical Industries Ltd., the world's largest generic drugmaker, set a target of revenue of $31 billion in 2015, more than double its current annual amount as it sees major growth in overall use of generics.Athersys Inc. Shares More Than Double on Pfizer Agreement to Develop Inflammatory Bowel Disease Therapy Biopharmaceutical company Athersys Inc. is in line for up to $111 million in revenue through an agreement with a Pfizer Inc. unit to develop and market a stem cell therapy for patients who suffer from inflammatory bowel disease.Michigan Biotech Startup Lycera Moving Headquarters to Massachusetts The University of Michigan spinoff, which raised a $36 million Series A round of funding last year, plans to maintain its research and development operations in Plymouth, according to a statement from the company. POLICY NEWS Cancellation of Notice NOT-CA-09-017, Clarification of Page Limitations for Research Plan in PAR-09-026 Collaborative Research in Integrative Cancer Biology and the Tumor Microenvironment (U01) (NOT-OD-10-029) [National Institutes of Health, United States]Notice on P40 Page Limits (NOT-RR-10-002) [National Institutes of Health, United States] Academic Research Enhancement Award (Parent R15) (PA-10-070) [National Institutes of Health, United States]Technologies and Software to Support Integrative Cancer Biology Research (R43/R44) [Small Business Innovation Research, United States] EVENTS9th International Conference on New Trends in Immunosuppression and Immunotherapy February 4-6, 2010Geneva, Switzerland13th International Conference on Lymphocyte Activation and Immune Regulation: Inflammasome and InflammationFebruary 5-7, 2010Newport Beach, United States Keystone Symposia: Tolerance and AutoimmunityFebruary 21-26, 2010Taos, United StatesWorld Immune Regulation Meeting – IVMarch 29-April 1, 2010Davos, Switzerland IMMUNOLOGY 2010May 7-11, 2010Baltimore, United States14th International Congress of ImmunologyAugust 22-27, 2010Kobe, JapanVisit our events page to stay up to date with the latest events in the cell, gene and immunotherapy community. JOB OPPORTUNITIES Research & Development Scientist – Pancreatic Cell Biology (STEMCELL Technologies)Research & Development Scientist – Endothelial Cell Biology (STEMCELL Technologies) Research & Development Scientist – Epithelial Cell Biology (STEMCELL Technologies)Data Entry Assistant – Marketing (STEMCELL Technologies)Recruit Top Talent Reach more than 11,000 potential candidates by posting your organization's career opportunities with Cell Therapy News.Visit here to post your career opportunities. Have we missed an important article or publication in Immune Regulation News? Click here to submit! Learn more about Immune Regulation News: Archives | Events | Subscribe | Contact Us


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As everyone is probably aware the Supreme Court is taking arguments on the most important gun rights case of all time, Heller v City of DC. The main argument has been decided by nearly every judge on the Court has given evidence they believe in the individual right to own firearms, the difference being in the "reasonable restriction." I'm glad to hear that this "collective right" BS is gone, as Marko demonstrates. I see no reasoning behind this idea that the Second Amendment, alone among all other rights granted in the Constitution, is not individual. Luckily the Court seems to be ready to move against this.
Next on the antigun argument is the "well regulated militia" which they take to mean the National Guard, which coincidentally didn't exist for a hundred plus years after the Bill of Rights was conceived. The main problem with this fallacy is the fact that it is totally against what the Founding Fathers believed in. They had just escaped the terrible hand of a despot and did not want concentrated power to ever threaten the freedom of its citizenry. The Second Amendment is precisely put in place to protect individuals from not only other individuals but also from the threat of overreaching government. This is why the National Guard argument fails: the National Guard is a governmental organization and therefore is the antithesis of what the Amendment was designed for.
I'm anxious to see the ultimate outcome of Heller. It will either be back to square one or the biggest victory for gun rights in this country's history. I hope that enough of the Justices have the same opinion of the issue, and I'm too afraid we're about to go right back to square one. This time we'll have a much more Sisyphean task on our hands.


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Skip to 0 minutes and 9 secondsTo conclude, scholars have begun to delineate the distinct domain of social entrepreneurship by identifying different typologies, by examining the potential to address context-specific social problems, and by exploring its implications for total wealth creation. These entrepreneur typologies vary in how they discover social opportunities, determine their impact on the broader social system, and assemble the resources needed to pursue these opportunities. This approach further increases our understanding as to why social entrepreneurship offers solutions to complex social issues by applying business market oriented models. These solutions reflect the unique values that social entrepreneurs hold, and the search processes they follow in identifying, evaluating, and exploiting opportunities in a complex and uncertain world.
Concluding social entrepreneurship
This video concludes the section on social entrepreneurship.
© University of Groningen


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THE team @ THE salon
"A visit to the hairdressers is more than a haircut alone.
It's about a total experience."
Kane Choo
Creative Director
Kane Choo
Creative Director
His Salon Experience
Kane single-handedly dispels the notion that good hairstylists require and early head-start in the business – this late bloomer started in his late twenties!
Learning the Craft
Having trained at Toni & Guy in London, Kane also attended London's L'Oreal Academy for advanced coloring studies.
Always on the Move…..
A normal day finds Kane booked solid from morning to evening by clients from both Singapore and the region. It's also not uncommon to find Kane not just traveling within the country from one
local fashion shoot to another, but flying to various foreign locations for overseas assignments, too!
Celebrity Connections
Kane has styled the famous locks of Gigi Leung and is still styling and taking care of the glamorous Lisa Ang's hair.
When he does manage to find some time to himself, the house-proud Kane can be found pottering around his apartment or out shopping.
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|← 1242 →
March 19, 1971
|We have 3 images of Dark Shadows 1242
Daphne's funeral is held, and Morgan lures Bramwell into the locked room.
- The great house at Collinwood in the year 1841. A time when terror held sway over the Collins family. Terror brought on by a one hundred and sixty year old curse. Once again a member of the family has been chosen to go into the legendary locked room that has brought death or madness to all who have spent a night in it. The time it is Kendrick Young, related through his sudden marriage to Melanie Collins. Tomorrow night he will enter its doors to survive or perish. Meanwhile, another drama is unfolding in the gazebo... a drama that will lead to attempted murder.
Morgan contemplates murder. Daphne's funeral is held. Morgan writes a note asking Bramwell to go to the remote part of Collinwood. Kendrick waits for night so he can go in to the locked room. Morgan knocks out Bramwell and locks him in the room. Morgan announces that he has locked Bramwell in the room. Catherine runs to Bramwell.
Memorable quotes
Dramatis personae
- ← Jonathan Frid as Bramwell Collins →
- ← Grayson Hall as Julia Collins →
- ← John Karlen as Kendrick Young →
- ← Lara Parker as Catherine Harridge Collins →
- ← Keith Prentice as Morgan Collins →
Background information and notes
- Final appearance of the Gazebo and Old House sets.
- Closing credits scene: Collinwood foyer.
- Morgan has known Catherine all his life.
- INTERNAL MONOLOGUE: Bramwell: (reads letter) / The child; Kendrick: I must break the curse.
- TIMELINE: Kendrick will enter the locked room tomorrow night. Daphne's funeral is tomorrow. Day 454 begins, and will end in 1244. Morgan arranges to meet Bramwell at 5pm. 4pm: Three hours until nightfall. 4:50pm: Bramwell enters Collinwood. 5:30pm: Thirty minutes until Kendrick enters the locked room.
Bloopers and continuity errors
- Near the end of the episode while Kendrick is pacing in the drawing room, a crew member can be heard whispering, "Here she comes," right before the clock chimes 5:30.
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Besblock Limited01952 685000
The Natural Lightweight Building Block - Pumice - The Pumice used in the manufacture of this range of building blocks was formed B.C. by volcanic action.
The low density of the material is due to its cellular structure created by air bubbles which formed when the dissolved gasses in the volcanic lava were released during cooling, effectively turning the lava into foam.
This raw material conforming to BS 3797 : 1990 has a class one fire resistance rating and is chemically inert.
Besblock Pumice products are produced to BS 6073 : Part 1 : 1981
Dense Aggregate Building Blocks - Bescrete - Natural Aggregate Building Blocks
Bescrete building blocks from Besblock, are manufactured from the earth's natural aggregates. Proven over millions of years, these are acknowledged to be amongst the most durable materials known to man.
The composition of this raw material, which conforms to
BS 882 : 1992, gives the finished product (manufactured to BS 6073 : Part 1 : 1981) a superior and consistent chemical stability free from deleterious substances.
This consistency would be difficult to equal with products manufactured from FBA (furnace bottom ash), as the chemical stability is wholly dependent on efficient combustion of the primary raw material within the furnace which may give variable results.
Lightweight Aggregate Building Blocks - Insulit - Insulite is a range of lightweight (density 1400 kg's / m3) load bearing building blocks manufactured in the main from re claimed and re cycled raw materials conforming to BS 3797. The products themselves are manufactured to BS 6073: Part 1.
The blocks are cast in steel moulds onto steel pallets and compacted by vibration, following which they are placed in specially designed chambers where the curing process of the green concrete is accelerated by means of humidity.
The compaction process of the concrete is vital to the quality of the finished product.
At Besblock we employ Columbia presses. These are internationally acclaimed as the very finest in their field and their unicontrolled vibration system ensures even density and compaction within the mould, giving a consistent test quality on every block.
"Star Performer." So named because of it's truly universal application characteristics within the general purpose housingmarket (flats, masonettes etc.) When used as directed in this section, this
100mm building block will conform with the mandatory requirements of sound and thermal insulation, beam and block flooring and most other aspects required of a 100mm block.
Think of the on site benefits, particularly in construction supervision, that the use of only one block could bring to you
Getting in contact with Besblock Limited
Halesfield Industrial Estate
Tel: 01952 685000
Besblock Limited Products & Services


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Join us for the closing cocktail of the 4th edition of MUTEK_IMG!
Mingle with the grand Montréal community of digital creators, meet our speakers and the participants of the German-Canadian VR:RV project, and experience a fine selection of digital and virtual reality works.
We encourage you to bring your own creations (on Google cardboard, tablet etc.) and take advantage of this opportunity to get precious feedback and spark inspiring discussions.
The cocktail includes the 14th edition of Composite, an initiative of the Conseil des arts de Montréal.
Each edition of Composite features the presentation of 8 projects from the digital arts and industries by their creators, in front of an audience composed of professionals in culture, marketing, design and the business world. The aim is to connect artists, cultural workers and entrepreneurs in order to inspire new projects and collaborations.
Here are the projects selected for Composite #14:
Timothy Thomasson (CA) AQUARIUM
Shirin Anlen (IL) Marrow
Eric Marradi – Spoken Adventures (CA)
RYBN.ORG (FR) Offshore Tour Operator
Philippe Lambert – Office national du film du Canada (CA) Rêve
Camille Barbotteau – Pulsarium (CA) HOROS
Mathieu Le Sourd – MAOTIK (CA) AERYON
Isabelle L'Italien – CQAM (CA) and Marc Beaudet – Turbulent (CA) CQAM - Turbulent Residency
For more information about Composite and previous editions, visit the website.
Phi Centre - Space D407 St-Pierre StreetMontréal, Canada


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Friday, June 27, 2008
The last lighthouse-keeper
It looks as if the struggle is definitively lost: from 2010 on there won't be any keepers more on the Dutch lighthouses. Unmanned lighthouses is the new policy of the Dutch Traffic Department - economization and modernization. Camera's and computers will take over the tasks of the keepers.
Yesterdays' broadcast on Netwerk.tv (click for the full report - in Dutch) about this item is not an impressive piece of press-work: cheap and suggestive. Nevertheless I agree it's a bad development. The Dutch lighthouse keepers always offered the perfect local coastguard service and are since centuries an essential contribution to safety at sea. I don't believe computerization nor a central traffic-management centre will replace that.
Not only the Seakayakers are going miss the daily contact, the local knowledge and the good advises of the lighthouse keepers ;-) And I don't like the idea how kayak-incidents like this and this would have ended without the local coastguard support of the lighthouse crews!
Geplaatst door Hans Heupink op 23:27
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The Domestic Status of the Human Rights Clauses of the United Nations Charter
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Presents an essay on the issues of universal nature of human rights. Efforts of women leaders from several African countries in dealing with human rights violations; Tensions between developing and developed countries in tackling these issues; Areas that call for coalitions among different...


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Youth unemployment is a major challenge as millions of young people – the talent base of society – are left without jobs and without hope.
Enormous as our challenge is, we are not alone. Youth unemployment is a growing crisis across the world.
The International Labour Organisation estimates that, internationally, 75-million people between the ages of 15 and 24 cannot find work.
The Organisation for Economic Co-operation and Development finds that high levels of youth unemployment have persisted in the wake of the financial crisis and threaten to scar young people, affecting their career paths and future income.
It is important to remember this because, regrettably, a blame game has infused much of the discussion – as if we have policies perversely designed to keep young people out of jobs.
There is a saying: beware those who come with simplistic answers to complex problems. I have listened to the opposition's somewhat glib prescriptions, the praise song to one silver bullet that will solve all our youth employment problems.
They promise everyone everything from one simple intervention and then seek to demonise and delegitimise anyone who disagrees – in this case, most of the main youth organisations as well as major unions.
It is, of course, the luxury of opposition to ignore practical realities. If only our world was that easy.
This is not to say there is no place for subsidies to work seekers or work creators aimed at bringing young people into jobs. However, in contrast to the Democratic Alliance, the government is working on an inclusive, multipronged strategy suited to the scale of the challenge we face.
In June this year, 185 governments assembled with the world's major business and trade union representatives to discuss youth unemployment. Their call for action says young people's voices should be heard, their creativity engaged and their rights respected in dealing with the youth employment crisis. Youth organisations have rightly said: no solution for us without us.
We need to forge a consensus on how to address youth employment, instead of a kragdadige [forceful]approach to push through a single measure in the face of opposition from youth organisations and trade unions.
We have now ramped up social dialogue on youth employment.
Youth unemployment
From June this year we elevated the discussions to leadership level, called in the leaders of Business Leadership South Africa, the Black Business Council and Business Unity South Africa, the representatives of women, disabled persons, civic organisations and, above all, youth representatives drawn from the South African Youth Council and the leaders of trade union federations Cosatu, Fedusa and Nactu.
The parties have agreed on the importance of a compact on youth employment and identified core principles and programmes.
They include current and potential private sector initiatives on skills, internships, work-readiness programmes and bridging measures to draw young people into employment on a large scale.
They recognised the value of a public sector programme of youth brigades focused on rural, environmental, literacy, health and other activities and drawing on the experience of current programmes.
Again, no single mechanism can address the challenge of youth unemployment. As the International Labour Organisation says, we require an "integrated approach that combines macroeconomic policies and targeted measures which address labour demand and supply, as well as the quantity and quality of employment".
This is particularly true in South Africa, where we face both a demographic bulge and the extraordinary levels of joblessness and exclusion that apartheid left behind.
That is why we need a new growth path. But structural change takes time. For that reason, we are looking to short- as well as long-term measures and a package that is proportionate to the scale of the problem.
Skills accord
We are taking action as the government. We have set targets for public employment schemes that should have a real effect on youth joblessness.
The expanded public works programme has about 800 000 participants. Moreover, Cabinet decided on substantially growing the community work programme tenfold to a million participants by 2015.
Youth brigades let young people serve their communities and work together. We are talking to our social partners and youth organisations about setting up brigades for health, education, rural development and green issues, among others.
The skills accord lays the basis for expanding apprenticeships and internships. The public service committed to 60 000 new internships, about seven times as many as today. The private sector and state-owned enterprises also set themselves stretch targets.
Finally, we are exploring work-seeker and work-creator subsidies with stakeholders.
For the longer run, the new growth path identifies key jobs drivers in the economy with the national infrastructure plan and the industrial policy action plan as key levers to support them.
Government support
Where jobs drivers benefit from government support, we will set targets for new entrants – for instance, in the green economy and infrastructure, business-process services and the digital migration in broadcasting.
We have tabled a proposal for a youth employment committee under the National Economic and Development Labour Council to consider specific proposals to boost employment for young workers. By building in collaboration, it would avoid unnecessary conflict as well as exploitative programmes.
In addition, where young people want to start their own business, the government will help them. The government is establishing specific programmes for youth entrepreneurs and co-ops, including a role in installing solar water heaters.
Education counts. Today, less than 10% of young people with a tertiary degree are unemployed, compared with 35% of those with matric and 40% of those who never finished secondary school.
Expanded counselling
The skills and education accords capture strong commitments by the social partners. We will also accelerate work on the second-chance matric. Moreover, the green paper on post-secondary education and training foresees a tenfold expansion in further education and training institutions to four million by 2030, whereas higher education doubles to 1.5-million. And we are working with social partners to improve the transition from school to work, including through expanded counselling and support for job searches.
We cannot address joblessness on the scale left by apartheid through tax incentives alone, although the full range of measures, including carefully structured subsidies and grants, have a role to play.
We can also not help young people through measures that displace older workers. That approach would deepen social divisions and poverty. It has already failed the youth of the Western Cape.
Ultimately, we need collective action and solidarity to bring about systemic changes so that the economy can provide opportunities for all. That is the path foreseen in the Freedom Charter. That is the aim of the new growth path, the national infrastructure plan and the national development plan. That is why we need a multipronged strategy to deal with youth unemployment.
Ebrahim Patel is Economic Development Minister.


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|Sighting Report
|Occurred : 3/2/2013 16:00 (Entered as : 3/2/2013 16:00)
Reported: 3/2/2013 8:02:03 PM 20:02
Posted: 3/3/2013
Location: Princeton, MN
Shape: Other
Duration:7 minutes
|Saw a string of many lights.
As we got closer the seem to disappear.
We think the angle we were at made made them visable because as the toad curved the were gradually disappearing.
((NUFORC Note: Witness elects to remain totally anonymous; provides no contact information. PD))


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Is dabbing liquids on canvas (
To admire an old picture is to pour our sensibility into a funeral urn instead of casting it forward with violent spurts of creation and action. Do you want to waste the best part of your strength in a useless admiration of the past, from which you will emerge exhausted, diminished, trampled on?
Futurist Manifesto, Filippo Tomasso Marinetti, 1909The art of this century is digital, interactive and open-source. It is so new, so different, so unlike the past that the art establishment hasn't grokked the sea-change yet.
The art of this century is still so new that half the artists don't even know they are doing it yet.
The time has come for us to be the masters.
Aesthetic Meditations, Guillaume Apollinaire, 1913So what is this new art?
Hello! It's the internet, doh! But with a twist.
We use many verbs after the word "Internet". The Internet sells..., teaches..., preaches...
But can the Internet beautify? Or inspire? Or move you?
Can there be web sites that you go to just as you might go visit a museum or attend a concert - so that you might be moved or feel connected to something greater than yourself?
Can there be web sites that are simply and purely works of art?
Is the destiny of the Web to be a method of sending out commercial advertising, learned treatises and pornography? Or are there other possibilities?
The imagination is perhaps on the point of reasserting itself, of reclaiming its rights. If the depths of our mind contain within it strange forces capable of augmenting those on the surface, or of waging a victorious battle against them, there is every reason to seize them.There is beginning to be evidence that the Internet is as valid a media as paper, film or piano. There are occasional glimpses of exquisite forms embedded in pop-up animations. From time to time web pages appear that are profoundly moving though we may not be able to say why.
Manifesto of Surrealism, André Breton (1924)
But these experiences are still few and far between.
What we do see are hundreds of pages where you can buy the art of previous centuries. Many artist's have put their Artists Statements on-line and have photo galleries of their archaic works. Great artists also build the sets on online games and create stunning visual effects for advertising. Artists cannot starve all the time after all.
These antique uses for the Internet are almost the work of an enemy. It's as if piano's were only used to create jingles for commercials. It's as if canvas paintings could only depict images of products to be sold.
With the Internet we have phenomenal creativity engine at our disposal and yet we are still using only a fraction of its potential power. The Internet will eventually be the essential repository for form and meaning. We who love art must begin to call to artists to learn to harness this power.
And we must make this call not merely for the sake of art itself. There is the dark side to be dealt with as well. Is your world a happy place? Are the people well fed (neither too little nor too much)? Is your world free of religious war and mayhem. Are your tax dollars being well spent. Is there enough money for your retirement. No, no and more no's. We live in a world with thousands of complex issues that need sorting out. The politicians, the CEO's, the economists, the religious leaders of our times send out so many mixed messages that it seems impossible to have a clear vision about where the world will be 100 years from now.
Did you just hear the words "clear vision"? So what sort of people have clear visions that can transform civilizations? Artists, of course. People like Giotto, Manet, Picasso or Mozart and Beethoven or Dante, Shakespeare or Joyce have been able to clarify their eras.
We now have a splendid new media to create the art of this century and the people of this century have an urgent need for the clarity of vision that great art engenders.
Artists, now is time for you to weave your selves, your brains, your visions into the world wide web.


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The Northwest Florida Transportation Corridor Authority approved its seventh, and possibly last, master plan Thursday to help ease traffic congestion on U.S. Highway 98.
DESTIN � The Northwest Florida Transportation Corridor Authority approved its seventh, and possibly last, master plan Thursday to help ease traffic congestion on U.S. Highway 98.
Its goals in 2013 are far more modest than the ones laid out in 2007, when the newly minted organization had money to spend and an incoming House Speaker in Destin�s Ray Sansom to lean on.
Gone is the talk of a billion dollar bypass running the length of Eglin Air Force Base. In its place, this group of authority members debated the merits of a 1.67-mile project they call the Destin Cross Town Connector.
The $6.6 million project would provide an east-west alternative to U.S. 98 and help open what most members believe to be the most congested area between Pensacola and Franklin County.
�Destin is the biggest bottleneck on the whole corridor,� said Jim Anders, Walton County�s delegate to the eight-member board and one of four remaining original members.
But after board members and their advisers approved the strategy and praised its list of 10 priority projects, discussion turned to potentially lethal legislation.
Specifically, it is a bill to be voted on Monday in the state Senate that could do away with the Corridor Authority.
SB 1132 �completely revamps this authority� said HDR Engineering representative Bob Kellner, who has been following the legislation.
Under the bill, Escambia, Santa Rosa, Wakulla and Franklin counties would be dropped from the authority and Okaloosa, Walton, Bay and Gulf counties would be absorbed into something called a regional transportation finance authority.
The same legislation �turns governance and control of the Mid-Bay Bridge Authority� over to the new regional authority, said Jim Vest, the Bridge Authority�s executive director.
Only the Senate bill includes language that would break up the transportation authority, Kellner told the board, so even if it passes that chamber it still could die in a conference committee of the Senate and House.
A signature from Gov. Rick Scott also would be required.
�I don�t know what the realities of the discussion are,� Kellner told the board. �We just need to stay focused and see what the governor signs.�
Contact Daily News Staff Writer Tom McLaughlin at 850-315-4435 or firstname.lastname@example.com. Follow him on Twitter @TomMnwfdn.


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Belt and Road Fuels Fujian Mawei Shipbuilding Growth
China's Fujian Mawei Shipbuilding, the largest shipyard in Fujian province, has reported a dramatic increase in orders as a result of the Belt and Road Initiative.
Orders from places such as Singapore, Malaysia and the Middle East have increased to 53 ships over the past five years, over half of the yard's total orders for that time. For example, China Daily reports that the company has received an order for 12 container ships from Malaysian and German shipowners, two of which will be delivered by the end of this year.
In January, Singapore-based Ocean Tankers ordered six handy-sized tankers, and earlier this year, the Marshall Islands-based subsidiary of Chinese deep sea fishing company Shanghai Kaichuang Marine International ordered three new tuna purse seiners from the yard.
Fujian Mawei Shipbuilding was established in 1866 and has played a key role in China's shipbuilding industry. The yard continues to expand into new vessel types, for example seafood processing and delivery vessels, which are expected to be in demand in emerging and Belt and Road markets.
The Fujian province has boosted transport and trade infrastructure to support the Belt and Road initiative. China Daily reports the province's economy grew 8.1 percent last year, with foreign trade contributing to one third of its gross domestic product.
Chinese shipyards won 35 percent of the world's orders last year, but the nation has suffered from over-capacity. Dalian Shipbuilding Industry completed just 12 ships in 2017, and the government decided to merge its top two shipbuilders: China State Shipbuilding and China Shipbuilding Industry.
However, Tug Technology & Business reports that there has been an increase in tug construction this year for both domestic and international owners, with Chinese shipyards securing contracts for at least 15 tugs in the first six months of the year.