Pseudomonas sp. to Sphingobium indicum: a journey of microbial degradation and bioremediation of Hexachlorocyclohexane.

The unusual process of production of hexachlorocyclohexane (HCH) and extensive use of technical HCH and lindane has created a very serious problem of HCH contamination. While the use of technical HCH and lindane has been banned all over the world, India still continues producing lindane. Bacteria, especially Sphingomonads have been isolated that can degrade HCH isomers. Among all the bacterial strains isolated so far, Sphingobium indicum B90A that was isolated from HCH treated rhizosphere soil appears to have a better potential for HCH degradation. This conclusion is based on studies on the organization of lin genes and degradation ability of B90A. This strain perhaps can be used for HCH decontamination through bioaugmentation.

Localization of HCH catabolic genes (lin genes) in Sphingobium indicum B90A.

The locations of hexachlorocyclohexane (HCH) catabolic (lin) genes were investigated in HCH degrading sphingomonad, Sphingobium indicum B90A (that was isolated from India). Southern blot analysis revealed the presence of linA1, linC, linDER and linX (linX1 and linX2) on the plasmid DNA in Sphingobium indicum B90A.

Non-point source pollution: determination of replication versus persistence of Escherichia coli in surface water and sediments with correlation of levels to readily measurable environmental parameters.

Racine, Wisconsin, located on Lake Michigan, experiences frequent recreational water quality advisories in the absence of any identifiable point source of pollution. This research examines the environmental distribution of Escherichia coli in conjunction with the assessment of additional parameters (rainfall, turbidity, wave height, wind direction, wind speed and algal presence) in order to determine the most probable factors that influence E. coli levels in surface waters. Densities of E. coli were highest in core samples taken from foreshore sands, often exceeding an order of magnitude greater than those collected from submerged sands and water. Simple regression and multivariate analyses conducted on supplementary environmental data indicate that the previous day's E. coli concentration in conjunction with wave height is significantly predictive for present-time E. coli concentration. Genetic fingerprinting using repetitive element anchored PCR and cellular fatty acid analysis were employed to assess the presence of clonal isolates which indicate replication from a common parent cell. There were relatively few occurrences of clonal patterns in isolates collected from water, foreshore and submerged sands, suggesting that accumulation of E. coli, rather than environmental replication, was occurring in this system. Non-point source pollution, namely transport of accumulated E. coli from foreshore sands to surface waters via wave action, was found to be a major contributor to poor recreational water quality at the Lake Michigan beaches involved in this study.

Evaluation of hexachlorocyclohexane contamination from the last lindane production plant operating in India.

PURPOSE: α-Hexachlorocyclohexane (HCH), ß-HCH, and lindane (γ-HCH) were listed as persistent organic pollutants by the Stockholm Convention in 2009 and hence must be phased out and their wastes/stockpiles eliminated. At the last operating lindane manufacturing unit, we conducted a preliminary evaluation of HCH contamination levels in soil and water samples collected around the production area and the vicinity of a major dumpsite to inform the design of processes for an appropriate implementation of the Convention. METHODS: Soil and water samples on and around the production site and a major waste dumpsite were measured for HCH levels. RESULTS: All soil samples taken at the lindane production facility and dumpsite and in their vicinity were contaminated with an isomer pattern characteristic of HCH production waste. At the dumpsite surface samples contained up to 450 g kg(-1) Σ HCH suggesting that the waste HCH isomers were simply dumped at this location. Ground water in the vicinity and river water was found to be contaminated with 0.2 to 0.4 mg l(-1) of HCH waste isomers. The total quantity of deposited HCH wastes from the lindane production unit was estimated at between 36,000 and 54,000 t. CONCLUSIONS: The contamination levels in ground and river water suggest significant run-off from the dumped HCH wastes and contamination of drinking water resources. The extent of dumping urgently needs to be assessed regarding the risks to human and ecosystem health. A plan for securing the waste isomers needs to be developed and implemented together with a plan for their final elimination. As part of the assessment, any polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF) generated during HCH recycling operations need to be monitored.

Sphingobium ummariense sp. nov., a hexachlorocyclohexane (HCH)-degrading bacterium, isolated from HCH-contaminated soil.

A hexachlorocyclohexane (HCH)-degrading bacterial strain (RL-3T) was isolated from an HCH dump site located in the northern part of India. Resting cell assays and analytical GC studies confirmed the ability of strain RL-3T to degrade HCH isomers. Southern blot hybridization studies revealed the presence of lin genes, which are involved in the HCH degradation pathway, in this bacterium. The 16S rRNA gene sequence of strain RL-3T showed that it was most closely related to Sphingobium cloacae JCM 10874T (97.3 %) and Sphingobium fuliginis MTCC 7295T (96.4 %). Phylogenetic analysis based on 16S rRNA gene sequences placed strain RL-3T between S. cloacae JCM 10874T and S. fuliginis MTCC 7295T. The DNA G+C content of strain RL-3T was 62 mol%. The DNA-DNA relatedness values of strain RL-3T with S. cloacae JCM 10874T and S. fuliginis CCM 7327T were 8.65 and 7.47 %, respectively. SGL1 was the major sphingolipid and spermidine was the major polyamine in strain RL-3T. The major fatty acids in strain RL-3T were C(18 : 1)omega7c (56.6 %), C(16 : 0) (14 %) and C(14 : 0) 2-OH (7.4 %). Ubiquinone Q-10 was the major respiratory quinone. Phylogenetic distinctiveness, DNA-DNA relatedness values, biochemical and physiological characterization and unique phenotypic characteristics suggest that strain RL-3T represents a novel species of the genus Sphingobium, for which the name Sphingobium ummariense sp. nov. is proposed. The type strain is RL-3T (=MTCC 8599T=CCM 7431T).

Identification of human enteric pathogens in gull feces at Southwestern Lake Michigan bathing beaches.

Ring-billed (Larus delawarensis Ord, 1815) and herring (Larus argentatus Pontoppidan, 1763) gulls are predominant species of shorebirds in coastal areas. Gulls contribute to the fecal indicator burden in beach sands, which, once transported to bathing waters, may result in water quality failures. The importance of these contamination sources must not be overlooked when considering the impact of poor bathing water quality on human health. This study examined the occurrence of human enteric pathogens in gull populations at Racine, Wisconsin. For 12 weeks in 2004 and 2005, and 7 weeks in 2006, 724 gull fecal samples were examined for pathogen occurrence on traditional selective media (BBL CHROMagar-Salmonella, Remel Campy-BAP, 7% horse blood agar) or through the use of novel isolation techniques (Campylobacter, EC FP5-funded CAMPYCHECK Project), and confirmed using polymerase chain reaction (PCR) for pathogens commonly harbored in gulls. An additional 226 gull fecal samples, collected in the same 12-week period in 2004, from a beach in Milwaukee, Wisconsin, were evaluated with standard microbiological methods and PCR. Five isolates of Salmonella (0.7%), 162 (22.7%) isolates of Campylobacter, 3 isolates of Aeromonas hydrophila group 2 (0.4%), and 28 isolates of Plesiomonas shigelloides (3.9%) were noted from the Racine beach. No occurrences of Salmonella and 3 isolates of Campylobacter (0.4%) were found at the Milwaukee beach. A subset of the 2004 samples was also examined for Giardia and Cryptosporidium and was found to be negative. Information as to the occurrence of human pathogens in beach ecosystems is essential to design further studies assessing human health risk and to determine the parameters influencing the fate and transport of pathogens in the nearshore environment.

Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation with Sphingobium indicum B90A.

Soil pollution with hexachlorocyclohexane (HCH) has caused serious environmental problems. Here we describe the targeted degradation of all HCH isomers by applying the aerobic bacterium Sphingobium indicum B90A. In particular, we examined possibilities for large-scale cultivation of strain B90A, tested immobilization, storage and inoculation procedures, and determined the survival and HCH-degradation activity of inoculated cells in soil. Optimal growth of strain B90A was achieved in glucose-containing mineral medium and up to 65% culturability could be maintained after 60 days storage at 30 degrees C by mixing cells with sterile dry corncob powder. B90A biomass produced in water supplemented with sugarcane molasses and immobilized on corncob powder retained 15-20% culturability after 30 days storage at 30 degrees C, whereas full culturability was maintained when cells were stored frozen at -20 degrees C. On the contrary, cells stored on corncob degraded gamma-HCH faster than those that had been stored frozen, with between 15 and 85% of gamma-HCH disappearance in microcosms within 20 h at 30 degrees C. Soil microcosm tests at 25 degrees C confirmed complete mineralization of [(14)C]-gamma-HCH by corncob-immobilized strain B90A. Experiments conducted in small pits and at an HCH-contaminated agricultural site resulted in between 85 and 95% HCH degradation by strain B90A applied via corncob, depending on the type of HCH isomer and even at residual HCH concentrations. Up to 20% of the inoculated B90A cells survived under field conditions after 8 days and could be traced among other soil microorganisms by a combination of natural antibiotic resistance properties, unique pigmentation and PCR amplification of the linA genes. Neither the addition of corncob nor of corncob immobilized B90A did measurably change the microbial community structure as determined by T-RFLP analysis. Overall, these results indicate that on-site aerobic bioremediation of HCH exploiting the biodegradation activity of S. indicum B90A cells stored on corncob powder is a promising technology.

Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors.

Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/mul or 25 ng of T4 gene 32 protein/mul to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.

Distribution of Giardia duodenalis genotypes and subgenotypes in raw urban wastewater in Milwaukee, Wisconsin.

Giardia cysts in 131 raw wastewater samples from Milwaukee, Wis., were genotyped by sequence analysis of the triosephosphate isomerase gene which showed the presence of two distinct genotypes (assemblages A and B) of Giardia duodenalis. Of the 131 samples, 111 belonged to assemblage A, and the remaining samples belonged to assemblage B. A high degree of genetic polymorphism was evident within the assemblage B cluster, with 10 distinct subgenotypes identified, eight of which have not been reported before.

Molecular surveillance of Cryptosporidium spp. in raw wastewater in Milwaukee: implications for understanding outbreak occurrence and transmission dynamics.

Six Cryptosporidium spp. were found in 50 of 179 Milwaukee wastewater samples collected weekly over a year. Of the eight subtypes of Cryptosporidium hominis and Cryptosporidium parvum present, allele Ib was found in 14 of 16 samples, and its sequence was identical to that of the subtype in human samples from the 1993 Milwaukee outbreak of cryptosporidiosis.

Decision analysis: point-of-care Chlamydia testing vs. laboratory-based methods.

OBJECTIVE: To evaluate and compare the performance of several different methods available for detection of Chlamydia trachomatis (Ct) infection, and to explore possible testing and treatment strategies incorporating point-of-care testing versus laboratory-based tests. DESIGN: Prospective trial and decision analysis. SETTING: Large, urban, publicly funded sexually transmitted disease clinic. PARTICIPANTS: 1,384 female patients. METHODS: Each subject was tested for Ct infection by direct fluorescent antibody (DFA, Sanofi/Kallestad, Chaska, MN), optical immunoassay (OIA, Thermo Electron, Point of Care and Rapid Diagnostics, Louisville CO), McCoy cell culture (in-house method), and polymerase chain reaction (microwell PCR, microwell assay, Roche, Branchburg NJ). RESULTS: Performing a rapid in-clinic test on women who did not meet empiric treatment criteria would have increased the overall proportion of infected persons receiving same-day treatment from 48.6% to 79.1% using DFA or 78.4% using OIA. CONCLUSIONS: Use of empiric treatment criteria and same-day point-of-care testing for patients not meeting the empiric treatment threshold appears to be an appropriate, useful, and cost-effective strategy for increasing same-day treatment of Ct infections in this population.