Fecal indicators and bacterial pathogens in bottled water from Dhaka, Bangladesh

Forty-six bottled water samples representing 16 brands from Dhaka, Bangladesh were tested for the numbers of total coliforms, fecal indicator bacteria (i.e., thermotolerant Escherichia coli and Enterococcus spp.) and potential bacterial pathogens (i.e., Aeromonas hydrophil, Pseudomonas aeruginos, Salmonella spp., and Shigella spp.). Among the 16 brands tested, 14 (86%), ten (63%) and seven (44%) were positive for total coliforms, E. coil and Enterococcus spp., respectively. Additionally, a further nine (56%), eight (50%), six (37%), and four (25%) brands were PCR positive for A. hydrophila lip, P. aeruginosa ETA, Salmonella spp. invA, and Shigella spp. ipaH genes, respectively. The numbers of bacterial pathogens in bottled water samples ranged from 28 ± 12 to 600 ± 45 (A. hydrophila lip gene), 180 ± 40 to 900 ± 200 (Salmonella spp. invA gene), 180 ± 40 to 1,300 ± 400 (P. aeruginosa ETA gene) genomic units per L of water. Shigella spp. ipaH gene was not quantifiable. Discrepancies were observed in terms of the occurrence of fecal indicators and bacterial pathogens. No correlations were observed between fecal indicators numbers and presence/absence of A. hydrophila lip (p = 0.245), Salmonella spp. invA (p = 0.433), Shigella spp. ipaH gene (p = 0.078), and P. aeruginosa ETA (p = 0.059) genes. Our results suggest that microbiological quality of bottled waters sold in Dhaka, Bangladesh is highly variable. To protect public health, stringent quality control is recommended for the bottled water industry in Bangladesh. Key words: bottled water, fecal indicator bacteria, quantitative PCR, bacterial pathogens, public health risk.

Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species

Three native freshwater crayfish Cherax species are farmed in Australia namely; Redclaw (Cherax quadricarinatus), Marron (C. tenuimanus), and Yabby (C. destructor). Lack of appropriate data on specific nutrient requirements for each of these species, however, has constrained development of specific formulated diets and hence current use of over-formulated feeds or expensive marine shrimp feeds, limit their profitability. A number of studies have investigated nutritional requirements in redclaw that have focused on replacing expensive fish meal in formulated feeds with non-protein, less expensive substitutes including plant based ingredients. Confirmation that freshwater crayfish possess endogenous cellulase genes, suggests their potential ability to utilize complex carbohydrates like cellulose as nutrient sources in their diet. To date, studies have been limited to only C. quadricarinatus and C. destructor and no studies have compared the relative ability of each species to utilize soluble cellulose in their diets. Individual feeding trials of late-juveniles of each species were conducted separately in an automated recirculating culture system over 12 week cycles. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch. Water temperature, conductivity and pH were maintained at constant and optimum levels for each species. Animals were fed at 3% of their body weight twice daily and wet body weight was recorded bi-weekly. At the end of experiment, all animals were harvested, measured and midgut gland extracts assayed for alpha-amylase, total protease and cellulase activity levels. After the trial period, redclaw fed with RD showed significantly higher (p<0.05) specific growth rate (SGR) compare with animals fed the TD while SGR of marron and yabby fed the two diets were not significantly different (p<0.05). Cellulase expression levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD. Amylase and protease activity in all three species were significantly higher in the animals fed with RD (Table 1). These results indicate that test animals of all species can utilize starch better than dietary soluble cellulose in their diet and inclusion of 20% soluble cellulose in diets does not appear to have any significant negative effect on their growth rate but survival was impacted in C. quadricarinatus while not in C. tenuimanus or C. destructor.

Effects of soluble dietary cellulose on specific growth rate, survival and digestive enzyme activities in three freshwater crayfish (Cherax) species

The current study evaluated the effect of soluble dietary cellulose on growth, survival and digestive enzyme activity in three endemic, Australian freshwater crayfish species (redclaw: Cherax quadricarinatus, marron: C. tenuimanus, yabby: C. destructor). Separate individual feeding trials were conducted for late-stage juveniles from each species in an automated recirculating freshwater, culture system. Animals were fed either a test diet (TD) that contained 20% soluble cellulose or a reference diet (RD) substituted with the same amount of corn starch, over a 12 week period. Redclaw fed with RD showed significantly higher (p<0.05) specific growth rates (SGR) compared with animals fed the TD, while SGR of marron and yabby fed the two diets were not significantly different. Expressed cellulase activity levels in redclaw were not significantly different between diets. Marron and yabby showed significantly higher cellulase activity when fed the RD (p<0.05). Amylase and protease activity in all three species were significantly higher in the animals fed with RD (p<0.05). These results indicate that test animals of all three species appear to utilize starch more efficiently than soluble dietary cellulose in their diet. The inclusion of 20% soluble cellulose in diets did not appear, however, to have a significant negative effect on growth rates.

Bacterial kinetics-controlled shape-directed biosynthesis of silver nanoplates using Morganella psychrotolerans

We show for the first time that by controlling the growth kinetics of Morganella psychrotolerans, a silver-resistant psychrophilic bacterium, the shape anisotropy of silver nanoparticles can be achieved. This is particularly important considering that there has been no report that demonstrates a control over shape of Ag nanoparticles by controlling the growth kinetics of bacteria during biological synthesis. Additionally, we have for the first time performed electrochemistry experiments on bacterial cells after exposing them to Ag(+) ions, which provide significant new insights about mechanistic aspects of Ag reduction by bacteria. The possibility to achieve nanoparticle shape control by using a "green" biosynthesis approach is expected to open up new exciting avenues for eco-friendly, large-scale, and economically viable shape-controlled synthesis of nanomaterials.

Aqueous phase synthesis of copper nanoparticles : a link between heavy metal resistance and nanoparticle synthesis ability in bacterial systems

We demonstrate aqueous phase biosynthesis of phase-pure metallic copper nanoparticles (CuNPs) using a silver resistant bacterium Morganella morganii. This is particularly important considering that there has been no report that demonstrates biosynthesis and stabilization of pure copper nanoparticles in the aqueous phase. Electrochemical analysis of bacterial cells exposed to Cu2+ ions provides new insights into the mechanistic aspect of Cu2+ ion reduction within the bacterial cell and indicates a strong link between the silver and copper resistance machinery of bacteria in the context of metal ion reduction. The outcomes of this study take us a step closer towards designing rational strategies for biosynthesis of different metal nanoparticles using microorganisms.

Does a 10-valent pneumococcal-Haemophilus influenzae protein D conjugate vaccine prevent respiratory exacerbations in children with recurrent protracted bacterial bronchitis, chronic suppurative lung disease and bronchiectasis : protocol for a randomised controlled trial

Background Recurrent protracted bacterial bronchitis (PBB), chronic suppurative lung disease (CSLD) and bronchiectasis are characterised by a chronic wet cough and are important causes of childhood respiratory morbidity globally. Haemophilus influenzae and Streptococcus pneumoniae are the most commonly associated pathogens. As respiratory exacerbations impair quality of life and may be associated with disease progression, we will determine if the novel 10-valent pneumococcal-Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) reduces exacerbations in these children. Methods A multi-centre, parallel group, double-blind, randomised controlled trial in tertiary paediatric centres from three Australian cities is planned. Two hundred six children aged 18 months to 14 years with recurrent PBB, CSLD or bronchiectasis will be randomised to receive either two doses of PHiD-CV or control meningococcal (ACYW(135)) conjugate vaccine 2 months apart and followed for 12 months after the second vaccine dose. Randomisation will be stratified by site, age (<6 years and >= 6 years) and aetiology (recurrent PBB or CSLD/bronchiectasis). Clinical histories, respiratory status (including spirometry in children aged >= 6 years), nasopharyngeal and saliva swabs, and serum will be collected at baseline and at 2, 3, 8 and 14 months post-enrolment. Local and systemic reactions will be recorded on daily diaries for 7 and 30 days, respectively, following each vaccine dose and serious adverse events monitored throughout the trial. Fortnightly, parental contact will help record respiratory exacerbations. The primary outcome is the incidence of respiratory exacerbations in the 12 months following the second vaccine dose. Secondary outcomes include: nasopharyngeal carriage of H. influenzae and S. pneumoniae vaccine and vaccine-related serotypes; systemic and mucosal immune responses to H. influenzae proteins and S. pneumoniae vaccine and vaccine-related serotypes; impact upon lung function in children aged >= 6 years; and vaccine safety. Discussion As H. influenzae is the most common bacterial pathogen associated with these chronic respiratory diseases in children, a novel pneumococcal conjugate vaccine that also impacts upon H. influenzae and helps prevent respiratory exacerbations would assist clinical management with potential short- and long-term health benefits. Our study will be the first to assess vaccine efficacy targeting H. influenzae in children with recurrent PBB, CSLD and bronchiectasis.

Firmicutes dominate the bacterial taxa within sugar-cane processing plants

Sugar cane processing sites are characterised by high sugar/hemicellulose levels, available moisture and warm conditions, and are relatively unexplored unique microbial environments. The PhyloChip microarray was used to investigate bacterial diversity and community composition in three Australian sugar cane processing plants. These ecosystems were highly complex and dominated by four main Phyla, Firmicutes (the most dominant), followed by Proteobacteria, Bacteroidetes, and Chloroflexi. Significant variation (p , 0.05) in community structure occurred between samples collected from ‘floor dump sediment’, ‘cooling tower water’, and ‘bagasse leachate’. Many bacterial Classes contributed to these differences, however most were of low numerical abundance. Separation in community composition was also linked to Classes of Firmicutes, particularly Bacillales, Lactobacillales and Clostridiales, whose dominance is likely to be linked to their physiology as ‘lactic acid bacteria’, capable of fermenting the sugars present. This process may help displace other bacterial taxa, providing a competitive advantage for Firmicutes bacteria.

Simultaneous expression of the bacterial Dictyoglomus thermophilum xynB gene under three different Trichoderma reesei promoters

Multiple copies of expression cassettes driven by the Trichoderma reesei xylanase 2 (xyn2) and cellobiohydrolase 2 (cbh2) promoters were introduced into the recombinant T. reesei EC-21 generated to express a thermostable Dictyoglomus thermophilum xylanase (XynB) under the egl2 promoter for further improvement of the enzyme yield. The transformants were screened based on increased XynB activity only. Multiple promoter transformant MPP-4 expressing the xynB gene under all the three promoters was found to be the highest producer of XynB, giving a 65% increase in yield compared to the parental single-promoter recombinant EC-21. The multiple-promoter transformant strains harboured six to nine copies of the xynB gene. Amongst the three promoters, egl2 seemed to have the strongest effect on XynB expression. The shotgun approach we used proved to be effective for rapid enhancement of protein expression using three promoters active at the near-neutral pH of the cultivation medium.

Using the Gini coefficient with BIOLOG substrate utilisation data to provide an alternative quantitative measure for comparing bacterial soil communities

A measure quantifying unequal use of carbon sources, the Gini coefficient (G), has been developed to allow comparisons of the observed functional diversity of bacterial soil communities. This approach was applied to the analysis of substrate utilisation data obtained from using BIOLOG microtiter plates in a study which compared decomposition processes in two contrasting plant substrates in two different soils. The relevance of applying the Gini coefficient as a measure of observed functional diversity, for soil bacterial communities is evaluated against the Shannon index (H) and average well colour development (AWCD), a measure of the total microbial activity. Correlation analysis and analysis of variance of the experimental data show that the Gini coefficient, the Shannon index and AWCD provided similar information when used in isolation. However, analyses based on the Gini coefficient and the Shannon index, when total activity on the microtiter plates was maintained constant (i.e. AWCD as a covariate), indicate that additional information about the distribution of carbon sources being utilised can be obtained. We demonstrate that the Lorenz curve and its measure of inequality, the Gini coefficient, provides not only comparable information to AWCD and the Shannon index but when used together with AWCD encompasses measures of total microbial activity and absorbance inequality across all the carbon sources. This information is especially relevant for comparing the observed functional diversity of soil microbial communities.

Capacity of fatty acid profiles and substrate utilization patterns to describe differences in soil microbial communities associated with increased salinity or alkalinity at three locations in South Australia

Fatty acid methyl ester (FAME) profiles, together with Biolog substrate utilization patterns, were used in conjunction with measurements of other soil chemical and microbiological properties to describe differences in soil microbial communities induced by increased salinity and alkalinity in grass/legume pastures at three sites in SE South Australia. Total ester-linked FAMEs (EL-FAMEs) and phospholipid-linked FAMEs (PL-FAMEs), were also compared for their ability to detect differences between the soil microbial communities. The level of salinity and alkalinity in affected areas of the pastures showed seasonal variation, being greater in summer than in winter. At the time of sampling for the chemical and microbiological measurements (winter) only the affected soil at site 1 was significantly saline. The affected soils at all three sites had lower organic C and total N concentrations than the corresponding non-affected soils. At site 1 microbial biomass, CO 2-C respiration and the rate of cellulose decomposition was also lower in the affected soil compared to the non-affected soil. Biomarker fatty acids present in both the EL- and PL-FAME profiles indicated a lower ratio of fungal to bacterial fatty acids in the saline affected soil at site 1. Analysis of Biolog substrate utilization patterns indicated that the bacterial community in the affected soil at site 1 utilized fewer carbon substrates and had lower functional diversity than the corresponding community in the non-affected soil. In contrast, increased alkalinity, of major importance at sites 2 and 3, had no effect on microbial biomass, the rate of cellulose decomposition or functional diversity but was associated with significant differences in the relative amounts of several fatty acids in the PL-FAME profiles indicative of a shift towards a bacterial dominated community. Despite differences in the number and relative amounts of fatty acids detected, principal component analysis of the EL- and PL-FAME profiles were equally capable of separating the affected and non-affected soils at all three sites. Redundancy analysis of the FAME data showed that organic C, microbial biomass, electrical conductivity and bicarbonate-extractable P were significantly correlated with variation in the EL-FAME profiles, whereas pH, electrical conductivity, NH 4-N, CO 2-C respiration and the microbial quotient were significantly correlated with variation in the PL-FAME profiles. Redundancy analysis of the Biolog data indicated that cation exchange capacity and bicarbonate-extractable K were significantly correlated with the variation in Biolog substrate utilization patterns.

Atmospheric pressure plasmas : infection control and bacterial responses

Cold atmospheric pressure plasma (APP) is a recent, cutting-edge antimicrobial treatment. It has the potential to be used as an alternative to traditional treatments such as antibiotics and as a promoter of wound healing, making it a promising tool in a range of biomedical applications with particular importance for combating infections. A number of studies show very promising results for APP-mediated killing of bacteria, including removal of biofilms of pathogenic bacteria such as Pseudomonas aeruginosa. However, the mode of action of APP and the resulting bacterial response are not fully understood. Use of a variety of different plasma-generating devices, different types of plasma gases and different treatment modes makes it challenging to show reproducibility and transferability of results. This review considers some important studies in which APP was used as an antibacterial agent, and specifically those that elucidate its mode of action, with the aim of identifying common bacterial responses to APP exposure. The review has a particular emphasis on mechanisms of interactions of bacterial biofilms with APP.

Pulmonary innate immunity in children with protracted bacterial bronchitis

Objective To determine bronchoalveolar lavage (BAL) levels of 3 innate immunity components (human alpha-defensin-2 [hBD2], mannose-binding lectin [MBL], and surfactant protein-A [SP-A], the relationship with airway neutrophilia and infection, and cytokine production of stimulated BAL cells in children with current protracted bacterial bronchitis (PBB), children with resolved PBB (PBB well), and controls. Study design BAL of 102 children (mean age 2.8 years) fulfilling predefined criteria of current PBB (n=61), PBB well (n=20), and controls (n=21) was cultured (quantitative bacteriology) and viruses examined by polymerase chain reaction. hBD2, MBL, and SP-A were measured, and cytokine production of lipopolysaccharide-stimulated BAL cells were determined. Results Median hBD2 and MBL levels were significantly higher in the current PBB group (hBD2 = 164.4, IQR 0-435.5pg/mL; MBL = 1.7, 0.4-4ng/mL) than in the PBB well group (hBD2 = 0, IQR 0-85.2; MBL = 0.6, IQR 0.03-2.9) and controls (hBD2 = 3.6, IQR 0-126; MBL = 0.4, IQR 0.02-79). hBD2 was significantly higher in children with airway infection (n = 54; median 76.9, IQR 0-397.3) compared with those without (n = 48; 0, IQR 0-236.3), P=0.04. SP-A levels and cytokine production of stimulated BAL cells were similar between groups. Conclusion In children's airways, hBD2, but not MBL and SP-A, relates to inflammation and infection. In children with PBB, mechanisms involving airway hBD2 and MBL are augmented. These pulmonary innate immunity components and the ability of BAL cells to respond to stimuli are unlikely to be deficient.

The antigen 43 structure reveals a molecular velcro-like mechanism of autotransporter-mediated bacterial clumping

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure–function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped β-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular “Velcro-like” mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.

Discovery of an archetypal protein transport system in bacterial outer membranes

Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.

Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.