Conventional wastewater treatment and reuse site practices modify bacterial community structure but do not eliminate some opportunistic pathogens in reclaimed water.

Water recycling continues to expand across the United States, from areas that have access to advanced, potable-level treated reclaimed water, to those having access only to reclaimed water treated at conventional municipal wastewater treatment plants. This expansion makes it important to further characterize the microbial quality of these conventionally-treated water sources. Therefore, we used 16S rRNA gene sequencing to characterize total bacterial communities present in differentially-treated wastewater and reclaimed water (n = 67 samples) from four U.S. wastewater treatment plants and one associated spray irrigation site conducting on-site ultraviolet treatment and open-air storage. The number of observed operational taxonomic units was significantly lower (p < 0.01) in effluent, compared to influent, after conventional treatment. Effluent community structure was influenced more by treatment method than by influent community structure. The abundance of Legionella spp. increased as treatment progressed in one treatment plant that performed chlorination and in another that seasonally chlorinated. Overall, the alpha-diversity of bacterial communities in reclaimed water decreased (p < 0.01) during wastewater treatment and spray irrigation site ultraviolet treatment (p < 0.01), but increased (p < 0.01) after open-air storage at the spray irrigation site. The abundance of Legionella spp. was higher at the sprinkler system pumphouse at the spray irrigation site than in the influent from the treatment plant supplying the site. Legionella pneumophila was detected in conventionally treated effluent samples and in samples collected after ultraviolet treatment at the spray irrigation site, while Legionella feeleii persisted throughout on-site treatment at the spray irrigation site, and, along with Mycobacterium gordonae, was also detected at the sprinkler system pumphouse at the spray irrigation site. These data could inform the development of future treatment technologies and reuse guidelines that address a broader assemblage of the bacterial community of reclaimed water, resulting in reuse practices that may be more protective of public health.

Smokeless tobacco products harbor diverse bacterial microbiota that differ across products and brands.

Smokeless tobacco products contain numerous chemical compounds, including known human carcinogens. Other smokeless tobacco constituents, including bacteria, may also contribute to adverse health effects among smokeless tobacco users. However, there is a lack of data regarding the microbial constituents of smokeless tobacco. Our goal was to characterize the bacterial microbiota of different smokeless tobacco products and evaluate differences across product types and brands. DNA was extracted from 15 brands of smokeless tobacco products (including dry snuff, moist snuff, snus, and Swedish snus) and 6 handmade products (e.g., toombak) using an enzymatic and mechanical lysis approach. Bacterial community profiling was performed using PCR amplification of the V1-V2 hypervariable region of the 16S rRNA gene, followed by 454 pyrosequencing of the resulting amplicons and sequence analysis using the QIIME package. Total viable counts were also determined to estimate the number of viable bacteria present in each product. Average total viable counts ranged from 0 to 9.35 × 107 CFU g-1. Analysis of the 16S rRNA gene sequences revealed high bacterial diversity across the majority of products tested: dry snuff products where characterized by the highest diversity indices compared to other products. The most dominant bacterial phyla across all products were Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. Significant differences in both bacterial community composition and in silico predicted gene content were observed between smokeless tobacco product types and between brands of specific smokeless tobacco products. These data are useful in order to comprehensively address potential health risks associated with the use of smokeless tobacco products.

Mentholation affects the cigarette microbiota by selecting for bacteria resistant to harsh environmental conditions and selecting against potential bacterial pathogens.

BACKGROUND: There is a paucity of data regarding the microbial constituents of tobacco products and their impacts on public health. Moreover, there has been no comparative characterization performed on the bacterial microbiota associated with the addition of menthol, an additive that has been used by tobacco manufacturers for nearly a century. To address this knowledge gap, we conducted bacterial community profiling on tobacco from user- and custom-mentholated/non-mentholated cigarette pairs, as well as a commercially-mentholated product. Total genomic DNA was extracted using a multi-step enzymatic and mechanical lysis protocol followed by PCR amplification of the V3-V4 hypervariable regions of the 16S rRNA gene from five cigarette products (18 cigarettes per product for a total of 90 samples): Camel Crush, user-mentholated Camel Crush, Camel Kings, custom-mentholated Camel Kings, and Newport Menthols. Sequencing was performed on the Illumina MiSeq platform and sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package. RESULTS: In all products, Pseudomonas was the most abundant genera and included Pseudomonas oryzihabitans and Pseudomonas putida, regardless of mentholation status. However, further comparative analysis of the five products revealed significant differences in the bacterial compositions across products. Bacterial community richness was higher among non-mentholated products compared to those that were mentholated, particularly those that were custom-mentholated. In addition, mentholation appeared to be correlated with a reduction in potential human bacterial pathogens and an increase in bacterial species resistant to harsh environmental conditions. CONCLUSIONS: Taken together, these data provide preliminary evidence that the mentholation of commercially available cigarettes can impact the bacterial community of these products.

Occurrence of vancomycin-resistant and -susceptible Enterococcus spp. in reclaimed water used for spray irrigation.

Reclaiming municipal wastewater for agricultural, environmental, and industrial purposes is increasing in the United States to combat dwindling freshwater supplies. However, there is a lack of data regarding the microbial quality of reclaimed water. In particular, no previous studies have evaluated the occurrence of vancomycin-resistant enterococci (VRE) in reclaimed water used at spray irrigation sites in the United States. To address this knowledge gap, we investigated the occurrence, concentration, and antimicrobial resistance patterns of VRE and vancomycin-susceptible enterococci at three U.S. spray irrigation sites that use reclaimed water. We collected 48 reclaimed water samples from one Mid-Atlantic and two Midwest spray irrigation sites, as well as their respective wastewater treatment plants, in 2009 and 2010. Samples were analyzed for total enterococci and VRE using standard membrane filtration. Isolates were purified and then confirmed using biochemical tests and PCR. Antimicrobial susceptibility testing was conducted using the Sensititre® microbroth dilution system. Data were analyzed by two-sample proportion tests and one-way analysis of variance. We detected total enterococci and VRE in 71% (34/48) and 4% (2/48) of reclaimed water samples, respectively. Enterococcus faecalis was the most common species identified. At the Mid-Atlantic spray irrigation site, UV radiation decreased total enterococci to undetectable levels; however, subsequent storage in an open-air pond at this site resulted in increased concentrations of enterococci. E. faecalis isolates recovered from the Mid-Atlantic spray irrigation site expressed intrinsic resistance to quinupristin/dalfopristin; however, non-E. faecalis isolates expressed resistance to quinupristin/dalfopristin (52% of isolates), vancomycin (4%), tetracycline (13%), penicillin (4%) and ciprofloxacin (17%). Our findings show that VRE are present in low numbers in reclaimed water at point-of-use at the sampled spray irrigation sites; however, resistance to other antimicrobial classes is more prevalent, particularly among non-E. faecalis isolates.

Detection of vancomycin-resistant enterococci (VRE) at four U.S. wastewater treatment plants that provide effluent for reuse.

Vancomycin-resistant enterococci (VRE), a leading cause of hospital-acquired infections, can occur in wastewater. However, to date, no previous studies have evaluated the occurrence of VRE at wastewater treatment plants (WWTPs) that send their treated effluent to reuse sites. We evaluated the occurrence, concentration, and antimicrobial resistance patterns of VRE at U.S. WWTPs associated with reuse sites. We collected 44 wastewater samples, representing treatment steps from influent to effluent, from two Mid-Atlantic and two Midwest WWTPs between October 2009 and October 2010. Samples were analyzed for total enterococci and VRE using membrane filtration. Isolates were confirmed using biochemical tests and PCR. Antimicrobial susceptibility testing was performed by Sensititre microbroth dilution. Data were analyzed by two-sample proportion tests and analysis of variance. We detected VRE in 27% (12/44) of all wastewater samples collected and VRE represented 3% of total enterococci detected at all WWTPs. More samples were VRE-positive from the Mid-Atlantic compared to the Midwest WWTPs (p=0.008). VRE concentrations decreased as treatment progressed at all WWTPs, except at Mid-Atlantic WWTP1 where there was an increase in VRE concentrations in activated sludge reactor samples. VRE were not detected in chlorinated effluent, but were detected in one un-chlorinated effluent sample. All unique VRE isolates were multidrug resistant. Fifty-five percent (12/22) of the isolates displayed high-level aminoglycoside resistance. Our findings show that chlorination reduces the occurrence of VRE in wastewater. However, WWTP workers could be exposed to VRE during wastewater treatment. Our data also raise potential concerns about VRE exposure among individuals who come into contact with un-chlorinated reclaimed water.