Study of Legionella pneumophila treatment with copper in drinking water by single cell-ICP-MS.

Legionella pneumophila is a persistent opportunistic pathogen that poses a significant threat to domestic water systems. Previous studies suggest that copper (Cu) is an effective antimicrobial in water systems. A rapid and sensitive quantification method is desired to optimize the conditions of L. pneumophila treatment by Cu and to better understand the interaction mechanisms between Cu and cells. In this study, we developed a highly sensitive single cell (SC)-ICP-MS method to monitor L. pneumophila cell concentration and track their uptake of Cu. The SC-ICP-MS method showed excellent sensitivity (with a cell concentration detection limit of 1000 cells/mL), accuracy (good agreement with conventional hemocytometry method), and precision (relative standard deviation < 5%) in drinking water matrix. The cupric ions (Cu2+) treatment results indicated that the total L. pneumophila cell concentration, Cu mass per cell, colony-forming unit counting, and Cu concentration in supernatant all exhibited a dose-dependent trend, with 800-1200 µg/L reaching high disinfection rates in drinking water. The investigation of percentages of viable and culturable, viable but nonculturable (VBNC), and lysed cells suggested there always were VBNC present at any Cu concentration. Experimental results of different Cu2+ treatment times further suggested that L. pneumophila cells developed an antimicrobial resistant mechanism with the prolonged Cu exposure. This is the first quantification study on the interactions of Cu and L. pneumophila in drinking water using SC-ICP-MS.

Fatty acids, esters, and biogenic oil disinfectants: novel agents against bacteria.

For hundreds of years, disinfectants have comprised a variety of active chemical agents that destroy microorganisms through a wide spectrum of mechanisms. In recent years, there has been growing interest in novel disinfectants. One novel method for disinfectant is aerosols. Since the beginning of the 20th century, aerosols produced by the volatilization and subsequent recondensation of oil vapors have been utilized as obscurants (smoke) screens during military operations. Specifically, a petroleum middle distillate, known as the FOG oil, has been used in the US military battlefield to create obscurant smoke screens. Biogenic oils are non-petroleum-based oils that resemble FOG oil in terms of their physical characteristics. Furthermore, FOG and biogenic oils have characteristics that make them preferable to other disinfectants that are frequently employed. In this review, we examine the antimicrobial activities of mineral oils and biogenic oil esters aerosols/vapors as novel disinfectants against bacteria and other microorganisms.

An Initial Report of the Antimicrobial Activities of Volatiles Produced during Rapid Volatilization of Oils.

Aerosols generated through volatilization and subsequent recondensation of oil vapors have been used as obscurant (smoke) screens during military operations since the early twentieth century. Specifically, a petroleum middle distillate known as the fog oil (FO) has been used in US military battlefields to create obscurant smoke screens. During a study on the feasibility of replacing petroleum-derived FO with vegetable oil-derived esters such as methyl soyate (MS), it was observed that that FO and MS aerosols and vapors did not exhibit detectable mutagenic activity but were lethal to Ames strains bacteria even after very short exposure periods. These results opened the potential use of oil-derived vapors as antimicrobial agents. Subsequent studies showed that optimal aerosol/vapor production conditions could further enhance disinfectant efficiency. For this purpose, we examined the antimicrobial activities of mineral oils and biogenic oil ester aerosols/vapors against a wide range of Gram-positive and Gram-negative bacteria. The results of the study showed that the aerosols/vapors obtained from mineral oil or vegetable oil ester under proper conditions can serve as an excellent antibacterial disinfectant.

Tuning Polymorphs and Morphology of Microbially Induced Calcium Carbonate: Controlling Factors and Underlying Mechanisms.

Microbially precipitated calcium carbonate (CaCO3) has drawn broad attention due to its potential applications in various areas, for example, biocementation, medicine, and soil reinforcement. Sporosarcina pasteurii (S. pasteurii), formerly known as Bacillus pasteurii, has been investigated for CaCO3 biomineralization due to its high ureolytic activity. A high degree of supersaturation with respect to the presence of bacterial cell wall, extracellular polymeric substances, and organic byproducts of bacterial activity plays an important role in the formation and stabilization of CaCO3 polymorphs. Although microbially induced CaCO3 and its polymorphs have been investigated broadly, the mechanisms of polymorph selection and morphological evolution are not well understood. This study employs ex situ approaches to address the complication of biomineralization in the presence of living organisms and to elucidate how solution chemistry, bacterial activity, and precipitation kinetics alter the polymorphism and morphology of CaCO3 induced by S. pasteurii. The results indicate that in the presence of enough calcium ions and urea (as a carbonate source), the bacterial activity favors the formation and stabilization of vaterite. The morphological observations also provide valuable information on the particles' microstructure. The morphology of calcite evolves from single crystal to polycrystalline structures, and the morphology of vaterite evolved from spherical to oval-shaped structures on increasing the organic material concentration. Specific functional groups also exert morphological control on CaCO3 polymorphs. However, the sensitivity of the calcite polymorph to the composition and orientation of these functional groups is higher compared to that of the vaterite polymorph. These findings offer important insights that can be used to constrain a set of experimental conditions for synthesizing a certain polymorph ratio for vaterite/calcite or a particular morphology of each polymorph and shed light on the crystallization and phase transformation mechanisms in such complicated bioenvironments.

Quantitative real-time PCR assay with immunohistochemical evaluation of HER2/neu oncogene in breast cancer patients and its correlation with clinicopathological findings.

Human epidermal growth factor receptor HER2/neu status is an important prognostic factor for breast cancer as it is crucial in stimulating growth and cellular motility. Overexpression of HER2/neu is observed in 10%-35% of the human breast cancer and is associated with prognosis and response to treatment. The magnitude of amplification must be determined to facilitate better prognosis and personalized therapy in the affected patient. This study aims to investigate the HER2/neu status in breast cancer by concurrent HER2/neu protein overexpression immunohistochemically with HER2/neu DNA amplification by quantitative real-time polymerase chain reaction (PCR), allowing accurate and precise quantification of HER2/neu amplification after a follow-up period. A total of 54 paired tissue samples from formalin-fixed paraffin-embedded (FFPE) breast cancer patients enrolled in this study were collected to evaluate tumor and normal tissues. Only cases with 80% and more tumor cells were included. For confirmation of immunohistochemistry (IHC) results, qPCR was used to determine the HER2/neu amplification. The association between clinicopathological variables like age, tumor size, histological grade, stage, lymph node status, hormone receptor status, family history, recurrence rate, and vital status was evaluated. We observed that 11/54 (20.4%) of the tumor tissues are positive for HER2/neu protein overexpression by IHC. A total of 8 out of these 11 cases (72.7%), which presented a score of 3+, showed gene amplification of HER2/neu. The concordance rate between IHC and qPCR was 94.4%. HER2/neu gene amplification was found to be significantly associated with recurrence, increased risk of death, and progesterone receptor status, supporting a negative prognostic role of HER2/neu in breast cancer survival. In conclusion, IHC can be used as an initial screening test to detect HER2/neu protein overexpression, and the use of qPCR can verify the IHC results and establish HER2/neu status in routine clinical practice.

Phytoremediation removal rates of benzene, toluene, and chlorobenzene.

Phytoremediation is a sustainable remedial approach, although performance efficacy is rarely reported. In this study, we assessed a phytoremediation plot treating benzene, toluene, and chlorobenzene. A comparison of the calculated phytoremediation removal rate with estimates of onsite contaminant mass was used to forecast cleanup periods. The investigation demonstrated that substantial microbial degradation was occurring in the subsurface. Estimates of transpiration indicated that the trees planted were removing approximately 240,000 L of water per year. This large quantity of water removal implies substantial removal of contaminant due to large amounts of contaminants in the groundwater; however, these contaminants extensively sorb to the soil, resulting in large quantities of contaminant mass in the subsurface. The total estimate of subsurface contaminant mass was also complicated by the presence of non-aqueous phase liquids (NAPL), additional contaminant masses that were difficult to quantify. These uncertainties of initial contaminant mass at the site result in large uncertainty in the cleanup period, although mean estimates are on the order of decades. Collectively, the model indicates contaminant removal rates on the order of 10-2-100 kg/tree/year. The benefit of the phytoremediation system is relatively sustainable cleanup over the long periods necessary due to the presence of NAPL.

The Engaged Microbiologist: Bringing the Microbiological Sciences to the K-12 Community.

Exposing K-12 students to cutting edge science that impacts their daily lives can bring classroom lessons to life. Citizen-science projects are an excellent way to bring high-level science to the classroom and help satisfy one of the cornerstone concepts of the Next Generation Science Standards (NGSS), "engaging in practices that scientists and engineers actually use." This can be a daunting task for teachers who may lack the background or resources to integrate these projects into the classroom. This is where scientific societies such as the American Society for Microbiology (ASM) can play a critical role. ASM encourages its members to engage with the K-12 community by providing networking opportunities and resources for ASM members and K-12 teachers to work together to bring microbiology into the classroom. Journal of Microbiology & Biology Education.

Functional analysis of the nifQdctA1y4vGHIJ operon of Sinorhizobium fredii strain NGR234 using a transposon with a NifA-dependent read-out promoter.

Rhizobia are a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes (Fix phenotype). Synthesis of the nitrogenase and its accessory components is under the transcriptional control of the key regulator NifA and is generally restricted to the endosymbiotic forms of rhizobia known as bacteroids. Amongst studied rhizobia, Sinorhizobium fredii strain NGR234 has the remarkable ability to fix nitrogen in association with more than 130 species in 73 legume genera that form either determinate, indeterminate or aeschynomenoid nodules. Hence, NGR234 is a model organism to study nitrogen fixation in association with a variety of legumes. The symbiotic plasmid pSfrNGR234a carries more than 50 genes that are under the transcriptional control of NifA. To facilitate the functional analysis of NifA-regulated genes a new transposable element, TnEKm-PwA, was constructed. This transposon combines the advantages of in vitro mutagenesis of cloned DNA fragments with a conditional read-out promoter from NGR234 (PwA) that reinitiates NifA-dependent transcription downstream of transposition sites. To test the characteristics of the new transposon, the nifQdctA1y4vGHIJ operon was mutated using either the Omega interposon or TnEKm-PwA. The symbiotic phenotypes on various hosts as well as the transcriptional characteristics of these mutants were analysed in detail and compared with the ineffective (Fix(-)) phenotype of strain NGRΔnifA, which lacks a functional copy of nifA. De novo transcription from inserted copies of TnEKm-PwA inside bacteroids was confirmed by qRT-PCR. Unexpectedly, polar mutants in dctA1 and nifQ were Fix(+) on all of the hosts tested, indicating that none of the six genes of the nifQ operon of NGR234 is essential for symbiotic nitrogen fixation on plants that form nodules of either determinate or indeterminate types.