A critical opportunity: Detecting and Reducing Lead in Drinking Water at child care facilities.

There is no safe level of lead exposure. As exposure from point sources like lead paint have decreased, non-point sources such as drinking water have become a greater proportional source of total lead exposure. Even at low levels, lead exposure is shown to harm children, contributing to impaired development as well as learning and behavioral issues. This paper summarizes the key results of an Environmental Defense Fund (EDF) pilot study conducted at 11 child care facilities in 4 US states to evaluate approaches to testing and remediating lead in water at child care facilities. Over 75% of first draw samples contained lead levels under the 1 µg/L level recommended by the American Academy of Pediatrics (AAP). However, 10 of 11 child care facilities produced at least one sample above 1 µg/L. Fixture flushing, aerator cleaning, and fixture replacement were evaluated as remediation strategies. Fixture replacement was effective when initial lead was above 5 µg/L. Aerator cleaning did not have a measurable effect on lead levels for most fixtures but unexpectedly significantly increased lead levels in approximately 30% of fixtures. The 2021 Lead and Copper Rule (LCR) revision was applied to study data to determine whether updates would flag cases of low-level lead in child care settings and was found insufficient to prompt mitigation unless high lead was present at most taps.

Improving state-level emergency well disinfection strategies in the United States.

After flooding events, well users are encouraged to disinfect their private wells. However, well disinfection strategies are not consistently applied or proven effective. This study examines the science-based evidence that disinfection procedures reduce microbial loading in well water; reviews inclusion of disinfection principles in state-level emergency protocols; and explores research gaps potentially hindering disinfection efficacy. Emergency well disinfection protocols from 34 states were reviewed based on instructions for creating chlorine solutions; circulating chlorine solutions throughout the distribution system; achieving effective CT disinfection (chlorine dose*contact time); and post-disinfection guidance. Many protocols were missing key information about fundamentals of disinfection. Only two protocols instructed well users to verify chlorine residuals and three protocols instructed users to measure water pH. Most protocols recommended that high chlorine doses be introduced into the well, circulated throughout the system, and stagnated for several hours. A CT value estimated to inactivate at least 99.9% (3-log removal) of Cryptosporidium (255 mg-hr/L) was predicted to be achieved by 72.7% of protocols, and estimated CT values ranged from 35 to 16,327 mg-hr/L. Two research gaps identified were determining whether chlorine doses should differ based on well water chemistries and evaluating the appropriate chlorine dose that should be recommended for inactivating pathogens. This effort underscores a need for consistent, evidence-based messaging in emergency well disinfection protocols.

An evaluation of security measures implemented to address physical threats to water infrastructure in the state of Mississippi.

The events of September 11, 2001, increased and intensified domestic preparedness efforts in the United States against terrorism and other threats. The heightened focus on protecting this nation's critical infrastructure included legislation requiring implementation of extensive new security measures to better defend water supply systems against physical, chemical/biological, and cyber attacks. In response, municipal officials have implemented numerous safeguards to reduce the vulnerability of these systems to purposeful intrusions including ongoing vulnerability assessments, extensive personnel training, and highly detailed emergency response and communication plans. This study evaluates fiscal year 2010 annual compliance assessments of public water systems with security measures that were implemented by Mississippi's Department of Health as a response to federal requirements to address these potential terrorist threats to water distribution systems. The results show that 20 percent of the water systems in this state had at least one security violation on their 2010 Capacity Development Assessment, and continued perseverance from local governments is needed to enhance the resiliency and robustness of these systems against physical threats.

Nitric oxide in chemostat-cultured Escherichia coli is sensed by Fnr and other global regulators: unaltered methionine biosynthesis indicates lack of S nitrosation.

We previously elucidated the global transcriptional responses of Escherichia coli to the nitrosating agent S-nitrosoglutathione (GSNO) in both aerobic and anaerobic chemostats, demonstrated the expression of nitric oxide (NO)-protective mechanisms, and obtained evidence of critical thiol nitrosation. The present study was the first to examine the transcriptome of NO-exposed E. coli in a chemostat. Using identical conditions, we compared the GSNO stimulon with the stimulon of NO released from two NO donor compounds {3-[2-hydroxy-1-(1-methyl-ethyl)-2-nitrosohydrazino]-1-propanamine (NOC-5) and 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7)} simultaneously and demonstrated that there were marked differences in the transcriptional responses to these distinct nitrosative stresses. Exposure to NO did not induce met genes, suggesting that, unlike GSNO, NO does not elicit homocysteine S nitrosation and compensatory increases in methionine biosynthesis. After entry into cells, exogenous methionine provided protection from GSNO-mediated killing but not from NO-mediated killing. Anaerobic exposure to NO led to up-regulation of multiple Fnr-repressed genes and down-regulation of Fnr-activated genes, including nrfA, which encodes cytochrome c nitrite reductase, providing strong evidence that there is NO inactivation of Fnr. Other global regulators apparently affected by NO were IscR, Fur, SoxR, NsrR, and NorR. We tried to identify components of the NorR regulon by performing a microarray comparison of NO-exposed wild-type and norR mutant strains; only norVW, encoding the NO-detoxifying flavorubredoxin and its cognate reductase, were unambiguously identified. Mutation of norV or norR had no effect on E. coli survival in mouse macrophages. Thus, GSNO (a nitrosating agent) and NO have distinct cellular effects; NO more effectively interacts with global regulators that mediate adaptive responses to nitrosative stress but does not affect methionine requirements arising from homocysteine nitrosation.

Transcriptional responses of Escherichia coli to S-nitrosoglutathione under defined chemostat conditions reveal major changes in methionine biosynthesis.

Nitric oxide and nitrosating agents exert powerful antimicrobial effects and are central to host defense and signal transduction. Nitric oxide and S-nitrosothiols can be metabolized by bacteria, but only a few enzymes have been shown to be important in responses to such stresses. Glycerol-limited chemostat cultures in defined medium of Escherichia coli MG1655 were used to provide bacteria in defined physiological states before applying nitrosative stress by addition of S-nitrosoglutathione (GSNO). Exposure to 200 microm GSNO for 5 min was sufficient to elicit an adaptive response as judged by the development of NO-insensitive respiration. Transcriptome profiling experiments were used to investigate the transcriptional basis of the observed adaptation to the presence of GSNO. In aerobic cultures, only 17 genes were significantly up-regulated, including genes known to be involved in NO tolerance, particularly hmp (encoding the NO-consuming flavohemoglobin Hmp) and norV (encoding flavorubredoxin). Significantly, none of the up-regulated genes were members of the Fur regulon. Six genes involved in methionine biosynthesis or regulation were significantly up-regulated; metN, metI, and metR were shown to be important for GSNO tolerance, because mutants in these genes exhibited GSNO growth sensitivity. Furthermore, exogenous methionine abrogated the toxicity of GSNO supporting the hypothesis that GSNO nitrosates homocysteine, thereby withdrawing this intermediate from the methionine biosynthetic pathway. Anaerobically, 10 genes showed significant up-regulation, of which norV, hcp, metR, and metB were also up-regulated aerobically. The data presented here reveal new genes important for nitrosative stress tolerance and demonstrate that methionine biosynthesis is a casualty of nitrosative stress.

Genome-wide transcriptional response of chemostat-cultured Escherichia coli to zinc.

Zinc is an essential trace metal ion for growth, but an excess of Zn is toxic and microorganisms express diverse resistance mechanisms. To understand global bacterial responses to excess Zn, we conducted transcriptome profiling experiments comparing Escherichia coli MG1655 grown under control conditions and cells grown with a toxic, sublethal ZnSO4 concentration (0.2 mM). Cultures were grown in a defined medium lacking inorganic phosphate, permitting maximum Zn bioavailability, and in glycerol-limited chemostats at a constant growth rate and pH. Sixty-four genes were significantly up-regulated by Zn stress, including genes known to be involved in Zn tolerance, particularly zntA, zraP, and hydG. Microarray transcriptome profiling was confirmed by real-time PCR determinations of cusF (involved in Ag and Cu efflux), ais (an Al-inducible gene), asr (encoding an acid shock-inducible periplasmic protein), cpxP (a periplasmic chaperone gene), and basR. Five up-regulated genes, basR and basS [encoding a sensor-regulator implicated in Salmonella in Fe(III) sensing and antibiotic resistance], fliM (flagellar synthesis), and ycdM and yibD (both with unknown functions), are important for growth resistance to zinc, since mutants with mutations in these genes exhibited zinc sensitivity in liquid media and on metal gradient plates. Fifty-eight genes were significantly down-regulated by Zn stress; notably, several of these genes were involved in protection against acid stress. Since the mdt operon (encoding a multidrug resistance pump) was also up-regulated, these findings have important implications for understanding not only Zn homeostasis but also how bacterial antibiotic resistance is modulated by metal ions.