Genomic Epidemiology of Salmonella enterica Circulating in Surface Waters Used in Agriculture and Aquaculture in Central Mexico.

Salmonella enterica can survive in surface waters (SuWa), and the role of nonhost environments in its transmission has acquired increasing relevance. In this study, we conducted comparative genomic analyses of 172 S. enterica isolates collected from SuWa across 3 months in six states of central Mexico during 2019. S. enterica transmission dynamics were assessed using 87 experimental and 112 public isolates from Mexico collected during 2002 through 2019. We also studied genetic relatedness between SuWa isolates and human clinical strains collected in North America during 2005 through 2020. Among experimental isolates, we identified 41 S. enterica serovars and 56 multilocus sequence types (STs). Predominant serovars were Senftenberg (n = 13), Meleagridis, Agona, and Newport (n = 12 each), Give (n = 10), Anatum (n = 8), Adelaide (n = 7), and Infantis, Mbandaka, Ohio, and Typhimurium (n = 6 each). We observed a high genetic diversity in the sample under study, as well as clonal dissemination of strains across distant regions. Some of these strains are epidemiologically important (ST14, ST45, ST118, ST132, ST198, and ST213) and were genotypically close to those involved in clinical cases in North America. Transmission network analysis suggests that SuWa are a relevant source of S. enterica (0.7 source/hub ratio) and contribute to its dissemination as isolates from varied sources and clinical cases have SuWa isolates as common ancestors. Overall, the study shows that SuWa act as reservoirs of various S. enterica serovars of public health significance. Further research is needed to better understand the mechanisms involved in SuWa contamination by S. enterica, as well as to develop interventions to contain its dissemination in food production settings. IMPORTANCE Surface waters are heavily used in food production worldwide. Several human pathogens can survive in these waters for long periods and disseminate to food production environments, contaminating our food supply. One of these pathogens is Salmonella enterica, a leading cause of foodborne infections, hospitalizations, and deaths in many countries. This research demonstrates the role of surface waters as a vehicle for the transmission of Salmonella along food production chains. It also shows that some strains circulating in surface waters are very similar to those implicated in human infections and harbor genes that confer resistance to multiple antibiotics, posing a risk to public health. This study contributes to expand our current knowledge on the ecology and epidemiology of Salmonella in surface waters.

Development of an automated ballast water treatment verification system utilizing fluorescein diacetate hydrolysis as a measure of treatment efficacy.

Methods for verifying ballast water treatments in foreign vessels are needed to protect the Great Lakes from the discharge of live non-native organisms or pathogens. A prototype automated viability test system using fluorescein diacetate (FDA), a membrane permeable fluorogen, to differentiate live from dead bacteria and algae is described. The automated fluorescence intensity detection device (AFIDD) captures cultured algae or organisms in Detroit River water (simulated ballast water) on 0.2 µm filters, backwashes them from the filter into a cuvette with buffer and FDA for subsequent fluorescence intensity measurements, and washes the filters with sterile water for serial automated reuse. Preliminary manual versions of these procedures were also tested. Tests of various buffers determined N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N,N-Bis(2-hydroxyethyl)taurine (BES) and 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.0 to be the best buffers, causing the least spontaneous FDA breakdown without inhibiting enzymatic activity. Fluorescence in the presence of live organisms increased linearly over time, and the rate of increase was dependent on the sample concentration. Following simulated ballast water treatments with heat or chlorine, the fluorescence produced by Detroit River samples decreased to near control (sterile water) levels. Automated measurements of FDA hydrolysis with a reusable filter backwash system should be applicable to near real-time remote-controlled monitoring of live organisms in ballast water.

Ninety-day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette: DNA adducts and alveolar macrophage cytogenetics.

To study the genotoxic effects of subchronic exposure to environmental tobacco smoke, Sprague-Dawley rats were exposed to 0, 0.1, 1.0, and 10 mg total particulate matter (TPM)/m3 of aged and diluted sidestream smoke (ADSS) from 1R4F reference cigarettes 6 hr per day, 5 days a week for 13 weeks. DNA from lung, heart, larynx, bladder, and liver was tested for adduct formation by the 32P-postlabeling assay after 28 (except bladder) and 90 days of exposure and 90 days after cessation of exposure. In addition, alveolar macrophages from animals exposed for 28 or 90 days were examined for chromosomal aberrations. Exposure-related DNA adducts were not observed in any tissue in any of the animals exposed to 0.1 or 1.0 mg TPM/m3. However, increased levels of DNA adducts with diagonal radioactive zones were observed in lung, heart, and larynx DNA of animals exposed to the highest concentration of ADSS (10 mg TPM/m3). Adduct analyses with varying amounts of DNA from lungs of mid- and high-exposure animals clearly indicate that the dose-response for DNA adduct formation is nonlinear. The adduct levels were highest after 90 days of exposure and were significantly reduced in all target tissues 90 days after cessation of exposure. Chromosomal aberrations in alveolar macrophages were not elevated in any group after 28 or 90 days of exposure. These results indicate a no-observed-effect-level (NOEL) of at least 1.0 mg/m3 for DNA adduct formation in lung, heart, and larynx, and a NOEL of at least 10 mg/m3 for the induction of chromosome aberrations in alveolar macrophages, under the conditions of this study.

DNA adduct formation in mice following dermal application of smoke condensates from cigarettes that burn or heat tobacco.

A prototype cigarette that heats tobacco (test cigarette), developed by R.J. Reynolds Tobacco Company, has yielded consistently negative results in several in vivo and in vitro genetic toxicology tests. The objective of the present study was to evaluate the potential of cigarette smoke condensate (CSC) from the test cigarette to induce DNA adducts in mouse tissues and compare the results with those obtained with CSC from a reference tobacco-burning cigarette (1R4F). CD-1 mice were skin-painted with CSC from reference and test cigarettes three times a week for 4 weeks. The highest mass of CSC applied was 180 mg "tar" per week per animal for both reference and test cigarette. DNA adducts were analyzed in skin and lung tissues using the 32P-postlabeling method with the P1 nuclease modification. Distinct diagonal radioactive zones (DRZ) were observed in the DNA from both skin and lung tissues of animals dosed with reference CSC, whereas no corresponding DRZ were observed from the DNA of animals dosed with either test CSC or acetone (solvent control). The relative adduct labeling (RAL) values of skin and lung DNA from reference CSC-treated animals were significantly greater than those of the test CSC-treated animals. The RAL values of the test CSC-treated animals were no greater than those of solvent controls. The negative results in DNA adduct assays with test CSC are consistent with all previous results of in vivo and in vitro genetic toxicology testing on this cigarette and provide additional evidence that smoke condensate from the test cigarette is not genotoxic.

Analysis of cytogenetic effects in bone-marrow cells of rats subchronically exposed to smoke from cigarettes which burn or only heat tobacco.

The genotoxic effects of 90-day nose-only exposures to smoke from new cigarettes, which heat but do not burn tobacco (New), or from reference cigarettes, which burn tobacco, were evaluated in Sprague-Dawley rats by examining the cytogenetic endpoints of sister-chromatid exchanges (SCE), chromosome aberrations, and micronuclei in bone-marrow cells. The concentrations of wet total particulate matter (WTPM) and carbon monoxide in the smoke from both cigarette types were similar. The mainstream smoke from both New and reference cigarettes was adjusted to WTPM concentrations of approx. 200 and 400 micrograms/l for low and high smoke exposure. Rats were exposed to smoke 1 h per day, 5 days per week for 13 consecutive weeks. Inhalation of smoke by the exposed animals was confirmed by analysis of blood carboxyhemoglobin and plasma nicotine. Examination of bone-marrow cells following the final day of exposure showed that smoke from neither the New nor reference cigarette induced a positive response in the SCE, chromosome aberration, or micronucleus assays in rats.