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Abstract Background Abnormal birthweights are critical public health challenges accountable for most non-communicable diseases and perinatal mortalities. Regardless of the myriad of mixed evidence on maternal factors responsible for abnormal birthweight globally, most of these findings are attained from urban and rural settings. This study serves as one of the key pieces of evidence in view of the increasing prevalence of abnormal birthweight particularly in some parts of semi-rural Ghana. The study, therefore, aims to estimate the prevalence of abnormal birthweight and identify some possible maternal risk factors for abnormal birthweight in Northern Ghana. Methods A retrospective cross-sectional study was conducted in Savelugu municipality from February–March 2022. A total of 356 mothers aged 16–46 years, having a neonate and attending postnatal care service, were recruited as study participants. Data were collected from maternal and child health record books and through structured interviews. To identify the maternal risk factors for abnormal birthweight, chi-square/Fischer’s exact test and multinomial logistic regression were employed as bivariate and multivariate analyses, respectively, at 95% confidence level. Results Prevalence rates of low birthweight and macrosomia were 22.2% and 8.7%, respectively. Maternal anaemia in first trimester (AOR 3.226; 95% CI 1.372–7.784) and third trimester (AOR 23.94; 95% CI 7.442–70.01) of gestation was strong predictors for low birthweight. Mothers belonging to minority ethnic groups (AOR 0.104; 95% CI 0.011–0.995); mothers who had ≥ 8 antenatal care visits (AOR 0.249; 95% CI 0.103–0.602); and mothers having neonates whose birth length > 47.5 cm (AOR 0.271; 95% CI 0.113–0.651) had reduced odds for low birthweight. Alternatively, mothers with gestational weeks ≥ 42 (AOR 23.21; 95% CI 4.603–56.19) and mothers from the richest households (highest socioeconomic homes) (AOR 14.25; 95% CI 1.638–23.91) were more likely to birth to macrosomic infants. Conclusion The prevalence rates of low birthweight and macrosomia were relatively high. Anaemia in the first and third trimesters was strong determinants of low birthweight. Being minority ethnic group, frequency of antenatal visits, and childbirth length reduced the risk of low-weight births. Advanced gestational age and socioeconomic status of mothers were also predictors of macrosomia. Hence, nutrition counselling, community health education, and promotion of lifestyle improvement coupled with strengthening of health service delivery are recommended interventions.

Introduction: Breeding profiles at the periparturient stage in red foxes which mated naturally or were subjected to artificial insemination were retrospectively surveyed using 130 vixens during their reproductive seasons of 2012–2017 in Japan. Material and Methods: Natural mating vixens were encouraged a maximum of three times with the same male, while artificial insemination was conducted using frozen-thawed semen with the bovine semen extender as a diluent. Results: With natural mating, conception rates after one, two, and three copulations were 55.8%, 68.0%, and 85.7%, respectively, showing a significant difference between the rates for one and three copulations. Conception rates with artificial insemination were 82.4%. Mean gestation periods were between 52.1 and 53.3 days in all groups. Mean litter sizes were 3.7–4.3 cubs with natural mating, and 4.4 cubs with artificial insemination. Although some sporadic and inconsistent changes in litter sizes were noted between primiparous and multiparous groups, these were of doubtful clinical importance. Conclusion: This is the first report from Japan concerning basic breeding events of red fox vixens in captivity.

We investigated the possibility to reduce the usage of Blasin®Flowable (BF), a disinfectant inhibiting the germination and appressorium formation of Pyricularia oryzae Cavara conidia, by using carbon dioxide microbubbles (CO2MB). Germination was significantly inhibited by 10 000-fold diluted BF solution containing CO2MB generated by the decompression-type generator compared to CO2 millibubbles (CO2MMB) and CO2MB generated by the gas-water circulating-type generator. Appressorium formation in the 10 000-fold diluted BF solution containing both CO2MBs was less than that in CO2MMB. Scanning electron microscopy showed wrinkles and dents on the surface of conidia treated with 5 000-fold diluted BF solution containing both CO2MBs. Via transmission electron microscopy, we observed the expansion of the vacuole and the intracellular space and bloated or absent lipid granules in the conidia treated with BF solution containing both CO2MBs. Our results show that inhibition of the conidium germination and appressorium formation of P. oryzae Cavara by 10 000-fold diluted BF solution could be achieved by using the decompression-type CO2MB.

Abstract Background In Japan, there is a large increase in the number of elderly persons who potentially need home-visit nursing services (VNS). However, the number of persons using the VNS has increased only little in comparison to the number of individuals who use home social services, which are also covered by the Long-Term Care Insurance (LTCI) system. This cross-sectional study investigated the predictors of the VNS used under the LTCI system in Japan. Methods We used 1,580 claim data from all the users of community-based services and 1,574 interview survey data collected in 2001 from the six municipal bodies in Japan. After we merged the two datasets, 1,276 users of community-based services under the LTCI were analyzed. Multiple logistic regression models stratified by care needs levels were used for analysis. Results Only 8.3% of the study subjects were VNS users. Even among study participants within the higher care-needs level, only 22.0% were VNS users. In the lower care level group, people with a higher care level (OR: 3.50, 95% CI: 1.50–8.93), those whose condition needed long term care due to respiratory or heart disease (OR: 4.31, 95% CI: 1.88–89.20), those whose period of needing care was two years or more (OR: 2.01, 95% CI: 1.14–3.48), those whose service plan was created by a medical care management agency (OR: 2.39, 95% CI: 1.31–4.33), those living with family (OR: 1.86, 95% CI: 1.00–3.42), and those who use home-help services (OR: 2.12, 95% CI: 1.17–3.83) were more likely to use the VNS. In the higher care level group, individuals with higher care level (OR: 3.63, 95% CI: 1.56–8.66), those with higher income (OR: 3.79, 95% CI: 1.01–14.25), and those who had regular hospital visits before entering the LTCI (OR: 2.36, 95% CI: 1.11–5.38) were more likely to use the VNS. Conclusions Our results suggested that VNS use is limited due to management by non-medical care management agencies, due to no caregivers being around or a low income household. The findings of this study provide valuable insight for LTCI policy makers: the present provision of VNS should be reconsidered.

Abstract Background Carnosine is a dipeptide that improves exercise performance. The carnosine synthesis mechanism through carnosine and ß-alanine ingestion remains unclear. Therefore, we investigated the tissue distribution of carnosine synthase, ATP-grasp domain-containing protein-1 (ATPGD1) mRNA, and ATPGD1 and carnosine specific dipeptidase (CN1) gene expression profiles in mice that were given carnosine or ß-alanine orally. Methods ddY mice (7-week-old) were randomly divided into three groups (n = 6 to 8 animals per group) and were orally given 2 g/kg body weight of carnosine, ß-alanine, or water. After 15, 30, 60, 120, 180, or 360 min of treatment, the tissues (brain, blood, liver, kidneys, olfactory bulbs, hindleg muscles) were collected. The obtained tissues measured the expression of ATPGD1 and CN1 genes using quantitative PCR methods. Results The ATPGD1 gene was expressed in muscle and to a lesser extent in brain. The expression of ATPGD1 in the vastus lateralis muscle increased significantly at 180 min (P = 0.023) after carnosine ingestion and 60 (P = 0.023) and 180 min (P = 0.025) after ß-alanine ingestion. Moreover, the carnosine group showed a significantly increased renal expression of the CN1 gene 60 min after ingestion (P = 0.0015). Conclusions The ATPGD1 gene showed high expression levels in brain and muscle. The ß-alanine or carnosine administration significantly increased ATPGD1 and CN1 expression in mice.

Abstract Background Daughters-in-law have played an important role in informal care-giving arrangements within East Asian traditional norms. The aim of this study was to measure the impact of daughter-in-law care-giving on the survival of care recipients. We prospectively examined the associations between different types of kinship relationship between the main family caregiver and the care recipient in relation to survival among care recipients. Methods A questionnaire was administered to Japanese community-dwelling seniors who were eligible to receive national long-term care insurance (LTCI) community-based services. Among 191 individuals whose informal care-giving arrangement was definitively determined, we observed 58 care recipients receiving care from spouses, 58 from daughters-in-law, 27 from biological daughters, 25 from other relatives, and 23 care recipients living alone. Results During 51 months of follow-up from December 2001, 68 care recipients died, 117 survived, and 6 moved. Hazard ratios of each care-giving arrangement were estimated by Cox proportional hazard models adjusted for care recipients' demographic factors, their care needs level based on their physical and cognitive functioning and their service use, caregivers' demographic factors, and household size. The highest risk of mortality was found for female elders receiving care from daughters-in-law (HR 4.15, 95% CI 1.02-16.90) followed by those receiving care from biological daughters (HR 1.64, 95% CI 0.37-7.21), compared to women receiving spousal care. By contrast, male elders receiving care from daughters-in-law tended to live longer than those receiving care from their spouses. Conclusions Our finding suggests that there may be a survival "penalty" for older Japanese women who are cared for by their daughters-in-law.

Abstract Occupational cases with allergic reaction to fragrance substances, which refer to various chemicals providing aroma characteristics, are arising with its recent usage diversification from pharmaceutical, perfume industry to aromatic remedies. However, chemicals responsible for fragrance allergy have hardly been identified because its component is complex and its sensitization is not frequent. This report will present a case of acute allergic dermatitis that is likely induced by 3-hexylthiophene, one of aromatic compounds often contained in fragrance substances. The case, who was a 27-year male researcher engaged in organic chemical synthesis for six years, was exposed to 3-hexylthiophene and its product (2-bromo-3-hexylthiophene) through an experiment in May 2004 and itching, swelling and eczema immediately developed from face to back. This case of sensitization to 3-hexylthiophene suggests that it be a possible allergen for fragrance allergy.

Abstract Background With the introduction of long-term care insurance (LTCI) in Japan, more home care services are available for the community-dwelling elderly. To deliver effective home care services, it is important to know the effects of service use. In this study, as the first step to determine this, we sought to describe different home service use in the sustained/improved group and deteriorated group in their care needs levels, and to report the relationship between the use of home care services and changes in care needs levels. Methods The participants included 624 of a total of 1,474 users of LTCI services in one city in Japan. Home care service users were stratified into a 'lower care needs level subgroup' and a 'higher care needs level subgroup' based on the baseline care needs level. Simple statistical comparison and multiple logistic regression analyses in which the change in care needs level was set as a dependent variable were performed. Gender, age, and baseline care needs level were designated as control variables. Home based services were treated as independent variables. In this study, home care services consisted of home help, home bathing services, a visiting nurse, home rehabilitation, nursing home daycare, health daycare, loan of medical devices, respite stay in a nursing home, respite stay in a health care facility, respite stay in a sanatorium-type medical care facility, and medical management by a physician. Results In the lower care needs level subgroup, age (OR = 1.04, CI, 1.01-1.08), use of respite stay in a nursing home (OR = 2.55; CI, 1.43-4.56), and the number of types of long-term care services (OR = 1.33; CI, 1.02-1.74) used during an 11 month period were significantly related to a deterioration of the user's care needs level. In the higher care needs level subgroup, use of medical management by a physician (OR = 6.99; CI, 1.42-41.25) was significantly related to a deterioration of the user's care needs level. There were no home based services significantly related to sustaining or improving the user's care needs level. Conclusion There were different home service use in two groups (the sustained/improved group and the deteriorated group). Respite stay in a nursing home service use and more types of service use were related to experiencing a deterioration of care needs level in lower care needs level community-dwelling elderly persons in Japan. Further, medical management by a physician service was related to experiencing a deterioration of care needs level in higher care needs level community-dwelling elderly persons.

Abstract Background The relationship between bed days and benzodiazepine prescription (BDZ) in Western countries is inconclusive, and no hospital-based report has documented this phenomenon in Japan. This study was done to assess the association between bed days and BDZ in a Japanese hospital. Methods 21,489 adult patients (55.1% men, mean age 59.9 years old) hospitalized between April, 2005 and December, 2006 were enrolled in the study. Patient age, sex, ICD-10 diagnosis, prescription profile, and days of hospital stay were assessed in 13 non-psychiatric departments using a computer ordering system. Patients prescribed a benzodiazepine during hospitalization were defined as positive. Results Of the total sample, 19.9% were allocated to the benzodiazepine (+) group. Female sex and older age were significant factors associated with benzodiazepine prescription. The median number of bed days was 13, and the likelihood of BDZ significantly increased with the number of bed days, even after controlling for the effects of age, gender, and ICD-10 diagnosis. For example, when the analysis was limited to patients with 50 bed days or longer, the percentage of BDZ (32.7%) was equivalent to that of a report from France. Conclusion Irrespective of department or disease, patients prescribed benzodiazepine during their hospital stay tended to have a higher number of bed days in the hospital. The difference in the prevalence of BDZ between this study and previous Western studies might be attributed to the relatively short length of hospital stay in this study. Because BDZs are often reported to be prescribed to hospitalized patients without appropriate documentation for the indications for use, it is important to monitor the rational for prescriptions of benzodiazepine carefully, for both clinical and economical reasons.

Abstract Background The tumour suppressor phosphatase and tensin homolog (PTEN) is an important negative regulator of cell-survival signaling. To evaluate the correlation between PTEN expression and clinicopathological characteristics of colorectal cancer patients with and without liver metastases, we investigated PTEN expression in primary colorectal cancer and colorectal cancer liver metastases. Methods Sixty-nine pairs of primary colorectal cancer and corresponding liver metastasis specimens were analyzed immunohistochemically, and the correlation between immunohistochemical findings and clinicopathological factors was investigated. Seventy primary colorectal cancer specimens from patients without liver metastases were used as controls. Results PTEN was strongly expressed in 44 (62.9%) colorectal cancer specimens from patients without liver metastases. In contrast, PTEN was weakly expressed in 52 (75.4%) primary colorectal cancer specimens from patients with liver metastases, and was absent in liver metastases. Weak PTEN expression in colorectal cancer tissues was significantly associated with advanced TNM stage (p < 0.01) and lymph node metastasis (p < 0.05). PTEN expression was significantly stronger in primary colorectal cancer specimens from patients without liver metastases. Furthermore, among colorectal cancer patients with liver metastases, the 5-year survival rate was significantly higher in patients with positive PTEN expression compared to those with negative PTEN expression (p = 0.012). Conclusion Our results suggest that loss of PTEN expression is involved with colorectal cancer aggressive capacity and that diagnostic evaluation of PTEN expression may provide valuable prognostic information to aid treatment strategies for colorectal cancer patients.

Abstract Background Alpha-fetoprotein (AFP)-producing gastric cancer is known to frequently cause multiple liver metastases and to have an extremely poor prognosis. Case presentation A 64-year-old Japanese man admitted to our hospital was diagnosed with gastric cancer with liver metastases. He underwent a total gastrectomy with splenectomy, and pathological stage IV disease according to the classification proposed by the Japanese Gastric Cancer Association was assigned. The histological diagnosis was poorly differentiated adenocarcinoma, and tumor production of AFP was confirmed by immunohistochemical staining. Following surgery, the patient received combination chemotherapy consisting of TS-1 and paclitaxel. Initially, AFP levels decreased dramatically and computed tomography (CT) revealed regression of liver metastases. However, multiple new liver metastases appeared and serum AFP levels increased after 5 months. A regimen of 5-FU plus paclitaxel followed by paclitaxel monotherapy was used next. Serum AFP levels once again decreased and CT showed regression or disappearance of liver metastases. The patient currently has a very good quality of life, and is receiving weekly paclitaxel monotherapy as an outpatient. No progression of liver metastases has been observed to date. Conclusion We consider this rare case to have significant value with respect to treatment of AFP-producing gastric cancer with multiple liver metastases, and propose that combining surgery with chemotherapeutic agents such as paclitaxel may lead to a better prognosis in such cases.

Abstract Background The transmembrane protein c-kit is a receptor tyrosine kinase (KIT) and KIT is expressed in solid tumors and hematological malignancies such as gastrointestinal stromal tumor (GIST), small-cell lung cancer and chronic myelogenous leukemia (CML). KIT plays a critical role in cell proliferation and differentiation and represents a logical therapeutic target in GIST and CML. In pancreatic cancer, c-kit expression has been observed by immunohistochemical techniques. In this study, we examined the influence of c-kit expression on proliferation and invasion using five pancreatic cancer cell lines. In addition, the inhibitory effect of imatinib mesylate on stem cell factor (SCF)-induced proliferation and invasion was evaluated. Finally, we also analyzed KIT and SCF expression in pancreatic cancer tissues using immunohistochemistry and correlated the results with clinical features. Results RT-PCR revealed that two pancreatic cancer cell lines, PANC-1 and SW1990, expressed c-kit mRNA. By Western blot analysis, c-kit protein was also present in those lines. In KIT-positive pancreatic cancer cell lines, proliferation and invasion were significantly enhanced by addition of SCF. In contrast, SCF did not enhance proliferation and invasion in the three KIT-negative lines (BxPC-3, Capan-2 and MIA PaCa-2). 5 μM imatinib mesylate significantly inhibited SCF-enhanced proliferation to the same extent compared with the control. Similarly, SCF-enhanced invasive ability was significantly inhibited by 5 μM imatinib mesylate. KIT was expressed in 16 of 42 clinical specimens by immunohistochemistry, and KIT expression was significantly related to venous system invasion. Furthermore, patients expressing both KIT and SCF had a somewhat lower survival. Conclusion Our results demonstrated that the SCF-KIT pathway enhanced the proliferation and invasiveness in KIT-positive pancreatic cancer cell lines and that the enhanced proliferation and invasion were inhibited by imatinib mesylate. We propose that inhibitors of c-kit tyrosine kinase receptor have the potential to slow the progression of KIT-positive pancreatic cancers.

Recreational water epidemiology studies are rare in settings with minimal wastewater treatment where risk may be highest, and in tropical settings where warmer temperature influences the ecology of fecal indicator bacteria commonly used to monitor recreational waters. One exception is a 1999 study conducted in São Paulo Brazil. We compared the risk and exposure characteristics of these data with those conducted in the United Kingdom (UK) in the early 1990s that are the basis of the World Health Organization's (WHO) guidelines on recreational water risks. We then developed adjusted risk difference models (excess gastrointestinal illness per swimming event) for children (<10 years of age) and non-children (≥10 years of age) across five Brazil beaches. We used these models along with beach water quality data from 2004 to 2015 to assess spatial and temporal trends in water quality and human risk. Risk models indicate that children in Brazil have as much as two times the risk of gastrointestinal illness than non-children. In Brazil, 11.8% of the weekly water samples from 2004 to 2015 exceeded 158 enterococci CFU/100 ml, the highest level of fecal streptococci concentration measured in the UK study. Risks associated with these elevated levels equated to median NEEAR-Gastrointestinal Illness (NGI) risks of 53 and 96 excess cases per 1000 swimmers in non-children and children, respectively. Two of the five beaches appear to drive the overall elevated NGI risks seen during this study. Distinct enteric pathogen profiles that exist in tropical settings as well as in settings with minimal wastewater treatment highlight the importance of regionally specific guideline development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Edwardsiella tarda infections are frequent causes of severe outbreaks in the fish farming industry besides representing possible zoonotic risks. However, naturally occurring outbreaks that affect various species besides fishes are seldom described., Aim: To report an outbreak of acute mortality caused by E. tarda affecting multiple species that inhabited a natural pond in the state of São Paulo, Brazil., Materials and Methods: Three adult tilapias, three Mallard ducks and one Snow egret were necropsied and subjected to further microbiological tests. Gross and microscopic lesions were documented. The antibiotic susceptibility and phylogenetic similarities among fish and avian strains were also determined. The E. tarda species was confirmed through MALDI-TOF, partial sodB sequencing and phylogenetic analysis., Results: Macroscopical findings between the three species included intestinal dilatation, mucosal hyperaemia and mucous to liquid contents. Common histopathology findings included acute enteritis, increased number of intraepithelial lymphocytes with bacteria adhered to the intestinal epithelium and lymphoid depletion in the spleen. E. tarda was isolated from several organs from all affected species. The phylogeny employing amplified fragment length polymorphism (AFLP) of eleven strains revealed high similarity (>90%) among the isolates regardless of the affected species or sampled organs. Ten isolates of E. tarda showed susceptibility to all tested antibiotics., Conclusions: E. tarda was identified as the cause of death of the species examined. Further studies would be necessary to determine the virulence of these strains and the possible risks regarding public health.

Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750-4 400 000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log 10 7.2-8.0 marker equivalents (ME) 100 mL -1 ) and biologically treated wastewater samples (median log 10 4.6-6.0 ME 100 mL -1 ) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.

Background: The Globicatella genus comprises Gram-positive, facultative anaerobic, α-hemolytic and catalase negative cocci morphologically and phenotypically very similar to Streptococcus and Aerococcus genus which can lead to misidentification and underestimation of this pathogen. Globicatella species have already been isolated from human and animals with heart and brain disorders. Their clinical relevance in animals, and its zoonotic potential, remains unknown due to the difficulty in their identification., Objective: To present the isolation, phenotypic and molecular characterization of G. sulfidifaciens from urinary tract infection in sows., Materials and Methods: Urine samples from 140 sows of two swine herds located in São Paulo State (Brazil) yielded the isolation of three presumptive G. sulfidifaciens strains. Identification and species confirmation were done by MALDI-TOF MS and 16S rRNA sequencing. Strains were further characterized by single enzyme amplified fragments length polymorphism (SE-AFLP) and broth microdilution techniques., Results: All three isolates were confirmed as G. sulfidifaciens. The SE-AFLP genotyping resulted in distinct fingerprint patterns for each strain. All isolates presented high MIC values to tetracycline, sulphonamides, aminoglycosides and tylosin tartrate, which present high usage in human and animal medicine., Conclusions: Globicatella sulfidifaciens could be related to sporadic urinary tract infections in swine and appear to present alarming antimicrobial susceptibility profile. It is necessary to differentiate Streptococcus-like microorganisms in routine laboratory diagnostics for the correct identification of underestimated species potentially pathogenic to animals.

The aim of this study was to perform the identification and molecular characterization of Arcobacter cryaerophilus and Arcobacter butzleri isolated from caiman (Caiman yacare), kept at a production farm, in Brazil. Forty fecal samples were analyzed. After isolation and identification, 21/40 strains of A. butzleri and 19/40 strains of A. cryaerophilus were subjected to PCR for potential virulence gene detection. The results of the PCR showed 38/40 strains positive for the cadF, cj1349, ciaB, and tlyA genes, 39/40 strains positive for the pldA gene, and 40/40 strains positive for the mviN gene. None of the strains presented the irgA gene. Hemagglutinin (hecA gene) and hemolysin (hecB) genes were detected in 21/40 and 16/40 strains, respectively. The SE-AFLP showed a great genetic diversity, but some clonally groups were disseminated in various tanks. These data reveal that the strains presented the same virulence traits described from Arcobacter isolated from food-borne disease in humans.

Traditional microbiological methods enable genus-level identification of Streptococcus spp. isolates. However, as the species of this genus show broad phenotypic variation, species-level identification or even differentiation within the genus is difficult. Herein we report the evaluation of protein spectra cluster analysis for the identification of Streptococcus species associated with disease in swine by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 250 S. suis-like isolates obtained from pigs with clinical signs of encephalitis, arthritis, pneumonia, metritis, and urinary or septicemic infection were studied. The isolates came from pigs in different Brazilian states from 2001 to 2014. The MALDI-TOF MS analysis identified 86% (215 of 250) as S. suis and 14% (35 of 250) as S. alactolyticus, S. dysgalactiae, S. gallinaceus, S. gallolyticus, S. gordonii, S. henryi, S. hyointestinalis, S. hyovaginalis, S. mitis, S. oralis, S. pluranimalium, and S. sanguinis. The MALDI-TOF MS identification was confirmed in 99.2% of the isolates by 16S rDNA sequencing, with MALDI-TOF MS misidentifying 2 S. pluranimalium as S. hyovaginalis. Isolates were also tested by a biochemical automated system that correctly identified all isolates of 8 of the 10 species in the database. Neither the isolates of the 3 species not in the database ( S. gallinaceus, S. henryi, and S. hyovaginalis) nor the isolates of 2 species that were in the database ( S. oralis and S. pluranimalium) could be identified. The topology of the protein spectra cluster analysis appears to sustain the species phylogenetic similarities, further supporting identification by MALDI-TOF MS examination as a rapid and accurate alternative to 16S rDNA sequencing.

The persistent organic pollutants (POPs), such as organochlorine pesticides and PCBs, are ordinarily monitored in the aquatic environment or in soil in the environmental quality monitoring programs in São Paulo, Brazil. One of the core matrices proposed in the POPs Global Monitoring Plan (GMP) from the Stockholm Convention list is the ambient air, which is not a usual matrix for POPs monitoring in the country. In this study POP levels were evaluated in the air samples from an urban site in São Paulo City over five years, starting in 2010 as a capacity building project for Latin America and the Caribbean region for POP monitoring in ambient air using passive samplers. Furthermore, after the end of the Project in 2012, the monitoring continued in the same sampling site as means to improving the analytical capacity building and contribute to the GMP data. The POPs monitored were 17 congeners of 2,3,7,8 chloro-substituted PCDDs and PCDFs, dioxin-like PCBs, indicator PCBs, organochlorine pesticides and toxaphene. The results show a slight decrease in PCDD/F, dl-PCBs and indicator PCBs levels along the five years. The organochlorine pesticide endosulfan was present at its highest concentration at the beginning of the monitoring period, but it was below detection level in the last year of the monitoring. Some other organochlorine pesticides were detected close to or below quantitation limits. The compounds identified were dieldrin, chlordane, α-HCH, γ-HCH, heptachlor, heptachlor epoxide, hexachlorobenzene and DDTs. Toxaphene congeners were not detected. These results have confirmed the efficacy of passive sampling for POP monitoring and the capacity building for POP analysis and monitoring was established. However more needs to be done, including expansion of sampling sites, new POPs and studies on sampling rates to be considered in calculating the concentration of POPs in ambient air using a passive sampler., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The objectives of the study were to detect and genotype Cryptosporidium spp. and Giardia intestinalis in wastewater samples obtained from five cities with high transit of people in the State of São Paulo, Brazil, and at the entrance of a Wastewater Treatment Plant (WWTP) in Lima, Peru. Samples were collected and concentrated by centrifugation. The genomic DNA was extracted for molecular characterization by nested PCR for Cryptosporidium and double nested PCR for Giardia, followed by sequencing and phylogenetic analysis. G. intestinalis was found in 63.6 % of the samples, and the human assemblages A and B were identified. Cryptosporidium sp. was found in 36.4 % of the samples, and the species were corresponding to Cryptosporidium hominis, Cryptosporidium cuniculus, and Cryptosporidium muris. Results revealed the presence of human pathogenic Cryptosporidium species and G. intestinalis human pathogenic assemblages. Molecular tools highlight the importance to map the genetic diversity of these parasites, as well as to detect their epidemiological circulation pathway in the environment.

This study reports the presence of hospital-associated high-risk lineages of OXA-23-producing ST79 Acinetobacter baumannii and SPM-1-producing ST277 Pseudomonas aeruginosa in urban rivers in Brazil. These findings indicate that urban rivers can act as reservoirs of clinically important multidrug-resistant bacteria, which constitute a potential risk to human and animal health., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Understanding the diversity and metal removal ability of microorganisms associated to contaminated aquatic environments is essential to develop metal remediation technologies in engineered environments. This study investigates through 16S rRNA deep sequencing the composition of a biostimulated microbial consortium obtained from the polluted Tietê River in São Paulo, Brazil. The bacterial diversity of the biostimulated consortium obtained from the contaminated water and sediment was compared to the original sample. The results of the comparative sequencing analyses showed that the biostimulated consortium and the natural environment had γ-Proteobacteria, Firmicutes, and uncultured bacteria as the major classes of microorganisms. The consortium optimum zinc removal capacity, evaluated in batch experiments, was achieved at pH=5 with equilibrium contact time of 120min, and a higher Zn-biomass affinity (KF=1.81) than most pure cultures previously investigated. Analysis of the functional groups found in the consortium demonstrated that amine, carboxyl, hydroxyl, and phosphate groups present in the consortium cells were responsible for zinc uptake., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A type 2 vaccine-derived poliovirus (VDPV), differing from the Sabin 2 strain at 8.6% (78/903) of VP1 nucleotide positions, was isolated from seawater collected from a seaport in São Paulo State, Brazil. The P1/capsid region is related to the Sabin 2 strain, but sequences within the 5'-untranslated region and downstream of the P1 region were derived from recombination with other members of Human Enterovirus Species C (HEV-C). The two known attenuating mutations had reverted to wild-type (A481G in the 5'-UTR and Ile143Thr in VP1). The VDPV isolate had lost the temperature sensitive phenotype and had accumulated amino acid substitutions in neutralizing antigenic (NAg) sites 3a and 3b. The date of the initiating OPV dose, estimated from the number of synonymous substitutions in the capsid region, was approximately 8.5 years before seawater sampling, a finding consistent with a long time of virus replication and possible transmission among several individuals. Although no closely related type 2 VDPVs were detected in Brazil or elsewhere, this VDPV was found in an area with a mobile population, where conditions may favor both viral infection and spread. Environmental surveillance serves as an important tool for sensitive and early detection of circulating poliovirus in the final stages of global polio eradication.

The release of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae to the environment is a public health issue worldwide. The aim of this study was to investigate the genetic background of genes encoding ESBLs in wastewater treatment plants (WWTPs) in São Paulo, southeastern Brazil. In 2009, during a local surveillance study, seven ESBL-producing Enterobacteriaceae strains were recovered from five WWTPs and screened for ESBL genes and mobile genetic elements. Multilocus sequence typing (MLST) was carried out, and wild plasmids were transformed into electrocompetent Escherichia coli. S1-PFGE technique was used to verify the presence of high molecular weight plasmids in wild-type strains and in bla ESBL-containing E. coli transformants. Strains harbored bla CTX-M-8, bla CTX-M-15, and/or bla SHV-28. Sequencing results showed that bla CTX-M-8 and bla CTX-M-15 genes were associated with IS26. MLST revealed new sequence types for E. coli (ST4401, ST4402, ST4403, and ST4445) and Klebsiella pneumoniae (ST1574), except for one K. pneumoniae from ST307 and Enterobacter cloacae from ST131. PCR and S1-PFGE results showed CTX-M-producing E. coli transformants carried heavy plasmids sizing 48.5-209 kb, which belonged to IncI1, IncF, and IncM1 incompatibility groups. This is the first report of CTX-M-8 and SHV-28 enzymes in environmental samples, and the present results demonstrate the plasmid-mediated spread of CTX-M-encoding genes through five WWTPs in São Paulo, Brazil, suggesting WWTPs are hotspots for the transfer of ESBL genes and confirming the urgent need to improve the management of sewage in order to minimize the dissemination of resistance genes to the environment.

Aerococcus viridans has been reported as a human and animal pathogen causing urinary tract infection, arthritis, pneumonia, meningitis and endocarditis. Routinely, A. viridans is not surveyed in clinical diagnosis laboratories and commonly is misidentified as other bacteria. There is no concrete data on the prevalence and impact of the pathogen to both human and animal health. In the present study, we report the isolation and molecular and antibiotic susceptibility characterization of A. viridans strains from porcine urinary infections. A total of 22 isolates were identified as A. viridans by MALDI-TOF MS and confirmed by 16S rRNA gene sequencing. Isolates were genotyped by single enzyme amplified fragments length polymorphism (SE-AFLP) that resulted in 19 clusters of which 81.2% were composed by single isolates. The high genetic heterogeneity corroborates previous studies and appears to be a particularity of A. viridans. The minimal inhibitory concentration (MIC) values also presented variability especially for ceftiofur, fluoroquinolones and aminoglycosides. The high MICs of aminoglycosides, tetracyclines and macrolides seen among the A. viridans corroborate previous reports and the widespread veterinary usage of these antibiotics demand attention for the implication of A. viridans infection to both human and animal health., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Regulating recreational water exposure to pathogens within the tropics is a major public health and economic concern. Although numerous epidemiological studies estimating the risk to recreational marine water exposure have been conducted since the 1950s, few studies have been done in the tropics. Furthermore, many have suggested that the use of fecal indicator bacteria for monitoring recreational water quality in temperate regions is not appropriate in the tropics. We analyzed a large cohort study of five beaches in Sao Paulo, Brazil, conducted during consecutive weekends in the summer of 1999 that estimated risk to water, sand, and food exposures. Enterococci and Escherichia coli concentrations were measured each day of the study. Elevated risks were estimated for both swimming (OR = 1.36 95% CI: 1.05-1.58) and sand contact (OR = 1.29 95% CI 1.05-1.58). A 1 log increase in enterococci concentration was associated with an 11% increase in risk (OR = 1.11 95% CI: 1.04-1.19). For E. coli a 1-log increase in concentration was associated with 19% increase in risk (OR = 1.19 95% CI: 1.14-1.28). Most countries with beaches in the tropics are lower or middle income countries (LMIC) and rely on tourism as a major source of income. We present data that suggests fecal indicator bacteria such as enterococci are an appropriate indicator of risk in tropical urban settings where contamination is coming from predominantly human sources. Additional studies in tropical settings could help inform and refine guidelines for safe use of recreational waters., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Vibrio cholerae O1 is the causative agent of cholera and is ubiquitous in the aquatic environment, while V. cholerae strains non-O1 and non-O139 are recognized as causative agents of sporadic and localized outbreaks of diarrhea. Here, we report the complete sequence of a non-O1 and non-O139 V. cholerae strain (VCC19), which was isolated from the environment in Brazil. The sequence includes the integrative conjugative element (ICE). This paper is the first report of the presence of such an element in a V. cholerae strain isolated in Brazil., (Copyright © 2014 de Sá et al.)

Different types of water bodies, including lakes, streams, and coastal marine waters, are often susceptible to fecal contamination from a range of point and nonpoint sources, and have been evaluated using fecal indicator microorganisms. The most commonly used fecal indicator is Escherichia coli, but traditional cultivation methods do not allow discrimination of the source of pollution. The use of triplex PCR offers an approach that is fast and inexpensive, and here enabled the identification of phylogroups. The phylogenetic distribution of E. coli subgroups isolated from water samples revealed higher frequencies of subgroups A1 and B23 in rivers impacted by human pollution sources, while subgroups D1 and D2 were associated with pristine sites, and subgroup B1 with domesticated animal sources, suggesting their use as a first screening for pollution source identification. A simple classification is also proposed based on phylogenetic subgroup distribution using the w-clique metric, enabling differentiation of polluted and unpolluted sites.

Pseudomonas aeruginosa is an important cause of infection, especially in immunocompromised patients. In this regard, strains producing carbapenemases, mainly metallo-β-lactamases (MBLs), have become a significant public health concern. Here, we present the complete annotated genome sequence (65.7 kb) of an F8-related lytic myovirus (Pbunalikevirus genus) that infects MBL-producing P. aeruginosa strains.

A survey of Giardia and Cryptosporidium was conducted in surface water used as drinking water sources by public water systems in four densely urbanized regions of Sao Paulo State, Brazil. A Quantitative Microbial Risk Assessment, based on protozoa concentrations, was performed to estimate the probability of protozoa infection associated with drinking water ingestion. A total of 206 source water samples were analyzed over a 24 month period using the USEPA Method 1623. The risk of infection was estimated using an exponential dose response model, children and adults exposure and a gamma distribution for (oo)cyst concentrations with three scenarios for treating censored data. Giardia was detected in 102 of the samples, and 19 of them were also positive for Cryptosporidium, with maximum concentrations of 97.0 cysts/L and 6.0 oocysts/L, respectively. Risk distributions were similar for the three scenarios. In the four regions, the estimated risk of Giardia infection per year, for adults and children, ranged from 0.29% to 2.47% and from 0.08% to 0.70%, respectively. Cryptosporidium risk infection varied from 0.15% to 0.29% for adults and from 0.04% to 0.08% for children. In both cases, the calculated risk surpassed the risk of infection of 10(-4) (1:10,000) defined as tolerable by USEPA for a yearly exposure. The probability of Giardia infection was very close to the rates of acute diarrheic disease for adults (1% to 3%) but lower for children (2% to 7%). The daily consumption of drinking water was an important contributing factor for these differences. The Microbiological Risk Assessment carried out in this study provides an indication of infection risks by Giardia and Cryptosporidium in the population served by these source waters. Strategies for source water protection and performance targets for the water treatment should be established to achieve the required level of public health risk., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Brazilian regulations for nonpotable reuse are being established using World Health Organization guidelines, however, they should be developed based on local monitoring studies. This study intended to analyze enteroviruses, protozoa and viable Ascaris sp. eggs in raw (24) and treated (24) effluents from four Wastewater Treatment Plants of São Paulo State, Brazil. The protozoa were detected with the US Environmental Protection Agency (USEPA) Method 1623 in the treated effluents and by centrifugation/Immunomagnetic Separation in the raw influent samples. Viable Ascaris sp. eggs were analyzed according to a modified USEPA method. Enteroviruses were quantified by using human rhabdomyosarcoma cells after adequate concentration procedures. All wastewater influents were positive for Giardia sp. whereas Cryptosporidium sp. was detected in 58.3% of the samples. Giardia sp. and Cryptosporidium sp. were present in 79.2 and 25.0% respectively, of the treated wastewater samples. Viable Ascaris sp. eggs were detected in 50.0 and 12.5% of influent and treated wastewater samples. Enteroviruses were isolated in the 24 raw influent samples and in 46% of the treated samples. Taking into account the densities of Giardia sp. in some treated wastewaters intended to be used as reclaimed water, Quantitative Microbial Risk Assessment studies should be conducted to establish pathogen quantitative criteria for a future Brazilian regulation for water reuse.

Fecal bacterial indicator analyses have been widely used for monitoring the water quality. This study was designed to determine the ratio between the density of Escherichia coli and other Thermotolerant Coliforms (TtC) bacteria from freshwater samples collected for a two-year period of monitoring. TtC were enumerated by membrane filtration on mFC agar. E. coli enumeration was done by two methods: TtC colonies identified in mFC were inoculated in EC-MUG or water samples were filtered and inoculated in modified mTEC agar media, and both methods were compared for quantitative recovery of E. coli. The results pointed out a mean percentage of E. coli among other thermotolerant coliforms (E. coli/TtC ratio) of 84.3% in mFC media. Taking these results into account, a mandatory standard of 1000 thermotolerant coliforms would correspond to 800 E. coli and the adoption of these E. coli based standards will represent a major improvement for the monitoring of freshwater quality.

This study aimed to assess the sanitary quality of water, and wet and dry sand from three beaches located in the South Coast region of São Paulo State, Brazil, selected taking into account the frequency of tourists and the water quality (good, fair and poor). Thirty-six water samples each of wet and dry sand and seawater were collected monthly over a period of one year and analyzed for fecal indicator bacteria (FIB: thermotolerant coliforms, Escherichia coli, and enterococci), presumptive Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans and dermatophytes. The results revealed FIB concentrations more elevated in dry sand followed by wet sand and water. P. aeruginosa and presumptive S. aureus were detected with a similar frequency in water and sand samples, but maximum concentrations and geometric means were higher in dry sand. C. albicans was detected only in water samples whereas the dermatophyte Microsporum sp. was isolated exclusively from dry and wet sand samples. This evaluation showed also that the environment had a significant influence on P. aeruginosa but not on presumptive S. aureus concentrations. According to threshold values proposed in the literature for E. coli and enterococci dry sand densities, none of the beaches would be considered of sufficient quality for recreational activities.

Repetitive element sequence-based polymerase chain reaction (rep-PCR) is one of the commonest methods used to identify sources of fecal contamination of water systems. In this work, BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) was used to discriminate Escherichia coli strains originating from different animals and water sources, and the suitability of the technique for bacterial source tracking (BST) was evaluated. A total of 214 strains from humans, 150 strains from animals, 55 strains from sewage and 77 strains from water bodies were analyzed by the BOX-PCR technique. When maximum similarity between the fingerprints was used, a correct classification rate of 84% was achieved for strains from human and animal sources. Furthermore, 95% of the strains found in sewage were classified as being from human sources by at least one of the four classification tools used. Classification of the strains found in water bodies in the State of São Paulo was based on the fingerprints obtained for human and animal sources. Most of the sampling sites appeared to be affected by mixed sources of fecal contamination. The use of BOX-PCR for BST could be especially valuable in developing countries, where simplicity and cost are important considerations., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The protozoan parasite Cryptosporidium has emerged as one of the most important water contaminants, causing waterborne outbreaks of diarrheal diseases worldwide. The small size of oocysts under the microscope and the possibility of changes in characteristics of oocysts, mainly in environmental samples, make the taxonomy of the genus difficult if morphologic characteristics are considered. This limitation encouraged the application of molecular methods to identify this microorganism. The aim of this study was to detect and identify by nested-polymerase chain reaction oocysts of Cryptosporidium present in water samples in the state of São Paulo, Brazil. Water samples were concentrated through a membrane filter, DNA was extracted by using a standard technique, and both amplification reactions used forward and reverse oligonucleotides that were complementary to Cryptosporidium 18S ribosomal RNA gene sequences. Thirty water samples from different sites of collection in the state of São Paulo were evaluated. Cryptosporidium oocysts were detected in 30% of the samples. By genoptyping, C. hominis and Cryptosporidium sp. were identified in recreational water and C. meleagridis was identified in surface water samples. This is the first report of C. hominis in environmental samples in Brazil. Although identification of Cryptosporidium is still a difficult task, molecular methods are essential for specific identification and are a helpful tool to aid to understand the epidemiology of this parasite in Brazil.

Giardia duodenalis is a protozoan that parasitizes humans and other mammals and causes giardiasis. Although its isolates have been divided into seven assemblages, named A to G, only A and B have been detected in human faeces. Assemblage A isolates are commonly divided into two genotypes, Al and All. Even though information about the presence of this protozoan in water and sewage is available in Brazil, it is important to verify the distribution of different assemblages that might be present, which can only be done by genotyping techniques. A total of 24 raw and treated sewage, surface and spring water samples were collected, concentrated and purified. DNA was extracted, and a nested PCR was used to amplify an 890 bp fragment of the gdh gene of G. duodenalis, which codes for glutamate dehydrogenase. Positive samples were cloned and sequenced. Ten out of 24 (41.6%) samples were confirmed to be positive for G. duodenalis by sequencing. Phylogenetic analysis grouped most sequences with G. duodenalis genotype All from GenBank. Only two raw sewage samples presented sequences assigned to assemblage B. In one of these samples genotype All was also detected. As these assemblages/genotypes are commonly associated to human giardiasis, the contact with these matrices represents risk for public health.

The aim of this work was to verify the presence of seven virulence factors (ST, LT, eae, stx(1), stx(2), INV and EAEC) among Escherichia coli strains isolated from healthy humans, bovines, chickens, sheep, pigs and goats, from two sewage treatment plants and from the Tietê River. We have found a high prevalence of eae, stx(1) and stx(2) in ruminants. The EAEC gene was only found in humans and sewage. No strains presented ST, LT or INV. BOX-PCR fingerprints revealed a high diversity among the strains analysed and a non-clonal origin of strains that presented the same virulence factors. Therefore, we concluded that ruminants may constitute an important reservoir of most diarrheagenic E. coli in Brazil, except for EAEC strains. These results emphasize the importance of the identification of the animal source of fecal contamination for the correct water risk assessment.

Background: Escherichia coli strains are commonly found in the gut microflora of warm-blooded animals. These strains can be assigned to one of the four main phylogenetic groups, A, B1, B2 and D, which can be divided into seven subgroups (A0, A1, B1, B22, B23, D1 and D2), according to the combination of the three genetic markers chuA, yjaA and DNA fragment TspE4.C2. Distinct studies have demonstrated that these phylo-groups differ in the presence of virulence factors, ecological niches and life-history. Therefore, the aim of this work was to analyze the distribution of these E. coli phylo-groups in 94 human strains, 13 chicken strains, 50 cow strains, 16 goat strains, 39 pig strains and 29 sheep strains and to verify the potential of this analysis to investigate the source of fecal contamination., Results: The results indicated that the distribution of phylogenetic groups, subgroups and genetic markers is non-random in the hosts analyzed. Strains from group B1 were present in all hosts analyzed but were more prevalent in cow, goat and sheep samples. Subgroup B23 was only found in human samples. The diversity and the similarity indexes have indicated a similarity between the E. coli population structure of human and pig samples and among cow, goat and sheep samples. Correspondence analysis using contingence tables of subgroups, groups and genetic markers frequencies allowed the visualization of the differences among animal samples and the identification of the animal source of an external validation set. The classifier tools Binary logistic regression and Partial least square--discriminant analysis, using the genetic markers profile of the strains, differentiated the herbivorous from the omnivorous strains, with an average error rate of 17%., Conclusions: This is the first work, as far as we are aware, that identifies the major source of fecal contamination of a pool of strains instead of a unique strain. We concluded that the analysis of the E. coli population structure can be useful as a supplementary bacterial source tracking tool.

Surface water contaminated by domestic sewage discharges is a potential source of pathogens, including protozoa. During 2005-2006, the source water (Atibaia River) of the Surface Water Treatment Plant (WTP) of Campinas city, São Paulo, Brazil was sampled to obtain an assessment of Cryptosporidium oocyst and Giardia cyst concentrations. Calcium carbonate flocculation (CCF) and membrane filtration (MF) concentration techniques, with and without purification by immunomagnetic separation (IMS) were evaluated. The cysts and oocysts were detected by immunofluorescence assay (IFA) and confirmed by differential interference contrast (DIC). Membrane filtration method generally produced higher recovery efficiency. Giardia spp. was detected in 87.5% of the water samples analyzed with densities ranging from 2.5 to 120 cysts per L. Cryptosporidium spp were detected in 62.5% and the concentrations ranged from 15 to 60 oocysts per L. Cryptosporidium oocyst and Giardia cyst concentrations detected in this study were elevated and are associated with discharge of untreated sewage in Atibaia River. Measures should be taken to protect surface water from sources of contamination.

Aeromonads are inhabitants of aquatic ecosystems and are described as being involved in intestinal disturbances and other infections. A total of 200 drinking water samples from domestic and public reservoirs and drinking fountains located in São Paulo (Brazil), were analyzed for the presence of Aeromonas. Samples were concentrated by membrane filtration and enriched in APW. ADA medium was used for Aeromonas isolation and colonies were confirmed by biochemical characterization. Strains isolated were tested for hemolysin and toxin production. Aeromonas was detected in 12 samples (6.0%). Aeromonas strains (96) were isolated and identified as: A. caviae (41.7%), A. hydrophila (15.7%), A.allosacharophila (10.4%), A. schubertii (1.0%) and Aeromonas spp. (31.2%). The results revealed that 70% of A. caviae, 66.7% of A. hydrophila, 80% of A. allosacharophila and 46.6% of Aeromonas spp. were hemolytic. The assay for checking production of toxins showed that 17.5% of A. caviae, 73.3% of A. hydrophila, 60% of A. allosacharophila, 100% of A. schubertii, and 33.3% of Aeromonas spp. were able to produce toxins. The results demonstrated the pathogenic potential of Aeromonas, indicating that the presence of this emerging pathogen in water systems is a public health concern.

The presence of Escherichia coli in drinking water is an indication of fecal contamination and can represent a risk of waterborne diseases. Forty-nine E. coli strains isolated from different sources of drinking water (distribution system, well, spring and mineral water) were placed into the phylogenetic groups A (15 strains), B1 (19 strains), B2 (2 strains) and D (13 strains). Approximately 30% of the strains analyzed belonged to groups B2 and D, which usually include potentially extraintestinal pathogenic strains. Moreover, the assignment of the strains to different phylogenetic groups indicates that different contamination events occurred in these waters. These results were compared with the distribution of E. coli strains isolated from two rivers and two dams into the phylogenetic groups. A significant difference was observed when the distribution of drinking water strains into the phylogenetic groups was compared to the results obtained from the Guarapiranga Dam and the Jaguari and Sorocaba Rivers. The results obtained in this work suggest that PCR-based methods can be used for a rapid assessment of potentially pathogenic E. coli strains in water samples.

Contamination of recreational waters and public water supplies by Escherichia coli represents a risk for public health, since some strains can be pathogenic or propagated with other pathogenic microorganisms. In this study, two reservoirs, Billings and Guarapiranga (São Paulo metropolitan area, Brazil), were investigated in order to assess E. coli diversity. Genetic typing using rep-PCR completely differentiated all strains and enabled the determination of their genetic variability. Although the same level of genetic variability was observed for strains originating from both reservoirs, randomization procedures showed that isolates from the same reservoir were more closely related to each other. Phylogenetic group frequencies in each reservoir suggested that contamination in the Billings reservoir was mostly from humans, whereas contamination in the Guarapiranga reservoir was mostly from animals. Colony blot experiments using probes from several virulence factor genes showed that both reservoirs contained potential pathogenic strains and may represent a risk to recreational or household usage of these water resources.

Restriction fragment length polymorphism (RFLP) and sequence analyses of the PCR-amplified 16S-23S rDNA intergenic spacer (ITS) were used for differentiating Acidithiobacillus thiooxidans strains from other related acidithiobacilli, including A. ferrooxidans and A. caldus. RFLP fingerprints obtained with AluI, DdeI, HaeIII, HinfI and MspI enabled the differentiation of all Acidithiobacillus reference strains into species groups. The A. thiooxidans strains investigated (metal mine isolates) yielded identical RFLP patterns to the A. thiooxidans type strain (ATCC 19377(T)), except for strain DAMS, which had a distinct pattern for all enzymes tested. Fourteen A. ferrooxidans mine strains were assigned to 3 RFLP groups, the majority of which were grouped with A. ferrooxidans ATCC 23270(T). The spacer region of one representative strain from each of the RFLP groups obtained was subjected to sequence analysis, in addition to eleven additional A. thiooxidans strains isolated from sediment and water samples, and A. caldus DSM 8584(T). The tRNA(IIe) and tRNA(Ala) genes, present in all strains analyzed, showed high sequence similarity. Phylogenetic analysis of the ITS sequences differentiated all three Acidithiobacillus species. Inter- and infraspecific genetic variations detected were mainly due to the size and sequence polymorphism of the ITS3 region. Mantel tests showed no significant correlation between ITS sequence similarity and the geographical origin of strains. The results showed that the 16S-23S rDNA spacer region is a useful target for the development of molecular-based methods aimed at the detection, rapid differentiation and identification of acidithiobacilli.

In the routine São Paulo state (Brazil) surface water quality-monitoring program, which includes the Salmonella microsome mutagenicity assay as one of its parameters, a river where water is taken and treated for drinking water purposes has repeatedly shown mutagenic activity. A textile dyeing facility employing azo-type dyes was the only identifiable source of mutagenic compounds. We extracted the river and drinking water samples with XAD4 at neutral and acidic pH and with blue rayon, which selectively adsorbs polycyclic compounds. We tested the industrial effluent, raw, and treated water and sediment samples with YG1041 and YG1042 and compared the results with the TA98 and TA100 strains. The elevated mutagenicity detected with YG-strains suggested that nitroaromatics and/or aromatic amines were causing the mutagenicity detected in the samples analyzed. Positive responses for the blue rayon extracts indicated that mutagenic polycyclic compounds were present in the water samples analyzed. The mutagen or mixture of mutagens present in the effluent and water samples cause mainly frameshift mutations and are positive with and without metabolic activation. The Salmonella assay combined with different extraction procedures proved to be very useful in the identification of the origin of the pollution and in the identification of the classes of chemical compounds causing the mutagenic activity in the river analyzed.

Giardia lamblia and Cryptosporidium parvum are two protozoan intestinal parasites responsible for many drinking-water-related disease outbreaks in recent years. They are very resistant to conventional water treatment processes, can persist for long times in the environment and are, therefore, of great concern for public health. This work aimed to evaluate the presence of Giardia and Cryptosporidium in water sources from São Paulo State, Brazil, as part of the "Evaluation of Inland Waters from São Paulo State" project from CETESB. Over a period of 19 months, 278 water samples from 28 sites located in 10 watersheds were analysed. The immunofluorescence assay was used after concentration of the samples by the calcium carbonate flocculation technique. Thermotolerant (faecal) coliforms, faecal streptococci and Clostridium perfringens were also determined in order to verify the existence of correlation between these bacterial indicators and the protozoa. Giardia and Cryptosporidium were detected in 27% and 2.5% of the samples, respectively, a lower figure compared with the results reported by other authors, especially for Cryptosporidium. A Spearman rank correlation test demonstrated a significant correlation between Giardia and faecal indicator concentrations. According to the American Regulation of Monitoring (ICR), treated water from 16 of these 28 collection sites should also be analysed to evaluate whether the treatment process could remove the parasites. Some technical deficiencies of these methods still limit the utilisation of the monitoring results for public health decisions, but the data here reported will help to improve the quality of drinking water in São Paulo State.

Since 1979, the Environmental Agency of São Paulo State in Brazil, CETESB, has been using the Salmonella mutagenicity assay to assess the quality of natural waters. This paper is a compilation of data obtained during the last 20 years from more than a thousand samples. Potencies up to 30,000 revertants/l were observed in 137 positive samples. The Salmonella typhimurium strain TA98 was more sensitive than TA100; 79% of the mutagenicity was detected by this strain, regardless of the presence of S9-mix. A classification of the mutagenic response was proposed to facilitate in the dissemination of the information to the public. The classification was low, moderate, high and extreme for samples with mutagenic potency (revertants/l equivalent) of < 500, 500-2500, 2500-5000 and > 5000, respectively. As a result of this effort to standardize methodologies, compile and classify the mutagenic effect of water pollution, in 1998, the Salmonella mutagenicity assay was officially and systematically included in the São Paulo State Water Quality Monitoring Program. This assay has proven to be a useful tool in the identification of important pollution sources. Correction and prevention actions in Water Pollution Control Programs were generated as a result.