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We describe a cross-sectional survey to identify risk factors for colonisation of neonates by extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae. This occurred following exposure to a colonised healthcare worker during an outbreak in an intermediate-risk neonatal unit. In total, 120 neonates admitted consecutively during a three-month period were screened for ESBL-producing K. pneumoniae by rectal swabbing and 27 were identified as colonised. Multivariate analysis showed colonisation to be independently associated with use of antibiotics and absence of breastfeeding. Previous use of antibiotics presented an odds ratio (OR) of 12.3 [95% confidence interval (CI): 3.66-41.2, P<0.001]. The most commonly used antibiotics were penicillin and amikacin. Breastfeeding was associated with reduced risk for colonisation (OR: 0.22; 95% CI: 0.05-0.99; P=0.049). Nine isolates recovered during the first stage of the outbreak and 27 isolates from surveillance cultures were typed thereafter by pulsed-field gel electrophoresis, revealing six different profiles (A-F). Clones A, C, and E were implicated in the first stage of the outbreak, whereas among the 27 strains recovered from surveillance cultures, all six clones were identified. Clone A was also found on the hand of a nursing auxiliary with onychomycosis. We concluded that prior antimicrobial use predisposed to colonisation. The possible role of breastfeeding as a protective factor needs to be further elucidated. Detection of different genotypes of ESBL-producing K. pneumoniae suggests that dissemination of mobile genetic elements bearing the ESBL gene may have been superimposed on the simple dissemination of a clone during the outbreak. [ABSTRACT FROM AUTHOR]

Clin Microbiol Infect 2010; 16: 630–632 A clinical Klebsiella pneumoniae isolate carrying the extended-spectrum β-lactamase gene variants blaSHV-40, blaTEM-116 and blaGES-7 was recovered. Cefoxitin and ceftazidime activity was most affected by the presence of these genes and an additional resistance to trimethoprim-sulphamethoxazole was observed. The blaGES-7 gene was found to be inserted into a class 1 integron. These results show the emergence of novel blaTEM and blaSHV genes in Brazil. Moreover, the presence of class 1 integrons suggests a great potential for dissemination of blaGES genes into diverse nosocomial pathogens. Indeed, the blaGES-7 gene was originally discovered in Enterobacter cloacae in Greece and, to our knowledge, has not been reported elsewhere. [ABSTRACT FROM AUTHOR]

The overuse of antimicrobials in livestock has contributed to the emergence and selection of clinically relevant multidrug-resistant bacteria. In Brazil, there is no conclusive information on the occurrence of Escherichia coli producing extended-spectrum β-lactamase (ESβL) in cattle breeding, which is an important sector of agribusiness in this country. Herein, we investigated the presence of ESβL-positive E. coli strains in dairy cattle from a commercial farm with routine practice of therapeutic cephalosporins. Ninety-five rectal swab samples were collected from healthy dairy calves and cows under treatment with ceftiofur. Samples were screened for the presence of ESβL producers, and positive isolates were identified by MALDI-TOF, with subsequent screening for genes encoding ESβL variants by PCR and sequencing. The presence of ESβL (CTX-M-15)-producing E. coli was confirmed in calves, and lactating and dry cows. Most ESβL strains with genetic homologies ≥ 90% were grouped into two major PFGE clusters, confirming the suscessful expansion of clonally related lineages in animals from different lactating cycles, on the same property. Four representatives CTX-M-15-positive E. coli strains had their genomes sequenced, belonging to the clonal complex (CC) 23 and sequence type (ST) 90. A phylogeographical landscape of ST90 was performed revealing a global One Health linkage. Our results highlight the intestinal microbiota of dairy cattle as a hotspot for the spread of critical priority ESβL-producing E. coli and demonstrate that ST90 is an international clone genomically adapted to human and animal hosts, which deserve additional investigation to determine its zoonotic potential and impact in food chain., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The spread of antimicrobial resistance (AMR) through multiple reservoirs is a global concern. Wastewater is a critical AMR dissemination source, so this study aimed to assess the persistence of resistance genetic markers in wastewater using a culture-independent approach. Raw and treated wastewater samples (n = 121) from a wastewater treatment plant (WWTP), a human hospital, a veterinary hospital, and a pig farm were monthly collected and concentrated by filtration. DNA was extracted directly from filter membranes, and PCR was used in the qualitative search of 32 antimicrobial resistance genes (ARGs). Selected genes (bla CTX-M , bla KPC , qnrB, and mcr-1) were enumerated by quantitative real-time PCR (qPCR). Twenty-six ARGs were detected in the qualitative ARGs search, while quantitative data showed a low variation of the ARG's relative abundance (RA) throughout the months, especially at the human hospital and the WWTP. At the WWTP, despite significantly reducing the absolute number of gene copies/L after each treatment stage (p < 0.05), slight increases (p > 0.05) in the RAs of genes bla CTX-M , qnrB, and mcr-1 were observed in reused water (tertiary treatment) when compared with secondary effluent. Although the increase is not statistically significant, it is worth noting that there was some level of ARGs concentration after the disinfection process. No significant absolute or relative after-treatment quantification reductions were observed for any ARGs at the veterinary hospital or the pig farm. The spread of ARGs through sewage needs to be continuously addressed, because their release into natural environments may pose potential risks of exposure to resistant bacteria and impact local ecosystems., (© 2024 John Wiley & Sons Ltd.)

The unbridled dissemination of multidrug-resistant pathogens is a major threat to global health and urgently demands novel therapeutic alternatives. Antimicrobial photodynamic therapy (aPDT) has been developed as a promising approach to treat localized infections regardless of drug resistance profile or taxonomy. Even though this technique has been known for more than a century, discussions and speculations regarding the biochemical mechanisms of microbial inactivation have never reached a consensus on what is the primary cause of cell death. Since photochemically generated oxidants promote ubiquitous reactions with various biomolecules, researchers simply assumed that all cellular structures are equally damaged. In this study, biochemical, molecular, biological and advanced microscopy techniques were employed to investigate whether protein, membrane or DNA damage correlates better with dose-dependent microbial inactivation kinetics. We showed that although mild membrane permeabilization and late DNA damage occur, no correlation with inactivation kinetics was found. On the other hand, protein degradation was analyzed by three different methods and showed a dose-dependent trend that matches microbial inactivation kinetics. Our results provide a deeper mechanistic understanding of aPDT that can guide the scientific community toward the development of optimized photosensitizing drugs and also rationally propose synergistic combinations with antimicrobial chemotherapy., (© 2022 American Society for Photobiology.)

Antimicrobial blue light (aBL) offers efficacy and safety in treating infections. However, the bacterial targets for aBL are still poorly understood and may be dependent on bacterial species. Here, we investigated the biological targets of bacterial killing by aBL (λ = 410 nm) on three pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Initially, we evaluated the killing kinetics of bacteria exposed to aBL and used this information to calculate the lethal doses (LD) responsible for killing 90 and 99.9% of bacteria. We also quantified endogenous porphyrins and assessed their spatial distribution. We then quantified and suppressed reactive oxygen species (ROS) production in bacteria to investigate their role in bacterial killing by aBL. We also assessed aBL-induced DNA damage, protein carbonylation, lipid peroxidation, and membrane permeability in bacteria. Our data showed that P. aeruginosa was more susceptible to aBL (LD 99.9 = 54.7 J/cm 2 ) relative to S. aureus (LD 99.9 = 158.9 J/cm 2 ) and E. coli (LD 99.9 = 195 J/cm 2 ). P. aeruginosa exhibited the highest concentration of endogenous porphyrins and level of ROS production relative to the other species. However, unlike other species, DNA degradation was not observed in P. aeruginosa. Sublethal doses of blue light (90 ) could damage the cell membrane in Gram-negative species but not in S. aureus. In all bacteria, oxidative damage to bacterial DNA (except P. aeruginosa), proteins, and lipids occurred after high aBL exposures (>LD 99.9 ). We conclude that the primary targets of aBL depend on the species, which are probably driven by variable antioxidant and DNA-repair mechanisms. IMPORTANCE Antimicrobial-drug development is facing increased scrutiny following the worldwide antibiotic crisis. Scientists across the world have recognized the urgent need for new antimicrobial therapies. In this sense, antimicrobial blue light (aBL) is a promising option due to its antimicrobial properties. Although aBL can damage different cell structures, the targets responsible for bacterial inactivation have still not been completely established and require further exploration. In our study, we conducted a thorough investigation to identify the possible aBL targets and gain insights into the bactericidal effects of aBL on three relevant pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. This research not only adds new content to blue light studies but opens new perspectives to antimicrobial applications.

Introduction: Non-typhoidal Salmonella (NTS), are frequently found in sewage and are one of the main causes of diarrhea in developed and developing countries due to poor sanitation conditions. In addition, NTS can potentially act as reservoirs and vehicles for the transmission of antimicrobial resistance (AMR), which can be facilitated by the discharge of sewage effluents into environmental matrices. This study aimed to analyze a NTS Brazilian collection, focusing on their antimicrobial susceptibility profile and the presence of clinically relevant AMR-encoding genes., Methodology: Forty-five non-clonal NTS strains from serotypes Salmonella enteritidis (n = 6), Salmonella enterica serovar 1,4,[5],12:i:- (S. 1,4,[5],12:i:-) (n = 25), Salmonella cerro (n = 7), Salmonella typhimurium (n = 3) and Salmonella braenderup (n = 4) were studied. Antimicrobial susceptibility testing was done using the Clinical and Laboratory Standards Institute guidelines (2017) and genes encoding resistance to beta-lactams, fluoroquinolones and aminoglycosides were identified by polymerase chain reaction and sequencing., Results: Resistance to β-lactams, fluoroquinolones, tetracyclines and aminoglycosides was frequent. The highest rates were observed for nalidixic acid (89.0%), followed by tetracycline (67.0%), ampicillin (67.0%), amoxicillin + clavulanic acid (64.0%); ciprofloxacin (47.0%) and streptomycin (42.0%). The AMR-encoding genes detected were qnrB, oqxAB, blaCTX-M and rmtA., Conclusions: Raw sewage has been considered a valuable tool to evaluate epidemiological population patterns and this study supports the view that NTS with pathogenic potential and resistance to antimicrobials are circulating in the studied region. This is worrisome due to the dissemination of these microorganisms throughout the environment., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Solange Martone-Rocha, Milena Dropa, Beatriz Moreira Calixto da Cruz, Débora Belisário Mendes Oliveira Leite, Talita Pereira dos Santos, Maria Tereza Pepe Razzolini.)

Psittacine birds are commonly kept as companion birds and the maintenance of these birds in captivity may represent a zoonotic risk and contribute to the propagation of multidrug-resistant and β-lactamase extended-spectrum (ESBLs)-producing pathogens. This study aimed to identify and characterize strains of the Klebsiella pneumoniae complex isolated from diseased psittacine birds, determining virulence and resistance profiles. K. pneumoniae strains were isolated from 16 birds (16/46). All strains carried more than three virulence genes, with a high frequency of fimH and kpn (93.75%), uge (87.52%), and irp-2 (81.25%) genes. The antimicrobial susceptibility revealed that 3/16 strains were ESBL producers. Genomic analysis revealed that CTX-M-15-positive strains belonged to sequence types (STs) ST15, ST147, and ST307, characterized as international clones associated with outbreaks of healthcare-associated infections (HAIs) worldwide.

The quality of aquatic ecosystems is a major public health concern. The assessment and management of a freshwater system and the ecological monitoring of microorganisms that are present in it can provide indicators of the environment and water quality to protect human and animal health. with bacteria is. It is a major challenge to monitor the microbiological bacterial contamination status of surface water associated with anthropogenic activities within rivers and freshwater reservoirs. Understanding the composition of aquatic microbial communities can be beneficial for the early detection of pathogens, improving our knowledge of their ecological niches, and characterizing the assemblages of microbiota responsible for the degradation of contaminants and microbial substrates. The present study aimed to characterize the bacterial microbiota of water samples collected alongside the Madeira River and its small tributaries in rural areas near the Santo Antonio Energia hydroelectric power plant (SAE) reservoir in the municipality of Porto Velho, Rondonia state, Western Brazil. An Illumina 16s rRNA metagenomic approach was employed and the physicochemical characteristics of the water sample were assessed. We hypothesized that both water metagenomics and physicochemical parameters would vary across sampling sites. The most abundant genera found in the study were Acinetobacter , Deinococcus , and Pseudomonas . PERMANOVA and ANCOM analysis revealed that collection points sampled at the G4 location presented a significantly different microbiome compared to any other group, with the Chlamidomonadaceae family and Enhydrobacter genus being significantly more abundant. Our findings support the use of metagenomics to assess water quality standards for the protection of human and animal health in this microgeographic region.

The absence of a standardized method for detecting oocysts in water samples makes it difficult to characterize them, including in water for reuse. This study aimed to detect Toxoplasma gondii oocysts using two extraction methods. Using method 1693/2014 USEPA, 30 L of water for reuse from two wastewater treatment plants (WWTPs) in the city of São Paulo, Brazil, was concentrated, totaling 20 samples. The supernatant generated from the immunomagnetic separation (IMS) step was collected for detection of T. gondii oocysts. For DNA extraction, two techniques were used: the commercial kit DNeasy PowerSoil Kit® optimized with the enzyme Zymolyase® and with freeze-thaw steps. DNA quantification was performed with the target sequence of gene B1. From 16 samples submitted to enzymatic extraction, four were positive. In freeze-thaw extraction, no DNA was detected. DNA extraction was the essential step for oocyst detection given the resistant nature of their wall., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The current COVID-19 pandemic has emphasized the vulnerability of communities living in the urban outskirts and informal settlements. The lack of reliable COVID-19 case data highlights the importance and application of wastewater-based epidemiology. This study aimed to monitor the COVID-19 trends in four vulnerable urban communities (slums and low-income neighborhoods) in metropolitan São Paulo by assessing the SARS-CoV-2 RNA viral load in wastewater. We analyzed 160 samples from May 2020 to June 2021 with weekly or fortnightly samplings. The samples were ultracentrifuged with glycine elution and quantified by N1/N2 SARS-CoV-2 RT-qPCR. The results of positivity were 100% (Paraisópolis, Heliópolis and Cidade Tiradentes) and 76.9% (Vila Brasilândia). The new case numbers of COVID-19, counted from the onset of symptoms, positively correlated with SARS-CoV-2 N1 viral loads from the two largest communities (p<0.001). SARS-CoV-2 infectivity was tested in Vero E6 cells after concentration with the two techniques, ultrafiltration (Centricon ® Plus-70 10 kDa) and sucrose cushion ultracentrifugation, but none of the evaluated samples presented positive results. Next-generation sequencing (NGS) analysis from samples collected in March and August 2021 revealed the presence of the clade 20 J (lineage P.1) belonging to the most prevalent circulating variant in the country. Our results showed that wastewater surveillance data can be used as complementary indicators to monitor the dynamics and temporal trends of COVID-19. The infectivity test results strengthened the evidence of low risk of infection associated with SARS-CoV-2 in wastewater.

COVID-19 pandemic has led to concerns on the circulation of SARS-CoV-2 in the environment, its infectivity from the environment and, the relevance of transmission via environmental compartments. During 31 weeks, water samples were collected from a heavily contaminated stream going through an urban, underprivileged community without sewage collection. Our results showed a statistically significant correlation between cases of COVID-19 and SARS in the community, and SARS-CoV-2 concentrations in the water. Based on the model, if the concentrations of SARS-CoV-RNA (N1 and N2 target regions) increase 10 times, there is an expected increase of 104% [95%CI: (62-157%)] and 92% [95%CI: (51-143%)], respectively, in the number of cases of COVID-19 and SARS. We believe that differences in concentration of the virus in the environment reflect the epidemiological status in the community, which may be important information for surveillance and controlling dissemination in areas with vulnerable populations and poor sanitation. None of the samples were found infectious based cultures. Our results may be applicable globally as similar communities exist worldwide., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we isolated a KPC-2-producing ST2787 Klebsiella quasipneumoniae subsp. quasipneumoniae (WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all β-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly, bla KPC-2 was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel non-typable/IncP-6 hybrid plasmid in a newly assigned ST1407 Enterobacter asburiae (WW19C) also harboring bla KPC-2 . Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (~11 kb) in both plasmids contained a Tn 3 transposon disrupted by a Tn 3 -IS Apu -flanked element and the core sequence was composed by ΔIS Kpn6 / bla KPC-2 /Δ bla TEM-1 /IS Kpn27 . Both strains also carried genes conferring resistance to heavy metals (e.g., arsenic, mercury, lead, cadmium, copper), pesticides (e.g., glyphosate), disinfectants, and several virulence-related genes, posing a potential pathogenic risk in the case of infections. This is the first study documenting bla KPC-2 associated with IncP-6 plasmids in K. quasipneumoniae and Enterobacter cloacae complex from wastewater in Argentina and highlights the circulation of IncP-6 plasmids as potential reservoirs of bla KPC-2 in the environment., Competing Interests: Author MVH is employed by Agua y Saneamientos Argentinos S.A. (AySA), in Buenos Aires, Argentina. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ghiglione, Haim, Penzotti, Brunetti, D´Amico González, Di Conza, Figueroa-Espinosa, Nuñez, Razzolini, Fuga, Esposito, Vander Horden, Lincopan, Gutkind, Power and Dropa.)

The presence of Staphylococcus aureus in drinking water is a concern because of its potential to cause human infection and also because of its multiple antimicrobial resistance. This study evaluated the water quality of drinking water fountains and mist makers in four municipal parks of São Paulo for 13 months. Although all samples met bacteriological water quality criteria according to Brazilian regulations, the absence of residual chlorine (<0.1 mg/L) was observed. These data were significantly correlated with the frequency of S. aureus that was found in 25.2% of the samples. The mecA gene was detected in 36.7% of the isolates demonstrating its potential for resistance to several antimicrobials. Furthermore, 27.3% isolates carrying the mecA gene had methicillin-resistant Staphylococcus aureus (MRSA) phenotypic potential. The presence of S. aureus with characteristics of microbial resistance in water for human consumption is an unprecedented finding. Hence, conducting surveillance for opportunistic bacteria, such as staphylococci in drinking water, is reasonable to take control measures and to protect human health, especially in public places with high attendance.

Critical priority pathogens have globally disseminated beyond clinical settings, thereby threatening wildlife. Andean Condors (Vultur gryphus) are essential for ecosystem health and functioning, but their populations are globally near threatened and declining due to anthropogenic activities. During a microbiological and genomic surveillance study of critical priority antibiotic-resistant pathogens, we identified pandemic lineages of multidrug-resistant extended-spectrum β-lactamase (ESBL)-producing Escherichia coli colonizing Andean Condors admitted at two wildlife rehabilitation centres in South America. Genomic analysis revealed the presence of genes encoding resistance to hospital and healthcare agents among international E. coli clones belonging to sequence types (STs) ST162, ST602, ST1196 and ST1485. In this regard, the resistome included genes conferring resistance to clinically important cephalosporins (i.e., CTX-M-14, CTX-M-55 and CTX-M-65 ESBL genes), heavy metals (arsenic, mercury, lead, cadmium, copper, silver), pesticides (glyphosate) and domestic/hospital disinfectants, suggesting a link with anthropogenic environmental pollution. On the other hand, the presence of virulence factors, including the astA gene associated with outbreak of childhood diarrhoea and extra-intestinal disease in animals, was identified, whereas virulent behaviour was confirmed using the Galleria mellonella infection model. E. coli ST162, ST602, ST1196 and ST1485 have been previously identified in humans and food-producing animals worldwide, indicating that a wide resistome could contribute to rapid adaptation and dissemination of these clones at the human-animal-environment interface. Therefore, these results highlight that Andean Condors have been colonized by critical priority pathogens, becoming potential environmental reservoirs and/or vectors for dissemination of virulent and antimicrobial-resistant bacteria and/or their genes, in associated ecosystems and wildlife., (© 2020 John Wiley & Sons Ltd.)

The natural diversification of CTX-M β-lactamases led to the emergence of Asp240Gly variants in the clinic that confer reduced susceptibility to ceftazidime (CAZ). In this study, we compared the impact of this substitution on CAZ and ceftazidime-avibactam (CZA) MICs against isogenic Escherichia coli strains with different porin deficiencies. Our results show a noticeable increase in CAZ resistance in clones expressing Asp240Gly-harboring CTX-M when combined with OmpF porin deficiency. Kinetic analysis revealed that the k cat / K m selection of CAZ-resistant clones yielded nonsusceptible CTX-M producers (MIC of >16 μg/ml) only after overnight incubation; the addition of avibactam (AVI) decreased MICs to a susceptible range against these variants. In contrast, the use of CZA as a selective agent did not yield resistant clones. AVI inactivated both CTX-M-12 and CTX-M-96, with an apparent inhibition constant comparable to that of SHV-2 and 1,000-fold greater than that of PER-2 and CMY-2, and In vitro for CTX-M-12 was 24- and 35-fold higher than that for CTX-M-96 and CTX-M-15, respectively. Molecular modeling suggests that AVI interacts similarly with CTX-M-96 and CTX-M-15. We conclude that the impact of Asp240Gly in resistance may arise when other mechanisms are also present (i.e., OmpF deficiency). Additionally, CAZ selection could favor the emergence of CAZ-resistant subpopulations. These results define the role of Asp240 and the impact of the -Gly substitution and allow us to hypothesize that the use of CZA is an effective preventive strategy to delay the development of resistance in this family of extended-spectrum β-lactamases.k 2 /K for CTX-M-12 was 24- and 35-fold higher than that for CTX-M-96 and CTX-M-15, respectively. Molecular modeling suggests that AVI interacts similarly with CTX-M-96 and CTX-M-15. We conclude that the impact of Asp240Gly in resistance may arise when other mechanisms are also present (i.e., OmpF deficiency). Additionally, CAZ selection could favor the emergence of CAZ-resistant subpopulations. These results define the role of Asp240 and the impact of the -Gly substitution and allow us to hypothesize that the use of CZA is an effective preventive strategy to delay the development of resistance in this family of extended-spectrum β-lactamases., (Copyright © 2018 American Society for Microbiology.)

Cryptosporidium and Giardia are associated with cases of water and foodborne outbreaks in the world. This study included 50 samples of surface raw water collected from two watersheds in the state of São Paulo, Brazil. The isolation of (oo)cysts was performed in accordance with the U.S. Environmental Protection Agency's methods 1623 and genotypic characterization and quantification were carried out by Nested PCR and qPCR assays based on 18S rRNA and gdh genes, respectively. U.S. EPA 1623 method showed the presence of (oo)cysts in 40% ([Formula: see text] = 0.10 oocysts/L) and 100% ([Formula: see text] = 7.6 cysts/L) of samples from São Lourenço River, respectively, and 24% ([Formula: see text] = 0.8 oocysts/L) and 60% ([Formula: see text] = 1.64 cysts/L) of Guarapiranga Reservoir, respectively. The qPCR assay detected C. hominis/parvum in 52% (0.06 to 1.85 oocysts/L) of São Lourenço River and 64% (0.09 to 1.4 oocysts/L) of Guarapiranga Reservoir samples. Presence/absence test for Giardia intestinalis was positive in 92% of São Lourenço River and 8% of Guarapiranga Reservoir samples. The assemblage A was detected in 16% (0.58 to 2.67 cysts/L) in São Lourenço River and no positive samples were obtained for assemblage B in both water bodies. The characterization of anthroponotic species C. parvum/hominis, G. intestinalis, and assemblage A was valuable in the investigation of possible sources of contamination in the watersheds studied confirming the need of expanding environmental monitoring measures for protection of these water sources in our country.

Spatial and temporal collections provide important data on the distribution and dispersal of species. Regional-scale monitoring invariably involves hundreds of thousands of samples, the identification of which is costly in both time and money. In this respect, metabarcoding is increasingly seen as a viable alternative to traditional morphological identification, as it eliminates the taxonomic bottleneck previously impeding such work. Here, we assess whether terrestrial arthropods collected from 12 pitfall traps in two farms of a coffee (Coffea arabica L.) growing region of Sao Paulo State, Brazil could differentiate the two locations. We sequenced a portion of the cytochrome oxidase 1 region from minimally processed pools of samples and assessed inter- and intraspecific parameters across the two locations. Our sequencing was sufficient to circumscribe the overall diversity, which was characterized by few dominant taxa, principally small Coleoptera species and Collembola. Thirty-four operational taxonomic units were detected and of these, eight were present in significantly different quantities between the two farms. Analysis of community-wide Beta diversity grouped collections based on farm provenance. Moreover, haplotype-based analyses for a species of Xyleborus beetle showed that there is significant population genetic structuring between the two farms, suggesting limited dispersal. We conclude that metabarcoding can provide important management input and, considering the rapidly declining cost of sequencing, suggest that large-scale monitoring is now feasible and can identify both the taxa present as well as contribute information about genetic diversity of focal species., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The emergence of high-risk clones of multidrug-resistant (MDR) bacteria in aquatic environments has generated an important public health problem, creating an urgent need to strengthen surveillance. This study reports the occurrence of clinically significant MDR Enterobacteriaceae and non-fermentative bacteria carrying carbapenemases (KPC-2), extended-spectrum β-lactamases (CTX-M) and plasmid-mediated quinolone resistance (PMQR) genes in urban lakes and reservoirs, in Southeastern Brazil. In this regard, the detection of hospital-associated lineages of KPC-2-producing Klebsiella pneumoniae belonging to the international clonal complex CC258 (ST11) and CTX-M-15-producing Escherichia coli belonging to the international CC10 (ST617), in an urban lake, is reported for the first time. Whole genome sequencing (WGS) analysis of KPC-2-producing K. pneumoniae ST11 revealed that bla KPC-2 gene was carried by an IncN plasmid on a Tn4401b element. This study support that aquatic environments with public access can act as reservoirs of clinically important MDR bacteria, constituting a potential risk to human and animal health. On the other hand, the detection of high-risk clones highlights the extra-hospital spread of clinically significant bacteria into urban aquatic environments., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nontyphoidal Salmonella (NTS) is a relevant pathogen involved in gastroenteritis outbreaks worldwide. In this study, we determined the capacity to combine the most probable number (MPN) and multiplex polymerase chain reaction (PCR) methods to characterize the most important Salmonella serotypes in raw sewage. A total of 499 isolates were recovered from 27 raw sewage samples and screened using two previously described multiplex PCR methods. From those, 123 isolates were selected based on PCR banding pattern-identical or similar to Salmonella Enteritidis and Salmonella Typhimurium-and submitted to conventional serotyping. Results showed that both PCR assays correctly serotyped Salmonella Enteritidis, however, they presented ambiguous results for Salmonella Typhimurium identification. These data highlight that MPN and multiplex PCR can be useful methods to describe microbial quality in raw sewage and suggest two new PCR patterns for Salmonella Enteritidis identification.

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.

A colistin-resistant Escherichia coli strain was recovered from a patient with a diabetic foot infection in Brazil. Whole-genome analysis revealed that the E. coli isolate belonged to the widespread sequence type (ST) 101 and harbored the mcr-1 gene on an IncX4 plasmid that was highly similar to mcr-1-bearing IncX4 plasmids that were recently identified in Enterobacteriaceae from food, animal, and human samples recovered on different continents. These results suggest that self-transmissible IncX4-type plasmids may represent promiscuous plasmids contributing to the intercontinental spread of the mcr-1 gene., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

During a Brazilian multicentric antimicrobial resistance surveillance study, colistin resistance was investigated in 4,620 Enterobacteriaceae isolated from human, animal, food and environmental samples collected from 2000 to 2016. We present evidence that mcr-1-positive Escherichia coli has been emerging in South America since at least 2012, supporting a previous report on the possible acquisition of mcr-1-harbouring E. coli by European travellers visiting Latin American countries.

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.

Introduction: CTX-M enzymes are the most prevalent extended-spectrum beta-lactamases (ESBLs) in Brazil and around the world. The spread of CTX-M lies in their ability to be mobilized by insertion sequences and integrons. This study aimed to identify the mobile genetic structures associated with bla(CTX-M) genes from clinical Enterobacteriaceae strains., Methodology: Twenty-eight clinical non-clonal Enterobacteriaceae were screened by PCR for the presence of bla(CTX-M) genes and class 1 integrase (int1), and for the association of bla(CTX-M) with class 1 integrons. Plasmid incompatibility groups were assessed by PBRT. Wild-type plasmids were transformed into electrocompetent E. coli, and the S1-PFGE technique was used to verify the presence of high-molecular-weight plasmids in both wild-type strains and E. coli transformants., Results: Sequencing showed that strains carried bla(CTX-M-2) (n = 25) and bla(CTX-M-59) (n = 3) genes inserted into the 3'-end of complex class 1 integrons. Thirteen strains also carried bla(TEM) and bla(SHV) genes. CTX-M-2/59-containing complex class 1 integrons were also present in E. coli transformants. The most frequent Inc groups were IncA/C (n = 10) and IncF (n = 8). Heavy plasmids were observed in both wild-type strains and E. coli transformants., Conclusions: The presence of the same bla(CTX-M-2-group)-containing genetic structure in seven Enterobacteriaceae species isolated at seven hospital wards shows the great mobility potential of complex class 1 integrons. Also, this is the first report of TEM-15, SHV-45, and SHV-55 in Latin America. The genetic environment of bla(CTX-M-2) accounts for their maintenance and spread among Gram-negative bacteria.

Diversification of the CTX-M β-lactamases led to the emergence of variants responsible for decreased susceptibility to ceftazidime, like the Asp240Gly-harboring "ceftazidimases". We solved the crystallographic structure of the Asp240Gly variant CTX-M-96 at 1.2 Å and evaluated the role of Asp240 in the activity toward oxyimino-cephalosporins through simulated models and kinetics. There seem to be subtle changes in the conformation of the active site cavity of CTX-M-96, compared to enzyme variants harboring the Asp240, and these small rearrangements could be due to localized shifts in the environment of the β3 strand. According to the crystallographic evidence, CTX-M-96 presents a "compact" active site, which in spite of its reduced cavity seems to allow the proper interaction with oxyimino-cephalosporins, as suggested by simulated models. The term "ceftazidimases" that is currently applied for the Asp240Gly-harboring CTX-M variants should be used carefully. Structural differences between CTX-M harboring the Asp240Gly mutation (and also probably others like those at Pro167) do not seem to be conclusive to determine the "ceftazidimase" behavior observed in vivo, which is in turn partially supported by the mild improvement in the catalytic efficiency toward ceftazidime by CTX-M-96 and similar enzymes, compared to "parental" Asp240-harboring variants. In addition, it is observed that alterations in OmpF expression could act synergistically with CTX-M-96 for yielding clinical resistance toward ceftazidime. We therefore propose that the observed resistance in vivo is due to the sum of synergic mechanisms, and the term "cefotaximases associated with ceftazidime resistance" could be conveniently used to describe CTX-M harboring the Asp240Gly substitution.

CTX-M-type extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have been increasingly identified in humans and animals, and their potential transmission by contaminated food has been highlighted. In this study, we report for the first time the isolation of multidrug-resistant (MDR) Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis strains harboring blaCTXM-2 or blaCTXM-8 gene variants in chicken meat sold in markets in southeast Brazil. In this regard, the genetic environment of the blaCTX-M-2 gene is composed of a complex class 1 integron and an ISCR1-associated sequence with dfr and/or aadA gene cassettes located within the variable region. In summary, chicken meat may be a reservoir of MDR Enterobacteriaceae harboring blaCTX-M-type genes, which is a public health concern., (Copyright © 2014 Elsevier B.V. All rights reserved.)

CTX-M-131 is a natural Asp240Gly variant from the CTX-M-2 group detected in a Providencia rettgeri clinical strain from Brazil. Molecular analysis showed that blaCTX-M-131 was inserted in a complex class 1 integron harbored by a 112-kb plasmid, which has not been previously described as a platform for CTX-M-encoding genes with the Asp240Gly mutation. Steady-state kinetic parameters showed that the enzyme has a typical cefotaximase catalytic profile and an enhanced activity against ceftazidime., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: This study aims to scrutinize Salmonella spp. and its serotypes in sewage sludge samples from wastewater treatment plants, and assesses the presence of virulence genes and antibiotics resistant to the profile. Samples (n = 54) were collected and analyzed in accordance with the EPA Method 1682/2006. For positive serological reaction, 40 strains were selected for PCR analyses and detection of spvC, invA and sseL virulence genes, plasmid presence and resistance to antibiotics., Results: Salmonella spp. was detected in 38.9% of the samples collected (<0.006473 to 12.19 MPN/gTS). The most prevalent serotype was Salmonella Infantis. All Salmonella spp. (n = 35) presented at least one of the three virulence genes mentioned above and 40% harboured plasmids. Salmonella Typhimurium strains were isolated harbouring at least one of the following virulence genes: spvC, invA or sseL. Four Salmonella spp. isolates were resistant to tetracycline; three were resistant to trimethoprim-sulfamethoxazole, and one isolate was resistant to ciprofloxacin. Two Salmonella spp. strains presented multi resistance to antimicrobial agents., Conclusions: The results obtained demonstrated that Salmonella spp. have been found in sewage sludge, thus it is essential to set measures to mitigate human health risks when it is intended to be applied on agricultural soils.

Multiple papers have been published regarding the bacterial resistance theme over the last years. A variety of information has reached general and scientific public, daily bringing up data on new resistant microorganisms, new drugs, outbreaks, epidemiological news, resistance gene dissemination, and the lack of information in a particular field has caught our attention: the public health department. Most of researchers, physicians and government employees interpret the public health field as a separate department, not linked to this antibiotic resistance era that we are living nowadays. In this paper we carefully tried to fill in the blanks between public health and the bacteria resistance issue, also considering historical, social, economical and biological problematic that come with this possible pre-antibiotic era.

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.