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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]

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.)

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.

We describe two patients with HIV/AIDS who presented pulmonary and intestinal infection caused by Cryptosporidium parvum, with a fatal outcome. The lack of available description of changes in clinical signs and radiographic characteristics of this disease when it is located in the extra-intestinal region causes low prevalence of early diagnosis and a subsequent lack of treatment.

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.

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.)

Vibrio fluvialis is a halophilic bacterium found in many environments and is mainly associated with sporadic cases and outbreaks of gastroenteritis in humans. Here, we describe the genome sequences of environmental strains of V. fluvialis 560 (Vf560) and V. fluvialis 539 (Vf539) possessing a variant of the integrative and conjugative element (ICE) SXT for the first time in Brazil and South America., (Copyright © 2015 de Oliveira Veras et al.)

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.)

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.

Background: In the last decade, non-multiresistant methicillin-resistant Staphylococcus aureus (NM-MRSA) has been described as an important agent in bloodstream infections in our hospital., Methods: This prospective cohort study, conducted from February 2009 through January 2010 in the neonatal unit, evaluated 403 newborns (NB), their 382 mothers and 148 health care workers (HCW)., Results: Approximately 217 NB (54%), 187 mothers (48%) and 87 HCW (59%) were colonized by S. aureus (SA). MRSA colonization was greater among NB (15%) than mothers (4.7%) and HCW (3.4%). Although mother-to-NB transmission occurred, in most cases mothers were not responsible for NB colonization. There were 2 predominant PFGE patterns among the NB and some mothers and HCW became colonized by them. Factors significantly associated with MRSA carriage by NB were lower level of maternal schooling (risk factor: odds ratio: 2.99; 95% confidence interval: 1.10-8.07) and maternal rhinosinusitis (protective factor: odds ratio: 0.33; 95% confidence interval:0.12-0.88). Among NB who remained hospitalized for more than 72 hours, breast feeding was protective (odds ratio: 0.22; 95% confidence interval: 0.05-0.98). All the isolates were NM-MRSA, carried few virulence factors and SCCmec types IVa and type IVd predominated., Conclusions: Although there were no cases of infection, nosocomial transmission of MRSA clearly occurred in the neonatal unit, and this highlights the need for infection control practices such as hand hygiene to prevent cross-dissemination. Other healthcare practices, which are very basic but also ample in scope, may play a role, such as general education of women and breast feeding.

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.

Introduction: Listeria species are susceptible to most antibiotics. However, over the last decade, increasing reports of multidrug-resistant Listeria spp. from various sources have prompted public health concerns. The objective of this study was to characterize the antibiotic susceptibility of Listeria spp. and the genetic mechanisms that confer resistance., Methodology: Forty-six Listeria spp. isolates were studied, and their minimal inhibitory concentrations of antibiotics were determined by microdilution using Sensititre standard susceptibility MIC plates. The isolates were screened for the presence of gyrA, parC, lde, lsa(A), lnu(A), and mprF by PCR, and the amplified genes were sequenced., Results: All isolates were susceptible to penicillin, ampicillin, tetracycline, erythromycin, and carbapenems. Resistance to clindamycin, daptomycin, and oxacillin was found among L. monocytogenes and L. innocua, and all species possessed at least intermediate resistance to fluoroquinolones. GyrA, parC, and mprF were detected in all isolates; however, mutations were found only in gyrA sequences. A high daptomycin MIC, as reported previously, was observed, suggesting an intrinsic resistance of Listeria spp. to daptomycin., Conclusions: These results are consistent with reports of emerging resistance in Listeria spp. and emphasize the need for further genotypic characterization of antibiotic resistance in this genus.

In the last decade, atypical Listeria monocytogenes and L. innocua strains have been detected in food and the environment. Because of mutations in the major virulence genes, these strains have different virulence intensities in eukaryotic cells. In this study, we performed phenotypic and genotypic characterization of atypical L. monocytogenes and L. innocua isolates obtained from swine slaughterhouses and meat markets. Forty strains were studied, including isolates of L. monocytogenes and L. innocua with low-hemolytic activity. The isolates were characterized using conventional phenotypic Listeria identification tests and by the detection and analysis of L. monocytogenes-specific genes. Analysis of 16S rRNA was used for the molecular identification of the Listeria species. The L. monocytogenes isolates were positive for all of the virulence genes studied. The atypical L. innocua strains were positive for hly, plcA, and inlC. Mutations in the InlC, InlB, InlA, PI-PLC, PC-PLC, and PrfA proteins were detected in the atypical isolates. Further in vitro and transcriptomic studies are being developed to confirm the role of these mutations in Listeria virulence.

Yersinia enterocolitica is an important foodborne pathogen that causes illness in humans and animals. Y. enterocolitica is also the most heterogeneous species of the genus and is divided into distinct serotypes and over six biotypes. Y. enterocolitica biotype 1A strains are classically considered as nonpathogenic; however, some biotype 1A isolates have been considered as causative of gastrointestinal disease, yielding symptoms indistinguishable from those produced by pathogenic biotypes. Even after decades of isolation of clinical strains, the pathogenic mechanisms of these isolates are still not fully understood. In the present study, 122 Yersinia enterocolitica biotype 1A strains isolated from swine slaughterhouses and meat markets in Sao Paulo, Brazil, were characterized according to the presence of the virulence genes ail, virF, and ystA. A total of 94 strains were positive to at least one virulence gene (77.05%), and 67 were positive to all of them (54.92%). Twenty-two strains were submitted to PFGE genotyping resulting in 22 distinct pulsotypes, varying from 50% to 84% of genetic similarity. Any clustering tendency among pulsotypes related to origin, isolation site, or even virulence profile was not observed. The present study reports an important contamination of the environment in swine slaughterhouses, meat markets, and pork, by potentially virulent Y. enterocolitica biotype 1A.

Yersinia enterocolitica is a foodborne pathogen that causes illness in humans and animals. The biotype 4/O:3 has been commonly associated with yersiniosis and is characterized by the presence of chromosomal and extra-chromosomal virulence genes. Molecular typing methods have been successfully used to characterize Y. enterocolitica genetic heterogeneity and to study the epidemiology of the bacteria from different origins. In this study, 320 Y. enterocolitica biotype 4/O:3 isolates originating in pigs and slaughterhouses were characterized according to the virulence profile, and 61 isolates were typified through SE-AFLP, ERIC-PCR, and PFGE techniques. The majority of the isolates originated from pigs, and the predominant virulence profile was ail+ virF+ rfbC+ ystA+, representing 83.4% of the tested isolates. All of the Y. enterocolitica 4/O:3 isolates were positive for at least ystA gene. The SE-AFLP and ERIC-PCR patterns were highly homogeneous. The SE-AFLP was more discriminative than the ERIC-PCR and tended to cluster isolates according to the slaughterhouse. Despite the limited genetic diversity of Y. enterocolitica 4/O:3, PFGE was shown to be the most discriminative technique considering one band of difference. Fattening pigs proved to be an important reservoir of Y. enterocolitica biotype 4/O:3 carrying virulence genes.

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.

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.

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

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.

Since Staphylococcus aureus can cause several types of diseases, the development of antibiotic resistance poses an even greater threat to public health. S. aureus is known to possess the adaptive capability to promptly respond to antibiotics, making it resistant and increasingly difficult to treat; methicillin-resistant strains of S. aureus are a major concern with regard to this species. Previous studies reported the identification of methicillin-resistant S. aureus in food, demonstrating that this can represent a source of S. aureus which may carry the mecA gene. Fifty-seven S. aureus isolates, previously obtained from different types of food, were screened by polymerase chain reaction with specific primers for the mecA gene, which mediates methicillin resistance. Five (9%) isolates showed the presence of mecA gene, demonstrating that food may contain microorganisms possessing resistance genes. This study emphasizes the need to include food as a possible source of S. aureus carrying mecA gene and the need to monitor these products. Moreover, this is the first report of the presence of mecA genes in S. aureus isolated from ready-to-eat food in Brazil and Latin America., (© Mary Ann Liebert, Inc.)

A sustainable option for nitrogen removal is the anaerobic ammonium-oxidizing (anammox) process in which ammonium is oxidized to nitrogen gas with nitrite as electron acceptor. Application of this process, however, is limited by the availability of anammox biomass. In this study, two Brocadia-like anammox phylotypes were successfully enriched, detected and identified from an activated sludge taken from a domestic wastewater treatment plant (Minas Gerais, Brazil) employing a Sequencing Batch Reactor (SBR). The dominant phylotype was closely related to 'Candidatus Brocadia sinica', but one clone seemed to represent a novel species for which we propose the name 'Candidatus Brocadia brasiliensis'. Based on Fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 52.7% (±15.6) anammox bacteria after 6 months of cultivation. The cultivation process can be divided into three phases: phase 1 (approximately 25 days) was characterized by heterotrophic denitrification metabolism, phase 2 was the propagation phase and phase 3 (from the 87th day onwards), in which significant anammox activity was detected. A long-term performance of the SBR showed a near perfect removal of nitrite based on the influent NO(2)(-)-N concentration of 61-95 mg L(-1). The average ammonia removal efficiency was 90% with the influent NH(4)(+)-N concentration of 55-82 mg L(-1). Therefore, anammox cultivation and enrichment from activated sludge was possible under a controlled environment within 3 months.

Aeromonas are widely distributed in the aquatic environment, and are considered to be emerging organisms that can produce a series of virulence factors. The present study was carried out in a sanitary sewage stabilization pond treatment system, located in Lins, State of São Paulo, Brazil. Most probable number was applied for estimation of the genus Aeromonas. Colony isolation was carried out on blood agar ampicillin and confirmed by biochemical characterization. Aeromonas species were isolated in 72.4% of influent samples, and in 55.2 and 48.3% of effluent from anaerobic and facultative lagoons, respectively. Thirteen Aeromonas species were isolated, representing most of the recognized species of these organisms. Even though it was possible to observe a tendency of decrease, total elimination of these organisms from the studied system was not achieved. Understanding of the pathogenic organism's dynamics in wastewater treatment systems with a reuse potential is especially important because of the risk it represents.

It is known that Aeromonas spp. possess different chromosomal β-lactamase genes. Presence and phenotypic expression of bla TEM, bla SHV, and bla CTX-M ESBL-encoding genes were investigated in environmental water isolates of Aeromonas hydrophila and Aeromonas jandaei. Presence of bla SHV and bla CTX-M genes was not observed, and bla TEM gene was verified in 91% of the isolates. Sequencing of 10 fragments showed the occurrence of bla TEM-116.

A clinical Klebsiella pneumoniae isolate carrying the extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and bla(GES-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 bla(GES-7) gene was found to be inserted into a class 1 integron. These results show the emergence of novel bla(TEM) and bla(SHV) genes in Brazil. Moreover, the presence of class 1 integrons suggests a great potential for dissemination of bla(GES) genes into diverse nosocomial pathogens. Indeed, the bla(GES-7) gene was originally discovered in Enterobacter cloacae in Greece and, to our knowledge, has not been reported elsewhere.

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hlyA/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hlyA/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.

Extended-spectrum beta-lactamases (ESBL) in enterobacteria are recognized worldwide as a great hospital problem. In this study, 127 ESBL-producing Enterobacteriaceae isolated in one year from inpatients and outpatients at a public teaching hospital at São Paulo, Brazil, were submitted to analysis by PCR with specific primers for bla SHV, bla TEM and bla CTX-M genes. From the 127 isolates, 96 (75.6%) Klebsiella pneumoniae, 12 (9.3%) Escherichia coli, 8 (6.2%) Morganella morganii, 3 (2.3%) Proteus mirabilis, 2 (1.6%) Klebsiella oxytoca, 2 (1.6%) Providencia rettgeri, 2 (1.6%) Providencia stuartti, 1 (0.8%) Enterobacter aerogenes and 1 (0.8%) Enterobacter cloacae were identified as ESBL producers. Bla SHV, bla TEM and bla CTX-M were detected in 63%, 17.3% and 33.9% strains, respectively. Pulsed field gel eletrophoresis genotyping of K. pneumoniae revealed four main molecular patterns and 29 unrelated profiles. PCR results showed a high variety of ESBL groups among strains, in nine different species. The results suggest the spread of resistance genes among genetically different strains of ESBL-producing K. pneumoniae in some hospital wards, and also that some strongly related strains were identified in different hospital wards, suggesting clonal spread in the institutional environment.

Aims: To determine the prevalence and expression of metallo-beta-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil., Methods and Results: Eighty-seven Aeromonas isolates belonging to Aeromonas hydrophila (n = 41) and Aer. jandaei (n = 46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97.6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla(IMP), bla(VIM) and bla(SPM-1) were not detected. On the other hand, production of MBL activity was detectable in 87.8% and 10.9% of the cphA-positive Aer. hydrophila and Aer. jandaei isolates respectively., Conclusions: Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity., Significance and Impact of the Study: These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.

Tramp ants are an outstanding group of organisms that have a definitive and important role in carrying pathogens. The purpose of this study was to develop a microbiological sterile technique of collecting ants from contaminated areas. The traps were composed of Petri dishes containing culture media, and were tested to verify the applicability of the system. A positive correlation between the microorganism growth and the presence of ants inside or around traps was observed. The technique described demonstrated to be useful to collect ants from different environments, helping the surveillance of pathogenic microorganisms that are of public health concern.

Molecular characterization of Cryptosporidium spp.oocysts in clinical samples is useful for public health since it allows the study of sources of contamination as well as the transmission in different geographical regions. Although widely used in developed countries, in Brazil it is restricted to academic studies, mostly using commercial kits for the extraction of genomic DNA, or in collaboration with external reference centers, rendering the method expensive and limited. The study proposes the application of the modifications recently introduced in the method improving feasibility with lower cost. This method was efficient for clinical samples preserved at -20 degrees C for up to six years and the low number of oocysts may be overcome by repetitions of extraction.

Wastewater reuse has become an important alternative to agricultural irrigation; on the other hand, it poses concern with regard to public health. Total coliform and Escherichia coli concentration, presence of helminth eggs and Salmonella, and physical-chemical parameters were evaluated in raw and treated wastewater. Chemical and biochemical oxygen demand removal efficiency was 74.6 and 77.9%, respectively. As for organic nitrogen, total phosphorus, and total suspended solids, total efficiency removal was 17.4, 12.5, and 32.9%, respectively. The average density of total coliforms and E. coli was 3.5 x 10(9) and 1.8 x 10(8) MPN/100 mL and 1.1 x 10(7) MPN/100 mL and 3.9 x 10(5) MPN/100 mL for raw and treated wastewater, respectively. Ascaris eggs were observed in 80.8% of the samples collected, and viable eggs in 42.3% of the samples. Salmonella was detected in 36.4% of the samples. The values observed in treated wastewater did not show the adequate bacteriological quality, as recommended by World Health Organization (Geneva, Switzerland). Therefore, additional measures should be taken to achieve an improved microbiological and parasitological quality.

A polyphasic taxonomy study was undertaken of three strains of Pseudoalteromonas haloplanktis subsp. tetraodonis (Simidu et al. 1990) Gauthier et al. 1995. DNA was prepared from each of the strains and genomic relatedness was measured by DNA-DNA hybridization. Strains KMM 458T and IAM 14160T shared 99% genetic relatedness, but were only 48-49% related to the type strain of Pseudoalteromonas haloplanktis subsp. haloplanktis, IAM 12915T. The third strain, P. haloplanktis subsp. tetraodonis A-M, showed 83% genetic similarity with P. haloplanktis subsp. haloplanktis IAM 12915T and 32% with KMM 458T. From these results, it is concluded that strains KMM 458T and IAM 14160T comprise a separate species, originally described as Alteromonas tetraodonis, whereas strain A-M belongs to the species Pseudoalteromonas haloplanktis. Based on phenotypic and chemotaxonomic data, genomic fingerprint patterns, DNA-DNA hybridization data and phylogenetic analysis of 16S rRNA, it is proposed that the species Alteromonas tetraodonis be retrieved and recognized as Pseudoalteromonas tetraodonis comb. nov. (type strain IAM 14160T = KMM 458T).

Oysters ( Crassostrea gigas ) originating from the southern coast of the State of São Paulo-Brazil were analyzed for Vibrio species. Most Probable Numbers (MPN/100 g) were obtained for Vibrio alginolyticus (<3-1,500), Vibrio parahaemolyticus (<3-1,200), Vibrio fluvialis (<3-150), Vibrio cholerae non 01 (<3-40), Vibrio furnissii (<3-40), Vibrio mimicus (<3-40) and Vibrio vulnificus (<3-30). The highest incidence was observed for V. alginolyticus (81%), followed by V. parahaemolyticus (77%), V. cholerae non 01 (31 %), V. fluvialis (27%), V. furnissii (19%), V. mimicus (12%), and V. vulnificus (12%). Forty-eight percent of the isolates tested were positive for enterotoxins in the rabbit ileal loop (RIL) test and 11.1% in the suckling mice test. Vibrio parahaemolyticus (1.1 %) was positive in the Kanagawa test. Vibrio vulnificus (25%) showed lethality in young adult mice. During the field inspection it was observed that generally the conditions of storage were not adequate as 65% of the samples were maintained in temperatures ranging from 25 to 40°C, 19% were frozen, 12% refrigerated and 4% was immersed in contaminated seawater. These results emphasize the great potential for food poisoning by inadequately preserved seafood, and the necessity to upgrade the standards for food quality assessment.

Mussels (Perna perna) harvested on the coast of Ubatuba, in three different stations in the State of São Paulo, Brazil, were examined for Vibrio spp. over a 1 year period. The ranges of most probable number (MPN 100 g-1) were: Vibrio alginolyticus (< 3-24,000), V. parahaemolyticus (< 3-24,000), V. fluvialis (< 3-1100), V. cholerae non-O1 (< 3-23), V. furnissii (< 3-30), V. mimicus (< 3-9) and V. vulnificus (< 3-3). The highest incidence was observed for V. alginolyticus (92-100%), followed by V. parahaemolyticus (67-92%), V. fluvialis (34-67%), V. vulnificus (8-17%), V. furnissii (0-17%), V. mimicus (0-17%) and V. cholerae non-O1 (0-8%). Tests for virulence factors were positive in 34.1% of the vibrios in the rabbit ileal loop and 31.7% in the Dean test. Positive results in the Kanagawa test were obtained with 0.51% of V. parahaemolyticus strains. The mean values (MPN 100 g-1) of faecal coliforms in mussels from the three regions varied from 1100 to 44,000, and seawater collected at the same stations gave average values for faecal coliforms in the range 18-3300 MPN 100 ml-1. These results highlight the potential risks of food poisoning associated with raw or undercooked seafood.