Characterization of Escherichia coli Isolated from Sows Presenting Purulent Vulvar Discharge.

Purulent vulvar discharge is a clinical sign of genitourinary tract infections, which are a significant concern in swine facilities, leading to sow culling and mortality. Escherichia coli is one of the main agents involved in these diseases. This study aimed to characterize the virulence and antimicrobial resistance profiles as well as the phylotype of Escherichia coli strains isolated from sows with purulent vulvar discharge. The results showed that at least 2 of the 29 tested virulence genes related to extraintestinal pathogenic E. coli were present in all strains tested. The most frequent gene was iutA, present in all strains, followed by the genes iucD, csgA, iss2, and irp2. Associations between iron uptake genes, genes related to adhesion, attachment, and serum resistance, as well as genes related to toxin release and bacteriocin, were frequent. The most prevalent phylotype was B1 (40.0%), followed by A (18.5%), D (11.9%), C (9.6%), B2 (7.4%), E (4.4%), F (1.5%), and Clade I (0.7%), with B2 being related to highly virulent traits. The strains presented elevated resistance to antimicrobials such as ciprofloxacin, streptomycin, cephalexin, florfenicol, and ampicillin. More than 90% of the strains were identified as multidrug-resistant, indicating the selection that is induced by the high use of antimicrobials in swine farming.

Vaginal microbiota signatures in healthy and purulent vulvar discharge sows.

Purulent vulvar discharges, primarily caused by genito-urinary tract infections, are an important source of economic loss for swine producers due to sow culling and mortality. However, the agents that compose the vaginal microbiota of sows and their changes during infections are not well understood. The first goal of this study was to characterize and compare the vaginal bacterial content of healthy (HE, n = 40) and purulent vulvar discharge sows (VD, n = 270) by a culture-dependent method and MALDI-TOF MS identification. Secondly, we performed 16S rRNA targeted metagenomic approach (n = 72) to compare the vaginal microbiota between these groups. We found a wide variety of bacteria, with Proteobacteria, Firmicutes, and Bacteroidota being the most abundant phyla in both groups, as well as Escherichia-Shigella, Streptococcus, and Bacteroides at the genus level. Most agents identified in the sequencing method also grew in the culture-dependent method, showing the viability of these bacteria. Alpha diversity did not differ between HE and VD sows, regarding sample richness and diversity, but a beta-diversity index showed a different microbiota composition between these groups in two tested herds. ANCOM analysis revealed that Bacteroides pyogenes were more abundant in VD females and can be a marker for this group. Other agents also require attention, such as the Streptococcus dysgalactiae and Staphylococcus hyicus found in remarkably greater relative abundance in VD sows. Network analysis revealed important positive correlations between some potentially pathogenic genera, such as between Escherichia-Shigella, Trueperella, Streptococcus, Corynebacterium, and Prevotella, which did not occur in healthy sows. We conclude that the alteration of the vaginal microbiota between healthy and purulent vulvar discharge sows, although not extreme, could be due to the increase in the relative abundance of specific agents and to associations between potentially pathogenic bacteria.

Main bacterial species causing clinical disease in ornamental freshwater fish in Brazil.

Bacterial diseases are common in ornamental fish, more frequently associated with ubiquitous bacteria from the aquarium environment. The disease can lead to fish mortality and cause high economic losses if not rapidly controlled. The aim of this study was to identify the main causative bacterial agents of infection in ornamental fish with different clinical signs. A total of 126 freshwater fish, from 12 families and 38 species, with clinical signs were collected in a wholesaler in São Paulo, SP, Brazil. Samples were taken from the eye, skin ulcers, kidneys, and gills, plated on MacConkey, CHROMagar Orientation, and blood agar and incubated under aerobic and anaerobic conditions. Bacterial identification was performed by MALDI-TOF mass spectrometry. From the 126 studied animals, 112 were positive for bacterial isolation. Among the positive animals, 32.1% presented infection caused by a single bacterial species, while in the remaining 67.9%, two to six different bacterial species were identified. A total of 259 bacterial strains were obtained and classified among 46 bacterial species. The species of higher frequency were Aeromonas veronii (26.3%), Aeromonas hydrophilla (16.2%), Shewanella putrefaciens (7.3%), Citrobacter freundii (8.1%), Vibrio albensis (5.8%), and Klebsiella pneumoniae (4.2%). MALDI-TOF MS showed to be a rapid method for diagnosis of bacterial disease outbreaks in ornamental fish establishments.

Companion psittacine birds as reservoir of gentamicin and vancomycin-resistant Enterococcus spp / Psitacídeos de companhia como reservatórios de Enterococcus spp. gentamicina e vancomicina-resistentes

Enterococcus are recognized worldwide as significant nosocomial agents that have been continuously envolving to adapt to different niches and acquire resistance to several antibiotic classes. Vancomycin and gentamicin-resistant strains of E. faecalis and E. faecium have been associated with nosocomial human infections. Some epidemiological studies suggest the participation of pets as reservoirs of vancomycin and gentamicin-resistant Enterococcus strains. However, the role of companion birds as reservoirs of these strains has been poorly studied. In this study, 126 psittacine birds were evaluated and 26.9% carried Enterococcus spp., including the species E. faecalis, E. faecium, E. hirae, E. phoeniculicola, E. gallinarum and E. casseliflavus. The antibiotic resistance profile showed four high-level gentamicin-resistance (HLGR) strains. In addition, two strains presented intermediate levels of vancomycin resistance. Resistant strains were isolated from fecal and oropharynx samples of sick and clinically healthy birds, suggesting that psittacine birds may act as reservoirs of HLGR Enterococcus spp. However, sick birds appear to be more implicated in the enterococci transmission than healthy birds.(AU)

Enterococcus são reconhecidos mundialmente como significantes agentes nosocomiais, que têm continuamente se adaptado a diferentes nichos e adquirido resistência a várias classes de antibióticos. Cepas de E. faecalis e E. faecium vancomicina e gantamicina-resistentes têm sido associadas a infecções nosocomiais em humanos. Alguns estudos epidemiológicos sugerem a participação de aves como reservatórios de cepas de Enterococcus vancomicina e gentamicina-resistentes. Entretanto, a relação das aves de companhia como reservatórios destas cepas tem sido pouco estudada. Neste estudo, 126 psitacídeos foram avaliados, e 26,9% destes eram portadores de Enterococcus spp., incluindo as espécies E. faecalis, E. faecium, E. hirae, E. phoeniculicola, E. gallinarum e E. casseliflavus. O perfil de resistência antibiótica mostrou quatro cepas com alto nível de resistência a gentamicina (ANRG). Além de duas cepas com nível intermediário de resistência a vancomicina. As cepas resistentes foram isoladas de amostras fecais e de orofaringe de aves doentes e clinicamente saudáveis, sugerindo que psitacídeos podem estar atuando como reservatórios para Enterococcus spp. com ANRG. Contudo, Aves doentes parecem estar mais relacionadas à transmissão de enterococcus, do que aves saudáveis.(AU)

Persistence of Fecal Contamination Indicators and Pathogens in Class B Biosolids Applied to Sugarcane Fields.

Agricultural recycling of human Class B biosolids in sugarcane ( spp.) crop is a promising alternative to reduce the costs of biosolids disposal. However, the presence of fecal contamination indicators such as thermotolerant coliforms and pathogenic organisms such as enterovirus and spp. in biosolids impose barriers to effective and widespread use of biosolids as fertilizer. In addition, there is a scarcity of studies that investigate the persistence of these organisms in tropical soils. This study aimed to evaluate the persistence of pathogenic and fecal indicators for 258 d in a tropical clayey soil amended with human Class B biosolids and cultivated with sugarcane. Treatments were immediate incorporation of biosolids into soil after application (T1) or superficial application of biosolids followed by incorporation after 35 d (T2), emulating the typical procedure in sugarcane fields. Thermotolerant coliforms were estimated to persist for 437 d in T1 and 398 d in T2. For enterovirus, mean estimated persistence time in soil was 26 d for T1, but the sampling frequency was insufficient in T2 for persistence analysis. After 35 d, no enterovirus was detected in any sample. Mean estimated persistence time for viable spp. eggs in soil was 22 d in T1 and 41 d in T2.

Streptococcus suis in Brazil: Genotypic, Virulence, and Resistance Profiling of Strains Isolated from Pigs between 2001 and 2016.

Streptococcus suis remains an important challenge for the worldwide swine industry. Considering that Brazil is a major pork producer and exporter, proper monitoring of the pathogen and resistance rates are required. We present here the characterization of Brazilian S. suis strains isolated over a 15 year period by pulsed-field gel electrophoresis (PFGE) typing, capsular, virulence, and antimicrobial resistance profiling. Serotype prevalence revealed a predominance of serotype 2/½ followed by 3, 7, 1/14, 6, 8, 18, 28, and 27; the latter had not yet been reported in Brazil. Resistance profiling enabled the differentiation of nine profiles presenting resistance to three and up to eight antimicrobial classes. Even though an association between the most resistant strains and isolation year starting from 2009 was observed, a high frequency of multidrug-resistant strains isolated from 2001 to 2003 was also detected. This suggests that despite the isolation period, S. suis strains already presented high resistance selection pressure. A slight association of serotype 2/½ with some virulence profiles and PFGE pulsotypes was also identified. Nevertheless, no clonal dispersion or persistency of clones over the analyzed years and herds was detected.

Correction for Ichiwaki et al., "Genome Sequence of Micromonospora sp. NBS 11-29, an Antibiotic and Hydrolytic Enzyme Producer, Isolated from River Sediment in Brazil".

[This corrects the article DOI: 10.1128/genomeA.00552-17.].

Identification through MALDI-TOF mass spectrometry and antimicrobial susceptibility profiling of bacterial pathogens isolated from sow urinary tract infection.

BACKGROUND: Urinary tract infection (UTI) is a common disease in sows due to intensification of pig production. Despite direct economic losses, UTI prevalence and respective microbial identification are still poorly studied. OBJECTIVE: The aims of this study were to identify the causative agents of UTI in sows through MALDI-TOF MS and to characterize their antimicrobial resistance profiles. MATERIALS AND METHODS: Urine samples from 300 sows of three herds from São Paulo State (Brazil) were screened for UTI; suggestive samples were submitted to bacterial isolation. Species identification was performed by MALDI-TOF MS and susceptibility profiles were determined using disc diffusion method. RESULTS: 128 samples suggestive of UTI were analyzed; 48% of the animals presented UTI caused by a single pathogen, while the remaining 52% presented mixed infection. Escherichia coli stood out with the highest frequency among both single and mixed infections. The Gram-positive were exclusively associated with 27% of single infections. The mixed infections were further classified into 49 profiles. The high frequency of multiresistant profiles stood out for most of the studied isolates. CONCLUSIONS: MALDI-TOF MS enabled the identification of rare pathogens related to UTI which may represent higher risk for porcine health, especially considering high frequency of multiresistant profiles.

Characterization of Corynebacterium diphtheriae, C. confusum and C. amycolatum isolated from sows with genitourinary infection.

Porcine Corynebacterium infection is still poorly studied, even though the pig has been described as an asymptomatic carrier of Corynebacterium species, including the zoonotic species C. ulcerans, C. confusum and C. amycolatum. Here we present the identification, molecular and antimicrobial susceptibility characterization of coryneform bacteria isolated from sows with urinary tract infection. C. diphtheriae, C. confusum and C. amycolatum were isolated from sows with urinary infection and metritis. Corynebacterium species were identified by MALDI-TOF MS and confirmed by 16S rRNA and rpoB sequencing. All porcine C. diphtheriae strains were further characterized as non-toxigenic (tox-). SE-AFLP genotyping was also performed and enabled not only Corynebacterium species differentiation but also the assessment of C. amycolatum genetic heterogeneity. All studied porcine Corynebacterium strains presented alarming resistance profiles with high MIC values for macrolides/lincosamide, tetracyclines and quinolones, which can be related with high usage in both veterinary and human medicine. Isolation of zoonotic Corynebacterium species from commercial swine is important for assessing the potential zoonotic risk for farmers and further implication for both human and animal treatment.

Genome Sequence of Micromonospora sp. NBS 11-29, an Antibiotic and Hydrolytic Enzyme Producer, Isolated from River Sediment in Brazil.

The genus Micromonospora comprises actinomycetes with high biotechnological potential, due to their ability to produce secondary metabolites and enzymes. In this study, we report the draft genome sequence of Micromonospora sp. NBS 11-29, which showed antibacterial, cellulolytic, and xylanolytic activities under in vitro conditions.

Persistence of Ascaris spp. Ova in Tropical Soil Cultivated with Eucalyptus and Fertilized with Municipal Biosolids.

In many countries, the main reason for severely restricting or outright banning the land application of class B biosolids is the lack of risk assessment for adverse human health impacts. Among pathogens that are not often studied are helminth ova, including that of the spp. Almost all of the knowledge about the persistence of spp. ova in soils fertilized with biosolids is based on studies developed in North America, Europe, and Asia. These studies have almost always been conducted under temperate climate conditions, which may cause erroneous interpretations when the conclusions are extrapolated to tropical regions such as those found in Brazil. This team evaluated the persistence of viable spp. ova in a sandy Quartzipsamment tropical soil, previously planted with × hybrid () and fertilized with biosolids, over a 52-wk period. During the reporting period, the average temperature of soil and biosolids fluctuated between 15 and 30°C, and the average moisture of biosolids fluctuated between 60 and 90%. The estimated persistence time of viable spp. ova after land application was estimated at close to 7 wk, indicating that ova may not be viable for as long as it has been shown to be in studies of more temperate areas. The relationship of temperature with persistence of viable spp. ova in a tropical soil was stronger than moisture content, suggesting that temperature substantially contributed to their nonviability over the course of the experiment.

Characterization of porcine Trueperella pyogenes by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), molecular typing and antimicrobial susceptibility profiling in Sao Paulo State.

Trueperella pyogenes can be found as a commensal or pathogenic bacterium among animals causing a variety of pyogenic infections in several species. The agent appears to act primarily as an opportunistic pathogen but may disseminate and produce metastatic abscesses accompanied or not by mastitis, metritis or pneumonia. In this study, 30 porcine T. pyogenes strains were identified by MALDI-TOF MS and 16S rRNA sequencing and further evaluated in relation to their resistance and genetic profiles through broth microdilution and single enzyme AFLP. All strains were susceptible to ß-lactams, florfenicol, gentamicin, spectinomycin and tiamulin. The highest resistance rates were observed for sulfonamides, tetracyclines and clindamycin. All isolates could be characterized by SE-AFLP presenting more than 80% of similarity, despite their distinct origins. Four genotypes were detected with the segregation of T. pyogenes ATCC 19411 from Brazilian T. pyogenes strains. No correlation between genotypes and isolates origins and susceptibility profile was observed.

Molecular and antimicrobial susceptibility characterization of Globicatella sulfidifaciens isolated from sow's urinary tract infection.

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.

Evaluation of protein spectra cluster analysis for Streptococcus spp. identification from various swine clinical samples.

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.

Molecular and antimicrobial susceptibility profiling of atypical Streptococcus species from porcine clinical specimens.

The Streptococcus species present broad phenotypic variation, making identification difficult using only traditional microbiological methods. Even though Streptococcus suis is the most important species for the worldwide swine industry, other Streptococcus species appear to be able to cause disease in swine and could represent a higher underestimated risk for porcine health. The aim of this study was to identify Streptococcus-like isolates by MALDI-TOF MS and 16S rRNA sequencing and further molecular and antibiotic susceptibility characterization of the atypical Streptococcus species capable of causing disease in swine. Fifty presumptive Streptococcus isolates from diseased pigs isolated from different Brazilian States between 2002 and 2014 were evaluated. Among the studied isolates, 26% were identified as Streptococcus hyovaginalis, 24% as Streptococcus plurianimalium, 12% as Streptococcus alactolyticus, 10% as Streptococcus hyointestinalis, and the remaining isolates belonged to Streptococcus henryi (6%), Streptococcus thoraltensis (6%), Streptococcus gallolyticus (6%), Streptococcus gallinaceus (4%), Streptococcus sanguinis (4%), and Streptococcus mitis (2%). The Streptococcus isolates were successfully identified by spectral cluster analysis and 16S rRNA sequencing with 96% of concordance between the techniques. The SE-AFLP analysis also supported Streptococcus species distinction and enabled further observation of higher genetic heterogeneity intra-species. The identified Streptococcus species presented variable MIC values to ß-lactams, enrofloxacin and florfenicol, and high resistance rates to tetracyclines and macrolides, which appear to be directly related to the industry's antimicrobial usage and resistance selection.

Molecular and antibiotic susceptibility characterization of Aerococcus viridans isolated from porcine urinary infection.

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.

Pathogenic parasites and enteroviruses in wastewater: support for a regulation on water reuse.

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.

Assessing the infection risk of Giardia and Cryptosporidium in public drinking water delivered by surface water systems in Sao Paulo State, Brazil.

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.

Comparison of thermotolerant coliforms and Escherichia coli densities in freshwater bodies

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.