Methylene blue@silver nanoprisms conjugates as a strategy against Candida albicans isolated from balanoposthitis using photodynamic inactivation.

Balanoposthitis can affect men in immunocompromised situations, such as HIV infection and diabetes. The main associated microorganism is Candida albicans, which can cause local lesions, such as the development of skin cracks associated with itching. As an alternative to conventional treatment, there is a growing interest in the photodynamic inactivation (PDI). It has been shown that the association of photosensitizers with metallic nanoparticles may improve the effectiveness of PDI via plasmonic effect. We have recently shown that the association of methylene blue (MB), a very known photosensitizer, with silver prismatic nanoplatelets (AgNPrs) improved PDI of a resistant strain of Staphylococcus aureus. To further investigate the experimental conditions involved in PDI improvement, in the present study, we studied the effect of MB concentration associated with AgNPrs exploring spectral analysis, zeta potential measurements, and biological assays, testing the conjugated system against C. albicans isolated from a resistant strain of balanoposthitis. The AgNPrs were synthesized through silver anisotropic seed growth induced by the anionic stabilizing agent poly(sodium 4-styrenesulfonate) and showed a plasmon band fully overlapping the MB absorption band. MB and AgNPrs were conjugated through electrostatic association and three different MB concentrations were tested in the nanosystems. Inactivation using red LED light (660 nm) showed a dose dependency in respect to the MB concentration in the conjugates. Using the highest MB concentration (100 µmol⋅L-1) with AgNPr, it was possible to completely inactivate the microorganisms upon a 2 min irradiation exposure. Analyzing optical changes in the conjugates we suggest that these results indicate that AgNPrs are enhancers of MB photodynamic action probably by a combined mechanism of plasmonic effect and reduction of MB dimerization. Therefore, MBAgNPrs can be considered a suitable choice to be applied in PDI of resistant microorganisms.

Effect of rumen-protected choline on dairy cow metabolism, immunity, lactation performance, and vaginal discharge microbiome.

Rumen-protected choline (RPC) promotes benefits in milk production, immunity, and health in dairy cows by optimizing lipid metabolism during transition period management and early lactation. However, the RPC success in dairy cows depends on choline bioavailability, which is affected by the type of protection used in rumen-protected choline. Therefore, our objectives were to determine the effects of a novel RPC on dry matter intake (DMI), identify markers of metabolism and immunity, and evaluate lactation performance. Dry Holstein (n = 48) cows at 245 ± 3 d of gestation were blocked by parity and assigned to control or RPC treatment within each block. Cows enrolled in the RPC treatment received 15 g/d of CholiGEM (Kemin Industries, Cavriago RE, Italy) from 21 d prepartum and 30 g/d of CholiGEM from calving to 21 d postpartum. During the transition period, DMI was measured daily, and blood was sampled weekly for energy-related metabolites such as ß-hydroxybutyrate (BHB), glucose, and nonesterified fatty acids (NEFA), as well as immune function markers such as haptoglobin (Hp) and lipopolysaccharide-binding protein (LPB). Vaginal discharge samples were collected at the calving and 7 d postpartum and stored in microcentrifuge tubes at -80°C until 16S rRNA sequencing. The main responses of body condition score, body weight, DMI, milk yield, milk components, and immune function markers were analyzed using the GLIMMIX procedure of SAS with the effects of treatment, time, parity, and relevant covariates added to the models. The relative abundance of microbiome α-diversity was evaluated by 3 indexes (Chao1, Shannon, and Simpson) and ß-diversity by principal coordinate analysis and permutational multivariate ANOVA. We found no differences in DMI in the pre- and postpartum periods. Cows fed RPC increased the yields of energy- and 3.5% fat-corrected milk and fat yield in primiparous and multiparous cows, with an interaction between treatment and parity for these lactation variables. However, we found no differences in milk protein and lactose up to 150 DIM between treatments. Glucose, NEFA, and BHB had no differences between the treatments. However, RPC decreased BHB numerically (control = 1.07 ± 0.13 vs. RPC = 0.63 ± 0.13) in multiparous on the third week postpartum and tended to reduce the incidence of subclinical ketosis (12.7% vs. 4.2%). No effects for Hp and LPB were found in cows fed RPC. Chao1, Shannon, and Simpson indexes were lower at calving in the RPC treatment than in the Control. However, no differences were found 7 d later for Chao1, Shannon, and Simpson indexes. The vaginal discharge microbiome was altered in cows fed RPC at 7 d postpartum. Fusobacterium, a common pathogen associated with metritis, was reduced in cows fed RPC. Rumen-protected choline enhanced lactation performance and health and altered the vaginal discharge microbiome which is a potential proxy for uterine healthy in dairy cows. The current study's findings corroborate that RPC is a tool to support adaptation to lactation and shed light on opportunities for further research in reproductive health.

Transmission of tuberculosis in an endemic urban setting in Brazil.

SETTING: Two out-patient facilities in São Paulo, Brazil. OBJECTIVE: To study the transmission pattern of tuberculosis (TB) among human immunodeficiency virus (HIV) infected and uninfected persons in a setting endemic for TB. DESIGN: A prospective study comparing HIV-seropositive and -seronegative TB patients identified consecutively between 1 March 1995 and 1 April 1997. The patients were stratified according to their Mycobacterium tuberculosis isolate IS6110 RFLP patterns. Risk factors were sought for infection with an RFLP cluster pattern strain, inferred to represent recent transmission. RESULTS: Fifty-eight (38%) of 151 HIV-seropositive patients and 36 (25%) of 142 HIV-seronegative patients were infected with M. tuberculosis isolates that belonged to cluster patterns (OR 1.84, 95% CI 1.08-3.13). Multidrug-resistant (MDR) strains were isolated from 19 patients, all of whom were HIV seropositive; 12 (63%) of these, and 46 (35%) of 132 drug-susceptible isolates had cluster patterns (OR 3.20, 95% CI 1.08-9.77). CONCLUSION: In a TB-endemic urban setting in Brazil, the proportion of cases resulting from recent transmission appears to be greater among HIV-seropositive than among HIV-seronegative patients. A large proportion of MDR-TB (63%) cases was caused by strains that had cluster RFLP patterns, suggesting recent transmission of already resistant organisms. This type of knowledge regarding TB transmission may help to improve locally appropriate TB control programs.

Association between alpha-hemolysin production and HeLa cell-detaching activity in fecal isolates of Escherichia coli.

Escherichia coli isolates that cause detachment of cell monolayers during in vitro adherence assays (cell-detaching E. coli [CDEC]) were recently reported as a potential new group of enteropathogenic bacteria. In the present study, 269 E. coli isolates from feces of children 1 to 5 years of age were identified as CDEC in a detaching assay developed with HeLa cells. The great majority of these isolates were hemolytic within 3 h of growth on blood agar plates and hybridized with a DNA probe for alpha-hemolysin (93.7%), while most of the non-detaching isolates were hemolytic within 24 h (3.6%) or nonhemolytic (94.8%). E. coli isolates that produced alpha-hemolysin were found in 60 (30%) of 200 children with diarrhea and 47 (24%) of 200 age-matched controls. No statistical significance was found for the differences in alpha-hemolysin production among the matched pairs (P = 0.2). These data suggest that CDEC isolates are not associated with diarrhea in the population studied.

Catalase expression, katG, and MIC of isoniazid for Mycobacterium tuberculosis isolates from São Paulo, Brazil.

The MIC of isoniazid, peroxidase-catalase expression, and the presence of the katG gene for 102 Mycobacterium tuberculosis isolates from patients in São Paulo were compared. Fifty-three isoniazid-resistant and 49 isoniazid-sensitive isolates were analyzed by polymerase chain reaction (PCR) for the presence of katG sequences. All isoniazid-sensitive and 43 (81%) isoniazid-resistant isolates expressed catalase (P = .001). None of isoniazid-sensitive and 4 (7%) of 53 isoniazid-resistant isolates lacked katG sequences. Among 6 isolates with MICs > 50 micrograms/mL, 5 (83%) did not express catalase and 2 lacked katG sequences; only 1 had complete gene deletion shown by Southern blot analysis. These findings indicate a correlation between loss of catalase and isoniazid resistance among highly resistant isolates, but these isolates were a small proportion of resistant clinical M. tuberculosis isolates from São Paulo.

Survey of cytotoxin production among Escherichia coli strains characterized as enteropathogenic (EPEC) by serotyping and presence of EPEC adherence factor (EAF) sequences.

A total of 108 Escherichia coli strains characterized as enteropathogenic (EPEC) by serotyping and the presence of EPEC adherence factor (EAF) sequences were examined for cytotoxin production by cell line assays and colony hybridization with Shiga-like toxin (SLT) probes. Cytolethal distending toxin (CLDT) production was found in three (2.8%) strains belonging to serotype O86:H34, while one O111ab:NM strain hybridized with a SLT-II probe but did not express any cytotoxic activity. All four strains showed localized adherence to HeLa cells and hybridized to an E. coli attaching-effacing gene (eae) probe. The CLDT-producing strains had multiple plasmids and some were present in all strains, including a plasmid of approximately 54 MDa that hybridized with the EAF probe.

Vibrios among patients of good socioeconomic conditions during the cholera epidemic in Recife, Brazil.

Between March and July, 1992, we screened for Vibrio all fecal samples submitted for bacteriologic diagnosis at a private clinical laboratory in Recife. Of 1435 cultures examined only 1 (0.07%) was positive for V. cholerae 01, biovar Eltor, serovar Inaba, but 17 (1.2%) yielded non-cholera Vibrio (V. cholerae non-01; V. fluvialis; V. furnissii, V. parahaemolyticus and Vibrio spp). Thus, V. cholerae 01, differently of other enteropathogenic vibrios, spared individuals of good socioeconomic conditions even during the cholera epidemic, which made hundreds of victims in the neighboring slums.

Use of probes to detect virulence factor DNA sequences in Escherichia coli strains isolated from foods.

Escherichia coli strains were isolated from 96 food samples (32 milks, 4 dairy products, 36 raw meats, 7 meat products, 7 sandwiches and 10 ready-to-eat meals). A total of 306 colonies was submitted to hybridization assays with DNA probes for the following virulence factors: heat-labile toxins (LT-I and LT-II), heat-stable toxins (ST-h and ST-p). Shiga-like toxins (SLT-I and SLT-II), adherence factor of enteropathogenic E. coli (EAF) and invasive factor (INV). Six colonies isolated from 4 food samples hybridized with the probes for LT-II (3 colonies isolated from a milk sample), SLT-I and SLT-II (1 colony isolated from raw bovine meat) or EAF (2 colonies isolated from two raw chicken meat samples).

Shiga-like toxin converting phage of enterohemorrhagic Escherichia coli strain 933.

Production of Shiga-like toxin (SLT) by enterohemorrhagic Escherichia coli (EHEC) is controlled by phage conversion, and specific phages carry either the SLT-I or SLT-II operon. EHEC strain 933 produces both SLT-I and SLT-II. Previous studies demonstrated that the vast majority of phages recovered from strain 993 have hexagonal heads with short tails and encode SLT-II. However, conflicting results were obtained concerning the properties of SLT-I converting phages from strain 933. The present study reexamined the recovery of phages from 933 by various methods and characterized the restriction fragments from strain 933 DNA that hybridized with radiolabeled DNA from the SLT-I converting phage 933J, which has an elongated head with a long tail, and the SLT-II converting phage 933W. In the present study, only SLT-II converting phages like 933W were recovered from strain 933. A set of restriction fragments that hybridized with DNA from phage 933J but not 933W was present both in wild type strain 933 and in the variant 933D, which produces only SLT-I and was shown here to be cured of phage 933W. The sizes of the restriction fragments in strain 933 that were homologous with phage 933J differed, however, from those of phage 933J. These data indicate that the phage we isolated and named 933J probably did not originate from strain 933 as we originally reported. The present evidence demonstrates that strain 933 contains both the SLT-II converting phage 933W and other sequences of DNA homologous with phage 933J that probably represent a defective SLT-I converting phage.

Isolation and characterization of monoclonal antibodies to Shiga-like toxin II of enterohemorrhagic Escherichia coli and use of the monoclonal antibodies in a colony enzyme-linked immunosorbent assay.

The major obstacle in large-scale epidemiological investigations of the incidence of Shiga-like toxin (SLT)-producing Escherichia coli in diarrheal stools is the lack of a rapid, specific test to detect toxin. Enterohemorrhagic E. coli produces elevated levels of SLT-I, SLT-II, or both cytotoxins (also called Verotoxins). SLT-I but not SLT-II can be neutralized by antiserum to purified Shiga toxin and by monoclonal antibodies to the B subunit of SLT-I. In this study, monoclonal antibodies were generated against a crude preparation of SLT-II produced by an E. coli K-12 strain lysogenized with the 933W toxin-converting phage of enterohemorrhagic E. coli 933. Hybridoma culture supernatants were screened for anti-SLT-II antibodies by a cytotoxicity neutralization assay and by an enzyme-linked immunosorbent assay (ELISA). Of 53 ELISA-positive lines, 5 were capable of neutralizing the cytotoxicity of SLT-II but not of SLT-I, Shiga toxin, or a variant of SLT-II produced by E. coli that causes edema disease of swine. All five monoclonal antibodies immunoprecipitated the isolated A subunit of SLT-II but not the B subunit. Of these five neutralizing monoclonal antibodies, four were of the immunoglobulin M class and one belonged to the immunoglobulin G1 subclass. All five lines had kappa light chains. These neutralizing monoclonal antibodies have been used as probes in a colony ELISA to detect SLT-II-positive bacterial colonies. The colony ELISA with these monoclonal antibodies is a specific, sensitive test with potential diagnostic value.

Shiga-like toxin production and attaching effacing activity of Escherichia coli associated with calf diarrhea.

Four hundred twenty-nine isolates of Escherichia coli from calves were tested for the production of HeLa cell cytotoxin(s). Isolates that produced enough cytotoxin to be detected in culture supernatants of iron-depleted broth were considered to produce increased amounts of cytotoxins. Isolates also were tested for homology with a DNA probe for a gene that encodes localized adherence of human enteropathogenic E coli. Four isolates produced increased amounts of cytotoxin that was neutralized by Shiga antitoxin (toxin designated as Shiga-like toxin-I [SLT-I]). A 5th isolate produced increased amounts of cytotoxin (SLT+) that was not neutralized by the Shiga antitoxin, but was neutralized by antitoxin against a variant of SLT (toxin designated as SLT-II). None of the isolates hybridized with the probe for the localized adherence gene. Three of the SLT+ isolates belonged to human enteropathogenic E coli serogroups O26 and O111. All 5 of the SLT+ isolates were from calves with diarrhea, but none of the 5 SLT+ isolates contained genes for classic heat-labile or heat-stable enterotoxins, for K99 fimbriae, or for invasiveness; neither did any of them adhere to HeLa cells in culture. Three of the 5 SLT+ isolates had attaching and effacing activities when inoculated into ligated intestinal loops of rabbits. One of the isolates with attaching and effacing activity in rabbits was originally isolated from a calf with lesions characteristic of those produced by attaching effacing E coli (AEEC). Calves inoculated with this SLT+ AEEC isolate developed focal colonic lesions characteristic of those produced by AEEC, but did not develop diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS)

Two toxin-converting phages from Escherichia coli O157:H7 strain 933 encode antigenically distinct toxins with similar biologic activities.

Escherichia coli O157:H7 strain 933 contains two distinct toxin-converting phages (933J and 933W). The biologic activities and antigenic relationship between the toxins produced by 933J and 933W lysogens of E. coli K-12, as well as the homology of the genes that encode the two toxins, were examined in this study. The 933J and 933W toxins, like Shiga toxin produced by Shigella dysenteriae type 1, were cytotoxic for the same cell lines, caused paralysis and death in mice, and caused fluid accumulation in rabbit ileal segments. The cytotoxic activity of 933J toxin for HeLa cells was neutralized by anti-Shiga toxin, whereas the activity of 933W toxin was not neutralized by this antiserum. In contrast, an antiserum prepared against E. coli K-12(933W) neutralized 933W toxin but not 933J toxin or Shiga toxin. For E. coli 933, most of the cell-associated cytotoxin was neutralized by anti-Shiga toxin, whereas most of the extracellular cytotoxin was neutralized by anti-933W toxin. However, a mixture of these antisera indicated the presence of both toxins in cell lysates and culture supernatants. Among 50 elevated cytotoxin-producing strains of E. coli, we identified 11 strains isolated from cases of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome that produced cell-associated cytotoxins which were neutralized by the 933W antitoxin. Southern hybridization studies showed that the cloned toxin structural genes from phage 933J hybridized with DNA from phage 933W under conditions estimated to allow no more than 26% base-pair mismatch. These findings indicate that E. coli produces two genetically related but antigenically distinct cytotoxins with similar biologic activities which we propose to name Shiga-like toxins I and II. Strains of E. coli that produce elevated levels of Shiga-like toxin I or Shiga-like toxin II, or both, have been associated with the clinical syndromes of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome.

Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli.

Three monoclonal antibodies, designated MAb 16E6, MAb 13C4, and MAb 19G8, were produced which recognize Shiga-like toxin (SLT) from Escherichia coli. All three monoclonal antibodies neutralized the cytotoxicity of E. coli SLT and were able to immunoprecipitate intact labeled toxin with Staphylococcus aureus protein A. The three antibodies were of the G1 heavy and kappa light chain classes. MAb 16E6 bound to the B subunit of SLT in Western blots and also neutralized the lethality of the toxin for mice and the enterotoxicity of the toxin in ligate rabbit ileal loops. The ability of MAb 16E6 to neutralize the cytotoxicity, lethality, and enterotoxicity of E. coli confirms the hypothesis that all three activities are associated with a single toxin. MAb 16E6 and MAb 13C4 also neutralized the cytotoxicity of purified Shiga toxin from Shigella dysenteriae type 1 and Shiga-like toxic activities in crude cell extracts from Shigella flexneri, Vibrio cholerae, Vibrio parahaemolyticus, and Salmonella typhimurium. Thus, Shiga toxin and the SLTs from E. coli, Shigella flexneri, V. cholerae, V. parahaemolyticus, and Salmonella typhimurium share a common B subunit epitope that is involved in neutralization. MAb 13C4 has been successfully used in a colony blot assay for the detection of bacterial colonies which produce high levels of SLT. Sixty-two different strains of bacteria were tested by both the cytotoxicity and colony blot assays for the presence of SLT. The colony blot assay detected all strains of bacteria which produce greater than or equal to 10(5) 50% cytotoxic doses of SLT per ml of sonic lysate. There were no false-positive results among the 62 samples tested.

Atividade comparada da cefoperazona e cinco cefalosporinas frente a 628 amostras de enterobacterias, Pseudomonas e Staphylococcus. / Comparative activity of cefoperazone and five other cephalosporins against 628 strains of Enterobacteriaceae, Pseudomonas and Staphylococcus

CIM50 e CIM90 da cefoperazona e outras cinco cefalosporinas (cefoxitina, cefamandol, cefazolina, cefalexina e cefaloridina) foram analisadas frente a 628 amostras de: Escherichia coli, K. pneumoniae S. enteritidis, Shigella, Enterobacter, P. microbilis, P. morgani, Serratia, Citrobacter, Pseudomonas, S. aureus e S. epidermidis. Este estudo confirmou o amplo espectro de acao da cefoperazona que, ao contrario das outras cefalosporinas, apresentou excelente atividade contra P. aeruginosa. Em relacao as demais amostras, sua atividade foi superior ou semelhante as outras cefalosporinas, com excecao das amostras de S. enteritidis, que foram relativamente resistentes a cefoperazona e as cefalosporinas, com excecao da cefoxitina