Maximum inhibitory dilution of mouthwashes containing chlorhexidine and polyhexamethylene biguanide against salivary staphylococcus aureus

OBJECTIVE: The aim of the present study was to determine the in vitro maximum inhibitory dilution (MID) of two chlorhexidinebased oral mouthwashes (CHX): Noplak®, Periogard®, and one polyhexamethylene biguanide-based mouthwash (PHMB): Sanifill Premium® against 28 field Staphylococcus aureus strains using the agar dilution method. MATERIALS AND METHODS: For each product, decimal dilutions ranging from 1/10 to 1/655,360 were prepared in distilled water and added to Mueller Hinton Agar culture medium. After homogenization, the culture medium was poured onto Petri dishes. Strains were inoculated using a Steers multipoint inoculator and dishes were incubated at 37ºC for 24hours. For reading, MID was considered as the maximum dilution of the mouthwash still capable of inhibiting microbial growth. RESULTS: Sanifill Premium® inhibited the growth of all strains at 1/40 dilution and of 1 strain at 1/80 dilution. Noplak® inhibited the growth of 23 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Periogard® showed inhibited growth of 7 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Data were submitted to Kruskal-Wallis statistical test, showing significant differences between the mouthwashes evaluated (p<0.05). No significant difference was found between Noplak® and Periogard® (p>0.05). Sanifill Premium® was the least effective (p<0.05). CONCLUSION: It was concluded that CHX-based mouthwashes present better antimicrobial activity against S. Aureus than the PHMB-based mouthwash.

Determination of the maximum inhibitory dilution of cetylpyridinium chloride-based mouthwashes against staphylococcus aureus: an in vitro study

The aim of this in vitro study was to determine the maximum inhibitory dilution (MID) of four cetylpyridinium chloride (CPC)-based mouthwashes: CPC+Propolis, CPC+Malva, CPC+Eucaliptol+Juá+Romã+Propolis (Natural Honey®) and CPC (Cepacol®), against 28 Staphylococcus aureus field strains, using the agar dilution method. Decimal dilutions ranging from 1/10 to 1/655,360 were prepared and added to Mueller Hinton Agar. Strains were inoculated using Steers multipoint inoculator. The inocula were seeded onto the surface of the culture medium in Petri dishes containing different dilutions of the mouthwashes. The dishes were incubated at 37ºC for 24 h. For readings, the MID was considered as the maximum dilution of mouthwash still capable of inhibiting microbial growth. The obtained data showed that CPC+Propolis had antimicrobial activity against 27 strains at 1/320 dilution and against all 28 strains at 1/160 dilution, CPC+Malva inhibited the growth of all 28 strains at 1/320 dilution, CPC+Eucaliptol+Juá+Romã+Propolis inhibited the growth of 2 strains at 1/640 dilution and all 28 strains at 1/320 dilution, and Cepacol® showed antimicrobial activity against 3 strains at 1/320 dilution and against all 28 strains at 1/160 dilution. Data were submitted to Kruskal-Wallis test, showing that the MID of Cepacol® was lower than that determined for the other products (p<0.05). In conclusion, CPC-mouthwashes showed antimicrobial activity against S. aureus and the addition of other substances to CPC improved its antimicrobial effect.

Antibacterial activity of four mouthrinses containing triclosan against salivary Staphylococcus aureus

The maximum inhibitory dilution (MID) of triclosan-based mouthwashes against 28 Staphylococcus aureus strains was evaluated. Dilutions ranging from 1/10 to 1/655,360 were prepared. Strains were inoculated using a Steers multipoint inoculator. The MID was considered as the maximum dilution capable of inhibiting microorganism growth. The mouthwashes presented different MIDs.

Occurrence of Staphylococcus aureus in raw milk produced in dairy farms in São Paulo state, Brazil

The objective of the present study was to evaluate the occurrence of Staphylococcus aureus in milk produced in 37 farms located in the regions of Ribeirão Preto and São Carlos, state of São Paulo, Brazil. Two-hundred and eight samples of milk from individual cows showing subclinical mastitis, and 37 samples of bulk tank milk were analyzed. S. aureus strains were detected in 18 (7.3%) milk samples: 14 (6.7%) from samples of individual cows, and 4 (10.8%) from bulk tank milk. Two individual milk samples (14.3%) and two bulk milk samples contained enterotoxigenic S. aureus. PFGE analysis revealed the genetic heterogeneity of the strains isolated from raw milk, which presented to 13 S. aureus patterns. Results confirmed the potential transmission of staphylococcal food poisoning to consumers via milk of cows affected by subclinical mastitis, mainly when raw milk is ingested.

Perfil de sensibilidade microbiana in vitro de linhagens de Staphylococcus spp. isoladas de vacas com mastite subclínica

Objetivou-se com este estudo avaliar a sensibilidade antimicrobiana in vitro de 291 isolados de Staphylococcus spp. recuperados de amostras de leite de vacas com mastite subclínica, em 15 propriedades rurais localizadas na Região Metropolitana do Recife (A), Agreste (B) e Zona da Mata (C) do estado de Pernambuco. Dos 291 isolados, 170(58,4%) foram classificados como Staphylococcus coagulase negativa (SCN), 84(28,9%) como Staphylococcus aureus e 37(12,7%) como Staphylococcus coagulase positiva (SCP). Para o estudo do perfil de sensibilidade a antimicrobianos empregou-se a técnica de difusão em discos, foram avaliadas 16 drogas antimicrobianas utilizadas no tratamento das mastites. O antibiótico que apresentou melhor eficácia in vitro foi a associação entre neomicina + bacitracina + tetraciclina com percentuais de 98,4%, 99,3%, 89,7% para as regiões A, B e C, respectivamente. O antibiótico menos eficaz foi a ampicilina que apresentou 56,5% de resistência para as amostras da região A, 72,8% para a região B e 71,8% na região C. Os resultados obtidos mostram a necessidade da realização periódica de testes de sensibilidade in vitro, pois existem variações no perfil de sensibilidade e resistência que podem comprometer o tratamento do animal bem como os programas de controle da mastite bovina causada pelo Staphylococcus spp.

Seroprevalence of Rickettsia bellii and Rickettsia felis in dogs, São José dos Pinhais, State of Paraná, Brazil

Brazilian spotted fever (BSF) is a vector-borne zoonosis caused by Rickettsia rickettsii bacteria. Dogs can be host sentinels for this bacterium. The aim of the study was to determine the presence of antibodies against Rickettsia spp. in dogs from the city of São José dos Pinhais, State of Paraná, Southern Brazil, where a human case of BSF was first reported in the state. Between February 2006 and July 2007, serum samples from 364 dogs were collected and tested at 1:64 dilutions by indirect immunofluorescence assay (IFA) against R. rickettsii and R. parkeri. All sera that reacted at least to one of Rickettsia species were tested against the six main Rickettsia species identified in Brazil: R. rickettsii, R. parkeri, R. bellii, R. rhipicephali, R. amblyommii and R. felis. Sixteen samples (4.4%) reacted to at least one Rickettsia species. Among positive animals, two dogs (15.5%) showed suggestive titers for R. bellii exposure. One sample had a homologous reaction to R. felis, a confirmed human pathogen. Although Rickettsia spp. circulation in dogs in the area studied may be considered at low prevalence, suggesting low risk of human infection, the present data demonstrate for the first time the exposure of dogs to R. bellii and R. felis in Southern Brazil.

Avaliação in vitro da eficácia de desinfetantes comerciais utilizados no pré e pós-dippingfrente amostras de Staphylococcus spp. isoladas de mastite bovina

Objetivou-se com este estudo avaliar a sensibilidade in vitro de Staphylococcus spp.frente a alguns desinfetantes comerciais utilizados no pré e pós-dipping em vacas leiteiras. Foram testados um total de 60 isolados de Staphylococcus spp. identificados como S. aureus (50) e Staphylococcus coagulase positiva (10) recuperados de glândulas mamárias de vacas com mastite subclínica procedentes das regiões Metropolitana do Recife, Agreste e Zona da Mata do Estado de Pernambuco. O estudo da eficácia dos desinfetantes utilizados no pré e pós-dipping foi realizado utilizando-se os seguintes princípios ativos: cloro (2,5%), iodo (0,57%), clorexidine (2,0%), amônia quaternária (4,0%) e ácido lático (2,0%) em quatro tempos distintos (15", 30", 60" e 300"). Observou-se que 100% de S. aureus foram sensíveis ao iodo, 93,3% sensíveis a clorexidine, 80% sensíveis a amônia, 35,6% sensíveis ao ácido lático e 97,8% resistentes ao cloro no tempo de 60". Com relação a Staphylococcus coagulase positiva (SCP), 100% dos isolados foram sensíveis ao iodo, 81,8% sensíveis a amônia quaternária, 99,9% sensíveis ao ácido lático, 72,7% sensíveis a clorexidine e 100% resistentes ao cloro no tempo de 60". Conclui-se que a maior atividade desinfetante in vitro foi verificada para o iodo e clorexidine frente a S. aureus e do iodo e ácido lático frente aos SCP e que há necessidade de avaliação periódica dos desinfetantes utilizados nas propriedades leiteiras nas regiões estudadas, pois, existem variações no perfil de sensibilidade e resistência aos desinfetantes que podem comprometer os programas de controle da mastite bovina causada por Staphylococcus spp.

First Report of the Isolation and Molecular Characterization of Rickettsia amblyommii and Rickettsia felis in Central America

During 2010, 15 adult ticks, identified as Amblyomma cajennense, were collected from horses in Cahuita and Turrialba districts, whereas 7 fleas, identified as Ctenocephalides felis, were collected from a dog in San Jose city, Costa Rica. In the laboratory, three A. cajennense specimens, two from Cahuita and one from Turrialba, were individually processed for rickettsial isolation in cell culture, as was a pool of seven fleas. Rickettsiae were successfully isolated and established in Vero cell culture from the three ticks and from a pool of seven fleas in C6/36 cell culture. The three tick isolates were genotypically identified as Rickettsia amblyommii, and the flea isolate was identified as Rickettsia felis through DNA sequencing of portions of the rickettsial genes gltA, ompA, and ompB of each isolate. In addition, other seven ticks were shown to contain rickettsial DNA. Polymerase chain reaction products of at least two of these ticks were sequenced and also showed to correspond to R. amblyommii. Overall, 66.7% (10/15) of the A. cajennense adult ticks were found to be infected with rickettsiae. This is the first report of a successful isolation in cell culture of R. amblyommii and R. felis from Central America.

Experimental Infection of the Opossum Didelphis aurita by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense and Amblyomma dubitatum

This work evaluated the infection of opossums (Didelphis aurita) by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and their role as amplifier hosts for horizontal transmission to Amblyomma cajennense and/or Amblyomma dubitatum ticks. Infection in D. aurita was induced by intraperitoneal inoculation with R. felis (n = 4 opossums), R. bellii (n = 4), and R. parkeri (n = 2). Another group of six opossums were inoculated intraperitoneally with Leibovitz-15 sterile culture medium, representing the uninfected groups (n = 2 opossums simultaneously to each infected group). Opossum blood samples collected during the study were used for DNA extraction, followed by real-time polymerase chain reaction targeting the rickettsial gene gltA, hematology, and detection of Rickettsia spp.-reactive antibodies by indirect immunofluorescence assay. Opossums were infested with uninfected A. cajennense and/or A. dubitatum for 30 days postinoculation (DPI). Flat ticks molted from ticks fed on opossums were allowed to feed on uninfected rabbits, which were tested for seroconversion by immunofluorescence assay. Samples of flat ticks were also tested by real-time polymerase chain reaction. Inoculated opossums showed no clinical abnormalities. Antibodies to Rickettsia spp. were first detected at the second to fourth DPI, with detectable titers until the 150th DPI. Rickettsemia was detected only in one opossum inoculated with R. parkeri, at the eighth DPI. Only one A. cajennense tick (2.0%) previously fed on a R. parkeri-inoculated opossum became infected. None of the rabbits infested with opossum-derived ticks seroconverted. The study demonstrated that R. felis, R. bellii, and R. parkeri were capable to produce antibody response in opossums, however, with undetectable rickettsemia for R. felis and R. bellii, and very low rickettsemia for R. parkeri. Further studies must be done with different strains of these rickettsiae, most importantly the strains that have never gone through in vitro passages.

Novel sequence types (STs) of Staphylococcus aureus isolates causing clinical and subclinical mastitis in flocks of sheep in the northeast of Brazil

Staphylococcus aureus is one of the most important infectious mastitis causative agents in small ruminants. In order to know the distribution of Staph. aureus strains associated with infectious mastitis in flocks of sheep in the northeast of Brazil and establish whether these clones are related to the strains distributed internationally, this study analysed the genetic diversity of Staph. aureus isolates from cases of clinical and subclinical mastitis in ewes by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In this research, 135 ewes with mastitis from 31 sheep flocks distributed in 15 districts were examined. Staph. aureus was isolated from sheep milk in 9 (29%) out of 31 herds located in 47% of the districts surveyed. MLST analysis allowed the identification of four STs (ST750, ST1728, ST1729 and ST1730). The last three with their respective novel alleles (g/p-220; pta-182 and yqil-180) were recently reported in the Staph. aureus MLST database (http://www.mlst.net). Each novel allele showed only a nucleotide different from those already described. The occurrence of CC133 (ST750 and ST1729) in this study is in agreement with other reports that only a few clones of Staph. aureus seem to be responsible for most cases of mastitis in dairy farms and that some of these clones may have broad geographic distribution. However, the prevalence of CC5 (ST1728 and ST1730)-an important group related to cases of colonization or infection in humans-differs from previous studies by its widespread occurrence and may suggest human contamination followed by selective pressures of the allelic diversifications presented for these STs.

Novel benzofuroxan derivatives against multidrug-resistant Staphylococcus aureus strains: Design using Topliss` decision tree, synthesis and biological assay

The aim of this study was the design of a set of benzofuroxan derivatives as antimicrobial agents exploring the physicochemical properties of the related substituents. Topliss` decision tree approach was applied to select the substituent groups. Hierarchical cluster analysis was also performed to emphasize natural clusters and patterns. The compounds were obtained using two synthetic approaches for reducing the synthetic steps as well as improving the yield. The minimal inhibitory concentration method was employed to evaluate the activity against multidrug-resistant Staphylococcus aureus strains. The most active compound was 4-nitro-3-(trifluoromethyl)[N`-(benzofuroxan-5-yl) methylene] benzhydrazide (MIC range 12.7-11.4 mu g/mL), pointing out that the antimicrobial activity was indeed influenced by the hydrophobic and electron-withdrawing property of the substituent groups 3-CF(3) and 4-NO(2), respectively. (C) 2011 Elsevier Ltd. All rights reserved.

Identification of Staphylococcus aureus Carrying the mecA Gene in Ready-to-Eat Food Products Sold in Brazil

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.

Design, synthesis, antimicrobial activity and molecular modeling studies of novel benzofuroxan derivatives against Staphylococcus aureus

Molecular modi. cation is a quite promising strategy in the design and development of drug analogs with better bioavailability, higher intrinsic activity and less toxicity. In the search of new leads with potential antimicrobial activity, a new series of 14 4-substituted [N`-(benzofuroxan-5-yl) methylene] benzohydrazides, nifuroxazide derivatives, were synthesized and tested against standard and multidrug-resistant Staphylococcus aureus strains. The selection of the substituent groups was based on physicochemical properties, such as hydrophobicity and electronic effect. These properties were also evaluated through the lipophilic and electrostatic potential maps, respectively, considering the compounds with better biological pro. le. Twelve compounds exhibited similar bacteriostatic activity against standard and multidrug-resistant strains. The most active compound was the 4-CF(3) substituted derivative, which presented a minimum inhibitory concentration (MIC) value of 14.6-13.1 mu g/mL, and a ClogP value of 1.87. The results highlight the benzofuroxan derivatives as potential leads for designing new future antimicrobial drug candidates. (C) 2009 Elsevier Ltd. All rights reserved.

Staphylococcus hominis subsp novobiosepticus strains causing nosocomial bloodstream infection in Brazil

To report the isolation of six Staphylococcus hominis subsp. novobiosepticus (SHN) strains from hospitalized patients with bloodstream infections in two Brazilian hospitals and to characterize their susceptibility profile to several antimicrobials. Species identification was performed by biochemical methods and sodA gene sequencing. The MICs of antimicrobials were determined by broth and agar dilution methods and by Etest. Isolates were typed by PFGE and PCR amplification was used to detect the ccr gene complex and the mec class. Morphometric evaluation of cell wall was performed by transmission electron microscopy (TEM). Susceptibility profiles indicated that the majority of isolates (five) were multidrug-resistant. Overlapping and multiplex PCR showed that five out of the six strains harboured SCCmec type III with class A mec and type 3 ccr. The initial vancomycin MIC value of 4 mg/L for these strains increased to 16-32 mg/L after growth for 10 days in BHI broth supplemented with this antimicrobial. TEM indicated that vancomycin resistance was associated with cell wall thickening and to another mechanism not fully elucidated. Only one SHN strain was oxacillin- and vancomycin-susceptible. The nosocomial infections in at least five of the patients from both hospitals were caused by a single clone of SHN. It is very important to consider SHN strains as the cause of nosocomial infections. The clinical implications resulting from the pattern of multidrug resistance in these strains may be complicated by the emergence of vancomycin resistance.