In vitro Time Kill of Trimethoprim/Sulfamethoxazole against Stenotrophomonas maltophilia versus Escherichia coli using Cation Adjusted Muller Hinton Broth and ISO-Sensitest Broth

Trimethoprim/sulfamethoxazole (TMP/SMZ) is considered the treatment of choice for infections caused by Stenotrophomonas maltophilia , but limited pharmacodynamic data are available to support current susceptibility breakpoints or guide optimal dosing. Time-kill studies using a TMP/SMZ concentration of 4/40 μg/mL were conducted to compare 4 S. maltophilia with 4 Escherichia coli having the same MICs (0.25/4.75-4/76 μg/mL) in cation adjusted Mueller Hinton Broth (CAMHB) and ISO-Sensitest™ broth (ISO). With the exception of the resistant isolates (4/76 μg/mL), which resulted in regrowth approaching control, TMP/SMZ displayed significantly greater killing for E. coli compared with S. maltophilia at each MIC. Against E. coli , mean changes at 24 hour were -4.49, -1.73, -1.59, and +1.83 log 10 colony forming units (CFU) for isolates with MICs of 0.25/4.75, 1/19, 2/39, and 4/74 μg/mL, respectively. The f AUC/MIC required for stasis, 1-log 10 , and 2-log 10 CFU reductions were 40.7, 59.5, and 86.3, respectively. In contrast, TMP/SMZ displayed no stasis or CFU reductions against any S. maltophilia regardless of MIC, and no pharmacodynamic thresholds were quantifiable. Observations were consistent in both CAMHB and ISO broth. These data add increasing evidence that current TMP/SMZ susceptibility breakpoints against S. maltophilia should be reassessed.

Quantification of Fosfomycin in Combination with Nine Antibiotics in Human Plasma and Cation-Adjusted Mueller-Hinton II Broth via LCMS

Fosfomycin-based combination therapy has emerged as an attractive option in our armamentarium due to its synergistic activity against carbapenem-resistant Gram-negative bacteria (CRGNB). The ability to simultaneously measure fosfomycin and other antibiotic drug levels will support in vitro and clinical investigations to develop rational antibiotic combination dosing regimens against CRGNB infections. We developed an analytical assay to measure fosfomycin with nine important antibiotics in human plasma and cation-adjusted Mueller–Hinton II broth (CAMHB). We employed a liquid-chromatography tandem mass spectrometry method and validated the method based on accuracy, precision, matrix effect, limit-of-detection, limit-of-quantification, specificity, carryover, and short-term and long-term stability on U.S. Food & Drug Administration (FDA) guidelines. Assay feasibility was assessed in a pilot clinical study in four patients on antibiotic combination therapy. Simultaneous quantification of fosfomycin, levofloxacin, meropenem, doripenem, aztreonam, piperacillin/tazobactam, ceftolozane/tazobactam, ceftazidime/avibactam, cefepime, and tigecycline in plasma and CAMHB were achieved within 4.5 min. Precision, accuracy, specificity, and carryover were within FDA guidelines. Fosfomycin combined with any of the nine antibiotics were stable in plasma and CAMHB up to 4 weeks at −80 °C. The assay identified and quantified the respective antibiotics administered in the four subjects. Our assay can be a valuable tool for in vitro and clinical applications.

Formulation of Anti Acne Sheet Mask from Bandotan Leaf Extract (Ageratum conyzoides L.) against Propionibacterium acnes

Acne can occur due to increased sebum excretion, inflammation of the skin triggered by the bacteria Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus. The purpose of this study was to determine the formulation of sheet mask preparations of bandotan leaf extract (Ageratum conyzoides L.) in inhibiting the growth of Propionibacterium acnes bacteria. This type of research is an experimental study which include plant identification, making ethanol extract of bandotan leaves, making sheet mask formulations, evaluating the characteristics of the preparation and testing antibacterial activity using disc paper method using Mueller Hinton Agar media. The extract was carried out by maceration using 70% ethanol as a solvent. Testing the effectiveness of antibacterial by measuring the diameter of the inhibition zone, after that the data were analyzed using the One Way ANOVA statistical test. The results of the evaluation of the preparation showed that the preparation was homogenous, with pH ranging from 4.5 to 6.5 and the preparation did not cause irritation on volunteers skin. The results of the inhibition zone measurements showed that the inhibition zones at 2.5% concentration is 4.7mm, 5% (6.83mm), 7.5% (10.2mm) and positive control (20.57mm). This means that the higher the concentration, the larger the diameter of the inhibition zone obtained. The conclusion in this study is bandotan leaf extract (Ageratum conyzoides L.) can be formulated into anti acne sheet mask which is stable during storage with a strong inhibitory power at 7.5% concentration, which is 10.2 mm. Keywords: Ageratum conyzoides L., bandotan leaf, Propionibacterium acnes, sheet mask

Diagnosis and Stratification of Pseudomonas aeruginosa Infected Patients by Immunochemical Quantitative Determination of Pyocyanin From Clinical Bacterial Isolates

The development of a highly sensitive, specific, and reliable immunochemical assay to detect pyocyanin (PYO), one of the most important virulence factors (VFs) of Pseudomonas aeruginosa, is here reported. The assay uses a high-affinity monoclonal antibody (mAb; C.9.1.9.1.1.2.2.) raised against 1-hydroxyphenazine (1-OHphz) hapten derivatives (PC1; a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids). Selective screening using PYO and 1-OHphz on several cloning cycles allowed the selection of a clone able to detect PYO at low concentration levels. The microplate-based ELISA developed is able to achieve a limit of detection (LoD) of 0.07 nM, which is much lower than the concentrations reported to be found in clinical samples (130 μM in sputa and 2.8 μM in ear secretions). The ELISA has allowed the investigation of the release kinetics of PYO and 1-OHphz (the main metabolite of PYO) of clinical isolates obtained from P. aeruginosa-infected patients and cultured in Mueller–Hinton medium. Significant differences have been found between clinical isolates obtained from patients with an acute or a chronic infection (~6,000 nM vs. ~8 nM of PYO content, respectively) corroborated by the analysis of PYO/1-OHphz levels released by 37 clinical isolates obtained from infected patients at different stages. In all cases, the levels of 1-OHphz were much lower than those of PYO (at the highest levels 6,000 nM vs. 300 nM for PYO vs. 1-OHphz, respectively). The results found point to a real potential of PYO as a biomarker of P. aeruginosa infection and the possibility to use such VF also as a biomarker for patient stratification[2] and for an effective management of these kinds of infections.

ANÁLISE DO EFEITO BACTERIOSTÁTICO DE SEMENTES DE MORINGA FRENTE A ESCHERICHIA COLI

Introdução: Estudos das propriedades da Moringa oleifera revelaram que a planta é rica em compostos bioativos relevantes, possuindo atividade antioxidante, anti-inflamatória e antibacteriana. Bactérias patogênicas como a Escherichia coli (EPEC) são uma das principais responsáveis por infecções alimentares e urinárias. Objetivos: analisar se o extrato aquoso obtido a partir de sementes de moringa é capaz de ter ação bacteriostática frente a bactéria gram-negativa Escherichia coli. Material e métodos: A partir do pó de semente de moringa, foi realizada a pesagem de 1g do mesmo diluído em 10ml de água destilada que foi filtrada em seguida. A partir desse extrato aquoso inicial, realizou-se diluições para adquirir as concentrações de 5%, 10% e 15% de moringa. A cepa microbiológica utilizada foi a Escherichia coli EPEC codificada: IOC 011ab CDC. Para análise do efeito antibacteriano, realizou-se o Teste de Suscetibilidade aos Antimicrobianos por meio da técnica disco-difusão. Para execução do mesmo os discos foram embebidos com 20 uL do extrato de moringa e posicionado em placa de petri contendo ágar Mueller Hinton já semeada com a bactéria. Além dos discos contendo o extrato, posicionou-se discos de antibióticos para análise comparativa. Todos os testes foram executados em triplicata e após o período de incubação (24h na estufa bacteriológica a 37 ºC), os halos de inibição formados ao redor dos discos foram medidos com halômetro e os dados obtidos a partir da média dos halos de cada triplicata tabelados em planilha do Excel. Resultados: As médias dos halos corresponderam a 10mm na diluição de 5%; 10,66mm na diluição de 10% e 0mm na diluição a 15%. E para o controle positivo utilizado (Gentamicina), apresentou halo de 24mm. Conclusão: O extrato aquoso de moringa mostrou um potencial efeito bacteriostático frente a Escherichia coli, apresentando melhores resultados na diluição a 10%.

ATIVIDADE ANTIMICROBIANA DO EXTRATO DE PÓLEN DA ABELHA MELIPONA SCUTELLARIS FRENTE A BACTÉRIA KLEBSIELLA PNEUMONIAE

Introdução: O pólen apícola é produzido pelas abelhas mediante a aglutinação do pólen das flores misturado ao néctar e às substâncias produzidas por suas glândulas salivares. O pólen pode ser considerado um alimento funcional devido ao seu alto valor nutritivo e teor de compostos fenólicos que lhe conferem atividades antioxidantes e são capazes de reduzir o risco de doenças crônicas, dentre outras propriedades bioativas. Objetivo: Diante do exposto, objetivou-se neste estudo in vitro investigar o comportamento antimicrobiano do pólen de M. scutellaris em ensaios microbiológicos frente a bactéria Klebsiella pneumoniae. Material e Métodos: As análises microbiológicas foram executadas no Laboratório de Microbiologia Clínica e Experimental do Polo Tecnológico Agroalimentar do município de Arapiraca – AL a partir do extrato hidroalcoólico de pólen apícola nas diluições de 100%, 15%, 10% e 5% que foram depositadas no volume de 20µL em discos de papel-filtro e sobrepostos em placas de Petri em triplicata semeadas com K. pneumoniae proveniente do Programa Nacional de Controle de Qualidade (PNCQ) e em meio ágar Mueller-Hinton solidificado, através da técnica de difusão em disco. Resultados: Os resultados foram obtidos mediante leitura dos halos de inibição formados com o desenvolvimento das colônias após o período de incubação das placas (24h a 37 ºC), os quais demonstraram atividade positiva bacteriostática do extrato frente a cepa bacteriana analisada. Conclusão: Desta forma, foi possível concluir que o pólen apícola de M. scutellaris pode ser uma nova alternativa na área farmacológica para o tratamento de patógenos microbianos de interesse clínico e sugere-se a realização de novos estudos com outras cepas bacterianas.

Effect of pH on the Electrochemical Behavior of Hydrogen Peroxide in the Presence of Pseudomonas aeruginosa

The influence of pH on the electrochemical behavior of hydrogen peroxide in the presence of Pseudomonas aeruginosa was investigated using electrochemical techniques. Cyclic and square wave voltammetry were used to monitor the enzymatic activity. A modified cobalt phthalocyanine (CoPc) carbon electrode (OPG), a known catalyst for reducing O2 to H2O2, was used to detect species resulting from the enzyme activity. The electrolyte was a sterilized aqueous medium containing Mueller-Hinton (MH) broth. The open-circuit potential (OCP) of the Pseudomonas aeruginosa culture in MH decreased rapidly with time, reaching a stable state after 4 h. Peculiarities in the E / I response were observed in voltammograms conducted in less than 4 h of exposure to the culture medium. Such particular E/I responses are due to the catalase’s enzymatic action related to the conversion of hydrogen peroxide to oxygen, confirming the authors’ previous findings related to the behavior of other catalase-positive microorganisms. The enzymatic activity exhibits maximum activity at pH 7.5, assessed by the potential at which oxygen is reduced to hydrogen peroxide. At higher or lower pHs, the oxygen reduction reaction (ORR) occurs at higher overpotentials, i.e., at more negative potentials. In addition, and to assess the influence of bacterial adhesion on the electrochemical behavior, measurements of the bacterial-substrate metal interaction were performed at different pH using atomic force microscopy.

ISOLATION METHODS FOR MOLECULAR DETECTION AND ANTIBIOTIC RESISTANCE PATTERN OF CAMPYLOBACTER SPP IN LAYER CHICKENS

This study was conducted to compare two culture methods for the isolation of Campylobacter spp from commercial layer chickens and subsequently confirmed by Polymerase Chain Reaction assays (PCR). Furthermore, the antimicrobial resistance profiles of PCR positive Campylobacter isolates were determined.Cloacal swab samples (550) from chickens randomly selected from five poultry farms in the four geographical zones in Ogun State were cultured for Campylobacter using modified charcoal Cefoperazone deoxycholate agar (MCCDA) and an improved culture method involving Preston broth pre-enrichment and subsequent subculture on Mueller Hinton agar with Campylobacter growth supplements. Putative isolates were later confirmed by PCR assay and sequencing analysis.Other isolates that grew on MCCDA and confirmed by sequencing analysis are Enterococcus faecalis, Escherichis coli, Comamonas kerstli and Pseudomonas aeroginusa . The antibiotic resistant profile of all the isolates were evaluated genotypically for resistance genes to tetracyclines (tetO), multiclasses (cmeB), aminoglycosides (aphA-3-1) and β-lactams (Blaoxa-61) using multiplex PCR (mPCR), and phenotypically for chlortetracycline, tylosin, streptomycin, ciprofloxacin and erythromycin resistance by microbroth dilution method which correspond to the antibiotic resistance genes. The apparent prevalence of Campylobacter was 16.8% by MCCDA while none of the isolates was positive to PCR. Meanwhile, prevalence rate of 26% was obtained using Preston broth pre-enrichment and Mueller Hinton agar with Campylobacter growth supplements, of which 11/50 (22%) of the isolates was confirmed positive by PCR. Genotypic characterization of PCR positive isolates showed 10/11(90%) were C. coli, 1/11(10%) other Campylobacter species and 0% C. jejuni. All the isolates carried both tetO and cmeB resistant genes. The results of minimum inhibitory concentration presented all PCR positive isolates had resistance of 10/10(100%), 9/10(90%), 6/10(60%), 9/10(90%), and 8/10(80%) to tetracycline, ciprofloxacin, erythromycin, spectinomycin and tylosin respectively. In addition, all isolates carried multiple resistance to most antibiotics tested which are commonly used in poultry practice in Nigeria. Campylobacter spp in the study areas showed diverse genotypic characteristics, and gene mediated multidrug resistance.

Homeopathy: a possible weapon against multidrug-resistant bacteria to antibiotics

Background: The antimicrobial resistance is a genetic phenomenon, related to the existence of the gens restrained in microorganism that codify different biochemical mechanisms that obstruct the drugs actions. Some species present resistance widespread in all over the world, like the case of Staphylococcus aureus. This is one of the main bacteria that, in a period of time, has got multiple resistance against the antibiotics, and it’s also, an important agent causative of the nosocomiais infections. The present report evaluated the action of the different homeopathic medicines about the growth of the bacteria Staphylococcus aureus and Staphylococcus aureus MRSA (Methicillin-resistant Staphylococcus aureus) “in vitro”. Methods: Doses of 150, 250 and 350 µL of the homeopathic medicines Silicea, Hepar sulfor, Belladona, Arnica montana, Mercurio solubilis and nosode of Stafilococcus aureus, in the dynamism 6cH, 12cH e 30cH had been placed in 3mL culture liquid Mueller Hinton. It was added to this blend 10 µL of a diluted bacterial solution 1/10, where of the solution in 0,5 of the Macfarley scale in 37°C, the growth in the tubes was evaluated in Spectrophotometric of 600 nm. Results: The results demonstrated that, for the Staphilococcus aureus, we have got significant bacteria inhibition in about 70 to 90% of the growth “in vitro”, provided by the homeopathic medicines Hepar sulfor in the dynamism of 30cH, Belladona in the dynamisms of 6cH and 30cH, in the Staphilococcus aureus nosode in the dynamisms 6cH and 30cH and Silicea in the dynamism of CH6, with regard to the control with alcohol 30%. The Staphilococcus aureus MRSA presented inhibition from 40% to 20% of the bacteria growth “in vitro” related to the control with alcohol 30%, with the same medicines used before. Conclusion: We can conclude that the homeopathic medicines have an inhibitory action in the bacteria growth, including in bacteria resistance to the antibiotics. This information can suggest that a concerted action of antibiotics and homeopathic medicines, could improve the action of the antibiotics in the bacteria causative of infections in the biological tissues.

In vivo Pharmacokinetic/Pharmacodynamic (PK/PD) Profiles of Tulathromycin in an Experimental Intraperitoneal Haemophilus parasuis Infection Model in Neutropenic Guinea Pigs

Tulathromycin is a semi-synthetic macrolide antimicrobial that has an important role in veterinary medicine for respiratory disease. The objective of the study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model to examine the efficacy and determine an optimal dosage of tulathromycin intramuscular (IM) treatment against Haemophilus parasuis infection induced after intraperitoneal inoculation in neutropenic guinea pigs. The PKs of tulathromycin in serum and lung tissue after intramuscular administration at doses of 1, 10, and 20 mg/kg in H. parasuis-infected neutropenic guinea pigs were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The tulathromycin minimum inhibitory concentration (MIC) against H. parasuis was ~16 times lower in guinea pig serum (0.03 μg/mL) than in cation-adjusted Mueller-Hinton broth (CAMHB) (0.5 μg/mL). The ratio of the 168-h area under the concentration-time curve (AUC) to MIC (AUC168h/MIC) positively correlated with the in vivo antibacterial effectiveness of tulathromycin (R2 = 0.9878 in serum and R2 = 0.9911 in lung tissue). The computed doses to achieve a reduction of 2-log10 CFU/lung from the ratios of AUC72h/MIC were 5.7 mg/kg for serum and 2.5 mg/kg for lung tissue, which lower than the values of 13.2 mg/kg for serum and 8.9 mg/kg for lung tissue with AUC168h/MIC. In addition, using as objective a 2-log10 reduction and an AUC0−72h as the value of the PK/PD index could be more realistic. The results of this study could provide a solid foundation for the application of PK/PD models in research on macrolide antibiotics used to treat respiratory diseases.