Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity

The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0-10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 hours. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (> 5 log) and more than 4 logs P. aeruginosa and Salmonella Typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine.

Caracteres culturais das bactérias dos nódulos de algumas leguminosas tropicais

23 isolates from root nodules of 13 leguminous species were tested in laboratory for cultural and physiological characteristics. The following conclusions can be drawn: 1 - The cultural and physiological characters were not enough to identify the different isolates. 2 - The data from cultural and physiological tests help the identification of the bacteria, when checked with data from cross-inoculation tests reported elsewhere (GALLI, 1958). The bacteria studied herein may be grouped as the following: a) nodule bacteria from Vigna sinensis, Calopogonium muconoides, Centrosema pubescens, Desmodium adscendens, D. discolor, Indigofera mucronata, I. subulata, I. sumatrana, Stylosanthes guyanensisA Tephrosia cândida and Teramnus uncinatus: bacteria with scanty growth; alcaline reaction in milk-brom cresol purple; no acid production in medium with any of the carbohydrates used. b) nodule bacteria from pea and from Vicia obscura: bacteria with moderate growth; alcaline reaction, with serum zone, in milk-brom cresol purple; acid production in medium with all the carbohydrates used. c) nodule bacteria from Leucaena glauca: bacteria with moderate growth; alcaline reaction, with serum zone, in milk-brom cresol purple; acid production from rafinose and manitol. d) nodule bacteria from Cratylia floribunda: bacteria with scanty growth; alcaline reaction in milk-brom cresol purple; acid production from mannose and ramnose.

Nematódeos e bactérias em fôlhas de alcachôfra

Diseased plants of artichoke (Cynara scolimus L.) from the Instituto Agronômico, Campinas, Brazil, harboured nematodes in the leaves. The nematodes proved to belong to the species Protorhabditis oxyuris (Claus, 1862) Dougherty, 1955. As it could not be charged as the primary agent of the disease, a study of the bacteria found in the material was made. This disclosed that 4 different bacterial forms were present, namely Bacillus subtilis, Bacterium udum, Xanthomonas beticola var. cynarae n. var., and Cory neb acterium fascians var. cynarae n. var. The typical forms of X. beticola and of C. fascians are known as pathogenic to a number of plants: but, in the present case, unfortunately no inoculation trial could be made as an attempt to clear up the possible pathogenicity of any of them also to Cynara scolimus. The nematode P. oxyuris is the secondary agent, which however possibly play an important part in the distribution of the bacteria, as it has been repeatedly pointed out by previous writers with reference to closely related species. The symptoms of the disease are described. The main injuries found on leaves were necrotic areas progressing from the distal part of the limb, resulting in a great reduction of the leaf superficies.

Fixação do dióxido de carbono por bactérias nitrificantes

During the oxidation of the substrate, both Nitrosomonas and Nitrobacter have part of the energy made available as high energy phosphate, mamely ADP and ATP. This chemical energy is used to fix CO2. The nature of the reducing power is unknown at present. Active cells of Nitrobacter were shown to fix CO2 along the same pathway as found in higher plant photosynthesis. Sonic extracts of Nitrosomonas and Nitrobacter when incubated with NaH14CO3 and cofactors showed two ports of entry of CO2 into organic compounds one being, as expected, the carboxidismutase reaction. On protein basis an equivalent amount of CO2 was, however, incorporated via the oxaloacetic carboxylase reation. It is clear then that both micoorganisms possess typical autotrophic and heterotrophic mechanisms for the fixation of CO2 which is required for the primary synthesis of cell material.

Estudos sôbre a possível ação mutagênica de antibióticos em bactérias

Foram estudados 4 antibióticos (penicilina, estreptomicina, cloranfenicol e aureomicina) com relação a indução de mutações em uma linguagem de S. tuphimurium deficiente para triptofano e uma linhagem de X. campestris deficiente para o mesmo amino-ácido. Não foi verificada atividade mutagênica em nenhum dos antibióticos, em relação aos dois fatores estudados.

Efeitos de doses crescentes de nitrogênio e da inoculação de bactérias fixadoras de nitrogênio atmosférico, na cultura da soja (Glycine max, Merril)

Para estudar os efeitos de doses crescentes de nitrogênio, na presença e ausencia de Rhizobium, na cultura da soja, foram instalados ensaios em dois solos arenosos pobres em mate ria orgânica dos municipios de Herculandia e de Regente Feijó. No ensaio de Herculandia constatou-se efeitos de Rhizobium, enquanto no de Regente Feijo houve efeitos linear e cúbico das doses de nitrogênio, nao sendo observado efeito de inoculação. Em ambos os casos notaram-se cloroses nos tratamentos sem nitrogênio, todavia no ensaio de Regente Feijo foi temporária enquanto no de Herculândia a clorose persistiu ate o fim do ciclo nas parcelas sem inoculaçao e sem nitrogênio.

Microbiologia da carne moida: 1. contagem total de bactérias

Como primeira etapa de um programa de estudos microbiológicos da carne moida comercializada em Piracicaba, quarenta amostras de carne bovina, provenientes de dois tipos de estabelecimento de venda a varejo (açougue e supermercado), correspondendo a dois sistemas de distribuição de carne (um antigo e outro recente), foram analisados quanto ao número total de bactérias, segundo as recomendações da "American Public Health Association". As amostras foram obtidas semanalmente pela manhã e à tarde, durante aproximadamente dois meses. A incubação das placas de Petri foi feita a 21º C (72 horas) e a 32º (48 horas). Os resultados podem ser resumidos como segue: 1) Contagens totais elevadas foram, em geral, observadas, com diversas amostras na faixa de 10(7) a 10(9) bactérias/grama; as médias obtidas foram 6,9 x 10(7) (21ºC) e 2,5 x 10(7) (32ºC) bactérias por grama. 2) A incubação feita a 21ºC resultou, em geral, em contagens mais elevadas que as correspondentes à temperatura de incubação de 32ºC; todavia, a diferença observada não foi estatìsticamente significativa. 3) As contagens obtidas para as amostras correspondentes ao sistema antigo de distribuição de carne foram significativamente mais elevadas que as correspondentes ao sistema recente, o que se relacionou com contagens mais elevadas obtidas para as amostras coletadas pela manhã no estabelecimento menor.

Contagem total de bactérias e enumeração de coliformes em cortes de varejo de carne bovina e em equipamentos de supermercados

No presente trabalho foi determinada a contagem total de bactérias (incubação a 32°C e a 5°C) existentes na superfície de alguns cortes de varejo de carne bovina comercializada por tres supermercados de Piracicaba, SP, bem como da superfície de diversos tipos de equipamento utilizado nas salas de desossa desses estabelecimentos. Foi determinado também o número mais provável (NMP) de coliformes totais e, para um grupo de amostras de carne, o NMP de Escherichia coli. Para um dos supermercados (o mais antigo e menos adequado sob o aspecto sanitário) foi possível constatar uma diferença significativa entre as contagens totais em cortes recebidos já desossados pelo estabelecimento, a favor destes, e as contagens em cortes preparados na sala de desossa. O equipamento, tanto no estado considerado limpo (após o dia de trabalho) e aquele em uso apresentaram contagens totais excessivas; não houve diferença significativa entre um e outro caso, e, em ambos, a temperatura de incubação 32°C resultou em valores estatisticamente superiores aos obtidos com a incubação a 5°C. As contagens provenientes de análises de superfícies de madeira foram significativamente mais elevadas que as verificadas em superfícies metálicas (serra elétrica, facas, afiadores). A ocorrência de coliformes totais foi geral, tanto na carne como no equipamento, muitas vezes em números elevados. Em amostras de carne, analisadas quanto à presença e ao número de coliformes totais e de Escherichia coli, verificou-se que 96% daquelas positivas para coliformes totais também o eram para esta bactéria. Atras de material em uso foram positivas quanto à ocorrência de coliformes totais. Tal como ocorreu a carne, o número mais possível (NMP) assumiu, em vários casos, elevados valores. Embora não comprovado estatisticamente, os resultados sugerem uma maior ocorrência desse grupo de microrganismos nas superfícies de madeira (mesa e ou cepo).

Caracterização de bactérias dos gêneros Mima e Herella (Tribo Mimeae, DeBord, 1942): 1) Propriedade morfo-bioquímicas e sensibilidade aos antibióticos

No presente trabalho, os autores estudaram as propriedades morfo-bioquímicas e a sinsibilidade aos antibióticos de 19 amostras de bactérias dos gêneros Mima e Herellea isoladas de material clínico e identificadas como Mima polymorpha variedade oxidans, Mima polymorpha e Herellea vaginicola. No estudo bioquímico observou-se que Herellea vaginicola foi oxidase negativa e em meio complexo nitrogenado, consistentemente ataca a glicose, galactose, manose, arabinose, xilose, lactose a 10% e irregularmente ataca a ramnose e a celobiose; em base sintética nitrogenada, além das atividades citadas, consistentemente produziu ácido a partir da lactose. Mima polymopha foi oxidase negativa, não apresentando atividade glicidolítica, quer em meio complexo nitrogenado, quer em base sintética nitrogenada. Mima polymorpha var. oxidans, foi oxidase positiva, não revelando nenhuma atividade glicidolítica. Herellea vaginicola e Mima polymorpha mostraram grande sensibilidade à gabromicina, knamicina, neomicina, colistin, sendo que a última também foi muito sensível ao cloranfenicol e rovamicina. Mima polymorpha var. oxidans, apresentou grande sensibilidade à knamicina, neomicina, colistin, cloranfenicol e wintomylon. A sensibilidade das amostras a 1 a 0,1 unidade de penicilina/ml, nas condições ensaiadas no presente trabalho, não foi absoluta, como a observada por Baumann, Doudoroff & Stanier (1968a) que permitisse uma separação entre amostras oxidase positiva e negativa ou uma diferenciação dentro do grupo das bactérias oxidase positiva.

The interaction of Gram negative bacteria and S. mansoni in mice with experimental Schistosomiasis

Animals (122 mice) were infected each with eighty cercariae of S. mansoni and subsequently challenged intravenously eight weeks later with the following gram-negative organisms. S. typhi, E. coli, Klebsiella-enterobacter species, Proteus mirabilis and Pseudomonas aeruginosa. Enumeration of bacteria in the liver, spleen and blood and S. mansoni from the portal sistem was performed from one to four weeks later in infected animals. A significant difference between infection produced by S. typhi and other gram negative organisms was observed: S. typhi persisted longer in the spleen and liver and could be recovered from S. mansoni worms up to three weeks following bacterial infection. Other gram negative bacteria disappeared from S. mansoni worms after two weeks of initial challenge. Additional animals (51 mice) infected with S. mansoni were given S. typhi, E. coli or sterile saline. After two weeks, animals were sacrificed and the recovery rate of worms from the portal system, and the mesenteric and hepatic oogram were determined. in animals infected with E. coli a significant decrease in the number of worms was observed compared to the saline control group; thirty worms were recovered in the control group compared to two worms in e. coli infected animals. In addition, the patterns of oviposition was significantly different in these latter animals suggesting complete inhibition of this process. Following S. typhi infection the difference in recovery of worms and pattern of oviposition was minimal. These findings suggest a difference in the interaction of various gram negative bacteria and S. mansoni and are consistent with the clinical observation of prolonged salmonella bacteremia in patients with schistosomiasis.

High-throughput hydrophilic interaction chromatography coupled to tandem mass spectrometry for the optimized quantification of the anti-Gram-negatives antibiotic colistin A/B and its pro-drug colistimethate.

Colistin is a last resort's antibacterial treatment in critically ill patients with multi-drug resistant Gram-negative infections. As appropriate colistin exposure is the key for maximizing efficacy while minimizing toxicity, individualized dosing optimization guided by therapeutic drug monitoring is a top clinical priority. Objective of the present work was to develop a rapid and robust HPLC-MS/MS assay for quantification of colistin plasma concentrations. This novel methodology validated according to international standards simultaneously quantifies the microbiologically active compounds colistin A and B, plus the pro-drug colistin methanesulfonate (colistimethate, CMS). 96-well micro-Elution SPE on Oasis Hydrophilic-Lipophilic-Balanced (HLB) followed by direct analysis by Hydrophilic Interaction Liquid Chromatography (HILIC) with Ethylene Bridged Hybrid - BEH - Amide phase column coupled to tandem mass spectrometry allows a high-throughput with no significant matrix effect. The technique is highly sensitive (limit of quantification 0.014 and 0.006μg/mL for colistin A and B), precise (intra-/inter-assay CV 0.6-8.4%) and accurate (intra-/inter-assay deviation from nominal concentrations -4.4 to +6.3%) over the clinically relevant analytical range 0.05-20μg/mL. Colistin A and B in plasma and whole blood samples are reliably quantified over 48h at room temperature and at +4°C (<6% deviation from nominal values) and after three freeze-thaw cycles. Colistimethate acidic hydrolysis (1M H2SO4) to colistin A and B in plasma was completed in vitro after 15min of sonication while the pro-drug hydrolyzed spontaneously in plasma ex vivo after 4h at room temperature: this information is of utmost importance for interpretation of analytical results. Quantification is precise and accurate when using serum, citrated or EDTA plasma as biological matrix, while use of heparin plasma is not appropriate. This new analytical technique providing optimized quantification in real-life conditions of the microbiologically active compounds colistin A and B offers a highly efficient tool for routine therapeutic drug monitoring aimed at individualizing drug dosing against life-threatening infections.

Macrophage Migration Inhibitory Factor Deficiency Is Associated With Impaired Killing of Gram-Negative Bacteria by Macrophages and Increased Susceptibility to Klebsiella pneumoniae Sepsis.

The cytokine macrophage migration inhibitory factor (MIF) is an important component of the early proinflammatory response of the innate immune system. However, the antimicrobial defense mechanisms mediated by MIF remain fairly mysterious. In the present study, we examined whether MIF controls bacterial uptake and clearance by professional phagocytes, using wild-type and MIF-deficient macrophages. MIF deficiency did not affect bacterial phagocytosis, but it strongly impaired the killing of gram-negative bacteria by macrophages and host defenses against gram-negative bacterial infection, as shown by increased mortality in a Klebsiella pneumonia model. Consistent with MIF's regulatory role of Toll-like 4 expression in macrophages, MIF-deficient cells stimulated with lipopolysaccharide or Escherichia coli exhibited reduced nuclear factor κB activity and tumor necrosis factor (TNF) production. Addition of recombinant MIF or TNF corrected the killing defect of MIF-deficient macrophages. Together, these data show that MIF is a key mediator of host responses against gram-negative bacteria, acting in part via a modulation of bacterial killing by macrophages.

Valoración del estudio vesical y del tracto urinario superior en pacientes con citología urinaria positiva y pruebas de imagen negativas

Introducció: La citologia urinària positiva en context de cistoscopia sense evidència de tumor macroscòpic obliga a continuar el seu estudi. En el nostre centre es realitzen biòpsies vesicals múltiples normatitzades (BMN) i citologia ureteral selectiva en aquest casos. Material i mètodes: Estudi retrospectiu de 70 pacients amb citologia urinària positiva en absència de tumor macroscòpic. Es van avaluar els resultats de totes les biòpsies vesicals múltiples i les citologies ureterals selectives realitzades. Resultats: Es va diagnosticar CIS vesical em 45 (64,3%) pacients. Es va detectar citologia ureteral positiva em 12: cinc (7,2%) en el costat esquerre i set (10%) en el dret. Es va observar CIS vesical concomitant em 2 de 5 pacients amb PAP ureteral positiu esquerra i em 5 de 7 amb PAP ureteral positiu dret. Conclusions: La biòpsia múltiple normatitzada (BMN) és útil per a aquest casos. Els pobres resultats obtinguts en citologia ureteral selectiva posen en dubte la rendibilitat de la seva utilització sistemàtica.

Expression of mosquito active toxin genes by a Colombian native strain of the gram-negative bacterium Asticcacaulis excentricus

Mosquito control with biological insecticides, such as Bacillus sp. toxins, has been used widely in many countries. However, rapid sedimentation away from the mosquito larvae feeding zone causes a low residual effect. In order to overcome this problem, it has been proposed to clone the Bacillus toxin genes in aquatic bacteria which are able to live in the upper part of the water column. Two strains of Asticcacaulis excentricus were chosen to introduce the B. sphaericus binary toxin gene and B. thuringiensis subsp. medellin cry11Bb gene cloned in suitable vectors. In feeding experiments with these aquatic bacteria, it was shown that Culex quinquefasciatus, Aedes aegypti, and Anopheles albimanus larvae were able to survive on a diet based on this wild bacterium. A. excentricus recombinant strains were able to express both genes, but the recombinant strain expressing the B. sphaericus binary toxin was toxic to mosquito larvae. Crude protease A. excentricus extracts did not degrade the Cry11Bb toxin. The flotability studies indicated that the recombinant A. excentricus strains remained in the upper part of the water column longer than the wild type Bacillus strains.