Toxicity of phenyl-mercuric lactate for fish

Phenyl-mercuric lactate is included in the pulp processing reagents by some paper mills to eliminate slime formation in the pulp. Small quantities of this chemical are added to the wet pulp in the beaters, particularly for the bactericidal action against Aerobacter aerogenes. Subsequently the mecurial is carried away in the wash waters. However, as the highly poisonous nature of many compounds of mercury is well known, questions have been raised concerning the pollution hazards created by phenyl-mercuric lactate in streams receiving effluents from mills using this substance.

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-Angstrom crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 Angstrom, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.

The beta-glucoside genes of Klebsiella aerogenes: conservation and divergence in relation to the cryptic bgl genes of Escherichia coli

The ability to metabolize aromatic beta-glucosides such as salicin and arbutin varies among members of the Enterobacteriaceae. The ability of Escherichia coli to degrade salicin and arbutin appears to be cryptic, subject to activation of the bgl genes, whereas many members of the Klebsiella genus can metabolize these sugars. We have examined the genetic basis for beta-glucoside utilization in Klebsiella aerogenes. The Klebsiella equivalents of bglG, bglB and bglR have been cloned using the genome sequence database of Klebsiella pneumoniae. Nucleotide sequencing shows that the K. aerogenes bgl genes show substantial similarities to the E. coli counterparts. The K. aerogenes bgl genes in multiple copies can also complement E. coli mutants deficient in bglG encoding the antiterminator and bglB encoding the phospho-beta-glucosidase, suggesting that they are functional homologues. The regulatory region bglR of K aerogenes shows a high degree of similarity of the sequences involved in BglG-mediated regulation. Interestingly, the regions corresponding to the negative elements present in the E. coli regulatory region show substantial divergence in K aerogenes. The possible evolutionary implications of the results are discussed. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.v. All rights reserved.

Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes

OBJECTIVES:
The intrinsically encoded ramA gene has been linked to tigecycline resistance through the up-regulation of efflux pump AcrAB in Enterobacter cloacae. The molecular basis for increased ramA expression in E. cloacae and Enterobacter aerogenes, as well as the role of AraC regulator rarA, has not yet been shown. To ascertain the intrinsic molecular mechanism(s) involved in tigecycline resistance in Enterobacter spp., we analysed the expression levels of ramA and rarA and corresponding efflux pump genes acrAB and oqxAB in Enterobacter spp. clinical isolates.

METHODS:
The expression levels of ramA, rarA, oqxA and acrA were tested by quantitative real-time RT-PCR. The ramR open reading frames of the ramA-overexpressing strains were sequenced; strains harbouring mutations were transformed with wild-type ramR to study altered ramA expression and tigecycline susceptibility.

RESULTS:
Tigecycline resistance was mediated primarily by increased ramA expression in E. cloacae and E. aerogenes. Only the ramA-overexpressing E. cloacae isolates showed increased rarA and oqxA expression. Upon complementation with wild-type ramR, all Enterobacter spp. containing ramR mutations exhibited decreased ramA and acrA expression and increased tigecycline susceptibility. Exceptions were one E. cloacae strain and one E. aerogenes strain, where a decrease in ramA levels was not accompanied by lower acrA expression.

CONCLUSIONS:
Increased ramA expression due to ramR deregulation is the primary mediator of tigecycline resistance in clinical isolates of E. cloacae and E. aerogenes. However, some ramA-overexpressing isolates do not show changes in ramR, suggesting alternate pathways of ramA regulation; the rarA regulator and the oqxAB efflux pump may also play a role in tigecycline resistance in E. cloacae.

Emended description of porcine [Pasteurella] aerogenes, [Pasteurella] mairii and [Actinobacillus] rossii

The aim of this study was to improve the definition and identification of a group of veterinarily important bacteria referred to as the [Pasteurella] aerogenes-[Pasteurella] mairii-[Actinobacillus] rossii complex. These organisms have mainly been isolated from the reproductive and intestinal tracts of pigs and in most cases have been considered as opportunistic pathogens. A collection of 87 strains were characterized by phenotypic analysis from which 41 strains were selected for 16S rRNA gene sequence comparison, out of which 23 have been sequenced in the present study. One group of 21 strains phenotyped as biovars 1, 3-5, 9-11, 19 and 25-27, including the type strain of [P.] aerogenes, showed 16S rRNA gene sequence similarities of 99.6 % or higher; another group of 18 strains including biovars 2, 6-8, 12-15, 21, 23, 24 and 26A and the type strain of [A.] rossii showed 97.8 % or higher 16S rRNA gene sequence similarity. Between the two groups, 93.8-95.7 % 16S rRNA gene sequence similarity was observed. Strains of [P.] mairii showed 99.5 % similarity, with 95.5-97.2 and 93.8-95.5 % similarity to strains of [P.] aerogenes and [A.] rossii, respectively. Four strains could not be classified with any of these groups and belonged to other members of Pasteurellaceae. Comparisons were also made to DNA-DNA hybridization data. Biovars 1, 9, 10, 11 and 19, including the type strain of [P.] aerogenes, linked at 70 % DNA reassociation, whereas strains identified as biovars 2, 6, 7, 8, 12, 15 and 21 of [P.] aerogenes linked at 81 %. The latter group most likely represents [A.] rossii based on the 16S rRNA gene sequence comparisons. DNA reassociation between the [P.] aerogenes and [A.] rossii groups was at most 37 %, whereas 47 % was the highest DNA reassociation found between [P.] aerogenes and [P.] mairii. The study showed that [P.] aerogenes, [P.] mairii and [A.] rossii can not be easily separated and may consequently be misidentified based on current knowledge of their phenotypic characteristics. In addition, these taxa are difficult to separate from other taxa of the Pasteurellaceae. A revised scheme for separation based upon phenotypic characters is suggested for the three species [P.] aerogenes emend., [P.] mairii emend. and [A.] rossii emend., with the respective type strains ATCC 27883T, NCTC 10699T and ATCC 27072T.

Characterization of PaxA and its operon: a cohemolytic RTX toxin determinant from pathogenic Pasteurella aerogenes

Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times.

Über einen Fall von embolischer, eitriger Nephritis und Pneumonie bei einem Saugfohlen, hervorgerufen durch das Bacterium lactis aerogenes.

Inaug.-diss.--Hannover, 1912.

Studies on "Sappe" disease of the silkworm, Bombyx mori : II. Effect of age of larvae on the manifestation of the disease

The effect of age of the larvae on the manifestation of the "Sappe" disease of the silkworm by oral inoculation of different pathogens, viz., Aerobacter cloacae, Pseudomonas boreopolis, Escherichia freundii, Achromobacter delmarvae, A. Superficialis, Pseudomonas ovalis, and Staphylococcus albus was tested. It was found that the reaction of the larva to the pathogen was influenced by its age. Some, e.g., Escherichia freundii, were more lethal when introduced at early stages whereas certain others, e.g., Aerobacter cloacae and Staphylococcus albus, caused maximum damage when invading older larvae. Irrespective of the age of infection, death of the worms mainly occurred during molting and before spinning. The studies also indicated that growth and mortality of the larvae were affected differentially by the pathogens.

Traditional medicinal uses and biological activities of some plant extracts of African Combretum Loefl., Terminalia L. and Pteleopsis Engl. species

In Africa various species of Combretum, Terminalia and Pteleopsis are used in traditional medicine. Despite of this, some species of these genera have still not been studied for their biological effects to validate their traditional uses. The aim of this work has been to document the ethnomedicinal uses of several species of Combretum and Terminalia in Mbeya region, south-western Tanzania, and to use this information for finding species with good antimicrobial and cytotoxic potential. During a five weeks expedition to Tanzania in spring 1999 sixteen different species of Combretum and Terminalia, as well as Pteleopsis myrtifolia were collected from various locations in the districts of Mbeya, Iringa and Dar-es-Salaam. Traditional healers in seven different villages in the Mbeya region were interviewed in Swahili and Nyakyusa on the medicinal uses of Combretum and Terminalia species shown to them. A questionnaire was used during the interviews. The results of the interviews correlated well between different villages, the same species being used in similar ways in different villages. Of the ten species shown to the healers six were frequently used for treatment of skin diseases, bacterial infections, diarrhea, oedema and wounds. The dried plants were most commonly prepared into hot water decoctions or mixed into maize porridge, Ugali. Infusions made from dried or fresh plant material were also common. Wounds and topical infections were treated with ointments made from the dried plant material mixed with sheep fat. Twenty-one extracts of six species of Combretum and four of Terminalia, collected from Tanzania, were screened for their antibacterial effects against two gram-negative and five gram-positive bacteria, as well as the yeast, Candida albicans, using an agar diffusion method. Most of the screened plants showed substantial antimicrobial activity. A methanolic root extract of T. sambesiaca showed the most potent antibacterial effects of all the plant species screened, and gave a MIC value of 0.9 mg/ml against Enterobacter aerogenes. Also root extracts of T. sericea and T. kaiserana gave excellent antimicrobial effects, and notably a hot water extract of T. sericea was as potent as extracts of this species made from EtOH and MeOH. Thus, the traditional way of preparing T. sericea into hot water decoctions seems to extract antimicrobial compounds. Thirty-five extracts of five species of Terminalia, ten of Combretum and Pteleopsis myrtifolia were screened for their antifungal effects against five species of yeast (Candida spp.) and Cryptococcus neoformans. The species differed from each other to their antifungal effects, some being very effective whereas others showed no antifungal effects. The most effective extracts showed antifungal effects comparable to the standard antibiotics itraconazol and amphotericin B. Species of Terminalia gave in general stronger antifungal effects than those of Combretum. The best effects were obtained with methanolic root extracts of T. sambesiaca, T. sericea and T. kaiserana, and this investigation indicates that decoctions of these species might be used for treatment of HIV-related fungal infections. Twenty-seven crude extracts of eight species of Combretum, five of Terminalia and Pteleopsis myrtifolia were evaluated for their cytotoxic effects against human cancer cell lines (HeLa, cervical carcinoma; MCF 7, breast carcinoma, T 24 bladder carcinoma) and one endothelial cell line (BBCE, bovine brain capillary endothelial cells). The most outstanding effects were obtained with a leaf extract of Combretum fragrans, which nearly totally inhibited the proliferation of T 24 and HeLa cells at a concentration of 25 ug/ml and inhibited 60 % of the growth of the HeLa cells at a concentration of 4.3 ug/ml. The species of Terminalia were less cytotoxically potent than the Combretum species, although T. sericea and T. sambesiaca gave good cytotoxic effects (< 30 % proliferation). In summary this study indicates that some of the species of Terminalia, Combretum and Pteleopsis, used in Tanzanian traditional medicine, are powerful inhibitors of both microbial and cancer cell growth. In depth studies would be needed to find the active compounds behind these biological activities.

Prevalencia de bacterias entéricas en leches crudas del departamento de Managua

Con el objetivo de investigar la prevalencia de bacterias entéricas en leches crudas del Departamento de Managua se llevo a efecto el presente trabajo entre Junio y Octubre de 1965. La investigación se llevó a efecto con 37 productores de leche del Departamento de Managua, los cuales la venden a las distintas pasteurizadoras del Departamento. Las muestras fueron tomadas en el momento en que los productores entregan la leche a las Pasteurizadoras, sometiéndolas luego a las pruebas bacteriológicas necesarias para el aislamiento e identificación de las bacterias entericas presentes. Se encontraron cinco géneros de enterobacterias, siendo los más frecuentemente encontrados: Aerobacter en un 62.1%, Escherichis 10.8% y los géneros Salmonella, Shinella y Proteus en un 2.7%. Se encontró que las muestras examinadas estaban contaminadas con germenes estericos en un 81.08%. No se encontró ninguna relación aparente entre la contaminación de los germenes entericos y la prueba de reducción de azul de metileno.

Microbiological evaluation and keeping quality of fishes reared in livestock sewage fed ponds without artificial feed

Livestock sewage has been utilized for fish culture. There is lack of information on microbiological evaluation and keeping quality of these fishes. This paper reveals the incidence, types of micro-organisms and keeping quality of fishes reared in livestock sewage fed ponds without artificial feed. These fishes revealed microbial incidence and keeping quality comparable to other fishes. Initial mesophilic and psychrophilic counts varied from 3.38 to 5.56 and 2.47 to 4.74. On an average, the counts reduced by about 40% after evisceration and washing. Whole as well as washed fishes had refrigerated (8 ± 1°C) life of not more than 4 days. The average psychrophilic and mesophilic counts of ice (0 to 1°C) stored whole fishes up to 10th day varied from 3.66 to 4.81, 4.61 to 5.24 and in eviscerated and washed fishes 2.17 to 3.69 and 2.78 to 4.41. Both remained acceptable till the 10th day. Qualitative study of surface slime and gills revealed presence of Aerobacter (Enterobacter), Aeromonas, Alcaligenes, Bacillus, Clostridia, E. coli, Klebsiella, Micrococci, Proteus and Pseudomonas.

Disseminação horizontal de genes que codificam para β-lactamases de espectro alargado em isolados de enterobacteriaceae de origem hospitalar

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

tolQ is required for cloacin DF13 susceptibility in Escherichia coli expressing the aerobactin/cloacin DF13 receptor IutA

We investigated the role of the tolQ gene in the import of cloacin DF13 across the outer membrane of Escherichia coli strains expressing the IutA receptor. The IutA outer-membrane protein is the receptor for the siderophore ferric aerobactin and also binds cloacin DF13, a bacteriocin produced by strains of Enterobacter aerogenes. In this report we present evidence that tolQ is required for the internalization of cloacin DF13 upon binding to IutA but it is not involved in the transport of ferric aerobactin.

Produção de biohidrogénio por bactérias a partir de resíduos fermentescíveis

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Engenharia Química e Bioquímica