Combinations of Clavulanic Acid and other β-lactam Antibiotics against Strains of Pseudomonas aeruginosa, Serratia marcescens and other Bacteria

Combinations of clavulanic acid, a new β-lactamase inhibitor, with five cephalosporins and one cephamycin were tested against cell-free β-lactamases obtained from Serratia marcescens, Pseudomonas aeruginosa and an Enterobacter strain, 265A. Cefotaxime was the most resistant antibiotic and cephalothin the most sensitive antibiotic to β-lactamases. Low concentrations of clavulanic acid gave some protection against the Serratia and Pseudomonas enzymes. The most active source of β-lactamase was the 265A strain, against which only cefotaxime was highly resistant. Clavulanic acid had only a slight inhibitory effect on this enzyme, which was confirmed by an agar method, and potentiated slightly the activity of cephalothin and cefoxitin against two β-lactamase producing strains of Staphylococcus aureus. Lysis by cephalothin of one strain of S. marcescens was potentiated in the presence of clavulanic acid.     

β-Lactamase production and in vitro antimicrobial susceptibility of Porphyromonas gingivalis

Abstract β-Lactamase production by 98 Porphyromonas strains was investigated by the nitrocefin (chromogenic cephalosporin) test. Human isolates of P. gingivalis (91), P. endodontalis (2), and P. asaccharolytica (1) were tested, with four closely related Porphyromonas spp. of animal origin and four reference strains. The in vitro susceptibility of 64 P. gingivalis strains was investigated on Brucella blood agar by the E test. None of the human Porphyromonas isolates tested produced β-lactamase, but one Porphyromonas strain of animal origin, most closely resembling P. endodontalis , produced β-lactamase. P. gingivalis was susceptible to almost all of the drugs tested: benzylpenicillin, ampicillin, cefaclor, cefuroxime, erythromycin, clindamycin, tetracycline, doxycycline, metronidazole and ciprofloxacin; all strains were inhibited at 0.016 μg/ml, 0.023 μg/ml, 0.315 μg/ml, 0.064 μg/ml, 0.19 μg/ml, 0.016 μg/ml, 0.094 μg/ml, 0.047 μg/ml, 0.023 μg/ml, and 0.75 μg/ml of these drugs, respectively. Cotrimoxazole exhibited variable efficacy against P. gingivalis ; the range of MICs was 0.1095-32.0 μg/ml. The results indicate that β-lactamase production is currently not a problem amongst clinical isolates of P. gingivalis and strains are susceptible to most antimicrobial agents.     

β-Lactam resistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins

The beta-lactams are by far the most widely used and efficacious of all antibiotics. Over the past few decades, however, widespread resistance has evolved among most common pathogens. Streptococcus pneumoniae has become a paradigm for understanding the evolution of resistance mechanisms, the simplest of which, by far, is the production of beta-lactamases. As these enzymes are frequently plasmid encoded, resistance can readily be transmitted between bacteria. Despite the fact that pneumococci are naturally transformable organisms, no beta-lactamase-producing strain has yet been described. A much more complex resistance mechanism has evolved in S. pneumoniae that is mediated by a sophisticated restructuring of the targets of the beta-lactams, the penicillin-binding proteins (PBPs); however, this may not be the whole story. Recently, a third level of resistance mechanisms has been identified in laboratory mutants, wherein non-PBP genes are mutated and resistance development is accompanied by deficiency in genetic transformation. Two such non-PBP genes have been described: a putative glycosyltransferase, CpoA, and a histidine protein kinase, CiaH. We propose that these non-PBP genes are involved in the biosynthesis of cell wall components at a step prior to the biosynthetic functions of PBPs, and that the mutations selected during beta-lactam treatment counteract the effects caused by the inhibition of penicillin-binding proteins.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Early Effects of β,β'-Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l -[³⁵S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.     

Some Morphological and Physiological Characteristics of Gram Negative Anaerobic Bacteria isolated from the Alimentary Tract of the Pig

S ummary . A collection of 100 strains of Gram negative anaerobic bacteria isolated from the alimentary tract of the pig has been divided into four groups on morphological and physiological grounds. The four groups resemble the genera Bacteroides, Sphaerophorus, Veillonella and Peptostreptococcus as described in Bergey's Manual (Breed, Murray & Smith, 1957).     

Lysis by β-lactam Antibiotics: Structure-activity Relationships in the Cephalosporins

The ability of several cephalosporins to lyse Staphylococcus aureus and Escherichia coli has been studied. Cephradine and cephalexin, which have a methyl substituent at C-3, did not lyse E. coli , but a third 3-methyl compound, deacetoxycephalothin, caused bacteriolysis. All the compounds tested (including cephalexin and cephradine) lysed Staph. aureus . The minimum lytic concentration of a cephalosporin for E. coli often exceeded its minimum inhibitory concentration, while for staphylococci this was never the case.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Effects of Mercuric Chloride on Growth and Morphology of Selected Strains of Mercury-Resistant Bacteria

A survey of the comparative cytological effects of growth in the presence of mercury by a group of mercury-resistant bacterial cultures and a characterization of the process of bacterial adaptation to Hg²⁺ ion was accomplished. Mercury resistance was found to be dependent upon the ability to volatilize mercury from the medium and upon the amount of mercury accumulated by the cells. The results indicate that most cultures which adapt to growth in the presence of HgCl₂ exhibit extensive morphological abnormalities. Significant effects are delay in the onset of growth and cell division and numerous structural irregularities associated with cell wall and cytoplasmic membrane synthesis and function. A detailed analysis of the adaptation process and the resulting effects on morphology was performed on an Enterobacter sp. During the period preceding active multiplication, a selection for mercury-resistant mutants occurred. It was also demonstrated that growth commenced only at a specific threshold concentration of Hg²⁺.     

The effects of βbT-lactams on the isoelectric focusing of βbT-lactamases

A range of concentrations of ceftazidime (4–64 mg I^-1) was shown to cause no induction of the TEM-1 and TEM-5 β-lactamases produced by Escherichia coli Nb. Increasing the concentration of ceftazidime in cultures of E. coli Nb caused a concomitant increase in the intensity of a satellite band of pI 5.2. The same increase in this satellite band was observed when ceftazidime was added to cell-free β-lactamase peparations from E. coli Nb and the separate addition of 11 different β-lactams to TEM-1 showed that each compound produced its own unique pattern of satellite bands. In addition, the mixing of ceftazidime with TEM-1 and 13 other TEM-derived β-lactamases caused a similar satellite band to be observed but ceftazidime did not have the same effect on PSE or SHV β-lactamases. Consequently, the addition of ceftazidime to a β-lactamase preparation prior to isoelectric focusing (IEF) may help to verify if a particular β-lactamase is TEM-derived. Purification of the satellite bands by electrodialysis and their subsequent re-focusing demonstrated that the ceftazidime-induced satellite bands can revert to a protein which has a pI similar to the parent band, illustrating the possible reversibility and dynamic nature of β-lactamase satellite bands on IEF. These results enable a better interpretation to be made of β-lactamase satellite bands observed on IEF.     

Plant-Derived Neurotoxic Amino Acids (β-N-Oxalylamino-l-Alanine and β-N-Methylamino-l-Alanine): Effects on Central Monoamine Neurons

In the present study the subacute effects of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high-affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5-HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH-immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P-immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10(-5) M) reduced high-affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high-affinity [3H]DA uptake and the cortical high-affinity [3H]5-HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.     

碱性纤维素酶革兰氏阴性菌株筛选及酶学性质研究

用CMC平板筛选方法,从造纸厂碱性淤泥中获得透明圈直径大于30mm的产碱性纤维素酶革兰氏阴性菌H8005。液体摇瓶培养产生碱性CMC酶活力高达4．2IU／mL。酶学性质初步研究显示,H8005产生的CMC酶反应的pH值以8．0左右为适;在碱性条件下具有较高的酶活和一定的稳定性;反应温度以55℃左右为宜;且具有较好的温度稳定性。Mn^2＋与Fe^3＋对酶反应有促进作用,Cu^2＋和Pb^2＋对酶反应有抑制作用。该菌产生的纤维素酶在棉织品的水洗整理及洗涤剂工业中具有非常良好的应用前景。     

Differentiation of Gram Positive and Gram Negative Bacteria in Transparent Acrylic Resin Emulsion Replicas of Surfaces of Plants

When acrylic resin emulsion (Rohm & Haas Primal AC-33) is allowed to dry on a leaf surface, it can be peeled off to give a transparent replica of the surface with bacteria and fungi embedded in it. The distribution of micro-organisms in the replica appears to reflect the patterns in which they occur naturally on the surface. The resin replicas may be stained by a variety of microscopical stains, the best of which is phenol-acetic-aniline blue, and are suitable for high power light microscopy.
Acrylic resin emulsion differs from widely used cellulose-based materials in that permanent differentiated preparations of Gram positive and Gram negative stained bacteria may be produced from a wide variety of types of surface.     

Purification and characterization of ββ-N-acetylhexosaminidase from the phytopathogenic fungus Bipolaris sorokiniana

N-acetylhexosaminidase (HEX) from the phytopathogenic fungus Bipolaris sorokiniana was isolated and characterized. The production of HEX by B. sorokiniana was not altered by growing on different carbon sources. Enzyme purification was carried out by sequential liquid chromatography on Sephacryl S-200 HR, and p-aminobenzyl-2-acetamido-2-deoxy-β- d -thioglucopyranoside agarose. The purification was about 70-fold, with a yield of 41%, determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme had pH and temperature optima of 4·5 and 55 °C, respectively. The molecular weight of non-denatured enzyme was estimated as 120 000 Da by gel filtration chromatography, and about 55 000 Da by SDS-PAGE. The fungal HEX had glycosylated residues as evidenced by binding to Concanavalin-A. Bipolaris sorokiniana enzyme was also active with p-nitrophenyl-chitobioside and p-nitrophenyl-N-acetylgalactosaminide as substrates.     

甲醇利用型吡咯喹啉醌产生菌的筛选及鉴定

从虎杖内生细菌和黏细菌中筛选吡咯喹啉醌(PQQ)产生菌。采用3种以甲醇为唯一碳源的培养基对160株供试菌株进行摇瓶培养发酵,发酵产物采用光谱学分析法及HPLC法筛选。结果显示,通过初筛和复筛共得到甲醇利用型菌134株,PQQ产生菌4株,其中菌株083114的PQQ产量为64.34 mg/L。菌株083114的16S rRNA基因序列分析结果显示,其序列与酸快生芽孢杆菌(Bacillus acidiceler)的系统发育关系最近。虎杖内生细菌及黏细菌中存在PQQ产生菌。     

Purification and partial amino acid sequence of plantaricin 1.25ααα and 1.25βββ, two bacteriocins produced by Lactobacillus plantarum TMW1.25

Two bacteriocins produced by Lactobacillus plantarum TMW1.25 have been purified by a four-step purification procedure, including ammonium sulphate precipitation and cation-exchange chromatography followed by hydrophobic-interaction chromatography on octyl sepharose. The final purification was performed by repeated reversed-phase chromatography steps which yielded two bacteriocin fractions designated plantaricin 1.25 alpha and plantaricin 1.25 beta. The molecular masses of the peptides in these fractions were 5979 and 5203 Da, respectively. Combination of the fractions did not have any synergistic effects on bacteriocin activity, indicating that they each contain a one-peptide bacteriocin. The major peptide in the alpha fraction was blocked at its N-terminus, and a partial sequence (25 residues) could only be obtained after cleavage with CNBr. This sequence did not show clear homologies with known bacteriocins. The beta peptide has been sequenced almost completely and consists, presumably, of 53 residues. This peptide displayed strong homology to the known N-terminal part of brevicin 27 produced by Lactobacillus brevis SB27. The results showed that the beta peptide contains as many as six consecutive lysine residues at the N-terminus.