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1.
V L Seligy R W Beggs J M Rancourt A F Tayabali 《Journal of industrial microbiology & biotechnology》1997,18(6):370-378
The redox dyes MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyl tetrazolium bromide, thiazolyl blue) and XTT (sodium 3′- {1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis{4-methoxy-6-nitro}
benzene sulphonic acid hydrate) were used as dosimetry reporters in liquid (multi-well) and solid support (membrane) assays
to estimate spore viability and content of commercial BT products derived from fermentation of Bacillus thuringiensis subsp kurstaki (Btk) and subsp israelensis (Bti). QC tests on five BT products were done using spore, protein and gene contents, and morphology (scanning electron microscopy)
as indicators. Spore levels (6–40 × 109 colony forming units (CFU) ml−1) were approximately equivalent when based on International Units (IU) of potency. Spore viability was highly stable over
a broad range of temperatures and pHs but germination and growth were restricted (optima: pH ≈ 7.5 and 37°C). Quantitative
bioreduction activity (QBA) of MTT and XTT correlated with vegetative cell production. Depending on manipulation of pre-assay
conditions, both dyes could discriminate doses from ∼2 to 109 spores (or 10−3 to 106 IU). Non-toxic effects of XTT and its formazan product enabled automated collection of data on growth and dose. Solid support
assays also reliably estimated product dosage by in situ detection of CFU. With appropriate reference dilutions of microbe-containing products the QBA assays can provide high throughput
QC monitoring of product comparisons and field release in aerial spray or water injection applications.
Received 18 December 1996/ Accepted in revised form 11 March 1997 相似文献
2.
A simple procedure is described for the rapid assay of β-lactamases suitable for use in the quantitation of β-lactamase inhibition
in crude culture filtrates of soil microbes. The proposed method is based on measuring the blue reaction product (λmax 750 nm) formed during reduction of phosphomolybdic acid with reducing products generated from β-lactam hydrolysis and is
not influenced significantly by the metabolites of microbial culture filtrates which often interfere with conventional β-lactamase
assay techniques. 相似文献
3.
4.
Ann Lövgren Cathrine R. Carlson Katarina Eskils Anne-Brit Kolstø 《Current microbiology》1998,37(4):245-250
The insect pathogen Bacillus thuringiensis (Bt) has earlier been shown to possess virulence factors in addition to the crystal toxins. Bt subsp. gelechiae strain Bt13 lacks crystals but is still virulent to lepidopteran insects. Among the virulence co-expressed genes are two
phospholipases; phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-degrading phospholipase C (PC-PLC),
flagellin, and β-lactamase I. In addition to these putative virulence factors the toxic neutral metalloprotease immune inhibitor
A (InA) has been identified. In this paper we report a circular 5.9 Mb combined physical and genetic map of the of the Bt
subsp. gelechiae chromosome. The genes encoding PI-PLC, PC-PLC, InA, flagellin, and β-lactamase I are shown to be scattered over the chromosome.
The PLC-encoding genes have been cloned from Bt13, and DNA sequencing showed that the Bt subsp. gelechiae PLC genes are >90% identical to their previously cloned equivalents from Bt or B. cereus. An HD-1 crystal toxin (cryIA) gene probe was found to hybridize to the Bt13 chromosome, but not to extrachromosomal elements.
Received: 26 March 1998 / Accepted: 6 May 1998 相似文献
5.
We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λ
max=390 nm) is converted to a red product (λ
max=486 nm). Since CA can irreversibly inhibit β-lactamase activity, the level of CA in a sample can be measured as a function
of the A
390/A
486 ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L−1 and 50 μg L−1 to 10 mg L−1, respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay
was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale
mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those
of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows
direct visual observation of CA levels on agar plates. 相似文献
6.
Species of the Bacteroides fragilis group are considered the most common anaerobe in human and animal infections and also harbor plasmids conferring resistance
to several antibiotics. In this study, resistance to cefoxitin, plasmid profile and β-lactamase production in species of the
B. fragilis group isolated from intestinal tracts of calves were evaluated. One hundred sixty-one B. fragilis group bacteria isolated from calves with and without diarrhea were analyzed. Cefoxitin susceptibility was performed using
an agar dilution method, β-lactamase production by using a nitrocefin method, and plasmid extraction by using a commercial
kit. Minimal inhibitory concentration values for cefoxitin ranged from 32 to > 512 μg/ml, and 47 bacteria (29.2%) were resistant
to cefoxitin (breakpoint 16 μl). Only seven isolates harbored plasmids varying from 6.0 to 5.0 kb, and a 5.5-kb plasmid in
B. vulgatus Bd26e and B. fragilis Bc5j might be related to cefoxitin resistance. β-lactamase was detected in 33 (70.2%) isolates. The cepA gene was observed in total DNA and in the 5.5-kb plasmid. The plasmid presence in organisms isolated from cattle may be important
in ecologic terms, and it needs further study. 相似文献
7.
For this study, 150 clinical isolates of Klebsiella pneumoniae were collected from one hospital in Beijing, China, and assayed for minimal inhibitory concentration (MIC) of imipenem. To
elucidate the mechanisms responsible for imipenem MIC variation among extended-spectrum β-lactamase (ESBL)-positive and -negative
K. pneumoniae strains, a variety of β-lactamase genes (bla
TEM, bla
CTX-M, bla
SHV, and bla
OXA) were screened by polymerase chain reaction (PCR). The outer membrane profile and expression of related genes (ompK35 and ompK36) then were analyzed and evaluated, respectively. None of the tested isolates were clinically resistant to imipenem, but the
range of MICs among ESBL-positive and -negative strains was significantly different. Deficiency in the expression of outer
membrane proteins (OmpK35,36) was observed in some of both ESBL-positive(17.6%) and -negative strains (10.9%), but only the
ESBL-positive strains depressed by the expression of ompK35/36 had an increased MIC of imipenem (≥0.5 mg/l). These results confirmed that the combination of SHV-1, CTX-M-3, CTX-M-14, TEM-1,
or OXA-11 production and reduced expression of ompK35/36 may not result in clinical resistance to imipenem but does correlate
with increasing imipenem MIC. 相似文献
8.
Liping Xie Mingfei Xu Tao Yang Chunbao Zhu Baoquan Zhu Youjia Hu 《Biochemistry. Biokhimii?a》2010,75(3):336-341
An SHV β-lactamase gene was amplified from a β-lactam resistant Klebsiella pneumoniae K-71 genomic DNA. After expression and purification, we demonstrated that peptide P1 could inhibit the hydrolysis activity
of both TEM-1 and SHV β-lactamase in vitro. Three mutations were introduced into P1 in which the first residue S was replaced by F, the 18th residue V was mutated to
Y, and the 15th residue Y was substituted with A, C, G, and R to obtain the mutants of P1-A, P1- C, P1-G, and P1-R, respectively.
The mutant peptides were purified and their inhibitory constants against TEM-1 and SHV β-lactamase were determined. All these
β-lactamase inhibitory peptides could inhibit the activity of both β-lactamases, while the mutant peptides showed stronger
inhibitory activities against TEM-1 β-lactamase than against SHV β-lactamase. Inhibition data suggested that P1-A improved
the β-lactamase inhibitory activity by over 3-fold compare to P1. When P1-A was incubated with K. pneumoniae K-71 in Luria-Bertani medium containing ampicillin, it showed a much stronger growth of inhibition ratio over P1. This study
gives us a good candidate for development of novel β-lactamase inhibitors. 相似文献
9.
V. Kumar J. L. de la Fuente A. L. Leitão P. Liras J. F. Martin 《Applied microbiology and biotechnology》1996,45(5):621-628
The bla gene of the cephamycin cluster of Nocardia lactamdurans has been subcloned in the shuttle plasmids pULVK2 and pULVK2A and amplified in N. lactamdurans LC411. The transformants showed two- to threefold higher β-lactamase activity. Formation of β-lactamase preceded the onset
of cephamycin biosynthesis. The β-lactamase of N. lactamdurans inactivated penicillins and, to a lesser extent, cephalosporin C but did not hydrolyse cephamycin C. This β-lactamase was
highly sensitive to clavulanic acid (50% inhibition was observed at 0.48 μg/ml clavulanic acid). The N. lactamdurans bla gene was disrupted in vivo by inertion of the kanamycin-resistance gene. Three bla-disrupted mutants, BD4, BD8 and BD12, were selected that lacked β-lactamase activity. Overexpresion of the bla gene resulted in N. lactamdurans transformants that were resistant to penicillin whereas mutants in which the bla gene was disrupted were supersensitive to this antibiotic. The three N. lactamdurans mutants with the bla gene disrupted showed a significant increase of cephamycin biosynthesis in solid medium, whereas transformants with the amplified
bla gene produced reduced levels of cephamycin. The cephamycin-overproducing Merck strain N. lactamdurans MA4213 showed no detectable levels of β-lactamase activity. The β-lactamase plays a negative role in cephamycin biosynthesis
in solid medium, but not in liquid medium.
Received: 26 July 1995/Received revision: 18 December 1995/Accepted: 8 January 1996 相似文献
10.
Two clinical strains of Klebsiella pneumoniae (K. pneumoniae) and one isolate of Escherichia coli (E. coli) were collected from two large general hospitals in China. Conjugation experiment, susceptibility testing, isoelectric focusing,
PCR, and sequencing techniques as well as clone, expression, purification and kinetics were carried out to describe the characterization
of the novel SHV-tpye enzyme. The analysis of plasmid profiling and pulsed-field gel electrophoresis of the novel enzyme were
performed to investigate epidemiology. These isolates had CTX-M-14 and SHV-89 β-lactamases. SHV-89 β-lactamase of pI 7.6 is
a novel variant with two substitutions compared with the sequence of SHV-1: Leu35Gln and Met129Val. Its gene also had two
silent mutations at positions 369 and 774, respectively. The results of substrate profiles and MIC determinations showed the
activity of the novel enzyme was insufficient for the enzyme to count as an extended-spectrum β-lactamase (ESBL). The substrates
of the enzyme were also characterized. Furthermore, the three novel SHV enzyme-producing strains were epidemiologically unrelated.
The emergence of a novel SHV-type β-lactamase is rarely described in other areas. This study illustrates the importance of
molecular survelliance in tracking SHV-producing strains in large teaching hospitals and emphasizes the need for epidemiological
monitoring.
J.-B. Li and J. Cheng contributed equally to this work. 相似文献
11.
Buatong Jirayu Phongpaichit Souwalak Rukachaisirikul Vatcharin Sakayaroj Jariya 《World journal of microbiology & biotechnology》2011,27(12):3005-3008
The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory
concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts
from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates
(61.3%) produced inhibitory compounds. Most of the extracts (28–32%) inhibited Staphylococcus aureus (MIC/MBC 4–200/64–200 μg ml−1). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml−1). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4–200/8–200 μg ml−1 and 8–200/8–200 μg ml−1, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32–200/32–200 μg ml−1). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8–32 μg ml−1against Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity
and could be suitable sources of new antimicrobial natural products. 相似文献
12.
Tejedor-Junco MT González-Martín M Lupiola P González-Lama Z 《Folia microbiologica》1998,43(6):683-686
The interaction of type-I β-lactamases fromEnterobacter cloacae with diverse β-lactam compounds was examined. The ability of penicillin and cefoxitin to induce β-lactamase production in
this strain was assessed. The effect of β-lactamase inhibitors was measured on β-lactamase extracts and on intact cells.E. cloacae 78 strain is a stably derepressed mutant showing limited susceptibility to a number of antibiotics except imipenem. Imipenem
would therefore be the appropiate choice for therapy of infections caused by stably derepressed mutants ofEnterobacter sp. producing type-I β-lactamases. 相似文献
13.
Bujdáková H Hanzen J Jankovicová S Klimácková J Moravcíková M Milosovic P Michálková-Papajová D Kallová J Jakab A Kettner M 《Folia microbiologica》2001,46(4):339-344
Occurrence and transferability of β-lactam resistance in 30 multi-resistantEscherichia coli, Klebsiella spp.,Enterobacter spp.,Pantoea agglomerans, Citrobacter freundii andSerratia marcescens strains isolated from children between 0 and 3 years of age is presented. The strains were resistant to ampicillin (30),
cefoxitin (22), cefotaxime (30), ceftriaxone (30), ceftazidime (30) and aztreonam (28), but susceptible to cefepime (30) and
imipenem (26). Twenty-eight of 30 isolates possessed a transferable resistance confirmed by conjugation and isolation of 79–89-kb
plasmids. The β-lactam resistance was due to production of β-lactamases and ceftazidime proved to be stronger β-lactamase
inductor than ceftriaxone. Twenty-five clinical isolates expressed transferable extended spectrum β-lactamases, and chromosomally
encoded AmpC β-lactamase. 相似文献
14.
15.
Francisella tularensis ssp. tularensis is a category A select agent and the causal organism for the zoonotic disease tularemia. The vast majority of F. tularensis isolates are β-lactamase-positive. β-lactamase production is widely believed to be responsible for the inefficacy of β-lactams in the treatment of tularemia. In this study, we report the cloning and characterization of the two chromosomally encoded F. tularensis ssp. holarctica live-vaccine strain (LVS) β-lactamases. The two LVS β-lactamases were homologous to F. tularensis Schu S4 open reading frames FTT0681c and FTT0611c and have been named bla1
LVS
and bla2
LVS
, respectively. Recombinant expression in Escherichia coli suggested that bla1
LVS
did not encode a functional β-lactamase, whereas bla2
LVS
encoded a functional β-lactamase that hydrolyzed penicillins but was inactive against third-generation cephalosporins, including cefprozil. As both LVS and Schu S4 were susceptible to cefprozil, we developed three new shuttle vectors based on selection for the production of the Blashv-2 extended-spectrum β-lactamase with cefprozil. The resulting shuttle vectors were suitable for recombinant gene expression and complementation studies in LVS and Schu S4. 相似文献
16.
Kupper M Bauvois C Frère JM Hoffmann K Galleni M Bebrone C 《Extremophiles : life under extreme conditions》2012,16(1):45-55
The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-β-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature
E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a β-lactamase
activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5′-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary
structures characteristic of the MBL αββα fold. 相似文献
17.
Chunyan Wang Hongyue Dang Yongsheng Ding 《World journal of microbiology & biotechnology》2008,24(12):2889-2896
Environmental microbiology investigation was performed to determine the molecular diversity of β-lactamase genes among ampicillin-resistant
bacteria from Jiaozhou Bay. β-lactamase genes were detected in 93.8% of the bacterial isolates identified as Enterobacteriaceae. The most frequently detected gene was bla
TEM, followed by bla
SHV, bla
OAX-1, bla
MOX and bla
CMY. Most of the isolates (68.8%) were positive for the intI1 integrase gene, and two isolates were also found for the intI2 gene. The dfr and aadA gene cassettes were predominant. Anthropogenic contamination from onshore sewage processing plants might contribute predominantly
to the β-lactamase gene reservoir in the studied coastal waters. Environmental antibiotic-resistant bacteria and resistance
genes may serve as bioindicators of coastal environmental quality or biotracers of the potential contamination sources. This
is the first report of the prevalence and characterization of β-lactamase genes and integrons in coastal Enterobacteriaceae from China. 相似文献
18.
Pseudomonas syringae isolate BR2R produces tabtoxin, a β-lactam-containing antibiotic, and the causative agent of wildfire disease of green bean
(Phaseolus vulgaris). β-Lactamase production has been suggested as the mechanism that protects P. syringae from tabtoxin. We sought to determine whether the organism produces β-lactamase and whether the enzyme plays a role in protection
from this antibiotic. P. syringae and mutants defective in tabtoxin production and resistance produce β-lactamase. Three distinct β-lactamases with molecular
weights of 41,000 were identified. The isoelectric points of the proteins were 6.1, 6.8, and 9.2. The enzymes preferentially
hydrolyze cephalosporin. This investigation demonstrates that the organism produces multiple β-lactamases and describes characteristics
of the proteins. 相似文献
19.
Mariachiara Conte Francesco Aliberti Laura Fucci Marina Piscopo 《World journal of microbiology & biotechnology》2007,23(12):1679-1683
Antibacterial effects of various arginine- and lysine-rich polycationic proteins and polymers were evaluated by broth and
solid dilution assay on a range of foodborne pathogens, Gram-positive and Gram-negative bacteria. The Minimum Inhibitory Concentration
(MIC) and the Minimum Bactericidal Concentration (MBC) of α-poly-l-lysine (poly-lys), α-poly-l-arginine (poly-arg) and protamines from herring sperm (clupeine sulphate) and salmon sperm (salmine sulphate) were determined
on Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella sonnei,
Escherichia coli O157:H7 and Pseudomonas aeruginosa. All these molecules showed antibacterial activity on all strains with different MIC and MBC values. The molecular mechanisms
underlying the effect of α-poly-l-arginine might be related to the entrance of the molecule into the cell. In fact α-poly-l-arginine labelled with 7-Diethylamino coumarin-3-carboxylic acid, succinimidyl ester (DEAC,SE) showed ability to permeate
the cell membrane of B. cereus and E. coli O157:H7. 相似文献
20.
Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli 总被引:4,自引:0,他引:4
Jan Maarten van Dijl Anne de Jong Hilde Smith Sierd Bron Gerard Venema 《Molecular & general genetics : MGG》1991,227(1):40-48
Summary The effects of 25-fold overproduction ofEscherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between
signal sequence functions selected from theBacillus subtilis chromosome and the mature part of TEM-β-lactamase, were studied inE. coli. One precursor (pre[A2d]-β-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of
the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[Al3i]-β-lactamase), which was not
processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction.
However, this did not result in efficient release of the mature β-lactamase into the periplasm. In contrast, the processing
rates of wild-type pre-β-lactamase and pre(A2)-β-lactamase, already high under standard conditions, were not detectably altered
by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein
export inE. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies. 相似文献