Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation

The effects of timentin on shoot regeneration of tobacco (Nicotiana tabaccum) and Siberian elm (Ulmus pumila L.) and its use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were determined. Timentin is a mixture of ticarcillin and clavulanic acid, and at concentrations of 200–500 mg/l with ratios of ticarcillin:clavulanic acid of 50:1 and 100:1, it had little effect on shoot regeneration of tobacco or Siberian elm. Timentin was as effective in suppressing A. tumefaciens as carbenicillin and cefatoxime at concentrations commonly used in transformation. The disarmed A. tumefaciens strain LBA4404 in infected tobacco leaf tissues was visually undetectable after three subcultures on medium with 500 mg/l of timentin and 250 mg/l carbenicillin. Timentin was stable in solid agar medium and remained effective for at least 70 days, but was unstable when stored as a mixed stock solution or as separate ticarcillin and clavulanic acid stock solutions at –20°C or –80°C freezer for 4 weeks. Timentin may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation. Received: 8 September 1997 / Revision received: 19 November 1997 / Accepted: 2 December 1997     

The inhibition of staphylococcal beta-lactamase by clavulanic acid.

Clavulanic acid inhibited both the extracellular and cell-extract beta-lactamases of the four Staphylococcus aureus strains tested. The inhibition of S. aureus Russell cell-extract enzyme appeared to be active-site-directed and proceeded in a first-order fashion consistent with the formation of a covalent intermediate. Inhibited enzyme free of excess clavulanic acid was shown to regenerate enzyme activity slowly at pH 7.0, but the rate of reactivation increased at acid pH. When the enzyme was incubated with excess clavulanic acid complete inhibition was rapidly obtained, during further incubation clavulanic acid was shown to disappear slowly and complete loss of clavulanic acid from the reaction mixture coincided with the onset of the return of enzyme activity. A reactive enamine resulting from enzymic hydrolysis of the beta-lactam ring of clavulanic acid has been proposed as a possible intermediate in the inhibitory mechanism.     

Pseudomonas aeruginosa and beta-lactam resistance phenotypes (Tunisia 1989-1990)]

366 strains of P. aeruginosa were studied. More than half of samples were issued from urology (40%) and general surgery (21%) and samples are mostly urine specimens (46%). The phenotypes of resistance to 3 beta-lactams (respectively ticarcillin, cefotaxime and ceftazidime) showed that 73% of the strains were wild phenotype (SSS-SIS-SRS). The MIC of ticarcillin +/- clavulanic acid, azlocillin, cefoperazone, cefsulodin, cefotaxime and ceftazidime against 93 strains chosen among different phenotypes of resistance showed that ceftazidime is the most active beta-lactam, 93% of strains being inhibited by 16 micrograms/ml. These MIC and iodometric test performed on 33 strains with ticarcillin MIC > 128 mg/l showed that acquired resistance was non enzymatic in 21% of cases and was related to beta-lactamase production in 79% of cases.     

Structure-activity relationships amongst beta-lactamase inhibitors

Using a variety of beta-lactamases including those from Escherichia coli (TEM-1), Enterobacter cloacae P99 and Staphylococcus aureus the inhibition profiles (I50 values) were determined for various groups of compounds including penicillins, penicillanic acid derivatives (sulphone and beta-halo substitutions), olivanic acids and clavulanic acid derivatives including substituted ethers and amines. Some of the latter compounds had higher activity than clavulanic acid with and without preincubation of enzyme with inhibitor but they still had poor activity against the P99 enzyme. Improvements in activity against Class I cephalosporinases were obtained with some derivatives of clavulanic acid but this was usually achieved at the expense of activity against clavulanate susceptible beta-lactamases. The olivanic acids had the highest activity against the widest range of beta-lactamases.     

TRC-1: Emergence of a clavulanic acid-resistant TEM β-lactamase in a clinical strain

A novel TEM-derived plasmid-encoded beta-lactamase, resistant to inhibition by clavulanic acid, has been identified in a clinical strain of Escherichia coli found in Scotland. The beta-lactamase gene was carried on an 81-kb plasmid that conferred no other resistances. The novel enzyme conferred resistance to the amoxycillin/clavulanic acid combination on the host bacterium. The beta-lactamase has a pI of 5.25 and lies between the PSE-4 and SAR-1 beta-lactamases on an isoelectric focusing gel. This beta-lactamase has a Mr value of 25,000, similar to the TEM-1 enzyme and a comparable substrate profile. Its most significant difference is that it is inhibited by clavulanic acid 100-fold less efficiently than the TEM-1 enzyme. The enzyme was confirmed to be derived from the TEM enzymes by probing the plasmid DNA with an intragenic gene probe for TEM-1. This is the first report of a clinical bacterium carrying a TEM-enzyme that confers resistance to clavulanic acid combinations and we have designated the beta-lactamase as TRC-1.     

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight.Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice.     

Concerning the sensitivity of Acinetobacter calcoaceticus

The strains of Acinetobacter calcoaceticus, var. anitratus (A. c. a.) were isolated in the nosocomial environment as an opportune pathogen. The therapy of choice may be determined after in vitro tests. Our results show following therapeutical possibilities: beta-lactam antibiotics--cephalosporins of IIIrd generation (cefotaxime), also combinations of antimicrobials have shown good results: amoxycillin or ticarcillin with clavulanic acid. Best synergistic effect was found in combination ticarcillin-amikacin.     

Structure-Activity Relationships Amongst β-Lactamase Inhibitors

Abstract

Using a variety of β-lactamases including those from Escherichia coli (TEM-1), Enterobacter cloacae P99 and Staphylococcus aureus the inhibition profiles (I₅₀ values) were determined for various groups of compounds including penicillins, penicillanic acid derivatives (sulphone and β-halo substitutions), olivanic acids and clavulanic acid derivatives including substituted ethers and amines. Some of the latter compounds had higher activity than clavulanic acid with and without preincubation of enzyme with inhibitor but they still had poor activity against the P99 enzyme. Improvements in activity against Class I cephalosporinases were obtained with some derivatives of clavulanic acid but this was usually achieved at the expense of activity against clavulanate susceptible β-lactamases.The olivanic acids had the highest activity against the widest range of β-lactamases.     

Mutants of Streptomyces clavuligerus with disruptions in different genes for clavulanic acid biosynthesis produce large amounts of holomycin: possible cross-regulation of two unrelated secondary metabolic pathways

A Streptomyces clavuligerus ccaR::aph strain, which has a disruption in the regulatory gene ccaR, does not produce cephamycin C or clavulanic acid, but does produce a bioactive compound that was identified as holomycin by high-performance liquid chromatography (HPLC) and infrared and mass spectrometry. S. clavuligerus strains with disruptions in different genes of the clavulanic acid pathway fall into three groups with respect to holomycin biosynthesis. (i) Mutants with mutations in the early steps of the pathway blocked in the gene ceaS (pyc) (encoding carboxyethylarginine synthase), bls (encoding a beta-lactam synthetase), or open reading frame 6 (ORF6; coding for an acetyltransferase of unknown function) are holomycin nonproducers. (ii) Mutants blocked in the regulatory gene ccaR or claR or blocked in the last gene of the pathway encoding clavulanic acid reductase (car) produce holomycin at higher levels than the wild-type strain. (iii) Mutants with disruption in cyp (coding for cytochrome P450), ORF12, and ORF15, genes that appear to be involved in the conversion of clavaminic acid into clavaldehyde or in secretion steps, produce up to 250-fold as much holomycin as the wild-type strain. An assay for holomycin synthetase was developed. This enzyme forms holomycin from holothin by using acetyl coenzyme A as an acetyl group donor. The holomycin synthase activities in the different clavulanic acid mutants correlate well with their production of holomycin.     

OHIO-1 β-lactamase resistant to mechanism-based inactivators

Abstract Spontaneous mutants of OHIO-1 β-lactamase, an SHV-1 family enzyme, resistant to inactivation by clavulanic acid, sulbactam and tazobactam, have been isolated. The resistant mutant (M4) was inhibited by 100 μg/ml ampicillin plus 32 μg/ml clavulanic acid compared to ≤2 μg/ml clavulanic acid required for the parent strain. The pI of the mutant beta-lactamase was 7.0, identical to the parent enzyme. Kinetic parameters showed that the M4 enzyme had an increased V_max/K_m ratio for all beta-lactam substrates compared to the parent enzyme. The apparent K _i for clavulanic acid, sulbactam and tazobactam was 15.1, 182 and 18 μM, respectively, up to 70-fold higher than the parent enzyme. Partial nucleotide sequencing revealed that the mutant enzyme had a predicted methionine₆₉→ isoleucine₆₉ substitution accounting for the observed changes in substrate specificity.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

洋葱伯克霍尔德菌感染家兔抗感染疗效与体外药物敏感性的比较

目的使用无CLSI完整解释标准的药物,哌拉西林/他唑巴坦、替卡西林/棒酸、头孢吡肟治疗洋葱伯克霍尔德菌感染的家兔模型,观察比较何种体外药物敏感试验更能准确反应临床疗效。方法 57株临床分离的洋葱伯克霍尔德菌,感染57只家兔腹腔。将感染家兔分成3组(A、B、C组),每组19只;分别在48、72、96、120和144 h注射药物:A组每只家兔注射1 mL浓度为2 g/mL哌拉西林/他唑巴坦;B组每只家兔注射1 mL浓度为0.1g/mL头孢吡肟;C组每只家兔注射1 mL浓度为0.6 g/mL替卡西林/棒酸。比较肉汤稀释法(MIC法)、K-B法及VITEK法检测的药物敏感性结果与体内药物治疗的疗效关系。结果哌拉西林/他唑巴坦治疗组(A组),K-B法检测结果与家兔感染模型治疗过程相符率为72.2%,MIC法66.7%,VITEK法为66.7%。替卡西林/棒酸治疗组(B组),MIC法83.3%,VITEK法为77.8%,K-B法检测结果与家兔感染模型治疗过程相符率为50.0%。头孢吡肟治疗组(C组),MIC法88.9%,VITEK法为77.8%,K-B法检测结果与家兔感染模型治疗过程相符率为72.2%。结论在3种药物敏感性检测法中,哌拉西林/他唑巴坦以K-B法检测结果与感染家兔的抗感染疗效最为接近;替卡西林/棒酸及头孢吡肟均以MIC法与感染家兔的抗感染疗效最为接近;针对洋葱伯克霍尔德菌的感染建议临床使用不同的方法检测不同的药物体外敏感性。     

Chemical studies on the inactivation of Escherichia coli RTEM beta-lactamase by clavulanic acid.

Incubation of clavulanic acid with the beta-lactamase from Escherichia coli RTEM leads to enzyme-catalyzed depletion of clavulanic acid, to transient inhibition, and to irreversible inactivation of the enzyme. Both the transiently inhibited and the irreversibly inactivated species show a marked increase in the absorbance at 281 nm that is proportional to the decrease in enzyme activity. Hydroxylamine treatment of irreversibly inactivated enzyme restores about one-third of the catalytic activity, with a concomitant decrease in absorbance at 281 nm. Polyacrylamide isoelectric focusing of the irreversibly inactivated enzyme shows three bands of approximately equal intensity, different from native enzyme. Upon hydroxylamine treatment, one of the three bands disappears and now focuses identically with native enzyme. It is evident that the irreversible inactivation of enzyme by an excess of clavulanic acid generates three products, one of which can be reactivated by hydroxylamine.     

Following the reactions of mechanism-based inhibitors with beta-lactamase by Raman crystallography

The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase (EC 3.5.2.6) have been followed in single crystals using a Raman microscope. The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species. A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group. By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation. The Raman data show that all three inhibitors form an enamine-type acyl enzyme reaching maximal populations at 10, 22, and 29 min for sulbactam, clavulanic acid, and tazobactam, respectively. The enamine intermediate exhibits a characteristic and relatively intense band near 1595 cm(-1) due to a stretching motion of the O=C-C=C-NH moiety that shifts to lower frequency upon NH <--> ND exchange. This feature was used to follow the kinetics of enamine buildup and decay in the crystal. Quantum mechanical calculations support the assignment of the 1595 cm(-1) band, as well as several other bands, to a trans-enamine species. The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl (C=O) peak disappears prior to the appearance of the enamine 1595 cm(-1) band. Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design.     

Identification and susceptibility to antimicrobial agents of strictly anaerobic bacteria isolated from hospitalized patients

The aim of this study was to identify anaerobic strains isolated in 2001 from clinical specimens obtained from patients of Warsaw hospital and to evaluate a susceptibility of these strains to antimicrobial agents. In 2001 two hundred and twenty five clinical strains of obligate anaerobes were cultured, which were identified in the automatic ATB system (bioMérieux, France) using biochemical tests API 20 A. Drug-susceptibility of strains was determined also in ATB system with the use of ATB ANA strips. C. difficile strains were isolated on selective CCCA medium. Toxins A/B of C. difficile directly in stool specimens were detected by means of ELISA test (TechLab, USA). Fifty four strains of Gram-negative anaerobes (B. fragilis strains dominated) and 171 strains of Gram-positive anaerobes (the greatest number of strains belonged to genus Peptostreptococcus) were cultured from clinical specimens. In the cases of antibiotic-associated diarrhea 28 C. difficile strains were isolated and C. difficile toxins A/B were detected in 39 stool samples. The most active in vitro antimicrobials against Gram-negative anaerobes were metronidazole, imipenem, ticarcillin combined with clavulanic acid and piperacillin with tazobactam. Gram-positive, clinical strains of anaerobes were the most susceptible in vitro to beta-lactam antibiotics combined with beta-lactamase inhibitors (amoxicillin/clavulanate, piperacillin/tazobactam, ticarcillin/clavulanate) and imipenem.     

Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus

Summary Streptomyces clavuligerus produced simultaneously cephamycin C and clavulanic acid in defined medium in long-term fermentations and in resting-cell cultures. Biosynthesis of cephamycin by phosphate-limited resting cells was dissociated from clavulanic acid formation by removing either glycerol or sulphate from the culture medium. In absence of glycerol no clavulanic acid was formed but cephamycin production occurred, whereas in absence of sulphate no cephamycin was synthesized but clavulanic biosynthesis took place. Sulphate, sulphite and thiosulphate were excellent sulphur sources for cephamycin biosynthesis while l-methionine and l-cysteine were poor precursors of this antibiotic. Increasing concentrations of sulphate also stimulated clavulanic acid formation. The biosynthesis of clavulanic acid was much more sensitive to phosphate (10–100 mM) regulation than that of cephamycin. Therefore, the formation of both metabolites was pertially dissociated at 25 mM phosphate. By contrast, nitrogen regulation by ammonium salts or glutamic acid strongly reduced the biosynthesis of both cephamycin and clavulanic acid.     

Antibiotic resistance among anaerobic Gram-negative bacilli: lessons from a French multicentric survey

Temporal changes of antibiotic susceptibilities among anaerobes in France are followed in our laboratory since 1992. For Bacteroides strains, resistance increased from 1992 to 1998 for amoxicillin-clavulanic acid, cefotetan and clindamycin. The present study evaluates the situation in 2000 for 434 Gram-negative anaerobic clinical isolates (obtained from 9 large university hospitals) by testing amoxicillin and ticarcillin alone or combined with clavulanic acid, cefoxitin, cefotetan, imipenem, clindamycin and metronidazole (using the NCCLS-approved method for MIC determination. The main genera tested included Bacteroides (359 strains of the fragilis group), Prevotella (40 strains), Fusobacterium (23 strains) and miscellaneous species (8 strains). Resistance rates within the B. fragilis group were: amoxicillin-clavulanic acid 5.6%, ticarcillin 33%, ticarcillin-clavulanic acid 2%, cefoxitin 13%, cefotetan 44%, clindamycin 33%, imipenem 1% and metronidazole <1%, respectively. Only one strain of B. fragilis was resistant to metronidazole (MIC=64 mg/L); due to the presence of the nimA gene on the chromosome. Resistance to imipenem or metronidazole was only found among the B. fragilis species. These two former drugs excepted, B. fragilis was less resistant to antibiotics than the other species. beta-lactamase production was detected for 357/359 strains of the fragilis group, 26/40 stains of Prevotella and 3/23 strains of Fusobacterium. Dynamic changes of antibacterial resistance are occurring within the B. fragilis group: decreased resistance to amoxicillin-clavulanic acid, ticarcillin-clavulanic acid, imipenem while resistance for cefoxitin, cefotetan, clindamycin continues to increase. Regular antibiotic surveys are needed as a source of information to guide the empirical therapy of anaerobic infections.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid.

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.