Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis

Real-time PCR-based assays specific for Brucella abortus, Brucella melitensis and Brucella suis were developed. The assays utilize an upstream primer that is derived from 3' end of the genetic element IS 711, whereas the downstream primers and probes are designed from signature sequences specific to a species or a biovar. The PCR reactions were monitored for fluorescence resonance energy transfer by including two adjacent labeled probes that hybridize to the amplicons as they are formed. The upstream probes were labeled with fluorescein at 3' end while Cy5 was attached to the 5' end of the downstream probes. An increase in the ratio of fluorescein to Cy5 fluorescence during the cycling was indicative of positive amplification event. The assays were accomplished in less than 30 min using a LightCycler in real-time mode. The assays were tested on known strains as well as field isolates and were found to be specific for all known biovars of B. abortus, B. melitensis and biovar 1 of B. suis. Therefore, specificity, sensitivity, speed and real-time detection make these assays attractive for use in epidemiological and ecological studies.     

Clinical findings, diagnostic approach, and outcome of Brucella melitensis epididymo-orchitis

We have studied 912 patients with brucellosis. Of these, 631 (69.2%) were male and 48 had epididymo-orchitis, giving an incidence of epididymo-orchitis of 7.6%. The duration of symptoms before diagnosis was 52.5 +/- 70 days. All the patients had fever, swelling, and scrotal pain, but only 2 (4.2%) reported urinary symptoms. Seven patients (14.5%) had leukocyte figures above 11 x 10(9)/L, and urine analysis was normal in 69% of the patients. Blood cultures were positive in 65.8% of cases. A total of 33 patients (68.8%) received a combination of doxycycline plus streptomycin and 13 (27.1%) doxycycline plus rifampin. The overall percentage of failure or relapse was 8.8%: 7.1% in the doxycycline plus streptomycin group and 20% in the doxycycline plus rifampin group. None of the patients required surgery. Pending clinical trials to confirm the results, conservative management with a combination of doxycycline for 2 months and streptomycin for 14 to 21 days appears to be adequate and could avoid unnecessary orchiectomy.     

Change in the Cell Envelope of Escherichia coli Carrying the Thermosensitive Drug Resistance Factor, Rts 1, at the Nonpermissive Temperature

Escherichia coli, harboring the temperature-sensitive drug-resistant factor Rts 1, formed filaments on solid medium at the nonpermissive temperature (42 C). In addition, the rate of adsorption of T4D phage progressively decreased during growth at 42 C. Susceptibility to a variety of antibiotics increased suggesting that the permeability barrier to these antibiotics may be disrupted at the nonpermissive temperature. These observations were interpreted to suggest that the target of the temperature-sensitive Rts 1 gene product responsible for altering host growth may be the cell envelope.     

Susceptibilities of Brucella melitensis isolates to clinafloxacin and four other new fluoroquinolones.

The susceptibilities of 120 clinical isolates of Brucella melitensis and 3 reference strains of the same species to six fluoroquinolones (clinafloxacin, PD 117596, PD 131628, PD 138312, PD 140248, and ciprofloxacin) were examined by agar dilution MIC methodology. Clinafloxacin was the most active compound tested (MIC at which 50% of strains tested were inhibited [MIC50] and MIC90 of 0.06 micrograms/ml). Its level of activity was slightly higher than that of PD 117596 (MIC50 and MIC90 of 0.12 micrograms/ml). PD 131628 and ciprofloxacin were less active than clinafloxacin, with MIC50s ranging from 0.12 to 0.25 micrograms/ml and MIC90s of between 0.25 and 0.5 micrograms/ml for the two compounds. The activity levels of PD 138312 and PD 140248, with MIC50s ranging from 1 to 2 micrograms/ml and MIC90s of 4 to 8 micrograms/ml, were lower than those of the other fluoroquinolones tested.     

Functional characterization of Brucella melitensis NorMI,an efflux pump belonging to the multidrug and toxic compound extrusion family

Two putative proteins (NorMI and NorMII) similar to the multidrug efflux protein NorM of Vibrio parahaemolyticus are encoded by the Brucella melitensis 16 M genome. We show that a drug-hypersusceptible Escherichia coli strain overexpressing NorMI displays increased resistance to norfloxacin, ciprofloxacin, gentamicin, tetraphenylphosphonium ion, acriflavine, and berberine. This elevated resistance was proven to be mediated by an energy-dependent efflux mechanism. NorMI belongs to the multidrug and toxic compound extrusion family and is the first multidrug efflux protein identified in Brucella spp.     

In vitro susceptibility of Brucella melitensis to new cephalosporins crossing the blood-brain barrier.

The in vitro susceptibilities of 83 clinical isolates of Brucella melitensis to seven cephalosporins and a monobactam were determined. Ceftizoxime, ceftriaxone, and cefotaxime were the most effective agents tested, with MICs ranging from 0.25 to 2 micrograms/ml. Moxalactam, cefoperazone, cefuroxime, and ceftazidime showed MICs between 4 and 64 micrograms/ml, with moxalactam being the most active agent in this group. Aztreonam showed poor activity, with MICs higher than 64 micrograms/ml.     

耐碳青霉烯铜绿假单胞菌耐药基因筛查及对多粘菌素异质性耐药的研究

[目的]分析耐碳青霉烯铜绿假单胞菌(CRPA)耐药基因特征及对多粘菌素异质性的耐药情况.[方法]收集76株CRPA,PCR检测耐药相关基因,检测菌株对多粘菌素的敏感性.选15株行群体谱分析多粘菌素异质性,应用联合用药实验筛选用药方案.[结果]76株CRPA,碳青霉烯酶相关基因IMP阳性15株(19.74％),VIM阳性...     

Resistance of Spheroplasts and Whole Cells of Pseudomonas cepacia to Polymyxin B

Sucrose-lysozyme spheroplasts were prepared from two strains of Pseudomonas cepacia and tested for susceptibility to polymyxin B and benzalkonium chloride. Spheroplasts were more susceptible than whole cells to benzalkonium chloride but not to polymyxin B. Disruption of the outer membrane layer was not by itself sufficient to render P. cepacia susceptible to polymyxin B.     

Incompatible Natures of the HIV-1 Envelope in Resistance to the CCR5 Antagonist Cenicriviroc and to Neutralizing Antibodies

Cenicriviroc is a CCR5 antagonist which prevents human immunodeficiency virus type 1 (HIV-1) from cellular entry. The CCR5-binding regions of the HIV-1 envelope glycoprotein are important targets for neutralizing antibodies (NAbs), and mutations conferring cenicriviroc resistance may therefore affect sensitivity to NAbs. Here, we used the in vitro induction of HIV-1 variants resistant to cenicriviroc or NAbs to examine the relationship between resistance to cenicriviroc and resistance to NAbs. The cenicriviroc-resistant variant KK_652-67 (strain KK passaged 67 times in the presence of increasing concentrations of cenicriviroc) was sensitive to neutralization by NAbs against the V3 loop, the CD4-induced (CD4i) region, and the CD4-binding site (CD4bs), whereas the wild-type (WT) parental HIV-1 strain KK_WT from which cenicriviroc-resistant strain KK_652-67 was obtained was resistant to these NAbs. The V3 region of KK_652-67 was important for cenicriviroc resistance and critical to the high sensitivity of the V3, CD4i, and CD4bs epitopes to NAbs. Moreover, induction of variants resistant to anti-V3 NAb 0.5γ and anti-CD4i NAb 4E9C from cenicriviroc-resistant strain KK_652-67 resulted in reversion to the cenicriviroc-sensitive phenotype comparable to that of the parental strain, KK_WT. Resistance to 0.5γ and 4E9C was caused by the novel substitutions R315K, G324R, and E381K in the V3 and C3 regions near the substitutions conferring cenicriviroc resistance. Importantly, these amino acid changes in the CCR5-binding region were also responsible for reversion to the cenicriviroc-sensitive phenotype. These results suggest the presence of key amino acid residues where resistance to cenicriviroc is incompatible with resistance to NAbs. This implies that cenicriviroc and neutralizing antibodies may restrict the emergence of variants resistant to each other.     

In-vitro activity of ciprofloxacin, ceftriaxone and five other antimicrobial agents against 95 strains of Brucella melitensis

We studied the in-vitro activity of seven antibiotics against 95 strains of Brucella melitensis isolated in blood cultures of 95 patients with brucellosis. The minimum inhibitory concentration (MIC) was measured by the agar dilution method. All strains of B. melitensis were inhibited by doxycycline at 0.25 mg/l, tetracycline at 0.5 mg/l, ciprofloxacin at 0.5 mg/l, streptomycin at 1 mg/l, ceftriaxone at 1 mg/l, rifampicin at 4 mg/l and by co-trimoxazole at 0.5/9.5 mg/l. We did not find strains resistant to any of the antibiotics studied. All antibiotics, including ciprofloxacin and ceftriaxone, showed a good in-vitro activity against B. melitensis.     

Comparative in vitro activities of ofloxacin, difloxacin, ciprofloxacin, and other selected antimicrobial agents against Brucella melitensis.

The in vitro activities of three quinolones (ofloxacin, difloxacin, and ciprofloxacin) were compared with those of trimethoprim-sulfamethoxazole, streptomycin, tetracycline, and rifampin against 47 Brucella melitensis strains. Ofloxacin was the most active of the test antimicrobial agents. It inhibited 90% of B. melitensis strains at a concentration of 0.02 micrograms/ml.     

Phase-Variable Expression of lptA Modulates the Resistance of Neisseria gonorrhoeae to Cationic Antimicrobial Peptides

Phosphoethanolamine (PEA) decoration of lipid A produced by Neisseria gonorrhoeae has been linked to bacterial resistance to cationic antimicrobial peptides/proteins (CAMPs) and in vivo fitness during experimental infection. We now report that the lptA gene, which encodes the PEA transferase responsible for this decoration, is in an operon and that high-frequency mutation in a polynucleotide repeat within lptA can influence gonococcal resistance to CAMPs.     

转染VMAT2基因的中国仓鼠卵巢细胞对毒物抵抗作用的研究

目的:研究毒物鱼藤酮和1-甲基-4-苯基吡啶离子(MPP+)对野生型及转染囊泡单胺转运蛋白(VMAT2)基因的中国仓鼠卵巢细胞(CHO)的毒性作用。方法:将不同浓度的MPP+、鱼藤酮与野生型CHO细胞(WT-CHO)和转染VMAT2基因的CHO细胞(VMAT2-CHO)共同培养,采用细胞MTT比色法和形态学观察,探讨毒物对细胞的毒性作用,通过激光共聚焦显微镜观察毒物对2种细胞的细胞内钙浓度([Ca2+]i)的影响。结果:VMAT2-CHO在一定时间(72 h)内对MPP+(0.2-2.0 mmol/L)和鱼藤酮(0.05-1.0μmol/L)的毒性有抵抗作用(P<0.05)。结论:VMAT2可以抵抗一定浓度的MPP+和鱼藤酮引起的神经细胞毒性作用。     

KpnEF,a New Member of the Klebsiella pneumoniae Cell Envelope Stress Response Regulon,Is an SMR-Type Efflux Pump Involved in Broad-Spectrum Antimicrobial Resistance

Klebsiella pneumoniae has been frequently associated with nosocomial infections. Efflux systems are ubiquitous transporters that also function in drug resistance. Genome analysis of K. pneumoniae strain NTUH-K2044 revealed the presence of ∼15 putative drug efflux systems. We discuss here for the first time the characterization of a putative SMR-type efflux pump, an ebrAB homolog (denoted here as kpnEF) with respect to Klebsiella physiology and the multidrug-resistant phenotype. Analysis of hypermucoviscosity revealed direct involvement of kpnEF in capsule synthesis. The ΔkpnEF mutant displayed higher sensitivity to hyperosmotic (∼2.8-fold) and high bile (∼4.0-fold) concentrations. Mutation in kpnEF resulted in increased susceptibility to cefepime, ceftriaxone, colistin, erythromycin, rifampin, tetracycline, and streptomycin; mutated strains changed from being resistant to being susceptible, and the resistance was restored upon complementation. The ΔkpnEF mutant displayed enhanced sensitivity toward structurally related compounds such as sodium dodecyl sulfate, deoxycholate, and dyes, including clinically relevant disinfectants such as benzalkonium chloride, chlorhexidine, and triclosan. The prevalence of kpnEF in clinical strains broadens the diversity of antibiotic resistance in K. pneumoniae. Experimental evidence of CpxR binding to the efflux pump promoter and quantification of its expression in a cpxAR mutant background demonstrated kpnEF to be a member of the Cpx regulon. This study helps to elucidate the unprecedented biological functions of the SMR-type efflux pump in Klebsiella spp.     

In vitro activity of N-formimidoyl thienamycin against 98 clinical isolates of Brucella melitensis compared with those of cefoxitin, rifampin, tetracycline, and co-trimoxazole.

IN vitro susceptibilities of 98 isolates of Brucella melitensis to N-formimidoyl thienamycin, tetracycline, co-trimoxazole, rifampin, and cefoxitin were determined. N-Formimidoyl thienamycin showed good activity which was similar to those of tetracycline and rifampin and different from that of the other beta-lactam antibiotic tested (cefoxitin), which showed poor activity. Co-trimoxazole showed good activity.