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    Effect of plasmid curing on the 2, 3-dihydroxybenzoic acid production and antibiotic resistance of Acinetobacter sp. B-W
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    Abstract:
    시드로포어인 2, 3-dihydroxybenzoic acid (DHB)를 생산하는 Acinetobacter sp. B-W의 플라스미드를 분석한 결과, 20 kb 크기의 플라스미드를 함유하였다. 배양 온도 $43^{\circ}C$ 가 플라스미드가 제거된 돌연변이체 생산에 효과적이었다. 이 돌연변이체는 2,3-DHB 생산 능력을 소실하였으며, chrome azurol S (CAS) 아가 배지에서 시드로포어 생산이 검출되지 않았다. 포도당과 황산 망간을 함유한 배지에서 $28^{\circ}C$로 3일간 배양한 B-W 원 균주와 돌연변이체의 배양 상등액의 pH는 각각 4.5와 8.5로 나타났다. 돌연변이체에서는 ampicillin, actinomycin D, bacitracin, lincomycin과 vancomycin 같은 항생제에 대한 저항성이 사라졌으며, 이러한 항생제에 대한 최소 억제 농도(MIC)가 급격하게 감소하였다. B-W 균주에서 분리한 플라스미드로 대장균을 형질전환시킨 결과, 원 균주와 같은 크기의 플라스미드가 이 형질전환 대장균에서 발견되었다. 플라스미드가 제거된 돌연변이체에서는 플라스미드가 발견되지 않았다. 20 kb 크기의 플라스미드에 2,3-DHB 생산 유전자와 여러 항생제 저항성 유전자가 자리잡고 있는 것으로 추정된다. Acinetobacter sp. B-W producing siderophore, 2, 3-dihydroxybenzoic acid (DHB) was analyzed for plasmid content. Strain B-W harbored plasmid of 20 kb in size. Growth at $43^{\circ}C$ was effective in producing mutant cured of plasmid of strain B-W. This mutant lost the ability to produce 2, 3-DHB. Formation of siderophore halos on the chrome azurol S (CAS) agar medium was not detected by cured strain B-W. pHs of supernatants of wild type strain B-W and cured mutant grown in glucose and $MnSO_4$ containing medium at $28^{\circ}C$ for 3 days were 4.5 and 8.5, respectively. Antibiotic resistance against ampicillin, actinomycin D, bacitracin, lincomycin, and vancomycin was lost in cured mutant. Plasmid curing of strain B-W resulted in drastic reduction of minimal inhibitory concentration (MIC) of several antibiotics. E. coli $DH5{\alpha}$ was transformed with plasmid isolated from strain B-W. The transformant E. coli $DH5{\alpha}$ harbored a plasmid of the same molecular size as that of the donor plasmid. Transformant E. coli $DH5{\alpha}$ produced 2, 3-DHB and contained antibiotic resistant ability. Thus a single plasmid of 20 kb seemed to be involved in 2, 3-DHB production. Genes encoding resistance to antibiotics were also supposed to be located on this plasmid.
    Keywords:
    Lincomycin
    Amp resistance
    Objective To study the mechanism of ampicillin resistance in Haemophilus in fluenza(HI)strains.Methods A to- tal of 300 strains of HI isolated from children with respiratory tract infection were collected.Their resistance to ampicillin was detected by E-test.β-lactamase production was determined by chromogenic cepha[osporin(nitrocefin)test.TEM-1 and ROB-1 gene conferring ampicillin resistance were detected by polymerase chain reaction(PCR).Results β-lactamase was produced in all the 32 ampicillin-resistant HI strains.TEM-1 gene was identified in 31 of the 32 ampicillin-resistant strains.ROB-1 gene was detected in only 1 ampicillin-resistant strain.Conclusions TEM-1 β-lactamase is the major mechanism of anlpicillin resist- ance in HI isolated from children with respiratory tract infection.ROB-1 gene should be carefully monitored for a long time.
    Amp resistance
    Citations (0)
    Once a Hemophilus influenzae isolate is identified as the cause of a respiratory tract infection in an adult, it should be tested for beta-lactamase production, ie, for ampicillin resistance. The incidence of ampicillin-resistant strains of H influenzae is increasing. The Centers for Disease Control in Atlanta estimates an average incidence nationwide of 18% to 22%; the rate varies considerably from community to community. Thus, practitioners should be aware of the ampicillin-resistance rate in their community and should keep this rate in mind especially when treating patients empirically. Patients with H influenzae infections who are acutely ill, who fail to respond to ampicillin, or who are known to have an ampicillin-resistant infection on the basis of laboratory findings should receive therapy designed to combat ampicillin-resistant strains.
    Amp resistance
    Objective To study the resistance mechanism of ampicillin-resistant Haemophilus influenzae strains collected from hospitals in Beijing,Shanghai and Guangzhou during the past four years.Methods During 2000—2003,899 strains of H.influenzae isolated from children with respiratory tract infection were collected to detect their resistance to ampicillin.The susceptibility testing was determined by E test.β-lactamase production was detected by chromogenic cephalosporin (nitrocefin) test.TEM-1 and ROB-1 gene conferring ampicillin resistance were detected by polymerase chain reaction (PCR).Results β-lactamase was produced in all the 74 ampicillin-resistant H.influenzae strains.They all contained TEM-1 gene.No ROB-1 gene was detected in these isolates.Conclusions TEM-1 β-lactamase is the principal mechanism of ampicillin resistance of H.influenzae isolates from the upper respiratory tract of children.
    Amp resistance
    Citations (0)
    Ampicillin-resistant Hemophilus influenzae had not been identified in Fresno, California, before June 1976. In the 12 months that followed, eight resistant type B strains and three resistant nontypable isolates were cultured from patients treated at two hospitals that provide nearly all of the acute pediatric inpatient care for the area. Two of the resistant strains were obtained from patients with invasive infections and represented 4.2 percent of Hemophilus influenzae isolated from blood, cerebrospinal fluid or joint aspirates during the 12 months. The remaining six resistant type B strains were obtained from 117 patients, and were the predominant organism in cultures of other sites, primarily respiratory secretions. In two of three patients infected with nontypable organisms, resistance appeared to emerge during therapy with ampicillin. Measurement of beta lactamase was a practical and accurate method for differentiating between ampicillin-sensitive and resistant strains. All ten of the beta lactamase-positive isolates tested had minimal inhibitory concentrations (MIC) for ampicillin of 15 mug per ml, or less. In contrast 30 beta lactamase-negative strains had MIC's of 1.5 mug per ml, or less, of ampicillin. Our results indicate that ampicillin resistance has become a significant problem in the Central Valley of California and probably the entire state.
    Amp resistance
    Citations (4)
    Ampicillin-resistant Haemophilus influenzae does occur now in the FRG. In one isolate a plasmid with resistance genes (R-factor) could be demonstrated as cause of the ampicillin resistance. This R-factor influences production of a beta-lactamase of the TEM type which destroys ampicillin. The infectious nature of the ampicillin resistance was proven by the fact that it was transferable to other bacterial species through cocultivation. Parallel to ampicillin resistance tetracycline resistant Haemophilus influenzae has occurred in the FRG. Here the resistance was equally bound to plasmids. These R-factors are infectious as well. Molecular analysis of the 3 isolated resistance factors in Haemophilus influenzae showed that they carry the same resistance genes which are known from R-factors of Enterobacteriaceae. In the therapy of purulent infections due to Haemophilus influenzae such as childhood meningitis one can no longer rely on general ampicillin sensitivity of the offender. Apart from ampicillin and tetracycline resistant Haemophilus influenzae chloramphenicol resistance has been observed in a few cases.
    Amp resistance
    Citations (1)
    The properties of donor deoxyribonucleic acid (DNA) from three clinical isolates and its ability to mediate the transformation of competent Rd strains to ampicillin resistance were examined. A quantitative technique for determining the resistance of individual Haemophilus influenzae cells to ampicillin was developed. When this technique was used, sensitive cells failed to tolerate levels of ampicillin greater than 0.1 to 0.2 mug/ml, whereas three resistant type b beta-lactamase-producing strains could form from the colonies in 1- to 3-mug/ml levels of the antibiotic. DNA extracted from the resistant strains elicited transformation of the auxotrophic genes in a multiply auxotrophic Rd strain. For two of the donors, transformation to ampicillin resistance occurred after the uptake of a single DNA molecule approximately 104-fold less frequently than transformation of auxotrophic loci and was not observed to occur at all with the third. The frequency of transformation to ampicillin resistance was two- to fivefold higher in strain BC200 (Okinaka and Barnhart, 1974), which was cured of a defective prophage. All three clinical ampicillin-resistant strains were poor recipients, but the presence of the ampicillin resistant genes in strain BC200 did not reduce its competence. Sucrose gradients of DNA from ampicillin-resistant transformants of BC200 and from the original ampicillin-resistant strains showed that: (i) all the DNA preparations had high molecular weights; (ii) donor activity for ampicillin resistance sedimented heterogeneously and in parallel with genome DNA up to the highest molecular weights observed (100 x 106 to 200 x 106); and (iii) genetic transformation of ampicillin resistance from strain BC200-amp90383 required the physical integrity of a linearly integrated segment of DNA having a molecular weight of about 25 x 106 to 30 x 106.
    Amp resistance
    Prophage
    A total of 1,102 clinical isolates of Gram-negative bacilli was obtained from four hospitals during 1967 and these cultures were tested for sensitivity to ampicillin. Approximately 80% of the strains of Escherichia coli and 90% of the strains of Proteus mirabilis, the two organisms most frequently isolated, were sensitive to ampicillin. Klebsiella-Enterobacter species and Pseudomonas aeruginosa were generally insensitive. Comparison of these results with data obtained in an earlier study with Gram-negative organisms isolated in 1961 showed that there had been no significant increase in the incidence of resistance of Gram-negative bacilli to ampicillin during the period 1961-67. The majority of ampicillin-resistant strains of E. coli isolated in 1967 transferred ampicillin resistance to a sensitive strain of E. coli K12. Only four ampicillin-resistant strains of E. coli isolated in 1961 were available for transferable resistance tests but all four strains transferred ampicillin resistance. Infective or transferable resistance was therefore a feature of ampicillin resistance of certain Gram-negative bacteria before ampicillin became generally available for clinical use.
    Amp resistance
    Klebsiella
    Gram-Negative Bacteria
    Citations (16)
    A 1-year-old boy with recurrent otitis media had been repeatedly treated with antibiotics. A few days after withdrawal of administered ampicillin he again contracted otitis media and ampicillin-resistant Haemophilus influenzae was isolated. The strain was serologically untypable. No ampicillin-resistant H. influenzae was found in his family or at the day-care centre that he attended. The ability to produce the β-lactamase elaborated from this strain could be transferred to ampicillin-sensitive strains of H. influenzae and Escherichia coli in frequencies of 0.7 × 10–7 and 4.1 × 10–4 respectively. The transcipients obtained were ampicillin-resistant and β-lactamase producing. In the transcipients of E. coli, however, the marker for ampicillin resistance was quite unstable.
    Amp resistance
    Objective This study was designed to investigate ampicillin resistance in H.influenzae strains isolated from infected children in Suzhou,and the mechanism of resistance.Methods The susceptibility of 135 of clinical isolates of H.influenzae were tested by Kirby-Bauer disc method.Chromogenic nitrocephin disc was used to detect beta-lactamase.TEM and ROB beta-lactamase genes were detected by PCR.Results The rate of ampicillin resistance was 17.8% in H.influenzae isolated from children in Suzhou.Beta-lactamase was produced by all ampicillin-resistant H.influenzae isolates.No beta-lactamase negative ampicillin-resistant H. influenzae (BLNAR) was identified.Beta-lactamase gene TEM was detected in 27.4% of H.influenzae isolates (TEM gene was also present in 10.4% of ampicillin-sensitive strains).ROB gene was detected in 0.7% of H.influenzae isolates (This ROB-type strain also carried TEM gene).One strain of non-TEM-type,and non-ROB-type beta-lactamse-producing H.influenzae was identified.Conclusions Ampicillin resistance in H.influenzae isolates from children in this region is challenging.The resistance mechanism of ampicillin resistant H.influenzae is production of beta-lactamase,mainly TEM-type enzyme.It should be noted that the emergence of ampicillin-sensitive strain carrying TEM gene,which may predicts the development of new ampicillin-resistant strains.
    Amp resistance
    Beta-lactamase
    Strain (injury)
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