Ultrastructural study of polymyxin-resistant isolates of Pseudomonas aeruginosa.

Upon exposure to 6,000 U of polymyxin B sulfate per ml, cells of the polymyxin-sensitive PAO 1 strain of Pseudomonas aeruginosa displayed in thin sections long projections arising from the outer membrane of the cell wall and extensive cytoplasmic degradation with accumulation of cytoplasmic membrane infoldings. Polymyxin-resistant isolates derived from the PAO 1 strain, however, grew well in the presence of 6,000 U of polymyxin per ml and exhibited none of these effects, having instead the appearance of a typically healthy cell. Freeze-etching of cells of the sensitive strain grown in basal medium without polymyxin revealed a concave cell wall layer studded with numerous particles. Freeze-etching of cells of the resistant isolates grown in basal medium containing 6,000 U of polymyxin per ml revealed a concave cell wall layer (i.e., the outer half of the outer membrane) in which most of these particles were absent. Thus, acquisition of resistance to polymyxin was correlated with an alteration in the architecture of the outer membrane. When the resistant isolates were grown in the basal medium lacking polymyxin and then freeze-etched, the particle distribution in the concave cell wall layer resembled that of the sensitive parent strain. The cells had regained sensitivity to polymyxin upon suspension in medium containing 6,000 U/ml as determined by their failure to grow and by internal damages seen in thin sections. These cells also had acquired increased sensitivity to ethylenediaminetetraacetate, whereas the polymyxin-resistant cells grown in the presence of polymyxin were resistant to lysis by ethylenediaminetetraacetate. The polymyxin-resistant isolates were not stable mutants but instead represented an adaptive response to the presence of polymyxin in the medium.     

Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism

The isolated outer membrane from cells of a Pseudomonas aeruginosa strain exhibiting adaptive resistance to polymyxin was not affected by polymyxin treatment, as monitored by electron microscopy of negatively stained preparations. This was in sharp contrast with extensive disruption by polymyxin of the outer membranes of the parent polymyxin-sensitive strain and the resistant strain following reversion to greater polymyxin sensitivity. The isolated cytoplasmic membrane of the polymyxin-resistant strain, on the other hand, remained sensitive to the disruptive effects of polymyxin treatment. The permeability characteristics of the resistant strains appear to be altered, as indicated by differences in minimal inhibitory concentrations for a variety of antibiotics between the polymyxin-sensitive and polymyxin-resistant strains. No evidence was found for a polymyxin-inactivating enzyme in osmotic shock fluid from the polymyxin-resistant strain. No evidence for a cytoplasmic membrane repair mechanism was found in the polymyxin-resistant strain. These observations suggest that the mechanism of adaptive polymyxin resistance in this model system is the alteration of the outer membrane so that it excludes polymyxin from reaching the still sensitive cytoplasmic membrane.     

Comparison of the sensitivity of Pseudomonas aeruginosa cultures that synthesize melanin and other pigments to 12 antibiotics and 5-nitro-8-quinolinol]

Sensitivity of 80 Pseudomonas aeruginosa cultures forming melanin and 80 cultures synthesizing other pigments to amikacin, gentamicin, kanamycin, carbenicillin, levomycetin, monomycin, 5-NOK, polymyxin M, rifampicin, streptomycin, tetracycline, tobramycin and erythromycin was determined. It was found that the cultures of Ps. aeruginosa synthesizing melanin were less resistant to most of the antibiotics that the other representatives of this species.     

Antibiotic sensitivity and pathogenetic factors of hospital strains of Pseudomonas aeruginosa isolated from patients treated in a resuscitation unit]

Strains of Pseudomonas aeruginosa were isolated from patients treated in the Centre of Thermal Affections in 1985-1989. It was shown that 72.9, 59.3, 33.8 and 54.2 per cent of the isolates were sensitive to cefotaxime, tobramycin, gentamicin and polymyxin, respectively. The study of pathogenicity factors of the isolates revealed that 83 per cent of the strains produced thermolabile enterotoxin, 79.6 per cent of the strains had adhesive activity and 71.1 per cent of the strains produced hemolysin. The study detected combinations of various pathogenicity factors. 42.3 per cent of the isolates had both adhesive and enterotoxigenic properties. Adhesiveness and hemolytic activity were shown by 13.5 per cent of the strains. 16.9 per cent of the strains produced both enterotoxin and hemolysin. Adhesive activity, enterotoxigenicity and hemolysin production were observed in 6.7 per cent of the strains. It was noted that the strains of P. aeruginosa resistant to polymyxin mainly produced enterotoxin (18.6 per cent) and those resistant to cefotaxime had adhesive activity (34.0 per cent).     

Antibiotic resistance of Pseudomonas aeruginosa strains isolated from patients with infected burns

Sensitivity to 15 drugs of 248 P. aeruginosa strains isolated from patients with infected burns was studied by the method of agar dilution. All of the strains were resistant to polymyxin M, ceporin, erythromycin and oleandomycin. Most of the strains were resistant to streptomycin, monomycin, ampicillin and rifadin. Moderate resistance of the strains to carbenicillin, morphocycline, vibramycin, kanamycin, tetraolean and tetracycline was observed: the maximum concentrations of these antibiotics (128 microgram/ml) inhibited the growth of 85, 69, 63, 51.8, 43.6 and 41.2 per cent of the strains respectively. Gentamicin proved to be most active against the strains of P. aeruginosa and inhibited 87 per cent of the strains when used in the therapeutic doses. The study provided recomendation of the drugs for parenteral and local use in treatment of burns infected with P. aeruginosa.     

多重耐药铜绿假单胞菌的耐药性及耐药基因检测分析

目的探讨呼吸内科病房分离的多重耐药铜绿假单胞菌相关耐药基因。方法采用纸片扩散法进行体外药敏试验,聚合酶链反应（PCR）对其中32株多重耐药铜绿假单胞菌进行相关基因TEM、SHV、CTX-M-9、DHA、VIM、PER、OXA、IMP和OprD2检测,并对耐药基因进行测序。结果 32株多重耐药铜绿假单胞菌对多黏菌素B耐药率是3.1%,对其余抗菌药物耐药率为53%～100%,β-内酰胺酶基因OXA-10、TEM-1、DHA-1、PER-1和IMP-1基因阳性率分别为46.9%、21.9%、15.6%、12.5%和31.3%,未检出SHV基因、CTX-M-9基因和VIM基因;另外OprD2基因缺失率达68.8%。结论呼吸内科病房多重耐药的铜绿假单胞菌携带多种β-内酰胺酶基因,应引起高度重视。     

The Influence of Calcium or Manganese on the Resistance to EDTA, Polymyxin B or Cold Shock, and the Composition of Pseudomonas aeruginosa Grown in Glucose- or Magnesium-depleted Batch Cultures

Cultures were batch grown in simple salts media in which growth was limited either by depletion of glucose and magnesium (C/Mg-dep) or by glucose alone (C-dep). Cultures were also grown in these media supplemented by calcium and/or manganese.
All cultures grown in the C-dep media were sensitive to ethylenediaminetetraacetic acid (EDTA), polymyxin and also to cold shock but were relatively resistant to ethyleneglycol-bis(2-aminoethyl ether)-N, N-tetraacetic acid (EGTA). Inclusion of calcium or manganese in the growth medium enhanced lysis by EDTA. Cultures grown in the basic C/Mg-dep media were resistant to EDTA, EGTA, polymyxin and to cold shock. Sensitivity to these agents was retained by cultures grown in C/Mg-dep media supplemented with Ca²⁺ and/or Mn²⁺. Cells grown in C/Mg-dep media with added Mn²⁺ were more sensitive to EDTA and polymyxin than those from the unsupplemented C/Mg-dep media but still resistant compared with C-dep cultures. All cultures from supplemented C/Mg-dep media were more sensitive to EGTA than those from any of the C-dep media.
Whole cells and cell walls from these various media had differing amounts of cell wall, phosphorus, amino sugar, carbohydrates, readily extractable lipid (REL), total phospholipid (PL), and especially differences in cell wall divalent metal cation content.
The differences in PL, REL and amino sugars and carbohydrate did not correlate with the response of C-dep and C/Mg-dep bacteria to EDTA, EGTA or polymyxin. The results are discussed in relation to the hypothesis that the sensitivity of Pseudomonas aeruginosa to polymyxin and EDTA is more dependent on outer membrane cation content rather than on other components, e.g. PL and lipopolysaccharide.     

铜绿假单胞菌的分布特点耐药性分析

目的分析3年来我院患者铜绿假单胞菌的耐药特点,为临床合理用药提供依据,并有助于医院感染预防与控制。方法对2010年至2012年我院各类感染患者标本中分离获得的铜绿假单胞菌,采用纸片扩散法（K-B法）检测抗菌药物的敏感性,并用WHONET5．3软件对药敏结果进行统计分析。结果3年分离的铜绿假单胞菌共计369株,其对多黏菌素B无耐药,对亚胺培南、美罗培南、头孢哌酮／舒巴坦、哌拉西彬他唑巴坦和阿米卡星的耐药率较低（6．81％～22．73％）,对其他抗菌药物的耐药率较高（32．47％～73．38％）。结论铜绿假单胞菌对多种抗菌药物均具有较高的耐药性,临床上治疗该菌感染时应根据药敏检测结果选择抗菌药物。     

Antibiotic sensitivity of nonfermenting gram-negative bacteria

Antibiotic resistance of 132 strains of nonfermenting gram-negative bacteria (NGNB) was studied. 43, 20, 17, 14 and 12 of them belonged to Acinetobacter calcoaceticus (anitratus and lwoffi), Pseudomonas cepacia, Alcaligenes faecalis, P. stutzeri and P. maltophilia, respectively. With rare exceptions all the strains were resistant to benzylpenicillin, oxacillin, lincomycin, ampicillin and cephaloridine. Sensitivity to the other antibiotics varied within wide ranges. Amikacin (94.3 per cent) and tobramycin (90.8 per cent), as well as polymyxin, rifampicin and gentamicin (71.7-66.9 per cent) had the highest effect. The majority of the antibiotics had higher activity (p less than 0.01) against the tested NGNB as compared to their activity against P. aeruginosa. Antibioticograms of every of the tested species of NGNB revealed that P. cepacia and P. stutzeri were the most resistant species. The biovars of Acinetobacter varied in their antibiotic resistance: A. subsp. lwoffi was more sensitive to the majority of the antibiotics though some of them, i.e. doxycycline, carbenicillin, and polymyxin were more active against A. subsp. anitratus.     

In Vitro Effects of Carbenicillin Combined with Gentamicin or Polymyxin B Against Pseudomonas aeruginosa

Disodium carbenicillin and gentamicin sulfate have both shown promise in the treatment of infections caused by Pseudomonas aeruginosa. This study was designed to explore possible synergistic relationships among the new as well as the established antimicrobial agents used to treat such infections. With an agar dilution technique, minimum inhibitory concentrations of 27 strains of P. aeruginosa were determined in two-dimensional tests. Graphs of equal biological activity (isobolograms) demonstrated moderate synergistic effects of the carbenicillin-gentamicin combination over therapeutically feasible concentration ranges. In contrast, the combination of carbenicillin and polymyxin B showed only additive or slightly antagonistic effects. Tests of bacterial killing confirmed the presence of carbenicillin-gentamicin synergy in 3 of 6 strains of P. aeruginosa, but did not show true antagonism between carbenicillin and polymyxin B. Clinical trials of both drug combinations are advisable to determine whether therapeutic results can be improved, and whether the dosages of gentamicin or polymyxin B can thereby be reduced to lessen their toxic hazards.     

Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.     

影响多粘菌素B对绿脓杆菌杀菌效力的主要环境因素的研究

本文通过改变温度,水活度,气体条件和营养含量等影响绿脓杆菌生长的主要环境因素,测定多粘菌素B对绿脓杆菌的最小杀菌浓度(MBC)。结果表明环境因素导致或显著影响绿脓杆菌对抗生素的生态耐受性。实验表明多粘菌素B对绿脓杆菌的杀菌效力,除药物对细菌特有的药理学作用外,还取决于细菌的生长环境。结合冷休克率试验表明,环境影响细菌群体处于分裂状态的菌数。若分裂状态菌数下降表明生长速度减慢。提示了多粘菌素B对绿脓杆菌的效力指数,定量分析可以作为其综合效力作用的表现。以同步培养法确定在单个细胞周期中的抗生素敏感阶段。同时以冷休克率试验资料证明细菌处于分裂状态和幼龄期是其敏感阶段。初步阐述了生长速度缓慢与药物的生态耐受性密切相关。     

多粘菌素耐药性的研究进展

多粘菌素因在多重耐药革兰氏阴性菌上的治疗效果良好,再度被应用于临床,其耐药水平在多种抗菌药中曾一度较低,但目前有研究表明多粘菌素的耐药率有增加趋势。作为抗击多重耐药革兰氏阴性菌的最后一道防线,如何抑制其耐药的发生就显得尤为重要。本文就多粘菌素的耐药性现状、产生机制及防控措施三个方面进行了综述,为指导临床科学合理使用多粘菌素及革兰氏阴性菌耐药菌株传播和蔓延的防控措施提供理论依据。     

Influence of Serum and Calcium on the Bactericidal Activity of Gentamicin and Carbenicillin on Pseudomonas aeruginosa

Because calcium was found to be antagonistic in vitro to the activity of colistin and polymyxin B on Pseudomonas aeruginosa, the effects of calcium and serum on gentamicin and carbenicillin were also examined. Serum was antagonistic to gentamicin in antibiotic tube dilution tests on five strains of P. aeruginosa. Serum was not antagonistic to carbenicillin in tube dilution tests. Physiologic concentrations of calcium antagonized the activity of gentamicin but not carbenicillin. The antagonism observed with gentamicin was less than that previously seen with colistin. The antagonistic effect of calcium and serum was removed by a chelating agent. Gentamicin and carbenicillin may be more active in vivo against P. aeruginosa than colistin or polymyxin B.     

Sensitivity of Pseudomonas aeruginosa to Normal Serum and to Polymyxin

Strains of Pseudomonas aeruginosa isolated from clinical material were very variable in their sensitivity to the bactericidal action of normal serum mediated by the complement system. Fifty per cent killing end points ranged from 0.015 ml to greater than 0.4 ml. Most of the strains with relatively greater sensitivity to serum were isolated from patients with cystic fibrosis. Immunization of rabbits resulted in antisera with enhanced levels of bactericidal antibody, except with one strain which was resistant to the bactericidal action of normal serum and antiserum. When P. aeruginosa was cultivated at 41 C instead of at 37 C, it was significantly more sensitive to serum and to several antibiotics, thereby implicating fever as a host defense mechanism in Pseudomonas infections. In contrast to their heterogeneity to serum bactericidal activity, the strains were relatively homogeneous in their sensitivity to polymyxin, with no apparent association between their sensitivity to the two antimicrobial agents.     

Electron microscope study of the effect of benzalkonium, chlorhexidine and polymyxin on Pseudomonas cepacia

Electron micrographs of Pseudomonas cepacia cell grown in nutrient broth show an external membrane which is distinctly wavy, when compared with similar preparations of Pseudomonas aeruginosa, and which is not affected by growing in the presence of broth containing benzalkonium (10 microgram/ml), chlorhexidine (10 microgram/ml) or polymyxin (25 units/ml). Both benzalkonium (10 microgram/ml) and chlorhexidine (10 microgram/ml) damage the cytoplasmic membrane of P. cepacia cell grown in the presence of the chemicals. Contrasts are shown between the effect of polymyxin (chlorhexidine and benzalkonium) on the outer membrane of P. cepacia and P. aeruginosa.     

Pyocin sensitivity of pseudomonas aeruginosa pretreated with antibiotics.

Strains of Pseudomonas aeruginosa exposed to subinhibitory concentrations of either polymyxin B, gentamicin, or carbenicillin were compared with untreated organisms for changes in sensitivity to pyocin. Pretreatment with polymyxin B or gentamicin resulted in a decreased sensitivity to pyocin, while carbenicllin pretreatment did not alter pyocin sensitivity.     

Effectiveness of Acetic Acid, Betadine, Amphyll, Polymyxin B, Colistin, and Gentamicin Against Pseudomonas aeruginosa

The in vitro effectiveness of three germicides and three chemotherapeutic drugs against hospital-isolated strains of Pseudomonas aeruginosa was determined. Solutions of 1% acetic acid, 0.5% Amphyll, and 0.1% Betadine were bactericidal for six strains tested within 15 min at room temperature. Over a 3-year period at the Albany Medical Center Hospital, the percentages of strains of P. aeruginosa susceptible to polymyxin B, colistin, and gentamicin did not increase. During this 3-year period, polymyxin B and colistin were administered at the hospital but gentamicin was not.     

Multi-drug resistant Pseudomonas aeruginosa: towards a therapeutic dead end?

Pseudomonas aeruginosa is a major hospital-associated pathogen that can cause severe infections, most notably in patients with cystic fibrosis or those hospitalized in intensive care units. In this context, the current increase in incidence of multi-drug resistant (MDR) isolates of P. aeruginosa (MDRPA) raises serious concerns. MDR in P. aeruginosa is defined as the resistance to 3 or 4 of the following antibiotic classes: penicillins/cephalosporins/monobactams, carbapenems, aminoglycosides, and fluoroquinolones. These strains constantly cumulate several resistance mechanisms as a consequence of multiple genetic events, i.e., chromosomal mutations or horizontal transfers of resistance genes. Involved mechanisms may include active efflux, impermeability resulting from porins loss, plasmid-encoded b-lactamases/carbapenemases or aminoglycosides-modifying enzymes, and enzymatic or mutation-associated changes in antibiotics targets. Antibiotic selection pressure represents the leading risk factor for MDRPA acquisition. Colistin (polymyxin E) remains active on virtually all MDRPA isolates, and increasingly appears as the last available option to treat infections caused by these strains. However, the emergence of colistin resistance has been reported in P. aeruginosa, which may announce the spread of pan-resistant strains in a close future.     

Microsatellite and minisatellite markers based DNA fingerprinting and genetic diversity of blast and ufra resistant genotypes

A total of 78 alleles and 29 loci were detected from nine microsatellite and three minisatellite markers, respectively across 26 blast and ufra disease resistant genotypes. For blast resistant genotypes, the Polymorphic Information Content (PIC) values ranged from 0.280 to 0.726 and RM21 was considered as the best marker. PIC values ranged from 0.5953 to 0.8296 for ufra resistant genotypes and RM23 was the best marker for characterization of ufra resistant genotypes. The genetic similarity analysis using UPGMA clustering generated nine clusters with coefficient of 0.66 for blast resistant genotypes while five genetic clusters with similarity coefficient of 0.42 for ufra resistant genotypes. In order to develop resistant varieties of two major diseases of rice, hybridisation should be made using the parents, BR29 and NJ70507, BR36 and NJ70507 for blast, while BR11 and Aokazi, BR3 and Aokazi, Rayda and BR3 and Rayda and BR11 for ufra.