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.     

广州儿童医院重症监护病房感染病原菌的分布及耐药性分析

目的探讨儿科重症监护病房（PICU）感染病原菌的分布及耐药情况,为临床合理选用抗菌药提供参考。方法对广州市儿童医院PICU病房2003年11月-2005年10月各类感染标本所分离的病原菌的分布及耐药性进行回顾性分析。结果共检出295株病原菌,其中革兰阴性杆菌213株（72．2％）,主要为铜绿假单胞菌、不动杆菌等非发酵菌;革兰阳性球菌58株（19．7％）,主要为葡萄球菌;真菌24株（8．1％）。药敏结果提示铜绿假单胞菌及不动杆菌对亚胺培南、头孢哌酮／舒巴坦、环丙沙星及阿米卡星较为敏感,铜绿假单胞菌对头孢噻肟耐药率较高,而不动杆菌对头孢哌酮、氨曲南、庆大霉素耐药严藿。肠杆菌科细菌对氨苄西林、氨苄西林／舒巴坦、哌拉西林及多种头孢菌素耐药率较高而对亚胺培南、头孢哌酮／舒巴坦、阿米卡星等较敏感。葡萄球菌对青霉素、红霉素严重耐药,但对万古霉素、替考拉宁及阿米卡星敏感性高。结论铜绿假单胞菌等非发酵菌已成为PICU病房感染的主要病原菌。根据病原菌种类及药敏结果合理应用抗菌药是有效控制危重病患儿感染和减少耐药菌株产生的重要手段。     

In vitro effect of subinhibitory concentrations of ceftazidime and meropenem on the serum sensitivity of Pseudomonas aeruginosa strains

The aim of this study was to determine the effect of subminimal inhibitory concentrations (subMICs) of ceftazidime, meropenem and gentamicin on the in vitro serum sensitivity of Pseudomonas aeruginosa strains isolated from a variety of isolation sites at two medical wards and an intensive care unit in a government university hospital in Croatia. A total of 20 serum-resistant P aeruginosa strains isolated from different clinical specimens were selected. Bacteria were exposed to 1/2, 1/4, 1/8, 1/16, and 1/32 x MIC of each antibiotic tested. Sensitivity of P. aeruginosa strains to bactericidal activity of normal human serum before and after bacterial exposure to subMICs was determined. Significant difference in serum sensitivity of the strains was observed after the bacteria were exposed to subMICs of ceftazidime and meropenem (p < 0.01), while the exposure to subMICs of gentamicin did not affect significantly the resistance of tested strains to the serum bactericidal activity. Comparing the number of serum-resistant strains before and after exposure to subMICs of antibiotics, statistically significant differences were determined (p < 0.01) after exposure of the strains to 1/2, 1/4, 1/8 and 1/16 x MIC of meropenem, and after exposure to 1/2, 1/4 and 1/8 x MIC of ceftazidime. SubMICs of ceftazidime and meropenem affected not only the resistance to serum bactericidal activity of bacteria, but also their morphology. The alterations in bacterial morphology caused by subMICs of ceftazidime and meropenem could be connected with consecutive bacterial serum sensitivity.     

Impaired bactericidal activity of serum of a child suffering from focal proliferative glomerulonephritis.

The serum of a child with focal proliferative glomerulonephritis was found to exhibit a weaker bactericidal activity against Pseudomonas aeruginosa, Salmonella typhimurium, Salmonella enteritidis and Escherichia coli strains as compared with sera of the child's parents. The child's serum showed a low haemolytical activity of complement as well as a low C3 concentration. The authors believe that the abnormal complement concentration could cause the impaired bactericidal activity of the patient serum.     

Biphasic effect of gingipains from Porphyromonas gingivalis on the human complement system

Periodontitis is an inflammatory disease of the supporting structures of the teeth and is caused by, among other agents, Porphyromonas gingivalis. P. gingivalis is very resistant to killing by human complement, which is present in a gingival fluid at 70% of the serum concentration. We found that the incubation of human serum with purified cysteine proteases of P. gingivalis (gingipains) or P. gingivalis wild-type strains W83 and W50 resulted in a drastic decrease of the bactericidal activity of the serum. In contrast, serum treated with P. gingivalis mutants lacking gingipains (particularly strains without HRgpA) maintained significant bactericidal activity. To understand in detail the mechanism by which gingipains destroy the serum bactericidal activity, we investigated the effects of gingipains on the human complement system. We found that all three proteases degraded multiple complement components, with arginine-specific gingipains (HRgpA and RgpB) being more efficient than lysine-specific gingipain (Kgp). Interestingly, all three proteases at certain concentrations were able to activate the C1 complex in serum, which resulted in the deposition of C1q on inert surfaces and on bacteria themselves. It is therefore plausible that P. gingivalis activates complement when present at low numbers, resulting in a local inflammatory reaction and providing the bacteria with a colonization opportunity and nutrients. At later stages of infection the concentration of proteases is high enough to destroy complement factors and thus render the bacteria resistant to the bactericidal activity of complement.     

The relation between the bactericidal activity of complement and the character of the bacterial surfaces

The bactericidal activity of sera not containing antibodies (sera from precolostral piglets and calves) was tested with strains of gram-negative bacteria with different surfaces. The accuracy of the method of bactericidal test was evaluated statistically the bactericidal unit of complement was defined for comparing the activity of sera of different animals and different species. Various methods used for estimating the character of bacterial surface were compared. It was found that the bactericidal activity of piglet sera is directly dependent on the content of complement in the sera tested and the character of the bacterial surface (in the R-form). In selected strains there is a correlation in all criteria characterizing the surfaces of bacteria, and their susceptibility to bactericidal activity of sera; in a group of 37 strains selected at random, correlation with only one of the surface characteristics (stability in solution after heating to 100°C for 1 hour) was found. In calf sera a component was found which increases the effect of complement to some strains (e.g.Shigella shigae). This component may by absorbed from the serum only in the presence of complement. The nature of this factor is discussed.     

Characteristics of gram-negative microflora on environmental objects in puerperal wards and their resistance to antibacterial preparations and disinfectants

The species composition of gram-negative opportunistic bacteria isolated from different objects at three puerperal wards of a maternity clinic was studied. Escherichia coli and Acinetobacter were found to have a fairly wide circulation. The objects most contaminated by these bacteria were determined. The study showed that up to 33.3% of the isolated hospital strains of gram-negative bacteria were characterized by multiple resistance to antibiotics used in medical practice and to sulfathiazole. The strains showed the highest sensitivity to gentamicin and kanamycin. Most of the hospital strains were sensitive to chloramine and nirtan, but 4-13% of Klebsiella and Pseudomonas aeruginosa strains showed enhanced resistance to 0.1% chloramine solution.     

Bactericidal activity of normal human serum to gram-negative rods with sialic acid containing lipopolysaccharide

The complement plays the most important role in eliminating bacterial invasion of the host, by facilitating phagocytosis of potential pathogens and by participating in the direct essential role in protecting gram-negative bacteria against bactericidal activity of serum. Sialic acids which are important constitutes of animal tissue glycoconjugates are also present in antigens of some bacterial strains. The susceptibility of gram-negative strains with sialic acid--containing lipopolysaccharides to bactericidal action sera was examined.     

Occurrence of Chloramphenicol-acetylating Enzymes in Various Gram-negative Bacilli

The occurrence of a chloramphenicol-acetylating enzyme, similar to that found in Escherichia coli, carrying an R factor was investigated in various gram-negative bacilli. The acetylated products of chloramphenicol were identified by chromatography and quantitatively assayed after benzene extraction. The investigated strains were of the Salmonella-Arizona group, the Klebsiella-Aerobacter group, Serratia marcescens, the Proteus group, and Pseudomonas aeruginosa, most of which were isolated from 1947 to 1957. Both chloramphenicol-sensitive and -resistant strains were included, but none of them was able to transfer chloramphenicol resistance by conjugation. In the Proteus group, a significant level of a chloramphenicol-acetylating enzyme was found in most strains, whether they were sensitive or resistant to chloramphenicol; the resistant strains showed higher levels of the enzyme. Some chloramphenicol-sensitive strains lacked this enzyme. Only the sensitive strains containing the enzyme could easily produce chloramphenicol-resistant mutants with higher enzyme activity. Thus, the chloramphenicol resistance of this group can be reasonably explained on the basis of the chloramphenicol-acetylating enzyme. All of the Pseudomonas aeruginosa strains were resistant to chloramphenicol, and most strains showed low levels of the enzyme (which, however, did not appear sufficient to explain their resistance). All of the strains of the other groups (except one strain of Enterobacter cloacae) lacked the enzyme, although most strains of the Klebsiella-Aerobacter group and of S. marcescens were resistant to chloramphenicol. With respect to the origin of the resistance gene of the R factor, it is noteworthy that the strains of Proteus mirabilis isolated in 1947 possessed this enzyme before the discovery of chloramphenicol.     

Drug susceptibility of Pseudomonas aeruginosa strains isolated from patients of a hospital and specialistic outpatients clinics of the SP ZOZ in Nidzica

The aim of this study was to evaluate a frequency of isolation and antimicrobial susceptibility testing (AST) of Pseudomonas aeruginosa strains cultured from clinical specimens collected from patients hospitalized in wards and specialistic outpatients clinics of a hospital in Nidzica (01. 09. 2000 -31. 12. 2003). During over three years 392 Pseudomonas aeruginosa strains were cultured from 16346 clinical samples provided to bacteriological laboratory. P. aeruginosa strains were isolated from 2.5% of examined specimens. Susceptibility of Pseudomonas aeruginosa strains to antimicrobial agents was tested. The highest in vitro activity against clinical P. aeruginosa strains demonstrated imipenem. One strain was resistant to imipenem. This strain was isolated from a patient of a surgical department. Metalo-beta-lactamase was not detected (MBL-negative strain).Twenty nine strains were ESBL producer (7.4% of all strains). The contribution of Pseudomonas aeruginosa strains to the etiology of nosoconial and ambulatory infections increases. In vitro activity of antibacterial agents against P. aeruginosa strains should be monitored during therapy of infections. Resistance to antibiotics/chemothe-rapeutics may be acquired during treatment with antibacterial agent to which P. aeruginosa strain was susceptible according to the antibiogram.     

Adherence of Pseudomonas aeruginosa strains to solid surfaces

The aim of this study was to evaluate adherence of 59 strains of Pseudomonas aeruginosa to nitric-acid cleansed glass surfaces. There were differences in adherence between the investigated strains. The highest adherence was noticed among human strains (the average percentage was 13.3 +/- 7.51%) and the lowest adherence was determined among swine strains (the average percentage amounted 6 +/- .37%). We conclude that strains of Pseudomonas aeruginosa isolated from humans colonise glass surfaces better than strains isolated from animals.     

Enhanced killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes in presence of subinhibitory concentrations of carbenicillin and ticarcillin

The effect of carbenicillin and ticarcillin on the killing of Pseudomonas aeruginosa was studied with an in vitro system using peripheral blood polymorphonuclear (PMN) leukocytes collected from human donors. No corticosteroid was given to the donor prior to leukocytes collection by a continuous flow cell separator. The assay was carried out with or without serum. P. aeruginosa yield after a 4 hour-incubation was estimated by colony counting. In Hanks' balanced salt solution, P. aeruginosa strains 74 and 78 were resistant to human PMN leukocytes. The presence of subinhibitory concentrations of carbenicillin or ticarcillin (1/10th the minimal inhibitory concentration (MIC) for P. aeruginosa 74, 1/4th the MIC for P. aeruginosa 78) enhanced the bactericidal activity of human leukocytes. Difference between the numbers of bacteria recovered with PMN cells and without cells increased with concentration of carbenicillin or ticarcillin. The synergistic effect was not observed when serum (heated fetal calf serum or heated pooled human serum) was used. The mode of action of carbenicillin and ticarcillin on bactericidal activity of phagocytic cells was not elucidated, but we suggest the effect is due not to action on the phagocytic cells themselves but on the microorganisms.     

Frequency and susceptibility patterns of non-fermenting gram-negative rods isolated from patients hospitalised in intensive care units of a tertiary care hospital

The aim of the study was to assess frequency and susceptibility to antimicrobial agents of non-fermenting gram-negative rods isolated from clinical specimens obtained from patients requiring intensive care, with emphasis on profile of the unit. Identification of cultured isolates was done using automated VITEK and API systems (bioMerieux, France). Susceptibility to antimicrobial agents was tested by a disk-diffusion method according to the NCCLS recommendations. In total the analysis comprised 425 strains of non-fermenting gram-negative rods, constituting 58.9% of all isolates of gram-negative bacteria. In blood cultures predominated strains of A. baumannii (46.8%) and P. aeruginosa (40.4%), while in cultures of other clinical specimens these bacteria comprised 42.9% and 43.9% of isolates. Major differences were observed in frequency of these species on both ICU units. Strains of non-fermenting rods isolated from blood cultures comprised a lower percentage of strains susceptible to antimicrobials (particularly cefepime and carbapenems) than isolates cultured from other specimens. Strains of A. baumannii resistant to imipenem and meropenem were detected with a frequency of 12.5% and 26.7%, respectively. Resistance of P. aeruginosa strains to carbapenems was 62.2% and 44.3%, respectively. There was a relatively high percentage of strains susceptible to cefepime (82.0%), ceftazidime (78.9%), amikacin (77.8%) and piperacillin/tazobactam (69.7%). Conclusions: 1. There was a predominance (58.9%) of strains of gram-negative non-fermenting rods. 2. Isolates from blood cultures were characterised by a much higher percentage of resistant strains in comparison to other specimens. 3. Strains of A. baumannii resistant to carbapenems were recorded. 4. There were differences in frequency and antimicrobial susceptibility among the strains of P. aeruginosa and A. baumannii depending on the type of clinical specimen and ICU profile.     

Sialic Acid-Containing Lipopolysaccharides of Salmonella O48 Strains—Potential Role in Camouflage and Susceptibility to the Bactericidal Effect of Normal Human Serum

Sialic acid (N-acetylneuraminic acid, NeuAc) plays an essential role in protecting gram-negative bacteria against the bactericidal activity of serum and may contribute to the pathogenicity of bacteria by mimicking epitopes that resemble host tissue components (molecular mimicry). The role of sialic acid (NeuAc)-containing lipopolysaccharides (LPS) of Salmonella O48 strains in the complement activation of normal human serum (NHS) was investigated. NeuAc-containing lipooligosaccharides cause a downregulation of complement activation and may serve to camouflage the bacterial surface from the immunological response of the host. Serotype O48 Salmonella strains have the O-antigen structure containing NeuAc while its serovars differ in outer membrane protein composition. In this study, the mechanisms of complement activation responsible for killing Salmonella O48 serum-sensitive rods by NHS were established. Four of such mechanisms involving pathways, which are important in the bactericidal mechanism of complement activation, were distinguished: only the classical/lectin pathways, independent activation of the classical/lectin or alternative pathway, parallel activation of the classical/lectin and alternative pathways, and only the alternative pathway important in the bactericidal action of human serum. To further study the role of NeuAc, its content in bacterial cells was determined by gas-liquid chromatography-mass spectrometry in relation to 3-deoxy-D-manno-2-octulosonic acid (Kdo), an inherent constituent of LPS. The results indicate that neither the presence of sialic acid in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of complement and that the presence of sialic acid in the structure of LPS is not sufficient to block the activation of the alternative pathway of complement. We observed that for three strains with a very high NeuAc/Kdo ratio the alternative pathways were decisive in the bactericidal action of human serum. The results indicated that those strains are not capable of inhibiting the alternative pathway very effectively. As the pathogenicity of most Salmonella serotypes remains undefined, research into the interactions between these bacterial cells and host organisms is indispensable.     

Comparison of bacteriostatic and bactericidal activity of 13 essential oils against strains with varying sensitivity to antibiotics

Aims:  To compare the bacteriostatic and bactericidal activity of 13 chemotyped essential oils (EO) on 65 bacteria with varying sensitivity to antibiotics.
Methods and Results:  Fifty-five bacterial strains were tested with two methods used for evaluation of antimicrobial activity (CLSI recommendations): the agar dilution method and the time-killing curve method. EO containing aldehydes ( Cinnamomum verum bark and Cymbopogon citratus ), phenols ( Origanum compactum , Trachyspermum ammi , Thymus satureioides , Eugenia caryophyllus and Cinnamomum verum leaf) showed the highest antimicrobial activity with minimum inhibitory concentration (MIC) <2% (v/v) against all strains except Pseudomonas aeruginosa . Alcohol-based EO ( Melaleuca alternifolia , Cymbopogon martinii and Lavandula angustifolia ) exhibited varying degrees of activity depending on Gram status. EO containing 1·8-cineole and hydrocarbons ( Eucalyptus globulus , Melaleuca cajeputii and Citrus sinensis ) had MIC_90% ≥ 10% (v/v). Against P. aeruginosa , only C. verum bark and O. compactum presented MIC ≤2% (v/v). Cinnamomum verum bark, O. compactum , T. satureioides , C. verum leaf and M. alternifolia were bactericidal against Staphylococcus aureus and Escherichia coli at concentrations ranging from to 0·31% to 10% (v/v) after 1 h of contact. Cinnamomum verum bark and O. compactum were bactericidal against P. aeruginosa within 5 min at concentrations <2% (v/v).
Conclusions:  Cinnamomum verum bark had the highest antimicrobial activity, particularly against resistant strains.
Significance and Impact of the Study:  Bacteriostatic and bactericidal activity of EO on nosocomial antibiotic-resistant strains.     

Occurrence of beta-lactamase type ESBL and IBL in Pseudomonas aeruginosa rods]

The aim of the study was to determine extended spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) among Pseudomonas aeruginosa strains. A total of 43 strains isolated from humans (6), hospital sink (1), fish (15), cattle (5), swine (5), dog (1), redder (1) fur animals (9) were studied. ESBL-producing strains were detected with double disc diffusion test according to Jarlier et al. (8). Clavulonate and tazobactam were used as the inhibitors of ESBL. Inducible beta-lactamases were determined using double disc method according to Sanders (15). Cefoxitin was the inductor of these beta-lactamases. The susceptibility study was carried out using the disc diffusion method according to NCCLS standards. A total of 8 ESBL (18.6% of all strains) and 31 (72%) IBL producing strains were detected. The obtained results indicate the necessity of monitoring of ESBL- and IBL-producing strains of Pseudomonas aeruginosa.     

医院内4种主要革兰阴性菌分布及耐药率分析

摘要：目的 分析大连某三甲医院4种主要革兰阴性病原菌的分布及细菌耐药性变迁,为抗菌药物的合理使用提供依据。方法 回顾性分析2012?2015年住院患者中分离的4种革兰阴性菌临床分布及每年的耐药率,利用WHONET 5.6软件进行统计分析。结果 4年分离致病菌28 485株,其中大肠埃希菌2 994株,肺炎克雷伯菌1 375株,鲍曼不动杆菌1 079株,铜绿假单胞菌1 998株,共占分离致病菌总数的26.1%。4种菌对哌拉西林的耐药率最高（37.2%~78.2%）,对头孢哌酮/苏巴坦耐药率最低（6.7%~39.1%）。2012?2015年,大肠埃希菌、肺炎克雷伯菌和铜绿假单胞菌的抗生素耐药率有下降趋势,鲍曼不动杆菌对多数抗生素耐药率都高,且逐年上升。结论 本院4年感染中4种主要革兰阴性菌占主导地位。4种菌对不同抗生素耐药率和变化不同,鲍曼不动杆菌耐药最严重。4种菌对于头孢哌酮/苏巴坦均比较敏感,对哌拉西林耐药严重。在临床治疗中应有针对性加强管理和用药。     

Amlodipine: a cardiovascular drug with powerful antimicrobial property

Ten cardiovascular drugs were procured in pure form from their manufacturers in India and screened for antimicrobial property against fifteen known bacteria belonging to both gram-positive and gram-negative types. These bacteria were inhibited by the common antibiotics at 1-5 mg ml(-1) level through our earlier studies. Since most of the bacteria were moderate to highly responsive to amlodipine, this compound was further tested in vitro against 504 bacteria comprising 4 genera of gram-positive and 15 genera of gram-negative bacteria. Most of these were inhibited by the drug at 50-200 microg ml(-1) level and few strains were sensitive even at lower concentrations (10 microg ml(-1)). The bacteria could be arranged in the decreasing order of sensitivity towards amlodipine in the following manner: Staphylococcus aureus, Vibrio cholerae, Vibrio parahemolyticus, Shigella spp., Salmonella spp., Bacillus spp., whereas Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa were found to be resistant to the lower concentrations of the drug. Amlodipine was found to be bactericidal in nature when its mode of action was studied against S. aureus 6571, V. cholerae 14035 and Sh boydii 8 NCTC 254/66. The antibacterial activity of amlodipine could also be confirmed in vivo. When it was given to Swiss strain of white mice at different dosages (30 and 60 microg/mouse), it could significantly protect the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74. According to Chi square test the in vivo data were highly significant (p<0.001).     

急性化脓性骨髓炎患者病原菌分布及耐药性分析

目的:分析急性化脓性骨髓炎患者病原菌的分布特点及耐药情况。方法:取急性化脓性骨髓炎患者窦道深部分泌物或病灶组织做细菌培养及药敏试验。结果:80例患者共培养出病原菌18种110株:其中7例同时培养出3种细菌,15例同时培养出2种细菌,58例培养出1种细菌。110株细菌中,革兰氏阳性(G+)菌55株,占50.0%,主要为金黄色葡萄球菌14株,占25.5%;革兰氏阴性(G-)菌52株,占47.3%,主要为铜绿假单胞菌13株,占25.0%。真菌3株,占2.7%。金黄色葡萄球菌对抗菌药物万古霉素最敏感,耐药率为7.1%,对青霉素耐药率最高,耐药率为92.9%;铜绿假单胞菌对抗菌药物头孢哌酮最敏感,耐药率为7.7%,对亚胺培南的耐药率最高,为92.3%。结论:化脓性骨髓炎的致病菌中革兰氏阳性菌和革兰氏阴性菌的的占比基本持平,大多数病原菌对常用的抗菌药物均具有耐药性。     

Analysis of the etiological structure and dynamics of antibiotic resistance in the causative agents of suppurative inflammatory diseases in the patients at a large general hospital

The structure of the causative agents isolated from patients with pyoinflammatory infections in 1980-1983 was analysed. It was shown that the surgical and urological infections were mainly caused by gram-negative bacteria. The other pyoinflammatory infections were mainly due to gram-positive cocci. A relatively high frequency of the strains of gram-negative bacteria, especially among Pseudomonas spp. and Enterobacter spp., resistant to aminoglycoside antibiotics, such as gentamicin, sisomycin and tobramycin with preserved sensitivity to amikacin and netilmicin in the majority of the strains was shown. Among the beta-lactam antibiotics cephotaxim and cephalotin were most active against gram-negative bacteria and staphylococci, respectively. The majority of the antibiotic resistant strains of gram-negative bacteria had analogous structures and levels of resistance to 7-12 antibiotics which might indicate the occurrence of 1-2 resistance plasmids among the clinical strains.