Development of antibodies to OEP, exotoxin A and exoenzymes of Pseudomonas aeruginosa in man

Antibody titers to OEP, exotoxin A and exoenzymes (protease and elastase) in normal sera of 256 specimens of cord blood, children and adults were measured by passive hemagglutination test. Serum antibody to exotoxin A was detected in cord blood samples. The level of mean antibody titers dropped during the first year of age, then rose and reached a plateau at the age of 2 to 5 years and dropped thereafter. Mean antibody titers to OEP by age groups were similar to those of exotoxin A. Antibodies to exoenzymes were not detectable initially, but the level rose during the second year of age and reached a plateau during childhood. Positive antibody titers (1:20) to exotoxin A and OEP were found in all sera belonging to some age groups between 11 to 30 years. The rate of acquisition of antibodies to exoenzymes was low. As for the antibodies to exotoxin A, the disappearance of detectable antibody by treatment with 2-mercaptoethanol was observed during the age of 1 to 4 years. Initial pseudomonas colonization may be common and asymptomatic in infants and young children.     

铜绿假单胞菌外毒素A对淋巴细胞转化的抑制作用

目的探讨铜绿假单胞菌外毒素A（PEA）对人淋巴细胞转化的抑制作用及抗．PEA血清的中和作用。方法采集健康成年人的静脉血,密度梯度离心分离外周血单个核细胞（PBMC）。采用台盼蓝染色排除法及四甲基偶氮唑蓝（MTT）法确定PEA对淋巴细胞的非毒性剂量范围。抑制实验分为对照组、4个不同浓度PEA组及抗血清预处理组6个组。非毒性剂量范围内不同浓度的PEA、植物血凝素（PHA）与淋巴细胞37℃、5％CO2共同温育48h,MTT法测定淋巴细胞转化。结果PEA对淋巴细胞的非毒性剂量范围为0～800ng／ml。PEA浓度在50—400ng／ml,对淋巴细胞转化存在显著抑制作用（P〈0．05）;而抗．PEA血清可完全中和PEA射淋巴细胞转化的抑制作用,与对照组比较差异无显著性（P〉0．05）。结论非毒性剂量的PEA对淋巴细胞转化存在抑制作用,抗-PEA血清具有保护性中和作用。     

铜绿假单胞菌外毒素A对淋巴细胞转化的抑制作用

目的探讨铜绿假单胞菌外毒素A(PEA)对人淋巴细胞转化的抑制作用及抗-PEA血清的中和作用。方法采集健康成年人的静脉血,密度梯度离心分离外周血单个核细胞(PBMC)。采用台盼蓝染色排除法及四甲基偶氮唑蓝(MTT)法确定PEA对淋巴细胞的非毒性剂量范围。抑制实验分为对照组、4个不同浓度PEA组及抗血清预处理组6个组。非毒性剂量范围内不同浓度的PEA、植物血凝素(PHA)与淋巴细胞37°C、5%CO2共同温育48 h,MTT法测定淋巴细胞转化。结果PEA对淋巴细胞的非毒性剂量范围为0~800 ng/m l。PEA浓度在50~400 ng/m l,对淋巴细胞转化存在显著抑制作用(P<0.05);而抗-PEA血清可完全中和PEA对淋巴细胞转化的抑制作用,与对照组比较差异无显著性(P>0.05)。结论非毒性剂量的PEA对淋巴细胞转化存在抑制作用,抗-PEA血清具有保护性中和作用。     

Pseudomonas aeruginosa antibodies in human plasma.

Plasma samples from healthy adults were examined for antibodies to the common protective antigen (OEP) [1] by passive hemagglutination (HA) reaction [2] with the finding that the majority of them had an antibody titer of 60 or lower. 2-Mercaptoethanol (2-ME) treatment, however, caused a decrease in HA titer down to 16 or lower in most of the cases, suggesting that the OEP antibody resides mostly in IgM and slightly in IgG. The finding was corroborated by gel filtration on Sephadex G-200. It appears likely that IgA is not implicated in the HA titer since IgA was HA negative. However, the detection of enzymatic activity by enzyme-linked immunosorbent assay (ELISA) [9] still suggests a possibility that OEP antibody resides in IgA. On the other hand, antibodies to proteolytic enzymes, especially protease and elastase [3], elaborated by Pseudomonas aeruginosa, were found to be present in most cases at a titer of 16 or lower by HA reaction. It was also found that they reside mostly in IgM.     

Pseudomonas aeruginosa exotoxin A: its role in retardation of wound healing: the 1992 Lindberg Award.

Bacterial concentrations greater than 10(5) colony-forming units/gm of tissue prevent wound healing. However, it has not been determined whether it is the number of bacteria or a toxin produced by these organisms that impedes the wound healing process. Pseudomonas aeruginosa (PSAR), a burn wound pathogen, produces a dermonecrotic toxin, exotoxin A. Studies have indicated a role for exotoxin A in the pathogenicity of PSAR. We investigated the role of exotoxin A in the retardation of contraction. Acute granulating wounds were created on 90 Sprague-Dawley rats. The animals were equally divided into six groups and were treated topically as follows: group 1, sham: no infection, no treatment; group 2, exotoxin A; group 3, exotoxin A and antiexotoxin; group 4, autoclaved PSAR 10(6); group 5, 10(6) viable PSAR inoculated in the wound; group 6, 10(6) viable PSAR and antiexotoxin. Wound contraction was measured with the use of planimetry twice a week. Serial biopsies were performed on all wounds. Contraction rates revealed significantly (p < 0.05) retarded closure in the animals treated with exotoxin A and in the viable PSAR group when compared with the rates of the noninfected control groups. Animals treated with exotoxin A plus antiexotoxin A and those treated with live PSAR and antiexotoxin showed contraction rates identical to the control groups. These data suggest that exotoxin A in PSAR infections retards wound healing and that neutralization of the toxin restores the normal healing process.     

铜绿假单胞菌外毒素A基因DNA片段的克隆及序列测定

目的 建立含有铜绿假单胞菌外毒素A基因DNA片段的克隆株。方法 根据已报道的序列设计铜绿假单胞菌外毒素A基因的特异引物,用PCR方法从标本扩增出预期大小的DNA片段,并将该片段纯化后与T载体连接,转化到大肠埃希菌,筛选出含有插入片段的克隆。结果 阳性克隆株经DNA序列测定,证实与已报道的铜绿假单胞菌外毒素A基因DNA序列,在399个碱基中仅有3个碱基不同,但编码的氨基酸完全一致。结论 成功构建含有铜绿假单胞菌外毒素A基因的克隆,为大量制备探针和用PCR方法检测铜绿假单胞菌提供了基础。     

Epithelial application of Pseudomonas aeruginosa exotoxin A results in a selective targeting to cells in the liver, spleen and lymph node.

Pseudomonas aeruginosa exotoxin A (PE) is a 67-kDa protein expressed under the selective pressure of a low iron environment. Previous studies using non-toxic PE chimeras containing a viral surface antigen, the V3 loop of MN gp120 from human immunodeficiency virus type 1 (HIV-1), resulted in not only an effective mucosal immunization but also a striking systemic immune response following epithelial application. Presently, we have examined the possibility that such a strong dual immune response was generated by the efficient targeting of critical cells of the immune system. Mice were dosed with 10 microg of toxic PE or a non-toxic mutant of PE (ntPE) by intratracheal instillation. Examination of lung, liver and spleen tissues isolated 4, 8 and 12 h following intratracheal instillation with PE demonstrated specific cell damage in these tissues which was not observed in mice dosed with ntPE. Based upon the location and characteristics of observed responses, the cells targeted by PE appear to be involved in the antigen presentation arm of the immune response. Since ntPE chimeras with inserted peptide antigen epitopes from a wide variety of pathogens are easy to prepare and administer, these results support this approach for mucosal immunization.     

Release of exotoxin A, peptidoglycan and endotoxin after exposure of clinical Pseudomonas aeruginosa isolates to carbapenems in vitro

BACKGROUND: Production of several cell-associated components and extracellular enzymes can play important roles in the pathogenesis of Pseudomonas aeruginosa infections. METHODS: We characterized the time course of morphological changes, production of exotoxin A (ETA) and release of peptidoglycan (PG) and endotoxin (ET) in clinical P. aeruginosa isolates after exposure to carbapenems including imipenem, panipenem, meropenem and biapenem at 0.5, 2, 8 and 32 microg/ml. RESULTS: The amount of ETA in the supernatant of bacterial cultures exposed to carbapenems correlated with the number of viable cells, independently of morphological changes. Formation of ovoid cells and rapid cell lysis induced by carbapenems above the minimal inhibitory concentrations (MICs) decreased ETA production and ET release, while filamentation and prolonged cell lysis increased ETA production and/or ET release. Neither the number of viable cells nor bacterial morphology was related to the amount of PG released throughout the 6-hour observation period. CONCLUSION: Exposure to concentrations of carbapenems above the MIC resulted in rapid cell lysis of P. aeruginosa and decreased ETA levels and ET release, while filamentation and prolonged cell lysis induced by exposure to sub-MICs of carbapenems were associated with increased ETA production and/or greater ET release.     

Distribution of porin and lipopolysaccharide antigens on a Pseudomonas aeruginosa permeability mutant.

Pseudomonas aeruginosa common surface antigens were compared in a permeability mutant (PCC118) and its parent (PAO503). The distribution of lipopolysaccharide and porin antigens in the mutant supports the conclusion that beta-lactam permeability was affected by lipopolysaccharide-side chain presentation rather than by a change in porin number.     

Interaction of polycationic antibiotics with Pseudomonas aeruginosa lipopolysaccharide and lipid A studied by using dansyl-polymyxin.

A fluorescent derivative of polymyxin B (dansyl-polymyxin) was used to study the interaction of polycations with lipopolysaccharide (LPS) and lipid A from Pseudomonas aeruginosa. Dansyl-polymyxin became bound to LPS and lipid A sites, including Mg2+-binding sites, resulting in a 20-fold enhancement of fluorescence. A Hill plot of the binding data showed that the binding of dansyl-polymyxin to LPS was cooperative (n = 1.98) and of high affinity (S0.5 = 0.38 microM). The maximal binding capacity of LPS was approximately four molecules of dansyl-polymyxin per mol of LPS. The dansyl-polymyxin interaction with lipid A displayed similar kinetics (n = 2.26; S0.5 = 0.38 microM), and the maximal binding capacity was approximately 2 mol of dansyl-polymyxin per mol of lipid A. A variety of polycationic compounds, including gentamicin, streptomycin, and polymyxin B, as well as Mg2+, were able to displace dansyl-polymyxin bound to LPS or to lipid A. Marked differences both in terms of the degree of displacement and in terms of the amount of competing polycation required to displace a given amount of dansyl-polymyxin were observed. Addition of excess polymyxin B resulted in displacement of all of the dansyl-polymyxin, demonstrating that only polymyxin-binding sites were being probed. Our data demonstrate that polymyxin B binds to multiple sites on LPS, including sites which bind aminoglycoside antibiotics and other polycationic compounds.     

Correlation between lipopolysaccharide structure and permeability resistance in beta-lactam-resistant Pseudomonas aeruginosa

Four beta-lactam-resistant permeability mutants of Pseudomonas aeruginosa PAO503 were studied. The resistance phenotypes were correlated to changes within the lipopolysaccharide. Two of the mutants, PCC1 and PCC19, were shown to differentiate between beta-lactams on the basis of relative hydrophobicity. The more hydrophilic antibiotics were less effective at inhibiting these strains. This phenotype was correlated to the presence of mannose, in measurable quantities, in lipopolysaccharide isolated from these strains. The other two strains, PCC23 and PCC100, differentiated between cephem antibiotics on the basis of electrical charge. The presence of a positive charge markedly increased the relative efficiency of an antibiotic. This correlation did not hold for penam derivatives, with the lower-molecular-weight, dianionic molecules being the most effective. Mutants of this type were changed in the amount of "side chain" sugars or, to minor extent, in their outer membrane protein profiles.     

Chemical and chromatographic analysis of lipopolysaccharide from an antibiotic-supersusceptible mutant of Pseudomonas aeruginosa.

Lipopolysaccharides extracted from Pseudomonas aeruginosa strain K799 and its antibiotic-supersusceptible derivative Z61 were analyzed chemically and chromatographically. The side-chain polysaccharides purified by gel exclusion chromatography were compositionally identical, being composed of fucosamine (2-amino-2,6-dideoxygalactose), quinovosamine (2-amino-2,6-dideoxyglucose), and an unidentified amino sugar. In addition, low amounts of the core-specific components (glucose, rhamnose, alanine, and galactosamine) were found associated with the side chains from both strains. An average molecular weight of 38,000 to 50,000 was calculated for this fraction based on the glucose and rhamnose levels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the lipopolysaccharides from these two strains were microheterogeneous. Qualitative analysis of the lipopolysaccharide neutral sugars, using a series of single-step revertants of mutant Z61, demonstrated that full revertants showed patterns indistinguishable from those of the wild-type strain K799, whereas partial revertants had intermediate levels and mutant Z61 low levels of neutral sugars. Quantitative analysis revealed that the core oligosaccharide fraction from the wild-type strain had a glucose/rhamnose/galactosamine ratio of 4:1:1, whereas the core from Z61 exhibited major deficiencies in both glucose and rhamnose. The lipid A from both strains contained five fatty acids, namely, 3-hydroxydecanoate, dodecanoate, 2- and 3-hydroxydodecanoate, and hexadecanoate. Whereas the overall fatty acid content was equal, the mutant strain showed markedly lower levels of dodecanoate and hexadecanoate and increased levels of 2-hydroxydodecanoate. Results of whole-cell fatty acid analyses were consistent with this observation. Evidence for an additional alteration of the lipid A of strain Z61 was obtained from acid hydrolysis studies and infrared spectra of isolated lipid A, although the actual chemical basis could not be determined by a variety of techniques. It is suggested that the state of the lipopolysaccharide is able to influence the number of open functional protein F pores in the outer membrane of P. aeruginosa.     

Ionic binding of 3H-gentamicin and short-time bactericidal activity of gentamicin against Pseudomonas aeruginosa isolates with different lipopolysaccharide structures.

The clinical isolates of Pseudomonas aeruginosa can be roughly classified into long- and short-LPS strains and LPS-deficient strains. Ionic binding of 3H-gentamicin was the highest in the long-LPS strains followed in descending order by short-LPS and LPS-deficient strains. However, PAC605 strain, completely lacking in the repeated units of O-polysaccharide and also lacking in some of the neutral sugar residues of the core oligosaccharide region, highly bound to 3H-gentamicin and it is suggested that the negatively charged sites on the deep-core oligosaccharide region and/or on lipid A participated in the binding to 3H-gentamicin. This manner of binding may be also applied to P. aeruginosa No. 45 (LPS-deficient), a clinical isolate.     

Enhanced survival in Pseudomonas aeruginosa septicemia associated with high levels of circulating antibody to Escherichia coli endotoxin core.

We studied the relationship between serum antibodies to the cross-reactive endotoxin core of Escherichia coli and survival following Pseudomonas aeruginosa septicemia. Core glycolipid was purified from the outer cell membrane of a uridine diphosphate galactose 4-epimerase-deficient rough mutant E. coli (J5 strain), characterized, and used as the antigen in a quantitative enzyme-linked immunosorbent assay (ELISA) to measure core-specific IgG and IgM antibodies. 43 patients with Pseudomonas septicemia, among whom there was a mortality of 42%, were evaluated. Core-specific antibody concentrations in acute sera ranged from 1 to 49 micrograms/ml in the case of IgG and from 1 to 200 micrograms/ml for IgM. Core-specific antibodies of both isotypes were higher in patients who survived compared with those who succumbed to their septicemias (mean, microgram/ml +/- SEM, 26 +/- 3 vs. 14 +/- 4, P = 0.005 for IgG, and 55 +/- 12 vs. 18 +/- 5, P = 0.009 for IgM). Although total IgG levels were also higher in acute sera from survivors compared with nonsurvivors (mean, mg/dl +/- SEM, 1,120 +/- 99 vs. 694 +/- 119, P = 0.004), total IgM levels were virtually identical in the two groups (146 +/- 23 vs. 148 +/- 48, P = 0.52). Conversely, patients with core-specific IgG levels greater than 10 micrograms/ml at the onset of septicemia had better survival than those with levels less than 10 micrograms/ml (79 vs. 14%, P less than 0.001), and patients with core-specific IgM levels greater than 30 micrograms/ml had better survival than those with levels less than 30 micrograms/ml (81 vs. 44%, P = 0.01). In comparison, patients with total IgG levels greater than 1,000 mg/dl also had better survival than those with levels less than 1,000 mg/dl (82 vs. 42%, P = 0.01), while those with total IgM levels greater than 150 mg/dl showed somewhat less improvement in survival compared with those with levels less than 150 mg/dl (71 vs. 50%, P = 0.12). Core-specific IgM was highly correlated with core-specific IgG (r = 0.52), but not with type-specific anti-lipopolysaccharide (r = 0.13) or anti-toxin A (r = 0.12) antibodies, or with total IgG (r = 0.28) or IgM (r = 0.31). In contrast, core-specific IgG correlated somewhat more closely with type-specific antibodies (r = 0.36), and with total IgG (r = 0.51) and IgM (r = 0.52). Stepwise linear discriminant analysis indicated that type-specific antibody levels were the best predictor of outcome, among those antibodies examined, followed by anti-core IgM. Although anti-core IgG, anti-toxin A, and total IgG levels all correlated individually with survival, none augmented the prognostic power of type-specific antibodies in combination with anti-core IgM, which together predicted outcome accurately 73.5% of the time. Host factors not significantly associated with anti-core antibody levels included rapidly fatal underlying disease, age, sex, leukopenia, and prior treatment with cytotoxic drugs. In contrast, prior steroid therapy was associated with low levels of both core-specific IgG and IgM (P < 0.05). These data suggest cross-protective activity against P. aeruginosa septicemia of naturally occurring antibodies to the endotoxin core of E. coli. Anti-core antibodies, particularly of the IgM isotype appear to augment the more specific protective immunity engendered by antibodies to the O-specific side chains of Pseudomonas lipopolysaccharides. This cross-protective immunity likely applies to other Gram-negative pathogens as well.     

Characterization of the onset and consequences of pneumonia due to fluoroquinolone-susceptible or -resistant Pseudomonas aeruginosa

OBJECTIVES: This study was conducted to identify and compare the microbiological and clinical outcomes among hospitalized adults with pneumonia caused by fluoroquinolone-susceptible or -resistant strains of Pseudomonas aeruginosa. Antibiotic regimens used prior to, as well as those used to treat, the infections were characterized. PATIENTS AND METHODS: This non-randomized multicentre study included 100 consecutively identified patients with pneumonia caused by fluoroquinolone-susceptible (n = 50) or fluoroquinolone-resistant (n = 50) strains of P. aeruginosa. Medical records were examined for demographic, clinical and treatment variables including antibiotics received in the 30 days before the index respiratory or blood culture; AUICs were calculated for each patient using reported or derived MICs. Multivariate logistic and linear regressions were used to identify factors associated with successful clinical and microbiological outcomes. RESULTS: The study population was primarily elderly, frequently in a critical care unit, with low serum albumin and with a high probability of failure and mortality. Patients with pneumonia caused by fluoroquinolone-resistant P. aeruginosa were more likely to have received antibiotics within 7 days before the infection (P = 0.027); the antibiotic regimen was more likely to be of a weak potency (mean AUIC of 58 versus 169, P = 0.001) and to include levofloxacin (P < 0.0001) than what was administered to patients who became infected with a fluoroquinolone-susceptible strain. Regardless of susceptibility, a mean of between 2 and 3 weeks of directed antibiotic therapy was administered to each patient. CONCLUSIONS: Pneumonia caused by fluoroquinolone-resistant P. aeruginosa is frequently associated with prior exposure to levofloxacin. Treatment of P. aeruginosa pneumonia is difficult and usually consists of combination regimens with multiple modifications.