Antibiotic sensitivity of different antigenic variants of Pseudomonas aeruginosa isolated in a hospital

The serological typing of 96% of P. aeruginosa strains isolated in hospitals has been carried out by means of agglutinating sera, and the tendency towards the prevalence of the strains of group V (25.3%) and serovar O11 (18.1%) has been revealed. The determination of the antibiotic sensitivity of different variants has shown that the strains of groups I, V and serovar O11 possess pronounced resistance to a wide spectrum of antibiotics, their resistance to gentamycin and polymixin being higher than in the strains of other serotypes. The determination of antibiotic resistance can serve only as an additional test in the serological typing of P. aeruginosa.     

Influence of growth temperature on the development of Escherichia coli polysaccharide K antigens

The established seventy-one Escherichia coli polysaccharide capsular K antigenic test strains were examined for the development of the K antigen after growth at 37 degrees C and 18 degrees C. Twenty-eight K antigens were not detectable after growth of bacteria at 18 degrees C, while the remaining 43 antigens were developed at both temperatures. Most of the temperature-dependent antigens belonged to electrophoretically fast-moving K polysaccharides of rather low molecular weight, characteristically found among the common K antigens from extraintestinal disease isolates. Lipopolysaccharide O antigens were developed at both growth temperatures.     

Serological Studies of the T and Tumor Antigens of the Oncogenic Simian Adenoviruses

Tumor-specific antigens and antisera were prepared for eight of the oncogenic simian adenoviruses. Complement-fixation tests revealed three distinct serological subgroups. This grouping was maintained in studies of virus-infected cells (T antigens) although high titered preparations were obtained for only the major subgroup I. The current grouping is as follows: (I) SV1, SV11, SV25, SV33, SV34, SV38; (II) SV20, SV23; (III) SA 7. Antigens from each subgroup were rapidly inactivated at 56 C, and group II and III antigens were also markedly inactivated at 37 C. One of the tumors (SV1) also contained SV40 T antigen, suggesting origin from a simian adenovirus-SV40 "hybrid."     

The rfb locus from Pseudomonas aeruginosa strain PA103 promotes the expression of O antigen by both LPS-rough and LPS-smooth isolates from cystic fibrosis patients

Summary
Strains of Pseudomonas aeruginosa initially isolated from patients with cystic fibrosis (CF) often express a smooth lipopolysaccharide (LPS) containing many long O side-chain antigens, but once a chronic infection is established, strains recovered from these patients express little or no LPS O antigen. The genetic basis for this loss of O antigen expression by P. aeruginosa CF isolates is unknown. We report here that 20 CF isoiates of P. aeruginosa , 13 of which are LPS-rough, were each capable of expressing serogroup 011 antigen when provided with the rfb iocus from P. aeruginosa serogroup 011 strain PA103 on the recombinant plasmid pLPS2. Eight of the thirteen LPS-rough isolates co-expressed another, presumably endogenous, O antigen when they contained pLPS2. Different subcloned regions of pLPS2 complemented distinct strains to restore endogenous O antigen expression. These data suggest that the loss of O antigen expression by P. aeruginosa CF isolates results from alterations specific to the rfb region, and is not due to mutations involving other loci or ancillary LPS genes.     

Genetic determination of the O antigens of Salmonella groups B (4,5,12) and C1(6,7)

M?kel?, P. Helena (University of Helsinki, Helsinki, Finland). Genetic determination of the O antigens of Salmonella groups B (4,5,12) and C(1) (6,7). J. Bacteriol. 91:1115-1125. 1966.-The genetic determination of the O antigens of Salmonella was studied by Hfr or F' crosses between strains of groups B (antigens 4,5,12) and C(1) (antigens 6,7). The main genetic determinants of the specificities 4 of group B and 7 of group C(1) behaved as alleles of one locus, called O or O-4/7. This is probably identical with O-4/9, responsible for the serological difference between groups B and D, and with the "rough" locus rouB. At least parts of the antigens 12 of group B and 6(2) of group C are determined at the same locus. The gene O-5 is closely linked to O-4/7, both mapping in the approximately 2 minutes distance between his and metG in the order his - O-4/7 - O-5 - metG. In crosses of group B donors with group C(1) recipients, a serologically new type, called semirough (SR), appeared in most recombinants that had inherited the O-4 allele of the group B donor. These SR forms are serologically intermediate between smooth and rough forms, showing poor stability in saline but possessing the specificities 4 and 12 and (some of them) 5. On the basis of previous biochemical studies, the hypothesis has been put forward that the side chains of their lipopolysaccharide are much shorter than normal group B side chains, probably containing only one repeating unit per side chain. A gene SR-4 responsible for the elongation of group B side chains beyond the first repeating unit was mapped between gal and try, group B and D bacteria being Sr-4(+), and group C being SR-4(-).     

The structure and serologic distribution of an extracellular neutral polysaccharide from Pseudomonas aeruginosa immunotype 3

Previous work has described small molecular weight neutral polysaccharides from isolates of Pseudomonas aeruginosa that appear to be associated with the lipopolysaccharide (LPS) and distributed across serologic barriers defined by antibody to the O side chain. We have isolated and characterized another of these structures obtained from culture supernatants of an immunotype 3 strain of P. aeruginosa. The isolated neutral polysaccharide has a tetrasaccharide repeat unit: (formula; see text) where Rha is rhamnose. The structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments, acid hydrolysis, methylation analysis, Smith degradation, and optical rotation determinations. Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 degrees C, 2 h) LPS from the seven Fisher immunotype strains of P. aeruginosa bound well to the neutral polysaccharide. Antibodies affinity purified from these sera using immobilized neutral polysaccharide as well as a neutral polysaccharide-specific monoclonal antibody, E87, reacted with an antigenically similar structure found among many isolates of different LPS serotypes in a colony blot and with LPS from the seven Fisher immunotypes in an immunoblot. In an immunoblot assay, the neutral polysaccharide inhibited binding of the monoclonal antibody, E87, to material present in LPS preparations from a variety of serotypes. This structure may represent another P. aeruginosa neutral polysaccharide variant found associated with the LPS.     

Immunochemistry of Yersinia enterocolitica O3 grown at different temperatures.

Comparative agglutinations of homogeneous stable suspensions prepared with Yersinia enterocolitica growth at 37 degrees C and at 25 degrees C were performed with anti-sera prepared in rabbits with the bacteria grown at both these temperatures. Sera prepared with live Y. enterocolitica grown at 37 degrees C agglutinated both suspensions at a much lower titre than the sera prepared with formaldehyde-treated bacteria is grown at 25 degrees C. All the sera in which strongly precipitating antibodies were induced reacted, in agar-gel, against native and heated proteins. The small amounts of antipolysaccharides induced in all the sera reacted only in the ring test against the bacterial polysaccharides. The absorption of the sera prepared with live Y. enterocolitica grown at 37 degrees C, with antigens synthesized at 25 degrees C did not remove all the homologous antibodies; apparently, some determinants are specific for the bacteria grown at 37 degrees C. Morphological changes of the small rods to elongated bacilli and filamentous forms were observed in most cultures of the Y. enterocolitica grown at 37 degrees C; these changes coincided with a low yield of proteins and point to an inhibitory effect of the 37 degrees C temperature.     

Development of an O-antigen serotyping scheme for Cronobacter sakazakii

Cronobacter sakazakii is an opportunistic pathogen that can cause severe infections. Serotyping provides a basis for the categorization of bacterial strains and is an important tool for epidemiological and surveillance purposes. In this study, of the 135 Cronobacter strains tested initially, 119 were identified as C. sakazakii and used. A serotyping scheme for C. sakazakii that classifies strains based on their different O antigens was developed. Seven antisera that exhibited high agglutinin titers (>640) were produced. O2 and O6 antisera were specific for their homologous strains, O4 and O7 antisera gave heterologous titers with O1 and O6 antigens, respectively, and O1, O3, and O5 antisera cross-reacted with each other and require preabsorption with the other two antigens. All of these 119 C. sakazakii strains were clearly assigned to these seven serotypes. O1 and O2 are the dominant serotypes, comprising 69.7% of the isolates. We also characterized the O-antigen gene clusters using restriction fragment length polymorphism (RFLP). The grouping of C. sakazakii strains based on their RFLP banding patterns correlated well with the grouping of strains based on our serotyping scheme. The serotype scheme presented here could prove to be a useful tool for serotyping C. sakazakii isolates.     

Evaluation of the sensitivity of selected serological tests and activity of Coxiella burnetii antigens in the diagnosis of Q fever]

Sensitivity of three serological tests: indirect immunofluorescence assay (If), complement fixation test (CF), and microagglutination test (MA) was evaluated. Sera (118 samples) of humans suspected of C. burnetii infection were tested. Phase II antibodies were detected in 68.6% of sera and phase I antibodies--in 38.2% of sera. Among seropositive to phase II antigen--93.8% of sera reacted in IF, 62.9% in MA, and 32.1% in CF; among seropositive to phase I antigens--100% of samples reacted in IF, 2.6% in MA and 2.6% in CF. Calculated sensitivity of above tests was as followed: IF-93.8%, MA-67.1%, CF-34.2%. Some human sera (6.1%) reacted with hen egg antigens in CF. Reactivity of diagnostic antigens prepared from reference Henzerling strain and four others isolated in Poland with rabbit immune sera and sera of individuals suspected of C. burnetii infection in IF was compared. Generally, the immune sera reacted in highest titres with homologous antigens derived from homologous strains. Human sera showed differentiated activity to particular antigens. The titres of phase I antibodies fluctuated from 0 to 16 depending on the antigen applied. Because of that fact diagnostic antigens should be prepared from the mixture of reference strains and isolates from a region under study.     

Adherence ability of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis to two different epithelial cell lines

The ability of 59 wild-type strains of Pseudomonas aeruginosa to adhere to the HeLa and Buffalo Green Monkey Kidney (BGMK) cells was investigated. Twenty strains were isolated from sputa of cystic fibrosis patients, while 19 strains were isolated from tracheal aspirates and 20 from bronchial secretions of patients without cystic fibrosis, and they were used as a control group of strains. The statistically significant difference between adherence ability of strains was observed (p < 0.01). While most of the tracheal and bronchial isolates were hyperadhesive (51-110 bacteria per cell) most of the cystic fibrosis isolates adhered poorly to the HeLa and BGMK cells (1-10 bacteria per cell). The bacterial binding to the cells was blocked when bacteria were incubated at 80 degrees C for 20 min before the adherence assay. These results indicate that alginate is not involved in the adherence of P. aeruginosa to the used epithelial cell lines, and, because of that, mucoid strains isolated from persistently colonized cystic fibrosis patients showed poor adherence ability.     

Immunologic study of artificial complex antigens obtained from P. aeruginosa lipopolysaccharides]

Artificial antigens were obtained on the basis of the polysaccharide component of P. aeruginosa complexed with an indifferent protein. Immunological study indicated that the specific polysaccharide of P. aeruginosa lipopolysaccharide contained two structures, high molecular and low molecular, having qualitative and quantitative differences in their hydrocarbon composition. Artificial complex antigens possessed serological and immunogenic properties, the low molecular polysaccharide fraction complexed with protein having less pronounced serological and immunogenic activity than polysaccharide and the high molecular fraction complexed with protein. Antificial complex antigens exerted no protective effect in generalized P. aeruginosa infection in rats.     

Geographical distribution of milky disease bacteria in the eastern United States based on phylogeny

A phylogenetic grouping of 48 different isolates of milky disease bacteria isolated in the United States was determined using genomic RFLP analysis and 16S rDNA sequence comparison. A clear distinction between Paenibacillus popilliae isolates and Paenibacillus lentimorbus isolates was evident from the results of each procedure. The P. popilliae isolates segregated into two phylogenetic groups and the P. lentimorbus isolates segregated into three phylogenetic groups. In the United States, P. popilliae group 1 was generally isolated from insects collected west of the Appalachian Mountains. P. popilliae group 2 was only isolated from insects collected east of the Appalachian Mountains. P. lentimorbus groups 1 and 2 were obtained from insects collected west and south of the Appalachians. P. lentimorbus group 3 was identified in insects collected east of the mountains. From five different locations in Connecticut, 12 milky disease bacterial isolates were classified as P. popilliae and three were classified as P. lentimorbus. Except for one isolate, all P. popilliae isolates were of phylogenetic group 2. The three P. lentimorbus strains were isolated from diseased insects that had been collected from a localized area in the state. These three strains formed a separate phylogenetic grouping (i.e., group 3) of P. lentimorbus and, based on 16S rDNA sequence comparisons, were most similar to the newly identified P. lentimorbus Semadara strain recently isolated in Japan. All milky disease bacteria that had been isolated from commercially available insecticide preparations were identified as P. popilliae group 1.     

Phenotypic characterization and description of two major O-serotypes in Tenacibaculum maritimum strains from marine fishes

Tenacibaculum maritimum is the etiological agent of marine flexibacteriosis disease, with the potential to cause severe mortalities in various cultured marine fishes. The development of effective preventive measures (i.e. vaccination) requires biochemical, serological and genetic knowledge of the pathogen. With this aim, the biochemical and antigenic characteristics of T. maritimum strains isolated from sole, turbot and gilthead sea bream were analysed. Rabbit antisera were prepared against sole and turbot strains to examine the antigenic relationships between the 29 isolates and 3 reference strains. The results of the slide agglutination test, dot-blot assay and immunoblotting of lipopolysaccharides (LPS) and membrane proteins were evaluated. All bacteria studied were biochemically identical to the T. maritimum reference strains. The slide agglutination assays using O-antigens revealed cross-reaction for all strains regardless of the host species and serum employed. However, when the dot-blot assays were performed, the existence of antigenic heterogeneity was demonstrated. This heterogeneity was supported by immunoblot analysis of the LPS, which clearly revealed 2 major serological groups that were distinguishable without the use of absorbed antiserum: Serotypes O1 and O2. These 2 serotypes seem to be host-specfic. In addition, 2 sole isolates and the Japanese reference strains displayed cross-reaction with both sera in all serological assays, and are considered to constitute a minor serotype, O1/O2. Analysis of total and outer membrane proteins revealed that all strains share a considerable number of common bands that are antigenically related.     

The hemagglutinating activities of Pseudomonas aeruginosa lectins PA-IL and PA-IIL exhibit opposite temperature profiles due to different receptor types

The two Pseudomonas aeruginosa lectins PA-IL and PA-IIL, which are very similar in subunit size, composition and properties, but differ in carbohydrate specificity, were shown to exhibit opposite temperature profiles in hemagglutination tests. The galactophilic PA-IL, which interacts with the erythrocyte I antigen (together with B or P system antigens), resembles Ii system-specific 'cold hemagglutinins' (including antibodies and lectins of animals and plants) in low (4 degrees C) temperature optimum, while the hemagglutination by the fucose- and mannose-binding PA-IIL (like that of antibodies and lectins which do not bind to these antigens) increases on raising the temperature from 4 to 37 degrees C and even to 42 degrees C. The preferential production of both P. aeruginosa lectins at 28 degrees C and their much stronger interaction with enzyme (protease or sialidase)-damaged cells, as well as the lower temperature optimum (4 degrees C) of PA-IL-binding to the host cells, may be associated with the saprophytic rather than parasitic designation of this bacterium.     

Iodine sensitivity of bacteria isolated from iodinated water systems

Fourteen bacterial isolates, predominantly Pseudomonas sp., from two water systems disinfected by iodinated anion-exchange resins were studied and compared with an isolate of Pseudomonas aeruginosa from a povidone-iodine solution and four other isolates. Pseudomonas cepacia and P. aeruginosa grown in brain heart infusion were 3 to 5 logs less sensitive to 1 mg/L I2 (pH 7.2, 1 min) when compared with cultures grown in phosphate buffer. Another P. cepacia isolate was the least sensitive culture when grown in brain heart infusion (1 log decrease) but was more sensitive after cultivation in phosphate buffer (5 logs). Isolates from an iodinated potable water system, including P. cepacia, Staphyloccus warneri, and a Bacillus sp., were all less sensitive to iodine than a "resistant" P. aeruginosa and three other isolates when grown in brain heart infusion. A clinical isolate of P. aeruginosa exhibited intermediate sensitivity. The sensitivity of bacteria to iodine is thus highly variable, depending on the organism as well as the growth conditions.     

The Pseudomonas aeruginosa lipid A deacylase: selection for expression and loss within the cystic fibrosis airway

Lipopolysaccharide (LPS) is the major surface component of gram-negative bacteria, and a component of LPS, lipid A, is recognized by the innate immune system through the Toll-like receptor 4/MD-2 complex. Pseudomonas aeruginosa, an environmental gram-negative bacterium that opportunistically infects the respiratory tracts of patients with cystic fibrosis (CF), can synthesize various structures of lipid A. Lipid A from P. aeruginosa strains isolated from infants with CF has a specific structure that includes the removal of the 3 position 3-OH C10 fatty acid. Here we demonstrate increased expression of the P. aeruginosa lipid A 3-O-deacylase (PagL) in isolates from CF infants compared to that in environmental isolates. PagL activity was increased in environmental isolates by growth in medium limited for magnesium and decreased by growth at low temperature in laboratory-adapted strains of P. aeruginosa. P. aeruginosa PagL was shown to be an outer membrane protein by isopycnic density gradient centrifugation. Heterologous expression of P. aeruginosa pagL in Salmonella enterica serovar Typhimurium and Escherichia coli resulted in removal of the 3-OH C14 fatty acid from lipid A, indicating that P. aeruginosa PagL recognizes either 3-OH C10 or 3-OH C14. Finally, deacylated lipid A species were not observed in some clinical P. aeruginosa isolates from patients with severe pulmonary disease, suggesting that loss of PagL function can occur during long-term adaptation to the CF airway.     

N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Usefulness of a microimmunodiffusion test for the detection of Penicillium marneffei antigenemia,antibodies, and exoantigens

Eight sera from culturally-proven cases of penicilliosis marneffei and their corresponding isolates were examined for circulating antibody(ies) and antigen, and exoantigens, respectively, using a microimmunodiffusion (MID) test. Two of the 8 sera produced strong precipitins (1-2) when reacted against control Penicillium marneffei antigen (5-week-old shaken cultures at 25 C) in the presence of control rabbit anti-P. marneffei serum. Five of the 8 sera produced a strong precipitin line when reacted against control hyperimmune serum to P. marneffei. These five sera, and one additional serum, which tested negative for antibody to P. marneffei, demonstrated the presence of antigen by reacting only against the anti-P. marneffei serum. Serological evaluations of the sera revealed that the MID test is capable of detecting antibody and antigen in AIDS patients having penicilliosis marneffei infections. Exoantigen analysis of the 8 P. marneffei isolates, which were previously identified using this conventional and time-consuming macro- and micro-morphological characteristics, showed the presence of 1 to 4 specific exoantigens in MID. With the exoantigen analysis, the identity of all of the isolates was confirmed as P. marneffei. Our studies indicated that the serological tests are useful for detecting circulating antibody and/or antigen in patients' sera, and that the exoantigen test is reliable for confirming the identity of P. marneffei cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of antigens present in the extracellular products and cell surface of Vibrio anguillarum serotypes O1, O2, and O3.

Antigens present in the extracellular products (ECP) and cell walls of strains of Vibrio anguillarum of serotypes O1, O2, and O3 isolated from different fish species in distinct geographic areas were characterized. The usefulness of slide agglutination, dot blot assay, and quantitative agglutination for subtyping V. anguillarum serovars was also evaluated. The three serological assays used to establish the serogroups within V. anguillarum isolates demonstrated that serotype O1 constitutes a homogeneous group, whereas within serotypes O2 and O3, two different patterns of serological reactions were detected. Among the three serological methods used, only dot blot and quantitative agglutination assays differentiated subgroups within serotypes O2 and O3 with unabsorbed sera. Electrophoretic analysis and immunoblot assays of cell envelope and ECP components showed that strains belonging to serotype O1 possessed immunologically related lipopolysaccharide (LPS) and proteins, while V. anguillarum isolates grouped in serotypes O2 and O3 exhibited internal heterogeneity in their LPS and protein banding patterns. On the other hand, although the LPS present in the ECP and those obtained from cell envelopes of V. anguillarum strains showed apparently different gel patterns, a strong relationship between both types of LPS was seen by immunoblot assay. From these results, it can be concluded that V. anguillarum strains representative of each of the antigenic groups (O1, O2 alpha, O2 beta, O3A, and O3B) and their ECPs should be included in the formulation of vaccines against vibriosis in areas where the three serotypes coexist.     

Characterization of pseudomonads isolated from diseased fleece.

A total of 59 Pseudomonas isolates was obtained from 11 samples of diseased fleece taken from live sheep. All but four of the isolates could be assigned to one of nine Pseudomonas species, of which P. aeruginosa, P. alcaligenes, P. mendocina , and P. putida were the most common. P. aeruginosa was found in four of the fleece samples and, when present, appeared to predominate. Although several of the isolates of P. aeruginosa lacked the ability to produce pyocyanin (and some produced neither pyocyanin nor fluorescein), nearly all produced several virulence factors. Of the other pseudomonads, many produced proteinase, esterase, and catalase, several were able to grow at 42 degrees C and reduce nitrate, and some also produced lipase and hemolysin and, like P. aeruginosa, might serve to initiate (or sustain) the dermatitis frequently associated with fleece rot in sheep.