Identification of infectious Pseudomonas aeruginosa strains in an occupational saturation diving environment

OBJECTIVES: Occupational saturation divers have various skin disorders, of which skin infections are the most serious and frequent. Pseudomonas aeruginosa is the microbe most often isolated from skin infections in divers. The purpose of the present work was (a) to report the occurrence of P aeruginosa in skin infections in operational saturation diving in the North Sea from 1987 to 1995; (b) to report the environmental occurrence of P aeruginosa in saturation diving systems, and finally (c) to identify possible relations between infection related to strains of P aeruginosa and environmental isolates of the microbe. RESULTS: During the period 1987-95, P aeruginosa was isolated from 257 skin infections in operational saturation divers. Most of the isolates related to infection by P aeruginosa show a unique growth inhibition pattern towards the normal skin flora, and the serotype pattern of P aeruginosa from skin infections is limited compared with similar infections in non-divers. In a mini-epidemiological study on board one diving vessel during one operational diving period, five significantly different DNA fragment profiles were found among the 12 isolates related to infection by P aeruginosa obtained from the saturation system. In two cases the infectious genotypes were detected in the fresh water for the saturation chambers weeks before the arrival of the infected diver. CONCLUSIONS: The most commonly used epidemiological marker for P aeruginosa world wide, also used in earlier studies, is serotyping, but with pulsed field gel electrophoresis (PFGE) miniepidemiology it was shown to be insufficient for epidemiological purposes in saturation environments. PFGE analyses were shown to be superior both to antibacterial factor and to serotyping in epidemiological analyses of P aeruginosa infections in saturation diving.     

An inexpensive infrared growth sensor array for detection of bacterial antibiotic susceptibility

An inexpensive infrared sensor was constructed and used for the rapid testing of bacterial antibiotic susceptibility by detection of changes in absorbance at 950 nm. By comparing cultures of clinical isolates together with control strains (Escherichia coli NCTC 10418, Staphylococcus aureus NCTC 6571 or Pseudomonas aeruginosa NCTC 10662) after addition of an antibiotic, results on susceptibility were obtained within 3-5 h from the original plate culture. Representative strains of E. coli, P. aeruginosa, and S. aureus were tested successfully against ampicillin, penicillin, gentamicin or ciprofloxacin.     

Identification of Neisseria gonorrhoeae in cultures from tonsillo-pharyngeal specimens by means of a slide co-agglutination test (Phadebact Gonococcus Test)

A slide co-agglutination test (Phadebact Gonococcus Test) for the serological identification of Neisseria gonorrhoeae was assessed on gonococcal-like, oxidase positive colonies from 120 cultures, originating from about 6,500 consecutive tonsillo-pharyngeal specimens received at the Neisseria Department, Statens Seruminstitut. The test was performed after subculture on a serum-free medium, since this procedure was found to reduce the number of strains showing inconclusive reactions (pseudo co-agglutination). If this pseudo co-agglutination does occur, however, the test can be repeated with the addition of trypsin to the test system. This causes the previously inconclusive reactions to be reverted to clearly positive reactions in the case of gonococci, and to clearly negative reactions in more than half of the previously inconclusive reactions with other bacterial strains. The results obtained by the Phadebact Gonococcus Test were compared with those obtained by bacteriological identification procedures. Fifty-six of the 120 cultures examined contained gonococci, and all strains were identified by the slide co-agglutination test (five strains with the addition of trypsin). The remaining 64 cultures were negative or exhibited consistently pseudo co-agglutination (eight strains). The specificity and sensitivity of the reagent was further confirmed by the examination of 53 strains of Neisseria gonorrhoeae and 50 strains representing Neisseria species commonly occurring in tonsillo-pharyngeal specimens. The Phadebact Gonococcus Test was considered to be a reliable alternative to routine bacteriological identification of Neisseria gonorrhoeae.     

Effect of climatic-weather conditions on the incidence of miscarriage

Two new methods for serological grouping of beta-hemolytic streptococci, the nitrous acid extraction procedure of El Kholy et al. and the slide agglutination method of Christensen et al., were compared with the Lancefield hot-hydrochloric acid extraction method in classifying 92 strains of groups A, B, C, and G. The nitrous acid extraction method was easily performed, specific, and sensitive when highly potent antisera were used. For the Christensen method these highly potent antisera had to be diluted to avoid cross-reactions between groups A and C and groups B and G, respectively. A few strains, most of them group B, could not be grouped by the latter method. Using these three grouping methods, two sets of commercial sera were compared with the more potent sera supplied by R. C. Lancefield. The low antibody content of these commercial sera, especially anti-group B and G sera, contributed to the inferior results obtained in some of the grouping reactions.     

Comparative study of the antibiograms as well as serotype and virulence of Ps. aeruginosa cultures

Seven antibiotics, such as gentamycin, kanamycin, streptomycin, polymyxin M, carbenicillin, tetracycline and rifampicin were studied with respect to their effect on 260 strains of Ps. aeruginosa isolated from various sources within 1945-1971. Gentamycin and polymyxin M proved to be most active in vitro. Carbenicillin showed moderate activity. The sensitivity levels of the cultures of Ps. aeruginosa isolated from various sources within 30 years were identical. Serological typing of 59 cultures of Ps. aeruginosa was performed and their virulence was studied in parallel with their antibiotic sensitivity testing. No correlation between the antibiograms, serotypes and virulence of Ps. aeruginosa was found.     

Plasmid-mediated gentamicin resistance of Pseudomonas aeruginosa and its lack of expression in Escherichia coli

We isolated 11 nonconjugative plasmids mediating resistance to aminoglycoside antibiotics, including gentamicin, from Pseudomonas aeruginosa strains. Their genetic properties were investigated in both P. aeruginosa and Escherichia coli transformants. The plasmid molecular weights ranged from 11 x 10(6) to 24 x 10(6). A low level or complete absence of gentamicin resistance was observed when these plasmids were introduced into E. coli, but gentamicin resistance was restored when the plasmids were transferred back to P. aeruginosa from E. coli. Aminoglycoside-modifying enzyme activity was detected in P. aeruginosa harboring these plasmids, but was absent or greatly reduced in E. coli strains. This lack of expression may explain the observed decrease in aminoglycoside resistance.     

Lysogenic conversion as a factor influencing tolerance to vancomycin in Staphylococcus aureus

The standard, non-lysogenic, bacteriophage-free S. aureus NCTC 8325-4 strain was lysogenized with 15 different, obtained in our laboratory staphylokinase-converting bacteriophages belonging to serological groups A, B and F. MIC and MBC of vancomycin as well as the ratio of MBC to MIC were evaluated for all 15 lysogenic derivatives. The obtained results were compared with those for maternal strain. In the case of eight strains the ratio MBC/MIC showed the presence of tolerance to vancomycin (MBC/MIC > or = 32). Four of the vancomycin-tolerant derivatives were lysogenized with bacteriophages belonging to the serological group A, two were members of group B and two belonged to group F.     

Evaluation of serotyping, biotyping, plasmid banding pattern analysis, and HEp-2 vacuolation factor assay in the epidemiological investigation of Bacillus cereus emetic-syndrome food poisoning

To assess the value of the plasmid banding patterns, the vacuolation factor (VF) assay, biotyping, and serological typing as epidemiological markers for strains of Bacillus cereus causing emetic-syndrome illness, 43 isolates from five outbreaks and an additional 76 strains isolated in food-poisoning outbreaks caused by other enteric pathogens were examined by these techniques, and the results were compared. Thirty-eight (88%) of the 43 outbreak strains produced vacuolation responses in HEp-2 cells and were all starch-hydrolysis negative. The other 76 strains associated with outbreaks caused by other food-poisoning bacteria gave all negative VF production results except four strains, and 56 (74%) of these strains produced positive reactions in starch hydrolysis tests. Starch hydrolysis emerged as a convenient screen for VF production, because no starch hydrolysis-positive strains produced VF. With the exception of one isolate, all 38 VF-positive isolates from emtic-syndrome outbreaks were serotype H.1. Isolates from four of the five outbreaks revealed identical plasmid banding patterns in each outbreak, whereas only three of eight serotype H.1 strains from the fifth outbreak exhibited indistinguishable plasmid banding patterns. These results suggest that the plasmid banding pattern analysis may be of value in discriminating between isolates of the same serotype, and establishing if an outbreak arises from a common food source. In conclusion, the vacuolation factor assay combined with the plasmid banding patterns proved to be a valuable tool for the epidemiological investigation of emetic-syndrome outbreaks caused by B. cereus. Moreover, these methods are particularly useful for laboratories that do not have ready access to serotyping facilities.     

Identification and typing of hospital strains of Acinetobacter calcoaceticus-Acinetobacter baumanni complex

A collection of 95 strains of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, isolated between 1991 and 1993 in the Prague Burn Center (BC), was studied. Ninety-one strains were isolated from 43 patients: 50 of them from burnt sites, 22 from endotracheal tube, 13 from urine, 3 from blood and 3 from venous catheter, and 4 strains were isolated from the hospital environment and the nursing staff. The strains were classified by restriction endonuclease fingerprinting of total DNA, plasmid profile analysis, ribotyping, comparison of antibiograms, biotyping and according to epidemiological data, into 31 relatedness groups each of them including 1 to 29 strains, likely to be isolates of the same strain. None of the methods used enabled to distinguish all groups. The importance of the polyphasic approach is emphasized since three multiresistant strains, isolated almost simultaneously in the BC, needed at least two methods to be distinguished (e.g. ribotyping and biotyping). Twenty-eight representative strains of different groups were identified by ribotyping: 18 of them were allocated to genomospecies 2 (A. baumannii), 5 to genomospecies 3 and 5 to genomospecies 13 sensu Tjernberg and Ursing. Only A. baumannii was found to spread among patients. Strains of two multiresistant groups persisted in the BC throughout the period studied and strains of one of these groups were responsible for an outbreak in the autumn of 1993. The methods mentioned above were used to describe 12 multiresistant strains isolated in three hospital wards in other localities. When ribotyped these strains were identified as A. baumannii. The strains of the same origin were identical in their typing profiles while the strains of different origins were easy to differentiate using any of the above methods; nevertheless, 2 of these groups were almost identical to 2 groups of multiresistant strains isolated in the BC.     

Effect of cyclopentolate on human plasma cholinesterase

The beta-lactamase isoelectric focusing patterns of 37 strains of Yersinia enterocolitica from various serological and biochemical groups were examined. Strains of different serological groups generally had different patterns, but those of serological groups 1, 2, 3 and 9 were identical.     

Demonstration that anti-phospholipid auto-antibodies react with both anionic and zwitterionic phospholipids

Mice that had been treated with cyclophosphamide and ampicillin were fed with Pseudomonas aeruginosa. These procedures induced an endogenous septicaemia under conditions mimicking the pathophysiology of the disease in man. This model was used to compare the mortality rates in mice infected with P. aeruginosa isolates from various clinical sources. Mortality rates in mice given isolates from blood cultures had a broad range (0-100%), but the mean rate was significantly higher than with isolates from other infection sites. Moreover, blood isolates persisted in the intestines of mice after oral inoculation, whereas most isolates from other sources were gradually eliminated. Most P. aeruginosa isolates from blood culture produced significantly higher levels of exotoxin A and total proteases than isolates from other infection sites. Amongst the blood isolates, all but one of the lethal strains produced large quantities of exotoxin A or total proteases or both. Taken together, the results suggest that the ability of P. aeruginosa to adhere to the intestinal tract and to produce high levels of exo-enzymes may contribute to the development of fatal septicaemia.     

Loss of CD28 expression on T lymphocytes: a marker of replicative senescence

A massive accumulation of neutrophils, mainly due to enhanced interleukin-8 (IL-8) levels, is believed to contribute to the deleterious effects of Pseudomonas aeruginosa lung infection, e.g., in cystic fibrosis (CF). Antibodies to phospholipase C, an exoenzyme of P. aeruginosa, are detected early and at high levels in CF patients. However, P. aeruginosa produces at least two types of phospholipase C (PLC), one haemolytic (PLC-H) and the other non-haemolytic (PLC-N), both with mol.wts of c. 77 kDa. Experiments were performed to evaluate the potential contribution of P. aeruginosa PLC to neutrophil accumulation during infection. Therefore, P. aeruginosa PLC-H and PLC-N were compared with regard to IL-8 generation from human monocytes. Purified PLC-H as well as culture supernates (mol.wt > 50 kDa) of a P. aeruginosa strain capable of producing both PLC-H and PLC-N, and mutant strains deficient in the production of one or other phospholipase, or both, were examined. Purified PLC-H (only at low concentrations up to 1 unit/4 x 10(5) monocytes), induced a dose-dependent increase in IL-8 release and IL-8-specific mRNA expression over that of unstimulated cells (at 4-, 12- and 24-h incubation times). Higher concentrations of PLC-H led to a decrease in IL-8 release and IL-8-specific mRNA expression. These findings were confirmed by the results obtained with the supernates of cultures of mutant strains of P. aeruginosa PAO1 that produced either a PLC-H or PLC-N or neither. Stimulation and inhibition of IL-8 release and mRNA expression were associated with a culture supernate fraction of mol. wt > 50 kDa and containing PLC-H. These results contribute to the understanding of the role of both P. aeruginosa PLC in IL-8 generation during their interaction with human monocytes.     

In vitro activity of temocillin, a new beta-lactamase-stable penicillin active against enterobacteria

The activity of temocillin was investigated in vitro against 523 clinical isolates of enterobacteria and Pseudomonas aeruginosa. The minimum inhibitory concentration of the new compound for all ampicillin-susceptible enterobacteria and for 90% of ampicillin-resistant enterobacteria was 16 micrograms/ml or less, a concentration readily achieved in plasma. P. aeruginosa strains were uniformly resistant to temocillin. All but 3 of a separate group of 48 enterobacteria exhibiting resistance to the combination of clavulanic acid and amoxicillin were found to be inhibited by 16 micrograms or less of temocillin per ml. The new compound also displayed good activity against a group of laboratory stock cultures selected on the basis of differential resistance to presently available beta-lactam agents. Two of these strains were cefotaxime resistant.     

In vitro inhibition of the growth of Gardnerella vaginalis by bacteriocins produced by strains of Pseudomonas aeruginosa

BACKGROUND: Pseudomonas aeruginosa with inhibitory capacity in vitro was studied on Gardnerella vaginalis strains. METHODS: Antimicrobial activity was demonstrated by inhibitory halos of bacterial growth on solid media by two methods: crossed streak and agar well diffusion. The inhibitory activity of this substance produced by P. aeruginosa was characterized as bacteriocin by: activity spectrum sensitivity proteolytic enzyme, chloroform, heat, pH, ultraviolet, irradiation effect and molecular weight. RESULTS: Four strains of P. aeruginosa producers of bacteriocins were chosen for this study and contacted with 40 strains of G. vaginalis. The producing strain D inhibited 70% of these G. vaginalis strains. The strains B and C inhibited 55% and 52.5%, respectively. The 3 strains presented a wide rank of action but the strain A had effect on a few strains of G. vaginalis. CONCLUSIONS: This work showed the inhibitory in vitro effect of bacteriocins of P. aeruginosa on strains of G. vaginalis. The results obtained suggest the probable topic application of bacteriocins as an alternative of conventional therapeutic on this infection biological control.     

Widespread detection of PER-1-type extended-spectrum beta-lactamases among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey: a nationwide multicenter study

We studied the prevalence and molecular epidemiology of PER-1-type beta-lactamases among Acinetobacter, Klebsiella, and Pseudomonas aeruginosa strains isolated over a 3-month period in eight university hospitals from distinct regions of Turkey. A total of 72, 92, and 367 Acinetobacter, Klebsiella, and P. aeruginosa isolates were studied, respectively. The presence of blaPER was determined by the colony hybridization method and later confirmed by isoelectric focusing. We detected PER-1-type beta-lactamases in 46% (33/72) of Acinetobacter strains and in 11% (40/367) of P. aeruginosa strains but not in Klebsiella strains. PER-1-type enzyme producers were highly resistant to ceftazidime and gentamicin, intermediately resistant to amikacin, and susceptible or moderately susceptible to imipenem and meropenem. Among PER-1-type-beta-lactamase-positive isolates, five Acinetobacter isolates and six P. aeruginosa isolates from different hospitals were selected for ribosomal DNA fingerprinting with EcoRI and SalI. The EcoRI-digested DNAs were later hybridized with a digoxigenin-labelled PER-1 probe. The ribotypes and the lengths of blaPER-carrying fragments were identical in four Acinetobacter strains. A single isolate (Ac3) harbored a PER gene on a different fragment (approximately 4.2 kbp) than the others (approximately 3.4 kbp) and showed a clearly distinguishable ribotype. Ribotypes of P. aeruginosa strains obtained with EcoRI showed three patterns. Similarly, in Pseudomonas strains two different EcoRI fragments harbored blaPER (approximately 4.2 kbp in five isolates and 3.4 kbp in one isolate). PER-1-type beta-lactamases appear to be restricted to Turkey. However, their clonal diversity and high prevalence indicate a high spreading potential.     

Norepinephrine loss selectively enhances chronic nigrostriatal dopamine depletion in mice and rats

A total of 3,700 Pseudomonas aeruginosa isolates were collected from 17 general hospitals in Japan from 1992 to 1994. Of these isolates, 132 carbapenem-resistant strains were subjected to DNA hybridization analysis with the metallo-beta-lactamase gene (blaIMP)-specific probe. Fifteen strains carrying the metallo-beta-lactamase gene were identified in five hospitals in different geographical areas. Three strains of P. aeruginosa demonstrated high-level imipenem resistance (MIC, > or = 128 micrograms/ml), two strains exhibited low-level imipenem resistance (MIC, < or = 4 micrograms/ml), and the rest of the strains were in between. These results revealed that the acquisition of a metallo-beta-lactamase gene alone does not necessarily confer elevated resistance to carbapenems. In several strains, the metallo-beta-lactamase gene was carried by large plasmids, and carbapenem resistance was transferred from P. aeruginosa to Escherichia coli by electroporation in association with the acquisition of the large plasmid. Southern hybridization analysis and genomic DNA fingerprinting profiles revealed different genetic backgrounds for these 15 isolates, although considerable similarity was observed for the strains isolated from the same hospital. These findings suggest that the metallo-beta-lactamase-producing P. aeruginosa strains are not confined to a unique clonal lineage but proliferated multifocally by plasmid-mediated dissemination of the metallo-beta-lactamase gene in strains of different genetic backgrounds. Thus, further proliferation of metallo-beta-lactamase-producing strains with resistance to various beta-lactams may well be inevitable in the future, which emphasizes the need for early recognition of metallo-beta-lactamase-producing strains, rigorous infection control, and restricted clinical use of broad-spectrum beta-lactams including carbapenems.     

Relative importance of enterotoxigenic and invasive enteropathogenic bacteria in infantile diarrhoea

Swedish children and adults (648 patients) with acute diarrhoea were investigated for enterotoxigenic strains in stool cultures. A total number 74 strains were isolated from 28 patients and assayed in the rabbit intestinal loop test and the adrenal cell test. Only three of the enterotoxigenic E. coli (ETEC) isolates belonged to classical enteropathogenic serotypes of E. coli (EPEC). Two enterotoxigenic strains of Proteus morganii, two of Enterobacter hafniae and one of Citrobacter freundii were isolated. None of 67 EPEC strains were found to produce a heat-labile enterotoxin (LT) in either of the two test systems. A number of Yersinia enterocolitica and Pseudomonas aeruginosa strains from stool cultures often produced toxic effects in the cell test but no enterotoxin activity was detected for any of the strains investigated either in the adrenal cell test for heat-labile enterotoxin (LT) or the suckling mouse test for heat-stable enterotoxin (ST). All EPEC isolates were also tested for ST and for invasive properties in the Sereny test; each isolate was negative in both test systems. It is concluded that production of LT and ST enterotoxin were common in stool isolates from Ethiopian children but a rare phenomenon among Swedish children with acute infantile diarrhoea. Isolation of aerobic stool bacteria with invasive properties seems to be uncommon both in Ethiopian and Swedish children. However, since both LT and ST as well as invasive properties seem to be very unstable genetic properties in many of these stool isolates improved sensitive methods for the last two properties will probably change this picture in the future.     

Genotyping of Shigella flexneri 2a strains isolated from patients with acute dysentery in St. Petersburg

Polymerase chain reaction with universal primers (UP-PCR) was used for the genotyping of 76 S.flexneri 2a cultures isolated from patients with acute dysentery in infectious and psychoneurological hospitals of St. Petersburg. 9 types were determined, and each of them included cultures with identical UP-PCR patterns. The population of the infective agent was more heterogeneous in psychoneurological hospitals where the change of types was registered in May-September 1995. Some of these types were probably epidemic strains. UP-PCR was found to be a promising method increasing the efficiency of traditional epidemiological analysis.     

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.     

Evaluation of multiple Pseudomonas aeruginosa strains with different characteristics in clinical specimens; combined results from serotyping, drug susceptibility and enzyme production

Multiple Pseudomonas aeruginosa strains with different characteristics are occasionally isolated from a clinical specimen. Therefore, more than five isolated colonies of P. aeruginosa obtained at random from each clinical specimen (47 sputa, 18 urine, 10 pus and 8 others). These were investigated for serotype, drug susceptibility to eight antimicrobial agents and productivity of enzymes, such as protease and elastase. The specimens with multiple serotype colonies were shown in 17% of the sputa, 11% of the urine and 10% of the pus. 45.7% of the specimens with single serotype colonies exhibited more than two different patterns of enzyme productivity and so did 47.1% different patterns of drug susceptibility. Single serotype strains of P. aeruginosa with different characteristics of these tests were demonstrated in 81.3% of the urine, 73.6% of the sputum, 50.0% of the pus and 66.7% of others. We conclude that it is important to recognize the possible existence of multiple P. aeruginosa strains with different patterns of the enzyme productivity and drug susceptibility, regardless of single serotype, in clinical specimens.