Pyocin Typing Research Articles

Prevalence of bacteriocins and their co-association with virulence factors within Pseudomonas aeruginosa catheter isolates

Urinary tract infections represent common nosocomial infectious diseases. Bacteriocin production has been recently described as a putative virulence factor in these infections but studies focusing particularly on Pseudomonas aeruginosa are not available. Therefore, we assessed the prevalence of the bacteriocin genes, their co-occurrence and their co-association with previously detected virulence factors in a set of 135 P. aeruginosa strains from catheter-associated urinary tract infections (CAUTIs). The overall bacteriocinogeny reached 96.3 % with an average of 3.6 genes per strain. The most frequently detected determinants were the encoded pyocins S4 (76.3 %), R (69.6 %), and S2 (67.4 %). A statistically significant co-occurrence and a negative relationship were observed between several pyocin types. Particular pyocins exhibited associations with biofilm formation, production of pyochelin, pyocyanin, antibiotic-degrading enzymes, overall strain susceptibility and resistance, and motility of the strain. Co-occurrence of the pyocins S2 and S4 (p<<0.0001; Z = 13.15), both utilizating the ferripyoverdine receptor FpvAI, was found but no relation to pyoverdine production was detected. A negative association (p = 0.0047; Z=-2.83) was observed between pyochelin and pyocin S5 utilising the ferripyochelin receptor FptA. Pairwise assays resulted in 52.1 % inhibition which was equally distributed between soluble and particle types of antimicrobials. In conclusion, pyocin determinants appear to be important characteristics of CAUTI-related P. aeruginosa isolates and could contribute to their urovirulence.

Investigating the uptake mechanism of S type pyocins

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen with a high mortality and morbidity rate. It has many mechanisms of resistance to antibiotics, which makes it hard to treat. Pyocin is a protein that is produced by P.aeruginosa that kills related strain bacteria. pyocin type S bind to ferrisiderophore receptors that uses the TonB system to translocate into the bacterial cell. The aim of this study was to express and purify receptor and translocation (R+T) domains of pyocin S1, S2 and S3 plus the TonB1 receptor and ToLAIII receptor of P.aeruginosa to determine if pyocins S uses these to transverse the inner membrane of target cells. IPTG was used to induce the protein in the expression and analyzed by SDS-PAGE gel, giving fragment size of 11 kDa (ToLAIII), 23 kDa (TonB1), 45 kDa (S2 R+T) and 79 kDa (S3 R+T). Protein purification was cried on the four proteins using affinity chromatography technique by His-tag in C-terminal of S2 and S3 R+T domain, N-terminal of TonB1 and ToLAIII proteins. In addition, Gel Filtration chromatography was used to further purify the proteins so that the interaction between them could be tested. This study confirmed that the proteins used is expressed and purified, so in the future study the gel filtration would be carried out for R+T domains of the other S pyocins and TonB1 protein so that the interaction would be tested between the proteins in the study.

Epidemiology of Pseudomonas aeruginosa in agricultural areas.

A prevailing opinion is that the strains of Pseudomonas aeruginosa that infects both plants and humans are two separate species. This study strongly disputes that notion until the modern molecular technology proves otherwise. This paper examines a spectrum of strains occurring in nature, their habitats, dissemination, their relationship to clinical strains, and the environmental conditions that favor their colonization of plants. The isolates were obtained from clinical specimens, plants, soil, and water. The identity of these strains was confirmed using pyocin typing and biochemical assays. The data reveal that agricultural soils, potted ornamental plants, hoses, fountains, and faucets frequently harbored P. aeruginosa. However, it was not commonly found in semi-arid areas, suggesting that moisture and high humidity is necessary for colonization and survival. Though found in soil, P. aeruginosa was seldom isolated on edible plant parts. The pathogenicity of various strains on plants was tested by inoculating vegetables, lettuce slices (Lactuca sativa L. "Great Lakes"), celery stalks (Apium graveolens L. var. Dulce], potato tuber slices (Solanum tuberosum L. "Whiterose"), tomato (Lycopersicon esculentum L. Mill), cucumber (Cucumis sativus L.), rutabaga (Brassica campestris L.), and carrot (Daucus carota L. var sativa). There was considerable variation in the strains' ability to cause rot, but no difference was observed between clinical isolates and others from agricultural fields, water, and soil. Two of the clinical isolates from burn patients, P. aeruginosa PA13 and PA14, exhibited the greatest virulence in causing rot in all the plants that were tested, especially on cucumber, lettuce, potato, and tomato. The study discusses how closely the epidemiology of P. aeruginosa relates to many plant pathogens, and the ability of human isolates to colonize plants and food material under favorable conditions. The biochemical and phenotypic similarity among strains from the clinical and agricultural material is strongly indicative that they are the same species and that plants and soil are natural reservoirs for P. aeruginosa.

Prevalance Pseudomonas aeruginosa Among Libyan Patients and its Association with Hospital's Environment in Benghazi

A cross sectional study was carried out which included 300 clinical specimens such as urine, pus, throat swabs, ear swabs, blood, taken from patients admitted to different departments, in addition to 300 samples from the surrounding environment in four hospitals in Benghazi. The aim is to identify the major strains of P aeruginosa isolated from various sources in different hospitals from both the patients and surrounding environment, also to determine the level of resistance to the widely used antibiotics in clinical isolates of P aeruginosa. P aeruginosa were isolated from 91 clinical specimens and 18 from environmental sources. All were Gram negative motile bacilli, oxidase positive and grew at 42oC. All isolates were confirmed by Analytical Profile Index (API 20 NE) for their biochemical activity, all were positive for arginine dihydrogenase, citrate utilization and gelatin hydrolysis, while 37.4% were positive for aerobic glucose utilization. The antibiotic sensitivity tests were carried out according to the disc diffusion method (modified kirby bauer technique). Out of 91 strains of P aeruginosa isolated from clinical specimens and 18 strains from the environment, the most effective antibiotics were respectively Ciprofloxacin (82.2%, 83.3%), Imipenem (80.2%, 72.2%), Amikacin (72.5%, 77.8%) and Tobramycin (49.5%, 50%) Gentamicin showed lowest rate of sensitivity (42.2%, 50%). Other antibiotics tested: ampicillin, chloramphenicol, augmentin, pipemidic acid, colistin sulphate and nalidixic acid. The strains isolated were found to have high or total resistance to them. Pyocin typing was used for the characterization of 109 isolates of Pseudomonas aeruginosa isolated. The scheme of Gillies and Govan was adopted and the procedure gave 86.8% of clinical isolates were typable, while 50% of environmental isolates were typable, 12 (15%) of 79 typable strains isolates from clinical specimens were classifiable while 67 (85%) were nonclassifiable. More over all typable isolates from environment were nonclassifiable. The study concludes that Pseudomonas infection is high among our patients in Benghazi hospitals, and reflects the hospital environment as source of infection. This study also recommends that there should be a review in the current antibiotics policy.

Bacteriocin-mediated competition in cystic fibrosis lung infections.

Bacteriocins are toxins produced by bacteria to kill competitors of the same species. Theory and laboratory experiments suggest that bacteriocin production and immunity play a key role in the competitive dynamics of bacterial strains. The extent to which this is the case in natural populations,especially human pathogens, remains to be tested. We examined the role of bacteriocins in competition using Pseudomonas aeruginosa strains infecting lungs of humans with cystic fibrosis (CF). We assessed the ability of different strains to kill each other using phenotypic assays, and sequenced their genomes to determine what bacteriocins (pyocins) they carry. We found that(i) isolates from later infection stages inhibited earlier infecting strains less,but were more inhibited by pyocins produced by earlier infecting strains and carried fewer pyocin types; (ii) this difference between early and late infections appears to be caused by a difference in pyocin diversity between competing genotypes and not by loss of pyocin genes within a lineage overtime; (iii) pyocin inhibition does not explain why certain strains outcompete others within lung infections; (iv) strains frequently carry the pyocin-killing gene, but not the immunity gene, suggesting resistance occurs via other unknown mechanisms. Our results show that, in contrast to patterns observed in experimental studies, pyocin production does not appear to have a major influence on strain competition during CF lung infections.

Prevalence of Multidrug-Resistant Pseudomonas aeruginosa in Patients with Nosocomial Infections at a University Hospital in Egypt, with Special Reference to Typing Methods

Multidrug resistant (MDR) P.aeruginosa had emerged in Egypt and seen mainly in nosocomial infections due to the selective pressure by overusage of antibiotics. This study was planned to delineate prevalence of MDR P.aeruginosa in nosocomial infection patients, and to screen for ESβLs producing P.aeruginosa with typing of P.aeruginosa isolates in Menofia University Hospitals (MUH), Egypt. Our study included 287 inpatients admitted to Menoufia University Hospital and having different nosocomial infections. Samples from medical staff and from hospital environment were collected. Antibiotyping of P. aeruginosa isolates were determined. MDR and ESBLs P. aeruginosa were detected. Plasmid DNA analysis and pyocin typing were done. In our study, a total of 57 P. aeruginosa strains were isolated. P. aeruginosa and its MDR phenoype accounted for 19 % and 9.5% respectively of nosocomial infections. MDR and ESβLs producer P. aeruginosa infections were commonly isolated from burn wound specimens followed by urinary tract, respiratory tract and wound infections. We detected high rates of MDR P. aeruginosa (52%) and of ESβLs producer strains (45.6%) and those ESBLs strains were all MDR. Amikacin and imipenem were the most effective drugs against P. aeruginosa. 23 different resistance patterns were identified, profiles from (1 8) were prevalent. The most prevalent antibiotype (2) included 12 MDR isolates, 9 clinical and 3 environmental isolates having same patterns. 61.5% of ESβLs isolates harbor plasmids. Five groups could be demonstrated among our P. aeruginosa isolates. Each had the same antibiotype and plasmid profile. In conclusions, our results clarified that threats of MDR and ESBLs P. aeruginosa become of major concern in our hospital and implementation of infection control strategies are major concerns to avoid the spread of this threat. Eenvironmental sources may have a significant role in spread of MDR P. aeruginosa among hospitalized patients. Combination of Journal of Virology & Microbiology 2 simple typing methods as antibiotyping with plasmid profiling may be the cheapest and effective method for tracing source of infection.

Phenotypic stress response of Pseudomonas aeruginosa following culture in water microcosms

The purpose of the present study was to explore the potential behavioural changes of Pseudomonas aeruginosa following growth in different aquatic environmental conditions. To achieve this, P. aeruginosa was cultured in various water microcosms for 12 months under fixed (pH, nutrients and temperature) factors. P. aeruginosa responses to these conditions were investigated using colony morphotype, biochemical and enzymatic characterisation, pyocin typing, serotyping, sensitivity to different classes of antibiotics and molecular identification. Results show that starvation in water microcosms lead to unusual phenotypes. Of interest is that the pyocin changed from 24/n in the wild type to 83/a following culture in the water microcosms, and the serotype changed from O6 in the wild type to O1 in microcosm-cultured P. aeruginosa. Furthermore, the starvation period in various aquatic microcosms enhanced the resistance of P. aeruginosa against beta-lactam antibiotics. Compared to the other aquatic environments, the seawater microcosm produced the greatest amount of variations in P. aeruginosa. Overall, data demonstrated a high adaptability of P. aeruginosa to environmental changes. This may explain the unusual antibiotic-resistant phenotypes belonging to P. aeruginosa species, and their capacity for spreading that leads to human infections.

Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines.

Pyocin typing of 82 Pseudomonas aeruginosa strains, collected from different Iranian clinical sources, revealed that one isolate, P. aeruginosa 42A, produced pyocin S2, a protease-sensitive bacteriocin. Pyocin S2 production was induced by mitomycin C (2 micro g/mL) in the pyocin S2 producer P. aeruginosa 42A. Pyocin S2 was purified using ion exchange chromatography with CM-Sepharose CL-6B and sodium phosphate buffer (pH 8) from an 80% ammonium sulfate precipitate of whole-cell lysates. Pyocin activity of the fractions was detected using the Govan spot testing method. The purity of the active fraction was confirmed by SDS-PAGE, where a single band with a molecular mass of 74 kDa was detected. Cytotoxic effects of purified pyocin S2 and partially purified pyocin from P. aeruginosa 42A on the human tumor cell lines HepG2 and Im9 and the normal human cell line HFFF (Human Foetal Foreskin Fibroblast) were studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results demonstrated that partially purified pyocin and pyocin S2 exhibited substantial inhibitory effects on the growth of the tumor cell lines HepG2 and Im9, while no inhibitory effects were observed on the normal cell line HFFF. Pure lipopolysaccharide was used as a control and was found to have no inhibitory effect on any of the cell lines tested.

Antibiotic resistance and molecular epidemiology of Shigella spp. in northeastern Argentina

To evaluate the antibiotic resistance of strains of Shigella spp. isolated from feces samples from northeastern Argentina and to characterize the strains in terms of their molecular epidemiology. We studied 132 isolates of Shigella spp. obtained from feces samples from 132 patients with diarrhea who were seen at various private and public laboratories in the Argentine provinces of Chaco and Corrientes during the period of 1998 to 2002. Each strain was characterized according to its serotype, its resistance to 13 individual or combination antibiotics, and its sensitivity to pyocins. With 52 strains selected in relation to their antimicrobial susceptibility profiles we conducted plasmid profile analysis using alkaline lysis, and the repetitive extragenic palindromic sequences were determined by amplifying repetitive DNA segments using polymerase chain reaction. The chi-square test was used to compare proportions, with a level of statistical significance of 0.05. Shigella flexneri was the most common species (78%), followed by S. sonnei (22%). In general, the resistance of S. flexneri to the antibiotics studied was greater than that of S. sonnei, and this difference was statistically significant (P < 0.001) for ampicillin, tetracycline, chloramphenicol, and the combination of ampicillin and sulbactam. The S. flexneri strains also showed multiple resistance more often than S. sonnei strains (84.5% vs. 31.0%; P < 0.001). The strains isolated from S. flexneri were grouped into five pyocin types, three plasmid profiles, and five patterns of repetitive palindromic sequences. The strains of S. sonnei formed three pyocin types, two plasmid profiles, and three patterns of repetitive palindromic sequences. Given that the Shigella species that were studied showed a high level of resistance to the most frequently used antibiotics, surveillance activities should be implemented in order to detect and control the appearance of new resistant strains. Applying epidemiological typing techniques can provide more precise information about the distribution and evolution of resistant strains of circulating microorganisms.

Random Amplification of Polymorphic DNA Based Typing of Pseudomonas Aeruginosa.

Pseudomonas aeruginosa was isolated from various sources during the course of an epidemic outbreak of bacterial endophthalmitis following an eye camp at Sangli, Maharashtra. 15 distinct isolates were obtained from clinical samples. Typing of the 15 isolates was performed by random amplified polymorphic DNA (RAPD) analysis, pyocin typing and antibiogram. RAPD typing was rapid, labour friendly and could be done within six hours. RAPD analysis produced reproducible electrophoretic band patterns on the basis of which three distinct amplification patterns could be visualised. The conventional typing methods were labour intensive and took about 48 hours. However, the results of RAPD typing, pyocin typing and antibiogram did not correlate with each other. This study suggests that RAPD typing could be an additional rapid typing method for studying the epidemiology of infectious disease outbreaks due to P aeruginosa.

Spread of a multiresistant strain of Pseudomonas aeruginosa in an adult cystic fibrosis clinic

We initiated a prospective surveillance study to investigate possible Pseudomonas aeruginosa cross-infection in our cystic fibrosis centre. We characterised isolates by pyocin typing and pulsed-field gel electrophoresis. 22 (14%) of 154 patients with chronic P aeruginosa had isolates with similar and new pyocin and pulsed-field gel electrophoresis types. The shared isolates showed unusual phenotypic features: they were non-pigmented, non-motile, and resistant to a number of antipseudomonal antibiotics. Cross-infection by a multiresistant P aeruginosa strain has therefore occurred in patients attending our cystic fibrosis centre. We recommend microbiological surveillance in other cystic fibrosis centres.

Impact of heavy metals on the selective phenotypical markers of Pseudomonas aeruginosa

The aim of this study was to characterize the impact of heavy metals on phenotypical markers of Pseudomonas aeruginosa. Twenty-two isolates of P. aeruginosa, either clinical (20) or secondary treated wastewater (2), were used to inoculate micro-ecosystems of sterile distilled water or secondary waste effluent in the presence of subminimal inhibitory concentrations of a variety of heavy metals commonly encountered in the aquatic naturally habitat (Ca2+, Co2+, Cr3+, Cu2+, Hg2+, Ni2+, Zn2+). Micro-ecosystems were exposed to visible light at laboratory temperature and individual strains were reisolated after a 1-, 3-, or 6-month period. The re-isolates (129) were characterized using hierarchical classification analysis in order to define affinities among variants of P. aeruginosa. Subsequently, discriminant analysis was used to detect eventual relationships among the different phenotypical markers studied. Results of the hierarchical classification, based on qualitative or quantitative approaches, showed clearly that incubation of P. aeruginosa in the presence of heavy metals altered the studied phenotypical markers, namely serotype, phage type, MIC of metals, and pyocin type. Discriminant analysis showed that the studied phenotypical markers could be classified into four clusters: C1 (L1 and L2 phage types, Hg tolerance and/or resistance, S2 serotype), C2 (P2 pyocin type, Cd tolerance and/or resistance, S1 serotype), C3 (Co and Cr tolerance and/or resistance) and C4 (P1 pyocin type, Ni, Zn, and Cu tolerance and/or resistance).

Heterogeneity among infecting strains of Pseudomonas aeruginosa in diverse departments of a large Tunisian hospital

Clinical isolates of Pseudomonas aeruginosa were obtained during a half-year screening period of five different wards of the La Rabta Hospital (Tunis). Distinct clinical isolates (N= 82) were obtained from patients, 40 (48%) of which originated from the Department of Otolaryngology. In order to define the local epidemiology of this opportunistic organism, all strains were serotyped, analysed for pyocin production and genetically characterized with the help of pulsed-field gel electrophoresis (PFGE). The data show that, despite the frequent occurrence of identical serotypes, most of the isolates represent unique pyocin types (N= 53) and genotypes (N= 64). A combination of the pyocin and PFGE data showed that nearly all strains were of unique types, except for two pairs of strains. A limited number of strain clusters was observed on the basis of DNA typing data alone. This involved eight genotypes, some of which were clustered with respect to clinical environment or time. Genotype 22 occurred most frequently (6/83, 7%) and independently of time and locale, indicating that it may represent either a clonal type constituting a major fraction of all P. aeruginosa isolates in the region or a more prevalent organism. Despite a relatively high incidence of P. aeruginosa infections, the polyclonality of these strains shows that, in La Rabta Hospital, pseudomonal infections are not primarily due to excessive spread of a single bacterial genotype.

Epidemiologic characterization of Pseudomonas aeruginosa in patients with cystic fibrosis

To determine persistence and variability of colonization with Pseudomonas aeruginosa in cystic fibrosis patients over long time periods, and to look for possible cross-colonization. In total, 469 Pseudomonas aeruginosa isolates were obtained from 30 patients during the period from April 1994 to April 1996. The sources were mainly sputum and a few deep throat swabs. All grown strains dissimilar in macromorphology were processed separately. Typing with PFGE was carried out by contour-clamped homogeneous electric field electrophoresis. Genomic DNA was subjected to the rare-cutting restriction enzyme SpeI. For pyocin typing, the procedure described by Fyfe was applied. After typing with PFGE, we observed 40 restriction profiles. Eighteen different pyocin types were found. The most frequent pyocin type was type 3, followed by types 1 and 5. Twenty-two patients were persistently colonized by one clone specific and different for each patient, and four were co-colonized by a second clone also different for each of these patients. Cross-colonization had apparently been rare in the cystic fibrosis center of Leipzig. Typing with PFGE is well suited for detailed investigations of colonization with Pseudomonas aeruginosa in cystic fibrosis patients. Pyocin typing can provide additional information for epidemiologic purposes.

The use of SDS polyacrylamide gel electrophoresis of periplasmic proteins to subtype Pseudomonas aeruginosa pyocin type 10/b clinical isolates.

The periplasmic protein banding patterns (PPBPs) of thirteen strains of Pseudomonas aeruginosa pyocin type 10/b implicated in two nosocomial outbreaks in the neonatal unit of Rafeidia Hospital, Nablus, Palestine, were examined. In addition, five strains from sporadic cases from the same unit occurring in 1996 and 1997 were also studied. Despite different sources of the strains, PPBPs generated by PAGE suggested a clonal nature of the strains obtained during each of the two outbreaks. Although they had very similar PPBPs, the two outbreak clones were not identical. In contrast, sporadic strains of P. aeruginosa pyocin type 10 appeared to be much more heterogeneous than those of the two outbreaks. PPBP analysis appeared to be a useful tool that may be of value for epidemiological purposes.

EVALUATION OF HOSPITAL ACQUIRED PSEUDOMONAS INFECTION IN PARAPLEGIC AND ORTHOPAEDIC CASES

One hundred isolates of Pseudomonas aeruginosa from patients of the spinal cord injury and orthopaedic centre were evaluated on the basis of 2 laboratory assays; antibiotic sensitivity and pyocin typing. Antibiotic drug resistance was found in 54 per cent of the isolates. The urinary tract, pressure sores and wound swabs were the most common clinical specimens. Spinal cord injury patients showed a high rate of nosocomial colonisation. Ceftazidime, amikacin, norflox and ciprofloxacin were the most active antimicrobial agents. Gentamicin, carbenicillin and tobramycin showed high resistance. Pyocin typing with 22 strains did yield information to establish a clinico - epidemiological relationship.

Multiresistant Pseudomonas aeruginosa serogroup O:11 outbreak in an intensive care unit.

OBJECTIVE: To determine whether 15 multiresistant Pseudomonas aeruginosa isolates from an intensive care unit (ICU) outbreak were related, were endemic, and belonged to the O:12 European clone. METHODS: Forty-six P. aeruginosa isolates from a large hospital were investigated with respect to their antibiotic resistance profiles, serogroups, bacteriocin types and DNA fingerprints obtained by pulsed-field gel electrophoresis (PFGE) of genomic DNA digested with Xbal. RESULTS: Fourteen of the ICU outbreak isolates were indeed identical with respect to their serogroup, O:11, pyocin type, 10/a, and PFGE type, A. Clone A was endemic and dominant throughout the hospital, even though, within the ICU, it underwent phenotypic alterations, such as loss of cell wall lipopolysaccharide side-chains, or acquisition of ceftazidime and imipenem resistance. Bacteriocin typing was more discriminatory than serotyping, but PFGE could differentiate further among phenotypically identical strains. It also allowed the tracking of an O:6 strain, as it was becoming gradually more resistant and undergoing a bacteriocin-type conversion while remaining genotypically unaltered. CONCLUSIONS: Using three typing methods, a nosocomial multiresistant strain distinct from the previously described dominant European O:12 clone was characterized, and the ability of PFGE to identify clonal isolates even when these appear phenotypically distinct was demonstrated.

Susceptibility of Pseudomonas aeruginosa of various pyocin types to the newly synthesized ampicillin derivative, N-(6,7-difluoroquinolonyl)ampicillin.

Six hundred and thirty-two isolates of Pseudomonas aeruginosa of 17 pyocin types were collected in 1993 in Taiwan. Types 1, 10, 3, 35 and 12 were the most common pyocin types identified in Taiwan with isolation frequencies of 47.3%, 24.4%, 7.6%, 3.6% and 2.2%, respectively. Several pyocin subtypes were determined. All pyocin types (one isolate of each tested) were resistant to ampicillin and nalidixic acid, but sensitive to fluoroquinolone antibiotics, such as norfloxacin and enoxacin, indicating that cross-resistance to quinolone antibiotics of nalidixic acid and fluoroquinolone derivatives has not developed. A new ampicillin derivative of 6,7-difluoroquinolonic acid, N-(6,7-difluoroquinolonyl)-ampicillin (AU-1), was synthesized by coupling ampicillin with 6,7-difluoroquinolonic acid (FP-3). Compound AU-1 was much more active than either ampicillin or FP-3 alone against all pyocin types of P. aeruginosa and induced filamentation in most growing cells.

Typing of Pseudomonas aeruginosa strains from patients with cystic fibrosis: phenotyping versus genotyping

OBJECTIVE: To assess the discriminatory power of two genotypic and two phenotypic techniques by analysis of Pseudomonas aeruginosa sputum isolates obtained with long term intervals from 29 independent cystic fibrosis (CF) patients. METHODS: Fifty-eight strains of P. aeruginosa were subjected to serotyping and pyocin production was assessed. Arbitrarily primed polymerase chain reaction (AP PCR) and pulsed-field gel electrophoresis (PFGE) were applied in order to detect genetic polymorphisms. RESULTS: From the results of different typing techniques, it appeared that the numbers of separate types varied between 11 and 43, and the percentage of identical P. aeruginosa pairs from individual patients varied between 51% and 72%, depending on the test system used. AP PCR and PFGE displayed enhanced resolution when compared to serotyping and pyocin typing; both DNA typing techniques generated concordant results, although differences in resolution are apparent. This results in 15% discordance, which may be the result of differences in the definitions of (sub)clonal relatedness as applied for AP PCR and PFGE, respectively. CONCLUSIONS: Molecular typing techniques are superior to phenotyping where P. aeruginosa is concerned. AP PCR is a fast and useful technique for determining clonality among P. aeruginosa strains from chronically colonized CF patients. It is clear, however, that the interpretation of data and comparative analysis of PFGE and AP PCR results necessitates additional (international) standardization and the development of practical guidelines.

Imipenem and cephem resistant Pseudomonas aeruginosa carrying plasmids coding for class B beta-lactamase.

From October 1988 to January 1992, nine isolates of Pseudomonas aeruginosa carrying transferable plasmids encoding imipenem-hydrolyzing beta-lactamase (pI = c. 9.5) were recovered from nine different patients in a neurosurgical ward of a hospital in Japan. The beta-lactamase activities of the sonicated extracts from the transconjugants were inhibited by EDTA and this was partially reversible by the addition of zinc cation. The substrate specificity and pI of the beta-lactamase were similar to those of the metallo beta-lactamases from P. aeruginosa and Serratia marcescens TN9106. All strains were resistant to imipenem, carbenicillin and antipseudomonal cephems including ceftazidime, cefsulodin, cefpirome, while four and five strains were susceptible to piperacillin and aztreonam, respectively. Both low level imipenem resistance and high level cephem resistance were co-transferred with the production of metallo beta-lactamase, while resistance to piperacillin, aztreonam, and high level imipenem-resistance were not selected. Production of chromosomal cephalosporinase in piperacillin resistant strains was derepressed, and production of outer membrane protein of D2 was diminished in highly imipenem resistant strains. Six strains were isolated in 1991, and the amounts of antipseudomonal agents, especially imipenem, used in the neurosurgical ward increased markedly in this year. Only three of the nine isolates had the same serotype, pyocin type and phage type. Our results suggest that the repeated isolation of imipenem and cephem-resistant P. aeruginosa producing metallo beta-lactamase was related to the high usage of antipseudomonal beta-lactam antibiotics such as imipenem, and was exacerbated by the dissemination of a plasmid.