How and why to monitor Pseudomonas aeruginosa infections in the long term at a cystic fibrosis centre.

BACKGROUND: Pseudomonas aeruginosa is a major cystic fibrosis (CF) pathogen causing chronic respiratory infections and posing a risk for cross-infection between patients with CF. AIM: To propose an algorithm for long-term surveillance of P. aeruginosa and assess its suitability for monitoring the epidemiological situation at a CF centre with approximately 300 patients. METHODS: Over a nine-year period, over 300 P. aeruginosa isolates from 131 infected patients were tested by multi-locus sequence typing (MLST) and/or random amplified polymorphic DNA (RAPD) assay. FINDINGS: MLST analysis led to the identification of 97 different sequence types which were distributed among 17 RAPD-generated (pseudo)clusters. This indicates that the easy-to-perform RAPD assay is only suitable for intra-individual, not interindividual, strain analyses. No epidemic strains were observed. Longitudinal analysis revealed that 110 of the 131 patients were infected with the same strain over the observation period, whereas 21 patients had a strain replacement or a new infection. Chronic infection was found in 99 of the 131 patients, and the remaining 32 patients met the criteria for intermittent infection (as defined by the Leeds criteria). Eighteen of the 32 patients (56%) with intermittent infection were infected with the same strain for up to nine years. CONCLUSION: The strain type only changed in 16% of 131 patients with chronic or intermittent infection. As many as 56% of patients considered to have intermittent infection were actually chronically infected with the same strain for many years.

[Small colony variants Staphylococcus aureus]. / Trpaslicí kmeny--small colony variants Staphylococcus aureus.

The phenomenon of dwarf colonies of S. aureus, the so-called small colony variants (SCVs), is associated with chronic and recurrent staphylococcal infections. Most frequently, these phenotypic variants differ from normal strains of S. aureus in colony size, morphology, pigmentation and other characteristics as well as molecular genetic changes. SCVs frequently emerge as a result of mutations in metabolically important and regulatory genes. The mutations are a cause of SCVs auxotrophy. From a clinical point of view, an increased ability of SCVs to resist antibiotic therapy and also an ability to persist within eukaryotic host cells are of importance.

Small colony variants of Staphylococcus aureus--review.

Bacterial variants of Staphylococcus aureus called small colony variants (SCVs) originate by mutations in metabolic genes, resulting in emergence of auxotrophic bacterial subpopulations. These variants are not particularly virulent but are able to persist viable inside host cells. SCVs show their characteristic auxotrophic growth deficiency and depressed α-cytotoxin activity. Environmental pressure such as antibiotics, select for isogenic SCV cells that are frequently found coexisting with their parent wild-type strains in a mixed bacterial culture. SCV strains often grow on blood agar as non-pigmented or pinpoint pigmented colonies and their key biochemical tests are often non-reactive. Their altered metabolism or auxotrophism can result in long generation time and thus SCV phenotype, more often than not SCV can be overgrown by their wild-type counterparts and other competitive respiratory flora. This could affect laboratory detection. Thus, molecular methods, such as 16S rRNA partial sequencing or amplification of species-specific DNA targets (e.g. coagulase, nuclease) directly from clinical material or isolated bacterial colonies, become the method of choice. Patients at risk of infection by S. aureus SCVs include cystic fibrosis patients (CF), patients with skin and foreign-body related infections and osteomyelitis, as they suffer from chronic staphylococcal infections and are subject to long-term antibiotic therapy. Molecular evidence of SCV development has not been found except for some random mutations of the thymidylate synthase gene (thyA) described in SCV S. aureus strains of CF patients. These variants are able to bypass the antibiotic effect of folic acid antagonists such as sulfonamides and trimethoprim. Resistance to gentamicin and aminoglycosides in the hemin or menadione auxotrophic SCVs was hypothesized as being due to decreased influx of the drugs into cells as a result of decreased ATP production and decreased electrochemical gradient on cell membranes.

Delineation of the endemic and sporadic methicillin-resistant Staphylococcus aureus clones in a Czech hospital.

The aim of this study was to define the endemic clones of methicillin-resistant Staphylococcus aureus (MRSA) among strains collected between September, 2001, and February, 2003, at the regional hospital of Nový Jicín, Czech Republic. The isolates were characterized by susceptibility tests, HindIII ribotyping, and pulsed-field gel electrophoresis. Representatives of each clonal type were analyzed by multilocus sequence typing and staphylococcal cassette chromosome mec (SCCmec) typing. The prevalence of the most important macrolide (ermA, ermB, ermC, and msrA) and aminoglycoside (aac6'-aph2", aph3', and ant4') resistance genes was evaluated as well. Our results document the existence of two international MRSA clones: (1) the Iberian clone (ST247:SCCmec IA:PFGE A:ribotype H2), endemic in the hospital and associated to a single multiresistant phenotype; and (2) clone EMRSA-15 (ST22:SCCmec IV:PFGE H-ribotype H7), appearing in the beginning of 2002 and associated with three phenotypes. These two clones could be distinguished by antibiogram, distribution of macrolide and aminoglycoside resistance genes (ermA, aac6'-aph2", ant4' versus ermC and msrA in a few isolates), production of beta-lactamase, and presence of enterotoxin A (in the Iberian clone).

[Detection of MRSA in a group of 752 strains of S. aureus using a cefoxitin disk]. / Odlisení MRSA v souboru 752 kmenu S. aureus pomocí disku s cefoxitinem.

A set of 752 S. aureus strains including 665 fresh clinical isolates, 82 collection strains from the NRL for staphylococci and three control strains for external quality assessment were tested for susceptibility to oxacillin by three routine phenotypic methods with oxacillin (agar screening method, dilution micromethod and disk diffusion method) and a new method with a 30 micrograms cefoxitin disk. Gene mecA coding for oxacillin resistance was detected by PCR, PBP2a gene product was detected by latex agglutination. All of 218 oxacillin resistant strains--MRSA (methicillin resistant S. aureus)--gave inhibition zones of 6-19 mm around the cefoxitin disk, i.e. zones within the range set up for oxacillin resistant strains, eight out of these strains showing false oxacillin susceptibility in one or more phenotypic tests. It can be stated that the presence of an inhibition zone of < 20 mm around the 30 micrograms cefoxitin disk allows for reliable differentiation between MRSA and oxacillin susceptible S. aureus.

Update on the major clonal types of methicillin-resistant Staphylococcus aureus in the Czech Republic.

The purpose of our study was the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 21 hospitals in the Czech Republic in the period 2000-2002 and comparison with previous results from 1996-1997. Strains were analyzed by pulsed-field gel electrophoresis (PFGE) of SmaI digests and ribotyping of HindIII digests hybridized with a 16S-23S DNA probe. The prevalence of the most clinically important macrolide (ermA, ermB, ermC, and msrA) and aminoglycoside (aph3', ant4', and aac6'-aph2") resistance genes was evaluated as well. Selected isolates representative of each clonal type were analyzed by multilocus sequence typing and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec) carried by the bacteria. Our results document the displacement of the Brazilian clone (ST239, SCCmec type IIIA, PFGE type B, ribotype H1) by a new clone that we named "Czech clone" (ST239, SCCmec type IIIA, PFGE type F, ribotype H6) and the maintenance of the Iberian clone (ST247, SCCmec type IA, PFGE type A, ribotype H2) exclusively in one hospital in the Czech Republic. In addition, we found a correlation between the distribution of aminoglycoside resistance genes and MRSA clonal types.

Detection and characterization of feline Bartonella henselae in the Czech Republic.

The aims of the study were to characterize isolates of Bartonella henselae and to determine the prevalence of bacteremic domestic cats in urban and suburban parts of Prague, Czech Republic. Five (18%) gram-negative fastidious bacterial single-cat isolates were recovered from 27 hemocultures incubated without previous freezing. Four of these isolates originated from flea infested stray cats (n=6) and one from a shelter cat without any ectoparasites (n=21). None of the 34 previously frozen specimens from flea free pet cats yielded any bacteria. All five isolates were catalase and oxidase negative. Their enzymatic activity, RFLP profile of citrate synthetase gene (gltA) and DNA-DNA hybridization results were typical of B. henselae. According to their PvuII and BglI ribotypes the isolates could be allocated to two homogeneous groups. Ribotype HindIII and RFLP of 16S-23S rRNA spacer region analysis gave unique profiles different from those of Bartonella quintana, Bartonella elizabethae and Bartonella clarridgeiae. The 16S rRNA type-specific amplification revealed an identical profile typical of B. henselae genotype II for all the cat isolates studied. Pulsed-field gel electrophoresis (PFGE) assigned a different profile to each of the isolates studied. Determination of the enzymatic activity, RFLP of gltA gene, RFLP of 16S-23S rRNA spacer region, and HindIII ribotype could be efficient tools for identification of B. henselae isolates. Ribotyping (PvuII, BglI), 16S rRNA typing and PFGE may be useful methods to prospect ecology and epidemiology of the agent.

In situ detection of Bartonella henselae cells.

A new in situ hybridization technique was developed for identification of Bartonella henselae cells in cell suspension or in tissue sections. Use of highly specific probes labeled with either fluorescein or digoxigenin allows discrimination between B. henselae and closely related B. quintana cells. No cross-hybridization with other Bartonella or non-Bartonella species was observed. Besides its specificity it showed higher sensitivity as compared to PCR based detection methods. Moreover, its application allows direct observation of B. henselae in infected tissues.

Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains.

Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus--MRSA--clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.

Methicillin-resistant Staphylococcus aureus clonal types in the Czech Republic.

Molecular surveillance studies have documented the extensive spread of methicillin-resistant Staphylococcus aureus (MRSA) clones. Studies carried out by Centro de Epidemiologia Molecular-Network for Tracking Gram-Positive Pathogenic Bacteria (CEM/NET) led to the identification of two international multidrug-resistant strains, which were designated as the Iberian and Brazilian MRSA clones and which were defined by multiple genomic typing methods; these included ClaI restriction digests hybridized with mecA- and Tn554-specific DNA probes and pulsed-field gel electrophoresis (PFGE). The genotypic characteristics of these clones are distinct: the Iberian clone is defined as mecA type I, Tn554 type E (or its variants), and PFGE pattern A (I:E:A), whereas the Brazilian clone is defined as mecA type XI (or its variants), Tn554 type B, and PFGE pattern B (XI:B:B). In this study, we characterized 59 single-patient isolates of MRSA collected during 1996 and 1997 at seven hospitals located in Prague and five other cities in the Czech Republic by using the methodologies mentioned above and by using ribotyping of EcoRI and HindIII digests hybridized with a 16S-23S DNA probe. The Brazilian MRSA clone (XI:B:B) was the major clone (80%) spread in two hospitals located in Prague and one located in Brno; the Iberian MRSA clone (I:E:A or its variant I:DD:A), although less representative (12%), was detected in two hospitals, one in Prague and the other in Plzen. Almost all the strains belonging to clone XI:B:B (45 of 47) corresponded to a unique ribotype, E1H1, whereas most strains of the I:E:A and I:DD:A clonal types (6 of 7) corresponded to ribotype E2H2.

Genotypic and phenotypic similarity of multiresistant Acinetobacter baumannii isolates in the Czech Republic.

The diversity of 103 clinical isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex obtained between 1991 and 1997 from 17 Czech hospitals was studied by ribotyping, biotyping, plasmid profiling and antibiotic susceptibility testing. According to the EcoRI ribotypes, all but one of these isolates were identified to the DNA group level: 77 isolates were allocated to DNA group 2 (A. baumannii), 14 to DNA group 3, 10 to DNA group 13 sensu Tjernberg and Ursing and one to DNA group 1 (A. calcoaceticus). In total, 50 different EcoRI ribotypes and 10 biotypes were observed. Plasmids were found in 92% of the isolates and a high variability in plasmid profiles was found in isolates of the same DNA group. The combination of typing profiles allowed two predominant groups (termed A and B) to be distinguished among the A. baumannii isolates (37 and eight isolates, respectively) that shared a specific ribotype and were highly similar in other properties. These two groups comprised both sporadic and outbreak isolates and were found in most localities. Group A and B isolates were markedly more resistant to antibiotics than most of the remaining isolates, thus representing 85% of all multiresistant isolates. The features of groups A and B corresponded to those of two epidemic clones identified recently among hospital strains in north-western Europe.

Plesiomonas shigelloides in water and fish.

Plesiomonas shigelloides strains isolated from aquariums, surface water and fish were serotyped using the international antigenic scheme and a provisional scheme designed for environmental strains, called the "Schubert" scheme. A remarkable variety of serovars represented in both schemes was found, namely in surface water samples and in fish.

[Identification and typing of hospital strains of Acinetobacter calcoaceticus-Acinetobacter baumanni complex]. / Identifikace a typizace nemocnicních kmenu komplexu Acinetobacter calcoaceticus-Acinetobacter baumannii.

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.