A highly discriminatory multi-typing scheme for Proteus mirabilis and Proteus vulgaris

Strains of Proteus mirabilis and P. vulgaris isolated in England, Scotland and Sweden were characterised by proticine production-proticine sensitivity (P-S) typing, O serotyping and Dienes typing methods. The determinants of O antigenicity were independent of those determining proticine production and proticine sensitivity. Because of this independence, the combination of P-S typing and O serotyping for the analysis of the 133 serotypable strains separated them into 81 distinct types whereas P-S typing and O serotyping methods alone separated them into only 56 and 19 types respectively. There was a relationship between the Dienes type and the P-S type; the determinants of Dienes compatibility were the proticine production-proticine sensitivity characters. The determinants of O antigenicity appeared to play no role in the Dienes reaction. Some strains that were indistinguishable by P-S typing and O serotyping methods were distinguished by Dienes typing.     

Bacteriocine typing of Proteus.

A method of typing isolates of Proteus mirabilis and Proteus vulgaris is described based on the sensitivity of the organisms to bacteriocine. Twelve standard proteocine producing strains were selected from a large number of isolates tested, and from these liquid proteocine preparations were prepared. The sensitivities of 1805 isolates to these 12 preparations were then determined and none was found to be untypable. From the results a bacteriocine typing system has been developed.     

An evaluation of two methods of bacteriocine typing of organisms of the genus Proteus.

An assessment of two simple methods of typing Proteus isolates using bacteriocines is described. The methods chosen were those of Cradock-Watson and Al-Jumaili. It has been shown that the use of liquid bacteriocine preparations is more satisfactory than methods involving proteocine production in solid media. For example, no non-typable isolates were encountered and a large number of strains were demonstrated. This method is therefore proposed for use in a routine diagnostic laboratory.     

Resistotyping of Proteus mirabilis and a comparison with other methods of typing

Resistotyping of P. mirabilis using 10 compounds is reported. The method was tested for reproducibility and specificity and results were compared with those obtained by serological, bacteriophage, and proticine typing methods and the Dienes test. The possible relationship between resistance to the chemicals used in the test and antibiotics was also studied.The method was found to be simple, reproducible, show good specificity, and compare favourably with other typing methods. No linkage between resistance to the chemicals and antibiotic resistance was detected.     

Modification of dienes mutual inhibition test for epidemiological characterization of Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa is an important cause of community-associated and nosocomial infections related to exposure to aqueous environments. Such infections often occur in the setting of a common-source outbreak, in which case epidemiological characterization of isolates may be necessary. In this preliminary study, a modification of the Dienes mutual inhibition test, ordinarily used to assess the relatedness of swarming Proteus mirabilis strains, was used to study 15 P. aeruginosa isolates, with the results compared to those obtained by ribotype analysis. Complete concordance was noted between the results of the Dienes test and those of ribotyping. These observations suggest that further studies are warranted to assess the utility of the modified Dienes test as a simple, inexpensive, and reliable means for epidemiological typing of P. aeruginosa.     

Pyocine-typing of hospital strains of Pseudomonas pyocyanea

A method of typing Pseudomonas pyocyanea is described which makes use of the property of bacteriocine production. The method was applied in an epidemiological study of Ps. pyocyanea infection in a general hospital. It was found that the majority of strains believed on epidemiological grounds to be of common origin were allotted to the same pyocine type.     

Differentiation of Proteus mirabilis by bacteriophage typing and the Dienes reaction.

A provisional typing schema based on sensitivity to 23 bacteriophages has been established for Proteus mirabilis. Seventy-three bacteriophages were isolated on strains of P. mirabilis (64), P. vulgaris (1), P. morganii (7), and P. rettgeri (1), but those isolated on P. mirabilis were the most useful in differentiating other strains of . mirabilis. From the 73 phages studied, the best 23 were chosen by computer analysis for the provisional system, which was then used to study P. mirabilis infections in a 500-bed general hospital. All patient isolates for 19 months were saved and then compared by bacteriophage typing and the Dienes reaction in a retrospective study. There was evidence for only three instances of cross-infection or -colonization during this time. Bacteriophage typing was very sensitive in differentiating strains, since 200 strains were differentiated into 113 different lysis patterns and 94% were typable. The Dienes reaction was useful at times but often gave reactions that were difficult to read or that changed when the tests were repeated. The bacteriophages described by Schmidt and Jeffries were also evaluated and proved useful in combination with ours. The value of bacteriophage typing was clearly established, and work toward a standardized schema for P. mirabilis should continue.     

Evaluation of the discriminatory powers of the Dienes test and ribotyping as typing methods for Proteus mirabilis

A total of 63 clinical isolates of Proteus mirabilis collected over a 19-month period were typed by the Dienes test and ribotyping. Ribotyping was performed using the fully automated RiboPrinter Microbial Characterization System (Qualicon, Wilmington, Del.). Isolates that were indistinguishable by the Dienes test and/or ribotyping were characterized further by pulsed-field gel electrophoresis (PFGE). Most of the isolates represented unique strains as judged by the Dienes test and ribotyping. Forty isolates represented 40 different ribotypes and Dienes types. The remaining 23 isolates were grouped into 13 Dienes types, 12 ribotypes, and 14 PFGE types. The index of discrimination was 0.980 for the Dienes test, 0.979 for ribotyping, and 0.992 for PFGE. Both the Dienes test and ribotyping are useful methods for identifying individual strains of P. mirabilis. The Dienes test is simple, inexpensive, and easy to perform. It can be performed in virtually any laboratory and should be used in the initial epidemiologic characterization of P. mirabilis isolates.     

Immunogenetics of the HLA system

The study of the HLA system was primarily initiated to understand the basis for the histocompatibility between recipients and tissue donors. HLA typing methods are being continuously improved and biochemical and molecular typing, in particular, are expected to provide precise typing of the HLA system. Conventional HLA typing methods can define antigen specificities, while biochemical and molecular methods will provide direct allele typing that is based on the actual sequence polymorphism. The precise tissue typing will definitely improve the outcome of transplantation. Structural studies have revealed the highly polymorphic nature of the HLA system and given insight to understanding the molecular basis of the HLA polymorphism. One big immunological puzzle remaining to be answered is how T-cell receptor molecules recognize peptide antigen in conjunction with the HLA molecule. The crystallization of the T-cell receptor molecule, an experiment currently underway, will eventually reveal the structural basis of the trimolecular interaction.     

Molecular epidemiology of Proteus mirabilis infections of the catheterized urinary tract

Proteus mirabilis compromises the care of many patients undergoing long-term indwelling bladder catheterization. It forms crystalline bacterial biofilms in catheters which block the flow of urine, causing either incontinence due to leakage or painful distention of the bladder due to urinary retention. If it is not dealt with, catheter blockage can lead to pyelonephritis and septicemia. We have examined the epidemiology of catheter-associated P. mirabilis infections by use of pulsed-field gel electrophoresis (PFGE) of NotI restriction enzyme digests of bacterial DNA. This technique was shown to be more discriminatory than the classical phenotypic Dienes typing technique. We demonstrated that each of 42 isolates from diverse environmental sources and 10 of 12 isolates from blood, wound swabs, and mid-stream urine samples of hospitalized patients had distinct genotypes. Examination of a set of 55 isolates of P. mirabilis, each from a different clinical or environmental source, identified 49 distinct genotypes and 43 Dienes types. The index of discrimination was 0.993 for the PFGE method and 0.988 for the Dienes method. Applying the PFGE method to isolates from catheter-associated urinary tract infections confirmed that the strains present in the crystalline catheter biofilms were identical to those isolated from the same patient's urine. An analysis of samples taken during a prospective study of infections in catheterized nursing home patients revealed that a single genotype of P. mirabilis can persist in the urinary tract despite many changes of catheter, periods of noncatheterization, and antibiotic therapy.     

Comparison of epidemiological marker methods for identification of Salmonella typhimurium isolates from an outbreak caused by contaminated chocolate.

Plasmid profile analysis, restriction endonuclease analysis, and multilocus enzyme electrophoresis were used in conjunction with serotyping, bacteriophage typing, and biochemical fingerprinting to trace epidemiologically related isolates of Salmonella typhimurium from an outbreak caused by contaminated chocolate products in Norway and Finland. To evaluate the efficiency of the epidemiological marker methods, isolates from the outbreak were compared with five groups of control isolates not known to be associated with the outbreak. Both plasmid profile analysis and phage typing provided further discrimination over that produced by serotyping and biochemical fingerprinting. Plasmid profile analysis and phage typing were equally reliable in differentiating the outbreak isolates from the epidemiologically unrelated controls and were significantly more effective than multilocus enzyme electrophoresis and restriction enzyme analysis of total DNA. The greatest differentiation was achieved when plasmid profile analysis and phage typing were combined to complement serotyping and biochemical fingerprinting. However, none of the methods employed, including restriction enzyme analysis of plasmid DNA, were able to distinguish the outbreak isolates from five isolates recovered in Norway and Finland over a period of years from dead passerine birds and a calf.     

A critical review of typing methods for Candida albicans and their applications.

During the 1980s, a large number of typing methods for the strain differentiation of Candida albicans were described in the literature. Although these methods have been based on a variety of physiological and genetic markers, none is ideal. This review discusses the characteristics of an ideal typing method in terms of its typability, reproducibility, and discriminatory power. Ways of determining these characteristics are presented so that the available typing methods for Candida albicans can be objectively compared. Available typing methods for C. albicans include serotyping, morphotyping, resistotyping, biotyping, and killer yeast typing. Electrophoretic methods include immunoblotting, isoenzyme analysis, analysis of DNA restriction fragment length polymorphism, karyotyping, and the use of DNA probes. The application of these methods to epidemiological research, the investigation of outbreaks of disease, and the study of virulence is described. The potential impact of the phenomenon of phenotypic switching on the reproducibility of these typing methods is discussed. It is concluded that many of the available typing methods have not been adequately assessed by their developers and that several have only poor discriminatory power or reproducibility.     

The association of particular types of Proteus with chronic suppurative otitis media

During a period of 12 months, 57 strains of Proteus were isolated from the ears of 38 unrelated patients with chronic suppurative otitis media. Each strain was identified, typed for bacteriocin production and sensitivity, and tested for Dienes compatibility. The majority of the strains (79%) were P. mirabilis; all but one of the remainder were P.vulgaris. Although 19 different bacteriocin production/sensitivity types were found, two rare types, P. mirabilis P7/S5,12 and P. vulgaris P0/S9, were associated with 47% of these infections. This was confirmed by Dienes typing. Patients with bilateral ear disease carried a different strain in each ear. There was no evidence that persistence of infection had arisen because of the development of antibiotic resistance. Although there was some evidence that persistence may have been the result of reinfection, the isolation of these rare types of Proteus from so many patients with chronic suppurative otitis media may indicate that they play an important role in the pathogenesis of the disease. Most of the Proteus isolates were of "non-faecal" types and it is believed that infection had arisen by a route other than the faecal-aural one.     

Evaluation of the PhP system for biochemical-fingerprint typing of strains of Salmonella of serotype Typhimurium.

The Phene Plate (PhP) system of biochemical fingerprinting of bacteria is a computerised typing system, based on quantitative measurements of the kinetics of several biochemical reactions of bacteria grown in liquid medium in microtitration plates. For each isolate tested, it yields a biochemical fingerprint comprising several kinds of quantitative data which are useful for establishing similarities among strains with a personal-computer program. In this study, a set of 16 specific substrates was chosen to differentiate strains of Salmonella of serotype Typhimurium. The system was evaluated for its typability, reproducibility and discriminatory power in tests with a collection of 100 epidemiologically unrelated Typhimurium strains and results were compared with those obtained by phage typing. At an identity level of 0.980, strains were assigned by this method to 51 biochemical phenotypes (BPTs), giving a diversity index of 0.963 and a resolution index of 0.210. In contrast, 24 phage types (PTs) were identified among these isolates (a diversity index of 0.901). The combined use of biochemical fingerprinting by the PhP system and phage typing discriminated 82 phenotypes (a diversity index of 0.994). Stability of markers in each of the methods was also evaluated after subculture of 20 strains for 21 consecutive days. Only nine biochemical reactions were found that were subject to small, but measurable, changes for at least one isolate. These changes slightly decreased the mean similarity coefficients among strains but the overall BPTs of the strains showed changes in four strains (20%). In contrast, eight strains (40%) showed changes in their PTs after this treatment. It is concluded that the PhP system is a highly discriminatory and reproducible method for typing Typhimurium strains. It is easy to perform, and may be used alone or in combination with phage typing in epidemiological studies of Typhimurium strains.     

A Critical Review of Typing Methods for Candida albicans and Their Applications

Abstract

During the 1980s, a large number of typing methods for the strain differentiation of Candida albicans were described in the literature. Although these methods have been based on a variety of physiological and genetic markers, none is ideal. This review discusses the characteristics of an ideal typing method in terms of its typability, reproducibility, and discriminatory power. Ways of determining these characteristics are presented so that the available typing methods for Candida albicans can be objectively compared. Available typing methods for C. albicans include serotyping, morphotyping, resis-totyping, biotyping, and killer yeast typing. Electrophoretic methods include immunoblotting, isoenzyme analysis, analysis of DNA restriction fragment length polymorphism, karyotyping, and the use of DNA probes. The application of these methods to epidemiological research, the investigation of outbreaks of disease, and die study of virulence is described. The potential impact of the phenomenon of phenotypic switching on the reproducibility of these typing methods is discussed. It is concluded that many of the available typing methods have not been adequately assessed by their developers and that several have only poor discriminatory power or reproducibility.     

Epidemiologic typing of international collections of Klebsiella spp.: computerized biochemical fingerprinting compared with serotyping,phage typing and pulsed-field gel electrophoresis

ObjectiveThe Phene Plate (PhP) biochemical fingerprinting system is based on measurements of the kinetics of selected biochemical reactions performed in microtiter plates, and computerized data-processing. This study compared the performance of the PhP system as an epidemiologic tool with other commonly used typing systems.MethodsPhP typing was applied to 107 nosocomial Klebsiella spp. isolates from 10 collections, mostly representing outbreaks. The results were compared with those obtained by capsular (K) serotyping, phage typing and, for a subset of isolates (n=33), pulsed-field gel electrophoresis (PFGE).ResultsClusters of identical or closely related isolates based on serotype, phage type, PhP type and PFGE type were found in most collections. The typeability was 100%, 95%, 94% and 68% for PhP, K, PFGE and phage typing, respectively. The agreement between the typing methods was high (88–96%). The discriminatory power was high for PhP and PFGE (diversity index 0.95 and 0.97, respectively), but lower for phage typing (diversity index 0.91) and K typing (diversity index 0.87).ConclusionsLike serotyping and PFGE, PhP typing is useful in studies of the nosocomial epidemiology of Klebsiella spp. Combining PhP typing with PFGE or K typing rarely yielded additional information when comparing isolates within each collection, but PFGE sometimes discriminated between isolates of similar PhP type derived from different collections.     

Two-laboratory collaborative study on identification of mycobacteria: molecular versus phenotypic methods.

Previous studies have indicated that the conventional tests used for the identification of mycobacteria may (i) frequently result in erroneous identification and (ii) underestimate the diversity within the genus Mycobacterium. To address this issue in a more systematic fashion, a study comparing phenotypic and molecular methods for the identification of mycobacteria was initiated. Focus was given to isolates which were difficult to identify to species level and which yielded inconclusive results by conventional tests performed under day-to-day routine laboratory conditions. Traditional methods included growth rate, colonial morphology, pigmentation, biochemical profiles, and gas-liquid chromatography of short-chain fatty acids. Molecular identification was done by PCR-mediated partial sequence analysis of the gene encoding the 16S rRNA. A total of 34 isolates was included in this study; 13 of the isolates corresponded to established species, and 21 isolates corresponded to previously uncharacterized taxa. For five isolates, phenotypic and molecular analyses gave identical results. For five isolates, minor discrepancies were present; four isolates remained unidentified after biochemical testing. For 20 isolates, major discrepancies between traditional and molecular typing methods were observed. Retrospective analysis of the data revealed that the discrepant results were without exception due to erroneous biochemical test results or interpretations. In particular, phenotypic identification schemes were compromised with regard to the recognition of previously undescribed taxa. We conclude that molecular typing by 16S rRNA sequence determination is not only more rapid (12 to 36 h versus 4 to 8 weeks) but also more accurate than traditional typing.     

Biochemical fingerprinting compared with ribotyping and pulsed-field gel electrophoresis of DNA for epidemiological typing of enterococci.

The Phene Plate (PhP) biochemical fingerprinting system for bacteria is based on measurements of the kinetics of bacterial biochemical reactions. This system was modified for typing of enterococci and was compared with DNA typing by pulsed-field gel electrophoresis and with ribotyping by using 45 Enterococcus faecalis isolates from international collections. It was also used to study 170 fecal enterococcal isolates from healthy individuals and 28 isolates of E. faecalis from the blood of neonates. The PhP system showed a high degree of discriminatory power for unrelated enterococcal isolates. Among the 170 unrelated fecal isolates, 107 isolates from international collections, PhP typing discriminated 19 types, and ribotyping discriminated 5 types. In most cases, when isolates were of the same DNA type, they were also of the same PhP type, and the level of agreement between these two methods was high (96%). A combination of PhP typing and DNA typing identified 34 different types, but ribotyping did not yield any further discrimination. PhP typing of E. faecalis isolates from healthy individuals (n = 89) and from the blood of neonates with septicemia (n = 28) yielded a diversity of 0.93 for both populations and similar major PhP types in both populations. Thus, the isolates from blood seemed to consist of a normal E. faecalis population, without a dominance of certain strains associated with virulence. We conclude that the PhP system is useful for epidemiological studies of enterococcal isolates, yielding results similar to those obtained with DNA typing by pulsed-field gel electrophoresis. Since PhP typing is a method that is simple and rapid and that is based on automatic evaluation of the data, it is suitable for analyzing large numbers of isolates and can be used alone or in combination with DNA typing or epidemiological and ecological studies of enterococci.     

Variation in metabolism of biochemical test substrates by Klebsiella species: an epidemiological tool.

The variation in metabolism of biochemical test substrates by klebsiella isolates has been demonstrated. It is suggested that this variation is likely to result in false-negative reactions in biochemical tests incubated for short periods. The observations made may explain the reported difficulties in obtaining reproducible results in biotyping Klebsiella strains. Preliminary work suggests that differences in substrate metabolism will provide a means of increasing the sensitivity of methods for the biochemical typing of Klebsiella spp.     

Serotyping and the Dienes reaction on Proteus mirabilis from hospital infections

The serotype of 320 strains of Proteus mirabilis from clinical material was determined. Using 20 O antisera and four H antisera 61% of strains could be fully identified and 90% partially identified. A large number of serotypes were recognized but no difference was found between the serotype of organisms infecting the urinary tract and those from other infections. Biochemically identical organisms found in the same ward generally differed in serology. Proteus mirabilis was isolated from the faeces of 84.5% of 84 patients with urinary infection and from none of 20 normal controls. By serology and the Dienes test 61% of the organisms isolated from the urine and faeces of a single patient were identical, indicating that infection arose from the intestine.Most groups of serologically identical strains could, by the Dienes test, be further divided into a number of subtypes indicating that the strains were different and that cross infection had not been responsible for their spread. With three serological groups, however, the majority of strains belonged to a single Dienes type and it was concluded that these organisms had been spread from a common reservoir or carrier.Because of the unreliability of the Dienes test when carried out on random organisms it is suggested that reliable results can only be obtained by combining the Dienes test with serotyping.