Purification of the bacteriophage T4 type II DNA topoisomerase.
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Bacteriophage T4↗
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Use of a ribosomal RNA gene probe for the epidemiological study of methicillin and ciprofloxacin resistant Staphylococcus aureus.
Conventional bacteriophage typing was combined with ribotyping in the analysis of methicillin and ciprofloxacin resistant Staphylococcus aureus strains isolated in increasing frequency since the introduction of the new 4-quinolones as therapeutic agents in the Tel-Aviv Medical Center. Whole-cell DNA was digested with EcoRI and HindIII restriction endonucleases. Agarose gel electrophoresis, Southern blotting, and hybridization by biotinylated probe DNA coding for ribosomal RNA revealed 7 to 14 bands. Analysis of the patterns established a single DNA type in EcoRI as well as in HindIII digests for all strains except one. Control strains from other sources differed in their band patterns. Bacteriophage typing confirmed the results of DNA typing. Thus, the frequent occurrence of staphylococcal isolates resistant to 4-quinolones in the hospital was not due to mutational development of resistance in many strains, but to the spread of a resistant strain.
Anti-Infective Agents↗
STATEMENT on availability and uses of staphlococcal phage typing.
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Bacteriophage Typing↗
[Analysis of the plasmid profile of various Salmonella serotypes].
INTRODUCTION: Every year foodborne infections cause millions of illnesses but many of them go undiagnosed and unreported. The epidemiology of these illnesses is changing, new pathogens have emerged (Escherichia coli O157:H7, Cyclospora cayetanensis, Vibrio vulnificus). Salmonella spp. is the most common bacterial cause of acute enterocolitis with us. All over the world, as well as in our country the most often isolated serotype is Salmonella Enteritidis. A great problem in many countries is the multiresistant Salmonella Typhimurium, as well as other serotypes resistant to a great number of antimicrobial drugs (S. Hadar, S. Typhi). Clinical microbiologists are often asked to determine the relatedness of bacterial isolates. Recently, traditional methods of strain typing such as bacteriophage typing, resistotyping and serotyping, have been supplemented or replaced in many laboratories with newer molecular methods such as plasmid fingerprinting, ribotyping. PCR-based methods, etc. The goal of strain typing is to provide evidence that epidemiologically related isolates collected during an outbreak are also genetically related and thus represent the same strain. MATERIAL AND METHODS: In the laboratory for Enterobacteriaceae of the Institute of Public Health Novi Sad in the four year period (1995-1998) 3659 primary isolates of Salmonella spp. were isolated using standard bacteriological methods (cultural, biochemical and serological examination). For certain strain of Salmonella Enteritidis, S. Typhimurium and S. Hadar susceptibility to antimicrobial agents was tested by disc-diffusion test (Kirby-Bauer) and plasmid profiles were analyzed. Plasmid DNA was extracted by Birnboim and Doly alkaline lysis method and plasmid bands were separated by electrophoresis in agarose gel. RESULTS: In the period of 1995-1998 the most common serotype isolated was Salmonella Enteritidis with 3017 (82.5%) of the total number of isolated Salmonellas; S. Typhimurium 203 (5.5%), S. Hadar 118 (3.2%). Plasmid profiles were tested in 10 S. Enteritidis isolates that originated from patients with sporadic cases of diarrhea. All investigated strains had one plasmid band with molecular weight of 38 MDa. All isolates were susceptible to all antibiotics tested. Ten isolates of S. Hadar originated from one outbreak from food samples and stools of patients with diarrheal disease and from the worker in the restaurant. All isolates were resistant to ampicillin and streptomycin, and plasmid profile analysis showed 5 plasmid bands with molecular weights of 13, 5.4, 4.2, 2.0 and 1.7 MDa. Chosen strains of S. Typhimurim were not epidemically related. Strains number 1, 3, 4, 5 were susceptible to all antibiotics tested, and had only one plasmid of 50 MDa, strain number 7 was resistant to streptomycin and had 2 plasmid bands of 50 and 1.7 MDa, other strains were multiresistant and had different plasmid profiles with 4-7 plasmid bands with molecular weights ranging from 50-1.4 MDa. CONCLUSION: Plasmid profile analysis is not a sufficient method for examination of Salmonella Enteritidis which is the most common cause of enterocolitis with us. It is, however, a helpful method for proving epidemiological and clonal relatedness of Salmonella isolates that are resistant to antimicrobial agents and have a great number of plasmids (such as some strains of S. Typhimurim and S. Hadar).
Enterocolitis↗
[Determination of the serological type of Pasteurella pseudotuberculosis cultures using type Pasteurella pseudotuberculosis bacteriophages].
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Bacteriophage Typing↗
Automation of Salmonella typhi phage typing.
Two steps in the procedure for bacteriophage typing of Salmonella typhi have been automated. The culture inoculum was applied by flooding the surface of phage agar in a 150x20 mm petri dish and removing the excess liquid with a safety pipettor. This step replaced the older method of manually preparing up to 100 individual areas for inoculation. The number of bacteria per unit area was the same with both methods, so the automated method involved no change in the technique. The second automated step involved use of a bacteriophage applicator which simultaneously dispensed 59 uniform drops of bacteriophages onto the inoculated plate. The automated procedure has reduced personnel time by about 90% with no less in sensitivity or accuracy.
Automation↗
Effect of extracellular protease production on bacteriophage sensitivity of Vibrio cholerae.
The effect of varying levels of extracellular protease production on the bacteriophage type of Vibrio cholerae 1621 serotype 01, biotype E1 Tor, has been investigated. It has been shown that the production of high levels of exoprotease can alter the apparent type of the strain by rendering it insensitive to infection by a number of bacteriophages. Prevention of a productive infection appears to be due to altered surface characteristics rather than to specific damage to the bacteriophages. Such altered surface characteristics may be due to auto-digestion. The importance of this observation to epidemiological studies of V. cholerae is noted and discussed.
Bacteriophage Typing↗
Staphylococcus aureus nasal carriage in rheumatoid arthritis: antibody response to toxic shock syndrome toxin-1.
OBJECTIVE: To determine the prevalence of Staphylococcus aureus nasal carriage and to compare antibody responses to two superantigens, staphylococcal toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA), in rheumatoid arthritis patients and normal subjects. METHODS: 88 rheumatoid arthritis patients and 110 control subjects were cultured for nasal carriage of S aureus; 62 isolates were bacteriophage typed. Twenty five patients and 11 spouses were tested for antibodies to TSST-1, SEA, and sonicate extracts of Bacteroides fragilis and Escherichia coli; 19 patients were HLA-DR typed. RESULTS: 50% of patients and 33% of normal subjects were S aureus carriers. Bacteriophage typing of isolates suggested significant differences between strains isolated from the two groups. Patients showed higher IgG (P = 0.0025) and IgA (P = 0.0372) antibody levels to TSST-1 than normal spouses and these responses were not related to rheumatoid factor titres or HLA-DR type. CONCLUSION: When compared to normals, rheumatoid arthritis patients more often carry S aureus in their nasal vestibule, carry a distinct subpopulation of S aureus strains, and have higher average antibody levels to TSST-1.
Adult↗
Mutations of the bacteriophage T4 type II DNA topoisomerase that alter sensitivity to antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide and an antibacterial quinolone.
Various antitumor and antibacterial agents target type II DNA topoisomerases, stabilizing a cleaved DNA reaction intermediate and thereby converting topoisomerase into a cellular poison. Two 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)-resistant bacteriophage T4 topoisomerases have previously been characterized biochemically, and we have now determined the sequence of the causative mutations. In one case, a mutation (E457K) in a conserved domain of gp39 (ATPase subunit) causes resistance to antitumor agent m-AMSA but hypersensitivity to the quinolone oxolinic acid. In the second case, a combination of two amino acid substitutions (S79F and G269V) in gp52 (DNA-cleaving subunit) causes resistance to both m-AMSA and oxolinic acid. The S79F mutation is responsible for drug resistance, whereas the G269V mutation suppresses a topoisomerase deficiency caused by S79F. Surprisingly, the G269V mutation by itself causes a dramatic hypersensitivity to both inhibitors, defining a new class of topoisomerase mutants. Because S79 and the adjacent N78 are homologous to two key residues of DNA gyrase that affect quinolone sensitivity, we generated additional amino acid substitutions at these two positions. The substitutions alter sensitivity to m-AMSA and to oxolinic acid, sometimes in opposite directions. Furthermore, the quinolone sensitivities of the various mutants paralleled those of corresponding gyrase mutants. These results support models in which both quinolones and antitumor agents bind to a conserved site that overlaps the active site of the enzyme.
Amino Acid Sequence↗
Interspecies conversion of Clostridium botulinum type C to Clostridium novyi type A by bacteriophage.
When Clostridium botulinum type C is cured of its prophage it simultaneously ceases to produce toxin. This nontoxigenic culture can then be converted to another toxigenic bacterial species, Clostridium novyi type A or to toxigenic Clostridium botulinum types C or D, by specific bacteriophages. The toxigenicity and type of toxin produced by these cultures depends upon the continued presence of these bacteriophages.
Bacteriophages↗
Typing of methicillin-resistant Staphylococcus aureus by antibiotic resistance phenotypes.
The identification of new epidemic strains of methicillin-resistant Staphylococcus aureus is essential for rapid, effective infection control. We have developed a typing method which uses antibiotic sensitivity patterns to differentiate methicillin-resistant S. aureus and which is faster and more cost-effective than biochemical analysis or bacteriophage typing. Characterisation of phenotypes which are chromosomally-encoded, plasmid- or chromosomally-encoded or exclusively plasmid-mediated has enabled us to separate Australian strains of methicillin-resistant S. aureus into 11 classes, representatives of which were indistinguishable by bacteriophage type, or plasmid profile alone. The value of this procedure is thus clearly shown.
Acriflavine↗
First evidence of lysogeny in Propionibacterium freudenreichii subsp. shermanii.
Dairy propionic acid bacteria, particularly the species Propionibacterium freudenreichii, play a major role in the ripening of Swiss type cheese. Isometric and filamentous bacteriophages infecting P. freudenreichii have previously been isolated from cheese. In order to determine the origin of these bacteriophages, lysogeny of P. freudenreichii was determined by isometric bacteriophage type analysis. The genomic DNA of 76 strains were hybridized with the DNA of nine bacteriophages isolated from Swiss type cheeses, and the DNA of 25 strains exhibited strong hybridization. Three of these strains released bacteriophage particules following UV irradiation (254 nm) or treatment with low concentrations of mitomycin C. A prophage-cured derivative of P. freudenreichii was readily isolated and subsequently relysogenized. Lysogeny was therefore formally demonstrated in P. freudenreichii.
Bacteriophages↗
Molecular relationships among serogroup B bacteriophages of Staphylococcus aureus.
The typing bacteriophages 55, 80, 83A, and 85 of Staphylococcus aureus, representative of the three major lytic groups of serological group B aureophages, have been examined for relatedness of their genomes and virion proteins. Phages 11 and 80 alpha were also examined to determine the relationship of phage 80 alpha to phages 11 and 80. Total genome hybridization measurements divided the phages into two groups. Phages 55 and 80, in the first group, had DNA homology of 50%. Phages 11, 80 alpha, 83A, and 85 formed a second group with 27 to 65% homology. Homology between the two groups was in the range of 14 to 22%. Phage 80 alpha is more closely related to phage 11 than to phage 80, though it is probably not a simple recombinant of phages 11 and 80. Restriction enzyme digestion and phage [32P]DNA hybridization analysis of the endonuclease-generated fragments from each phage DNA confirmed the findings of the DNA homology measurements. The endonuclease fragment patterns generated by EcoRI and HindIII were distinctive for each phage, confirming that none of the phages are closely related. Common sequences were present in most fragments from the phage DNAs when the labeled probe DNA was from a different phage in the same group. Cross-group probing of endonuclease fragments revealed both a diminished level of homology when similar sequences were present and the probable absence of some sequences. Virion proteins, examined by polyacrylamide gel electrophoresis, were similar in number and molecular weight for phages 11, 80 alpha, 83A, and 85, reflecting the DNA homology analyses. The virion proteins from phages 55 and 80, however, were more distinctive, and both differed from the phages in the other group.
DNA Restriction Enzymes↗
A comparison of flagellar typing and phage typing as means of subdividing the O groups of Pseudomonas aeruginosa.
Strains of Pseudomonas aeruginosa from hospitals were typed by O and H serology and bacteriophage typing. The H sera were prepared against purified flagella of six type strains. The most common O serogroups were O6, O11, O16 and O10, and together these groups represented more than half of the total number of strains. O subgrouping proved useful for the further division of groups O2 and O6. Percentage H typability was high, and many H patterns were found. Comparison of H typing and phage typing as a means of making subdivisions within O groups showed that, although the general discriminatory power of the two methods was similar, H typing performed better than phage-typing in the more common O serogroups.
Bacteriophage Typing↗
Defective particle assembly in wild type P2 bacteriophage and its correction by the lg mutation.
The mutation lg of phage P2 has been located on the genetic map of P2 to the right of, and closely linked to, the del2 deletion, probably within tail gene F. The lg mutation causes larger burst sizes, compared with the wild type, especially at high incubation temperatures. The frequency of defective particles is lower in preparations of P2 lg than in those of wild type P2. It seems that the mutation lg improves the efficiency of particle assembly.
Coliphages↗
Latex agglutination-negative methicillin-resistant Staphylococcus aureus recovered from neonates: epidemiologic features and comparison of typing methods.
An unusual strain of methicillin-resistant Staphylococcus aureus (MRSA) was repeatedly isolated from infants in a newborn special care unit (NBSC) and a newborn intensive care unit. Between January 1989 and March 1990, approximately 100 isolates from infected or colonized infants were recovered. Surveillance cultures taken during this time revealed a 20% colonization rate, which was defined as recovery of MRSA from the nares, umbilicus, or groin. Isolates were identified as S. aureus by tube coagulase reactivity and heat-stable nuclease production but were unreactive in a latex agglutination assay. Representative isolates that were collected during the outbreak and that were found to share the latex agglutination assay-negative phenotype were compared by antibiogram (12 isolates), bacteriophage typing (20 isolates), capsular polysaccharide typing (30 isolates), and plasmid as well as chromosomal DNA analyses (20 isolates). All isolates known to be associated with the outbreak had nearly identical antibiograms and were notably susceptible to clindamycin. Staphylococcal bacteriophage typing was not useful in determining the relatedness of the isolates, since the majority were nontypeable. Plasmid pattern analysis revealed one large plasmid (approximately 100 kb) of equivalent size among the isolates. Capsular polysaccharide typing revealed that 14 of 30 isolates tested were type 5. Isolates identified in children at two other hospitals in the city which were also unreactive by the latex agglutination assay and clindamycin susceptible had plasmid and antibiogram patterns identical to those of isolates from the NBSC. Pulsed-field gel electrophoresis of restriction enzyme-digested genomic DNAs from the outbreak isolates demonstrated identical patterns which could be clearly differentiated from those of other unrelated MRSA. The strain from the NBSC is, therefore, unique and underscores the need for caution in interpreting the latex agglutination reactivities of MRSA isolates.
Bacterial Typing Techniques↗
Multidrug resistant Salmonella.
A total of 326 salmonella strains was isolated and studied from suspected enteric fever cases in Mumbai (Bombay) during a 2 year period from May 1992 to July 1994. These were identified using standard biochemical and serological tests. Bacteriophage typing, antibiotic sensitivity and conjugation experiments were also carried out. S. typhi was the most common serotype accounting for 75.46% of the strains. Among S. typhi strains 87% were biotype I and 13% were biotype II. 9.5% strains were of S. paratyphi A, 5.52% of S. typhimurium, 4.60% of S. worthington, 4.30% of S. havana and 0.62% of S. enteritidis. The commonest bacteriophage type of S. typhi was E1, and of S. paratyphi A type 1, whereas 88.88% strains of S. typhimurium were untypable. Most of the strains were multidrug resistant including commonly used antibiotics such as chloramphemicol, ampicillin, and cotrimaxazole. Quinolone derivatives such as Ciprofloxacin were found to be the most effective drugs. In the conjugation experiments there was direct transfer of resistance pattern and enbloc transfer of resistance was observed in most strains. Salmonella typhi is still the most commonly encountered species. There is an alarming increase in multidrug resistance.
Anti-Bacterial Agents↗
Imipenem and cefotaxime resistance: transduction by wild-type phages in hospital strains of Pseudomonas aeruginosa.
A wild-type bacteriophage appeared and was isolated from a Pseudomonas aeruginosa strain resistant to imipenem, cefotaxime, kanamycin and streptomycin (susceptible to carbenicillin, aztreonam, amikacin and fluoroquinolones). The best transducing properties were obtained with phage lysates prepared from bacteria growing on cefotaxime or imipenem. Transducing properties were found specific for individual recipient strain(s) susceptible to all drugs. A high-frequency of transduction was recorded for kanamycin and particularly for cefotaxime resistance determinants, followed by an imipenem determinant. This is now the fourth published wild-type bacteriophage, isolated from lysogenic nosocomial P. aeruginosa resistant to imipenem which was found to transduce this resistance determinant to susceptible pseudomonads.
Cefotaxime↗