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Amplification methods for the detection of bacterial resistance genes
Abstracts /Journal
of Microbiological Methods 27 (1996) 97-107
gion of six patients of group 1, two patients of group 2 and further two of the blood donors were sequenced. In comparison with the published HBVDNA sequences diverse mutations of variable frequency were found. In group 1, the rare point mutations which didn’t result in amino substitutions were mainly found in the pre Sl-region. On the contrary, the mutations on groups 2 and 3 were clustering in the S-region, especially in the 2 loops of the “a’‘-determinate. In all 4 sequences a mutation at amino acid 145 was found lated to evolve during vaccination.
which is postuAdditionally, in
one blood donor three of the four cysteins which are necessary to form the SHBs loops are substituted. Surprisingly, just the persons without clinical symptoms have an increased rate of mutations which have tainted effects on the amino acid sequence in contrast to the persons with clinical manifestations. 14
Detection of uncultured bacterial range PCR and sequencing D. Goldenberger and M. Alhvegg
pathogens
by broad-
Department of Medical Microbiology, University of Ziirich, Gloriastrasse 30, 8028 Ziirich, Switzerland
The comparison of 16s rRNA sequences is a well-known tool for taxonomic and phylogenetic investigations and numerous sequences are filed in different data bases which are accessible via on-line search. This data pool can also be used for diagnostic purposes. We have developed a rapid, simple and Ireliable method to generate partial 16s rRNA gene sequences (ca. 250-400 nucleotides) of bacteria directly from normally sterile clinical specimens. The procedure includes, measures to reduce the contamination of reagents with bacterial DNA and is also compatible with the use of dUTP and uracil-N-glycosylase for the prevention of laboratory contamination. The method was evaluated with specimens containing Bartonella henselae or B. quintana (as shown by speciesspecific PCR) and with removed heart valves of patients with endocarditis containing various bacteria (as shown by a positive culture of the valves or by previous positive blood culture). It was then successfully applied to demonstrate the presence of bacterial DNA in culture-negative specimens, e.g. Kmgella kingae in a joint fluid, and Tropheryma whippelii, the agent of Whipple’s disease, in heart valves (2 patients) and in a patient with spondylodiscitis. 15 Amplification metlhods for the detection of bacterial resistance genes M.R. Visser and J. Verhoef Eijkman- Winkler Inst. of Med. Microbiology, Uniu. Hospital Utrecht, Netherlands
With the explosion of infections caused by multi-resistant microbes, I here is an urgent need for rapid detection of resistant microorganisms. Studies comparing classical methods such as disc diffusion and MIC determinations with PCR for the detection of resistance have been performed for a number of antibiotics or specific organisms. The detection of methicillin resistance in S. aureus is
101
one of the best studied applications of PCR for the detection of antibiotic resistance. The problems caused by multiple genes leading to the same resistance phenotype are illustrated by,aminoglycoside resistance detection by PCR. Detection of antibiotic resistance in M. tuberculosis and M. lepme demonstrate the potential value of gene amplification in slow growing microorganisms. Finally, the detection of vancomycin resistance in enterococci shows the importance of PCR in monitoring emerging antibiotic resistance. In conclusion: PCR techniques have a potential for the detection of antibiotic resistance, but many problems have to be overcome before gene amplification techniques will find a widespread use in this field. 16 Detection of Bartonella quintana by PCR in an HIVseropositive patient with bacillary angiomatosis D. Pierard, A. De Coninck, G. Muyldermans, P. Lacer, J. Andre and S. Lauwers, et al. Department of Microbiology, Akademisch Ziekenhuis Universiteit Brussel, 81090 Brussels, Belgium
Vrije
A 33 years-old IV-drug user was admitted for regression of his general condition and multiple red nodular skin lesions, one of them being ulcerated and another causing osteolysis of the scapula. He was treated with radiotherapy for suspicion of Kaposi sarcoma. HIV infection had been diagnosed two years earlier when he was hospitalised in another institution for skin lesions considered as psoriasis. At that time he rejected further medical attention. Histological examination of a skin biopsy disclosed lesions of bacillary angiomatosis CBA) both by light and electron microscopy. PCR for a 60-kDa Bartonella protein and for the intergenic spacer region between 16 and 23 S RNA were both positive and restriction analysis of the PCR product of the latter identified B. quintana DNA. Attempts to grow the bacteria remained unsuccessful. In spite of initiation of therapy with erythromycin, the patient’s condition worsened. He rejected further therapy, left the hospital and died a few days later. Although no lice were found on this patient, they are probably the vector of transmission, since his personal hygiene was very poor. Re-examination of histological material of the biopsy taken two years earlier showed discrete lesions of BA, already at that time. This case report shows that BA can progress slowly even in immunocompromised patients. PCR could be used as a rapid diagnostic tool for this disease that can be treated with antibiotics. 17 Cats are probably not the only reservoir for infections due to Bartonella hensekae A.T.A. Box, A. Sander, I. Perschil, D. Goldenberger and M. Altwegg Depatiment of Medical Microbiology, Uniuersity of Ziirich, Glonbstrasse 30, CH-8028 Ziirich, Switzerland
Bartonella henselae (B.h.1 is an emerging bacterial pathogen causing cat scratch disease (CSD) and bacillary angiomatosis CBA). Cats bacteremic with B.h. are considered to be the reservoir from which humans become infected. We and others (1.2) have previously observed that there are two variants of B.h. with respect to the
of Microbiological Methods 27 (1996) 97-107
gion of six patients of group 1, two patients of group 2 and further two of the blood donors were sequenced. In comparison with the published HBVDNA sequences diverse mutations of variable frequency were found. In group 1, the rare point mutations which didn’t result in amino substitutions were mainly found in the pre Sl-region. On the contrary, the mutations on groups 2 and 3 were clustering in the S-region, especially in the 2 loops of the “a’‘-determinate. In all 4 sequences a mutation at amino acid 145 was found lated to evolve during vaccination.
which is postuAdditionally, in
one blood donor three of the four cysteins which are necessary to form the SHBs loops are substituted. Surprisingly, just the persons without clinical symptoms have an increased rate of mutations which have tainted effects on the amino acid sequence in contrast to the persons with clinical manifestations. 14
Detection of uncultured bacterial range PCR and sequencing D. Goldenberger and M. Alhvegg
pathogens
by broad-
Department of Medical Microbiology, University of Ziirich, Gloriastrasse 30, 8028 Ziirich, Switzerland
The comparison of 16s rRNA sequences is a well-known tool for taxonomic and phylogenetic investigations and numerous sequences are filed in different data bases which are accessible via on-line search. This data pool can also be used for diagnostic purposes. We have developed a rapid, simple and Ireliable method to generate partial 16s rRNA gene sequences (ca. 250-400 nucleotides) of bacteria directly from normally sterile clinical specimens. The procedure includes, measures to reduce the contamination of reagents with bacterial DNA and is also compatible with the use of dUTP and uracil-N-glycosylase for the prevention of laboratory contamination. The method was evaluated with specimens containing Bartonella henselae or B. quintana (as shown by speciesspecific PCR) and with removed heart valves of patients with endocarditis containing various bacteria (as shown by a positive culture of the valves or by previous positive blood culture). It was then successfully applied to demonstrate the presence of bacterial DNA in culture-negative specimens, e.g. Kmgella kingae in a joint fluid, and Tropheryma whippelii, the agent of Whipple’s disease, in heart valves (2 patients) and in a patient with spondylodiscitis. 15 Amplification metlhods for the detection of bacterial resistance genes M.R. Visser and J. Verhoef Eijkman- Winkler Inst. of Med. Microbiology, Uniu. Hospital Utrecht, Netherlands
With the explosion of infections caused by multi-resistant microbes, I here is an urgent need for rapid detection of resistant microorganisms. Studies comparing classical methods such as disc diffusion and MIC determinations with PCR for the detection of resistance have been performed for a number of antibiotics or specific organisms. The detection of methicillin resistance in S. aureus is
101
one of the best studied applications of PCR for the detection of antibiotic resistance. The problems caused by multiple genes leading to the same resistance phenotype are illustrated by,aminoglycoside resistance detection by PCR. Detection of antibiotic resistance in M. tuberculosis and M. lepme demonstrate the potential value of gene amplification in slow growing microorganisms. Finally, the detection of vancomycin resistance in enterococci shows the importance of PCR in monitoring emerging antibiotic resistance. In conclusion: PCR techniques have a potential for the detection of antibiotic resistance, but many problems have to be overcome before gene amplification techniques will find a widespread use in this field. 16 Detection of Bartonella quintana by PCR in an HIVseropositive patient with bacillary angiomatosis D. Pierard, A. De Coninck, G. Muyldermans, P. Lacer, J. Andre and S. Lauwers, et al. Department of Microbiology, Akademisch Ziekenhuis Universiteit Brussel, 81090 Brussels, Belgium
Vrije
A 33 years-old IV-drug user was admitted for regression of his general condition and multiple red nodular skin lesions, one of them being ulcerated and another causing osteolysis of the scapula. He was treated with radiotherapy for suspicion of Kaposi sarcoma. HIV infection had been diagnosed two years earlier when he was hospitalised in another institution for skin lesions considered as psoriasis. At that time he rejected further medical attention. Histological examination of a skin biopsy disclosed lesions of bacillary angiomatosis CBA) both by light and electron microscopy. PCR for a 60-kDa Bartonella protein and for the intergenic spacer region between 16 and 23 S RNA were both positive and restriction analysis of the PCR product of the latter identified B. quintana DNA. Attempts to grow the bacteria remained unsuccessful. In spite of initiation of therapy with erythromycin, the patient’s condition worsened. He rejected further therapy, left the hospital and died a few days later. Although no lice were found on this patient, they are probably the vector of transmission, since his personal hygiene was very poor. Re-examination of histological material of the biopsy taken two years earlier showed discrete lesions of BA, already at that time. This case report shows that BA can progress slowly even in immunocompromised patients. PCR could be used as a rapid diagnostic tool for this disease that can be treated with antibiotics. 17 Cats are probably not the only reservoir for infections due to Bartonella hensekae A.T.A. Box, A. Sander, I. Perschil, D. Goldenberger and M. Altwegg Depatiment of Medical Microbiology, Uniuersity of Ziirich, Glonbstrasse 30, CH-8028 Ziirich, Switzerland
Bartonella henselae (B.h.1 is an emerging bacterial pathogen causing cat scratch disease (CSD) and bacillary angiomatosis CBA). Cats bacteremic with B.h. are considered to be the reservoir from which humans become infected. We and others (1.2) have previously observed that there are two variants of B.h. with respect to the