Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Imipenem and Cefotaxime Resistance: Transduction by Wild-Type Phages in Hospital Strains of Pseudomonas aeruginosa J. Blahová, M. Hupková, V. Krčméry & V. Schäfer To cite this article: J. Blahová, M. Hupková, V. Krčméry & V. Schäfer (1992) Imipenem and Cefotaxime Resistance: Transduction by Wild-Type Phages in Hospital Strains of Pseudomonas aeruginosa, Journal of Chemotherapy, 4:6, 335-337, DOI: 10.1080/1120009X.1992.11739187 To link to this article: http://dx.doi.org/10.1080/1120009X.1992.11739187
Published online: 15 Jul 2016.
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Citing articles: 4 View citing articles
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Date: 19 August 2017, At: 05:24
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Journal of Chemotherapy
lmipenem and Cefotaxime Resistance: Transduction by Wild-Type Phages in Hospital Strains of Pseudomonas aeruginosa ]. BLAHOVA * - M. HUPKOV A * V. KRCMERY * - V. SCHAFER **
Summary ---------------- ---------------- -
A wild-type bacteriophage appeared and was isolated from 11 Pseudomonas seruginoss 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 reci· pient 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. seruginoss resistant to imipenem which was found to transduce this resistance determinant to susceptible pseudomonads. Key words: bacteriophage, imipenem, cefotaxime, phages, Pseudomonas aeruginosa.
Vol. 4 - n. 6 (335-337) - 1992
INTRODUCTION
Our previous publications 1 • 2 • 5 indicate that wild-type bacteriophages can be detected in, and isolated from, poly-resistant hospital strains of Pseudomonas aerugionsa when plating them, for conjugal transfer, on antibiotic-containing media (to pick up individual resistant colonies for donor culture preparations) . Such phages were found to transduce, in specific conditions and with suitable recipient strains, resistance determinants to certain newer betalactam drugs like aztreonam (AZA) and imipenem (IMI) . Two such pahges were found to have a certain specificity in that they transduce, with higher frequency, either AZA resistance (phage AP-2), for «Aeruginosa Phage», as recommended by B. Holliway) or IMI resistance (Phage AP-12), to a selected recipient strain susceptible to all drugs 3 • In this report we described the detection and subsequent isolation of a bacteriophage designated as AP-150, as it was detected in the 150th imipenem-resistant strain of P. aeruginosa isolated from a patient from Frankfurt University Clinic and tested for conjugal transfer of IMI resistance . The phage lysate was able to transduce, actively, either cefotaxime or IMIresistance, or both resistances, dependent on the recipient strain used. MATERIALS AND METHODS
Correspondence:
J.
Blahova. * Institute of Preventive and Clinical Medicine, Limbova 14, 83301 Bratislava, Czecho-Slovakia. ** Hygiene-Institut der Universitat, Paul Ehrlich Str. 40 Frankfurt/Main, Germany. © Edizioni Riviste Scientifiche - Firenze
Bacteriophage AP-150 was isolated from a wild-type, hospital-isolated strain of P. aeruginosa resistant to streptomycin (STR), kanamycin (KAN), cefazolin-cefalotin (CEF), cefotaxime (CTX) and imipenem (IMI). It was susceptible ISSN 1120-009X
Downloaded by [Australian Catholic University] at 05:24 19 August 2017
336
]. BLAHOVA - M. HUPKOVA -
to carbenicillin (CAR), gentamicin (GEN), amikacin (AMI), ceftazidime (CTZ), aztreonam (AZA) and fluoroquinolones. As usual in our procedure for conjugal transfer 4 , the collection of IMI-resistant P. aeruginosa strains from Frankfurt University Clinics was inoculated on MacConkey Agar (DIFCO) containing a single drug from the spectrum of its multiple drug resistance. The next step in the procedure was to pick up individual resistant colonies to prepare a donor-strain culture in Nutrient broth (DIFCO). In individual cases, however, one can observe a lytic reaction on the surface of the growth of a P. aeruginosa strain indicating the presence of a lysogenic wild-type bacteriophage. Plaques of the phage could then be picked up from the surface of the medium, and after propagation of the phage by a double layer softagar method ' a phage lysate can be prepared having the titer as high as 10 9 of p.f.u. '· 2 · ' . This lysate(s) can then be used for transduction using one of four recipient strains 3 • We received these strains from Professor Susumu Mitsuhashi (Maebashi, Japan) and they are designated PAO 1670, and, from the ML series of strains, ML 1008, 1292 and M-88. They all are auxotrophic mutants, and, thus can be differentiated from each other by amino acid requirements for their growth. This difference can also be used for experiments with conjugal transfer
v.
KRCMERY -
v.
SCHAFER
among them, using them alternatively as donor and recipient strains. The details of the procedure of transduction of antibiotic resistance and of the phenotypical expression as well as for phage lysate preparation have been described previously 2 • 3 • s.
RESULTS AND DISCUSSION
Results of transuductional transfer of the rsistance determinants by the AP-150 phage isolated from the P. aerugionsa strain No . 709, are given in Table 1. Transductants of the recipient strains PAO 16 70 selected on media with cefotaxime are depicted in Figure 1. From Table 1 it can be seen that three resistance determinants out of 5 determinants of resistance to anti-pseudomonadal antibiotics could be transduced to susceptible P. aeruginosa recipient strains, with frequencies of 5 ·10 - 7 to 2·10- 9 • The identity of transductants was verified by checking their amino acid requirement on minimal media. A representative number of transductants from each selection plate was tested for their eventual co-resistance to other drugs in the spectrum of the donor strain by an indirect selection method 5 • As can be seen in Table 1 while all IMI-selected transductants were resistant to imipenem only, CTX-selected transductants were also always resistant to kan-
TABLE 1 - Transduction of antibiotic resistance by the phage AP-150. Number of transductants in individual recipient strains and spectra of resistance transduced.
Number of transductants of recipient strain
Selection of transductants with
ML-1008
'KAN,.
173
CTX,.
182
69
62
CTX ,.
148
54
40
IMI1o
12
32
121
IMI"
4
10
32
PAO 1670
M-88
Resistance determinants transduced 1
ML-1292 80%, 20% KAN CTX In ML-1008: All CTX-selected are CTX KAN In PA0-1670: All CTX-selected are CTX KAN In M-88: All CTX-selected are CTX KAN
In ML-1008: In PA0-1670: In ML-1292:
All IMI-selected are IMI only All IMI-selected are IMI only All IMI-selected are IMI only
1 Demonstrated by the indirect selection method, see text. Abbreviations : !CAN-kanamycin, CTX-cefotaxime, IMI-imipenem. Numbers indicate concentration of the antibiotic used for selection, in mg. L- 1. 2
Downloaded by [Australian Catholic University] at 05:24 19 August 2017
IMIPENEM AND CEFOTAXIME RESISTANCE: TRANSDUCTION BY WILD-TYPE PHAGES, ETC.
Figure~! - Transductants of PAC1670 on media with cefotaxime, CTX.
amycin. In contrast, the majority of KAN-selected transductants have this resistance determinant separated from that of CTX. In no case was the fertility factor or transfer factor tra cotransduced, i.e. no conjugal transfer of any resistance determinant(s) from any type of transAs IMIductants could be demonstrated. selected transductants were only IMI-resistant, it might be concluded that in the donor hospital strain No. 709, this resistance is not associated with other resistances and is governed by a single gene, or a cluster of genes in the chromosome or on a plasmid. Transduction of antibiotic resistance by the phage AP-150 is characteristic in that a relatively high number of transductants accepting CTX-resistance could be isolated, as if this transduction would be somehow CTX-specific. This is analogous to the AP-2 and AP-12 bacteriophages studied previously 3 which rendered a considerably high number of transductants selected with aztreonam, or imipenem, respectively. It is remarkable that, while transductants that accepted a KAN -resistance determinant
337
could be obtained only in a single P. ae.ruginosa recipient strain, CTX and IMI-selected transductants were isolated on three recipient strains. It appeared that the most suitable and least «restrictive» recipient strain for transduction by the AP-150 phage was the ML-1008 strain, which accepted all three resistance determinants transduced, i.e. KAN, CTX and IMI. The KAN determinant, as said, was accepted by this strain only. The strains M-88 and ML1292, in contrast, each accepted a single determinant only, i.e. CTX or IMI. Thus, these experiments confirmed our previous conclusion that transductional transfer of antibiotic resistance determinants in P. aeruginosa seems to be strain-specific, and we discussed the epidemiologic significance of this restrictive behavior of bacteriophages 3 • s. By this systematic study of bacteriophages which could be isolated from polyresistant P. aeruginosa strains from Frankfurt University Clinic 2 • 3 it might be concluded that transduction by bacteriophages could play a significant role in disseminating resistance determinants, even to the newest drugs, among hospital strains of P. aeruginosa. REFERENCES ' Sagai H, Krcmery V, Hasuda K, Knothe H, Mitsuhashi S. R factor mediated resistance to aminoglycoside resistance in P aeruginosa. Jpn J Microbial 1975; 19, 3: 427-432 . 'Seginkova Z, Krcmery V, Knothe H . Ceftazidime resistance in P aerugionsa. Transduction by wild type phage. J Infect Dis 1986; 154, 6: 1049-1950. 'BlahovaJ, Hupkova M, Krcme.ry V, Schaefer V. Transduction of resistance to imipenem, aztreonam and ceftazidime in nosocomial strains of P aeruginosa. Infection, in press. • Knothe H, Shah H, Krcmery V, Mitsuhashi S. Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of K pneumoniae and S marescens. Infection 1983; 11, 6: 315-317. 'Knothe H, Lebek G, Krcmery V, Seginkova Z. Transduction of amikacin, gentamicin and tobramycin resistance in P aeruginosa with phage F116 and AP-19, Zbl BakterioL I Orig 1981; 150: 506-510.
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Imipenem and Cefotaxime Resistance: Transduction by Wild-Type Phages in Hospital Strains of Pseudomonas aeruginosa J. Blahová, M. Hupková, V. Krčméry & V. Schäfer To cite this article: J. Blahová, M. Hupková, V. Krčméry & V. Schäfer (1992) Imipenem and Cefotaxime Resistance: Transduction by Wild-Type Phages in Hospital Strains of Pseudomonas aeruginosa, Journal of Chemotherapy, 4:6, 335-337, DOI: 10.1080/1120009X.1992.11739187 To link to this article: http://dx.doi.org/10.1080/1120009X.1992.11739187
Published online: 15 Jul 2016.
Submit your article to this journal
View related articles
Citing articles: 4 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [Australian Catholic University]
Date: 19 August 2017, At: 05:24
Downloaded by [Australian Catholic University] at 05:24 19 August 2017
Journal of Chemotherapy
lmipenem and Cefotaxime Resistance: Transduction by Wild-Type Phages in Hospital Strains of Pseudomonas aeruginosa ]. BLAHOVA * - M. HUPKOV A * V. KRCMERY * - V. SCHAFER **
Summary ---------------- ---------------- -
A wild-type bacteriophage appeared and was isolated from 11 Pseudomonas seruginoss 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 reci· pient 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. seruginoss resistant to imipenem which was found to transduce this resistance determinant to susceptible pseudomonads. Key words: bacteriophage, imipenem, cefotaxime, phages, Pseudomonas aeruginosa.
Vol. 4 - n. 6 (335-337) - 1992
INTRODUCTION
Our previous publications 1 • 2 • 5 indicate that wild-type bacteriophages can be detected in, and isolated from, poly-resistant hospital strains of Pseudomonas aerugionsa when plating them, for conjugal transfer, on antibiotic-containing media (to pick up individual resistant colonies for donor culture preparations) . Such phages were found to transduce, in specific conditions and with suitable recipient strains, resistance determinants to certain newer betalactam drugs like aztreonam (AZA) and imipenem (IMI) . Two such pahges were found to have a certain specificity in that they transduce, with higher frequency, either AZA resistance (phage AP-2), for «Aeruginosa Phage», as recommended by B. Holliway) or IMI resistance (Phage AP-12), to a selected recipient strain susceptible to all drugs 3 • In this report we described the detection and subsequent isolation of a bacteriophage designated as AP-150, as it was detected in the 150th imipenem-resistant strain of P. aeruginosa isolated from a patient from Frankfurt University Clinic and tested for conjugal transfer of IMI resistance . The phage lysate was able to transduce, actively, either cefotaxime or IMIresistance, or both resistances, dependent on the recipient strain used. MATERIALS AND METHODS
Correspondence:
J.
Blahova. * Institute of Preventive and Clinical Medicine, Limbova 14, 83301 Bratislava, Czecho-Slovakia. ** Hygiene-Institut der Universitat, Paul Ehrlich Str. 40 Frankfurt/Main, Germany. © Edizioni Riviste Scientifiche - Firenze
Bacteriophage AP-150 was isolated from a wild-type, hospital-isolated strain of P. aeruginosa resistant to streptomycin (STR), kanamycin (KAN), cefazolin-cefalotin (CEF), cefotaxime (CTX) and imipenem (IMI). It was susceptible ISSN 1120-009X
Downloaded by [Australian Catholic University] at 05:24 19 August 2017
336
]. BLAHOVA - M. HUPKOVA -
to carbenicillin (CAR), gentamicin (GEN), amikacin (AMI), ceftazidime (CTZ), aztreonam (AZA) and fluoroquinolones. As usual in our procedure for conjugal transfer 4 , the collection of IMI-resistant P. aeruginosa strains from Frankfurt University Clinics was inoculated on MacConkey Agar (DIFCO) containing a single drug from the spectrum of its multiple drug resistance. The next step in the procedure was to pick up individual resistant colonies to prepare a donor-strain culture in Nutrient broth (DIFCO). In individual cases, however, one can observe a lytic reaction on the surface of the growth of a P. aeruginosa strain indicating the presence of a lysogenic wild-type bacteriophage. Plaques of the phage could then be picked up from the surface of the medium, and after propagation of the phage by a double layer softagar method ' a phage lysate can be prepared having the titer as high as 10 9 of p.f.u. '· 2 · ' . This lysate(s) can then be used for transduction using one of four recipient strains 3 • We received these strains from Professor Susumu Mitsuhashi (Maebashi, Japan) and they are designated PAO 1670, and, from the ML series of strains, ML 1008, 1292 and M-88. They all are auxotrophic mutants, and, thus can be differentiated from each other by amino acid requirements for their growth. This difference can also be used for experiments with conjugal transfer
v.
KRCMERY -
v.
SCHAFER
among them, using them alternatively as donor and recipient strains. The details of the procedure of transduction of antibiotic resistance and of the phenotypical expression as well as for phage lysate preparation have been described previously 2 • 3 • s.
RESULTS AND DISCUSSION
Results of transuductional transfer of the rsistance determinants by the AP-150 phage isolated from the P. aerugionsa strain No . 709, are given in Table 1. Transductants of the recipient strains PAO 16 70 selected on media with cefotaxime are depicted in Figure 1. From Table 1 it can be seen that three resistance determinants out of 5 determinants of resistance to anti-pseudomonadal antibiotics could be transduced to susceptible P. aeruginosa recipient strains, with frequencies of 5 ·10 - 7 to 2·10- 9 • The identity of transductants was verified by checking their amino acid requirement on minimal media. A representative number of transductants from each selection plate was tested for their eventual co-resistance to other drugs in the spectrum of the donor strain by an indirect selection method 5 • As can be seen in Table 1 while all IMI-selected transductants were resistant to imipenem only, CTX-selected transductants were also always resistant to kan-
TABLE 1 - Transduction of antibiotic resistance by the phage AP-150. Number of transductants in individual recipient strains and spectra of resistance transduced.
Number of transductants of recipient strain
Selection of transductants with
ML-1008
'KAN,.
173
CTX,.
182
69
62
CTX ,.
148
54
40
IMI1o
12
32
121
IMI"
4
10
32
PAO 1670
M-88
Resistance determinants transduced 1
ML-1292 80%, 20% KAN CTX In ML-1008: All CTX-selected are CTX KAN In PA0-1670: All CTX-selected are CTX KAN In M-88: All CTX-selected are CTX KAN
In ML-1008: In PA0-1670: In ML-1292:
All IMI-selected are IMI only All IMI-selected are IMI only All IMI-selected are IMI only
1 Demonstrated by the indirect selection method, see text. Abbreviations : !CAN-kanamycin, CTX-cefotaxime, IMI-imipenem. Numbers indicate concentration of the antibiotic used for selection, in mg. L- 1. 2
Downloaded by [Australian Catholic University] at 05:24 19 August 2017
IMIPENEM AND CEFOTAXIME RESISTANCE: TRANSDUCTION BY WILD-TYPE PHAGES, ETC.
Figure~! - Transductants of PAC1670 on media with cefotaxime, CTX.
amycin. In contrast, the majority of KAN-selected transductants have this resistance determinant separated from that of CTX. In no case was the fertility factor or transfer factor tra cotransduced, i.e. no conjugal transfer of any resistance determinant(s) from any type of transAs IMIductants could be demonstrated. selected transductants were only IMI-resistant, it might be concluded that in the donor hospital strain No. 709, this resistance is not associated with other resistances and is governed by a single gene, or a cluster of genes in the chromosome or on a plasmid. Transduction of antibiotic resistance by the phage AP-150 is characteristic in that a relatively high number of transductants accepting CTX-resistance could be isolated, as if this transduction would be somehow CTX-specific. This is analogous to the AP-2 and AP-12 bacteriophages studied previously 3 which rendered a considerably high number of transductants selected with aztreonam, or imipenem, respectively. It is remarkable that, while transductants that accepted a KAN -resistance determinant
337
could be obtained only in a single P. ae.ruginosa recipient strain, CTX and IMI-selected transductants were isolated on three recipient strains. It appeared that the most suitable and least «restrictive» recipient strain for transduction by the AP-150 phage was the ML-1008 strain, which accepted all three resistance determinants transduced, i.e. KAN, CTX and IMI. The KAN determinant, as said, was accepted by this strain only. The strains M-88 and ML1292, in contrast, each accepted a single determinant only, i.e. CTX or IMI. Thus, these experiments confirmed our previous conclusion that transductional transfer of antibiotic resistance determinants in P. aeruginosa seems to be strain-specific, and we discussed the epidemiologic significance of this restrictive behavior of bacteriophages 3 • s. By this systematic study of bacteriophages which could be isolated from polyresistant P. aeruginosa strains from Frankfurt University Clinic 2 • 3 it might be concluded that transduction by bacteriophages could play a significant role in disseminating resistance determinants, even to the newest drugs, among hospital strains of P. aeruginosa. REFERENCES ' Sagai H, Krcmery V, Hasuda K, Knothe H, Mitsuhashi S. R factor mediated resistance to aminoglycoside resistance in P aeruginosa. Jpn J Microbial 1975; 19, 3: 427-432 . 'Seginkova Z, Krcmery V, Knothe H . Ceftazidime resistance in P aerugionsa. Transduction by wild type phage. J Infect Dis 1986; 154, 6: 1049-1950. 'BlahovaJ, Hupkova M, Krcme.ry V, Schaefer V. Transduction of resistance to imipenem, aztreonam and ceftazidime in nosocomial strains of P aeruginosa. Infection, in press. • Knothe H, Shah H, Krcmery V, Mitsuhashi S. Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of K pneumoniae and S marescens. Infection 1983; 11, 6: 315-317. 'Knothe H, Lebek G, Krcmery V, Seginkova Z. Transduction of amikacin, gentamicin and tobramycin resistance in P aeruginosa with phage F116 and AP-19, Zbl BakterioL I Orig 1981; 150: 506-510.
