World Journal of Microbiology and Biotechnofogy 6, 117-120
Characterization of plQ5 plasmid originating from Klebsiella pneumoniae
Zainab AI-Doori, Areal Clor and Norryai AIi
plQ5 plasmid consists of a group of genes specifying resistance to amplclllin, chloramphenicol, carbencillln, kanamycin and trimethoprim-sulphamethoxazole. It is isolated in Klebslella pneumoniae ZD532, is about 26.8 Kb and is freely transmissible to various bacterial species of Gram-negative bacteria. Physical characterization revealed that plQ5 plasmld has a single site for Hindlll, BamHI, EcoRI and two sites for Bglll restriction enzyme. Le plasm|de plQ5 conslate en un groupe de g~nes codant pour la r6sistance ~, I'ampicllline, au r & la carbencllline, la kanamycine et au trlm6thoprlmsulpham(~thoxazole. II eat isol~ dans Klebslella pneumonlae ZD532; sa Iongueur est d'envlron 26.8kb et il est Iibrement transmissible ~ diverses esp~ces bact6rlennes Gram-n6gatlves. La carect6rlsatlon physique r6vble que le plasmlde plQ5 a un site unique pour ies enzymes de restriction Hindlll, BamHI, EcoRI et deux sites pour
BgAI. Zainab AI-Doori is with the Biological Research Centre, Jadiriyah, P.O. Box 2371, Baghdad, Iraq; Amal Clor and Norryai All are with the Genetic Engineering and Biotechnology Research Centre, Jadiriyah, P.O. Box 2250, Baghdad, Iraq. Zainab AI-Doori is the corresponding author.
(~ 1990Rapid Communications of Oxford Ltd.
Klebsiella is an opportunistic pathogen and is a causative agent of several kinds of infections in humans and animals. Facinelli & Calegari (1984) reported that multiple-resistance of Klebsiella pneumoniae to antibiotics may cause a hospital epidemic which is due to a self transmissible plasmid found in several Klebsiella strains at the hospital. A single plasmid, however, may also become distributed through many different bacterial strains and may spread over a large territory (Tietze & Tschape 1983). In earlier studies, we demonstrated that the dominant pattern of muhiple-antibiotic-resistant strains among clinical isolates of Klebsiella pneumoniae is resistant to ampicillin, chloramphenicol, carbencillin, kanamycin and trimethoprim-sulphamethoxazole (A1-Doori eta]. 1986). Further investigation of these multiple-resistant clinical isolates led to the discovery of many plasmids. In this investigation we describe the genetical and physical characterization of pIQ5 plasmid by demonstrating the conjugal transfer of the antibiotic resistance determinants between pIQ5 plasmid and many bacterial strains of diverse origin.
Materials and Methods Bacterial Strains Klebsiellapneumoniae ZD532 is the source of pIQ5. All other bacterial strains which are used as recipients in this study are listed (see Table 1). Conjugation The millipore-mating technique (Towner & Vivian 1976) was adapted for testing the transferability of pIQ5 plasmid from Klebsiellapneumoniae ZD532 into different recipient strains which belong to different families. Transconjugants were selected on glucose minimal media (Clowes & Hayes 1968) supplemented with one or all of the following antibiotics: ampicillin (50 I.tg/ml), chloramphenicol (30 Ixg/ml), carbencillin (100 ~tg/ml) and kanamycin (39 I.tg/ml). However, glucose minimal media is not employed in all mating experiments. For Acinetobacter mating, sodium glutamate was used as a sole carbon source at 0.2% (w/v) and conjugation was carried out at 30~
117
Z. ALDoori, A . C/or and N. A l i In the case of Proteus mating, nicotinic acid (5 I.tg/ml) was added as a growth requirement to glucose minimal medium. TCBS medium was used for Vibrio cholerae mating. Finally, in the case of Pseudomonas conjugation, selection was for kanamycinl000 resistance only due to its natural resistance to other antibiotics.
Selection of Spontaneous Rifampicin-Resistant Recipient Strains Overnight nutrient broth cultures of recipients were diluted 1:10 (v/v) and incubated for 2 to 3 h. A sample of 0.1 ml of each culture was plated on glucose minimal media supplemented with rifampicin (100 ~tg/ml). Spontaneous rifampicin-resistant mutants were picked and purified on the same selective plate. As described above, special media were used for A cinetobacter, Proteus and Vibrio supplemented with the same concentration of rifampicin. Preparation ofplasmid D N A . The boiling method of Maniatis et a/. (1982) was followed to prepare pIQ5 plasmid DNA. Molecular weight determination of pIQ5 plasmid D N A . Horizontal agarose minigel electrophoresis was carried out in Tris/borate buffer (Maniatis et aL 1982) in 0.8% (w/v) agarose. Plasmid DNA, ~, HindlII digest (Boehringer Mannheim) with the loading buffer were subjected to 100 V at 40 mA for 2 h. After staining the minigel with ethidium bromide at 0.5 ~tg/ml, photography under ultraviolet light (300 nm) was carried out. However, the relative mobilities of DNA bands were plotted versus 1/log molecular weight of standard ~. DNA (see Figure 1). Restriction endonudease digestion. The restriction endonucleases HindIII, BamHI, EcoRI and BglI1 were used for pIQ5 digestion under conditions recommended by the supplier (Boehringer Mannheim). DNA fragments were subjected to electrophoresis conditions and photography as described above.
Results Genetical characterization of the plasmid pIQ5 revealed that it is a conjugative plasmid; pIQ5 plasmid is transmissible to many bacterial species (see Table 1). Some of the bacterial species are members of the Enterobacteriaceae such as Escherichia coli ZD546, Shigella boydii and Proteus mirabilis. The plasmid pIQ5 transferred to members of other families such as A cinetobacter calcoaceticus,A ZOtObatter vinelandii, Pseudomonas aeruginosa and Vibrio cholera. The frequencies of transfer of pIQ5 ranged between 10 -2 and 10 -6 (see Table 1). The size of plasmid pIQ5 is 26.8 Kb (Figure 1). Restriction analysis revealed that this plasmid has a single site for each of HindIII, BamHI and EcoRI enzymes and two sites for BgllI enzyme; two fragments are generated after BgllI digestion with molecular weights of 19 Kb and 7.6 Kb respectively (see Figure 2).
Table 1. Transferability of plQ5 plasmid from Klebslella pneumonlae ZD532 into various bacterial species. Recipients Escherichia coil ZD546 (metB B1 recA rift). Shigella boydii Ray12 Azotobacter vinelandii Acinetobacter calcoaceticus Proteus mirabilis Pseudomonas aeruginosa PAO Vibrio cholerae ZDV101
118
Source
Average frequency of transfer
Z. AI-Doori
0.3 x 10 -2
A. Clor J. Flemming J. Flemming
0.1 x 10 -4 1 • 10 -6 4 x 10 -6
J. Flemming B. Holloway Z. AI-Doori
0.2 x 10 a 7.2 x 10 -6 1.24 x 10 -2
Characterization of pIQ5 plasmid 3.5
2.5
-j
1.5
o.s I
10
i
20
i
30
40
50
M ig r a t i o n ( m m ) Figure 1. Demonstration of plasmid plQ5 molecular weight using 2 DNA Hindlll digest as standard.
Discussion Many antibiotic-resistant strains of K. pneumoniae responsible for outbreaks of systemic infection in a hospital nursery have been studied. For example (and similar to pIQ5), K. pneumoniae RO106 contains plasmids which confer resistance to ampicillin, carbencillin, chloramphenicol and streptomycin (O'Callaghan et al. 1978). These plasmids are similar in size to that of pIQS; restriction analysis of pIQ5 DNA with BglII confirmed two fragments are generated with a total a b c d e f molecular size of 26.6 Kb. RO16 plasmids are conjugative (O'Callaghan et al. 1978) and transfer at a frequency of 10 -6. However, transferability of pIQ5 is higher (see Table 1). It 9 it z' 9 9 9 9 ] seems likely that pIQ5 was responsible for the spread of multiple antibiotic resistance among clinical isolates of K. pneumoniae because it represents the dominant pattern of multiple antibiotic resistance in this pathogen (A1-Doori et al. 1986; A1-Doori 1988). Moreover, all antibiotic-resistant determinants were m m w expressed in all tested recipients, i.e. there was no lack of expression of these markers in any recipient which confirms that pIQ5 is a broad host range plasmid or a promiscuous plasmid. This transferability of pIQ5 plasmid makes it similar to other plasmids such as RP4, RK2 and R68. Similarity of pIQ5 to RP4 includes restriction analysis, in that pIQ5 DNA has only a single site for EcoRl, HindIII and BamHI restriction endonuclease enzymes (Figure 2). Recently, the occurrence of R plasmids in Klebsiella strains derived from a hospital infection was demonstrated (Casewell & Philips 1981), but to our knowledge no further work on the genetic characterization of these R plasmids was carried out. It has been reported that large R plasmids (76 to 104 Md) belong Figure 2. Restriction analysis of plQ5 to Inc FIV group and lower molecular weight R plasmids (25 to 36 Md) belonging plasmid DNA. Lane (a) ,~ Hindlll digest to the Inc N grouping. It would be interesting to test whether pIQ5 belong to (23606, 9636, 6636, 4333, 2257, 1985 base N incompatibility or another group. Finally, plasmid pIQ5 as a small naturally pairs); (b) undigested plQ5 plasmid DNA, lanes c to f plQ5 digested with Hindlll, occurring self-transmissible plasmid could be used for introducing foreign (heteroBamHI, Bglll and EcoRI, respectively. logous) DNA into novel hosts. m
m m
m
m
119
Z. Al-Doori, A . C/or and N. A l i
References AL-DOORI, Z. 1988 Multiple antibiotic resistance in isolates from Baghdad. A P U A (WHO) Newsletter 6 (2), 6-8. AL-DOORI, Z., NAOM, I.S., CLOR, A.M., NOURI, V.F., KADDOURI,M., NAIMI, I., AL-ANI, Z. & MURAD, K. 1986 Multiple antibiotic resistance of Klebsidla pneumoniae. Baghdad Journal of BiologicalScience Research 17, 267-278. CASEWELL,M.W. &: PHILIPS,I. 1981 Aspects of the plasmid mediated antibiotic resistance and epidemiology of Klebsidla species American Journal of Medicine70, 459-462. CLOWES, R.C. & HAYES, W. 1968 Experiments in Microbial Genetics. Oxford: Blackwell Scientific. FACINELLI, B. & CALEGARI,L. 1984 A hospital epidemic caused by a multiple antibiotic resistant Klebsidlapneumoniae implication for a conjugative R plasmid. Bollettinodell'lstituto Sieroterapico Milanese 63, 111-117. MANIATIS,T., FRITSCH E.F. & SAMBROOK,J. Eds. 1982 Molecular Cloning Cold Spring Harbor: Cold Spring Harbor Laboratory. O'CALLAGHAN, R.S., ROUSSET, K.M., HARKESS, N.H., MURRAY, M.L., LEWIS, A.C. & WILLIAMS,W.L. 1978 Analysis of increasing antibiotic resistance of Klebsidla pneumoniae relative to changes in chemotherapy. Journal of Infectious Diseases138, 293-298. TIETZE, E. & TSCHAPE, H. 1983 Plasmic pattern analysis of natural bacterial isolates and its epidemiological implication. Journal of Hygiene90, 475--488. TOWNER, K.J. & VIVIAN,A. 1976 RP4-mediated conjugation in Acinetobacter calcoaceticus. Journal of General Microbiology93, 355-360.
(Received as revised 9 January 1990; accepted 2 February t990)
120
Characterization of plQ5 plasmid originating from Klebsiella pneumoniae
Zainab AI-Doori, Areal Clor and Norryai AIi
plQ5 plasmid consists of a group of genes specifying resistance to amplclllin, chloramphenicol, carbencillln, kanamycin and trimethoprim-sulphamethoxazole. It is isolated in Klebslella pneumoniae ZD532, is about 26.8 Kb and is freely transmissible to various bacterial species of Gram-negative bacteria. Physical characterization revealed that plQ5 plasmld has a single site for Hindlll, BamHI, EcoRI and two sites for Bglll restriction enzyme. Le plasm|de plQ5 conslate en un groupe de g~nes codant pour la r6sistance ~, I'ampicllline, au r & la carbencllline, la kanamycine et au trlm6thoprlmsulpham(~thoxazole. II eat isol~ dans Klebslella pneumonlae ZD532; sa Iongueur est d'envlron 26.8kb et il est Iibrement transmissible ~ diverses esp~ces bact6rlennes Gram-n6gatlves. La carect6rlsatlon physique r6vble que le plasmlde plQ5 a un site unique pour ies enzymes de restriction Hindlll, BamHI, EcoRI et deux sites pour
BgAI. Zainab AI-Doori is with the Biological Research Centre, Jadiriyah, P.O. Box 2371, Baghdad, Iraq; Amal Clor and Norryai All are with the Genetic Engineering and Biotechnology Research Centre, Jadiriyah, P.O. Box 2250, Baghdad, Iraq. Zainab AI-Doori is the corresponding author.
(~ 1990Rapid Communications of Oxford Ltd.
Klebsiella is an opportunistic pathogen and is a causative agent of several kinds of infections in humans and animals. Facinelli & Calegari (1984) reported that multiple-resistance of Klebsiella pneumoniae to antibiotics may cause a hospital epidemic which is due to a self transmissible plasmid found in several Klebsiella strains at the hospital. A single plasmid, however, may also become distributed through many different bacterial strains and may spread over a large territory (Tietze & Tschape 1983). In earlier studies, we demonstrated that the dominant pattern of muhiple-antibiotic-resistant strains among clinical isolates of Klebsiella pneumoniae is resistant to ampicillin, chloramphenicol, carbencillin, kanamycin and trimethoprim-sulphamethoxazole (A1-Doori eta]. 1986). Further investigation of these multiple-resistant clinical isolates led to the discovery of many plasmids. In this investigation we describe the genetical and physical characterization of pIQ5 plasmid by demonstrating the conjugal transfer of the antibiotic resistance determinants between pIQ5 plasmid and many bacterial strains of diverse origin.
Materials and Methods Bacterial Strains Klebsiellapneumoniae ZD532 is the source of pIQ5. All other bacterial strains which are used as recipients in this study are listed (see Table 1). Conjugation The millipore-mating technique (Towner & Vivian 1976) was adapted for testing the transferability of pIQ5 plasmid from Klebsiellapneumoniae ZD532 into different recipient strains which belong to different families. Transconjugants were selected on glucose minimal media (Clowes & Hayes 1968) supplemented with one or all of the following antibiotics: ampicillin (50 I.tg/ml), chloramphenicol (30 Ixg/ml), carbencillin (100 ~tg/ml) and kanamycin (39 I.tg/ml). However, glucose minimal media is not employed in all mating experiments. For Acinetobacter mating, sodium glutamate was used as a sole carbon source at 0.2% (w/v) and conjugation was carried out at 30~
117
Z. ALDoori, A . C/or and N. A l i In the case of Proteus mating, nicotinic acid (5 I.tg/ml) was added as a growth requirement to glucose minimal medium. TCBS medium was used for Vibrio cholerae mating. Finally, in the case of Pseudomonas conjugation, selection was for kanamycinl000 resistance only due to its natural resistance to other antibiotics.
Selection of Spontaneous Rifampicin-Resistant Recipient Strains Overnight nutrient broth cultures of recipients were diluted 1:10 (v/v) and incubated for 2 to 3 h. A sample of 0.1 ml of each culture was plated on glucose minimal media supplemented with rifampicin (100 ~tg/ml). Spontaneous rifampicin-resistant mutants were picked and purified on the same selective plate. As described above, special media were used for A cinetobacter, Proteus and Vibrio supplemented with the same concentration of rifampicin. Preparation ofplasmid D N A . The boiling method of Maniatis et a/. (1982) was followed to prepare pIQ5 plasmid DNA. Molecular weight determination of pIQ5 plasmid D N A . Horizontal agarose minigel electrophoresis was carried out in Tris/borate buffer (Maniatis et aL 1982) in 0.8% (w/v) agarose. Plasmid DNA, ~, HindlII digest (Boehringer Mannheim) with the loading buffer were subjected to 100 V at 40 mA for 2 h. After staining the minigel with ethidium bromide at 0.5 ~tg/ml, photography under ultraviolet light (300 nm) was carried out. However, the relative mobilities of DNA bands were plotted versus 1/log molecular weight of standard ~. DNA (see Figure 1). Restriction endonudease digestion. The restriction endonucleases HindIII, BamHI, EcoRI and BglI1 were used for pIQ5 digestion under conditions recommended by the supplier (Boehringer Mannheim). DNA fragments were subjected to electrophoresis conditions and photography as described above.
Results Genetical characterization of the plasmid pIQ5 revealed that it is a conjugative plasmid; pIQ5 plasmid is transmissible to many bacterial species (see Table 1). Some of the bacterial species are members of the Enterobacteriaceae such as Escherichia coli ZD546, Shigella boydii and Proteus mirabilis. The plasmid pIQ5 transferred to members of other families such as A cinetobacter calcoaceticus,A ZOtObatter vinelandii, Pseudomonas aeruginosa and Vibrio cholera. The frequencies of transfer of pIQ5 ranged between 10 -2 and 10 -6 (see Table 1). The size of plasmid pIQ5 is 26.8 Kb (Figure 1). Restriction analysis revealed that this plasmid has a single site for each of HindIII, BamHI and EcoRI enzymes and two sites for BgllI enzyme; two fragments are generated after BgllI digestion with molecular weights of 19 Kb and 7.6 Kb respectively (see Figure 2).
Table 1. Transferability of plQ5 plasmid from Klebslella pneumonlae ZD532 into various bacterial species. Recipients Escherichia coil ZD546 (metB B1 recA rift). Shigella boydii Ray12 Azotobacter vinelandii Acinetobacter calcoaceticus Proteus mirabilis Pseudomonas aeruginosa PAO Vibrio cholerae ZDV101
118
Source
Average frequency of transfer
Z. AI-Doori
0.3 x 10 -2
A. Clor J. Flemming J. Flemming
0.1 x 10 -4 1 • 10 -6 4 x 10 -6
J. Flemming B. Holloway Z. AI-Doori
0.2 x 10 a 7.2 x 10 -6 1.24 x 10 -2
Characterization of pIQ5 plasmid 3.5
2.5
-j
1.5
o.s I
10
i
20
i
30
40
50
M ig r a t i o n ( m m ) Figure 1. Demonstration of plasmid plQ5 molecular weight using 2 DNA Hindlll digest as standard.
Discussion Many antibiotic-resistant strains of K. pneumoniae responsible for outbreaks of systemic infection in a hospital nursery have been studied. For example (and similar to pIQ5), K. pneumoniae RO106 contains plasmids which confer resistance to ampicillin, carbencillin, chloramphenicol and streptomycin (O'Callaghan et al. 1978). These plasmids are similar in size to that of pIQS; restriction analysis of pIQ5 DNA with BglII confirmed two fragments are generated with a total a b c d e f molecular size of 26.6 Kb. RO16 plasmids are conjugative (O'Callaghan et al. 1978) and transfer at a frequency of 10 -6. However, transferability of pIQ5 is higher (see Table 1). It 9 it z' 9 9 9 9 ] seems likely that pIQ5 was responsible for the spread of multiple antibiotic resistance among clinical isolates of K. pneumoniae because it represents the dominant pattern of multiple antibiotic resistance in this pathogen (A1-Doori et al. 1986; A1-Doori 1988). Moreover, all antibiotic-resistant determinants were m m w expressed in all tested recipients, i.e. there was no lack of expression of these markers in any recipient which confirms that pIQ5 is a broad host range plasmid or a promiscuous plasmid. This transferability of pIQ5 plasmid makes it similar to other plasmids such as RP4, RK2 and R68. Similarity of pIQ5 to RP4 includes restriction analysis, in that pIQ5 DNA has only a single site for EcoRl, HindIII and BamHI restriction endonuclease enzymes (Figure 2). Recently, the occurrence of R plasmids in Klebsiella strains derived from a hospital infection was demonstrated (Casewell & Philips 1981), but to our knowledge no further work on the genetic characterization of these R plasmids was carried out. It has been reported that large R plasmids (76 to 104 Md) belong Figure 2. Restriction analysis of plQ5 to Inc FIV group and lower molecular weight R plasmids (25 to 36 Md) belonging plasmid DNA. Lane (a) ,~ Hindlll digest to the Inc N grouping. It would be interesting to test whether pIQ5 belong to (23606, 9636, 6636, 4333, 2257, 1985 base N incompatibility or another group. Finally, plasmid pIQ5 as a small naturally pairs); (b) undigested plQ5 plasmid DNA, lanes c to f plQ5 digested with Hindlll, occurring self-transmissible plasmid could be used for introducing foreign (heteroBamHI, Bglll and EcoRI, respectively. logous) DNA into novel hosts. m
m m
m
m
119
Z. Al-Doori, A . C/or and N. A l i
References AL-DOORI, Z. 1988 Multiple antibiotic resistance in isolates from Baghdad. A P U A (WHO) Newsletter 6 (2), 6-8. AL-DOORI, Z., NAOM, I.S., CLOR, A.M., NOURI, V.F., KADDOURI,M., NAIMI, I., AL-ANI, Z. & MURAD, K. 1986 Multiple antibiotic resistance of Klebsidla pneumoniae. Baghdad Journal of BiologicalScience Research 17, 267-278. CASEWELL,M.W. &: PHILIPS,I. 1981 Aspects of the plasmid mediated antibiotic resistance and epidemiology of Klebsidla species American Journal of Medicine70, 459-462. CLOWES, R.C. & HAYES, W. 1968 Experiments in Microbial Genetics. Oxford: Blackwell Scientific. FACINELLI, B. & CALEGARI,L. 1984 A hospital epidemic caused by a multiple antibiotic resistant Klebsidlapneumoniae implication for a conjugative R plasmid. Bollettinodell'lstituto Sieroterapico Milanese 63, 111-117. MANIATIS,T., FRITSCH E.F. & SAMBROOK,J. Eds. 1982 Molecular Cloning Cold Spring Harbor: Cold Spring Harbor Laboratory. O'CALLAGHAN, R.S., ROUSSET, K.M., HARKESS, N.H., MURRAY, M.L., LEWIS, A.C. & WILLIAMS,W.L. 1978 Analysis of increasing antibiotic resistance of Klebsidla pneumoniae relative to changes in chemotherapy. Journal of Infectious Diseases138, 293-298. TIETZE, E. & TSCHAPE, H. 1983 Plasmic pattern analysis of natural bacterial isolates and its epidemiological implication. Journal of Hygiene90, 475--488. TOWNER, K.J. & VIVIAN,A. 1976 RP4-mediated conjugation in Acinetobacter calcoaceticus. Journal of General Microbiology93, 355-360.
(Received as revised 9 January 1990; accepted 2 February t990)
120
