World Journal
of Microbiology
& Biotechnology
12, 585-588
Plasmid distribution in Escherichia co/i urinary isolates with special reference to aerobactin and colicin production K. Harjai, S. Pajni, S. Chhibber and S. Sharma” Plasmids were detected in 31 out of 35 strains of Escherichia coli isolated from unclassified tract infection at a median value of 1.88 plasmid bands per isolate. The isolates showed aerobactin and colicin production with the distribution of plasmid bands having a median value (plasmid bands per isolate) in aerobactin-positive and aerobactin-negative strains respectively. For the median plasmid bands per isolate was 3.66 compared to 1.80 for colicin-negative strains. Key words: Aerobactin,
colicin, Escherichia coli, growth,
plasmid profile.
Association between the number of virulence attributes of Escherichia coli and its ability to establish in the urinary tract has shown that many of these characteristics are plasmidmediated (Johnson 1991). Colicin production, one of the plasmid-mediated characters, provides an advantage to the organism in infections of the urinary tract (Montgomerie et al. 1973; Davies et al. 1981; Chhibber et al. 1988). Although possession of the colicin V plasmid markedly enhances virulence, colicin V activity itself may not be the only factor responsible (Quakenbush & Falkow 1979) since this plasmid also carries genes for aerobactin production (Smith & Huggins 1976; Williams 1979). The latter characteristic has also been shown to play an important role in the pathogenicity of the infecting organism especially in the urinary tract (Carbonetti ef al. 1986; Harjai et al. 1994). The genes for aerobactin production are known to be present either on the chromosome or the plasmid (Valvano & Crosa 1984; Valvano et al. 1986). Emphasis has mainly been on the incidence of production in clinical isolates (Carbonetti et al. 1986; Orskov et ul. 1988; Harjai ef al. 1990) and the genetics of this characteristic (Valvano & Crosa 1984; Bindereif & Neilands 1985; Johnson ef al. 1988). However, only limited information is available on the distribution of plasmids in urinary isolates of E. coli with reference to aerobactin production. The purpose of
the present investigation was to study the plasmid profiles of Escherichiu coli uroisolates and to correlate them with their ability to grow and produce aerobactin in iron depleted conditions which prevails in the urinary tract in viuo (Shand ef al. 1985).
Materials Bacterial
@ 1996 Rapid Science
Panjab University. author.
and Methods
Strains
Thirty five clinical isolates of Escherichiu cob, were isolated from the urine of patients with unclassified urinary tract infection. Detection of Colicin Production production was tested using the agar overlay method (Ctowes & Hayes 1968). Nutrient agar plates containing 0.5 gg/ml Colicin
mitomycin
C were
prepared,
air dried
and inoculated
with
selected
strains. Plates were incubated overnight at 37°C and subsequently exposed to chloroform for 10-15 min. The indicator strain E. cali Kl2 was grown in nutrient broth at 37°C for 4 h. At the end of the incubation, the broth was mixed 50°C and overlaid on plates seeded
with with
soft nutrient agar at the test strains. The
plates were incubated at 37°C overnight. Clear zones around the stabbed strains indicated colicin production. E. coli BM21 (pCoIVK30) and E. Co/i BM2l were used as positive and negative controls respectively. Bacterial Growth Iron
restricted
dipyridyl The authors are with the Department of Microbiology, Chandigarh-160014, India; fax: 541409. * Corresponding
cases of urinary an association of of 2.33 and 1.72 colicin producers,
Sansonetti
conditions
were
created
by adding
to synthetic medium M9 according (1986).
The
growth
medium and in M9 medium
profile containing
150 pmol a,a’to Nassif and
was monitored a,a’-dipyridyl.
in M9 The seed
Publishers World]oumaf
of Mmobiology 6 Biotechnology. Vol 12, 1996
585
K. Harjai et al. Table 1: Plasmid uroisolates.
distribution
Number of plasmid bands per isolate
Number plasmld bands
of
1
13
2
7
Total number of isolates
Colicin
6
3
that relative
molecular number
production
aer+
aer-
2
2 9 4
0
4 13 9
4 3
3
6
3
3
5
1
4 5
2 3
1 0
1 3
2 0
0 3
1.88
3.66
1.80
2.33
1.72
value
of
and
Relative
HMW’
(11);
their
relative
plasmid
mass
mass
(kb)
19.0 (1); 5.2 (1)t
7.0, 4.1 (1); 17.0,
HMW, HMW,
6.0, 5.2,
in
3.4 3.4
(1); (1);
HMW, HMW,
4.6 (1);
(1);
4.5, 9.5,
HMW,
3.7 4.8
(1); (1);
5.9, 4.8 (1); 11.7, 5.93, 5.54 (1).
19.0. 7.52, 1.5 (1).
5.0, 4.0 (1); HMW,
17.0, 4.8, 4.2, 3.0, 4.6
(1);
HMW,
1.5 (1); 20.0, HMW,
5.9,
22.0,
3.0,
9.5, 6.5, 5.5.
weight bands. t Number in parentheses of isolates containing plasmid bands
5.2
of
culture (prepared by inoculating strains into either M9 or M9 containing dipyridyl) was added to medium at 2% (v/v). Incubation was continued and growth was assayed at 610 nm (Harjai et al. 1990).
Detection of Aerabactin Prodwtion Aerobactin was detected by a cross-feeding test as described by Carbonetti and Williams (1985) using E. coli LGI522 as the indicator strain (this mutant is impaired in the uptake of enterochelin and synthesis of aerobactin, but has an intact receptor for the ferric-aerobactin complex). A lawn of the indicator strain (I x IO6 bacteria per plate) was made on plates of Luria-agar containing 200 pmol a,cr’-dipyridyl. A single colony of the test strains was stabbed in one of the sectors of this plate. Production of aerobactin was visualised as a halo of growth surrounding the aerobactinproducer strain. E. cali 294 (pColV-K30) and E. cob HBlOl were used as positive and negative controls respectively. These two strains were obtained from the Institute of Microbial Technology, Chandigarh, India.
Humid Analysis plasmid
DNA
was prepared
fied overnight cultures using the alkaline lysis method (Bimboin & Doly 1979). DNA samples were resolved on 0.7% agarose gels at 6 V/cm and bands were observed at 260 nm using U.V. transilluminator. Lambda DNA digested with Hind111 and EcoRl were used as molecular size markers.
Results
4.8 (1); HMW,
mass.
For small scale isolation,
Aerobactin
0 0
(1).
the
35 E. co/i
4
5.0,
* HMW-high represents
col -
among
13 7
HMW. 2
production
production
1 2
HMW, 7.52 (1); 11.7, 7.52 5.54 (1); HMW, HMW (1). 3
and colicin
co1+
of plasmids of E. co/i.
Number isolates tested
to aerobactln
0
median
Table 2: Frequency different uroisolates
in relation
from
unampli-
Plasmid profiles of 35 Escherichiu co/i strains revealed the presence of one, or more than one plasmid band in 31 isolates. The median value of plasmid bands isolated was 1.88 plasmid bands per isolate. The number of plasmid bands detected in an isolate varied from o to 5 (Table 1) and their relative plasmid mass is shown in Table 2. Plasmid profiles were correlated with aerobactin production by these isolates. No relationship between plasmids and aerobactin could be inferred due to the presence of a large number of plasmid bands of different sizes in the producer strains. However, the aerobactin producing strains were found to have a higher number of plasmid bands as compared to non-aerobactin producers. The median value was 2.33 for the aer+ strains and 1.72 for aer- strains (Table I). Similarly, for colicin production a higher median value was observed for co1 ’ strains as compared to colstrains (3.66 and 1.80 plasmid bands per isolate respectively) (Table I). Growth of the E. co/i strains also correlated with the number of plasmid bands. Maximum growth inhibition in iron depleted medium was observed in strains having more than one plasmid band (Figure I). Percentage growth inhibition was lower in strains possessing smaller numbers of plasmid bands, while strains with no plasmid bands showed minimal growth inhibition.
Discussion The presence of a higher number of plasmids in clinical isolates of E. coli as compared to environmental isolates has been observed previously (Platt et al. 1984). The present study shows that uropathogens are likely to possess plas-
Plasmid
distribution
in urinary
Escherichia coli
plasmids cannot effectively compete with other less encumbered organisms. Aerobactin production may thus serve an important role in these circumstances (Castignetti & Smarda 1986). In the urinary tract, in which there is iron limitation (Warner et al. 1981), the ability to produce aerobactin would be of special value in the growth and establishment of a uropathogen. This indicates that while the presence of a higher number of plasmids may not be enough to provide an ecological advantage to the bacteria, properties encoded by plasmids will determine multiplication and ultimate survival in such an environment.
J-*
* *
References Bindereif, A. & Neilands, J.B. 1985 Aerobactin isolates
of Escherichia
cd. ]ourmd
of Bacteriology
genes in clinical 161, 727-735.
Bimboim, H. & Doly J. 1979 A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Research 7, 1513-1523.
I0
T
1
NUMBER
3
4
5
OF PLASM10
BANDS Figure 1. Extent of growth inhibition in relation to the number of plasmid
in the presence bands detected.
of dipyridyl
mids in higher numbers with a median value of 1.88 plasmid bands per isolate. Co1 V plasmid encodes both aerobactin synthesis and colicin production (Williams 1979). However, in our study there was no correlation between these two properties since only 4 strains producing aerobactin elaborated colicin as well. This confirms the earlier observations of Martinez et al (1989) who failed to find an association between colicin production and the number of plasmids in aerobactin-positive strains. We have observed that the majority of aerobactin-positive strains carried more than one plasmid band (median value 2.33) indicating that strains exhibiting a larger number of plasmid bands are more likely to acquire aerobactin producing ability. These findings are in accordance with the report of Gonzalo et al. (1988) who concluded that an aerobactin character somehow contributes towards maintenance of extrachromosomal elements. The presence of plasmids has also been shown to affect the evolution of the bacterial chromosome which in turn aids the survival of the bacterial host (Bouma & Lenski 1988). Growth studies in the present investigation indicate that strains harbouring more plasmid bands show maximum growth inhibition when grown in medium M9 containing dipyridyl in comparison to growth in M9 medium alone. Zund and Lebek (1980) and Cooper et al. (1987) suggested that possession of plasmids leads to decreased bacterial growth. In most natural environmental conditions where there is iron starvation, growth rate is likely to be slow, and under these conditions strains harbouring different
Bouma, J.E. & Lenski, R.E. 1988 Evolution of a bacteria/plasmid association. Nature 335, 351-352. Carbonetti, N.H. & Williams, P.H. 1985 Detection of aerobactin production. In Virulence of Escherichiu co/i Reviews und Methods, Volume 13, ed Sussmann, M. pp. 419-424. London: Academic Press. Carbon&i, N.H., Boonchai, S., Parry, S.H., Vaisanen-Rhen, V., Korhonen, T.K. & Williams, P.H. 1986 Aerobactin mediated iron uptake by Escherichiu coli isolates from human extraintestinal infections. Infection and Immunity 5 1, 96&968. Castignetti, D. & Smarda, Jr. J. 1986 Siderophores, the iron nutrition of plants, and nitrate reductase. FEBS Letters 209, 147-151.
Chhibber, S., Harjai, K.K., Sharma, S., Banerjee, S.K. & Vadehra, D.V. 1988 Preliminary observation on the role of klebocin in experimental hematogenous pyelonephritis in mice. Indian ]ownul of Microbiology 28, 306-309. Clowes, R.C. & Hayes, W. 1968 Experiments in Microbial Genetics. Oxford and Edinburgh: Blackwell Scientific Publications. Cooper, N., Brown, M. & Caulcott, C.A. 1987 A mathematical method for analyzing plasmid stability in micro-organisms. ]ournu~ of General Microbiology 133, 1871-1880. Davies, D.L., Falkiner, F.R. & Hardy, K.G. 1981 Colicin V production by clinical isolates of Escherichiu co/i. Infection and Immunity 31, 574-579. Gonzalo, M.R., Martinez, J.L., Baquero, F., Gomez-Lus, R. & Perez-Diaz, J.C. 1988 Aerobactin production linked to transferable antibiotic resistance in Escherichiu coli strains isolated from sewage. FEMS Microbiology Letters 50, 57-59. Harjai, K., Saxena, M., Chhibber, S. & Sharma, S. 1990 In vitro growth of urinary Escherichiu co/i related to siderophore production. Folia Microbiologicu 35, 149-154. Harjai, K., Chhibber, S., Rao Bhau, L.N. & Sharma, S. 1994 Introduction of plasmid carrying an incomplete set of genes for aerobactin production alters virulence of Escherichiu coli HBlOl. Microbial Puthogenesis 17, 261-270. Johnson, J.R., Moseley, S.L., Roberts, P.L. & Stamm, W.E. 1988 Aerobactin and other virulence factor genes among strains of Escherichiu coli causing urosepsis: association with patients characteristics. Infection and lmmwzify 56, 405-412. Johnson, J.R. 1991 Virulence factors in Escherichiu coli urinary tract infection. Clinical Microbiology Reviews 4, 80-128. Martinez, J.L., Cercenado, E. & Baquero, F. 1989 Aerobactin
K. Hmjui et al. production and plasmid distribution in Escherichia co/i clinical isolates. FEMS Microbiology Leffers 60, 41-44. Montgomerie, J.R., Kalmanson, G.M., Harwick, H.J. & Guze, L.B. I973 Relation between bacteriocin production and virulence of Streptococcus faecalis var. liquefaciens. Proceedings of the Society for Experimenfal Biology and Medicine 144, 868-870. Nassif, S. & Sansonetti, PJ. Correlation of the virulence of Klebsiela pneumoniae Kl and KZ with the presence of a plasmid encoding aerobactin. Infection and Immunity 54, 603-608. Orskov, I., Svanborg Eden, C. & Orskov, F. 1988 Aerobactin production of serotyped Escherichia coli from urinary tract infections. Medical Microbiology and Immunology 177, 9-14. Platt, D.J., Sommerville, J.S., Kraft, C.A. & Timbury, M.C. 1984 Antimicrobial resistance and the ecology of Escherichia coli plasmids. ]oumal of Hygiene 93, 181-188. Quakenbush, R.L. & Falkow, S. 1979 Reiationship between colicin V activity and virulence in Escherichia co/i. Infection and Immunity
24,562-564. Shand, G.H., Silverman, bacteria in conditions. Smith, H.W. association
588
Anwar, H., Kadurugamuwa, S.H. & Melling, J. 1985. In urinary tract infection grow Infection and Imm~nify 48, 35-39. & Huggins, M.B. 1976 Further of the colicin V plasmid of
World ~ouml
of Microbmlogy
J., Brown, M.R.W., viva evidence that under iron-restricted observations Escherichia
6 Biofechnology. Vol 12, 1996
on the co2i with
pathogenicity and with survival in the alimentary tract. Journal of General Microbiology 92, 335-350. Valvano, M.A. & Crosa, J.H. 1984 Aerobactin iron transport genes commonly encoded by certain Co1 V plasmids occur in the chromosome of human invasive strain of Escherichia coli KI. Infecfion and Immunity 46, 159-167. Valvano, M.A., Silver, R.P. & Crosa, J.H. 1986 Occurrence of chromosome or plasmid mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Escherichiu coli KI. Infection and Immunify 52, 192-199. Warner, P.J., Williams, P.H., Bindereif, A. & Neilands, J.B. 1981 Co1 V plasmid-specified aerobactin synthesis by invasive strains of Escherichia coli. Infection and Immunity 33, 540-545. Williams, P.H. I979 Novel iron uptake system specified by co1 V plasmids: an important component in the virulence of invasive strains of Escherichia co/i. Infection and Immunify 26, 925-932. Zund, P. & Lebek, G. I980 Generation time prolonging R plasmids: correlation between increase in the generation time of Escherichiu coli by R plasmids and their molecular size. Plasmid
3, 65-69. (Received 1996)
in revised
form
27 March
1996;
accepted
28 March
of Microbiology
& Biotechnology
12, 585-588
Plasmid distribution in Escherichia co/i urinary isolates with special reference to aerobactin and colicin production K. Harjai, S. Pajni, S. Chhibber and S. Sharma” Plasmids were detected in 31 out of 35 strains of Escherichia coli isolated from unclassified tract infection at a median value of 1.88 plasmid bands per isolate. The isolates showed aerobactin and colicin production with the distribution of plasmid bands having a median value (plasmid bands per isolate) in aerobactin-positive and aerobactin-negative strains respectively. For the median plasmid bands per isolate was 3.66 compared to 1.80 for colicin-negative strains. Key words: Aerobactin,
colicin, Escherichia coli, growth,
plasmid profile.
Association between the number of virulence attributes of Escherichia coli and its ability to establish in the urinary tract has shown that many of these characteristics are plasmidmediated (Johnson 1991). Colicin production, one of the plasmid-mediated characters, provides an advantage to the organism in infections of the urinary tract (Montgomerie et al. 1973; Davies et al. 1981; Chhibber et al. 1988). Although possession of the colicin V plasmid markedly enhances virulence, colicin V activity itself may not be the only factor responsible (Quakenbush & Falkow 1979) since this plasmid also carries genes for aerobactin production (Smith & Huggins 1976; Williams 1979). The latter characteristic has also been shown to play an important role in the pathogenicity of the infecting organism especially in the urinary tract (Carbonetti ef al. 1986; Harjai et al. 1994). The genes for aerobactin production are known to be present either on the chromosome or the plasmid (Valvano & Crosa 1984; Valvano et al. 1986). Emphasis has mainly been on the incidence of production in clinical isolates (Carbonetti et al. 1986; Orskov et ul. 1988; Harjai ef al. 1990) and the genetics of this characteristic (Valvano & Crosa 1984; Bindereif & Neilands 1985; Johnson ef al. 1988). However, only limited information is available on the distribution of plasmids in urinary isolates of E. coli with reference to aerobactin production. The purpose of
the present investigation was to study the plasmid profiles of Escherichiu coli uroisolates and to correlate them with their ability to grow and produce aerobactin in iron depleted conditions which prevails in the urinary tract in viuo (Shand ef al. 1985).
Materials Bacterial
@ 1996 Rapid Science
Panjab University. author.
and Methods
Strains
Thirty five clinical isolates of Escherichiu cob, were isolated from the urine of patients with unclassified urinary tract infection. Detection of Colicin Production production was tested using the agar overlay method (Ctowes & Hayes 1968). Nutrient agar plates containing 0.5 gg/ml Colicin
mitomycin
C were
prepared,
air dried
and inoculated
with
selected
strains. Plates were incubated overnight at 37°C and subsequently exposed to chloroform for 10-15 min. The indicator strain E. cali Kl2 was grown in nutrient broth at 37°C for 4 h. At the end of the incubation, the broth was mixed 50°C and overlaid on plates seeded
with with
soft nutrient agar at the test strains. The
plates were incubated at 37°C overnight. Clear zones around the stabbed strains indicated colicin production. E. coli BM21 (pCoIVK30) and E. Co/i BM2l were used as positive and negative controls respectively. Bacterial Growth Iron
restricted
dipyridyl The authors are with the Department of Microbiology, Chandigarh-160014, India; fax: 541409. * Corresponding
cases of urinary an association of of 2.33 and 1.72 colicin producers,
Sansonetti
conditions
were
created
by adding
to synthetic medium M9 according (1986).
The
growth
medium and in M9 medium
profile containing
150 pmol a,a’to Nassif and
was monitored a,a’-dipyridyl.
in M9 The seed
Publishers World]oumaf
of Mmobiology 6 Biotechnology. Vol 12, 1996
585
K. Harjai et al. Table 1: Plasmid uroisolates.
distribution
Number of plasmid bands per isolate
Number plasmld bands
of
1
13
2
7
Total number of isolates
Colicin
6
3
that relative
molecular number
production
aer+
aer-
2
2 9 4
0
4 13 9
4 3
3
6
3
3
5
1
4 5
2 3
1 0
1 3
2 0
0 3
1.88
3.66
1.80
2.33
1.72
value
of
and
Relative
HMW’
(11);
their
relative
plasmid
mass
mass
(kb)
19.0 (1); 5.2 (1)t
7.0, 4.1 (1); 17.0,
HMW, HMW,
6.0, 5.2,
in
3.4 3.4
(1); (1);
HMW, HMW,
4.6 (1);
(1);
4.5, 9.5,
HMW,
3.7 4.8
(1); (1);
5.9, 4.8 (1); 11.7, 5.93, 5.54 (1).
19.0. 7.52, 1.5 (1).
5.0, 4.0 (1); HMW,
17.0, 4.8, 4.2, 3.0, 4.6
(1);
HMW,
1.5 (1); 20.0, HMW,
5.9,
22.0,
3.0,
9.5, 6.5, 5.5.
weight bands. t Number in parentheses of isolates containing plasmid bands
5.2
of
culture (prepared by inoculating strains into either M9 or M9 containing dipyridyl) was added to medium at 2% (v/v). Incubation was continued and growth was assayed at 610 nm (Harjai et al. 1990).
Detection of Aerabactin Prodwtion Aerobactin was detected by a cross-feeding test as described by Carbonetti and Williams (1985) using E. coli LGI522 as the indicator strain (this mutant is impaired in the uptake of enterochelin and synthesis of aerobactin, but has an intact receptor for the ferric-aerobactin complex). A lawn of the indicator strain (I x IO6 bacteria per plate) was made on plates of Luria-agar containing 200 pmol a,cr’-dipyridyl. A single colony of the test strains was stabbed in one of the sectors of this plate. Production of aerobactin was visualised as a halo of growth surrounding the aerobactinproducer strain. E. cali 294 (pColV-K30) and E. cob HBlOl were used as positive and negative controls respectively. These two strains were obtained from the Institute of Microbial Technology, Chandigarh, India.
Humid Analysis plasmid
DNA
was prepared
fied overnight cultures using the alkaline lysis method (Bimboin & Doly 1979). DNA samples were resolved on 0.7% agarose gels at 6 V/cm and bands were observed at 260 nm using U.V. transilluminator. Lambda DNA digested with Hind111 and EcoRl were used as molecular size markers.
Results
4.8 (1); HMW,
mass.
For small scale isolation,
Aerobactin
0 0
(1).
the
35 E. co/i
4
5.0,
* HMW-high represents
col -
among
13 7
HMW. 2
production
production
1 2
HMW, 7.52 (1); 11.7, 7.52 5.54 (1); HMW, HMW (1). 3
and colicin
co1+
of plasmids of E. co/i.
Number isolates tested
to aerobactln
0
median
Table 2: Frequency different uroisolates
in relation
from
unampli-
Plasmid profiles of 35 Escherichiu co/i strains revealed the presence of one, or more than one plasmid band in 31 isolates. The median value of plasmid bands isolated was 1.88 plasmid bands per isolate. The number of plasmid bands detected in an isolate varied from o to 5 (Table 1) and their relative plasmid mass is shown in Table 2. Plasmid profiles were correlated with aerobactin production by these isolates. No relationship between plasmids and aerobactin could be inferred due to the presence of a large number of plasmid bands of different sizes in the producer strains. However, the aerobactin producing strains were found to have a higher number of plasmid bands as compared to non-aerobactin producers. The median value was 2.33 for the aer+ strains and 1.72 for aer- strains (Table I). Similarly, for colicin production a higher median value was observed for co1 ’ strains as compared to colstrains (3.66 and 1.80 plasmid bands per isolate respectively) (Table I). Growth of the E. co/i strains also correlated with the number of plasmid bands. Maximum growth inhibition in iron depleted medium was observed in strains having more than one plasmid band (Figure I). Percentage growth inhibition was lower in strains possessing smaller numbers of plasmid bands, while strains with no plasmid bands showed minimal growth inhibition.
Discussion The presence of a higher number of plasmids in clinical isolates of E. coli as compared to environmental isolates has been observed previously (Platt et al. 1984). The present study shows that uropathogens are likely to possess plas-
Plasmid
distribution
in urinary
Escherichia coli
plasmids cannot effectively compete with other less encumbered organisms. Aerobactin production may thus serve an important role in these circumstances (Castignetti & Smarda 1986). In the urinary tract, in which there is iron limitation (Warner et al. 1981), the ability to produce aerobactin would be of special value in the growth and establishment of a uropathogen. This indicates that while the presence of a higher number of plasmids may not be enough to provide an ecological advantage to the bacteria, properties encoded by plasmids will determine multiplication and ultimate survival in such an environment.
J-*
* *
References Bindereif, A. & Neilands, J.B. 1985 Aerobactin isolates
of Escherichia
cd. ]ourmd
of Bacteriology
genes in clinical 161, 727-735.
Bimboim, H. & Doly J. 1979 A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Research 7, 1513-1523.
I0
T
1
NUMBER
3
4
5
OF PLASM10
BANDS Figure 1. Extent of growth inhibition in relation to the number of plasmid
in the presence bands detected.
of dipyridyl
mids in higher numbers with a median value of 1.88 plasmid bands per isolate. Co1 V plasmid encodes both aerobactin synthesis and colicin production (Williams 1979). However, in our study there was no correlation between these two properties since only 4 strains producing aerobactin elaborated colicin as well. This confirms the earlier observations of Martinez et al (1989) who failed to find an association between colicin production and the number of plasmids in aerobactin-positive strains. We have observed that the majority of aerobactin-positive strains carried more than one plasmid band (median value 2.33) indicating that strains exhibiting a larger number of plasmid bands are more likely to acquire aerobactin producing ability. These findings are in accordance with the report of Gonzalo et al. (1988) who concluded that an aerobactin character somehow contributes towards maintenance of extrachromosomal elements. The presence of plasmids has also been shown to affect the evolution of the bacterial chromosome which in turn aids the survival of the bacterial host (Bouma & Lenski 1988). Growth studies in the present investigation indicate that strains harbouring more plasmid bands show maximum growth inhibition when grown in medium M9 containing dipyridyl in comparison to growth in M9 medium alone. Zund and Lebek (1980) and Cooper et al. (1987) suggested that possession of plasmids leads to decreased bacterial growth. In most natural environmental conditions where there is iron starvation, growth rate is likely to be slow, and under these conditions strains harbouring different
Bouma, J.E. & Lenski, R.E. 1988 Evolution of a bacteria/plasmid association. Nature 335, 351-352. Carbonetti, N.H. & Williams, P.H. 1985 Detection of aerobactin production. In Virulence of Escherichiu co/i Reviews und Methods, Volume 13, ed Sussmann, M. pp. 419-424. London: Academic Press. Carbon&i, N.H., Boonchai, S., Parry, S.H., Vaisanen-Rhen, V., Korhonen, T.K. & Williams, P.H. 1986 Aerobactin mediated iron uptake by Escherichiu coli isolates from human extraintestinal infections. Infection and Immunity 5 1, 96&968. Castignetti, D. & Smarda, Jr. J. 1986 Siderophores, the iron nutrition of plants, and nitrate reductase. FEBS Letters 209, 147-151.
Chhibber, S., Harjai, K.K., Sharma, S., Banerjee, S.K. & Vadehra, D.V. 1988 Preliminary observation on the role of klebocin in experimental hematogenous pyelonephritis in mice. Indian ]ownul of Microbiology 28, 306-309. Clowes, R.C. & Hayes, W. 1968 Experiments in Microbial Genetics. Oxford and Edinburgh: Blackwell Scientific Publications. Cooper, N., Brown, M. & Caulcott, C.A. 1987 A mathematical method for analyzing plasmid stability in micro-organisms. ]ournu~ of General Microbiology 133, 1871-1880. Davies, D.L., Falkiner, F.R. & Hardy, K.G. 1981 Colicin V production by clinical isolates of Escherichiu co/i. Infection and Immunity 31, 574-579. Gonzalo, M.R., Martinez, J.L., Baquero, F., Gomez-Lus, R. & Perez-Diaz, J.C. 1988 Aerobactin production linked to transferable antibiotic resistance in Escherichiu coli strains isolated from sewage. FEMS Microbiology Letters 50, 57-59. Harjai, K., Saxena, M., Chhibber, S. & Sharma, S. 1990 In vitro growth of urinary Escherichiu co/i related to siderophore production. Folia Microbiologicu 35, 149-154. Harjai, K., Chhibber, S., Rao Bhau, L.N. & Sharma, S. 1994 Introduction of plasmid carrying an incomplete set of genes for aerobactin production alters virulence of Escherichiu coli HBlOl. Microbial Puthogenesis 17, 261-270. Johnson, J.R., Moseley, S.L., Roberts, P.L. & Stamm, W.E. 1988 Aerobactin and other virulence factor genes among strains of Escherichiu coli causing urosepsis: association with patients characteristics. Infection and lmmwzify 56, 405-412. Johnson, J.R. 1991 Virulence factors in Escherichiu coli urinary tract infection. Clinical Microbiology Reviews 4, 80-128. Martinez, J.L., Cercenado, E. & Baquero, F. 1989 Aerobactin
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World ~ouml
of Microbmlogy
J., Brown, M.R.W., viva evidence that under iron-restricted observations Escherichia
6 Biofechnology. Vol 12, 1996
on the co2i with
pathogenicity and with survival in the alimentary tract. Journal of General Microbiology 92, 335-350. Valvano, M.A. & Crosa, J.H. 1984 Aerobactin iron transport genes commonly encoded by certain Co1 V plasmids occur in the chromosome of human invasive strain of Escherichia coli KI. Infecfion and Immunity 46, 159-167. Valvano, M.A., Silver, R.P. & Crosa, J.H. 1986 Occurrence of chromosome or plasmid mediated aerobactin iron transport systems and hemolysin production among clonal groups of human invasive strains of Escherichiu coli KI. Infection and Immunify 52, 192-199. Warner, P.J., Williams, P.H., Bindereif, A. & Neilands, J.B. 1981 Co1 V plasmid-specified aerobactin synthesis by invasive strains of Escherichia coli. Infection and Immunity 33, 540-545. Williams, P.H. I979 Novel iron uptake system specified by co1 V plasmids: an important component in the virulence of invasive strains of Escherichia co/i. Infection and Immunify 26, 925-932. Zund, P. & Lebek, G. I980 Generation time prolonging R plasmids: correlation between increase in the generation time of Escherichiu coli by R plasmids and their molecular size. Plasmid
3, 65-69. (Received 1996)
in revised
form
27 March
1996;
accepted
28 March
