Materials and Methods
Bacterial Strains
Outbreak causing as well as the sporadic MDR blood
culture isolates of S. typhi (resistance pattern ACCoT)
obtained in between 1991-2001, and the other enteric
bacteria (E. coli, K. pneumoniae and P. vulgaris from
urinary tract infection cases) that had the common
resistance pattern ACCoT during 1995-2001 were
used to study the transferability of their multidrug
resistance. The MDR S. typhi isolates showed either
sensitivity or resistance to nalidixic acid (Nx). The
antibiotic sensitive S. typhi and the E. coli C600 (F—
NxR) strains were also included in the study, as the
recipient strains. The S. typhi recipient strain was
obtained by culturing blood from enteric fever patient
attending the Calcutta School of Tropical Medicine for
treatment. E. coli V517 strain was used as the
molecular marker for plasmid.
In Vitro Transferability of Antibiotic Resistance
Transfer of antibiotic resistance of the MDR bacterial
strains (having the common resistance pattern
ACCoT): S. typhi (n=70), E. coli (n=42), K. pneumoniae
(n=13) and P. vulgaris (n=6) was carried out by
conjugation experiments following the standard
protocols (12,13), with slight modification described
elsewhere.(14) The minimum inhibitory
concentrations (MICs) for antibiotics against which
resistance was transferred from donors to the
recipients were checked by agar dilution method.(15)
Isolation of Plasmid DNA and Agarose Gel
Electrophoresis
The MDR isolates of S. typhi (n=70), E. coli (n=42), K.
pneumoniae (n=13) and P. vulgaris (n=6) having the
common resistance pattern ACCoT, the
corresponding transconjugant strains acquiring
ACCoT-resistance, and the antibiotic sensitive S. typhi
isolates (n=20) were subjected for plasmid DNA
isolation following the protocol of Birnboim and Doly
(16), and Kado and Liu (17), with some modifications.
According to the protocol of Birnboim and Doly, from
10 ml of bacterial culture (in nutrient broth, Hi-Media,
India) plasmid DNAs were isolated using 0.48 ml, 1
ml and 0.8 ml of solution I, II and III, respectively.
After the addition of solution III, the lysate was kept
in ice for 30 minutes and centrifuged for 15 minutes.
Phenol-chloroform (1:1) treatment was followed with
the clear supernatant, plasmid DNA was precipitated
with equal volume of chilled isopropyl alcohol, and
DNA pellet was dissolved in 100 μl of TE buffer. In
case of Kado and Liu method, we used chilled
isopropyl alcohol to precipitate the plasmid DNA
instead of diethyl ether.
Agarose gel electrophoresis of the isolated plasmid
DNAs was carried out in tris-borate buffer system
(18), using 0.8% agarose, for 4 h at 50v. The gel was
stained with ethidium bromide and results were
documented in gel-doc system. Electrophoretic
separation of plasmid by molecular weight and
subsequent size estimations were accomplished using
reference strain of E. coli V517.
Results
Transfer of resistance from S. typhi to E. coli C600
The MDR S. typhi strains showing the resistance
pattern ACCoT or ACCoTNx transferred ACCoT-
resistance to E. coli C600 recipient strain, and thus the
recipient strains acquired the resistance traits for A, C,
Co and T. The transfer frequencies among the
epidemic causing S. typhi strains, namely BS13, AS12,
M54 obtained respectively from Bagnan, Asansol and
Khardah in the year 1991, were 0.83 x 10-5, 0.73 x 10-5,
0.78 x 10-5, respectively (Table 1). MDR S. typhi
strains, viz. B2/92, isolated in the year 1992, also
transferred ACCoT-resistance with a frequency of
0.89 x 10-5. In the secondary transfer studies, the E. coli
C600 transconjugants obtained from the primary
conjugation experiments were used as the donors,
and the antibiotic sensitive S. typhi was the recipient
strain. Here also, the transfer of ACCoT-resistance
was noticed (Table 1).