Molecular characterization of cholera outbreak isolates in South Africa, 2008-2009
Date
2016
Authors
Ismail, Husna
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Abstract
Cholera is an acute diarrhoeal disease that generally presents as abrupt watery diarrhoea and
vomiting. For the years 2008 to 2009, South Africa experienced two major outbreaks of
cholera. The first outbreak was reported from May to July 2008 (Chapter Three) and the
second outbreak from November 2008 to April 2009 (Chapter Four). Within both events,
Vibrio cholerae (V. cholerae) O1 identified at peripheral laboratories displayed resistance to
three or more routinely tested antimicrobial agents. The molecular epidemiology and
mechanism of antimicrobial resistance of V. cholerae O1 isolates was investigated. This was
achieved by using various molecular techniques, which included pulsed-field gel
electrophoresis (PFGE) analysis, polymerase chain reaction (PCR), nucleotide sequencing,
identification of plasmid DNA and Southern blot hybridization analysis.
Methods
As part of routine characterization of V. cholerae isolates at the Centre for Enteric Diseases
(CED), isolates underwent serological and biochemical confirmatory identification as well as
antimicrobial susceptibility testing using the Etest method. PFGE analysis was performed on
V. cholerae O1 isolates digested with NotI restriction enzyme. One-hundred V. cholerae O1
isolates, ten isolates characterized in Chapter Three and 90 isolates characterized in Chapter
Four were selected for further analysis to ensure that all PFGE banding patterns were
represented. Three probable mechanisms of antimicrobial resistance were investigated.
Firstly, PCR was used to detect for the presence of class 1 integrons (3’-CS and 5’-CS), class
2 integrons (intI2), plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrS,
qnrC and qepA), quinolone resistance determinant (qnrVC3), ESBL producing genes (blaTEM,
blaSHV and blaCTX-M), genes coding for the quinolone resistance-determining region (QRDR)
of DNA gyrase (gyrA/gyrB) and topoisomerase IV (parC/parE), SXT element-integrase gene
(SXTint) and associated SXT resistance genes (floR, sul2, dfrA1, dfr18, strA and strB) and
the class A tetracycline resistance determinant (tetA).
The presence of resistance plasmids was investigated by isolation of intact bacterial plasmid
DNA. Southern blotting and DNA probing was used to investigate the location of resistance
genes on the plasmids. Secondly, nucleotide sequencing was used to detect amino acid
mutations in the QRDR of DNA gyrase and topoisomerase IV respectively. Thirdly, to
determine the role of an active efflux pump in quinolone resistance, susceptibility testing to
nalidixic acid was investigated in ten V. cholerae O1 isolates characterised in Chapter Three
using agar dilution in the presence and absence of two efflux pump inhibitors, reserpine and
phenylalanyl arginine- -naphthylamide. PCR analysis was used to detect for virulence
determinants, which included the enzymatic A subunit of the cholera toxin (CT), ctxA and the
gene encoding for the toxin co-regulated pilus (TCP), tcpA respectively. In addition, the
complete coding region of the ctxAB gene was amplified and sequenced from four V.
cholerae O1 isolates, two isolates characterized in Chapter Three and two isolates
characterized in Chapter Four as several V. cholerae O1 atypical El Tor isolates have been
described in Africa. Minimum inhibitory concentration (MIC) values for azithromycin were
determined for all 100 V. cholerae O1 isolates using both the Etest and agar dilution methods
(Chapter Five). PCR-analysis was used to determine the presence of seven macrolide
resistance determinants (mefA, ereA, ereB, ermB, mphA, mphB and mphD) in all 100 V.
cholerae O1 isolates.
Results
For both cholera outbreaks, a total of 751 isolates were received and available for analysis.
All 31 isolates recovered from the first outbreak (Chapter Three) were characterized as V.
cholerae O1 serotype Ogawa. For the second outbreak (Chapter Four) 708 isolates were
characterized as serotype Ogawa, while the remaining 12 isolates were characterized as
serotype Inaba. All isolates analyzed from both outbreaks were susceptible to ciprofloxacin
and imipenem, but resistant to six or more antimicrobial agents tested for surveillance
purposes. All V. cholerae O1 isolates were shown to be resistant to nalidixic acid, cotrimoxazole,
trimethoprim, sulfamethoxazoleand streptomycin. Extended-spectrum -
lactamase (ESBL) activity was observed in V. cholerae O1 isolates (MIC 64 μg/ml) from
both outbreaks. In the second outbreak reduced susceptibility to ampicillin, tetracycline,
kanamycin, chloramphenicol, erythromycin and furazolidone were observed. Dendrogram
analysis produced two main PFGE clusters. PFGE fingerprint patterns from V. cholerae O1
isolates recovered from the first outbreak clustered away from V. cholerae O1 isolates
recovered from the second outbreak (data not shown in this study). Class 1 integrons, class 2
integrons and PMQR genes were not detected by PCR.All 100 V. cholerae O1 isolates were
PCR-positive for the SXTint gene and five of the six associated SXT resistance genes
encoding for chloramphenicol (floR), sulfamethoxazole (sul2), trimethoprim (dfrA1) and
streptomycin (strA and strB). Seventeen V. cholerae O1 isolates (ten isolates characterized in
Chapter Three and seven isolates characterized in Chapter Four) were PCR-positive for the
tetA resistance determinant. Nucleotide sequencing of the QRDR, showed that all nalidixic
acid-resistant isolates harboured the same mutations in GyrA (S83-I) and ParC (S85-L) but
none were observed in GyrB and ParE.There was no involvement of an active efflux pump in
quinolone resistance in ten isolates characterised in Chapter Three. Sixteen V. cholerae O1
isolates (ten isolates characterized in Chapter Three and six isolates characterized in Chapter
Four) harboured a single plasmid of approximately 140 kilobase pairs in size and showed to
harbour the blaTEM gene, which produced the TEM-63 -lactamase.PCR analysis showed that
all 100 V. cholerae O1 isolates were positive for the CT, and all were PCR-positive for the El
Tor variant of the TCP. Nucleotide sequencing of the ctxAB gene of the four selected isolates
showed that all four isolates expressed the encoded ctxB allele for the CT of the classical
biotype and were defined as “altered El Tor”. A mobilome is characterized by related genome
sequences that differ by combinations of genomic islands, prophages and integrative
conjugative elements. All four isolates contained an identical mobilome profile pattern,
profile B. Comparative analysis using both the Etest and agar dilution methods (Chapter
Five) showed that all V. cholerae O1 isolates were susceptible to azithromycin provided that
the tentative breakpoint of 16μg/ml is applied. All 100 isolates were PCR-negative for all
seven macrolide resistance determinants, which are commonly associated in the family
Enterobacteriaceae respectively.
Conclusion
This is the first incidence of TEM-63 -lactamase-producing, antimicrobial-resistant,
toxigenic V. cholerae O1 altered El Tor isolates in South Africa. This study highlights the
need to further analyze antimicrobial resistance and track emerging epidemic isolates of V
cholerae O1. The MIC values and PCR results reported in this study for azithromycin
provides a foundation for the surveillance of azithromycin susceptibility and to determine
MIC breakpoints in V. cholerae O1 isolates circulating in South Africa.
Description
A thesis submitted to the Faculty of Health Science, University of the Witwatersrand,
Johannesburg, South Africa, in fulfilment of the requirements for the degree, Doctorate of
Philosophy in Medicine, 2009-2015