Vibrio cholerae O1 outbreak isolates in Mozambique and South Africa in 1998 are multiple-drug resistant, contain the SXT element and the aadA2 gene located on class 1 integrons

A. Dalsgaarda,*, A. Forslunda, D. Sandvanga, L. Arntzenb and K. Keddyb

a Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark; b Division of Epidemic Enteric Diseases, South African Institute for Medical Research, PO Box 1038, Johannesburg 2000, South Africa


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The characteristics of Vibrio cholerae O1 biotype El Tor, serotype Ogawa isolates from outbreaks of cholera in 1998 amongst migrant workers in the South African provinces of Gauteng and Mpumalanga, on the border of Mozambique, are reported. The isolates seem to have originated from the same clone since they are of two closely related BglI ribotypes. These ribotypes had a high similarity to ribotypes of V. cholerae O1 recently found in three South-east Asian countries. Isolates were resistant to furazolidone, streptomycin, sulfamethoxazole, trimethoprim and tetracycline. Only two isolates contained plasmids of 54 and 63 kb in size. PCR and DNA sequencing revealed that the chromosomally located resistance determinants present included an aadA2 gene cassette contained in a class 1 integron; the SXT element, which is a transposon-like element containing resistance genes; and the tetA gene. A co-transfer of chromosomal closely located genes encoding the SXT element and tetA was shown by mating experiments, PCR and pulsed-field gel electrophoresis analyses. Our study shows for the first time that multiple-resistant V. cholerae O1 isolates containing class 1 integrons and the SXT element were responsible for cholera outbreaks in Southern Africa. Studies are needed to determine the spread of this multiple-resistant O1 strain and further genetic details of the association of the SXT element, tetA and class 1 integrons, including their means of transfer.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
African countries have in recent years experienced more epidemics and cases of cholera than countries in South-east Asia.1,2 After the seventh pandemic caused by Vibrio cholerae O1 biotype El Tor reached and initially spread in West African countries in the 1970s, the majority of countries in the Eastern and Southern parts of Africa have experienced major cholera epidemics, occasionally with unusually high mortalities ranging from 2 to 20%, in rare cases.1,2 A total of 293 121 cholera cases and 10 586 deaths were reported to the World Health Organization (WHO) in 1998, with Africa accounting for the largest part with 72% of the global total.1 In contrast to the V. cholerae strains causing outbreaks in South-east Asia, which have been extensively characterized, little information is available about the characteristics of the recent epidemic strains implicated in cholera outbreaks in Africa.

Mozambique was hit in August 1997 by an epidemic caused by V. cholerae O1 biotype El Tor with the number of cases totalling more than 10 000 by the end of 1997. The epidemic continued into 1998 and 1999, with V. cholerae O1 being introduced into South Africa in 1998 with migrant workers from Mozambique, in particular to the provinces of Gauteng and Mpumalanga, the latter bordering Mozambique.3 Although all the cases in Gauteng were identified in migrant labourers, many of those in Mpumalanga were acquired in South Africa through contamination of local water sources. In addition to fluid and electrolyte replacement, antibiotics were found to be effective in the treatment of cholera. However, resistance to co-trimoxazole and tetracycline was reported. Subsequently, 27 cases of cholera were identified in children in an informal settlement in Kwazulu-Natal in 1999, the third province to have been affected by cholera (communication from the Department of Health of South Africa). However, only limited information has been provided about the characteristics of the V. cholerae O1 strains responsible for these outbreaks, including their genotype(s) and the genetic mechanisms encoding the antibiotic resistance reported.

Although there may often be only scarce information reported about the antibiotic susceptibility of epidemic V. cholerae strains, particularly in African countries, it appears that antibiotic-resistant V. cholerae strains are increasingly being found worldwide.4–8 Plasmids have encoded and transferred resistance in V. cholerae O1,7,9,10 but rarely in V. cholerae O139,11 the other serotype causing cholera. A novel genetic element, termed the SXT element, which has properties similar to those of a conjugative transposon, was found to carry genes encoding resistance to sulfamethoxazole, trimethoprim and streptomycin in V. cholerae O139 and O1 strains isolated in India, but was not present in O1 strains obtained in 1994 from Rwandan refugees in Goma, Zaire.12 A recent report showed that gene cassettes contained in class 1 integrons were distributed among different V. cholerae O-serotypes of mainly clinical origin in Thailand.13 Such cassettes also contained resistance genes to aminoglycosides in V. cholerae O1 strains isolated after 1990 in Vietnam14 as well as in O1 strains isolated during a cholera outbreak in Albania and Italy in 1994.15 Other studies indicate that class 1 integrons are commonly found in multiple antibiotic-resistant clinical isolates belonging to different bacterial species.16–18 Class 1 integrons are gene expression elements that have been described as vehicles for a possible horizontal acquisition of resistance gene cassettes. The genetic composition and reviews of class 1 integrons have been provided elsewhere.19–23

The current study was initiated to determine characteristics of V. cholerae O1 isolated in 1998 from migrant workers during cholera outbreaks in two South African provinces, one of which is situated along the border with Mozambique. We have analysed the clonal relationship between these isolates and the genetic mechanisms encoding antibiotic resistance in them. In particular we have studied the presence and transfer of the SXT element and resistance genes in class 1 integrons.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sources and characterization of V. cholerae strains

A total of 20 V. cholerae isolated between 30 December 1997 and 29 May 1998 were included in the study (Table 1Go). Fourteen isolates were from migrant workers in the province of Mpumalanga, a predominantly rural area of South Africa bordering Mozambique and Swaziland. Many Mozambican workers pass through and settle in Mpumalanga on their way to seek work in Gauteng, which is a main centre for the gold mining industry. Five isolates were from migrant workers in Gauteng. One isolate (703M) was from a South African woman who had gone to Botswana, Mozambique and Tanzania on holiday.


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Table 1. Characteristics of V. cholerae O1 isolated in Mozambique and South Africa
 
Each isolate was from stool or rectal swabs collected and cultured at local hospitals on to thiosulphate–citrate–bile salts–sucrose agar (Eiken Ltd, Tokyo, Japan) before and after pre-enrichment in alkaline peptone water (pH 8.6) and on to blood agar. Bacterial specimens were sent for confirmation of the identification to the South African Institute for Medical Research (SAIMR) in Johannesburg where they were identified as V. cholerae using criteria described by Sakazaki.24 Isolates were tested for agglutination in polyvalent O1 and mono-specific Ogawa and Inaba antisera (Denka Seiken, Tokyo, Japan). Faecal specimens were not cultured for enteric pathogens other than V. cholerae. Further characterization of the isolates, including colony hybridization, plasmid analysis, mating experiments, ribotyping, PCR, pulsed-field gel electrophoresis (PFGE) and DNA sequencing, was conducted in Denmark.

Isolates were tested by the colony hybridization technique for DNA sequences encoding cholera toxin (CT) using an alkaline phosphatase-labelled 23 bp oligonucleotide probe and appropriate controls.25,26

Antibiotic susceptibility testing

The MICs were tested for each isolate of the following antibiotics supplied by different commercial companies: ampicillin, chloramphenicol, ciprofloxacin, erythromycin, furazolidone, nalidixic acid, streptomycin, tetracycline, trimethoprim and trimethoprim/sulfamethoxazole. Standard powders, with the exception of furazolidone, were dissolved according to the instructions of the manufacturer to give a full concentration of 128 mg/L. Furazolidone was diluted in 0.1 M phosphate buffer at pH 8 in a water bath at 56°C. Volumes of 0.1 mL of each antibiotic were added to the microtitre tray. Two-fold dilutions were prepared in the microtitre tray to a final dilution of 0.125 mg/L. Test and control organisms were suspended directly in cation-adjusted Mueller–Hinton broth (SAIMR) to a 0.5 McFarland standard and diluted 1:10. One drop from the suspension was added to each well containing antibiotics. The plates were incubated aerobically overnight at 37°C and examined for visual growth the following day. For comparison purposes, six V. cholerae O1 isolates from an earlier outbreak in South Africa in 1982 were included in the MIC testing.27 The MIC was defined as the lowest concentration of antibiotic giving no visible growth according to NCCLS guidelines.28 As there are no guidelines for erythromycin, these values were calculated according to Olsson-Liljequist & Möllby.29

Isolation of plasmid DNA and ribotyping

Plasmid preparation was carried out by the method of Kado & Liu30, modified by incubating the cells at elevated pH (12.75) for 30 min at 56°C during the lysis step. Following electrophoresis, the plasmids were visualized as described.31 V. cholerae O1 isolate 1075/25 containing an approximately 150 kb plasmid was used as the control strain.9 Plasmid sizes were estimated from the migration distance in the agarose gels relative to the migration distance of reference plasmids in Escherichia coli strains V517 and 39R86132,33 by the method of Rochelle et al.34 Repeated extraction of plasmid DNA was carried out for all isolates.

Total bacterial DNA was extracted by the method of Murray & Thompson.35 On the basis of previous studies,36,37 the restriction enzyme BglI was used to digest chromosomal DNA. Ribotyping was performed by the procedure described by Dalsgaard et al.37 with digoxigenin-labelled 16S and 23S rRNA probes. A 1 kb DNA molecular size standard (Gibco-BRL, Gaithersburg, MD, USA) was used as a size marker. Ribotype patterns were considered to be different when there was a difference of one or more bands between isolates. Each ribotype was designated VCSA (V. cholerae, South Africa) followed by an arbitrary number. The following isolates were included in the ribotyping studies as well as PCR analysis (see below) for comparison purposes: V. cholerae O1 isolate VC-VN-62 from Vietnam,14 isolate 33/97 from Thailand,38 VC20 and CO840 from India,39 and isolate F2107, isolated during a cholera epidemic in Guinea-Bissau in 1994/1995.40

PCR amplification and DNA sequence analysis

Initially, we selected a specific class 1 primer set qacE{Delta}1 and sul1 directed at the 3'-CS conserved segment of class 1 integrons.22,41 It should be noted that a few class 1 integrons are known not to contain the qacE{Delta}1 and sul1 genes. Thus, such strains would not be detected using these primers. Isolates that did not yield an amplicon using the qacE{Delta}1 and sul primers were tested with another class 1 integron-specific primer set, inDS-F and inDS-B, targeted at the 5'-CS end (Figure 1Go and Table 2Go). A schematic presentation of the general structure of class 1 integrons with the gene cassette found in our study is shown in Figure 1Go. PCR was carried out as described by Dalsgaard et al.37 Isolates yielding a PCR amplicon with the class 1 primers were further amplified with the integron primers in-F and in-B (F, forwards; B, backwards), which amplify the region between the 5'-CS and 3'-CS conserved segments, giving products of variable size depending on the number and length of inserted gene cassettes (Figures 1 and 3GoGo; Table 2Go). Based on the recent findings of class 1 integrons containing aminoglycoside resistance gene cassettes in V. cholerae O1 isolates isolated in Vietnam14 and Thailand,13 the primers in-F and aadA-B were used to determine whether the integron contained a gene cassette encoding resistance to streptomycin and spectinomycin.14,42 All PCR primers used for the detection of class 1 integrons and gene cassettes are listed in Table 2Go. Salmonella enterica serotype Typhimurium strain 9720921 and Acinetobacter spp. strain R4-96 were included as positive and negative controls, respectively.43 A 100 bp molecular weight standard (Gibco-BRL) was used as a size marker during electrophoresis of all PCR products.



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Figure 1. Association between integron structure and PCR products modified from Sandvang et al.3 The lines below the integron structure represent amplicons and the bold line represents the sequenced amplicon. 5'-CS and 3'-CS represent the 5' and 3' conserved segments of the integron; attI the attenuation site; and qacE{Delta}1 and sul1 encode resistance to disinfectant and sulphonamide, respectively. The individual gene cassettes are shown in relative sizes. The recombination site (59 bp element) is shown as a black circle.

 

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Table 2. PCR primers for identification and characterization of integrons and gene cassettes in V. cholerae O1 isolated in Mozambique and South Africa
 


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Figure 3. Examples of PCR products of V. cholerae O1 isolates isolated in Mozambique and South Africa using the primers qacE{Delta}1-F and sul1-B (lanes B and E); primers in-F and in-B (lanes F–H); primers in-F and aadA-B (lanes I–K); SXT primers int1-F and int1-B (lanes M–O); and primers tetA1 and tetA2 (lanes P–R). Unless indicated otherwise, the lanes indicate the isolate designation. Lanes: A, 100 bp molecular weight standard; B, 531D; C, 703M; D, R4-96; E, 9720921; F, 556S; G, R4-96; H, 9720921; I, 711A; J, R4-96; K, 9720921; L, 100 bp molecular weight standard; M, 808M; N, K-12; O, MO10; P, 796E; Q, R4-96; R, NCTC 50078; S, 100 bp molecular weight standard.

 
Tetracycline resistance determinant classes A to E were detected by multiplex PCR.44 The specific sets of primers used for PCR amplification are listed in Table 3Go. Based on the melting temperatures of the primers and the molecular size of the amplification products, three separate PCRs were used, one for TetA and TetE, one for TetB and TetD, and one for TetC. Crude lysates were obtained by heating 100 µL of bacterial suspensions for 5 min at 95°C. After centrifugation at 13 000g for 20 min, 5 µL of the supernantant were used as DNA template for PCR amplification. PCRs used AmpliTag Gold polymerase (Perkin Elmer, Foster City, CA, USA). The conditions were as follows for each specific PCR: (i) TetA and TetE, 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 62°C for 1 min and 72°C for 1 min, followed by a final extension at 72°C for 10 min; (ii) TetB and TetD, 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 57°C for 1 min and 72°C for 1 min, followed by a final extension at 72°C for 10 min; (iii) TetC, 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 62°C for 30 s and 72°C for 45 s, followed by a final extension at 72°C for 10 min. Reference E. coli strains containing the A, B, C, D and E tetracycline resistance determinants were used as positive controls in the multiplex PCR.45


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Table 3. PCR primer sets used for the detection of tetracycline determinant classes A to E in V. cholerae O1
 
Waldor et al.12 identified in V. cholerae O1 and O139 an approximately 62 kb self-transmissible, chromosomally integrating genetic element, which they termed the SXT element. The SXT element was found to contain genes encoding resistance to sulphonamides, trimethoprim and streptomycin.12 As all isolates in our study were resistant to each of these antimicrobials, we decided to investigate whether our isolates contained the SXT element by using PCR primers that produce a 592 bp internal fragment of the integrase of the SXT element. DNA sequences and information about the primers were kindly provided by Dr Matthew K. Waldor at the Harvard Medical School in Boston (Table 2Go). Briefly, PCR conditions were as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60°C for 1 min and 72°C for 2 min, followed by a final extension at 72°C for 10 min. V. cholerae O139 strain MO10 and an E. coli K-12 no. J53-1 strain were used as positive and negative controls, respectively.12

Amplified DNA was purified before sequencing using Microspin S-400HR columns purchased from Pharmacia Biotech (Hørsholm, Denmark) and the nucleotide sequence was determined by cycle sequencer using the AmplitaqFS dye terminator kit and a 373A automatic sequencer (Applied Biosystems/Perkin Elmer, Foster City, CA, USA).46 To analyse the identity of determined sequences, a comparison was made with gene banks using the BLAST software.47

Transfer of class 1 integron gene cassettes, tetracycline resistance determinants and the SXT element

The nalidixic acid-resistant mutant strain E. coli K-12, no. J53-1 (lac+, pro, met and NalR)48 and a streptomycin- and rifampicin-resistant mutant strain of V. cholerae O1 strain 1407 isolated during the epidemic in Guinea-Bissau in 198740 were used as recipients in conjugation experiments using isolates 556S, 711A, 796E, 808M and 995V as donors. After mating on non-selective L agar (Difco, Detroit, MI, USA) incubated at 37°C for 4–6 h growth, transconjugants were harvested and appropriate dilutions were spread on plates of Fluorocult E. coli O157:H7 agar (Merck, Virum, Denmark) supplemented with nalidixic acid 40 mg/L and adequate concentrations of selected drugs. In conjugation experiments with E. coli K-12 as the recipient, tetracycline 10 mg/L, streptomycin 25 mg/L and trimethoprim 32 mg/L were used for selection together with nalidixic acid 40 mg/L, whereas in conjugation experiments with V. cholerae 1407 as the recipient, tetracycline 10 mg/L, spectinomycin 10 mg/L, streptomycin 25 mg/L and trimethoprim 32 mg/L were used for selection together with rifampicin 50 mg/L. Phenotypic appearance was used to differentiate possible spontaneous nalidixic acid-resistant donors from the transconjugants. Transconjugants were studied for transfer of class 1 integron gene cassettes, tetracycline resistance determinants and the SXT element by PCR as described earlier in this article.

PFGE analysis and co-transfer of tetracycline resistance determinants and the SXT element

As tetracycline resistance determinants and the SXT element were found to be transferable in the conjugation experiments (see below), we decided to investigate a possible co-transfer of these resistance genetic elements by the use of PFGE analysis and Southern blotting using SXT and tetA probes. PFGE analysis was carried out using the restriction enzyme NotI as described previously, using a modified contour-clamped homogeneous electric field (CHEF-DR3) system (Bio-Rad, Alleroed, Denmark).49 The running conditions were 6 V/cm at 14°C for 22 h. The pulse times were 15–25 s for 3 h and 8–25 s for 19 h. NotI is the preferred restriction enzyme used in PFGE typing of V. cholerae O1. Furthermore, analysis of the published sequences of the SXT element and the tetA gene using BLAST software revealed no restriction sites within the two gene elements. PCR products of tetA and SXT were amplified as described earlier, labelled with digoxigenin and used as probes in Southern blot analysis. Phage {gamma} DNA (Amersham Pharmacia, Hørsholm, Denmark) was used as molecular size marker with bands separated by increments of 48.5 kb.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Active surveillance for V. cholerae was undertaken in South Africa at the start of the Mozambican epidemic in 1997, complemented by localized campaigns by the Department of Health in high risk areas, such as informal settlements in Mpumalanga and Gauteng and mine hostels in Gauteng. As a result, the first strains of V. cholerae in this country were isolated from swabs of sewage. Unfortunately, none of these isolates was stored. Despite the campaign, a number of cases, both local and imported, did occur, including one death in a child in whom treatment was delayed because of late presentation at the local clinic. Before the outbreak, doxycycline was regarded as the treatment of choice for V. cholerae in South Africa. It only became apparent after the V. cholerae isolates had been sent to the SAIMR reference laboratory that many of the isolates were resistant to tetracyclines. Use of antibiotics was halted once susceptibility patterns became known. Characteristics of the isolates are shown in Table 1Go.

The 20 clinical isolates studied all showed typical biochemical and serological reactions of V. cholerae and were identified as V. cholerae O1 biotype El Tor, serotype Ogawa. All isolates hybridized with the CT probe yielding dark blue or purple colonies.

The MIC results were similar for most of the isolates, confirming that the current outbreak was probably caused by a single V. cholerae O1 strain showing multiple-drug resistance to clinically important drugs including chloramphenicol, trimethoprim, tetracycline and furazolidone (Table 4Go). The MIC testing confirmed susceptibility only to ciprofloxacin for the majority of the strains isolated from the current outbreak. Notable exceptions were isolate 703M, which retained ampicillin and tetracycline susceptibility but showed elevated resistance to nalidixic acid. Also, isolate 1105Y retained tetracycline and chloramphenicol susceptibility. With the exception of isolate 82/31502, the six isolates from the earlier outbreak in 1982 were all fully susceptible to the antibiotics tested (Table 4Go).


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Table 4. MICs for V. cholerae isolates from the recent cholera outbreak in 1997 and that in 1981/1982 in South Africa
 
Repeated plasmid analysis showed that only isolates 796E and 808M contained plasmids of approximately 63 and 54 kb in size, respectively, indicating that the multiple antibiotic resistance found was probably not associated with the presence of plasmids, but was located on the chromosome. The control strain contained an approximately 150 kb plasmid (results not shown).

Ribotyping with BglI produced three closely related ribotype patterns. The majority of isolates were of ribotypes VCSA1 and VCSA2, with the latter type containing a 5.7 kb size fragment that was absent in type VCSA1 (Figure 2Go and Table 1Go). Ribotype VCSA1 showed a high degree of similarity to the identical type shown by isolate VC-VN-62 from Vietnam, isolate 33/97 from Thailand and isolate VC20 from India, as VCSA1 isolates only lacked a 2.2 kb fragment shown by the three control strains. Isolate 703M showed a unique type VCSA3, which was indistinguishable from the typing patterns of isolate CO840 isolated in India in 1995 and isolate F2107 isolated during the cholera epidemic in Guinea-Bissau in 1994/1995.40 Although these three isolates showed an identical ribotype, repeated susceptibility testing to nalidixic acid showed a MIC of 64 mg/L for isolate CO840, whereas for isolate F2107 the MIC was only 0.5 mg/L. Thus, different MICs of nalidixic acid were found for the three isolates. No specific relationship was found between the place of isolation and the ribotypes VCSA1 and VCSA2.



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Figure 2. Examples of BglI ribotypes of V. cholerae O1 isolates associated with disease outbreaks in Mozambique and South Africa in 1998. Explanation of lanes includes isolate designation unless otherwise stated. Lanes: A, 1 kb molecular weight standard; B, VCSA1, 870X; C, VC-VN-62; D, 33/97; E, VC20; F, VCSA2, 1105; G, VCSA3, 703M; H, CO 840; I, F2107; J, 1 kb molecular weight standard.

 
An approximately 800 bp 3'-CS PCR product was obtained from 19 of the 20 V. cholerae isolates studied using the class 1 integron primers qacE{Delta}1-F and sul1-B. Only isolate 703M did not yield an amplicon with these primers. Also, PCR with the class 1 specific primers targeted at the 5'-CS end did not yield any amplicons for isolate 703M. Testing of isolates CO840 and F2107 with the PCR primer sets targeted at the 3'-CS and 5'-CS ends did not yield any amplicons. Thus, none of the three isolates seems to contain class 1 integrons. A PCR product of approximately 1000 bp was obtained from all the isolates that contained class 1 integrons using the in-F and in-B primers, whereas isolate 703M did not yield an amplicon (Figure 3Go). PCR with in-F and aadA-B primers yielded an approximately 750 bp amplicon in all isolates that yielded a product with the in-F and in-B primers, and thus confirmed the presence of the aminoglycoside resistance gene cassette aadA, which confers resistance to streptomycin and spectinomycin (Figure 3Go and Table 2Go).42,50 DNA sequencing of the 1000 bp amplicon from isolate 870X confirmed the presence of the streptomycin/spectinomycin gene cassette aadA2. Following sequence alignment and comparison with other GenBank sequences using the BLAST software this nucleotide sequence showed 100% identify to an aadA2 gene cassette integrated within a class 1 integron (GenBank accession no. AF071555, base pair no. 1855– 2863). Control strains VC-VN-62, 33/97 and VC20 yielded the 800 bp amplicon using the qacE{Delta}1-F and sul1-B primers and each isolate was found to contain the aadA gene as shown by PCR using in-F and aadA-B primers.

Each of the 18 tetracycline-resistant V. cholerae O1 tested in multiplex PCR yielded an amplicon of 957 bp identical to the product of the reference strain for tetracycline resistance determinant A (E. coli NCTC 50078) (Figure 3Go).45 None of these isolates tested yielded PCR products with the primers for tetracycline determinants B, C, D and E. Isolates 703M and 1105Y, which were sensitive to tetracycline, did not yield amplicons with any of the tetracycline primers studied. None of the reference isolates from Vietnam, Thailand, India or Guinea-Bissau contained tetA.

PCR for the SXT element showed that all 20 V. cholerae O1 isolates tested yielded a 592 bp amplicon of an identical size to the positive control V. cholerae O139 strain MO10 (Figure 3Go). The reference strains CO840 and F2107, which showed a ribotype identical to VCSA3, contained the SXT element, whereas the the isolates from Vietnam, Thailand and India (VC20), which all showed a ribotype closely related to VCSA1, did not contain the SXT element.

Repeated conjugation mating experiments by the agar plating method with the recipient E. coli K-12 revealed growth of transconjugants following mating with the plasmid-containing donor strains 796E and 808M at transfer frequencies of 2.2 x 10–5 and 1.0 x 10–4 transconjugants/recipient, respectively. Transconjugants were not found following mating with the other donor strains. PCR analysis of transconjugants showed that the genes encoding the tetracycline resistance determinant A and the SXT element were transferable, whereas the class 1 integron and aadA2 could not be transferred. In mating experiments with the recipient V. cholerae strain 1407, only the donor strains 711A, 796E and 808M yielded numbers of transconjugants at transfer frequencies of 8.3 x 10–5, 7.8 x 10–4 and 1.8 x 10–3 transconjugants/recipient, respectively. As was found for E. coli K-12 transconjugants, PCR analysis of transconjugants showed that tetA and the SXT element were transferable from each of the three isolates, whereas the class 1 integron and aadA2 could not be transferred. Plasmid analysis of transconjugants showed that both the 54 and 63 kb plasmids were transferable into V. cholerae, whereas none of the transconjugants contained plasmids following mating with E. coli K-12. Overall, antimicrobial susceptibility testing of the V. cholerae transconjugants showed that the resistance genes contained in the SXT element and tetA were transferred and expressed in each of the transconjugants. One important exception was found in a transconjugant obtained from mating with isolate 808M. This transconjugant contained the 54 kb plasmid, was sensitive to sulphonamides and tetracycline, and did not yield an amplicon in the PCR analysis for tetA. This suggests that the tetA gene was not located on the plasmid.

PFGE, Southern blot analysis and hybridization experiments showed that the SXT probe hybridized with two fragments of approximately 48 and 88 kb in each of the isolates 711A, 796E and 808M (results not shown). The tetA probe also hybridized with these two fragments, suggesting that the SXT element and tetA are closely located on the chromosome. The co-transfer of the SXT element and tetA was confirmed as probes for both gene elements hybridized with identical 83 and 105 kb fragments in the V. cholerae transconjugants obtained from mating with each of the three donor strains. Southern blot analysis and hybridization experiments with plasmid preparations of the transconjugants showed that in 10/11 transconjugants, the tetA probe hybridized with the transferred plasmid, whereas in all 11 transconjugants the transferred plasmid hybridized with the SXT probe. Following PFGE and Southern blot analysis, the recipient control strain V. cholerae 1407 did not hybridize with either of the two probes.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the past 20 years, there have been two cholera outbreaks in South Africa. In both outbreaks, V. cholerae O1 was introduced into the country from Mozambique.3,27 In the early 1980s, Mozambique was in the throes of a civil war, and as a result, medical supplies were poor and disease was rife. An influx of refugees into the South African provinces of Mpumalanga and Kwazulu-Natal introduced cholera into this country. The epidemic in South Africa in 1980–1987 included 25251 culture-confirmed cases. The province of Kwazulu-Natal (then known as Natal) was particularly affected.27 As a result of the rapid response by the local Departments of Health in Gauteng, Mpumalanga and Kwazulu-Natal, the recent outbreak in 1997/1998 was far smaller with only a total of 20 culture confirmed cases occurring in Mpumalanga and another five cases in Gauteng.3 However, following the completion of our study the cholera outbreak spread to most areas of Mozambique and parts of South Africa. In Mozambique alone, more than 44000 cases were reported to the WHO in 1999.51 Although some cases in our study were undoubtedly acquired in South Africa, others were imported from Mozambique (Table 1Go), as a large population of Mozambican migrants settled in this country during and after the Mozambican war. Economic migrants continue to enter South Africa in search of work, and cross-border traffic between the two countries is high. A policy of antibiotic treatment of cases and contacts was introduced in the earlier epidemic,27 although contact treatment was discouraged in the more recent epidemic (communication from the Department of Health). Previous use of antibiotics in earlier outbreaks may be in part responsible for the extensive increase in antibiotic resistance that we have observed between the two cholera epidemics.52 It is unknown whether the isolates responsible for earlier and recent epidemics are of clonal origin. The association between the development of resistance to tetracycline, chloramphenicol and co-trimoxazole with large-scale use of antibiotics for treatment and prophylaxis of cholera is well recognized.9,10,53 Still, our demonstration of multiple-drug resistant V. cholerae O1 isolates showing resistance to all the antibiotics traditionally used to treat cholera is disturbing and has a direct impact on the treatment of current and future cholera cases in South Africa and other countries to which this isolate may spread.

The co-transfer of apparently closely located chromosomal genes encoding the SXT element and tetracycline resistance suggests that these genes and their mechanisms of transfer may be important in the maintenance and transfer of resistance among V. cholerae and other enteric pathogens. Our findings corroborate those of Waldor et al.,12 who also found that the SXT element and its resistance genes to sulphonamides, trimethoprim and streptomycin were transferable. We have further shown for the first time co-transfer of tetA and the SXT element. In the initial report of the SXT element, it was suggested that the element was approximately 62 kb in size and had a preferential integration site in the chromosome.12 Sequencing the ends of the SXT element mapped the insertion site to the 5' end of the prfC gene, a gene encoding a peptide release factor 3 that functions in the termination of protein synthesis.54 Furthermore, it was shown by PCR with primers that only recognized the joined ends of the SXT element, that when excised from the chromosome, it was found in an extrachromosomal circular form similar to conjugative transposons.54 Additional sequencing showed an integrase gene with high sequence similarity to the integrase family of recombinases found near the 5' end of the SXT element. Recent mutational studies showed that the integrase gene plays a role in the excision or circularization of the element as well as the integration of SXT into the host chromosome.55 The co-transfer of the SXT element and tetA in our study indicates that SXT may be able to transfer chromosomal DNA from donor to recipient strains. Indeed, Hochhut et al.55 showed that Tn10 insertions downstream of SXT, but not upstream, were transferred. Further studies of the transfer mechanisms of SXT and tetA, e.g. transposons, are planned. Also, it cannot be ruled out that tetracycline resistance genes may be located on the SXT element.

In our study, transfer of class 1 integrons and the aadA2 gene was not shown and further work is needed to study the transfer of class 1 integrons and their resistance gene cassettes. As in our study, only one resistance gene cassette (aadA2) was found in chromosomally located class 1 integrons in the studies of V. cholerae O1 isolates from Vietnam and O1 isolates from Thailand.14,56 However, several mainly chromosomally located gene cassettes encoding resistance to different aminoglycosides, ß-lactams and trimethoprim were identified among V. cholerae isolates of different O-serotypes in Thailand.13 Bissonnette & Roy57 proposed that integrons evolve by the insertion of gene cassettes in an empty integron named In0. Currently, little is known about the stability, acquisition and mode of possible transfer of class 1 integron gene cassettes in V. cholerae. It could be feared that chromosomal resistance genes on class 1 integrons and in the SXT element are more stable in V. cholerae than plasmid-encoded resistance genes, which appear unstable in V. cholerae and disappear in the absence of selective pressure. Future studies are needed to determine whether the V. cholerae O1 isolate in our study has or will spread to neighbouring countries of South Africa and countries elsewhere on the African continent, most of which have experienced cholera recently.1 Also, it could be speculated that recent occasional unusually high mortality rates experienced during cholera outbreaks in some African countries could be associated with multiple-drug resistant O1 isolates carrying resistance genes located in SXT elements and class 1 integrons. A recent study in our laboratory indicated that this was indeed the case in the cholera epidemic in Guinea-Bissau in 1996/1997, where a significant increase in mortality was found associated with the emergence of an O1 isolate carrying a large conjugative resistance plasmid harbouring a class 1 integron.58

The V. cholerae O1 outbreak isolates from Mozambique and South Africa showed ribotypes closely related to the ribotypes of recent epidemic isolates isolated in India, Thailand and Vietnam (Figure 2Go). In Vietnam, the ribotype shown by isolate VN-62 (designated R1 in the original study) was the predominant type after 1990,14 whereas an identical ribotype, as shown by isolate 33/97 (designated RS1 in the original study), was the only V. cholerae O1 ribotype demonstrated after the O139 serotype disappeared in Thailand in 1993.59 Furthermore, the V. cholerae O1 isolates from India, Vietnam and Thailand also carried class 1 integrons containing the aadA gene cassette found among the Mozambican and South African O1 isolates.13,14 Although the V. cholerae O1 isolates from India, Thailand and Vietnam did not contain the SXT element and the tetA gene, our molecular findings suggest that the V. cholerae O1 outbreak isolate in the present study may have originated from the same clone as did the South-east Asian O1 isolate. However, clonality of these O1 isolates needs further confirmation, e.g. by combined PFGE analysis and hybridization with selected gene probes. Also, it remains to be determined why this possible clonal isolate seems to have replaced other V. cholerae isolates in the countries mentioned. Furthermore, the time and mode of spread of the isolate between the two continents are unknown.

It will be interesting to investigate in future studies to what degree antibiotic resistance in V. cholerae may evolve through integron-mediated acquisition of additional gene cassettes and the SXT element. Also, additional studies are needed to determine further genetic details of the association of the SXT element, tetA and class 1 integrons, including their means of transfer, e.g. by transposons.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful for the technical assistance provided by Naseema Aithma at the South African Institute for Medical Research in Johannesburg, South Africa and Anne-Mette Petersen of the Royal Veterinary and Agricultural University, Copenhagen, Denmark. We thank Matthew Waldor for the sequence data of the SXT PCR primers. This study was supported by the Danish Council for Development Research, DANIDA grant no. 90928.


    Notes
 
* Corresponding author. Tel: +45-35-282720; Fax: +45-35-282757; E-mail: ad{at}kvl.dk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Received 29 May 2001; returned 7 August 2001; revised 17 September 2001; accepted 19 September 2001