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Antimicrobial Agents and Chemotherapy, November 2004, p. 4263-4270, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4263-4270.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Infections with Nontyphoidal Salmonella Species Producing TEM-63 or a Novel TEM Enzyme, TEM-131, in South Africa

Tersia Kruger,1 Dora Szabo,2 Karen H. Keddy,1* Kathleen Deeley,2 Jane W. Marsh,2 Andrea M. Hujer,4 Robert A. Bonomo,4 and David L. Paterson2*

Enteric Diseases Reference Unit, National Institute for Communicable Diseases (NHLS), and University of the Witwatersrand, Johannesburg, Republic of South Africa,1 Division of Infectious Diseases, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania,2 Institute for Medical Microbiology, Semmelweis University, Budapest, Hungary,3 Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio4

Received 18 November 2003/ Returned for modification 13 February 2004/ Accepted 7 July 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Salmonella spp. producing extended-spectrum beta-lactamases (ESBLs) have been reported in many countries, but there is no information on their prevalence in Africa. ESBL-producing Salmonella enterica serotype Isangi and S. enterica serotype Typhimurium strains have been noted in South Africa since 2001. A total of 160 consecutive isolates of Salmonella spp. were collected from 13 hospitals located in different cities in South Africa over a 5-month period from December 2002 to April 2003. All strains were screened for production of ESBLs by the double disk diffusion test and for AmpC production by assessing resistance to cefoxitin. blaSHV, blaTEM, blaCTX-M, and blaCMY-2 were sought from all ESBL-positive and cefoxitin-resistant isolates. A total of 15.6% (25 of 160) isolates produced SHV or TEM ESBLs, and 1.9% (3 of 160) produced CMY-2. Nine S. enterica serotype Typhimurium, eight S. enterica serotype Isangi, and three S. enterica serotype Muenchen strains produced either TEM-63 or a derivative of TEM-63 designated TEM-131. Both TEM-63 and TEM-131 have an isoelectric point of 5.6, and their sequences have the following amino acid substitutions compared to the TEM-1 sequence: Leu21Phe, Glu104Lys, Arg164Ser, and Met182Thr. Additionally, TEM-131 has an Ala237Thr substitution. ESBL-producing Salmonella spp. have become a significant public health problem in South Africa with particular implications for the treatment of serious nontyphoidal Salmonella infections in children, for whom extended-spectrum cephalosporins were the preferred treatment.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Resistance to the extended-spectrum cephalosporins among members of the family Enterobacteriaceae has become a growing worldwide problem subsequent to the occurrence of extended-spectrum beta-lactamases (ESBLs) and AmpC-type beta-lactamases (9). Although reports of ESBLs associated with Salmonella spp. are relatively rare compared to those for other species in the family Enterobacteriaceae, the number of reported cases in this organism has been increasing in recent years. Salmonellae have been found to express a wide variety of ESBL types, including TEM, SHV, PER, OXA, and CTX-M enzymes (1, 6, 10, 12, 21, 39, 44). Additionally, Salmonella strains have been detected which produce plasmid-mediated AmpC-type beta-lactamases (21, 35, 38).

The advent of ESBLs and AmpC beta-lactamases in Salmonella species is of considerable therapeutic importance, especially in developing nations where infections with these organisms are numerous. Resistance to ampicillin, trimethoprim-sulfamethox-azole, and chloramphenicol is now exceedingly common, necessitating use of fluoroquinolones or extended-spectrum cephalosporins as treatment of extraintestinal infections (13). Widespread fluoroquinolone use in children has been discouraged because of the potential adverse effects on cartilage development. Therefore, extended-spectrum cephalosporins (especially cefotaxime or ceftriaxone) are the mainstay of treatment of serious infections due to nontyphoidal Salmonella spp. in children. The production of ESBLs or AmpC beta-lactamases consequently has considerable implications for clinical microbiology laboratories and physicians in areas in which infections with nontyphoidal Salmonella spp. are common.

ESBLs have been found in many enterobacterial species in South Africa (7, 14, 22, 36). Since 2000, the Enteric Diseases Reference Unit of the National Institute for Communicable Diseases in South Africa has noted increasing numbers of nontyphoidal Salmonella isolates, particularly S. enterica serotype Typhimurium and S. enterica serotype Isangi, with positive screening tests for ESBLs. The aim of this study was to determine the genetic basis for antibiotic resistance in these isolates and to briefly describe the epidemiology of infections with these organisms.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains. Salmonella isolates of human origin are sent to the Enteric Diseases Reference Unit of the National Institute for Communicable Diseases in Johannesburg, South Africa, from clinical microbiology laboratories across the country as part of national surveillance for enteric pathogens. A total of 160 consecutive Salmonella isolates arriving in this laboratory, collected from thirteen different hospitals in South Africa between December 2002 and March 2003, were selected for further analysis. The identification of the isolates as being of Salmonella species was confirmed at the Enteric Diseases Reference Unit by conventional biochemical tests. Serotyping of all isolates was performed, using the method of slide agglutination on the basis of lipopolysaccharide (O) and flagellar (H) antigens and commercially available antisera (Bio-Rad, Marnes-la-Coquette, France), according to the Kauffman-White scheme for Salmonella serotyping (25, 37). Double disk diffusion testing was performed according to the method of Jarlier et al. (23), with ceftriaxone, cefotaxime, ceftazidime, and aztreonam disks 30 mm (center to center) away from a disk containing amoxicillin-clavulanic acid. Further analysis of the isolates was undertaken in Pittsburgh and Cleveland.

Antibiotic susceptibility. Susceptibility tests were performed using the Kirby-Bauer disk diffusion method and following NCCLS guidelines (32). Antimicrobials used were ampicillin, aztreonam, cefixime, cefepime, cefoxitin, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, amoxicillin-clavulanic acid, ertapenem, imipenem, meropenem, norfloxacin, nalidixic acid, ofloxacin, levofloxacin, gatifloxacin, ciprofloxacin, moxifloxacin, tetracycline, chloramphenicol, streptomycin, and trimethoprim-sulfamethoxazole.

The MICs of cefotaxime, cefotaxime plus clavulanic acid, ceftazidime, ceftazidime plus clavulanic acid, cefepime, cefoxitin, nalidixic acid, ciprofloxacin, trimethoprim-sulfamethoxazole, imipenem, and meropenem were determined by E-test for strains with positive double disk diffusion tests (as an indicator of ESBL production) or cefoxitin resistance (as an indicator of possible plasmid-mediated AmpC beta-lactamases). The strips were used according to the manufacturer's instructions (AB Biodisk, Solna, Sweden).

Escherichia coli ATCC 25922 was used as the reference strain for antimicrobial susceptibility testing (32).

IEF. Analytical isoelectric focusing (IEF) was performed in duplicate on all isolates with positive double disk diffusion test results or cefoxitin resistance by the use of methods that have been previously described (34). Enzyme activity was detected by placing filter paper soaked in nitrocefin (Becton Dickinson, Sparks, Md.) (500 µg/ml) over the focused gel.

PFGE. Pulsed field gel electrophoresis (PFGE) analysis was performed according to the Centers for Disease Control and Prevention PulseNet protocol (41). Briefly, genomic DNA was isolated and digested with XbaI (New England Biolabs, Beverly, Mass.). PFGE was performed with a CHEF III system (Bio-Rad, Hercules, Calif.) and the following run parameters: for block I, a switch time of 3 to 65 s and a run time of 17 h; for block II, a switch time of 15 to 30 s and a run time of 6 h. Dendrograms were created with Molecular Analyst (Bio-Rad) by using the Dice coefficient, unweighted pair group method with arithmetic means (UPGMA), and a position tolerance of 1.3%. Relatedness of the isolates was also determined by the criteria of Tenover et al. (42).

Plasmid profiles. Plasmids were extracted and electrophoresed by the method of Kado and Liu (24). Transformation assays were performed by electroporation (Gene Pulser; Bio-Rad) with E. coli strain DH10B (Bio-Rad) as the recipient. Transformants were cultivated on nutrient agar containing ampicillin (100 µg/ml).

PCR screening. A 10-µl aliquot of an overnight culture of the test isolate was diluted 1:10 with water and boiled for 15 min. PCR amplification was then performed with 10 µl of this dilution as the DNA template. The primer sets are shown in Table 1. Four primer pairs were used: blaTEM-1 (14), blaSHV-1 (14), blaCTX-M (8), and blaCMY-2 (33, 34). PCR conditions were as previously described (8, 14, 33).


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  		TABLE 1. Nucleotide sequences of the oligonucleotides used for amplification and sequencing

 
Sequencing. The primers used for DNA sequencing are shown in Table 1. Numbering follows the scheme of Ambler et al. (3). The nucleotide sequences of the amplified products were determined using ABI3700 and ABI3100 genetic analyzers at a core facility at the University of Pittsburgh. Data collection and analysis were performed using Lasergene DNASTAR sequencing analysis software.

Nucleotide sequence accession number. The DNA sequence and deduced amino acid sequence of TEM-131, the novel beta-lactamase, has been deposited in GenBank and assigned accession number AY436361.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Epidemiology. From 1999 to 2003, the Enteric Diseases Reference Unit of the National Institute for Communicable Diseases in the Republic of South Africa received annually, from all provinces of the country, between 500 and 1,500 nontyphoidal Salmonella strains for serotyping. Prior to 2000, S. enterica serotype Isangi was a rare isolate, but in 2002 was second only to S. enterica serotype Typhimurium in frequency of isolates received by the Enteric Diseases Reference Unit. In 2002, S. enterica serotype Isangi accounted for approximately 20% of all isolates received. Furthermore, the majority of S. enterica serotype Isangi isolates were found to have positive double disk diffusion test results, implying the presence of ESBLs. Since 2000, isolates of other serotypes (most notably S. enterica serotype Typhimurium) have also been found to produce ESBLs.

Strains. A total of 160 consecutive Salmonella isolates from the Enteric Diseases Reference Unit, National Institute for Communicable Diseases, South Africa, were selected for further analysis. These isolates were collected from 13 hospitals in South Africa from December 2002 to March 2003. The sources of the isolates were blood cultures (94 isolates), feces (48 isolates), urine (8 cultures), pleural fluid (3 isolates), cerebrospinal fluid (1 isolate), pericardial fluid (1 isolate), and miscellaneous (5 isolates). The serotypes of the strains were as follows: 115 S. enterica serotype Typhimurium, 14 S. enterica serotype Enteritidis, 10 S. enterica serotype Isangi, 6 S. enterica serotype Dublin, 5 S. enterica serotype Muenchen, 3 S. enterica serotype Hadar, 2 S. enterica serotype Newport, and 1 each of S. enterica serotype Anatum, S. enterica serotype Bovismorbificans, S. enterica serotype Infantis, S. enterica serotype Molade, and S. enterica serotype Schwarzengrund. A total of 25 (15.6%) isolates (14 S. enterica serotype Typhimurium, 8 S. enterica serotype Isangi, and 3 S. enterica serotype Muenchen) were screen positive for ESBL production. Two S. enterica serotype Typhimurium and the one S. enterica serotype Schwarzengrund were cefoxitin resistant. For purposes of further epidemiologic analysis, a further 23 S. enterica isolates of serotypes Typhimurium, Isangi, and Muenchen which were ampicillin resistant but not screen positive for ESBL production or cefoxitin resistant were chosen for further analysis.

IEF. Each strain which was screen positive for ESBL production or which was cefoxitin resistant produced one or two beta-lactamases with pI values of 5.4, 5.6, 8.2, and greater than 8.2 in various combinations (Table 2). Beta-lactamase enzymes with pI values in the range of 5.4 to 5.6 (consistent with the pI range of TEM enzymes) were identified in all isolates screen positive for ESBLs. Enzymes with a pI value of 8.2 (consistent with the pI range of SHV enzymes) were detected in 12 S. enterica serotype Typhimurium and 1 S. enterica serotype Isangi isolate. Two S. enterica serotype Typhimurium and the one S. enterica serotype Schwarzengrund that produced beta-lactamase enzymes with cefoxitin resistance had pI values greater than 8.2 (consistent with the pI range of AmpC-like beta-lactamases). More than one beta-lactamase was identified in 12 S. enterica serotype Typhimurium and 1 S. enterica serotype Isangi strains.


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  		TABLE 2. Molecular epidemiology of ESBL- and non-ESBL-producing isolates of S. enterica serotype Typhimurium, S. enterica serotype Isangi, S. enterica serotype Muenchen, and S. enterica serotype Schwarzengrund

 
Electroporation and plasmid analysis. Large (>10-kb) plasmids were isolated from all the strains (data not shown). The MICs of the transformants with different plasmids are shown in Table 3.


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  		TABLE 3. Antibiotic susceptibility (MIC values [in micrograms per milliliter]) of the E. coli DH10B strain without (Tf–) and with (Tf+) plasmid

 
PCR for detection of blaTEM, blaSHV, blaCTX-M, and blaCMY resistance genes and sequencing results. blaTEM amplification was achieved for all the isolates screen positive for ESBL production. Thus, 14 S. enterica serotype Typhimurium isolates, the 8 S. enterica serotype Isangi isolates, and the 3 S. enterica serotype Muenchen isolates were found to have blaTEM genes. Among the TEM-type ESBLs, TEM-63 and one novel TEM-type, TEM-131, were identified. The amino acid substitutions of the sequence of TEM-63 compared to the TEM-1 beta-lactamase sequence were Leu21Phe, Glu104Lys, Arg164Ser, and Met182Thr. The new TEM beta-lactamase (TEM-131) differed from TEM-63 by a single substitution (Ala237Thr) (Table 4). This protein has been designated TEM-131 (http://www.lahey.org/studies/webt.htm).


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  		TABLE 4. Sequence analysis of the blaTEM genes from the isolates with phenotypic evidence of ESBL productiona

 
Three S. enterica serotype Muenchen and six S. enterica serotype Isangi isolates carried the gene for TEM-63, and two S. enterica serotype Isangi and nine S. enterica serotype Typhimurium isolates carried the new TEM-131 gene (Table 2). Seven S. enterica serotype Typhimurium strains and one S. enterica serotype Isangi strain produced the SHV-5 enzyme, and five S. enterica serotype Typhimurium strains produced the SHV-12 enzyme. Under the experimental conditions in this study, we were unable to detect any blaCTX-M genes. Of the cefoxitin-resistant isolates, two S. enterica serotype Typhimurium and one S. enterica serotype Schwarzengrund carried the CMY-2 gene.

Antibiotic susceptibility testing. Isolates producing TEM-131 had ceftazidime MICs of >256 µg/ml, cefotaxime MICs in the range of 6 to 64 µg/ml, and cefepime MICs in the range of 4 to 16 µg/ml (Table 5). These ranges were also seen in the transformant E. coli DH5{alpha} strains producing this enzyme (Table 3). While the isolates producing TEM-63 also had ceftazidime MICs > 256 µg/ml, the cefotaxime MICs were somewhat lower than those observed with strains producing TEM-131 (cefotaxime MICs of 1.5 µg/ml in the TEM-63-producing transformant E. coli DH5{alpha} strain compared to 6 µg/ml in the TEM-131-producing strain) (Tables 3 and 5).


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  		TABLE 5. Antibiotic susceptibility (MIC values [in micrograms per milliliter]) of the resistant Salmonella strains

 
As expected, isolates producing CMY-2 had elevated MICs for ceftazidime (range 198 to >256 µg/ml), cefotaxime (32 µg/ml), and cefoxitin (>256 µg/ml) while retaining susceptibility to the carbapenems.

All but one of the ESBL-producing or CMY-2-producing isolates were resistant to trimethoprim-sulfamethoxazole. All isolates were resistant to chloramphenicol. Although no isolates were ciprofloxacin resistant, all seven S. enterica serotype Typhimurium isolates producing TEM-131 and SHV-5 and all six S. enterica serotype Isangi isolates producing TEM-63 were resistant to nalidixic acid. All isolates were susceptible to carbapenems.

PFGE. PFGE results of the S. enterica serotype Typhimurium isolates are presented in Table 2. A total of 29 isolates of S. enterica serotype Typhimurium were possibly related by PFGE (PFGE type A). These isolates were found in four provinces (Gauteng, Eastern Cape, Free State, and Western Cape). SHV-12-producing isolates which were indistinguishable from one another (PFGE type A1) were found in two hospitals in Gauteng and a hospital in the Eastern Cape. These hospitals are more than 1,000 km from one another (Fig. 1). TEM-131-producing isolates which were indistinguishable from one another (PFGE type A3) were found in a hospital in Gauteng and in a hospital in the Free State. TEM-131-producing S. enterica serotype Isangi isolates, unrelated to each other by PFGE, were found in two different hospitals in Gauteng.



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  		FIG. 1. Locations of the cities in which ESBL- or AmpC-producing salmonellae were obtained.

 
TEM-63-producing S. enterica serotype Isangi isolates, possibly related by PFGE (Table 2), were found in two different hospitals in Gauteng province, while isolates of TEM-63-producing S. enterica serotype Muenchen, indistinguishable from one another by PFGE, were found in two different hospitals in the Eastern Cape. The three CMY-2 producers (all different PFGE types) were found in patients in three different hospitals in Gauteng and in the Western Cape.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The advent of resistance of nontyphoidal Salmonella to extended-spectrum cephalosporin antibiotics is of significant public health importance. The treatment of choice for Salmonella meningitis or bacteremia in neonates is cefotaxime, and extended-spectrum cephalosporins are widely used in the treatment of bacteremia or osteomyelitis due to nontyphoidal Salmonella infections in both infants and older children. We have found that 25 of 160 (15.6%) nontyphoidal Salmonella isolates from South Africa produced ESBLs and 3 of 160 (1.9%) produced CMY-2. Furthermore, these isolates were frequently multiply resistant, lacking susceptibility to inexpensive agents such as ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol. Some isolates were also nalidixic acid resistant—infections with such isolates may not respond as well as expected to fluoroquinolones (13).

We identified TEM-63, or a novel TEM enzyme (with the same isoelectric point), TEM-131, in S. enterica serotype Muenchen, S. enterica serotype Isangi, and S. enterica serotype Typhimurium isolates. According to previously published reports, TEM-type beta-lactamases have been rarely found in ESBL-producing salmonellae. The TEM-3 enzyme was found in S. enterica serotype Typhimurium in Casablanca (1), TEM-4 was found in S. enterica serotype Mbandaka in Tunisia (28), and a TEM-52 strain was found in a nontyphoidal Salmonella isolate in Korea (27). It is noteworthy that TEM-63 has been noted in several previous reports of ESBL-producing Klebsiella, Proteus, and Enterobacter strains in South Africa but never previously in Salmonella strains (22). We speculate that TEM-63 is prevalent throughout members of the Enterobacteriaceae family across South Africa. It is not certain to us whether the origin of blaTEM-63 was in Salmonella spp. or whether it originated in other organisms and was then transferred to Salmonella spp.

In all, six S. enterica serotype Isangi and three S. enterica serotype Muenchen isolates produced the TEM-63 enzyme. TEM-131, the novel TEM beta-lactamase, was produced by an additional two S. enterica serotype Isangi and nine S. enterica serotype Typhimurium strains. The sequence of TEM-63 has four amino acid changes compared with the sequence of TEM-1, and TEM-131 has an additional change compared to TEM-63. This change (alanine to threonine at position 237) also occurs in TEM-5, TEM-24, and TEM-86. Of potential interest is that we observed somewhat higher cefotaxime MICs for TEM-131-producing transformant strains compared to TEM-63-producing transformant strains (Tables 3 and 5). Ceftazidime MICs were greatly elevated (>256 µg/ml) for both TEM-63- and TEM-131-producing strains.

One S. enterica serotype Isangi and seven S. enterica serotype Typhimurium strains produced not only the novel TEM-131 enzyme but also SHV-5. Additionally, five S. enterica serotype Typhimurium isolates produced TEM-1 and SHV-12. Although SHV-5 has previously been found in South Africa (14), SHV-12 has not. SHV-type beta-lactamases have been more frequently found in ESBL-producing salmonellae worldwide than TEM-type ESBLs (34). A variety of SHV-type ESBLs have been previously noted in salmonellae (5, 6, 20, 21, 28-30, 39, 40, 43, 44, 46). Under the experimental conditions we used, we did not find any isolates which produced a CTX-M-type beta-lactamase, in contrast to the rising significance of these ESBLs in Klebsiella and other species (34). CTX-M-type ESBLs have been previously found in nontyphoidal Salmonella spp. (10).

Two S. enterica serotype Typhimurium and one S. enterica serotype Schwarzengrund isolates produced CMY-2 beta-lactamase, a beta-lactamase of the AmpC type. CMY-2-producing S. enterica serotype Typhimurium has been observed in Taiwan (47), Romania (29), and the United States (16, 45). More notably in North America, S. enterica serotype Newport has been found to be a producer of CMY-2 (2, 4). However other serovars which have been found to produce CMY-2 include S. enterica serotype Hadar (46), S. enterica serotype Senftenberg (26, 39), S. enterica serotype Mikawasima (31), and S. enterica serotype Montevideo (31). To our knowledge, this is the first report of a CMY-2-producing S. enterica serotype Schwarzengrund strain. It is possible that use of primers specific for CMY-2 did not allow us to detect other AmpC beta-lactamases which may have been present in our strains. However, with the exception of one TEM-63-producing S. enterica serotype Isangi strain, which showed intermediate resistance to cefoxitin, all other strains were cefoxitin susceptible, making clinically significant AmpC production unlikely in these strains.

South Africa has the highest number of human immunodeficiency virus (HIV)-infected people in the world, with an estimated 5 million infected by the virus (15). Patients with HIV infection have an increased risk of invasive salmonellosis (11, 17-19). More than half of the isolates (59% [94 of 160]) in this series were blood culture isolates. Given the high rate of invasive infection, antibiotic resistance in Salmonella isolates in South Africa is potentially of great clinical significance. Ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol, and extended-spectrum cephalosporins are widely utilized therapies for serious Salmonella infections and yet are ineffective antibiotic options in the ESBL- and CMY-2-producing isolates we described. Some of the isolates were also nalidixic acid resistant, potentially limiting the effectiveness of fluoroquinolones. Disturbingly, we found ESBL- or CMY-2-producing isolates in four geographically distant provinces. Our molecular epidemiologic analysis shows that some isolates in different provinces were indistinguishable by PFGE (Table 2), indicating a common source or person-to-person spread. Preliminary epidemiologic data suggest that the multiresistant strains actually originated in a nosocomial setting, but we speculate that there has now been spread into the community.

We recommend that resources be utilized so that clinicians in southern Africa can collect relevant sterile site cultures and that clinical microbiology laboratories perform appropriate testing for susceptibility to extended-spectrum cephalosporins. Examples of relevant infections include neonatal Salmonella meningitis and Salmonella bacteremia or osteomyelitis. We, and others, are presently investigating the clinical impact of ESBL production by isolates that are not resistant to cefotaxime or ceftriaxone by conventional standards (that is, isolates for which drug MICs are less than 64 µg/ml).


   ACKNOWLEDGMENTS
 
We thank Elias Khomane, Lloyd Clarke, and John Kolano for technical assistance.

This work was supported by a grant from the Centers for Disease Control and Prevention (RS1/CCR 322404-02) to David Paterson. The work of Tersia Kruger was supported in part by a grant from the National Health Laboratory Service and the University of Witwatersrand. The Veterans Affairs Department Merit Review Program supported Robert Bonomo.


   FOOTNOTES
 
* Corresponding author. Mailing address for David Paterson: UPMC Division of Infectious Diseases, Suite 3A Falk Medical Building, 3601 5th Ave., Pittsburgh, PA 15213. Phone: (412) 648-6478. Fax: (412) 648-6399. E-mail: patersond@msx.dept-med.pitt.edu. Mailing address for Karen Keddy: Enteric Diseases Reference Unit, National Institute for Communicable Diseases, NHLS, P.O. Box 1038, 2000 Johannesburg, Republic of South Africa. Phone: 27-11-489-9151. Fax: 27-11-489-9357. E-mail: karen.keddy{at}nhls.ac.za. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Antimicrobial Agents and Chemotherapy, November 2004, p. 4263-4270, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4263-4270.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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