Integron-Associated Antibiotic Resistance in Salmonella enterica Serovar Typhi from Asia

Abstract

Eighteen of 25 isolates of Salmonella enterica serovar Typhi were multidrug resistant and contained class 1 integrons with a single cassette, dfrVII or aadA1. The dfrVII-containing integron was likely borne on an IncHI1 plasmid. Salmonella serovar Typhi could become resistant to broad-spectrum cephalosporins by integrating cassettes, such as veb-1, a common cassette in Asia.

Integrons are genetic elements that can integrate, by site-specific recombination, gene cassettes, which usually confer antibiotic resistance. Three classes of integrons have been characterized in detail and are involved in antibiotic resistance.

The occurrence of multidrug resistance (MDR) (to amoxicillin, chloramphenicol, sulfonamides, streptomycin, tetracycline, and trimethoprim) in Salmonella enterica serovar Typhi has been increasing (C. Rodrigues, A. Mehta, and V. R. Joshi, Letter, Clin. Infect. Dis. 34:126, 2002), and MDR strains have been responsible for numerous outbreaks on the Asian continent (22). Integrons have been found in different serovars of S. enterica subsp. enterica but never in serovar Typhi. The aim of our study was to evaluate the contribution of integrons to the antibiotic resistance of serovar Typhi strains in Asia.

Twenty-five strains from Vietnam (23) and India (1) between 1999 and 2000 were chosen for this study. They included 18 strains resistant to at least one of the following antibiotics: a sulfonamide, chloramphenicol, streptomycin, trimethoprim, and beta-lactams (Table 1). Susceptibility testing was performed by the disk diffusion method on Mueller-Hinton agar (bioMérieux, Marcy-l'Etoile, France) according to the recommendations of the Comité de l'Antibiogramme of the Société Française de Microbiologie (CA/SFM). Total DNA was prepared by using the Qiamp tissue kit (Qiagen, Inc., Chatsworth, Calif.). Transfer of antibiotic resistance from S. enterica serovar Typhi to Escherichia coli C1a (Nal^r) was achieved on selective medium containing 50 μg of nalidixic acid per ml plus either 5 μg of trimethoprim per ml or 25 μg of streptomycin per ml. Genomic diversity was studied by pulsed-field gel electrophoresis (PFGE) and analysis of XbaI restriction fragments as described previously (12). Plasmids of the H1 incompatibility group were detected by amplification of a 365-bp region of the RepHI1A replicon as described previously (19).

TABLE 1.

Properties and cassette content of S. enterica serovar Typhi clinical isolates and transconjugants

Strain	Pulsotype	Antibiotic resistancea	intI gene	Gene cassette	IncH1 plasmidb		Source
					CI	T
LIM5	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM6	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM7	C		None		−		Vietnam
LIM8	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM9	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM10	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM11	B	Cm Sm Sp Su	intI1	aadA1	+	−	Vietnam
LIM12	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM13	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM14	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM15	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM16	B	Cm Sm Sp Su	intI1	aadAI	+	−	Vietnam
LIM17	A		None		−		Vietnam
LIM18	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM19	A		None		−		Vietnam
LIM20	A		None		−		Vietnam
LIM21	B	Cm Sm Sp Su	intI1	aadA1	+	−	Vietnam
LIM22	A		None		−		Vietnam
LIM23	A		None		−		Vietnam
LIM24	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM25	A		None		−		Vietnam
LIM26	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM27	C	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM28	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	Vietnam
LIM29	A	Am* Cm* Su* Tc* Te* Tp*	intI1	dfrVII	+	+	India

^aAbbreviations: Am, ampicillin; Cm, chloramphenicol; Sm, streptomycin; Sp, spectinomycin; Su, sulfonamide; Tc, ticarcillin; Te, tetracycline, Tp, trimethoprim. Antibiotic resistance characteristics of the transconjugants are indicated by asterisks.

^bAbbreviations: CI, clinical isolate; T, transconjugant.

Mapping of integrons.

Strains were screened for the presence of class 1, 2, and 3 integrons as described previously (18). The intI1 gene was detected in the 18 resistant strains but not in the susceptible isolates (Table 1). Class 2 or 3 integrons were not detected.

Cassette assortment in class 1 integrons was determined as described previously (18). The 18 strains had an integron containing a single cassette (Table 1): dfrVII conferring resistance to trimethoprim (15 strains) or aadA1 conferring resistance to spectinomycin and streptomycin (3 strains). The dfr and aadA cassettes were shown to be common in integrons in members of the family Enterobacteriaceae (15). The dfrVII gene cassette was reported in integrons in gram-negative bacteria and was found in S. enterica serovar Typhi associated with an IncH1 plasmid (19), but the presence of integrons was not investigated.

Class 1 integrons were previously found in multiresistant S. enterica serovar Typhimurium phage type DT104 (1). Integrons were also detected in other phage types (5) and in other nontyphoidal serovars: Agona, Brandenburg, Enteritidis, Goldcoast, Hadar, Infantis, Ohio, Panama, Poona, Saintpaul, Virchow, Worthington, and serovar [4,5,12:i:−] (2, 4, 6, 9, 10, 14). All these integrons except that of serovar Infantis contained a streptomycin-spectinomycin resistance determinant alone or in combination with other gene cassette; this determinant could be aadA2 or aadA1, but it was aadA1 in most cases.

Moreover, class 1 integrons have been detected in other gram-negative bacteria from Asia: Acinetobacter (17), Pseudomonas (7, 13), and members of the family Enterobacteriaceae (8). Many of these integrons carried the veb-1 or bla_VIM-2 cassette encoding resistance to broad-spectrum beta-lactams.

Distribution of integrons among the PFGE types.

Three PFGE patterns or pulsotypes were obtained (Table 1). The three strains containing the integron with the aadA1 cassette exhibited pattern B. Two strains exhibited pattern C; one strain was susceptible, and one contained the dfrVII-associated integron cassette. The single strain from India, LIM29, exhibited pattern A, which was recovered in most of the strains from Vietnam, suggesting a clonal dissemination in Asia. The integron containing the dfrVII cassette was found in pulsotypes A and C, suggesting a horizontal transfer of this integron via plasmids and/or transposons.

Previous studies on the molecular epidemiology of S. enterica serovar Typhi strains from Asia showed a genetic diversity among susceptible strains (20), whereas MDR isolates were more homogeneous (19, 21). In our study, pattern A was observed in resistant and susceptible strains.

Plasmid analysis.

In S. enterica serovar Typhi, MDR is mainly due to large (approximately 180-kb) conjugative plasmids of the H1 incompatibility group that is found throughout Southeast Asia (11, 19). However, a recent study showed that at least six different plasmids of different incompatibility groups could encode the MDR phenotype in S. enterica serovar Typhi in Asia (16). A majority of the IncFI plasmids are also involved in the MDR phenotype of serovar Typhimurium isolates (3). In our study, all 18 resistant strains contained a 365-bp region of the RepHI1A replicon, suggesting that each isolate contained a plasmid of the H1 incompatibility group.

Resistance to ampicillin, chloramphenicol, tetracycline, trimethoprim, and sulfonamides was transferred en bloc from each of the strains harboring the integron containing the dfrVII cassette to E. coli. All the transconjugants contained the class 1 integron and an IncH1 plasmid as detected by PCR, indicating that the integron was borne on this element. By contrast, transfer of streptomycin-spectinomycin resistance was not obtained from the three isolates carrying the aadA1 cassette, although these strains contained an IncH1 plasmid (Table 1). Resistance to chloramphenicol or sulfonamides was also not transferred, suggesting that all the resistance determinants are physically linked.

Conclusions.

To our knowledge, this is the first report of the presence of integrons in S. enterica serovar Typhi. In our study all the isolates were susceptible to fluoroquinolones and to broad-spectrum cephalosporins. In the past decade, fluoroquinolones were considered very effective in treating typhoid fever. However, strains resistant to fluoroquinolones have been reported and are now endemic in India and Pakistan (23). Genes encoding resistance to broad-spectrum cephalosporins, such as veb-1, were part of integron cassettes. Thus, the presence of integrons in S. enterica serovar Typhi is of clinical significance, because the strains might easily become resistant to broad-spectrum cephalosporins, which remain the basis of therapy against fluoroquinolone-resistant and MDR strains. This acquisition of resistance might happen by capture of gene cassettes, such as veb-1 or bla_VIM-2, in one of the integrons described in this study. Otherwise, the acquisition of another plasmid able to replicate in Salmonella serovar Typhi could result in resistance to broad-spectrum cephalosporins.