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Letter
Integrons in Salmonella
Keurmassar, Senegal
Amy Gassama-Sow,*† Awa Aïdara-Kane,† Nabil Raked,* François Denis,*
and Marie-Cécile Ploy*
*Laboratoire de Bactériologie-Virologie-Hygiène, Limoges, France; and
†Institut Pasteur, Dakar, Sénégal
Suggested citation
for this article:
Gassama-Sow A, Aïdara-Kane A, Raked N, Denis F, Ploy M-C. Integrons
in Salmonella Keurmassar, Senegal [letter]. Emerg Infect Dis
[serial on the Internet] 2004 Jul [date cited]. Available from:
http://www.cdc.gov/ncidod/EID/vol10no7/03-0666.htm
To the Editor: Infections caused by Salmonella are the
primary cause of foodborne diseases; multidrug resistance to Salmonella
enterica subsp. enterica is increasing. The selective pressure
created by the widespread use of antimicrobial agents in animals and humans
as prophylactic and therapeutic agents may have contributed to the dissemination
of resistant bacterial strains. In 2000, the new serovar Keurmassar (35:c:1,2)
of S. enterica, was described in Senegal (1).
Integrons are efficient gene-capture systems by site-specific recombination
and are involved in antimicrobial-drug resistance in gram-negative bacteria
(2). Three classes of integrons are well characterized
and are involved in antimicrobial resistance. Integrons have been found
in different nontyphoidal serovars of S. enterica and recently
in serovar Typhi (3).
We evaluated the contribution of integrons to the antimicrobial drug
resistance of eight isolates of S. enterica serovar Keurmassar
sent to the Senegalese National Salmonella and Shigella
Reference Laboratory at the Pasteur Institute in Dakar from March to May
2000. One strain was isolated from poultry flesh, and seven strains were
isolated from human stool or blood samples. Susceptibility testing was
performed by disk diffusion method on Mueller-Hinton agar according to
the Comité de l'antibiogramme, Société Française de Microbiologie, recommendations.
The eight strains expressed an extended-spectrum β-lactamase, which
was previously identified as SHV-12 (1). The strains
were also resistant to aminoglycosides (amikacin, gentamicin, netilmicin,
spectinomycin, streptomycin, and tobramycin), chloramphenicol, sulfamethoxazole,
tetracycline, and trimethoprim. Genomic diversity was studied by pulsed-field
gel electrophoresis (PFGE) and analysis of XbaI restriction fragments
as described previously (3). The eight strains isolated
from poultry and humans specimens showed identical PFGE patterns, which
suggested that all strains of S. enterica serovar Keurmassar isolated
until May 2000 in Senegal belonged to the same clone.
Strains were screened for the integrons by polymerase chain reaction
by using three sets of primers specific for the intI1, intI2,
and intI3 genes coding for the integrase as described previously
(3). The intI1 gene was detected in all strains.
Class 2 or 3 integrons were not detected. Cassette assortment in class
1 integrons was determined by using the primers 5´CS and 3´CS
complementary to the 5´ and 3´ segments as described previously
(3). With these primers, we obtained two amplification
products of 1 kb and 1.7 kb for each strain, which suggested that all
strains contained at least two class 1 integrons. Sequencing of these
amplification products showed that the first product of 1 kb contained
the aadA2 cassette, which confers resistance to streptomycin and
spectinomycin. The second amplicon of 1.7 kb carried a new arrangement
of two cassettes: aac(6´)-IIc, which confers resistance to
gentamicin, netilmicin, and tobramycin; and ereA2, which encodes
resistance to erythromycin. Class 1 integrons were previously found in
strains of S. enterica of different serovars: Agona, Albany, Brandenburg,
Enteritidis, Goldcoast, Hadar, Infantis, Ohio, Panama, Poona, Saintpaul,
Typhi, Typhimurium, Virchow, and Worthington (3–7). All
these integrons, except that of serovar Infantis, contained a streptomycin-spectinomycin
resistance determinant, aadA2 or mostly aadA1, alone or
in combination with other gene cassettes. The cassette aac(6´)-IIc
was previously described in a single class 1 integron in Pseudomonas
aeruginosa (AF162771). The ereA2 cassette was first described
in Providencia stuartii (8) in a class 1 integron.
This cassette was since described in class 1 integrons of clinical gram-negative
isolates and recently in a class 2 integron in Escherichia coli
(9). To determine whether the resistance determinants
carried by the integrons were transferable, we performed a conjugation
experiment from S. enterica serovar Keurmassar to an E. coli
strain resistant to nalidixic acid. We first used a selective medium containing
nalidixic acid 50 µg/mL plus 25 µg/mL of streptomycin, one
of the two integrons carrying the aadA2 cassette. All antimicrobial
drug resistances were transferred at once from each strain to E. coli.
The analysis of plasmid content prepared by alkaline lysis method from
all transconjugants showed a single plasmid of >30 kb. The polymerase
chain reaction analysis of the plasmid DNA confirmed the transfer of the
two integrons, which suggested that the integrons were borne by a conjugative
plasmid.
The multidrug resistance of these strains could be explained by the fact
that antimicrobial agents are used extensively in the poultry industry
in Senegal to reduce deaths and to increase productivity (1).
Moreover, in Senegal, as in many countries in Africa, antimicrobial agents
are sold over the counter, which leads to self-medication, thus increasing
the selective pressure.
This is the first finding of integrons in the newly described serovar
Keurmassar of S. enterica. One integron contained two cassettes,
aac(6´)-IIc and ereA2. This was also the first finding
of such an integron with a new arrangement of these two cassettes in a
clonal strain of S. enterica serovar Keurmassar that had recently
emerged. Indeed, the aac(6´)-IIc cassette was described only
once in P. aeruginosa. Moreover, aminoglycosides are not used extensively
in Africa because they are very expensive. Therefore, determining how
this cassette combination was selected is difficult. The strains studied
were resistant to multiple antimicrobial agents, including broad-spectrum
cephalosporins by production of the extended-spectrum β-lactamase
SHV-12. The two class 1 integrons described here could account for the
resistance to only a few drugs. The blaSHV-12 gene was
not carried by an integron. Otherwise, ampicillin, trimethoprim, and tetracycline
are the antimicrobial agents commonly used to treat diarrheal diseases
in Africa. All of the strains studied were resistant to these antimicrobials
agents. Trimethoprim resistance dfr genes are frequently found
in integrons (2). However, in this study, we were not
able to detect integrons containing dfr cassettes.
The presence of a conjugative plasmid and integrons in this serovar is
of clinical importance. Indeed, the spread of the multiple antimicrobial
agent resistance to other Salmonella serovars or gram-negative
bacteria might easily occur by the transfer of such a plasmid. Moreover,
integrons could allow the acquisition of new genes.
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