• View in gallery

    Analysis of resistant Salmonella strains by PFGE. DNA was digested with XbaI and separated by PFGE on a 1% agarose gel. The designation number of strains tested are reported above each lane. Lane 1320–2841: S. typhimurium DT104; 2033–2821: S. hadar.

  • 1

    Murray CJL, López AD, 1997. Mortality by cause for eight regions of the world: Global burden of disease study. Lancet 349 :1969–1976.

  • 2

    Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, 1995. Manual of Clinical Microbiology. Washington, DC: ASM Press.

  • 3

    Cabrera R, Ruiz J, Marco F, Oliveira I, Arroyo M, Aladueña A, Usera MA, Jiménez de Anta MT, Gascón J, Vila J, 2004. Characterization of the mechanism of resistance to several antimicrobial agents in Salmonella clinical isolates causing traveler’s diarrhea. Antimicrob Agents Chemother 48 :3934–3939.

    • Search Google Scholar
    • Export Citation
  • 4

    Stock I, Wiedemann B, 2000. Natural antibiotic susceptibility of Salmonella enterica strains. Int J Antimicrob Agents 16 :211– 217.

  • 5

    Cabrera R, Ramírez M, Bravo L, García B, Fernández A, 1998. Serotipaje de los microorganismos pertenecientes al género Salmonella. Enf Infec Microbiol 18 :150–152.

    • Search Google Scholar
    • Export Citation
  • 6

    Estevez Touzard M, Díaz González M, Monte Boada RJ, Toledo Rodríguez I, Ramón Bravo J, 1993. The infectious etiology of acute diarrheal diseases in the Republic of Cuba, 1991. Rev Cub Med Trop 45 :139–145.

    • Search Google Scholar
    • Export Citation
  • 7

    Llop A, 2002. Antimicrobial resistance and microbiological surveillance in Cuba. In: Salvatierra-Gonzalez R, Benguigui Y, eds. Antimicrobial Resistance in the Americas: Magnitude and Containment of the Problem. Washington, DC: Pan American Health Organization; 111–118.

  • 8

    Sabaté M, Prats G, 2002. Estructura y función de los integrones. Enferm Infecc Microbiol Clin 20 :341–345

  • 9

    Guerra B, Soto S, Cal S, Mendoza MC, 2000. Antimicrobial resistance and spread of Class 1 integrons among Salmonella serotypes. Antimicrob Agents Chemother 44 :2166–2169.

    • Search Google Scholar
    • Export Citation
  • 10

    Roe MT, Vega E, Pillai SD, 2003. Antimicrobial resistance markers of Class 1 and Class 2 integron-bearing Escherichia coli from irrigation water and sediments. Emerg Infect Dis 9 :822–826.

    • Search Google Scholar
    • Export Citation
  • 11

    Severino P, Magalhaes VD, 2004. Integrons as tools for epidemiological studies. Clin Microbiol Infect 10 :156–162.

  • 12

    Boop CA, Brenner FW, Wells JG, Strockbine NA, 1999. Escherichia, Shigella and Salmonella. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, eds. Manual of Clinical Microbiology. 7th ed. Washington, DC: ASM Press; 459–474.

  • 13

    National Committee for Clinical Laboratory Standards, 2003. Performance Standards for Antimicrobial Susceptibility Testing. 9th ed. Wayne, PA: National Committee for Clinical Laboratory Standards.

  • 14

    Guerra B, Malorny B, Schroeter A, Helmuth R, 2003. Multiple resistance mechanisms in fluoroquinolone-resistant Salmonella isolates from Germany. Antimicrob Agents Chemother 47 :2059.

    • Search Google Scholar
    • Export Citation
  • 15

    Navia MM, Ruiz J, Sánchez Céspedes J, Vila J, 2003. Detection of dihydrofolate reductase genes by PCR and RFLP. Diagn Microbiol Infect Dis 46 :295–298.

    • Search Google Scholar
    • Export Citation
  • 16

    Gautom RK, 1997. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other Gram-negative organisms in 1 day. J Clin Microbiol 35 :2977–2980.

    • Search Google Scholar
    • Export Citation
  • 17

    Lawson AJ, Desai M, O’Brien SJ, Davies RH, Ward LR, Therfall EJ, 2004. Molecular characterization of an outbreak strain of multiresistant Salmonella enterica serovar Typhimurium DT104 in the UK. Clin Microbiol Infect 10 :143–147.

    • Search Google Scholar
    • Export Citation
  • 18

    Threlfall EJ, 2000. Epidemic Salmonella typhimurium DT 104-a truly international multirresistant clone. J Antimicrob Chemother 46 :7–10.

    • Search Google Scholar
    • Export Citation
  • 19

    Baggesen DL, Sandvang D, Aarestrup FM, 2000. Characterization of Salmonella enterica serovar Typhimurium DT104 isolated from Denmark and comparison with isolates from Europe and the United States. J Clin Microbiol 38 :1581–1586.

    • Search Google Scholar
    • Export Citation
  • 20

    Martin LJ, Fyfe M, Dore K, Buxton JA, Pollari F, Henry B, Middleton D, Ahmed R, Jamieson F, Ciebin B, McEwen SA, Wilson JB, 2004. Increased burden of illness associated with antimicrobial-resistant Salmonella enterica serotype Typhimurium infections. J Infect Dis 189 :377–384.

    • Search Google Scholar
    • Export Citation
  • 21

    Meunier D, Boyd D, Mulvey MR, Baucheron S, Mammina C, Nastasi A, Chaslus-Dancla E, Cloeckaert A, 2002. Salmonella enterica serotype Typhimurium DT104 antibiotic resistance genomic island I in serotype paratyphi B. Emerg Infect Dis 8 :430–433.

    • Search Google Scholar
    • Export Citation
  • 22

    Espinase F, Gheorghiu R, Poiata A, Labia R, Nicolas-Chanoine MH, 1997. Reduced susceptibility to co-amoxiclav in Escherichia coli, Salmonella Typhimurium and Klebsiella pneumoniae isolated in Romania between 1985 and 1993. J Antimicrob Chemother 39 :103–106.

    • Search Google Scholar
    • Export Citation
  • 23

    Ruiz J, Navia MM, Marco F, Vila J, 2004. Mecanismos de resistencia a betalactámicos y ácido nalidíxico en aislados clínicos de Salmonella enterica serotipo Hadar y Bsilla. Enferm Infecc Microbiol Clin 22 :252–253.

    • Search Google Scholar
    • Export Citation
  • 24

    Valdezate S, Echeíta A, Díez R, Usera MA, 2000 Evaluation of phenotypic and genotypic markers for characterization of the emerging gastroenteritis pathogen Salmonella Hadar. Eur J Clin Microbiol Infect Dis 19: 275–281.

    • Search Google Scholar
    • Export Citation
  • 25

    Reguera JA, Baquero F, Pérez-Díaz JC, Martínez JL, 1991. Factors determining resistance to β-lactam combined with β-lactamase inhibitors in Escherichia coli. J Antimicrob Chemother 27 :569–573.

    • Search Google Scholar
    • Export Citation
  • 26

    Carattoli A, Filetici E, Villa L, Dionisi AM, Ricci A, Luzzi I, 2002. Antibiotic resistance genes and Salmonella genomic island 1 in Salmonella enterica serovar Typhimurium isolated in Italy. Antimicrob Agents Chemother 46 :2821–2828.

    • Search Google Scholar
    • Export Citation
  • 27

    Poirel L, Guibert M, Bellais S, Naas T, Nordmann P, 1999. Integron-and Carbenicillinase-mediated reduced susceptibility to amoxicillin-clavulanic acid in isolates of multidrug-resistant Salmonella enterica serotype Typhimurium DT 104 from French patients. Antimicrob Agents Chemother 43 :1098–1104.

    • Search Google Scholar
    • Export Citation
  • 28

    Doublet B, Lailler R, Meunier D, Brisabois A, Boyd D, Mulvey MR, Chaslus-Dancla E, Cloeckaert A, 2003. Variant Salmonella genomic island 1 resistance gene cluster in Salmonella enterica serovar Albany. Emerg Infect Dis 5 :585–591.

    • Search Google Scholar
    • Export Citation
  • 29

    Tavechio AT, Ghilardi AC, Peresi JT, Fuzihara TO, Yonamine EKM, Fernández SA, 2002. Salmonella serotypes isolated from nonhuman sources in Salmonella in Brazil, from 1996 through 2000. J Food Prot 65 :1041–1044.

    • Search Google Scholar
    • Export Citation
  • 30

    Cruchaga S, Echeíta A, Aladueña A, García-Peña J, Frias N, Usera MA, 2001. Antimicrobial resistance in Salmonella from humans, food and animals in Spain in 1998. J Antimicrob Chemother 47 :315–321.

    • Search Google Scholar
    • Export Citation
  • 31

    Centers for Disease Control, 1998. Multistate outbreak of Salmonella serotype Agona infections linked to toasted oats cereal-United States. JAMA 280 :411.

    • Search Google Scholar
    • Export Citation
  • 32

    Eiguer T, Butta N, 1983. Annual distribution of serotypes of Salmonella, Shigella, and infantile enteropathogenic Escherichia coli in the Republic of Argentina 1979–1981. Rev Argent Microbiol 15 :19–24.

    • Search Google Scholar
    • Export Citation
  • 33

    Gutiérrez-Cogco L, Montiel-Vázquez E, Aguilera-Pérez P, González-Andrade MC, 2000. Serotipos de Salmonella identificados en los servicios de salud de México. Salud Pública Méx 42 :490–495.

    • Search Google Scholar
    • Export Citation
  • 34

    Boyd D, Cloeckaert A, Chaslus-Dancla E, Mulvey MR, 2002. Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob Agents Chemother 46 :1714–1722.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DISSEMINATION OF SALMONELLA ENTERICA SEROTYPE AGONA AND MULTIDRUG-RESISTANT SALMONELLA ENTERICA SEROTYPE TYPHIMURIUM IN CUBA

View More View Less
  • 1 Servicio de Microbiología, IDIBAPS, Hospital Clínic, Barcelona, Spain; Centro de Salud Internacional, Universidad de Barcelona, Hospital Clínic, Barcelona, Spain; Instituto de Medicina Tropical “Pedro Kourí,” Ciudad Habana, Cuba; Instituto de Salud Carlos III, Majadahonda, Madrid, Spain

The molecular epidemiology, antimicrobial susceptibility, and mechanisms of resistance of 34 Salmonella spp. strains causing acute gastroenteritis, isolated from different provinces in Cuba, were determined. Sixty-four percent of the strains showed multiresistance. Salmonella typhimurium was the most frequent with 15 strains (44%), 13 of which belonged to phagotype 104 and presented similar genetic profiles of pulsed field gel electrophoresis. High levels of resistance to tetracycline (53%), spectinomycin (50%), ampicillin (44%), and chloramphenicol (41%) were found. Resistance to tetracycline was associated with the tet G and tet A genes. Resistance to ampicillin was caused by the presence of β-lactamases, mainly the CARB type. The floR gene was the main mechanism of resistance to chloramphenicol. Our results showed an antimicrobial susceptible clone of Salmonella enterica serotype Agona in two separate regions. This is the first report of the widespread dissemination of a multiresistant clone of S. enterica serotype Typhimurium definitive phage type 104 in Cuba.

INTRODUCTION

In both developing and developed countries, acute gastroenteritis causes high levels of morbidity and mortality.1 One of the main etiological agents of this disease is Salmonella spp. The Salmonella genus is composed of approximately 2,400 serotypes with different phenotypic and genotypic characteristics.2 One of these characteristics is antimicrobial resistance. The Salmonella genus has been traditionally susceptible to antimicrobial agents, but an increase in the levels of resistance worldwide has been observed.3,4 Salmonella infections in Cuba is the third more frequent causes of acute gastroenteritis in adults and children after rotavirus and Entamoeba histolytica.5,6

In Cuba, several studies have reported a high percentage of Salmonella spp. strains resistant to several antimicrobial agents such as ampicillin, chloramphenicol, nalidixic acid, and trimethoprim/sulphamethoxazole.7

An efficient route of acquisition of resistance is through genetic elements including plasmids, transposons, and integrons. Currently, nine classes of integrons have been described, with the class 1 integron being the most frequently detected in clinical strains.8 Integrons have been described as an important cause of the spread of antimicrobial resistance in a variety of enteric bacteria including Salmonella.9,10 Two scenarios can be envisaged in antimicrobial resistance: 1) dissemination of an antimicrobial-resistant clone or 2) dissemination of a resistant gene carried in a genetic element. Integrons have also been considered as a tool for studying molecular epidemiology.11

The aim of this study was to determine the antimicrobial susceptibility and the molecular mechanisms of resistance of Salmonella spp. strains causing acute gastroenteritis in Cuba and to determine the potential dissemination of a resistant clone.

MATERIALS AND METHODS

Bacterial isolates.

A total of 34 sporadic Salmonella strains isolated from feces of patients with acute gastroenteritis isolated from different regions of Cuba in 2002 were analyzed. The strains were isolated by the different Provincial Reference Laboratories of each region of the country (the laboratories belong to a National Surveillance Network) and sent to the National Reference Laboratory of Cuba. The strains were further studied in the Laboratory of Clinical Microbiology at the Clínic Hospital in Barcelona, Spain. The strains were isolated and identified by conventional methods.12

Serotyping and phage typing.

Serotyping and phage typing were performed in the Carlos III Institute (Madrid, Spain), taking into account the Manual of Kauffman and White and following the recommendations of Edward and Ewing.2,12

Antimicrobial susceptibility testing.

The antimicrobial susceptibility test to nine antimicrobial agents, ampicillin (AMP), amoxicillin/clavulanic acid (AMC), nalidixic acid (NAL), tetracycline (TET), trimethoprim/sulphamethoxazole (SXT), chloramphenicol (CHL), gentamicin (GEM), ciprofloxacin (CIP), and spectinomycin (SPT), was performed using the Kirby-Bauer method. Interpretation of results was carried out according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines.13 Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Escherichia coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853 were used as controls. Multiresistance was defined as resistance to four or more unrelated antimicrobial agents.

Detection of the mechanisms of resistance.

To determine the quinolone-resistance mechanisms, mutations in the quinolone-resistant determining region (QRDR) of the gyrA gene were detected by polymerase chain reaction (PCR) and DNA sequencing. A previously described colorimetric assay was performed to detect the presence of chloramphenicol acetyl transferase activity (CAT).14 The presence of the cmlA and floR genes associated with chloramphenicol resistance, the genes encoding β-lactamases (tem-like, carb-like, shv-like, and oxa 1-like), and the tet A, tet B, and tet G genes related to tetracycline resistance acquisition were detected by PCR. To determine the mechanism of trimethoprim resistance, the presence of dihydrofolate reductases was detected by PCR with generic primers and posterior restriction fragment length polymorphism (RFLP) with the appropriate restriction enzyme according to previously described methodology.15 The PCR products were detected by electrophoresis in 2% agarose gels. All the primers and PCR conditions used in this study have been previously described.3

Detection of integrons.

In all the resistant Salmonella strains, the presence of Class 1 integrons was detected by PCR with specific primers described previously.14

DNA sequencing of the PCR products.

The purified PCR products visualized in gels were processed for DNA sequencing and analyzed in an automatic DNA sequencer (ABI 377; Perkin Elmer, Emeryville, CA) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Perkin Elmer).

Low-frequency restriction analysis of chromosomal DNA and pulsed field gel electrophoresis.

The analysis of chromosomal DNA by digestion with low frequency of cleavage restriction enzymes and separation of the fragments by pulsed field gel electrophoresis (PFGE) was performed as follows. Salmonella strains were cultivated in brain-heart infusion (BHI) and incubated overnight. They were resuspended in buffer TE-1. The plugs containing the DNA were elaborated with agarose insert 1.8%.16 Chromosomal DNA contained in the agarose plugs was digested with XbaI (Roche Diagnostic, Mannheim, Germany), and PFGE was performed with the CHEF DR II system (Biorad Laboratories, Hercules, CA) in 0.5× Tris-Borate-EDTA buffer (Severn Biotech, Kidderminster, UK). DNA restriction fragments were resolved in agarose (Bio-Rad) 1% wt/vol gels, and a low-range PFGE marker (New England Biolabs, Beverly, MA) was used as a size standard. Electrophoresis conditions were of 2.0–64 seconds for 20 hours.17

RESULTS

Of the 34 Salmonella strains studied from different regions around the country, Salmonella typhimurium was the most frequent, with 15 strains (44%), 13 of which belonged to phagotype 104, with 2 strains showing a non-recognized phagotype pattern. Salmonella agona was the second most frequently isolated serotype with eight strains (23%), followed by Salmonella hadar with two strains. Only one strain was found in other serotypes, such as Salmonella senftenberg, Salmonella infantis, Salmonella branderburg, Salmonella orion, Salmonella saintpaul, and one monophasic. Three strains were non-typable.

In the 34 Salmonella strains studied, 22 strains presented resistance to at least one antimicrobial agent, with S. typhimurium with 15 strains (68%) being the most prevalent among the resistant Salmonella. High levels of resistance were found mainly to tetracycline in 18 strains (53%), spectinomycin in 17 strains (50%), ampicillin in 15 strains (44%), and to chloramphenicol in 14 strains (41%). In addition, three strains presented resistance to amoxicillin/clavulanic acid, and eight strains (24%) showed intermediate resistance to amoxicillin/clavulanic acid (Table 1).

Molecular epidemiologic analysis was performed in S. typhimurium and S. hadar because of their multiresistance and in S. agona because of the fact that it was the second most prevalent serotype isolated. S. typhimurium showed five patterns of PFGE, being the pattern number 1 the most frequent with 11 isolates of 15 (Table 2; Figure 1). Although showing a different phagotype, the two strains corresponding to serotype Hadar presented an identical pattern of PFGE (Table 2). Among the eight Salmonella strains belonging to serotype Agona, six of the seven susceptible isolates belonged to the same PFGE pattern, whereas the remaining susceptible isolate and the resistant isolate each showed a different pattern. The geographical distribution of the resistant clones is shown in Table 2, which shows that the main S. typhimurium clone (clone 1) was detected in eight different provinces, distributed throughout the island, and the S. agona clone was disseminated in two provinces: Cienfuegos and Holguín.

Analysis of the molecular mechanisms of resistance showed that resistance to tetracycline was caused by the presence of the tet G gene (13 of 18 tetracycline-resistant strains) and the tet A gene (6 of 18 tetracycline-resistant strains). One strain presented both resistant determinants. The main mechanism of ampicillin resistance was the presence of CARB type β-lactamases, observed in 13 of 15 ampicillin-resistant strains, with only one strain of S. hadar showing a TEM type β-lactamases. No strain presented OXA type and SHV type β-lactamases. All chloramphenicol-resistant isolates presented the floR gene, and only the S. senftenberg strain showed CAT activity. None of the isolates presented the cmlA gene. One strain presented resistance to trimethoprim/sulphamethoxazole because of the presence of the dfrA1 gene (Table 3). The strain with intermediate susceptibility to nalidixic acid did not show any mutation in the gyr A or par C genes.

Two Class 1 integrons were present in 11 of the 22 resistant strains (Table 4): one integron of 1,200 bp containing the carb2 gene, and the other with a size of approximately 1,000 bp containing the aadA2 gene. Another Class 1 integron (1,500 bp) was detected in the S. senftenberg strain and contained the aadB and aadA2 genes. Finally, two S. typhimurium and one S. agona with resistance to spectinomycin showed one Class 1 integron with a size of 1,000 bp containing the aadA2 gene.

DISCUSSION

Although S. typhimurium definitive phage type (DT) 104 has been detected in human infections in England and Wales since the early 1960s, multidrug-resistant strains of this phage type were not identified until the early 1980s.18 In this study, despite only 34 Salmonella strains analyzed, we could detect the dissemination of S. typhimurium DT 104 in different provinces of Cuba. In addition, isolates belonging to the same clone were detected in the same geographical area, suggesting a local outbreak. The same scenario was reported in previous studies performed in Canada, United States, and Europe.1921 This phage type is commonly resistant to ampicillin, chloramphenicol/flufenisal, spectinomycin/ streptomycin, sulfonamides, and tetracyclines.21 In addition of this typical resistance pattern, some strains of S. typhimurium DT 104 also presented resistance to fluoroquinolones and trimethoprim. The high percentage of intermediate resistance to amoxicillin/clavulanic acid found among our strains is worthy of mention. Different reports have shown a decrease in the susceptibility of S. typhimurium and other serotypes to this antimicrobial agent. Espinase et al.22 reported a decreased susceptibility to amoxicillin/clavulanic acid in three S. typhimurium strains isolated from patients in Rumania. Similarly, in a French university hospital, many S. typhimurium strains with reduced susceptibility to amoxicillin/clavulanic acid were isolated.15 This fact has also been shown in S. hadar and S. bsilla.23,24 Theoretically, several mechanisms may explain this reduced susceptibility to amoxicillin/clavulanic acid, including a decrease in permeability to β-lactams, as is known for Escherichia coli, or the over-expression of specific β-lactamases.25 In our study, all strains with reduced susceptibility carried the β-lactamases type CARB, whose over-expression may explain the origin of this reduced susceptibility to amoxicillin/clavulanic acid.

The PFGE patterns generated with the XbaI enzyme found in our S. typhimurium DT 104 strain were similar to other described before by several authors using the same conditions.19,26,27 Baggesen et al.19 compared the PFGE profile of Danish S. typhimurium DT 104 strains with the profile of PFGE obtained in strains isolated in five different countries and found highly homogeneous profiles, indicating that the multidrug-resistant S. typhimurium DT 104 had probably been spread clonally in these countries.

All the strains analyzed belonging to S. typhimurium DT 104 presented the floR and tet G genes, being responsible for resistance to chloramphenicol and tetracycline, respectively. These genes have been previously described to be located in the Salmonella genomic island 1 (SGI1).26,28 In this island, resistance genes are clustered and are bracketed by two integron structures,18 identical to those found in our study. The first integron carried the aadA2 gene, which confers resistance to spectinomycin and streptomycin, and the second integron contained the carb2 gene, conferring resistance to ampicillin.28 All the S. typhimurium DT 104 strains but two analyzed in our study presented these two integrons. The two strains presenting only the integron carrying the aadA2 gene may have a truncated resistance island. Therefore, S. typhimurium strains with the same pattern by PFGE can present different integrons, suggesting that this is not a discriminate tool to be used in epidemiologic analysis, as has been suggested.11

Three strains were not typable using the traditional serotyping techniques. Two of these three strains presented a similar mechanism of resistance profile as S. typhimurium DT 104. Other serotypes that showing a multiresistant phenotype were S. hadar and S. senftenberg, the latter of which is not frequently found. However, in some countries such as Brazil, it is a frequent serotype, occupying the third place of Salmonella isolates (10.3%) behind S. typhimurium and S. enteritidis.29 According to the network of human Salmonella surveillance in Europe, S. hadar is widely distributed across the European continent.24 Cruchaga et al.30 reported high levels of resistance, mainly to spectinomycin, ampicillin, tetracycline, and nalidixic acid, in strains of S. hadar isolates from humans and food, respectively. Analysis of PFGE profiles of the two resistant S. hadar was similar, but the PFGE in this serotype is of limited value, and the combination of different epidemiologic methods is recommended depending of the phage type studied.23 In our study, these two strains belonged to two different phage types and also showed different resistance phenotypes. Therefore, they may be considered two different clones, although those differences in the resistant phenotypes and phagotypes might reflect the geographical distribution of a single clonal type.

Epidemiologic analysis by PFGE of the S. agona strains showed a predominant clone disseminated mainly in two separate provinces: Cienfuegos and Holguín. In fact, although no molecular epidemiologic analysis have been performed, previous reports described this serotype among one of the main Salmonella serotypes isolated throughout the country and in several countries of American continent.4,3133 In 1998 in the United States, a total of 11 states reported an increase in cases of S. agona.31 In Argentina and Mexico, several authors reported this serotype among the most frequent Salmonella isolated.32,33 The presence of antimicrobial-resistant strains of this serotype has previously been reported.34 However, the prevalence of antimicrobial-resistant strains is lower than the antimicrobial resistance observed in S. typhimurium. In some S. agona–resistant strains, the same resistant island, SGI1, has been shown.34 However, in our study, the multiresistant S. agona strain did not show the resistant determinants located in this island, although the integron, carrying the aadA2 gene, was found.

Our results show the dissemination of a multiresistant clone of S. typhimurium DT 104 and an antimicrobial susceptible clone of S. agona in two separate regions in Cuba. This work presents the first description in Cuba of the multiresistant clone of S. typhimurium DT 104 since first being identified in the United Kingdom in the late 1980s.18

Table 1

Antimicrobial susceptibility of the Salmonella clinical isolates

ResistantIntermediateSusceptible
Antimicrobial agentN%N%N%
N, number of strains.
Ampicillin1544001956
Amoxicillin/clavulanic acid398242367
Spectinomycin1750001750
Nalidixic acid00133397
Ciprofloxacin000034100
Gentamicin26003294
Trimethoprim-sulphametoxazole13003397
Tetracycline1853001647
Chloramphenicol1441002059
Table 2

Serotype, phagetype, and PFGE pattern of the resistant Salmonella strains analyzed

StrainProvinceSerotypePhagetypePFGE
PNR, pattern not recognized; NT, non-typable; NP, non-performed.
1320GranmaTyphimurium104I
1837HolguínTyphimurium104I
2667Santiago de CubaTyphimurium104I
1273CamagüeyTyphimurium104II
2834HolguínTyphimurium104I
3098La HabanaTyphimurium104I
47CamagüeyTyphimurium104I
1822HolguínTyphimurium104I
1270CamagüeyTyphimurium104I
410Las TunasTyphimurium104I
2841HolguínTyphimurium104III
1946Pinar del RíoTyphimurium104I
3147Sancti SpiritusTyphimurium104I
885MatanzasTyphimuriumPNRIV
1958HolguínTyphimuriumPNRV
2821HolguínHadar17VI
2033MatanzasHadar22VI
1764Santiago de CubaAgonaNTVII
1708CamagüeySenftenbergNTNP
1818Holguín4.12NTNP
1829Holguín4.12NTNP
1989bPinar del Rio4.12NTNP
Table 3

Mechanisms of resistance of the resistant Salmonella spp.

Mechanism of resistance
StrainSerotypeSPTCHLAMPTETSXT
ND, not determined; SPT, spectinomycin; CHL, chloramphenicol; AMP, ampicillin; TET, tetracycline; SXT, trimethoprim/sulphamethoxazole.
2033S. hadartemtet A
18184, 12aadA2floRcarb2tet G
1320S. typhimuriumaadA2floRcarb2tet G
18294, 12aadA2floRcarb2tet G
1837S. typhimuriumaadA2floRcarb2tet G
2667S. typhimuriumaadA2floRcarb2tet G
1273S. typhimuriumaadA2floRcarb2tet A, tet G
2834S. typhimuriumaadA2floRcarb2tet G
3098S. typhimuriumaadA2floRcarb2tet G
47S. typhimuriumaadA2floRcarb2tet G
1989b4, 12tet A
1708S. senftenbergaadA2CATdfrA1
1958S. typhimuriumtet A
1822S. typhimuriumaadA2floRcarb2tet G
1270S. typhimuriumaadA2floRcarb2tet G
2821S. hadartet A
885S. typhimuriumND
410S. typhimuriumaadA2floRcarb2tet G
2841S. typhimuriumaadA2floRcarb2tet G
1946S. typhimuriumaadA2
3147S. typhimuriumaadA2
1764S. agonaaadA2
Table 4

Class 1 integrons observed

StrainsSerotypeCompositionSize
11S. typhimuriumcarb2/aadA21,200, 1,000 bp
2S. typhimuriumaadA21,000 bp
24, 12carb2/aadA21,200, 1,000 bp
1S. agonaaadA21,000 bp
1S. senftenbergaadB, aadA21,500 bp
Figure 1.
Figure 1.

Analysis of resistant Salmonella strains by PFGE. DNA was digested with XbaI and separated by PFGE on a 1% agarose gel. The designation number of strains tested are reported above each lane. Lane 1320–2841: S. typhimurium DT104; 2033–2821: S. hadar.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 6; 10.4269/ajtmh.2006.74.1049

*

Address correspondence to Jordi Vila, Servicio de Microbiología, Hospital Clínic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. E-mail: jvila@ub.edu

Authors’ addresses: Roberto Cabrera and Jordi Vila, Servicio de Microbiología, IDIBAPS, Hospital Clínic, Villarroel 170, Barcelona, Spain; Joaquim Ruiz, Joaquim Gascón, and Pedro L. Alonso, Centro de Salud Internacional, Universidad de Barcelona, Hospital Clínic, Barcelona, Spain; Margarita Ramírez, Laura Bravo, and Anabel Fernández, Instituto de Medicina Tropical “Pedro Kourí”, Autopista Novia del Medio día, Km 6, CP 601, Marianao 13, Ciudad Habana, Cuba. Ana Aladueña and Aurora Echeíta, Instituto de Salud Carlos III, Carretera de Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain.

Financial support: This study was funded by grant FIS02/0353 from the Spanish Ministry of Health, by grant 2005 SGR 00444 from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya, Spain. R. Cabrera has a fellowship from Fundación Carolina and BBVA, Spain, and J. Ruiz has a fellowship from RICET.

REFERENCES

  • 1

    Murray CJL, López AD, 1997. Mortality by cause for eight regions of the world: Global burden of disease study. Lancet 349 :1969–1976.

  • 2

    Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, 1995. Manual of Clinical Microbiology. Washington, DC: ASM Press.

  • 3

    Cabrera R, Ruiz J, Marco F, Oliveira I, Arroyo M, Aladueña A, Usera MA, Jiménez de Anta MT, Gascón J, Vila J, 2004. Characterization of the mechanism of resistance to several antimicrobial agents in Salmonella clinical isolates causing traveler’s diarrhea. Antimicrob Agents Chemother 48 :3934–3939.

    • Search Google Scholar
    • Export Citation
  • 4

    Stock I, Wiedemann B, 2000. Natural antibiotic susceptibility of Salmonella enterica strains. Int J Antimicrob Agents 16 :211– 217.

  • 5

    Cabrera R, Ramírez M, Bravo L, García B, Fernández A, 1998. Serotipaje de los microorganismos pertenecientes al género Salmonella. Enf Infec Microbiol 18 :150–152.

    • Search Google Scholar
    • Export Citation
  • 6

    Estevez Touzard M, Díaz González M, Monte Boada RJ, Toledo Rodríguez I, Ramón Bravo J, 1993. The infectious etiology of acute diarrheal diseases in the Republic of Cuba, 1991. Rev Cub Med Trop 45 :139–145.

    • Search Google Scholar
    • Export Citation
  • 7

    Llop A, 2002. Antimicrobial resistance and microbiological surveillance in Cuba. In: Salvatierra-Gonzalez R, Benguigui Y, eds. Antimicrobial Resistance in the Americas: Magnitude and Containment of the Problem. Washington, DC: Pan American Health Organization; 111–118.

  • 8

    Sabaté M, Prats G, 2002. Estructura y función de los integrones. Enferm Infecc Microbiol Clin 20 :341–345

  • 9

    Guerra B, Soto S, Cal S, Mendoza MC, 2000. Antimicrobial resistance and spread of Class 1 integrons among Salmonella serotypes. Antimicrob Agents Chemother 44 :2166–2169.

    • Search Google Scholar
    • Export Citation
  • 10

    Roe MT, Vega E, Pillai SD, 2003. Antimicrobial resistance markers of Class 1 and Class 2 integron-bearing Escherichia coli from irrigation water and sediments. Emerg Infect Dis 9 :822–826.

    • Search Google Scholar
    • Export Citation
  • 11

    Severino P, Magalhaes VD, 2004. Integrons as tools for epidemiological studies. Clin Microbiol Infect 10 :156–162.

  • 12

    Boop CA, Brenner FW, Wells JG, Strockbine NA, 1999. Escherichia, Shigella and Salmonella. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, eds. Manual of Clinical Microbiology. 7th ed. Washington, DC: ASM Press; 459–474.

  • 13

    National Committee for Clinical Laboratory Standards, 2003. Performance Standards for Antimicrobial Susceptibility Testing. 9th ed. Wayne, PA: National Committee for Clinical Laboratory Standards.

  • 14

    Guerra B, Malorny B, Schroeter A, Helmuth R, 2003. Multiple resistance mechanisms in fluoroquinolone-resistant Salmonella isolates from Germany. Antimicrob Agents Chemother 47 :2059.

    • Search Google Scholar
    • Export Citation
  • 15

    Navia MM, Ruiz J, Sánchez Céspedes J, Vila J, 2003. Detection of dihydrofolate reductase genes by PCR and RFLP. Diagn Microbiol Infect Dis 46 :295–298.

    • Search Google Scholar
    • Export Citation
  • 16

    Gautom RK, 1997. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other Gram-negative organisms in 1 day. J Clin Microbiol 35 :2977–2980.

    • Search Google Scholar
    • Export Citation
  • 17

    Lawson AJ, Desai M, O’Brien SJ, Davies RH, Ward LR, Therfall EJ, 2004. Molecular characterization of an outbreak strain of multiresistant Salmonella enterica serovar Typhimurium DT104 in the UK. Clin Microbiol Infect 10 :143–147.

    • Search Google Scholar
    • Export Citation
  • 18

    Threlfall EJ, 2000. Epidemic Salmonella typhimurium DT 104-a truly international multirresistant clone. J Antimicrob Chemother 46 :7–10.

    • Search Google Scholar
    • Export Citation
  • 19

    Baggesen DL, Sandvang D, Aarestrup FM, 2000. Characterization of Salmonella enterica serovar Typhimurium DT104 isolated from Denmark and comparison with isolates from Europe and the United States. J Clin Microbiol 38 :1581–1586.

    • Search Google Scholar
    • Export Citation
  • 20

    Martin LJ, Fyfe M, Dore K, Buxton JA, Pollari F, Henry B, Middleton D, Ahmed R, Jamieson F, Ciebin B, McEwen SA, Wilson JB, 2004. Increased burden of illness associated with antimicrobial-resistant Salmonella enterica serotype Typhimurium infections. J Infect Dis 189 :377–384.

    • Search Google Scholar
    • Export Citation
  • 21

    Meunier D, Boyd D, Mulvey MR, Baucheron S, Mammina C, Nastasi A, Chaslus-Dancla E, Cloeckaert A, 2002. Salmonella enterica serotype Typhimurium DT104 antibiotic resistance genomic island I in serotype paratyphi B. Emerg Infect Dis 8 :430–433.

    • Search Google Scholar
    • Export Citation
  • 22

    Espinase F, Gheorghiu R, Poiata A, Labia R, Nicolas-Chanoine MH, 1997. Reduced susceptibility to co-amoxiclav in Escherichia coli, Salmonella Typhimurium and Klebsiella pneumoniae isolated in Romania between 1985 and 1993. J Antimicrob Chemother 39 :103–106.

    • Search Google Scholar
    • Export Citation
  • 23

    Ruiz J, Navia MM, Marco F, Vila J, 2004. Mecanismos de resistencia a betalactámicos y ácido nalidíxico en aislados clínicos de Salmonella enterica serotipo Hadar y Bsilla. Enferm Infecc Microbiol Clin 22 :252–253.

    • Search Google Scholar
    • Export Citation
  • 24

    Valdezate S, Echeíta A, Díez R, Usera MA, 2000 Evaluation of phenotypic and genotypic markers for characterization of the emerging gastroenteritis pathogen Salmonella Hadar. Eur J Clin Microbiol Infect Dis 19: 275–281.

    • Search Google Scholar
    • Export Citation
  • 25

    Reguera JA, Baquero F, Pérez-Díaz JC, Martínez JL, 1991. Factors determining resistance to β-lactam combined with β-lactamase inhibitors in Escherichia coli. J Antimicrob Chemother 27 :569–573.

    • Search Google Scholar
    • Export Citation
  • 26

    Carattoli A, Filetici E, Villa L, Dionisi AM, Ricci A, Luzzi I, 2002. Antibiotic resistance genes and Salmonella genomic island 1 in Salmonella enterica serovar Typhimurium isolated in Italy. Antimicrob Agents Chemother 46 :2821–2828.

    • Search Google Scholar
    • Export Citation
  • 27

    Poirel L, Guibert M, Bellais S, Naas T, Nordmann P, 1999. Integron-and Carbenicillinase-mediated reduced susceptibility to amoxicillin-clavulanic acid in isolates of multidrug-resistant Salmonella enterica serotype Typhimurium DT 104 from French patients. Antimicrob Agents Chemother 43 :1098–1104.

    • Search Google Scholar
    • Export Citation
  • 28

    Doublet B, Lailler R, Meunier D, Brisabois A, Boyd D, Mulvey MR, Chaslus-Dancla E, Cloeckaert A, 2003. Variant Salmonella genomic island 1 resistance gene cluster in Salmonella enterica serovar Albany. Emerg Infect Dis 5 :585–591.

    • Search Google Scholar
    • Export Citation
  • 29

    Tavechio AT, Ghilardi AC, Peresi JT, Fuzihara TO, Yonamine EKM, Fernández SA, 2002. Salmonella serotypes isolated from nonhuman sources in Salmonella in Brazil, from 1996 through 2000. J Food Prot 65 :1041–1044.

    • Search Google Scholar
    • Export Citation
  • 30

    Cruchaga S, Echeíta A, Aladueña A, García-Peña J, Frias N, Usera MA, 2001. Antimicrobial resistance in Salmonella from humans, food and animals in Spain in 1998. J Antimicrob Chemother 47 :315–321.

    • Search Google Scholar
    • Export Citation
  • 31

    Centers for Disease Control, 1998. Multistate outbreak of Salmonella serotype Agona infections linked to toasted oats cereal-United States. JAMA 280 :411.

    • Search Google Scholar
    • Export Citation
  • 32

    Eiguer T, Butta N, 1983. Annual distribution of serotypes of Salmonella, Shigella, and infantile enteropathogenic Escherichia coli in the Republic of Argentina 1979–1981. Rev Argent Microbiol 15 :19–24.

    • Search Google Scholar
    • Export Citation
  • 33

    Gutiérrez-Cogco L, Montiel-Vázquez E, Aguilera-Pérez P, González-Andrade MC, 2000. Serotipos de Salmonella identificados en los servicios de salud de México. Salud Pública Méx 42 :490–495.

    • Search Google Scholar
    • Export Citation
  • 34

    Boyd D, Cloeckaert A, Chaslus-Dancla E, Mulvey MR, 2002. Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob Agents Chemother 46 :1714–1722.

    • Search Google Scholar
    • Export Citation

Author Notes

Reprint requests: Jordi Vila, Servicio de Microbiología,, Hospital Clínic de Barcelona, Villarroel 170, 08036 Barcelona, Spain. E-mail: jvila@ub.edu.
Save