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Chemotherapy 2007;53:392–396 DOI: 10.1159/000109768

Molecular Epidemiology and Antibiotic Resistance Patterns of Salmonella enterica from Southwestern Greece

Iris Spiliopoulou Sofia Zografou Amalia Goula George Dimitracopoulos

Myrto Christofidou

Department of Microbiology, School of Medicine, University of Patras, Patras , Greece

Introduction

The occurrence of food-borne diseases caused by non-typhoid Salmonella is an important public health prob-lem worldwide. The emergence and spread of antimicro-bial resistant strains including Salmonella species have reinforced the need for epidemiological studies describ-ing the prevalence and the patterns of resistance of these bacteria. In Europe, Salmonella enterica serotype Enter-itidis has emerged as the most prevalent serotype associ-ated with gastrointestinal infections [1–3] . The Centers for Disease Control and Prevention also reported that the two most frequent serovars since 1993 are Salmonella en-teritidis and Salmonella typhimurium [4] . Additionally S. typhimurium isolates express a multidrug-resistant phenotype [4–6] .

The application of pulsed-field gel electrophoresis (PFGE) has been proved to be useful for the discrimina-tion and epidemiological characterization of S. enterica strains [1, 3, 5, 6] . In a study conducted on the distribu-tion of S. enteritidis among Salmonella strains referred to the National Reference Center for Salmonella and Shi-gella from 1987 to 1993 in Greece, 3 major clones were identified by PFGE, including strains with different sus-ceptibility patterns [1] . In another study performed in the same center, 328 S. typhimurium isolates from different sources were tested and grouped into 26 resistance phe-notypes and five types of PFGE [5] .

Key Words

Salmonella enterica � Serotypes � Antibiotic resistance � Clones

Abstract

Background: A study was conducted at the University Hos-pital of Patras between January 2002 and December 2003 to investigate antibiotic resistance patterns and clonality of Salmonella enterica in southwestern Greece. Methods: Nine-ty-five isolates recovered from different outpatients were characterized by specific antisera and were tested for their susceptibility to various antimicrobial agents. Clones were identified by pulsed-field gel electrophoresis (PFGE) of Xba I chromosomal DNA digests. Results: Five serotypes were characterized among 95 isolates, recovered mainly from children, with the predominance of Salmonella enteritidis fol-lowed by Salmonella typhimurium . The strains were further distinguished by PFGE to 16 clones. The majority of S. enter-itidis (61 strains) belonged to clone A, already characterized in Greece, while S. typhimurium (18 isolates) belonged to 3 clones, exhibiting multiresistant phenotypes. Conclusions: One clone S. enteritidis , type A, circulates in southwestern Greece, mainly during summer, while clonality was also ob-served among S. typhimurium .

Copyright © 2007 S. Karger AG, Basel

Received: February 7, 2006 Accepted after revision: July 24, 2006 Published online: October 12, 2007

Myrto Christofidou, MD Department of Microbiology, School of Medicine University of Patras GR–26500 Patras (Greece) Tel. +30 2610 993 978, Fax +30 2610 994 922, E-Mail christof@med.upatras.gr

© 2007 S. Karger AG, Basel 0009–3157/07/0536–0392$23.50/0

Accessible online at: www.karger.com/che

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The aim of the present study was to obtain recent in-formation on the antibiotic resistance patterns and the epidemiological relationship among the nontyphoid Sal-monella isolates recovered from different outpatients with gastrointestinal infections during a 2-year period (2002–2003) in the University Hospital of Patras, in southwestern Greece. For this purpose phenotypes and genotypes of the isolates were characterized by serotyp-ing, antibiotic resistance testing and PFGE of Xba I DNA digests. Our results were compared with the 3 major S. enteritidis clones previously characterized in Greece.

Materials and Methods

A total of 219 fecal specimens from outpatients with gastroen-teritis attending the clinics at the University Hospital of Patras were tested for the presence of Salmonella species from January 2002 to December 2003. This hospital covers an area of 740,000 inhabitants, roughly 7% of the total population of Greece. After overnight incubation at 37 ° C in selenite enrichment broth (Bec-ton Dickinson, BD BBL, Le Pont de Claix, France) the samples were subcultered on Salmonella-Shigella and bismuth sulfite agar plates (BD BBL). Following a further incubation period overnight at 37 ° C, colonies were identified by their biochemical profile us-ing the Enterotube II system (BD BBL) and were serotyped for cell wall and flagellar antigens by the Kauffmann-White method us-ing agglutination with specific antisera (Difco, BD BBL) [7] .

Susceptibility tests to various antimicrobials, ampicillin, amoxicillin with clavulanic acid, ceftriaxone, chloramphenicol, ciprofloxacin, tetracycline, streptomycin and sulfamethoxazole/trimethoprim were performed both by the disk diffusion method (BD BBL) and by measuring minimum inhibitory concentrations (MICs) using Etest strips (AB BIODISK, Solna, Sweden). Disk dif-fusion susceptibility tests and MICs were interpreted according to the recommendations of the National Committee for Clinical Laboratory Standards and published breakpoints for fluoroqui-nolones (resistant if MIC 6 0.125 mg/l) [8–10] .

Molecular typing of the isolates was performed by PFGE. Preparation of chromosomal DNA, digestion with Xba I and elec-trophoresis in a CHEF-DRIII apparatus (Bio-Rad, Herculus, Ca-lif., USA) were carried out as described previously [1, 5, 6] . The banding patterns of the strains were compared visually following the criteria of Tenover et al. [11] . Isolates that differed in their PFGE fragment patterns by one to three bands were regarded as closely related and were classified in the same clone [5, 6, 11] . Three epidemic S. enterica strains characterized in the hospitals of Athens were included in the PFGE analysis [1] .

Results and Discussion

In the present study, 95 S. enterica isolates were recov-ered from different outpatients (1 isolate per patient). The serovars identified during the 2-year period were S. en-

teritidis (65 isolates or 68%) followed by S. typhimurium (18 isolates or 19%), Salmonella newport (7 isolates or 7%), Salmonella infantis (4 isolates or 4%) and 1 Salmo-nella paratyphi A. Nontyphoidal salmonellae are recog-nized as one of the principal causes of food poisoning worldwide [2–6] . An increased incidence of gastrointes-tinal infections caused by S. enterica serovar Enteritidis has been reported in Greece and in other countries [1–3] . The predominance of S. enteritidis is in accordance with data reported from Sweden, France and previous studies in Greece [1–3] .

In our study 69 isolates were recovered from children younger than 17 years of age, 50 males and 19 females. Twenty-six isolates were recovered from adults, 13 males and 13 females, 18–70 years old ( table 1 ). Fifty S. enteriti-dis , 12 S. typhimurium and all (7) S. newport were iso-lated from children, including isolates with a variable an-tibiotic resistance phenotype. No association of gender was observed among adults, while among children boys with salmonellosis predominated, a result that may rep-resent a more adventurous lifestyle, being exposed to contamination on a daily basis. In a surveillance study in Sweden, investigating the serotype epidemiology in re-turning travelers from various countries including Greece, it was shown that the risk of salmonellosis de-creased with increasing age, a risk that is associated with increased fecal/oral contamination and decreased im-munity, as well as ingestion of larger volumes of poten-tially contaminated food by children [2] .

The highest risk of salmonellosis in Europe exists dur-ing June to September [2, 3] . This is also supported byour study ( fig. 1 ). The majority of S. enteritidis as well as S. typhimurium isolates were collected during summer with a peak in July 2003. Surveillance data from Norway, Sweden and Finland of Salmonella infections among travelers returning from Greece have revealed the spread of S . enteritidis PT 14b especially in Crete [12] . Eating chicken was related to illness and the first peak of cases observed in mid-August might be associated with out-breaks in the country as well [12] .

In our collection, no isolate exhibited resistance to cef-triaxone and ciprofloxacin even though lower MIC break-points were used for ciprofloxacin [10] . Reduced suscep-tibility to fluoroquinolones has been reported to be as-sociated with the activation of efflux pump systems even among strains without gyrA mutations [13] . In another study, testing susceptibility of S . typhi and S . paratyphi A, no isolate exhibited resistance to ceftriaxone and cipro-floxacin [14] . Seventeen isolates (18%) were resistant to tetracycline, 13 (14%) to ampicillin, 10 (11%) to strepto-

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mycin, 7 (7%) to chloramphenicol, 6 (6%) to amoxicillin with clavulanic acid and two to sulfamethoxazole/trim-ethoprim. S. enteritidis exhibited a more sensitive pheno-type with only one multiresistant isolate (resistance to

ampicillin/amoxicilllin with clavulanic acid/streptomy-cin/tetracycline) ( table 2 ). Among the 18 S. typhimurium , 6 isolates were multiresistant while 3 additional ones were resistant to one or two compounds ( table 2 ). Our findings

Serotypes PFGE Children (n = 69) Adults (n = 26) Total

males females males females

S. enteritidis A 36 11 6 8 61E 1 – – – 1H – 1 – – 1K – – 1 – 1M 1 – – – 1

S. typhimurium G 5 3 1 1 10F 2 1 1 2 6P – 1 – 1 2

S. newport B 2 – – – 2J 1 1 – – 2N – 1 – – 1O 2 – – – 2

S. infantis D – – – 1 1I – – 1 – 1L – – 2 – 2

S. paratyphi A C – – 1 – 1

Total 16 50 19 13 13 95

Table 1. Serotypes and PFGE types of S. enterica , in relation to age and sex ofthe patients

Fig. 1. Seasonal distribution of serotypes of S. enterica isolated during the study pe-riod (2002–2003).

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are similar to previously published studies, where the ma-jority of S . enteritidis were susceptible to the antibiotics used and S . typhimurium multiresistant [1, 4–6] . It has been reported that Salmonella strains of human origin in Greece showed lower rates of resistance to antibiotics compared to those isolated from animal sources [1] . A decrease in the resistance rates during a 7-year study pe-riod was observed due to the introduction of restrictive measures on antibiotic usage in farming [1] . On the con-trary, increasing resistance rates among S . typhimurium from human sources are observed worldwide with com-bined resistance to more than four compounds, suggest-ing that the treatment of salmonellosis should be based on the antibiotic susceptibility results [4, 6] .

PFGE of Xba I DNA digests classified S . enteritidis strains into five types. Clone A, already identified in a large study in Greece [1] , included 61 strains, while the remaining 4 strains were classified into 4 different clones ( tables 1, 2 ). Ten S . typhimurium strains belonged to PFGE type G, 6 more strains to type F and 2 to type P. The ma-

jor clones were equally isolated during 2002 and 2003, mainly from July to September, associated with the sea-sonal variation of salmonellosis. Polyclonality was ob-served among the strains isolated from sporadic cases from October to June during the study period. A gener-ally poor genetic variability among S . enteritidis strains was already identified [1] . In a genomic analysis of S . en-teritidis and S . typhimurium isolated in Finland, espe-cially among travelers from south European countries, including Greece, a dominant PFGE type was identified among S . enteritidis (SENTXB.001) that seems to be re-lated to our main clone [15] . PFGE as a typing method has the disadvantage of interlaboratory variation in the interpretations of DNA fingerprints [16, 17] . Multilocus sequence typing that has recently been applied for Salmo-nella has the advantage that data are available on the In-ternet and can be easily compared [16, 17] . It has been proved that application of multilocus sequence typing of Salmonella housekeeping genes showed lower discrimi-natory power than PFGE because these bacteria lack di-

Table 2. Antibiotic resistance patterns and clones, characterized by PFGE, among the S. enterica serotypes isolated during the study period (2002–2003)

Serotypes PFGE Antibiotic resistance patterns Total

S Am Am/Amc

Am/SXT

Te Str/Te Am/Amc/Str/Te

Am/Amc/SXT/C/Str/Te

Am/C/Str/Te

Am/Amc/C/Str/Te

S. enteritidis A 54 – 2 – 4 1 – – – – 61(n = 65) E 1 – – – – – – – – – 1

H 1 – – – – – – – – – 1K – – – – – – 1 – – – 1M 1 – – – – – – – – – 1

S. typhimurium G 5 – – 1 1 – – 1 2 – 10(n = 18) F 3 – – – 1 – – – 2 – 6

P 1 – – – – – – – 1 – 2

S. newport B – – – – 1 – – – – 1 2(n = 7) J 1 1 – – – – – – – – 2

N 1 – – – – – – – – – 1O 1 – – – – 1 – – – – 2

S. infantis D 1 – – – – – – – – – 1(n = 4) I 1 – – – – – – – – – 1

L 2 – – – – – – – – – 2

S. paratyphi A(n = 1)

C – – 1 – – – – – – – 1

Total 16 73 1 3 1 7 2 1 1 5 1 95

S = Sensitive; Am = ampicillin; Amc = amoxicillin with clavulanic acid; SXT = sulfamethoxazole/trimethoprim; Te = tetracycline; Str = streptomycin; C = chloramphenicol.

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versity in the DNA sequences of the aforementioned genes [16] . S . typhimurium isolates were successfully characterized by multiple-locus variable-number tan-dem repeat analysis, that is a scheme facilitating exchange of typing data [17] .

In our collection of strains, resistant and sensitive S . enteritidis coexisted in clone A, which is the same iden-tified in Athens [1] . The presence of sensitive and resis-tant populations to different antibiotics in the same clones was characterized and has highlighted the potential of the public health problem by showing that indistinguish-able strains could be recovered from different sources [1, 3] . The majority of S . typhimurium isolates of our collection (10 strains) belonged to clone G followed by F (6 strains), suggesting that epidemiological links exist among these cases such as interfamily spread, even though we have not identified larger outbreaks. In a pre-vious study in Greece, 5 clones were characterized among S . typhimurium isolates including multiresistant strains

[5] . Outbreaks due to ampicillin-resistant S . typhimuri-um strains belonging to 2 main clones were identified in France, indicating that epidemics might also occur through this serotype [6] .

In the present study we have shown that S . enteritidis and S . typhimurium serotypes were isolated with an in-creasing rate during summertime of the study period (2002–2003), showing a predominance among children and especially boys. Clonality was observed among mul-tiresistant S . typhimurium isolates and a common clone, A, among S . enteritidis was identified, allowing us to con-clude that this is one of the main clones circulating in Greece.

Acknowledgments

The authors wish to thank Dr. Panayotis T. Tassios for kindly providing the representative S . enteritidis strains of different clonal types.

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