European Journal of Clinical Microbiology and Infectious Diseases

, Volume 21, Issue 12, pp 887–889

Retrospective Analysis of the First Clonal Outbreak of Nalidixic Acid-Resistant Shigella sonnei Shigellosis in Israel

Authors

  • D. Dagan
    • Department of Pediatrics A, Schneider Children's Medical Center of Israel, Petach Tikva, Israel
  • N. Orr
    • Army Health Branch Research Unit, Medical Corps, Israel Defense Force, Tel Hashomer, Israel
    • Department of Epidemiology and Preventive Medicine, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, P.O.B. 39040, Tel Aviv 69978, Israel
  • M. Yavzori
    • Army Health Branch Research Unit, Medical Corps, Israel Defense Force, Tel Hashomer, Israel
  • Y. Yuhas
    • Felsenstein Medical Research Center, Petach Tikva, Israel
  • D. Meron
    • Infectious Diseases Unit, Haemek Medical Center, Afula, Israel
  • S. Ashkenazi
    • Department of Pediatrics A, Schneider Children's Medical Center of Israel, Petach Tikva, Israel
    • Department of Epidemiology and Preventive Medicine, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, P.O.B. 39040, Tel Aviv 69978, Israel
    • Army Health Branch Research Unit, Medical Corps, Israel Defense Force, Tel Hashomer, Israel
    • Department of Epidemiology and Preventive Medicine, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv, P.O.B. 39040, Tel Aviv 69978, Israel
Concise Article

DOI: 10.1007/s10096-002-0843-9

Cite this article as:
Dagan, D., Orr, N., Yavzori, M. et al. Eur J Clin Microbiol Infect Dis (2002) 21: 887. doi:10.1007/s10096-002-0843-9
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Abstract

Reported here is a retrospective molecular analysis of the isolates recovered from the first outbreak of nalidixic acid (NA)-resistant Shigella sonnei shigellosis to occur in Israel. The outbreak affected 94 children. In the retrospective analysis, a total of 13 NA-resistant isolates and five NA-susceptible isolates recovered during the outbreak period were examined. Restriction fragment length polymorphism profiles obtained by digestion with BamHI, PvuI, HinfI or SmaI yielded identical profiles for all 18 isolates. All NA-resistant strains had an identical plasmid profile, but this profile differed from that displayed by the susceptible strains. In all of the NA-resistant strains a 304 bp fragment in the gyrA gene coding for a region associated with NA resistance was sequenced and showed a single point mutation, Ser83→Phe. In this outbreak, the isolates of NA-resistant Shigella sonnei belonged to a single clone and NA resistance was associated with a point mutation in the gyrA gene.

Introduction

Shigellosis is endemic throughout the world and is hyperendemic in developing countries. Appropriate antibiotic treatment can reduce the length of disease and the risk of complications [1, 2], but the spectrum of antibiotic treatments effective against shigellosis is becoming very limited due to the increasing antimicrobial resistance of the organism. While Shigella sonnei and Shigella flexneri are still susceptible to quinolones in industrialized countries, in developing regions Shigella dysenteriae type 1 and Shigella flexneri show increasing rates of resistance to these drugs [3, 4]. The genetic basis of Shigella resistance to quinolones has only rarely been investigated [4, 5].

Israel is a country that is highly endemic for shigellosis. The annual incidence of infection is approximately 1,000 cases per million residents, which is about 20 times higher than that in the USA [6, 7]. Shigella sonnei accounts for almost 90% of the cases of shigellosis in Israel [8]. Of 3,240 Shigella sonnei isolated in Israel between 1991 and 1996, 94.4%, 73.4% and 43.6% were found to be resistant to trimethoprim-sulfamethoxazole, ampicillin and tetracycline, respectively [9]. The proportion of strains exhibiting multiple drug resistance was higher for Shigella sonnei than for the other serotypes studied. However, the Shigella sonnei strains were sensitive to nalidixic acid and ofloxacin. Nalidixic acid, which is approved for use in children and has been found efficacious against childhood shigellosis [10], remains the primary oral therapy for shigellosis in Israel.

In the present report we describe the first outbreak of shigellosis caused by a nalidixic acid (NA)-resistant Shigella sonnei clone in Israel, and we analyze the molecular basis of the acquired resistance to nalidixic acid.

Materials and Methods

This investigation was conducted retrospectively to characterize the isolates obtained during the first outbreak of NA-resistant shigellosis to occur in northern Israel. Records of the bacteriological laboratories serving the population of northern Israel were searched and the medical files pertaining to cases of NA-resistant Shigella sonnei shigellosis identified between December 1996 and September 1997 were scrutinized. Thirteen NA-resistant Shigella sonnei strains and five NA-sensitive Shigella sonnei strains isolated during the outbreak were available for reassessment of antimicrobial susceptibility and for molecular characterization.

In all of the laboratories in which NA-resistant Shigella sonnei strains were isolated during the outbreak, antimicrobial susceptibility testing was performed using the Kirby & Bauer disk diffusion method following the guidelines of the National Committee for Clinical Laboratory Standards [11]. The following agents were investigated: amoxicillin, amoxicillin-clavulanate, ciprofloxacin, norfloxacin, ampicillin, trimethoprim-sulfamethoxazole, tetracycline and nalidixic acid. In the reassessment of the 13 isolates selected for retrospective analysis the same procedure was used.

Restriction fragment length polymorphism (RFLP) analysis was performed as described previously [12]. Briefly, chromosomal DNA was cleaved with BamHI, PvuI, HinfI or SmaI. The cleaved DNA was then separated onto 0.8% agarose gel and transferred to GeenScreen plus membranes (DuPont, New England Nuclear, USA) according to the supplier's instructions. Lambda DNA digested with HindIII was used as a molecular mass marker on each gel. The 2.5 Kb EcoRI-HindIII restriction fragment of plasmid PKK3535 encoding for the 16S rRNA and part of the 23S rRNA was used as a probe and the membranes were hybridized with boiled denaturated 32P-labeled probe. Membranes were exposed to x-ray film and the hybridization banding patterns were analyzed. Plasmids were extracted from selected strains using a NucleoSpin plasmid purification kit (Macherey-Nagel, Germany) and separated onto 0.8% agarose.

A 304 bp fragment in the gyrA gene coding for a region associated with NA resistance was amplified from selected strains using the following primers: (a) upper 5′AAG CCG GTA CAC CGT CGC GTA CTT TAC G3′; (b) lower 5′ACC GTC TCT TTT TCG AGA TCG GCC ATC AG3′. The amplified fragments were sequenced and their sequences were compared to the published sequences of gyrA.

Results and Discussion

Between January and June 1997 a total of 94 cases of NA-resistant Shigella sonnei shigellosis were identified among children living in northern Israel (Fig. 1). Sixty-six of the 94 cases of NA-resistant Shigella sonnei shigellosis occurred during February and March 1997, and 155 cases of NA-sensitive Shigella sonnei shigellosis were identified at the same laboratories during the period January–June 1997 (Fig. 1).
Fig. 1.

Identified cases of NA-resistant and NA-sensitive Shigella sonnei shigellosis in northern Israel between December 1996 and September 1997

For our retrospective analysis of the outbreak, personal data were available for 88 of the 94 patients with NA-resistant Shigella sonnei shigellosis. Among them, 45 were male and 43 were female; the average age of the patients was 4.74 years (range, 1–13 years) for males and 5.62 years (range, 1–16 years) for females. Data on signs and symptoms of disease were available for 65 patients. All of them suffered from diarrhea, with an average of 6.4 watery stools per day. Forty-four percent had bloody stools, 43% suffered abdominal cramps, 80% had fever above 38°C, and 53% had vomited.

In susceptibility testing at the time of the outbreak, all of the 94 NA-resistant Shigella sonnei strains isolated were also resistant to trimethoprim-sulfamethoxazole and amoxicillin but sensitive to ciprofloxacin. A similar antimicrobial susceptibility pattern was noted during the reassessment of the 13 NA-resistant isolates tested in the retrospective analysis. At the original time of testing, 37 of the isolates were sensitive to both amoxicillin-clavulanate and tetracycline, 20 were resistant to both amoxicillin-clavulanate and tetracycline, 36 were resistant to amoxicillin-clavulanate but sensitive to tetracycline, and one isolate was sensitive to amoxicillin-clavulanate but resistant to tetracycline.

The RFLP profiles obtained by digestion with either BamHI, PvuI, HinfI or SmaI were identical for all of the 13 NA-resistant and five NA-susceptible strains used in the retrospective analysis (Fig. 2). The plasmid profiles of all NA-resistant strains were identical. However, they differed from those displayed by the NA-sensitive strains of Shigella sonnei, showing an additional band located close to the 6.5 kb band of the marker. A 304 bp fragment in the gyrA gene coding for a region associated with NA resistance was sequenced and showed a single point mutation, Ser83→Phe in all NA-resistant strains.
Fig. 2.

Restriction fragment length polymorphism analysis of Shigella sonnei strains isolated from patients involved in the outbreak. Chromosomal extracts were digested with SmaI. R, NA-resistant strains; S, Na-sensitive strains; L, laboratory strain

This is the first report of an epidemic NA-resistant Shigella sonnei strain being isolated in Israel. The outbreak affected mostly young children in both urban and rural settlements of northern Israel. The clinical presentation of the disease was similar to that usually observed among cases of shigellosis.

The source of the epidemic strain remains unclear due to the lack of epidemiological-analytical investigation at the time of the outbreak. According to the epidemic curve, however, it appears that the infection most probably spread via person-to-person contact, with a peak attack rate occurring between January and March 1997. The RFLP profiles of the NA-resistant Shigella sonnei isolates indicate that they probably originated from a single clone. The outbreak did not extend to additional regions in Israel, and the NA-resistant Shigella sonnei clone disappeared after June 1997.

To date, the genetic basis of resistance to quinolones has been defined mainly in regard to Escherichia coli and Salmonella enterica serotype Typhimurium where the gyrA mutation encoding Ser83→Phe substitution is frequently associated with resistance to nalidixic acid [13, 14]. Among Shigella flexneri strains isolated in Hong Kong between 1986 and 1995, 59.6% were resistant to nalidixic acid and the resistance was associated with a gyrA mutation encoding for Ser83 alteration [5]. High-level fluoroquinolone resistance of Shigella dysenteriae type 1 may also occur in the absence of gyrA mutations and involve a proton motive force-dependent efflux system [4].

To the best of our knowledge, our study represents the first attempt to uncover the genetic basis for quinolone resistance in Shigella sonnei isolates. The sequence of a 304 bp fragment of gyrA gene coding for the region associated with NA resistance revealed a single point mutation, Ser83→Phe, in all NA-resistant Shigella sonnei strains involved in the outbreak, which is similar to that reported previously for Shigella flexneri [5]. Furthermore, our comparison of this region with published sequences of Shigella flexneri, Escherichia coli and Salmonella typhimurium [5, 15] disclosed very high homology with Shigella flexneri and Escherichia coli, while the homology with Salmonella typhimurium was less evident, especially within the region associated with NA resistance.

In the outbreak presented here, the isolated strains of NA-resistant Shigella sonnei most probably originated from a single clone and NA resistance was associated with a single point mutation in the gyrA gene.

Acknowledgements

This study was supported in part by grant no. DAMD17-98-2-8001 from U.S. Army Medical Research & Material Command, Fort Detrick, Frederick, MD, USA, provided to DC.

Copyright information

© Springer-Verlag 2003