Emerging Infectious Diseases
[Volume 6 No.1 / January - February 2000]

Dispatches

Molecular Typing of Multidrug-Resistant Salmonella
Blockley Outbreak Isolates from Greece

Panayotis T. Tassios,* Christos Chadjichristodoulou,
Maria Lambiri, Athina Kansouzidou-Kanakoudi, Zannina
Sarandopoulou,* Jenny Kourea-Kremastinou, Leonidas S.
Tzouvelekis,* Nicholas J. Legakis*
*University of Athens, Athens, Greece; National Center
for Surveillance and Intervention, Athens, Greece;
National School of Public Health, Athens, Greece; and
Salmonella Reference Center for Macedonia and Thrace,
Thessaloniki, Greece

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 During 1998, a marked increase (35 cases)
 in human gastroenteritis due to Salmonella
 Blockley, a serotype rarely isolated from
 humans in the Western Hemisphere, was noted
 in Greece. The two dominant
 multidrug-resistance phenotypes (23 of the
 29 isolates studied) were associated with
 two distinct DNA fingerprints, obtained by
 pulsed-field gel electrophoresis of genomic
 DNA.

Salmonella Blockley is rarely isolated in the Western
Hemisphere. According to Enter-net, the international
network for surveillance of Salmonella and
verocytotoxin-producing Escherichia coli infections, S.
Blockley represented 0.6% of all Salmonella serotypes
isolated in Europe during the first quarter of 1998, a
full 100-fold lower than the dominant serotype, S.
Enteritidis (67.1%) (1). However, S. Blockley is among
the five most frequently isolated serotypes from both
avian and human sources in Japan (2,3), Malaysia (4,5),
and Thailand (6). A single foodborne outbreak in the
United States (7) and sporadic human infections in
Europe associated with travel to the Far East (8),
animal infection (9) or carriage (10,11), and
environmental isolates have also been reported (12,13).

Regardless of the frequency of S. Blockley isolation,
its rates of resistance to antibiotics have been high.
Among Spanish salmonellae isolated from natural water
reservoirs, S. Blockley and S. Typhimurium had the
highest rates of multidrug resistance (12). Comparing
1980-1989 with 1990-1994, researchers from Tokyo noted
an increase in the number of S. Blockley isolates
resistant to one or more antibiotics, from 92.0% to
98.2% for imported cases and from 57.4% to 88.7% for
domestic cases (3,14). In Thailand, isolates from human
or other sources also had high rates of resistance to
streptomycin, tetracycline, kanamycin, and
chloramphenicol and lower rates to ampicillin and
trimethoprim/sulfamethoxazole (15).

Nevertheless, few attempts at typing S. Blockley
isolates with molecular methods have been described,
and these have been limited to the characterization of
plasmid content (2,16).

During the second and third quarters of 1998, Enter-net
reported higher numbers of S. Blockley isolates than
during the same period of the previous year in several
European countries (1). The epidemiologic
investigations conducted in Germany, England and Wales,
and Greece did not confirm a source for this increase
(17-19).

In this study, we characterized the Greek outbreak
isolates further, both with respect to their antibiotic
resistance phenotypes and DNA fingerprints obtained by
pulsed-field gel electrophoresis of genomic DNA.

The Study

The study sample consisted of 28 of 35 S. Blockley
strains isolated from May to December 1998 (19), one
strain from February 1999, and four epidemiologically
unrelated control strains: one from 1996 and three from
1997. All isolates were from human cases of enteritis.
Identification was performed by the API 20E system
(BioMerieux S.A., Marcy l'Etoile, France) and
serotyping with commercially obtained antisera
(BioMerieux) (20).

Susceptibility to kanamycin, streptomycin, ampicillin,
amoxicillin/clavulanic acid, cefepime, tetracycline,
chloramphenicol, trimethoprim/sulfamethoxazole,
gentamicin, nalidixic acid, and ciprofloxacin was
tested by a disk diffusion assay according to National
Committee for Clinical Laboratory Standards guidelines
(21). Genomic DNA was prepared and digested with XbaI
(New England Biolabs) (22). Chi-square tests or Fisher
exact tests were used to calculate two-tailed
probabilities.

S. Blockley accounted for seven of the 13,199
salmonella isolates identified in Greece from 1976 to
1997. However, 35 gastroenteritis cases due to this
serotype were reported from May to December 1998 (19).
Twenty-nine S. Blockley strains isolated from fecal
specimens of patients with gastroenteritis during May
1998 to February 1999, along with four epidemiologically unrelated clinical isolates from 1996 and 1997, were therefore studied for susceptibility to antibiotics. The 1998 outbreak isolates were scattered throughout Greece; S. Blockley was isolated later, starting in August 1998, in northern Greece.

All isolates were susceptible to 
trimethoprim/sulfamethoxazole, ampicillin,
amoxicillin/clavulanic acid, gentamicin, and
ciprofloxacin (Table). High resistance rates were
observed to tetracycline (100%), streptomycin and
kanamycin (90%), chloramphenicol (83%), and nalidixic
acid (52%). Six resistance phenotypes could be
distinguished (Table) with the two major phenotypes of
outbreak isolates being resistant to kanamycin,
streptomycin, tetracycline, and chloramphenicol (ATC)
or kanamycin, streptomycin, tetracycline,
chloramphenicol, and nalidixic acid (ATCN). Most (76%)
strains isolated after August 24, 1998, were nalidixic
acid-resistant (resistance phenotypes ATCN, TCN, ATN),
unlike strains isolated up to August 17, 1998 (17%)
(1.29 <RR = 3.03 <7.11, p = 0.005).

Table. Resistant phenotypes for isolates of
Salmonella Blockley in Greece (Table not 
available in ascii)

When pulsed-field gel electrophoresis was used
to obtain DNA fingerprints for these isolates
(Figure 1), all belonged to the same type, A,
although eight subtypes, A1-A8, could be
distinguished on the basis of one to three DNA
fragment differences (Figures 1, 2). Two of the
four isolates from previous years belonged to
unique subtypes A6 and A7; the other two
belonged to subtypes A2 and A8, shared by
outbreak isolates (Table). In contrast, 93% of
the 1998 outbreak strains yielded PFGE patterns
common to two or more isolates. Indeed, most
outbreak isolates were grouped in subtypes A2
and A4, consisting of 11 and 12 isolates,
respectively (Table).

Figure 1. Sample pulsed-field gel electrophoresis
(PFGE) gel of representative Salmonella Blockley
isolates, indicating common and unique DNA
fingerprints.... (Figures not available in ascii)

Figure 2. Dendrogram of similarity among the observed
pulsed-field gel electrophoresis patterns.
(Figures not available in ascii)

Pulsed-field gel electrophoresis subtypes were
associated with resistance phenotypes. Most
resistance phenotype ATC isolates belonged to
subtype A2 (3.03. <RR = 11.86 <46.5, p =
0.0000098), while most resistance phenotype
ATCN isolates belonged to A4 (2.76 <RR = 7.11
<18.30, p = 0.0000416). In addition, most
isolates in the two major subtypes appearing
before August 17, 1998, belonged to the A2
group, while most isolates appearing after
August 24, 1998, belonged to the A4 group (1.47
<RR = 9.82 <65.45, p = 0.0006). Finally, unlike
A4, the earlier A2 subtype was not isolated in
northern Greece.

Conclusions

Our results indicate that PFGE is useful in
distinguishing epidemiologically related S.
Blockley isolates since two of the four
nonoutbreak isolates displayed unique PFGE
patterns, A7 and A8, while PFGE patterns A2 and
A4 grouped most of the 29 outbreak isolates (11 and 12,
respectively).

These two chromosomal fingerprints, differing by two DNA fragments, were associated with two distinct resistance phenotypes. The resistance phenotype of A4 isolates, ATCN, was identical to the earlier resistance phenotype of A2 isolates, ATC, except for the resistance to nalidixic acid. Nevertheless, these two PFGE/antibiotic resistance types, A2/ATC and A4/ATCN, displayed a clear distribution both in time and space.

The data may, therefore, indicate two main sources for the outbreak. Alternatively, and perhaps more likely, these two closely related types may together constitute the outbreak clone, evolved with time to acquire resistance to nalidixic acid. Resistance may well have originated in the food source, since several antibiotic classes are
used as feed supplements in animal rearing and aquaculture in Greece: sulfonamides (trimethoprim/sulfathiazine), tetracyclines (oxytetracycline), and quinolones (oxolinic acid). However, as in other European countries (17,18), the epidemiologic investigation did not locate a common source to account for the wide geographic spread of cases (19). Although travel was not mentioned in the Greek
patients' questionnaire responses, the possibility that the source was an imported food cannot be ruled out. The association with smoked eel of Italian origin in the German outbreak has not been microbiologically confirmed (17). The only other previous European report of a human outbreak attributed to S. Blockley, probably from vegetables contaminated by this organism, which was prevalent in
irrigation water in the Spanish region of Granada, is anecdotal (13). A documented S. Blockley enteritis epidemic in a U.S. hospital in 1966 was attributed to contaminated ice cream; however, this was also not microbiologically confirmed (7).

While this serotype may remain important in Europe, its high rates of resistance to kanamycin, streptomycin, tetracycline, and chloramphenicol, which were in agreement with studies from the Far East (3) and Spain (12), are cause for concern. Unlike the Far Eastern strains, no resistance to -lactam antibiotics or cotrimoxazole was observed in our study. The two dominant resistant phenotypes of S. Blockley from natural polluted waters in Spain were sulfonamides, streptomycin, and tetracycline; and neomycin, streptomycin, kanamycin, tetracycline, and chloramphenicol (12), as
in the Greek strains, except for the absence of resistance to nalidixic acid.

In agreement with differences in animal reservoirs and transmission routes and therefore the mechanism of resistance acquisition among different Salmonella serotypes, the main patterns of resistance observed in S. Blockley were distinct from those predominating in the two major serotypes from isolates of both human and animal food origin in Greece. In S. Enteritidis, the most frequent resistance
phenotype was resistance to ampicillin (24), while in S.
Typhimurium, the most frequent resistant phenotype was resistance to sulfonamides and streptomycin (A. Markogiannakis, P.T. Tassios, N.J. Legakis, unpub. obs.). Furthermore, the considerably high rate of resistance to nalidixic acid is equally unprecedented in both the
Far Eastern and Spanish S. Blockley isolates and in other salmonella serotypes from Greece. Since resistance to nalidixic acid can be a precursor of resistance to fluoroquinolones, one of the two drug classes of choice for invasive salmonella disease, this feature of
these S. Blockley strains is particularly disturbing. S. Blockley, previously a prevalent serotype in the Far East but rare elsewhere, nevertheless posed a public health problem in several European countries. The source of the European outbreaks, however, remains unclear. Given the increased international commerce in food, a collaborative study would be useful in identifying potential similarities between the recent European strains and established
strains from the Far East.

Acknowledgments

We thank Veneta Lukova and Soula Christou for assistance with antibiotic susceptibility testing.

This work was funded in part by the Ministry of Health and Welfare.

Dr. Legakis is professor and head of the department of Microbiology of the Medical School of the National University of Athens. His research interests include molecular typing, mechanisms of antibiotic resistance of bacterial pathogens, and molecular diagnosis of infections.

Address for correspondence: Nicholas J. Legakis, Department of Microbiology, Medical School, University of Athens, M. Asias 75, 115 27 Athens, Greece; fax: 301-7709-180; e-mail: nlegakis@cc.uoa.gr

References

  1. Fisher IST, on behalf of Enter-net participants. Enter-net Quarterly Salmonella Report - 98/3 (1998 Jul-Sep). Available from: URL:  http://www2.phls.co.uk/reports/latest.htm
  2. Limawongpranee S, Hayashidani H, Okatani AT, Ono K, Hirota C, Kaneko K, et al. Prevalence and persistence of Salmonella in broiler chicken flocks. J Vet Med Sci 1999;61:255-9.
  3. Matsushita S, Yamada S, Sekiguchi K, Kusunokui J, Ohta K, Kudoh Y. Serovar-distribution and drug-resistance of Salmonella strains isolated from domestic and imported cases in 1990-1994 in Tokyo. Kansenshogaku Zasshi 1996;70:42-50.
  4. Yasin RM, Jegathesan MM, Tiew CC. Salmonella serotypes isolated in Malaysia over the ten-year period 1983-1992. Asia Pac J Public Health 1996-97;9:1-5.
  5. Rusul G, Khair J, Radu S, Cheah CT, Yassin RM. Prevalence of Salmonella in broilers at retail outlets, processing plants and farms in Malaysia. Int J Food Microbiol 1996;33:183-94.
  6. Sasipreeyajan J, Jerngklinchan J, Koowatananukul C, Saitanu K. Prevalence of salmonellae in broiler, layer and breeder flocks in Thailand. Trop Anim Health Prod 1996;28:174-80.
  7. Morse LJ, Rubenstein AD. A food-borne institutional outbreak of enteritis due to Salmonella blockley. JAMA 1967;202:939-40.
  8. Albert S, Weber B, Schafer V, Rosenthal P, Simonsohn M, Doerr HW. Six enteropathogens from a case of acute gastroenteritis. Infection 1990;18:381-2.
  9. van Duijkeren E, Sloet van Oldruitenborgh-Oosterbaan MM, Houwers DJ, van Leeuwen WJ, Kalsbeek HC. Equine salmonellosis in a Dutch veterinary teaching hospital. Vet Rec 1994;135:248-50.
 10. Mallaret MR, Turquand O, Blatier JF, Croize J, Gledel J, Micoud M, et al. Human salmonellosis and turtles in France. Rev Epidemiol Sante Publique 1990;38:71-5.
 11. Levre E, Valentini P, Brunetti M, Sacchelli F. Stationary and migratory avifauna as reservoirs of Salmonella, Yersinia and Campylobacter. Ann Ig 1989;1:729-40.
 12. Morinigo MA, Cornax R, Castro D, Jimenez-Notaro M, Romero P, Borrego JJ. Antibiotic resistance of Salmonella strains isolated from natural polluted waters. Journal of Applied Bacteriology 1990;68:297-302.
 13. Garcia-Villanova Ruiz B, Cueto Espinar A, Bolanos Carmona MJ. A comparative study of strains of salmonella isolated from irrigation waters, vegetables and human infections. Epidemiol Infect 1987;98:271-6.
 14. Matsushita S, Yamada S, Inaba M, Kusunoki J, Kudoh Y, Ohashi M. Serovar distribution and drug resistance of Salmonella isolated from imported and domestic cases in 1980-1989 in Tokyo. Kansenshogaku Zasshi 1992;66:327-39.
 15. Bangtrakulobth A, Suthienkul O, Kitjakara A, Pornrungwong S, Siripanichgon K. First isolation of Salmonella blockley in Thailand. Southeast Asian J Trop Med Public Health 1994;25:688-92.
 16. Atanassova V, Matthes S, Muhlbauer E, Helmuth R, Schroeter A, Ellendorff F. Plasmid profiles of different Salmonella serovars from poultry flocks in Germany. Berl Munch Tierarztl Wochenschr 1993;106:404-7.
 17. Hamouda O. Outbreak of Salmonella blockley infections in Germanypreliminary investigation results.  Eurosurveillance Weekly 1998;2:980924. Available at URL:
http://www.eurosurv.org/1998/980924.htm
 18. Benons L. Salmonella blockley infections in England and Wales, 1998. Eurosurveillance Weekly 1998;2:980924. Available at URL: http://www.eurosurv.org/1998/980924.htm
 19. Vassilogiannakopoulos A, Tassios P, Lampiri M. Salmonella blockley infection in Greece. Eurosurveillance Weekly 1999;3:990408. Available at URL:
http://www.eurosurv.org/1999/990408.htm
 20. Kauffman F. Serological diagnosis of Salmonella species. Copenhagen (Denmark): Munksgaard; 1972.
 21. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard M7-A2. Wayne (PA): The Committee; 1997.
 22. Tassios PT, Vatopoulos AC, Mainas E, Gennimata D, Papadakis J, Tsiftsoglou A, et al. Molecular analysis of
ampicillin-resistant sporadic Salmonella typhi and Salmonella paratyphi B clinical isolates. Clin Microbiol Infect 1997;3:317-23.
 23. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9.
 24. Tassios PT, Markogiannakis A, Vatopoulos AC, Katsanikou E, Velonakis EN, Kourea-Kremastinou K, et al. Molecular
epidemiology of antibiotic resistance of Salmonella enteritidis during a 7-year period in Greece. J Clin Microbiol 1997;35:1316-21.

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