N. NÓGRÁDY1, A. IMRE2, Á. KOSTYÁK3, J. PÁSZTI1 AND B. NAGY2

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N. NÓGRÁDY1, A. IMRE2, Á. KOSTYÁK3, J. PÁSZTI1 AND B. NAGY2 PHENO- AND GENOTYPIC CHARACTERIZATION OF RECENT SALMONELLA INFANTIS ISOLATES FROM HUMAN AND ANIMAL SOURCES N. NÓGRÁDY1, A. IMRE2, Á. KOSTYÁK3, J. PÁSZTI1 AND B. NAGY2 1Department of Phage typing and Molecular Epidemiology, National Center for Epidemiology, Budapest 2Veterinary Medical Research Institute of the HAS, Budapest 3Microbiology Department, National Food Investigation Institute, Budapest MMT Congress, Keszthely, 18-20. Oct. 2006.

Top five human serotypes in 2004 INTRODUCTION Top five human serotypes in 2004 Serotype Total Freq (%) Enteritidis 5 501 72,8 Typhimurium 573 7,6 Infantis 448 5,9 Blockley 59 0,8 Saintpaul 53 0,7 Others 923 12,2 7 557 100 Aim: to investigate the genetic diversity and the possible genetic relationship among the Hungarian S. Infantis isolates from different sources. (Germany: 5.72%, Norway: 5.47%, Japan: 8,12%, New-Zealand: 5.13%, + the third-fourth most frequent serotype in 16/34 countries who reported data to the EnterNet in 2004)

MATERIALS AND METHODS 1. 132 S. Infantis isolates (2004-2005): Human stool (HS) n=56 Broiler chicken faeces (CF) n=29, Broiler chicken meat (CM) n=31, Other animals (OA) n=16, PHENOTYPIC METHODS: Sero-typing (Kauffman-White scheme) Phage-typing (GV Laszlo et al., 1988) Antibiotic susceptibility testing (AMP, CHL, GEN, KAN, STR, SXT, TET, NAL, CIP, CTX)

MATERIALS AND METHODS 2. GENOTYPIC METHODS: - PCRs: Antibiotic resistance genes: - tetA (Guerra et al., 2001) - Class 1 integron carriage (Daly et al., 2000), (sequencing) Virulence associated genes: - spvC (Salmonella plasmid virulence Haneda et al., 2001) - sipA (SPI1, T3SS-1, entry into cells) - sopB (SPI5, T3SS-1, effector protein, invasion) - Molecular fingerprinting by - PFGE (XbaI, PulseNet protocol, Fingerprinting II software) UPGMA, Dice - ERIC2 PCR (Zaher and Cimolai, 1997) - Plasmid profiling (Kado and Liu, 1981)

Other PTs:111,214,451,481,617,627,637,653,683,687,817, phage-resistant Other R-types: Str, Nal, StrTet, TetNal, StrTetNalAmp, StrTetNalAmpSxt, StrTetNalCip, (Km,Cm,Gm)

Antibiotic resistance genes Virulence associated genes Isolates Antibiotic resistance genes Virulence associated genes class 1 integron (1.0 kb, aadA1) tetA spvC sipA sopB HS (n=56) 45 (80%) 46 (82%) 100% CM (n=31) 27 (87%) 21 (68%) CF (n=29) 22 (76%) 25 (86%) OA (n=16) 10 (62%) 7 (44%) Σ (n=132) 104 (79%) 99 (75%)

Dendrogram generated by Fingerprinting II software showing the results of cluster analysis on the basis of ERIC2 PCR fingerprinting of 132 S. Infantis isolates Name PT R-type Name PT R-type 85% 85% HS CM CF OA cluster A clusterB

Dendrogram generated by Fingerprinting II software showing the results of cluster analysis on the basis of PFGE fingerprinting of 132 S. Infantis isolates Name PT R-type Name PT R-type 83% 83% HS CM CF OA

Plasmid profiles HS CM CF Isolate <170 kb 2 or more No HS (n=56) 38 (68%) 8 7 CM (n=31) 27 (87%) 4 - CF (n=29) 19 (65%) 10 OA (n=16) 10 (62%) 6 Σ (n=132) 94 (73%) 22 (17%) 13 (10%)

CONCLUSIONS S. Infantis strains with the following characteristics: PT213 or PT217, StrTetNal, (addA1, tetA) one large (< 170 kb) plasmid, spvC negative, sipA and sopB positive became widely spread in the Hungarian broiler industry and in the food chain and appeared in the human population as well.

FURTHER QUESTIONS What makes these strains capable of surviving in the broiler chickens and their environment? What is the role of the large plasmid? (conjugation, plasmid RFLP, southern hybridization for Tn21, merA, integron (sul1 and qacΔE1), tetA) What S. Infantis genotypes are present in other countries?

ACKNOWLEDGEMENT The excellent technical assistance of Mrs. Margit Király, Mrs. Judit Orbán and Mrs. Vera Koppány is gratefully acknowledged. The authors thank dr. Gábor Kardos for the initial ERIC2 PCR analysis and dr. Katalin Krisztalovics for the human epidemiological data. This work was partially supported by the NKFP 4/040/2001 project. N. Nógrády is the holder of a Bolyai János stipend from the Hungarian Academy of Sciences.