Emma Filetici

Evaluation of DNA Extraction Methods for Use in Combination with SYBR Green I Real-Time PCR To Detect Salmonella enterica Serotype Enteritidis in Poultry

ABSTRACT The objective of this study was to develop a rapid, reproducible, and robust method for detecting Salmonella enterica serotype Enteritidis in poultry samples. First, for the extraction and purification of DNA from the preenrichment culture, four methods (boiling, alkaline lysis, Nucleospin, and Dynabeads DNA Direct System I) were compared. The most effective method was then combined with a real-time PCR method based on the double-stranded DNA binding dye SYBR Green I used with the ABI Prism 7700 system. The specificity of the reaction was determined by the melting temperature (Tm) of the amplicon obtained. The experiments were conducted both on samples of chicken experimentally contaminated with serotype Enteritidis and on commercially available poultry samples, which were also used for comparisons with the standard cultural method (i.e., ISO 6579/2001). The results of comparisons among the four DNA extraction methods showed significant differences except for the results from the boiling and Nucleospin methods (the two methods that produced the lowest threshold cycles). Boiling was selected as the preferred extraction method because it is the simplest and most rapid. This method was then combined with SYBR Green I real-time PCR, using primers SEFA-1 and SEFA-2. The specificity of the reaction was confirmed by the Tm, which was consistently specific for the amplicon obtained; the mean peak Tm obtained with curves specific for serotype Enteritidis was 82.56 ± 0.22°C. The standard curve constructed using the mean threshold cycle and various concentrations of serotype Enteritidis (ranging from 103 to 108 CFU/ml) showed good linearity (R2 = 0.9767) and a sensitivity limit of less than 103 CFU/ml. The results of this study demonstrate that the SYBR Green I real-time PCR constitutes an effective and easy-to-perform method for detecting serotype Enteritidis in poultry samples.

Salmonella enteritidis in Italy

During the period 1982-1992 the percentages of Salmonella enteritidis isolations in Italy have increased from 2.4 to 57.1% from human beings and from 0.5 to 22.8% from food. Seven hundred and fifty-seven isolates, 702 from man and 55 from food, were characterized. Phage type 4 accounted for the 76.8% of these isolates. The majority of strains were sensitive to the antibiotics tested; only 18 (2.4%) showed resistance to three or more antibiotics by nine different patterns.

Antibiotic Resistance Genes and Salmonella Genomic Island 1 in Salmonella enterica Serovar Typhimurium Isolated in Italy

ABSTRACT Fifty-four epidemiologically unrelated multidrug-resistant Salmonella enterica serovar Typhimurium isolates, collected between 1992 and 2000 in Italy, were analyzed for the presence of integrons. Strains were also tested for Salmonella genomic island 1 (SGI1), carrying antibiotic resistance genes in DT104 strains. A complete SGI1 was found in the majority of the DT104 strains. Two DT104 strains, showing resistance to streptomycin-spectinomycin and sulfonamides, carried a partially deleted SGI1 lacking the flost, tetR, and tetA genes, conferring chloramphenicol-florfenicol and tetracycline resistance, and the integron harboring the pse-1 gene cassette, conferring ampicillin resistance. The presence of SGI1 was also observed in serovar Typhimurium strains belonging to other phage types, suggesting either the potential mobility of this genomic island or changes in the phage-related phenotype of DT104 strains.

Foodborne outbreaks caused by salmonella in Italy, 1991–4

This report summarizes studies on 1699 foodborne outbreaks, in Italy, reported to the Istituto Superior di Sanità (ISS) (the National Institute of Health of Italy, Rome) during the period 1991-4. The most frequently reported foodborne outbreaks were caused by salmonellae (81%), in particular by Salmonella enteritidis and non-serotyped group D salmonella (34% and 33% of the total salmonella outbreaks, respectively). A vehicle was implicated in 69% of the salmonella outbreaks; eggs were implicated in 77% of the outbreaks for which a vehicle was identified or suspected. Salmonella strains isolated in 54 outbreaks were studied for phenotypic and genotypic characteristics. The isolates belonged to S. enteritidis (50 outbreaks), S. typhimurium (three outbreaks) and S. hadar (one outbreak). In the S. enteritidis outbreaks, phage type 4 was most frequently isolated (64.8%), followed by phage type 1 (14.8%). The virulence plasmid of 38 megadaltons was found in many different phage types of S. enteritidis.

Molecular Characterization of Multidrug-Resistant Strains of Salmonella enterica Serotype Typhimurium and Monophasic Variant (S. 4,[5],12:i:–) Isolated from Human Infections in Italy

Salmonella enterica serovar Typhimurium (STM) represents the prevalent cause of foodborne gastroenteritis in Italy with the majority of isolates exhibiting multidrug resistance. A resistant pattern that includes ampicillin (A), streptomycin (S), sulfonamide (Su), and tetracycline (T) (ASSuT) but lacks resistance to chloramphenicol (C) has recently emerged in Italy among strains of STM and of its monophasic variant, S. enterica subspecies enterica serovar S. 4,[5],12:i:-. With the aim to evaluate their clonal relationships, 553 strains of STM and S. 4,[5],12:i:- with the ASSuT and ACSSuT resistance patterns isolated in Italy from human infections between 2003 and 2006 were characterized by pulsed-field gel electrophoresis (PFGE) according to the PulseNet-Europe protocol and nomenclature. Among both the STM and S. 4,[5],12:i:- ASSuT strains, the predominant PFGE profile was STYMXB.0079 (53.2-73.0% of strains, respectively), while the STM ACSSuT strains belonged to the STYMXB.0061 (37.2% of strains) and STYMXB.0067 (29.9% of strains). Bionumerics cluster analysis of the nonunique PFGE profiles showed that more than 90% of ASSuT and ACSSuT-resistant strains were included in two distinct clusters with a genetic homology of 73% each other, suggesting that the ASSuT-resistant strains belong to a same clonal lineage different from that of the ACSSuT strains. Phage typing showed that 23% of the ASSuT STM strains were not typeable and 22.3% were U302. The same phage types were observed among the ASSuT strains of S. 4,[5],12:i:-. A different figure was observed for the ACSSuT strains: the STM isolates mostly belonged to DT104 (70.2%), while none of the S. 4,[5],12:i:- strains belonged to this phage type. This study indicates that the tetra-resistant ASSuT strains of STM and S. 4,[5],12:i:-, increasingly isolated in Italy, belong to a same clonal lineage and that the S. 4,[5],12:i:- strains circulating in our country mainly derive from this STM clonal lineage.

Nucleotide sequence of the chromosomal region conferring multidrug resistance (R-type ASSuT) in Salmonella Typhimurium and monophasic Salmonella Typhimurium strains

OBJECTIVES The aim of this study was to sequence the chromosomal region conferring resistance to ampicillin, streptomycin, sulphonamides and tetracycline (R-type ASSuT) in a Salmonella Typhimurium (STM) monophasic strain (4,[5],12:i:-) belonging to the PFGE profile STYMXB.0079. The presence of this resistance region and the analysis of its genetic environment was investigated in a selection of strains. METHODS A Sau3A1 genomic library was used to determine the nucleotide sequence of the genomic resistance region. PCRs were performed on 10 epidemiologically unrelated Salmonella strains, both STM and monophasic STM, with R-type ASSuT and PFGE profile STYMXB.0079, in order to investigate the presence of the resistance genes, the left and right junctions and the internal regions of the resistance region, as well as the genetic environment. RESULTS The genomic resistance region consisted of two regions, resistance region 1 (RR1), conferring resistance to ampicillin, streptomycin and sulphonamides, and resistance region 2 (RR2), conferring tetracycline resistance. These resistance regions were both surrounded by IS26 elements and sequence comparative analysis showed 99% sequence identity with a region of plasmid pO111_1 from an Escherichia coli strain. All 10 strains were positive for the four resistance genes, the left and right junctions and the internal regions of RR1 and RR2. Concerning the genetic environment, all the strains lacked the STM1053-1997 and STM2694 genes, while only monophasic STM strains showed deletion of the fljA-fljB operon. CONCLUSIONS This study describes two resistance regions localized on the bacterial chromosome of a clonal lineage of STM and monophasic STM that are widespread in Italy.

Conventional and molecular approaches to isolates of Salmonella hadar from sporadic and epidemic cases.

In September 1994 an outbreak of gastroenteritis occurred in 437 people who had consumed lunch in the canteen of a factory in Central Italy. Salmonella sp. was isolated from stools of 99 patients and in 73 of them Salmonella hadar was identified. This is the first outbreak caused by this serotype described in Italy. In order to examine the genotypic basis of the epidemic strains, molecular typing was applied to sporadic strains isolated before and after the outbreak episode. For this purpose phage type, resistance to antibiotics, DNA plasmid profile and sites of insertion of the mobile element of IS200 were determined. The epidemic strains were genetically distinct from the non‐epidemic isolates; they were shown to be phage type 26, harbouring four small plasmids, were resistant to nalidixic acid and showed a unique characteristic IS200 fingerprint. The typing methods used in this study allowed the identification and discrimination of the outbreak strains from related isolates. They can thus be considered as a tool for epidemiological purposes. In addition we should point out the emerging resistance to nalidixic acid, largely used in veterinary medicine, in Salm. hadar.

Italian experience in Salmonella enteritidis 1978–1988: characterization of isolates from food and man

Salmonella enteritidis accounted for 5.45% of the 118.685 Salmonella isolates from man and for 2.65% of the 3.315 Salmonella isolates from food in Italy in the eleven year period 1978 to 1988. In the years 1978-1982 no S. enteritidis strain was isolated from eggs and poultry; in the years 1983-1988 the 53% of S. enteritidis isolates from food were from eggs and poultry. In 1989 S. enteritidis accounted for 744 isolates from man and 22 from food of which 80% were from eggs and poultry (partial data). In that year 18 outbreaks caused by S. enteritidis were reported to the National Centre of Enteric Pathogens in Rome. Characteristics of 81 S. enteritidis isolates were examined of which 27 were from sporadic cases involving humans and 40 from outbreaks in humans; 14 isolates were from food, all but one connected with the outbreaks. All the isolates studied were sensitive to the antibiotics tested; plasmid profile analysis showed a predominant profile pattern in both epidemic and non-epidemic strains; lysine decarboxylase was present in all the strains tested. Although in at least three epidemics a common supplier of eggs was proved, the source was not identified. Unfortunately it was not possible to determine the phage type of isolates because of the unavailability of specific phages.

Egg-related Salmonella enteritidis, Italy, 1991.

In recent years, Salmonella enteritidis has become an increasingly important public health problem in Italy. In some parts of the country, the fraction of total human salmonella isolates accounted for by S. enteritidis has risen from 3-4% in the mid-1980s to more than 30% in 1990. Between 1990 and 1991, the number of reported S. enteritidis outbreaks increased more than sixfold. The 33 outbreaks reported in 1991 occurred in seven contiguous regions in northern and central Italy and were clustered in time between June and October: in the majority, products containing raw or undercooked shell eggs were implicated. Five of the egg-related outbreaks that occurred within a 30 kilometre radius over a 7-week period were investigated in detail. A phage type 1 strain containing a 38.9 MDa plasmid appeared responsible for three of the outbreaks, while in the remaining two a phage type 4 strain, also with a 38.9 MDa plasmid was isolated. Efforts are being made to enhance epidemiological surveillance and laboratory evaluation, and the use of pasteurized eggs has been recommended for high-risk populations.

Serotype and phage type distribution of salmonellas from human and non-human sources in Italy in the period 1973–1995

According to the data collected at the Rome-based National Reference Centre for Enterobacteria, 266,547 Salmonella strains isolated from human sources (226,513) and from non human sources(40,034) were characterised in Italy during the period 1973–1995. The serotype of all isolates, and the antibiotype and phage type of selected isolates were determined. Human-source isolations grew from 4372 in 1973 to 12,310 in 1995; non-human source isolations, from 339 in 1973 to 3459 in 1995. Salm.Typhimurium ranked first in the list of the most common serotypes isolated from both sources in the period 1973–1988 except in the years 1975 and 1976 when it was overtaken by Salm. Wien. Since 1983 Salm. Enteritidis has been among the top ten isolates from animals, and ranked first in the list of isolates from humans in 1988 and from non human sources in 1991. During the last years the number of multidrug-resistant strains, mostly belonging to phage types 104 and 193 of Salm. Typhimurium has been rising. Salmonella strains have also been isolated from numerous extraintestinal infections, almost exclusively caused by Salm. Enteritidis and Typhimurium.