Optimization of Eva Green real-time mPCR for differentiating C. jejuni/coli directly from feces.

OBJECTIVE: To develop and optimize a rapid molecular method for diagnosing campylobacteriosis directly from a clinical fecal sample and at the same time for determining the most common causing agents - C. jejuni/coli. MATERIALS AND METHODS: 38 clinical fecal samples from hospitalized patients with diarrheal syndrome were tested using a rapid immunochromatographic test. All positive samples were tested for confirmation by culturing in a microaerophilic atmosphere. The Eva Green real-time mPCR reaction of a direct fecal sample was conducted using the "IQ5TM Real-Time PCR System" apparatus. RESULTS: Out of 38 clinical fecal samples which were ICT positive, 18 strains were isolated by culture, namely, 17 of C. jejuni and 1 of C. coli. The Eva Green real-time mPCR reaction also reported 18 positive samples for Campylobacter, out of which 17 were of C. jejuni and only one of C.coli. CONCLUSION: We developed and optimized the Eva Green real-time mPCR for the detection and species differentiation of C. jejuni/coli directly from a clinical fecal sample. The molecular analysis we described has a 100% sensitivity and specificity when comparing the results obtained by it to those of the culture method, which is currently the "gold standard" in the diagnosis of campylobacteriosis (Tab. 2, Fig. 1, Ref. 6).

Systemic and extraintestinal forms of human infection due to non-typhoid salmonellae in Bulgaria, 2005-2010.

The purpose of this investigation was to review the clinical cases diagnosed as systemic or extraintestinal salmonellosis between 2005 and 2010 in Bulgaria, to determine the antimicrobial resistance of the causative salmonellae, and to analyze the pulsed field gel electrophoresis (PFGE) profiles of extraintestinal Salmonella Corvallis isolates. Culture, biochemical tests, and serotyping were performed. Resistance to 12 antimicrobial agents was studied with the Bauer-Kirby disk diffusion method. The double-disk synergy method was used for the screening of the presence of extended-spectrum beta-lactamases (ESBLs). PFGE typing and analysis of the dendrogram was performed for the comparative investigation of Salmonella Corvallis isolates. Between 2005 and 2010, 2,227 human non-typhoid Salmonella isolates were investigated at the National Reference Laboratory of Enteric Pathogens, Sofia, Bulgaria. Thirty-three strains (1.48 %) from nine national regions were isolated from patients with systemic and extraintestinal forms of salmonellosis. The serotype distribution was as follows: S. enteritidis (n = 21), S. choleraesuis (diphasic n = 3; monophasic n = 3), S. typhimurium (n = 2), Salmonella Corvallis (n = 2), Salmonella Montevideo (n = 1), and S. javiana (n = 1). Eight patients developed severe forms of infections: sepsis (n = 2), septic shock (n = 1 with fatal outcome), meningitis (n = 3), and acute renal failure (n = 2). Twenty-two percent of isolates were resistant to ampicillin and gentamicin, 17.64 % to tetracycline, 14.28 % to nalidixic acid, and 10 % to chloramphenicol. All isolates were susceptible to ciprofloxacin. One Salmonella Corvallis isolate recovered from a patient with chronic hemolytic anemia produced an ESBL and its PFGE profile demonstrated less than 96 % similarity to fecal and wound Salmonella Corvallis with susceptible phenotypes. S. enteritidis was the most common cause of systemic and extraintestinal forms of human salmonellosis in Bulgaria. Resistance to ampicillin and gentamicin were the predominant profiles, although one Salmonella Corvallis isolate produced an ESBL. The ESBL-producing Salmonella Corvallis isolate clustered separately from the susceptible Salmonella Corvallis isolates.

Salmonellosis cases caused by a rare Salmonella Enteritidis PT6c associated with travel to Bulgaria, June-July 2008.

In June 2008 an outbreak of gastroenteritis was registered in Sunny Beach resort situated on the Black Sea coast in Bulgaria, affecting 14 employees of a hotel, five of whom tested positive for Salmonella Enteritidis. During June-July 2008 four sporadic S. Enteritidis cases were also reported and two of them were foreign tourists. In the same period S. Enteritidis cases connected with travel to Bulgaria were reported to the European Centre for Disease Prevention and Control (ECDC) from Finland, United Kingdom, Sweden, Germany and Norway. We describe a study performed to find out relatedness between Bulgarian and Finnish S. Enteritidis isolates using phage typing (PT) and pulse-field gel electrophoresis (PFGE). Fifteen S. Enteritidis isolates from Bulgaria and 195 from Finland (including 28 from travellers to Bulgaria) were phage typed. Within Bulgarian isolates four different PTs were found and PT6c with eight strains was predominant. Nineteen out of 28 strains isolated from the Finns visiting Bulgaria belonged also to PT6c. PFGE typing (with one enzyme) of all S. Enteritidis PT6c strains (8 Bulgarian and 19 Finnish isolates) showed indistinguishable PFGE profile. The typing results thus demonstrated a link between Bulgarian and Finnish S. Enteritidis isolates. We conclude that S. Enteritidis PT6c was the cause of salmonella outbreak in Sunny Beach and was exported to Finland, and likely to the United Kingdom, Norway, Sweden and Germany.

Occurrence and characterization of Salmonella enterica subspecies enterica serovar 9,12:l,v:- strains from Bulgaria, Denmark, and the United States.

In 2006, Salmonella enterica serovar I 9,12:l,v:- emerged in Bulgaria. The aim of this study was to characterize Salmonella serovar I 9,12:l,v:- isolates from Bulgaria, Denmark, and the United States. We compared isolates of Salmonella I 9,12:l,v:- and diphasic serovars with similar antigenic formulas by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility. The phase 2 flagellin gene (fljB) was also sequenced for selected isolates. By PFGE, the Salmonella I 9,12:l,v:- isolates from Bulgaria were indistinguishable from the isolate from the United States and distinct from isolates from Denmark; furthermore, several Salmonella I 9,12:l,v:- were indistinguishable from an isolate of Salmonella serovar Goettingen. Sequence analysis showed 100% sequence identity with known H:e,n,z15 sequences of Salmonella Goettingen, which has the antigenic formula I 9,12:l,v:e,n,z15. The study indicated that Salmonella I 9,12:l,v:- is a monophasic variant of Salmonella Goettingen and is present in different countries and on different continents.

Molecular characterization and occurrence of extended-spectrum beta-lactamase resistance genes among Salmonella enterica serovar Corvallis from Thailand, Bulgaria, and Denmark.

Fifty nine Salmonella Corvallis isolates from humans and food products in Bulgaria, Denmark, and Thailand were examined for antimicrobial susceptibility and characterized by pulsed-field gel electrophoresis (PFGE). Cephalosporin-resistant isolates were examined for the presence of genes encoding beta-lactamases by PCR and sequencing. Ten different PFGE types were observed. One type (30 isolates) was recovered in all three countries; three types were found only in Bulgaria, two only in Denmark, two only in Thailand, and two both in Denmark and Thailand. Ten isolates were susceptible to all antimicrobial agents tested, whereas 41 were resistant to three or more antimicrobials. Most resistance was observed among the isolates from Bulgaria. Of the 25 isolates from Bulgaria, 20 displayed resistance to ampicillin and the cephalosporins ceftiofur and cephalothin. All 20 isolates tested negative for bla (CMY-1), bla (CMY-2), and bla (ACC), but positive for bla (SHV), of which five were sequenced to bla (SHV-2). Plasmid profiling and hybridization revealed that the bla (SHV) gene was located on plasmids of approximately 70 kb. Five plasmid profiles were found among these 20 isolates. The plasmid profiling confirmed the PFGE-type and was able to further subdivide the strains. Seventeen of these 20 isolates contained also bla (TEM), of which nine representatives were sequenced to bla (TEM-1B), or bla (TEM-1H). One isolate contained bla (CTX-M-15), bla (SHV-2), and bla (TEM-1H), with the bla (CTX-M-15), and bla (TEM-1H) genes located on a 63-kb transferable plasmid. This study showed a high frequency of resistance among S. Corvallis isolated from humans and food products in Bulgaria, with a lower frequency in Thailand and Denmark. The clonal relatedness among the isolates from three countries could indicate a recent spread of this serovar.

Surveillance of human salmonellosis in Bulgaria, 1999-2004: trends, shifts and resistance to antimicrobial agents.

This article analyses the distribution of resistant salmonella and resistance mechanisms among the most frequently encountered serotypes in Bulgaria. Culture, biochemical tests and serotyping were used for identification. Screening for resistance to 14 antimicrobial agents with the standard Bauer-Kirbi disk-diffusion method. The double disk synergy method was used to determine production of extended-spectrum a-lactamases (ESBL). Transfer of genes coding for ESBLs with experimental conjugation. Specific primers were used for PCR detection of bla-CTX-M, bla-SHV and bla-TEM. 245 resistant salmonella strains were determined in our study; the majority originated from sporadic cases of human illness or asymptomatic infection and the remaining 23 were isolated from outbreaks. 79 producers of ESBL were detected: 5 S. Enteritidis, 1 S. Typhimurium, 9 S. Isangi and 62 S. Corvallis with types of enzymes: CTX-M3, TEM and SHV. Gene coding for extended-spectrum a- lactamases were successfully transferred into a recipient Escherichia coli C1A strain simultaneously with genes coding for resistance to aminoglycosides and sulphonamides (for bla-CTX-M3) and gene coding for resistance to aminoglycosides and chloramphenicol (for bla-SHVand bla-TEM). PCR amplification revealed bla-CTX-M3 genes in S. Enteritidis, and bla-SHV and bla-TEM in S. Corvallis. Salmonellae have revealed increasing resistance to all clinically important groups of antimicrobial agents. Bulgaria is the first country in the world where ESBL in serotype Corvallis has been reported. A wide diversity of resistance genes is found among the leading serotypes of salmonella causing human disease in Bulgaria.