Clonal spread of Salmonella enterica serovar Infantis in Serbia: acquisition of mutations in the topoisomerase genes gyrA and parC leads to increased resistance to fluoroquinolones.

Quinolone-resistant Salmonella Infantis (n = 64) isolated from human stool samples, food and poultry during the years 2006-2011 were analysed for their resistance phenotypes, macrorestriction patterns and molecular mechanisms of decreased susceptibility to fluoroquinolones. Minimum inhibitory concentrations (MICs) of nalidixic acid (NAL) and ciprofloxacin (CIP) were determined by the agar dilution procedure, and the susceptibility to additional antimicrobial agents was determined by the disc diffusion method. To assess the influence of enhanced efflux activity, MICs were determined in the presence and absence of the inhibitor PAßN. The results of pulsed-field gel electrophoresis (PFGE) typing revealed that quinolone-resistant S. Infantis in Serbia had similar or indistinguishable PFGE profiles, suggesting a clonal spread. All S. Infantis showed combined resistance to NAL and tetracycline, whereas multiple drug resistance to three or more antibiotic classes was rare (2 isolates of human origin). The MICs ranged between 512 and 1024 µg/mL for NAL and 0.125-2 µg/mL for CIP. A single-point mutation in the gene gyrA leading to a Ser83→Tyr exchange was detected in all isolates, and a second exchange (Ser80→Arg) in the gene parC was only present in eight S. Infantis isolates exhibiting slightly higher MICs of CIP (2 µg/mL). The inhibitor PAßN decreased the MIC values of CIP by two dilution steps and of NAL by at minimum 3-6 dilution steps, indicating that enhanced efflux plays an important role in quinolone resistance in these isolates. The plasmid-mediated genes qnr, aac(6')-lb-cr and qepA were not detected by PCR assays.

A one-year prospective study on the antibiotic resistance of E. coli strains isolated in urinary specimens of children hospitalized at the University Pediatric Medical Center in Novi Sad, Serbia.

PURPOSE: Urinary tract infections (UTIs), the most common serious bacterial infections in children, are frequently caused by Escherichia coli. The purpose of this study was to investigate E. coli resistance/multidrug resistance to antibiotics most frequently used for UTIs. METHODS: Children 0-18 years of age, hospitalized at the University Pediatric Hospital in Novi Sad, Serbia, were included in a 1-year observational prospective study. The microbiological analysis was performed using the standard Kirby-Bauer disk diffusion method. The results were analyzed using WHONET 5.4 software. RESULTS: E. coli was isolated from 61.7 % of positive urine specimens. In general, higher average E. coli antibiotic resistance was found in infants and toddlers compared to children and adolescents (33.4 vs. 25.0 %) (p < 0.0001). Furthermore, it was observed that the average resistance to all the tested antibiotics was higher in boys than in girls (37.0 vs. 25.1 %) (p < 0.0001). E. coli was highly susceptible to piperacillin/tazobactam (>93.1 %), amikacin (86.3 %), quinolones (>75.0 %), and penems (>96.6 %). The prevalence of multiresistant E. coli strains was significantly higher in infants and toddlers (72.3 vs. 36.8 %) (p < 0.0001). CONCLUSIONS: E. coli, a common cause of UTIs in children admitted to pediatric hospitals, is highly resistant/multidrug-resistant to commonly used antibiotics. Higher average resistance is found in infants and toddlers than in children and adolescents, as well as in boys compared to girls. These findings are important for the regional empiric therapy of UTIs and call for actions to decrease E. coli antibiotic resistance.

Molecular typing and antimicrobial resistance of Salmonella Enteritidis isolated from poultry, food, and humans in Serbia.

Molecular typing and resistotyping coupled with gyrA single nucleotide polymorphism (SNP) of 60 Salmonella Enteritidis (SE) isolates originated from poultry, food, and humans in Serbia is described. Molecular fingerprinting was performed by randomly amplified polymorphic DNA (RAPD) using four primers, and the diversity index (D) was 0.688. In combination with resistotyping and gyrA SNP, D increased to 0.828. A total of 23 genetic groups were obtained. When four RAPD primers were combined, epidemic isolates from a fast-food restaurant outbreak were clustered in a distinctive genetic group. Among 60 SE strains, three had multiple resistances to three or more antibiotics. Nine strains were resistant to nalidixic acid (NAL; a non-fluorinated quinolone). The mutations in quinolone resistance-determining region (QRDR) found in NAL-resistant strains were attributed to Asp(87) → Asn in six strains, Asp(87) → Gly in one strain, and Ser(83) → Phe in one strain. One NAL-resistant strain had no mutations in QRDR, suggesting another mechanism of resistance.

[Salmonella bacteremia]. / Salmonela bakteriemije.

INTRODUCTION: Non-typhi salmonellae are invasive bacteria, which can, in certain conditions, get into the blood stream, and course bacteriemia or localized infections in different organ systems. Incidence of salmonella bacteriemia varied in different studies between 5.2-13.7%. The incidence is higher in patients younger than 1 year and the elderly. Among different serotypes, the most invasive are Salmonella typhimurium, Salmonella choleraesuis and Salmonella Virchow. The aim of our study was to determine the incidence of salmonella bacteriemia among patients hospitalized at the Clinic of Infectious Diseases Novi Sad during 1991-1998, and to point to certain risk factors for salmonella bacteriemia. RESULTS: During 1991-1998, 1309 patients were hospitalized with the diagnosis of Salmonella gastroenteritis. 12 patients (0.99%) had positive blood culture. The average age of salmonella bacteriemia patients was 17 (1-54 year), but 50% of them were younger than 10. 4/12 patients (33.33%) had positive stool and blood culture, but in 8/12 (66.66%) only positive blood culture had been established. The main serotype was Salmonella enteritidis (83.33%) and after that Salmonella Virchow (16.66%). In more than 50% of patients there was at least one risk factor responsible for dissemination: sideropenic anemia (25%), cerebral palsy (8.33%) and chronic cardiac disease (8.33%). Average duration of fever was 13.16 days and diarrhea 8.83. All patients presented with hepatosplenomegaly. One patient had a localized salmonella infection (periappendicular abscess) and she had undergone successful surgery. All our patients had been under cephalosporins of the third generation for 2 weeks and they recovered completely without sequelae. CONCLUSIONS: A relatively low incidence of salmonella bacteriemia (0.99%) in our patients can be explained with small number of children younger than 1 year and patients older than 70 years of age in whom bacteriemia is most common. Positive blood culture doesn't mean obligatory positive stool culture. Routine laboratory analyses are relatively nonspecific for the diagnosis of generalized infection. Cephalosporins of the third generation are considered to be the drug of choice in treatment of salmonella bacteriemia.

[Analysis of the plasmid profile of various Salmonella serotypes]. / Analiza plazmidskih profila razlicitih serotipova Salmonela.

INTRODUCTION: Every year foodborne infections cause millions of illnesses but many of them go undiagnosed and unreported. The epidemiology of these illnesses is changing, new pathogens have emerged (Escherichia coli O157:H7, Cyclospora cayetanensis, Vibrio vulnificus). Salmonella spp. is the most common bacterial cause of acute enterocolitis with us. All over the world, as well as in our country the most often isolated serotype is Salmonella Enteritidis. A great problem in many countries is the multiresistant Salmonella Typhimurium, as well as other serotypes resistant to a great number of antimicrobial drugs (S. Hadar, S. Typhi). Clinical microbiologists are often asked to determine the relatedness of bacterial isolates. Recently, traditional methods of strain typing such as bacteriophage typing, resistotyping and serotyping, have been supplemented or replaced in many laboratories with newer molecular methods such as plasmid fingerprinting, ribotyping. PCR-based methods, etc. The goal of strain typing is to provide evidence that epidemiologically related isolates collected during an outbreak are also genetically related and thus represent the same strain. MATERIAL AND METHODS: In the laboratory for Enterobacteriaceae of the Institute of Public Health Novi Sad in the four year period (1995-1998) 3659 primary isolates of Salmonella spp. were isolated using standard bacteriological methods (cultural, biochemical and serological examination). For certain strain of Salmonella Enteritidis, S. Typhimurium and S. Hadar susceptibility to antimicrobial agents was tested by disc-diffusion test (Kirby-Bauer) and plasmid profiles were analyzed. Plasmid DNA was extracted by Birnboim and Doly alkaline lysis method and plasmid bands were separated by electrophoresis in agarose gel. RESULTS: In the period of 1995-1998 the most common serotype isolated was Salmonella Enteritidis with 3017 (82.5%) of the total number of isolated Salmonellas; S. Typhimurium 203 (5.5%), S. Hadar 118 (3.2%). Plasmid profiles were tested in 10 S. Enteritidis isolates that originated from patients with sporadic cases of diarrhea. All investigated strains had one plasmid band with molecular weight of 38 MDa. All isolates were susceptible to all antibiotics tested. Ten isolates of S. Hadar originated from one outbreak from food samples and stools of patients with diarrheal disease and from the worker in the restaurant. All isolates were resistant to ampicillin and streptomycin, and plasmid profile analysis showed 5 plasmid bands with molecular weights of 13, 5.4, 4.2, 2.0 and 1.7 MDa. Chosen strains of S. Typhimurim were not epidemically related. Strains number 1, 3, 4, 5 were susceptible to all antibiotics tested, and had only one plasmid of 50 MDa, strain number 7 was resistant to streptomycin and had 2 plasmid bands of 50 and 1.7 MDa, other strains were multiresistant and had different plasmid profiles with 4-7 plasmid bands with molecular weights ranging from 50-1.4 MDa. CONCLUSION: Plasmid profile analysis is not a sufficient method for examination of Salmonella Enteritidis which is the most common cause of enterocolitis with us. It is, however, a helpful method for proving epidemiological and clonal relatedness of Salmonella isolates that are resistant to antimicrobial agents and have a great number of plasmids (such as some strains of S. Typhimurim and S. Hadar).

[Shigellae isolated in 1997--plasmid profiles and antibiotic resistance]. / Sigele izolovane u 1997. godini--plazmidski profil i rezistencija na antibiotike.

INTRODUCTION: Shigella spp is one of the most frequently isolated bacteria causing acute diarrhea with us. Genetics of pathogenicity of Shigella spp. includes chromosomal and plasmid genes. Most virulence factors are coded by invasion plasmid antigen genes residing on a 180-230 MDa plasmid. There is a big problem with multiple resistance of Shigella spp. strains, which is mostly plasmid-borne. Genetic analysis of bacterial cells, that is plasmid profile analysis, is important for investigation of sources and ways of spreading of the infection. All isolates originating from the same clone have identical plasmid profiles, i.e. number and size of plasmids. The aim of the investigation was: comparing the type of resistance to antimicrobical agents found in epidemic and nonepidemic. Shigella strains isolated in 1997, analyzing plasmid profiles of these isolates and confirming their epidemic connection. MATERIAL AND METHODS: Susceptibility to antibiotics was examined by a standard disc-diffusion method. Plasmid profiles of 40 strains (20 from the outbreak and 20 from sporadic cases) were tested using a method of alkaline lysis by Birnboim and Doly followed by electrophoresis in agarose gel. RESULTS: Shigella strains were resistant to antimicrobial agents which are most commonly used. Epidemic isolates shared the same resistance type, they were resistant to cephalexin, streptomycin and co-trimoxazole. The dominant type of resistance of nonepidemic strains was to ampicillin, streptomycin and co-trimoxazole. Strains isolated during the outbreak had identical plasmid profiles (2 plasmid bands of 55 and 1.5 MDa). Non-epidemic isolates had different plasmid profiles as well as type of resistance. CONCLUSION: Strains of Shigella spp. isolated during an outbreak had the same type of resistance and the same plasmid profiles, which indicated their origin from the same clone. The plasmid profile analysis is a reliable and precise method for determination of epidemic connection of Shigella isolates.

[Virulence of plasmids of diarrhea-causing strains of Salmonellae wien and Escherichia coli]. / Ispitivanje plazmida virulencije u sojeva Salmonellae wien i Escherichia coli uzrocnika dijereje.

Use of molecular genetic methods in the study of sources and ways of spreading the infection provides precise data on the mutual clonal origin of the strains, on the basis of the determination of plasmid DNK maps. Results of the investigation of Salmonellae Wien strain show the same type of resistance to antimicrobial drugs and the impact of resistotypization as an epidemiologic marker. The analysis of the plasmid profile in these strains has confirmed the presence of FIme virulence plasmid of 80 megadalton in size and resistance plasmid of 60 megadalton which was by conjugation transferred to the recipient strain (in vitro experiment). The presence of antigen factors of colonization in escherichial coli, a cause of diarrhea, was confirmed: CFA-I in the serogroup 078, and CFA-II in the serogroup 06.