Phylo-typing of clinical Escherichia coli isolates originating from bovine mastitis and canine pyometra and urinary tract infection by means of quadruplex PCR.

BACKGROUND: Escherichia coli is one of the major causative agents of bovine mastitis worldwide, and is typically associated with acute, clinical mastitis. Besides this, E. coli strains which belong to the extra-intestinal pathogenic group are also the major cause of urinary tract infections and pyometra in dogs. OBJECTIVES: In this study, it was aimed to investigate phylo-groups/subgroups in 155 E. coli isolates obtained from acute bovine mastitis, 43 from urinary tract infections of dogs and 20 from canine pyometra by a formerly described triplex PCR and recently described new quadruplex polymerase chain reaction (PCR) method. RESULTS: Group A1 (n = 118; 76%) and B1 (n = 71; 46%) were found to be the most prevalent groups by triplex and quadruplex PCR assays in mastitis isolates, respectively. Phylo-typing of 43 urinary tract isolates also revealed that most of the isolates belonged to A1 (n = 23; 54%) by triplex and B2 (n = 36; 84%) by quadruplex PCR assays. The isolates assigned as group A1 (n = 17; 85%) by triplex PCR could not be classified by quadruplex PCR in pyometra isolates. CONCLUSIONS: The results support the hypothesis that E. coli strains isolated from bovine mastitis cases are environmental. Also, groups C, E and F were identified as new phylo-groups for the first time in acute bovine mastitis cases. The comparison of triplex PCR with quadruplex PCR results revealed that most of the groups assigned in triplex PCR were altered by quadruplex PCR assay.

[The value of PCR method in the species-level identification of a mucoid Salmonella sp. strain isolated from the urine culture of a case with asymptomatic nephrolithiasis]. / Nefrolitiazisli asemptomatik bir olgunun idrar kültüründen izole edilen mukoid Salmonella sp. susunun tür düzeyinde tanimlanmasinda PCR yönteminin degeri.

Colonies of the Salmonella strains usually show a smooth (S) character. Therefore, Salmonella strains producing mucoid colony are very rarely encountered in the literature. Identification of the mucoid Salmonella strains to the species level is difficult via conventional methods, since the mucus layer does not allow the bacterium to respond to the antigenic reactions. In this study we aimed to emphasize the identification of Salmonella serotypes by the polymerase chain reaction (PCR) when rough (R) or mucoid (M) Salmonella isolates are encountered in the laboratory. The urine culture of a 17-year-old female patient revealed growth of 100.000 cfu/mL gram-negative bacilli in mucoid colony morphology. The isolate was identified as Salmonella sp. by biochemical tests and Vitek 2 (bioMérieux, France) automated identification system. Agglutination tests showed negative reaction with the known antiserums. Absence of agglutination was attributed to the mucoid character of the isolate. Identification of the Salmonella sp. was confirmed by Vitek MS MALDI-TOF (bioMérieux, France) analysis method, however, the serotype of the strain could not be identified. In order to verify that the mucoid colony was Salmonella spp., species-specific PCR was performed using invA primers, and Salmonella sp. identification was verified by observing a 284 base-pair (bp) PCR amplicon. Subsequently, serogrouping was done by multiplex-PCR (mPCR), which could identify the O:B (O:4), O:C1 (O:7), O:C2-C3 (O:8), O:D (O:9, O:9,46, O:9,46,27), and O:E (O:3,10, O:3,19) somatic antigens. It was detected that the mucoid Salmonella sp. formed a band of approximately 615 bp in size and took place in group D. Another mPCR directed towards O:D1(O:9) and O:E1(3,10) somatic antigens to detect subgroups of group D mucoid Salmonella spp., revealed that the isolate formed a DNA band of approximately 624 bp in size and took place in group D1 which is usually isolated from human. Modified version of another mPCR was used to determine phase-1 flagellar antigen of common Salmonella serovars, as well as to determine the phase-1 flagellar antigen of mucoid Salmonella spp. in group D1. Thus, the isolate was serotyped as Salmonella Enteritidis (1.9,12:g,m:-). Antibiotic susceptibility test performed by disc diffusion method in line with the recommendations of CLSI, revealed that the isolate was susceptible to ampicillin, ciprofloxacin, ceftriaxone, trimethoprim-sulfamethoxazole and chloramphenicol. In conclusion, PCR is a reliable and rapid alternative method that contributes to the conventional serotyping of Salmonella when rough or mucoid strains that lack somatic and flagellar antigens, are isolated.

Salmonella profile in chickens determined by real-time polymerase chain reaction and bacteriology from years 2000 to 2003 in Turkey.

From years 2000 to 2003, Salmonella was investigated from a total of 1785 samples comprised of chicken intestinal samples, cloacal swabs, drag swabs, litter samples and chick dust samples collected from 191 poultry breeding flocks belonging to 15 different chicken breeding stock companies in the Marmara region, Turkey by a SYBR green-based real-time polymerase chain reaction (SGBRT-PCR), by a probe-specific real-time polymerase chain reaction (PSRT-PCR) and by standardized bacteriology as described in the manual of National Poultry Improvement Plan and Auxillary Provisions, United States Department of Agriculture. Between January 2000 and July 2001, Salmonella was detected at the rates of 5.87% and 4.10% out of a total of 1242 samples by SGBRT-PCR and bacteriology, respectively. From July 2001 until December 2003, Salmonella was found at rates of 11.42% and 5.52% from a total of 543 samples by PSRT-PCR and bacteriology, respectively. The dominant Salmonella serovar was determined as Salmonella enterica subsp. enterica Serovar Enteritidis (S. Enteritidis), while serogroup C1 and C2 in 2001 and serogroup E1 in 2002 were isolated as additional serovars. As a conclusion, S. Enteritidis seems to be the major problem in poultry breeding flocks in Turkey, and both of the real-time polymerase chain reaction methods were found more sensitive than standard bacteriology for the detection of Salmonella from poultry samples.