Comparative study of pathogenicity tests for Shigella spp. and enteroinvasive Escherichia coli strains.

The purpose of this study was to optimize an in vitro pathogenicity model, as an alternative to Sereny test (on Guinea pigs). The study was performed on 13 Shigella spp. and 3 enteroinvasive Escherichia coli (EIEC) strains isolated in Romania between 2005 and 2007. The investigation implied the comparative evaluation between the Sereny test and Cravioto's adapted method for the assessment of the adherence and invasion capacity of the studied bacterial strains on HeLa cells, as well as the PCR detection of ipaH gene presence. The Sereny test was positive for all the strains tested. All strains adhered to the cellular layer, with a prevalent diffuse pattern for EIEC and an aggregative diffuse one for Shigella strains. The quantitative assessment of the invasion potential proved a high intracellular multiplication rate in 100% of the tested strains, with a progressive increase correlated with the incubation time. The present study has proven the existence of a good correlation between in vivo Sereny test and in vitro determination of the invasive capacity on eukaryotic HeLa cells, pleading for the utility of this test in identifying the invasive strains, taking into consideration the rigid regulations concerning the use of laboratory animals.

Identification of plasmid-mediated quinolone resistance qnr-like genes in Romanian clinical isolates of Escherichia coli and Klebsiella pneumoniae.

A collection of putative ESBL-producing Escherichia coli (119 isolates) and Klebsiella pneumoniae (122 isolates) originating from extraintestinal human specimens was screened for qnrA, qnrB, and qnrS-like genes by PCR. Seven K. pneumoniae isolates, which were resistant to ciprofloxacin, were detected as carrying qnrA1-like genes, while one K. pneumoniae and one E. coli isolate were positive for qnrS-like determinant. The latter isolates were susceptible to ciprofloxacin. This is the first study identifying qnr-like genes in our area. Further studies are needed to document the contribution of the plasmid mediated quinolone resistance to the increase in bacterial resistance to fluoroquinolones in Romania.

Laboratory diagnosis of infectious diarrhoea syndrome; a three years study in two hospitals of infectious diseases.

Infectious diarrhoea is a syndrome caused by a variety of bacterial, viral and parasitic organisms which represents a major cause of morbidity and mortality all over the world. The wide diversity of etiological agents impairs the surveillance and the diagnosis and affects the correct treatment applied to reduce the long-term complications. Besides well known enteric pathogens such as Salmonella, Shigella and Yersinia, a high number of emergent and re-emergent aetiologies are now recognised to be at the origin of diarrhoea. The lack of a correct diagnostic algorithm and adequate methods of analyses leads to under-evaluation and incertitude in an important number of clinical cases. Our study was designed as a complex analysis of the stool specimens collected from the patients, in the purpose to improve the laboratory diagnostic and to enhance the number of confirmed cases of infectious diarrhoea. A number of 756 samples from inpatients with diarrhoea were tested targeting pathogenic and opportunistic bacteria, viruses and parasites by classical and molecular methods. We documented that, in case of non-Salmonella, non-Shigella, non-Yersinia diarrhoea, the quality of diagnostic was improved by increasing the percentage of positive specimens to 22.49% compared to 11.12% when only bacteria, 5.56% when only viruses and 4.10% when only parasites were investigated. The laboratory data are of great value in evaluating the diarrhoea syndrome offering the documentation for an accurate epidemiological response and an adequate treatment.

Pulsed-field gel electrophoresis associated to phage typing improves the discrimination of epidemiologically unrelated Salmonella enterica serovar typhimurium isolates.

A combination of phage typing and pulsed-field gel electrophoresis (PFGE) of Xbal- and Blnl-digested chromosomal DNA has been used to study 18 epidemiologically unrelated human Salmonella enterica serovar Typhimurium isolates, which were collected during 2007 within a single Romanian county. Phage typing could assign only four of the isolates to three definitive phage types (DT41, DT86, and DT116), the rest being untypable by this classical method. PFGE analysis of the double enzyme-digested DNA, performed in an attempt to further discriminate the strains, allowed the typing of all the studied isolates. Xbal-digested genomic DNA segregated the isolates into 7 X-types and Blnl restriction differentiated them into 8 B-types. Our PFGE results documented the circulation of a rather homogeneous population of S. Typhimurium strains within the same county. As in the case of other human pathogens, epidemiological conclusions might be more accurate if based on both phenotypic and genotypic methods, therefore molecular typing should be added within the national laboratory-based surveillance of Salmonella infections.

Application of pulsed-field gel electrophoresis to laboratory surveillance of small community outbreaks of Salmonella enterica serovar Typhimurium.

Infectious diarrhea syndrome is an important cause of human morbidity around the world, and Salmonella genus remains one of the most prevalent etiology. Salmonella enterica serovar Typhimurium outbreak-associated isolates received by the Laboratory for Enteric Pathogens from N.I.R.D.M.I. "Cantacuzino" for confirmation and typing were analyzed by genomic pulsed-field gel electrophoresis (PFGE) and phage susceptibility testing to establish their relatedness. Both typing methods proved to have similar discriminatory power. The isolates originating from the same outbreak belonged to the same phage type and showed indistinguishable PFGE profiles. The molecular characterization of autochthonal Salmonella enterica Typhimurium outbreak human isolates provided laboratory evidence that epidemiologically related isolates collected from community outbreaks of disease were also genetically related. In order to improve the national and international surveillance of major foodborne pathogens the reference laboratory centers are required to establish and maintain the capacity to perform a wide range of both phenotypic and genotypic methods to support outbreak investigations.