A Snapshot of the Genetic Diversity of Salmonella Enteritidis Population Involved in Human Infections in Romania Taken in the European Epidemiological Context.

In the absence of consistent national molecular typing data to enhance the surveillance of Salmonella Enteritidis, it was considered useful to collect baseline information on the genetic diversity and antibiotic susceptibility of strains isolated in Romania between January 2016 and April 2020 and compare them to strains described in major international outbreaks of the same period. A collection of 245 clinical isolates were genotyped by a standardised multiple-locus variable-number of tandem repeats analysis (MLVA) 5-loci protocol and screened for antimicrobial resistance against 15 compounds. Twenty strains were further subjected to whole genome sequencing (WGS) and compared to epidemiologically relevant high-throughput sequencing data available in European databases. Twenty-seven MLVA genotypes were identified, of which three, commonly reported in Europe between 2016-2020, covered 72% of the collection. Antibiotic resistance was detected in 30% of the strains, with resistance to nalidixic acid and ciprofloxacin as the most common phenotype, and also associated with two prevalent MLVA clones. WGS-derived multilocus sequence typing (MLST) revealed a single sequence type (ST11) further resolved into 10 core-genome MLST complex types. The minimum spanning tree constructed from the cgMLST data clustered Romanian and international strains, which shared more than 95% of the core genes, revealing links with a contemporaneous multi-country outbreak. This study could be regarded as a forerunner to the advent of using this integrative approach in the public health practice at a national level and thus contribute to the concerted actions at a European level to stop outbreaks.

Molecular Typing of Escherichia coli O157 Isolates from Romanian Human Cases.

Verocytotoxin-producing Escherichia coli (VTEC) of serogroup O157 are among the most important causes of severe cases of foodborne disease and outbreaks worldwide. As little is known about the characteristic of these strains in Romania, we aimed to provide reference information on the virulence gene content, phylogenetic background, and genetic diversity of 7 autochthonous O157 strains collected during 2016 and 2017 from epidemiologically non-related cases. These strains were typed by a combination of phenotypic and molecular methods routinely used by the national reference laboratory. Additionally, 4 of them were subjected to whole-genome sequencing (WGS), and public web-based tools were used to extract information on virulence gene profiles, multilocus sequence types (MLST), and single nucleotide polymorphism (SNP)-based phylogenetic relatedness. Molecular typing provided evidence of the circulation of a polyclonal population while distinguishing a cluster of non-sorbitol-fermenting, glucuronidase-negative, phylogenetic group E, MLST 1804 strains, representing lineage II and clade 7, which harbored vtx2c, eae-gamma, and ehxA genes. A good correlation between the routine typing methods and WGS data was observed. However, SNP-based genotyping provided a higher resolution in depicting the relationships between the O157:H7 strains than that provided by Pulse-field gel electrophoresis. This study should be a catalyst for improved laboratory-based surveillance of autochthonous VTEC.

Molecular characterisation of human Shiga toxin-producing Escherichia coli O26 strains: results of an outbreak investigation, Romania, February to August 2016.

IntroductionAt the beginning of 2016, an increase in paediatric haemolytic uremic syndrome (HUS) cases was observed in Romania. The microbiological investigations allowed isolation of Shiga toxin-producing Escherichia coli (STEC) O26 as the causative agent from most cases. Methods: An enhanced national surveillance of HUS and severe diarrhoea was established across the country following the identification of the first cases and was carried out until August 2016. A total of 15 strains were isolated from 10 HUS and five diarrhoea cases. Strains were characterised by virulence markers (i.e. stx type/subtype, eae, ehxA genes), phylogroup, genetic relatedness and clonality using PCR-based assays, PFGE and multilocus sequence typing (MLST). The first six strains were further characterised by whole genome sequencing (WGS). Results: Five PCR-defined genotypes were distinguished. All strains from HUS cases harboured stx2a and eae, with or without stx1a, while strains from diarrhoea cases carried exclusively stx1a and eae genes. PFGE resolved strains into multiple pulsotypes, compatible with a certain geographic segregation of the cases, and strains were assigned to phylogroup B1 and sequence type (ST) 21. WGS confirmed the results of conventional molecular methods, brought evidence of O26:H11 serotype, and complemented the virulence profiles. Discussion/conclusion: This first description of STEC O26 strains from cases in Romania showed that the isolates belonged to a diverse population. The virulence content of most strains highlighted a high risk for severe outcome in infected patients. Improving the national surveillance strategy for STEC infections in Romania needs to be further considered.

Molecular characterization of bacteremic Escherichia coli isolates in Romania.

The increasing prevalence of invasive infections caused by antibiotic resistant Escherichia coli strains in Romanian patients, already mentioned in the European reports, requires better knowledge of their specific traits. Thus, a set of 38 E. coli blood isolates, collected between 2010 and 2012 at one of the local hospitals participating into the European Antimicrobial Resistance Surveillance Network, was investigated retrospectively with respect to the phylogenetic origin, extraintestinal virulence-associated markers (i.e. fimH, papC, papG alleles, sfa/foc, afa/dra, hly, cnf1, sat, iucC, fyuA, ibeA), and beta-lactamase encoding genes (i.e. bla CTX-M, bla TEM, and bla SHV alleles). The isolates with extended-spectrum beta-lactamase (ESBL) phenotypes were further characterized using PCR-based replicon typing and multilocus sequencing typing. For ST131 members, pulsed-field gel electrophoresis (PFGE) and PCR-based detection of fimH30 allele were performed. Overall, the isolates were more likely members of the major phylogenetic group A (53 %) and to a lesser extent of groups B2 (29 %), D (10 %), and B1 (8 %). All but three of the virulence markers sought (i.e. papGI, hly, cnf1) were detected with prevalence ranging from 3 % (i.e. ibeA, papGIII) to 87 % (fimH). As expected, the most complex genotypes (four to seven virulence markers) defined the isolates derived from phylogenetic groups B2 and D. ESBL producers were bla CTX-M-15-positive, mostly of phylogroup A (67 %), harboured IncF multireplicon plasmids, and belonged to six sequence types (i.e. ST10, ST131, ST167, ST410, ST540, ST1275). Members of ST10 clonal complex (i.e. ST10, ST167) were the most common. The ST131 isolates belonged to H30 subclone and displayed 74 % similarity at PFGE analysis.

Carbapenemase-Producing Klebsiella pneumoniae in Romania: A Six-Month Survey.

This study presents the first characterization of carbapenem-non-susceptible Klebsiella pneumoniae isolates by means of a structured six-month survey performed in Romania as part of an Europe-wide investigation. Klebsiella pneumoniae clinical isolates from different anatomical sites were tested for antibiotic susceptibility by phenotypic methods and confirmed by PCR for the presence of four carbapenemase genes. Genome macrorestriction fingerprinting with XbaI was used to analyze the relatedness of carbapenemase-producing Klebsiella pneumoniae isolates collected from eight hospitals. Among 75 non-susceptible isolates, 65 were carbapenemase producers. The most frequently identified genotype was OXA-48 (n = 51 isolates), eight isolates were positive for blaNDM-1 gene, four had the blaKPC-2 gene, whereas two were positive for blaVIM-1. The analysis of PFGE profiles of OXA-48 and NDM-1 producing K. pneumoniae suggests inter-hospitals and regional transmission of epidemic clones. This study presents the first description of K. pneumoniae strains harbouring blaKPC-2 and blaVIM-1 genes in Romania. The results of this study highlight the urgent need for the strengthening of hospital infection control measures in Romania in order to curb the further spread of the antibiotic resistance.

MECHANISMS OF RESISTANCE TO CIPROFLOXACIN AND GENETIC DIVERSITY OF ESCHERICHIA COLI STRAINS ORIGINATING FROM URINE CULTURES PERFORMED FOR ROMANIAN ADULTS.

Urinary tract infections (UTI) with Escherichia coli are among the most common infections presenting in general practice. Fluoroquinolones (FQs) are relied on for their empirical therapy but recent reports indicate a concerning increase in the percentage of FQ-resistant E. coli isolates in many countries, including Romania. Sixty E. coli strains with ciprofloxacin resistance and cephalosporin susceptibility isolated from urine specimens of non-hospitalized patients during a five-month period (October 2014 - February 2015) were further analyzed to determine the molecular basis of FQ resistance (i.e. mutations in chromosomal gyrA, gyrB, parC genes and presence of plasmid-borne qnrA, qnrB, qnrS, and aac(6'-Ib-cr genes), the phylogenetic background (i.e. phylogenetic groups A, B1, B2, C, D, E, F or clade I), O25b/ST131 status, and genetic relatedness inferred from the XbaI pulsed-field gel electrophoresis (PFGE) profiles as a measure of isolate-specific genetic composition. The PCR-based phylotyping showed that most strains were assigned to non-B2 phylogenetic groups (i.e. group A/21 strains, group B1/14 strains, group B2/10 strains, group C/8 strains, group D/3 strains, group F/4 strains). Already described chromosomal mutations associated to FQ resistance were found, the strains being double gyrA mutants (i.e. Ser83Leu, Asp87Asn) with one or two parC mutations (e.g. Ala56Thr, Ser80Ile, Glu84Gly). Seven percent of the strains harboured plasmid-borne genes qnrS1 (2 strains) and aac(6'-Ib-cr (2 strains). Based on the PCR results, 15% of the strains were members of the O25b/ST131 clone and possessed the gyrA/parC allele combination which is considered as hallmark of H30 subclone. PFGE genotyping revealed a genetically diverse population of FQ-resistant E. coli. ST131 strains displayed more homogeneous PFGE profiles than non-ST131. The ST131 cluster extended to 77.74% similarity versus 60% overall. These findings underscore the need for ongoing surveillance to capture the complexity of the emerging population of FQ-resistant strains disseminated across our community.

ANTIMICROBIAL RESISTANCE AND GENETIC DIVERSITY OF SHIGELLA SONNEI ISOLATES FROM ROMANIA.

Although not endemic in the European Union and European Economic Area, shigellosis is included among the priority food- and waterborne diseases carefully surveyed by the European Centre for Disease Prevention and Control. From 2010 to 2012, 1018 cumulated confirmed shigellosis cases were reported to the European Surveillance System by Romania. This retrospective study aimed to provide insights into the antibiotic resistance and genetic diversity of a set of Shigella sonnei isolates recovered during that period. A total of 59 S. sonnei isolates were subjected to antimicrobial susceptibility testing (ampicillin, cefotaxim, chloramphenicol, streptomycin, gentamicin, kanamycin, tetracycline, sulphonamides compound, trimethoprim, trimethoprim-sulfamethoxazole, nalidixic acid, and ciprofloxacin), biotyping, and molecular characterization of resistance to third-generation cephalosporins, fluoroquinolones, and integron content. Pulsed-field gel electrophoresis (PFGE) was performed in order to assess the genetic relatedness of the isolates. Thirty-eight (64%) of the studied isolates displayed multidrug-resistant (MDR) phenotypes, the most common resistance profile comprising resistance to ampicillin, streptomycin, sulphonamides compound, trimethoprim, and trimethoprim-sulfamethoxazole. Resistance to cefotaxim was detected in a biotype g blaCTX-M-15 and a biotype e blaCMY-2-positive isolate, respectively. Resistance to ciprofloxacin and/or nalidixic acid was detected in three MDR isolates and was due to known mutations in gyrA gene leading to aminoacid substitutions (i.e. Ser83Leu, Asp87Tyr, Asp87Gly). Either class 1 or class 2 integrons were identified in 10 isolates. Comparisons of XbaI PFGE patterns of S. sonnei isolates revealed 9 clonal groups and 6 unique patterns. The genotyping results suggested that dissemination of clonal groups of S. sonnei may have persisted over the years in Romania.

Escherichia coli ST131 causing invasive infections in Romanian patients--a threat we can no longer ignore.

Escherichia coli sequence type ST131 is a major pandemic clonal group of drug-resistant extraintestinal pathogenic E. coli (ExPEC) involved in community-onset and healthcare-associated infections. Thus far, its presence in our area has been paid little attention. This is a preliminary study intended to detect ST131 among 87 clinical isolates retrieved from a larger and unpublished E. coli collection. The study isolates originated from various specimens associated with invasive infections (blood, deep surgical wounds/abscesses, tracheal aspirates, pleural fluid, cerebrospinal fluid, and peritoneal fluid) and were collected between 2010 and 2014. Based on the main inclusion criteria, resistance to extended-spectrum cephalosporins (ESCs) and/or fluoroquinolones (FQs), the isolates were distributed in three categories: isolates with resistance to FQs (20 isolates), to ESCs (8 isolates), and to FQs and ESCs (59 isolates), respectively. Polymerase chain reaction (PCR) -based assays were performed to determine the major phylogenetic groups, to predict the MLST ST131 status, and to detect the bla(CTX-M) content of the ESC-resistant isolates. Overall, the studied isolates derived from phylogenetic groups B2 (42 isolates), A (30 isolates), B1 (11 isolates), and D (4 isolates). Thirty-five isolates, originating from blood (26 isolates), deep wounds (6 isolates), tracheal aspirates (2 isolates), and cerebrospinal fluid (1 isolate), were identified as members of O25b:H4 ST131. Most of them displayed resistance to both ESCs and FQs and harboured group 1 bla(CRX-M) genes. The emergence of ST131 in our region can no longer be ignored. Focused attention to this lineage could reduce infection-related morbidity and antibiotic resistance.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area.

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Recurrent Infective endocarditis of the native aortic valve due to ESBL producing Escherichia coli (e coli) after therapeutic ERCP.

UNLABELLED: We describe the first case of ESBL producing E coli endocarditis of the native aortic valve in which prophylaxis was performed according to currently available guidelines. A 75 year-old woman presented at our emergency department with a two week complaint of fever, fatigue, anorexia, diffuse abdominal pain after ERCP therapeutic. The initial laboratory examinations showed increased levels of ESR, CRP, fibrinogen, alkaline phosphatase, gammaGT, aspartate aminotransferase, alanine aminotransferase and direct fraction of bilirubin. Two blood cultures were positive for ESBL-producing E coli. The abdominal sonography revealed intrahepatic biliary tree dilation, cholecystectomy and minimal aerobilia in the left liver lobe. A transthoracic echocardiogram showed a small vegetation adherent to the aortic valve and a moderate amount of aortic and mitral regurgitation. Treatment with imipenem/cilastatin for 35 days was performed with a favorable outcome. During this period she underwent endoscopic metal stenting to replace the plastic tube. The patient was discharged after 40 days of hospitalization. CONCLUSION: Patients with high risk also for endocarditis and infections or bowel colonization with multiple drug resistant Enterobacteriaceae such as ESBL- producing E coli that have undergone multiple and repetitive therapeutic procedures are at risk for endocarditis with this type of bacteria. Prophylaxis and therapy with appropriate antibiotics must be considered in these patients.

[Bacterial enteric pathogens' resistance to fluoroquinolones and last generation cephalosporines]. / Rezistenta patogenilor enterici bacterieni la fluoroquinolone si cefalosporine de ultima generatie.

The increase of incidence of resistance to the antibiotics became the most worrisome subject within the clinical and research communities in the medical fields. Intrinsic resistance genetic mutations, horizontal transfer of mobile structures carrying genes coding for resistance to the antibiotics within the pan-microbial genome are representing the bacterial resistome which is bearing the genetic information regarding the defensive mechanisms developed by micro-organisms to protect themselves against antibiotics. Rice in the resistance of enteric bacteria, pathogens involved in a large number of human infections, to the cephalosporin of last generation and to the fluoroquinolones is a very actual subject in the medical area. Production of beta-lactamases with extended spectrum is the most important enzymatic defence system, developed by micro-organisms, consisting in the inactivation of beta-lactam antibiotics by destroying the beta-lactam ring. Enterobacteria are able to produce beta-lactamases of type TEM, SHV and/or CTX-M. Punctual mutations in nucleotide structure of bla genes, coding for beta-lactamases synthesis, are leading on production of a large diversity of enzymes with enlarged spectrum of activity (ESBL). At the beginning of 90's the first beta-lactamases resistance to clavulanic acid were detected and in our days more then 170 TEM, 120 SVH and 90 CTX-MESBLs are known. Escherichia coli strains are producing, firstly, TEM ESBLs, Klebsiella pneumoniae SHV ESBLs. and both are producing CTX-M type ESBLs, are resistant to the fluoroquinolones due to punctual mutations in nucleotide structure of gyr gene coding for gyrases production, enzymes involved in nucleic acids replication. Resistance to the antibiotics with extended activity is a public health threat due to their capacity of large spreading within bacterial population, when the coding structures are located on mobile genetic structures. The menace increase when genes coding for fluoroquinolones resistance (qnr) are identified on such of structures.

A multiple typing approach is recommended for the laboratory-based surveillance of salmonellosis in Romania.

In Romania, Salmonella enterica serovar Typhimurium isolates are currently typed by antimicrobial resistance profiles and phage typing, as part of the national laboratory-based surveillance system of human enteric infections. The aim of the present study was to assess the added value of complementing this approach with molecular fingerprinting, namely pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeats analysis (MLVA). Thirty-six S. Typhimurium isolates received by the Reference Center for Human Salmonella Infections for confirmation and typing from the Microbiology Departments of three Public Health Authorities, were selected for this study. Phage typing revealed that 14 isolates (39%) were nontypeable (NT). Twenty-two isolates were assigned to 5 phage types: DT193 (11 isolates), U302 (7 isolates), DT116 (2 isolates), DT41 (1 isolate) and DT86 (1 isolate). Antimicrobial susceptibility testing showed that all the NT and DT116 isolates were multidrug resistant and extended-spectrum betalactamase producers. All the examined isolates were typeable when using the molecular approach. Both methods gave conclusive and comparable results, documenting the genetic relatedness and discriminating the outbreak isolates from sporadic cases. We conclude that in order to improve outbreak investigation and surveillance of salmonellosis in Romania, the current routine typing of Salmonella isolates should be complemented with at least one of these DNA fingerprinting methods.

Escherichia coli pathotypes associated with diarrhea in Romanian children younger than 5 years of age.

To document the association of pathogenic Escherichia coli with diarrhea in Romanian children, 250 E. coli fecal isolates from children under 5 years of age were PCR-screened for well-recognized virulence determinants, as well as for their phylogenetic background. The putative diarrheagenic E. coli (DEC) were investigated for susceptibility to various antibiotics. Overall, 61 E. coli isolates were classified as enteroaggregative E. coli (29 isolates), atypical enteropathogenic E. coli (22 isolates), enterotoxigenic E. coli (8 isolates), and verotoxin-producing E. coli (1 isolate), and one isolate was categorized as unconventional DEC. Only 8 of the PCR-positive isolates would have been assumed to be pathogenic based on their O antigenicity, which highlights the limited effectiveness of serotyping. More than a half (51%) of the pathogenic isolates expressed a multidrug-resistant phenotype, which raises concerns about the therapeutic pediatric approach. The DEC isolates were heterogeneous phylogenetically, deriving from all four major groups: A (31 isolates), B2 (14 isolates), B1 (10 isolates), and D (6 isolates), respectively. Thus, the phylogenetic descent was less significant than the virulence gene content. Our findings document the importance of DEC as a cause of childhood diarrhea in Romania, providing evidence that efforts should be made to estimate the burden of infections by etiology for a better medical approach.

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.

Group B streptococcus colonization of Romanian women: phenotypic traits of isolates from vaginal swabs.

In the attempt to enrich the local contemporary laboratory data regarding the group B streptococcus (GBS) colonization, isolates obtained from the vaginal swab cultures were characterized for their serotype distribution and antibiotic susceptibility. The 100 GBS isolates analyzed were collected during a four-month period of year 2009 from women screened in ambulatory for vaginal carriage of GBS. The GBS isolates were classified based on their capsular polysaccharide structures using commercially available antisera. Susceptibility to penicillin, ampicillin, erithromycin, clindamycin, tetracycline, ofloxacin, and chloramphenicol was initially tested using antibiotic disk diffusion technique according to CLSI guidelines. Minimum inhibitory concentrations of erythromycin and tetracycline for the isolates with reduced susceptibility were evaluated according to the CLSI criteria and macrolide-lincosamide-streptogramin B (MLSB) resistance was investigated by a double-disk test with erythromycin and clindamycin disks. All the GBS isolates were serotypeable. Their distribution comprised six different serotypes of which serotypes II (26%), III (26%), and Ia (19%) prevailed and no serotype VI, VII, and VIII isolates were found. Overall, the GBS isolates were fully susceptible to penicillin and ampicillin, but the rates of susceptibility to the other antimicrobial agents tested were decreased, ranging from 87% for chloramphenicol to 5% for tetracycline. Reduced susceptibility to clindamycin and erythromycin was detected in 18% and 19% of isolates, respectively. For the latter, 84% displayed a constitutive MLSB phenotype, 11% had an inducible MLSB phenotype, and M phenotype was expressed by 5% of them. Erythromycin-resistant GBS isolates displayed concurrently resistance to at least one more antibiotic. In conclusion, according to our study the most frequent GBS serotypes isolated from the vaginal microflora were II and III, followed by serotype Ia. While the GBS isolates remain susceptible to beta-lactams, resistance to alternative antimicrobial drugs such as erythromycin and clindamycin seems to be an increasing concern for our region. Further phenotypic and genotypic studies are required to identify specific aspects of GBS strains colonizing or infecting the local population.

[Specifics of urinary tract infection investigated in hospitalized patients]. / Particularitati ale infectiei tractului urinar investigate în mediu spitalicesc.

Urinary tract infections investigated in hospital show a series of clinical and etiological features, especially concerning antibiotic sensitivity, determined by the recurrent pattern of these disease, which often determines severe urinary or general complications. We considered 36,136 urine samples belonging to patients admitted in hospital with history or symtomatology of urinary infection. We noted the following: Urine bacteriological investigation is one of the most asked for laboratory examinations in hospital, having a diagnostic significance (more than 10(5) cfu/ml) in only 15,45% of cases. The etiological patterns, though similar to those registered in ambulatory investigations, present a series of characteristics like: decreased percent of isolation of Enterobacteriaceae - 76.14%, compared to 98.90%, increased proportion of some groups of Gram-negative bacilli highly-resistant to antibiotics, such as Pseudomonas and Acinetobacter and of some groups of Gram-positive cocci--especially group D streptococcus. Accordingly, the sensitivity patterns denote the decrease of the proportion of strains that are sensitive to "regular" antibiotics, this phenomenon implying the use of recent antibiotics, more costly. Once again we point out to the necessity of conducting antibiotherapy by means of evaluating sensitivity during therapy.

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.

Molecular detection of verotoxin-producing Escherichia coli isolates in stool samples from patients with diarrhea.

Despite its occurence as a commensal in the human intestine, Escherichia coli is also known as a versatile gastrointestinal pathogen. Identification of diarrheagenic E. coli (DEC) requires the accurate discrimination of pathogenic strains from commensal flora, and this is not an easy task if the diagnostic tools are inadequate. As the information regarding the relative contribution of DEC among other identifiable causes of infectious diarrhea in Romanian patients is scarce, a prospective study was conducted to evaluate the prevalence of enteropathogenic Escherichia coli (EPEC) and verotoxin-producing Escherichia coli (VTEC) isolates in the diarrheagenic stool specimens of 120 children and 270 adults. PCR-based detection of the eae, bfp, vtx1 and vtx2 genes was added to the conventional culture and slide agglutination with 12 commercial EPEC antisera and O157:H7 antisera for identifying EPEC and VTEC isolates. Even though E. coli colonies belonging to traditional EPEC serogroups were isolated from 35 children and 17 adults, only the isolates recovered from 16 children and 2 adults harboured at least one of the targeted pathogenicity-associated genes. The children shedding EPEC outnumbered the adults (16.7% vs. 7.4%). Based on the virulence genotype identified, the prevalence of atypical EPEC (eae+) was higher than of typical EPEC (eae+ bfpA+), and typical EPEC identification was restricted to children. Owing to the molecular analysis 5 children and 10 adults that could have been overlooked by the routine microbiological investigation were diagnosed as infected with VTEC. Considering that none of the screened stool specimens was positive for E. coli O157:H7, this study reports for the first time the presence of VTEC nonO157 in local patients with diarrhea. Our results bring evidence that both EPEC and VTEC isolates are circulating as agents of local sporadic cases of human diarrhea. Further studies are needed to evaluate the contribution of DEC to the human disease burden in Romania, based on improved diagnostic tools targeting the main virulence traits of E. coli clinical isolates.

Characteristics of Romanian fluoroquinolone-resistant human clinical Escherichia coli isolates.

Alarming progressive increase in the prevalence of antimicrobial resistance in Escherichia coli has been documented worldwide. Previous studies have suggested that many E. coli clinical isolates are actually low-virulence opportunists whose success derives more from antibiotic resistance than from pathogenic capability. The co-existence of ESBL production and fluoroquinolone resistance was reported as a major therapeutic challenge for E. coli infections. Considering the sparse information regarding the genetic background of virulence and antibiotic resistance of local isolates, a collection of ciprofloxacin-resistant E. coli isolates from human extraintestinal specimens was analyzed using PCR, PCR-sequencing, and PFGE, in order to clarify some aspects regarding their mechanisms of antimicrobial resistance, phylogenetic origin, the content of virulence-encoding determinants, and clonal relatedness. The tested fluoroquinolone resistant E. coli (FQREC) isolates, which displayed genetic heterogeneity, carried double mutations in the QRDR of gyrA previously described, which could explain their high resistance to ciprofloxacin. More than half of them (69%) possessed group 1 blaCTX. like genes, and with one exception, all these isolates were ESBL producers. The FQREC isolates belonging to non B2 phylogenetic groups outnumbered the isolates derived from B2 group (60 versus 27 isolates), and their overall content of virulence-encoding genes (fim, pap, sfa/foc, afa, hly, cnf and aer) was reduced. Regardless of the phylogenetic origin, the most prevalent virulence-associated genes possessed by the FQREC isolates were aer and fim determinants, while none of these isolates carried hly and cnf genes. In the case of weakened patients, the E. coli isolates do not need a robust virulence repertoire in order to overcome the host defense systems. The co-resistance of many FQREC isolates to extended-spectrum cephalosporins may provide a substantial advantage to their survival and spreading within the hospital environment.