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.

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 This is a preliminary study intended to detect ST131 among 87 clinical isolates retrieved from a larger and unpublished E. coli collection.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.

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.

[Trends of antimicrobial resistance in microbial strains isolated from invasive infections in Romania. Experience of participation in EARSS (European Antimicrobial Resistance Surveillance System), 2002-2008]. / Tendintele rezistentei la antibiotice a izolatelor din infectii invazive în România. Experienta participarii în Sistemul European de Supraveghere a Rezistentei la Antibiotice (EARSS) în perioada 2002-2008.

EARSS (European Antimicrobial Resistance Surveillance System) is the biggest antimicrobial resistance surveillance project in the world financed from public finds, aiming to provide validated and comparable official data on antimicrobial resistance of invasive microbial strains (isolated from blood and CSF), belonging to 6 indicator bacterial species, i.e.: S. aureus, E. coli, E. faecium/faecalis, Str. pneumoniae, Ps. aeruginosa, K. pneumoniae. Romania reported data to EARSS since 2002 so far. Though the number of participating laboratories increased progressively from 12 to 35, the number of hospitals which reported for EARSS. as the number of strains included in the data base remained steady and relatively low. This issue is related to the particular position of Romania in the European context, in respect of the very low number of blood cultures performed in hospitals. Our paper is presenting the trends of antimicrobial resistance in the indicator strains in the 2002-2008 interval. During the 2002-2008 interval, Romania reported to EARSS a total number of 1276 bacterial strains, distributed by species as follows: 513 S aureus, 369 E. coli, 128 Streptococcus pneumoniae, 127 Enterococcus spp.. 71 Klebsiella pneumoniae, 68 Pseudomonas aeruginosa. Klebsiella pneumoniae and Pseudomonas aeruginosa were reported, according to the EARSS protocol, only for the 2005-2008 interval. It is difficult to describe trends, specially in Enterococcus, Streptococcus pneumonaie and the 2 species collected only since 2005, because of the low number of isolates, but there are several results that are supporting us to claim that antimicrobial resistance in invasive isolates is a real problem in Romanian hospitals, like in other Central, Southern and South Eastern European countries: more than 25% of S. aureus strains resistant to methicilline, with more than 50% in some years, high aminoglycozides resistance in more than 70-80% of Enterococcus faecium invasive strains, more than 80% of strains resistant to 3rd generation cephalosporines etc.

[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.

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.

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.

Virulence genotypes of Escherichia coli strains from bacteremia in relation to phylogeny.

Bacteremia is the principal way of dissemination of local infections to distant organs. Escherichia coli bacteremia is almost always clinically significant, suggesting an increased risk of developing sepsis syndrome. Fifty-one E. coli bloodstream human isolates were analyzed using PCR technique for several molecular markers associated with extraintestinal virulence, and their phylogenetic group assignment, taking into account the link between the phylogenetic background and the intrinsic virulence of this species. Sixteen virulence genotypes have been identified, the majority of the blood isolates carrying the association of two genes. The genes encoding type 1 fimbria and aerobactin had the highest prevalence. As a confirmation of other studies, the strains assigned to E. coli phylogenetic group B2 exhibited the highest concentration of virulence genes, and represented almost half of the clinical blood isolates. The multifactorial virulence of E. coli strains isolated from invasive infections reflects a phylogenetic inheritance, and supports the concept of ExPEC pathotype as a subset of E. coli population involved in human infectious diseases. The surveillance of geographical variation of E. coli pathogenic clones is useful for epidemiological analysis.

Incidence of virulence-encoding genes among enteric Escherichia coli strains isolated from healthy subjects.

Escherichia coli, heterogeneous species consisting of commensal and pathogenic strains, is causing a broad spectrum of intestinal and extra intestinal diseases, ranging from asymptomatic infections to septicaemia, according to its capacity to produce different virulence factors. The incidence of different virulence-associated genes among the strains isolated from healthy subjects, taking into account that the human gastrointestinal tract is considered an important source for spreading E. coli strains, was evaluated. A total of 241 E. coli strains isolated from 41 healthy subjects, working in the food chain and coming to the laboratory for periodical medical control, were investigated for harbouring patogenicity factors--encoding genes. Extra intestinal virulence-associated genes, pap, sfa/foc, afa, hly, cnf and intestinal ones eaea, bfp, agg, It, st, vtx1 (stx1), vtx2 (stx2) and ipaH, were targeted by PCR using cellular lysate for total DNA. Genes encoding for adherence were the most prevalent. A number of 67 strains (27.80%) were positive for pap genes and 34 strains (14.11%) presented PCR positive results when afa genes were targeted, but sfa/foc genes were identified in only 10 strains (4.15%). Genes encoding for toxigenesis were less prevalent. A total of 9 strains amplified hly genes, 2.49% were positive for cnf genes and only 2 strains presented vtx1(stx1) gene. The results are in concordance with those which demonstrate that healthy subjects carrying strains possessing virulence-encoding genes could represent a reservoir for environmental circulation of such strains, considered life-threatening when a receptive host is encountered.

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.

Comparison of genomic profiles of Escherichia coli isolates from urinary tract infections.

Formally included in the larger category of extraintestinal pathogenic Escherichia coli (ExPEC), the uropathogenic E. coli remains the most frequent cause of urinary tract infection (UTI), an important endemic health problem. The genomic DNA of E. coli urinary isolates from adults diagnosed with urinary tract infections and of E. coli fecal isolates from healthy subjects was analysed by PCR for the presence of virulence factor encoding genes pap, sfa/foc, afa, hly and cnf and by field inversion gel electrophoresis (FIGE) fingerprinting of XbaI DNA macrorestriction fragments. The aim was to obtain more detailed microbiological data regarding the community circulating strains in respect of their virulence potential and genetic relatedness. Almost 70% of the urinary strains carried at least one of the target virulence genes, and only 35.5% of the fecal E. coli strains were positive in the PCR screening. Taking into account the virulence genotypes exhibited, a part of the strains isolated from the urinary tract could be defined as belonging to the ExPEC pathotype. A unique FIGE profile was obtained for each of the selected isolates and the dendrogram generated by Taxotron software package analysis suggested a polyclonal population of potential uropathogenic strains clustered into 14 groups of only 60% similarity. For better understanding the epidemiology of UTIs, diseases commonly caused by such a heterogeneous species like E. coli, molecular analysis methods could be essential due to their increased power of identification and fingerprinting.