Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates

Molecular typing of Klebsiella species has become important for monitoring dissemination of β-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)₅- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)₅ and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)₅ and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)₅ and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.     

Polyphasic taxonomic characterization of Lactobacillus rossiae isolates from Belgian and Italian sourdoughs reveals intraspecific heterogeneity

(GTG)₅-PCR fingerprinting and pheS sequence analysis of 18 Lactobacillus rossiae isolates, mainly originating from Belgian and Italian artisan sourdoughs, revealed intraspecies grouping as evidenced by the delineation of three and two subgroups, respectively. On the other hand, 16S rRNA and rpoA gene sequence analysis and DNA–DNA hybridizations supported the accommodation of all isolates in a single species. No correlation between genetic and phenotypic heterogeneity was observed. Collectively, these data do not warrant taxonomic division of L. rossiae. On the other hand, the considerable differences in intraspecies sequence variation of L. rossiae isolates displayed by the pheS (9.8%) and rpoA (1.1%) genes highlight that the discriminatory power of housekeeping genes as alternative genomic markers for the 16S rRNA gene in the identification of Lactobacillus species may significantly differ from gene to gene. In conclusion, this study has demonstrated that a polyphasic approach remains highly useful for identification of isolates belonging to genotypically heterogeneous species such as L. rossiae.     

Differentiation of Fecal <Emphasis Type="Italic">Escherichia coli</Emphasis> from Human,Livestock, and Poultry Sources by rep-PCR DNA Fingerprinting on the Shellfish Culture Area of East China Sea

The rep-PCR DNA fingerprinting performed with REP, BOX A1R, and (GTG)₅ primers was investigated as a way to differentiate between human, livestock, and poultry sources of fecal pollution on the area of Xiangshan Bay, East China Sea. Of the three methods, the BOX-PCR DNA fingerprints analyzed by jack-knife algorithm were revealed high rate of correct classification (RCC) with 91.30, 80.39, 89.39, 86.14, 93.24, 87.72, and 89.28% of human, cattle, swine, chicken, duck, sheep, and goose E. coli isolates classified into the correct host source, respectively. The average rate of correct classification (ARCC) of REP-, BOX-, and (GTG)₅-PCR patterns was 79.88, 88.21, and 86.39%, respectively. Although the highest amount of bands in (GTG)₅-PCR fingerprints could be observed, the discriminatory efficacy of BOX-PCR was superior to both REP- and (GTG)₅-PCR. Moreover, the similarity of 459 isolates originated from shellfish and growing water was compared with fecal-obtained strains. The results showed that 92.4 and 96.2% E. coli strains isolated from midstream and downstream shellfish samples, respectively, had a ≥80% similarity with corresponding strains isolated from fecal samples. It was indicated that E. coli in feces could spread from human sewage or domestic farms to the surrounding shellfish culture water, and potentially affect the quality of shellfish. This work suggests that rep-PCR fingerprinting can be a promising genotypic tool applied in the shellfish growing water management on East China Sea for source identification of fecal pollution.     

Investigation of Mannheimia haemolytica bacteriophages relative to host diversity

Aims

This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle.

Methods and Results

Strictly lytic phages were not detected from bovine nasopharyngeal (n = 689) or water trough (n = 30) samples, but Myoviridae‐ or Siphoviridae‐like phages were induced from 54 of 72 M. haemolytica strains by mitomycin C, occasionally from the same strain. Phages with similar restriction fragment length polymorphism profiles (RFLP ≥70% relatedness) shared common host serotypes 1 or 2 (P < 0·000 1). Likewise, phages with similar RFLP tended to occur in genetically related host bacteria (70–79% similarity). Host range assays showed that seven phages from host serotypes 1, 2 and 6 lysed representative strains of serotypes 1, 2 or 8. The genome of vB_MhM_1152AP from serotype 6 was found to be collinear with P2‐like phage φMhaA1‐PHL101.

Conclusions

Prophages are a significant component of the genome of M. haemolytica and contribute significantly to host diversity. Further characterization of the role of prophage in virulence and persistence of M. haemolytica in cattle could provide insight into approaches to control this potential respiratory pathogen.

Significance and Impact of the Study

This study demonstrated that prophages are widespread within the genome of M. haemolytica isolates and emphasized the challenge of isolating lytic phage as a therapeutic against this pathogen.     

Trinucleotide repeat sequence-based PCR as a potential approach for genotyping Mycobacterium gordonae strains

Diseases that are caused by non-tuberculous mycobacteria (NTM) continue to pose difficult clinical problems, and the epidemiological aspect of NTM-caused diseases is of great importance. In the case of Mycobacterium gordonae there is no adequate genotyping scheme. Here we present a potential rapid and reproducible genetic assay that uses trinucleotide repeat sequence-based PCR (TRS-PCR) for genotyping M. gordonae. The proposed method constitutes a useful single-primer PCR screen for genotyping this species. Among 10 TRS-containing primers, after applying (CAC)₄-based PCR to 36 strains of M. gordonae, we found a discriminatory index of 0.975. The accuracy of this analysis was supported by a reasonable reproducibility of 92%. These results were compared with the Enterobacterial Repetitive Intergenic Consensus Sequences (ERIC)-PCR typing scheme which had lower discriminatory index of 0.93 and its reproducibility was only 86.3%.     

Comparison of five rep-PCR genomic fingerprinting methods for differentiation of fecal Escherichia coli from humans, poultry and wild birds

The development of a methodology to identify the origin of fecal pollution is important both for assessing the degree of risk posed to public health and for developing strategies to mitigate the environmental loading of pathogens associated with waterborne disease transmission. Five rep-PCR genomic fingerprinting methods, such as rep-PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR, ERIC2-PCR, BOX-PCR and (GTG)(5)-PCR, were assessed for their potential in differentiation of 232 fecal Escherichia coli isolates obtained from humans, poultry (chicken, duck and turkey) and wild birds (Canada goose and gull). Based on the results of cluster analysis and discriminant function analysis, (GTG)(5)-PCR was found to be the most suitable method for molecular typing of fecal E. coli, followed by BOX-PCR, REP-PCR, ERIC-PCR and ERIC2-PCR. A discriminant function analysis of (GTG)(5)-PCR fingerprints showed that 94.1%, 79.8%, 80.5%, 74.4%, 86.7% and 88.6% of turkey, chicken, duck, Canada goose, gull and human E. coli isolates were classified into the correct host group, respectively. Subsequently, (GTG)(5)-PCR was tested for its ability to track the origin of 113 environmental E. coli isolated from natural pond water. In conclusion, the (GTG)(5)-PCR genomic fingerprinting method can be considered as a promising genotypic tool for epidemiological surveillance of fecal pollution in aquatic environments.     

Identification of Staphylococcus spp. using (GTG)₅-PCR fingerprinting

A group of 212 type and reference strains deposited in the Czech Collection of Microorganisms (Brno, Czech Republic) and covering 41 Staphylococcus species comprising 21 subspecies was characterised using rep-PCR fingerprinting with the (GTG)₅ primer in order to evaluate this method for identification of staphylococci. All strains were typeable using the (GTG)₅ primer and generated PCR products ranging from 200 to 4500 bp. Numerical analysis of the obtained fingerprints revealed (sub)species-specific clustering corresponding with the taxonomic position of analysed strains. Taxonomic position of selected strains representing the (sub)species that were distributed over multiple rep-PCR clusters was verified and confirmed by the partial rpoB gene sequencing. Staphylococcus caprae, Staphylococcus equorum, Staphylococcus sciuri, Staphylococcus piscifermentans, Staphylococcus xylosus, and Staphylococcus saprophyticus revealed heterogeneous fingerprints and each (sub)species was distributed over several clusters. However, representatives of the remaining Staphylococcus spp. were clearly separated in single (sub)species-specific clusters. These results showed rep-PCR with the (GTG)₅ primer as a fast and reliable method applicable for differentiation and straightforward identification of majority of Staphylococcus spp.     

Development of a Pulsed-Field Gel Electrophoresis (PFGE) method for molecular typing of clinical isolates of Arcanobacterium haemolyticum

We developed a two-block PFGE method to study molecular variation among clinical isolates of Arcanobacterium haemolyticum, an often overlooked human pathogen. Three main macrorestriction profiles were defined among 15 isolates. PFGE was an objective method for characterizing A. haemolyticum and may be useful in molecular epidemiological studies of this organism.     

Genotypic characterization of Enterobacter sakazakii isolates by PFGE, BOX-PCR and sequencing of the fliC gene

Aims:  Enterobacter sakazakii is an emerging food-borne pathogen that can cause rare but severe forms of neonatal meningitis, bacteraemia and necrotizing enterocolitis. A rapid typing method at the strain level is needed to determine the monoclonality or polyclonality of the isolates during outbreaks.
Methods and Results:  The BOX-PCR fingerprinting technique, which targets the repetitive BOX sequences, and sequencing of the flagellin gene, fliC , were evaluated against a panel of 27 Ent. sakazakii strains from clinical and environmental sources. The typeability and discriminatory power of the techniques were compared with those of pulsed-field gel electrophoresis (PFGE), the reference genotyping method. BOX-PCR results yielded 92% agreement with PFGE results, whereas fliC gene sequencing was poorly discriminative.
Conclusions:  In our study, BOX-PCR and PFGE were similarly discriminatory to type Ent. sakazakii strains. The weak variability of the Ent. sakazakii fliC gene was related to the absence of the variable central domain present in most fliC genes of Enterobacteriaceae.
Significance and Impact of the Study:  The BOX-PCR typing provides an accurate discrimination and a rapid answer to identify clonal isolates of Ent. sakazakii .     

<Emphasis Type="Italic">Mannheimia haemolytica</Emphasis> OmpP2-like is an amyloid-like protein,forms filaments,takes part in cell adhesion and is part of biofilms

Mannheimia haemolytica causes respiratory disease in cattle. Amyloid proteins are a major component of biofilms; they aid in adhesion and confer resistance against several environmental insults. The amyloid protein curli is highly resistant to protease digestion and physical and chemical denaturation and binds Congo red (CR) dye. The purpose of this study was to characterize an approximately 50-kDa CR-binding amyloid-like protein (ALP) expressed by M. haemolytica. This protein resisted boiling and formic acid digestion and was recognized by a polyclonal anti-Escherichia coli curli serum, suggesting its relationship with amyloid proteins. Immunolabeling and transmission electron microscopy showed that antibodies bound long, thin fibers attached to the bacterial surface. Mass spectrometry analysis indicated that these fibers are M. haemolytica OmpP2-like proteins. The purified protein formed filaments in vitro, and antiserum against it reacted positively with biofilms. An in silico analysis of its amino acid sequence indicated it has auto-aggregation properties and eight amyloid peptides. Rabbit polyclonal antibodies generated against this ALP diminished the adhesion of ATCC 31612 and BA1 M. haemolytica strains to A549 human epithelial cells, indicating its participation in cell adhesion. ALP expressed by M. haemolytica may be important in its pathogenicity and ability to form biofilms.     

Pseudomonas cichorii as the causal agent of midrib rot,an emerging disease of greenhouse-grown butterhead lettuce in Flanders

Bacterial midrib rot of greenhouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) is an emerging disease in Flanders (Belgium) and fluorescent pseudomonads are suspected to play an important role in the disease. Isolations from infected lettuces, collected from 14 commercial greenhouses in Flanders, yielded 149 isolates that were characterized polyphasically, which included morphological characteristics, pigmentation, pathogenicity tests by both injection and spraying of lettuce, LOPAT characteristics, FAME analysis, BOX-PCR fingerprinting, 16S rRNA and rpoB gene sequencing, as well as DNA–DNA hybridization. Ninety-eight isolates (66%) exhibited a fluorescent pigmentation and were associated with the genus Pseudomonas. Fifty-five of them induced an HR⁺ (hypersensitive reaction in tobacco leaves) response. The other 43 fluorescent isolates were most probably saprophytic bacteria and about half of them were able to cause rot on potato tuber slices. BOX-PCR genomic fingerprinting was used to assess the genetic diversity of the Pseudomonas midrib rot isolates. The delineated BOX-PCR patterns matched quite well with Pseudomonas morphotypes defined on the basis of colony appearance and variation in fluorescent pigmentation. 16S rRNA and rpoB gene sequence analyses allowed most of the fluorescent isolates to be allocated to Pseudomonas, and they belonged to either the Pseudomonas fluorescens group, Pseudomonas putida group, or the Pseudomonas cichorii/syringae group. In particular, the isolates allocated to this latter group constituted the vast majority of HR⁺ isolates and were identified as P. cichorii by DNA–DNA hybridization. They were demonstrated by spray-inoculation tests on greenhouse-grown lettuce to induce the midrib rot disease and could be re-isolated from lesions of inoculated plants. Four HR⁺ non-fluorescent isolates associated with one sample that showed an atypical midrib rot were identified as Dickeya sp.     

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

Introduction

Acquired AmpC enzymes, classified as miscellaneous extended-spectrum β-lactamase (ESBL_M) enzymes according to a recently proposed β-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBL_M are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla_AmpC detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes.

Material and methods

Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/− cloxacillin at Malmö University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla_AmpC real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing.

Results and discussion

The real-time PCR assay was able to detect and sub-classify all acquired bla_AmpC genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla_AmpC real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated.     

A Comparison of BOX-PCR and Pulsed-Field Gel Electrophoresis to Determine Genetic Relatedness of Enterococci from Different Environments

Genetic relatedness of enterococci from poultry litter to enterococci from nearby surface water and groundwater in the Lower Fraser Valley regions of British Columbia, Canada was determined. A new automated BOX-PCR and Pulsed-Field Gel Electrophoresis (PFGE) were used to subtype enterococcal isolates from broiler and layer litter and surface and groundwater. All surface water samples (n = 12) were positive for enterococci, as were 11% (3/28) of groundwater samples. Enterococcus faecium (n = 90) was isolated from all sources, while Enterococcus faecalis (n = 59) was isolated from all sources except layer litter. The majority of E. faecalis originated from broiler litter (28/59; 47.5%) while the majority of E. faecium were isolated from layer litter (29/90; 32.2%). E. faecalis grouped primarily by source using BOX-PCR. Isolates from water samples were dispersed more frequently among PFGE groups containing isolates from poultry litter. E. faecium strains were genetically diverse as overall clustering was independent of source by both molecular methods. Subgroups of E. faecium isolates based upon source (layer litter) were present in BOX-PCR groups. Three individual E. faecalis groups and two individual E. faecium groups were 100% similar using BOX-PCR; only one instance of 100% similarity among isolates using PFGE was observed. Although enterococci from litter and water sources were grouped together using BOX-PCR and PFGE, isolates originating from water could not be definitively identified as originating from poultry litter. Automation of BOX-PCR amplicon separation and visualization increased the reproducibility and standardization of subtyping using this procedure.     

PCR-Based Detection of Extended-Spectrum β-Lactamases (bla CTX-M-1 and bla TEM ) in Escherichia coli, Salmonella spp. and Klebsiella pneumoniae Isolated from Pigs in North Eastern India (Mizoram)

Cephalosporins are major antimicrobials used to treat serious infections. However, their effectiveness is being compromised by the emergence of extended-spectrum β-lactamases (ESBLs). A total of 138 enteric bacteria were isolated from 53 faecal samples of pigs collected from different districts of Mizoram, of which 102 (73.91 %) were Escherichia coli, 26 (18.84 %) were Salmonella spp. and 10 (7.25 %) were Klebsiella pneumoniae. Phenotypic confirmatory test (Double Discs Synergy Test) showed that 8 (5.80 %) E. coli isolates were ESBLs producer. PCR analysis confirmed that out of the eight isolate, 7 (5.07 %) harboured bla _CTX-M-1 gene and/or bla _TEM gene. Of the eight positive isolates, 7 (5.07 %) and 3 (2.17 %) were found to be positive for bla _CTX-M-1 gene and bla _TEM gene, respectively, of which 3 (2.17 %) isolates were positive for both the genes. Only 4 (2.90 %) E. coli isolates carried bla _CTX-M-1 gene alone. Agarose gel electrophoresis showed that all the isolates were carrying plasmids ranging between 0.9 and ~30 kb. Out of the seven isolates positive for bla _CTX-M-1 and/or bla _TEM , 2 (1.84 %) isolates were confirmed for bla _CTX-M-1 gene in their plasmid. Only one E. coli isolate was found to be positive for both the genes in its plasmid. The resistance plasmid could not be transferred to a recipient by in vitro horizontal gene transfer method.     

Photobacterium piscicola sp. nov., isolated from marine fish and spoiled packed cod

Five isolates from marine fish (W3^T, WM, W1S, S2 and S3) and three isolates misclassified as Photobacterium phosphoreum, originating from spoiled modified atmosphere packed stored cod (NCIMB 13482 and NCIMB 13483) and the intestine of skate (NCIMB 192), were subjected to a polyphasic taxonomic study. Phylogenetic analysis of 16S rRNA gene sequences showed that the isolates were members of the genus Photobacterium. Sequence analysis using the gapA, gyrB, pyrH, recA and rpoA loci showed that these isolates formed a distinct branch in the genus Photobacterium, and were most closely related to Photobacterium aquimaris, Photobacterium kishitanii, Photobacterium phosphoreum and Photobacterium iliopiscarium. The luxA gene was present in isolates W3^T, WM, W1S, S2 and S3 but not in NCIMB 13482, NCIMB 13483 and NCIMB 192. AFLP and (GTG)₅-PCR fingerprinting indicated that the eight isolates represented at least five distinct genotypes. DNA–DNA hybridizations revealed 89% relatedness between isolate W3^T and NCIMB 192, and values below 70% with the type strains of the phylogenetically closest species, P. iliopiscarium LMG 19543^T, P. kishitanii LMG 23890^T, P. aquimaris LMG 26951^T and P. phosphoreum LMG4233^T. The strains of this new taxon could also be distinguished from the latter species by phenotypic characteristics. Therefore, we propose to classify this new taxon as Photobacterium piscicola sp. nov., with W3^T (=NCCB 100098^T = LMG 27681^T) as the type strain.     

Typing of Campylobacter jejuni and Campylobacter coli isolated from live broilers and retail broiler meat by flaA-RFLP, MLST, PFGE and REP-PCR

We analyzed 100 Campylobacter spp. isolates (C. jejuni and C. coli) from Grenada, Puerto Rico and Alabama, which were collected from live broilers or retail broiler meat. We analyzed these isolates with four molecular typing methods: restriction fragment length polymorphism of the flaA gene (flaA-RFLP), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and automated repetitive extragenic palindromic polymerase chain reaction (REP-PCR) using the DiversiLab system. All methods performed similarly for the typing of C. jejuni and C. coli. The DNA extraction method appears to influence the results obtained with REP-PCR. This method was better for the typing of C. jejuni than C. coli, however both REP-PCR and flaA-RFLP generated types that were indistinguishable between C. jejuni and C. coli and appeared to be random, without any relationship to species, location, or source of isolates. PFGE and MLST generated typing results that had a better correlation with the geographic location of the isolates and showed higher concordance with the Wallace coefficient. The adjusted Rand coefficient did not show higher concordance among the methods, although the PFGE/MLST combination exhibited the highest concordance. PFGE and MLST revealed a better discriminatory power for C. coli isolates than REP-PCR or flaA-RFLP. The use of readily available online tools to calculate the confidence interval of the Simpson's index of diversity and the adjusted Rand and Wallace coefficients helped estimate the discriminatory power of typing methods. Further studies using different C. jejuni and C. coli strains may expand our understanding of the benefits and limitations of each of these typing methods for epidemiological studies of Campylobacter spp.     

Staphylococcus petrasii sp. nov. including S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov., isolated from human clinical specimens and human ear infections

Thirteen coagulase-negative, oxidase-negative, and novobiocin-susceptible staphylococci were isolated from human clinical specimens. The isolates were differentiated from known staphylococcal species on the basis of 16S rRNA, hsp60, rpoB, dnaJ, tuf, and gap gene sequencing, automated ribotyping, (GTG)₅-PCR fingerprinting, and MALDI-TOF MS analysis. Phylogenetic analysis based on the 16S rRNA gene sequence indicated phylogenetic relatedness of the analyzed strains to Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus devriesei, and Staphylococcus lugdunensis. DNA–DNA hybridization experiments between representative strains CCM 8418^T, CCM 8421^T, and the closest phylogenetic neighbors confirmed that the isolates represent novel Staphylococcus species, for which the name Staphylococcus petrasii sp. nov. is proposed. Genotypic and phenotypic analyses unambiguously split the strains into two closely related subclusters. Based on the results, two novel subspecies S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov. are proposed, with type strains CCM 8418^T (=CCUG 62727^T) and CCM 8421^T (=CCUG 62728^T), respectively.     

The DiversiLab system versus pulsed-field gel electrophoresis: Characterisation of extended spectrum β-lactamase producing Escherichia coli and Klebsiella pneumoniae

Fast and reliable epidemiological typing methods for identifying outbreaks and epidemic strains of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae are urgently needed. The DiversiLab system (DL) has been proposed for these purposes. We compared DL to pulsed-field gel electrophoresis (PFGE) on a national collection of ESBL-producing Escherichia coli (n = 258; of which 226 isolates were typeable with PFGE) and Klebsiella pneumoniae (n = 48) isolated in 2007. For E. coli the Wallace coefficients showed that the probability of two isolates of the same DL type having the same PFGE type was only 19.8% and the probability of two isolates of the same PFGE type having the same DL type was 90.4%. For K. pneumoniae the Wallace coefficients showed that the probability of two isolates of the same DL type having the same PFGE type was 100% and the probability of two isolates of the same PFGE type having the same DL type was 79%, indicating that for this K. pneumoniae strain collection DL was slightly more discriminatory. Only four of 48 isolates had discordant results with the two methods. In E. coli 42% of the isolates were sequence type 131 and these isolates were related at > 95% similarity with DL and at ≥ 60% similarity with PFGE. In summary, for E. coli DL performed well in identifying isolates related by PFGE, but overestimated the genetic relatedness in the studied collection. This indicates that DL could be a primary screening method for excluding unrelated isolates. Isolates shown to be related must be confirmed with a more discriminatory method. For K. pneumoniae, DL discriminated well but overestimated the diversity of the isolates compared to PFGE, assuming a risk of missing possible genetic relatedness.