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Poland's first Enterobacteriaceae isolate producing the New Delhi metallo-β-lactamase (NDM) was identified in August 2011. Escherichia coli sequence type ST410 NDM-1 was cultured from a critically ill patient who had been transferred directly from the Congo. The bla NDM-1 gene was carried by conjugative IncFII-type plasmid pMC-NDM (87,619 bp), which showed structural similarity to plasmid pGUE-NDM, which was identified earlier in France in an E. coli ST131 isolate of Indian origin.

CTX-M-producing Escherichia coli isolates from three Croatian hospitals were analyzed. All bla CTX-M-15 genes and one bla CTX-M-3a gene resided in widely spread IS Ecp1 transposition modules, but other bla CTX-M-3a genes were in a new configuration with two IS 26 copies, indicating a new event of gene mobilization from a Kluyvera ascorbata genome. The study confirmed the role of the E. coli ST131 clonal group with IncFII-type plasmids in the spread of bla CTX-M-15 and of IncL/M pCTX-M3-type plasmids in the dissemination of bla CTX-M-3a .

Penicillin-binding proteins (PBPs) in representatives of two Streptococcus pneumoniae clonal groups that are prevalent in Poland, Poland 23F -16 and Poland 6B -20, were investigated by PBP profile analysis, antibody reactivity pattern analysis, and DNA sequence analysis of the transpeptidase (TP) domain-encoding regions of the pbp2x , pbp2b , and pbp1a genes. The isolates differed in their MICs of β-lactam antibiotics. The majority of the 6B isolates were intermediately susceptible to penicillin (penicillin MICs, 0.12 to 0.5 μg/ml), whereas all 23F isolates were penicillin resistant (MICs, ≥2 μg/ml). The 6B isolates investigated had the same sequence type (ST), determined by multilocus sequence typing, as the Poland 6B -20 reference strain (ST315), but in the 23F group, isolates with three distinct single-locus variants (SLVs) in the ddl gene (ST173, ST272, and ST1506) were included. None of the isolates showed an identical PBP profile after labeling with Bocillin FL and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and only one pair of 6B isolates and one pair of 23F isolates (ST173 and ST272) each contained an identical combination of PBP 2x, PBP 2b, and PBP 1a TP domains. Some 23F isolates contained PBP 3 with an apparently higher electrophoretic mobility, and this feature also did not correlate with their STs. The data document a highly variable pool of PBP genes as a result of multiple gene transfer and recombination events within and between different clonal groups.

This study presents the results of a survey of the in-vitro susceptibility to antimicrobial agents of major pathogens responsible for community-acquired respiratory tract infections in Poland during 2002-2004. The collection of 1184 bacterial isolates comprised 398 Streptococcus pneumoniae, 344 Haemophilus influenzae, 302 Streptococcus pyogenes and 140 Moraxella catarrhalis. Among the pneumococcal isolates, 16.8% were penicillin-non-susceptible (PNSP), of which 80.6% were identified as multidrug-resistant. Overall, 9.0% of H. influenzae isolates were beta-lactamase-positive, although this percentage increased noticeably in the third year of the study. Based on PCR results, 12.8% of H. influenzae isolates were identified as low-level beta-lactamase-negative, ampicillin-resistant (BLNAR), and one isolate as low-level beta-lactamase-positive, amoxycillin-clavulanic acid-resistant (BLPACR). Pulsed-field gel electrophoresis (PFGE) classified 45 H. influenzae isolates with altered penicillin-binding proteins into 15 PFGE types, including two predominant types (with four and six sub-types) containing 15 and ten isolates, respectively. Resistance to tetracycline, erythromycin and clindamycin was found in 20.9%, 8.9% and 4.6% of S. pyogenes isolates, respectively. The production of beta-lactamase characterised 91.4% of M. catarrhalis isolates. In summary, the overall occurrence of PNSP in Poland remains stable, although there was a noticeable increase in the proportion of fully-resistant isolates. A rising trend in the prevalence of beta-lactamase producers and low-level BLNAR isolates was observed among Polish isolates of H. influenzae.

Objectives To perform the first multinational Enterobacter cloacae clonality study, using the MLST scheme newly developed in Japan. Methods The analysis included 195 rectal carriage E. cloacae isolates resistant to expanded-spectrum cephalosporins (ESCs), collected from patients in 12 hospital units across Europe and Israel. All of the isolates were typed by PFGE and 173 isolates were subjected to MLST. ESC resistance was analysed phenotypically; genes encoding ESBLs and carbapenemases were identified by PCR and sequencing. Results MLST distinguished 88 STs, which correlated with the PFGE data. PFGE was more discriminatory, producing 129 pulsotypes (169 patterns). Numerous STs were observed in several countries each. The most widespread were ST66, ST78, ST108 and ST114, each having at least 10 isolates from three to five countries, diversified into multiple pulsotypes, with clusters of related isolates in one or more centres. Analysis of the STs against the MLST database revealed several epidemic clonal complexes, such as those with central genotypes ST74 (including ST78) or ST114 (including ST66). ESC resistance was equally related to overexpression of the AmpC cephalosporinase and to ESBL production. Among ESBL producers some spreading subclones were identified, including specific ST66, ST78 and ST114 pulsotypes, associated with CTX-M-15 production. Several isolates produced carbapenemase VIM-1 or KPC-2. Conclusions Together with the information available in the MLST database, our results suggest that, like Escherichia coli and Klebsiella pneumoniae, E. cloacae harbours clonal lineages of increased epidemic potential that may be associated with resistance spread.

Meningitis caused by Streptococcus pneumoniae represents an important factor of morbidity and mortality in humans. In a significant number of cases, this disease is associated with specific clones of the organism, the so-called invasive pneumococcal clones. The aim of the study was to analyze 156 S. pneumoniae isolates identified as etiological agents of meningitis in Poland in the years 1997 through 2002. The isolates were characterized by multilocus sequence typing (MLST), and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE) and with the MLST data on invasive pneumococci from other countries. Eighty-nine different sequence types were found in the group of isolates, 50 of which had been known before including 19 of the major invasive clones. However, a significant fraction of the isolates possessed novel combinations of known and new MLST alleles. The majority of penicillin-nonsusceptible isolates belonged to the group of international multiresistant clones (Spain 23F -1, Spain 6B -2, Spain 9V -3, Poland 23F -16, and Poland 6B -20), which underlined the importance of these in the dissemination of antimicrobial resistance. The results of the MLST analysis correlated well with the PFGE data, thus again demonstrating good congruence between the two typing methods for S. pneumoniae . The majority of the isolates (95.5%) belonged to families 1 or 2 of the surface protein PspA, confirming its potential usefulness as the vaccine antigen candidate.

Nasopharyngeal carriage of Streptococcus pneumoniae , Haemophilus influenzae , and Moraxella catarrhalis in 226 children in different settings (in a crèche [day care center], in an orphanage, and at home) during two seasons (winter and spring) was studied. The rates of carriage of S. pneumoniae and H. influenzae were markedly higher in the crèche and in the orphanage than in the home setting (e.g., 56.5, 63.3, and 25.9%, respectively, for S. pneumoniae in winter). Approximately 80% of the S. pneumoniae isolates identified in the crèche and in the orphanage belonged to the serotypes represented in the seven-valent pneumococcal vaccine, and 4.4% of the children were colonized by H. influenzae type b. Almost all H. influenzae isolates were fully susceptible to the antimicrobial agents tested, and only five (3.6%) produced β-lactamase; in contrast, 100% of the M. catarrhalis isolates were β-lactamase positive. Among S. pneumoniae isolates, 36.2% were nonsusceptible to penicillin (PNSP) and 11.8% were fully resistant to penicillin (PRP). All PNSP isolates were obtained from children at the crèche and at the orphanage but not among children brought up at home, and all PRP isolates showed a multiresistant phenotype. Colonization by PRP isolates correlated well with prior treatment with β-lactams. For the majority of children colonized at both sampling times, strain replacement of S. pneumoniae and H. influenzae was observed; long-term colonization by a single strain was rare.

Background: Lyme disease is a multisystem, multistage infection caused by three genospecies of the Borrelia burgdorferi sensu lato species. The diagnosis of Lyme disease is based on a history of tick-bite, physical examination, and serological tests. In the seronegative patients with Lyme borreliosis symptoms, additional testing should be introduced. Methods: The study group was composed of 240 hospitalized patients presented with various clinical symptoms suggesting Lyme borreliosis: 221 of the patients with neurological abnormalities and 19 with oligoarticular arthritis. Citrated blood and serum samples were collected from the patients for culture and serological examination, respectively. Moreover, 173 cerebrospinal and 6 synovial fluid samples were tested. New oligonucleotide primers based on B. burgdorferi sensu lato 16SrRNA gene sequences were designed for the detection of the bacteria in blood, cerebrospinal, and synovial fluid specimens with PCR. Levels of specific antibodies were measured in serum, cerebrospinal fluid and synovial fluid samples using ELISA and Western blot. B. burgdorferi spirochetes from blood, cerebrospinal fluid, and synovial fluid samples were cultured in cell line. Extracted and purified B. burgdorferi DNA was identified by PCR with new oligonucleotide primers. Then three genospecies were identified by PCR amplification with other primer sets specific for 16S rDNA and/or by the restriction fragment length polymorphism of 23S(rrl)-5S(rrf). Results: Bacterial DNA were found in samples from 32 patients, including 28 patients with neuroborreliosis and 4 with Lyme arthritis. B. burgdorferi-sipecific IgM and/or IgG serum antibodies were detected in 14 of these patients. Fourteen strains of Borrelia garini, 4 strains of Borrelia afzelii and 1 strain of B. burgdorferi sensu stricto were identified by PCR. Genospecies were not recognized in 13 specimens. Conclusions: The procedure can be a rapid and sensitive diagnostic method for the detection of etiological agents in clinical materials derived from patients with the clinical symptoms of Lyme borreliosis. It can be utilized for both basic research as well as routine laboratory diagnosis.

The CTX-M-15 extended-spectrum beta-lactamase (ESBL) was recently identified in Enterobacteriaceae isolates in India, and demonstrated significant hydrolytic activity against ceftazidime, in contrast to the majority of CTX-M enzymes. CTX-M-15 differs from CTX-M-3, which is one of the most prevalent ESBLs in Poland, by only a single amino acid change (Asp-240-->Gly). Three cefotaxime- and ceftazidime-resistant Enterobacteriaceae isolates, recovered during 1998-2000 in two Polish hospitals, were found to produce CTX-M-15. Similar to those from India, the isolates contained the ISEcp1 insertion sequence located upstream of the bla(CTX-M-15) gene, which has been recently demonstrated to mobilize 3'-adjacent genes to transfer between DNA replicons. However, its different position with respect to the beta-lactamase gene indicated the independent selection of the ESBL gene in the two countries.

Twenty-two Klebsiella pneumoniae and two K. oxytoca extended-spectrum β-lactamase (ESBL)-producing isolates were collected in 1996 from patients in two pediatric wards of the University Hospital in Wrocław, Poland. Molecular typing has revealed that the K. pneumoniae isolates represented four different epidemic strains. Three kinds of enzymes with ESBL activity (pI values of 5.7, 6.0, and 8.2) were identified. The pI 6.0 β-lactamases belonged to the TEM family, and sequencing of the bla TEM genes amplified from representative isolates revealed that these enzymes were TEM-47, previously identified in K. pneumoniae isolates from pediatric hospitals in Łódź and Warsaw. One of the TEM-47-producing strains from Wrocław was very closely related to the isolates from the other cities, and this indicated countrywide spread of the epidemic strain. The pI 5.7 β-lactamase was produced by a single K. pneumoniae isolate for which, apart from oxyimino-β-lactams, the MICs of β-lactam–inhibitor combinations were also remarkably high. Sequencing revealed that this was a novel TEM β-lactamase variant, TEM-68, specified by the following combination of mutations: Gly238Ser, Glu240Lys, Thr265Met, and Arg275Leu. The new enzyme has most probably evolved from TEM-47 by acquiring the single substitution of Arg275, which before was identified only twice in enzymes with inhibitor resistance (IR) activity. TEM-68 was shown to be a novel complex mutant TEM β-lactamase (CMT-2) which combines strong ESBL activity with relatively weak IR activity and, when expressed in K. pneumoniae , is able to confer high-level resistance to a wide variety of β-lactams, including inhibitor combinations. This data confirms the role of the Arg275Leu mutation in determining IR activity and documents the first isolation of K. pneumoniae producing the complex mutant enzyme.

The study aims were: (i) to define the prevalence of and risk factors for colonization by extended spectrum β-lactamase (ESBL) -producing Enterobacteriaceae (EPE) among healthcare workers (HCWs) and family members (FMs) of EPE-colonized patients in rehabilitation units and (ii) to compare EPE isolates from these three groups. The study included 286 FMs of 194 EPE-carrying patients identified in five rehabilitation units located in Israel, Italy, France and Spain. The EPE were detected in rectal swabs from 26 (9%) of 286 FMs screened. In multivariate analyses, older age of FM, greater mean number of hours spent with the patient, being a daughter or a female spouse of a patient, and chronic lung disease of the patient were significantly associated with carriage in the FM. Escherichia coli was the most common organism (76%), followed by Klebsiella pneumoniae (19%). Isolates were typed by pulsed field gel electrophoresis and multilocus sequence typing, and ESBLs were identified by PCR sequencing. A comparison of paired species isolates from FMs and their respective patient showed that 17 of 23 strains were indistinguishable. EPE were detected in 35 (3.5%, E. coli = 34) of the 1001 HCWs screened. Feeding patients was associated with EPE carriage by HCWs. Only 7 of 23 E. coli subclones cultured from HCWs were also represented among 376 patient-derived ESBL-producing E. coli isolates from the same rehabilitation units. In Spain, a higher proportion of HCWs and FMs were ESBL carriers than elsewhere (p

Acinetobacter baumannii is currently one of the key nosocomial pathogens causing severe infections; of special concern is its resistance to expanded-spectrum cephalosporins (ESCs) and carbapenems, often associated with the few so-called European clones ([6][1], [7][2], [19][3]). It has two natural

Increasing resistance due to the production of ESBL in Escherichia coli (ESBL-E. coli) has become a major threat to public health. Our aims were to study the incidence of ESBL-E. coli acquisition during hospitalization and the transmission rates of different ESBL-E. coli clones. This was a prospective case-control study, conducted in two geriatric rehabilitation wards in Tel-Aviv. Serial rectal cultures were collected from admission till discharge. All patient-unique ESBL-E. coli isolates were subjected to molecular typing by PFGE, MLST and determination of ESBL genes. An acquisition of ESBL-E. coli was defined as traceable when a patient with the same ST, PFGE type and ESBL gene was hospitalized in the same ward in parallel to the acquisition case. ESBL-E. colis were recovered from 125 patients out of 492 enrolled: 52 were recovered upon admission, 59 acquired ESBL-E. coli during their stay, and there was undetermined status in 14 patients. A low Norton's score was associated with acquisition (O.R. 1.14 for each point, 95% C.I. 1.01–1.29, p < 0.05). ESBL-E. coli infections (n = 9) had occurred only in ESBL-E. coli carriers. The pandemic ST131 clone was the most common (48/125). The majority of the isolates (101/125) produced CTX-M-type ESBL. Of the 59 acquisition cases, 32 were traced to another patient. In-hospital dissemination was highest in the CTX-M-27-producing ST131 and the SHV-5-producing ST372 sub-clones (acquisition/admission ratios of 17/11 and 9/3, respectively), with almost all cases traced to other patients. In conclusion, most ESBL-E. coli acquisition cases were traceable to other patients. The transmission potential varied significantly between ESBL-E. coli clones.

After the first report in May 2008, the National Reference Center for Susceptibility Testing confirmed 113 cases of infection or colonization by KPC-producing members of the family Enterobacteriaceae in Poland by the end of 2009. The vast majority of patients were found in 18 hospitals; three patients were diagnosed at outpatient clinics. Most of the institutions were in the Warsaw area, including three hospitals with the highest numbers of cases. When available, the data on previous hospitalizations often indicated that these hospitals were the probable acquisition sites; one patient arrived from New York. The group of 119 unique isolates consisted of Klebsiella pneumoniae ( n = 114), followed by Klebsiella oxytoca ( n = 3), and Escherichia coli ( n = 2). The K. pneumoniae isolates were dominated by the clone sequence type 258 (ST258) ( n = 111); others were ST11 and ST23. The ST258 group was heterogeneous, with 28 pulsed-field gel electrophoresis (PFGE) subtypes, ∼25 plasmid profiles, and nine β-lactamase patterns differing by KPC variants (KPC-2 mainly), and SHV-12, CTX-M-3, and TEM-1-like enzymes. Plasmids carrying bla KPC genes varied in size (∼48 to 250 kb), structure, and conjugation potential. Transferable IncFII K plasmids of ∼110 to 160 kb, probably pKpQIL or its derivatives, were observed in all K. pneumoniae clones and in K. oxytoca . Also prevalent were nontypeable pETKp50-like plasmids of ∼50 kb, found in K. pneumoniae ST258 and E. coli isolates (ST93 and ST224). Two K. pneumoniae-E. coli pairs from single patients might represent the in vivo transfer of such plasmids. The striking diversity of KPC producers at the early stage of dissemination could result from several introductions of these bacteria into the country, their multidirectional evolution during clonal spread, and transfer of the plasmids.

Plasmidic KPC β-lactamases in gram-negative pathogens are of the highest clinical and epidemiologic concern, conferring resistance to all β-lactams, including carbapenems (15, 18). The main KPC producer is Klebsiella pneumoniae, but other species are being identified as well (15). Strains with KPCs spread rapidly and cause outbreaks; in hospitals on the east coast of the United States and in Israel, they have already become endemic (2, 3, 10, 14, 17). Recently, more countries have reported the presence of these organisms, in some cases as a result of importation (15).

The frequency of tetracycline resistance in Streptococcus pneumoniae isolates in Poland is one of the highest in Europe. The aim of this study was to analyze the clonal diversity and resistance determinants of tetracycline-nonsusceptible S. pneumoniae isolates identified in Poland and to investigate the effect of tetracycline resistance on their susceptibilities to tigecycline, doxycycline, and minocycline. We have analyzed 866 pneumococcal isolates collected from 1998 to 2003 from patients with respiratory tract diseases, and 242 of these (27.9%) were found to be resistant to tetracycline. All of the resistant isolates were characterized by testing of their susceptibilities to other antimicrobials, serotyping, pulsed-field gel electrophoresis (PFGE), and identification of tetracycline resistance genes and transposons. Selected isolates representing the main PFGE types were analyzed by multilocus sequence typing. Among the isolates investigated, 27 serotypes and 146 various PFGE patterns, grouped into 90 types, were discerned. The most common PFGE type, corresponding to serotype 19F and sequence type 423, was represented by 22.3% of all of the tetracycline-resistant isolates. The tet (M) gene was the sole resistance gene in the group of isolates studied, and in over 96% of the isolates, the Tn 916 family of tet (M)-containing conjugative transposons was detected. Several isolates contained specific variants of the transposons, the Tn 1545 -like, Tn 3872 -like, or Tn 2009 -like element. The correlation between the MICs of tetracycline, doxycycline, and minocycline was revealed, whereas no cross-resistance to tetracycline and tigecycline was observed.

We have analyzed 40 metallo-β-lactamase (MBL)-producing isolates of Pseudomonas aeruginosa ( n = 38), Pseudomonas putida ( n = 1), and Acinetobacter genospecies 3 ( n = 1) from 17 hospitals in 12 cities in Poland that were identified in 2000 to 2004. Pulsed field gel electrophoresis typing classified the P. aeruginosa isolates into eight types, with two types differentiated further into subtypes. Each of the types was specific either to a given center or to several hospitals of the same or neighboring geographic area. Almost all of the organisms produced β-lactamase VIM-2; the only exceptions were several P. aeruginosa isolates from two centers which expressed VIM-4. The bla VIM genes resided exclusively within class 1 integrons, and these were located in either chromosomal or plasmid DNA. PCR-restriction fragment length polymorphism study of the variable regions of the integrons, followed by DNA sequencing, revealed the presence of eight different, mostly novel gene cassette arrays, six of which contained bla VIM-2 and two of which contained bla VIM-4 . The occurrence of the integron variants correlated well with the geographic distribution of the MBL-producing organisms, and this suggested that their emergence in particular parts of the country had been likely due to a number of independent events. The following regional dissemination of MBL producers could be attributed to various phenomena, including their clonal spread, horizontal transmission of resistance determinants, or both. All of the data collected in this study revealed that even at this early stage of detection, the epidemiological situation concerning MBL producers in Poland has already been complex and very dynamic.

The reproducibility of the binary typing (BT) protocol developed for epidemiological typing of Staphylococcus aureus was analyzed in a biphasic multicenter study. In a Dutch multicenter pilot study, 10 genetically unique isolates of methicillin-resistant S. aureus (MRSA) were characterized by the BT assay as presented by van Leeuwen et al. [J. Clin. Microbiol. 2001 39 (1) 328]. The BT assay, including a standardized DNA extraction protocol was performed in duplicate in eleven medical microbiology laboratories. Two different hybridization detection procedures were applied and a prelabeled DNA sample as process control was included. Only three laboratories accurately identified all strains. Divergence in technical procedures resulted in misinterpretation due to an increasing number of faint or absent hybridization signals in combination with high background staining. The binary type of the process control was determined correctly by all participating laboratories. The feasibility of the BT protocol was related directly to the skill of the laboratory personnel. On the basis of the national study, we concluded that the DNA extraction protocol needed modification to improve DNA yield and purity. Subsequently, seven European laboratories participated in an international study to determine the reproducibility of the modified BT protocol. Each center was asked to analyze 10 DNA samples previously extracted from 10 MRSA strains (phase 1) and, additionally, to analyze 10 MRSA strains, using the standardized or their in-house DNA isolation protocol (phase 2). A prelabeled DNA process control sample was included again. The binary types of all DNA samples were identified correctly by all but one laboratories. This latter laboratory diverged from the protocol by adding an excess of labeled DNA to the hybridization mixture, resulting in a high background and, therefore, noninterpretable BT results. All centers produced identical BT results for the process control. Five of the seven centers correctly identified the binary types of all 10 MRSA strains in phase 2 of the international study. Three of these centers used their in-house DNA extraction protocol. Divergence from the standard BT protocol in the remaining two centers resulted in no interpretable BT data for the 10 MRSA strains. The study demonstrated that each center that followed the BT protocol to the letter could generate reproducible results, irrespective whether or not an in-house DNA isolation protocol was used. The current BT protocol thus represents a simple method generating robust, reproducible genotype data for S. aureus strains.

Eighty-four clinical isolates of the family Enterobacteriaceae , recovered from 1998 to 2000 in 15 hospitals in 10 Polish cities, were analyzed. All the isolates produced β-lactamases with pIs of 8.4 and 5.4, and the pI 8.4 enzymes were demonstrated to hydrolyze cefotaxime but not ceftazidime in the in vitro bioassay. PCR analysis and DNA sequencing have revealed that in all cases the pI 8.4 β-lactamase was probably the CTX-M-3 extended-spectrum β-lactamase (ESBL) variant, which was originally identified in 1996 in Praski Hospital in Warsaw. In the majority of isolates, bla CTX-M-3 genes resided within large conjugative plasmids with similar fingerprints, which, in the context of the high degree of diversity of the randomly amplified polymorphic DNA types of the isolates, suggested that horizontal transfer of plasmids was likely the main mechanism of CTX-M-3 spread. The dissemination of plasmids was probably preceded by the center-to-center transmission of several strains, as indicated by the identification by pulsed-field gel electrophoresis of closely related or possibly related Klebsiella pneumoniae , Escherichia coli , and Citrobacter freundii isolates in five different hospitals. CTX-M-3-producing organisms revealed a very high degree of diversity in β-lactam resistance levels and patterns. This was attributed to several factors, such as the production of other β-lactamases including additional ESBLs, possible quantitative variations in CTX-M-3 expression, segregation of AmpC derepressed mutants, and permeability alterations.

Vancomycin-resistant enterococci (VRE) have recently become an increasing problem in hospitals in Poland, being responsible for a growing number of nosocomial outbreaks. In this work, we have analyzed the second outbreak of VRE with the VanB phenotype to be identified in the country. It was caused by clonal dissemination of a single strain of vancomycin-resistant Enterococcus faecalis (VRES) and horizontal transmission of vancomycin resistance genes among several vancomycin-resistant Enterococcus faecium (VREM) strains. Two similar restriction fragment length polymorphism types of the vanB gene cluster characterized VRES and VREM isolates, and they both contained the same vanB2 variant of the vanB gene. Two vancomycin-susceptible E. faecium (VSEM) isolates, recovered from the same wards during the outbreak, proved to be related to certain VREM isolates and could represent endemic strains that had acquired vancomycin resistance. One VSEM and four VREM isolates, all identified in the same patient, belonged to a single clone, although they revealed remarkable diversity in terms of susceptibility, PFGE patterns, plasmid content, and number of vanB gene cluster copies. Most probably they reflected the dynamic evolution of an E. faecium strain in the course of infection of a single patient. One of the VREM isolates turned out to be resistant to teicoplanin, which coincided with the use of this antibiotic in the patient's therapy. Its vanB gene variant differed by a single mutation from that found in other isolates; however, it also lacked a large part of the vanB gene cluster, including the regulatory genes vanR B and - S B , and the vancomycin-inducible promoter P YB . Expression of the resistance genes vanH B , - B , and - X B was constitutive in the mutant, and this phenomenon was responsible for its unusual phenotype.