Clonal Distribution of Methicillin-Resistant Staphylococcus aureus in Poland

We report on a study of 158 methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates obtained from 1990 to 1996 in 18 different hospitals in Poland. All isolates were recovered from infection and carriage sites of patients, carriage sites of health care personnel, and hospital environment samples. Fifty-seven MRSA strains described here were studied previously and these were divided into two different clusters according to the degree of heterogeneity of methicillin resistance expression. The aim of this study was to extend the correlation between the two clusters and identify the clonal identities among all isolates by a combination of different methodologies: (i) analysis of mecA polymorphs and Tn554 insertion patterns and (ii) determination of pulsed-field gel electrophoresis patterns of chromosomal SmaI digests. Ninety-seven of 158 strains showed a heterogeneous expression of resistance to methicillin. Among these, 75 (77.3%) were ClaI-mecA type I, ClaI-Tn554 type NH (NH, no homology with transposon Tn554), and pulsed-field gel electrophoresis (PFGE) pattern A (I::NH::A); 10 isolates were III::B::M (10.3%); and the remaining clones included a few or single isolates. The isolates with homogeneous expression of resistance to methicillin (n = 61) were predominantly ClaI-mecA type III (49 of 61 [80.3%]) but had great variability in their ClaI-Tn554 and PFGE patterns. This study confirmed the existence of two main clusters of MRSA in Poland.     

Epidemiological Analysis of Salmonella enteritidis Isolates from Humans and Broiler Chickens in Thailand by Phage Typing and Pulsed-Field Gel Electrophoresis

To determine the phage types (PT) of Salmonella enteritidis found in Thailand and to clarify the potential for human infection by S. enteritidis in broiler chicken meat, human and poultry isolates taken from Thailand between 1990 and 1997 were phage typed and analyzed by pulsed-field gel electrophoresis (PFGE). Ten different PT were found among the 302 isolates phage typed, with PT 4 being the most frequent in human (73.9%) and poultry (76.2%) isolates, followed by PT 1 (8.0%), 8 (3.6%), and 7a (2.2%) in human isolates and by PT 7a (4.9%), 1 (3.7%), and 12 (2.4%) in poultry isolates. Of the 53 isolates analyzed by PFGE, 45 showed an indistinguishable pattern (pattern A) by BlnI-digested PFGE and the other 8 isolates showed a very similar pattern that differed by only a few bands. These results indicate the spread of a genetically identical clone of S. enteritidis in humans and poultry in Thailand.     

Epidemiology of a Dominant Clonal Strain of Vancomycin-Resistant Enterococcus faecium at Separate Hospitals in Boston, Massachusetts

In 1996, the dominant (43%) strain of vancomycin-resistant enterococci (VRE; type A) at Massachusetts General Hospital was identified at Brigham and Women’s Hospital (BWH). To characterize the epidemiology of infection with type A isolates of VRE at BWH, we collected demographic and clinical data for all patients from whom VRE were isolated from a clinical specimen through September 1996. The first clinical isolates from all BWH patients from whom VRE were isolated were typed by pulsed-field gel electrophoresis of SmaI digests of chromosomal DNA. Among patients hospitalized after the first patient at BWH infected with a type A isolate of VRE was identified, exposures were compared between patients who acquired type A isolates of VRE and those who acquired other types of VRE. Isolates from 99 patients identified to have acquired VRE were most commonly from blood (n = 27), urine (n = 19), or wounds (n = 19). Three months after the index patient arrived at BWH and at a time when ≥12 types of strains of VRE were present, type A isolates of VRE became dominant; 39 of 75 (52%) of the study cohort had acquired type A isolates of VRE. We found no association between the acquisition of type A isolates of VRE and transfer from another institution or temporal overlap by service, ward, or floor with patients known to have acquired type A isolates of VRE. By multivariate analysis, only residence in the medical intensive care unit (adjusted odds ratio [OR], 3.2; 95% confidence interval [CI], 1.4 to 107) and the receipt of two or more antibiotics per patient-day (adjusted OR, 12.2; 95% CI, 1.2 to 9.0) were associated with the acquisition of strain A. This strain of VRE, dominant at two Boston hospitals, was associated with intensity of antibiotic exposures (i.e., two or more antibiotics per patient-day). We hypothesize that this strain may have unidentified properties providing a mechanism favoring its spread and dominance over other extant isolates, and further studies are needed to define these properties.     

Molecular Analysis of Glycopeptide-Resistant Enterococcus faecium Isolates Collected from Michigan Hospitals over a 6-Year Period

The purpose of this study was to evaluate the molecular relatedness of clinical isolates of glycopeptide-resistant Enterococcus faecium isolates collected from hospitals in Michigan. A total of 379 isolates used in this study were all vancomycin-resistant E. faecium isolates collected from 28 hospitals and three extended-care facilities over a 6-year period from 1991 to 1996. For the 379 isolates, there were 73 pulsed-field gel electrophoresis (PFGE) strain types. Within strain types, there were as many as six restriction fragment differences. Most isolates (70%) belonged to six strain types, which were designated M1 (36%), M2 (3%), M3 (18%), M4 (6%), M10 (4%), and M11 (3%). PFGE strain M1 was cultured from 135 patients in 13 hospitals during the period 1993 to 1996. Strain type M2 was cultured from 11 patients in two hospitals during the period 1991 to 1992 and was not observed after 1992. Strain type M3 was cultured from 70 patients in 10 hospitals during the period of 1994 to 1996. Both M4 and M10 were cultured from 23 patients in three hospitals and from 15 patients in two hospitals, respectively, during 1995 to 1996. M11 was cultured from 13 patients in four hospitals during 1996. A total of 23 of 28 hospitals had evidence of clonal dissemination of some isolates. Plasmid content and hybridization analysis done on 103 isolates from one hospital and two affiliated extended-care facilities indicated that the strains contained from one to eight plasmids. Mating experiments indicated transfer of vancomycin resistance from 94 of these isolates into plasmid-free E. faecium GE-1 at transfer frequencies of <10⁻⁹ to 10⁻⁴. Gentamicin resistance and erythromycin resistance were cotransferred at various frequencies. A probe for the vanA gene hybridized to the plasmids of 23 isolates and to the chromosomes of 72 isolates. A probe for the vanB gene hybridized to the chromosomes of 8 isolates. The results of this study suggest inter- and intrahospital dissemination of vancomycin-resistant E. faecium strains over a 6-year period in southeastern Michigan. The majority of isolates studied belonged to the same few PFGE strains, indicating that clonal dissemination was responsible for most of the spread of resistance that occurred.     

Virulence Properties of Shiga Toxin-Producing Escherichia coli (STEC) Strains of Serogroup O118, a Major Group of STEC Pathogens in Calves

Shiga toxin-producing Escherichia coli (STEC) strains of serogroup O118 are the most prevalent group among STEC strains in diarrheic calves in Germany (L. H. Wieler, Ph.D. thesis, University of Giessen, 1997). To define their virulence properties, 42 O118 (O118:H16 [n = 38] and O118:H− [n = 4]) strains were characterized. The strains displayed three different Stx combinations (Stx1 [36 of 42], Stx1 and Stx2 [2 of 42], and Stx2 [4 of 42]). A total of 41 strains (97.6%) harbored a large virulence-associated plasmid containing hly_EHEC (hly from enterohemorrhagic E. coli). The strains’ adhesive properties varied in relation to the eukaryotic cells tested. Only 28 of 42 strains (66.7%) showed localized adhesion (LA) in the human HEp-2 cell line. In contrast, in bovine fetal calf lung (FCL) cells, the number of LA-positive strains was much higher (37 of 42 [88.1%]). The locus of enterocyte effacement (LEE) was detected in 41 strains (97.6%). However, not all LEE-positive strains reacted positively in the fluorescence actin-staining (FAS) test, which indicated the attaching and effacing (AE) lesion. In HEp-2 cells, only 22 strains (52.4%) were FAS positive, while in FCL cells, the number of FAS-positive strains was significantly higher (38 of 42 [90.5%; P < 0.001]). In conclusion, the vast majority of the O118 STEC strains from calves (41 of 42 [97.6%]) have a high virulence potential (stx, hly_EHEC, and LEE). This virulence potential and the high prevalence of STEC O118 strains in calves suggest that these strains could be a major health threat for humans in the future. In addition, the poor association between results of the geno- and phenotypical tests to screen for the AE ability of STEC strains calls the diagnostic value of the FAS test into question.     

Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases

The vacuolating cytotoxin and the cytotoxin-associated protein, encoded by vacA and cagA, respectively, are important virulence determinants of Helicobacter pylori. Sixty-five H. pylori strains were isolated from dyspeptic patients (19 with peptic ulcer disease, 43 with chronic gastritis, and 3 with gastric cancer) and studied for differences in the vacA and cagA genes and their relationship to VacA and CagA expression, cytotoxin activity, and the clinical outcome of infection. By PCR, fifty-four (83.1%) of 65 strains had the vacA signal sequence genotype s1 and only 10 (15.4%) had the type s2. After primer modification, the vacA middle-region types m1 and m2 were detected in 24 (36.9%) and 41 (63.1%) strains, respectively. The combinations s1-m2 (31 [47.7%]) and s1-m1 (23 [35.4%]) occurred more frequently than s2-m2 (10 [15.4%]) (P = 0.01). No strain with the combination s2-m1 was found. All 19 patients with peptic ulcers harbored type s1 strains, in contrast to 32 (74.4%) of 43 patients with gastritis (P = 0.02). The vacA genotype s1 was associated with the presence of cagA (P < 0.0001), VacA expression (P < 0.0001), and cytotoxin activity (P = 0.003). The cagA gene was detectable in 48 (73.8%) of 65 isolates and present in 16 (84.2%) of 19 ulcer patients and 29 (67.4%) of 43 patients with gastritis (P = 0.17). The vacA genotypes of German H. pylori isolates are identical to those previously reported. H. pylori strains of vacA type s1 are associated with the occurrence of peptic ulceration and the presence of cagA, cytotoxin activity, and VacA expression.     

Survival of Vancomycin-Resistant and Vancomycin-Susceptible Enterococci on Dry Surfaces

We compared the abilities of Enterococcus faecium strains (three vancomycin-resistant enterococci [VRE] and five vancomycin-susceptible enterococci [VSE]) and Enterococcus faecalis strains (one VRE and 10 VSE) to survive under dry conditions. Bacterial suspensions of the strains were inoculated onto polyvinyl chloride and stored under defined conditions for up to 16 weeks. All strains survived for at least 1 week, and two strains survived for 4 months. A statistical model was used to distribute the 19 resulting survival curves between two types of survival curves. The type of survival curve was not associated with the species (E. faecalis versus E. faecium), the source of isolation (patient versus environment), or the susceptibility to vancomycin (VRE versus VSE). Resistance to dry conditions may promote the transmissibility of a strain, but VRE have no advantages over VSE with respect to their ability to survive under dry conditions.     

Antimicrobial susceptibility and genetic characteristics of Streptococcus pneumoniae isolates indicating possible nosocomial transmission routes in a community hospital in Japan

A clinical study was designed to study Streptococcus pneumoniae isolates recovered from a community hospital in Japan from April 2001 to November 2002. A total of 73 isolates were defined as derived from inpatient, outpatient, and hospital staff groups. The MIC results showed that 20 strains (27.4%) were susceptible to penicillin G, 39 strains (53.4%) had intermediate resistance, and 14 strains (19.2%) had full resistance. Low susceptibility to macrolides was also detected: 32.9%, 32.9%, and 34.2% of all strains were resistant to erythromycin, clarithromycin, and azithromycin, respectively. Thirty strains (41%) were resistant to at least two different kinds of antibiotics. Nineteen disparate serotypes were detected besides two nontypeable strains, and the predominant serotypes were 19F and 23F. Pulsed-field gel electrophoresis (PFGE) pattern A was dominant in the serotype 19F group; this pattern was similar to that of the international clone Taiwan 19F. A total of 10 different patterns were detected in the 23F group and were distinguishable from those of the international clones Spain 23F and Taiwan 23F. Pattern b strains were identified in the same ward, and pattern d strains were found both in patients with nosocomial pneumococcal infections (NPI) and in outpatients. In conclusion, drug-resistant S. pneumoniae was spreading rapidly, especially isolates of the serotype 19F and 23F groups. PFGE data revealed interpatient transmission and suggested that there might be some association between NPI patient strains and outpatient strains.     

Molecular Epidemiological Study of Nosocomial Enterobacter aerogenes Isolates in a Belgian Hospital

In 1995, the rate of isolation of Enterobacter aerogenes in the Saint-Pierre University Hospital in Brussels, Belgium, was higher than that in the preceding years. A total of 45 nosocomial E. aerogenes strains were collected from 33 patients of different units during that year, and they were isolated from 19 respiratory specimens, 13 pus specimens, 7 blood specimens, 4 urinary specimens, 1 catheter specimen, and 1 heparin vial. The strains were analyzed to determine their epidemiological relatedness and were characterized by their antibiotic resistance pattern determination, plasmid profiling, and genomic fingerprinting by macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). The majority of the strains (82%) were multiply resistant to different commonly used antibiotics. Two major plasmid profiles were found: most strains (64%) harbored two plasmids of different sizes, whereas the others (20%) contained a single plasmid. PFGE with SpeI and/or XbaI restriction enzymes revealed that a single clone (80%) was responsible for causing infections or colonizations throughout the year, and this result was concordant with those obtained by plasmid profiling, with slight variations. By comparing the results of these three methods, PFGE and plasmid profiling were found to be the techniques best suited for investigating the epidemiological relatedness of E. aerogenes strains, and they are therefore proposed as useful tools for the investigation of nosocomial outbreaks caused by this organism.     

Comparison of Genomic Methods for Differentiating Strains of Enterococcus faecium: Assessment Using Clinical Epidemiologic Data

Genomic DNA extracted from 45 vancomycin-resistant Enterococcus faecium (VRE) isolates was cleaved with HindIII and HaeIII and subjected to agarose gel electrophoresis. The ability of this method (restriction endonuclease analysis [REA]) to distinguish strains at the subspecies level was compared with results previously determined by pulsed-field gel electrophoresis (PFGE). Chart reviews were performed to provide a clinical correlation of possible epidemiologic relatedness. A likely clinical association was found for 29 patients as part of two outbreaks. REA found 21 of 21 isolates were the same type in the first outbreak, with PFGE calling 19 strains the same type. In the second outbreak with eight patient isolates, HindIII found six were the same type and two were unique types. HaeIII found three strains were the same type, two strains were a separate type, and three more strains were unique types, while PFGE found three were the same type and five were unique types. No single “ideal” method can be used without clinical epidemiologic investigation, but any of these techniques is helpful in providing focus to infection control practitioners assessing possible outbreaks of nosocomial infection.     

Genetic Diversity of Borrelia burgdorferi in Lyme Disease Patients as Determined by Culture versus Direct PCR with Clinical Specimens

Two hundred seventeen isolates of Borrelia burgdorferi originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of B. burgdorferi performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [P < 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of B. burgdorferi patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.     

Phenotypic and genotypic characterization of verotoxin-producing Escherichia coli O103:H2 isolates from cattle and humans

Characterization of important non-O157 verotoxin-producing Escherichia coli (VTEC) has lagged considerably behind that of O157:H7 strains. This study characterized 91 VTEC O103:H2 strains from bovine and human sources and of North American and European origins by virulence or putative virulence genes, pulsed-field gel electrophoresis (PFGE) patterns, plasmid profiles, antimicrobial resistance, and colicin production. All strains were positive for vt1 and eae-; 97% were positive for ehxA; and all were negative for hlyA. Two strains carried vt2. There were 66 PFGE patterns grouped in six clusters, and there were 25 different plasmid profiles. Plasmid-encoded katP and etp genes were significantly more frequent in European than in North American human strains. The distribution of selected phenotypes was as follows: enterohemorrhagic E. coli (EHEC) hemolysin, 95%; colicin production, 38%; antimicrobial resistance, 58%. All the strains were negative for the alpha-hemolytic phenotype. In conclusion, the VTEC O103:H2 strains were diverse, as shown by PFGE, plasmid profiles, virulence markers, and antimicrobial resistance patterns, and all strains showed an EHEC hemolytic phenotype instead of the alpha-hemolytic phenotype that has been shown previously.     

Pulsed-field gel electrophoresis of Staphylococcus aureus isolates from atopic patients revealing presence of similar strains in isolates from children and their parents

Skin colonization with Staphylococcus aureus is often associated with atopic dermatitis, and staphylococcal enterotoxins have been implicated in the etiology of atopic disease. In this study, the colonization of patients with atopic dermatitis and their parents was investigated in order to evaluate the possibility of intrafamiliar transmission. S. aureus strains were isolated from 30 of 45 patients (66%). In 19 of 29 families (65%), at least one parent carried S. aureus, and the overall colonization rate of the parents was 48%. All strains were typed by pulsed-field gel electrophoresis (PFGE), and the presence of enterotoxin genes in the strains was assayed by multiplex PCR. A high percentage (84%) of the isolates present on the children and on at least one of their parents displayed identical PFGE and enterotoxin patterns as well as identical antibiotic resistance profiles, indicating intrafamiliar transmission. Forty-five percent of the strains did not carry any enterotoxin gene. The most frequently found enterotoxin genes were seg and sei, which were present in 36% of the strains, and seb, which was found in 24% of the strains. The other toxin genes occurred only in low frequencies. Most strains were resistant to penicillin (82%), and 15% showed resistance to more than one antibiotic. Intermediately-vancomycin-resistant S. aureus or methicillin-resistant S. aureus strains were not detected. In conclusion, this study indicates that the colonization rate of parents of atopic children is rather high and may increase the risk of recolonization of the child.     

Use of highly discriminatory fingerprinting to analyze clusters of Clostridium difficile infection cases due to epidemic ribotype 027 strains

We compared multilocus variable-number tandem-repeat analysis (MLVA) and macrorestriction endonuclease analysis using pulsed-field gel electrophoresis (PFGE) to determine their utility to identify clusters of Clostridium difficile infection (CDI) among 91 isolates of PCR ribotype 027 (NAP1, for North American pulsed-field type 1) from nine hospitals (and 10 general practitioners associated with one institution) in England. We also examined whether mortality in CDI cases was associated with specific MLVA subtypes. PFGE discriminated between ribotype 027 strains at >98% similarity, identifying five pulsovars (I to V) of 1 to 53 isolates. MLVA was markedly more discriminatory, identifying 23 types of 1 to 15 isolates (>71% similarity). PFGE pulsovars I and IV contained 14 and 8 MLVA types, respectively. Twenty-one of twenty-three (91%) of MLVA types were specific to individual PFGE pulsovars. Four CDI clusters were identified in institution A by conventional epidemiological analysis. MLVA typing identified two enlarged and two additional clusters. Thirty of forty-four (68%) patients in institution A with CDI caused by ribotype 027 strains were assigned to seven distinct clusters by a combination of MLVA typing and epidemiological records. Of 33 patients, comprising 14 different MLVA types, nine (27%) died by day 30 (early deaths). Eight of nine (89%) were associated with PFGE type IV C. difficile ribotype 027. Five of nine early deaths were associated with MLVA type 16, which was the dominant type in this cohort (10/33 cases); 4 other distinct MLVA types accounted for the other early deaths. MLVA was far superior to PFGE for analyzing clusters of CDI both within and between institutions. Further study is needed to examine whether subtypes of C. difficile ribotype 027 affect outcome.     

Characterization of Gentamicin-Susceptible Strains of Methicillin-Resistant Staphylococcus aureus Involved in Nosocomial Spread

We report an outbreak of epidemic Staphylococcus aureus strains characterized by an unusual heterogeneous resistance to methicillin and resistance to tobramycin but susceptibility to gentamicin (gentamicin-susceptible methicillin-resistant S. aureus [GS-MRSA]), contrasting with gentamicin-resistant homogeneous MRSA (GR-MRSA) that have been endemic in our hospital since the 1970s. A total of 97 GS-MRSA strains, which were shown by DNA hybridization to carry the mecA and ant(4′)-Ia genes, were studied. The 40 GS-MRSA strains isolated at the beginning of the outbreak (January 1992 to June 1993) were typed by using resistance patterns, phage typing, serotyping, and pulsed-field gel electrophoresis and were compared with GR-MRSA and methicillin-susceptible S. aureus (MSSA) strains isolated during the same period. Two dominant clones, A::1 and B::3, and one minor clone, C::5, were identified among the 40 GS-MRSA strains, according to pulsotypes (A to C) and their resistance patterns (1, 3, and 5), which were distinguishable from those of GR-MRSA and MSSA strains. A selection of 57 GS-MRSA strains, isolated from 1994 to 1996, were clustered in the same three clones. However, their distribution had changed in comparison with that in the 1992 to 1993 period: clone A::1 remained dominant (47 versus 42.5%), whereas clone B::3 progressively declined (5 versus 35%) and clone C::5, the most susceptible to antibiotics, spread (44 versus 2.5%). Epidemiological investigations revealed that some clones had been introduced via patients transferred from other hospitals and that cross-infection occurred within and between wards. Major changes in the use of antibiotics, especially aminoglycosides, cyclines, and macrolides, likely played a role in the emergence and spread of GS-MRSA strains.     

Characterization of IS1245 for Strain Typing of Mycobacterium avium

IS1245 is an insertion element widely prevalent among isolates of Mycobacterium avium. We used PvuII Southern blots to analyze IS1245 polymorphisms among 159 M. avium isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct M. avium strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS1245 and thus were typeable. Twenty-five (63%) strains had ≥10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS1245 typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS1245, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS1245 patterns. Within eight (25%) sets, one or more isolates had IS1245 patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS1245 typing those isolates would have been designated distinct strains. IS1245 was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS1245 provides a useful tool for molecular strain typing of M. avium but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships.     

Pulsed-Field Gel Electrophoresis Is More Efficient than Ribotyping and Random Amplified Polymorphic DNA Analysis in Discrimination of Pasteurella haemolytica Strains

One hundred thirty-three strains of Pasteurella haemolytica of both biotypes (90 and 43 strains of biotypes A and T, respectively) and almost all the serotypes were subjected to ribotyping, random amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE) analysis for epidemiological purposes. A total of 15 patterns recorded as ribotypes HA to HO were found for the P. haemolytica biotype A strains, with ribotypes HA, HC, and HD being encountered most often (66 strains [74%]); and 20 ribotypes, designated HA′ to HT′, that were clearly distinct from those observed for biotype A strains were observed for strains of biotype T. RAPD analysis generated a total of 44 (designated Rp1 to Rp44) and 15 (designated Rp1′ to Rp 15′) unique RAPD patterns for biogroup A and biogroup T, respectively. Analysis of the data indicated that a given combined ribotype-RAPD pattern could be observed for biotype A strains of different serotypes, whatever the zoological or geographic origin, whereas this was not the case for biotype T strains. PFGE appeared to be more efficient in strain discrimination since selected strains from various zoological or geographical origins harboring the same ribotype-RAPD group were further separated into unique entities.     

Determination of Helicobacter pylori Virulence by Simple Gene Analysis of the cag Pathogenicity Island

Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.     

Increasing rates of vancomycin resistance among Enterococcus faecium isolated from German hospitals between 2004 and 2006 are due to wide clonal dissemination of vancomycin-resistant enterococci and horizontal spread of vanA clusters

Results of national and international surveillance studies revealed increasing rates of vancomycin-resistant Enterococcus faecium (VREF) among German hospital patients since 2003. To investigate the molecular background of vanA-type glycopeptide resistance, 51 clinical VREF isolated between 2004 and 2006 and originating from 19 German hospitals representing 10 Federal States have been investigated. Isolates were characterised by multi-locus sequence typing (MLST), SmaI macrorestriction analysis in pulsed-field gel electrophoresis (PFGE), and multiple-locus variable-number tandem repeat analysis (MLVA). Phylogenetic relatedness between strains was identified using BioNumerics and eBURST software. Distribution of virulence markers esp and hyl_Efm was investigated by PCR. The structure of the vanA gene clusters was investigated by PCR, long-template PCR, sequencing and Southern hybridisations. The 51 VREF were rather diverse constituting different strain types, different virulence markers and vanA clusters. Within this diversity we found supportive data for a dissemination of related – already vancomycin-resistant – E. faecium among various hospitals and Federal States and for spread of identical vanA gene clusters among clonally different strain types within single hospitals. In conclusion, the increase in the rates of VREF among German hospital patients within the last 2 years might be rather complex and due to different molecular events and scenarios.     

Antimicrobial Resistance and Serotype Distribution of Streptococcus pneumoniae Strains Causing Childhood Infections in Bangladesh, 1993 to 1997

Three hundred sixty-two Streptococcus pneumoniae strains were isolated from children under 5 years of age at Dhaka Shishu (Children) Hospital from 1993 to 1997. The strains were isolated from blood (n = 105), CSF (n = 164), ear swab (n = 61), eye swab (n = 20), and pus (n = 12). Of the 362 isolates, 42 (11.6%) showed intermediate resistance (MIC, <0.1 μg/ml) and only 4 (1.1%) showed complete resistance (MIC, >2.0 μg/ml) to penicillin. Penicillin resistance exhibited a strong relationship with serotype 14; 47.8% of the penicillin-resistant strains belonged to this type. A remarkably high (64.1%) resistance to co-trimoxazole was observed, along with a significant increase during the time period studied; there was no relationship to capsular type. By way of contrast, penicillin resistance did not show any significant change during the study period. Resistance to chloramphenicol (2.2%) and erythromycin (1.1%) was rare. The high resistance to co-trimoxazole and its increasing trend demand elucidation of the clinical impact of pneumonia treatment by this antimicrobial and reconsideration of the World Health Organization recommendation for co-trimoxazole administration to children with community-acquired pneumonia at the health care worker level in Bangladesh.