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Frequent genetic recombination in natural populations of the marine cyanobacterium Microcoleus chthonoplastes.

A culture-independent method for multilocus sequence typing of Microcoleus chthonoplastes was developed based on mechanical separation of individual cyanobacterial filaments from natural microbial mat populations through micromanipulation, subsequent polymerase chain reaction (PCR) amplification and sequence analysis of three genetic loci (kaiC, petB/D, rDNA-ITS). Among 81 individuals sampled from intertidal sand flats of the North Sea and Baltic Sea, we found 8-14 different sequences (alleles) per genetic locus, resulting in 36 distinct genotypes with unique allele profiles. Non-congruent phylogenetic gene trees for the three loci analysed and split decomposition analysis indicated the occurrence of horizontal genetic exchange. The index of association determined for the entire population was 0.096, indicating that recombination occurs frequently enough to cause almost random association (linkage equilibrium) among alleles. Analysing individuals from three different locations in the North Sea and Baltic Sea, we did not find evidence for geographic subdivisions between populations.

Alleles↗

Outbreak of vancomycin-resistant Enterococcus spp. in an Italian general intensive care unit.

Following the identification of two clinical isolates of vancomycin-resistant enterococci (VRE) from intensive care unit (ICU) patients, a surveillance programme detected that six of eight ICU patients were colonised by VRE. Standard epidemic control measures were instituted in the ICU. During a 16-month period, 13 (2.5%) of 509 ICU patients had VRE-positive swabs upon admission, and 43 (8.7%) of 496 VRE-negative patients were colonised by VRE in the ICU. Patients who acquired VRE in the ICU had a longer ICU stay (p < 0.0001). No other statistically significant differences were demonstrated. Two patients had documented infection (infection/colonisation index, 3.6%; overall VRE infection frequency, 0.4%), but both recovered and were discharged. VRE colonisation did not increase the mortality rate. Automated ribotyping identified three clusters containing, respectively, the first 52 Enterococcus faecium isolates, two Enterococcus faecalis isolates, and two further isolates of E. faecium. Multilocus sequence typing demonstrated that two E. faecium isolates representative of the two ribotypes belonged to sequence types 78 and 18, and that these two isolates belonged to the epidemic lineage C1, which includes isolates with a wide circulation in northern Italy. The outbreak was controlled by continuous implementation of the infection control programme, and by the opening of a new unit with an improved structural design and hand-washing facilities.

Adult↗

Identification of macrolide-resistant clones of Streptococcus pyogenes in Portugal.

Although the overall level of macrolide resistance (27%) has remained stable in Portugal, a rapid inversion in the dominant phenotypes has been noted, with a sharp decrease in the MLS(B) phenotype paralleled by an increase in the M phenotype. To gain further insight into these changes, 325 macrolide-resistant isolates were characterised using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The use of Cfr9I, an isoschizomer of SmaI, to digest M phenotype isolates that were refractory to SmaI digestion allowed direct comparison of MLS(B) and M isolates. The results from PFGE and MLST were highly concordant and identified eight major clones, accounting for 92% of the isolates, each of which was associated exclusively with a single macrolide resistance phenotype. Two major clones were found among MLS(B) isolates, characterised by sequence types (ST) 46 (T12/emm22) and ST52 (T28/emm28), whereas clones characterised by ST39 (T4/emm4) and ST28 (T1/emm1) dominated among M isolates. The clone defined by ST52 corresponded to a bacitracin-resistant clone circulating in Europe, and a novel variant expressing other surface antigens (T12/emm22) was detected. The presence of the four major clones has been reported previously in other European countries, suggesting Europe-wide dissemination of a few macrolide-resistant lineages.

Bacitracin↗

Increased incidence of methicillin-resistant Staphylococcus aureus ST80, novel ST125 and SCCmecIV in the south-eastern part of Norway during a 12-year period.

A retrospective population-based study of genotypes of methicillin-resistant Staphylococcus aureus (MRSA) was performed during the period 1991-2003 in two counties in the south-eastern part of Norway. Isolates of MRSA from all individuals in the two counties in whom MRSA was detected were genotyped by means of multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A gene (spa) typing and amplified fragment length polymorphism (AFLP) analysis. Until 1999, only sporadic cases of MRSA infection were reported in these counties, but the incidence increased during the following years. Nine new MLST types were identified in this study. The predominant strains were ST239-MRSA-III, the novel ST125-MRSA-IV, and the central European community-acquired strain ST80-MRSA-IV reported previously. ST80-MRSA-IV was introduced into the two counties in 1997, and the incidence of infections has increased since 2000, so that ST80-MRSA-IV is now the commonest MRSA strain in the region. An increase in MRSA clones carrying SCCmecIV has occurred during recent years, which could indicate a shift in the MRSA population in Norway from hospital-acquired MRSA to community-acquired-MRSA.

Bacterial Proteins↗

Conventional and molecular investigation of meningococcal isolates in relation to two outbreaks in the area of Athens, Greece.

Two local outbreaks caused by serogroup B Neisseria meningitidis occurred in the Athens area of Greece during 2003. In total, 30 N. meningitidis isolates from patients and carriers, as well as sporadic cases, were investigated by conventional techniques (serogroup, serotype and serosubtype), multilocus sequence typing (MLST), analysis of variable number tandem repeats (VNTR) and random amplified polymorphic DNA (RAPD) analysis. Compared with the two other molecular techniques, VNTR analysis was a simple, reliable and highly discriminatory method for fine typing of meningococcal isolates, showing a good correlation with the epidemiological data for the two outbreaks analysed.

Adolescent↗

Molecular characterization of Enterococcus faecium isolated from hospitalized patients in Korea.

AIMS: Multilocus sequence typing (MLST) was performed for vancomycin-resistant Enterococcus faecium (VREF) from diverse geographical areas in Korea to obtain insights into the genetic relationships with other molecular profiles. To understand the diversity of lineages, vancomycin-susceptible E. faecium (VSEF) were included. METHODS AND RESULTS: A total of 60 E. faecium isolates were analysed by MLST and esp profile. Molecular typing of Tn1546 of 30 VREF strains was evaluated by overlapping PCR of Tn1546 and DNA sequencing. Seven sequence types (ST) were found among 30 VSEF isolates, and four STs were found among 30 VREF isolates. The types most frequently encountered were ST 78 (26 isolates) and ST 203 (16 isolates). Of the 60 E. faecium isolates, 35 isolates were positive for the esp gene. On molecular typing of Tn1546, all VREF isolates were divided into four main types. Strains with the same ST showed divergence in Tn1546 types and strains with the same Tn1546 type represented different STs. CONCLUSIONS: An association between Tn1546 typing and MLST was not found. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the horizontal spread of Tn1546 between strains plays a major role in the dissemination of vancomycin resistance in Korea.

Bacterial Typing Techniques↗

Standardized nonculture techniques recommended for European reference laboratories.

Culture-confirmed diagnosis of meningococcal invasive infections is often hindered by early antibiotic treatment. Nonculture molecular standardized methods are now essential tools for the immediate management of meningococcal infections. The European Monitoring Group on Meningococci (EMGM) recommends the following measures. (1) The implementation of standardized protocols of extraction methods for DNA isolation from clinical specimens for PCR-based identification and genogrouping of Neisseria meningitidis. (2) The use of molecular approaches (sequencing of target genes) for the determination of meningococcal susceptibility to antibiotics, such as sequencing of penA and rpoB genes for susceptibility to penicillin G and rifampicin, respectively. (3) The use of nonculture strain characterization by multilocus sequence typing (MLST) and sequence typing of porA and fetA. These approaches can be implemented either by individual reference laboratories or through collaboration and referral between centres.

Bacterial Outer Membrane Proteins↗

Lessons from meningococcal carriage studies.

Neisseria meningitidis, an obligate commensal of humans, normally colonizes the mucosa of the upper respiratory tract without affecting the host, a phenomenon known as carriage. In Europe, as much as 35% of young adults are carriers at a given time. Recent studies using molecular methods for clone identification have demonstrated the extensive genetic diversity of the strains isolated from carriers, in comparison with a limited number of hypervirulent strains associated with invasive disease. Published studies and new data generated through the framework of the EU-MenNet clearly indicated significant differences in pathogenicity between meningococcal clones and in the distribution of multilocus sequence types among isolates from asymptomatic carriers among European countries; simultaneous carriage of more than one meningococcal strain in the throat is rare, but occasionally occurs; and the commensal association of particular clones with a host is a long-term relationship, often lasting several months. Further investigations of the carrier state are warranted to improve our understanding of the epidemiology and pathogenesis of meningococcal disease, as well as to support the introduction and to measure the impact of mass vaccination.

Carrier State↗

Molecular characterisation of group A Neisseria meningitidis isolated in Sudan 1985-2001.

A total of 33 group A Neisseria meningitidis (Mc) isolates, collected in Sudan between 1985 and 2001, were studied in order to describe the changes over time in a country within the meningitis belt of Africa. The isolates were characterised by traditional phenotypic methods (serogrouping, serotyping, serosubtyping and antibiogram) and molecular techniques (genosubtyping, pulsed-field gel electrophoresis [PFGE] with restriction endonucleases SpeI and NheI, and multilocus sequence typing [MLST]). Three clones of group A Mc were identified: one before 1988 (sulphadiazine sensitive, serotype 4, genosubtype P1.7,13-1,35-1, sequence type 4 [ST-4]); another during and after the 1988 epidemic (sulphadiazine resistant, serotype 4, genosubtype P1.20,9,35-1, ST-5); and a third causing the 1999 epidemic (sulphadiazine resistant, serotype 4, genosubtype P1.20,9,35-1, ST-7). The first clone showed major differences compared to the other two. The second and third clones had many similarities with differences in only a single gene (pgm) in the MLST (47 of the 450 bp) but significant other differences according to the PFGE patterns. Within the clones, genosubtyping and MLST gave identical information (except one base substitution in the aroE gene in one isolate). However, the PFGE patterns showed changes over time within the clones, where SpeI revealed somewhat more diversity than NheI.

Anti-Infective Agents↗

A link between virulence and ecological abundance in natural populations of Staphylococcus aureus.

Staphylococcus aureus is a major cause of severe infection in humans and yet is carried without symptoms by a large proportion of the population. We used multilocus sequence typing to characterize isolates of S. aureus recovered from asymptomatic nasal carriage and from episodes of severe disease within a defined population. We identified a number of frequently carried genotypes that were disproportionately common as causes of disease, even taking into account their relative abundance among carriage isolates. The existence of these ecologically abundant hypervirulent clones suggests that factors promoting the ecological fitness of this important pathogen also increase its virulence.

Carrier State↗

In vitro activities of ceftobiprole, tigecycline, daptomycin, and 19 other antimicrobials against methicillin-resistant Staphylococcus aureus strains from a national survey of Belgian hospitals.

The in vitro activities of 22 antimicrobial agents, including ceftobiprole, daptomycin, and tigecycline, against 511 methicillin-resistant Staphylococcus aureus (MRSA) isolates from 112 Belgian hospitals were studied by using the CLSI agar dilution method. Isolates were characterized by pulsed-field gel electrophoresis (PFGE) analysis and by PCR detection of determinants of resistance to aminoglycosides, macrolides-lincosamides-streptogramins, and tetracyclines. A representative set of isolates with different PFGE genotypes was further characterized by multilocus sequence typing, determination of staphylococcal cassette chromosome mec (SCCmec) type, and multiplex PCR for toxic shock syndrome type 1 (TSST-1) and Panton-Valentine leukocidin genes. MRSA isolates belonged to nine epidemic MRSA clones, of which sequence type 45 (ST45)-SCCmec IV and ST8-SCCmec IV were predominant, accounting for 49 and 20% of isolates, respectively. The distribution of antimicrobial resistance and TSST-1 genes was strongly linked to clonal types. Ceftobiprole, daptomycin, and tigecycline showed high activity against all isolates of these sporadic and epidemic MRSA clones, as indicated by MIC(90)s of 2 mg/liter, 0.5 mg/liter, and 0.25 mg/liter, respectively. The MIC distribution of daptomycin and tigecycline was not different in isolates with decreased susceptibility to glycopeptides or tetracyclines, respectively. Ceftobiprole MICs were not correlated with oxacillin and cefoxitin MICs. These data indicate excellent activity of the newly developed agents ceftobiprole, daptomycin, and tigecycline against MRSA isolates recently recovered from hospitalized patients in Belgium, supporting their therapeutic potential for nosocomial MRSA infections.

Anti-Bacterial Agents↗

Phenotypic and molecular analysis of penicillin-nonsusceptible Streptococcus pneumoniae isolates in Poland.

beta-Lactams are the drugs of choice for the treatment of infections caused by the important bacterial pathogen Streptococcus pneumoniae. The recent growth of resistance of this organism to penicillin observed worldwide is of the highest concern. In this study, using 887 surveillance pneumococcal isolates recovered in Poland from 1998 to 2002, we observed the increase in penicillin nonsusceptibility from 8.7% to 20.3%. All of the 109 penicillin-nonsusceptible S. pneumoniae (PNSP) isolates identified, together with 22 archival PNSP isolates from 1995 to 1997, were subsequently analyzed by susceptibility testing, serotyping, profiling of pbp genes, pulsed-field gel electrophoresis, and multilocus sequence typing (MLST). Four predominant serotypes, serotypes 6B, 9V, 14, and 23F, characterized 85.5% of the isolates. MLST revealed the presence of 34 sequence types, 15 of which were novel types. Representatives of seven multiresistant international clones (Spain(23F)-1, Spain(6B)-2, Spain(9V)-3, Taiwan(23F)-15, Poland(23F)-16, Poland(6B)-20, and Sweden(15A)-25) or their closely related variants comprised the majority of the study isolates. The spread of Spain(9V)-3 and its related clone of serotype 14/ST143 has remarkably contributed to the recent increase in penicillin resistance in pneumococci in the country.

Anti-Bacterial Agents↗

Emergence in Vietnam of Streptococcus pneumoniae resistant to multiple antimicrobial agents as a result of dissemination of the multiresistant Spain(23F)-1 clone.

Surveillance for Streptococcus pneumoniae resistant to penicillin and other antimicrobial agents is necessary to define the optimal empirical antibiotic therapy for meningitis in resource-poor countries such as Vietnam. The clinical and microbiological features of 100 patients admitted to the Centre for Tropical Diseases in Ho Chi Minh City, Vietnam, between 1993 and 2002 with invasive pneumococcal disease were studied. A penicillin-nonsusceptible pneumococcus (MIC, > or =0.1 micro g/ml) was isolated from the blood or cerebrospinal fluid of 8% of patients (2 of 24) between 1993 and 1995 but 56% (20 of 36) during 1999 to 2002 (P < 0.0001). Pneumococcal isolates resistant to penicillin (MIC, > or =2.0 micro g/ml) increased from 0% (0 of 24) to 28% (10 of 36) (P = 0.002). Only one isolate was ceftriaxone resistant (MIC, 2.0 micro g/ml). Penicillin-nonsusceptible pneumococci were isolated from 78% of children younger than 15 years (28 of 36) compared with 25% of adults (16 of 64) (P = 0.0001). Isolation of a penicillin-nonsusceptible pneumococcus in adults with meningitis was independently associated with referral from another hospital (P = 0.005) and previous antibiotic therapy (P = 0.025). Multilocus sequence typing showed that 86% of the invasive penicillin-resistant pneumococcus isolates tested (12 of 14) were of the Spain(23F)-1 clone. The serotypes of >95% of the penicillin-nonsusceptible pneumococci were included in the currently available pneumococcal vaccines. Our findings point to the recent introduction and spread of the Spain(23F)-1 clone of penicillin-resistant pneumococci in Vietnam. Simple clinical predictors can be used to guide empirical antibiotic therapy of meningitis. Pneumococcal vaccination may help to control this problem.

Adolescent↗

Mutations in the DNA mismatch repair proteins MutS and MutL of oxazolidinone-resistant or -susceptible Enterococcus faecium.

Mutations in mutS and mutL, which encode DNA mismatch repair (MMR) proteins, can confer hypermutator phenotypes and may facilitate the emergence of mutational antibiotic resistance in bacteria. Linezolid-resistant enterococci (LRE) rarely emerge during therapy and contain mutations in 23S rRNA genes. As enterococci with defective MMR could be prone to the development of oxazolidinone resistance mutations, we investigated 13 clinical isolates of Enterococcus faecium, including 2 LRE, for mutations in mutSL. A 4,944-bp fragment spanning mutSL was sequenced from two pairs of linezolid-resistant (MICs, 64 micro g/ml) and linezolid-susceptible (MICs, 2 micro g/ml) E. faecium isolates (one pair from Austria and one pair from the United Kingdom) identical by pulsed-field gel electrophoresis. The pairs represented distinct strains in which linezolid resistance had emerged during therapy. The MutSL peptides of all four isolates had amino acid substitutions compared with the sequence of E. faecium strain DO (used for genome sequencing). These were Val352Ile (one pair of isolates only) and Met628Leu in MutS and Leu387Pro, Tyr406Phe, Thr415Ser, Phe427Leu, and Phe565Ile in MutL. The significance of these changes remains unknown; these isolates did not show a demonstrable hypermutator phenotype. The same substitutions were found in two of nine geographically diverse linezolid-susceptible enterococcal isolates; the other seven isolates had MutSL sequences identical to that of strain DO. Multilocus sequence typing revealed that all isolates with alternate MutSL peptides belonged to a distinct lineage of a prevalent E. faecium clonal complex, designated CC17. Further studies are needed to investigate the prevalence of these MutSL mutations and their possible roles in the emergence of E. faecium strains resistant to oxazolidinones and other antibiotic classes.

Acetamides↗

Derivatives of a vancomycin-resistant Staphylococcus aureus strain isolated at Hershey Medical Center.

Antimicrobial susceptibilities and genetic relatedness of the vancomycin-resistant Staphylococcus aureus strain (VRSA) isolated at Hershey, Pa. (VRSA Hershey), and its vancomycin-susceptible and high-level-resistant derivatives were studied and compared to 32 methicillin-resistant S. aureus strains (MRSA) isolated from patients and medical staff in contact with the VRSA patient. Derivatives of VRSA were obtained by subculturing six VRSA colonies from the original culture with or without vancomycin. Ten days of drug-free subculture caused the loss of vanA in two vancomycin-susceptible derivatives for which vancomycin MICs were 1 to 4 microg/ml. Multistep selection of three VRSA clones with vancomycin for 10 days increased vancomycin MICs from 32 to 1,024 to 2,048 microg/ml. MICs of teicoplanin, dalbavancin, and oritavancin were also increased from 4, 0.5, and 0.12 to 64, 1, and 32 microg/ml, respectively. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing analysis indicated that VRSA Hershey was the vanA-acquired variety of a common MRSA clone in our hospital with sequence type 5 (ST5). Three of five vancomycin-intermediate S. aureus strains tested from geographically different areas were also ST5, and the Michigan VRSA was ST371, a one-allele variant of ST5. Derivatives of VRSA Hershey had differences in PFGE profiles and the size of SmaI fragment that carries the vanA gene cluster, indicating instability of this cluster in VRSA Hershey. However induction with vancomycin increased glycopeptide MICs and stabilized the resistance.

Alleles↗

High prevalence of antimicrobial resistance among clinical Streptococcus pneumoniae isolates in Asia (an ANSORP study).

A total of 685 clinical Streptococcus pneumoniae isolates from patients with pneumococcal diseases were collected from 14 centers in 11 Asian countries from January 2000 to June 2001. The in vitro susceptibilities of the isolates to 14 antimicrobial agents were determined by the broth microdilution test. Among the isolates tested, 483 (52.4%) were not susceptible to penicillin, 23% were intermediate, and 29.4% were penicillin resistant (MICs >/= 2 mg/liter). Isolates from Vietnam showed the highest prevalence of penicillin resistance (71.4%), followed by those from Korea (54.8%), Hong Kong (43.2%), and Taiwan (38.6%). The penicillin MICs at which 90% of isolates are inhibited (MIC(90)s) were 4 mg/liter among isolates from Vietnam, Hong Kong, Korea, and Taiwan. The prevalence of erythromycin resistance was also very high in Vietnam (92.1%), Taiwan (86%), Korea (80.6%), Hong Kong (76.8%), and China (73.9%). The MIC(90)s of erythromycin were >32 mg/liter among isolates from Korea, Vietnam, China, Taiwan, Singapore, Malaysia, and Hong Kong. Isolates from Hong Kong showed the highest rate of ciprofloxacin resistance (11.8%), followed by isolates from Sri Lanka (9.5%), the Philippines (9.1%), and Korea (6.5%). Multilocus sequence typing showed that the spread of the Taiwan(19F) clone and the Spain(23F) clone could be one of the major reasons for the rapid increases in antimicrobial resistance among S. pneumoniae isolates in Asia. Data from the multinational surveillance study clearly documented distinctive increases in the prevalence rates and the levels of antimicrobial resistance among S. pneumoniae isolates in many Asian countries, which are among the highest in the world published to date.

Anti-Infective Agents↗

Levofloxacin-resistant invasive Streptococcus pneumoniae in the United States: evidence for clonal spread and the impact of conjugate pneumococcal vaccine.

The emergence of fluoroquinolone resistance in sterile-site isolates of Streptococcus pneumoniae is documented in this study characterizing all invasive levofloxacin-resistant (MIC, > or = 8 mg/liter) S. pneumoniae isolates (n = 50) obtained from the Centers for Disease Control and Prevention Active Bacterial Core Surveillance from 1998 to 2002. Resistance among all isolates increased from 0.1% in 1998 to 0.6% in 2001 (P = 0.008) but decreased to 0.4% in 2002, while resistance among vaccine serotypes continued to increase from 0.3% in 1998 to 1.0% in 2002, suggesting that fluoroquinolones continue to exert selective pressure on these vaccine serotypes. Only 22% of resistant isolates were not covered by the conjugate vaccine serogroups. Multilocus sequence typing revealed that 58% of resistant strains were related to five international clones identified by the Pneumococcal Molecular Epidemiology Network, with the Spain(23F)-1 clone being most frequent (16% of all isolates). Thirty-six percent of the isolates were coresistant to penicillin, 44% were coresistant to macrolides, and 28% were multiresistant to penicillin, macrolides, and fluoroquinolones. Fifty percent of the isolates were resistant to any three drug classes. Ninety-four percent of the isolates had multiple mutations in the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes. In 16% of the isolates, there was evidence of an active efflux mechanism. An unusual isolate was found that showed only a single parE mutation and for which the ciprofloxacin MIC was lower (2 mg/liter) than that of levofloxacin (8 mg/liter). Our results suggest that invasive pneumococcal isolates resistant to levofloxacin in the United States show considerable evidence of multiple resistance and of clonal spread.

Anti-Bacterial Agents↗

Molecular evolution perspectives on intraspecific lateral DNA transfer of topoisomerase and gyrase loci in Streptococcus pneumoniae, with implications for fluoroquinolone resistance development and spread.

Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread.

Amino Acid Sequence↗