Molecular epidemiology of macrolide-resistant isolates of Streptococcus pneumoniae collected from blood and respiratory specimens in Norway

Norway has a low prevalence of antimicrobial resistance, including macrolide-resistant Streptococcus pneumoniae (MRSP). In a nationwide surveillance program, a total of 2,200 S. pneumoniae isolates were collected from blood cultures and respiratory tract specimens. Macrolide resistance was detected in 2.7%. M-type macrolide resistance was found in 60% of resistant isolates, and these were mainly mef(A)-positive, serotype-14 invasive isolates. The erm(B)-encoded macrolide-lincosamide-streptogramin B (MLS(B)) type dominated among the noninvasive isolates. One strain had an A2058G mutation in the 23S rRNA gene. Coresistance to other antibiotics was seen in 96% of the MLS(B)-type isolates, whereas 92% of the M-type isolates were susceptible to other commonly used antimicrobial agents. Serotypes 14, 6B, and 19F accounted for 84% of the macrolide-resistant isolates, with serotype 14 alone accounting for 67% of the invasive isolates. A total of 29 different sequence types (STs) were detected by multilocus sequence typing. Twelve STs were previously reported international resistant clones, and 75% of the macrolide-resistant isolates had STs identical or closely related to these clones. Eleven isolates displayed 10 novel STs, and 7/11 of these "Norwegian strains" coexpressed MLS(B) and tetracycline resistance, indicating the presence of Tn1545. The invasive serotype-14 isolates were all classified as ST9 or single-locus variants of this clone. ST9 is a mef-positive M-type clone, commonly known as England(14)-9, reported from several European countries. These observations suggest that the import of major international MRSP clones and the local spread of Tn1545 are the major mechanisms involved in the evolution and dissemination of MRSP in Norway.     

Molecular epidemiology of penicillin-susceptible non-beta-lactam-resistant Streptococcus pneumoniae isolates from Greek children

A total of 128 Streptococcus pneumoniae isolates that were susceptible to penicillin but resistant to non-beta-lactam agents were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and antibiotic resistance genotyping. The serotypes 6A/B (49%), 14 (14%), 19A/F (11%), 11A (9%), 23A/F (4%), 15B/C (2%), and 21 (2%) were most prevalent in this collection. Of the isolates, 65% were erythromycin resistant, while the remaining isolates were tetracycline and/or trimethoprim-sulfamethoxazole resistant. Fifty-nine distinct RFEL types were identified. Twenty different RFEL clusters, harboring 2 to 19 strains each, accounted for 76% of all strains. Confirmatory multilocus sequence typing analysis of the genetic clusters showed the presence of three international clones (Tennessee(23F)-4, England(14)-9, and Greece(6B)-22) representing 30% of the isolates. The erm(B) gene was present in 70% of the erythromycin-resistant isolates, whereas 18 and 8% contained the mef(A) and mef(E) genes, respectively. The pneumococci representing erm(B), erm(A), and mef genes belonged to distinct genetic clusters. In total, 45% of all isolates were tetracycline resistant. Ninety-six percent of these isolates contained the tet(M) gene. In conclusion, penicillin-susceptible pneumococci resistant to non-beta-lactams are a genetically heterogeneous group displaying a variety of genotypes, resistance markers, and serotypes. This suggests that multiple genetic events lead to non-beta-lactam-resistant pneumococci in Greece. Importantly, most of these genotypes are capable of disseminating within the community.     

Molecular epidemiology of erythromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites

Erythromycin-resistant isolates of Streptococcus pneumoniae from blood cultures and noninvasive sites were studied over a 3-year period. The prevalence of erythromycin resistance was 11.9% (19 of 160) in blood culture isolates but 4.2% (60 of 1,435) in noninvasive-site isolates. Sixty-two of the 79 resistant isolates were available for study. The M phenotype was responsible for 76% (47 of 62) of resistance, largely due to a serotype 14 clone, characterized by multilocus sequence typing as ST9, which accounted for 79% (37 of 47) of M phenotype resistance. The ST9 clone was 4.8 times more common in blood than in noninvasive sites. All M phenotype isolates were PCR positive for mef(A), but sequencing revealed that the ST9 clone possessed the mef(A) sequence commonly associated with Streptococcus pyogenes. All M phenotype isolates with this mef(A) sequence also had sequences consistent with the presence of the Tn1207.1 genetic element inserted in the celB gene. In contrast, isolates with the mef(E) sequence normally associated with S. pneumoniae contained sequences consistent with the presence of the mega insertion element. All MLS(B) isolates carried erm(B), and two isolates carried both erm(B) and mef(E). Fourteen of the 15 MLS(B) isolates were tetracycline resistant and contained tet(M). However, six M phenotype isolates of serotypes 19 (two isolates) and 23 (four isolates) were also tetracycline resistant and contained tet(M). MICs for isolates with the mef(A) sequence were significantly higher than MICs for isolates with the mef(E) sequence (P < 0.001). Thus, the ST9 clone of S. pneumoniae is a significant cause of invasive pneumococcal disease in northeast Scotland and is the single most important contributor to M phenotype erythromycin resistance.     

Molecular epidemiology of penicillin-non-susceptible Streptococcus pneumoniae isolates from children with invasive pneumococcal disease in Germany

A population-based nationwide surveillance of antibiotic resistance associated with invasive pneumococcal disease (IPD) in children and adolescents (aged<16 years) was performed in Germany between 1997 and 2004. In total, 1517 isolates were collected, of which 5.1% and 1.1% were intermediately- or fully-resistant, respectively, to penicillin G. During the 8-year study period, an increase in resistance to both penicillin G and erythromycin A was observed, and the frequency of isolates exhibiting reduced susceptibility to penicillin G or erythromycin A increased from 1.4% and 11.1%, respectively, in 1997, to 8.7% and 29.0%, respectively, in 2004. Among the penicillin non-susceptible pneumococcal isolates, serotypes 14 (24.5% of isolates), 23F (16.0%) and 6B (16.0%) were found most frequently. Multilocus sequence typing of 58 (62%) penicillin G non-susceptible isolates revealed that sequence type (ST) 156 (Spain9V-3 clone) and its single-locus variant ST 557 were widespread in Germany. Moreover, 17 new penicillin G non-susceptible STs were defined for the first time. The study illustrated the genetic heterogeneity of antibiotic-resistant pneumococcal isolates in Germany.     

Molecular epidemiology of penicillin-nonsusceptible Streptococcus pneumoniae among children in Greece

A total of 145 penicillin-nonsusceptible Streptococcus pneumoniae strains were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and penicillin-binding protein (PBP) genotyping. The serotypes 23A and 23F (54%), 19A and 19F (25%), 9V (5%), 15A, 15B, and 15C (4%), 6A and 6B (4%), and 21 (4%) were most prevalent in this collection. Fifty-three distinct RFEL types were identified. Sixteen different RFEL clusters, harboring 2 to 32 strains each, accounted for 82% of all strains. Eight of these genetic clusters representing 60% of the strains were previously identified in other countries. A predominant lineage of 66 strains (46%) harboring five RFEL types and the serotypes 19F and 23F was closely related to the pandemic clone Spain(23F)-1 (genetic relatedness of > or =85%). Another lineage, representing 11 strains, showed close genetic relatedness to the pandemic clone France(9V)-3. Another lineage of 8 serotype 21 strains was Greece specific since the RFEL types were not observed in an international collection of 193 genotypes from 16 different countries. Characterization of the PBP genes pbp1a, pbp2b, and pbp2x revealed 20 distinct PBP genotypes of which PBP type 1-1-1, initially observed in the pandemic clones 23F and 9V, was predominantly present in 11 RFEL types in this Greek collection of penicillin-nonsusceptible strains (55%). Sixteen PBP types covering 52 strains (36%) were Greece specific. This study underlines the strong contribution of penicillin-resistant international clones to the prevalence and spread of penicillin-nonsusceptible pneumococci among young children in Greece.     

Characterization of ermB gene transposition by Tn1545 and Tn917 in macrolide-resistant Streptococcus pneumoniae isolates

In Streptococcus pneumoniae, the ermB gene is carried by transposons, such as Tn917 and Tn1545. This study investigated the relationship between macrolide resistance and the presence of the ermB gene on Tn917 or Tn1545 in 84 Japanese pneumococcal isolates. Macrolide-resistant strains were classified into two groups as follows. Group 1 (19 strains) showed a tendency to high resistance to erythromycin (MIC at which 50% of isolates are inhibited, 4 mg/liter; MIC at which 90% of isolates are inhibited [MIC(90)], 128 mg/liter) but susceptibility to rokitamycin (MIC(90), 1 mg/liter), with the ermB gene located on Tn1545. Group 2 (65 strains) showed a tendency to high resistance to both antibiotics (MIC(90)s for both erythromycin and rokitamycin, >128 mg/liter), with the ermB gene located on Tn917. There were no strains with constitutive macrolide resistance in either group. All of the strains in group 2 had a deletion in the promoter region of ermB and an insertion of the TAAA motif in the leader peptide. The results of pulsed-field gel electrophoresis and serogrouping showed that Tn1545 spread clonally while Tn917 spread both horizontally and clonally. In conclusion, in Japanese macrolide-resistant S. pneumoniae isolates, the ermB gene is carried and spread primarily by Tn917.     

Molecular epidemiology of clinical isolates of ampc producing Klebsiella pneumoniae

Purpose: AmpC producing K. pneumoniae have been increasingly reported from India but epidemiological studies are lacking. In the present study, molecular epidemiology of extended-spectrum AmpC beta-lactamases (ESACs) producing clinical isolates of K. pneumoniae prevalent in our hospital was studied. Methods: Fifty-one non-repeat, consecutive, clinical isolates of K. pneumoniae producing AmpC enzymes, were subjected to whole cell protein profile analysis (SDS-PAGE) and ribotyping. The antimicrobial susceptibility was determined using standard disk diffusion technique. The isolates showing decreased susceptibility to cefoxitin (<18 mm) or cefotetan (<16 mm) were subjected to modified three-dimensional test for detection of AmpC enzyme. Results: Six different types of protein profiles were observed. Ribotyping could further discriminate between the strains that were clustered by protein fingerprinting. Twelve different ribo-patterns were identified. Ribotyping was found to have a better Discriminatory Index (0.98) than that of SDS-PAGE (0.78). Of the 26 isolates that showed decreased susceptibility to cefoxitin and/or cefotetan 13 isolates were found to harbour AmpC enzyme. Conclusions: The study demonstrated the usefulness of SDS-PAGE whole cell protein profile analysis and ribotyping to identify the clonality of the ESACs isolates, the latter having a higher discriminatory power. The presence of ESACs isolates in the community as well as in hospital settings emphasizes the need for regular monitoring of antimicrobial resistance.     

Decline in macrolide-resistant Streptococcus pyogenes isolates from French children

We studied the macrolide resistance and serotypes of 585 group A streptococcus (GAS) isolates collected from French children with pharyngitis. Nineteen isolates (3.2%) were erythromycin-resistant and harbored the following resistance genes: 31.6% mef(A), 15.8% erm(A), and 52.6% erm(B). The 19 isolates included 7 different emm types (4, 1, 11, 2, 28, 12, and 77) and 7 corresponding multilocus sequence types. The current fall in macrolide consumption has led to a very low rate of GAS macrolide resistance.     

Molecular epidemiology of Streptococcus uberis isolates from dairy cows with mastitis

Pulsed-field gel electrophoresis and antimicrobial sensitivity testing were used as tools to investigate the epidemiology of Streptococcus uberis mastitis in dairy cows. A total of 62 different strains were found among 138 isolates from the four herds investigated, and between 10 and 26 different strains were found in each herd. There was no strain common to all four herds. Identical strains of S. uberis were detected from different quarters of individual cows and from cows within the same herd, suggesting that transmission from quarter to quarter and cow to cow had occurred. Despite the great variation in S. uberis strains, persistent infection with the same strain within a lactation was observed in most cows. Predominant strains were present in two herds. Preliminary investigations could not clarify why these particular strains might predominate, but in one herd there was a significant difference between the prevalence of clinical mastitis in quarters infected with the predominant strain and that in quarters infected with other strains, suggesting the greater virulence of the predominant strain. The wide variety of S. uberis strains found is consistent with an environmental source of S. uberis. However, evidence of direct transmission, the persistence of infection, and the predominance of particular strains in some herds indicate that S. uberis infections are epidemiologically complex and that the relative importance of these factors in the occurrence of mastitis may differ between herds.     

Molecular epidemiology of extended-spectrum beta-lactamase-producing,fluoroquinolone-resistant isolates of Klebsiella pneumoniae in Taiwan

Strains of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) have emerged worldwide. Concomitant ciprofloxacin resistance with ESBL production in K. pneumoniae isolates would severely restrict treatment options. Among 39 (18.5%) of 211 ESBL-KP isolates resistant to ciprofloxacin (MIC, >/=4 micro g/ml), 37 (95%) were high level resistant (MIC, >/=16 micro g/ml). These isolates were also cross resistant to the newer fluoroquinolones, including levofloxacin, gatifloxacin, gemifloxacin, and garenoxacin (BMS 284756). Sitafloxacin was most active against these ciprofloxacin-resistant ESBL-KP isolates with MICs for 67% of the isolates being 相似文献   

Dissemination of macrolide-resistant Streptococcus pneumoniae isolates containing both erm(B) and mef(A) in South Korea

Macrolide resistance was detected in 64 of 77 (83.1%) Streptococcus pneumoniae isolates from South Korea. Seven (10.9%) isolates contained only mef(A), 32 (50%) contained only erm(B), and 25 (39.1%) contained mef(A) and erm(B). Nineteen isolates containing mef(A) and erm(B) belonged to serotype 19F, and seven isolates were identical to the Taiwan(19F)-14 clone.     

Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae in a university hospital, Ankara, Turkey

Penicillin-resistant Streptococcus pneumoniae isolates (n = 76) from clinical samples of patients admitted to Hacettepe University Hospital between January 1997 and December 2001 were included in the study. MICs of penicillin G, erythromycin A, clindamycin, cefaclor, cefotaxime, vancomycin, chloramphenicol, tetracycline, ciprofloxacin and rifampicin were determined by agar dilution. The isolates were serogrouped on the basis of the Neufeld Quellung reaction and were typed by BOX-PCR. Genetic polymorphism of the penicillin resistance genes pbp2b and pbp2x was investigated by restriction fragment length polymorphism (RFLP) analysis. Of the 76 isolates tested, 64 (84.2%) showed intermediate resistance to penicillin, while 12 (15.8%) were resistant to higher levels of penicillin (MIC > or = 2 mg/L). The resistance patterns of the isolates revealed six different resistance profiles. There were 22 different serotypes, with c. 55% of the isolates belonging to serotypes 23B, 19A, 19F, 14, 6 A and 9V. Five distinct patterns for pbp2b and 12 distinct patterns for pbp2x were obtained by RFLP analysis of penicillin-binding protein genes. The combination of these patterns allowed isolates to be classified into 22 fingerprint subgroups. BOX-PCR analysis showed that the isolates fell into 14 distinct BOX genotypes, with 33 subtypes. Serotype 9V isolates with pbp genotype 2-6 and BOX-PCR type 4, 4.1 or 4.2 were related to the pandemic clone Spain(9V)-3. No relatedness to other international clones was detected among the other study strains, but genetic relatedness was observed among some of the serotype 19A and 23B isolates. Overall, the results demonstrated that most of the penicillin-resistant pneumococcal isolates in Turkey, other than those belonging to serotypes 9V, 19A and 23B, were derived from several independent clones, possibly resulting from multiple importation of strains originating from outside the country. Differences in pbp patterns, serotypes and resistance profiles among isolates that showed similar BOX-PCR patterns supported the hypothesis that horizontal transfer of capsular genes, pbp genes and other genetic determinants between S. pneumoniae and viridans group streptococci may have occurred.     

Macrolide-resistance mechanisms in Streptococcus pneumoniae isolates from Belgium

Of 233 erythromycin-resistant pneumococcal isolates collected in Belgium in 1999-2000, 89.7% carried the erm(B) gene, 6% the mef(A) gene, and 3.5%erm(B) plus mef(A). Two isolates contained neither erm(B) nor mef(A); one contained an erm(A) subclass erm(TR) gene, while the other contained an A2058G mutation in domain V of the 23S rRNA gene. Of 209 erm(B)-positive isolates, 191 had clindamycin MICs > 16 mg/L and 18 had MICs < or = 16 mg/L. Mef(A)-positive isolates all displayed the M resistance phenotype. Telithromycin remained active against erythromycin-resistant isolates, with the highest telithromycin MIC50 being found in mef(A)-positive isolates. No difference in the prevalence of different resistance mechanisms was observed compared to isolates collected in 1995-1997.     

Molecular characterization of clinical Streptococcus pneumoniae isolates with reduced susceptibility to fluoroquinolones emerging in Italy

Fifteen Streptococcus pneumoniae clinical isolates with reduced fluoroquinolone susceptibility (defined as a ciprofloxacin MIC of > or = 4 microg/ml), all collected in Italy in 2000-2003, were typed and subjected to extensive molecular characterization to define the contribution of drug target alterations and efflux mechanisms to their resistance. Serotyping and pulsed-field gel electrophoresis analysis indicated substantial genetic unrelatedness among the 15 isolates, suggesting that the new resistance traits arise in multiple indigenous strains rather than through clonal dissemination. Sequencing of the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE demonstrated that point mutations producing single amino acid changes were more frequent in topoisomerase IV (parC mutations in 14 isolates and parE mutations in 13) than in DNA gyrase subunits (gyrA mutations in 7 isolates and no gyrB mutations observed). No isolate displayed a quinolone efflux system susceptible to carbonyl cyanide m-chlorophenylhydrazone; conversely, four-fold or greater MIC reductions in the presence of reserpine were observed in all 15 isolates with ethidium bromide, in 13 with ulifloxacin, in 9 with ciprofloxacin, in 5 with norfloxacin, and in none with five other fluoroquinolones. The effect of efflux pump activity on the level and profile of fluoroquinolone resistance in our strains was minor compared with that of target site modifications. DNA mutations and/or efflux systems other than those established so far might contribute to the fluoroquinolone resistance expressed by our strains. Susceptibility profiles to nonquinolone class antibiotics and resistance-associated phenotypic and genotypic characteristics were also determined and correlated with fluoroquinolone resistance. A unique penicillin-binding protein profile was observed in all five penicillin-resistant isolates, whereas the same PBP profile as S. pneumoniae R6 was exhibited by all six penicillin-susceptible isolates. This is the first attempt to molecularly characterize clinical isolates of S. pneumoniae with reduced susceptibility to fluoroquinolones emerging in Italy.     

Clinical and microbiological epidemiology of Streptococcus pneumoniae bacteraemia

A survey of Streptococcus pneumoniae bacteraemia in the Grampian region of Scotland was carried out over a 2-year period. One hundred and four bacteraemic episodes were identified in 103 patients, an incidence of 9.8/100,000 population/year, and the mortality was 24%. Clinical information was abstracted from 92 sets of patient notes and 98 isolates of S. pneumoniae were available for further study. The incidence of S. pneumoniae bacteraemia was highest at the extremes of age and peaked at 78 cases/100,000 population/year in those over 80 years old. Many patients had predisposing conditions, of which chronic lung disease (23%), chronic alcohol abuse (10%) and malignant disease (10%) were the commonest. Age was the highest risk factor for mortality, with 20 of the 22 deaths in those over 65 years old. The commonest serotype of S. pneumoniae isolated was serotype 14 (23.5%). Only one isolate (serotype 6A) showed intermediate resistance to penicillin, but 12 isolates (12.2%) were resistant to erythromycin. Nine of these 12 isolates were of serotype 14 and had MICs clustered in the range 12-24 mg/L. Examination of all serotype 14 isolates by pulsed-field gel electrophoresis (PFGE) showed the presence of two distinct genetic clusters, with all the erythromycin-resistant isolates in the same cluster. These isolates had similar PFGE profiles to erythromycin-resistant serotype 14 strains isolated elsewhere in the UK and they were positive for the mefE gene by PCR, confirming that resistance was of the M phenotype. The recent increase in erythromycin resistance in S. pneumoniae may be due, at least in part, to the spread of a serotype 14 clone of the M phenotype which appears to be an important cause of invasive disease.     

Molecular epidemiology of Streptococcus pneumoniae with decreased susceptibility to penicillin in a Paris children's hospital.

Among pneumococci with decreased susceptibility or pneumococci resistant to penicillin (PRP) isolated at Armand-Trousseau children's hospital, those expressing capsular serotypes 23F, 9V, and 14 were the most frequently isolated. We compared 53 clinical isolates (14 type 9V, 26 type 23F, and 13 type 14) by analysis of chromosomal macrorestriction patterns and DNA restriction patterns of the penicillin-binding protein (PBP) genes pbp 2b, pbp 2x, and pbp 1a. All 9V isolates originated from the same clone. Five 23F clones were distinguished, the largest of which comprised 20 isolates. The main type 14 clone comprised nine isolates; three other type 14 strains were closely related to the 9V clone, probably by horizontal transfer of capsular biosynthesis genes. Most 23F and type 14 isolates shared the same PBP gene restriction patterns as the 9V clone, suggesting horizontal transfer of altered PBP genes.     

Multiclonal origin of macrolide-resistant Mycoplasma pneumoniae isolates as determined by multilocus variable-number tandem-repeat analysis

There was a high percentage of macrolide resistance in Mycoplasma pneumoniae clinical isolates in China. The genetic relatedness of macrolide-resistant M. pneunomiae strains was investigated using the multilocus variable-number tandem-repeat assay (MLVA). Among 152 M. pneunomiae isolates, the 137 macrolide-resistant strains were clustered into 15 MLVA types, indicating that the high macrolide resistance rate in M. pneumoniae is a result of the dissemination of the multiple resistant clones.     

Antimicrobial susceptibility of invasive isolates of Streptococcus pneumoniae in Ireland

Between January 1999 and June 2002, 646 invasive isolates of Streptococcus pneumoniae were collected in Ireland. MICs of penicillin, ciprofloxacin, cefotaxime, moxifloxacin and linezolid were determined by Etest methodology. Eighty-seven (13.5%) isolates showed intermediate resistance to penicillin, while seven (1.1%) showed high-level resistance. Eighty-seven (13.5%) isolates were resistant to erythromycin, but all isolates were susceptible to cefotaxime, moxifloxacin and linezolid. The prevalence of pneumococcal isolates non-susceptible to penicillin in Ireland is worryingly high, but currently there are alternative agents available to treat invasive infection.     

Molecular characterization of non-penicillin-susceptible Streptococcus pneumoniae in Norway

A total of 125 non-penicillin-susceptible Streptococcus pneumoniae isolates were received at the Norwegian Institute of Public Health in the period from 1995 to 2001. The strains were tested for antimicrobial susceptibility, serotyped, and genotyped by multilocus sequence typing (MLST); and their penicillin-binding proteins (PBPs) were typed by restriction fragment length polymorphism analysis of their pbp genes. Of the 125 strains, 48 (38%) were fully resistant to penicillin and 77 (62%) were intermediately resistant to penicillin. Most of the strains resistant to penicillin were also resistant to one or several additional antibiotics. The most frequent serotypes among the non-penicillin-susceptible strains were 14, 9V, 19F, 23F, and 6B. MLST analysis showed a high degree of genetic diversity among the 119 strains tested, with a total of 74 different sequence types. Six of the 26 internationally known resistant clones were present; the Spain(9V)-3 clone was the most frequent, with 19 isolates. A total of 74 (62%) of the isolates were related to 1 of the 26 international clones. Restriction enzyme analyses of the pbp1a, pbp2b, and pbp2x genes revealed 12, 12, and 19 different patterns, respectively; and a total of 43 different PBPs types were demonstrated. Our data indicate that the non-penicillin-susceptible strains in Norway are highly diverse genetically and that limited spread of the internationally known resistant strains occurred in the country in the period examined.