Natural history of drug-resistant clones of Streptococcus pneumoniae colonizing healthy children in Portugal

A total of 3,539 Streptococcus pneumoniae (Pn) were recovered from 4,969 nasopharyngeal samples of children attending 13 day-care centers (DCCs) located in Lisbon, Portugal, during a surveillance study from January, 2001, through March, 2003, integrated in the European intervention project (EURIS, European Resistance Intervention Study). All Pn isolates were tested for anti-biotyping and drug-resistant pneumococci (DRPn) were further tested by serotyping and pulsed-field gel electrophoresis (PFGE). Overall carriage of Pn was very high (71.2%) and 39.9% of the isolates were resistant to antimicrobials (22.5% with decreased susceptibility to penicillin and 17.4% susceptible to penicillin and resistant to other antimicrobials). Serotypes 6B, 14, 23 F, 19F, and 19 A were prevalent among the 1,287 DRPn and 5.8% of the isolates were non-typeable. Eighty PFGE patterns were identified among 1,285 DRPn, and 93.1% of the DRPn belonged to 26 major clonal types that comprised: Pneumococcal Molecular Epidemiology Network (PMEN) clones (76.3%), Portuguese (PT)-DCC clones, previously detected in 1996-1999 (14.3%), and EURIS PT-DCC new clones, identified for the first time in the EURIS study, during 2001-2003 (9.4%). Comparing with previous Portuguese surveillance studies carried out since 1996, we observed that carriage increased from 47% to 71%, but no major changes were detected on the prevalence of pneumococcal serotypes. Moreover, although PMEN clones were predominant in all DCCs, in the present study the majority of them were gradually decreasing in time whereas several PT-DCC and new clones seemed to be increasing.     

Antimicrobial resistance and serotypes of nasopharyngeal strains of Streptococcus pneumoniae in Brazilian adolescents

The aim of this study was to describe the frequency of antimicrobial-resistance and serotypes of nasopharyngeal pneumococcal isolates from adolescents. Clinical data and nasopharyngeal specimens for culture were collected from 1,013 adolescents as a part of a population-based study. A total of 83 isolates of Streptococcus pneumoniae were identified (8.2%). Seventy-four of the 83 isolates were serotyped. The median age of the 83 adolescents colonized by pneumococci was 14 years (mean 14 +/- 2.2 yrs); 55.4% were males. Intermediate resistance to penicillin was detected in 7.2% (6/83). No strain showed high resistance to penicillin. All isolates were susceptible to clindamycin, chloramphenicol, rifampin, and vancomycin; 37.3%, 18.1%, and 4.8% were resistant to trimethoprim-sulfamethoxazole, tetracycline, and erythromycin, respectively. The most frequent serotypes (5-10% of strains each) were 6B, 6A, 23F, and 18C among 28 serotypes/serogroups identified; 18.9% of the strains were nontypeable (NT). Intermediate resistance to penicillin was detected in serotypes 6B, 14, and NT. The rate of resistance to penicillin of nasopharyngeal isolates is low considering data from other studies about invasive strains recovered from children in Brazil. Serotype patterns are similar, except for type 14, which was unusually infrequent.     

Genetic diversity and clonal patterns among antibiotic-susceptible and -resistant Streptococcus pneumoniae colonizing children: day care centers as autonomous epidemiological units

Characterization by antibiotype of the 1,096 Streptococcus pneumoniae recovered from 2,111 nasopharyngeal samples of children attending 16 day care centers (DCCs) in Lisbon, Portugal, and molecular typing of 413 drug-resistant pneumococci (DRPn) and 89 fully drug-susceptible pneumococci (DSPn) has allowed several conclusions. (i) There was an increase in the frequency of DRPn colonizing children in DCCs from 40% in 1996 to 45% in 1997 to 50% in 1998. (ii) Drug resistance spread by cross-transmission of DRPn clones. A few (8 out of 57) DRPn clones were repeatedly isolated from a large number of children in several DCCs and during each period of surveillance, suggesting the epidemic nature of these clones, which included lineages representing internationally spread S. pneumoniae clones. (iii) Dissemination of resistance determinants among pneumococci colonizing the nasopharynx occurred. Association of identical pulsed-field gel electrophoresis patterns with diverse antibiotypes among pneumococci colonizing children suggests that the high prevalence of DRPn involves not only cross-transmission of resistant strains but also dispersal of resistance genes through recombinational mechanisms. (iv) DCCs are autonomous epidemiological units. Among the 413 DRPn, 57 different lineages were detected; these lineages were dispersed among the 16 DCCs to produce unique microbiological profiles for each of the DCCs. Higher genetic diversity and less sharing of clonal types were observed among the DSPn.     

Genetic relatedness of resistant and multiresistant Streptococcus pneumoniae strains,recovered in the Athens area,to international clones

The prevalence of resistance to antibiotics was examined among 318 Streptococcus pneumoniae strains isolated during 1998 and 1999 in a children's hospital in Athens. The rate of resistance to penicillin was 25.8% (intermediate 22%, resistant 3.8%); 42.5% of the strains were resistant to > or = 1 antibiotic and 20% were multidrug resistant. Resistance to penicillin was lowest in invasive strains (8.3%) and highest in ear isolates (31%). A review of the same microbiology laboratory's records revealed that there has been a gradual increase in penicillin resistance since 1988-1989, when it was 5%. Capsular types were determined for 77 strains resistant to > or = 1 antibiotic, and 69 (90%) of them belonged to the following five serotypes: 19F, 14, 9V, 23F, and 6B. Seventy-five strains were analyzed by pulsed-field gel electrophoresis (PFGE) and 59/75 (79%) shared five electrophoretic types. The largest cluster consisted of 19 serotype 19F strains, of which 18 were nonsusceptible to penicillin and most were multidrug resistant and shared a common and distinct electrophoretic pattern not resembling any known clone. A group of 17 strains that were nonsusceptible to penicillin belonged to serotypes 9V (10), 14 (6), and 19F (1) and shared a common PFGE type similar to the international clone Spain9V-3. Seven serotype 23F strains, of which five were multidrug resistant, belonged to the international clone Spain23F-1. Among the strains susceptible to penicillin but resistant to non-beta-lactam antibiotics, the largest cluster consisted of 13 isolates resistant to erythromycin that belonged to serotype 14 and shared an electrophoretic pattern characteristic of the clone England14-9. Finally, three serotype 6B strains were penicillin susceptible and multidrug resistant and had features similar to the Mediterranean 6B clone. The introduction and spread of several antibiotic-resistant international clones accounts at least in part for the increase in pneumococcal resistance observed in recent years in the Athens metropolitan area.     

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.     

Prevalence of serotypes and molecular epidemiology of Streptococcus pneumoniae strains isolated from children in Beijing, China: identification of two novel multiply-resistant clones

Three-hundred and seventy-six strains of Streptococcus pneumoniae isolated from clinical specimens and nasopharyngeal swabs from children at daycare centers and hospitals in Beijing China, between January 1997 and March 1998, were serotyped. Twenty-seven different serotypes were identified. The most prevalent serotypes in the carriage isolates were 6A, 19F, 23F, and 15 and were found in 66.8% of cases. Serotype data indicate that 51.8% of carrier strains would be included in the 11-valent conjugate vaccine formulation, while inclusion of vaccine-related serotypes, increased the potential vaccine coverage to 79.4%. Serotypes 7, 6B, 23F, 19F, 15, and 3 accounted for 62% of clinical strains, with 70% vaccine-related serotypes. DNA fingerprinting of 47 penicillin resistant and 71 penicillin-susceptible/macrolide-resistant strains by BOX polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), and penicillin binding protein (PBP)-fingerprinting identified two novel clones: one a serotype 23F multiresistant clone resistant to penicillin, tetracycline, erythromycin, clindamycin, and variably resistant to chloramphenicol and trimethoprim-sulphamethoxazole; and the second a multiresistant penicillin-susceptible, macrolide-resistant serotype 6A clone, highly resistant also to tetracycline, clindamycin, and trimethoprim-sulphamethoxazole. The macrolide resistance determinant in 89% of erythromycin-resistant strains tested (penicillin-susceptible and penicillin-resistant) was the erm gene, both the erm and mef genes were simultaneously found in 6%, and mef alone in 3.4%. The data demonstrates that macrolide resistant strains in China include clonal strains and strains with dual mef and erm resistance determinants.     

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.     

Prevalence and patterns of resistance of Streptococcus pneumoniae strains isolated from carriers attending day care centers in the area of Athens

The prevalence and patterns of Streptococcus pneumoniae resistance to antibiotics was examined in 146 nasopharyngeal carrier strains obtained during April and May, 1997, from 382 healthy children attending eight day care centers (DCCs) in the area of Athens. Reduced susceptibility to at least one antibiotic was found in 32.6% as follows: penicillin 11.4% (intermediate), cefotaxime 0.8% (intermediate), trimethoprim-sulfamethoxazole 22.7%, erythromycin 13.6%, tetracycline 11.4%, chloramphenicol 8.3%. Most of the nonsusceptible to penicillin isolates belonged to serogroups 23, 9, and 19. Multidrug resistance was detected in 11.4% of S. pneumoniae isolates including five penicillin nonsusceptible serogroup 23 strains. More than half of the multidrug resistant strains were susceptible to penicillin and belonged to serogroups 6 (4), 23 (1), 19 (1), and 1(1). Strains that belonged to the same serogroup/serotype and had identical resistance patterns appeared to cluster in some DCCs. Antibiotic use in the previous month was associated with reduced susceptibility to penicillin (p = 0.007) and multidrug resistance (p = 0.012). In conclusion, a moderate prevalence of reduced susceptibility to penicillin in pneumococcal carrier strains was found in our community. Multidrug resistance was common and was often associated with susceptibility to penicillin. Several distinct patterns of resistance were observed, suggesting the spread of resistant clones to our country.     

Persistence of two invasive Streptococcus pneumoniae clones of serotypes 1 and 5 in comparison to that of multiple clones of serotypes 6B and 23F among children in southern Israel

We conducted a study to examine the clonal distribution of invasive serotype 1 and 5 isolates as representatives of serotypes that are rarely carried by healthy individuals compared to that of invasive serotype 6B and 23F isolates as representatives of serotypes often carried by young children for prolonged periods. All invasive serotype 1, 5, 6B, and 23F isolates recovered from blood cultures during January 1995 to May 1999 were analyzed; these included 66 serotype 1, 30 serotype 5, 11 serotype 6B, and 15 serotype 23F isolates. One hundred thirty-three nasopharyngeal (NP) isolates of the indicated four serotypes from healthy children were also studied. The strains were characterized using serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis profiling. We found that both invasive and NP serotype 1 and 5 isolates were susceptible to penicillin and that each serotype showed only one clonal type. In contrast, serotype 6B and 23F strains showed different phenotypic characteristics as well as multiple clonal types; 10 clones were identified among 6B isolates, and 11 clones were identified among 23F isolates.     

The Three Major Spanish Clones of Penicillin-Resistant Streptococcus pneumoniae Are the Most Common Clones Recovered in Recent Cases of Meningitis in Spain

One hundred six isolates of Streptococcus pneumoniae recovered in Spain from patients with meningitis in 1997 and 1998 were characterized by multilocus sequence typing. A heterogeneous collection of genotypes was associated with meningitis in Spain: 65 different sequence types were resolved and, even at a genetic distance of 0.43, there were 37 distinct lineages. Thirty-eight percent of the isolates, including all isolates of serotypes 6B, 9V, 14, and 23F, were resistant to penicillin, and 24% of the isolates were members of the three major Spanish penicillin-resistant or multidrug-resistant clones of serotypes 6B, 9V, and 23F or serotype variants of these clones. These three clones (MICs, 1 to 2 microg of penicillin/ml) were the most common clones associated with pneumococcal meningitis in Spain during 1997 and 1998. Only two of the other clones associated with meningitis were penicillin resistant (MICs, 0.12 to 0.5 microg/ml). One of the two most prevalent penicillin-susceptible clones causing meningitis (serotype 3) has not been detected outside of Spain, whereas the other (serotype 18C) has been recovered from patients with meningitis in the United Kingdom, The Netherlands, and Denmark. The prevalence of meningitis caused by isolates of the three major Spanish penicillin-resistant or multiply antibiotic-resistant clones, which are now globally distributed, is disturbing and clearly establishes their ability to cause life-threatening disease.     

Properties of novel international drug-resistant pneumococcal clones identified in day-care centers of Lisbon, Portugal

In this study, 61 drug-resistant Streptococcus pneumoniae strains were characterized by multilocus sequence typing (MLST). These strains were representatives of 26 major clones (defined using pulsed-field gel electrophoresis) accounting for 93% of the 1,285 drug-resistant Streptococcus pneumoniae isolates recovered from the nasopharynges of healthy children attending day-care centers in Lisbon during 2001 to 2003. Using MLST, 13 of the 26 clones were found to be identical or closely related to 11 Pneumococcal Molecular Epidemiology Network (PMEN) clones, 4 clones were found to be unique as there were no identical or highly related allelic profiles deposited in the MLST database, and the remaining 9 clones had sequence types that matched or differed at a single or double locus from allelic profiles available in the MLST database. These nine clones were of serotypes 33F, 10A, 19A, 19F, 6A, 20, 24F, and 3, one was nontypeable, and, by MLST, they were found to be identical or highly related to isolates from disease origin that were dispersed internationally. Since the majority of these clones had serotypes that are not included in the 7-valent conjugate pneumococcal vaccine, monitoring of these clones is important for surveying their possible spread in the future. We propose the inclusion of these novel international clones in the PMEN.     

Antibiotics resistance of Streptococcus pneumoniae. Results from the Brittany registry for 1999

Throughout 1999, clinical microbiology laboratories of 13 hospitals in Brittany have recovered Streptococcus pneumoniae isolates in 832 patients, 312 (37.5%) female and 518 (62.2%) male. Two hundred fifty five of them (30.6%) were children. One hundred eighty eight isolates were recovered from blood cultures (22.6%), 16 from CSF (1.9%), 449 from lungs (54%), and 88 from ear exsudates (10.6%).A 5 microgram oxacillin-disk test was used to detect isolates with reduced susceptibility to penicillin G. Determination of MICs of penicillin G, amoxicillin and cefotaxime were then performed by agar dilution method on 402 strains previously categorized resistant or intermediate.Five hundred forty six isolates were PSDP, 33.5% of them were resistant to penicillin G, 2.2% to amoxicillin and 0.2% to cefotaxime. As expected, a decreased susceptibility to beta-lactamins was frequently associated with resistance to macrolides, cotrimoxazole and tetracycline. Among PSDP, the most prevalent serotypes were 23 (23.7%), 14 (23.5%) and 19 (19.1%).In Brittany, the constant rise of PSDP (1993-1994: 28.5%; 1997: 56.4%; 1999: 65.6 %) could be perhaps explain by analysis of social and demographic data.     

Population structure of invasive <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> isolates in Italy prior to the implementation of the 7-valent conjugate vaccine (1999–2003)

A total of 773 pneumococcal isolates were collected from a nationwide surveillance of invasive pneumococcal diseases during 1999–2003 prior to the implementation of the 7-valent conjugate vaccine (PCV7) in Italy. The isolates included vaccine serotypes (VS, 393 isolates), vaccine-related serotypes (VRS, 93), and nonvaccine serotypes (NVS, 279). The ten most prevalent serotypes were: 14 (16.4%), 3 (8.4%), 23F (8%), 19F (7.4%), 4 (5.9%), 7F (5.8%), 9V (5.3%), 6B (4.9%), 19A (4.7%), and 1 (3.7%). VRS or NVS isolates showed a lower rate of penicillin or drug resistance than VS. Representative isolates of the major VS, VRS, and NVS were genotyped by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The isolates examined were found to belong to 18 international clones and to eight newly described clones. VS isolates sharing clonal groups with VRS or NVS were also detected. Evidence of a past history of capsular switching events was observed in five clones.     

Clonal analysis of Streptococcus pneumoniae nonsusceptible to penicillin at day-care centers with index cases, in a region with low incidence of resistance: emergence of an invasive type 35B clone among carriers

The nasopharyngeal carriage rate of potential respiratory pathogens was studied in 36 index children with a pneumococci nonsusceptible to penicillin (PNSP), in 595 healthy children, and in 123 personnel at 16 day-care centers (DCCs) with index cases in the Stockholm area, an urban area with a low incidence of antibiotic resistant pneumococci, during the winter of 1997-1998. The spread and clonality of PNSP, Haemophilus influenzae and Moraxella catarrhalis, were studied by analyzing antibiotic susceptibility and serotype, and for PSNP also by using pulsed-field electrophoresis (PFGE) and multilocus sequence typing (MLST). In contrast to the low carriage rate found among the adult contacts (2%), 40% of the children harbored pneumococci, of which 20% were PNSP. Nasopharyngeal colonization decreased with age. The 49 PNSP isolates consisted of 20 clones, of which 10 could be identified in more than one child attending the same or different DCCs. In five DCCs, we observed a spread of PNSP from the index case. A novel PNSP clone of type 35B, found to cause invasive disease in several states in the United States, was found to emerge among several carriers at two DCCs . A high proportion of PNSP isolates were multiresistant to antibiotics (34%), which has implications for treatment regimens, even in a country like Sweden where the proportion of PNSP currently is low (3-4%). One PNSP clone of type 9V found among the carriers, has been shown to cause invasive disease in Sweden as well as in other countries, suggesting that one reason for the occurrence of invasive PNSP clones may be their ability to colonize and spread among healthy carriers. Other internationally spread antibiotic resistant pneumococcal clones found were of types 9V, 19F, and 23F.     

Occurrence and Characteristics of Erythromycin-Resistant Streptococcus pneumoniae Strains Isolated in Three Major Brazilian States

We investigated the occurrence and phenotypic and genotypic characteristics of erythromycin-resistant Streptococcus pneumoniae strains isolated in three major states in Brazil, from 1990 to 1999. Of the 931 pneumococcal strains evaluated, 40 (4.3%) were erythromycin-resistant (Ery-R). Among the 40 Ery-R strains, 90.0%, 80.0%, 27.5%, 5.0%, and 2.5% were resistant to tetracycline, trimethoprim-sulfamethoxazole, penicillin, chloramphenicol, and rifampin, respectively. None of the strains were resistant to ofloxacin or to vancomycin. Most [37 (92.5%)] of the 40 Ery-R isolates presented the MLS(B) phenotype and 3 (7.5%) strains showed the M phenotype. PCR testing indicated that all MLS(B) phenotype isolates harbored the erm(B) gene only, whereas the mef(A/E) gene was present in all isolates presenting the M phenotype. The tet(M) gene was the most frequent (86.1%) among Ery-R isolates that were also resistant to tetracycline. Pulsed-field gel electrophoresis (PFGE) analysis after SmaI digestion revealed the occurrence of clonal relationships within groups of strains belonging to serotypes 14, 19A, and 23F. All Ery-R isolates belonging to serotype 14 were susceptible to penicillin and were included in a single clonal group (named Ery(14)-A) related to the England(14-)9 internationally spread clone.     

Serotypes and clonal types of penicillin-susceptible streptococcus pneumoniae causing invasive disease in children in five Latin American countries

We used multilocus sequencing typing (MLST) to determine the genetic backgrounds of 185 recent penicillin susceptible Streptococcus pneumoniae isolates with serotypes that most frequently cause invasive disease in preschool age children in five Latin American countries-Argentina, Brazil, Colombia, Mexico, and Uruguay. Most of the isolates were associated with pneumonia (90/185), meningitis (74/185), and bacteremia (17/185). The collection of strains included seven serotypes-14, 6B, 5, 1, 23 F-which represent the serotypes of S. pneumoniae most frequently associated with sterile site infections in children. Also included were strains expressing serotypes 7F and 3. Comparison of serotype and multilocus sequence type allowed division of the isolates into two groups: strains expressing serotypes 1, 5, 3, and 7 were represented by a relatively few sequence types while strains expressing serotypes 6B, 14, and 23 F showed great genetic diversity. The genetic diversity of serotypes 14, 6B, and 23 F may be related to the capacity of these serotypes to colonize the nasopharynx of healthy carriers during which opportunities for diversification through genetic exchanges can occur. The findings present an interesting contrast with the results of an earlier study in which over 80% of invasive penicillin- resistant serotype 14 and 23 isolates from the same countries were found to belong to as few as two pandemic clones of S. pneumoniae.     

Novel penicillin-, cephalosporin-, and macrolide-resistant clones of Streptococcus pneumoniae serotypes 23F and 19F in Taiwan which differ from international epidemic clones

A cluster (14 of 18) of Streptococcus pneumoniae serotype 23F isolates that were resistant to penicillin (PEN), cephalosporin, and macrolide was found in one day care center in Kaohsiung, Taiwan. We analyzed the 18 isolates by pulsed field gel electrophoresis (PFGE). All but one serotype 23F isolate demonstrated identical PFGE patterns, which were different from the established pattern of the internationally spread Spanish 23F clone. The three strains of serotype 19F also showed a uniform pattern. These data strongly suggest that two novel clones of PEN-, cephalosporin-, and macrolide-resistant S. pneumoniae serotypes 23F and 19F are present in Taiwan.     

Novel antibiotic-resistant pneumococcal strains recovered from the upper respiratory tracts of HIV-infected adults and their children in Kisumu, Kenya

In a survey of genetic diversity within penicillin-nonsusceptible pneumococcal isolates in Kenya, we examined 162 upper respiratory isolates from 104 human immunodeficiency virus (HIV)-infected adults and 46 children in a cotrimoxazole prophylaxis study. Antibiotic resistance levels were high; 152 (94.4%) were cotrimoxazole nonsusceptible (134 fully resistant) and 124 (77%) were intermediately penicillin resistant. Isolates nonsusceptible to penicillin and cotrimoxazole (PNCNP) were found among 24 of the 29 serotypes encountered, 15 of which have rarely or never had documented nonsusceptibility to penicillin. These included serotypes 3, 4, 7C, 7F, 10A, 11A, 13, 15A, 15B, 16F, 17F, 19B, 21, 35A, and 35B. Segments of pbp2b genes from 9 PNCNP (serotypes 3, 13, 15A, 16F, 20, and 35A) were typical of resistance-conferring alleles in that they were highly divergent and contained two substitutions thought to be critical for resistance. Similarly, the dhfr genes from 3 PNCNP were divergent and contained a substitution required for cotrimoxazole resistance. Multilocus sequence typing (MLST) of 48 PNCNP revealed 33 sequence types (STs), none of which were previously recorded at http://www.mlst.net. Comparisons with all known STs revealed that 23 of these STs were unrelated to other known STs, whereas 10 STs were highly related to STs from internationally disseminated strains, including 2 of the 26 antibiotic-resistant clones recognized by the Pneumococcal Molecular Epidemiology Network. Based upon differing serotypes expressed by strains of identical or closely similar genotypes, there has been an extensive history of capsular switching within seven genetic clusters represented by these 10 STs and related STs described at http://www.mlst.net.     

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.     

Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae and molecular characterization of multidrug-resistant serotype 19F, 6B,and 23F Pneumococci in northern Thailand

Penicillin-resistant Streptococcus pneumoniae is widely spread worldwide. Our study was undertaken to examine the susceptibility and serotypes of S. pneumoniae in northern Thailand. Ninety-three S. pneumoniae strains were isolated from 93 patients at Chiang Mai University Hospital, Chiang Mai, Thailand, from September 1999 to June 2000. The strains were isolated from sputum (n = 51), blood (n = 15), nasopharynges (n = 14), and other sources (e.g., pus, ears, ascites, and cerebrospinal fluid) (n = 13). Of the 93 isolates, 29 (31.2%) were susceptible, 24 (25.8%) showed intermediate resistance (MIC, 0.12 to 1.0 micro g/ml), and 40 (43.0%) were fully resistant (MIC, >/=2.0 micro g/ml) to penicillin G. Seven (46.7%) from blood, 5 (35.7%) from nasopharynges, 15 (29.4%) from sputum, and 2 (15.4%) from other sources were susceptible isolates. Serotyping with the use of antiserum revealed differences in the predominant types that were susceptible (6A, 11A, and 19A), intermediately resistant (6B and 23F), and fully resistant (6B, 19F, and 23F). Molecular typing by pulsed-field gel electrophoresis of multidrug-resistant pneumococci showed four patterns (A, B, C, and D) for 16 isolates of serotype 19F, with pattern B being predominant (12 isolates). This finding was different from that with the Taiwan multidrug-resistant serotype 19F clone. Eleven isolates of serotype 6B all showed pattern E, and nine isolates of serotype 23F showed two patterns (F and G), with pattern F being predominant (seven isolates). This finding was similar to that with the Spanish multidrug-resistant serotype 23F clone. Our results indicated that the resistance of pneumococci to antibiotics in northern Thailand is progressing rapidly and that effort should be intensified to prevent any spread of pandemic multidrug-resistant serotypes 19F, 6B, and 23F.