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1.  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 (MIC90s) 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 MIC90s 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 Taiwan19F clone and the Spain23F 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.
doi:10.1128/AAC.48.6.2101-2107.2004
PMCID: PMC415617  PMID: 15155207
2.  Emergence in Vietnam of Streptococcus pneumoniae Resistant to Multiple Antimicrobial Agents as a Result of Dissemination of the Multiresistant Spain23F-1 Clone 
Antimicrobial Agents and Chemotherapy  2002;46(11):3512-3517.
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, ≥0.1 μg/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, ≥2.0 μg/ml) increased from 0% (0 of 24) to 28% (10 of 36) (P = 0.002). Only one isolate was ceftriaxone resistant (MIC, 2.0 μg/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 Spain23F-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 Spain23F-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.
doi:10.1128/AAC.46.11.3512-3517.2002
PMCID: PMC128725  PMID: 12384358
3.  Prevalence of antimicrobial drug-resistant Streptococcus pneumoniae in Washington State. 
Western Journal of Medicine  1998;169(6):364-369.
We conducted a survey to assess the prevalence and geographic distribution of antimicrobial drug resistance among invasive isolates of Streptococcus pneumoniae in Washington State. Sequential sterile-site pneumococcal isolates were submitted from 13 hospital laboratories between 1 October 1995 and 30 January 1997. We serotyped 275 isolates from adults and children and determined minimum inhibitory concentrations (MIC) for commonly used antimicrobial drugs. Data were abstracted from medical records to compare differences in outcome and risk factors for infection. Of the 275 isolates, 73 (26.5%) were nonsusceptible to one or more antimicrobial drugs. Penicillin-nonsusceptible pneumococci (PNSP, MIC > or = 0.1 microgram/ml) accounted for 42 (15.3%) of the 275 isolates including 4 (1.5%) resistant strains (MIC > or = 2 micrograms/ml). The 42 PNSP included serogroups 6, 9, 14, 19, and 23, all of which are represented in the 23-valent pneumococcal vaccine. PNSP were also nonsusceptible to trimethoprim/sulfamethoxazole (92.9%), erythromycin (38.1%), imipenem (28.6%), and ceftriaxone (23.8%). Forty-seven (17.1%) of the 275 isolates were multiple drug-nonsusceptible pneumococci (MDNSP). A significantly greater number of patients < or = 12 years of age were infected with MDNSP compared with those > 12 years. Prior use of antimicrobial drugs and an immunosuppressive disorder were risk factors for infection with PNSP. In summary, pneumococci nonsusceptible to penicillin and other antimicrobial drugs are prevalent among adults with invasive pneumococcal disease in Washington State. A large proportion of PNSP are resistant to other commonly used antimicrobial drugs. Local antibiotic susceptibility data should be considered when designing empiric treatment regimens.
PMCID: PMC1305404  PMID: 9866434
4.  In Vitro Antimicrobial Susceptibilities of Streptococcus pneumoniae Clinical Isolates Obtained in Canada in 2002 
Empirical treatment is best guided by current surveillance of local resistance patterns. The goal of this study is to characterize the prevalence of antimicrobial nonsusceptibility within pneumococcal isolates from Canada. The Canadian Bacterial Surveillance Network is comprised of laboratories from across Canada. Laboratories collected a defined number of consecutive clinical and all sterile site isolates of S. pneumoniae in 2002. In vitro susceptibility testing was performed by broth microdilution with NCCLS guidelines. Rates of nonsusceptibility were compared to previously published reports from the same network. A total of 2,539 isolates were tested. Penicillin nonsusceptibility increased to 15% (8.5% intermediate, 6.5% resistant) compared to 12.4% in 2000 (P ≤ 0.025, χ2). Only 32 (1.3%) isolates had an amoxicillin MIC of ≥4 μg/ml and only 2 of 32 cerebrospinal fluid isolates had an intermediate susceptibility to ceftriaxone by meningeal interpretive criteria (MIC = 1 μg/ml). A total of 354 (13.9%) isolates were macrolide nonsusceptible (46.3% MLSB, 56.7% M phenotype), increasing from 11.4% in 2000 (P ≤ 0.0075, χ2). Only 13 (<1%) isolates had a telithromycin MIC of >1 μg/ml. Ciprofloxacin nonsusceptibility (defined as an MIC of ≥4 μg/ml) increased to 2.7% compared to 1.4% in 2000 (P ≤ 0.0025, χ2) and was primarily found in persons ≥18 years old (98.5%). Nonsusceptibility to penicillin, macrolides, and fluoroquinolones is increasing in Canada. Nonsusceptibility to amoxicillin and ceftriaxone remains uncommon. Newer antimicrobials such as telithromycin and respiratory fluoroquinolones have excellent in vitro activity.
doi:10.1128/AAC.48.9.3305-3311.2004
PMCID: PMC514745  PMID: 15328089
5.  Nasopharyngeal Carriage of Streptococcus pneumoniae in Healthy Children: Implications for the Use of Heptavalent Pnemococcal Conjugate Vaccine 
Emerging Infectious Diseases  2002;8(5):479-484.
We assessed the prevalence of Streptococcus pneumoniae serotypes in the nasopharynx of healthy children, antimicrobial susceptibility patterns, risk factors for carriage, and the coverage of heptavalent pneumococcal conjugate vaccine. In 2,799 healthy infants and children, the S. pneumoniae carrier rate was 8.6% (serotypes 3, 19F, 23F, 19A, 6B, and 14 were most common). Most pneumococci (69.4%) were resistant to one or more antimicrobial classes. The rate of penicillin resistance was low (9.1%); macrolide resistance was high (52.1%). Overall, 63.2% of the isolates belonged to strains covered by the heptavalent pneumococcal vaccine. This percentage was higher in children <2 years old (73.1%) and in those >2-5 years old(68.9%). Sinusitis in the previous 3 months was the only risk factor for carrier status; acute otitis media was the only risk factor for the carriage of penicillin-resistant S. pneumoniae. Most the isolated strains are covered by the heptavalent conjugate vaccine, especially in the first years of life, suggesting that its use could reduce the incidence of pneumococcal disease.
doi:10.3201/eid0805.010235
PMCID: PMC2732490  PMID: 11996682
Streptococcus pneumoniae; nasopharyngeal carriage; epidemiology; conjugate vaccine; children
6.  Phenotypic and Genotypic Characterization of Streptococcus pneumoniae Strains Colonizing Children Attending Day-Care Centers in Norway▿  
Journal of Clinical Microbiology  2008;46(8):2508-2518.
A cross-sectional study of nasopharyngeal colonization with Streptococcus pneumoniae was performed among 573 children attending 29 day-care centers (DCCs) in Norway prior to the start of mass vaccination with the heptavalent pneumococcal conjugate vaccine (PCV-7). A sensitive sampling method was employed, including transport in an enrichment broth and serotyping of pneumococci directly from the broth, in addition to traditional single-colony isolation from blood agar plates. The prevalence of carriage was high, peaking at 88.7% in 2-year-olds. More than one serotype was isolated from 12.7% of the carriers. Of 509 isolates obtained, 227 (44.6%) belonged to the PCV-7 serotypes. Penicillin nonsusceptibility was rare (1.8% of the isolates). Nonsusceptibility to erythromycin (5.9%), clindamycin (2.0%), and tetracycline (5.5%) was associated with PCV-7 serotypes (P < 0.001). Multilocus sequence typing was performed on the whole strain collection, revealing 102 sequence types (STs), of which 31 (30.4%) were novel. Eleven isolates (2.2%) belonged to the England14-9 clone, and 19 isolates (3.7%) belonged to, or were single-locus variants of, the Portugal19F-21 clone. The pneumococcal populations within the DCCs were composed of a majority of isolates with STs shared between the DCCs and a minority of isolates with STs unique for each DCC. The highest numbers of different STs, including novel STs, were found within the most frequent serotypes. Our study indicates that carriage of S. pneumoniae is highly prevalent among children in Norwegian DCCs, with a genetically diverse pneumococcal population consisting of unique microepidemic DCC populations.
doi:10.1128/JCM.02296-07
PMCID: PMC2519506  PMID: 18524970
7.  In Vitro Activities of Broad-Spectrum Cephalosporins against Nonmeningeal Isolates of Streptococcus pneumoniae: MIC Interpretation Using NCCLS M100-S12 Recommendations 
Journal of Clinical Microbiology  2002;40(2):669-674.
Publication of the NCCLS M100-S12 document in January 2002 introduced ceftriaxone and cefotaxime MIC interpretative breakpoints of ≤1 μg/ml (susceptible), 2 μg/ml (intermediate), and ≥4 μg/ml (resistant) for nonmeningeal isolates of Streptococcus pneumoniae. To estimate the effect of these breakpoint changes on clinical laboratory susceptibility testing results, nonmeningeal pneumococcal isolate (blood and respiratory) data from The Surveillance Network Database-USA, an electronic surveillance database, for the years 1996 to 2000 were collated and studied. Of 9,863 nonmeningeal isolates tested against ceftriaxone, 82.7% were susceptible, 13.2% were intermediate, and 4.1% were resistant by the M100-S11 NCCLS breakpoints (2001); by M100-S12 breakpoints, 95.9% of the isolates were susceptible, 3.1% were intermediate, and 1.0% were resistant. Of 10,777 nonmeningeal isolates tested against cefotaxime, 79.2% were susceptible, 14.3% were intermediate, and 6.5% were resistant by M100-S11 breakpoints; by M100-S12 breakpoints, 93.5% were susceptible, 4.2% were intermediate, and 2.3% were resistant. Overall, the new M100-S12 ceftriaxone and cefotaxime interpretative breakpoints for nonmeningeal isolates of S. pneumoniae decreased the number of isolates interpreted as intermediate by 10% and as resistant by 3 to 4%.
doi:10.1128/JCM.40.2.669-674.2002
PMCID: PMC153417  PMID: 11825993
8.  Highly Penicillin-Resistant Multidrug-Resistant Pneumococcus-Like Strains Colonizing Children in Oeiras, Portugal: Genomic Characteristics and Implications for Surveillance▿ † 
Journal of Clinical Microbiology  2009;48(1):238-246.
While performing surveillance studies in Oeiras, Portugal, designed to describe the impact of pneumococcal conjugate vaccine on colonization, we observed an increase from 0.7% in 2003 to 5% in 2006 in the prevalence of penicillin resistance (MIC of 2 to 6 mg/liter) among presumptively identified pneumococcal isolates. Although 15 of the 22 penicillin-resistant isolates obtained in 2006 were optochin resistant, they were bile soluble and thus considered to be bona fide pneumococci. This study aimed to clarify the nature of these isolates by using a combination of phenotypic and genotypic approaches that included routine strategies for pneumococcal identification, multilocus sequence analysis (MLSA), and comparative genomic hybridization (CGH). By MLSA, all isolates were classified as “streptococci of the mitis group” that, however, were distinct from typical Streptococcus pneumoniae or Streptococcus mitis. A single isolate was identified as Streptococcus pseudopneumoniae. CGH confirmed these findings and further indicated that a considerable part of the proposed pneumococcal core genome is conserved in these isolates, including several pneumococcal virulence genes (e.g., pavA, spxB, cbpE, and cbpD). These results suggest that among pneumococci and closely related streptococci, universal unique phenotypic and genetic properties that could aid species identification are virtually impossible to define. In pneumococcal colonization studies, when atypical strains are found, MLSA and CGH are informative tools that can be used to complement routine tests. In our study, after correct identification of the penicillin-resistant true pneumococci, we found that penicillin resistance levels among pneumococci remained stable from 2003 to 2006.
doi:10.1128/JCM.01313-09
PMCID: PMC2812262  PMID: 19906899
9.  Nasal Carriage in Vietnamese Children of Streptococcus pneumoniae Resistant to Multiple Antimicrobial Agents 
Resistance to antimicrobial agents in Streptococcus pneumoniae is increasing rapidly in many Asian countries. There is little recent information concerning resistance levels in Vietnam. A prospective study of pneumococcal carriage in 911 urban and rural Vietnamese children, of whom 44% were nasal carriers, was performed. Carriage was more common in children <5 years old than in those ≥5 years old (192 of 389 [49.4%] versus 212 of 522 [40.6%]; P, 0.01). A total of 136 of 399 isolates (34%) had intermediate susceptibility to penicillin (MIC, 0.1 to 1 mg/liter), and 76 of 399 isolates (19%) showed resistance (MIC, >1.0 mg/liter). A total of 54 of 399 isolates (13%) had intermediate susceptibility to ceftriaxone, and 3 of 399 isolates (1%) were resistant. Penicillin resistance was 21.7 (95% confidence interval, 7.0 to 67.6) times more common in urban than in rural children (35 versus 2%; P, <0.001). More than 40% of isolates from urban children were also resistant to erythromycin, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline. Penicillin resistance was independently associated with an urban location when the age of the child was controlled for. Multidrug resistance (resistance to three or more antimicrobial agent groups) was present in 32% of isolates overall but in 39% of isolates with intermediate susceptibility to penicillin and 86% of isolates with penicillin resistance. The predominant serotypes of the S. pneumoniae isolates were 19, 23, 14, 6, and 18. Almost half of the penicillin-resistant isolates serotyped were serotype 23, and these isolates were often multidrug resistant. This study suggests that resistance to penicillin and other antimicrobial agents is common in carriage isolates of S. pneumoniae from children in Vietnam.
PMCID: PMC89715  PMID: 10681307
10.  The multidrug-resistant PMEN1 pneumococcus is a paradigm for genetic success 
Genome Biology  2012;13(11):R103.
Background
Streptococcus pneumoniae, also called the pneumococcus, is a major bacterial pathogen. Since its introduction in the 1940s, penicillin has been the primary treatment for pneumococcal diseases. Penicillin resistance rapidly increased among pneumococci over the past 30 years, and one particular multidrug-resistant clone, PMEN1, became highly prevalent globally. We studied a collection of 426 pneumococci isolated between 1937 and 2007 to better understand the evolution of penicillin resistance within this species.
Results
We discovered that one of the earliest known penicillin-nonsusceptible pneumococci, recovered in 1967 from Australia, was the likely ancestor of PMEN1, since approximately 95% of coding sequences identified within its genome were highly similar to those of PMEN1. The regions of the PMEN1 genome that differed from the ancestor contained genes associated with antibiotic resistance, transmission and virulence. We also revealed that PMEN1 was uniquely promiscuous with its DNA, donating penicillin-resistance genes and sometimes many other genes associated with antibiotic resistance, virulence and cell adherence to many genotypically diverse pneumococci. In particular, we describe two strains in which up to 10% of the PMEN1 genome was acquired in multiple fragments, some as long as 32 kb, distributed around the recipient genomes. This type of directional genetic promiscuity from a single clone to numerous unrelated clones has, to our knowledge, never before been described.
Conclusions
These findings suggest that PMEN1 is a paradigm of genetic success both through its epidemiology and promiscuity. These findings also challenge the existing views about horizontal gene transfer among pneumococci.
doi:10.1186/gb-2012-13-11-r103
PMCID: PMC3580495  PMID: 23158461
11.  Nasopharyngeal Carriage of Penicillin-Resistant Streptococcus pneumoniae among Children with Acute Respiratory Tract Infections in Thailand: a Molecular Epidemiological Survey 
Journal of Clinical Microbiology  1999;37(6):1832-1838.
The prevalence of penicillin-resistant Streptococcus pneumoniae in Thailand has dramatically increased over the last decade. During a national survey, which was conducted from 1992 to 1994, 37.2% of the pneumococci isolated from the nasopharynges of children with acute respiratory tract infections were penicillin resistant (MIC, ≥0.1 μg/ml). In order to investigate the prevalence and clonal relatedness of nasopharyngeal carriage of penicillin-resistant S. pneumoniae in Thailand, a molecular epidemiological survey was undertaken. To this end, 53 penicillin-resistant pneumococcal isolates from children who suffered from acute respiratory tract infections and who originated from five distinct regions of the country were characterized in detail. DNA fingerprint analysis demonstrated 13 clusters, i.e., genotypes shared by two or more strains, and 14 unique genotypes. The cluster size varied from 2 (nine clusters) to 11 strains (one cluster). Six of the 13 restriction fragment end labeling clusters consisted of two or more distinct serotypes, indicating frequent horizontal transfer of capsular genes. Geographical distribution of the genotypes among the five regions of Thailand demonstrated that only four genetic clusters were restricted to single areas of the country, whereas the other nine clusters represented isolates collected in two or more districts. These observations demonstrate that the majority of the genetic clusters are spread throughout the country. The most predominant genetic cluster, representing 21% of the isolates, was identical to the Spanish pandemic clone 23F. In addition, the second largest cluster matched the Spanish-French international clone 9V. These data indicate that the genetic clones 23F and 9V, which are widely spread throughout the world, are the most predominant multidrug-resistant pneumococcal clones in Thailand. Therefore, we conclude that these pandemic clones are primarily responsible for the increase in the prevalence of pneumococcal penicillin resistance in Thailand.
PMCID: PMC84963  PMID: 10325333
12.  Aggregated Antibiograms and Monitoring of Drug-Resistant Streptococcus pneumoniae 
Emerging Infectious Diseases  2003;9(9):1089-1095.
Community-specific antimicrobial susceptibility data may help monitor trends among drug-resistant Streptococcus pneumoniae and guide empiric therapy. Because active, population-based surveillance for invasive pneumococcal disease is accurate but resource intensive, we compared the proportion of penicillin-nonsusceptible isolates obtained from existing antibiograms, a less expensive system, to that obtained from 1 year of active surveillance for Georgia, Tennessee, California, Minnesota, Oregon, Maryland, Connecticut, and New York. For all sites, proportions of penicillin-nonsusceptible isolates from antibiograms were within 10 percentage points (median 3.65) of those from invasive-only isolates obtained through active surveillance. Only 23% of antibiograms distinguished between isolates intermediate and resistant to penicillin; 63% and 57% included susceptibility results for erythromycin and extended-spectrum cephalosporins, respectively. Aggregating existing hospital antibiograms is a simple and relatively accurate way to estimate local prevalence of penicillin-nonsusceptible pneumococcus; however, antibiograms offer limited data on isolates with intermediate and high-level penicillin resistance and isolates resistant to other agents.
doi:10.3201/eid0909.020620
PMCID: PMC3016770  PMID: 14519245
Streptococcus pneumoniae; antibiogram; pneumococcal infections; drug resistance; population surveillance; epidemiology
13.  In Vitro Evaluation of the Antimicrobial Activity of Ceftaroline against Cephalosporin-Resistant Isolates of Streptococcus pneumoniae▿  
Increasing pneumococcal resistance to extended-spectrum cephalosporins warrants the search for novel agents with activity against such resistant strains. Ceftaroline, a parenteral cephalosporin currently in phase 3 clinical development, has demonstrated potent in vitro activity against resistant gram-positive organisms, including penicillin-resistant Streptococcus pneumoniae. In this study, the activity of ceftaroline was evaluated against highly cefotaxime-resistant isolates of pneumococci from the Active Bacterial Core surveillance program of the Centers for Disease Control and Prevention and against laboratory-derived cephalosporin-resistant mutants of S. pneumoniae. The MICs of ceftaroline and comparators were determined by broth microdilution. In total, 120 U.S. isolates of cefotaxime-resistant (MIC ≥ 4 μg/ml) S. pneumoniae were tested along with 18 laboratory-derived R6 strains with known penicillin-binding protein (PBP) mutations. Clinical isolates were characterized by multilocus sequence typing, and the DNAs of selected isolates were sequenced to identify mutations affecting pbp genes. Ceftaroline (MIC90 = 0.5 μg/ml) had greater in vitro activity than penicillin, cefotaxime, or ceftriaxone (MIC90 = 8 μg/ml for all comparators) against the set of highly cephalosporin-resistant clinical isolates of S. pneumoniae. Ceftaroline was also more active against the defined R6 PBP mutant strains, which suggests that ceftaroline can overcome common mechanisms of PBP-mediated cephalosporin resistance. These data indicate that ceftaroline has significant potency against S. pneumoniae strains resistant to existing parenteral cephalosporins and support its continued development for the treatment of infections caused by resistant S. pneumoniae strains.
doi:10.1128/AAC.01324-08
PMCID: PMC2630653  PMID: 19015339
14.  Trends in Drug Resistance, Serotypes, and Molecular Types of Streptococcus pneumoniae Colonizing Preschool-Age Children Attending Day Care Centers in Lisbon, Portugal: a Summary of 4 Years of Annual Surveillance 
Journal of Clinical Microbiology  2005;43(3):1285-1293.
Of the nasopharyngeal cultures recovered from 942 day care center (DCC) attendees in Lisbon, Portugal, 591 (62%) yielded Streptococcus pneumoniae during a surveillance performed in February and March of 1999. Forty percent of the isolates were resistant to one or more antimicrobial agents. In particular, 2% were penicillin resistant and 20% had intermediate penicillin resistance. Multidrug resistance to macrolides, lincosamides, and tetracycline was the most frequent antibiotype (17% of all isolates). Serotyping and molecular typing by pulsed-field gel electrophoresis were performed for 202 out of 237 drug-resistant pneumococci (DRPn). The most frequent serotypes were 6B (26%), 14 (22%), 19F (16%), 23F (10%), and nontypeable (12%). The majority (67%) of the DRPn strains were representatives of nine international clones included in the Pneumococcal Molecular Epidemiology Network; eight of them had been detected in previous studies. Fourteen novel clones were identified, corresponding to 26% of the DRPn strains. The remaining 7% of the strains were local clones detected in our previous studies. Comparison with studies conducted since 1996 in Portuguese DCCs identified several trends: (i) the rate of DRPn frequency has fluctuated between 40 and 50%; (ii) the serotypes most frequently recovered have remained the same; (iii) nontypeable strains appear to be increasing in frequency; and (iv) a clone of serotype 33F emerged in 1999. Together, our observations highlight that the nasopharynxes of children in DCCs are a melting pot of successful DRPn clones that are important to study and monitor if we aim to gain a better understanding on the epidemiology of this pathogen.
doi:10.1128/JCM.43.3.1285-1293.2005
PMCID: PMC1081291  PMID: 15750097
15.  Antimicrobial Susceptibility of Streptococcus pneumoniae in Eight European Countries from 2001 to 2003 
Susceptibility testing results for Streptococcus pneumoniae isolates (n = 2,279) from eight European countries, examined in the PneumoWorld Study from 2001 to 2003, are presented. Overall, 24.6% of S. pneumoniae isolates were nonsusceptible to penicillin G and 28.0% were resistant to macrolides. The prevalence of resistance varied widely between European countries, with the highest rates of penicillin G and macrolide resistance reported from Spain and France. Serotype 14 was the leading serotype among penicillin G- and macrolide-resistant S. pneumoniae isolates. One strain (PW 158) showed a combination of an efflux type of resistance with a 23S rRNA mutation (A2061G, pneumococcal numbering; A2059G, Escherichia coli numbering). Six strains which showed negative results for mef(A) and erm(B) in repeated PCR assays had mutations in 23S rRNA or alterations in the L4 ribosomal protein (two strains). Fluoroquinolone resistance rates (levofloxacin MIC ≥ 4 μg/ml) were low (Austria, 0%; Belgium, 0.7%; France, 0.9%; Germany, 0.4%; Italy, 1.3%; Portugal, 1.2%; Spain, 1.0%; and Switzerland, 0%). Analysis of quinolone resistance-determining regions showed eight strains with a Ser81 alteration in gyrA; 13 of 18 strains showed a Ser79 alteration in parC. The clonal profile, as analyzed by multilocus sequence typing (MLST), showed that the 18 fluoroquinolone-resistant strains were genetically heterogeneous. Seven of the 18 strains belonged to new sequence types not hitherto described in the MLST database. Europe-wide surveillance for monitoring of the further spread of these antibiotic-resistant S. pneumoniae clones is warranted.
doi:10.1128/AAC.49.7.2903-2913.2005
PMCID: PMC1168634  PMID: 15980367
16.  Serotype and Genotype Distribution among Invasive Streptococcus pneumoniae Isolates in Colombia, 2005–2010 
PLoS ONE  2014;9(1):e84993.
In Colombia, a laboratory-based surveillance of invasive Streptococcus pneumoniae isolates as part of SIREVA II PAHO has been conducted since 1994. This study describes the serotype distribution, antimicrobial resistance, and genetic relationships of pneumococcal isolates recovered in Colombia from 2005 to 2010. In this study, demographic data of invasive S. pneumoniae isolates were analyzed, and antimicrobial susceptibility patterns were determined. Pulse field gel electrophoresis (n = 629) and multilocus sequence typing (n = 10) were used to determine genetic relationship of isolates with minimal inhibitory concentration to penicillin ≥0.125 µg/mL. A total of 1775 isolates of S. pneumoniae were obtained. Fifteen serotypes accounted for 80.7% of isolates. Serotype 14 (23.1%) was the most frequent in the general population. Penicillin resistance was 30.7% in meningitis and 9.0% in non-meningitis. Clones Spain6BST90, Spain9VST156, Spain23FST81, and Colombia23FST338 were associated to isolates. Additionally, serotype 6A isolates were associated with ST460 and ST473, and 19A isolates with ST276, ST320, and ST1118. In conclusion, the surveillance program provided updated information of trends in serotype distribution, antimicrobial resistance and the circulation of clones in invasive pneumococcal diseases. These results could be helpful to understand the epidemiology of S. pneumoniae in Colombia, and provide a baseline to measure the impact of vaccine introduction.
doi:10.1371/journal.pone.0084993
PMCID: PMC3885649  PMID: 24416330
17.  Tracking the implementation of NCCLS M100-S12 expanded-spectrum cephalosporin MIC breakpoints for nonmeningeal isolates of Streptococcus pneumoniae by clinical laboratories in the United States during 2002 and 2003 
Background
The Performance Standards for Antimicrobial Susceptibility Testing, Twelfth Informational Supplement, M100-S12, published by the National Committee for Clinical Laboratory Standards (NCCLS) in January 2002 introduced distinct minimum inhibitory concentration (MIC) interpretative breakpoints for ceftriaxone, cefotaxime, and cefepime for nonmeningeal isolates of Streptococcus pneumoniae. Previously, a single set of interpretative breakpoints was used for both meningeal and nonmeningeal isolates.
Methods
To estimate the rate of adoption of the M100-S12 interpretive breakpoints by clinical laboratories, antimicrobial susceptibility test results for ceftriaxone and cefotaxime from nonmeningeal S. pneumoniae isolates were studied using data collected from January 2002 to June 2003 by The Surveillance Network® Database – USA (TSN®), an electronic surveillance database.
Results
Of the 262 laboratories that provided data that could be evaluated, 67.6% had adopted the M100-S12 breakpoints one and one-half years after they were published.
Conclusions
The NCCLS M100-S12 recommendation to interpret MICs to expanded-spectrum cephalosporins using two distinct sets of breakpoints for meningeal and nonmeningeal isolates of S. pneumoniae was steadily implemented by clinical microbiology laboratories in the United States following their initial publication in January 2002. The use of these new breakpoints more accurately reflects the clinical activities of expanded-spectrum cephalosporins than did the single set of interpretative breakpoints previously used for both meningeal and nonmeningeal isolates.
doi:10.1186/1476-0711-3-1
PMCID: PMC324563  PMID: 14713319
18.  In Vitro Activity of Ceftaroline against Clinical Isolates of Streptococcus pneumoniae Recovered in 43 U.S. Medical Centers during 2010-2011 
The in vitro activity of ceftaroline, a recently introduced parenteral cephalosporin, was assessed versus 1,750 isolates of Streptococcus pneumoniae recovered from patients with a variety of pneumococcal infections in 43 U.S. medical centers during 2010-2011. Using a breakpoint of ≤0.5 μg/ml for susceptibility, all of the isolates were found to be susceptible to ceftaroline. Ceftaroline MICs were consistently 16-fold lower than ceftriaxone MICs. Among the isolates characterized in this investigation, 38.9% were found to be nonsusceptible to penicillin (oral penicillin breakpoints) and 9.1% were nonsusceptible to ceftriaxone (nonmeningitis breakpoints).
doi:10.1128/AAC.00582-12
PMCID: PMC3370772  PMID: 22491687
19.  Temporal Trends of Antimicrobial Resistance and Clonality of Invasive Streptococcus pneumoniae Isolates in Finland, 2002 to 2006▿  
The antimicrobial resistance of Streptococcus pneumoniae, or pneumococcus, is a growing global problem. In our study, 3,571 invasive pneumococcal isolates, recovered from blood and cerebrospinal fluid samples from patients in Finland between the years 2002 and 2006, showed an increase in erythromycin nonsusceptibility from 16% to 28% (P < 0.0001) over the 5-year study period, as well as a doubling of penicillin nonsusceptibility from 8% to 16% (P < 0.0001). Erythromycin nonsusceptibility increased especially in isolates derived from 0- to 2-year-old children and was 46% for this age group in 2006. Although multiresistance, defined as nonsusceptibility to penicillin, erythromycin, and tetracycline, was fairly rare (5.1% in 2006), 38% of the erythromycin-nonsusceptible isolates were also penicillin nonsusceptible, while 74% of the penicillin-nonsusceptible isolates were nonsusceptible to erythromycin. In contrast to the situation in continental Europe, but mirroring that in North America, the most frequent macrolide resistance determinant carried by 56% of the tested macrolide-resistant pneumococci was the mef gene. Serotypes 14, 9V, 19A, 6B, and 19F were most frequently nonsusceptible to erythromycin or penicillin. The penicillin-resistant invasive isolates (n = 88) were genotyped by multilocus sequence typing, which revealed the presence of 25 sequence types, 9 of which were novel. The majority of the isolates were related to one of several globally disseminated penicillin- or multiresistant clones, most importantly the rlrA adhesion pilus carrying clones Spain9V ST156 and Taiwan19F ST236. The penicillin-resistant pneumococcal population in Finland is therefore a combination of internationally recognized genotypes as well as novel ones.
doi:10.1128/AAC.01464-08
PMCID: PMC2681517  PMID: 19273677
20.  Phenotypic and Molecular Analysis of Penicillin-Nonsusceptible Streptococcus pneumoniae Isolates in Poland▿  
β-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 (Spain23F-1, Spain6B-2, Spain9V-3, Taiwan23F-15, Poland23F-16, Poland6B-20, and Sweden15A-25) or their closely related variants comprised the majority of the study isolates. The spread of Spain9V-3 and its related clone of serotype 14/ST143 has remarkably contributed to the recent increase in penicillin resistance in pneumococci in the country.
doi:10.1128/AAC.01072-06
PMCID: PMC1797676  PMID: 17043125
21.  Laboratory survey of drug-resistant Streptococcus pneumoniae in New York City, 1993-1995. 
Emerging Infectious Diseases  1998;4(1):113-116.
Wide geographic variation in the prevalence of drug-resistant Streptococcus pneumoniae demonstrates the importance of tracking antimicrobial resistance locally. This survey of hospital microbiology laboratories in New York City found that penicillin resistance (MIC > or = 2.0 micrograms/ml) increased from 1.5% of S. pneumoniae isolates in 1993 to 6.3% in 1995 and that in 1995, one-third of isolates nonsusceptible to penicillin (MIC > or = 0.1 microgram/ml) were also nonsusceptible to an extended-spectrum cephalosporin (MIC > or = 1 microgram/ml).
PMCID: PMC2627676  PMID: 9452405
22.  In Vitro Activities of Cethromycin (ABT-773), a New Ketolide, against Streptococcus pneumoniae Strains That Are Not Susceptible to Penicillin or Macrolides 
Pneumococcal resistance to antimicrobials presents problems to physicians for empirical treatment of acute otitis media (AOM). Three hundred thirty-three isolates of Streptococcus pneumoniae selected for nonsusceptibility to penicillin (MIC >0.1 μg/ml) from the middle ear (n = 325) or mastoid (n = 8) of children seen between 1994 and 2000 at four children's hospitals in the United States were tested by broth microdilution for susceptibility to nine antibiotics. Using NCCLS 2002 breakpoints, resistance to the following drugs was as indicated: amoxicillin, 1%; azithromycin, 71%; cefprozil, 71%; ceftriaxone, 2%; cefdinir, 98%; erythromycin, 70%; levofloxacin, 0%; and trimethoprim-sulfamethoxazole, 93%. Of the penicillin- and erythromycin-nonsusceptible isolates, 97% were inhibited by cethromycin (ABT-773) and 83% were inhibited by telithromycin at a concentration of ≤0.125 μg/ml. Macrolide resistance among penicillin-nonsusceptible pneumococci increased from 44 to 80% in the 6 years of the study from which the isolates were selected; however, the proportion of isolates with M or MLSB phenotypes remained constant over the time period (53 and 18%, respectively). Prior treatment with a macrolide or clindamycin alone or in combination with a β-lactam resulted in 94 or 85% of isolates causing infections being macrolide and or clindamycin resistant. No prior individual macrolide (azithromycin, erythromycin, or clarithromycin) resulted in more macrolide resistance or in a more prevalent resistance phenotype. The ketolides appear to be active antimicrobials against penicillin- and macrolide-resistant pneumococci.
doi:10.1128/AAC.47.1.166-169.2003
PMCID: PMC149018  PMID: 12499186
23.  Survey of Susceptibilities of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis Isolates to 26 Antimicrobial Agents: a Prospective U.S. Study 
Antimicrobial Agents and Chemotherapy  1999;43(11):2612-2623.
An antimicrobial susceptibility surveillance study of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolates was performed during the winter of 1996–1997 in order to determine their susceptibilities to 5 fluoroquinolones and 21 other antimicrobial agents. Broth microdilution MICs were determined for 2,752 isolates from 51 U.S. medical centers. Of the 1,276 S. pneumoniae isolates, 64% were susceptible, 17% were intermediate, and 19% were highly resistant to penicillin. On the basis of the MICs at which 90% of isolates are inhibited and modal MICs, the hierarchy of the five fluoroquinolones from most to least active was grepafloxacin > sparfloxacin > levofloxacin = ciprofloxacin > ofloxacin. For S. pneumoniae isolates for which penicillin MICs were elevated, the MICs of the cephalosporins, macrolides, clindamycin, tetracycline, and trimethoprim-sulfamethoxazole were also elevated, but the MICs of the fluoroquinolones, vancomycin, and rifampin were not. The prevalence of penicillin-susceptible pneumococci varied by U.S. Bureau of the Census region (range, 44% in the East South Central region to 75% in the Pacific region). In addition, S. pneumoniae isolates from blood were significantly more susceptible to penicillin than those from respiratory, ear, or eye specimens, and pneumococci from patients ≤2 years old were significantly more resistant to penicillin than those from older patients (by chi-square analysis, P < 0.05). β-Lactamase was produced by 35% of H. influenzae isolates and 93% of M. catarrhalis isolates, resulting in increased MICs of amoxicillin and certain cephalosporins. We noted that the antimicrobial resistance patterns of S. pneumoniae isolates, which correlate with the penicillin susceptibility phenotype, vary by site of infection, age group of the patient, and geographic source of the isolate.
PMCID: PMC89533  PMID: 10543737
24.  Clonal Evolution Leading to Maintenance of Antibiotic Resistance Rates among Colonizing Pneumococci in the PCV7 Era in Portugal ▿  
Journal of Clinical Microbiology  2011;49(8):2810-2817.
The introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) in Portugal led to extensive serotype replacement among carriers of pneumococci, with a marked decrease of PCV7 types. Although antimicrobial resistance was traditionally associated with PCV7 types, no significant changes in the rates of nonsusceptibility to penicillin, resistance to macrolides, or multidrug resistance were observed. This study aimed to investigate the mechanisms leading to maintenance of antimicrobial resistance, despite marked serotype replacement. We compared, through molecular typing, 252 antibiotic-resistant pneumococci recovered from young carriers in 2006 and 2007 (era of high PCV7 uptake) with collections of isolates from 2002 and 2003 (n=374; low-PCV7-uptake era) and 1996 to 2001 (n=805; pre-PCV7 era). We observed that the group of clones that has accounted for antimicrobial resistance since 1996 is essentially the same as the one identified in the PCV7 era. The relative proportions of such clones have, however, evolved substantially overtime. Notably, widespread use of PCV7 led to an expansion of two Pneumococcal Molecular Epidemiology Network (PMEN) clones expressing non-PCV7 capsular variants of the original strains: Sweden15AST63 (serotypes 15A and 19A) and Denmark14ST230 (serotypes 19A and 24F). These variants were already in circulation in the pre-PCV7 era, although they have now become increasingly abundant. Emergence of novel clones and de novo acquisition of resistance contributed little to the observed scenario. No evidence of capsular switch events occurring after PCV7 introduction was found. In the era of PCVs, antimicrobial resistance remains a problem among the carried pneumococci. Continuous surveillance is warranted to evaluate serotype and clonal shifts leading to maintenance of antimicrobial resistance.
doi:10.1128/JCM.00517-11
PMCID: PMC3147772  PMID: 21632898
25.  Testing of Streptococcus pneumoniae for resistance to penicillin. 
Journal of Clinical Microbiology  1993;31(5):1246-1250.
The increasing prevalence of penicillin-resistant Streptococcus pneumoniae requires antibiotic susceptibility tests that can be done with greater ease and reliability. We measured the MIC of penicillin for pneumococci by the tube macrodilution method with Mueller-Hinton broth (MHB), Haemophilus Test Medium (HTM), Todd-Hewitt broth with 0.5% yeast extract (THY), and MHB with 3% lysed horse blood (LHB). Eight (19%) and 6 (14%) of 42 pneumococcal isolates failed to generate turbid growth in MHB and HTM, respectively, whereas all pneumococcal isolates did so in THY and LHB. For those strains that replicated to turbidity, the mean MICs of penicillin were lower in MHB and HTM than in THY and LHB, with differences being significant (P < 0.05) for comparisons with LHB. Four isolates appeared to be penicillin susceptible in HTM but were actually moderately resistant in THY and LHB, and two isolates appeared to be moderately resistant but were resistant. A similar failure to detect resistance was seen with MHB. S. pneumoniae ATCC 49619, a moderately penicillin-resistant strain that has been proposed for quality control testing, gave variable results in MHB or THM and appeared to be susceptible to penicillin in some assays, whereas the MICs for S. pneumoniae ATCC 49619 in THY or LHB fell within a twofold dilution range, with geometric means of 0.16 and 0.18 micrograms/ml, respectively. Pneumococcal isolates thus may appear falsely susceptible to penicillin when tested in MHB or HTM. LHB remains the standard medium; however, because THY is an easily prepared clear medium that can be used in automated systems and appears to yield results similar to those obtained with LHB, THY deserves consideration for routine use.
PMCID: PMC262912  PMID: 8501225

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