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Background: Introduction of pneumococcal conjugate vaccines (PCVs) has reduced the disease caused by vaccine serotypes in children, providing herd protection to adults. However, the emergence of nonvaccine serotypes is of great concern worldwide., Methods: This study includes national laboratory data from invasive pneumococcal disease (IPD) cases that affected pediatric and adult populations during 2009-2019. The impact of implementing different vaccine strategies for immunocompetent adults by comparing Spanish regions that used the 13-valent PCV (PCV13) vs regions that used the 23-valent pneumococcal polysaccharide vaccine (PPV23) was also analyzed for 2017-2019., Results: The overall reductions in IPD cases by PCV13 serotypes in children and adults were 88% and 59%, respectively, during 2009-2019, with a constant increase in serotype 8 in adults since 2015. IPD cases by additional serotypes covered by PPV23 increased from 20% in 2009 to 52% in 2019. In children, serotype 24F was the most frequent in 2019, whereas serotypes 3 and 8 accounted for 36% of IPD cases in adults. Introduction of PCV13 or PPV23 in the adult calendar of certain Spanish regions reduced the IPD cases by PCV13 serotypes by up to 25% and 11%, respectively, showing a decrease of serotype 3 when PCV13 was used., Conclusions: Use of PCV13 in children has affected the epidemiology, reducing the burden of IPD in children but also in adults by herd protection; however, the increase in serotype 8 in adults is worrisome. Vaccination with PCV13 in adults seems to control IPD cases by PCV13 serotypes including serotype 3., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Serotypes/genotypes causing invasive pneumococcal disease (IPD) in adults are determined by vaccination strategies. The aim of this study was to assess the epidemiology of IPD in adults (≥18 years) after PCV13 introduction for children: serotypes, clonal complexes, antibiotic non-susceptibility and clinical presentations. We performed a prospective, clinical surveillance of hospitalized culture-confirmed IPDs in adults in nine Spanish hospitals (August 2010-June 2015). A total of 1087 culture-confirmed IPD episodes were included, of which 772 (71.0%) had bacteremic pneumonia (401 complicated/371 uncomplicated pneumonia), 122 (11.2%) meningitis, 102 (9.4%) non-focal bacteremia, 34 (3.1%) peritonitis and 57 (5.3%) others. The most common serotypes were: 3 (12.7%), 19A (8.5%), 8 (7.7%), 7F (6.3%), 1 (4.2%), 6C (4.2%), 11A (4.2%), 22F (4.2%) and 14 (4.0%). Vaccine types (PCV13 + 6C) caused 49.8% of IPD episodes, with a significant decrease over the 5-year period, and significant decreases in serotypes 6C and 7F. The most common genotypes were: CC180 (8.4%), CC191 (6.0%), and CC53 (5.0%). Vaccine types caused 53.9% (414/768) pneumonia episodes and 58.9% (235/399) complicated pneumonia, 53.4% IPD in adults <50 years (143/268), and 54.7% IPD in immunocompetent patients (337/616). Overall non-susceptibility was 25.9% to penicillin (1.1% for parenteral criteria), 24.9% to erythromycin and 2.7% to levofloxacin. CONCLUSIONS: Although the percentage of vaccine-types causing IPDs in adults significantly decreased, it remained high. Associations of vaccine types with pneumonia (with complicated pneumonia for specific serotypes), and immunocompetent patients point to the burden of IPD caused by PCV13 serotypes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A prospective laboratory-based multicenter study that collected all adult invasive pneumococcal disease (IPD) episodes from 6 Spanish hospitals before (2008-2009) and after (2012-2013). The 13-valent pneumococcal conjugate vaccine (PCV13) licensure was conducted in order to analyze the impact of PCV13 introduction for children on adult IPD. A total of 1558 IPD episodes were detected. The incidence of IPD decreased significantly in the second period by -33.9% (95% CI, -40.3% to -26.8%). IPD due to PCV7 serotypes (-52.7%; 95% CI, -64.2% to -37.5%) and to PCV13 additional serotypes (-55.0% 95% CI, -62.0% to -46.7%) significantly decreased whereas IPD due to non-PCV13 serotypes remained stable (1.0% 95% CI, -12.9% to 17.2%). IPD due to all PCV13 additional serotypes significantly declined with the exception of serotype 3 (-11.3%; 95%CI -35.0% to 21.1%). IPD due to two non-PCV13 serotypes varied: serotype 6C that rose (301.6%; 95%CI, 92.7% to 733.3%, p<0.001), related to the expansion of ST3866C, and serotype 8 that decreased (-34.9%, 95%CI, -57.1 to -1.2, p = 0.049), related to a decline of the ST638. The recombinant clone ST652111A (variant of ST1569V) increased in frequency. The decrease of serotype 19A IPD was linked to a fall in those antibiotic susceptible clones. In the last period, rates of penicillin- and cefotaxime-resistance remained under 10% and 4%, respectively. Adult IPD decreased after the PCV13 introduction in Spain due to herd protection. The spread of multidrug resistant clones (ST3866C, ST652111A) related to non-PCV13 serotypes needs further surveillance.

The cell wall glucosaminidase LytB of Streptococcus pneumoniae is a surface exposed protein involved in daughter cell separation, biofilm formation and contributes to different aspects of the pathogenesis process. In this study we have characterized the antibody responses after immunization of mice with LytB in the presence of alhydrogel as an adjuvant. Enzyme-linked immunosorbent assays measuring different subclasses of immunoglobulin G, demonstrated that the antibody responses to LytB were predominantly IgG1 and IgG2b, followed by IgG3 and IgG2a subclasses. Complement-mediated immunity against two different pneumococcal serotypes was investigated using sera from immunized mice. Immunization with LytB increased the recognition of S. pneumoniae by complement components C1q and C3b demonstrating that anti-LytB antibodies trigger activation of the classical pathway. Phagocytosis assays showed that serum containing antibodies to LytB stimulates neutrophil-mediated phagocytosis against S. pneumoniae. Animal models of infection including invasive pneumonia and sepsis were performed with two different clinical isolates. Vaccination with LytB increased bacterial clearance and induced protection demonstrating that LytB might be a good candidate to be considered in a future protein-based vaccine against S. pneumoniae., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study analyzed temporal trends of non-susceptibility/serotypes in invasive pneumococci from Spanish regions where pneumococcal conjugate vaccines (PCVs) were not included in paediatric immunization programmes. All invasive pneumococcal isolates voluntarily sent to the Spanish Reference Laboratory for Pneumococci (January 1990-December 2013) from hospitals located in target study regions were analyzed by age group. The PCV estimated coverage in children <24 months was correlated with 13-valent PCV (PCV13) serotypes trends. A total of 28,124 invasive isolates were analyzed: 3138 (11.2%) from children <24 months, 2161 (7.7%) from children 24-59 months, 781 (2.8%) from children 5-14 years, and 22,044 (78.4%) from adults. The estimated coverage increased from 17.6% (2002) to around 40% (2010-2013). The percentage of PCV13 serotypes among all isolates over time followed a cubic significant trend (R(2)=0.884), with an increasing trend up to 2001 followed by a decrease (more prominent from 2010 onwards). The estimated PCVs coverage was significantly correlated with the decrease in the percentage of PCV13 isolates in children <24 months (r(2)=0.824) and in adults (r(2)=0.786), mainly due to decreases in serotypes 1 and 7F in adults, and in serogroup 6 and serotypes 7F and 19A in children <24 months. None of the non-PCV13 serotypes stood out with substantial increases in the last period. This study showed that the different serotypes (and its associated non-susceptibility trends) were not equally affected by low PCVs disposition. Lack of impact in certain serotypes as serotype 1 (in children 24-59 months), 6C (in all age groups), and 19A (in adults) suggests the need for increasing vaccine coverage in the target vaccine population to increase direct and indirect protection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Capsular switching allows pre-existing clones of Streptococcus pneumoniae expressing vaccine serotypes to escape the vaccine-induced immunity by acquisition of capsular genes from pneumococci of a non-vaccine serotype. Here, we have analysed the clonal composition of 492 clinical isolates of serotype 11A causing invasive disease in Spain (2000-2012), and their ability to evade the host immune response. Antibiograms, serotyping and molecular typing were performed. The restriction profiles of pbp2x, pbp1a and pbp2b genes were also analysed. Interaction with the complement components C1q, C3b, C4BP, and factor H was explored whereas opsonophagocytosis assays were performed using a human cell line differentiated to neutrophils. Biofilm formation and the polymorphisms of the major autolysin LytA were evaluated. The main genotypes of the 11A pneumococci were: ST62 (447 isolates, 90.6%), followed by ST6521 (35 isolates, 7.3%) and ST838 (10 isolates, 2.1%). Beta lactam resistant serotype 11A variants of genotypes ST838 and ST6521 closely related to the Spain9V-ST156 clone were first detected in 2005. A different pattern of evasion of complement immunity and phagocytosis was observed between genotypes. The emergence of one vaccine escape variant of Spain9V-ST156 (ST652111A), showing a high potential to avoid the host immune response, was observed. In addition, isolates of ST652111A showed higher ability to produce biofilms than ST83811A or ST6211A, which may have contributed to the emergence of this PEN-resistant ST652111A genotype in the last few years. The emergence of penicillin-resistant 11A invasive variants of the highly successful ST156 clonal complex merits close monitoring.

Background: Since the use of pneumococcal conjugate vaccines PCV7 and PCV13 in children became widespread, invasive pneumococcal disease (IPD) has dramatically decreased. Nevertheless, there has been a rise in incidence of Streptococcus pneumoniae non-vaccine serotypes (NVT) colonising the human nasopharynx. Nasopharyngeal colonisation, an essential step in the development of S. pneumoniae-induced IPD, is associated with biofilm formation. Although the capsule is the main pneumococcal virulence factor, the formation of pneumococcal biofilms might, in fact, be limited by the presence of capsular polysaccharide (CPS)., Methodology/principal Findings: We used clinical isolates of 16 emerging, non-PCV13 serotypes as well as isogenic transformants of the same serotypes. The biofilm formation capacity of isogenic transformants expressing CPSs from NVT was evaluated in vitro to ascertain whether this trait can be used to predict the emergence of NVT. Fourteen out of 16 NVT analysed were not good biofilm formers, presumably because of the presence of CPS. In contrast, serotypes 11A and 35B formed ≥45% of the biofilm produced by the non-encapsulated M11 strain., Conclusions/significance: This study suggest that emerging, NVT serotypes 11A and 35B deserve a close surveillance.

Since 2004, a total of 131 isolates of Streptococcus pneumoniae multidrug-resistant invasive serotype 8 have been detected in Spain. These isolates showed resistance to erythromycin, clindamycin, tetracycline, and ciprofloxacin. All isolates were obtained from adult patients and shared a common genotype (sequence type [ST]63; penicillin-binding protein 1a [pbp1a], pbp2b, and pbp2x gene profiles; ermB and tetM genes; and a ParC-S79F change). Sixty-eight isolates that required a ciprofloxacin MIC ≥16 μg/mL had additional gyrA gene changes. Serotype 8-ST63 pbp2x sequences were identical with those of antimicrobial drug-susceptible serotype 8-ST53 isolates. Serotype 8-ST63 pbp2b sequences were identical with those of the multidrug-resistant Sweden 15A-ST63 clone. Recombination between the capsular locus and flanking regions of an ST53 isolate (donor) and an ST63 pneumococcus (recipient) generated the novel 15A-ST63 clone. One recombination point was upstream of pbp2x and another was within pbp1a. A serotype 8-ST63 clone was identified as a cause of invasive disease in Spain.

In Spain, rates of ciprofloxacin resistance in pneumococci were low during the last decade (2.6% in 2002 and 2.3% in 2006). In 2012, the rate remained at 2.3%, equivalent to 83 of 3,621 isolates. Of the 83 resistant isolates, 15 showed a low level (MIC of 4 to 8 μg/ml) and 68 a high level (MIC of 16 to 128 μg/ml) of ciprofloxacin resistance. Thirteen low-level-resistant isolates had single changes in ParC, one had a single ParE change, and one did not present any mutations. High-level-resistant isolates had GyrA changes plus additional ParC and/or ParE changes: 51, 15, and 2 isolates had 2, 3, or 4 mutations, respectively. Although 24 different serotypes were observed, 6 serotypes accounted for 51.8% of ciprofloxacin-resistant isolates: 8 (14.5%), 19A (10.8%), 11A (7.2%), 23A (7.2%), 15A (6.0%), and 6B (6.0%). A decrease in pneumococcal 7-valent conjugate vaccine (PCV7) serotypes was observed from 2006 (35.7%) to 2012 (16.9%), especially of serotype 14 (from 16.3% to 2.4%; P<0.001). In comparison with findings in 2006, multidrug resistance was greater in 2012 (P=0.296), mainly due to the increased presence and/or emergence of clonal complexes associated with non-PCV7 serotypes: CC63 expressing serotypes 8, 15A, and 19A; CC320 (with serotype 19A); and CC42 (with serotype 23A). Although rates of ciprofloxacin resistance remained low and stable throughout the last decade, changes in serotype and genotype distributions were observed in 2012, notably the expansion of a preexisting multidrug-resistant clone, CC63, and the emergence of the CC156 clone expressing serotype 11A.

In this study, we analyzed the clinical and molecular epidemiology of invasive serotype 5 (Ser5) pneumococcal isolates in four teaching hospitals in the Barcelona, Spain, area (from 1997 to 2011). Among 5,093 invasive pneumococcal isolates collected, 134 (2.6%) Ser5 isolates were detected. Although the overall incidence of Ser5-related invasive pneumococcal disease (IPD) was low (0.25 cases/100,000 inhabitants), three incidence peaks were detected: 0.63/100,000 in 1999, 1.15/100,000 in 2005, and 0.37/100,000 in 2009. The rates of Ser5 IPD were higher among young adults (18 to 64 years old) and older adults (>64 years old) in the first two peaks, whereas they were higher among children in 2009. The majority (88.8%) of the patients presented with pneumonia. Comorbid conditions were present in young adults (47.6%) and older adults (78.7%), the most common comorbid conditions being chronic obstructive pulmonary disease (20.6% and 38.3%, respectively) and cardiovascular diseases (11.1% and 38.3%, respectively). The mortality rates were higher among older adults (8.5%). All Ser5 pneumococci tested were fully susceptible to penicillin, cefotaxime, erythromycin, and ciprofloxacin. The resistance rates were 48.5% for co-trimoxazole, 6.7% for chloramphenicol, and 6% for tetracycline. Two major related sequence types (STs), ST1223 (n = 65) and ST289 (n = 61), were detected. The Colombia(5)-ST289 clone was responsible for all the cases in the Ser5 outbreak in 1999, whereas the ST1223 clone accounted for 73.8% and 61.5% of the isolates in 2005 and 2009, respectively. Ser5 pneumococci are a frequent cause of IPD outbreaks in the community and involve children and adults with or without comorbidities. The implementation of the new pneumococcal conjugated vaccines (PCV10 and PCV13) might prevent such outbreaks.

We aimed to obtain insights on the nature of a collection of isolates presumptively identified as atypical Streptococcus pneumoniae recovered from invasive and non-invasive infections in Spain. One-hundred and thirty-two isolates were characterized by: optochin susceptibility in ambient and CO(2)-enriched atmosphere; bile solubility; PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802, aliB-like ORF2, and a specific 16S rRNA region; multilocus sequence analysis; and antimicrobial susceptibility. By multilocus sequence analysis, 61 isolates were S. pseudopneumoniae, 34 were pneumococci, 13 were S. mitis, and 24 remained unclassified as non-pneumococci. Among S. pseudopneumoniae isolates, 51 (83.6%) were collected from respiratory tract samples; eight isolates were obtained from sterile sources. High frequency of non-susceptibility to penicillin (60.7%) and erythromycin (42.6%) was found. Only 50.8% of the S. pseudopneumoniae isolates displayed the typical optochin phenotype originally described for this species. None harbored the cpsA gene or the pneumococcal typical lytA restriction fragment length polymorphism. The Spn9802 and the specific 16S rRNA regions were detected among the majority of the S. pseudopneumoniae isolates (n = 59 and n = 49, respectively). The ply and pspA genes were rarely found. A high genetic diversity was found and 59 profiles were identified. Among the S. pneumoniae, 23 were capsulated and 11 were non-typeable. Three non-typeable isolates, associated to international non-capsulated lineages, were recovered from invasive disease sources. In conclusion, half of the atypical pneumococcal clinical isolates were, in fact, S. pseudopneumoniae and one-fourth were other streptococci. We identified S. pseudopneumoniae and non-typeable pneumococci as cause of disease in Spain including invasive disease.

This study explored the serotype distribution and antibiotic susceptibility of adult non-invasive Streptococcus pneumoniae isolates received in the Spanish Reference Laboratory for Pneumococci immediately prior to introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in June 2001 (May 2000-May 2001) and 10 years afterwards (May 2010-May 2011). Serotyping was performed by Quellung reaction and/or dot-blot assay, and minimum inhibitory concentrations (MICs) were determined by agar dilution. Clinical and Laboratory Standards Institute (CLSI) breakpoints were used for susceptibility interpretation. A total of 1274 isolates were identified (650 in the first period and 624 in the second period). PCV7 serotypes (as a group) showed a decrease (P<0.001) from 43.2% in the first period to 13.9% in the second period, with MIC(90) values (MIC for 90% of the organisms) of levofloxacin for the remaining PCV7 serotypes of 16 μg/mL. Inversely, non-PCV7 serotypes (as a group) increased from 56.8% to 86.1% (P<0.001), mainly due to increases in serotypes 19A (294.1% increase; P<0.001) and 15A (180.0% increase; P=0.005). Globally, non-susceptibility to penicillin decreased from 54.2% in the first period to 36.9% in the second period (P<0.001). Serotype 19A became the most worrisome, with an increase (at least five dilutions) in MIC(90) for all β-lactams in the second period, with non-susceptibility increasing from 18.2% to 71.4% (P=0.003) for penicillin and from 0.0% to 38.1% (P=0.022) for amoxicillin. Cefditoren showed the highest intrinsic activity (lowest MIC(50)/MIC(90)) overall and also against serotype 19A. Continuous surveillance of serotype distribution and antibiotic susceptibility among adult non-invasive isolates is necessary to detect emerging serotypes and to continuously assess the intrinsic activity of highly active oral antibiotics such as levofloxacin and cefditoren and of parenteral antibiotics such as cefotaxime., (Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Introduction: The aim of this investigation was to study the epidemiology of nasopharyngeal (NP) colonization with Streptococcus pneumoniae after the introduction of the heptavalent pneumococcal conjugate vaccine (PCV7)., Methods: NP swabs were obtained from 848 children aged 6 months to six years seen in four primary care centres (healthy children) and in two emergency depeartments (sick children) from Seville. The study was conducted between February 2005 and June 2008., Results: A total of 278 (33%) children carried S. pneumoniae. Pneumococcal colonization was independently predicted by school attendance or child care participation (OR 2.21; 95% CI 1.54- 3.15; P=.0001) and younger age. Recent antibiotic use was protective (OR 0.68; 95% CI 0.48-0.94; P=.02). PCV7 uptake was 41%. Risk of carriage of PCV7- type pneumococci was lower among children who had received ≥1 dose of PCV7 (7% vs 29%; [OR 0.21; 95% CI 0.09-0.49; P=.0001]). The proportion of pneumococcal isolates with oral penicillin non-susceptibility and amoxicillin resistance were 33% and 3%, respectively. Amoxicillin resistance in colonized children was associated with prior antibiotic usage (OR 4.29; 95% CI 1.09-20.02)., Conclusions: NP colonization rates with PCV7- type pneumococci were low compared to those found in studies prior to PCV7 introduction, both in vaccinated and unvaccinated subjects. Factors related to age and overcrowding increased the prevalence of pneumococcal carriage. Use of antibiotics reduced the overall carriage of pneumococci, but was a risk factor for colonization with amoxicillin resistant pneumococci., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

Background: Conjugate vaccines, such as the 7-valent conjugate vaccine (PCV7), alter serotype nasopharyngeal carriage, potentially increasing cases of otitis media by non-vaccine serotypes., Methods: All paediatric middle ear fluid (MEF) isolates received in the Spanish Reference Laboratory for Pneumococci through a passive, laboratory-based surveillance system from January 1997 to June 2009 were analysed. Data from 1997 to 2000 were pooled as pre-vaccination period. Trends over time were explored by linear regression analysis., Results: A total of 2,077 isolates were analysed: 855 belonging to PCV7 serotypes, 466 to serotype 19A, 215 to serotype 3, 89 to serotype 6A and 452 to other serotypes (< 40 isolates each). Over time, there has been a decreasing trend for PCV7 serotypes (R(2) = 0.944; p < 0.001, with significant decreasing trends for serotypes 19F, 14, 23F and 9V), and increasing trends for serotype 19A (R(2) = 0.901; p < 0.001), serotype 3 (R(2) = 0.463; p = 0.030) and other non-PCV7 serotypes (R(2) = 0.877; p < 0.001), but not for serotype 6A (R(2) = 0.311; p = 0.094). Considering all isolates, amoxicillin non-susceptibility showed an increasing trend (R(2) = 0.528; p = 0.017). Regarding serotype 19A, increasing trends in non-susceptibility to penicillin (R(2) = 0.726; p = 0.001), amoxicillin (R(2) = 0.804; p < 0.001), cefotaxime (R(2) = 0.546; p = 0.005) and erythromycin (R(2) = 0.546; p = 0.009) were found, with amoxicillin non-susceptibility firstly detected in 2003 (7.4%) and increasing up to 38.0% in 2009. In PCV7 serotypes (which prevalence decreased from 70.7% during 1997-2000 to 10.6% in 2009) amoxicillin non-susceptibility rates showed an increasing trend (R(2) = 0.702; p = 0.002). However, overall, amoxicillin non-susceptibility (≈25% in 2008-9) could be mainly attributed to serotype 19A (> 35% isolates) since PCV7 strains represented < 11% of total clinical isolates., Conclusions: In contrast to reports on invasive pneumococcal strains, in MEF isolates the reduction in the prevalence of PCV7 serotypes was not associated with decreases in penicillin/erythromycin non-susceptibility. The high prevalence of serotype 19A among paediatric MEF isolates and the amoxicillin non-susceptibility found in this serotype are worrisome since amoxicillin is the most common antibiotic used in the treatment of acute otitis media. These data suggest that non-PCV7 serotypes (mainly serotype 19A followed by serotypes 3 and 6A) are important etiological agents of acute otitis media and support the added value of the broader coverage of the new 13-valent conjugate vaccine.

Objectives: To analyse the epidemiology of isolates of serotype 6C among invasive pneumococci isolated from children and adults in Spain between 1997 and 2009, and to characterize serotype 6C clones and macrolide and quinolone resistance mechanisms., Methods: Antimicrobial susceptibility was determined following CLSI guidelines. Phenotypic characterization of macrolide-resistant isolates was performed by the double disc diffusion method. Genes associated with resistance to erythromycin and tetracycline were sought by PCR, while quinolone resistance was analysed by restriction fragment length polymorphism of the quinolone resistance-determining region. Isolates were typed by multilocus sequence typing., Results: Seven hundred and eighty-nine of 866 serotype 6A pneumococci collected from 1997 to 2009 were available. Of these, 213 (27.0%) were serotype 6C; 16/163 (9.8%) in the 1997-2001 (pre-PCV7) period, 37/322 (11.5%) in the 2002-05 (early-PCV7) period and 160/381 (42.0%) in the 2006-09 (late-PCV7) period. The overall proportions of serotype 6C increased from 0.1% (pre-PCV7) to 1% (late-PCV7) for paediatric isolates and from 0.3% to 1.7% among adult isolates. A major serotype 6C lineage (ST224/ST1150/ST4821), accounting for 66.7% of the isolates, was identified across the whole period. In the late-PCV7 period the antimicrobial non-susceptibility of serotype 6C increased in association with the emergence of the ST386/ST4310/ST4825 lineage, which carried a Tn6002 transposon [erm(B) and tet(M) genes]., Conclusions: Serotype 6C pneumococci were identified in Spain during the period 1997-2009. The increase in serotype 6C in the late-PCV7 period was associated with the spread of the ST224/ST1150/ST4821 lineage and the emergence of the ST386/ST4310/ST4825 lineage.

A total of 91 of 1,480 invasive isolates (6.1%) collected from adults in Barcelona, Spain, in the period of 1994 to 2008 were of serogroup 6 (6B, 47 isolates; 6A, 28; and 6C, 16). Throughout this period, serotype 6B (Spain(6B)-ST90) decreased, and serotype 6A remained stable. An increase in serotype 6C (ST224) in the 2004-2008 period was observed.

The genetic structure and antibiotic nonsusceptibility of all serotype 19A Streptococcus pneumoniae pediatric pneumococcal isolates received at the Spanish Pneumococcal Reference Laboratory (1990 to 2008) were analyzed. Of them, 410 (79.8%) isolates belonged to 14 sequence types (STs) with >10 isolates each, and 104 to 73 STs (with 21 new STs, ST5141 to ST5161, with one isolate each). Time trends in 2000 to 2008 (n=471) were explored by lineal regression. Serotype 19A increased from 5.7% in 2000 to 16.8% in 2008 (R2=0.872; P=0.001). Decreasing trends (P<0.03) were found for ST202 (R2=0.774) and ST81 (R2=0.559), and increasing trends (P<0.03) for ST878 (R2=0.544) and ST320 (R2=0.530), both belonging to the clonal complex (CC) Denmark(14)-32 and first detected in 2003 and 2007, respectively, and ST2013 (R2=0.704) and ST4461 (R2=0.707), both appearing in 2004. Penicillin nonsusceptibility was clustered in ST81, ST276, ST320, ST878, ST2013, and ST4461 (>90% nonsusceptibility), and amoxicillin and cefotaxime nonsusceptibility in ST320: 87% amoxicillin (MIC50/MIC90=8/8 μg/ml) and 43.5% cefotaxime (MIC50/MIC90=1/2 μg/ml) nonsusceptibility. No trends were found for erythromycin nonsusceptibility (ranging from 38.5% to 66.7%) and cefotaxime nonsusceptibility (ranging from 0.0% to 7.8%), but increasing trends (P<0.02) were found for oral penicillin (from 16.7% in 2000 to 56.3% in 2008; R2=0.628) and amoxicillin (from 0.0% before 2007 to 13.8% in 2008; R2=0.628) nonsusceptibility. This study warns about the emergence of serotype 19A STs associated with high-level antibiotic nonsusceptibility, with a role for ST320 and ST878 occupying the niche left by some pneumococcal 7-valent conjugate vaccine (PCV7)-related resistant STs. The rapid expansion of serotype 19A and STs related to antibiotic resistance indicates that vaccines covering serotype 19A present advantages in countering invasive disease.

The susceptibilities of pneumococci recently collected (up to June 2009) in Spain (500 isolates nonsusceptible to oral penicillin and 150 susceptible isolates) from serotypes not included in the conjugate vaccine were determined. Most nonsusceptible isolates (53.6%) belonged to serotype 19A. Susceptibility rates in serotype 19A penicillin-intermediate (n = 201)/penicillin-resistant (n = 67) isolates were <33%/< or =6.0% (erythromycin and oral cephalosporins with defined breakpoints), 85.1%/11.9% (amoxicillin), and 96.0%/52.2% (cefotaxime), respectively. Low susceptibility to common oral beta-lactams was also found in serotypes 11A (95.5% susceptibility to cefotaxime and erythromycin) and 35B.

Objectives: Erythromycin resistance in Streptococcus pneumoniae is still increasing worldwide. All 78 erythromycin-resistant S. pneumoniae isolates collected from blood cultures in our hospital (2000-07) were studied and the population structure was analysed by using different mathematical diversity indexes., Methods: Erythromycin resistance determinants were screened by PCR. The population structure, including multilocus sequence typing, was analysed by using quantitative clonal diversity (diversity ratio, Simpson, Selander-Levin and Shannon mathematical indexes)., Results: The leading resistance gene was erm(B) (74.3% of the isolates), followed by the erm(B) plus mef(A) combination (17.9%) and mef(A) alone (7.7%). The most frequent serotypes were 14 (18%), 19A (15.4%) and 6B (11.5%). A polyclonal structure was detected in resistant strains, including the Spain(9V)-3, Spain(6B)-2 and Denmark(14)-32 international clones. Both genetic diversity and genetic distribution were high, particularly among clones containing erm(B) and erm(B) plus mef(A) determinants., Conclusions: The resistance determinants erm(B) and the combination of erm(B) plus mef(A) were observed within multiple S. pneumoniae bacteraemic clones. The preservation of a polyclonal structure might provide a suitable background for further evolution of antibiotic resistance.

Objectives: Multidrug-resistant Streptococcus pneumoniae isolates of serotype 19A have emerged all over the world in recent years. The aim of this study was to characterize highly penicillin-resistant pneumococcal strains of the 19A serotype, collected in Spain from 1997 to 2007 from patients with invasive disease., Methods: Antibiotic susceptibility was studied by microdilution. All penicillin-resistant pneumococci were typed by PFGE and selected strains were studied by multilocus sequence typing (MLST). The presence of genes related to the Tn916 family of transposons was investigated by PCR., Results: From a total of 1197 invasive pneumococcal isolates of serotype 19A received at the Spanish Reference Laboratory between 1997 and 2007, 51 (4.3%) strains showed high-level resistance to penicillin (MICs of 2-4 mg/L). These 51 isolates belonged to three multiresistant clones related to sequence type (ST)81 (n = 21), ST320 (n = 19) and ST276 (n = 11). All 51 serotype 19A pneumococci were tetracycline-resistant and had the tet(M) gene, and 41 strains were macrolide-resistant, harbouring the erm(B) gene. The presence of int and xis genes was detected in all strains associated with other genes of the Tn916 family., Conclusions: The rise in penicillin-resistant serotype 19A invasive pneumococci in Spain was associated with the emergence and clonal spread of two worldwide-disseminated multiresistant clones (ST276 and ST320). The Spain(23F)-1-19A (ST81) clone remained stable throughout the study period. Multidrug resistance was associated with transposons of the Tn916 family.

Among 4,215 Streptococcus pneumoniae isolates obtained in Spain during 2006, 98 (2.3%) were ciprofloxacin resistant (3.6% from adults and 0.14% from children). In comparison with findings from a 2002 study, global resistance remained stable. Low-level resistance (30 isolates with MIC 4-8 microg/mL) was caused by a reserpine-sensitive efflux phenotype (n = 4) or single topoisomerase IV (parC [n = 24] or parE [n = 1]) changes. One isolate did not show reserpine-sensitive efflux or mutations. High-level resistance (68 isolates with MIC >or=16 microg/mL) was caused by changes in gyrase (gyrA) and parC or parE. New changes in parC (S80P) and gyrA (S81V, E85G) were shown to be involved in resistance by genetic transformation. Although 49 genotypes were observed, clones Spain9V-ST156 and Sweden15A-ST63 accounted for 34.7% of drug-resistant isolates. In comparison with findings from the 2002 study, clones Spain14-ST17, Spain23F-ST81, and ST8819F decreased and 4 new genotypes (ST9710A, ST57016, ST43322, and ST71733) appeared in 2006.

All patients < or =14 years old with a diagnosis of pneumococcal meningitis (n = 160) were included (2001, 2004-2006). Incidence per 100,000 child <5 years old varied from 6.14 in 2001 to 2.83 in 2006 (54% of reduction, P < 0.01). There was a significant correlation (R2 = 0.9506) with PCV7 distribution increase. No significant changes in the incidence by nonvaccine serotypes were found when comparing 2001 and 2006. The case fatality rate was 7.5%.

Background: Although protein binding is a reversible phenomenon, it is assumed that antibacterial activity is exclusively exerted by the free (unbound) fraction of antibiotics., Methodology/principal Findings: Activity of cefditoren, a highly protein bound 3(rd) generation cephalosporin, over 24h after an oral 400 mg cefditoren-pivoxil bid regimen was studied against six S. pneumoniae strains (penicillin/cefditoren MICs; microg/ml): S1 (0.12/0.25), S2 (0.25/0.25), S3 and S4 (0.5/0.5), S5 (1/0.5) and S6 (4/0.5). A computerized pharmacodynamic simulation with media consisting in 75% human serum and 25% broth (mean albumin concentrations = 4.85+/-0.12 g/dL) was performed. Protein binding was measured. The cumulative percentage of a 24h-period that drug concentrations exceeded the MIC for total (T > MIC) and unbound concentrations (fT > MIC), expressed as percentage of the dosing interval, were determined. Protein binding was 87.1%. Bactericidal activity (> or = 99.9% initial inocula reduction) was obtained against strains S1 and S2 at 24h (T > MIC = 77.6%, fT > MIC = 23.7%). With T > MIC of 61.6% (fT > MIC = 1.7%), reductions against S3 and S4 ranged from 90% to 97% at 12h and 24h; against S5, reduction was 45.1% at 12h and up to 85.0% at 24h; and against S6, reduction was 91.8% at 12h, but due to regrowth of 52.9% at 24h. Cefditoren physiological concentrations exerted antibacterial activity against strains exhibiting MICs of 0.25 and 0.5 microg/ml under protein binding conditions similar to those in humans., Conclusions/significance: The results of this study suggest that, from the pharmacodynamic perspective, the presence of physiological albumin concentrations may not preclude antipneumococcal activity of highly bound cephalosporins as cefditoren.

Objectives: To study the influence of resistance phenotypes (based on sentinel antibiotics: penicillin and amoxicillin with/without clavulanate) on the cefuroxime versus cefditoren susceptibility of Streptococcus pneumoniae and Haemophilus influenzae recovered from children with acute otitis media., Methods: Middle ear isolates (193 S. pneumoniae and 114 H. influenzae) received in the Spanish Reference Laboratory (Instituto de Salud Carlos III) were tested. Antimicrobial susceptibility to penicillin, amoxicillin with/without clavulanate, cefuroxime and cefditoren was determined by agar dilution using Mueller-Hinton agar supplemented with 5% sheep blood for S. pneumoniae and Haemophilus Test Medium for H. influenzae. Strains were classified according to penicillin susceptibility (S. pneumoniae) or beta-lactamase production (H. influenzae)., Results: The decrease in penicillin susceptibility of S. pneumoniae (from the susceptible to the resistant category) decreased amoxicillin and cefuroxime susceptibility rates from 100% to 34% and 0%, respectively. All pneumococcal strains were inhibited by 0.5 mg/L cefditoren, including those from penicillin-resistant serotypes 14, 23F, 6B and 9V with higher amoxicillin versus penicillin MICs. Susceptibility rates of beta-lactamase-positive H. influenzae strains were 93.8% and 85.4% to amoxicillin/clavulanate and cefuroxime, respectively. Resistance to amoxicillin/clavulanate (MIC>or=8/4 mg/L) was 12.1% (8 out of 66) and 6.3% (3 out of 48) in beta-lactamase-negative and -positive strains, respectively. All H. influenzae strains were inhibited by

Most studies reporting Streptococcus pneumoniae (Sp) infections select either specific populations/diseases, or refer to a single or a low number of institutions. A nationwide point-prevalence study including 147 hospitals from which we collected all the isolates of Sp reported in a single week (February 16-22, 2004). Workload and clinical data were studied, and susceptibility testing and serotyping of all isolates were performed. The participating institutions had an estimated catchment population of 37,534,750 inhabitants. During the study week, microbiology laboratories received 224,956 samples of which 34,647 were positive and 360 had S. pneumoniae. Overall, 69% of the isolates were from adults (> or = 15 years). Most of the isolates (89%) were considered clinically significant. Infection was community-acquired in 88% of infections and monomicrobial in 79%. We calculated that S. pneumoniae was isolated 38 times per 100,000 inhabitants/year and it was present in 1% of all samples with one or more bacterial isolates. We also calculated that pneumococcal disease (invasive and noninvasive) was present in 34 patients per 100,000 inhabitants/year. Penicillin resistance (I+R) was 42%, and erythromycin resistance was 35%. The most frequent serotypes were 3, 19F, and 19A. Considering only invasive isolates, the estimated coverage of the 7-valent vaccine was 61% in children and 41% in adults, and the estimated coverage of the 23-valent vaccine in adults was 79%. A nationwide point-prevalence study is an efficient tool for surveying pneumococcal infection in a large population.

Eight optochin-susceptible (Opt(s)) alpha-hemolytic (viridans) streptococcus isolates were characterized at the molecular level. These isolates showed phenotypic characteristics typical of both viridans streptococci and Streptococcus pneumoniae. Comparison of the sequence of housekeeping genes from these isolates with those of S. pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae suggested that the Opt(s) isolates corresponded to streptococci of the mitis group. Besides, the Opt(s) streptococci were negative by a Gen-Probe AccuProbe pneumococcus test and hybridized with specific pneumococcal probes (lytA and ply) but also with ant, a gene not present in most S. pneumoniae strains. Moreover, the isolates were insoluble in 1% sodium deoxycholate but completely dissolved in 0.1% deoxycholate. Sequence analysis of the lytA gene revealed that the Opt(s) streptococci carried lytA alleles characteristic of those present in nonpneumococcal streptococci of the mitis group. The determination of the partial nucleotide sequence embracing the atp operon encoding the F(o)F(1) H(+)-ATPase indicated that the optochin susceptibility of the isolates was due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae by horizontal gene transfer.

Objectives: To investigate antimicrobial effects on a mixed culture of five Streptococcus pneumoniae serotypes (S) as an approach to ecology of population dynamics., Methods: A computerized pharmacodynamic model simulating concentrations obtained after levofloxacin, ciprofloxacin and azithromycin doses was used. Resistance patterns were S12, susceptible to study drugs; S31, low-level macrolide-resistant (efflux phenotype); S11, high-level macrolide-resistant (erm genotype); S9V, low-level quinolone-resistant; and S3, high-level quinolone-resistant. Initial mixed inocula (time 0) included similar percentages of each serotype., Results: At 24 h of control drug-free experiments, dominant strains were S9V (57.4%) and S12 (41.8%) with marginal populations of S31, S3 and S11. Azithromycin selected to a much higher extent the strain with low-level resistance to macrolides (S31) rather than the strain with high-level resistance (S11) (accounting for 99.9% versus 0.1% of the total population at 24 h). Ciprofloxacin selected to a higher extent low-level (S9V) rather than high-level (S3) quinolone resistance (72.4% versus 27.6%). Levofloxacin decreased the proportion of the predominant S9V in controls to 22.2% (an intermediate-resistant strain with MIC = 4 mg/L) and unmasked the high-level resistant strain (MIC = 32 mg/L) up to 77.8%., Conclusions: Strain distribution in an antibiotic-free environment depends on bacterial fitness in mono- and multi-strain niches. Selective pressure of antimicrobial regimens eradicate some populations and unmask minor populations, thus redistributing the whole population. Selective potential only for resistance phenotypes with very low prevalence (such as high-level quinolone resistance) in the community should be preferred to that selecting more prevalent resistance phenotypes.

Objectives: High-level penicillin resistance in Streptococcus pneumoniae requires extensive re-modulation of the penicillin-binding proteins (PBPs), and murM gene function is also required for the expression of resistance. In this work, we determined whether specific changes in PBPs were associated with specific MurM variants., Methods: Two collections of highly penicillin-resistant (MIC 2-8 mg/L) isolates, including 10 early (1997-1998) and 23 contemporary (2002-2004) isolates, were studied., Results: Most of the isolates belonged to clones Spain(6B)-2 (13 strains), Spain(23F)-1 (10 isolates) and Spain(14)-5 (20 isolates). Different protein variants of MurM (MA, MB5, MB6, MB9 and MB10), PBP1A (A-C), PBP2B (A-D) and PBP2X (A-C) were recognized, including two murM alleles not previously described. Particular [MurM-PBP1A-2B-2X] allelic combinations were predominant among the different clones, including [MA-B-B-B] for old (MIC 2 mg/L) and [MB10-C-A-B] for recent (MIC 4-8 mg/L) Spain(6B)-2 isolates, [MA-A-C-A] for Spain(23F)-1 and [MB5-A-A-A] in Spain(14)-5 isolates., Conclusions: Although S. pneumoniae has a basic recombinational population structure, our results indicate remarkable conservation of PBPs and MurM protein types within each clone. This suggests that particular PBPs-MurM combinations tend to be preserved and may have an independent evolutionary history in particular clones.

Objective: To analyse the molecular structure of several antimicrobial resistance determinants in isolates of the Spain(14)-5 clone to better understand its emergence and spread., Methods: The distinct genes and mechanism of resistance to penicillin, erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim were studied in an apparently homogeneous group of 117 isolates of the multidrug-resistant Spain(14)-5 major clone isolated in Spain between 1981 and 2004., Results: Several genotyping techniques such as PFGE, BOX-PCR and multilocus sequence typing (MLST) revealed a high degree of homogeneity among these isolates over time. Nevertheless, distinct variants of the clone could be established according to the restriction fragment length polymorphism (RFLP) patterns of the penicillin-binding protein (pbp) genes and the sequences of the dihydrofolate reductase (dhfr) gene. In addition, an association between the pbp2b RFLP patterns, the ddl alleles identified by MLST and the dhfr alleles was found. The emergence of variants of the Spain(14)-5 clone, which had lost macrolide and tetracycline resistance, while harbouring the ins and xis genes of the Tn916-Tn1545 family of conjugative transposons, was documented. Two different tet(M) alleles were detected in isolates of the clone, one of them with a mosaic structure., Conclusions: The finding of different patterns or alleles of the genes responsible for antibiotic resistance among isolates of the Spain(14)-5 clone from different Spanish cities indicates different evolutionary events within isolates of a unique Streptococcus pneumoniae clone.

Forty-eight Streptococcus pneumoniae isolates recovered from sputum samples from 26 cystic fibrosis (CF) patients attending our CF unit (1995 to 2003) were studied. Mean yearly incidence of isolation was 5.5%, and all were strains recovered from young patients (< or = 12 years). The isolation was linked to clinical exacerbation in 35% of the cases, but only 27% of these were not accompanied by other CF pathogens. Fifty percent of the patients presented with two to four isolates over the studied period. Pulsed-field gel electrophoresis-SmaI digestion revealed a high heterogeneity (32 pulsotypes among 48 isolates) and the persistence over a 6-month period of a single clone (clone A) in two patients. This clone, presenting a varied multiresistance phenotype, was identified as the Spain23F-1 clone and was also recognized in six other patients, including two out of nine patients from the CF unit of Sant Joan de Deu Hospital, Barcelona, Spain. In our isolates, 16 different serotypes were recognized, the most frequent being 23F (33.3%), 19F (18.8%), 6A (6.2%), and 6B (6.2%). High overall resistance rates were observed: to penicillin, 73%; to cefotaxime, 33%; to erythromycin, 42%; to tetracycline, 58%; to chloramphenicol, 48%; and to trimethoprim-sulfamethoxazole, 67%. Resistance to fluoroquinolones was not detected. Multiresistance was a common feature (60%). The percentage of S. pneumoniae strains with increased frequencies of mutation to rifampin resistance (> or = 7.5 x 10(-8)) was significantly higher (P = 0.02) in CF (60%) than among non-CF (37%) isolates in the same institution (M. I. Morosini et al., Antimicrob. Agents Chemother. 47:1464-1467, 2003). Even though a clear association with acute exacerbations could not be observed, long-term clonal persistence and variability, high frequency of antibiotic resistance, and hypermutability indicate the plasticity for adaptation of S. pneumoniae to the CF lung environment.

More than 50% of the nontypeable (NT) pneumococcal strains received in our laboratory for reference purposes are isolated in sporadic cases of conjunctivitis. To determine the genetic structure of the population of these NT conjunctival strains, we analyzed 75 pneumococci (40 NT and 35 typeable) isolated from conjunctivas and 30 (15 NT and 15 typeable) isolated from other sources. The NT and typeable conjunctival strains grouped in separate clusters, whereas NT and typeable pneumococci isolated from other sources were similarly distributed. NT conjunctival strains belonged to two well-differentiated clonal lineages. The first, represented by three newly described sequence types, featured fully antibiotic susceptible strains and appeared to be characteristic of conjunctival tissue; the second, represented by the previously described ST344, had a pattern of multiresistance to penicillin, tetracycline, and erythromycin and shared a genetic background with some NT strains isolated from other sources.

Among 2,882 Streptococcus pneumoniae sent to the Spanish Reference Laboratory during 2002, 75 (2.6%) were ciprofloxacin-resistant. Resistance was associated with older patients (3.9% in adults and 7.2% in patients > or =65 years of age), with isolation from noninvasive sites (4.3% vs. 1.0%), and with penicillin and macrolide resistance. Among 14 low-level resistant (MIC 4-8 microg/mL) strains, 1 had a fluoroquinolone efflux phenotype, and 13 showed single ParC changes. The 61 high-level ciprofloxacin-resistant (MIC > or =16 microg/mL) strains showed either two or three changes at ParC, ParE, and GyrA. Resistance was acquired either by point mutation (70 strains) or by recombination with viridans streptococci (4 strains) at the topoisomerase II genes. Although 36 pulsed-field gel electrophoresis patterns were observed, 5 international multiresistant clones (Spain23F-1, Spain6B-2, Spain9V-3, Spain14-5 and Sweden15A-25) accounted for 35 (46.7%) of the ciprofloxacin-resistant strains. Continuous surveillance is needed to prevent the dissemination of these clones.

Objectives: To investigate the association between geographical differences in antibiotic consumption and resistance of Streptococcus pneumoniae to penicillin and erythromycin in 15 provinces of Spain, taking into account the potential influence of a series of social and climatological factors., Methods: Possible correlations between prevalence of resistance to penicillin and erythromycin of S. pneumoniae, as determined in the national reference laboratory, and antibiotic consumption, and socio-economic and climatological variables were investigated. Partial correlations and multivariate linear regression were performed to assess the relative importance of variables predicting resistance and to investigate explicative factors for antibiotic consumption, respectively., Results: A correlation was found between resistance and educational level, the proportion of young people in the population and climate, but was explained by their effects on differences in antibiotic use, which appeared to be the basic and only force behind resistance patterns in different geographical areas. Antibiotic use was found to be determined by the interplay of adult illiteracy, rainfall and GDP per capita., Conclusions: Interventions aimed at improving educational level and economic growth might therefore be followed by a noticeable reduction in overall antibiotic consumption, which might in turn be followed by a reduction in penicillin and erythromycin resistance in clinical isolates of S. pneumoniae.

A multicenter study of 638 cases of community-acquired pneumonia due to Streptococcus pneumoniae (SP-CAP) was performed to assess current levels of resistance. Of the pneumococcal strains, 35.7% had an minimum inhibitory concentration (MIC) of penicillin of > or =0.12 microg/mL (3 isolates had an MIC of 4 microg/mL), 23.8% had an MIC of erythromycin of 128 microg/mL, and 22.2% were multidrug resistant. Logistic regression determined that chronic pulmonary disease (odds ratio [OR], 1.44], human immunodeficiency virus infection (OR, 1.98), clinically suspected aspiration (OR, 2.12), and previous hospital admission (OR, 1.69) were related to decreased susceptibility to penicillin, and previous admission (OR, 1.89) and an MIC of penicillin of MIC > or =0.12 microg/mL (OR, 15.85) were related to erythromycin resistance (MIC, > or =1 microg/mL). The overall mortality rate was 14.4%. Disseminated intravascular coagulation, empyema, and bacteremia were significantly more frequent among patients with penicillin-susceptible SP-CAP. Among isolates with MICs of penicillin of > or =0.12 microg/mL, serotype 19 was predominant and was associated with a higher mortality rate. In summary, the rate of resistance to beta -lactams and macrolides among S. pneumoniae that cause CAP remains high, but such resistance does not result in increased morbidity.

Objectives: Phage-coded lysins, i.e. murein hydrolases, are enzymes that destroy the cell wall of bacteria. A rapid killing of Streptococcus pneumoniae in the nasopharynx of mice has been described recently using a phage-coded murein hydrolase (enzybiotic). The in vivo effects of a dose-ranging treatment, using either of the phage-coded lytic enzymes Cpl-1 lysozyme or the Pal amidase, have been investigated here in a murine sepsis model., Methods: Purified Pal amidase and/or Cpl-1 lysozyme were used alone or in combination. These enzymes were injected intraperitoneally at different times after challenge with 5 x 10(7) cfu of a type 6B, antibiotic-resistant S. pneumoniae clinical isolate., Results: Animals challenged with 5 x 10(7) cfu of this strain alone died within 72 h, whereas a single intraperitoneal injection of Cpl-1 or Pal (200 microg; 1100 U) administered 1 h after the bacterial challenge was sufficient to effectively protect the mice, according to unpaired t-test (P<0.0001). Bacteraemia in unprotected mice reached colony counts >10(7) cfu/mL, whereas the mean colony count in lysin-protected animals was <10(6) cfu/mL over time and ultimately became undetectable. Interestingly, a synergic effect in vivo was observed with the combined use of 2.5 microg each of Cpl-1 and Pal., Conclusions: Our findings suggest strongly that phage lysins protect animals from bacteraemia and death. Moreover, the simultaneous attack of the pneumococcal peptidoglycan by a lysozyme and an amidase leads to a remarkable effect through enhanced destruction of the bacterial cell wall. The benefits of therapy with enzybiotics against pneumococcus reported here might warrant the examination of alternative strategies for the treatment of diseases caused by clinically relevant pathogens.

Two clinical isolates of viridans group streptococci (VS) with different degrees of susceptibility to optochin (OPT), i.e., fully OPT-susceptible (Opt(s)) VS strain 1162/99 (for which the MIC was equal to that for Streptococcus pneumoniae, 0.75 micro g/ml) and intermediate Opt(s) VS strain 1174/97 (MIC, 6 micro g/ml) were studied. Besides being OPT susceptible, they showed characteristics typical of VS, such as bile insolubility; lack of reaction with pneumococcal capsular antibodies; and lack of hybridization with rRNA (AccuProbe)-, lytA-, and pnl-specific pneumococcal probes. However, these VS Opt(s) strains and VS type strains hybridized with ant, a gene not present in S. pneumoniae. A detailed characterization of the genes encoding the 16S rRNA and SodA classified isolates 1162/99 and 1174/97 as Streptococcus mitis. Analysis of the atpCAB region, which encodes the c, a, and b subunits of the F(0)F(1) H(+)-ATPase, the target of optochin, revealed high degrees of similarity between S. mitis 1162/99 and S. pneumoniae in atpC, atpA, and the N terminus of atpB. Moreover, amino acid identity between S. mitis 1174/97 and S. pneumoniae was found in alpha helix 5 of the a subunit. The organization of the chromosomal region containing the atp operon of the two Opt(s) VS and VS type strains was spr1284-atpC, with spr1284 being located 296 to 556 bp from atpC, whereas in S. pneumoniae this distance was longer than 68 kb. In addition, the gene order in S. pneumoniae was IS1239-74 bp-atpC. The results suggest that the full OPT susceptibility of S. mitis 1162/99 is due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae and that the intermediate OPT susceptibility of S. mitis 1174/97 correlates with the amino acid composition of its a subunit.

The autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical Streptococcus pneumoniae) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent lytA alleles (pairwise evolutionary distances of about 20%) compared to the lytA alleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to S. pneumoniae. The clinical importance of these findings is underlined by the role of the lytA gene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the Streptococcus mitis group.