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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.)

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.

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.

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

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.

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.