Competitive Dominance within Biofilm Consortia Regulates the Relative Distribution of Pneumococcal Nasopharyngeal Density.

Streptococcus pneumoniae is a main cause of child mortality worldwide, but strains also asymptomatically colonize the upper airways of most children and form biofilms. Recent studies have demonstrated that ∼50% of colonized children carry at least two different serotypes (i.e., strains) in the nasopharynx; however, studies of how strains coexist are limited. In this work, we investigated the physiological, genetic, and ecological requirements for the relative distribution of densities, and spatial localization, of pneumococcal strains within biofilm consortia. Biofilm consortia were prepared with vaccine type strains (i.e., serotype 6B [S6B], S19F, or S23F) and strain TIGR4 (S4). Experiments first revealed that the relative densities of S6B and S23F were similar in biofilm consortia. The density of S19F strains, however, was reduced to ∼10% in biofilm consortia, including either S6B, S23F, or TIGR4, in comparison to S19F monostrain biofilms. Reduction of S19F density within biofilm consortia was also observed in a simulated nasopharyngeal environment. Reduction of relative density was not related to growth rates, since the Malthusian parameter demonstrated similar rates of change of density for most strains. To investigate whether quorum sensing (QS) regulates relative densities in biofilm consortia, two different mutants were prepared: a TIGR4ΔluxS mutant and a TIGR4ΔcomC mutant. The density of S19F strains, however, was similarly reduced when consortia included TIGR4, TIGR4ΔluxS, or TIGR4ΔcomC Moreover, production of a different competence-stimulating peptide (CSP), CSP1 or CSP2, was not a factor that affected dominance. Finally, a mathematical model, confocal experiments, and experiments using Transwell devices demonstrated physical contact-mediated control of pneumococcal density within biofilm consortia.IMPORTANCEStreptococcus pneumoniae kills nearly half a million children every year, but it also produces nasopharyngeal biofilm consortia in a proportion of asymptomatic children, and these biofilms often contain two strains (i.e., serotypes). In our study, we investigated how strains coexist within pneumococcal consortia produced by vaccine serotypes S4, S6B, S19F, and S23F. Whereas S6B and S23F shared the biofilm consortium, our studies demonstrated reduction of the relative density of S19F strains, to ∼10% of what it would otherwise be if alone, in consortial biofilms formed with S4, S6B, or S23F. This dominance was not related to increased fitness when competing for nutrients, nor was it regulated by quorum-sensing LuxS/AI-2 or Com systems. It was demonstrated, however, to be enhanced by physical contact rather than by a product(s) secreted into the supernatant, as would naturally occur in the semidry nasopharyngeal environment. Competitive interactions within pneumococcal biofilm consortia regulate nasopharyngeal density, a risk factor for pneumococcal disease.

Development of a TaqMan Array Card for Pneumococcal Serotyping on Isolates and Nasopharyngeal Samples.

Streptococcus pneumoniae is both a commensal and a major pathogen that causes invasive disease in people of all ages. The introduction of serotype-specific pneumococcal vaccines has reduced the burden of disease but has also led to replacement with new strains; thus, serotyping remains important for vaccine-related disease surveillance. Conventional serotyping methods are laborious and expensive. We developed an easy-to-perform genotypic TaqMan array card (TAC) to identify S. pneumoniae strains, including lytA-based sequences, and 53 sequence-specific PCRs to identify 74 serotypes/serogroups covering all current vaccine types as well as prevalent nonvaccine types. The TAC method was evaluated on 146 clinical S. pneumoniae isolates and 13 nonpneumococcal species that naturally inhabit the upper respiratory tract and yielded 97% (142/146) sensitivity and 100% (13/13) specificity versus results of standard Quellung serotyping. The calculated limit of detection was 20 to 200 fg (∼8 to 84 genome equivalents) per reaction. On 23 blinded nasopharyngeal specimens that were pneumococcus culture positive, the TAC pan-pneumococcus lytA assay was positive in 21 (91% sensitivity versus culture). On TAC lytA-positive specimens, a serotype result was obtained on 86%, and the result was 95% accurate versus the subsequent culture's Quellung result. TAC also detected mixed serotypes in two specimens where Quellung detected only the predominant serotype. This TAC method yields fast and comprehensive serotyping compared to the standard method and may be useful on direct specimens.

Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan.

Limited use of linezolid for treating methicillin-resistant Staphylococcus aureus (MRSA) infection was approved in Japan in 2006. We report here the status of linezolid-resistant MRSAs in Japan. Eleven linezolid-resistant clinical isolates from 11 patients at six hospitals were collected from 2006 through 2008. The minimal inhibitory concentration (MIC) of linezolid in these strains varied from 8 to 64 µg/ml. All strains had at least one G2576T mutation in the chromosomal gene(s) encoding domain V of the 23S ribosomal RNA (rRNA). Chromosomal DNA encoding five copies of the domain V region was analyzed by polymerase chain reaction (PCR). Strains with the linezolid MICs of 64, 32, 16, and 8 µg/ml had the G2576T mutation(s) in four, three (or four), two, and one copy of the 23S rRNA genes, respectively. These results suggest that the level of linezolid resistance seems to be roughly correlated with the number of mutations in the genes encoding 23S rRNA. DNA samples from all 11 strains were subjected to pulsed-field gel electrophoresis and were classified into seven independent clones having >92% identity. Among the 11 patients, five had been treated with linezolid and the remainder, in two hospitals, had no history of prior linezolid use. The results suggested possible nosocomial infections by linezolid-resistant MRSA.

Ultrastructural, metabolic and genetic determinants of the acquisition of macrolide resistance by Streptococcus pneumoniae.

Aim: Streptococcus pneumoniae (Spn) acquires genes for macrolide resistance, MEGA or ermB, in the human host. These genes are carried either in the chromosome, or on integrative conjugative elements (ICEs). Here, we investigated molecular determinants of the acquisition of macrolide resistance. Methods and Results: Whole genome analysis was conducted for 128 macrolide-resistant pneumococcal isolates to identify the presence of MEGA (44.5%, 57/128) or ermB (100%), and recombination events in Tn916-related elements or in the locus comCDE encoding competence genes. Confocal and electron microscopy studies demonstrated that, during the acquisition of macrolide resistance, pneumococcal strains formed clusters of varying size, with the largest aggregates having a median size of ~1600 µm2. Remarkably, these pneumococcal aggregates comprise both encapsulated and nonencapsulated pneumococci, exhibited physical interaction, and spanned extracellular and intracellular compartments. We assessed the recombination frequency (rF) for the acquisition of macrolide resistance by a recipient D39 strain, from pneumococcal strains carrying MEGA (~5.4 kb) in the chromone, or in large ICEs (>23 kb). Notably, the rF for the acquisition of MEGA, whether in the chromosome or carried on an ICE was similar. However, the rF adjusted to the acquisition of the full-length ICE (~52 kb), compared to that of the capsule locus (~23 kb) that is acquired by transformation, was three orders of magnitude higher. Finally, metabolomics studies revealed a link between the acquisition of ICE and the metabolic pathways involving nicotinic acid and sucrose. Conclusions: Extracellular and intracellular pneumococcal clusters facilitate the acquisition of full-length ICE at a rF higher than that of typical transformation events, involving distinct metabolic changes that present potential targets for interventions.

Molecular epidemiologic characteristics of Streptococcus pneumoniae isolates from children with meningitis in Japan from 2007 through 2009.

We examined associations of serotypes with multilocus sequence typing (MLST) data for 7 housekeeping genes and the genotype concerning penicillin resistance based on penicillin-binding protein (PBP) alterations in Streptococcus pneumoniae isolates from children with meningitis. From throughout Japan, we collected 115 pneumococcal isolates from the cerebrospinal fluid of patients 15 years old or younger from January 2007 to December 2009. We then carried out serotyping, MLST, and genotypic classification. Isolates included 24 serotypes and 52 sequence types (STs) according to MLST, of which 18 were novel. The 4 predominant serotypes included a variety of STs: 14 STs in serotype 6B (n = 24), 2 STs in 19F (n = 17), 6 STs in 23F (n = 14), and 5 STs in 14 (n = 11). Resistance genotypes included 6 types: 44.3% for gPRSP (pbp1a + 2x + 2b), 13.9% for gPISP (pbp1a + 2x), 9.6% for gPISP (pbp2x + 2b), 19.1% for gPISP (pbp2x), 3.5% for gPISP (pbp2b), and 9.6% for gPSSP. Interestingly, the most prevalent serotype of 6B included 7 newly identified STs and a variety of genotypes for resistance. STs in serotypes 23F and 14 were highly diverse, but not in 19F. These results suggest that various genetic elements in S. pneumoniae might be intrinsically susceptible to genetic mutations and recombination, with acceleration of emergence reflecting selection pressures such as antibiotic overuse.

Trends in empirical chemotherapy of bacterial meningitis in children aged more than 4 months in Japan: a survey from 1997 through 2008.

Bacterial meningitis is a serious problem in pediatric clinics and, therefore, needs urgent and empirical chemotherapy. We investigated 1,116 cases of empirical chemotherapy of patients aged older than 4 months from 1997 through 2008 by sending questionnaires. A single antibiotic treatment was carried out in less than 30% of the cases throughout the years, whereas the combination of two antibiotics had been practiced in more than 70% of the cases. The main antibiotics used were cephalosporins, carbapenems, and ampicillin. Combinatory use of ampicillin and cephalosporin was carried out in 74.7-82.7% of cases in 1997-2000, but sharply declined thereafter to 0-13.8% in 2004-2008. However, the combination of carbapenem and cephalosporin compensated for the decline, increasing from 3.8-6.6% in 1998-1999 to 79.5-89.9% in 2005-2008. The breakdown in the use of cephalosporins, carbapenems, and ampicillin in two-drug combinatory therapy was as follows. (i) Use of cefotaxime was 61.8-75.3% in 1997-2001, but decreased to nearly 50%, equivalent to the level of ceftriaxone use in 2003-2008. (ii) Use of ampicillin dropped from 74.7-92.3% in 1997-2000 to 4.6% in 2008, and this decreased level was compensated for by the use of carbapenems. Overall, combinatory chemotherapy of the third-generation cephalosporins and carbapenems seems to be practical. The discussion in this report includes the difference between Japan and the United States in the prevalence of the causative agents and the use of antibiotics. These studies provide information on trends in the treatment of children's meningitis in Japan and will be useful for the design of future empirical chemotherapy.

Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan.

Limited use of linezolid for treating methicillin-resistant Staphylococcus aureus (MRSA) infection was approved in Japan in 2006. We report here the status of linezolid-resistant MRSAs in Japan. Eleven linezolid-resistant clinical isolates from 11 patients at six hospitals were collected from 2006 through 2008. The minimal inhibitory concentration (MIC) of linezolid in these strains varied from 8 to 64 µg/ml. All strains had at least one G2576T mutation in the chromosomal gene(s) encoding domain V of the 23S ribosomal RNA (rRNA). Chromosomal DNA encoding five copies of the domain V region was analyzed by polymerase chain reaction (PCR). Strains with the linezolid MICs of 64, 32, 16, and 8 µg/ml had the G2576T mutation(s) in four, three (or four), two, and one copy of the 23S rRNA genes, respectively. These results suggest that the level of linezolid resistance seems to be roughly correlated with the number of mutations in the genes encoding 23S rRNA. DNA samples from all 11 strains were subjected to pulsed-field gel electrophoresis and were classified into seven independent clones having >92% identity. Among the 11 patients, five had been treated with linezolid and the remainder, in two hospitals, had no history of prior linezolid use. The results suggested possible nosocomial infections by linezolid-resistant MRSA.

Emergence of the community-acquired methicillin-resistant Staphylococcus aureus USA300 clone in a Japanese child, demonstrating multiple divergent strains in Japan.

In 2008 we isolated methicillin-resistant Staphylococcus aureus (MRSA) from an 11-month-old Japanese girl who lived in Saitama, Japan, and suffered from cellulitis of the lower thigh and sepsis. The MRSA (strain NN47) belonged to multilocus sequence type (ST) 8 and exhibited spa363 (t024), agr1, staphylococcal cassette chromosome mec (SCCmec) type IVa, and coagulase type III. It was positive for Panton-Valentine leukocidin (PVL) and the arginine catabolic mobile element (ACME). Pulsed-field gel electrophoresis (PFGE) demonstrated that the MRSA was the USA300 clone, which is the predominant community-acquired MRSA (CA-MRSA) in the US. Strain NN47 was divergent, in terms of the spa type and patterns of PFGE and plasmids, from the USA300-0114 type strain or USA300 strain NN36, previously isolated from a visitor (Indian girl) from the US. Strain NN47 was resistant to erythromycin, in addition to beta-lactam agents (e.g., oxacillin). These data demonstrate the first emergence of the USA300 clone in Japanese children who have never been abroad and have had no contact with foreigners (and therefore, the first USA300 spread in Japan), and also emergence of multiple divergent strains of the USA300 clone in Japan. Because the USA300 clone is highly transmissible and virulent, surveillance of the USA300 clone is needed.

[Childhood bacterial meningitis trends in Japan from 2007 to 2008].

We surveyed pediatrics bacterial meningitis epidemiology from January 2007 to December 2008 in Japan, with the following results: Cases numbered 287-160 male and 127 female-equivalent to 1.54-1.62 of 1,000 pediatric hospitalization per year. Children under 1-year-old accounted for the highest number of cases, which decreased with increasing age. Haemophilus influenzae was the most common cause of infection, followed by Streptococcus pneumoniae, group B streptococcus (GBS), and Escherichia coli. GBS and E. coli were major pathogens in children under 4 months of age, while H. influenzae and S. pneumoniae mainly accounted for those over 4 months of age. Susceptibility tests showed that 51% of H. influenzae isolates and 56.5% of S. pneumoniae isolates in 2008 were drug-resistant. Ampicillin combined with cephem antibiotics effective against GBS, E. coli, and Listeria, were mainly used to initially treat those under 4 months of age. In those over 4 months of age, carbapenem antibiotics are effective against PRSP and cephem antibiotics against H. influenza.

A Mechanism of Unidirectional Transformation, Leading to Antibiotic Resistance, Occurs within Nasopharyngeal Pneumococcal Biofilm Consortia.

Streptococcus pneumoniae acquires genes for resistance to antibiotics such as streptomycin (Str) or trimethoprim (Tmp) by recombination via transformation of DNA released by other pneumococci and closely related species. Using naturally transformable pneumococci, including strain D39 serotype 2 (S2) and TIGR4 (S4), we studied whether pneumococcal nasopharyngeal transformation was symmetrical, asymmetrical, or unidirectional. Incubation of S2Tet and S4Str in a bioreactor simulating the human nasopharynx led to the generation of SpnTet/Str recombinants. Double-resistant pneumococci emerged soon after 4 h postinoculation at a recombination frequency (rF) of 2.5 × 10-4 while peaking after 8 h at a rF of 1.1 × 10-3 Acquisition of antibiotic resistance genes by transformation was confirmed by treatment with DNase I. A high-throughput serotyping method demonstrated that all double-resistant pneumococci belonged to one serotype lineage (S2Tet/Str) and therefore that unidirectional transformation had occurred. Neither heterolysis nor availability of DNA for transformation was a factor for unidirectional transformation given that the density of each strain and extracellular DNA (eDNA) released from both strains were similar. Unidirectional transformation occurred regardless of the antibiotic-resistant gene carried by donors or acquired by recipients and regardless of whether competence-stimulating peptide-receptor cross talk was allowed. Moreover, unidirectional transformation occurred when two donor strains (e.g., S4Str and S19FTmp) were incubated together, leading to S19FStr/Tmp but at a rF 3 orders of magnitude lower (4.9 × 10-6). We finally demonstrated that the mechanism leading to unidirectional transformation was due to inhibition of transformation of the donor by the recipient.IMPORTANCE Pneumococcal transformation in the human nasopharynx may lead to the acquisition of antibiotic resistance genes or genes encoding new capsular variants. Antibiotics and vaccines are currently putting pressure on a number of strains, leading to an increase in antibiotic resistance and serotype replacement. These pneumococcal strains are also acquiring virulence traits from vaccine types via transformation. In this study, we recapitulated multiple-strain colonization with strains carrying a resistance marker and selected for those acquiring resistance to two or three antibiotics, such as would occur in the human nasopharynx. Strains acquiring dual and triple resistance originated from one progenitor, demonstrating that transformation was unidirectional. Unidirectional transformation was the result of inhibition of transformation of donor strains. Unidirectional transformation has implications for the understanding of acquisition patterns of resistance determinants or capsule-switching events.

Nosocomial, Multidrug-Resistant Klebsiella pneumoniae Strains Isolated from Mexico City Produce Robust Biofilms on Abiotic Surfaces but Not on Human Lung Cells.

BACKGROUND: Klebsiella pneumoniae (Kpn) strains are a leading cause of hospital-acquired infections, including ventilator-associated pneumonia. Resistance to antibiotics, biofilm formation, and the production of certain fimbriae play an important role in the pathogenesis. AIM: We investigated the genetic relatedness, antibiotic resistance, virulence potential, and ability to form biofilms of Kpn strains isolated from hospital-acquired infections (n = 76). Strains were isolated at three major hospitals serving the largest metropolitan urban area in Mexico City, Mexico. RESULTS: Enterobacterial repetitive intergenic consensus (ERIC)-PCR demonstrated that clonal groups predominate in each hospital. Selected strains chosen from clonal groups (n = 47) were multidrug resistant (MDR, 83%), although the majority (∼70%) were susceptible to carbapenems. All strains produced robust biofilms on abiotic surfaces, and ∼90% harbored adhesin genes fimH, mrkA, and ecpA. The ultrastructure of biofilms was further studied by high-resolution confocal microscopy. The average height of Kpn biofilms on abiotic surfaces was ∼40 µm. We then assessed formation of biofilms on human lung cells, as a surrogate of lung infection. While Kpn strains formed robust biofilms on abiotic surfaces, studies on lung cells revealed attachment to human cells but scarce formation of biofilms. Gene expression studies revealed a differential temporal expression of an adhesin (ecpA) and a capsule (galF) gene when biofilms were formed on different substrates. CONCLUSIONS: Kpn strains isolated from nosocomial infections in Mexico City are MDR, although the majority are still susceptible to carbapenems and form more robust biofilms on polystyrene in comparison to those formed on human cells.

Development and characterization of a synthetic DNA, NUversa, to be used as a standard in quantitative polymerase chain reactions for molecular pneumococcal serotyping.

Identification of Streptococcus pneumoniae and its more than 90 serotypes is routinely conducted by culture and Quellung reactions. Quantitative polymerase chain reactions (qPCRs) have been developed for molecular detection, including a pan-pneumococcus lytA assay, and assays targeting 79 serotypes. Reactions require genomic DNA from every target to prepare standards, which can be time consuming. In this study, we have developed a synthetic DNA molecule as a surrogate for genomic DNA and present new single-plex qPCR reactions to increase molecular detection to 94 pneumococcal serotypes. Specificity of these new reactions was confirmed with a limit of detection between 2 and 20 genome equivalents/reaction. A synthetic DNA (NUversa, ∼8.2 kb) was then engineered to contain all available qPCR targets for serotyping and lytA. NUversa was cloned into pUC57-Amp-modified to generate pNUversa (∼10.2 kb). Standards prepared from pNUversa and NUversa were compared against standards made out of genomic DNA. Linearity [NUversa (R2 > 0.982); pNUversa (R2 > 0.991)] and efficiency of qPCR reactions were similar to those utilizing chromosomal DNA (R2 > 0.981). Quantification with plasmid pNUversa was affected, however, whereas quantification with synthetic NUversa was comparable to that of genomic DNA. Therefore, NUversa may be utilized as DNA standard in single-plex assays of the currently known 94 pneumococcal serotypes.

Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact.

Staphylococcus aureus (Sau) strains are a main cause of disease, including nosocomial infections which have been linked to the production of biofilms and the propagation of antibiotic resistance strains such as methicillin-resistant Staphylococcus aureus (MRSA). A previous study found that Streptococcus pneumoniae (Spn) strains kill planktonic cultures of Sau strains. In this work, we have further evaluated in detail the eradication of Sau biofilms and investigated ultrastructural interactions of the biofilmicidal effect. Spn strain D39, which produces the competence stimulating peptide 1 (CSP1), reduced Sau biofilms within 8 h of inoculation, while TIGR4, producing CSP2, eradicated Sau biofilms and planktonic cells within 4 h. Differences were not attributed to pherotypes as other Spn strains producing different pheromones eradicated Sau within 4 h. Experiments using Transwell devices, which physically separated both species growing in the same well, demonstrated that direct contact between Spn and Sau was required to efficiently eradicate Sau biofilms and biofilm-released planktonic cells. Physical contact-mediated killing of Sau was not related to production of hydrogen peroxide as an isogenic TIGR4ΔspxB mutant eradicated Sau bacteria within 4 h. Confocal micrographs confirmed eradication of Sau biofilms by TIGR4 and allowed us to visualize ultrastructural point of contacts between Sau and Spn. A time-course study further demonstrated spatial colocalization of Spn chains and Sau tetrads as early as 30 min post-inoculation (Pearson's coefficient >0.72). Finally, precolonized biofilms produced by Sau strain Newman, or MRSA strain USA300, were eradicated by mid-log phase cultures of washed TIGR4 bacteria within 2 h post-inoculation. In conclusion, Spn strains rapidly eradicate pre-colonized Sau aureus biofilms, including those formed by MRSA strains, by a mechanism(s) requiring bacterium-bacterium contact, but independent from the production of hydrogen peroxide.

Single-plex quantitative assays for the detection and quantification of most pneumococcal serotypes.

Streptococcus pneumoniae globally kills more children than any other infectious disease every year. A prerequisite for pneumococcal disease and transmission is colonization of the nasopharynx. While the introduction of pneumococcal conjugate vaccines has reduced the burden of pneumococcal disease, understanding the impact of vaccination on nasopharyngeal colonization has been hampered by the lack of sensitive quantitative methods for the detection of >90 known S. pneumoniae serotypes. In this work, we developed 27 new quantitative (q)PCR reactions and optimized 26 for a total of 53 qPCR reactions targeting pneumococcal serotypes or serogroups, including all vaccine types. Reactions proved to be target-specific with a limit of detection of 2 genome equivalents per reaction. Given the number of probes required for these assays and their unknown shelf-life, the stability of cryopreserved reagents was evaluated. Our studies demonstrate that two-year cryopreserved probes had similar limit of detection as freshly-diluted probes. Moreover, efficiency and limit of detection of 1-month cryopreserved, ready-to-use, qPCR reaction mixtures were similar to those of freshly prepared mixtures. Using these reactions, our proof-of-concept studies utilizing nasopharyngeal samples (N=30) collected from young children detected samples containing ≥2 serotypes/serogroups. Samples colonized by multiple serotypes/serogroups always had a serotype that contributes at least 50% of the pneumococcal load. In addition, a molecular approach called S6-q(PCR)2 was developed and proven to individually detect and quantify epidemiologically-important serogroup 6 strains including 6A, 6B, 6C and 6D. This technology will be useful for epidemiological studies, diagnostic platforms and to study the pneumobiome.

Expression of Streptococcus pneumoniae Virulence-Related Genes in the Nasopharynx of Healthy Children.

Colonization and persistence in the human nasopharynx are prerequisites for Streptococcus pneumoniae disease and carriage acquisition, which normally occurs during early childhood. Animal models and in vitro studies (i.e. cell adhesion and cell cytotoxicity assays) have revealed a number of colonization and virulence factors, as well as regulators, implicated in nasopharyngeal colonization and pathogenesis. Expression of genes encoding these factors has never been studied in the human nasopharynx. Therefore, this study analyzed expression of S. pneumoniae virulence-related genes in human nasopharyngeal samples. Our experiments first demonstrate that a density of ≥10(4) CFU/ml of S. pneumoniae cells in the nasopharynx provides enough DNA and RNA to amplify the lytA gene by conventional PCR and to detect the lytA message, respectively. A panel of 21 primers that amplified S. pneumoniae sequences was designed, and their specificity for S. pneumoniae sequences was analyzed in silico and validated against 20 related strains inhabitants of the human upper respiratory tract. These primers were utilized in molecular reactions to find out that all samples contained the genes ply, pavA, lytC, lytA, comD, codY, and mgrA, whereas nanA, nanB, pspA, and rrgB were present in ∼91-98% of the samples. Gene expression studies of these 11 targets revealed that lytC, lytA, pavA and comD were the most highly expressed pneumococcal genes in the nasopharynx whereas the rest showed a moderate to low level of expression. This is the first study to evaluate expression of virulence- and, colonization-related genes in the nasopharynx of healthy children and establishes the foundation for future gene expression studies during human pneumococcal disease.

Novel role for the Streptococcus pneumoniae toxin pneumolysin in the assembly of biofilms.

UNLABELLED: Streptococcus pneumoniae is an important commensal and pathogen responsible for almost a million deaths annually in children under five. The formation of biofilms by S. pneumoniae is important in nasopharyngeal colonization, pneumonia, and otitis media. Pneumolysin (Ply) is a toxin that contributes significantly to the virulence of S. pneumoniae and is an important candidate as a serotype-independent vaccine target. Having previously demonstrated that a luxS knockout mutant was unable to form early biofilms and expressed less ply mRNA than the wild type, we conducted a study to investigate the role of Ply in biofilm formation. We found that Ply was expressed in early phases of biofilm development and localized to cellular aggregates as early as 4 h postinoculation. S. pneumoniae ply knockout mutants in D39 and TIGR4 backgrounds produced significantly less biofilm biomass than wild-type strains at early time points, both on polystyrene and on human respiratory epithelial cells, cultured under static or continuous-flow conditions. Ply's role in biofilm formation appears to be independent of its hemolytic activity, as S. pneumoniae serotype 1 strains, which produce a nonhemolytic variant of Ply, were still able to form biofilms. Transmission electron microscopy of biofilms grown on A549 lung cells using immunogold demonstrated that Ply was located both on the surfaces of pneumococcal cells and in the extracellular biofilm matrix. Altogether, our studies demonstrate a novel role for pneumolysin in the assembly of S. pneumoniae biofilms that is likely important during both carriage and disease and therefore significant for pneumolysin-targeting vaccines under development. IMPORTANCE: The bacterium Streptococcus pneumoniae (commonly known as the pneumococcus) is commonly carried in the human nasopharynx and can spread to other body sites to cause disease. In the nasopharynx, middle ear, and lungs, the pneumococcus forms multicellular surface-associated structures called biofilms. Pneumolysin is an important toxin produced by almost all S. pneumoniae strains, extensively studied for its ability to cause damage to human tissue. In this paper, we demonstrate that pneumolysin has a previously unrecognized role in biofilm formation by showing that strains without pneumolysin are unable to form the same amount of biofilm on plastic and human cell substrates. Furthermore, we show that the role of pneumolysin in biofilm formation is separate from the hemolytic activity responsible for tissue damage during pneumococcal diseases. This novel role for pneumolysin suggests that pneumococcal vaccines directed against this protein should be investigated for their potential impact on biofilms formed during carriage and disease.

A 25-year trace of methicillin-resistant Staphylococcus aureus dissemination in a geriatric hospital in Japan.

We analyzed 218 strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated from the septicemia patients in a geriatric hospital for 25 years. These strains were classified into 11 major DNA types, A through K, and 27 minor types. The strains belonging to group A and B isolated before 1990 were susceptible to imipenem (IPM), fluoroquinolone, and most other antibiotics tested, except that they were markedly resistant to gentamicin. Strains mostly isolated in 1985 and thereafter were classified into group C through K, and they were mainly resistant to IPM, fluoroquinolones, and clindamycin. Analysis of the MRSA marker gene, staphylococcal cassette chromosome mec (SCCmec), of these strains revealed that the strains in groups A and B had mainly type IV and type I, respectively, and that strains in groups C through J had mainly type II. These results suggested that the strains holding type II SCCmec were resistant to IPM, fluoroquinolone, and clindamycin and they were dominant-resistant type after late 1980s. The antibiotic resistance profiles of MRSA dramatically changed during late 1980s, and these were correlated with the SCCmec types. The lesson from this study would be that consistent execution of surveillance study is needed to update the resistant profiles.