OBJECTIVETo investigate the relationship between bacterial biofilm and acute otitis media by observing the feature of bacterial biofilm formation in middle-ear mucosa in the rat model of acute otitis media and to study the possibility of application this rat model in bacterial biofilm research.

METHODSA total of 30 healthy, male SD rats were studied, 24 animals served as experimental group were bilaterally injected with 50 µl of Streptococcus pneumoniae suspension (1 × 10(8) CFU/ml) via a transbullar approach into the middle ear cavity after anesthesia and six animals were bilaterally inoculated equivalent saline account for control group. At day 1, 3, 5, 7, 10 and 14 after inoculation, bilateral middle-ear mucosal specimens were collected from three infected animals and one control animal for scanning electron microscopy (SME). Membranoid substance attached the bilateral middle ear mucosa were collected under the microscope from the other one infected animals, which were prepared for confocal laser scanning microscope (CLSM) with immunofluorescence in situ labeling technique and light microscopy using Gram staining.

RESULTSAt the early stage of infection (1 day, 3 days), lots of bacterial adhesion, permanent planting in the local regions of the middle ear cavity and microcolonies formation were found, with mixed phagocytic cells, showing a primary bacterial biofilms formation. In the middle term of infection (5 days, 7 days), mature bacterial biofilm scattered on the mucosal surface, formed characteristic three-dimensional structure of "mushroom-shaped" towers. At the late inflammatory period (10 days, 14 days), the bacterial biofilms presented signs of recession. CLSM with FITC-ConA and PI double staining in situ labeling and light microscopy using Gram staining indicated that bacteria and polysaccharide matrix within the biofilms were viable.

CONCLUSIONSThese preliminary findings provide evidence that bacterial biofilms form at the early phase of acute middle ear infection and it may be an important factor in the development of recurrent or persistent otitis media. The rat model of AOM established in this study may be an ideal animal model facilitating the bacterial biofilms research.

Animals ; Biofilms ; growth & development ; Disease Models, Animal ; Ear, Middle ; microbiology ; Male ; Otitis Media, Suppurative ; microbiology ; Pneumococcal Infections ; microbiology ; Rats ; Rats, Sprague-Dawley

Adult ; Female ; Humans ; Laparoscopy ; Pelvic Inflammatory Disease ; etiology ; microbiology ; Pneumococcal Infections ; etiology ; Postoperative Complications ; Salpingostomy ; Smoking ; adverse effects ; Streptococcus pneumoniae

OBJECTIVE: To investigate the expression of Clara cell 10-KDa protein (CC010) in sinonasal mucosa of murine bacterial chronic rhinosinusitis (BCRS) model. METHOD: A murine BCRS model was established by Streptococcus pneumoniae inoculation plus Merocel ostiomeatal obstruction. After 12 week's intervention, histological changes of sinonasal mucosa in BCRS model were examined by hematoxylin and eosin stain, periodic acid-schiff stain, and Masson-Trichrome stain. The mRNA and protein expression of CC10 in sinonasal mucosa were determined by reverse transcription polymerase chain reaction and immunohistochemistry methods. The number of CC10 positive cells in sinonasal epithelium was also counted. RESULT: In BCRS model group, polymorphonuclear neutrophils (PMN), subepithelial collagen deposition, goblet cells, and epithelial thickness were significantly increased, compared with control group (P<0.01). However, CC10 positive cells, CC10 mRNA and protein expression in sinonasal mucosa of BCRS model group were significantly decreased, compared with control group (P<0.01). Moreover, the number of CC10 positive cells was significantly negatively correlated with PMN (r=-0.734, P<0.01), subepithelial collagen deposition (r=-0.776, P<0.01), epithelial goblet cells (r=-0.841, P<0.01), and epithelial thickness (r=-0.805, P<0.01), respectively. CC10 average grayscale value was significantly positively correlated with PMN (r=0.771, P<0.01), subepithelial collagen deposition (r=0.802, P<0.01), epithelial goblet cells (r=0.887, P<0.01), and epithelial thickness (r=0.855, P<0.01), respectively. CONCLUSION The expression of CC10 is downregulated in sinonasal mucosa in BCRS model. As an important endogenous modulin, CC10 might play a crucial role in the pathogenesis of chronic rhinosinusitis.
Animals ; Chronic Disease ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred C57BL ; Pneumococcal Infections ; metabolism ; Sinusitis ; metabolism ; microbiology ; Uteroglobin ; metabolism

OBJECTIVETo develop a mouse model of bacterial rhinosinusitis superposed on allergic rhinitis (AR), and to explore whether ongoing allergic rhinitis enhance the acute sinus infection and inflammation associated with Streptococcus pneumoniae (SP).

METHODSFourty mice of C57BL6/J were randomly divided on average into 4 groups: A [ovalbumin (OVA) + SP], B [OVA + normal saline (NS)], C [phosphate buffered solution (PBS) + SP] and D (PBS + NS). (1) Group A and B were sensitized by intraperitoneal injection with 200 microl (10%) OVA on days 1 through 9, and exposed to OVA (6%) intranasally on days 10 through 17, to induce allergic inflammation. OVA was replaced with PBS in group C and D in the same way. (2) Subsequently, group A and C were inoculated with SP intranasally on day 13, and NS was used in group B and D. On the 6th day after inoculation, mice were killed. Blood was collected from the orbital venous sinus after anesthesia. The heads were embedded with paraffin and serial sections were followed and stained with hematoxylin-eosin and toluidine blue (0.5%) for histological analysis and inflammation cells count. The number of polymorphonuclear neutrophils (PMN) and eosinophils (EOS) per square millimeter of sinus mucosa were calculated by using a computer-aided special software under microscope.

RESULTSAR models were successfully established in 9 mice from group A and 8 from group B. Histologic examination of the sinus from group A and B revealed significant mucosal edema and dilated venules. The symptoms were mild in group C, and no symptom was observed in group D. PMN (x +/- s) in group A (139.3 +/- 26.5)/mm2 was significantly higher than that in group B (70.7 +/- 16.7)/mm2, C (63.0 +/- 14.7)/mm2 and D (40.2 +/- 14.1)/mm2 respectively (P < 0.01); EOS and serous IL-5 level in group A (134.6 +/- 25.5)/mm2, (48.2 +/- 13.9) pg/ml and B (116.2 +/- 25.2)/mm2, (40.8 +/- 7.8) pg/ml, were higher than that in group C (16.7 +/- 2.7)/mm2, (23.9 +/- 8.7) pg/ml (P < 0.05) and D (13.4 +/- 4.9)/mm2, (24.6 +/- 6.5) pg/ml (P < 0.05).

CONCLUSIONSThe data demonstrate that an ongoing local allergic response augments bacterial infection in mice, and allergic sensitization alone without SP does not induce the sinus infection.

Animals ; Disease Models, Animal ; Eosinophils ; immunology ; Interleukin-5 ; blood ; immunology ; Mice ; Mice, Inbred C57BL ; Neutrophils ; immunology ; Pneumococcal Infections ; Rhinitis, Allergic, Perennial ; microbiology ; Sinusitis ; microbiology

OBJECTIVEThe present study was designed to investigate the situation of serotype distribution and beta-lactam antibiotics resistance of Streptococcus pneumoniae (S. pneumoniae) isolated from Chinese children, and to further understand the significance of vaccine for preventing infection caused by the bactria and controlling the resistance to antibiotics.

METHODSNasopharageal swab specimens were collected from randomly selected less than 5-year-old out-patients with upper respiratory infection in Beijing, Shanghai and Guangzhou, 2000 - 2002. Capsular typing was performed by the Quellung reaction tested using a simplified chessboard system for typing of S. pneumoniae. The coverage rate of the 7-valent pneumococcal conjugate vaccine (4, 6B, 9V, 14, 18C, 19F and 23F) was calculated. Antibiotic susceptibility was determined by E-test MIC method for beta-lactam antibiotics (penicillin, amoxicillin/clavulanic acid, cefaclor, cefuroxime and ceftriaxone).

RESULTSTotally 625 pneumococcal strains were typed. Serogroup 19, including 121 strains, was the most frequent serogroup observed (19.4%). Other frequently observed serotypes/serogroups in decreasing order of frequency were serotype/serogroups 23 (15.4%), 6 (13.3%), 14 (6.6%) and 15 (4.3%). Of all these isolates, about 57.6% (360/625) were in the 7-valent conjugate vaccine. Only 1, 6 and 12 strains were serotypes/serogroups 4, 9 and 18, respectively. The coverage rate for the 7-valent vaccine of penicillin nonsusceptible S. pneumoniae (PNSP) was higher than penicillin susceptible S. pneumoniae (PSSP) (73.2% and 46.1%). Serogroups 19 and 23, without other serotypes/serogroups, were significantly associated with PNSP (serogroup 19 accounted for 29.1% of PNSP and 12.2% of PSSP; serogroup 23 accounted for 23.8% of PNSP to 9.2% of PSSP). Overall, 140 strains (22.4%) could not be typed by using the chessboard system, and 117 strains (18.7%) were identified as other 28 kinds of serotype/serogroup. The strains showed different resistance change for beta-lactam antibiotics according to different serotype/serogroup during the three years.

CONCLUSIONSSerotype/Serogroup 19, 23, 6, 14 and 15 were the common types among the pneumococcal strains isolated from Chinese children. Serogroups 19 and 23 were significantly associated with PNSP. The 7-valent pneumococcal conjugate vaccine could cover most of the islotes.

Child, Preschool ; China ; epidemiology ; Drug Resistance, Multiple, Bacterial ; Humans ; Pneumococcal Infections ; epidemiology ; Respiratory Tract Infections ; epidemiology ; microbiology ; Serotyping ; Streptococcus pneumoniae ; classification ; drug effects

This study was performed to measure early changes in the serotype distribution of pneumococci isolated from children with invasive disease during the 3-year period following the introduction of 10- and 13-valent pneumococcal conjugate vaccines (PCVs) in Korea. From January 2011 to December 2013 at 25 hospitals located throughout Korea, pneumococci were isolated among children who had invasive pneumococcal disease (IPD). Serotypes were determined using the Quellung reaction, and the change in serotype distribution was analyzed. Seventy-five cases of IPD were included. Eighty percent of patients were aged 3-59 months, and 32% had a comorbidity that increased the risk of pneumococcal infection. The most common serotypes were 19A (32.0%), 10A (8.0%), and 15C (6.7%). The PCV7 serotypes (4, 6B, 9V, 14, 18C, 19F, 23F, and 6A) accounted for 14.7% of the total isolates and the PCV13 minus PCV7 types (1, 3, 5, 7F and 19A) accounted for 32.0% of the total isolates. Serotype 19A was the only serotype in the PCV13 minus PCV7 group. The proportion of serotype 19A showed decreasing tendency from 37.5% in 2011 to 22.2% in 2013 (P = 0.309), while the proportion of non-PCV13 types showed increasing tendency from 45.8% in 2011 to 72.2% in 2013 (P = 0.108). Shortly after the introduction of extended-valent PCVs in Korea, serotype 19A continued to be the most common serotype causing IPD in children. Subsequently, the proportion of 19A decreased, and non-vaccine serotypes emerged as an important cause of IPD. The impact of extended-valent vaccines must be continuously monitored.
Adolescent ; Bacteremia/complications/diagnosis ; Child ; Child, Preschool ; Female ; Hospitals ; Humans ; Infant ; Male ; Pneumococcal Infections/microbiology/*prevention & control ; Pneumococcal Vaccines/*immunology ; Republic of Korea ; Serotyping ; Streptococcus pneumoniae/*classification/isolation & purification ; Vaccines, Conjugate/*immunology

OBJECTIVETo investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins.

METHODSSpecific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a(+)-pepO and pET28a(+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression. Highly purified target proteins PepO and PsaA were obtained after purification. Mucosal immunization was performed for BALB/c mice and specific antiserum was prepared. ELISA was used to measure the antibody titer, and Western blot was used to analyze the specificity of the antiserum of target proteins. The mice were randomly divided into negative control group, PepO group, PsaA group, and PepO+PsaA combined immunization group, with 18 mice in each group. The models of different serotypes of Streptococcus pneumoniae infection were established to evaluate the immunoprotective effect of target proteins used alone or in combination.

RESULTSThe target proteins PepO and PsaA were successfully obtained and Western blot demonstrated that the antiserum of these proteins had good specificity. There was no significant difference in the titers of IgA in saliva and IgG in serum between the PepO group and the combined immunization group (P>0.05); however, these two groups had significantly higher antibody titers than the PsaA group (P<0.05). The PepO, PsaA, and combined immunization groups had significantly higher protection rates for mice infected with Streptococcus pneumoniae D39 and CMCC31436 in the nasal cavity than the negative control group (P<0.05). The PepO and combined immunization groups had a significantly higher protection rate for mice infected with Streptococcus pneumoniae D39 than the PsaA group (P<0.05). The results of colonization experiment showed that compared with the control group, the PepO, PsaA, and combined immunization groups showed a significant reduction in the colonization of Streptococcus pneumoniae (CMCC31693 and CMCC31207) in the nasopharynx and lung (P<0.05). The combined immunization group showed a better effect on reducing the colonization of CMCC31207 in the lung than the PepO and PsaA alone groups.

CONCLUSIONSCombined PepO/PsaA vaccines may produce a better protective effect by mucosal immunization compared with the vaccine used alone in mice. The combined vaccines can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lung. Therefore, such protein vaccines may have a great potential for research and development.

Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; analysis ; Bacterial Proteins ; immunology ; Female ; Immunization ; Lipoproteins ; immunology ; Lung ; microbiology ; Metalloendopeptidases ; immunology ; Mice ; Mice, Inbred BALB C ; Pneumococcal Infections ; prevention & control ; Pneumococcal Vaccines ; immunology ; Saliva ; immunology

The purpose of this study was to develop pneumococcal typing by multiplex PCR and compare it with conventional serotyping by quellung reaction. Pneumococcal strains used in this study included 77 isolates from clinical specimens collected from children at Seoul National University Children's Hospital from 2006 to 2010. These strains were selected as they represented 26 different serotypes previously determined by quellung reaction. Molecular type was determined by 8 sequential multiplex PCR assays. Bacterial DNA extracted from cultured colonies was used as a template for PCR, and primers used in this study were based on cps operon sequences. Types 6A, 6B, 6C, and 6D were assigned based on the presence of wciNbeta and/or wciP genes in 2 simplex PCRs and sequencing. All 77 isolates were successfully typed by multiplex PCR assays. Determined types were as follows: 1, 3, 4, 5, 6A, 6B, 6C, 6D, 7C, 7F, 9V, 10A, 11A, 12F, 13, 14, 15A, 15B/15C, 19A, 19F, 20, 22F, 23A, 23F, 34, 35B, and 37. The results according to the PCR assays were in complete concordance with those determined by conventional quellung reaction. The multiplex PCR assay is highly reliable and potentially reduces reliance upon conventional serotyping.
Child ; DNA Primers/chemistry/metabolism ; DNA, Bacterial/chemistry/genetics ; Humans ; Multiplex Polymerase Chain Reaction ; Pneumococcal Infections/microbiology ; Serotyping ; Streptococcus pneumoniae/*classification/genetics/isolation & purification

Penicillin-binding proteins (PBPs) are the target of β-lactam antibiotics (the major treatment for Streptococcus pneumoniae infections), and mutations in PBPs are considered as a primary mechanism for the development of β-lactam resistance in S. pneumoniae. This study was conducted to investigate the mutations in the PBPs of clinical S. pneumoniae isolates in Hangzhou, China, in correlation with β-lactam resistance. Results showed that 19F was the predominant serotype (7/27) and 14 of the S. pneumoniae isolates were resistant to both penicillin G and cephalosporin. Genotyping results suggested that β-lactam-resistant isolates primarily exhibited single-site mutations in both the STMK and SRNVP motifs of pbp1a in combination with double-site mutations in the STMK motif of pbp2x, which might be the primary mechanisms underlying the β-lactam resistance of the isolates in this study.
Anti-Bacterial Agents ; pharmacology ; China ; epidemiology ; Drug Resistance, Bacterial ; Humans ; Pneumococcal Infections ; epidemiology ; microbiology ; Streptococcus pneumoniae ; drug effects ; genetics ; beta-Lactams ; pharmacology

OBJECTIVETo investigate nasopharyngeal carriage rate, antimicrobial resistance and serotype distribution of Streptococcus pneumoniae among children with upper respiratory infection.

METHODSNasopharygeal swabs were collected from children with upper respiratory infection visiting the outpatient department of Beijing Children′s Hospital between March 2013 and February 2014. The antibiotic susceptibility was tested by Etest method, and the serotype was determined by Quellung reaction.

RESULTSThe nasopharyngeal carriage rate for Streptococcus pneumoniae was 23.8% (699/2 941). One hundred isolates were randomly chosen for antimicrobial susceptiblity test and serotyping. Up to 98.0% isolates were susceptible to parenteral penicillin. The susceptible rate against oral penicillin, however, was 33.0%. The non-susceptible rate to erythromycin and azithromycin was 97.0%. The multi-drug resistance rate was up to 86.0%. The common serotypes were 6A(12.0%), 19F(12.0%), 6B(10.0%), 23F(9.0%) and 14(8.0%). The coverage rates of 7-, 10- and 13-valent pneumococcal conjugate vaccine were 41.0%, 42.0% and 59.0% respectively.

CONCLUSIONSAbout 25% of children with upper respiratory infection are nasopharyngeal colonized by Streptococcus pneumoniae. The isolates show a high antimicrobial resistance. The 13-valent pneumococcal conjugate vaccine covers about 60.0% of the isolates.

Adolescent ; Carrier State ; epidemiology ; microbiology ; Child ; Child, Preschool ; Drug Resistance, Bacterial ; Female ; Humans ; Infant ; Male ; Nasopharynx ; microbiology ; Pneumococcal Vaccines ; immunology ; Respiratory Tract Infections ; microbiology ; Serotyping ; Streptococcus pneumoniae ; classification ; drug effects ; isolation & purification