Gamma-irradiation of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> for the use as an immunogenic whole cell vaccine

Streptococcus pneumoniae is a major respiratory pathogen that causes millions of deaths worldwide. Although subunit vaccines formulated with the capsular polysaccharides or their protein conjugates are currently-available, low-cost vaccines with wide serotype coverage still remain to be developed, especially for developing countries. Recently, gamma- irradiation has been considered as an effective inactivation method to prepare S. pneumoniae vaccine candidate. In this study, we investigated the immunogenicity and protective immunity of gamma-irradiated S. pneumoniae (r-SP), by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP), both of which were made by traditional inactivation methods. Intranasal immunization of C57BL/6 mice with r-SP in combination with cholera toxin as an adjuvant enhanced S. pneumoniaespecific antibodies on the airway mucosal surface and in sera more potently than that with h-SP or f-SP under the same conditions. In addition, sera from mice immunized with r-SP potently induced opsonophagocytic killing activity more effectively than those of h-SP or f-SP, implying that r-SP could induce protective antibodies. Above all, immunization with r-SP effectively protected mice against S. pneumoniae infection. Collectively, these results suggest that gamma- irradiation is an effective method for the development of a killed whole cell pneumococcal vaccine that elicits robust mucosal and systemic immune responses.     

The synthetic human beta-defensin-3 C15 peptide exhibits antimicrobial activity against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>, both alone and in combination with dental disinfectants

Streptococcus mutans is a major etiologic agent of human dental caries that forms biofilms on hard tissues in the human oral cavity, such as tooth and dentinal surfaces. Human β-defensin-3 (HBD3) is a 45-amino-acid natural antimicrobial peptide that has broad spectrum antimicrobial activity against bacteria and fungi. A synthetic peptide consisting of the C-terminal 15 amino acids of HBD3 (HBD3-C15) was recently shown to be sufficient for its antimicrobial activity. Thus, clinical applications of this peptide have garnered attention. In this study, we investigated whether HBD3-C15 inhibits the growth of the representative cariogenic pathogen Streptococcus mutans and its biofilm formation. HBD3-C15 inhibited bacterial growth, exhibited bactericidal activity, and attenuated bacterial biofilm formation in a dose-dependent manner. HBD3-C15 potentiated the bactericidal and anti-biofilm activity of calcium hydroxide (CH) and chlorhexidine digluconate (CHX), which are representative disinfectants used in dental clinics, against S. mutans. Moreover, HBD3-C15 showed antimicrobial activity by inhibiting biofilm formation by S. mutans and other dentinophilic bacteria such as Enterococcus faecalis and Streptococcus gordonii, which are associated with dental caries and endodontic infection, on human dentin slices. These effects were observed for HBD3-C15 alone and for HBD3-C15 in combination with CH or CHX. Therefore, we suggest that HBD3-C15 is a potential alternative or additive disinfectant that can be used for the treatment of oral infectious diseases, including dental caries and endodontic infections.     

Decrease of insoluble glucan formation in <Emphasis Type="Italic">Streptococcus mutans</Emphasis> by co-cultivation with <Emphasis Type="Italic">Enterococcus faecium</Emphasis> T7 and glucanase addition

Objectives

To develop preventive canine oral health bio-materials consisting of probiotics and glucanase to reduce insoluble glucan and volatile sulfur compound formation.

Results

Co-cultivation of Enterococcus faecium T7 with Streptococcus mutans at inoculation ratio of 3:1 (v/v) resulted in 25% reduction in the growth of Streptococcus mutans. Amounts of soluble and insoluble glucans produced by S. mutans were decreased to 70 and 55%, respectively. Insoluble glucan was decreased from 0.6 µg/ml in S. mutans culture to 0.03 µg/ml in S. mutans co-cultivated with E. faecium T7 in the presence of Lipomyces starkeyi glucanase. Volatile sulfur compound, a main component of halitosis produced by Fusobacteria nucleatum, was decreased by co-cultivating F. nucleatum with E. faecium.

Conclusion

E. faecium and glucanase can be combined as potentially active ingredients of oral care products for pets by reducing plaque-forming bacteria growth and their by-products that cause cavity and periodontal disease.

     

Effects of Lectins on initial attachment of cariogenic <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.     

Survey of potential factors involved in the low frequency of CP5 and CP8 expression in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from mastitis of dairy cattle from Argentina,Chile, and Uruguay

Staphylococcus aureus produces capsular polysaccharides (CPs) both in vivo and under defined culture conditions being serotypes 5 and 8 the most prevalent. S. aureus isolates that fail to produce CP5 or CP8 are defined as non-typeable (NT). Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. The prevalence of NT strains of S. aureus isolated from bovine mastitis varies according to the geographic origin of the strain. The aims of this work were to detect phenotypically and genotypically the capsular profile of 144 S. aureus isolated from bovine mastitis in Argentina, Chile, and Uruguay and explore the factors that are considered to be associated with capsule expression as presence of IS257, IScap, and agr typing of non-related collection. The detection of the IS257, IScap, cap genes, and agr typing was performed using PCR. The detection and quantification of capsular polysaccharide production were performed by ELISA assays. We found that 96% of the S. aureus isolates investigated carried cap5(8) genes but over 75% of strains do not express capsule in the three countries studied. However, only 6 isolates from Argentina carried the IScap element that totally suppressed the expression of the capsule, suggesting that other factors could influence on CP expression. Moreover, the agrI/NT association was statistically significant suggesting that this profile is a phenomenon observed not only in other parts of the world but also in our region.     

Virulence Factors in <Emphasis Type="Italic">Candida albicans</Emphasis> and <Emphasis Type="Italic">Streptococcus mutans</Emphasis> Biofilms Mediated by Farnesol

The aim of this study was to evaluate the effect of farnesol on the production of acids and hydrolytic enzymes by biofilms of Streptococcus mutans and Candida albicans. The present study also evaluated the time-kill curve and the effect of farnesol on matrix composition and structure of single-species and dual-species biofilms. Farnesol, at subinhibitory concentrations, showed a significant reduction in S. mutans biofilm acid production, but did not alter C. albicans hydrolytic enzyme production. The number of cultivable cells of both microorganisms was significantly reduced after 8 h of contact with farnesol. Extracellular matrix protein content was reduced for biofilms formed in the presence of farnesol. In addition, confocal laser scanning and scanning electron microscopy displayed structural alterations in all biofilms treated with farnesol, which included reduction in viable cells and extracellular matrix. In conclusion, farnesol showed favorable properties controlling some virulence factors of S. mutans and C. albicans biofilms. These findings should stimulate further studies using this quorum-sensing molecule, combined with other drugs, to prevent or treat biofilm-associated oral diseases.     

Characterization of a <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> strain with potential oral probiotic properties

Background

The microflora composition of the oral cavity affects oral health. Some strains of commensal bacteria confer probiotic benefits to the host. Lactobacillus is one of the main probiotic genera that has been used to treat oral infections. The objective of this study was to select lactobacilli with a spectrum of probiotic properties and investigate their potential roles in oral health.

Results

An oral isolate characterized as Lactobacillus brevis BBE-Y52 exhibited antimicrobial activities against Streptococcus mutans, a bacterial species that causes dental caries and tooth decay, and secreted antimicrobial compounds such as hydrogen peroxide and lactic acid. Compared to other bacteria, L. brevis BBE-Y52 was a weak acid producer. Further studies showed that this strain had the capacity to adhere to oral epithelial cells. Co-incubation of L. brevis BBE-Y52 with S. mutans ATCC 25175 increased the IL-10-to-IL-12p70 ratio in peripheral blood mononuclear cells, which indicated that L. brevis BBE-Y52 could alleviate inflammation and might confer benefits to host health by modulating the immune system.

Conclusions

L. brevis BBE-Y52 exhibited a spectrum of probiotic properties, which may facilitate its applications in oral care products.

     

Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> types 4 and 5 revised by NMR spectroscopy

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of non-stoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, theposition of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by ¹³C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner.

Open image in new window

where L-Rhap3Rlac2Ac is 2-O-acetyl-3-O-[(R-1-carboxyethyl]-L-rhamnose

     

Characteristic of Polysaccharide Complexes from <Emphasis Type="Italic">Centaurea scabiosa</Emphasis> L. and <Emphasis Type="Italic">Centaurea pseudomaculosa</Emphasis> Dobrocz.

We characterized polysaccharide complexes from Centaurea scabiosa L. and Centaurea pseudomaculosа Dobrocz. We proposed the technique of sequential selection of water-soluble polysaccharides and pectin substances from the aerial parts of studied objects. We have discovered that the content of water-soluble polysaccharides in the aerial parts of C. scabiosa was 2.8 times higher (2.7 ± 0.3%, n = 3) than in C. pseudomaculosа (0.97 ± 0.50%, n = 3). The content of pectin substances in the aerial parts of C. scabiosa was 2 times higher (7.6 ± 0.4%, n = 3) than in C. pseudomaculosа (3.9 ± 0.3%, n = 3). The residues of D-galacturonic acid, L-rhamnose, D-xylose, D-mannose, D-glucose, and D-galactose are the monomeric units of polysaccharide complexes from C. scabiosa and C. pseudomaculosa. Using ion-exchange chromatography, three polysaccharide fractions (molecular weights 667, 722, and 1027 kDa), whose monomer units are D-galacturonic acid, L-rhamnose, D-galactose, D-xylose, and D-glucose were isolated from the water-soluble polysaccharides of C. scabiosa.     

Isolation and Characterization of Antigenic Components of a New Heptavalent <Emphasis Type="Italic">Pseudomonas</Emphasis> Vaccine

ONE of the most serious opportunistic bacterial pathogens is Pseudomonas aeruginosa¹ and the inability of the common antimicrobial agents to combat such infections suggested the investigation of specific immunological prophylactic and therapeutic approaches. Therefore Fisher et al. began to develop a new serotype schema as a prerequisite for an immunizing preparation which would protect humans against the most prevalent strains of Pseudomonas. This communication describes the isolation, purification and preliminary characterization of seven new lipopolysaccharide antigens which were obtained from strains representative of each of the seven protective serotypes identified by Fisher et al.².     

Betulin inhibits cariogenic properties of <Emphasis Type="Italic">Streptococcus mutans</Emphasis> by targeting <Emphasis Type="Italic">vicRK</Emphasis> and <Emphasis Type="Italic">gtf</Emphasis> genes

Streptococcus mutans, a multivirulent pathogen is considered the primary etiological agent in dental caries. Development of antibiotic resistance in the pathogen has created a need for novel antagonistic agents which can control the virulence of the organism and reduce resistance development. The present study demonstrates the in vitro anti-virulence potential of betulin (lup-20(29)-ene-3β,28-diol), an abundantly available plant triterpenoid against S. mutans UA159. Betulin exhibited significant dose dependent antibiofilm activity without affecting bacterial viability. At 240 µg/ml (biofilm inhibitory concentration), betulin inhibited biofilm formation and adherence to smooth glass surfaces by 93 and 71 % respectively. It reduced water insoluble glucan synthesis by 89 %, in conjunction with down regulation of gtfBC genes. Microscopic analysis confirmed the disruption in biofilm architecture and decreased exopolysaccharide production. Acidogenicity and aciduricity, key virulence factors responsible for carious lesions, were also notably affected. The induced auto-aggregation of cells upon treatment could be due to the down regulation of vicK. Results of gene expression analysis demonstrated significant down-regulation of virulence genes upon betulin treatment. Furthermore, the nontoxic effect of betulin on peripheral blood mononuclear cells even after 72 h treatment makes it a strong candidate for assessing its suitability to be used as a therapeutic agent.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

Age does not influence the disease course in a mouse model of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> serotype 3 meningitis

In order to elucidate the causes for the increased mortality of aged patients with bacterial central nervous system (CNS) infections, we compared the course of Streptococcus pneumoniae (S. pneumoniae) meningitis in aged and young mice. Aged (21.2?±?3.1 months, n?=?40) and young (3.2?±?0.9 months, n?=?42) C57BL/6N and B6/SJL mice were infected by intracerebral injection of 50–70 CFU S. pneumoniae serotype 3 and monitored for 15 days. Aged and young mice did not differ concerning mortality (35% versus 38%), weight loss, development of clinical symptoms, bacterial concentrations in cerebellum and spleen as well as the number of leukocytes infiltrating the CNS. In contrast to results from our geriatric mouse model of Escherichia coli (E. coli) meningitis, where aged mice showed a higher mortality and an impaired elimination of bacteria, we did not find any differences between aged and young mice after intracerebral infection with S. pneumoniae serotype 3. This indicates that the increased susceptibility of aged mice to bacterial CNS infections is pathogen-specific: It appears less prominent in infections caused by hardly phagocytable pathogens with thick capsules like S. pneumoniae serotype 3, where the age-related decline of the phagocytic capacity of microglia and macrophages has a minor influence on the disease course.     

Anionic carbohydrate-containing cell wall polymers of <Emphasis Type="Italic">Streptomyces melanosporofaciens</Emphasis> and related species

The structures of cell wall anionic carbohydrate-containing polymers in Streptomyces melanosporofaciens VKM Ac-1864^T and phylogenetically close organisms—S. hygroscopicus subsp. hygroscopicus VKM Ac-831^T, S. violaceusniger VKM Ac-583^T, S. endus VKM Ac-1331^T, S. endus VKM Ac-129, and S. rutgersensis subsp. castelarensis VKM Ac-832^T—have been comparatively studied by chemical and NMR spectroscopic methods. The natural polymer of a new, previously unknown structure, Kdn (3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid) with β-galactose residues at C-9, has been found in the cell walls of all the strains under study. The cell walls of all the studied organisms contain three teichoic acids (TA): a predominant TA (1,3-poly(glycerol phosphate) with N-acetylated α-glucosaminyl substitutes by C-2 of glycerol, and minor TAs, 1,3-and 2,3-poly(glycerol phosphate) polymers without substitution. Their chains have O-acetyl and O-lysyl groups. Microorganisms of the above-mentioned species differ in the number of α-glucosaminyl substitutes and in the degree of their acetylation in the predominant teichoic acid.     

Heterologous expression of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> serotype 1 O-antigen analog in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 W3110 by transferring a minimum number of genes involved in O-polysaccharide biosynthesis

Objective

To heterologously produce the Shigella dysenteriae serotype 1 O-polysaccharide (O-PS, O-antigen) in Escherichia coli by transferring the minimum number of genes instead of the entire O-PS gene cluster.

Results

The three glycosyltransferase genes (rfbR, rfbQ and rfp) responsible for the formation of the O-repeat unit were introduced into E. coli K-12 W3110 to synthesize S. dysenteriae 1 O-PS. The specific O-antigen ladder type with different chain lengths of O-repeat units was observed in the recombinant E. coli strain by SDS-PAGE silver staining and western blotting using S. dysenteriae 1 lipopolysaccharide antiserum. Analysis by mass spectrometry and ion chromatography suggested generation of the specific S. dysenteriae 1 O-repeat unit structure with an extra glucose residue attached.

Conclusions

Recombinant E. coli expressing specific glycosyltransferase genes can generate the O-PS of S. dysenteriae 1 and might be able to synthesize heterologous O-antigens of various pathogenic bacteria for vaccine preparation.

     

Biofilm formation,antibiotic susceptibility and RAPD genotypes in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> clinical strains isolated from single centre intensive care unit patients

The aim of this study was to analyse genotypes, antimicrobial susceptibility patterns and serotypes in Pseudomonas aeruginosa clinical strains, including the clonal dissemination of particular strains throughout various intensive care units in one medical centre. Using random amplified polymorphic DNA (RAPD–PCR) and P. aeruginosa antisera, 22 different genotypes and 8 serotypes were defined among 103 isolates from 48 patients. No direct association between P. aeruginosa strain genotypes and serotypes was observed. RAPD typing in strains with the same serotype revealed different genotypes and, on the contrary, most strains with a different serotype displayed the same amplification pattern. The resulting banding patterns showed a high degree of genetic heterogeneity among all isolates from the patients examined, suggesting a non-clonal relationship between isolates from these patients. A higher degree of antibiotic resistance and stronger biofilm production in common genotypes compared to rare ones and genetic homogeneity of the most resistant strains indicated the role of antibiotic pressure in acquiring resistant and more virulent strains in our hospital. In conclusion, genetic characterisation of P. aeruginosa strains using RAPD method was shown to be more accurate in epidemiological analyses than phenotyping.     

Anionic polymers of the cell wall of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. VKM Ac-2534

The cell wall of Streptomyces sp. VKM Ac-2534, the causative agent of common scab in potato tubers, which does not synthesize thaxtomin and is phylogenetically close to phytopathogen Streptomyces setonii sp. ATCC 25497, contains two anionic carbohydrate-containing polymers. The major polymer is teichuronic acid, whose repeating unit is disaccharide → 4)-β-D-ManpNAc3NAcyA-(1 → 3)-α-D-GalpNAc-(1→, where Acy is a residue of acetic or L-glutamic acid. The polymer of such structure has been found in Gram-positive bacteria for the first time. The minor polymer is teichoic acid [1,5-poly(ribitol phosphate)], in which a part of the ribitol residues are glycosylated at C4 with β-D-Glcp and, probably, with β-D-GlcpNAc and some residues are O-acylated with Lys residues. The structures were proved by chemical and NMR spectroscopic methods. It is likely that the presence of acidic polysaccharides on the surface of the phytopathogenic streptomycete is necessary for its attachment to the host plant.     

Mortality in Captive Rhesus Monkeys (<Emphasis Type="Italic">Macaca mulatta</Emphasis>) in China Due to Infection with <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> Serotype O:1a

The most common serotypes of Yersinia pseudotuberculosis infecting non-human primates are serotypes O:1b, O:3, O:4, and O:7. The O:1a serotype has never been reported in non-human primates. The present study describes an outbreak of serotype O:1a with high fatality (6/18) in captive rhesus monkeys in China. Bacteria were isolated from different organs of the carcasses using standard microbiological procedures. The strain was identified using conventional and molecular techniques such as morphological and biochemical identification, serotype determination, PCR-sequence analysis based on the 16S rRNA gene, detection of virulence genes, and antimicrobial susceptibility testing. The pathogenicity was determined after experimental infection in mice. Taken together, the obtained data indicate that Y. pseudotuberculosis O:1a is a pathogen of concern and represents a potential threat to monkey conservation efforts.     

Evolutionary relationships in the genus <Emphasis Type="Italic">Secale</Emphasis> revealed by DArTseq DNA polymorphism

Till this day, there is not much known about the phylogeny of the Secale genus; therefore, in our research, we tried to shed some lights on the issue of rye (Secale genus) taxonomy. The genetic diversity and phylogenetic relationships were evaluated using 13,842 DArTseq? polymorphic markers. The model-based clustering (STRUCTURE software) separated our 84 samples into three main clusters: perennial cluster, annual cluster, and S. sylvestre cluster. The same result was obtained using Neighbour Joining tree and self-organizing maps. Secale sylvestre, S. strictum, and S. cereale are the three main species of the Secale genus. Three samples of rye are in basal positions in phylogenetic trees. These accessions share ancient morphological characters and are probably the ancestors of different lineages within Secale. Annual Secale taxa, with the exception of S. sylvestre, create one mutual taxon. We have found out that the semi-perennial taxa of S. cereale var. multicaule and S. strictum subsp. ciliatoglume are genetically closest to the annual species of S. cereale. Phylogenetic signals for semi-perennial and annual taxa are also present in S. strictum subsp. africanum. SNP-based analysis revealed that evolution of annual S. cereale has already begun in S. strictum subsp. africanum. The results showed that S. vavilovii cannot be considered as a separate species but a subspecies of S. cereale Secale cereale subsp. dighoricum is a hybrid. It is still not clear whether we can consider S. strictum subsp. strictum and S. strictum subsp. kuprijanovii as two separate species.     

Etiological characteristics of chlamydia trachoma conjunctivitis of Primary Boarding School students in the Qinghai Tibetan area

The aim of this study was to investigate the etiological characteristics of Chlamydia trachomatis conjunctivitis among resident students at primary schools in the Qinghai Tibetan area in order to understand the distribution of C. trachomatis and other pathogenic microorganisms, to detect the isolation rate of infectious pathogens, and to provide an evidence for further targeted efforts in the prevent of sporadic trachoma efforts. From two primary schools in Qinghai Province, ocular samples from 35 students who were clinically diagnosed as trachoma cases and 60 normal controls were obtained by swabbing their upper eyelids and lower conjunctival sacs. Samples were preserved at 4°C and airlifted to Beijing Tongren Hospital within 24 h. Real-time polymerase chain reaction(RT-PCR) was used to screen for C. trachomatis, and nested PCR was used to amplify a fragment of the omp A gene for serotype confirmation. Bacterial cultivation and sensitivity tests were conducted based on the 2015 version of the Clinical and Laboratory Standards Institute. Adenovirus, herpes simplex virus, cytomegalovirus, and Epstein-Barr virus were screened by RT-PCR. Among the 35 students with trachoma, 8 came from the Jianshetang Primary School and 27 came from the Central Primary School. Two novel C. trachomatis B serotypes(Gen Bank accession numbers KU737520 and KU737521) were detected based on a sequence analysis of the omp A gene. Single C. trachomatis infections accounted for 42.86%(9/21) of the cases, and infections with multiple bacteria, particularly Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, and Streptococcus pneumoniae, accounted for the remaining 57.14%(12/21). Of the 14 C. trachomatis-negative samples, one was positive for adenoviral infection(serotype D) and 13 were positive for bacterial infections(H. influenzae, M. catarrhalis, S. pneumoniae, S. aureus, streptococci other than S. pneumoniae, Staphylococcus epidermidis, Corynebacterium, and Arthrobacterium). In addition to C. trachomatis, the other bacteria and virus that were detected in the boarding students of primary schools in the Qinghai Tibetan area should be emphasized in trachoma prevention and control.