The prevalent Staphylococcus aureus clones and antibiotic susceptibility profiles are known to change dynamically and geographically; however, recent S. aureus strains causing infections in women and children in China have not been characterized. In this study, we analyzed the molecular epidemiology and antimicrobial resistance of S. aureus isolated from patients in four centers for women and children in Guangzhou, China. In total, 131 S. aureus isolates (100 from children and 31 from women) were analyzed by spa typing, multi-locus sequence typing, virulence gene and antimicrobial resistance profiling, staphylococcal chromosomal cassette mec typing, and mutation analyses of rpoB. A total of 58 spa types, 27 sequence types (STs), and 10 clonal complexes (CCs) were identified. While CC59 (ST59-IV, 48.8%; ST338-III, 35.7%) and CC45 (ST45-IV, 100%) were the major clones (84.4%) among MRSA isolates, CC5 (ST188, 24.3%; ST1, 21.6%) and CC398 (ST398, 70%) were the major ones (70.1%) among MSSA isolates. ST338-MRSA-III mostly found in pus but hardly in respiratory tract samples while ST45-MRSA-IV was on the opposite, even though they both found in blood and cerebrospinal fluid sample frequently. Staphylococcal enterotoxin genes seb-seq-sek were strongly associated with ST59 and ST338, while sec was associated with ST45, ST121, ST22, and ST30. All ST338, ST1232, and SCCmec III isolates carried lukF/S-PV genes. A total of 80% of ST338 isolates were resistant to erythromycin, clindamycin, and tetracycline. All ST45 isolates exhibited intermediate or complete resistance to rifampicin. In total, 481 HIS/ASN mutations in rpoB were found in rifampicin-resistant or intermediate-resistant isolates. ST338-III and ST45-IV emerged as two of three major clones in MRSA isolates from women and children in Guangzhou, China, though ST59-MRSA-IV remained the most prevalent MRSA clone. Clonal distribution of S. aureus varied, depending on the specimen source. Virulence genes and antibiograms were closely associated with the clonal lineage. These results clarified the molecular epidemiology of S. aureus from women and children in Guangzhou, China, and provide critical information for the control and treatment of S. aureus infections.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The limitations of current therapies for H. pylori infection include poor compliance and antibiotic resistance. Therefore, an effective anti-H. pylori vaccine would be an alternative or complement to antibiotic treatment. Urease B (UreB) is considered an ideal vaccine antigen against H. pylori infection. In this study, cholera toxin B subunit (CTB), a mucosal adjuvant, was used to enhance the immunogenicity of a novel Bacillus subtilis spore vaccine expressing CTB-UreB, along with the B. subtilis spore coat protein CotC as a fusion protein. Oral administration of B. subtilis spores expressing CotC-UreB or CotC-CTB-UreB led to increased levels of UreB-specific IgG in serum and UreB-specific IgA in faeces, as well as elevated levels of IL-10 and IFN-γ in splenocytes. In addition, oral administration of CotC-UreB or CotC-CTB-UreB spores induced significant reductions (80.0 and 90.5 %, respectively) in gastric H. pylori bacterial load (1.11±0.36×105 and 0.53±0.21×105 c.f.u., respectively) compared to that of the CotC control group (5.56±1.64×105 c.f.u., P<0.01). Moreover, CotC-CTB-UreB spores were significantly more effective at reducing the bacterial load than CotC-UreB spores (P<0.05). These results indicate that CotC-CTB-UreB-expressing B. subtilis spores are a potential vaccine candidate for the control of H. pylori infection.

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Methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization predisposes individuals for endogenous infections and is a major threat to children. Recently, oxacillin/cefoxitin-susceptible mecA-positive S. aureus (OS-MRSA) has been reported worldwide. Herein, a prospective, cross-sectional study was conducted across five schools, representing three educational stages, in Guangzhou, China. Nasal swabs from 2,375 students were cultured for S. aureus and all isolates were subjected to antibiotic susceptibility testing phenotypically and confirmed by femB and mecA genetic detection; all the isolates were classified as MSSA, MRSA, or OS-MRSA. All strains were also analyzed by multi-locus sequence typing. Among the 2,375 swabs, S. aureus was detected in 744 children (31.3%, 95% CI: 25.9–36.7%), of whom 72 had MRSA (3.0%, 95% CI: 0.6–5.4%) and 4 had OS-MRSA (0.2%, 95% CI: 0.1–0.3%), of which an oxacillin- and cefoxitin-susceptible MRSA strain was identified. The prevalence of S. aureus and MRSA was higher in younger children. The highest percentage of drug resistance of the S. aureus isolates (n = 744) was to penicillin (85.5%), followed by erythromycin (43.3%) and clidamycin (41.0%). The most prevalent sequence types (STs) were ST30, ST45, and ST188 in MSSA, accounting for 38.7% of the total isolates, whereas ST45, ST59, and ST338 accounted for 74.6% of the MRSA isolates and ST338 accounted for 50.0% of the OS-MRSA isolates. The MRSA and OS-MRSA isolates (n = 76) were grouped into three clades and one singleton, with clonal complex (CC) 45 as the most predominant linkage. The top nine multi-locus sequence typing-based CCs (CC30, CC45, CC5, CC1, CC15, CC944, CC398, CC59, CC7) represented 86.7% of all S. aureus isolates. All CC30 isolates were resistant to erythromycin and clidamycin, and almost all these isolates were also resistant to penicillin (99.2%). The CC45 and CC59 isolates exhibited high resistance rates to oxacillin at 31.5 and 59.0%, respectively. This study provides updated data valuable for designing effective control strategies to mitigate the burden of disease and to improve the adequacy of empirical antimicrobial treatments for potentially harmful infections.

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) has been identified as a modulator with anti-Th2 inflammation activity, and cholera toxin B (CTB) is a mucosal adjuvant that can also induce antigen tolerance. In this study, we constructed a CTB-NAP fusion protein on the surface of Bacillus subtilis spore and evaluate the efficiency of oral administration of the recombinant CTB-NAP spores in preventing asthma in mice. Oral administration of recombinant CTB or CTB-NAP spores significantly decreased serum ovalbumin (OVA)-specific IgE (p < 0.001) and increased fecal IgA (p < 0.01) compared to the treatment with non-recombinant spores. Oral administration of recombinant CTB or CTB-NAP spores induced IL-10 and IFN-γ expression and reduced IL-4 levels in bronchoalveolar lavage fluid (BALF). Moreover, CTB and CTB-NAP spores reduced the eosinophils in BALF and inflammatory cell infiltration in the lungs. Furthermore, CD4CD25Foxp3 Tregs in splenocytes were significantly increased in mice treated with recombinant CTB or CTB-NAP spores. The number of CD4CD25Foxp3 Tregs caused by CTB-NAP was higher than that by CTB alone. Our study indicated that B. subtilis spores with surface expression of subunit CTB or CTB-NAP could inhibit OVA-induced allergic inflammation in mice. The attenuated inflammation was attributed to the induction of CD4CD25Foxp3 Tregs and IgA. Moreover, the fusion protein CTB-NAP demonstrated a better efficiency than CTB alone in inhibiting the inflammation.

Background: Staphylococcus aureus (S. aureus) is a major pathogen of human infections. Its fecal carriage serves as a risk factor for nosocomial transmission and disease development. However, the rate of S. aureus fecal carriage among Chinese children has not yet been reported. Therefore, we sought to investigate the prevalence, characterization, and drug resistance of S. aureus isolated from pediatric patients' feces in Southern China. Methods: Fecal samples (2059) from pediatric patients in three centers in Guangzhou were cultured. From which, 412 S. aureus isolates were identified via selective mediums and automated VITEK Mass Spectrometer analysis. Antibiotic susceptibility was determined and DNA sequencing of seven housekeeping genes were used for multilocus sequence typing analysis. Results: The fecal carriage rates were 20.0% for S. aureus and 4.5% for methicillin-resistant S. aureus (MRSA). Moreover, S. aureus fecal carriage was positively correlated with outpatient status and gastroenteritis diagnosis. Moreover, age-related patterns were observed with respect to prevalence of S. aureus. Besides, a total of 76 sequence types (STs) were identified, including 25 newly assigned STs and 28 clonal complexes (CCs). ST188, ST6, and ST15 were the most prevalent methicillin-sensitive S. aureus (MSSA) clones, while ST59 and ST45 were the major MRSA clones. S. aureus isolates also exhibited high rates of penicillin (84.2%), erythromycin (38.8%), and clindamycin (35.9%) resistance. Specifically, all ST30 and ST338 isolates were resistant to erythromycin and clindamycin, 61% of ST7 were resistant to tetracycline, and 84% of ST45 exhibited resistance and intermediate resistance to rifampicin. Also, CC59 (ST338 and ST59) and CC45 exhibited different antibiotic resistance patterns. Conclusion: These results demonstrate the colonization dynamics and molecular epidemiology of S. aureus in child feces in Southern China. Further, they suggest an urgency for strengthening the surveillance programs in China and provide important information for the prevention and treatment of S. aureus infection.

Background Helicobacter pylori neutrophil‐activating protein (NAP) is an immune modulator with anti‐Th2 inflammation activity that can be used to prevent IgE‐mediated allergic reactions. Cholera toxin B (CTB) is a mucosal adjuvant that can induce antigen tolerance. Bacillus subtilis spores are an ideal vehicle for the oral delivery of heterologous antigens. Objective We investigated the therapeutic effect of recombinant NAP B subtilis spores on peanut allergies in a mouse model. Methods Female C3H/HeJ mice were sensitized and challenged with peanut extract by oral administration. Before challenge, recombinant NAP and CTB‐NAP (CNAP) spores were orally administered to sensitized mice for 4 weeks. Faecal peanut‐specific IgA and serum‐specific IgE, IgG1, and IgG2a levels were measured, and the intestinal microbiota was analysed. Mice were intraperitoneally injected with anti‐CD25 antibodies for regulatory T cell (Treg) depletion to evaluate the efficacy of Tregs in preventing peanut allergy. After challenge, anaphylactic reactions, plasma histamine, Tregs, and splenocyte interleukin (IL)‐10, IL‐4, IL‐5 and interferon‐γ (IFN‐γ) levels were evaluated. Results After 4 weeks of recombinant spore treatment, faecal IgA levels and serum IgG2a levels were increased, while serum IgG1 and IgE levels were reduced. Intestinal microbiota analysis revealed that CNAP spores increased the taxonomic abundance of Firmicutes at the phylum level and Clostridia at the class level. After challenge, the administration of NAP or CNAP spores to mice was found to ameliorate anaphylactic reactions and decrease plasma histamine levels. Administration of NAP or CNAP spores also enhanced IL‐10 and IFN‐γ secretion, and suppressed IL‐4 and IL‐5 secretion. The protective effect of CNAP spores was more pronounced than that of NAP spores; this therapeutic effect was lost after Treg depletion. Conclusions and Clinical Relevance Recombinant NAP spores successfully suppressed Th2 inflammation via the up‐regulation of Tregs; this may serve as a novel therapeutic approach for treating food allergies.

Abstract. Staphylococcus aureus (S. aureus) is one of the most frequently isolated pathogens in neonatal cases of early and late-onset sepsis. Drug resistance profiles and carriage of toxin genes may affect the treatment and outcome of an infection. The present study aimed to determine the antimicrobial resistance patterns and frequencies of the toxin-associated genes conserved virulence factor B (CvfB), staphylococcal enterotoxin Q (SEQ) and staphylococcal enterotoxin K (SEK) among S. aureus isolates recovered from paediatric patients with bloodstream infections (BSIs) in Guangzhou (China). Of the 53 isolates, 43.4% were methicillin-resistant S. aureus (MRSA), and resistance rates to penicillin, erythromycin, clindamycin, trimethoprim/sulfamethoxazole, tetracycline, and ciprofloxacin of 92.5, 66.0, 62.3, 13.2, 20.8 and 1.9% were recorded, respectively. However, no resistance to nitrofurantoin, dalfopristin/quinupristin, rifampicin, gentamicin, linezolid or vancomycin was detected. Resistance to erythromycin, clindamycin and tetracycline in the MRSA group was significantly higher than that in the methicillin-susceptible S. aureus (MSSA) group. No significant differences in antimicrobial resistance patterns were noted between two age groups (≤1 year and >1 year). The proportion of S. aureus isolates positive for CvfB, SEQ and SEK was 100, 34.0 and 35.8%, respectively, with 24.5% (13/53) of strains carrying all three genes. Compared with those in MSSA isolates, the rates of SEK, SEQ and SEK + SEQ carriage among MRSA isolates were significantly higher. Correlations were identified between the carriage of SEQ, SEK and SEQ + SEK genes and MRSA (contingency coefficient 0.500, 0.416, 0.546, respectively; P<0.01). In conclusion, MRSA isolated from the blood of paediatric patients with BSIs not only exhibited higher rates of antimicrobial resistance than MSSA from the same source, but also more frequently harboured SEK and SEQ genes. The combination of the two aspects influenced the dissemination of MRSA among children. The present study clarified the characteristics of BSI-associated S. aureus and enhanced the current understanding of the pathogenicity and treatment of MRSA.