Isolation and genome-wide analysis of the novel Acinetobacter baumannii bacteriophage vB_AbaM_AB3P2.

A lytic Acinetobacter baumannii phage, isolate vB_AbaM_AB3P2, was isolated from a sewage treatment plant in China. A. baumannii phage vB_AbaM_AB3P2 has a dsDNA genome that is 44,824 bp in length with a G + C content of 37.75%. Ninety-six open reading frames were identified, and no genes for antibiotic resistance or virulence factors were found. Genomic and phylogenetic analysis of this phage revealed that it represents a new species in the genus Obolenskvirus. Phage vB_AbaM_AB3P2 has a short latent period (10 min) and high stability at 30-70°C and pH 2-10 and is potentially useful for controlling multi-drug-resistant A. baumannii.

Fatal Necrotizing Enterocolitis in Neonate Caused by Cronobacter sakazakii Sequence Type 64 Strain of CRISPR Sublineage b.

We report fatal neonatal necrotizing enterocolitis in China caused by Cronobacter sakazakii capsular profile K1:CA1, sequence type 64, and CRISPR type 197. Phylodynamic analyses indicated that the strain originated from the ancient, widespread, and antimicrobial drug-sensitive CRISPR sublineage b. Enhanced surveillance and pathogenesis research on this organism are required.

Molecular characterisation of Staphylococcus aureus in school-age children in Guangzhou: associations among agr types, virulence genes, sequence types, and antibiotic resistant phenotypes.

BACKGROUND: Staphylococcus aureus, one of the most prevalent opportunistic pathogens, mainly colonizes the nasal cavity and is a risk factor for severe infections. Virulence factors and accessory gene regulator (agr) are key to the severity and diversity of staphylococcal infection. In this study, we aimed to characterise S. aureus agr-types and virulence genes and correlated them with genetic background and antibiotic-resistant phenotypes. RESULTS: Agr types were identified in 704 isolates (98.5%), with only 11 isolates were negative for agr type. Most of our isolates were classified as agr type I, followed by types III, II and IV. The enterotoxin c gene (sec) was detected in 48.6% of isolates, showing the highest prevalence among the five enterotoxin genes detected. The positivity rates for the lukS/F-PV and tsst genes were 4% and 2.2%, respectively, while neither sed nor SasX were detected. ST45, ST59, ST338, ST188, ST6, ST7, ST22, ST25, ST398, and ST944 belonged to agr I group, while ST5 and ST15 belonged to agr II group. ST30 and ST1 were classified into agr III group, and ST121 was assigned into agr IV group. The tsst gene was found exclusively within agr I and III types belonging to ST7 and ST30 isolates, while the lukS/F-PV was predominantly carried by agr I type isolates primarily within CC59 and CC22 clones. Among the methicillin-resistant S. aureus (MRSA) isolates, 89.7% belonged to agr I group, and 97.8% of rifampicin-resistant or intermediate isolates were assigned to agr I group. MRSA isolates harboured more tested virulence genes compared to methicillin-susceptible S. aureus isolates. CONCLUSIONS: We characterized the distributions of agr types and eight major virulence genes of 715 S. aureus isolates, and our findings revealed clear associations between agr types and STs, as well as virulence genes, and drug resistant phenotypes.

Molecular Characterization and Epidemiology of Carbapenem-Resistant Enterobacteriaceae Isolated from Pediatric Patients in Guangzhou, Southern China.

Background: Carbapenem-resistant Enterobacteriaceae (CRE) are spreading worldwide, posing a serious public health concern. However, the data on CRE strains that cause infections in children in Guangzhou remain limited. Therefore, this study aimed to investigate the epidemiology of CRE, drug resistance, and resistance mechanisms in children in Guangzhou, Southern China. Methods: In total, 54 nonrepetitive CRE strains were collected in pediatric patients at three centers in Guangzhou, Southern China, from January 2016 to August 2018. CRE isolates were used for further studies on antimicrobial susceptibility, the modified Hodge test (MHT), the modified carbapenem inactivation method (mCIM), and drug resistance genes. Multilocus sequence typing (MLST) was used to elucidate the molecular epidemiology of K. pneumoniae. Results: The isolated CRE strains include 34 K. pneumoniae (63.0%), 10 E. coli (18.5%), 4 Enterobacter cloacae (7.4%), and 6 Proteus mirabilis (11.1%) strains. The strains were isolated mainly from the blood (31.5%, n = 17), sputum (31.5%, n = 17), and urine (16.7%, n = 9). All CRE isolates were highly resistant to the third- or fourth-generation cephalosporins, carbapenems, and ß-lactam + ß-lactamase inhibitors (94.4%-96.3%). In addition, the resistance rates to amikacin, ciprofloxacin, levofloxacin, tigecycline, and colistin were 5.6%, 14.8%, 16.7%, 9.3%, and 0%, respectively. Carbapenemase was detected in 35 (64.8%) of the CRE isolates. The most dominant carbapenemase gene was bla NDM (n = 17, 48.6%), followed by bla IMP (n = 13, 37.1%) and bla OXA-23 (n = 4, 11.4%). Other carbapenemase genes (bla KPC, bla sim, bla Aim, bla GES, bla Gim, bla OXA-2 , and bla OXA-48 ) and the mcr-1 gene were not detected. MLST analysis showed high diversity among the K. pneumoniae, and ST45 (11.7%, 4/34) was the dominant sequence type. Conclusion: This study revealed bla NDM was the most dominant carbapenemase gene in children in Guangzhou. Infection control measures need to be taken for the prevention and treatment of CRE infections.

Oral administration of recombinant Bacillus subtilis spores expressing Helicobacter pylori neutrophil-activating protein suppresses peanut allergy via up-regulation of Tregs.

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.

Novel Multidrug-Resistant Cronobacter sakazakii Causing Meningitis in Neonate, China, 2015.

We report a case of meningitis in a neonate in China, which was caused by a novel multidrug-resistant Cronobacter sakazakii strain, sequence type 256, capsular profile K1:CA1. We identified genetic factors associated with bacterial pathogenicity and antimicrobial drug resistance in the genome and plasmids. Enhanced surveillance of this organism is warranted.

The recombinant fusion protein of cholera toxin B and neutrophil-activating protein expressed on Bacillus subtilis spore surface suppresses allergic inflammation in mice.

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, CD4+CD25+Foxp3+ Tregs in splenocytes were significantly increased in mice treated with recombinant CTB or CTB-NAP spores. The number of CD4+CD25+Foxp3+ 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 CD4+CD25+Foxp3+ Tregs and IgA. Moreover, the fusion protein CTB-NAP demonstrated a better efficiency than CTB alone in inhibiting the inflammation.

Automated classification of group B Streptococcus into different clonal complexes using MALDI-TOF mass spectrometry.

Objectives: To evaluate the performance of Matrix-Assisted Laser Desorption/Ionization Time-of Flight Mass Spectra (MALDI-TOF MS) for automated classification of GBS (Group B Streptococcus) into five major CCs (clonal complexes) during routine GBS identification. Methods: MALDI-TOF MS of 167 GBS strains belonging to five major CCs (CC10, CC12, CC17, CC19, CC23) were grouped into a reference set (n = 67) and a validation set (n = 100) for the creation and evaluation with GBS CCs subtyping main spectrum (MSP) and MSP-M using MALDI BioTyper and ClinProTools. GBS CCs subtyping MSPs-M was generated by resetting the discriminative peaks of GBS CCs subtyping MSP according to the informative peaks from the optimal classification model of five major CCs and the contribution of each peak to the model created by ClinProTools. Results: The PPV for the GBS CCs subtyping MSP-M was greater than the subtyping MSP for CC10 (99.21% vs. 93.65%), but similar for CC12 (79.55% vs. 81.06%), CC17 (93.55% vs. 94.09%), and CC19 (92.59% vs. 95.37%), and lower for CC23 (66.67% vs. 83.33%). Conclusion: MALDI-TOF MS could be a promising tool for the automated categorization of GBS into 5 CCs by both CCs subtyping MSP and MSP-M, GBS CCs subtyping MSP-M is preferred for the accurate prediction of CCs with highly discriminative peaks.

Co-occurrence of blaNDM-1, rmtC, and mcr-9 in multidrug-resistant Enterobacter kobei strain isolated from an infant with urinary tract infection.

OBJECTIVES: The co-emergence of mcr and carbapenem resistance genes in Gram-negative bacteria is a serious problem. This study aims to clarify the genetic characteristic of one novel multidrug-resistant Enterobacter kobei EC1382 with mcr-9 causing urinary tract inflammation in an infant. METHODS: Antimicrobial drug susceptibility testing was performed for this isolate using the broth microdilution method. Whole-genome sequencing was performed using the Illumina PacBio RS II platform and HiSeq platform, and the antimicrobial resistance genes, mobile elements, and plasmid replicon types were identified. Conjugation analysis was performed using Escherichia coli C600 as recipients. RESULTS: Enterobacter kobei EC1382 was resistant to carbapenem, aminoglycoside, and cephalosporin. Twenty-five antimicrobial resistance genes were identified, including genes conferring resistance to carbapenem (blaNDM-1), colistin (mcr-9), and aminoglycosides (rmtC). The blaNDM-1 gene, accompanied by bleMBL and rmtC located downstream of an ISCR14 element, was detected in the IncFII(Yp) type plasmid pEC1382-2. Interestingly, although E. kobei EC1382 was susceptible to colistin, it had three identical mcr-9 genes (two in the chromosome and one in the IncHI2-type plasmid pEC1382-1). The backbone (∼12.2-kb genetic fragment) of these mcr-9 (flanked by IS903B and IS481-IS26) regions were conserved in this strain, and they were found to be present in various bacteria as three types, implying a silent distribution. CONCLUSIONS: To the best of our knowledge, this is the first study to demonstrate the coexistence of blaNDM-1, rmtC, and mcr-9 in E. kobei. The silent prevalence of mcr-9 in bacteria may be a threat to public health.

Molecular characterization of Clostridium perfringens isolates from a tertiary children's hospital in Guangzhou, China, establishing an association between bacterial colonization and food allergies in infants.

BACKGROUND: Cow's milk protein allergy (CMPA) is one of the most common types of food allergy in infants. Faecal pathogen cultures showed that the positive rate of Clostridium perfringens was more than 30%, which was significantly higher than that for other bacteria. Therefore, it is speculated that Clostridium perfringens colonization may be one of the pathogenetic factors for CMPA in infants. We conducted a real-world evidence study. Infants aged 0-6 months with diarrhoea and mucoid and/or bloody stools were recruited from a large tertiary hospital in China. Faecal pathogen cultures for the detection of Clostridium perfringens were confirmed by flight mass spectrometry, and potential toxin genes were identified using PCR. After 12 months of follow-up, the diagnoses of CMPA and food allergy were recorded. The correlation was assessed by Pearson correlation analysis. RESULTS: In this study, 358 infants aged 0-6 months with gastrointestinal symptoms and faecal pathogen cultures were recruited. A total of 270 (44.07% girls; mean age, 2.78 ± 2.84 months) infants were followed up for 12 months. Overall, the rate of positivity for Clostridium perfringens in faecal pathogen cultures was 35.75% (128/358) in infants aged ≤ 6 months. The earliest Clostridium perfringens colonization was detected within 2 days after birth. The majority of Clostridium perfringens isolates were classified as type C in 85 stool samples. In the Clostridium perfringens-positive group, 48.21% (54/112) of infants were clinically diagnosed with food allergies after 12 months, including 37.5% (42/112) with CMPA, which was significantly higher than that of the negative group, with 7.59% (12/158) exhibiting food allergies and 5.06% (8/158) presenting CMPA (P < 0.0001). Faecal Clostridium perfringens positivity was significantly correlated with CMPA, food allergy, faecal occult blood, faecal white blood cells, antibiotic use, increased peripheral blood platelet counts, and decreased haemoglobin levels (P < 0.0001). CONCLUSIONS: This study demonstrates that intestinal colonization by Clostridium perfringens is common in infants. The majority of Clostridium perfringens isolates are classified as type C. Colonization of the intestine by Clostridium perfringens is associated with the development of CMPA and food allergy in infants.

Fecal carriage and molecular epidemiology of mcr-1-harboring Escherichia coli from children in southern China.

BACKGROUND: The increase of multidrug-resistant Enterobacteriaceae bacteria has led to the reintroduction of colistin for clinical treatments, and colistin has become a last resort for infections caused by multidrug-resistant bacteria. Enterobacteriaceae bacteria carrying the mcr-1 gene are majorly related to colistin resistance, which may be the main reason for the continued increase in the colistin resistance rate of Enterobacteriaceae. The study aimed to investigate the sequence type and prevalence of Escherichia coli (E. coli) harboring the mcr-1 gene in the gut flora of children in southern China. METHODS: Fecal samples (n = 2632) of children from three medical centers in Guangzhou were cultured for E. coli. The mcr-1-harboring isolates were screened via polymerase chain reaction (PCR). The colistin resistance transfer frequency was studied by conjugation experiments. DNA sequencing data of seven housekeeping genes were used for multi-locus sequence typing analysis (MLST). RESULTS: PCR indicated that 21 of the 2632 E. coli (0.80%) isolates were positive for mcr-1; these strains were resistant to colistin. Conjugation experiments indicated that 18 mcr-1-harboring isolates could transfer colistin resistance phenotypes to E. coli J53. MLST analysis revealed that the 21 isolates were divided into 18 sequence types (STs); E. coli ST69 was the most common (14.3%), followed by E. coli ST58 (9.5%). CONCLUSION: These results demonstrate the colonization dynamics and molecular epidemiology of E. coli harboring mcr-1 in the gut flora of children in southern China. The mcr-1 gene can be horizontally transmitted within species; hence, it is necessary to monitor bacteria that harbor mcr-1 in children.

Clinical and molecular epidemiology of carbapenem-resistant Enterobacteriaceae in pediatric inpatients in South China.

IMPORTANCE: This study assessed the clinical and molecular epidemiology of carbapenem-resistant Enterobacteriaceae in pediatric inpatients at three hospitals in South China by means of screening stool samples for carbapenem-resistant genes and a nested case-control study to determine risk factors for carriage of carbapenem-resistant Enterobacteriaceae. Of 4,033 fecal samples screened, 158 (3.92%) were positive for CRE, including Escherichia coli (51.27 %), Klebsiella pneumoniae (37.97%), and Enterobacter cloacae (6.96%). The most common carbapenemase genes harbored by gastrointestinal CRE strains were blaNDM-5, blaNDM-1, and blaIMP-4. Hematological malignancies, respiratory diseases, otolaryngological diseases, nervous system diseases, oral administration of third-generation cephalosporins, and the combined use of two or more antibiotics were independently associated with CRE colonization.

Genomic Basis of Occurrence of Cryptic Resistance among Oxacillin- and Cefoxitin-Susceptible mecA-Positive Staphylococcus aureus.

The oxacillin- and cefoxitin-susceptible mecA-positive Staphylococcus aureus is a novel "stealth" methicillin-resistant S. aureus (MRSA) type. Here, we sequenced the whole genome of two oxacillin- and cefoxitin-susceptible mecA-positive MRSA isolates from breast abscesses in a lactating woman and a nasal swab of a healthy student in Guangzhou for investigating the mechanism underlying its occurrence. The reversion of these isolates was selected by exposure to sub-MICs of cefoxitin with or without mupirocin. The mecA expression of both parental strains and their revertants was determined, and the whole genome of the revertants was sequenced. Comparative whole-genome analyses performed for both strains revealed that mecA of the clinical strain was mutated by a single-bp insertion at the 262nd position in the tandem repeat region of the gene, and this mutation that led to the formation of a premature stop codon. The colonizing strain was mutated by a novel G-to-A base substitution in the second promoter region (-35 bp) of mecA. The mecA expression level of strain 697 revertant was 37 times higher than that of the parental strain. Although the mecA expression level was even higher for parental strain 199 compared with that for its revertant, its cDNA sequence contained a single-bp insertion. Collectively, both the missense and single substitution mutations of the second promoter of mecA could render MRSA isolates as "stealth" MRSA, thereby emphasizing the importance of combining phenotype tests with mecA or penicillin-binding protein 2a detection for the identification of MRSA. IMPORTANCE The oxacillin- and cefoxitin-susceptible mecA-positive Staphylococcus aureus is a novel type of "stealth" methicillin-resistant S. aureus (MRSA), which is difficult to be detected using conventional methods. To investigate the genomic basis of their occurrence, we sequenced the whole genome of two previously recovered oxacillin- and cefoxitin-susceptible mecA-positive MRSA isolates from breast abscesses in a lactating woman and a nasal swab of a healthy student in Guangzhou. Complete SCCmec structure was absent except for mecA in clinical isolate 199. Additionally, a novel single-base pair insertion was observed in the clinical strain, which resulted in premature termination and a frameshift mutation. The colonizing isolate 697 had a Scc-mec-type IVa, and the second promoter region (-35 bp) of mecA was mutated by a novel G-to-A base substitution. The reversion of oxacillin- and cefoxitin-susceptible mecA-positive S. aureus to resistant MRSA isolates was selected by exposure to subminimum inhibitory cefoxitin with or without mupirocin.

Distribution of Biocide Resistance Genes and Association with Clonal Complex Genotypes in Staphylococcus aureus Isolated from School-Age Children in Guangzhou.

Purpose: Chlorhexidine and mupirocin are often prescribed to children in affected communities to prevent colonization and transmission of Staphylococcus aureus, but this has led to an increasing rate of biocide resistance. In this study, we aimed to determine the distribution of biocide resistance genes among S. aureus isolates from school-age children in Guangzhou, investigate chlorhexidine gluconate and mupirocin susceptibility and clonal complex (CC) genotypes in strains carrying biocide-resistance genes, and further explore the role of biofilms in this resistance. Patients and Methods: Antibiotic resistance and multilocus sequence genotyping were performed on 722 S. aureus isolates from previous study. The distribution of nine biocide genes (qacA/B, mupA, mepA, sepA, norA, lmrS, smr, mupB, qacG) was determined by PCR. Isolates carrying qacA/B or mupA genes were further tested for susceptibility to chlorhexidine gluconate (CHG) and mupirocin and biofilm formation abilities. Results: The most prevalent of the nine biocide resistance genes were mepA (95.57%), followed by norA (78.81%), lmrS (77.01%), and sepA (58.17%). The qacG gene was not detected. Distribution of sepA was significantly decreased in CC30 and CC45 genotypes, and presence of sepA was associated with resistance to antibiotics such as CLI, ERY, TCY, SXT, CIP, and LVX. In addition, 64 (94.1%, n=68) qacA/B+ isolates showed CHG resistance, 12 (100.0%, n=12) mupA+ isolates were mupirocin resistant, and 4 (80%, n=5) and 5 (100%, n=5) qacA/B+mupA+ isolates were CHG and mupirocin resistant, respectively. Of these 85 isolates, 98.8% (n=84) had different degrees of biofilm-forming abilities, which were positively associated with CHG and mupirocin resistance. Conclusion: The distribution of biocide resistance genes was associated with special CCs. The qacA/B and mupA genes are highly associated with resistance to CHG and mupirocin, and biofilm formation was found to contribute to this biocide resistance.

Active Surveillance, Drug Resistance, and Genotypic Profiling of Staphylococcus aureus Among School-Age Children in China.

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.

Oral administration of recombinant Bacillus subtilis spores expressing mutant staphylococcal enterotoxin B provides potent protection against lethal enterotoxin challenge.

Pathogenicity of Staphylococcus aureus is induced by staphylococcal enterotoxin B (SEB). A mutant form of SEB (mSEB) is immunogenic as well as less toxic. Recombinant mSEB and SEB were expressed in pET28a prokaryotic plasmids. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in mSEB-stimulated macrophages were lower than those in SEB-stimulated macrophages (p < 0.001, p < 0.01 respectively). Using CotC as a fusion protein, we constructed recombinant Bacillus subtilis spores expressing mSEB on the spore surface and evaluated their safety and protective efficacy via mouse models. Oral administration of mSEB-expressing spores increased SEB-specific IgA in feces and SEB-specific IgG1 and IgG2a in the sera, compared with mice in naïve and CotC spore-treated groups (p < 0.001, p < 0.01, p < 0.001 respectively). Six weeks following oral dosing of recombinant spores, significant differences were not found in the serum biochemical indices between the mSEB group and the naïve and CotC groups. Furthermore, oral administration of mSEB spores increased the survival rate by 33.3% in mice intraperitoneally injected with 5 µg of wild-type SEB plus 25 µg lipopolysaccharide (LPS). In summation, recombinant spores stably expressing mSEB were developed, and oral administration of such recombinant spores induced a humoral immune response and provided protection against SEB challenge in mice.

Prevalence, Characterization, and Drug Resistance of Staphylococcus Aureus in Feces From Pediatric Patients in Guangzhou, China.

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.

The Genomic Context for the Evolution and Transmission of Community-Associated Staphylococcus aureus ST59 Through the Food Chain.

Sequence type 59 (ST59) is a predominant clonal lineage of community-acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA) in Asia. Despite its increasing clinical relevance in China, the evolution and geographic expansion of ST59 has been relatively uncared for. Previous study has shown that ST59 was the predominant clone in food-related MRSA in China. This study compared the genomes of 87 clonal complex (CC) 59 S. aureus isolates sourced from food chain and infection cases to reconstruct the molecular evolution and geographical spread of ST59. Accordingly, three major sub-clades of ST59 were identified and these did not correlate with isolation source or location. Phylogenetic analysis estimated that ST59 in mainland China diverged from a most common recent ancestor around 1974, and most of the cases of cross-country transmission occurred between 1987 and 2000. Notably, two recent events of cross-country transmission through the food chain were observed, the isolates from these events diverged within relatively short time intervals. These isolates also showed high similarity in terms of their core genome, accessory genes, and antibiotic resistance patterns. These findings provide a valuable insight into the potential route of ST59 expansion in China and indicate a need for robust food chain surveillance to prevent the spread of this pathogen.

Prevalence, serotypes, and drug resistance of nontyphoidal Salmonella among paediatric patients in a tertiary hospital in Guangzhou, China, 2014-2016.

PURPOSE: Nontyphoidal Salmonella (NTS) is a common pathogen responsible for acute gastroenteritis among all ages; however, information on the prevalence, serotypes, and antibiotic susceptibility of NTS isolates is limited. We aimed to explore the characteristics of NTS isolated from paediatric patients in Guangzhou, China. METHODS: This was a retrospective study of 4586 stool culture collected at Guangzhou Women and Children's Medical Center from 2014 to 2016. RESULTS: We identified 220 (4.80%) NTS isolates in stool samples. Fourteen serotypes were identified among the 220 NTS isolates. Salmonella serotype Typhimurium was the most common serotype, representing 69.09%. The highest rate of resistance was recorded in relation to AMP (76.61%), followed by SXT (29.95%), CTX (29.93%), CHL (29.77%), CAZ (23.20%), CIP (7.51%), and CFS (7.18%). The resistance rates of NTS and serotype Typhimurium to CAZ in 2015 were significantly higher than those in 2014. The average hospitalisation duration of inpatients infected by NTS resistant to three or more clinically important agents was significantly longer than that of patients infected with NTS with less antibiotic resistance. CONCLUSION: NTS represents a major cause of paediatric gastroenteritis in Guangzhou, China, and the high level of resistance to third-generation cephalosporins coupled with increasing resistance to quinolones among isolated NTS from paediatric gastroenteritis is a serious public health concern that requires continued monitoring and rational usage of antibiotics.

Prevalence and Characterization of Staphylococcus aureus Isolated From Women and Children in Guangzhou, China.

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.