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.

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.

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.

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.

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.

Antimicrobial resistance and prevalence of CvfB, SEK and SEQ genes among Staphylococcus aureus isolates from paediatric patients with bloodstream infections.

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.

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.

Recombinant Bacillus subtilis spores expressing cholera toxin B subunit and Helicobacter pylori urease B confer protection against H. pylori in mice.

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.