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.

Evaluation of time to positivity for blood culture combined with immature granulocytes, neutrophil-to-lymphocyte ratio, and CRP in identifying bloodstream coagulase-negative Staphylococci infection in pediatric patients.

OBJECTIVE: To evaluate the application value of time to positivity (TTP) for blood culture combined with inflammatory parameters that included immature granulocyte percentage (IG%), immature granulocyte count (IG#), C-reactive protein (CRP), white blood cells (WBC) neutrophil percentage (NE%), and neutrophil-to-lymphocyte ratio (NLR), and to identify bloodstream infections from contamination with coagulase-negative staphylococci (CoNS) in pediatric patients. METHODS: Data of 12 897 inpatients with blood culture CoNS were retrospectively collected and analyzed from January-December 2019 at our hospital. According to pre-defined criteria, they were divided into a CoNS infection group (132 cases) and a CoNS contamination group (124 cases). Infection with Staphylococcus aureus (SA, 27 cases) at the same period was considered a positive control group. ROC curve analysis assisted in determining the value of applying TTP combined with the above-mentioned inflammatory parameters to distinguish CoNS infection from contamination. RESULTS: Among the 256 strains of CoNS, Staphylococcus hominis (55.1%), Staphylococcus epidermidis (32.0%), and Staphylococcus capitis (7.0%) were common. There was no significant difference in the subspecies distribution between the infection and contamination groups. The TTP of the CoNS infection group was significantly lower than the contamination group (P < .05). IG%, IG#, CRP, NE%, and NLR were all higher in the infected group as compared to the contaminated group (P < .05), while WBC was similar among groups. There was also no statistical difference in those parameters when comparing the CoNS infection and SA groups. ROC analysis showed that TTP value in identifying CoNS infection from contamination was the highest with area under the curve (AUC) of 0.913, and the sensitivity and specificity were 0.827 and 0.852, respectively, at the optimal cutoff value of 23.9 hours. This was followed by IG% (AUC = 0.712), with an optimal critical value of 0.55%, and a sensitivity of 0.519 and specificity of 0.797. All the AUC values of IG#, CRP, NE%, and NLR were <0.7. A combination of TTP with IG%, CRP, and NLR improved the AUC, sensitivity, specificity, accuracy, PPV, and NPV values to 0.977, 0.922, 0.957, 91.8%, 92.2%, and 91.3%, respectively. CONCLUSIONS: TTP within 24 hours indicates likelihood of CoNS as the pathogenic agent in pediatric patient blood culture. The combination of TTP with IG% CRP and NLR might improve the diagnostic accuracy.

Clinical and Molecular Epidemiology of Invasive Group B Streptococcus Disease among Infants, China.

Invasive group B Streptococcus (GBS) remains a leading cause of illness and death among infants globally. We conducted prospective and retrospective laboratory-based surveillance of GBS-positive cultures from infants <3 months of age in 18 hospitals across China during January 1, 2015-December 31, 2017. The overall incidence of GBS was 0.31 (95% CI 0.27-0.36) cases/1,000 live births; incidence was 0-0.76 cases/1,000 live births across participating hospitals. The case-fatality rate was 2.3%. We estimated 13,604 cases of GBS and 1,142 GBS-associated deaths in infants <90 days of age annually in China. GBS isolates were most commonly serotype III (61.5%) and clonal complex 17 (40.6%). Enhanced active surveillance and implementation of preventive strategies, such as maternal GBS vaccination, warrants further investigation in China to help prevent these infections.

Molecular characteristics of group B Streptococcus isolates from infants in southern mainland China.

BACKGROUND: Invasive group B Streptococcus (GBS) disease in Chinese infants has gradually gained attention in recent years, but the molecular epidemiology of the pathogen is still not well known. METHODS: This multicenter study retrospectively investigated distribution of capsular serotypes, sequence types (STs), and hypervirulent GBS adhesin gene (hvgA) in clinical GBS isolates that caused invasive disease in infants aged < 3 months of age in southern mainland China between January 2013 and June 2016. Genes for antibiotic resistance to tetracycline, erythromycin, and clindamycin were also examined. RESULTS: From a total of 93 GBS isolates taken from 34 early-onset disease (EOD, 0-6 days after birth) and 59 late-onset disease (LOD, 7-89 days after birth) cases, four serotypes were identified: serotypes III (79.6%), Ib (12.9%), Ia (4.3%), and V (3.2%). Serotype III accounted for 73.5% of EOD and 83.1% of LOD and was responsible for 75.5% of cases involving meningitis. Fifteen STs were found, with the majority being ST17 (61.3%), ST12 (7.5%), ST19 (7.5%), and others (23.7%). 96.8% of STs belonged to only five clonal complexes (CCs): CC17 (64.5%), CC10 (12.9%), CC19 (9.7%), CC23 (6.5%), and CC1 (3.2%). The hvgA gene was detected in 66.7% of GBS isolates and 95% of CC17 isolates, all of which were serotype III except one serotype Ib/CC17 isolate. A large proportion of GBS isolates were found to be resistant to tetracycline (93.5%), clindamycin (65.5%), and erythromycin (60.2%). Genes of tetO (74.7%) and tetM (46.0%) were found in tetracycline resistant isolates, linB (24.6%) in clindamycin resistant isolates, and ermB (87.5%) and mefA (3.6%) in erythromycin resistant isolates. CONCLUSION: Our results reveal higher prevalence of serotype III, ST17, CC17, hvgA expressing, and antibiotic resistant GBS isolates than previously reported in southern mainland China. This study provides guidance for appropriate measures of prevention and control to be taken in the future.

Virulence and resistance on various pathogens mediated by mobile genetic integrons via high flux assays.

BACKGROUND: Recognized as a resistance mechanism responsible for the emergence and prevalence of antimicrobial resistance, integron is widely distributed and spread among clinical microorganisms and play a key role in the dissemination of such antimicrobial resistance, which may eventually contribute to the unleashing of "Super Bugs" In this study, detection assays based on loop-mediated isothermal amplification (LAMP) methodologies targeting on class 1 to class 3 integrase genes was developed and evaluated. METHODS: LAMP methodology was employed to develop novel detection assays on class 1, 2 and 3 integrons. Firstly, this protocol was specifically designed to detect such integrons by targeting integrase genes intI1, intI2 and intI3. Development, evaluation and optimization of such LAMP assays was studied, including the reaction temperature, volumn, time, sensitivity and specificity of both primers and targets. A total of 1082 strains, including 397 integron positive and 685 integron negative microorganisms, were included for the application verification of the established LAMP assays. RESULTS: The indispensability of each primer was confirmed, and the optimal amplification was obtained under 63 °C for 45 min, with 25 µl reaction found to be the most cost-efficient volume. As application was concerned, all of the 397 integron-positive isolates yielded positive amplicons and other 685 integron-negative bacteria were negative for the integron-LAMP assays, revealing totaling 100% detection rate and specificity. CONCLUSIONS: The established integron-LAMP assays was demonstrated to be a valid and rapid detection method for integrons screening, which may aid in both the laboratory and clinical integron screening for microorganisms.

Epidemiology of invasive group B streptococcal disease in infants from urban area of South China, 2011-2014.

BACKGROUND: Group B Streptococcus (GBS) is a leading cause of morbidity and mortality in infants in both developed and developing countries. To our knowledge, only a few studies have been reported the clinical features, treatment and outcomes of the GBS disease in China. The severity of neonatal GBS disease in China remains unclear. Population-based surveillance in China is therefore required. METHODS: We retrospectively collected data of <3 months old infants with culture-positive GBS in sterile samples from three large urban tertiary hospitals in South China from Jan 2011 to Dec 2014. The GBS isolates and their antibiotic susceptibility were routinely identified in clinical laboratories in participating hospitals. Serotyping and multi-locus sequence typing (MLST) were also conducted for further analysis of the neonatal GBS disease. RESULTS: Total 70 cases of culture-confirmed invasive GBS infection were identified from 127,206 live births born in studying hospitals, giving an overall incidence of 0.55 per 1000 live births (95% confidence interval [CI] 0.44-0.69). They consisted of 49 with early-onset disease (EOD, 0.39 per 1000 live births (95% CI 0.29-0.51)) and 21 with late-onset disease (LOD, 0.17 per 1000 live births (95% CI 0.11-0.25)). The incidence of EOD increased significantly over the studying period. Five infants (4 EOD and 1 LOD) died before discharge giving a mortality rate of 7.1% and five infants (7.1%, 2 EOD and 3 LOD) had neurological sequelae. Within 68 GBS isolates from GBS cases who born in the studying hospitals or elsewhere, serotype III accounted for 77.9%, followed by Ib (14.7%), V (4.4%), and Ia (2.9%). MLST analysis revealed the presence of 13 different sequence types among the 68 GBS isolates and ST-17 was the most frequent sequence type (63.2%). All isolates were susceptible to penicillin, ceftriaxone, vancomycin and linezolid, while 57.4% and 51.5% were resistant to erythromycin and clindamycin, respectively. CONCLUSIONS: This study gains the insight into the spectrum of GBS infection in south China which will facilitate the development of the guidance for reasonable antibiotics usage and will provide evidence for the implementation of potential GBS vaccines in the future.

Virulence of thermolable haemolysi tlh, gastroenteritis related pathogenicity tdh and trh of the pathogens Vibrio Parahemolyticus in Viable but Non-Culturable (VBNC) state.

In the Viable but Non-Culturable (VBNC) state, microorganisms may survive under severe external environment. In this study, the specificity and sensitivity of PMA-LAMP assay on the detection of Vibrio Parahemolyticus (V. parahemolyticus) has been developed and evaluated, with further application on a number of food-borne V. parahemolyticus strains. Six primers were designed for recognizing 8 distinct targeting on tlh, tdh and trh gene. Through specific penetration through the damaged cell membrane of dead cells and intercalating into DNA, PMA could prevent DNA amplification of dead bacteria from LAMP, which enabled the differentiation of bacteria between VBNC state and dead state. The established PMA-LAMP showed significant advantage in rapidity, sensitivity and specificity, compared with regular PCR assay. The applicability had also been verified, demonstrating the PMA-LAMP was capable of detection on V. parahaemolyticus.

[Analysis of resistance tendency of bloodstream-infecting pathogens in China].

OBJECTIVE: To investigate the resistance profiles and the trend of bloodstream-infecting pathogens isolated from hospitalized patients during 2004-2010. METHODS: The bloodstream isolates were collected from 18 hospitals in 17 cities. Minimum inhibition concentrations (MIC) were determined using the agar dilution method recommended by CLSI (Clinical and Laboratory Standards Institute), and susceptibility results were analyzed according to the 2011 CLSI guideline. RESULTS: Among the 2004-2005, 2007-2008 and 2009-2010 periods, the proportions of clinical isolates were similar; 43.1% (149 isolates), 34.0% (151 isolates) and 47.5% (776 isolates) for Gram positive strains, 56.9% (197 isolates), 66.0% (293 isolates) and 52.5% (858 isolates) for Gram negative strains, respectively. The isolating rate of MRSA was 54.1% (20/37) in 2007-2008, which was the highest among the 3 periods during 2004 to 2010, while it decreased in 2009-2010 (36.5%, 62/170). The MRCNS proportions were similar across the 3 periods. One (1.8%) vancomycin-resistant Enterococcus faecium and 1 linezolid-resistant Enterococcus faecalis were found. Although the isolating rates of penicillin non-sensitive strains (oral) were similar between 2009-2010 and 2007-2008 [54.5% (6/11) and 53.9% (7/13), respectively], the resistant rates increased from 0% in 2007-2008 to 30.8% (4/13) in 2009-2010. The results were similar according to the non-meningitis criterion (IV), and the susceptibility rates decreased from 100.0% (11 isolates) in 2007-2008 to 84.6% (11/13) in 2009-2010. ESBL-harboring strains in E. coli were similar among the 3 periods during 2004 to 2010 [66.7% (30/45), 73.2% (71/97) and 67.9% (233/343), respectively]. ESBL-producing strains in Klebsilla pnuemoniae decreased year after year, 72.4% (21/29), 50.0% (18/36) and 41.1% (65/158) in 2004-2005, 2007-2008 and 2009-2010, respectively. Except that the sensitive rate of Enterobacter cloacae to ertapenem was 80% (32/40), the sensitive rates of other strains to carbapenems were still above 90% and the resistance rates were less than 5%. Acinetobacter baumannii had the highest multi-drug resistance rate (81.8%, 81/99). One strain (1.0%, 1/99) of Acinetobacter baumannii isolated in 2009-2010 was reported to be pan-resistant. CONCLUSIONS: We are facing a more serious situation of bacterial resistance. Acinetobacter baumannii resistance was most serious, usually with the characteristics of multiple drug resistance, and even pan-resistance. Carbapenems remain to be the most effective against enterobacteriaceae. Strains resistant to novel antibiotics (linezolid and tigecycline) have emerged.

Prevalence, serotype, and antimicrobial resistance profiles of children infected with Salmonella in Guangzhou, southern China, 2016-2021.

Purpose: Salmonella infection is a key global public health concern and has lead to an increased economic burden on society. We investigated the epidemiological characteristics and antimicrobial resistance profiles of clinically isolated Salmonella strains in Guangzhou Women and Children's Medical Center. Patients and methods: This was a retrospective study of 1,338 Salmonella strains collected from children in Guangzhou Women and Children's Medical Center during 2016 to 2021. Results: The results revealed that 1,338 cases of Salmonella were mainly isolated from feces and blood samples. The age distribution was dominated by infants under 3 years old. The seasonal distribution was high in summer and autumn. 48 serotypes were detected, and S. typhimurium (78.7%) was the predominant serogroup. The results of antimicrobial susceptibility showed that the highest resistance was observed in ampicillin (84.5%), while lower resistance was observed in piperacillin/tazobactam, cefoperazone/sulbactam and ciprofloxacin. The antimicrobial resistance rate of fecal isolates was higher than that of blood isolates. The five-year average detection rate of multi-drug resistant Salmonella was 8.5% (114/1338) and the MDR rate of S. typhimurium was the lowest (6.9%; 73/1053). Conclusion: We concluded that antibacterial treatment should be carefully selected according to serotype and antimicrobial sensitivity results in children. Antimicrobial resistance monitoring for multi-drug resistant Salmonella is still required.

Identification of New Delhi metallo-ß-lactamase gene (NDM-1) from a clinical isolate of Acinetobacter junii in China.

New Delhi metallo-ß-lactamase-1 (NDM-1) is a novel type of metallo-ß-lactamase (MBL) responsible for bacterial resistance to ß-lactam antibiotics. Acinetobacter junii was previously shown to possess a MBL phenotype; however, the genes responsible for this phenotype were not identified. In this study, we reported the identification of NDM-1 gene in a clinical isolate of A. junii from a child patient in China, which was resistant to all ß-lactams except aztreonam but sensitive to aminoglycosides and quinolones. The cloned NDM-1 gene contained an open reading frame of 813 bp and had a nucleotide sequence 99.9% identical (812/813) to reported NDM-1 genes carried by Acinetobacter baumannii , Enterococcus faecium , Escherichia coli , and Klebsiella pneumoniae . Recombinant NDM-1 protein was successfully expressed in E. coli BL21, and antibiotic sensitivities of the NDM-1-producing E. coli were largely similar to the A. junii 1454 isolate. The findings of this study raise attention to the emergence and spread of NDM-1-carrying bacteria in China.

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.

Impact of Platelet Glycoprotein Ia/IIa C807T Gene Polymorphisms on Coronary Artery Aneurysms of KD Patients.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis of unknown etiology in children. Coronary artery abnormalities are the most common complications of KD. Recent evidence showed that genetic polymorphisms may lead to susceptibility to KD. Genetic variants in platelet glycoprotein have been reported to be associated with coronary artery disease. The aim of the present study is to investigate the correlation between the role of platelet glycoprotein and coronary artery aneurysms in KD patients. METHODS: We did a case-control study that enrolled 818 KD patients and 1401 healthy children with the same age and sex from January 2013 to December 2016. Analysis of single-nucleotide polymorphism (rs1126643) of the platelet glycoprotein Ia/IIa C807T was performed by multiplex polymerase chain reactions in this study. RESULTS: A significant difference in the genotype distribution between KD cases and controls was observed for the glycoprotein Ia/IIa C807T (rs1126643) polymorphism (p=0.026). Compared with the healthy children, the rs1126643T allele carriers had odds ratio (OR) of 0.63 for developing KD (TT vs. CC: adjusted OR = 0.62, 95% confidence interval (CI) = 0.43-0.88,p=0.0078; TT vs. CT/CC: adjusted OR = 0.63, 95% CI = 0.44-0.889,p=0.0093). Furthermore, we also found that children less than 60 months of age and female patients with rs1126643 T allele carriers had an adjusted OR of 0.66 (95% CI = 0.46-0.95) for noncoronary artery aneurysm patients (p=0.0242). Single-nucleotide polymorphism rs1126643 TT seems to represent a protective factor against KD in coronary artery aneurysm formation in multivariate analysis. CONCLUSIONS: The platelet glycoprotein Ia/IIa T allele carriers may have a protective effect on the risk of coronary artery aneurysms of KD patients, especially in females and children aged less than 60 months. These results may provide evidence for platelet glycoprotein Ia/IIa gene polymorphisms in the pathogenesis of KD patients.

Diagnostic Performance of Various Methodologies for Group B Streptococcus Screening in Pregnant Woman in China.

Maternal vaginal/rectal colonization of group B streptococcus (GBS) is a main risk for neonatal invasive infection. Efficient determination of GBS colonization in pregnant women is crucial. This study aimed to investigate the prevalence of GBS carriage and evaluate the diagnostic performance of six methodologies for GBS screening conducted in China, including blood agar plate, liquid chromogenic medium, and loop-mediated isothermal amplification (LAMP) without pre-enrichment, chromogenic agar plate with pre-enrichment, and GBS antigen detection without and with pre-enrichment in comparison with the standard reference method (Lim broth-enriched subculture with plating on 5% sheep blood agar). Vaginal/rectal swabs were collected from 1,281 pregnant women at 35-37 weeks of gestation. Of them, 309 were taken in triplicate, one for Lim broth-enriched subculture, one for blood agar plate, and the third for GBS antigen detection (Reagent W); 177 were acquired in duplicate, one for Lim broth-enriched subculture and the other for GBS antigen detection (Reagent H); 502 were obtained in duplicate, one for Lim broth-enriched subculture and the other for liquid chromogenic medium; 158 were collected in duplicate, one for Lim broth-enriched subculture and the other for LAMP; and 135 were inoculated in Lim broth-enriched for GBS antigen detection (Reagent W) and subculture with chromogenic agar plate and 5% blood agar plate. The overall prevalence of GBS carriage was 10.1% (130/1,281, 95% CI: 8.5-12.1%) according to the standard reference method. Compared with the standard reference method, the LAMP had excellent performance of sensitivity (100%, 95%CI: 83.4-100%), specificity (94%, 95%CI: 88.1-97.1%), and Yoden index (0.940); as well as the blood agar plate with sensitivity (81.5%, 95%CI: 61.3-93.0%), specificity (100%, 95%CI: 98.3-100.0%), and Yoden index (0.815). The other four methods were not sufficient to reach the threshold in terms of sensitivity or specificity compared to the standard reference method. Furthermore, for LAMP, results can be obtained within 0.5-1 h, while for blood agar plate, which needed 24-48 h, and further identification was required. Our data suggested that the performance of LAMP was highly comparable to the standard Lim broth-enriched subculture and LAMP is considered as an alternative for fast and accurate GBS screening.

Integrin α2 gene polymorphism is a risk factor of coronary artery lesions in Chinese children with Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis, and the formation of coronary artery lesions(CAL) is its most common sequela. Both genetic and environmental factors are considered to be important factors of in KD. Integrin α2 (ITGA2) is a transmembrane receptor that is associated with susceptibility to several diseases, but its relevance to KD with CAL is unclear. METHODS: We genotyped ITGA2 rs1126643 in 785 KD patients with the CAL and no-CAL(NCAL) (300 patients with CAL, and 485 age- and sex-matched patients with NCAL). OR (95% CI) and adjusted OR (95% CI) were used to evaluate the intensity of the association. RESULTS: We found a significantly increased risk of KD with CAL associated with ITGA2 rs1126643 genotypes (CT vs CC: adjusted OR = 1.57, 95% CI = 1.16-2.12, P = 0.0032; CT/TT vs CC: adjusted OR = 1.49, 95% CI = 1.12-2.00, P = 0.0068; T vs C: adjusted OR = 1.66, 95% CI = 1.16-2.51, P = 0.0165). Moreover, we found that carriers of the CT/TT genotype had a significant risk of KD with coronary artery lesion susceptibility for children ≤60 months of age, and the CT/TT genotype was significantly associated with an increased risk of SCAL formation and MCAL formation when compared with the CC genotype. CONCLUSION: ITGA2 rs1126643 was associated with increased susceptibility and severity of CAL in KD.

Rapid Classification of Multilocus Sequence Subtype for Group B Streptococcus Based on MALDI-TOF Mass Spectrometry and Statistical Models.

Group B Streptococcus (GBS) is an important etiological agent of maternal and neonatal infections as well as postpartum women and individuals with impaired immunity. We developed and evaluated a rapid classification method for sequence types (STs) of GBS based on statistic models with Matrix-Assisted Laser Desorption/Ionization Time-of Flight Mass Spectrometry (MALDI-TOF/MS). Whole-cell lysates MALDI-TOF/MS analysis was performed on 235 well-characterized GBS isolates from neonatal invasive infections in a multi-center study in China between 2015 and 2017. Mass spectra belonging to major STs (ST10, ST12, ST17, ST19, ST23) were selected for model generation and validation. Recognition and cross validation values were calculated by Genetic Algorithm-K Nearest Neighbor (GA-KNN), Supervised Neural Network (SNN), QuickClassifier (QC) to select models with the best performance for validation of diagnostic efficiency. Informative peaks were further screened through peak statistical analysis, ST subtyping MSP peak data and mass spectrum visualization. For major STs, the ML models generated by GA-KNN algorithms attained highest cross validation values in comparison to SNN and QC algorithms. GA-KNN models of ST10, ST17, and ST12/ST19 had good diagnostic efficiency, with high sensitivity (95-100%), specificity (91.46%-99.23%), accuracy (92.79-99.29%), positive prediction value (PPV, 80%-92.68%), negative prediction value (NPV, 94.32%-99.23%). Peak markers were firstly identified for ST10 (m/z 6250, 3125, 6891) and ST17 strains (m/z 2956, 5912, 7735, 5218). Statistical models for rapid GBS ST subtyping using MALDI-TOF/MS spectrometry contributes to easier epidemical molecular monitoring of GBS infection diseases.

Predominance of III/ST19 and Ib/ST10 Lineages With High Multidrug Resistance in Fluoroquinolone-Resistant Group B Streptococci Isolates in Which a New Integrative and Conjugative Element Was Identified.

Fluoroquinolone (FQ)-resistant Group B Streptococcus (GBS) has been reported with considerable cross-resistance, worsening the crisis of multidrug-resistant (MDR) GBS in clinical settings. However, national epidemiological data on FQ-resistant GBS in mainland China have not been well-characterized. This study aimed to determine the prevalence of FQ resistance among GBS from neonatal invasive infections and maternal colonization in northern and southern China, to investigate the serotyping, multilocus sequence typing, and antibiotic cross-resistance, and to characterize the mutations in gyrA and parC genes in quinolone resistance-determining region (QRDR). In order to provide a comprehensive view of the location and structure of resistance genes, whole-genome sequencing on III/ST19 MDR isolates were performed. Among 426 GBS, 138 (32.4%) were FQ resistant, with higher prevalence in northern China than in southern China in both neonates (57.8%, 37/64 vs. 21.7%, 39/180) and pregnant women (50.9%, 29/57 vs. 26.4%, 33/125). Serotypes were distributed as III (48.5%), Ib (39.9%), V (6.5%), and Ia (5.1%). Sequence types were mainly ST19 (53.6%) and ST10 (39.1%), followed by ST12 (1.4%), ST17 (1.4%), ST23 (1.4%), and 0.7% each of ST27, ST188, ST197, and ST597. ST19 isolates were more prevalent in southern China than in northern China in both neonates (64.1%, 25/39 vs. 27.0%, 10/37) and pregnant women (81.8%, 27/33 vs. 41.4%, 12/29), whereas ST10 isolates were more common in northern China than in southern China in both neonates (64.9%, 24/37 vs. 20.5%, 8/39) and pregnant women (58.6%, 17/29 vs. 15.2%, 5/33). Serotype III isolates were mainly ST19 (89.6%, 60/67), while Ib isolates were largely ST10 (94.5%, 52/55). Sequencing data revealed several mutations in QRDR, including Ser81Leu in gyrA (99.2%, 130/131), Ser79Phe or Tyr in parC (76.2%, 48/63), and a previously unreported Ile218Thr and Ile219Phe double mutation pattern (49.2%, 31/63) in parC. ST10 isolates were associated with Ser79Phe (84%, 21/25), while ST19 isolates were limited to Ser79Tyr (95.7%, 22/23). A new integrative and conjugative element (ICE) harboring tetM and gyrA genes was identified in a III/ST19 isolate. This study investigates the molecular characteristics of FQ-resistant GBS in northern and southern China, emphasizing the need for continuous surveillance geographically and further research to characterize the mechanisms of ICE transfer.

[Investigation of campylobacter jejuni infection in children with diarrhea in Guangzhou].

OBJECTIVE: To investigate the incidence of campylobacter jejuni (CJ) infection and the drug resistance of CJ in children with diarrhea in Guangzhou. METHODS: The fecal samples of 3,351 children with diarrhea between July 2005 and June 2008 were collected for CJ culture. The species of CJ strains were identified by Lior methods. The drug susceptibility tests were performed by the Kirby-Bauer method. RESULTS: Two hundred and sixty-seven CJ strains (8.0%) were isolated from 3,351 samples. The children at age of 1 month to 1 year were susceptible to CJ, accounting for 91.0%. A higher incidence of CJ infection (76.8%) was found in summer and autumn. The CJ strains were susceptible to imipenem, amikacin, cefoperazone/sulbactam, chloramphenicol, macrolides and lincomycins. Parts of CJ strains (20%-40%) were resistant to ampicillin, quinolones and ambramycin. All CJ strains were resistant to sulfamethoxazole/trimethoprim and cefditoren. Two hundred and one strains (75.3%) were CJ biotype I. CONCLUSIONS: CJ is an important pathogen of diarrhea in children from Guangzhou. CJ is resistant to some antibiotics used often in clinical practice, and so it is thus important to use antibiotics based on the results of drug susceptibility tests in children with CJ infection.

[Distribution and antibiotic resistance of pathogens isolated from children with infectious diarrhea in Guangzhou].

OBJECTIVE: To study the distribution and antibiotic resistance of the isolated pathogens from children with infectious diarrhea in Guangzhou. METHODS: The fecal samples of 2 409 children with infectious diarrhea between January 2006 and December 2007 were collected and cultured. Pathogenic bacterium were isolated and identified by biochemical and serological methods. The antibiotic susceptibilities were tested by the Kirby-Bauer method. RESULTS: A total of 448 isolates of pathogenic bacterium (18.6%) were obtained, including Shigella (n=159), enteropathogenic Escherichia coli (n=141), Salmonella (n=76), Vibrion (n=11), fungus (n=41), and C jejuni (n=20). All of isolates of the three major pathogenic bacterium, Shigella, enteropathogenic Escherichia coli and Salmonella, were susceptible to imipenem and less than 10% of the isolates were resistant to the third generation cephalosporins and beta-lactamase inhibitors. However, the isolates showed a high resistance to ampicillin and sulfamethoxazole/trimethoprim (>75%). CONCLUSIONS: Shigella, enteropathogenic Escherichia coli and Salmonella were major pathogenic bacterium of diarrhea in children from Guangzhou. The major isolates were susceptible to imipenem, the third generation cephalosporins and beta -lactamase inhibitors, but were resistant to ampicillin and sulfamethoxazole/trimethoprim.

[Antimicrobial resistance and penicillin resistance-associated genes of Streptococcus pneumoniae isolated from children with respiratory tract infection].

OBJECTIVE: To investigate the antimicrobial resistance and penicillin resistance-associated genes (TEM and pbp2B) of Streptococcus pneumoniae (S. pneumoniae) isolated from sputum specimens of Guangzhou children with respiratory tract infection. METHODS: E-test and Kirby-Bauer methods were applied to detect the antibiotic susceptibility of 44 strains of S. pneumoniae. PCR was used to detect resistance genes pbp2B and TEM, followed by DNA sequence analysis of pbp2B gene. The sequence results were compared to those of penicillin-susceptible S. pneumoniae R6. RESULTS: Of the 44 isolates of S. pneumoniae, only 5 (11.4%) were susceptible to penicillin. All strains were resistant to erythromycin but susceptible to ofloxacin and vancomycin. The resistance rate of the isolates to clindamycin and trimoxazole was more than 90%. The S. pneumoniae isolates showed a high susceptibility to amoxicillin, imipenem and ceftriaxone, with a resistance rate of 0, 2.6% and 3.9%, respectively. The sequence analysis showed that more than 99% nucleotide sequence of pbp2B gene of five penicillin-susceptible isolates was the same as penicillin-susceptible S. pneumoniae R6, without any amino acid replacement. Site mutation was found in the remaining 39 penicillin-nonsusceptible isolates with a nucleotide mutation rate ranging from 13.2% to 23.1% and amino acid replacement rate from 6.5% to 10.9%. The 39 penicillin-nonsusceptible isolates were classified into 4 types according to the mutation site between Ser391 and Thr492 of pbp2B: type I (n=30), type II (n=7), type III (n=1) and type IV (n=1). No TEM gene was detected in all the 44 S. pneumoniae isolates. CONCLUSIONS: The S.pneumoniae isolates from Guangzhou children with respiratory tract infection are resistant to penicillin and erythromycin. Amoxicillin and the third generation cephalosporin may be recommended for treating S. pneumoniae infection. The mutation of pbp2B gene plays an important role in the development of S. pneumoniae resistance to penicillin.

[Cloning and sequence analysis of UreB of Helicobacter pylori isolated from children].

OBJECTIVE: To clone UreB gene of Helicobacter pylori (H. pylori) isolated from children to pGEX-4T-1 expression plasmid, and do sequence analysis. METHODS: A pair of specific primer was designed according to H. pylori UreB gene in the GenBank. Using H. pylori strains isolated from children as a template, a UreB gene was obtained by PCR. After EcoR I and Not I digestion, the PCR production was linked with pGEX-4T-1 which was digested with the same enzymes. The recombinant plasmid was transformed into E.coli BL21 and identified by double enzyme digestion and sequence analysis. The sequence results were compared with the gene sequence in the GenBank. RESULTS: A UreB gene was successfully amplified from children's H. pylori strain GZCH1. It was 1710 bp in size. The objective band was identified by double enzyme digestion. DNA sequence showed that UreB was in the correct open reading frame. The sequence comparison analysis showed that DNA and amino acid sequence identities of UreB gene with other strains were 98%. The sequence of UreB of H. pylori strain GZCH1 was submitted to GenBank (accession number:FJ455126). CONCLUSIONS: UreB of H. pylori strain GZCH1 is successfully cloned to pGEX-4T-1, which provides a basis for research of oral H. pylori vaccine.