A Shigella sonnei clone with extensive drug resistance associated with waterborne outbreaks in China.

Antimicrobial resistance of Shigella sonnei has become a global concern. Here, we report a phylogenetic group of S. sonnei with extensive drug resistance, including a combination of multidrug resistance, coresistance to ceftriaxone and azithromycin (cefRaziR), reduced susceptibility to fluoroquinolones, and even colistin resistance (colR). This distinct clone caused six waterborne shigellosis outbreaks in China from 2015 to 2020. We collect 155 outbreak isolates and 152 sporadic isolates. The cefRaziR isolates, including outbreak strains, are mainly distributed in a distinct clade located in global Lineage III. The outbreak strains form a recently derived monophyletic group that may have emerged circa 2010. The cefRaziR and colR phenotypes are attributed to the acquisition of different plasmids, particularly the IncB/O/K/Z plasmid coharboring the blaCTX-M-14, mphA, aac(3)-IId, dfrA17, aadA5, and sul1 genes and the IncI2 plasmid with an mcr-1 gene. Genetic analyses identify 92 accessory genes and 60 single-nucleotide polymorphisms associated with the cefRaziR phenotype. Surveillance of this clone is required to determine its dissemination and threat to global public health.

Molecular diagnostics and next-generation sequencing reveal real etiological characteristics of invasive Salmonella infection in febrile illness in Freetown, Sierra Leone.

Invasive Salmonella infection, which can cause typhoid/paratyphoid fever and invasive non-typhoidal salmonellosis, is a public health burden in Africa. Accurate diagnosis and etiological characterization are required to conduct prevalence and risk estimations for Salmonella infection; however, the utilization of optimal techniques and surveillance data are still insufficient. In this study, we performed a laboratory-based survey in Freetown, which is the biggest city in Sierra Leone with a high burden of typhoid fever, by using blood culture and molecular methods but not the Widal test, to estimate the prevalence and aetiology of invasive Salmonella infection among fever patients. We found a very low prevalence of typhoid fever in patients with fever during the investigation period, and this prevalence was clearly overestimated by the Widal test. Genome sequencing of the S. Typhi isolate from this work revealed that the strain carried multiple antibiotic resistance genes, and an epidemic clone that has existed in West Africa for years was also detected in Sierra Leone. By using metagenomic sequencing, one patient with invasive non-typhoidal salmonellosis was identified as having bacterial co-infections. Our data highlight that Salmonella surveillance based on accurate laboratory diagnosis and genome sequencing needs to be strengthened to provide a better estimation of the real epidemics and enable potential risk assessment by etiological analysis in Africa. Even in a laboratory with only basic equipment, it is possible to conduct next-generation sequencing for pathogen discovery in bloodstream infections and to determine the etiological characteristics of pathogene without complex combinations of laboratory methods.

The phenotypic and molecular characteristics of antimicrobial resistance of Salmonella enterica subsp. enterica serovar Typhimurium in Henan Province, China.

BACKGROUND: Salmonella enterica subsp. enterica serovar Typhimurium infections continue to be a significant public health threat worldwide. The aim of this study was to investigate antibiotic resistance among 147 S. Typhimurium isolates collected from patients in Henan, China from 2006 to 2015. METHODS: 147 S. Typhimurium isolates were collected from March 2006 to November 2015 in Henan Province, China. Antimicrobial susceptibility testing was performed, and the resistant genes of ciprofloxacin, cephalosporins (ceftriaxone and cefoxitin) and azithromycin were detected and sequenced. Clonal relationships were assessed by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). RESULTS: Of the 147 isolates, 91.1% were multidrug resistant (MDR), with 4.1% being resistant to all antibiotic classes tested. Of concern, 13 MDR isolates were co-resistant to the first-line treatments cephalosporins and ciprofloxacin, while three were also resistant to azithromycin. Seven PFGE patterns were identified among the 13 isolates. All of the isolates could be assigned to one of four main groups, with a similarity value of 89%. MLST assigned the 147 isolates into five STs, including two dominant STs (ST19 and ST34). Of the 43 ciprofloxacin-resistant isolates, 39 carried double gyrA mutations (Ser83Phe, Asp87Asn/Tyr/Gly) and a single parC (Ser80Arg) mutation, including 1 isolate with four mutations (gyrA: Ser83Phe, Asp87Gly; parC: Ser80Arg; parE: Ser458Pro). In addition, 12 isolates not only carried mutations in gyrA and parC but also had at least one plasmid-mediated quinolone resistance (PMQR) gene. Among the 32 cephalosporin-resistant isolates, the most common extended-spectrum ß-lactamase (ESBL) gene was blaOXA-1, followed by blaCTX-M, blaTEM-1, and blaCMY-2. Moreover, the mphA gene was identified in 5 of the 15 azithromycin-resistant isolates. Four MDR isolates contained ESBL and PMQR genes, and one of them also carried mphA in addition. CONCLUSION: The high level of antibiotic resistance observed in S. Typhimurium poses a great danger to public health, so continuous surveillance of changes in antibiotic resistance is necessary.

Prevalence of Yersinia enterocolitica Bioserotype 3/O:3 among Children with Diarrhea, China, 2010-2015.

Yersinia enterocolitica is thought to not significantly contribute to diarrheal disease in China, but evidence substantiating this claim is limited. We determined the prevalence of Y. enterocolitica infection and strain types present among children <5 years of age with diarrhea in China. The overall prevalence of pathogenic isolates was 0.59%. Prevalence of pathogenic bioserotype 3/O:3 varied geographically. In this population, the presence of fecal leukocytes was a characteristic of Y. enterocolitica infection and should be used as an indication for microbiological diagnostic testing, rather than for the diagnosis of bacillary dysentery. In contrast with Y. enterocolitica isolates from adults, which were primarily biotype 1A, isolates from children were primarily bioserotype 3/O:3. Most pathogenic isolates from children shared pulsed-field gel electrophoresis patterns with isolates from pigs and dogs, suggesting a possible link between isolates from animals and infections in children. Our findings underscore the need for improved diagnostics for this underestimated pathogen.

A large outbreak of acute gastroenteritis caused by the human norovirus GII.17 strain at a university in Henan Province, China.

BACKGROUND: Human noroviruses are a major cause of viral gastroenteritis and are the main etiological agents of acute gastroenteritis outbreaks. An increasing number of outbreaks and sporadic cases of norovirus have been reported in China in recent years. There was a large acute gastroenteritis outbreak at a university in Henan Province, China in the past five years. We want to identify the source, transmission routes of the outbreak by epidemiological investigation and laboratory testing in order to provide the effective control measures. METHODS: The clinical cases were investigated, and analysed by descriptive epidemiological methods according to factors such as time, department, grade and so on. Samples were collected from clinical cases, healthy persons, the environment, water, and food at the university. These samples were tested for potential bacteria and viruses. The samples that tested positive for norovirus were selected for whole genome sequencing and the sequences were then analysed. RESULTS: From 4 March to 3 April 2015, a total of 753 acute diarrhoea cases were reported at the university; the attack rate was 3.29%. The epidemic curve showed two peaks, with the main peak occurring between 10 and 20 March, accounting for 85.26% of reported cases. The rates of norovirus detection in samples from confirmed cases, people without symptoms, and environmental samples were 32.72%, 17.39%, and 9.17%, respectively. The phylogenetic analysis showed that the norovirus belonged to the genotype GII.17. CONCLUSIONS: This is the largest and most severe outbreak caused by genotype GII.17 norovirus in recent years in China. The GII.17 viruses displayed high epidemic activity and have become a dominant strain in China since the winter of 2014, having replaced the previously dominant GII.4 Sydney 2012 strain.

Molecular characterization and analysis of high-level multidrug-resistance of Shigella flexneri serotype 4s strains from China.

To conduct the first comprehensive analysis of Shigella flexneri serotype 4s, a novel serotype found in 2010, we identified 24 serotype 4s isolates from 1973 shigellosis cases in China (2002-2014). The isolates were characterized by single nucleotide polymorphism (SNP) phylogenetic analysis, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to determine their genetic relatedness, and analysed further for their antimicrobial susceptibilities and antimicrobial resistance determinants. The PFGE and SNP phylogenetic analyses suggest that S. flexneri serotype 4s strains are derived from multiple serotypes, including two predominant serotypes in China: serotype X variant and serotype II. Three new sequence types were identified by MLST. All isolates were resistant to ticarcillin, ampicillin and tetracycline, with high-level resistance to third-generation cephalosporins. Notably, all the isolates were multidrug resistant (MDR), with the highest levels of resistance observed for eight antimicrobials classes. Most isolates contain various antimicrobial resistance determinants. In conclusion, we found that serotype 4s isolates have multiple evolutionary sources, diverse biochemical characteristics and genomes, and highly prevalent multidrug resistance and antimicrobial-resistant determinants. With few clinical treatment options, continuous monitoring and timely intervention against this emerging MDR serotype is essential. The possibility that serotype 4s will become the next predominant serotype exists.

[Surveillance on the etiology and molecular epidemiology of Shigella sonnei isolated in Henan province from 2011 to 2014].

OBJECTIVE: To detect and analyze the distribution of virulence factors, antimicrobial resistance and pulsed field gel electrophoresis (PFGE) patterns of Shigella sonnei, isolated in Henan province from 2011 to 2014. METHODS: Samples of diarrhea patients were collected and isolated with SS selective culture medium in 37 ℃ for 18-24 hours. All strains were identified under the Kligler iron agar/motility-indol-urea biochemical action and API20E biochemical system. Serological typing and prepared DNA template were carried out with thermal cracking method and multiplex PCR, to detect the virulence genes of Shigella sonnei. According to the molecular typing method and K-B drug susceptibility testing method published by the international PulseNet bacterial infectious disease monitoring network and USA clinical and Laboratory Standards Institute, antimicrobial susceptibility tests and PFGE molecular characteristics of these positive strains isolated from sentinel hospitals patients stool samples were analyzed. RESULTS: Among the 98 strains of Sonnei type Ⅰ and 118 strains of Sonnei type Ⅱ, all the strains carried carry different virulence genes including SHET-1B, SHET-2, ial, ipaH genes, with 4 kinds of virulence gene combination types. All the 216 strains of Shigella sonnei belonged to the multi-drug resistant strains, including 34 isolates resistant to 2-4 kinds of antibiotics(15.7%), 147 isolates to 5-8 kinds of antibiotics (68.1%), 24 to 9-10 kinds of antibiotics (11.1%), 7 to 11 kinds of antibiotics (3.2%), and 4 to 13 kinds of antibiotics (1.9%). A total of 100 strains of Shigella sonnei were divided into 31 molecular patterns, digested by XbaⅠ and PFGE. Each pattern contained 1-13 strains with similarities ranged from 68.6%-100.0%. CONCLUSIONS: All the Shigella sonnei strains carried virulence pathogenic factors, presenting serious status on drug resistance. PFGE fingerprinting patterns showed high polymorphism and dominant characteristics. PFGE patterns of partial strains and corresponding antidrug spectrum presented certain relevance and with same aggregation relationship.

Emergence and Diversity of Salmonella enterica Serovar Indiana Isolates with Concurrent Resistance to Ciprofloxacin and Cefotaxime from Patients and Food-Producing Animals in China.

Salmonellosis is a major global foodborne infection, and strains that are resistant to a great variety of antibiotics have become a major public health concern. The aim of this study was to identify genes conferring resistance to fluoroquinolones and extended-spectrum ß-lactams in nontyphoidal Salmonella (NTS) from patients and food-producing animals in China. In total, 133 and 21 NTS isolates from animals and humans, respectively, exhibiting concurrent resistance to ciprofloxacin and cefotaxime were cultured independently from 2009 to ∼2013. All of the isolates were identified, serotyped, and subjected to antimicrobial susceptibility testing. Importantly, the isolates with concurrent resistance to ciprofloxacin and cefotaxime all were confirmed as S. enterica serovar Indiana. The presence of fluoroquinolone resistance genes and extended-spectrum ß-lactamases (ESBLs) was established by PCR and DNA sequencing. The occurrence and diversity of different genes conferring fluoroquinolone resistance [qepA, oqxAB, and aac(6')-Ib-cr] with mutations in topoisomerase-encoding genes (gyrA and parC) and several ESBLs (including CTX-M-65, CTX-M-27, CTX-M-15, CTX-M-14, and CTX-M-14/CTX-M-15) were noteworthy. Genes located on mobile genetic elements were identified by conjugation and transformation. Pulsed-field gel electrophoresis, used to determine the genetic relationships between these isolates, generated 91 pulsotypes from 133 chicken isolates and 17 pulsotypes from the 21 clinical isolates that showed considerable diversity. Analysis of the pulsotypes obtained with the isolates showed some clones appeared to have existed for several years and had been disseminating between humans and food-producing animals. This study highlights the emergence of ciprofloxacin- and cefotaxime-resistant S. enterica serovar Indiana, posing a threat to public health.

[Characteristics of drug resistance and molecular typing research for Salmonella Enteritidis isolated in Henan province from 2011 to 2013].

OBJECTIVE: To investigate the antimicrobial resistance status and pulsed field gel electrophoresis (PFGE) patterns of Salmonella Enteritidis (S.Enteritidis) strains in Henan province. METHODS: S. Enteritidis strains were isolated from seven sentinel hospitals from March 2011 to December 2013. According to molecular typing and Salmonella (Kirby-Bauer, K-B) drug susceptibility testing method published by the international PulseNet bacterial infectious disease monitoring network and USA Clinical and Laboratory Standards Institute (CLSI), we analyzed drug sensitivity of 8 kinds antibiotics and PFGE molecule characteristics of 120 S. Enteritidis isolates from seven sentinel hospitals. RESULTS: Among 120 strains of S. Enteritidis, 77 were isolated from male patients, 43 from female patients. A total of 78 strains S. Enteritidis were isolated from young children ranged from 0 to 5 years old (65.0%), including 57 strains isolated from 6 months to 2 years old (47.5%). The isolated time mainly centralized on May to October of the year, 11 strains isolated in March-April (9.2%), 48 were in May-July (40.0%),54 in August-October (45.0%), 7 in other months, with a typical summer seasonal characteristics. The resistance rate of 120 strains S. Enteritidis to ampicillin was 50.0% (n=60); to ceftazidime was 14.2% (n=17), to cefotaxime was 18.3% (n=22); to cefepime was 5.8% (n=7); to nalidixic acid was 61.7% (n=74); to ciprofloxacin was 8.3% (n=10), to norfloxacin was 5.8% (n=7); to gentamicin was 42.5% (n=51); to streptomycin was 21.7% (n=26); to chloramphenicol was 30.0% (n=36); resistance to methicillin benzyl ammonium was 11.7% (n=14), compound sulfamethoxazole resistance rate was 71.7% (n=86); the tetracycline resistant rate was 47.5% (n=57). All 120 strains of S. Enteritidis had different levels of resistance to 8 kinds of antibiotics, all strains were multidrug resistant strains, 28 isolates were resistant to 3-4 kinds of antibiotics (23.3%), 38 isolates were resistant to 5-6 kinds of antibiotics (31.7%), 39 isolates were resistant to 7-8 kinds of antibiotics (32.5%). All 120 strains of S. Enteritidis were divided into 44 molecular patterns by digestion with XbaI and pulsed field gel electrophoresis. each pattern contained 1-35 strains with similarity ranged from 54.3%-100%. EN14 and EN19 were the main PFGE types, including 35 and 29 strains respectively. CONCLUSION: The status of drug resistance of clinical isolates of Salmonella in Henan province was rather serious, PFGE patterns showed advantages and partial strain's corresponding resistant spectrum have certain relevance and the same aggregation relationship.

[Characteristics of drug resistance and molecular type of Salmonella typhi and Salmonella paratyphi isolated in Henan province, 2009-2011].

OBJECTIVE: To investigate the antibiotic resistance and pulsed field gel electrophoresis (PFGE) patterns of clinical isolates of Salmonella (S.) typhi and S. paratyphi in Henan province during 2009-2011. METHODS: According to molecular typing and Salmonella K-B drug susceptibility test method published by international PulseNet bacterial infectious disease monitoring network and USA Clinical and Laboratory Standards Institute (CLSI), the drug susceptibility and PFGE molecule characteristics of 78 S. typhi and S. paratyphi strains isolated from sentinel hospitals in Henan were analyzed. RESULTS: The 78 strains of S. typhi and S. paratyphi were resistant to 13 kinds of antibiotics, in which 62 were multidrug resistant (79.5%), 4 were resistant to 2-3 kinds of antibiotics (5.1%), 41 were resistant to 5-8 kinds of antibiotics (52.6%), 14 were resistant to 9-10 kinds of antibiotics (17.9%), 3 were resistant to 11-12 kinds of antibiotics (3.8%). The resistant rate to cephalosporins, quinolones and other 3 kinds of antibiotic showed an increase trends. Seventy two strains of S. typhi and S. paratyphi could be divided 14 molecular patterns by digestion with XbaⅠ and PFGE, each pattern contains 1-47 strains which shared the similarity of 66.03%-100.00%. CONCLUSIONS: The drug resistance of clinical isolates of S. typhi and S. paratyphi was serious in Henan. The PFGE patterns showed diversity, but the predominant patterns could be still found. The PFGE patterns of some strains were associated with their drug resistance.

[Investigation on distribution of Yersinia enterocolitica in Henan province between 2005 and 2011].

OBJECTIVE: To investigate the distribution of Yersinia enterocolitica in Henan province from 2005 to 2011. METHODS: A total of 6700 samples of stool specimen were collected from diarrhea patients and different domestic animals between 2005 and 2011 from Zhengzhou, Suixian and Dengfeng, as well as flies and the daub specimens of raw and cooked meat products. The bacteria were isolated by cold enrichment method, analyzed by the systematic biochemistry to determine the serotypes and bio-types, and tested the virulence genes by PCR method. RESULTS: A total of 216 strains of Yersinia enterocolitica were isolated from 11 kinds of animal hosts and foods, while 29.63% (64/216) of them were from swine. The dominant epidemic serotypes of the Yersinia enterocolitica were O: 5 and O: 8, accounted for 23.2% (50/216) and 20.4% (44/216), respectively; type 1A was the dominant bio-type, accounted for 84.7% (183/216). The dominant serotype and bio-type differed a lot among various hosts.16 pathogenic strains were isolated from swine, followed by diarrhea patients (6 strains) and dogs (6 strains). CONCLUSION: The distribution of the host of Yersinia enterocolitica was widespread, while swine was the dominant animal host.

[Analysis of etiological surveillance results of Shigella spp between 2009 and 2010 in Henan province].

OBJECTIVE: To explore the etiologic characteristics of bacillary dysentery found in Henan province, between year 2009 and 2010. METHODS: In order to explore the distribution of bacterial types, drug susceptibility and the virulence gene carrier situation, 482 strains of Shigella isolated in Henan province between 2009 and 2010 were pathogen-detected and analyzed by serotype screening, anti microbial sensitivity test and PCR methods. RESULTS: The 482 isolated strains were confirmed to be Shigella by both morphological and biochemical tests. The Shigella strains were divided into 13 serotypes in 2 groups, namely Shigella flexneri (B group) accounting for 72.0% (347/482) and Shigella sonnei (D group), accounting for 28.0% (135/482). The detection rate of Serotype F2a, as the dominant type of Shigella flexneri, decreased from 43.4% (106/245) in 2009 to 33.8% (80/237) in 2010; while the detection rate of Shigella sonnei increased from 13.1% (32/245) to 43.5% (103/237) in the same period. The results of microbial sensitivity tests carried out in year 2009 and 2010, both showed that over 98% of the 185 studied strains were resistant to ampicillin (AMP), trimethoprim-pyrimidine (TMP), tetracycline (TE), streptomycin (S) and nalidixic acid (NA).182 strains were recruited in the virulence factors detection, 67.6% (123/182) of which carried Shigella Enterotoxin 1B (set1B), Shigella Enterotoxin 2 (set2), invasive plasmid antigen H (ipaH) or invasion-related virulence factors (ial) and 24.2% (44/182) of which carried 3 virulence factors mentioned above. CONCLUSION: The prevalent serotypes of Shigella in Henan province have changed in recent years. The isolated strains showed high resistance to common antibacterial drugs and generally carried virulence factors.

[Effects of different frequencies of electroacupuncture on blood glucose level in impaired glucose tolerance patients].

OBJECTIVE: To Investigate the clinical efficacy of electroacupuncture (EA) at different frequencies for patients with impaired glucose tolerance (IGT). METHODS: A total of 120 IGT outpatients were randomly divided into control, EA-5 Hz, EA-50 Hz, and EA-100 Hz groups (n = 30/group). EA (1 mA) was applied to bilateral Pishu (BL 20), Shenshu (BL23), Zusanli (ST 36) and Sanyinjiao (SP6) for 20 min, once daily for 60 sessions. Body mass index (BMI), fasting blood glucose (FBG) and 2-hour post-prandial blood glucose (2 h PBG) contents were detected by using BAYER Blood Sugar Analyzer and glycosylated haemoglobin (HbA1c) content was detected by enzymatic assay. RESULTS: Following the treatment, both HbA1c and 2 h PBG levels in the EA-5 Hz group were significantly lower than those of the control group and those of pre-treatment in the same one group (P < 0.05, P < 0.01). No significant differences were found between the EA-5 Hz and control groups, between pre-treatment and post-treatment in the EA-5 Hz group in BMI and FBG levels; between the EA-50 Hz and control groups, between the EA-100 Hz and control groups, and between pre-treatment and post-treatment in the EA-50 Hz and EA-100 Hz groups in BMI, FBG,2 h PBG and HbA1c levels (P > 0.05). CONCLUSION: Lower frequency EA of BL 20, BL 23, ST 36 and SP 9 can reduce HbA1c and 2 h PBG levels in IGT patients, suggesting a helpful effect of EA in controlling the development of diabetes.

Prevalence and characterization of human Shigella infections in Henan Province, China, in 2006.

In 2006, 3,531 fecal samples were collected from patients with diarrhea in Henan Province, China. A total of 467 (13.2%) Shigella strains were isolated and serotyped. Seventy-one Shigella flexneri strains were characterized by MIC determination, pulsed-field gel electrophoresis (PFGE), and detection of genes encoding cephalosporin resistance. Most infections were caused by S. flexneri variant X [IV:(7),8] (27.6%), S. sonnei (24.2%), and S. flexneri 2a (20.8%). However, large regional differences were observed. Significantly higher odds (2.0) of females compared to males were infected with S. flexneri 2a. Untypeable S. flexneri (-:6) isolates were absent among males, as were untypeable S. flexneri [I:(7),8] isolates among females. Patient ages ranged from 2 months to 82 years, with 231 subjects (49.7%) <5 years of age. Most of the patients were male (62.1% [n = 290]). Infections peaked in July; week 27 with 38 cases (8.1%). All of the 71 S. flexneri conferred resistance to nalidixic acid; in addition, 21% (n = 15) and 79% (n = 56) were high- and low-level resistant to ciprofloxacin, respectively. Six S. flexneri isolates {serotype 2b [II:7,(8)] and 2b [II:(3),4;7,(8)]} harbored the bla(CTX-M-14) or bla(CTX-M-15) gene. A total of 52 unique XbaI PFGE patterns were observed among the 71 S. flexneri isolates with 11 distinct PFGE clusters. This study revealed a high prevalence of shigellosis with geographical differences in the distribution of serotypes in the distribution of serotypes and also differences in comparisons by gender. A high frequency of resistance, including 100% resistance to ciprofloxacin and resistance to extended-spectrum cephalosporins, was observed. We detected several isolates exhibiting the same PFGE type and MIC profile, indicating multiple undetected outbreaks.