Low prevalence of mcr-1 in Escherichia coli from food-producing animals and food products in China.

BACKGROUND: mcr-1-positive Escherichia coli has emerged as a significant threat to human health, veterinary health, and food safety in recent years. After the prohibition of colistin as a feed additive in animal husbandry in China, a noticeable reduction in both colistin resistance and the prevalence of mcr-1 was observed in E. coli from animals and humans. OBJECTIVES: To assess the prevalence of the colistin resistance gene mcr-1 and characterize its genetic context in E. coli strains derived from fecal and meat samples from food-producing animals in China. METHODS: A total of 1,353 fecal samples and 836 food samples were collected between 2019 and 2020 in China. E. coli isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their susceptibility to colistin were determined using the broth microdilution method. The colistin-resistant E. coli isolates were screened for the presence of mcr by PCR analysis and sequencing. The minimal inhibitory concentrations (MICs) of 15 antimicrobial agents against the mcr-1-positive strains were further tested using the agar dilution method, conjugation assays were performed, and whole genome sequencing was performed using Illumina HiSeq. RESULTS: In total, 1,403 E. coli strains were isolated. Thirteen isolates from chicken meat (n = 7), chickens (n = 3), and pigs (n = 3) were resistant to colistin with MIC values of 4 to 16 mg/L, and carried mcr-1. All mcr-1-positive strains, except for isolate AH20PE105, contained multiple resistance genes and exhibited multidrug-resistant phenotypes. They belonged to 10 sequence types (STs), including a novel ST (ST14521). mcr-1 was located on IncI2 (n = 9), IncX4 (n = 2), and IncHI2 (n = 2) plasmids, which were highly similar to other mcr-1-carrying plasmids sharing the same incompatibility type. Seven mcr-1-carrying plasmids could be successfully conjugally transferred to E. coli C600. CONCLUSIONS: While the low prevalence of mcr-1 (0.93%) identified in this study may not immediately seem alarming, the very emergence of this gene merits attention given its implications for colistin resistance and public health. Hence, ongoing surveillance of mcr-1 in E. coli remains crucial.

Low prevalence of mobilized resistance genes blaNDM, mcr-1, and tet(X4) in Escherichia coli from a hospital in China.

The emergence and spread of carbapenemase genes, colistin resistance genes mcr-1, and tigecycline resistance gene tet(X) represent a significant threat to clinical therapy and public health. In this study, we investigated the presence of carbapenemase genes, mcr-1, and tet(X) in 298 Escherichia coli strains obtained from a teaching hospital in China. In total, eight (2.68%), six (2.01%), and one (0.34%) E. coli isolates carried blaNDM, mcr-1, and tet(X4), respectively. The blaNDM gene was located on IncX3 (n = 4), F2:A-:B- (n = 3), and F2:A1:B1 (n = 1) plasmids, with high similarity to multiple plasmids belonging to the same incompatibility type from Enterobacteriaceae. Six MCR-producing strains contained mcr-1-carrying IncI2 plasmids, organized similarly to other mcr-1-bearing IncI2 plasmids from animals in China. The blaCTX-M-55/64/132/199 gene located within a typical transposition unit (ISEcp1-blaCTX-M-orf477Δ) was inserted near dnaJ to generate 5-bp direct repeats in four mcr-1-positive plasmids. The tet(X) and another four resistance genes [aadA2, tet(A), floR, and Δlnu(F)] were co-located on an IncX1 plasmid, highly similar to other tet(X4)-carrying IncX1 plasmids from Escherichia and Klebsiella of animal or food origin, except that the conjugative transfer region of IncX1 plasmids was absent in our plasmid. Although a low prevalence of blaNDM, mcr-1, and tet(X) was observed in E. coli from patients in this study, their dissemination associated with some successful pandemic plasmids is of great concern. The continued surveillance of these crucial resistance genes in patients should be strengthened.

Characterization of Extensively Drug-Resistant Salmonella enterica Serovar Kentucky Sequence Type 198 Isolates from Chicken Meat Products in Xuancheng, China.

The purpose of this study was to characterize extensively drug-resistant Salmonella enterica serovar Kentucky sequence type 198 (ST198) isolates from chicken meat products. Ten S. Kentucky strains obtained from chicken meat products in Xuancheng, China, carried 12 to 17 resistance genes, such as blaCTX-M-55, rmtB, tet(A), floR, and fosA3, combined with mutations within gyrA (S83F and D87N) and parC (S80I), resulting in resistance to numerous antimicrobial agents, including the clinically important antibiotics cephalosporin, ciprofloxacin, tigecycline, and fosfomycin. These S. Kentucky isolates shared a close phylogenetic relationship (21 to 36 single-nucleotide polymorphisms [SNPs]) and showed close genetic relatedness to two human clinical isolates from China. Three S. Kentucky strains were subjected to whole-genome sequencing using Pacific Biosciences (PacBio) single-molecule real-time (SMRT) technology. All antimicrobial resistance genes were located on their chromosomes and clustered in one multiresistance region (MRR) and Salmonella genomic island (SGI) SGI1-K. The MRRs in three S. Kentucky strains were bounded by IS26 at both ends and were inserted downstream of the bcfABCDEFG cluster with 8-bp direct repeats. The MRRs were related to those of IncHI2 plasmids but differed by insertions, deletions, and rearrangements of multiple segments involving resistance genes and plasmid backbones. This finding suggests that the MRR fragment possibly originates from IncHI2 plasmids. Four SGI1-K variants with slight differences were identified in 10 S. Kentucky strains. Mobile elements, particularly IS26, play an essential role in forming distinct MRRs and SGI1-K structures. In conclusion, the emergence of extensively drug-resistant S. Kentucky ST198 strains containing numerous chromosomally located resistance genes is alarming and needs continued surveillance. IMPORTANCE Salmonella spp. are important foodborne pathogens, and multidrug-resistant (MDR) Salmonella strains have become a serious threat to clinical therapy. MDR S. Kentucky ST198 strains have been increasingly reported from various sources and have become a global risk. In this study, we described extensively drug-resistant S. Kentucky ST198 strains from chicken meat products from a city in China. Numerous resistance genes are clustered in the chromosomes of S. Kentucky ST198 strains, possibly acquired with the help of mobile elements. This would facilitate the spread of numerous resistance genes as intrinsic chromosomal genes within this global epidemic clone, with the potential to capture more resistance genes. The emergence and dissemination of extensively drug-resistant S. Kentucky ST198 pose a severe clinical and public health threat; therefore, continuous surveillance is warranted.

Chromosomally and Plasmid-Located mcr in Salmonella from Animals and Food Products in China.

This study aimed to investigate the prevalence and genomic characteristics of the colistin resistance gene mcr in Salmonella enterica in China. In total, 445 S. enterica isolates from animals and food products were screened through PCR and sequencing for the presence of mcr. The mcr genes were detected in nine Salmonella strains (2.02%), with complete mcr-1 in S. enterica serovar Indiana (n = 1) and an S. Typhimurium monophasic variant (S. 4,[5],12:i:-; n = 1), mcr-4.3 in S. enterica serovar London (n = 1), and an incomplete mcr-1 in S. Indiana (n = 6). They exhibited MIC values of 0.25 to 8 mg/L to colistin and showed resistance to multiple antimicrobial agents. Whole-genome sequencing was performed on mcr-positive Salmonella strains using Illumina HiSeq or PacBio single-molecule real-time sequencing. The complete mcr-1 gene was located on conjugative IncN1-IncHI2 plasmid and IncX4 plasmid, respectively, with high similarity to other mcr-1-bearing plasmids belonging to the same incompatibility type. Together with an additional 13 antimicrobial resistance genes, the incomplete mcr-1 was embedded in an 81,442-bp multiresistance region on the chromosome in S. Indiana YZ20MCS6. The Δmcr-1-pap2 segment and a set of tellurite resistance determinants (terYXWZABCDEF) in six S. Indiana strains were similar to other IncHI2 plasmid backbones. The mcr-4.3 gene was located on an untyped plasmid pYULZMPS10. Although low prevalence of mcr was observed in Salmonella, continuous surveillance of this gene in Salmonella is required. Plasmids play an important role in mcr transmission, and mcr-1, although incomplete, can be captured by chromosomes with the help of mobile elements. IMPORTANCE Colistin is a last-resort antibiotic for severe infections caused by multidrug-resistant (MDR) Gram-negative pathogens. Colistin resistance genes mcr, particularly mcr-1, have been found in Enterobacteriaceae around the world, mainly in Escherichia coli and Salmonella. Salmonella enterica is a major foodborne pathogen, with MDR Salmonella being considered a "Serious Threat Level pathogen" by the Centers for Disease Control and Prevention. Therefore, the prevalence of mcr in Salmonella strains must be monitored. In this study, a low mcr prevalence (2.02%) was observed in Salmonella strains from animals and food products, with plasmid-borne mcr-1 in S. enterica serovar Indiana and an S. Typhimurium monophasic variant (S. 4,[5],12:i:-) and chromosomally located mcr-1 in S. Indiana. The mcr-4.3 gene was first identified in S. enterica serovar London associated with an untyped plasmid. Although this study reports a low mcr prevalence in Salmonella, the transmission ability of mcr-positive Salmonella strains to humans via the food chain is a public health concern.

Multiple Mechanisms of Tigecycline Resistance in Enterobacteriaceae from a Pig Farm, China.

We isolated eight tigecycline-resistant Enterobacteriaceae strains from a pig farm in Shanghai, China, including Escherichia coli (n = 1), Proteus cibarius (n = 1), and Enterobacter hormaechei (n = 6). Two of them (E. coli and P. cibarius) were positive for tet(X). E. coli SH19PTE6 contained an IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP6-tetX, highly similar to other tet(X)-bearing hybrid plasmids from E. coli in China. In P. cibarius SH19PTE4, tet(X) was located within a new chromosomal integrative and conjugative element (ICE), ICEPciChn2, belonging to the SXT/R391 ICE family. All tigecycline-resistant E. hormaechei isolates carried the tet(A) variant; cloning and transfer of this tet(A) variant into various hosts increased their MICs for tigecycline (4- to 8-fold). Tigecycline resistance observed on a pig farm is mediated by the tet(A) variant and tet(X) via a plasmid or ICE. The rational use of antibiotics such as doxycycline and surveillance of tigecycline resistance in livestock are warranted. IMPORTANCE As a last-resort antimicrobial agent to treat serious infections, the emergence and spread of tigecycline resistance in Enterobacteriaceae and Acinetobacter have raised global concerns. Multiple mechanisms mediate tigecycline resistance in Enterobacteriaceae, such as the monooxygenase Tet(X), mutations in Tet proteins, and overexpression of efflux pumps. Although tigecycline is not approved for animals, tigecycline resistance has been observed in Escherichia coli, Proteus cibarius, and Enterobacter hormaechei isolates on a pig farm, mediated by the tet(A) variant and tet(X) via a plasmid or ICE. The heavy use of tetracyclines such as doxycycline in food-producing animals in China may be the reason for the emergence and transmission of tigecycline resistance.

Chromosomally Located fosA7 in Salmonella Isolates From China.

This study aimed to investigate the prevalence of fosfomycin fosA7 in Salmonella enterica isolates from food animals and retail meat products in China and the impact of fosA7 on bacterial fitness. A total of 360 Salmonella isolates collected from 11 provinces and cities in China were detected for fosA7. All fosA7-positive Salmonella isolates were determined minimum inhibitory concentrations (MICs) and sequenced by Illumina Hiseq. The fosA7 gene of S. Derby isolate HA2-WA5 was knocked out. The full length of fosA7 was cloned into vector pBR322 and then transformed into various hosts. MICs of fosfomycin, growth curves, stability, and fitness of fosA7 were evaluated. The fosA7 gene was identified in S. Derby (ST40, n = 30) and S. Reading (ST1628, n = 5). MICs to fosfomycin of 35 fosA7-positive isolates were 1 to 32 mg/L. All fosA7 were located on chromosomes of Salmonella. The deletion of fosA7 in HA2-WA5 decreased fosfomycin MIC by 16-fold and slightly affected its fitness. The acquisition of plasmid-borne fosA7 enhanced MICs of fosfomycin in Salmonella (1,024-fold) and Escherichia coli (16-fold). The recombinant plasmid pBR322-fosA7 was stable in Salmonella Typhimurium, S. Pullorum, S. Derby, and E. coli, except for Salmonella Enteritidis, and barely affected on the growth of them but significantly increased biological fitness in Salmonella. The spread of specific Salmonella serovars such as S. Derby ST40 will facilitate the dissemination of fosA7. fosA7 can confer high-level fosfomycin resistance and enhance bacterial fitness in Salmonella if transferred on plasmids; thus, it has the potential to be a reservoir of the mobilized fosfomycin resistance gene.

Clinical observation on wheat grain moxibustion for chronic superficial gastritis of spleen-stomach deficiency and cold / 中国针灸

OBJECTIVE@#To compare the clinical effect of wheat grain moxibustion and warming acupuncture on chronic superficial gastritis of spleen-stomach deficiency and cold.@*METHODS@#A total of 200 patients with chronic superficial gastritis of spleen-stomach deficiency and cold were randomly divided into a wheat grain moxibustion group (100 cases, 13 cases dropped off ) and a warming acupuncture group (100 cases, 16 cases dropped off ). The acupoints of Zhongwan (CV 12), Tianshu (ST 25), Qihai (CV 6), Liangqiu (ST 34) and Zusanli (ST 36) were selected in both groups. The patients in the wheat grain moxibustion group were treated with wheat grain moxibustion, and 5 cone were given on every acupoint each time. Moxibustion was performed after acupuncture in the warming acupuncture group, and 3 cone each acupoint. Both groups were treated every other day for 15 times. The scores of clinical symptoms and signs, scores of physical component summary (PCS) and mental component summary (MCS) of quality of life in the two groups were recorded before and after treatment and during follow-up 1 month after treatment, and the clinical effects of the two groups were evaluated after treatment and during follow-up.@*RESULTS@#After treatment and during follow-up, the scores of clinical symptoms and signs of the two groups were lower than before treatment (@*CONCLUSION@#The wheat grain moxibustion can effectively improve the clinical symptoms, signs and quality of life in patients with chronic superficial gastritis of spleen-stomach deficiency and cold, and its long-term effect is better than warming acupuncture.

Screening Quorum-sensing-associated sRNA and construction of its overexpression and deletion mutant in Pseudomonas aeruginosa / 医学研究生学报

ObjectiveQuorum-sensing (QS) and small regulatory RNA (sRNA) play key regulatory roles in many signaling cascades of Pseudomonas aeruginosa. To investigate whether sRNA is involved in P. aeruginosa QS system, screening QS system-related sRNA, and to construct sRNA overexpression and deletion strains of Pseudomonas aeruginosa for further study of sRNA function.MethodsSRNA associated with the QS system was screened by qPCR and RNA-sequencing (RNA-seq). The target gene were amplified by PCR and inserted into the overexpression vector pROp200 or the homologous recombination vector pGSM-MR, respectively. The connection reaction solution of pROp200-sRNA and pGSM-ΔsRNA was transformed into Escherichia coli DH5a and SM10lp, respectively. The recombinant vectors were identified by PCR. The pROp200-sRNA was transformed into PAO1 by heat shock method, and the pGSM-ΔsRNA was transferred from SM10lp to PAO1 by conjugation. SRNA overexpression and deletion strains were identified by PCR, DNA sequencing and qPCR, the determination of the growth curves and the pyocyanin levels of strains.ResultsFive QS -associated sRNA P26, P5316.1, P30, P34 and AmiL were successfully screened by RNA-seq and qPCR. PCR, DNA sequencing and qPCR showed that sRNA of AmiL, P30 and P34 overexpression and knockout were successful. Compared with wild-type strain, sRNA overexpression and knockout had no significant effect on bacterial growth curve. It were notably that overexpression of AmiL and P30 inhibited and increase the production of pyocyanin, respectively (P0.05).ConclusionThe sRNA overexpression and deletion strains have been successfully constructed and can be used to study the regulatory relationship between sRNA and QS systems, and to further functional study.

Clinical Observations on Wheat-grain Size Cone Moxibustion plus Acupuncture for Mild to Moderate Carpal Tunnel Syndrome / 上海针灸杂志

Objective To investigate the effect of wheat-grain size cone moxibustion plus acupuncture on related high-frequency ultrasonographic indicators before and after its treatment of mild to moderate carpal tunnel syndrome and explore its mechanism of action. Method Sixty patients were randomized to an acupuncture group (20 cases) and a wheat-grain size cone moxibustion groups (40 cases). The acupuncture group received acupuncture at affected-side points Daling (PC7), Hegu (LI4), Neiguan (PC6) and Shousanli (LI10). The wheat-grain size cone moxibustion group received wheat-grain size cone moxibustion on point Daling and acupuncture at the other points. Treatment was given once every other day for a total of 10 times. The thickness of the transverse carpal ligament, the cross-sectional area of the median nerve at the level of the pisiform bone and the flattening ratio at the level of the hamate hook were measured by high frequency ultrasonography before and after treatment. The clinical therapeutic effect was evaluated using the SSS score. Result After treatment, the SSS score decreased in both groups compared with before (P<0.05) and was lower in the wheat-grain size cone moxibustion group than in the acupuncture group (P<0.05). The cure and marked efficacy rate was 71.1％ in the wheat-grain size cone moxibustion groupwhich was higher than 42.1％ in the acupuncture group (P<0.05). There were no statistically significant pre-/post-treatment differences in the thickness of the transverse carpal ligament and the flattening ratio at the level of the hamate hook in the two groups (P>0.05). After treatment, the cross-sectional area of the median nerve at the level of the pisiform bone decreased in both groups compared with before (P<0.05) and was smaller in the wheat-grain size cone moxibustion group than in the acupuncture group (P<0.05). Conclusion Wheat-grain size cone moxibustion plus acupuncture can markedly improve the clinical symptoms of carpal tunnel syndrome. Its mechanism of action is related to reducing the cross-sectional area and edema of the median nerve.

Association between cord blood IgE and toddler atopic diseases / 中华检验医学杂志

Objective To investigate the correlation between cord blood IgE level and allergic diseases (atopic dermatitis, food allergy, wheezing and allergic rhinitis) in 36 months old children and explore the susceptibility factors of allergy. Methods A cohort study was designed and Cord blood was collected during delivery of 779 women with full term, and the IgE level of the sample was detected by chemiluminescence immunoassay in Department of Laboratory Medicine, International Peace Maternity and Child Health Hospital, Shanghai Jiao Tong University School of Medicine between October 1, 2012 and May 31, 2014. 638 children were followed up at the age of 36 months. The incidence of atopic dermatitis, food allergy, wheezing and allergic rhinitis were investigated by questionnaire, and the relationship between cord blood IgE level and allergic diseases in children was analyzed by multiple logistic regression. Results A total of 638 pregnant women and children were included in this study. The age of pregnant women was (29.7± 3.3) years, and the IgE level in cord blood ranged from 0.1 to 8.43 IU/mL.In caesarean women, higher cord blood IgE was associated with increased risk of allergic dermatitis, wheezing and allergic rhinitis in children, OR=1.87, P<0.01;OR=1.86, P=0.01;OR=1.28, P=0.01;OR=1.98, P=0.01. Conclusion During cesarean section, the elevated IgE level of umbilical cord blood is correlated with the incidence of allergic diseases in children.