MALDI-TOF MS combined with AUC method for tigecycline susceptibility testing in Escherichia coli.

Objectives: The wide spread of tet(X4) gene orthologues in the environment, food, poultry and humans is causing serious tigecycline resistance. Consequently, developing a fast and universal method to detect tigecycline resistance has become increasingly important. Methods: During 2019-2022, 116 Escherichia coli isolates were obtained from nine provinces in China. All isolates were tested for their susceptibility to antimicrobial agents by the microdilution broth method and for the tet(X4) gene by PCR. Ten tet(X4)-positive E. coli isolates were used to confirm certain conditions, including the optimal incubation time, the optimal concentration of tigecycline, and the cut-off of the relative growth (RG) value. Results: The optimal time and concentration of tigecycline for separation of susceptible and resistant isolates was 2 h and 4 mg/L, and the RG cut-off value was 0.4. We validated whether the experiment was feasible using 116 isolates of E. coli. The method yielded a susceptibility of 94.9% (95% CI: 81.4%-99.1%) and a specificity of 96.1% (95% CI: 88.3%-99.0%). Conclusions: This research has shown that this optical antimicrobial susceptibility testing method can rapidly differentiate between susceptible and resistant phenotypes in isolates of E. coli. In the same range as the current gold-standard methods, the clinical turnaround time is reduced from 48 h to 2.5 h. The above results suggest that the method has splendid specificity and operationality.

Effective Motion Self-Learning Genre Using 360° Virtual Reality Content on Mobile Device: A Study Based on Taichi Training Platform.

Online fitness training, with its affordability and flexibility, offers a convenient way for individuals to engage in regular workouts that promote physical and mental health. Yet, learning fitness motions in this way presents various challenges and may not always be as effective as in-person training. To address the practical demands of motion learning, we conducted a systematic survey and accordingly proposed a four-stage self-learning genre that integrates immersive virtual reality (VR) environments with motion skill learning theories, strategies, and expert experience. Herein, we merged progressive structures and multi-level visual cues to enhance instruction, and proposed a fine-grained motion analysis method to provide adaptive correction feedback during training. Utilizing a Taichi training platform with the genre embedded, we systematically validated the effectiveness of the genre, and examined the potential impact of VR content presentation form on motion learning among different age groups, as well as their preferences and focus on VR fitness training genre design. Results from the quantitative analysis, qualitative evaluation, and case study showed that the 360° video-based VR content brought more positive motion learning performance and user experiences than the fully-simulated VR used in many previous studies. The proposed genre demonstrated outstandingly performance, experience, and usability, with each stage and design playing an effective role. Moreover, we offer several design considerations for VR fitness systems targeting diverse age groups, providing beneficial insights for VR development in the sports and health-related fields.

Phenotypic and genomic characteristics of clinical IMP-producing Klebsiella spp. Isolates in China.

BACKGROUND: IMP-producing Klebsiella spp. (IMPKsp) strains have spread globally, including in China. Currently, the prevalence and genomic characterization of IMPKsp is largely unknown nationwide. Here we aimed to provide a general overview of the phenotypic and genomic characteristics of IMPKsp strains. METHODS: 61 IMPKsp strains were obtained from 13 provinces in China during 2016-2021. All strains were tested for their susceptibility to antimicrobial agents by the microdilution broth method and sequenced with Illumina next-generation sequencing. We performed conjugation experiments on thirteen representative strains which were also sequenced by Oxford nanopore sequencing technology to characterize blaIMP-encoding plasmids. RESULTS: We find that all IMPKsp strains display multidrug-resistant (MDR) phenotypes. All strains belong to 27 different STs. ST307 emerges as a principal IMP-producing sublineage. blaIMP-4 is found to be the major isoform, followed by blaIMP-38. Seven incompatibility types of blaIMP-encoding plasmids are identified, including IncHI5 (32/61, 52.5%), IncN-IncR (10/61, 16.4%), IncFIB(K)-HI1B (7/61, 11.5%), IncN (5/61, 8.2%), IncN-IncFII (2/61, 3.3%), IncFII (1/61, 1.6%) and IncP (1/61, 1.6%). The strains carrying IncHI5 and IncN plasmids belong to diverse ST types, indicating that these two plasmids may play an important role in the transmission of blaIMP genes among Klebsiella spp. strains. CONCLUSIONS: Our results highlight that multi-clonal transmission, multiple genetic environments and plasmid types play a major role in the dissemination process of blaIMP genes among Klebsiella spp. IncHI5 type plasmids have the potential to be the main vectors mediating the spread of the blaIMP genes in Klebsiella spp.

Antibiotic resistance occurs when bacteria evolve to withstand antibiotic drugs. We are aware that a bacteria called Klebsiella is rapidly becoming resistant to carbapenems, a class of broad-spectrum antibiotics. In this study, we conducted a genetic and microbiological surveillance study across 13 provinces of China to understand factors that contribute to the growing bacterial drug resistance. We find that the way the multiple bacterial types interact with each other and swap certain genetic material may be the main cause of growing resistance. These findings call for close monitoring of genetic evolution as a matter of public health management strategy.

Escherichia coli high-risk clone ST410 harboring blaNDM-13 isolated from hospital wastewater in China.

A carbapenem-resistant Escherichia coli strain C-SRM-3 was isolated from hospital wastewater effluent in Hangzhou city, China in March 2022. Analysis of the whole genome sequence showed that this blaNDM-13-positive strain belonged to an internationally recognized high-risk clone ST410 responsible for the dissemination of carbapenem resistance in E. coli. This isolate displayed a multidrug-resistant phenotype and carried a cassette of antibiotic-resistant genes. blaNDM-13 gene was successfully transferred to the recipient E. coli C600 via conjugation. WGS results revealed that blaNDM-13 gene was located on an IncI1 type plasmid replicon. The phylogenetic reconstruction showed that wastewater-sourced C-SRM-3 strain was located in a single branch, far removed from human-derived and animal-sourced isolates. The detection of blaNDM-13 in hospital wastewater suggests that continuous monitoring of antibiotic-resistant genes in the environment is critical for the prevention of carbapenem-resistant bacteria spreading.

Emergence of the clinical rdar morphotype carbapenem-resistant and hypervirulent Klebsiella pneumoniae with enhanced adaption to hospital environment.

Klebsiella pneumoniae has evolved into strains of various phenotypes that pose a grave threat to human health in the past few decades. This study investigated a novel morphotype of K. pneumoniae with enhanced adaption to the hospital environment. Clinical K. pneumoniae were characterized by different genotypic and phenotypic tests. Gene knockout and complementation experiments were used to confirm the genetic changes that led to the morphological changes. ST15 carbapenem-resistant and hypervirulent (CR-hvKP) clinical strains with the "red, dry and rough" (rdar) morphotype were increasingly detected in hospitals in China. Strains with the rdar phenotype were found to be less virulent compared with that with typical morphologies but exhibit enhanced ability to adhere to the surface of various materials, and hence a dramatically increased rate of survival on various materials commonly found in the hospital environment. Comparative genomics analysis and gene function studies suggested the rdar morphotype was due to a G579D substitution in the BcsA protein which enabled the strain to produce a large amount of cellulose. These findings show evolutional phenotypic change enables K. pneumoniae strains to better survive both in human and hospital environments, facilitating its persistence and further dissemination.

Phenotypic and Genomic Characterization of ST133 Siderophore-Encoding Extensively Drug-Resistant Enterobacter hormaechei.

We identified an ST133 extensively drug-resistant Enterobacter hormaechei, C210017, with increased virulence in the Galleria mellonella infection model. Genomic analysis suggested it carried antibiotic resistance genes blaKPC-2 and mcr-9.1, and genes iutAiucABCD and iroBCDEN encoding the virulence factor, siderophores. Comparative genomics of C210017 and the 178 ST133 E. hormaechei strains in the database suggested they all belonged to serotype O3 and most strains (77.5%) carried the IncHI2 superplasmids associated with the resistance, virulence, and adaptation of the host strain.

Population genomic analysis reveals the emergence of high-risk carbapenem-resistant Escherichia coli among ICU patients in China.

OBJECTIVES: The increasing incidence of carbapenem-resistant Enterobacterales (CRE) mediated nosocomial infections has caused a significant public health burden globally. Currently, the prevalence and genomic characteristics of carbapenem-resistant Escherichia coli (CREC) in patients admitted to the intensive care unit (ICU) are unknown. METHODS: Herein, we present a nationwide genomic investigation of CREC isolates among ICU patients in China in 2018 and 2020. In total, 113 CREC isolates were identified from 1105 samples in 25 hospitals, and investigated with phenotyping and genomics approaches. RESULTS: Carbapenemases were produced in 94.69% (107/113) of CREC isolates, which comprise KPC-2 (n = 53, 49.53%), NDM (n = 51, 47.66%), IMP-4 (n = 2, 1.87%), and OXA-181 (n = 1, 0.93%). Notably, CREC isolates co-carrying mcr-9 and blaNDM-5 or tet(X4) and blaNDM-5 were first identified in clinical settings. The carbapenemase genes of most isolates were located on the plasmids. The blaKPC gene was mainly mediated by IncFII plasmids (n = 37, 69.81%), and blaNDM was located on the IncX3 plasmid (n = 36, 70.59%). CREC isolates belonged to diverse sequence types (STs) of which ST131 was the most prevalent blaKPC-positive CREC isolates (34/113, 30.09%), while blaNDM was associated with ST617 and ST410 isolates, thereby indicating that multiple CREC clones spread in Chinese ICU patients. CONCLUSIONS: This study highlights the emerging threat of high-risk CREC isolates such as ST131 circulating in the ICU in China. Hence, stringent monitoring of such high-risk clones should be performed.

Epidemiology and Molecular Characteristics of mcr-9 in Citrobacter spp. from Healthy Individuals and Patients in China.

With the globally prevailing carbapenemase-producing (CP) Citrobacter spp., polymyxin antibiotics have been reconsidered as one of the last-resort treatment options. Our study was conducted to investigate the prevalence of mcr-9 in Citrobacter species. From October to November 2021, 650 fecal samples and 215 Citrobacter isolates were collected from healthy individuals and infected patients, respectively. Isolates were screened for the presence of the mcr-9 gene by the PCR method. mcr-9-carrying strains were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Due to the susceptibility to colistin, Citrobacter spp. isolates were first induced to increase the expression of mcr-9 on China blue agar plates containing colistin and were then subjected to conjugation experiments. Whole-genome sequencing was performed on the Illumina NovaSeq PE150 system. The prevalence of mcr-9 in the Citrobacter genus from healthy guts and infected patients was 0.62% and 1.86%, respectively. In all mcr-9-positive strains, MICs of polymyxin B were observed at ≤2 µg/mL, displaying a nonresistant phenotype. As for conjugation experiments, only one isolate successfully transferred the mcr-9 gene to Escherichia coli C600. Whole-genome sequencing showed that eight mcr-9-positive Citrobacter isolates carried mcr-9 and genes encoding resistance to beta-lactam antibiotics, including blaCMY, blaDHA, blaSHV, blaTEM, and blaCTX-M. We also discovered that mcr-9 could be located on the pKPC-CAV1321 plasmid. Our study investigated the prevalence of mcr-9 in Citrobacter spp. in both healthy individuals and infected patients and described the carriage of mcr-9 on the pKPC-CAV1321 plasmid for the first time. IMPORTANCE The emergence of mcr homologues posed a serious threat to the therapeutic efficiency of polymyxin antibiotics. Citrobacter freundii is generally regarded as an opportunistic pathogen associated with a variety of nosocomial infections. In this study, we investigated the prevalence of mcr-9 in Citrobacter spp. isolates from healthy individuals and infected patients and highlighted the importance of the rational use of antibiotics. In addition, this epidemiological investigation is the first to describe the carriage of mcr-9 on plasmid pKPC-CAV1321 and confirms the horizontal transfer of this plasmid. Our research may shed new light on further studies of mcr-9 dissemination in humans.

Emergence of an Extensive Drug Resistant Pseudomonas aeruginosa Strain of Chicken Origin Carrying blaIMP-45, tet(X6), and tmexCD3-toprJ3 on an IncpRBL16 Plasmid.

This study reports an extensively drug resistant Pseudomonas aeruginosa strain PA166-2 which was of chicken origin and carrying blaIMP-45, tet(X6) and tmexCD3-toprJ3 on a single plasmid. The strain was characterized by antimicrobial susceptibility testing, resistance gene screening, conjugation assay, whole-genome sequencing, and bioinformatics analysis. Strain PA166-2 was resistant to tigecycline and carbapenems. It belonged to ST313 and carried a plasmid pPA166-2-MDR, which belongs to the incompatibility group IncpRBL16. pPA166-2-MDR harbored a 78 Kb multidrug resistance (MDR) region carrying an array of antimicrobial resistance genes, including blaIMP-45, tet(X6), and tmexCD3-toprJ3. The gene blaIMP-45 was inserted into the backbone of plasmid pPA166-2-MDR within a class 1 integron, In786. tmexCD3-toprJ3 in plasmid pPA166-2-MDR was inserted in umuC, constituting the genetic context of ISCfr1-tnfxB3-tmexC3-tmexD3-toprJ3-△umuC. The genetic context of tet(X6) in this plasmid was identical to that of other reported plasmid-borne tet(X) variants, namely, tet(X6)-abh-guaA-ISVsa3. To the best of our knowledge, this is the first report of the cooccurrence of blaIMP-45, tet(X6), and tmexCD3-toprJ3 in one plasmid in Pseudomonas sp. The emergence of plasmid-mediated tigecycline resistance genes tmexCD3-toprJ3 and tet(X6), as well as carbapenemase genes from chickens expanded the global transmission of vital resistance genes. Findings from us and from others indicate that plasmids of the incompatibility group IncpRBL16 may serve as a reservoir for carbapenem and tigecycline resistance determinants. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes infections that are difficult to treat. This study reported, for the first time, the occurrence of last-resort antibiotic resistance determinants blaIMP-45, tet(X6), and tmexCD3-toprJ3 on a single plasmid in P. aeruginosa from chickens. The P. aeruginosa strain belonged to ST313 and was resistant to last-line antibiotics, namely, carbapenems and tigecycline. The plasmid carrying the last-line resistance genes belonged to the incompatibility group IncpRBL16, which was reported to contain different profiles of accessory modules and thus carried diverse collections of resistance genes. The emergence of plasmid-mediated tigecycline resistance genes tmexCD3-toprJ3 and tet(X6), as well as carbapenemase genes, from chickens expanded the global transmission of vital resistance genes. The results in this study highlighted that IncpRBL16 plasmids may serve as a reservoir for the dissemination of resistance genes. Control measures should be implemented to prevent the further dissemination of such strains.

Distribution and spread of the mobilised RND efflux pump gene cluster tmexCD-toprJ in clinical Gram-negative bacteria: a molecular epidemiological study.

BACKGROUND: TMexCD1-TOprJ1, which is associated with phenotypic resistance to multiple classes of antibiotics, is a transmissible resistance-nodulation-division (RND) family efflux pump. However, the prevalence and genomic and phenotypic characteristics of clinical isolates with this important resistance determinant are poorly understood. In this study, we aimed to survey tmexCD-toprJ among clinical Gram-negative isolates collected from hospitals in China between 1991 and 2020 and characterise tmexCD-toprJ-positive clinical isolates. METHODS: We conducted online data retrieval and active nationwide surveillance in China to screen tmexCD-toprJ-positive strains. We characterised tmexCD-toprJ-positive clinical strains for their antimicrobial susceptibility, genetic and functional characteristics, and the potential inter-species transmission route of tmexCD-toprJ with whole genome sequencing and bioinformatics analyses. The function of tmexCD-toprJ in Pseudomonas sp and Proteus sp was investigated by tmexD gene knockdown using an isopropylthio-ß-galactoside-inducible CRISPR interference system. FINDINGS: Data retrieval obtained 53 strains carrying tmexCD-toprJ, comprising 32 Pseudomonas spp, 11 Klebsiella pneumoniae, one Aeromonas spp, one Citrobacter freundii, and one uncultured bacterium from diverse niches. 48 (0·64%) of 7517 clinical isolates from China, including seven Klebsiella spp, one Proteus mirabilis, and 40 Pseudomonas spp, carried tmexCD-toprJ. These isolates exhibited multidrug resistance phenotypes and co-harboured resistance genes, such as mcr and carbapenemases genes. tmexCD-toprJ was encoded on both plasmids and chromosomes in all Klebsiella spp that carried plasmid-borne tmexCD-toprJ (n=7), P mirabilis carried chromosome-borne tmexCD-toprJ, and Pseudomonas spp carried either plasmid-borne (n=19) or chromosome-borne (n=21) ones. tmexCD-toprJ had undergone clonal and horizontal transmission among clinical pathogens. Eight different types of genetic context of tmexCD-toprJ were identified, each of which was associated with different mobile elements, including IntI, IS6100, TnAs1-like, ISRor5, ISVsa3, ISCfr-like, Tn5393, and IS222-like, which might facilitate its transmission. Knockdown of tmexD led to a four times decrease in tigecycline minimum inhibitory concentrations in both Pseudomonas spp and Proteus spp. INTERPRETATION: Our study provides evidence to suggest that tmexCD-toprJ contributes to the antimicrobial resistance phenotypes in different bacterial species. tmexCD-toprJ has disseminated among diverse species of clinical pathogens, which warrants timely monitoring in clinical pathogens. FUNDING: National Natural Science Foundation of China, Guangdong Major Project of Basic and Applied Basic Research, Natural Science Foundation of Jiangsu Province.

The Rapid Emergence of Ceftazidime-Avibactam Resistance Mediated by KPC Variants in Carbapenem-Resistant Klebsiella pneumoniae in Zhejiang Province, China.

Ceftazidime-avibactam (CAV) is a new treatment option against carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. However, the rapid emergence of CAV resistance mediated by KPC variants has posed a severe threat to healthcare after its clinical application. The characteristics of CAV resistance in CRKP strains needs to be determined in China. A total of 477 CRKP isolates were collected from 46 hospitals in Zhejiang Province from 2018 to 2021. The results demonstrated that CAV had a potent activity against 94.5% of all CRKP (451/477, 95% CI: 93.0-96.1%) and 86.0% of CRKP strains carrying blaKPC genes (410/477, 95% CI: 83.5-88.4%). A total of 26 CAV-resistant strains were found. Among these strains, sixteen harbored metallo-ß lactamases, and two carried KPC-2 carbapenemase and mutated ompK35 and ompK36. Eight CRKP strains encoded KPC-33 or KPC-93, belonging to ST11, among which seven strains were detected in patients hospitalized in 2021 after exposure to CAV and one strain was associated with intra-hospital spread. CAV is a potent agent in vitro against CRKP strains. The rapid development of CAV resistance mediated by various KPC variants after a short period of CAV treatment has increased and brought difficulties in treating infections caused by CRKP strains, especially those belonging to ST11. The surveillance of bacterial resistance against CAV is highly recommended due to the steep development of CAV resistance and rapid evolution of KPC enzymes.

Co-transfer of last-line antibiotic resistance and virulence operons by an IncFIBk-FII-X3-ColKP3 hybrid plasmid in Klebsiella pneumoniae.

OBJECTIVES: To characterize a clinical Klebsiella pneumoniae isolate from China co-harbouring tet(X4), blaOXA-181 and the aerobactin operon on an IncFIBk-FII-X3-ColKP3 hybrid plasmid. METHODS: A tigecycline-resistant strain was recovered from the intestinal sample of a patient. It was subjected to antimicrobial susceptibility testing, conjugation assay, virulence testing, WGS, bioinformatics analysis, plasmid stability testing and fitness cost testing. RESULTS: The strain K. pneumoniae T877 was resistant to tigecycline, intermediate to piperacillin/tazobactam and ertapenem, and positive for tet(X), blaOXA-181 and the virulence-associated operon iutAiucABCD, which were located on the same plasmid, named pKPT877-hybrid. It was 99.96% identical to the IncFIBk-FII plasmid pSCH6109-Vir (accession number CP050860) from K. pneumoniae strain SCH6109 at 96% coverage with the absence of a 50 kb region on pKPT877-hybrid; this region was highly homologous to the 51 kb IncX3-ColKP3-type, blaOXA-181-carrying plasmid pOXA181-191773 (accession number CP080367). Plasmid pKPT877-hybrid was conjugatively transferable to the ST11 K. pneumoniae strains FJ8 and KP04. pKPT877-hybrid did not have a significant impact on the fitness cost and could be maintained stably in T877. CONCLUSIONS: We report for the first time (to the best of our knowledge) the co-transfer of last-line antibiotic resistance determinants [tet(X4) and blaOXA-181] and the aerobactin operon (iutAiucABCD) by a mobile IncFIBk-FII-X3-ColKP3 hybrid plasmid, which can be stably maintained in K. pneumoniae strains, even in the absence of antibiotic selective pressure. Once the plasmid transfers to a K. pneumoniae with porin deficiency, the strain might have high levels of resistance to carbapenems and tigecycline, which are the last line of defence against infections. Heightened and continuous efforts are needed to control its dissemination.

Emergence of an ST1326 (CG258) Multi-Drug Resistant Klebsiella pneumoniae Co-harboring mcr-8.2, ESBL Genes, and the Resistance-Nodulation-Division Efflux Pump Gene Cluster tmexCD1-toprJ1 in China.

CG258 is the dominant carbapenemase-producing Klebsiella pneumoniae clone worldwide and treatment of infections caused by this clone relies largely on the last-line antibiotics, colistin, and tigecycline. However, the emergence and global dissemination of mcr and tmexCD1-toprJ1 genes have significantly compromised their clinical applications. CG258 K. pneumoniae carrying both mcr and tmexCD1-toprJ1 have not been reported. A colistin-resistant strain T698-1 belonging to ST1326, a member of CG258, was isolated from the intestinal sample of a patient and characterized by the antimicrobial susceptibility testing, conjugation assay, WGS and bioinformatics analysis. It was resistant to colistin, tetracycline, aminoglycoside, fluoroqinolone, phenicols, sulfonamide, and some ß-lactams, and positive for mcr-8.2, tmexCD1-toprJ1, and ESBL genes (bla DHA-1 and bla CTX-M-15). The tmexCD1-toprJ1 gene cluster was located in an multi-drug resistant (MDR) region flanked by TnAs1 elements on an IncHI1B/FIB plasmid. The genetic context of tmexCD1-toprJ1 was slightly distinct from previously reported Tn5393-like structures, with an IS26 element disrupting the upstream Tn5393 and its adjacent genetic elements. The mcr-8.2 gene was inserted into the backbone of an IncFII/FIA plasmid with the genetic context of ISEcl1-mcr-8.2-orf-ISKpn26. To our knowledge, this is the first report of co-occurrence of mcr-8.2 and tmexCD1-toprJ1 in a CG258 K. pneumoniae strain. Though this strain is tigecycline sensitive, the acquisition of colistin and tigecycline resistance determinants by the endemic CG258 K. pneumoniae clone still poses a serious public health concern. CG258, which became resistant to multiple last resort antibiotics, would be the next emerging superbug.

Visuospatial Bias in Children with Autism Spectrum Disorder: Evidence from Line Bisection Tasks.

Previous studies have found reduced leftward bias of facial processing in individuals with Autism Spectrum Disorder (ASD). However, it is not clear whether they manifest a leftward bias in general visual processing. To shed light on this issue, the current study used the manual line bisection task to assess children 5 to 15 years of age with ASD as well as typically developing (TD) children. Results showed that children with ASD, similar to TD children, demonstrate a leftward bias in general visual processing, especially for bisecting long lines (≧ 80 mm). In both groups, participant performance in line bisection was affected by the hand used, the length of the line, the cueing symbol, and the location of the symbol. The ASD group showed a rightward bias when bisecting short lines (30 mm) with their left hands, which slightly differed from the TD group. These results indicate that while ASD individuals and TD individuals share a similar leftward bias in general visual processing, when using their left hands to bisect short lines, ASD individuals may show an atypical bias pattern.

Prevalence, transmission, and molecular epidemiology of tet(X)-positive bacteria among humans, animals, and environmental niches in China: An epidemiological, and genomic-based study.

Plasmid-mediated, transmissible, tigecycline-inactivating enzyme Tet(X) has attracted considerable public attention. However, so far studies have not addressed its impact on public health and the ecosystem. Herein, we report the prevalence and molecular epidemiology of tet(X)-positive bacteria (TPB) from diverse sources, investigate the host-specificity of TPB and the transferability of tet(X). Sample collection was conducted between 2018 and 2020 in 30 provinces in China. PCR screening suggested tet(X) was prevalent among freshwater fishes (24.7%, 95% CI 19.4-30.7%), followed by chickens (23.6%, 21.2-26.2%), cattle (19.3%, 16.4-22.5%), healthy individuals (6.2%, 5.4-7.1%), and patients (0.3%, 0.0-1.1%). Soil and freshwater samples all tested negative for tet(X). A total of 289 TPB were isolated from 7516 samples (120/1181 chicken, 82/669 cattle, 68/3229 healthy individual, 17/239 freshwater fish and 2/2121 clinical samples). TPB distributed in six major families of bacteria including Moraxellaceae (n = 99, 34.3%), Flavobacteriaceae (n = 95, 32.9%), Enterobacteriaceae (n = 83, 28.7%), Pseudomonadaceae (n = 9, 3.1%), Sphingobacteriaceae (n = 2, 0.7%) and unclassified Gammaproteobacteria (n = 1, 0.3%). Diverse tet(X) genes including tet(X2), tet(X3), tet(X4), tet(X5) and tet(X6) were identified from different TPB. The tet(X)-positive bacteria were highly diverse, with ST10 complex belonging to the dominant E. coli clone. Novel hosts of tet(X) including Enterobacter hormaechei, Ignatzschineria indica and Oblitimonas alkaliphila were identified. Isolates from different families exhibited different antimicrobial resistance profiles. Co-existence of tet(X) with other resistance genes such as floR (66.8%) and carbapenemase genes (33.2%) was commonly observed. tet(X) could be transferred among E. coli isolates at frequencies from 10-4 to 10-10. Species other than E. coli failed to transfer tet(X) gene to the E. coli recipient via conjugation. Discriminant analysis of principal components analysis suggested inter-host transmission of tet(X)-positive E. coli among diverse hosts was not observed. Future studies are needed to monitor the transmission trend as well as the impact of this resistance gene in clinical infection control.

A rapid MALDI-TOF mass spectrometry-based method for colistin susceptibility testing in Escherichia coli.

Colistin is recognized as a last-resort treatment option against multi-drug resistant bacteria including carbapenem-resistant Enterobacteriaceae (CRE). However, the plasmid-mediated colistin-resistance gene mcr-1 has been reported globally resulting in an increase of colistin-resistant bacteria. A quick and accurate method for determining the pathogen resistance of colistin is therefore crucial in the clinic. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a potential tool forto be applied for antimicrobial susceptibility testing. We compared the growth of Escherichia coli strains in the presence or absence of colistin. Automated analyses of the spectra were performed with a prototype software tool written with package R. Three mcr-1-positive and six mcr-1-negative E. coli were used for establishing the model to obtain the optimal incubation time, the breakpoint concentration of colistin and cut-off of the relative growth (RG) value. The distinction between susceptible and resistant strains was already noticeable after 2 h of incubation. The best separation between the susceptible and resistant strains was achieved at a concentration of 4 µg ml-1 and a relative growth cut-off value of 0.6. Application of the model for the analysis of 128 E. coli isolates, a sensitivity of 97.4% and a specificity of 88.2% were achieved compared with colistin MIC results. The rapid MALDI-TOF MS-based method approach is simple to set-up, uses a short incubation time, and had excellent outcomes with respect to sensitivity and specificity for colistin sensitivity testing in Escherichia coli.

Development of an In-House Rapid Antimicrobial Susceptibility Testing Protocol for Positive Blood Culture and Its Implementation in Routine Microbiology Laboratories.

Bloodstream infections (BSI) are associated with high morbidity and mortality and remain a leading cause of death. Blood culture (BC) including the identification and the antimicrobial susceptibility testing of the causative microorganisms should be performed as soon as possible. In this study, we developed an in-house rapid antimicrobial susceptibility testing (rAST) protocol for positive BC. First, the rAST was performed in the simulated positive BC of standard strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853) at three different times to assess the reproducibility and operability by dispensing four drops of BC broth onto a Mueller-Hinton agar plate after a positive signal. Furthermore, the rAST was performed in clinical positive BCs. The results of rAST at 4, 6, 8, and 18 h of incubation were compared with results of the standard 16- to 20-h disk diffusion method, and the preliminary breakpoints of the rAST method were established according to the inhibition diameter of sensitive strains and resistant strains. Finally, the rAST was performed in the simulated positive BC of clinical strains to evaluate the availability of the preliminary breakpoints. The rAST results of standard strains were distributed evenly at three different times. Among the 202 clinical strains used to establish the preliminary breakpoints, the number of zone diameters that could be read and interpreted (60, 87, 98, and 100%) increased with incubation time (4, 6, 8, and 18 h), and the categorical agreement was acceptable, with total error rates of 3.0, 2.3, 2.1, and 1.3% at 4, 6, 8, and 18 h of incubation, respectively. In conclusion, the in-house rAST protocol for positive BC can be implemented in routine laboratories. It provides reliable antimicrobial susceptibility testing results for BSI pathogens after 4-6 h of incubation.

How cognitive conflict affects judgments of learning: Evaluating the contributions of processing fluency and metamemory beliefs.

Previous research has documented that cognitive conflict affects basic cognitive processes such as memory, reasoning, and attention allocation. However, little research has explored whether its effect can be extended to higher cognitive processes such as metacognitive monitoring. The current study took a novel variant of a Stroop task that employed words presented in a color opposite to the color of the object itself (e.g., heart, presented in green) or same as the color of the object (e.g., forest, presented in green) as targets, an important form of metacognitive monitoring-judgments of learning (JOLs) was used as the measurement index to investigate the influence of cognitive conflict on metacognitive monitoring and to delineate the potential mechanisms underlying the cognitive conflict effect on JOLs. In Experiment 1, results showed that participants gave higher JOLs to consistent than to conflict words, even though cognitive conflict had little influence on memory recall. Experiment 2, employing a self-paced study task, found that conflict words were processed less rapidly than consistent ones, and the difference in processing fluency significantly mediated the cognitive conflict effect on JOLs. Experiment 3 employed an observer-learner task; the mediation analysis showed a complete mediation role of metamemory beliefs (observation JOLs) in the relationship between word type and JOLs. In Experiment 4, research results suggested that participants' beliefs about processing fluency played an important role in the cognitive conflict effect. To conclude, cognitive conflict is a reliable factor affecting higher cognitive processes (metamemory monitoring). Both processing fluency and metamemory beliefs tend to contribute to the cognitive conflict effect.

A method for screening tigecycline-resistant gene tet(X) from human gut.

OBJECTIVES: To develop an effective enrichment method for tet(X) detection, we performed PCR and Sanger sequencing to screen and confirm the presence of tet(X) gene. METHODS: Species were identified by MALDI-TOF MS analysis. The minimum inhibitory concentrations (MICs) of common antibiotics were determined by broth microdilution and interpreted according to the CLSI guidelines and EUCAST breakpoints. RESULTS: We obtained 29 (2.26%, 29/1284) tet(X4)-positive Escherichia coli, and 96.6% of those (28 isolates) exhibited resistance to tigecycline. CONCLUSION: This specific screening strategy for functional tet(X) mediating tigecycline resistance will be useful to facilitate development and advancement of our knowledge of tet(X).

Prevalence and mechanisms of fosfomycin resistance among KPC-producing Klebsiella pneumoniae clinical isolates in China.

The threat of antibiotic resistance has increased dramatically in recent years. Fosfomycin, an old antibiotic agent, has been re-introduced to fight infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP). However, the trend of fosfomycin resistance among KPC-KP strains is increasing. In this study, 80 KPC-KP clinical isolates were collected from three teaching hospitals during 2014-2017 in China and were subjected to whole-genome sequencing (WGS). The fosfomycin resistance phenotype and resistance mechanisms were investigated by antimicrobial susceptibility testing and carbon source growth test, respectively. Among all KPC-KP strains, 80.0% (64/80) were resistant to fosfomycin and 36.3% (29/80) were positive for the mobile fosfomycin resistance gene fosA3. Among the 63 strains that were unable to grow in M9 basic medium with glycerol-3-phosphate (G3P) as the sole carbon source (mediated by mutation of the target gene glpT), there was no significant difference regarding the MIC distribution of fosfomycin between fosA3-positive and fosA3-negative strains (P = 0.577). Among the 50 strains that were negative for fosA3 but positive for fosA, the fosfomycin MICs of strains unable to grow in M9 basic medium with G3P as the sole carbon source were significantly higher (P < 0.001) than in strains that were able to grow in M9 basic medium with G3P as the sole carbon source. Our findings indicate that fosfomycin resistance among KPC-KP in China is an emerging problem and the two major mechanisms of resistance identified were plasmid-mediated fosfomycin resistance gene fosA3 and mutation of the target gene glpT.