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Carbapenemase-producing Enterobacteriaceae (CPE) isolates are recognized as one of the most severe threats to public health. However, the population structure and genetic characteristics of CPE isolates among bloodstream infections (BSIs) are largely unknown. To address this knowledge gap, in this study, we included patients with clinically significant BSIs due to Enterobacterales isolates, recruited from 26 sentinel hospitals in China (2014–2015). CPE isolates were microbiologically and genomically characterized, including their susceptibility profiles, molecular typing, phylogenetic features, and genetic context analysis of carbapenemase-encoding genes. Of the 2569 BSI Enterobacterales isolates enrolled, 42 (1.6%) were carbapenemase-positive. Moreover, among the 2242 investigated isolates, 1111 (49.6%) extended-spectrum β-lactamase (ESBL)-producing isolates were identified in Escherichia (E.) coli, Klebsiella (K.) pneumoniae, Proteus (P.) mirabilis, and K. oxytoca. Whole genome sequencing analysis showed the clonal spread of K. pneumoniae carbapenemase (KPC)-2-producing K. pneumoniae sequence type 11 (ST11) and New Delhi metallo-β-lactamase (NDM)-5-producing E. coli ST167 in our collection. Plasmid analysis revealed that carbapenemase-encoding genes were located on multiple plasmids. A high prevalence of biofilm-encoding type 3 fimbriae clusters and yesiniabactin-associated genes was observed in K. pneumoniae isolates. This work demonstrates the high prevalence of ESBLs and the wide dissemination of CPE among BSI isolates in China, which represent real clinical threats. Moreover, our findings first illustrate a more comprehensive genome scenario of CPE isolates among BSIs. The clonal spread of KPC-2-producing K. pneumoniae ST11 and NDM-5-producing E. coli ST167 needs to be closely monitored.

Chen Huang,1,2 Qingyi Shi,2 Shuntian Zhang,2 Hongcheng Wu,1 Yonghong Xiao2 1Department of Respiratory Medicine, Ningbo Medical Center Lihuili Hospital, Ningbo, People’s Republic of China; 2State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of ChinaCorrespondence: Hongcheng WuDepartment of Respiratory Medicine, Ningbo Medical Center Lihuili Hospital, Ningbo, 315000, People’s Republic of ChinaEmail doctorwu1967@126.comYonghong XiaoState Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 310003, People’s Republic of ChinaTel +865 718 723 6421Email xiao-yonghong@163.comObjective: The spread of the plasmid-mediated colistin resistance gene mcr-1 poses a significant public health threat. Little information is available on the development of high-level colistin-resistant mutants (HLCRMs) in MCR-1-producing Escherichia coli (MCRPEC). The present study was designed to evaluate the impact of chromosomal modifications in pmrAB, phoPQ, and mgrB combined with mcr-1 on colistin resistance in E. coli.Methods: Five MCRPEC and three non-MCRPEC (E. coli ATCC25922 and two plasmid-curing) strains were used. The HLCRMs were selected through multi-stepwise colistin exposure. Moreover, two E. coli C600-pMCRs were constructed and used for selection of HLCRMs. Further analysis included mutation rates and DNA sequencing. Transcripts of pmrABC, phoP, mgrB, and mcr-1 were quantified by real-time quantitative PCR.Results: All tested HLCRMs were successfully isolated from their parental strains. Non-MCRPEC strains had higher minimum inhibitory concentrations (MICs) and mutation rates than MCRPEC strains. Nineteen amino acid substitutions were identified: seven in PmrA, six in PmrB, one in PhoP, three in PhoQ, and two in MgrB. Most were detected in non-MCRPEC strains. Sorting Intolerant From Tolerant predicted that four substitutions, PmrA Gly15Arg, Gly53Arg, PmrB Pro94Gln, and PhoP Asp86Gly, affected protein function. Two HLCRM isolates did not show amino acid substitutions in contrast to their parental MCRPEC isolates. No further mutations were detected in the second- and third-step mutants. Further transcriptional analysis showed that the up-regulation of pmrCAB expression was greater in the mutant of E. coli C600 than in E. coli C600-pMCR.Conclusion: Acquisition of the mcr-1 gene had a negative impact on the development of HLCRMs in E. coli, but was associated with low-level colistin resistance. Thus, colistin-based combination regimens may be effective against infections with MCR-1-producing isolates.Keywords: mutation, mcr-1, chromosomal resistance mechanisms, high-level colistin resistance, Escherichia coli

ST59 is the predominant pathotype of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in China. As a variant of ST59, there is relatively little known about the detailed information of ST338. To address this issue, here, we described thirteen ST338 CA-MRSA strains isolated from severe bloodstream infection cases, and focused on their epidemiology, genetic features and virulence potential. Phylogenetic analysis showed the earliest isolated strain of this study is likely a predecessor of recent ST338 lineage (after year of 2014). Furthermore, the phylogenetic reconstruction and time estimation suggested that ST338 evolved from ST59 in 1991. Notably, the carrying patten of virulence factors of all ST338 strains were similar, and the genomic islands νSaα, νSaγ and SaPI and the core virulence factors like hla and psm were detected in ST338 isolates. However, all ST338 isolates lacked some adhesion factors such as clfA, clfB, eap, cna and icaD. Additionally, among these ST338 strains, one PVL-negative ST338 isolate was detected. Experiment on mice nose and human alveolar epithelial cell showed that the nasal colonization ability of ST338 was weaker than that of CA-MRSA MW2. In a mouse bloodstream infection model and skin infection model, PVL+ and PVL− strains had the similar virulence, which was dependent on upregulation of toxin genes rather than the presence of mobile genetic elements such as ΦSa2 carrying PVL. Our findings provide important insight into the epidemiology and pathogenicity of the novel and highly virulent ST338-SCCmec Vb clone.

Background Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a major problem among nosocomial infections, and it is a serious threat to patients. The clinical characteristics and outcome of CRKP bloodstream infection (BSI) in nontransplant patients remains unelucidated. The aim of this study was as follows: identify the risk factors of CRKP infection; generate new ideas for prevention; and generate new ideas for the most effective therapeutic management in nontransplant patients. Methods The study retrospectively analyzed the clinical and microbiological data of nontransplant patients with K pneumoniae (KP) bacteremia from January 2013 to December 2015 to identify risk factors, clinical features, and outcomes using multivariate logistic regression analysis. Results Of the 371 patients with KP-BSI in nontransplant patients included in this study, 28.0% (N = 104) had CRKP. The 28-day mortality was higher in patients infected with CRKP (55.8%) than in those with carbapenem-susceptible KP (13.9%) (P < .001). Multivariate analysis showed previous gastric catheterization, previous use of carbapenems, hypoproteinemia, and high Acute Physiologic Assessment and Chronic Health Evaluation II scores as independent risk factors for CRKP-BSIs. Carbapenem-resistant KP infection, severe illness, and tigecycline therapy were independent risk factors for death from KP-BSIs. Taken together, inappropriate antibiotic treatment both in empirical and definitive therapy and imipenem minimum inhibitory concentrations (MICs) of >8 mg/L were associated with poor clinical outcome. Conclusions Nontransplant patients with CRKP-BSI had higher mortality. Carbapenems exposure was an independent risk factor for CRKP infection. Imipenem MICs of >8 mg/L, tigecycline therapy, and inappropriate treatments increased the 28-day mortality of KP-BSI patients.

Previous studies have shown that livestock (LA)-MRSA ST398 evolved from a human-adapted methicillin-susceptible S. aureus (MSSA) clone. However, detailed information regarding ST9 is still unclear. Here, we characterized a community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) ST9-SCCmec XII isolate that has not been previously reported to cause serious disease in China. We obtained whole-genome sequences of one ST9-t899-XII isolate—ZY462471—from a patient with bloodstream infection without livestock contact. The antibiotic susceptibilities of ZY462471 were determined and the clinical information was extracted from medical notes and compared with twenty-seven previously sequenced genomes. Phylogenetic reconstruction was performed to investigate the probable host evolutionary origins of ZY462471, and the difference in resistome and virulence factors were investigated. Virulence assay was performed to evaluate the high virulence potential of ZY462471 and compare the virulence between the closest ST9 MSSA neighbours. Clinical data suggested that ZY462471 is a CA-MRSA. Phylogenetic analysis showed a much closer relationship of ZY462471 with human-associated MSSA ST9 isolates than other LA-MRSA ST9 isolates, suggesting that ZY462471 probably evolved from ST9 MSSA predecessors by acquiring an SCCmec cassette. Importantly, virulence assays indicated that ZY462471 was highly virulent and compared with the MSSA ST9 predecessors, ZY462471 did not show attenuated virulence. Finally, we found that ZY462471 harboured an immune evasion cluster (IEC)-carrying βC-Φ, which is typically found in human clinical S. aureus rather than LA-MRSA isolates, suggesting that ZY4762471 obtained the IEC-carrying βC-Φs from human clinical S. aureus strains. Considering its high virulence potential, this strain should be monitored to prevent more widespread dissemination.

Tigecycline is one of the last-resort antibiotics to treat severe infections. Recently, tigecycline resistance has sporadically emerged with an increasing trend, and Tet(X) family represents a new ...

Carbapenem-resistantA prospective study was conducted on patients with mechanical ventilation from July 2018 to December 2019 in a tertiary hospital. Multi-genomics studies (16S rRNA amplicon, metagenomics, and whole-genome sequencing [WGS]) of endotracheal deep aspirate (ETA) were performed.Fifty-two ICU patients were enrolled, including 24 with CRAB VAP (CRAB-I), 22 with CRAB colonization (CRAB-C), and six CRAB-negative patients (infection-free) (CRAB-N). Diversity of pulmonary microbiota was significantly lower in CRAB-I than in CRAB-C or CRAB-N (mean Shannon index, 1.79 vs. 2.73 vs. 4.81,Lower respiratory tract microbiota dysbiosis including elevated relative abundance of

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a common cause of ventilator-associated pneumonia (VAP) in intensive care unit (ICU) patients, but infection and colonization are difficult to distinguish which aggravates the abuse of antimicrobial drugs and further accelerates the evolution of drug resistance. We sought to provide new clues for the diagnosis, pathogenesis and treatment of CRAB VAP based on lower respiratory tract (LRT) microbiota. Methods: A prospective study was conducted on patients with mechanical ventilation from July 2018 to December 2019 in a tertiary hospital. Multi-genomics studies (16S rRNA amplicon, metagenomics and whole-genome sequencing [WGS]) of endotracheal deep aspirate (ETA) were performed. Results: Fifty-two ICU patients were enrolled, including 24 with CRAB VAP (CRAB-I), 22 with CRAB colonization (CRAB-C), and six CRAB-negative patients (infection-free) (CRAB-N). Diversity of pulmonary microbiota was significantly lower in CRAB-I than in CRAB-C or CRAB-N (mean Shannon index, 1.79 vs. 2.73 vs. 4.81, PAcinetobacter was most abundant in CRAB-I (76.19%), moderately abundant in CRAB-C (59.14%), and least abundant in CRAB-N (11.25%), but its interactions with other genera exhibited the opposite pattern. Metagenomics and WGS analysis showed that virulence genes were more abundant in CRAB-I than in CRAB-C. Multi-locus sequence typing (MLST) of 46 CRAB isolates revealed that the main types were ST208 (30.43%) and ST938 (15.22%), with no difference between CRAB-I and CRAB-C. Conclusions: LRT microbiota dysbiosis including elevated relative abundance of Acinetobacter and reduced bacterial interactions, and virulence enrichment may lead to CRAB VAP.

Clostridioides difficile is the most common pathogen causing antibiotic-associated diarrhea. Previous studies showed that diverse sources, aside from C. difficile infection (CDI) patients, played a major role in C. difficile hospital transmission. This study aimed to investigate relationships and transmission potential of C. difficile strains from different sources. A prospective study was conducted both in the intensive care unit (ICU) and six livestock farms in China in 2018–2019. Ninety-eight strains from CDI patients (10 isolates), asymptomatic hospitalized carriers (55), the ICU environment (12), animals (14), soil (4), and farmers (3) were collected. Sequence type (ST) 3/ribotype (RT) 001, ST35/RT046, and ST48/RT596 were dominant types, distributed widely in multiple sources. Core-genome single-nucleotide polymorphism (cgSNP) analysis showed that hospital and farm strains shared several common clonal groups (CGs, strains separated by ≤ 2 cgSNPs) (CG4/ST3/RT001, CG7/ST35/RT046, CG11/ST48/RT596). CDI patients, asymptomatic carriers, and the ICU environment strains also shared several common CGs. The number of virulence genes was not statistically different between strains from different sources. Multi-source strains in the same CG carried identical virulence gene sequences, including pathogenicity genes at the pathogenicity locus and adhesion-related genes at S-layer cassette. Resistance genes (ermB, tetM, etc.) were widespread in multiple sources, and multi-source strains in the same CG had similar resistance phenotypes and carried consistent transposons and plasmid types. The study indicated that interspecies and cross-regional transmission of C. difficile occurs between animals, the environment, and humans. Community-associated strains from both farms and asymptomatic hospitalized carriers were important reservoirs of CDI in hospitals.

Previous studies on vancomycin-intermediate Staphylococcus aureus (VISA) have mainly focused on drug resistance, the evolution of differences in virulence between VISA and vancomycin-sensitive S. aureus (VSSA) requires further investigation. To address this issue, in this study, we compared the virulence and toxin profiles of pair groups of VISA and VSSA strains, including a series of vancomycin-resistant induced S. aureus strains—SA0534, SA0534-V8, and SA0534-V16. We established a mouse skin infection model to evaluate the invasive capacity of VISA strains, and found that although mice infected with VISA had smaller-sized abscesses than those infected with VSSA, the abscesses persisted for a longer period (up to 9 days). Infection with VISA strains was associated with a lower mortality rate in Galleria mellonella larvae compared to infection with VSSA strains (≥ 40% vs. ≤ 3% survival at 28 h). Additionally, VISA were more effective in colonizing the nasal passage of mice than VSSA, and in vitro experiments showed that while VISA strains were less virulent they showed enhanced intracellular survival compared to VSSA strains. RNA sequencing of VISA strains revealed significant differences in the expression levels of the agr, hla, cap, spa, clfB, and sbi genes and suggested that platelet activation is only weakly induced by VISA. Collectively, our findings indicate that VISA is less virulent than VSSA but has a greater capacity to colonize human hosts and evade destruction by the host innate immune system, resulting in persistent and chronic S. aureus infection.

China has been experiencing a rapid increase in the number of nosocomial infections caused by carbapenem-resistant Klebsiella pneumoniae ST11 (ST11-CRKP) for decades. The emergence of hypervirulent ST11-CRKP (ST11-CR-HvKP) strains is expected to become a serious public health issue in China, considering that carbapenem resistance and virulence have converged in an epidemic clone. K. pneumoniae strains that colonize the human intestinal tract may become a reservoir of virulence and carbapenemase-encoding genes. Here, we first characterized the genotypes and antimicrobial phenotypes of ST11-CR-HvKP strains isolated from diarrheal stool samples of inpatients in Zhejiang Province, China. Active surveillance approaches based on the findings of the present study should be implemented, particularly in intensive care units, to combat the spread of ST11-CR-HvKP and to improve treatment., The emergence and spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae sequence type 11 (ST11-CR-HvKP) in China are a great concern in the public health community. However, the underlying mechanism that enables its wide dissemination in China remains unclear. Here, we investigated the prevalence of carriage of carbapenemase-producing Enterobacteriaceae (CPE) among inpatients with diarrhea in a teaching hospital over 1 year to identify ST11-CR-HvKP reservoirs and to understand the genetic background and plasmid profiles of these pathogens. As assessed by stool analysis, the CPE colonization rate (12.4%) among the inpatients with diarrhea was high (12.4%). Antibiotic exposure, surgical history, and CPE positivity were correlated. Genomic investigation of 65 carbapenem-resistant K. pneumoniae isolates indicated a shared bacterial population in various wards. According to maximum likelihood phylogenetic tree analysis, these isolates were partitioned into three major clades. Analysis of the wzi locus revealed three different K types (KL105, KL47, and K64) among the ST11 isolates, indicating the genetic diversity of these isolates. Genetic and sequence mapping revealed the complexity of virulence and resistance plasmid sets harbored by the isolates. These observations indicate that the dissemination of resistant bacteria is more complex than initially anticipated and possibly involves multiple K. pneumoniae ST11 lineages and a variety of virulence plasmids. Collectively, we show for the first time that stool may be a source of ST11-CR-HvKP isolates. Furthermore, the findings reveal the silent dissemination of ST11-CR-HvKP bacteria in Zhejiang Province, China. Future investigations are warranted to determine the association between rectal colonization by ST11-CR-HvKP and clinical infections. IMPORTANCE China has been experiencing a rapid increase in the number of nosocomial infections caused by carbapenem-resistant Klebsiella pneumoniae ST11 (ST11-CRKP) for decades. The emergence of hypervirulent ST11-CRKP (ST11-CR-HvKP) strains is expected to become a serious public health issue in China, considering that carbapenem resistance and virulence have converged in an epidemic clone. K. pneumoniae strains that colonize the human intestinal tract may become a reservoir of virulence and carbapenemase-encoding genes. Here, we first characterized the genotypes and antimicrobial phenotypes of ST11-CR-HvKP strains isolated from diarrheal stool samples of inpatients in Zhejiang Province, China. Active surveillance approaches based on the findings of the present study should be implemented, particularly in intensive care units, to combat the spread of ST11-CR-HvKP and to improve treatment.

Tigecycline is one of the last-resort antibiotics to treat severe infections. Recently, tigecycline resistance has sporadically emerged with an increasing trend, and Tet(X) family represents a new resistance mechanism of tigecycline. In this study, a novel chromosome-encoded tigecycline resistance gene,tet(X14), was identified in a tigecycline-resistant and colistin-resistantEmpedobacter stercorisstrain ES183 recovered from a pig fecal sample in China. Tet(X14) shows 67.14-96.39% sequence identity to the other variants [Tet(X) to Tet(X13)]. Overexpression of Tet(X14) inEscherichia coliconfers 16-fold increase in tigecycline MIC (from 0.125 to 2 mg/L), which is lower than that of Tet(X3), Tet(X4) and Tet(X6). Structural modelling predicted that Tet(X14) shared a high homology with the other 12 variants with RMSD value from 0.003 to 0.055, and Tet(X14) can interact with tetracyclines by a similar pattern as the other Tet(X)s.tet(X14) and two copies oftet(X2) were identified on a genome island with abnormal GC content carried by the chromosome of ES183, and no mobile genetic elements were found surrounding, suggesting thattet(X14) might be heterologously obtained by ES183 via recombination. Blasting in Genbank revealed that Tet(X14) was exclusively detected on the chromosome ofRiemerella anatipestifer, mainly encoded on antimicrobial resistance islands.E. stercorisandR. anatipestiferbelong to the familyFlavobacteriaceae, suggesting that the members ofFlavobacteriaceaemaybe the major reservoir oftet(X14). Our study reports a novel chromosome-encoded tigecycline resistance genetet(X14). The expanded members of Tet(X) family warrants the potential large-scale dissemination and the necessity of continuous surveillance fortet(X)-mediated tigecycline resistance.

A tigecycline-resistance gene, tet(X6), was detected on a 159-kb novel plasmid carried by a tigecycline-susceptible livestock-associated Acinetobacter towneri isolate. The genetic context of tet(X6) (ΔISVsa3-tet(X6)-abh-guaA-ISVsa3) is highly similar with that of the other plasmid-borne tet(X) variants. The 23-Ala residue of the first FAD binding site conferred higher activity to Tet(X6) than the 23-Gly reside conserved in the other plasmid-borne tet(X)s. To our knowledge, this is the first report of tet(X6) carried by the plasmid.

Reaching to grasp movement is thought to rely upon two independent brain pathways. The dorsomedial one is involved in reaching while the dorsolateral one is dealing with grasping. However, some recent evidences suggested that the dorsomedial pathway might participate in grasp movement. Therefore, it is important to investigate whether PMd is involved in grasp planning, and if participating, what kind of role PMd played in grasp planning. In this study, two macaques monkeys were trained to grasp same object by instructing or freely choosing one of two grips, power grip or hook grip. A 96-channel microelectrode array was implanted to collect the population activity of PMd in each subject. Both single unit activity and population activity were analyzed. We found that nearly 21.0% and 26.8% units in PMd of two monkeys displayed grip selectivity during gesture planning in both instructing or freely choosing conditions. These units exhibit selectivity for different gestures when facing the identical visual stimuli (freely choosing condition). At the same time, similar activity patterns are displayed for the same gesture when faced with different selection strategies (freely choosing condition vs. instructing condition). These results show that some neurons of PMd are mainly involved in the hand shape preparation and have no obvious relationship with external visual stimuli and selection strategies.

We report the characterization of six carbapenem-resistant Raoultella spp. (CRRS) in our hospital and a genomic analysis of 58 publicly available isolates. CRRS isolates are sporadically identified around the world, and different transposons carrying carbapenemases were the resistant mechanisms. Mobile genetic elements play an important role in acquiring antibiotic resistance genes from the hospital., We report the characterization of six carbapenem-resistant Raoultella spp. (CRRS) in our hospital and a genomic analysis of 58 publicly available isolates. CRRS isolates are sporadically identified around the world, and different transposons carrying carbapenemases were the resistant mechanisms. Mobile genetic elements play an important role in acquiring antibiotic resistance genes from the hospital. An improved understanding of these transposon and targeted control measures will be very valuable to prevent CRRS dissemination.

Background: The emergence and spread of ST11 carbapenem-resistant, hypervirulent K. pneumonia (ST11-CR-HvKP) in China generated great concern from the public health community. The identification of ST11-CR-HvKP strain is expected to become a serious public health issue in China, considering the carbapenem resistance and virulence had converged in an epidemic clone. However, the underlying mechanism that enables its wide dissemination in China remains unclear.Results: Here, we investigate the prevalence of carbapenemase-producing Enterobacteriaceae (CPE) carriage by inpatients in a teaching hospital over a 1-year period, to identify ST11-CR-HvKP reservoirs, and to understand the transmission of these pathogens across healthcare networks. We identified a high colonization prevalence of CPE (12.4%) among inpatients with diarrhea. Correlations were detected between antibiotic exposure, surgical history, and being CPE positive. A genomic investigation of 65 CRKP isolates indicated a shared bacterial population among various wards. Maximum-likelihood phylogenetic tree demonstrated that these isolates were partitioned into three major clades. An analysis of the wzi locus revealed three different K types (KL105, KL47, and K64) among the ST11 isolates, indicating genetic diversity among these isolates. Our review of the cases showed that these patients had no contact with each other, indicating nosocomial transmission. Genetic and sequence mapping revealed complexity in the existence of virulence plasmids and resistance plasmids in the ST11-CRKP isolates. These data indicate that this process was more complicated than was earlier anticipated, as it may have involved multiple ST11 K. pneumoniae lineages and a variety of virulence plasmids. Conclusions: Collectively, this work represents the first evidence of gut microbiota may act as the source of ST11-CR-HvKP isolate. Active surveillance approaches, particularly in ICUs based on the results of this study, should be implemented to combat the spread of ST11-CR-HvKP and to improve patient outcomes. Key words: gut microbiota; hypervirulent; KPC-2; reservoir; genomic characterization

Table S1. Analysis of risk factors for 28â day mortality in 283 patients with ESBL- producing E. coli BSI. (DOCX 31 kb)

Figure S1. Kaplan-Meier survival estimates among non-transplant patients with BSIs caused by non-ESBL-producing E. coli and ESBL-producing E. coli. (A) Community-acquired infection. (B) Nosocomial-acquired infection. Figure S2. Kaplan-Meier 28 day survival estimates. (A) E. coli BSI patients (APACHE II score < 9) treated with carbapenem and β-lactam-β-lactamase inhibitor (BLBLI) combination antibiotics. (B) E. coli BSI patients (APACHE II score ≥ 9) treated with carbapenem and BLBLIs. (DOCX 122 kb)