Outer membrane vesicles mediating horizontal transfer of the epidemic blaOXA-232 carbapenemase gene among Enterobacterales.

OXA-232 is one of the most common OXA-48-like carbapenemase derivatives and is widely disseminated in nosocomial settings across countries. The blaOXA-232 gene is located on a 6-kb non-conjugative ColKP3-type plasmid, while the dissemination of blaOXA-232 into different Enterobacterales species and the polyclonal dissemination of OXA-232-producing K. pneumoniae revealed the horizontal transfer of blaOXA-232. However, it's still unclear how this non-conjugative ColKP3 plasmid could facilitate the mobilization of blaOXA-232. Here, we observed the in vivo intraspecies transfer of blaOXA-232 during a nosocomial outbreak of OXA-232-producing K. pneumoniae. We demonstrated the presence of ColKP3 OXA-232 plasmid in the outer membrane vesicles (OMVs) derived from clinical isolates, and OMVs could facilitate the horizontal transfer of blaOXA-232 among Enterobacterales. In contrast, for the most prevalent carbapenemase genes, including blaKPC-2 and blaNDM-1, though the presence of carbapenemase genes and plasmid backbones in the vesicular lumen was observed, OMVs couldn't promote effective transformation, probably due to the low copy number of plasmids in clinical isolates and the low number of plasmids loaded into vesicles. Conjugation assay revealed that the epidemic IncX3 NDM-1 and IncFII(pHN7A8)/IncR KPC-2 plasmids were conjugative and could be horizontally transferred via independent conjugation or with the help of a co-existent conjugative plasmid. For the large-size and low-copy number conjugative plasmids carrying carbapenemase genes, OMVs-mediated gene exchange may only serve as an alternative pathway for horizontal transfer. In conclusion, diverse mobilization strategies were employed by plasmids harboring carbapenemase genes, and plasmids display a proper choice of mobility pathway due to their individual properties.

Phenotypic and genetic characterization of hypervirulent Klebsiella pneumoniae in patients with liver abscess and ventilator-associated pneumonia.

Ventilator-associated pneumonia (VAP) and pyogenic liver abscess (PLA) due to Klebsiella pneumoniae infection can trigger life-threatening malignant consequences, however, there are few studies on the strain-associated clinical pathogenic mechanisms between VAP and PLA. A total of 266 patients consist of 129 VAP and 137 PLA were included for analysis in this study. We conducted a comprehensive survey for the two groups of K. pneumoniae isolates, including phenotypic experiments, clinical epidemiology, genomic analysis, and instrumental analysis, i.e., to obtain the genomic differential profile of K. pneumoniae strains responsible for two distinct infection outcomes. We found that PLA group had a propensity for specific underlying diseases, especially diabetes and cholelithiasis. The resistance level of VAP was significantly higher than that of PLA (78.57% vs. 36%, P < 0.001), while the virulence results were opposite. There were also some differences in key signaling pathways of biochemical processes between the two groups. The combination of iucA, rmpA, hypermucoviscous phenotype, and ST23 presented in K. pneumoniae infection is more important and highly prudent for timely treatment. The present study may contribute a benchmark for the K. pneumoniae clinical screening, epidemiological surveillance, and effective therapeutic strategies.

Nosocomial dissemination of blaIMP-4 among Klebsiella pneumoniae by horizontal gene transfer and clonal spread: the epidemic IncN plasmids and the emerging high-risk IMP-4-producing ST101 clone.

OBJECTIVES: To determine the genomic features of IMP-4-producing Klebsiella pneumoniae isolates recovered from paediatric patients and the transmission dynamics of blaIMP-4. METHODS: IMP-producing K. pneumoniae isolates were collected from paediatric patients in Shanghai Children's Medical Center from 2013 to 2020. WGS was performed for all isolates, and the complete genomes of three IMP-4-producing isolates were generated. The distribution of blaIMP-4-harbouring plasmids was determined, and a conjugation assay was employed to investigate the horizontal transfer of blaIMP-4-harbouring plasmids. RESULTS: We collected 21 blaIMP-carrying K. pneumoniae isolates, with IMP-4 (16/21, 76.2%) as the predominant subtype, followed by IMP-8 (n = 3) and IMP-26 (n = 2). IMP-4-producing isolates displayed a diverse population structure and all blaIMP-4 genes were located on plasmids, including IncN (n = 9), IncHI5 (n = 5), IncFII(K) (n = 1) and IncFII(pKP91) (n = 1), although only IncN plasmids were conjugative. Clonal transmission of ST101 strains carrying IncHI5 blaIMP-4-harbouring plasmids was observed, and the acquisition of blaIMP-4 by the international high-risk ST101 clone constituted a novel combination of ST101 clone and carbapenemase genes. Plasmid analysis demonstrated that the conjugal transfer of the IncHI5 blaIMP-4-harbouring plasmid might be blocked by the ST101 bacterial host. CONCLUSIONS: The horizontal transfer of IncN plasmids and clonal spread of the international high-risk ST101 clone facilitated the nosocomial dissemination of blaIMP-4 among K. pneumoniae. The emerging IMP-4-producing ST101 clone displays diverse combinations of carbapenemase genes, and this clone could be a continually evolving threat and warrants prospective monitoring.

Ceftazidime/Avibactam Resistance in Carbapenemase-Producing Klebsiella pneumoniae.

We identified a novel ceftazidime/avibactam resistance mechanism in sequence type 11 Klebsiella pneumoniae carbapenemase 2-producing K. pneumoniae. Plasmid recombination and chromosomal integration formed a novel virulence plasmid and provided an additional promoter for blaSHV-12, leading to blaSHV-12 overexpression and ceftazidime/avibactam resistance. Genetic rearrangement contributed to convergence of hypervirulence and ceftazidime/avibactam resistance.

Performance of Two Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) Systems for Identification of the Viridans Group Streptococci.

Background: Due to similar colony morphology among viridans group streptococci (VGS), the differentiation of VGS species remains difficult in routine clinical microbiology. Recently, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been described as a fast method for identifying various bacteria at species level, and also for the VGS strains. Methods: A total of 277 VGS isolates were identified with the two MALDI-TOF MS systems (VITEK MS and Bruker Biotyper). The tuf and rpoB gene sequencing was used as the reference identification method for comparison. Results: Based on tuf and rpoB gene sequencing, 84 isolates were S. pneumoniae and 193 strains were other VGS isolates including S. anginosus group (n=91, 47.2%), S. mitis group (n=80, 41.5%), S. bovis group (n=11, 5.7%), S. salivarius group (n=10, 5.2%), and S. mutans group (n=1, 0.5%). VITEK MS and Bruker Biotyper accurately identified 94.6% and 89.9% of all VGS isolates, respectively. VITEK MS showed better identification results than Bruker Biotyper for S. mitis group including S. pneumoniae and S. bovis group, but for other VGS isolates, two MALDI-TOF MS systems showed comparable identification performance. However, VITEK MS was able to identify S. gallolyticus to the subspecies level with high-confidence (S. gallolyticus ssp. pasteurianus), while the Bruker Biotyper system could not. While Bruker Biotyper system could be able to correctly differentiate the subspecies of S. salivarius from S. vestibularis, VITEK MS poorly identify. Conclusion: This study demonstrated that two MALDI-TOF MS systems allowed discrimination for most VGS isolates with different identification performance, but Bruker Biotyper could produce more misidentifications and VITEK MS system. It is crucial to be familiar with the performance of MALDI-TOF MS systems used in clinical microbiology.

Host Sorbitol and Bacterial Sorbitol Utilization Promote Clostridioides difficile Infection in Inflammatory Bowel Disease.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a widespread gastrointestinal inflammatory disorder with globally increasing incidence. Clostridioides difficile infection (CDI) often occurs in patients with intestinal dysbiosis, such as after antibiotic therapy. Patients with IBD have increased incidence of CDI and the clinical outcome of IBD is reportedly worsened by CDI. However, the underlying reasons remain poorly understood. METHODS: We performed a retrospective single-center and a prospective multicenter analysis of CDI in patients with IBD, including genetic typing of C difficile isolates. Furthermore, we performed a CDI mouse model to analyze the role of the sorbitol metabolization locus that we found distinguished the main IBD- and non-IBD-associated sequence types (STs). Moreover, we analyzed sorbitol concentration in the feces of patients with IBD and healthy individuals. RESULTS: We detected a significant association of specific lineages with IBD, particularly increased abundance of ST54. We found that in contrast to the otherwise clinically predominant ST81, ST54 harbors a sorbitol metabolization locus and was able to metabolize sorbitol in vitro and in vivo. Notably, in the mouse model, ST54 pathogenesis was dependent on intestinal inflammation-induced conditions and the presence of sorbitol. Furthermore, we detected significantly increased sorbitol concentrations in the feces of patients with active IBD vs patients in remission or healthy controls. CONCLUSIONS: Sorbitol and sorbitol utilization in the infecting C difficile strain play major roles for the pathogenesis and epidemiology of CDI in patients with IBD. CDI in patients with IBD may thus be avoided or improved by elimination of dietary sorbitol or suppression of host-derived sorbitol production.

Fluorescent metabolic labeling-based quick antibiotic susceptibility test for anaerobic bacteria.

Because of the advancements in medicine and science, the numbers of patients surviving complicated diseases are continuously increasing, which in turn leads to elevated chances of anaerobic infections by endogenous bacteria. Traditional growth yield-based antibiotic susceptibility tests (ASTs) against anaerobic bacteria are very time-consuming (≥48 h) and labor intensive, which delays the timely guidance of antibiotic prescription and increases the mortality of patients. Inspired by a fluorescent d-amino acid (FDAA) labeling-based AST (FaAST) that we recently developed for quick determination of aerobic bacteria's susceptibilities, here we report an accurate and fast AST method for anaerobic pathogens. Based on flow cytometry analysis of anaerobes that have been treated with various doses of antibiotics and metabolically labeled with FDAA, the intensities of which can reflect their affected metabolic status by the drugs, the MICs of each drug can then be determined. The whole process can be completed in 5 h. After testing 40 combinations of the representative anaerobic bacteria and antibiotics, our method demonstrates a high susceptibility category accuracy of 95.0%. This FaAST-based protocol is helpful in accurately and quickly guiding antibiotic decisions when treating critical infections caused by anaerobic bacteria.

Correction: Fluorescent metabolic labeling-based quick antibiotic susceptibility test for anaerobic bacteria.

[This corrects the article DOI: 10.1039/D2CB00163B.].

Ploidy Variation and Spontaneous Haploid-Diploid Switching of Candida glabrata Clinical Isolates.

The human fungal pathogen Candida glabrata is phylogenetically closely related to Saccharomyces cerevisiae, a model eukaryotic organism. Unlike S. cerevisiae, which has both haploid and diploid forms and a complete sexual cycle, C. glabrata has long been considered a haploid and asexual species. In this study, we analyzed the ploidy states of 500 clinical isolates of C. glabrata from four Chinese hospitals and found that approximately 4% of the isolates were in or able to spontaneously switch to an aneuploid (genomic DNA, 1N-2N), diploid (2N), or hyperdiploid (>2N) form under in vivo or in vitro conditions. Stable diploid cells were identified in 3% of the isolates (15/500). Of particular interest, one clinical strain existed only in the diploid form. Multilocus sequence typing (MLST) assays revealed two major genetic clusters (A and B) of C. glabrata isolates. Most of the isolates (70%) from China belonged to the A cluster, whereas most of the isolates from other countries (such as Iran, Japan, United States, and European countries) belonged to the B cluster. Further investigation indicated that C. glabrata cells of different ploidy forms differed in a number of respects, including morphologies, antifungal susceptibility, virulence, and global gene expression profiles. Additionally, C. glabrata could undergo spontaneous switching between the diploid and haploid forms under both in vitro and in vivo conditions. Given the absence of an apparent sexual phase, one would expect that the ploidy shifts could function as an alternative strategy that promotes genetic diversity and benefits the ability of the fungus to rapidly adapt to the changing environment. IMPORTANCE The human fungal pathogen Candida glabrata has long been thought to be a haploid organism. Here, we report the population structure and ploidy states of 500 clinical isolates of C. glabrata from China. To our surprise, we found that the ploidy of a subset of clinical isolates varied dramatically. Some isolates were in or able to switch to an aneuploid, diploid, or hyperdiploid form. C. glabrata cells with different ploidy differed in a number of biological respects, including morphology, antifungal susceptibility, virulence, and global gene expression profile. Given the absence of an apparent sexual phase in this fungus, we propose that ploidy switching could be a strategy for rapid adaptation to environmental changes and could function as an alternative to sexual reproduction.

Dissemination and Stability of the blaNDM-5-Carrying IncX3-Type Plasmid among Multiclonal Klebsiella pneumoniae Isolates.

NDM-5 carbapenemase was mainly identified in Escherichia coli, while the rapid transmission of blaNDM-5 among Enterobacteriaceae has raised serious public attention. This study identified 14 NDM-5-producing Klebsiella pneumoniae isolates from 107 carbapenem-resistant K. pneumoniae isolates, recovered from blood, urine, and normally sterile body fluids of pediatric patients from January 2016 to December 2018. All NDM-5-producing isolates were highly resistant to ß-lactams, while tigecycline and polymyxin B exhibited excellent antimicrobial activity. These 14 strains belonged to 9 different sequence types (STs) and displayed various pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they were not clonally related. S1-PFGE followed by Southern blotting showed that the blaNDM-5 gene was located on an ∼46-kb IncX3 plasmid in all strains. All blaNDM-5-carrying plasmids were successfully transferred into recipient E. coli J53. PCR-based sequencing demonstrated that all of the blaNDM-5-carrying plasmids shared highly similar backbones, with nucleotide sequence identity of >99%. Moreover, this plasmid displayed high sequence similarity to the previously reported epidemic IncX3 blaNDM-5-carrying plasmids, with dynamic changes observed only in blaNDM-5-surrounding elements. Interestingly, the IncX3 blaNDM-5-carrying plasmids showed strong stability in clinical isolates when cultured in antibiotic-free medium. However, after the conjugation inhibitor linoleic acid was added, a gradual increase in the level of IncX3 plasmid loss could be observed. Clinical isolates displayed 10% to 15% blaNDM-5-carrying plasmid loss after coculture with linoleic acid for 5 days. These results showed that the IncX3 plasmid facilitated the dissemination of blaNDM-5 among multiclonal K. pneumoniae strains in children and that conjugal transfer contributed significantly to IncX3 plasmid stability within K. pneumoniaeIMPORTANCE The emergence and spread of New Delhi metallo-ß-lactamase (NDM)-producing Enterobacteriaceae have been a serious challenge to public health, and NDM-5 shows increased resistance to carbapenems compared with other variants. NDM-5 has been identified mostly in E. coli but has rarely been described in K. pneumoniae and other Enterobacteriaceae isolates. Here, we present the dissemination of highly similar 46-kb IncX3 blaNDM-5-carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of blaNDM-5 among K. pneumoniae strains via the IncX3 plasmid. Moreover, the IncX3 blaNDM-5-carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and the plasmid maintenance was attributed partly to conjugal transfer. Plasmid conjugation is mediated by the type IV secretion system (T4SS), and T4SS is conserved among all epidemic IncX3 blaNDM-5-carrying plasmids. Therefore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugation-assisted persistence of IncX3 blaNDM-5-carrying plasmids.

Molecular epidemiology and risk factors of Stenotrophomonas maltophilia infections in a Chinese teaching hospital.

BACKGROUND: Stenotrophomonas maltophilia (S. maltophilia) is an important opportunistic pathogen that can be isolated in hospitals. With the abuse of broad spectrum antibiotics and invasive surgical devices, the rate of S. maltophilia infection is increasing every year. This study was an epidemiological analysis of the clinical and molecular characteristics of S. maltophilia infection in a Chinese teaching hospital. The goal was to obtain a comprehensive understanding of the status of S. maltophilia infection to provide strong epidemiological data for the prevention and treatment of S. maltophilia infection. RESULTS: A total of 93 isolates from Renji Hospital affiliated with the Shanghai Jiaotong University School of Medicine were included, in which 62 isolates were from male patients. In addition, 81 isolates were isolated from sputum samples. A total of 86 patients had underlying diseases. All patients received antibiotics. Multilocus sequence typing (MLST) analysis indicated that 61 different sequence types (STs) were found (including 45 novel STs), and MLST did not show significantly dominant STs. Pulsed field gel electrophoresis (PFGE) results showed that 93 isolates could be divided into 73 clusters, and they also showed weak genetic linkages between isolates. The resistant rates to trimethoprim/sulfamethoxazole (TMP/SMX) and levofloxacin were 9.7 and 4.3%, respectively, and all isolates were susceptible to minocycline. Four virulence gene's loci Stmpr1, Stmpr2, Smf-1, and Smlt3773 were positive in 79.6, 91.4, 94.6, and 52.7% of the isolates, respectively. Three biofilm genes rmlA, spgM, and rpfF were positive in 82.8, 92.5, and 64.5% of the isolates, respectively. Mean biofilm forming level of OD492 was 0.54 ± 0.49. We did not find any significant difference between different genders and different age-groups. We retrospectively analyzed data from patients in the intensive care unit (ICU) and the control group. The independent risk factors of those who were infected in the ICU included immunosuppression and the increased antibiotic usage. CONCLUSIONS: Most of the patients had prior medical usage histories and baseline diseases. The positive rate of virulence genes was high, the drug resistance rate of S. maltophilia was low, and the biofilm formation ability was strong. The increased use of antibiotics was an independent risk factor for S. maltophilia infection, which should receive more attention. No obvious clonal transmissions were found in the same departments.

Clinical and Molecular Epidemiology of Stenotrophomonas maltophilia in Pediatric Patients From a Chinese Teaching Hospital.

Objective: To study the molecular epidemiological characteristics of Stenotrophomonas maltophilia (SMA) isolated from patients in a pediatric teaching hospital in Shanghai so as to provide data for the prevention and treatment of SMA. Methods: Non-repetitive SMA strains were isolated from patients from January 2013 to December 2014. The cloning characteristics were analyzed using multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE), and the drug resistance was determined using the Kirby-Bauer disk method. Virulence genes and biofilm genes were detected using polymerase chain reaction (PCR). The biofilm forming ability was analyzed using the semi-quantitative biofilm formation test. Results: A total of 104 strains were collected, primarily from the pediatric intensive care unit and thoracic surgery, and these strains were isolated from sputum sources (n = 82). A majority of the patients were male (67/104), and the age range was between 6 days and 12 years old. A total of 95 patients had 1-3 baseline diseases. All of the patients had prior use of 1-4 antimicrobial agents. A total of 59 STs were detected using the MLST analysis, of which 45 were new. The sequence types of the SMA were scattered, with no trend in the clonal spread. The PFGE showed that the 104 strains could be divided into 93 clusters, with no obvious cluster aggregations. All of the strains were susceptible to levofloxacin, trimethoprim/sulfamethoxazole, and minocycline. The positive rates of the virulence genes stmPr1, stmPr2, smf-1, and smlt3773 locus were 98.1, 86.5, 100, and 91.3%, respectively. All of the strains had biofilm formation, and most of the strains had strong biofilm formation abilities. The positive rates of the three biofilm genes rmlA, spgM, and rpfF were 83.7, 100, and 45.2%, respectively. However, the point mutations of rmlA and spgM with strong biofilm formation abilities were significantly different from those with weak biofilm formation abilities. Conclusion: Most infected patients had prior use of antibiotics and underlying diseases, and the positive rate of the virulence gene was high. The strains were susceptible to three kinds of antibiotics and had strong biofilm formation abilities. The mutations of rmlA and spgM may be related to the biofilm formation ability, and no obvious clonal transmissions were found in the same clinical department.

Skin microbiota analysis-inspired development of novel anti-infectives.

BACKGROUND: The alarming spread of antimicrobial resistance requires the development of novel anti-infective drugs. Despite the recent research focus on the human microbiome and its likely value to understand and exploit inter-bacterial inhibitory phenomena as a source for antimicrobial strategies, the human microbiota has barely been investigated for the purpose of drug development. RESULTS: We performed a large screen analyzing over 3000 human skin isolates to evaluate bacterial competition within the human skin microbiota as a basis for the development of anti-infective therapeutics. We discovered a Staphylococcus hominis strain with strong and broad activity against Gram-positive pathogens that was mediated by the bacteriocin micrococcin P1 (MP1). In "probiotic" approaches, this strain led to reduced Staphylococcus aureus infection and accelerated closure of S. aureus-infected wounds. Furthermore, we used a nanoparticle strategy to overcome the physico-chemical limitations often encountered with natural substances such as MP1 and demonstrate a significant reduction of S. aureus infection by MP1-loaded nanoparticles. CONCLUSIONS: Our study gives examples of how analysis of bacterial interactions in the human microbiota can be explored for the development of novel, effective anti-infective strategies. Video Abstract.

Antimicrobial Resistance and Pathogenicity Determination of Community-Acquired Hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) can cause severe invasive infections in healthy and immunocompromised individuals. However, there is still lack of a consensus definition of hvKp. In this study, we solely focused on the clinical isolates cultured from subcutaneous drainage of community-acquired liver abscess cases, and an hvKp strain was defined on the basis of co-harboring virulence gene regulator of mucoid phenotype A (rmpA)/rmpA2, iucA, iroB, and peg-344. A total of 47 nonrepetitive hvKp isolates were collected from January 2015 to December 2017 in a tertiary teaching hospital in Shanghai, China. All isolates were susceptible to the commonly used antibiotics. Only one strain (RJ-Kp24) had the positive detection of blaCTX-M-14 and was resistant to ceftazidime, cefotaxime, and cefepime. S1-pulsed-field gel electrophoresis (PFGE) and southern hybridization confirmed the presence of a roughly 90 kb blaCTX-M-14-carrying plasmid and a roughly 240 kb virulence plasmid. Further analysis revealed that ST23 (n = 17) sequence type and K1 (n = 20) and K2 (n = 9) serotypes were dominant in hvKp, while only 31.9% (15/47) and 46.8% (22/47) of hvKp isolates displayed hypermucoviscosity and resistance to serum killing, respectively. For further evaluation of the pathogenicity of hvKp, six representative strains were randomly selected. Three strains, RJ-Kp10, RJ-Kp28, and RJ-Kp31, displayed a remarkable resistance to serum killing and neutrophil phagocytosis. Mouse lethality assay revealed that these strains had the 50% lethal dose (LD50) of 102-103 cell forming unit (CFU), while others had the LD50 of 104-105 CFU. These results demonstrated that strain virulence differed significantly within these defined hvKp. The convergence of multidrug resistance and enhanced virulence in K. pneumoniae has presented a major infection control challenge.

Increased Plasmid Copy Number Contributes to the Elevated Carbapenem Resistance in OXA-232-Producing Klebsiella pneumoniae.

A total of 345 unique (one isolate per patient) clinical carbapenem-resistant Klebsiella pneumoniae isolates were recovered in our hospital over the past 3 years (2016-2018), with 325, 14, and 6 isolates carried blaKPC-2, blaNDM-1, and blaOXA-232, respectively. The six OXA-232-Kp isolates were recovered in neurosurgery intensive care unit in 2018. All OXA-232-Kp belonged to ST15, differed by one or two pulsed-field gel electrophoresis (PFGE) bands, and belonged to the same clone. However, three isolates (RJ18-04-6) displayed elevated carbapenem resistance, with imipenem minimum inhibitory concentration (MIC) of 16-32 µg/mL. In contrast, other three isolates (RJ18-01-3) had imipenem MIC of 0.5-1 µg/mL. S1-PFGE revealed two different plasmid profiles and all strains had at least four plasmids. Strain RJ18-01 and RJ18-06 were selected for whole-genome sequencing, and a ColE2-type 6,141 bp blaOXA-232-carrying plasmid was identified. No blaOXA-232 gene was located on chromosome or other plasmids. Furthermore, no ß-lactamase resistance gene other than blaCTX-M-15 was identified. Polymerase chain reaction-based sequencing of blaOXA-232-carrying plasmid was performed, and all OXA-232-Kp shared identical blaOXA-232-carrying plasmid sequences. The expression and copy number of blaOXA-232 in RJ18-04-6 were significantly higher than those of other isolates. The hydrolysis activity of nitrocefin and carbapenems were about six-fold higher in RJ18-04-6. Since only one copy of blaOXA-232 gene was spotted on the plasmid, the elevated carbapenem resistance could be attributed to the increased copy number of blaOXA-232-carrying plasmids. OmpK35 porin deficiency was observed in one isolate with decreased carbapenem susceptibility and two isolates with elevated carbapenem resistance, suggesting that OmpK35 deficiency did not significantly alter carbapenem MICs in OXA-232-Kp.

Molecular Epidemiology and Risk Factors of Clostridium difficile ST81 Infection in a Teaching Hospital in Eastern China.

Background: The prevalence of Clostridium difficile causes an increased morbidity and mortality of inpatients, especially in Europe and North America, while data on C. difficile infection (CDI) are limited in China. Methods: From September 2014 to August 2019, 562 C. difficile isolates were collected from patients and screened for toxin genes. Multilocus sequence typing (MLST) and antimicrobial susceptibility tests by E-test and agar dilution method were performed. A case group composed of patients infected with sequence type (ST) 81 C. difficile was compared to the non-ST81 infection group and non CDI diarrhea patients for risk factor and outcome analyses. Results: The incidence of inpatients with CDI was 7.06 cases per 10,000 patient-days. Of the 562 C. difficile isolates, ST81(22.78%) was the predominant clone over this period, followed by ST54 (11.21%), ST3 (9.61%), and ST2 (8.72%). Toxin genotype tcdA+tcdB+cdt- accounted for 50.18% of all strains, while 29.54% were tcdA-tcdB+cdt- genotypes. Overall, no isolate was resistant to vancomycin, teicoplanin or daptomycin, and resistance rates to meropenem gradually decreased during these years. Although several metronidazole-resistant strains were isolated in this study, the MIC values decreased during this period. Resistance rates to moxifloxacin and clindamycin remained higher than those to the other antibiotics. Among CDI inpatients, longer hospitalization, usage of prednisolone, suffering from chronic kidney disease or connective tissue diseases and admission to emergency ward 2 or emergency ICU were significant risk factors for ST81 clone infection. All-cause mortality of these CDI patients was 4.92%(n=18), while the recurrent cases accounted for 5.74%(n=21). The 60-day mortality of ST81-CDI was significantly higher than non-ST81 infected group, while ST81 also accounted for most of the recurrent CDI cases. Conclusion: This study revealed the molecular epidemiology and risk factors for the dominant C. difficile ST81 genotype infection in eastern China. Continuous and stringent surveillance on the emerging ST81 genotype needs to be initiated.

Staphylococcus epidermidis Contributes to Healthy Maturation of the Nasal Microbiome by Stimulating Antimicrobial Peptide Production.

The composition of the human microbiome profoundly impacts human well-being. However, the mechanisms underlying microbiome maturation are poorly understood. The nasal microbiome is of particular importance as a source of many respiratory infections. Here, we performed a large sequencing and culture-based analysis of the human nasal microbiota from different age groups. We observed a significant decline of pathogenic bacteria before adulthood, with an increase of the commensal Staphylococcus epidermidis. In seniors, this effect was partially reversed. In vitro, many S. epidermidis isolates stimulated nasal epithelia to produce antimicrobial peptides, killing pathogenic competitors, while S. epidermidis itself proved highly resistant owing to its exceptional capacity to form biofilms. Furthermore, S. epidermidis isolates with high antimicrobial peptide-inducing and biofilm-forming capacities outcompeted pathogenic bacteria during nasal colonization in vivo. Our study identifies a pivotal role of S. epidermidis in healthy maturation of the nasal microbiome, which is achieved at least in part by symbiotic cooperation with innate host defense.

Emergence of an NDM-5-Producing Hypervirulent Klebsiella pneumoniae Sequence Type 35 Strain with Chromosomal Integration of an Integrative and Conjugative Element, ICEKp1.

Here, we report an NDM-5-producing sequence type 35 (ST35) hypervirulent Klebsiella pneumoniae strain, isolated from the blood of a male patient. It showed a remarkable resistance to serum killing and neutrophil phagocytosis and high virulence in a mouse peritonitis infection model. Instead of carrying a pLVPK-like virulence plasmid, chromosomal integration of ICEKp1 (∼76 kb) was identified in a specific asparagine-tRNA gene, harboring the iron acquisition system salmochelin genes (iroBCDN), a yersiniabactin gene, and a variant of the rmpA gene.

A versatile loop-mediated isothermal amplification microchip platform for Streptococcus pneumoniae and Mycoplasma pneumoniae testing at the point of care.

Community-acquired pneumonia (CAP) is the leading cause of mortality in children under five years of age, globally. Given that Streptococcus pneumoniae (S. pneumoniae) and Mycoplasma pneumoniae (M. pneumoniae) are the most common pathogens associated with CAP requiring hospital admission, a simple, low cost, highly sensitive method is in great need for immediate and early diagnosis of CAP. Herein, we report a versatile microfluidic chip platform integrated with loop-mediated isothermal amplification (LAMP) for simultaneous S. pneumoniae and M. pneumoniae testing at the point of care. The platform includes a polymer/paper microfluidic chip and a portable device. On-chip magnetic particle-based nucleic acid extraction is used for concentration of pathogens' genomic DNA and is followed by LAMP. The portable device has the function of heating the microfluidic chip, and photographing and transmitting the result to a smartphone. Complete extraction of the DNA using the microfluidic chip took ~15 min versus >1.5 h with a phenol-chloroform method. The analytical sensitivity of the assay was determined to be 20 fg by testing serial dilutions of target DNA ranging from 2 ng to 2 fg per reaction. We evaluated the clinical sensitivity and specificity of the IPµchip assay using 63 randomly selected oropharyngeal swabs and bronchoalveolar lavage fluid specimens from children. For comparison, these specimens were also tested against real-time PCR assay (M. pneumoniae), conventional PCR assay (S. pneumoniae), and culture tests (S. pneumoniae). These results yielded positive and negative predictive values for M. pneumoniae testing with the IPµchip platform of 96.9% and 100%, respectively. Compared with S. pneumoniae IPµchip, the clinical sensitivity of S. pneumoniae PCR and culture tests was 60% and 40%, respectively, while clinical specificity of the two tests was 100%. This versatile IPµchip platform has great potential for point of care testing of different kinds of pathogens, especially for developing nations.

Clinical application of a multiplex genetic pathogen detection system remaps the aetiology of diarrhoeal infections in Shanghai.

BACKGROUND: Culture-based diagnostic methods cannot achieve rapid and precise diagnoses for the identification of multiple diarrhoeal pathogens (DPs). A high-throughput multiplex genetic detection system (HMGS) was adapted and evaluated for the simultaneous identification and differentiation of infectious DPs and a broad analysis of DP infection aetiology. RESULTS: DP-HMGS was highly sensitive and specific for DP detection compared with culture-based techniques and was similar to singleplex real-time PCR. The uniform level of sensitivity of DP-HMGS for all DPs allowed us to remap the aetiology of acute diarrhoeal infections in Shanghai, correcting incidences of massively underdiagnosed DP species with accuracy approaching that of sequencing-based methods. The most frequent DPs were enteropathogenic Escherichia coli, rotavirus and Campylobacter jejuni. DP-HMGS detected two additional causes of infectious diarrhoea that were previously missed by routine culture-based methods: enterohemorrhagic E. coli and Yersinia enterocolitica. We demonstrated the age dependence of specific DP distributions, especially the distributions of rotavirus, intestinal adenovirus and Clostridium difficile in paediatric patients as well as those of dominant bacterial infections in adults, with a distinct "top 3" pattern for each age group. Finally, the multiplexing capability and high sensitivity of DP-HMGS allowed the detection of infections co-induced by multiple pathogens (approximately 1/3 of the cases), with some DPs preferentially co-occurring as infectious agents. CONCLUSIONS: DP-HMGS has been shown to be a rapid, specific, sensitive and appropriate method for the simultaneous screening/detection of polymicrobial DP infections in faecal specimens. Widespread use of DP-HMGS is likely to advance routine diagnostic and clinical studies on the aetiology of acute diarrhoea.