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IntroductionHypervirulent Klebsiella pneumoniae produce an increased amount of capsular substance and are associated with a hypermucoviscous phenotype. Capsule production is regulated by capsular regulatory genes and capsular gene cluster variations. In the present study, we focus on the effect of rmpA and wcaJon capsule biosynthesis.MethodsPhylogenetic trees were constructed to analyze wcaJ and rmpA sequence diversity in different serotypes hypervirulent strains. Then mutant strains (K2044ΔwcaJ, K2044K1wcaJ, K2044K2wcaJand K2044K64wcaJ) were used to verify the effects of wcaJ and its diversity on capsule synthesis and strain virulence. Furthmore, the role of rmpA in capsular synthesis and its mechanisms were detected in K2044ΔrmpA strain.ResultsRmpA sequences are conversed in different serotypes. And rmpA promoted the production of hypercapsules by simultaneously acting on three promoters in cps cluster. Whereas wcaJ, its sequences are different in different serotypes, and its loss result in the termination of capsular synthesis. Moreover, the results verified that K2 wcaJ could form hypercapsule in K2044 strains (K1 serotype), but K64 wcaJ could not.DiscussionThe interaction of multiple factors is involved in capsule synthesis, including wcaJ and rmpA. RmpA, an known conserved capsular regulator gene, acts on cps cluster promoters to promote the production of the hypercapsule. WcaJ as initiating enzyme of CPS biosynthesis, its presence determines the synthesis of capsule. Besides, different from rmpA, wcaJ sequence consistency is limited to the same serotype, which cause wcaJ functioning in different serotype strains with sequence recognition specificity.

PurposeTo investigate the mechanisms of Klebsiella pneumoniae-induced pyogenic liver abscess (PLA).MethodsForty-three K. pneumoniae strains from PLAs and 436 from non-PLAs were collected. Their differences were compared for virulence genes and factors, sequence types, and serotypes. Virulence genes wzi, wzy-K1, and wzi+wzy-K1 were deleted in K. pneumoniae NTUH-K2044. Various analyses, such as transmission electron microscopy, neutrophil killing tests, and mouse lethality tests, were used to confirm the consequent changes.ResultsDifferences were found between K. pneumoniae strains from PLA and non-PLA samples for virulence genes and factors, including metabolism genes (allS and peg-344), capsular polysaccharide (CPS)-synthesis channel gene (wzy-K1), CPS-regulating genes (p-rmpA, p-rmpA2, and c-rmpA), and siderophore genes (iucA and iroN). When wzy-K1 was positive, the difference between PLA and non-PLA samples was only observed with c-rmpA. Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 strains reverted to hypovirulence. In the Kupffer cell stimulation assay, interleukin (IL)-6, IL-12, IL-10, and transforming growth factor-β secretions were found to be equivalent in NTUH-K2044, Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 groups. Lower IL-1β and higher tumor necrosis factor-α secretions were observed for Δwzi, Δwzy-K1, and ΔwziΔwzy-K1.ConclusionsHypercapsule production is the cornerstone of hypervirulence, regardless of exopolysaccharides. K1 K. pneumoniae-induced PLA may decrease core inflammatory cytokines rather than increase anti-inflammatory cytokines. Exopolysaccharides could also attenuate the inflammatory response to aid in the immune escape of K. pneumoniae.

IntroductionThe objective of this study is to thoroughly analyze the detailed genomic characteristics of clinical strain 211703 of Aeromonas caviae, which co-carrying blaRSA-1 and blaNDM-1 genes. 211703 was isolated from the patient’s cerebrospinal fluid drainage sample in a Chinese tertiary hospital.MethodsCarbapenemase NDM was detected by the immunocolloidal gold technique. The MIC values were determined by VITEK2. The whole genome sequence of 211703 was analyzed using phylogenetics, genomic comparison, and extensive dissection.ResultsThis study revealed that 211703 only contained a single 4.78 Mb chromosome (61.8% GC content), and no plasmids were discovered in 211703. 15 different types of resistant genes were detected in the genome of 211703, including blaRSA-1 harbored on integrative and mobilizable element (IME) Tn7413a, and blaNDM-1 harbored on integrative and conjugative element (ICE). The ICE and IME were all carried on the chromosome of 211703 (c211703). Detailed comparison of related IMEs/ICEs showed that they shared similar conserved backbone regions, respectively. Comprehensive annotation revealed that blaRSA-1 was carried by the gene cassette of a novel integron In2148 on Tn7413a, and blaNDM-1 was captured by an insertion sequence ISCR14-like on the ICE of 211703. We speculated that mobile genetic elements (MGEs) such as ICE and IME facilitated the spread of resistance genes such as blaRSA-1 and blaNDM-1.DiscussionIn conclusion, this study provides an overall understanding of the genomic characterization of clinically isolated A. caviae 211703, and an in-depth discussion of multiple acquisition methods of drug resistance genes in Aeromonas. To the best of our knowledge, this is the first report of A. caviae carrying blaRSA-1 even both blaRSA-1 and blaNDM-1, and this is the first bacterium carrying blaRSA-1 isolated from the clinical setting.

Xinhua Luo,1,* Jin Zhang,1,* Min Yuan,2 Sihua Mou,1 Mengqiao Xu,1 Dakang Hu,1 Qinfei Ma,1 Lingfen Sun,1 Piaopiao Li,1 Zhiwei Song,1 Lianhua Yu,1 Kai Mu3,4 1Department of Clinical Laboratory Medicine, Taizhou Municipal Hospital Affiliated with Taizhou University, Taizhou, 318000, People’s Republic of China; 2State Key Laboratory for Infectious Diseases Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206, People’s Republic of China; 3Beijing Institute of Radiation Medicine, Beijing, 100850, People’s Republic of China; 4Beijing Key Laboratory of New Molecular Diagnosis Technologies for Infectious Diseases, Beijing, 100850, People’s Republic of China*These authors contributed equally to this workCorrespondence: Kai Mu, Beijing Key Laboratory of New Molecular Diagnosis Technologies for Infectious Diseases, Beijing, 100850, People’s Republic of China, Tel +86-010-66874794, Email kai_mu@outlook.com Lianhua Yu, Department of Clinical Laboratory Medicine, Taizhou Municipal Hospital affiliated with Taizhou University, Taizhou, 318000, People’s Republic of China, Email yulianhua64@126.comPurpose: This study aimed to explore the genomic characterization of multidrug-resistant IncHI5-carrying Klebsiella michiganensis strains and detailed genomic dissection of the IncHI5 plasmids.Materials and Methods: Through whole-genome sequencing, the IncHI5 plasmid pK92-qnrS was obtained from a single clinical K. michiganensis isolate K92. All complete genomes of K. michiganensis strains from the Genome database of NCBI were collected and used to construct a maximum likelihood (ML) phylogenetic tree. The epidemiology and geographic distribution of all the K. michiganensis strains were conducted. An extensive comparison of the seven IncHI5 plasmids of K. michiganensis (one from this study, six from GenBank) was applied.Results: This study revealed that all K. michiganensis strains carrying IncHI5 plasmids from different clonal groups were located in the southeast coastal area of China. The backbone regions of IncHI5 plasmids were composed of replicon (repHI5B and repFIB), partition (parABC), and conjugal transfer (tra1/tra2). The main accessory resistant regions of IncHI5 could be divided into two categories, Tn 1696-related region and Tn 6535-related region. These seven IncHI5 plasmids carried multiple drug-resistance genes which were all mediated by the mobile genetic elements (MGEs).Conclusion: Data presented here help to provide an overall in-depth understanding of epidemiology and geographic distribution of IncHI5-carrying K. michiganensis and the structure and evolutionary history of IncHI5 plasmids.Keywords: Klebsiella michiganensis, mobile genetic elements, Tn 1696, Tn 6535, antimicrobial resistance

This is the first report of extensively drug-resistant C. portucalensis harboring both bla KPC-2 and bla NDM-1 . This study will not only extend the understanding of the structural dissection of plasmids and chromosomes carried in C. portucalensis , but also expand knowledge of the genetic environment of the bla KPC-2 and bla NDM-1 genes. bla KPC-2 and bla NDM-1 genes have been suggested to facilitate the propagation and persistence of their host bacteria under different antimicrobial selection pressures.

Coronavirus disease 2019 (COVID-19) is an extremely contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early disease recognition of COVID-19 is crucial not only for prompt diagnosis and treatment of the patients, but also for effective public health surveillance and response. The reverse transcription-polymerase chain reaction (RT-PCR) is the most common method for the detection of SARS-CoV-2 viral mRNA and is regarded as the gold standard test for COVID-19. However, this test and those for antibodies (IgM and IgG) and antigens have certain limitations (e.g., by yielding false-negative and false-positive results). We have developed an RNA fluorescence in situ hybridization (FISH) method for high-sensitivity detection of SARS-CoV-2 mRNAs in HEK 293T cell cultures as a model. After transfection of HEK 293T cells with plasmids, Spike (S)/envelope (E) proteins and their mRNAs were clearly detected inside the cells. In addition, hybridization time could be reduced to 2 hours for faster detection when probe concentration was increased. Our approach might thus significantly improve the sensitivity and specificity of SARS-CoV-2 detection and be widely applied for the high-sensitivity single-molecular detection of other RNA viruses (e.g., Middle East respiratory syndrome coronavirus (MERS-CoV), Hepatitis A virus, all influenza viruses, and human immunodeficiency virus (HIV)) in various types of samples including tissue, body fluid, blood, and water. RNA FISH can also be utilized for the detection of DNA viruses (e.g., Monkeypox virus, human papillomavirus (HPV), and cytomegalovirus (CMV)) by detection of their mRNAs inside cells or body fluid.

Klebsiella pneumoniae is a notorious bacterium in clinical practice. Virulence, carbapenem-resistance and their convergence among K. pneumoniae are extensively discussed in this article. Hypervirulent K. pneumoniae (HvKP) has spread from the Asian Pacific Rim to the world, inducing various invasive infections, such as pyogenic liver abscess, endophthalmitis, and meningitis. Furthermore, HvKP has acquired more and more drug resistance. Among multidrug-resistant HvKP, hypervirulent carbapenem-resistant K. pneumoniae (Hv-CRKP), and carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) are both devastating for their extreme drug resistance and virulence. The hypervirulence of HvKP is primarily attributed to hypercapsule, macromolecular exopolysaccharides, or excessive siderophores, although it has many other factors, for example, lipopolysaccharides, fimbriae, and porins. In contrast with classical determination of HvKP, that is, animal lethality test, molecular determination could be an optional and practical method after improvement. HvKP, including Hv-CRKP and CR-HvKP, has been progressing. R-M and CRISPR-Cas systems may play pivotal roles in such evolutions. Hv-CRKP and CR-HvKP, in particular the former, should be of severe concern due to their being more and more prevalent.

Background To investigate the epidemiology of Klebsiella pneumoniae (K. pneumoniae) inducing pyogenic liver abscess (PLA) in east China and the role of hypervirulent carbapenem-resistant K. pneumoniae (Hv-CRKP). Methods Forty-three K. pneumoniae strains were collected from 43 patients with PLA at Hangzhou, China in 2017. Antimicrobial susceptibility tests, string test, multilocus sequence typing, pulsed-field gel electrophoresis, mobile genetic elements typing, regular PCR and sequencing, and Galleria mellonella (G. mellonella) lethality test were used to elucidate the epidemiology. Clinical data were collected. Results K. pneumoniae strains with serotypes K1 and K2 accounted for 69.8%, which shared 46.5% and 23.3% respectively. K. pneumoniae strains with clonal group 23 were predominant with a rate of 34.9%. Such antimicrobials showed susceptible rates over 80.0%: cefuroxime, cefotaxime, gentamycin, ticarcillin/clavulanate, ceftazidime, cefoperazone/tazobactam, cefepime, aztreonam, imipenem, meropenem, amikacin, tobramycin, ciprofloxacin, levofloxacin, doxycycline, minocycline, tigecycline, chloramphenicol, and trimethoprim-sulfamethoxazole. PFGE dendrogram showed 29 clusters for the 43 K. pneumoniae strains. Three Hv-CRKP strains were confirmed by G. mellonella lethality test, showing a constituent ratio of 7.0% (3/43). Totally three deaths were found, presenting a rate of 7.0% (3/43). The three died patients were all infected with Hv-CRKP. Conclusions K1 and K2 are the leading serotypes of K. pneumoniae causing PLA, which show highly divergent genetic backgrounds. Aminoglycosides, Generation 2nd to 4th cephalosporins, β-lactamase/β-lactamase inhibitors, carbapenems, fluoroquinolones are empirical choices. Hv-CRKP may confer an urgent challenge in the future.

K1/K2 hvKP strains acquire carbapenem-resistance plasmids, known as CR-hvKp, and carbapenem-resistant

ObjectiveTo investigate the epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP).MethodsTotally 436 K. pneumoniae strains were collected from 7 hospitals in mainland China between 2017.01 and 2018.02. Sequence types, serotypes, antimicrobial-resistance and virulence genes were analyzed. Additionally, string test, capsule stain, Periodic Acid Schiff stain, fitness analysis, quantitative real-time PCR and mouse lethality test were also performed. Molecular combinations were used to screen putative blaKPC(+)-HvKP and Hv-blaKPC(+)-KP, followed by the confirmation of mouse lethality test.ResultsDiverse detection rates were found for the virulence genes, ranging from c-rmpA (0.0%) to entB (100.0%). According to the molecular criteria, 127, 186, 9 and 26 strains were putatively denoted as HvKP, blaKPC(+)-KP, blaKPC(+)-HvKP and Hv-blaKPC(+)-KP. Mouse lethality test confirmed 2 blaKPC(+)-HvKP strains (JS184 and TZ20) and no Hv-blaKPC(+)-KP. JS184 showed K2 serotype, thin capsule, positive exopolysaccharid and string test. TZ20 presented K20 serotype, thin capsule, negative exopolysaccharide and string test. Compared with the positive control NTUH-K2044, equal galF expression and growth curves were confirmed for JS184 and TZ20.ConclusionsMolecular determination of CR-HvKP and Hv-CRKP brings remarkable bias compared with mouse lethality test. The exact prevalence of CR-HvKP is less than 1.0%, which of Hv-CRKP is much lower.

To demonstrate the detailed genetic characteristics of a blaNDM–1-carrying multidrug-resistant Aeromonas caviae strain, the complete genome of the A. caviae strain K433 was sequenced by Illumina HiSeq and Oxford nanopore platforms, and mobile genetic elements associated with antibiotic resistance genes were analyzed by a series of bioinformatics methods. A. caviae K433 which was determined to produce class B carbapenemase, was resistant to most antibiotics tested except amikacin. The genome of K433 consisted of a chromosome cK433 (6,482-kb length) and two plasmids: pK433-qnrS (7.212-kb length) and pK433-NDM (200.855-kb length), the last being the first investigated blaNDM-carrying plasmid from Aeromonas spp. By comparison of the backbone and MDR regions from the plasmids studied, they involved a highly homologous sequence structure. This study provides in-depth genetic insights into the plasmids integrated with blaNDM-carrying genetic elements from Aeromonas spp.

To investigate the epidemiology of carbapenem-resistant hypervirulentTotally 436Diverse detection rates were found for the virulence genes, ranging fromMolecular determination of CR-HvKP and Hv-CRKP brings remarkable bias compared with mouse lethality test. The exact prevalence of CR-HvKP is less than 1.0%, which of Hv-CRKP is much lower.

This study aimed to explore the genomic characterization of multidrug-resistant IncHI5-carryingThrough whole-genome sequencing, the IncHI5 plasmid pK92-qnrS was obtained from a single clinicalThis study revealed that allData presented here help to provide an overall in-depth understanding of epidemiology and geographic distribution of IncHI5-carrying

BackgroundCOVID-19 has been circulating worldwide since December 2019. However, its independent risk factors of mortality need further insights in elderly critical COVID-19 patients.MethodsTotally 48 elderly and critically ill COVID-19 patients with clear end point were enrolled when the data were collected, with 16 discharged and 32 died. Kaplan-Meier analysis and Cox regression were performed to identify the risk factors of mortality in elderly critical COVID-19 patients. Survival curve was conducted to present the impact of Diabetes Mellitus on mortality. Mann-Whitney U and t tests were used to 39 clinical variates between the survivor and non-survivor groups.ResultsAs Kaplan-Meier analysis confirmed, only three variates Diabetes Mellitus, chronic obstructive pulmonary disease and family aggregation showed significant difference between the survivor and non-survivor groups. However, only variate Diabetes Mellitus presented significance in Cox regression. Higher C-reaction protein, interleukin-2 (IL-2), IL-8 and creatinine were detected in survivor group than non-survivor group, which was reverse to estimated glomerular filtration rate. The other laboratory finding showed no significant difference between survivor and non-survivor groups.ConclusionsDiabetes Mellitus, chronic obstructive pulmonary disease and family aggregation can contribute to mortality by COVID-19 while Diabetes Mellitus also reduces the survival time of elderly and critically ill COVID-19 patients, highlighting the more important role of Diabetes Mellitus. Laboratory findings could not serve as good predictors of death for elderly and critically ill COVID-19 patients.

Virulence plasmids of hypervirulent Klebsiella pneumoniae (hvKp) have the potential to transfer to drug-resistant strains or integrate with other plasmids, facilitating the genome evolution of threatening pathogens. We conducted an in-depth analysis of the publicly available 156 complete genome sequences of hvKp together with a multi-region clinical cohort of 171 hvKp strains from China to provide evidence for the virulence plasmid evolution. Virulence plasmids were frequently detected in the ST23 and ST11 K. pneumoniae strains. Multidrug-resistant hvKp (MDR-hvKp) occupied a large proportion of hvKp, and the coexistence of virulence and resistance plasmids may be the major cause. Virulence plasmids commonly possessed multiple replicons, of which IncFIBK was the most prevalent (84.6%). We identified 49 IncFIBK alleles among 583 IncFIBK plasmids, and they could be divided into Clades I, II, and III. We further observed that conjugative and non-conjugative virulence plasmids could be distinguished by IncFIBK genetic diversity, and IncFIBK subtyping could also indirectly indicate a chimeric preference of conjugative virulence plasmids. On this basis, we developed an open-access web tool called KpVR for IncFIBK subtyping. In conclusion, the genetic diversity of IncFIBK virulence plasmids could be used for tracking the evolution of virulence plasmids, and further preventing the emergence of MDR-hvKp strains.

ObjectiveTo investigate the overall distributions of key virulence genes in Klebsiella pneumoniae, especially the hypervirulent blaKPC-positive K. pneumoniae (Hv-blaKPC(+)-KP).MethodsA total of 521 complete genomes of K. pneumoniae from GenBank were collected and analyzed. Multilocus sequence typing, molecular serotyping, antibiotic-resistance, virulence genes and plasmid replicon typing were investigated.ResultsPositive rates of virulence genes highly varied, ranging from 2.9 (c-rmpA/A2) to 99.6% (entB). Totally 207 strains presented positive fimH, mrkD, entB and wzi and 190 showed positive fimH, mrkD, entB, irp2 and wzi, which were the two primary modes. A total of 94, 165 and 29 strains were denoted as hypervirulent K. pneumoniae (HvKP), blaKPC(+)-KP and Hv-blaKPC(+)-KP. ST11 accounted for 17 among the 29 Hv-blaKPC(+)-KP strains; Genes iucA, p-rmpA2 and p-rmpA were positive in 28, 26 and 18 Hv-blaKPC(+)-KP strains respectively. Among the 29 Hv-blaKPC(+)-KP strains exhibiting four super clusters from GenBank, IncHI1B plasmids carrying virulence genes and IncFII ones with blaKPC were responsible for both 23 strains respectively.ConclusionsPositive rates of virulence genes vary remarkably in K. pneumoniae. Genes iucA, p-rmpA2 and p-rmpA were primary ones inducing Hv-blaKPC(+)-KP. IncHI1B plasmids carrying virulence genes and IncFII ones with blaKPC constitute the primary combination responsible for Hv-blaKPC(+)-KP. The making of Hv-blaKPC(+)-KP is mostly via blaKPC(+)-KP acquiring another plasmid harboring virulence genes.

Objective To investigate the overall distributions of key virulence genes in Klebsiella pneumoniae, especially the hypervirulent bla KPC-positive K. pneumoniae (Hv-bla KPC(+)-KP). Methods A total of 521 complete genomes of K. pneumoniae from GenBank were collected and analyzed. Multilocus sequence typing, molecular serotyping, antibiotic-resistance, virulence genes and plasmid replicon typing were investigated. Results Positive rates of virulence genes highly varied, ranging from 2.9 (c-rmpA/A2) to 99.6% (entB). Totally 207 strains presented positive fimH, mrkD, entB and wzi and 190 showed positive fimH, mrkD, entB, irp2 and wzi, which were the two primary modes. A total of 94, 165 and 29 strains were denoted as hypervirulent K. pneumoniae (HvKP), bla KPC(+)-KP and Hv-bla KPC(+)-KP. ST11 accounted for 17 among the 29 Hv-bla KPC(+)-KP strains; Genes iucA, p-rmpA2 and p-rmpA were positive in 28, 26 and 18 Hv-bla KPC(+)-KP strains respectively. Among the 29 Hv-bla KPC(+)-KP strains exhibiting four super clusters from GenBank, IncHI1B plasmids carrying virulence genes and IncFII ones with bla KPC were responsible for both 23 strains respectively. Conclusions Positive rates of virulence genes vary remarkably in K. pneumoniae. Genes iucA, p-rmpA2 and p-rmpA were primary ones inducing Hv-bla KPC(+)-KP. IncHI1B plasmids carrying virulence genes and IncFII ones with bla KPC constitute the primary combination responsible for Hv-bla KPC(+)-KP. The making of Hv-bla KPC(+)-KP is mostly via bla KPC(+)-KP acquiring another plasmid harboring virulence genes.

Streptococcus pneumoniae (S. pneumoniae) is a leading agent worldwide, which could cause community-acquired pneumonia, bacteraemia, and meningitis. However, the pathogeneses remain unclear. This study was conducted to investigate gene pneumococcal surface antigen A (psaA) expression and the adhesion differences of various S. pneumoniae strains. A total of 24 (N) S. pneumoniae strains were collected: 11 from blood (bd-SP), 12 from sputum (sd-SP) and one was ATCC49619. One millilitre of A549 pneumocytes (3.3×108/L) and 100 µl of each S. pneumoniae strain at 1.0 McFarland were mixed and incubated under 37 °C and 5% CO2 for three hours. The cells were centrifuged and extracted for psaA mRNA analysis. The former experiment was redone. After culture, the adherent cells were collected and cultured on blood agar plates. The ?CT values of psaA were 18.9, 29.9 (±2.5), 29.6 (±2.0) and 16.0, 17.0(±3.3), 18.6(±3.8) for ATCC49619, bd-SP and sd-SP before and after stimulation respectively, with the colony units of 23, 68.4 (±6.7) and 59.1 (±7.7), which showed equal adhesion between bd-SP and sd-SP. Moderate psaA expression and adhesion of S. pneumoniae might facilitate its pathogenesis, excess of which induces faster S. pneumoniae clearance. Key Words: Streptococcus pneumoniae; Alveolar epithelial cells; Virulence; Adhesion; pneumonia. Continuous...