Coexistence of a novel chromosomal integrative and mobilizable element Tn7548 with two blaKPC-2-carrying plasmids in a multidrug-resistant Aeromonas hydrophila strain K522 from China.

OBJECTIVES: Herein, we detected one multidrug-resistant Aeromonas hydrophila strain K522 co-carrying two blaKPC genes together with a novel chromosomal integrative and mobilizable element (IME) Tn7548 from China. To reveal the genetic characteristics of the novel reservoir of blaKPC-2 and IME in Aeromonas, a detailed genomic characterization of K522 was performed, and a phylogenetic analysis of Tn7412-related IMEs was carried out. METHODS: Carbapenemases were detected by using the immunocolloidal gold technique and antimicrobial susceptibility was tested by using VITEK 2. The whole genome sequences of K522 were analyzed using phylogenetics, detailed dissection and comparison. RESULTS: Strain K522 carried a Tn7412-related chromosomal IME Tn7548 and three resistance plasmids pK522-A-KPC, pK522-B-KPC, and pK522-MOX. A phylogenetic tree of 82 Tn7412-related IMEs was constructed and five families of IMEs were divided. These IMEs shared four key backbone genes int, repC, and hipAB, and carried various profiles of antimicrobial resistance genes (ARGs). pK522-A-KPC and pK522-B-KPC carried blaKPC-2 and belonged to IncG and unclassified type plasmid, respectively. The blaKPC-2 regions of these two plasmids were the truncated version derived from Tn6296, resulting in the carbapenem resistance of K522. CONCLUSIONS: We first reported A. hydrophila harboring a novel Tn7412-related IME Tn7548 together with two blaKPC-2 carrying plasmids and an MDR plasmid. Three of these four MGEs discovered in A. hydrophila K522 were novel. The emergence of novel MGEs carrying ARGs indicated the rapid evolution of the resistance gene vectors in A. hydrophila under selection pressure and would contribute to the further dissemination of various ARGs in Aeromonas.

Higher Virulence Renders K2 Klebsiella pneumoniae a Stable Share Among Those from Pyogenic Liver Abscess.

Objective: To explore why serotype K2 accounts for a stable share in Klebsiella pneumoniae from pyogenic liver abscess (PLA). Methods: Totally 15 K2 K. pneumoniae strains from PLA, 21 K2 from non-PLA, and 31 K1 from PLA were collected from China. Sequence typing, molecular serotyping, regular PCR, and Galleria mellonella lethality were performed. A total of 12 virulence genes were detected: peg-344, allS, p-rmpA, p-rmpA2, c-rmpA, fimH, mrkD, iucA, iroN, irp2, entB, and wzi. The differences between K2 K. pneumoniae strains from PLA and non-PLA were investigated along with K1 ones. Results: Significant differences were found between K2 strains from PLA and non-PLA for the rates of virulence genes peg-344 and iucA. The latter group also showed more diverse sequence types than the former. Significant differences were only found for virulence genes allS and irp2 between K1 and K2 strains from PLA. Based on the equal virulence factors backgrounds other than serotypes, K2 strain is more virulent than K1 as G. mellonella lethality confirmed. Gene p-rmpA only brings equal virulence to p-rmpA plus p-rmpA2 in K2 strain. Conclusion: Based on the same virulence factors backgrounds except serotypes, K2 K. pneumoniae is more virulent than K1 from PLA, which provides a survival advantage to maintain a stable share.

Coexistence of c-rmpA with p-rmpA and p-rmpA2 rather than excessive siderophores confers higher virulence in K1 Klebsiella pneumoniae.

Pyogenic liver abscess (PLA), which is particularly endemic in East Asia, is a relatively common and fatal infectious disease. Over the last 30-40 years, Klebsiella pneumoniae has replaced Escherichia coli as the dominant and overwhelming pathogen. To investigate the survival advantage of serotype K1 K. pneumoniae, we determined sequence types (STs), serotypes, and 11 virulence genes (allS, entB, irp2, iroN, iucA, fimH, mrkD, p-rmpA2, c-rmpA, p-rmpA, and peg-344). Virulence genes c-rmpA, p-rmpA, and p-rmpA2 in K. pneumoniae NTUH-K2044, which all confer hypercapsule and consequent hypervirulence, were deleted individually, and the consequent effects were evaluated. The lethality of various K1 K. pneumoniae strains was compared by using the Galleria mellonella model. In total, 31 K1 K. pneumoniae strains causing PLA and 30 causing non-PLA were identified. A significantly higher rate of c-rmpA was presented in PLA-derived K. pneumoniae strains than in non-PLA-derived strains. Similar ST23 (which dominates K1 strains) and string test-positive rates were observed in the two groups. Deletion of c-rmpA, p-rmpA, and p-rmpA2 individually did not confer significant effects on morphologies, such as positive string test, hypercapsule, and growth speed. Δc-rmpA presented weaker expressions of p-rmpA/p-rmpA2 than NTUH-K2044 and showed a higher expression of manC than Δp-rmpA and Δp-rmpA2. Three rmpAs conferred more virulence than one or two rmpAs, which presented an equally lethal effect in K1 K. pneumoniae. Klebsiella pneumoniae strains (H19 and H34) with the same genetic backgrounds except for siderophores showed equal virulence, but were less virulent than strain NTUH-K2044. Thus, the coexistence of c-rmpA with p-rmpA and p-rmpA2 enhances the lethality of K1 K. pneumoniae strains and the development of PLA. Excessive siderophores are not vital for the hypervirulence of K1 K. pneumoniae strains, although K1 strains usually harbour them on a molecular basis.

Coexistence of blaKPC-2 and blaNDM-1 in one IncHI5 plasmid confers transferable carbapenem resistance from a clinical isolate of Klebsiella michiganensis in China.

OBJECTIVES: This study firstly identified an IncHI5 plasmid pK254-KPC_NDM co-carrying two different class carbapenemase genes blaKPC-2 and blaNDM-1 in Klebsiella michiganensis K254. METHODS: The strain K254 was sequenced by high-throughput genome sequencing. A detailed genomic and phenotypic characterization of pK254-KPC_NDM was performed. RESULTS: pK254-KPC_NDM displayed the conserve IncHI5 backbone and carried a resistant accessory region: Tn1696-related transposon Tn7414 containing blaKPC-2 and blaNDM-1. A sequence comparison was applied to a collection of four Tn1696-related transposons (Tn7414-Tn7417) harbouring carbapenemase genes. For all these four transposons, the blaNDM-1 was carried by Tn125 derivatives within three different mobile genetic elements. Tn7414 further acquired another carbapenemase gene, blaKPC-2, because of the integration of the local blaKPC-2 genetic environment from Tn6296, resulting in the high-level carbapenem resistance of K. michiganensis K254. The conjugal transfer and plasmid stability experiments confirmed that pK254-KPC_NDM could be transferred intercellularly and keep the stable vertical inheritance in different bacteria, which would contribute to the further dissemination of multiple carbapenemase genes and enhance the adaption and survival of K. michiganensis under complex and diverse antimicrobial selection pressures. CONCLUSION: This study was the first to report the K. michiganensis isolate coharbouring blaKPC-2 and blaNDM-1 in the Tn1696-related transposon in IncHI5 plasmid. The emergence of novel transposons simultaneously carrying multiple carbapenemase genes might contribute to the further dissemination of high-level carbapenem resistance in the isolates of the hospital settings and pose new challenges for the treatment of nosocomial infection.

Epidemiology, molecular characterization, and drug resistance of IncHI5 plasmids from Enterobacteriaceae / Epidemiología, caracterización molecular y farmacorresistencia de plásmidos IncHI5 de Enterobacteriaceae

The increasingly frequent occurence of IncHI5 plasmids has attracted worldwide attention. The aim of this study was to perform an in-depth bioinformatics analysis to determine the genetic characteristics and global distribution of all IncHI5 plasmids. The geographic distribution and epidemiology of all IncHI5 plasmids from GenBank were analyzed based on relevant literature reports and background information from the National Center for Biotechnology Information (NCBI). Detailed annotation of antibiotic resistance genes was performed. A total of 65 IncHI5 plasmid genomes were collected in GenBank. All IncHI5 plasmids were carried by Enterobacteriaceae, of which Klebsiella pneumoniae accounted for the largest proportion (50%, 33/65). The host bacterium of IncHI5 plasmids was mainly isolated from Homo Sapiens (81%, 53/65). All strains carrying IncHI5 plasmids were mainly distributed in China (83%, 54/65). Evolutionary analysis can divide IncHI5 plasmids into two groups, namely Groups I/II, of which Group II was more widely distributed worldwide. This study showed that Enterobacteriaceae, especially Klebsiella, was the main host for IncHI5 plasmid. Almost all IncHI5 plasmids carried multiple types of antibiotic resistance genes, related to Tn1696 or Tn6535. The IncHI5 plasmids should be of continuing interest as good repositories for antibiotic resistance genes.(AU)

Genomic analysis of chromosomal cointegrated bla NDM-1-carrying ICE and bla RSA-1-carrying IME from clinical multidrug resistant Aeromonas caviae.

Introduction: The objective of this study is to thoroughly analyze the detailed genomic characteristics of clinical strain 211703 of Aeromonas caviae, which co-carrying bla RSA-1 and bla NDM-1 genes. 211703 was isolated from the patient's cerebrospinal fluid drainage sample in a Chinese tertiary hospital. Methods: Carbapenemase 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. Results: This 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 bla RSA-1 harbored on integrative and mobilizable element (IME) Tn7413a, and bla NDM-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 bla RSA-1 was carried by the gene cassette of a novel integron In2148 on Tn7413a, and bla NDM-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 bla RSA-1 and bla NDM-1. Discussion: In 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 bla RSA-1 even both bla RSA-1 and bla NDM-1, and this is the first bacterium carrying bla RSA-1 isolated from the clinical setting.

Epidemiology, molecular characterization, and drug resistance of IncHI5 plasmids from Enterobacteriaceae.

The increasingly frequent occurence of IncHI5 plasmids has attracted worldwide attention. The aim of this study was to perform an in-depth bioinformatics analysis to determine the genetic characteristics and global distribution of all IncHI5 plasmids. The geographic distribution and epidemiology of all IncHI5 plasmids from GenBank were analyzed based on relevant literature reports and background information from the National Center for Biotechnology Information (NCBI). Detailed annotation of antibiotic resistance genes was performed. A total of 65 IncHI5 plasmid genomes were collected in GenBank. All IncHI5 plasmids were carried by Enterobacteriaceae, of which Klebsiella pneumoniae accounted for the largest proportion (50%, 33/65). The host bacterium of IncHI5 plasmids was mainly isolated from Homo Sapiens (81%, 53/65). All strains carrying IncHI5 plasmids were mainly distributed in China (83%, 54/65). Evolutionary analysis can divide IncHI5 plasmids into two groups, namely Groups I/II, of which Group II was more widely distributed worldwide. This study showed that Enterobacteriaceae, especially Klebsiella, was the main host for IncHI5 plasmid. Almost all IncHI5 plasmids carried multiple types of antibiotic resistance genes, related to Tn1696 or Tn6535. The IncHI5 plasmids should be of continuing interest as good repositories for antibiotic resistance genes.

Emergence of Extensively Drug-Resistant ST170 Citrobacter portucalensis with Plasmids pK218-KPC, pK218-NDM, and pK218-SHV from a Tertiary Hospital, China.

The objective of this study is to characterize the molecular mechanism of a clinical carbapenem-resistant Citrobacter portucalensis strain K218, which coproduces KPC and NDM carbapenemases. K218 was isolated from a patient's blood sample in a Chinese tertiary hospital. Carbapenemases were detected by the immunocolloidal gold technique. The MIC values were determined by VITEK2. Whole-genome sequencing was performed on K218 and sequence data were analyzed using phylogenetics and extensive genomic comparison. This study reveals that K218 contains a single 5.08 Mb chromosome (51.8% GC content) and four plasmids, pK218-KPC (106 Kb), pK218-NDM (111 Kb), pK218-SHV (191 Kb), and pK218-NR (5 Kb). Twenty-nine types of antibiotic resistance genes were carried on K218, including blaKPC-2 harbored on pK218-KPC and blaNDM-1 harbored on pK218-NDM. Detailed comparison of related plasmids of pK218-KPC, pK218-NDM, and pK218-SHV showed that they shared similar conserved backbone regions, respectively. Comprehensive annotation revealed large accessory modules were recombined on the genome of K218. Further analysis speculated that mobile genetic elements bearing abundant resistance genes facilitated the formation of these accessory modules. In conclusion, this study provides an in-depth understanding of the genomic characterization of K218, an extensively drug-resistant C. portucalensis strain coproducing NDM and KPC carbapenemase. To the best of our knowledge, this is the first report of C. portucalensis strain coharboring blaKPC-2 and blaNDM-1 from the clinical setting. IMPORTANCE This is the first report of extensively drug-resistant C. portucalensis harboring both blaKPC-2 and blaNDM-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 blaKPC-2 and blaNDM-1 genes. blaKPC-2 and blaNDM-1 genes have been suggested to facilitate the propagation and persistence of their host bacteria under different antimicrobial selection pressures. Large accessory regions carrying blaKPC-2 and blaNDM-1 genes have become hot spots for transposition and integration, and their structural variation and evolution should receive attention. The multidrug-resistant plasmids pK218-KPC, pK218-NDM, and pK218-SHV with several multidrug resistance regions and the chromosome cK218 with two novel transposons Tn7410 and Tn7411 contribute to the formation of extensively drug-resistant C. portucalensis.

Emergence of bla NDM- 1-Carrying Aeromonas caviae K433 Isolated From Patient With Community-Acquired Pneumonia.

To demonstrate the detailed genetic characteristics of a bla NDM-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 bla NDM-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 bla NDM-carrying genetic elements from Aeromonas spp.

Prevalence of Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae and Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae in China Determined via Mouse Lethality Tests.

Objective: To investigate the epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP). Methods: Totally 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. Results: Diverse 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. Conclusions: Molecular 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.

Epidemiology of Klebsiella michiganensis Carrying Multidrug-Resistant IncHI5 Plasmids in the Southeast Coastal Area of China.

Purpose: 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, Tn1696-related region and Tn6535-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.

Characterization of Clinical and CT Manifestations of Pulmonary Cryptococcosis with Consolidation.

Considering the insufficient understanding of pulmonary cryptococcosis (PC) with consolidation, 22 patients were evaluated based on the chest computed tomography (CT) images and clinical manifestation. The clinical symptoms were mild, mainly manifesting as cough and sputum. Pulmonary lesions mostly involved a single lobe in a single lung with multiple lesions. Specifically, single lung involvement was observed in 17 cases, single lobe in 16 cases and multiple lesions in 14 case. Fifteen cases were mainly distributed in the periphery and 17 cases in the long axis in parallel to the pleura. Nineteen cases had air bronchograms. Eight cases displayed cavitation inside the lesions and 18 cases had surrounding halo signs. Seventeen cases had pleural thickening, of which 10 cases had "pasting wall" signs. The clinical symptoms of PC with consolidation were relatively mild. Comprehensive clinical and imaging performance could improve the diagnosis of the disease.

High-risk KPC-producing Klebsiella pneumoniae lack type I R-M systems.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) have disseminated worldwide and are a major threat to public health. The multidrug-resistant (MDR)-phenotype of KPC-KP are commonly associated with the presence of high molecular weight blaKPC plasmids. Restriction-modification (R-M) systems provide bacteria with innate defense against plasmids or other infectious gene elements. As blaKPC plasmids are favored by such MDR K. pneumoniae, it was of interest to examine the co-distribution of R-M and acquired blaKPC plasmids in KPC-KP. A total of 459 clinical K. pneumoniae isolates in China and 217 global whole-genome sequences in GenBank were collected to determine the prevalence of type I R-M systems. The type I R-M systems were scarce in the KPC-positive group and high-risk Klebsiella pneumoniae clonal group 258 (CG258). The polymorphisms of type I R-M observed in K. pneumoniae revealed the ubiquity of their recognition sequences in DNA; therefore, the type I R-M systems could attack most invading DNA elements, such as blaKPC genes. Overall, this work indicated the type I R-M systems may impact the acquisition of blaKPC genes in K. pneumoniae.

The type I-E CRISPR-Cas system influences the acquisition of blaKPC-IncF plasmid in Klebsiella pneumonia.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) have disseminated worldwide and emerged as major threats to public health. Of epidemiological significance, the international pandemic of KPC-KP is primarily associated with CG258 isolates and blaKPC-IncF plasmids. CRISPR-Cas system is an adaptive immune system that can hinder gene expansion driven by horizontal gene transfer. Because of blaKPC-IncF plasmids are favored by CG258 K. pneumoniae, it was of interest to examine the co-distribution of CRISPR and blaKPC-IncF plasmids in such isolates. We collected 459 clinical K. pneumoniae isolates in China and collected 203 global whole-genome sequences in GenBank to determine the prevalence of CRISPR-Cas systems. We observed that CRISPR-Cas system was significantly scarce in the CG258 lineage and blaKPC-positive isolates. Furthermore, the results of conjugation and plasmid stability assay fully demonstrated the CRIPSR-Cas system in K. pneumoniae could effectively hindered blaKPC-IncF plasmids invasion and existence. Notably, most blaKPC-IncF plasmids were also proved to be good targets of CRISPR owing to carry matched and functional protospacers and PAMs. Overall, our work suggests that type I-E CRISPR-Cas systems could impact the spread of blaKPC in K. pneumoniae populations, and the scarcity of CRISPR-Cas system was one of potential factors leading to the propagation of blaKPC-IncF plasmids in CG258 K. pneumoniae.

Absence of the type I-E CRISPR-Cas system in Klebsiella pneumoniae clonal complex 258 is associated with dissemination of IncF epidemic resistance plasmids in this clonal complex.

BACKGROUND: The pandemics caused by MDR Klebsiella pneumoniae are mostly due to the global dissemination of high-risk clonal complex 258 (CC258) and related IncF epidemic plasmids. However, the factors leading to the epidemiological advantages of CC258-IncF linkage remain obscure. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas) systems, providing adaptive immunity against invading DNA, play an important role in the interactions between plasmids and hosts. OBJECTIVES: To investigate the relationship between CRISPR-Cas systems and the high-risk linkage CC258-IncF. METHODS: CRISPR-Cas loci were detected among 381 collected K. pneumoniae clinical isolates and 207 K. pneumoniae complete genomes available in GenBank. MLST was used to determine the genetic relatedness of these isolates. Nucleotide BLAST was used to search for protospacers on K. pneumoniae plasmids. RESULTS: We observed an epidemic correlation between CRISPR-Cas loci, CC258 and IncF plasmids. Interestingly, most type I-E CRISPR-Cas systems identified carried spacers matching the backbone regions of IncF plasmids. CONCLUSIONS: Our results suggest that the absence of type I-E CRISPR-Cas systems in K. pneumoniae CC258 is strongly associated with the dissemination of IncF epidemic plasmids, contributing to the global success of the international high-risk linkage CC258-IncF. Our findings provide new information regarding the dissemination and evolution of the high-risk linkage of K. pneumoniae CC258-IncF and pave the way for new strategies to address the problem of antibiotic resistance.

RGS4 Regulates Proliferation And Apoptosis Of NSCLC Cells Via microRNA-16 And Brain-Derived Neurotrophic Factor.

PURPOSE: Regulator of G-protein signaling (RGS) proteins are GTPase-activating proteins that target the α-subunit of heterotrimeric G proteins. Many studies have shown that RGS proteins contribute to tumorigenesis and metastasis. However, the mechanism in which RGS proteins, especially RGS4, affect the development of non-small cell lung cancer (NSCLC) remains unclear. The aim of this study was to characterize the role of RGS4 in NSCLC. METHODS: RGS4 expression in NSCLC tissues was assessed using an immunohistochemistry tissue microarray. Additionally, RGS4 was knocked down using short-hairpin RNA to assess the regulatory function of RGS4 in the biological behaviors of human NSCLC cell lines. A xenograft lung cancer model in nude BALB/c mice was established to study whether RGS4 knockdown inhibits cancer cell proliferation in vivo. RESULTS: We observed an increase in RGS4 protein levels in NSCLC samples. RGS4 knockdown inhibited cell proliferation and induced apoptosis in H1299 and PC9 cell lines, but did not affect cell migration. Moreover, we found that RGS4 negatively regulated the expression of microRNA-16 (miR-16), a tumor suppressor. The inhibition of miR-16 resulted in upregulated RGS4 expression. We also found that RGS4 regulated the expression of brain-derived neurotrophic factor (BDNF) and activated the BDNF-tropomyosin receptor kinase B signaling pathway. CONCLUSION: This study revealed that RGS4 overexpression positively correlated with the development of NSCLC. TDownstream RGS4 targets (eg, miR-16 and BDNF) might be involved in the development of NSCLC and may serve as potential therapeutic targets for its treatment.

Impact of different Streptococcus pneumoniae on the secretion of interleukin and adhesin from THP-1 monocytes.

BACKGROUND: To investigate the secretion of interleukin-1ß (IL-1ß), IL-6, IL-10, IL-8, and soluble intercellular adhesin molecule 1 (sICAM-1) from THP-1 monocytes stimulated by different Streptococcus pneumoniae (S pneumoniae) strains. METHODS: Fifty-eight strains of S pneumoniae were collected: ATCC49619, 23 from sputum (sd-SP), 23 from blood (bd-SP), and 11 from cerebrospinal fluid (CSF; cd-SP). Such strains were cultured and suspended at 0.5 McFarland. THP-1 monocytes were cultured and resuspended at 5.0 × 108 /L, which were stimulated by S pneumoniae for 4, 8, and 12 hours, respectively. The suspensions were analyzed for IL-1ß, IL-6, IL-10, IL-8, and sICAM-1 using an ELISA method. The data were assayed with SPSS 19.0. RESULTS: Contrary to IL-10, the concentrations of IL-1ß, IL-6, IL-8, and sICAM-1 all increased first and then decreased. IL-1ß and sICAM-1 levels in the ATCC49619 group were both higher than all the clinical S pneumoniae groups (sd-SP, bd-SP, and cd-SP), IL-6 and IL-8 versa, and IL-10 equal. The difference among clinical S pneumoniae groups lay only in sICAM-1. cd-SP group showed lower sICAM-1 concentrations than sd-SP and bd-SP groups at both 4 and 8 hours. However, they became equal at 12 hours. CONCLUSIONS: The secretion summit is 8 hours for IL-1ß, IL-6, IL-8, and sICAM-1, bottom for IL-10. Different clinical S pneumoniae strains show similar ability to induce THP-1 cells secreting interleukins. However, cd-SP induces THP-1 cells secreting lower sICAM-1 than sd-SP and bd-SP, which may in turn facilitate its invasion into CSF.

Pandemic spread of blaKPC-2 among Klebsiella pneumoniae ST11 in China is associated with horizontal transfer mediated by IncFII-like plasmids.

OBJECTIVES: The aim of this study was to investigate the spread of the blaKPC-2 gene among Klebsiella pneumoniae and to illustrate the mechanism of dissemination of K. pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) ST11 in China. METHODS: A total of 354 K. pneumoniae isolates were collected from four hospitals in China and were characterized by multilocus sequence typing (MLST). Mobile genetic elements (MGEs) and pulsed-field gel electrophoresis (PFGE) analysis were used to identify the subtypes of K. pneumoniae ST11. Polymerase chain reaction (PCR)-based amplification and sequencing were performed to analyse Tn1721 transposons and IncFII-like plasmids. Electroporation experiments and whole-genome sequencing (WGS) were used to reveal the genetic environment of the blaKPC-2 gene. RESULTS: As the primary type (87.1%) of KPC-Kp, K. pneumoniae ST11 was not predominant in non-KPC-Kp (3.1%). ST11 KPC-Kp was clonally heterogeneous and could be further classified into 11 MGE types and 14 PFGE subtypes. Five Tn1721-blaKPC-2 variants were identified on IncFII-like plasmids. The detection rate of IncFII-like plasmids was much higher in ST11 KPC-Kp (100%) compared with non-ST11 KPC-Kp (16.0%) and the non-KPC-Kp group (7.5%). Moreover, the IncFII plasmid (with IIa replicon) was primarily detected on the MGE-F type (61.7%). The IncFIIk plasmid (with IIk replicon) was clustered into two subtypes: MGE-A (28.3%) and -F (41.5%). The detection of the IncFII and IncFIIk plasmids on MGE-A was 57.1% (20/35) and 42.9% (15/35), respectively. CONCLUSIONS: A close correlation was shown between ST11 KPC-Kp and IncFII-like plasmids. Horizontal transfer mediated by IncFII-like plasmids plays an important role in the pandemic expansion of blaKPC-2 among K. pneumoniae ST11 in China.

miR-296 inhibits the metastasis and epithelial-mesenchymal transition of colorectal cancer by targeting S100A4.

BACKGROUND: Dysregulation of microRNAs (miRNAs) is actively involved in the pathogenesis and tumorigenicity of colorectal cancer (CRC). miR-296 was found to play either oncogenic or tumor suppressive role in human cancers. However, the status of miR-296 and its function in CRC remain unknown. METHODS: The expression of miR-296 was confirmed by qRT-PCR in CRC tissues and cells, and its level was altered by corresponding miRNA vectors. Wound healing and Transwall assays were performed to detect the migration and invasion of CRC cells. The levels of proteins were measured using immunoblotting, immunohistochemistry and immunofluorescence. RESULTS: Underexpression of miR-296 was disclosed in CRC tissues and cells. Its decreased level was evidently correlated with adverse clinical parameters and poor prognosis of CRC patients. In vitro experiments indicated that miR-296 inhibited CRC cell migration and invasion. Mechanically, miR-296 inhibited the epithelial-mesenchymal transition (EMT) of CRC cells. A negative correlation between miR-296 and S100A4 expression was observed in CRC tissues. Luciferase reporter assays indicated that miR-296 inversely regulated the luciferase activity of 3'-UTR of S100A4. Herein, S100A4 was found to be a downstream molecule of miR-296 in CRC. Furthermore, S100A4 mediated the anti-metastatic effects of miR-296 on EMT, migration and invasion of CRC cells. CONCLUSIONS: miR-296 functions as an anti-metastatic factor mainly by suppressing S100A4 in CRC. It potentially acts as a prognostic predictor and a drug-target for CRC patients.

Drug Resistance Characteristics and Macrolide-Resistant Mechanisms of Streptococcus pneumoniae in Wenzhou City, China.

BACKGROUND Streptococcus pneumoniae (SP) is a Gram-positive, alpha-hemolytic, facultative anaerobic member of the genus Streptococcus. The erythromycin-resistant methylase (erm) gene and macrolide efflux (mef) gene are the 2 main genes that can mediate SP. Transposon (Tn) also plays an important role in the collection and metastasis of the gene. In the present study we investigated the drug resistance characteristics and the macrolide-resistant mechanisms of SP in Wenzhou City, China. MATERIAL AND METHODS Sixty-eight strains of SP were isolated from sputum samples of hospitalized children in the Second Affiliated Hospital of Wenzhou Medical University. These strains were analyzed using antimicrobial susceptibility tests to determine their drug resistance to 10 kinds of antibacterials. Macrolide-resistant phenotypes were identified using K-B method. PCR method was used to analyze the erm B gene, mef A gene, and int Tn gene. RESULTS Drug resistance rates of 68 strains of SP were 98.5%, 100.0%, 63.2%, 52.9%, 94.1%, 89.7%, 0.0%, 0.0%, 16.2%, and 14.7% for clindamycin, erythromycin, penicillin G, cefotaxime, tetracycline, sulfamethoxazole/trimethoprim, levofloxacin, vancomycin, chloramphenicol, and amoxicillin, respectively. Total detection rates of the erm B gene, mef A gene, and int Tn gene were 98.5%, 91.2%, and 100.0%, respectively. CONCLUSIONS SP shows significant multi-drug resistance in Wenzhou City, whereas there is no clinical value of macrolides antibiotics for SP. cMLSB mediated by erm B gene is the most predominant phenotype among macrolide-resistant SP. The int Tn gene may play an important role in horizontal transfer and clonal dissemination of SP drug resistance genes in Wenzhou City.