The roles of IL-19 and IL-20 in the inflammation of degenerative lumbar spondylolisthesis.

BACKGROUND: Degenerative lumbar spondylolisthesis (DLS) is a major cause of spinal canal stenosis and is often related to lower back pain. IL-20 is emerging as a potent angiogenic, chemotactic, and proinflammatory cytokine related to several chronic inflammatory bone disorders likes intervertebral disc herniation, rheumatoid arthritis (RA), osteoporosis, and bone fracture. IL-19 also acts as a proinflammatory cytokine in RA. The aim of the present study was to investigate whether IL-19 and IL-20 are involved in DLS and compare three different tissues including disc, facet joint, and ligamentum flavum of patients with DLS to verify which tissue is affected more by inflammation. METHODS: Disc, facet joint and ligamentum flavum from 13 patients with DLS was retrieved, and the expression pattern of IL-19, IL-20, IL-20R1, IL-20R2, TNF-α, IL-1ß, and MCP-1 was evaluated using immunohistochemical staining with specific antibodies. The disc cells were isolated and incubated with IL-19 and IL-20 under CoCl2-mimicked hypoxic conditions to analyze the proinflammatory cytokine expression pattern using real-time quantitative PCR with specific primers. RESULTS: IL-19 and IL-20 were positively stained and accompanied by abundant expression of TNF-α, IL-1ß, and MCP-1 in facet joints of DLS patients. IL-19 and IL-20's receptors (IL-20R1 and IL-20R2) were expressed on chondrocytes and fibrocytes/fibroblasts in facet joint and ligamentum flavum tissues from patients with DLS. There was a significant correlation between the expression of IL-20 and IL-1ß in facet joint. In vitro assay, IL-19 and IL-20 upregulated the expression of IL-1ß, IL-6, TNF-α, IL-8, VEGF, and MCP-1 in primary cultured DLS disc cells under CoCl2-mimicked hypoxic conditions. CONCLUSIONS: IL-19, IL-20, and their receptors as well as proinflammatory cytokines (TNF-α, IL-1ß, and MCP-1) were expressed more in facet joints than the other tissues in patients with DLS; therefore, the etiology of inflammation might be more facet-centric. IL-19 and IL-20 induced proinflammatory cytokine expression in disc cells and might play a role in the pathogenesis of DLS.

Characterization of CRISPR-Cas Systems in Clinical Klebsiella pneumoniae Isolates Uncovers Its Potential Association With Antibiotic Susceptibility.

Prokaryotic CRISPR-Cas systems limit the acquisition of genetic elements and provide immunity against invasive bacteriophage. The characteristics of CRISPR-Cas systems in clinical Klebsiella pneumoniae isolates are still unknown. Here, 97 K. pneumoniae genomes retrieved from the Integrated Microbial Genomes & Microbiomes genome database and 176 clinical isolates obtained from patients with bloodstream (BSI, n = 87) or urinary tract infections (UTI, n = 89) in Taiwan, were used for analysis. Forty out of ninety-seven genomes (41.2%) had CRISPR-Cas systems identified by the combination of CRISPRFinder and cas1 gene sequence alignment. The phylogenetic trees revealed that CRISPR-Cas systems in K. pneumoniae were divided into two types (type I-E, 23; subtype I-E∗, 17) based on the sequences of Cas1 and Cas3 proteins and their location in the chromosome. The distribution of type I-E and I-E∗ CRISPR-Cas systems was associated with the multilocus sequence typing and the pulsed-field gel electrophoresis results. Importantly, no CRISPR-Cas system was identified in published genomes of clonal complex 258 isolates (ST11 and ST258), which comprise the largest multi-drug resistant K. pneumoniae clonal group worldwide. PCR with cas-specific primers showed that 30.7% (54/176) of the clinical isolates had a CRISPR-Cas system. Among clinical isolates, more type I-E CRISPR-Cas systems were found in UTI isolates (BSI, 5.7%; UTI, 11.2%), and subtype I-E∗ CRISPR-Cas systems were dominant in BSI isolates (BSI, 28.7%; UTI, 15.7%) (p = 0.042). Isolates which had subtype I-E∗ CRISPR-Cas system were more susceptible to ampicillin-sulbactam (p = 0.009), cefazolin (p = 0.016), cefuroxime (p = 0.039), and gentamicin (p = 0.012), compared to the CRISPR-negative isolates. The strains containing subtype I-E∗ CRISPR-Cas systems had decreased numbers of plasmids, prophage regions, and acquired antibiotic resistance genes in their published genomes. Here, we first revealed subtype I-E∗ CRISPR-Cas system in K. pneumoniae potentially interfering with the acquisition of phages and plasmids harboring antibiotic resistance determinants, and thus maintained these isolates susceptible to antibiotics.

Comparative Genomics of Escherichia coli Sequence Type 219 Clones From the Same Patient: Evolution of the IncI1 blaCMY-Carrying Plasmid in Vivo.

This study investigates the evolution of an Escherichia coli sequence type 219 clone in a patient with recurrent urinary tract infection, comparing isolate EC974 obtained prior to antibiotic treatment and isolate EC1515 recovered after exposure to several ß-lactam antibiotics (ceftriaxone, cefixime, and imipenem). EC974 had a smooth colony morphology, while EC1515 had a rough colony morphology on sheep blood agar. RAPD-PCR analysis suggested that both isolates belonged to the same clone. Antimicrobial susceptibility tests showed that EC1515 was more resistant to piperacillin/tazobactam, cefepime, cefpirome, and ertapenem than EC974. Comparative genomic analysis was used to investigate the genetic changes of EC974 and EC1515 within the host, and showed three plasmids with replicons IncI1, P0111, and IncFII in both isolates. P0111-type plasmids pEC974-2 and pEC1515-2, contained the antibiotic resistance genes aadA2, tetA, and drfA12. IncFII-type plasmids pEC974-3 and pEC1515-3 contained the antibiotic resistance genes blaTEM-1, aadA1, aadA22, sul3, and inuF. Interestingly, blaCMY-111 and blaCMY-4 were found in very similar IncI1 plasmids that also contained aadA22 and aac(3)-IId, from isolates EC974 (pEC974-1) and EC1515 (pEC1515-1), respectively. The results showed in vivo amino acid substitutions converting blaCMY-111 to blaCMY-4 (R221W and A238V substitutions). Conjugation experiments showed a high frequency of IncI1 and IncFII plasmid co-transference. Transconjugants and DH5α cells harboring blaCMY-4 or blaCMY-111 showed higher levels of resistance to ampicillin, amoxicillin, cefazolin, cefuroxime, cefotaxime, cefixime, and ceftazidime, but not piperacillin/tazobactam, cefpime, or ertapenem. All known genes (outer membrane proteins and extended-spectrum AmpC ß-lactamases) involved in ETP resistance in E. coli were identical between EC974 and EC1515. This is the first study to identify the evolution of an IncI1 plasmid within the host, and to characterize blaCMY-111 in E. coli.

Role of class II P fimbriae and cytokine response in the pathogenesis of Escherichia coli kidney infection in diabetic mice.

BACKGROUND: The role of class II P fimbriae (P fimbriae II) in diabetic kidney infections is uncertain, although some genetic and epidemiological studies suggest a lower prevalence of P fimbriae II genes in Escherichia coli strains isolated from diabetic patients with complicated kidney infections. METHODS: We inoculated a P fimbriae II deficient E. coli (DH5αT) or an isogenic P fimbriae II expressing transformant (DH5αTP) into the bladders of diabetic and non-diabetic BALB/C mice, and sacrificed them after 3 days. The incidence of bladder or kidney infection (≥103 CFU of E. coli per bladder or kidney), bacteremia (≥102 CFU of E. coli on blood culture plate), kidney pathological score, immunoreactive Histo-score (H-score), and corrected H-score (H-score adjusted for Log10 CFU of bacteria in the kidney) were compared among groups. RESULTS: Diabetic mice were more susceptible to bladder infection than non-diabetic mice with both transformants. The geometric mean of bacteria counts in kidneys was significantly increased only when the diabetic mice were infected with DH5αTP. Among the 4 groups of mice, diabetic mice infected with DH5αTP had the highest incidence of kidney infection and bacteremia, and the highest renal pathology scores. The IL-8 H-score and the corrected IL-6 and IL-8 H-score were significantly lower in diabetic than non-diabetic mice. CONCLUSION: We concluded that P fimbriae II contribute to the pathogenesis and severity of E. coli kidney infections in diabetic mice. An impaired cytokine response may also contribute to the increased incidence and severity of kidney infections in diabetic hosts.

Complete Genome Sequence of Carbapenem-Resistant Klebsiella pneumoniae Strain 1756, Isolated from a Pus Specimen.

Carbapenem-resistant Klebsiella pneumoniae strain 1756 was isolated from a pus specimen from a Taiwanese patient. Here, the complete genome sequence of strain 1756 is presented.

Molecular characterization of antimicrobial susceptibility of Salmonella isolates: First identification of a plasmid carrying qnrD or oqxAB in Taiwan.

BACKGROUND/PURPOSE: The aim of this study is to characterize antibiotic-nonsusceptible Salmonella isolates in Taiwan. METHODS: A total of 76 Salmonella isolates showing lower susceptibility to cephalosporins or quinolones were identified from 1416 clinical isolates from 1999 to 2008. Minimal inhibitory concentrations for selected antimicrobial agents were tested by the agar dilution method. Antibiotic resistance-related genes were determined by polymerase chain reaction (PCR) combined with sequencing. Southern blotting, conjugation tests, and transformation tests were used to characterize plasmid-mediated quinolone resistance (PMQR) determinants. RESULTS: The observed nonsusceptible phenotypes of 76 isolates were against cefoxitin (57.9%), cefotaxime (43.4%), ceftazidime (40.8%), ceftriaxone (42.1%), cefepime (5.3%), ciprofloxacin (80.3%), and levofloxacin (81.6%). Among 44 cephalosporin-resistant isolates, TEM-1, CMY-2, CMY-14, CTX-M-3-like and CTX-M-15-like determinants were present in 31 (70.5%), 32 (72.7%), 1 (2.3%), 1 (2.3%), and 1 (2.3%) of isolates, respectively. PCR screening for PMQR genes of 62 quinolone-nonsusceptible isolates revealed the presence of qnrS, qnrD, aac(6')-Ib-cr, and oqxAB in 3 (4.8%), 2 (3.2%), 1 (1.6%), and 10 (16.1%) isolates, respectively. Among 36 isolates showing high resistance to quinolones, S83F/D87N and S83F/D87G amino acid substitutions of GyrA were found in 29 (80.6%) and 6 (16.7%) isolates, respectively. Moreover, among quinolone highly resistant isolates, eight (22.2%) of isolates showed over-expression of the PAßN-sensitive efflux pump. Transformants and transconjugants harboring qnrD- or oqxAB-plasmids showed decreased susceptibility to quinolones. CONCLUSION: GyrA mutations are the major mechanisms associated with quinolone-resistant Salmonella isolates in Taiwan. Overproduction of efflux pump genes and the presence of qnr and oqxAB play additional roles in reduced susceptibility to quinolones.

Plasmid-mediated quinolone resistance determinants in quinolone-resistant Escherichia coli isolated from patients with bacteremia in a university hospital in Taiwan, 2001-2015.

The aim of this study was to characterize fluoroquinolone (FQ)-resistant Escherichia coli isolates from bacteremia in Taiwan in 2001-2015. During the study period, 248 (21.2%) of 1171 isolates were identified as levofloxacin-resistant. The results of phylogenetic group analysis showed that 38.7% of the FQ-resistant isolates belonged to phylogenetic group B2, 23.4% to group B1, 22.6% to groupA, 14.9% to group D, and 0.4% belonged to group F. FQ-resistant isolates were highly susceptible to cefepime (91.5%), imipenem (96.0%), meropenem (98.8%), amikacin (98.0%), and fosfomycin (99.6%), as determined by the agar dilution method. ß-lactamases, including blaTEM (66.1%), blaCMY-2 (16.5%), blaCTX-M (5.2%), blaDHA-1 (1.6%), and blaSHV-12 (1.6%), were found in FQ-resistant isolates. The results of PCR and direct sequencing showed that 37 isolates (14.9%) harbored plasmid-mediated quinolone resistance (PMQR) genes. qnrB2, qnrB4, qnrS1, coexistence of qnrB4 and qnrS1, oqxAB, and aac(6')-Ib-cr were found in 1, 4, 4, 1, 15, and 14 isolates, respectively. PMQR genes were successfully transfered for 11 (29.7%) of the 37 PMQR-harboring isolates by conjugation to E. coli C600. These findings indicate that qnr genes remained rare in E. coli but demonstrate the potential spread of oqxAB and aac(6')-Ib-c in Taiwan.

Detection of Mycobacterium tuberculosis Complex in Paraffin-Embedded Tissues by the New Automated Abbott RealTime MTB Assay.

OBJECTIVE: The Abbott RealTime MTB assay, launched in June 2014, has been shown to have a competitive performance in the detection of the Mycobacterium tuberculosis (MTB) complex in respiratory specimens. The present study was conducted to investigate the usefulness of the Abbott MTB Realtime assay in the detection of MTB in formalin-fixed paraffin-embedded (FFPE) tissues. METHODS: A total of 96 FFPE specimens obtained from microbiologically proven MTB cases (N=60) and nontuberculous Mycobacterium cases (N=36) were analyzed. The performance of the Abbott MTB Realtime assay was compared with that of the Roche Cobas TaqMan MTB assay. RESULTS: The overall sensitivity and specificity of the Abbott assay were 63.3% and 97.2%, respectively, compared with 11.7% and 100% for the Cobas assay. The detection rate of the Abbott assay was much higher among 37 acid-fast-positive specimens than among 23 acid-fast-negative specimens (89.3% versus 21.7%, respectively). The detection rate of the assay was higher among 29 resection specimens than among 31 small biopsy specimens (86.2% versus 41.9%, respectively). CONCLUSION: Our results suggest that the Abbott RealTime MTB assay can be used to differentiate MTB from nontuberculous mycobacterial infections in acid-fast-positive FFPE tissues.

Overproduction of active efflux pump and variations of OprD dominate in imipenem-resistant Pseudomonas aeruginosa isolated from patients with bloodstream infections in Taiwan.

BACKGROUND: The emergence of imipenem-resistant Pseudomonas aeruginosa (IRPA) has become a great concern worldwide. The aim of this study was to investigate resistance mechanisms associated with bloodstream isolated IRPA strains in Taiwan. RESULTS: A total of 78 non-duplicated IRPA isolates were isolated from patients with bloodstream infection. The average prevalence of imipenem-resistance in those isolates was 5.9 % during a 10-year longitudinal surveillance in Taiwan. PFGE results showed high clonal diversity among the 78 isolates. VIM-2, VIM-3, OXA-10, and OXA-17 ß-lactamases were identified in 2 (2.6 %), 3 (3.8 %), 2 (2.6 %), and 1 (1.3 %) isolates, respectively. Active efflux pumps, AmpC ß-lactamase overproduction, and extended-spectrum AmpC cephalosporinases (ESACs) were found in 58 (74.4 %), 25 (32.1 %) and 15 (19.2 %) of IRPA isolates, respectively. oprD mutations with amino acid substitution, shortened putative loop L7, premature stop codon caused by point mutation, frameshift by nucleotide insertion or deletion, and interruption by insertion sequence were found in 19 (24.4 %), 18 (23.1 %), 15 (19.2 %), 14 (17.9 %), and 10 (12.8 %) of isolates, respectively. CONCLUSIONS: This study suggests that alterations in the OprD protein and having an active efflux pump are the main mechanisms associated with bloodstream isolated IRPA. Overproduction of AmpC, ESACs, and the presence of VIM- and OXA-type ß-lactamases play additional roles in reduced susceptibility to imipenem in P. aeruginosa isolates in Taiwan.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Accelerates Pathogen Identification and May Confer Benefit in the Outcome of Peritoneal Dialysis-Related Peritonitis.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and conventional standard methods were compared for time to pathogen identification and impact on clinical outcomes in peritoneal dialysis-related peritonitis patients. The MALDI-TOF MS method identified the causative microorganisms earlier (average time saved, 64 h for all pathogens), and patients had a shorter hospital stay (mean ± standard deviation, 5.2 ± 4.8 days versus 8.2 ± 4.5 days, P = 0.001).

Molecular characterization of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella spp. isolates in Mongolia.

BACKGROUND/PURPOSE: The aim of this study was to determine the molecular characteristics of ß-lactamase genes in extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae isolates from Mongolia. METHODS: Fifty-six ESBL-producing Enterobacteriaceae isolates were collected, of which 46 were Escherichia coli, seven were Klebsiella pneumoniae, and three were K. oxytoca. Minimum inhibitory concentrations for selected antibiotics were tested using the agar dilution method, and the ß-lactamase genes were determined using polymerase chain reaction combined with sequencing. Pulsed-field gel electrophoresis (PFGE) was used for genotyping all isolates, and phylogenetic grouping was performed on ESBL-producing E. coli isolates. Conjugation tests combined with plasmid digestion assays were used to determine whether there was a horizontal spread in Mongolia. RESULTS: Among the 56 ESBL-producing isolates, 43 isolates (76.8%) were resistant to fluoroquinolones, but all isolates were susceptible to carbapenems and amikacin. The polymerase chain reaction sequencing results showed that the dominant CTX-M genotype was CTX-M-15 (19/46, 41.3%) in the ESBL-producing E. coli isolates. By contrast, CTX-M-14 and CTX-M-3 were the major genotypes found in Klebsiella spp. Phylogenetic analysis revealed that 21 ESBL-producing E. coli isolates belonged to group D (21/46, 45.6%), followed by group A (13/46, 28.3%), group B2 (11/46, 23.9%), and group B1 (1/46, 2.2%). Only four E. coli isolates (4/46, 8.7%) belonged to the ST131 clone. PFGE showed that the ESBL-producing Enterobacteriaceae were genetically unrelated. The conjugation assay showed that two plasmids harboring CTX-M-15 in E. coli isolates were genetic unrelated, whereas seven plasmids harboring CTX-M-14 (5/7 and 2/7) and four plasmids harboring CTX-M-55 (4/4) showed genetic relatedness, indicating the dissemination of resistance plasmids in this area.

Allocation of Klebsiella pneumoniae Bloodstream Isolates into Four Distinct Groups by ompK36 Typing in a Taiwanese University Hospital.

The OmpK36 porin plays a role in carbapenem resistance and may contribute to bacterial virulence in Klebsiella pneumoniae. This study aimed to investigate the characteristics of different groups of K. pneumoniae separated by ompK36 typing. Among 226 nonduplicate K. pneumoniae bloodstream isolates collected at a Taiwanese hospital in 2011, four ompK36 types, designated types A, B, C, and D, were identified by PCR in 61, 28, 100, and 36 isolates, respectively; 1 isolate was untypeable. Statistical analysis showed significantly higher rates of antimicrobial resistance (all tested antibiotics except meropenem), extended-spectrum ß-lactamases or DHA-1 (47.5% together), Qnr-type quinolone resistance determinants (50.8%), and IncFIIA-type plasmids (49.2%) in group A than in others. Seventeen isolates were identified as belonging to 3 international high-risk clones (4 sequence type 11 [ST11], 10 ST15, and 3 ST147 isolates); all isolates but 1 ST15 isolate were classified in group A. The significant characteristics of group C were hypermucoviscosity (62.0%) and a higher virulence gene content. This group included all serotype K1 (n = 30), K2 (n = 25), and K5 (n = 3) isolates, 6 of 7 K57 isolates, all isolates of major clones associated with pyogenic liver abscesses (29 ST23, 11 ST65, 5 ST86, 7 ST373, and 1 ST375 isolates), and 16 (94.1%) of 17 isolates causing bacteremic liver abscesses. Twelve (42.9%) of the group B isolates were responsible for bacteremic biliary tract infections. Group D was predominant (83.3%) among 12 K20 isolates. This study suggests that most clinical K. pneumoniae isolates can be allocated into four groups with distinct characteristics based on ompK36 types.

Elevated cerebrospinal fluid endothelin 1 associated with neurogenic pulmonary edema in children with enterovirus 71 encephalitis.

OBJECTIVES: Neurogenic pulmonary edema (NPE) is a fatal complication in children with enterovirus 71 (EV71) encephalitis. Endothelin 1 (ET-1), a potent vasoconstrictor, can induce pulmonary edema in rats via intrathecal injections. Thus, it was hypothesized that ET-1 in the central nervous system may correlate with NPE in children with EV71 encephalitis. METHODS: Clinical data and ET-1 in the cerebrospinal fluid (CSF) were compared between three groups: (1) EV71 encephalitis with NPE; (2) EV71 encephalitis without NPE; and (3) non-EV71 aseptic meningitis. ET-1 immunostaining was performed on the brainstem of autopsy patients. RESULTS: The EV71 with NPE group showed significantly increased CSF levels of ET-1 compared to the EV71 without NPE and the non-EV71 aseptic meningitis groups (both p<0.01). The optimum cut-off point of ET-1 to predict NPE in EV71 patients, based on the receiver operating characteristic curve, was 0.5 pg/ml (sensitivity 83%, specificity 100%). Immunostaining in the brainstem showed increased ET-1 expression, mainly in the oligodendrocytes, in EV71 with NPE patients compared with control patients. CONCLUSION: ET-1 in the central nervous system may play a role in the development of NPE in children with EV71 infection and could be used as a biomarker or therapeutic target for NPE in EV71 encephalitis.

Deregulation of SLIT2-mediated Cdc42 activity is associated with esophageal cancer metastasis and poor prognosis.

INTRODUCTION: SLIT2, a secreted protein, has been found to inactivate Cdc42 GTPase to modulate neural cell migration. However, alteration of SLIT2-mediated Cdc42 in terms of migration regulation remains undefined in esophageal squamous cell carcinoma (ESCC). METHODS: We report here in ESCC cell, animal, and clinical models that SLIT2 acts as a migration suppressor and serves as a prognostic biomarker. RESULTS: The immunohistochemistry data indicated that 31.8% (49 of 154) of tumors from ESCC patients showed low expression of SLIT2 protein which correlated with poor overall survival and disease-free survival. DNA methylation analysis suggested that promoter hypermethylation is responsible for low expression of SLIT2 in ESCC. Knockdown of SLIT2 increased ESCC cell migration, while SLIT2 stable overexpression reduced cell migration. ESCC cells treated with conditioned media from cells overexpressing SLIT2 also suppressed cell migration. Importantly, silencing of SLIT2 decreased the complex formation, and thus induced Cdc42 activity and promoted membrane localization of focal adhesion kinase and Paxillin. Anti-metastatic effect of SLIT2 was confirmed in an experimental metastasis model of SLIT2 knockdown ESCC cells. CONCLUSION: Our results provide novel evidence that low expression of SLIT2 correlates with poor prognosis and promotes metastasis in ESCC, which may be regulated by the Cdc42-mediated pathways.

Early identification of microorganisms in blood culture prior to the detection of a positive signal in the BACTEC FX system using matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

BACKGROUND: Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a valuable method for rapid identification of blood stream infection (BSI) pathogens. Integration of MALDI-TOF MS and blood culture system can speed the identification of causative BSI microorganisms. MATERIALS AND METHODS: We investigated the minimal microorganism concentrations of common BSI pathogens required for positive blood culture using BACTEC FX and for positive identification using MALDI-TOF MS. The time to detection with positive BACTEC FX and minimal incubation time with positive MALDI-TOF MS identification were determined for earlier identification of common BSI pathogens. RESULTS: The minimal microorganism concentrations required for positive blood culture using BACTEC FX were >10(7)-10(8) colony forming units/mL for most of the BSI pathogens. The minimal microorganism concentrations required for identification using MALDI-TOF MS were > 10(7) colony forming units/mL. Using simulated BSI models, one can obtain enough bacterial concentration from blood culture bottles for successful identification of five common Gram-positive and Gram-negative bacteria using MALDI-TOF MS 1.7-2.3 hours earlier than the usual time to detection in blood culture systems. CONCLUSION: This study provides an approach to earlier identification of BSI pathogens prior to the detection of a positive signal in the blood culture system using MALDI-TOF MS, compared to current methods. It can speed the time for identification of BSI pathogens and may have benefits of earlier therapy choice and on patient outcome.