Distribution and antimicrobial resistance analysis of gram-negative bacilli isolated from a tertiary hospital in Central China: a 10-year retrospective study from 2012 to 2021

BackgroundGram-negative bacilli are one of the most common causes of various infections in clinical. The emergence and global spread of multi-drug resistant gram-negative bacilli has become a major challenge in the global public health field.MethodsA total of 51,189 non-repetitive strains of gram-negative bacilli were isolated in clinical settings. The antimicrobial susceptibility testing was conducted by using the automated VITEK 2 compact system and the matched AST susceptibility test card, complemented by the disk diffusion method. The antimicrobial susceptibility results were interpreted by CLSI. Rates of MDR and XDR in Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa were investigated. Used the chi-square test to determine whether the antimicrobial resistance rates of four major gram-negative bacilli isolated from ICU and non-ICU department have statistical differences.ResultsEscherichia coli (31.4%), Klebsiella spp. (21.2%), Acinetobacter spp. (13.8%), and P. aeruginosa (11.0%) were the most frequently isolated gram-negative bacilli. Escherichia coli was the top one organism isolated from urinary tract (68.4%), bloodstream (39.9%), body fluid (33.2%), wound and pus (37%), except for respiratory tract (8.8%). Whereas Acinetobacter baumannii and K. pneumoniae were the major isolated organisms from respiratory tract. Acinetobacter baumannii showed high resistance to fluoroquinolones, β-lactam/β-lactamase inhibitor combinations class, ceftazidime, cefepime, imipenem, and meropenem, the resistance rates reached more than 70%. Ceftazidime showed a lower resistance rate to E. coli than ceftriaxone. For E. coli, fluoroquinolones showed a high resistance rate (ciprofloxacin 61.36% and levofloxacin 53.97%), whereas amikacin, carbapenems exhibited a lower resistance rate fluctuating at 2%. Acinetobacter baumannii and K. pneumoniae showed rapid increases in carbapenem resistance whereas E. coli had the lowest resistance rate and remain stable at 2%. Acinetobacter baumannii exhibited the highest rate of MDR and XDR, reaching 60–80 and 45–55%, respectively. Compared to non-ICU departments, the resistance rates of four major gram-negative bacilli in the ICU department were much higher and the differences were statistically significant (p < 0.05).ConclusionAmikacin, carbapenems, and piperacillin/tazobactam exhibited relatively high sensitivity, whereas fluoroquinolones showed high resistance rate whether they can be the first-line antimicrobials for empirical treatment of UTI should take more consideration. The gram-negative bacilli in ICU were more resistance than that in non-ICU. These findings are helpful for clinicians using antimicrobials reasonably

The Correlation Mechanism between Dominant Bacteria and Primary Metabolites during Fermentation of Red Sour Soup

Chinese red sour soup is a traditional fermented product famous in the southwestern part of China owing to its distinguished sour and spicy flavor. In the present study, the effect of inoculation of lactic acid bacteria (LAB) on the microbial communities and metabolite contents of the Chinese red sour soup was investigated. Traditional red sour soup was made with tomato and red chilli pepper and a live count (108 CFU/mL) of five bacterial strains (including Clostridium intestinalis: Lacticaseibacillus rhamnosus: Lactiplantibacillus plantarum: Lacticaseibacillus casei: Lactobacillus paracei) was added and fermented for 30 days in an incubator at 37 °C. Three replicates were randomly taken at 0 d, 5 d, 10 d, 15 d, 20 d, 25 d and 30 d of fermentation, with a total of 21 sour soup samples. Metabolomic analysis and 16S-rDNA amplicon sequencing of soup samples were performed to determine microbial diversity and metabolite contents. Results revealed that fermentation resulted in the depletion of native bacterial strains as LAB dominated over other microbes, resulting in differences in the relative abundance of bacteria, and types or contents of metabolites. A decrease (p Lacticaseibacillus rhamnosus with differentially enriched metabolites including lactic acid, ascorbic acid, and chlorogenic acid, which can desirably contribute to the flavor and quality of the red sour soup

Syntaxin-3 Binds and Regulates Both R- and L-Type Calcium Channels in Insulin-Secreting INS-1 832/13 Cells.

Syntaxin (Syn)-1A mediates exocytosis of predocked insulin-containing secretory granules (SGs) during first-phase glucose-stimulated insulin secretion (GSIS) in part via its interaction with plasma membrane (PM)-bound L-type voltage-gated calcium channels (Cav). In contrast, Syn-3 mediates exocytosis of newcomer SGs that accounts for second-phase GSIS. We now hypothesize that the newcomer SG Syn-3 preferentially binds and modulates R-type Cav opening, which was postulated to mediate second-phase GSIS. Indeed, glucose-stimulation of pancreatic islet β-cell line INS-1 induced a predominant increase in interaction between Syn-3 and Cavα1 pore-forming subunits of R-type Cav2.3 and to lesser extent L-type Cavs, while confirming the preferential interactions between Syn-1A with L-type (Cav1.2, Cav1.3) Cavs. Consistently, direct binding studies employing heterologous HEK cells confirmed that Syn-3 preferentially binds Cav2.3, whereas Syn-1A prefers L-type Cavs. We then used siRNA knockdown (KD) of Syn-3 in INS-1 to study the endogenous modulatory actions of Syn-3 on Cav channels. Syn-3 KD enhanced Ca2+ currents by 46% attributed mostly to R- and L-type Cavs. Interestingly, while the transmembrane domain of Syn-1A is the putative functional domain modulating Cav activity, it is the cytoplasmic domain of Syn-3 that appears to modulate Cav activity. We conclude that Syn-3 may mimic Syn-1A in the ability to bind and modulate Cavs, but preferring Cav2.3 to perhaps participate in triggering fusion of newcomer insulin SGs during second-phase GSIS

Osteopontin, Bone Morphogenetic Protein-4, and Vitamin D Receptor Gene Polymorphisms in the Susceptibility and Clinical Severity of Spinal Tuberculosis

Background/Aims: Spinal tuberculosis (TB) is a common and dangerous form of extrapulmonary TB with unclear mechanisms in its occurrence and progression. This study investigated the clinical significances of bone morphogenetic protein-4 (BMP-4), osteopontin (OPN), and vitamin D receptor (VDR) gene polymorphism, mRNA and protein expression in spinal TB patients. Methods: BMP-4 and OPN gene polymorphisms were detected by direct DNA sequencing, while VDR-FokI polymorphisms were analyzed using PCR-RFLP. mRNA and protein expression was measured using real-time PCR and Western blot, respectively. Results: A significant lower frequency of TT genotype and T allele at 6007C>T polymorphism in BMP-4 gene; higher frequency of GG genotype and G allele at -66T>G polymorphism in OPN gene, and higher frequency of the ff genotype and f allele at the VDR-FokI polymorphism were observed in patients with spinal TB compared to controls. TT genotype of 6007C>T polymorphism correlated with a lower BMP-4 mRNA and protein expression, -66GG genotype correlated with a high OPN mRNA and protein expression, and ff genotype correlated with the lower VDR mRNA and protein levels in the intervertebral disc tissues. The TT genotype and low BMP-4 gene expression; the -66GG genotype and high OPN gene expression; and the ff genotype and low VDR gene expression significantly correlated with the clinical severity of spinal TB. Conclusion: The 6007C>T polymorphism of BMP-4, -66T>G polymorphism of OPN, and VDR-FokI polymorphism are the susceptible factors of spinal TB and indicators of the clinical severity. These three genes may collaborate in the development of spinal TB

Spectrin-based membrane skeleton supports ciliogenesis.

Cilia are remarkable cellular devices that power cell motility and transduce extracellular signals. To assemble a cilium, a cylindrical array of 9 doublet microtubules push out an extension of the plasma membrane. Membrane tension regulates cilium formation; however, molecular pathways that link mechanical stimuli to ciliogenesis are unclear. Using genome editing, we introduced hereditary elliptocytosis (HE)- and spinocerebellar ataxia (SCA)-associated mutations into the Caenorhabditis elegans membrane skeletal protein spectrin. We show that these mutations impair mechanical support for the plasma membrane and change cell shape. RNA sequencing (RNA-seq) analyses of spectrin-mutant animals uncovered a global down-regulation of ciliary gene expression, prompting us to investigate whether spectrin participates in ciliogenesis. Spectrin mutations affect intraflagellar transport (IFT), disrupt axonemal microtubules, and inhibit cilium formation, and the endogenous spectrin periodically distributes along cilia. Mammalian spectrin also localizes in cilia and regulates ciliogenesis. These results define a previously unrecognized yet conserved role of spectrin-based mechanical support for cilium biogenesis

Syn-3 co-immunoprecipitates (IP) distinct Ca_vs than Syn-1A in INS-1 cells.

<p>Syn-3 (A) and Syn-1A (C) interactions with the indicated Ca_vα1 subunits (Ca_v1.2, Ca_v1.3, Ca_v2.3 and Ca_v2.2) and auxiliary subunits (α₂δ-1 and β3) and SNAP25 in INS-1 cells. Densitometric analysis of Syn-3 co-IP (B) and Syn-1A co-IP (D), expressed as percent recovery of total lysate inputs (which showed equal protein loading in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147862#pone.0147862.s001" target="_blank">S1 Fig</a>), shows that high glucose (16.7 mM) plus GLP-1 (10 nM) increased the association of these syntaxins with the respective Ca_vs. Values are means±SEM, n = 3. *p<0.05, ***p<0.001, NS: not significant. As control (E) shows representative blots from five separate co-IP experiments with pre-immune IgG, which did not bring down syntaxins or Ca_vs (<i>left</i> lanes). <i>Righ</i>t lanes show the input lysates. All five experiments probed for the Ca_v α subunits and α₂δ-1, whereas β3, Syn-3 and Syn-1A were probed on two blots from separate experiments.</p

Syn-3 preferentially binds Ca_v2.3 while Syn-1A preferentially binds Ca_v1.3.

<p>Representative blots show HEK293 cells co-transfected with Ca_v1.3 (A) or Ca_v2.3 (C) with either Syn-1A or Syn-3, then subjected to co-IP with anti-Syn-1A or Syn-3 antibody. Co-precipitated proteins were identified with the indicated antibodies. Densitometric analysis of the co-precipitated Ca_v1.3 (B) or Ca_v2.3 (D), expressed as percent recovery of total lysate inputs. Values are means±SEM, n = 3; *p<0.05, **p<0.01.</p

Cytoplasmic Syn-3 domain but not cytoplasmic Syn-1A domain regulates Ca_v currents.

<p>(A) GST-Syn-3 cytoplasmic domain (a.a. 1–263) or GST-Syn-1A cytoplasmic domain (a.a. 1–265) or GST (control) was dialyzed into INS-1 cells, then Ca_v currents recorded. Shown are representative traces in the whole-cell mode with stimulation from −80–60 mV. (B) Current-voltage relationship of Ca_v channels. Currents were normalized to cell capacitance to yield current density. (C) Bar chart showing the maximum current density in INS-1 cells dialyzed with GST control (n = 9), GST-Syn-3 (n = 9), or GST-Syn-1A (n = 8). Values are means±SEM; *p<0.05; NS: no significant difference. (D and E) Representative blots show HEK293 cells transfected with Ca_v1.3 (D) or Ca_v2.3 (E) subjected to pull down with 300 pmol of GST-Syn-1A (a.a. 1–265), GST-Syn-3 (a.a. 1–263) or GST. (F) Summary analysis of four separate experiments. Data was expressed as means ± SEM; *p<0.05.</p

Depletion of Syntaxin 3 in INS-1 cells increased voltage-gated Ca²⁺ currents.

<p>(A) Representative traces showing Ca_v currents recorded in whole-cell mode from control and Syn-3 KD INS-1 cells. (B) Current-voltage relationship of Ca_vs from control (n = 16) and Syn-3 KD (n = 11) INS-1 cells. Currents were normalized to cell capacitance to yield current density. Values are means±SEM. (C) Bar chart shows the maximum increase in current density under stimulation of 10 mV voltage. *p<0.05 for control vs Syn-3 KD (D) Representative Ca_v currents from normal INS-1 cells before and after treatment with nifedipine (10 μM Nif; n = 8), SNX482 (400 nM SNX; n = 9) or ω-Conotoxin GVIA (100 nM Conotoxin; n = 10); their summary analysis (E) of the maximum increase in current densities normalized to the percentage of control (Con). ***p<0.001, **p<0.01 and *p<0.05 compared to control. We then performed another set of experiments (different from A-E) to compare the effects of nifedipine and SNX on Syn-3 KD (H and I) and Control INS-1 cells (F and G). (F) Representative Ca_v currents from control INS-1 cells before (control, n = 25) and after treatment with nifedipine (10 μM Nif) (n = 14) or SNX482 (400 nM SNX) (n = 6); their summary analysis (G) of the maximum increase in current densities normalized to the percentage of control (Con). ***p<0.001; *p<0.05 compared to Control. (H) Representative Ca_v currents of Syn-3 KD INS-1 cells before (Syn-3 KD, n = 11) and after treatment with nifedipine (n = 12) or SNX482 (n = 6); and their summary analysis (I) of the maximum increase in current densities normalized as percentages of the Syn-3 KD cells. *p<0.05 compared to Syn-3 KD. Here, Syn-3 KD Ca_v currents were 148% of Control cells, similar to A and B.</p