Restoring colistin sensitivity in colistin-resistant Salmonella and Escherichia coli: combinatorial use of berberine and EDTA with colistin.

The appearance and prevalence of multidrug-resistance (MDR) Gram-negative bacteria (GNB) have limited our antibiotic capacity to control bacterial infections. The clinical efficacy of colistin (COL), considered as the "last resort" for treating GNB infections, has been severely hindered by its increased use as well as the emergence and prevalence of mobile colistin resistance (MCR)-mediated acquired drug resistance. Identifying promising compounds to restore antibiotic activity is becoming an effective strategy to alleviate the crisis of increasing MDR. We first demonstrated that the combination of berberine (BBR) and EDTA substantially restored COL sensitivity against COL-resistant Salmonella and Escherichia coli. Molecular docking indicated that BBR can interact with MCR-1 and the efflux pump system AcrAB-TolC, and BBR combined with EDTA downregulated the expression level of mcr-1 and tolC. Mechanically, BBR combined with EDTA could increase bacterial membrane damage, inhibit the function of multidrug efflux pump, and promote oxidative damage, thereby boosting the action of COL. In addition, transcriptome analysis found that the combination of BBR and EDTA can accelerate the tricarboxylic acid cycle, inhibit cationic antimicrobial peptide (CAMP) resistance, and attenuate Salmonella virulence. Notably, the combination of BBR and EDTA with COL significantly reduced the bacterial load in the liver and spleen of a mice model infected with Salmonella. Our findings revealed that BBR and EDTA can be used as adjuvants collectively with COL to synergistically reverse the COL resistance of bacteria. IMPORTANCE: Colistin is last-resort antibiotic used to treat serious clinical infections caused by MDR bacterial pathogens. The recent emergence of transferable plasmid-mediated COL resistance gene mcr-1 has raised the specter of a rapid worldwide spread of COL resistance. Coupled with the fact of barren antibiotic development pipeline nowadays, a critical approach is to revitalize existing antibiotics using antibiotic adjuvants. Our research showed that berberine combined with EDTA effectively reversed COL resistance both in vivo and in vitro through multiple modes of action. The discovery of berberine in combination with EDTA as a new and safe COL adjuvant provides a therapeutic regimen for combating Gram-negative bacteria infections. Our findings provide a potential therapeutic option using existing antibiotics in combination with antibiotic adjuvants and address the prevalent infections caused by MDR Gram-negative pathogens worldwide.

Upregulation of outer membrane porin gene ompC contributed to enhancement of azithromycin susceptibility in multidrug-resistant Escherichia coli.

The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.

Higher Adalimumab Trough Levels Are Associated with Histologic Remission and Mucosal Healing in Inflammatory Bowel Disease.

(1) Many patients with inflammatory bowel disease (IBD) in endoscopic remission have persistent histologic activity, which is associated with worse outcomes. There are limited data on the association between adalimumab drug concentrations and histologic outcomes using validated histologic indices. We aimed to assess the relationship between adalimumab concentrations and the Robarts Histopathology Index (RHI). (2) Patients from a tertiary IBD center from 2013 to 2020 with serum adalimumab (ADA) trough concentrations measured during maintenance therapy (≥14 weeks) and a colonoscopy or flexible sigmoidoscopy with biopsies performed within 90 days of drug level were included. Blinded histologic scoring using the RHI was performed. Primary analysis assessed the relationship between adalimumab drug concentrations and histologic remission using receiver operating characteristic curve analysis. (3) In 36 patients (26 Crohn's Disease, 9 ulcerative colitis, 1 indeterminate), median adalimumab concentrations were higher (17.3 ug/mL, 12.2-24.0) in patients with histologic remission compared to those without (10.3 ug/mL, 6.8-13.9, p = 0.008). The optimal ADA concentration identified using the Youden threshold was ≥16.3 ug/mL (sensitivity 70%, specificity 90%). Patients with ADA ≥ 16.3 ug/mL had higher histologic remission rates (78%) compared to lower ADA concentrations (14%, p= 0.002), as well as higher mucosal healing rates (86%) compared to lower levels (12%, p = 0.001). Symptoms correlated weakly and non-significantly with both histologic (RHI) scores (r = 0.25, p = 0.2) and adalimumab concentrations (r = 0.05, p = 0.8). (4) The current study demonstrated that higher serum adalimumab concentrations (≥16.3 ug/mL) are needed for histologic remission and mucosal healing assessed using the RHI.

The Relationship Between the Endoscopic Healing Index, Fecal Calprotectin, and Magnetic Resonance Enterography in Crohn's Disease.

INTRODUCTION: The serum-based endoscopic healing index (EHI) test identifies endoscopic Crohn's disease (CD) activity. Data are lacking on the relationship between EHI with other endpoints. We assessed the relationship between EHI and the simplified Magnetic Resonance Index of Activity. MATERIALS AND METHODS: Data were prospectively collected on patients with CD with either an EHI or fecal calprotectin (FCAL) within 90 days of magnetic resonance enterography (MRE). Diagnostic accuracy was assessed using area under the receiver operator characteristics. Proportions with any, severe, and terminal ileum MR inflammation were compared above/below identified thresholds for both EHI and FCAL. RESULTS: A total of 241 MREs paired to either EHI or FCAL from 155 patients were included. Both EHI and FCAL had similar accuracy to diagnose inflammation (area under the receiver operator characteristics: EHI: 0.635 to 0.651, FCAL: 0.680 to 0.708). Optimal EHI values were 42 and 26 for inflammation on MRE and endoscopy, respectively. Patients with EHI ≥42 (100% vs. 63%, P=0.002), FCAL >50 µg/g (87% vs. 64%, P<0.001) and FCAL >250 µg/g (90% vs. 75%, P=0.02) had higher rates of simplified Magnetic Resonance Index of Activity ≥1 compared with lower values. EHI differentiated ileitis numerically more than FCAL (delta: 24% to 25% vs. 11% to 21%). Patients with FCAL ≥50 µg/g had higher rates of severe inflammation compared with FCAL <50 µg/g (75% vs. 47%, P<0.001), whereas smaller differentiation existed for EHI threshold of 42 (63% vs. 49%, P=0.35). CONCLUSION: Both EHI and FCAL were specific in their confirmation of inflammation and disease activity on MRE in patients with CD. However, MRE-detected inflammation was frequently present in the presence of low EHI and FCAL in similar proportions.

Impact of Race and Neighborhood Socioeconomic Characteristics on Liver Cancer Diagnosis in Patients with Viral Hepatitis and Cirrhosis.

Background: Concerning data have revealed that viral hepatitis and hepatocellular carcinoma (HCC) disproportionally impact non-White patients and those from lower socioeconomic status. A recent study found that HCC clusters were more likely to be in high poverty areas in New York City. Aims: We aim to investigate the impacts of neighborhood characteristics on those with viral hepatitis and cirrhosis, particularly with advanced HCC diagnosis. Methods: Patients with cirrhosis and viral hepatitis admitted to a New York City health system between 2012 and 2019 were included. Those with prior liver transplants were excluded. Neighborhood characteristics were obtained from US Census. Our primary outcome was HCC and advanced HCC diagnosis. Results: This study included 348 patients; 209 without history of HCC, 20 with early HCC, 98 with advanced HCC, and 21 patients with HCC but no staging information. Patients with advanced HCC were more likely to be older, male, Asian, history of HBV, and increased mortality. They were more likely to live in areas with more foreign-born, limited English speakers, and less than high school education. After adjusting for age, sex, and payor type, Asian race and low income were independent risk factors for advanced HCC. Neighborhood factors were not associated with mortality or readmissions. Conclusion: We observed that in addition to age and sex, Asian race, lower household income, lower education, and lower English proficiency were associated with increased risk of advanced HCC. These disparities likely reflect suboptimal screening programs and linkage to care among vulnerable populations. Further efforts are crucial to validate and address these concerning disparities.

Analysis of Regulatory Mechanism of AcrB and CpxR on Colistin Susceptibility Based on Transcriptome and Metabolome of Salmonella Typhimurium.

With the increasing and inappropriate use of colistin, the emerging colistin-resistant isolates have been frequently reported during the last few decades. Therefore, new potential targets and adjuvants to reverse colistin resistance are urgently needed. Our previous study has confirmed a marked increase of colistin susceptibility (16-fold compared to the wild-type Salmonella strain) of cpxR overexpression strain JSΔacrBΔcpxR::kan/pcpxR (simplified as JSΔΔ/pR). To searching for potential new drug targets, the transcriptome and metabolome analysis were carried out in this study. We found that the more susceptible strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels. The virulence-related genes and colistin resistance-related genes (CRRGs) were significantly downregulated in JSΔΔ/pR. There were significant accumulation of citrate, α-ketoglutaric acid, and agmatine sulfate in JSΔΔ/pR, and exogenous supplement of them could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. Additionally, we also demonstrated that AcrB and CpxR could target the ATP and reactive oxygen species (ROS) generation, but not proton motive force (PMF) production pathway to potentiate antibacterial activity of colistin. Collectively, these findings have revealed several previously unknown mechanisms contributing to increased colistin susceptibility and identified potential targets and adjuvants for potentiating colistin treatment of Salmonella infections. IMPORTANCE Emergence of multidrug-resistant (MDR) Gram-negative (G-) bacteria have led to the reconsideration of colistin as the last-resort therapeutic option for health care-associated infections. Finding new drug targets and strategies against the spread of MDR G- bacteria are global challenges for the life sciences community and public health. In this paper, we demonstrated the more susceptibility strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels and revealed several previously unknown regulatory mechanisms of AcrB and CpxR on the colistin susceptibility. Importantly, we found that exogenous supplement of citrate, α-ketoglutaric acid, and agmatine sulfate could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. These results provide a theoretical basis for finding potential new drug targets and adjuvants.

Prevalence of the optrA gene among Streptococcus suis isolates from diseased pigs and identification of a novel integrative conjugative element ICESsu988S.

Aim of this study was to investigate the prevalence and genetic environment of the oxazolidinone resistance gene optrA in Streptococcus suis (S. suis) isolates from diseased pigs in China. A total of 178 S. suis isolates were screened for the optrA gene by PCR. The phenotypes and genotypes of optrA-positive isolates were investigated by antimicrobial susceptibility testing, core genome Multilocus Sequence Typing (cgMLST), capsular serotypes determination and whole-genome sequencing (WGS). Fifty-one (28.7%) S. suis isolates were positive for optrA. Phylogenetic analysis indicated that the spread of the optrA among S. suis isolates was primarily due to horizontal transfer. Analysis of S. suis serotypes from diseased pigs revealed substantial diversity. The genetic environment of optrA was complex and diverse and could be divided into 12 different types. Interestingly, we identified a novel integrative and conjugative element ICESsu988S, carrying optrA and erm(T) genes. This is to the best of our knowledge the first report of the optrA and erm(T) co-located on an ICE in S. suis. Our results showed a high prevalence of optrA gene in S. suis isolates in China. Further research is needed to evaluate the importance of ICEs, as they horizontally propagate important clinical resistance genes.

Epidemic characteristics of the SXT/R391 integrated conjugative elements in multidrug-resistant Proteus mirabilis isolated from chicken farm.

This study was designed to depict prevalence and antimicrobial resistance characteristics of Proteus mirabilis (P. mirabilis) strains in 4 chicken farms and to probe the transfer mechanism of resistance genes. A total of 187 P. mirabilis isolates were isolated from 4 chicken farms. The susceptibility testing of these isolates to 14 antimicrobials showed that the multidrug resistance (MDR) rate was as high as 100%. The ß-lactamase resistance genes blaOXA-1, blaCTX-M-1G, blaCTX-M-9G and colistin resistance gene mcr-1 were highly carried in the P. mirabilis isolates. An MDR strain W47 was selected for whole genome sequencing (WGS) and conjugation experiment. The results showed that W47 carried 23 resistance genes and 64 virulence genes, and an SXT/R391 integrated conjugative elements (ICEs) named ICEPmiChn5 carrying 17 genes was identified in chromosome. ICEPmiChn5 was able to be excised from the chromosome of W47 forming a circular intermediate, but repeated conjugation experiments were unsuccessful. Among 187 P. mirabilis isolates, 144 (77.01%, 144/187) isolates carried ICEPmiChn5-like ICEs, suggesting that ICEs may be the major vector for the transmission of resistance genes among MDR chicken P. mirabilis strains in this study. The findings were conducive to insight into the resistance mechanism of chicken P. mirabilis strains and provide a theoretical basis for the use of antibiotics for the treatment of MDR P. mirabilis infections in veterinary clinic.

The Impact of Confounders on Symptom-Endoscopic Discordances in Crohn's Disease.

Background: Discordances between clinical and endoscopic Crohn's disease (CD) activity indices negatively impact the utility of clinic visits and efficacy assessments in clinical trials. Bile acid diarrhea (BAD) and small intestinal bacterial overgrowth (SIBO) mimic CD symptoms. This study quantified the impact of BAD and SIBO on the relationship between clinical and endoscopic disease activity indices. Methods: CD patients with 7α-hydroxy-4-cholesten-3-one (7C4) serum measurements and/or SIBO breath tests and matched clinical and endoscopic scores were included. Clinical remission (stool frequency [SF] ≤ 1 and abdominal pain score ≤ 1) rates were compared between those with and without (1) endoscopic remission, (2) BAD (7C4 > 55 ng/mL), and (3) SIBO. Results: Of 295 CD patients, 219 had SIBO testing and 87 had 7C4 testing. Patients with elevated 7C4 had lower proportions with clinical remission (14% vs 40%, P = .007) and SF ≤ 1 (14% vs 42%, P = .004) compared to those with normal 7C4. In patients with normal 7C4, higher rates of clinical remission (65% vs 27%, P = .01) and SF ≤ 1 (71% vs 27%, P = .003) existed in patients with endoscopic remission compared to those without endoscopic remission. Conversely, among the entire 295 patient cohorts, nearly identical clinical remission rates existed between those with and without endoscopic remission (25% vs 24%, P = .8), and the Crohn's Disease Patient-Reported Outcome-2 score was not accurate for predicting endoscopic remission (Area Under the Curve (AUC): 0.48; 95% CI, 0.42-0.55). SIBO status did not impact clinical remission rates (P = 1.0). Conclusions: BAD, but not SIBO, contributed to symptom scores. A relationship between endoscopic inflammation and clinical remission rates only existed in patients without 7C4 elevations.

Synergistic antibacterial activity of baicalin and EDTA in combination with colistin against colistin-resistant Salmonella.

The emergence and rapid spread of multidrug resistant (MDR) Gram-negative bacteria have posed a serious threat to global health and security. Because of the time-consuming, high cost and high risk of developing new antibiotics, a significant method is to use antibiotic adjuvants to revitalize the existing antibiotics. The purpose of the study is to research the traditional Chinese medicine baicalin with the function of inhibiting the efflux pump and EDTA whether their single or combination can increase the activity of colistin against colistin-resistant Salmonella in vitro and in vivo, and to explore its molecular mechanisms. In vitro antibacterial experiments, we have observed that baicalin and EDTA alone could enhance the antibacterial activity of colistin. At the same time, the combination of baicalin and EDTA also showed a stronger synergistic effect on colistin, reversing the colistin resistance of all Salmonella strains. Molecular docking and RT-PCR results showed that the combination of baicalin and EDTA not only affected the expression of mcr-1, but also was an effective inhibitor of MCR-1. In-depth synergistic mechanism analysis revealed that baicalin and EDTA enhanced colistin activity through multiple pathways, including accelerating the tricarboxylic acid cycle (TCA cycle), inhibiting the bacterial antioxidant system and lipopolysaccharide (LPS) modification, depriving multidrug efflux pump functions and attenuating bacterial virulence. In addition, the combinational therapy of colistin, baicalin and EDTA displayed an obvious reduction in bacterial loads cfus of liver and spleen compared with monotherapy and 2-drug combination therapy. In conclusion, our study indicates that the combination of baicalin and EDTA as a novel colistin adjuvant can provide a reliable basis for formulating the therapeutic regimen for colistin resistant bacterial infection.

Characterization of IncI1/ST71 and IncF18:A-:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate.

To characterize IncI1 and IncF18:A-:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate, antibiotic susceptibility testing, conjugation assays, transformation assays, S1-PFGE, and WGS analysis were performed. The 119,457-bp plasmid pEC014-1 with a multidrug-resistance region (MRR) containing four different segments interspersed with six IS26 elements, belonged to incompatibility group I1 and sequence type 71. The 154,516-bp plasmid pEC014-2 with two replicons, typed as FII-18 and FIB-1, carried 14 resistance determinants including blaTEM-1b, blaOXA-1, oqxAB, dfrA17, aac(6')-Ib-cr, sul1, sul2, tet(A), floR, catB3, hph(aph(4)-Ia), aacC4(aac(3)-IV), aadA5, arr-3, and a merEDACPTR loci in MRR, and additionally encoded three virulence loci: iroNEDCB, sitABCD, and iucABCD-iutA. Plasmid stability assays showed that pEC014-1 and pEC014-2 were stable in recipient E. coli C600 for at least 15 days of passage. Competition assays were carried out to evaluate the fitness impact of pEC014-2 carriage in vitro, revealing a decrease in host fitness. Growth kinetics showed that the growth rate for pEC014-1 or/and pEC014-2 bearing cells was significantly slower than that of the E. coli C600 host strain in the exponential stage (p < 0.01), with only cells carrying pEC014-1 sustaining rapid growth after 6 h of exponential growth. Our findings highlight the mosaic structures of epidemic plasmid IncI1/ST71 and F18:A-:B1 lineages and contribute to a better understanding of the evolution and dissemination of these multidrug resistance and virulence plasmids.

Genomic insight into the integrative conjugative elements from ICEHpa1 family.

Integrative conjugative elements (ICEs) are important carriers for disseminating resistance genes. We have previously reported a novel element ICEHpa1 carrying seven antibiotic resistance genes, which could be self-transmissible relying on the novel T4SS. To identify novel ICEHpa1 variants from 211 strains and novel T4SS encoded in ICEHpa1, and to explore the relationships in these ICEs, four complete sequences of ICEs were identified by WGS analysis and antimicrobial susceptibility testing was determined by broth microdilution. In addition, a comparative analysis of these ICEs was conducted with bioinformatic tools, and the transfer abilities of these ICEs were confirmed by conjugation. Four ICEHpa1 variants ICEGpa1818, ICEGpa1808, ICEGpa1807, and ICEGpa1815 with different resistance gene profiles were characterized, and their hosts showed different resistance spectrums. All ICEs shared the same backbone and were inserted into the tRNALeu site, and all resistance regions were inserted into the same target site between the accessory and integration regions. This study analyzed complete sequences of ICEs from the ICEHpa1 family and identified novel T4SS and insertion element ISGpa2. Diverse resistance genes extensively exist in these ICEs, serving as a reservoir for resistance genes and facilitating their dissemination.

CpxR negatively regulates IncFII-replicon plasmid pEC011 conjugation by directly binding to multi-promoter regions.

CpxAR is a global regulatory protein and has important roles in plasmid mating. However, except for traJ, the regulatory effect of CpxAR on other tra genes is unclear. The aim of this study was to explore the effects of CpxAR on conjugative transfer of the epidemic plasmid pEC011 (IncFII replicon) in Escherichia coli. The plasmid mating frequencies were significantly higher for the single deletion mutant strain FΔcpxR than for the parental strain F25922. Additionally, expression levels of traM, traJ and traY in FΔcpxR were significantly higher than those in F25922. Further investigations revealed that His6-CpxR protein could directly bind to the traM, traJ and traY promoter regions with the binding sites of 5'-TTTACATT-3' (PM), 5'-ATAAGAAT-3' (PJ), and 5'-AATTTTAT-3' (PY), respectively. Taken together, our results demonstrate that CpxAR can downregulate the expression of traM, traJ and traY by directly binding to the CpxR box-like sites of promoters, thus significantly reducing the mating rates of IncFII replicon plasmid pEC011.

Synergistic antibacterial activity of tetrandrine combined with colistin against MCR-mediated colistin-resistant Salmonella.

It has been recognized that colistin resistance is a growing problem that seriously impairs the clinical efficacy of colistin against bacterial infections. One strategy that has been proven to have therapeutic effect is to overcome the widespread emergence of antibiotic-resistant pathogens by combining existing antibiotics with promising non-antibiotic agents. In this work, antibiotic susceptibility testing, checkerboard assays and time-kill curves were used to investigate the antibacterial activity of the individual drugs and the potential synergistic activity of the combination. The molecular mechanisms of tetrandrine in combination with colistin were analyzed using fluorometric assay and Real-time PCR. To predict possible interactions between tetrandrine and MCR-1, molecular docking assay was taken. Finally, we evaluated the in vivo efficacy of tetrandrine in combination with colistin against MCR-positive Salmonella. Overall, the combination of tetrandrine and colistin showed significant synergistic activity. In-depth mechanistic analysis showed that the combination of tetrandrine with colistin enhances the membrane-damaging ability of colistin, undermines the functions of proton motive force (PMF) and efflux pumps in MCR-positive bacteria. The results of molecular docking and RT-PCR analyses showed that tetrandrine not only affects the expression of mcr-1 but is also an effective MCR-1 inhibitor. Compared with colistin monotherapy, the combination of tetrandrine with colistin significantly reduced the bacterial load in vivo. Our findings demonstrated that tetrandrine serves as a potential colistin adjuvant against MCR-positive Salmonella.

Utility of Therapeutic Drug Monitoring for Tumor Necrosis Factor Antagonists and Ustekinumab in Postoperative Crohn's Disease.

BACKGROUND: In postoperative Crohn's disease (POCD), data are lacking on relationships between serum biologic concentrations and treatment outcomes. We assessed if established threshold concentrations of infliximab (IFX), adalimumab (ADA), and ustekinumab (UST) impact outcomes in POCD. METHODS: Data were extracted from POCD patients with serum biologic concentration measurements using Weill Cornell Medicine biobanks. The primary outcome compared rates of deep remission (achieving both objective [endoscopic or biomarker] and clinical [Harvey-Bradshaw index or Crohn's Disease Patient Reported Outcome-2] remission), using established serum drug level cutoffs of IFX ≥3 µg/mL, ADA ≥7.5 µg/mL, and UST ≥4.5 µg/mL. RESULTS: In 130 patients, median IFX, ADA, and UST concentrations were 10 (interquartile range [IQR], 2.9-26.9) µg/mL, 10.5 (IQR, 4.9-14.9) µg/mL, and 6.9 (IQR, 5.1-10.2) µg/mL, respectively. In patients with IFX ≥3 µg/mL, higher rates of deep remission (39% vs 0%; P = .02) existed compared with those with IFX <3 µg/mL. Similar differences existed for clinical (44% vs 9%; P = .04) and objective (83% vs 62%; P = .1) remission. In patients with ADA ≥7.5 µg/mL, rates of deep (42% vs 0%; P = .02), clinical (42% vs 0%; P = .02), and objective (88% vs 40%; P = .007) remission were higher than patients with lower concentrations. For UST, rates of deep (28% vs 17%; P = 1.0), clinical (33% vs 33%; P = 1.0), and objective (70% vs 67%; P = 1.0) remission were similar between patients regardless of drug concentration. CONCLUSIONS: In POCD, established anti-tumor necrosis factor concentrations were associated with improved outcomes. No relationship between UST concentrations and postoperative outcomes existed.

Data are lacking on therapeutic drug monitoring in postoperative Crohn's disease. The current study found that tumor necrosis factor antagonist concentrations above established drug thresholds were associated with improved outcomes. In contrast, for ustekinumab, no relationship between drug thresholds and outcomes existed.

Spreading Advantages of Coresident Plasmids blaCTX-M-Bearing IncFII and mcr-1-Bearing IncI2 in Escherichia coli.

Two diverse conjugative plasmids can interact within bacterial cells. However, to the best of our knowledge, the interaction between blaCTX-M-bearing IncFII plasmid and mcr-1-carrying IncI2 plasmid colocated on the same bacterial host has not been reported. This study was initiated to explore the interaction and to analyze the reasons that these two plasmids are often coresident in multidrug-resistant Escherichia coli. To assess the interactions on plasmid stabilities, fitness costs, and transfer rates, we constructed two groups of isogenic derivatives, C600FII, C600I2, and C600FII+I2 of E. coli C600 and J53FII, J53I2, and J53FII+I2 of E. coli J53, respectively. We found that carriage of FII and I2 plasmids, independently and together, had not impaired the growth of the bacterial host. It was difficult for the single plasmid FII or I2 in E. coli C600 to reach stable persistence for a long time in an antibiotic-free environment, while the stability would be striking improved when they coresided. Meanwhile, plasmids FII and I2, whether together or apart, could notably enhance the fitness advantage of the host; moreover, E. coli coharboring plasmids FII and I2 presented more obvious fitness advantage than that carrying single plasmid FII. Coresident plasmids FII and I2 could accelerate horizontal cotransfer by conjugation. The transfer rates from a strain carrying coresident FII and I2 plasmids increased significantly when it mated with a recipient cell carrying one of them. Our findings highlight the advantages of coinhabitant FII and I2 plasmids in E. coli to drive the persistence and spread of plasmid-carried blaCTX-M and mcr-1 genes, although the molecular mechanisms of their coresidence warrant further study. IMPORTANCE More and more Enterobacteriaceae carry both blaCTX-M and mcr-1, which are usually located on IncFII-type and IncI2-type plasmids in the same bacterial host, respectively. However, the study on advantages of coresident plasmids in bacterial host is still sparse. Here, we investigated the stability, fitness cost, and cotransfer traits associated with coresident IncFII-type and IncI2-type plasmids in E. coli. Our results show that coinhabitant plasmids in E. coli are more stable, confer more fitness advantages, and are easier to transfer and cotransfer than a single plasmid IncFII or IncI2. Our findings confirm the advantages of coresident plasmids of blaCTX-M-bearing IncFII and mcr-1-bearing IncI2 in clinical E. coli, which will pose a serious threat to clinical therapy and public health.

IS1294 Reorganizes Plasmids in a Multidrug-Resistant Escherichia coli Strain.

The aims of this study were to elucidate the role of IS1294 in plasmid reorganization and to analyze biological characteristics of cointegrates derived from different daughter plasmids. The genetic profiles of plasmids in Escherichia coli strain C21 and its transconjugants were characterized by conjugation, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern hybridization, whole-genome sequencing (WGS) analysis, and PCR. The traits of cointegrates were characterized by conjugation and stability assays. blaCTX-M-55-bearing IncI2 pC21-1 and nonresistant IncI1 pC21-3, as conjugative helper plasmids, were fused with nonconjugative rmtB-bearing IncN-X1 pC21-2, generating cointegrates pC21-F1 and pC21-F2. Similarly, pC21-1 and pC21-3 were fused with nonconjugative IncF33:A-:B- pHB37-2 from another E. coli strain to generate cointegrates pC21-F3 and pC21-F4 under experimental conditions. Four cointegrates were further conjugated into the E. coli strain J53 recipient at high conjugation frequencies, ranging from 2.8 × 10-3 to 3.2 × 10-2. The formation of pC21-F1 and pC21-F4 was the result of host- and IS1294-mediated reactions and occurred at high fusion frequencies of 9.9 × 10-4 and 2.1 × 10-4, respectively. Knockout of RecA resulted in a 100-fold decrease in the frequency of plasmid reorganization. The phenomenon of cointegrate pC21-F2 and its daughter plasmids coexisting in transconjugants was detected for the first time in plasmid stability experiments. IS26-orf-oqxAB was excised from cointegrate pC21-F2 through a circular intermediate at a very low frequency, which was experimentally observed. To the best of our knowledge, this is the first report of IS1294-mediated fusion between plasmids with different replicons. This study provides insight into the formation and evolution of cointegrate plasmids under different drug selection pressures, which can promote the dissemination of MDR plasmids. IMPORTANCE The increasing resistance to ß-lactams and aminoglycoside antibiotics, mainly due to extended-spectrum ß-lactamases (ESBLs) and 16S rRNA methylase genes, is becoming a serious problem in Gram-negative bacteria. Plasmids, as the vehicles for resistance gene capture and horizontal gene transfer, serve a key role in terms of antibiotic resistance emergence and transmission. IS26, present in many antibiotic-resistant plasmids from Gram-negative bacteria, plays a critical role in the spread, clustering, and reorganization of resistance determinant-encoding plasmids and in plasmid reorganization through replicative transposition mechanisms and homologous recombination. However, the role of IS1294, present in many MDR plasmids, in the formation of cointegrates remains unclear. Here, we investigated experimentally the intermolecular recombination of IS1294, which occurred with high frequencies and led to the formation of conjugative MDR cointegrates and facilitated the cotransfer of blaCTX-M-55 and rmtB, and we further uncovered the significance of IS1294 in the formation of cointegrates and the common features of IS1294-driven cointegration of plasmids.

Double deletion of cpxR and tolC significantly increases the susceptibility of Salmonella enterica serovar Typhimurium to colistin.

BACKGROUND: The increasing use of colistin causes a serious breach in our last line of defence against MDR Gram-negative pathogens. Our previous study showed that CpxR overexpression increases the susceptibility of acrB and cpxR double-deleted Salmonella enterica serovar Typhimurium to colistin. OBJECTIVES: To identify the mechanism of CpxAR and efflux pumps that synergistically enhance the susceptibility of S. Typhimurium to colistin. METHODS: A series of cpxR- and tolC-deleted mutants and a cpxR-complemented strain from a multidrug-susceptible standard strain of S. Typhimurium (JS) were generated in our previous study. Herein, we investigated the susceptibility of these strains to colistin through the broth microdilution method, time-kill curves and survival assays. Growth curves were measured by OD600 in LB broth, tryptone-soy broth (TSB) and M9-glucose (0.2%) minimal media. Finally, molecular mechanisms underlying the mode of action were elucidated by transcriptomic analysis. RESULTS: We found that in contrast to JS (0.8 mg/L), the MIC of colistin for JSΔtolC::kan showed a 16-fold decrease (0.05 mg/L). Notably, JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were associated with a 256-fold decrease (0.0031 mg/L) compared with JS. Growth curves identified that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR displayed a markedly lower growth rate and poorer adaptability. In addition, time-kill curves and survival assays showed that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were more susceptible to colistin. Lastly, double deletion of cpxR and tolC enhanced oxidative damage through promoting oxidative phosphorylation, the tricarboxylic acid (TCA) cycle and trimethylamine N-oxide (TMAO) respiration. CONCLUSIONS: Our findings revealed that double deletion of cpxR and tolC significantly increases the susceptibility of S. Typhimurium to colistin.

The Formation of Two Hybrid Plasmids Mediated by IS26 and Tn6952 in Salmonella enterica Serotype Enteritidis.

To characterize the formation mechanism and characteristics of two cointegrate plasmids in Salmonella enterica serotype Enteritidis strain S13, plasmids from strain S13 and three corresponding transconjugants were subjected to whole genome sequencing and analyzed using bioinformatics tools. The traits of two fusion plasmids in transconjugants were characterized by stability and conjugation experiments. Sequence analysis indicated that strain S13 contained four plasmids, including mcr-1-bearing pS13-1, bla CTX-M-55-carrying pS13-2, tet(M)-bearing pS13-3, and floR-carrying pS13-4. IncN1-F33:A-:B- plasmid pS13-2, respectively, fused with IncFI:A-:B- plasmid pS13-3 and IncX1 plasmid pS13-4, which generated two cointegrate plasmids, designated pS13D and pS13F, which involved in two intermolecular replicative mechanisms mediated by IS26 and the novel transposon Tn6952 (ΔTnAS3-IS26-ΔISEcp1-ramA-ΔIS26-ΔTnAS1), respectively. This is the first report of the fusion of the IncN1-F33:A-:B- plasmid and IncFI:A-:B- plasmid mediated by IS26, and with IncX1 plasmid mediated by Tn6952. The formation and evolution of cointegrate plasmids could expand the resistance and host spectrum of fusion plasmids.