Colistin Research Articles

Effects of drinking water supplemented with apple vinegar, essential oils, or colistin sulfate on growth performance, blood lipids, antioxidant status, intestinal morphology, and gut microflora of broiler chickens.

With rising concerns about antibiotic resistance and its consequences on public health, the identification of safe and effective alternatives to antibiotics in the poultry industry has become increasingly critical. This study aimed to evaluate the effects of supplementing drinking water with apple vinegar and essential oils, compared to an antibiotic growth promoter, on the growth performance, serum lipid profile, antioxidant status, intestinal morphology, and gastrointestinal microflora population of broiler chickens. A total of 240 one-day-old male broiler chickens were randomly assigned to four treatments, each consisting of six replicate pens with ten birds per pen. The broiler chickens received the following experimental treatments: normal drinking water without additives (control) and drinking water supplemented with antibiotic (0.25 ml/L), apple vinegar (1 ml/L), and essential oils blend (0.5 ml/L). The results showed that adding antibiotic or essential oils blend to drinking water improved body weight gain and feed conversion ratio throughout the entire rearing period (P < 0.05). The serum levels of cholesterol and low-density lipoprotein in broilers that received apple vinegar or essential oils blend were lower than those in the control and antibiotic groups (P < 0.05). The essential oils blend treatment significantly increased the activity of glutathione peroxidase and superoxide dismutase in the serum of broiler chickens compared to the other treatments (P < 0.05). Furthermore, adding essential oils blend to the drinking water increased villus height and villus height to crypt depth ratio in the jejunum of broilers (P < 0.05). The broiler chickens in the apple vinegar and essential oil blend groups also exhibited a lower population of Enterobacteriaceae and a higher count of lactic acid bacteria in the ceca (P < 0.05). In summary, the addition of essential oils blend to the drinking water of broiler chickens has beneficial effects on production performance and can serve as an effective alternative to antibiotic growth promoters.

Cinnamic Acid Compounds (p-Coumaric, Ferulic, and p-Methoxycinnamic Acid) as Effective Antibacterial Agents Against Colistin-Resistant Acinetobacter baumannii.

Colistin-resistant Acinetobacter baumannii (COLR-Ab) is an opportunistic pathogen commonly associated with nosocomial infections, and it is difficult to treat with current antibiotics. Therefore, new antimicrobial agents need to be developed for treatment. Based on this information, we investigated the antimicrobial, antibiofilm, and combination activities of p-coumaric acid (p-CA), ferulic acid (FA), and p-methoxycinnamic acid (p-MCA) against five COLR-Ab isolates. p-CA, FA, and p-MCA exhibited antimicrobial activity against COLR-Ab isolates, with minimum inhibitory concentration (MIC) values in the range of 256-128 µg/mL, 1024-512 µg/mL, and 512-128 µg/mL, respectively. The combination effects of the compounds with colistin (COL) were evaluated using a checkerboard synergy test. The combinations exhibited a synergistic effect and caused a 128- to 16-fold decrease in COL MIC values. In addition, the biofilm production capacities of the COLR-Ab isolates and the antibiofilm activities of the compounds were determined using the microtitre plate-based crystal violet (CV) technique. The compounds showed effective antibiofilm activity against strong and moderate biofilm-producing isolates, inhibiting biofilm formation by 77.5% and 19.7%. Spectrometric measurements were used to examine the effect of compounds on membrane permeability; 1.9-, 1.66-, and 1.34-fold increases in absorbance values were observed at MIC concentrations of p-CA, FA, and p-MCA, respectively. Furthermore, morphological changes caused by the compounds in the isolate were observed using scanning electron microscopy (SEM) micrographs. According to the WST assay, the compounds did not show any statistically significant cytotoxic effect on the cells (p > 0.05). These findings indicate that p-CA, FA, and p-MCA may be potential new alternative candidates against resistant A. baumannii.

Nanoformulation of Polymyxin E Through Complex Coacervation: A Pharmacokinetic Analysis.

Objectives: Polymyxin E (PME), a polymyxin antibiotic, serves as a final resort against antibiotic resistance. Nephrotoxicity is the primary concern when employing PME. To alleviate this issue, researchers have explored strategies including dosing adjustments and innovative formulations. This study employed complex coacervation to create PME nanoformulations, capitalizing on PME's charge properties. The research question and hypothesis posed pertained to whether neutralization of PME's positive charge during formulation would reduce its antibiotic efficacy and alter its tissue distribution and other pharmacokinetic parameters. Our objective was to evaluate the capability of complex coacervation to mitigate the adverse effects of PME while preserving its antibacterial potency and therapeutic effectiveness. Methods: Three negatively charged polyions: potassium sucrose octasulfate, polytamic acid, and sodium hyaluronate, were used for formulation. We performed characterization on the nanocomplex formed by the polyions and PME. The nanoformulations underwent several tests, including minimum inhibitory concentration, in vivo efficacy on an infected mouse model, pharmacokinetic assessments, tissue distribution, and toxicity. Results: the three polyions formed coacervation complexes with PME at varying charge ratios, yielding nanoparticles smaller than 30 nm with low polydispersity (PDI < 0.3). The results demonstrated that complex coacervation-mediated PME nanoformulations exhibited equivalent or superior antibacterial activity, increased maximum tolerant dose, and fewer adverse reactions in animal tests. Conclusions: Utilizing complex coacervation, PME nanoformulations were developed, demonstrating efficacy in the formulation process. Pharmacokinetic assessments revealed absorption and distribution profiles akin to those of standalone PME. The positive charge inherent in PME causing its toxicity was mitigated after complex coacervation.

Efficacy, safety, and therapeutic drug monitoring of polymyxin B sulfate and colistin sulfate in critically ill patients: a real-world retrospective study.

Polymyxin B sulfate (PBS) and colistin sulfate (CS) are the last-line treatments for infections caused by multidrug-resistant Gram-negative bacteria, but their efficacy and safety have not been validated. The aims of the current study were to (1) determine their efficacy and safety among critically ill patients and the influencing factors, and (2) determine the relationships of drug exposure with efficacy and safety, to provide evidence for the precision dosing. This retrospective study included 100 critically ill patients treated with PBS and 80 treated with CS. The efficacy outcomes were clinical efficacy and 30-day mortality, while the safety indicator was acute kidney injury (AKI) incidence. There was no significant difference between the two drugs in clinical efficacy, 30-day mortality, or overall AKI incidence, but the incidence of stage 3 AKI was significantly higher in the PBS cohort than the CS cohort. Therapeutic drug monitoring (TDM) and trough concentration (Cmin) were significantly associated with clinical efficacy and AKI in both cohorts. Classification and regression tree analysis revealed that Cmin values of ≥0.91mg/L for PBS and Cmin ≥ 0.53mg/L for CS were associated with higher clinical efficacy. There is basically no significant difference in the efficacy and safety of PBS and CS. TDM can significantly improve the clinical efficacy of both drugs and reduce the incidence of AKI. TDM is therefore recommended to improve the clinical efficacy while reducing the adverse reactions.

Assessment of the impact of different SARS-CoV-2 inactivation operations on colistin sulfate plasma concentration results

ObjectiveTo evaluate the effects of different SARS-CoV-2 inactivation methods on the blood concentration of colistin sulfate.MethodsA colistin sulfate reference substance, a quality control plasma sample, and a clinically measured sample were transferred and heated in a 56 °C water batch for 30 min or irradiated under an ultraviolet (UV) lamp for 60 min to examine the stability of the reference solution and quality control plasma sample. Statistical analysis was conducted for the concentration of the clinically measured sample before and after inactivation with the intraclass correlation coefficient (ICC) method, the Passing–Bablok regression, and the Bland–Altman analysis.ResultsThe reference substance of colistin sulfate was unstable under UV inactivation. Under moist heat inactivation, the reference substance and quality control plasma sample of colistin sulfate were stable. The ICC of the concentration results of 27 clinical blood samples with and without moist heat inactivation was 0.995 (P < 0.001). The Passing–Bablok regression equation was Y = 0.01347 + 0.9861X (R2 = 0.994, n = 27). Finally, 96.30% (26/27) of test data was within the 95% limit of agreement. The results obtained with the three statistical analysis methods all showed a high correlation and agreement between the plasma concentration results of samples not inactivated and inactivated with moist heat inactivation.ConclusionThe clinical plasma concentration test samples for colistin sulfate can be processed with the moist heat virus inactivation method to reduce the risk of virus infection of medical staff.

Synergistic antimicrobial efficacy of glabrol and colistin through micelle-based co-delivery against multidrug-resistant bacterial pathogens.

Widespread bacterial infection and the spread of multidrug resistance (MDR) exhibit increasing threats to the public and thus require new antibacterial strategies. Coupled with the current slow pace of antibiotic development, the use of antibiotic adjuvants to revitalize existing antibiotics offers great potential. We aim to explore the synergistic antimicrobial mechanism of glabrol (GLA) and colistin (COL) while developing an innovative multifunctional micelle-based drug delivery system to enhance therapeutic efficacy. The synergy between GLA and COL was assessed through a combination of high-throughput screening and checkerboard analysis techniques. Moreover, we performed fluorescence-based assays to investigate the underlying mechanisms of action of the GLA and COL combination. We also developed a multifunctional drug delivery platform that integrates GLA and COL into co-loaded composite micelles, aimed at improving antibacterial efficacy against peritoneal sepsis and chronic bacterial wound infections caused by diverse microbial pathogens. We have discovered that natural flavonoids found in plants act synergistically with colistin against MDR bacterial infections, effectively improving its efficacy through a co-delivery strategy. The combination therapy consisting of GLA and COL exhibits enhanced antibacterial efficacy and is capable of clearing 99% of MDR Gram-positive and Gram-negative bacteria in 4 h. Mechanistic studies showed that COL increases the outer membrane permeability, which promotes the adhesion of GLA to the inner membrane, disrupting bacterial metabolism, and ultimately leading to bacterial death. Furthermore, a novel pH-responsive hydrogel system was developed and dispersed with GLA and COL co-loaded composite micelles to mitigate the selective pressure of antibiotics with fewer side effects. Lastly, such a system showed high efficacy in two animal models. Our findings provide a potential therapeutic option using a co-delivery system functionalized with combination therapy, to address the prevalent infections caused by complex bacterial infections and even MDR bacterial infections.

Development and validation of a UPLC-MS/MS method for simultaneous quantification of polymyxins and caspofungin in human plasma for therapeutic drug monitoring.

To develop a rapid, convenient, accurate, and low-residual-effect ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of polymyxin B sulfate and colistin sulfate in the blood of patients with multidrug-resistant bacterial infections, as well as caspofungin acetate in the blood of patients with fungal infections, thus facilitating the rational use of antibiotics in clinical applications. All analytes were diluted with 0.2% aqueous formic acid, and plasma proteins were precipitated using acetonitrile. The selected reaction monitoring (SRM) mode was used for measurement. Separation of all analytes was completed on a Hypersil GOLD C18 column (100×2.1mm, 3.0µm). They were quantitatively analyzed using electrospray ionization on a triple quadrupole mass spectrometer in the positive ion mode. The mobile phase consisted of water (containing 0.1% formic acid) and acetonitrile, which was delivered by gradient elution at a flow rate of 0.3ml/min. The internal standard was bacitracin zinc (BcZn), and the column temperature was maintained at 25°C. The runtime for each analysis was 3.5min. The procedure was validated following the recommendations of the U.S. Food and Drug Administration, which included measurements of accuracy (ranging from 83.27% to 105.86% for within-run and between-run accuracy), precision (with coefficients of variation from 2.50% to 16.51% for within-run precision and between-run precision), and matrix effects (ranging from 88.65% to 103.94%). The extraction recoveries ranged from 38.01% to 42.76 for polymyxin B1 (PMB1), polymyxin B2 (PMB2), polymyxin E1 (PME1), polymyxin E2 (PME2), and 88.65% to 89.84% for caspofungin (CPF). Plasma samples were stable under various storage conditions, including three freeze-thaw cycles at -80°C, 24-hour periods at room temperature and 4°C, and 30days of freezing at both -20°C and -80°C, with relative standard deviations (RSD) of less than 15%. In this study, a UPLC-MS/MS method was developed to simultaneously quantify PMB1, PMB2, PME1, PME2, and CPF in human plasma. The method was validated in blood samples from patients with multidrug-resistant bacteria combined with fungal infections and is suitable for therapeutic drug monitoring.

Efficacy and Safety of Aerosol Inhalation of Colistin Sulfate for the Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infection in the Peri-Operative Period of Liver Transplantation: A Single-Center Retrospective Study.

Objective: This study intended to evaluate the clinical efficacy and safety of colistin sulfate aerosol inhalation in combination with ceftazidime-avibactam for the treatment of pulmonary carbapenem-resistant Klebsiella pneumoniae (CRKP) infection during the peri-operative period of liver transplantation. Materials and Methods: A retrospective analysis was designed to investigate 52 patients who developed pulmonary CRKP infection after liver transplantation between December 1, 2019, and November 30, 2022. On the basis of whether they received colistin sulfate aerosol inhalation, the patients were divided into the treatment group (n = 29) and the control group (n = 23). The baseline information, infection status, CRKP enzyme type, inflammatory markers, liver and kidney function, and prognosis were compared and analyzed. Results: There were no significant differences in patient characteristics, infection status, and drug resistance enzyme type between the treatment group (treated with colistin sulfate aerosol inhalation and ceftazidime and avibactam sodium for injection) and the control group (treated with ceftazidime and avibactam sodium for injection alone). Colistin sulfate aerosol inhalation treatment reduced concentrations of inflammatory markers, with post-treatment white blood cell count, procalcitonin, and C-reactive protein significantly lower than pre-treatment levels (p < 0.05). Except for C-reactive protein at 14 days (p = 0.032), the two groups had no significant differences in other indicators. There were no significant differences in alanine aminotransferase, aspartate aminotransferase, total bilirubin, and glomerular filtration rate after treatment, indicating no discernible alteration in liver and kidney function. In addition, the treatment group took a significantly shorter time to normalize body temperature compared with the control group (p = 0.025), but there were no significant differences in the cure with no colonization rate and all-cause mortality rate between the two groups. Conclusions: The combination of colistin sulfate aerosol inhalation and ceftazidime and avibactam sodium for injection is effective in treating pulmonary CRKP infection during the peri-operative period of liver transplantation. It does not impose an additional burden on liver and kidney function, providing a new treatment option for this type of infection.

Rutin Synergizes with Colistin to Eradicate Salmonellosis in Mice by Enhancing the Efficacy and Reducing the Toxicity.

The wide dissemination of multidrug-resistant (MDR) Gram-negative bacteria poses a significant global health and security concern. As developing new antibiotics is generally costly, fastidious, and time-consuming, there is an urgent need for alternative therapeutic strategies to address the gap in antibiotic discovery void. This study aimed to investigate the activity of colistin (CS) in combination with a natural product, rutin (RT), to combat against Salmonella Typhimurium (S. Tm) in vitro and in vivo. The results showed that a combination with RT enabled the potentiation of CS efficacy. Further mechanistic analysis indicated that RT disrupted iron homeostasis to inactivate the PmrA/PmrB system, thereafter reducing the bacterial membrane modifications for enhancing CS binding. Besides enhancing bactericidal activity of CS, RT was also observed to mitigate the CS-induced nephrotoxicity, by which the dosing limitation of CS was overcome for better pathogen clearance. The animal trial eventually confirmed the in vivo synergistic interaction of RT with CS to treat the bacterial infection. To sum up, the present study uncovered the potential of RT as a viable adjuvant of CS to eradicate the infection and protect the hosts, which might serve as a promising alternative to combat infections caused by MDR Gram-negative bacteria.

Isolation and Molecular Detection of Bacteria from Frequently Touched Objects of Various Public Places at Sadar Upazila of Mymensingh District

The current research was conducted to investigate the magnitude of bacterial contamination in public buses, different wards of hospitals, and public toilets of bus stations and hospitals in Sadar Upazila, Mymensingh, Bangladesh. A total of 90 swab samples were collected aseptically from the most frequently touched surfaces of the sample area. Identification of the isolated bacteria was done by staining and biochemical tests, followed by molecular detection by PCR using genus or speciesspecific primers. Isolated organisms were then subjected to an antibiotic sensitivity test using disk diffusion techniques using 13 commonly available antibiotics. Among the samples, 76.67% (n=69/90) were positive for E. coli, 80% (n=72/90) were positive for Klebsiella spp., and 68% (n=61/90) were positive for Staphylococcus spp. Positive tetA and stx-1 genes were found in 40 and 19 E. coli isolates, respectively. 23 positive mecA genes, or MRSA, were found in Staphylococcus aureus isolates that pose a threat to public health. Toilets of the bus stations were the most contaminated place by the selected bacteria, with the prevalence of E. coli,Klebsiella spp., and Staphylococcus aureus being 81.48% (n=22/27), 77.78% (n=21/27), and 81.48% (n=22/27), respectively. In an antibiogram study, E. coli isolates showed 100% resistance against amoxicillin, azithromycin, tetracycline, and co-trimoxazole. Klebsiella spp. was revealed to be 100% resistant to amoxicillin, followed by colistin sulfate (60%). Staphylococcus aureus isolates were 100% resistant to methicillin, cefoxitin, and cefixime. The findings can be used to raise public awareness about the possible threat, hence preventing the spread of infectious disease in public places. J. of Sci. and Tech. Res. 6(1): 125-137, 2024

Web-Based Passive Surveillance: Multifactorial Assessment of Sonali Chicken Diseases and Antimicrobial Prescription Pattern in Bangladesh.

Despite the significant growth in Sonali chicken production across Bangladesh, inadequate disease surveillance and control measures along with indiscriminate antimicrobial use remain major challenges to the sector. In this study, we evaluated the disease burden and antimicrobial prescription patterns of Sonali chickens in Bangladesh using a web-based data recording system from 2020 to 2021 and analyzed 1690 cases. The diagnoses recorded in the system were presumptive, as they were based on clinico-epidemiological history, clinical signs, and gross necropsy findings noted by registered veterinarians. We conducted this study in Bogura, a district renowned for its high concentration of Sonali chicken farms. We estimated a higher prevalence of infection among grower chickens (69.0%) compared to starter chickens (31.0%). Small- to medium-sized flocks (63%) were more frequently infected than larger flocks (37.0%). Most disease cases occurred during the summer season (43.0%), followed by winter (27%), the rainy season (15%), and autumn (14%). Overall, climatic factors contributed to 51% of disease occurrence at temperatures below 25°C, 55% at high humidity (≥75%), and 57% during heavy rainfall (≥29 mm). The most prevalent disease was Newcastle disease (ND) (19.5%), followed by Marek's disease (9.8%), coccidiosis (7.4%), necrotic enteritis (4.7%), infectious bursal disease (3.2%), and infectious laryngotracheitis (3.2%). The odds of ND were 1.4 (grower chickens vs. starter chickens), 11.4 (summer vs. winter), 4.1 (autumn vs. winter), 3.9 (rainy vs. winter), 3.5 (≥25 °C vs. <25 °C), and 2.6 (≥75% vs. <75%). Tylvalosin (38.0%) was the most frequently prescribed antibiotic, followed by fluoroquinolones (9.0%), aminoglycosides (8.0%), and colistin sulphate (4.0%). These findings suggest that a web-based disease record could be an important tool for a centralized poultry disease surveillance system in low- and middle-income countries like Bangladesh.

The ameliorative effect of pioglitazone against colistin-induced nephrotoxicity is mediated by inhibition of NF-κB and restoration of Nrf2 signaling: An integrative bioinformatics prediction-guided in vitro study.

Pioglitazone, an anti-diabetic drug, has been previously shown to ameliorate kidney damage through anti-inflammatory and antioxidant effects. In this study, we employed an integrative bioinformatics approach to study the possible mechanisms involved in the mitigative effect of pioglitazone against colistin-induced nephrotoxicity. Next, we validated the results obtained from the bioinformatics study by pre-treating human kidney-2 (HK-2) cell line with pioglitazone 100 μM for 30 minutes and then treating the cells with colistin sulfate 1200 μM for 24 hours. Inflammatory signaling by cytokines and the nuclear factor erythroid 2 related factor 2 (Nrf2) signaling pathways were predicted to be involved in the ameliorative effect of pioglitazone against colistin-induced nephrotoxicity. The nuclear factor kappa B subunit p65 (NF-κB p65) and Nrf2 were among the predicted transcription factors regulating the hub genes. Moreover, miR-24, miR-16, and miR-21 were identified as potential pathogenic miRNAs regulating the hub genes. In contrast, miR-17, miR-27a, and miR-146a were identified as potential protective miRNAs. The in vitro study indicated that pioglitazone pre-treatment increased cell viability in HK-2 cells exposed to colistin. Pioglitazone pre-treatment reduced the expression of pro-inflammatory cytokine genes (IL6 and TNF). Moreover, pioglitazone reduced the protein expression of NF-κB p65 and increased the protein expression of Nrf2. The protective effect of pioglitazone against colistin-induced toxicity in HK-2 cells is related to its anti-inflammatory and antioxidant activity through modulating NF-κB-mediated inflammatory signaling and Nrf2-mediated antioxidative stress signaling.

Colistin-niclosamide effervescent dry suspension combats colistin-resistant Salmonella in vitro and in vivo

The increasing incidence of bacterial infections caused by multidrug-resistant (MDR) Gram-negative bacteria has deepened the need for new effective treatments. It has been reported that niclosamide (NIC) can restore the sensitivity of Gram-negative bacteria to colistin (COL). However, NIC is practically insoluble in water and sparingly soluble in organic solvents, leading to limited therapeutic applications. This study aims to prepare a COL-NIC effervescent dry suspension (CNEDS) and evaluate its antibacterial effect against COL-resistant Salmonella both in vitro and in broiler chickens. With the sedimentation volume ratio as an index, suitable suspending agent, wetting agent, filler and effervescent agent were screened through a single-factor method. The preparation conditions were optimized using the Box-Behnken response surface method to obtain the formulation for CNEDS. The quality evaluation results showed that the successfully prepared CNEDS had a sedimentation volume ratio of 0.99, a drying weight loss of 1.3%, and a re-dispersion capability of 1-2 times, all of which met pharmacopoeial requirements. In terms of pharmacological evaluation, we first demonstrated that CNEDS substantially restored COL sensitivity against COL-resistant bacteria. Subsequently, time-killing analysis, scanning electron microscopy (SEM) and live/dead assays confirmed the antibacterial activity of CNEDS against COL-resistant bacteria. Finally, a Salmonella infection model in broiler chickens was established to further assess the therapeutic effect of CNEDS in vivo. CNEDS improved the survival rate of broiler chickens, reduced the bacterial burden on organs. These findings suggest that CNEDS effectively overcome COL resistance, indicating its potential for the treatment of COL-resistant bacterial infections in broiler chickens.

A Day Saved is a Life Saved: Direct Antimicrobial Susceptibility Testing from Positively Flagged Blood Culture Bottles and their Concordance with the Routine Method.

Sepsis is a major health problem worldwide and is associated with high morbidity and mortality with every hour delay in initiation of therapy. A conventional method of blood culture and Antimicrobial Susceptibility Testing (AST) takes around 48-72 hours. Empirical antibiotics need to be administered until the sensitivity report is made available. It has been estimated that 20-50% of the empirical antibiotics are inappropriate, resulting in prolonged hospital stays, adverse effects, and emergence of drug resistance. Additionally, this also puts an extra financial burden on both the patients and healthcare settings. Performing direct Antimicrobial Sensitivity Testing (dAST) is an important tool to reduce turn-around time (TAT) by at least 18-24 hours, thus reducing morbidity and mortality among critically ill patients. Direct AST (dAST) was performed from the positively flagged blood culture bottles received between December, 2021 to May, 2022 from Intensive Care Units (ICUs) on Mueller- Hinton Agar (MHA) using four drops of withdrawn blood. dAST was performed for six drugs: Ceftriaxone-30 μg (CTR), Piperacillin/Tazobactam-100/10 μg (PIT), Meropenem-10 μg (MRP), Ciprofloxacin-5 μg (CIP), Aztreonam-30 μg (AT), and Colistin (CL). The zone of inhibition was interpreted as per CLSI M100 ed32, 2022 guidelines. A parallel conventional method was also performed to examine for categorical agreement and disagreement. Identification was carried out using MALDI-TOF MS from the colonies that appeared on the dAST plate on the subsequent day. A total of 162 positively flagged blood culture bottles were included in the study. The majority of the Gram-negative organisms were from Enterobacterales (n=109), followed by Acinetobacter spp. (n=28) and Pseudomonas aeruginosa (n=25). Out of the 972 isolate-antimicrobial combinations, overall Categorical Agreement (CA) was seen in 936 (96.3%), whereas disagreement was observed in 36 with minor error (mE) in 21 (2.2%), major error (ME) in 7 (0.7%), and very major error (VME) in 8 (0.8%) when compared to the routine method. Categorical agreement (CA) of > 99% was seen in ceftriaxone (CTR) and ciprofloxacin (CIP). In comparison, the lowest CA was observed with meropenem (MRP) at 92%. Colistin dAST was performed using the E-strip method, and the result obtained was highly convincing, with an overall disagreement of only 1.2%. Rapid dAST from positively flagged blood culture bottles proved to significantly reduce the TAT from the time of sample collection to the first availability of antimicrobial susceptibility report with excellent categorical agreement of > 95% using the conventional disc diffusion method. Results obtained were within the acceptance criteria set by U. S. Food and Drug Administration (FDA) guidelines of > 90% categorical agreement for a new method. We were able to obtain excellent concordance for colistin using the E-strip method. Performing dAST not only saves a "day", but its proper implementation would save a "life".

High antimicrobial resistance rates and multidrug resistance in Enterobacteriaceae isolates from poultry in Souk Ahras region, Algeria

Background and Aim: The spread of antimicrobial resistance (AMR) in the Enterobacteriaceae family represents a major global health problem for humans and animals. This study aimed to determine AMR levels and highlight the different resistance profiles of Enterobacteriaceae isolates collected from healthy broiler chickens in eastern Algeria. Materials and Methods: A total of 200 cloacal swabs of healthy broilers from several poultry farms located in the Souk Ahras region (eastern Algeria) were collected. Samples were inoculated on MacConkey agar, and the isolated bacteria were identified using the API 20E system. Antimicrobial susceptibility testing was conducted using the disk diffusion method in accordance with the Clinical and Laboratory Standards Institute guidelines. The broth microdilution method was used to determine the minimum inhibitory concentration of colistin (CT). Results: Two hundred and forty-one isolates of commensal Enterobacteriaceae were recovered, including: Escherichia coli (n = 194; 80.5%), Proteus mirabilis (n = 21; 8.71%), Escherichia fergusonii (n = 8, 3.32%), Salmonella spp. (n = 7, 2.9%), Enterobacter cloacae (n = 4, 1.66%), Klebsiella pneumoniae (n = 3, 1.25%), Serratia spp. (n = 3, 1.25%), and Kluyvera spp. (n = 1, 0.41%). High resistance rates were observed toward erythromycin (100%), doxycycline (96.68%), trimethoprim-sulfamethoxazole (95.85%), ciprofloxacin (94.19%), ampicillin (90.04%), kanamycin (78.01%), and amoxicillin-clavulanic acid (69.71%). However, moderate-to-low resistance rates were observed for CT (25.31%), ceftazidime (12.45%), and cefotaxime (8.71%). Interestingly, only two extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates were detected. All isolates (100%) were multidrug-resistant (MDR), among which 58.92% were resistant to six and seven antibiotics. Forty AMR profiles were identified, reflecting a wide diversity of resistance with combinations of three to ten antibiotics. Conclusion: Our findings revealed alarming rates of AMR, highlighting the need to take measures to combat the phenomenon of AMR to protect animals and public health. Keywords: Algeria, antibiotic resistance, Enterobacteriaceae, multidrug resistance, poultry.

Outer membrane permeability of mcr-positive bacteria reveals potent synergy of colistin and macromolecular antibiotics against colistin-resistant Acinetobacter baumannii.

Colistin (CT) is the last-resort of antibiotic against multidrug-resistance (MDR) Acinetobacter baumannii (A. baumannii) infection. However, colistin resistance is increasingly reported in A. baumannii isolates partially due to the global emergence and dissemination of plasmid-borne mobile colistin resistance (mcr) gene and is a threat to human health. Thus, available treatment strategies urgently required in the fight against colistin-resistant A. baumannii. Here, we showed that mcr confers damaged outer membrane (OM) permeability in A. baumannii, which could compromise the viability of A. baumannii. Consistently, A. baumannii with colistin resistance exhibits increased susceptibility to macromolecular antibiotics such as rifampicin (RIF) and erythromycin (ERY). Moreover, the combination therapy of colistin and rifampicin demonstrates efficacy against colistin-resistant A. baumannii, regardless of the presence of mcr. Altogether, our data suggest that the synergy of colistin in combination with macromolecular hydrophobic antibiotics poses a promising therapeutic alternative for colistin-resistant A. baumannii.

Commercial Day-Old Chicks in Nigeria Are Potential Reservoirs of Colistin- and Tigecycline-Resistant Potentially Pathogenic Escherichia coli.

Background: Frequent use of colistin (COL) and tetracyclines in the Nigerian poultry sector potentially triggers bacterial resistance against COL and tigecycline (TIG), which are last-line antibiotics used to treat multidrug-resistant infections. Aim/Objectives: This study aimed to isolate COL- and TIG-resistant E. coli from commercial day-old chicks distributed to poultry farmers in Nsukka Southeastern Nigeria, assess the production of extended-spectrum β-lactamase (ESBL) and carbapenemase by the isolates, and establish their pathogenic potentials. Materials and Methods: Non-duplicate cloacal swabs were systematically collected from 250 randomly selected day-old chicks. MacConkey agar with 1 µg/mL of COL and 16 µg/mL of tetracycline was used for the isolation of putative COL- and tetracycline-resistant E. coli, respectively. E. coli isolates were confirmed biochemically using the API20E Gram-negative identification kit and molecularly by polymerase chain reaction targeting the uidA gene. Phenotypic COL resistance was established using COL agar and COL disc elution tests, while TIG insusceptibility was determined with disc diffusion. ESBL and carbapenemase production was assessed by double-disc synergy and modified carbapenem inactivation methods, respectively. Pathogenic potentials were determined using phenotypic methods. Results: COL- and TIG-resistant E. coli was recovered from 95 (38.0%) and 62 (24.8%) swabs from the 250 chicks, respectively. None of the isolates were potential ESBL or carbapenemase producers. The COL-resistant isolates displayed pathogenic potentials such as biofilm formation, haemagglutination, cell surface hydrophobicity, surface layer, and gelatinase activities at rates of 30.7%, 8.4%, 33.7%, 23.5%, and 17.6%, respectively. Meanwhile, the TIG-resistant isolates exhibited their respective potentials at rates of 47.0%, 21.0%, 35.5%, 58.1%, and 43.6%. Red, dry, and rough (RDAR) was the predominant curli fimbriae, and the cellulose morphotype portrayed by both the COL- and TIG-unsusceptible potential biofilm-producing isolates. Conclusions: This study demonstrates that a significant percentage of commercial day-old chicks distributed to farmers in Nsukka, southeastern Nigeria, are colonized by potentially pathogenic COL- and TIG-resistant E. coli, which could spread to humans and the environment.

Colistin, Meropenem-Vaborbactam, Imipenem-Relebactam, and Eravacycline Testing in Carbapenem-Resistant Gram-Negative Rods: A Comparative Evaluation of Broth Microdilution, Gradient Test, and VITEK 2.

This study aimed to evaluate and compare the performance of different assays for antimicrobial susceptibility testing (AST) and minimum inhibitory concentration (MIC) determination for reserve antibiotics in carbapenem-resistant Enterobacterales (CREs), Pseudomonas aeruginosa (CRPAs), and Acinetobacter baumannii (CRABs). An analysis was conducted on 100 consecutive isolates: 50 CREs, 35 CRPAs, and 15 CRABs. Sensititre broth microdilution was used as a reference standard to evaluate the performance of VITEK 2 card AST-XN24 (bioMérieux), the respective gradient tests (bioMérieux), and UMIC colistin broth microdilution test strips (Bruker Daltonics). Errors, essential agreement (EA), and categorical agreement of MICs for colistin (COL), meropenem-vaborbactam (MVB), imipenem-relebactam (IRL), and eravacycline (ERV) were assessed. The agreement between both of the COL broth microdilution (BMD) methods was perfect (100/100). The gradient test and VITEK 2 analysis yielded comparable EA rates (92/100 and 72/79, respectively), with the latter not registering any very major errors (VMEs). The MVB gradient test achieved EA in 66 of 85 isolates and VITEK 2 in 70/85. For IRL, EA was reached in 69 and 64 of 85 cases by gradient test and VITEK 2 analysis, respectively. The ERV gradient test yielded false results in nearly all (12/15) CRABs but achieved EA in 46 of 50 CREs. The VITEK system recorded EA for ERV in 60 of 65 isolates. We observed substantial variability in the measured MICs between BMD and the alternative methods. In only a few constellations, VITEK 2 or gradient tests could substitute the reference method. BMD is the method of choice for COL analysis, with VITEK 2 representing an alternative method for CRPA testing. Alternative methods for MVB did not provide reliable results, except for Enterobacterales, when tested with the gradient test. However, resistant results need to be confirmed by BMD. Only BMD can be used for IRL MIC determination. VITEK 2 was mostly accurate in measuring ERV MICs, while the corresponding gradient test yielded reliable results exclusively in CREs. It is essential that laboratories are aware of which testing method provides reliable results for each combination of microorganisms and reserve antibiotics.

Virulence and Antimicrobial Resistance Patterns of Salmonella spp. Recovered From Migratory and Captive Wild Birds.

Salmonella spp., especially those are resistant to extended-spectrum β-lactamase (ESBL), are considered as major concern to global health due to their emergence and dissemination. The aim of this study was to investigate the virulence and antimicrobial resistance (AMR) profile of Salmonella spp. from migratory and captive wild birds. A total 262 faecal samples were collected, and the identification of Salmonella spp. was carried out using a standard culture and PCR as well as molecular detection of virulence and AMR genes. The overall prevalence of Salmonella was determined to be 30.92% (95% CI = 25.63-36.75). Migratory birds exhibited highest prevalence (38.10%), whereas wild birds in captivity showed a lower prevalence (23.40%). The agfA gene was detected at a higher rate at 24.69%. Salmonella spp. exhibited 100% resistance to tetracycline, followed by 58% ampicillin and 46% streptomycin. In addition, there was a resistance rate to ceftriaxone of 17% and to colistin sulphate of 25%. Interestingly, levofloxacin alone displayed 100% sensitivity across all isolates, while ciprofloxacin and azithromycin showed 73% and 64% sensitivity, respectively. The MAR index was 0.25 and 0.42, and 74.07% of all isolates showed multidrug resistance (MDR). It was shown that migratory and captive wild birds contained ESBL genes blaTEM (94.34% and 49.06%) and blaSHV (13.33% and 10%), respectively. Genes responsible for sulphonamide (sul1) resistance were detected in 13.33% and 79% of wild and migratory birds, respectively. Salmonella has been found in captive wild and migratory birds and could act as reservoirs for the transmission of MDR and ESBL bacteria.

A simple, robust and high-throughput LC-MS/MS method for the therapeutic drug monitoring of polymyxin B1, polymyxin B2, polymyxin B3, isoleucine-polymyxin B1, polymyxin E1 and polymyxin E2 in human plasma.

To facilitate clinical therapeutic drug monitoring (TDM) of polymyxin B (PB) and polymyxin E (PE), we developed and validated a simple LC-MS/MS method for simultaneous determination of PB (including polymyxin B1 (PB1), polymyxin B2 (PB2), polymyxin B3 (PB3) and isoleucine-polymyxin B1 (ile-PB1)) and PE (including polymyxin E1 (PE1) and polymyxin E2 (PE2)) in human plasma. PB or PE was extracted from 20.0μL plasma using a 5% (v/v) formic acid acetonitrile solution and separated on a BEH-C18 column (2.1 × 100 mm, 1.7μm) with a mobile phase consisting of 0.8% formic acid aqueous solution and 0.2% formic acid acetonitrile solution. Gradient elution was performed over 5.5min at a flow rate of 0.250 mL/min. Quantitative analysis was conducted in positive ion scanning mode by electrospray ionization and multiple reaction monitoring. The method validation was conducted based on bioanalytical method validation guidance, including specificity, calibration curve, precision, accuracy, recovery, matrix effect, stability and dilution integrity and all of the results satisfied the requirements. The method was simple, robust and high-throughput and is currently being used to provide a TDM service to enhancing therapeutic efficacy and safety use of the PB and PE.