Clinical analysis of colistin sulfate in the treatment of pneumonia caused by carbapenem-resistant Gram-negative bacteria

BACKGROUND Multidrug-resistant Gram-negative bacteria,exacerbated by excessive use of antimicrobials and immunosuppressants,are a major health threat.AIM To study the clinical efficacy and safety of colistin sulfate in the treatment of carbapenem-resistant Gram-negative bacilli-induced pneumonia,and to provide theoretical reference for clinical diagnosis and treatment.METHODS This retrospective analysis involved 54 patients with Gram-negative bacilli pneumonia admitted to intensive care unit of The General Hospital of the Northern Theater Command of the People's Liberation Army of China from August 2020 to June 2022.After bacteriological culture,the patients'airway secretions were collected to confirm the presence of Gram-negative bacilli.The patients were divided into the experimental and control groups according to the medication used.The research group consisted of 28 patients who received polymyxin sulfate combined with other drugs through intravenous,nebulization,or intravenous combined with nebulization,with a daily dosage of 1.5–3.0 million units.The control group consisted of 26 patients who received standard dosages of other antibiotics(including sulbactam sodium for injection,cefoperazone sodium sulbactam for injection,tigecycline,meropenem,or vaborbactam).RESULTS Of the 28 patients included in the research group,26 patients showed improvement,treatment was ineffective for two patients,and one patient died,with the treatment efficacy rate of 92.82%.Of the 26 patients in the control group,18 patients improved,treatment was ineffective for eight patients,and two patients died,with the treatment efficacy rate of 54.9%;significant difference was observed between the two groups(P<0.05).The levels of white blood cell(WBC),procalcitonin(PCT),and C-reactive protein(CRP)in both groups were significantly lower after treatment than before treatment(P<0.05),and the levels of WBC,PCT,and CRP in the research group were significantly lower than those in the control group(P<0.05).Compared with before treatment,there were no significant changes in aspartate aminotransferase,creatinine,and glomerular filtration rate in both groups,while total bilirubin and alanine aminotransferase decreased after treatment(P<0.05)with no difference between the groups.In patients with good clinical outcomes,the sequential organ failure assessment(SOFA)score was low when treated with inhaled polymyxin sulfate,and specific antibiotic treatment did not improve the outcome.Sepsis and septic shock as well as a low SOFA score were independent factors associated with good clinical outcomes.CONCLUSION Polymyxin sulfate has a significant effect on the treatment of patients with multiple drug-resistant Gram-negative bacilli pneumonia and other infections in the lungs and is safe and reliable.Moreover,the administration route of low-dose intravenous injection combined with nebulization shows better therapeutic effects and lower adverse reactions,providing new ideas for clinical administration.

Colistin Resistance Profiles, Molecular Investigation of mcr-1 and mcr-2 Plasmid Genes and Investigation of Carbapenemase Production in Pseudomonas and Acinetobacter Strains

Background and Purpose: The reintroduction of colistin as a last resort treatment against multi-resistant Gram-negative bacilli, is currently challenged by the emergence of colistin-resistant bacteria. The aim of this study was to assess the susceptibility of Pseudomonas and Acinetobacter strains to colistin, to identify carbapenemase production, and to investigate the plasmid genes involved in colistin resistance and carbapenemase production. Methodology: In order to establish the susceptibility profiles of 17 strains of Pseudomonas and Acinetobacter to colistin, their Minimum Inhibitory Concentrations (MICs) were determined using the liquid microdilution method. The possible production of carbapenemases was investigated with the modified Carbapenem Inactivation Method (mCIM). The search for genes encoding carbapenemases (blaOXA, blaIMP, blaCarba) and those responsible for plasmid resistance to colistin (mcr-1 and mcr-2) was performed by conventional PCR. Results and Conclusion: Ninety-four percent (94%) (16/17) of the strains were resistant to colistin. Intraspecies distribution was 50% (8/16), 31% (5/16), 13% (2/16) and 6% (1/16) for Acinetobacter baumannii, Pseudomonas aeruginosa, Pseudomonas luteola, and Pseudomonas fluorescens, respectively. Twenty-nine percent (29%) (6/17) of the strains produced carbapenemases. No mcr-1 and mcr-2 plasmid genes were detected. On the other hand, 17.6% (3/17) of the strains possessed the carbapenemase genes distributed as follows: Carba type (60%), OXA type (40%) and IMP type (0%). The results of this study highlight a high resistance to colistin in strains belonging to the genera Acinetobacter and Pseudomonas, and some of these strains produce carbapenemases.

硫酸粘杆菌素(Colistin sulfate)对大肠杆菌的抗生素后效应

用微量稀释法测定的硫酸粘杆菌素 ( colistin sulfate)对 4株大肠杆菌的最小抑菌浓度 ( MIC)和最低杀菌浓度( MBC) ,分别为 0 .2～ 0 .8m g/ L和 0 .8～ 1.6 mg/ L。 2 MIC硫酸粘杆菌素对 4株大肠杆菌作用 2 h后 ,10 0 0倍稀释去除药物 ,硫酸粘杆菌素对 4株大肠杆菌的抗生素后效应 ( PAE)在 1.0 6 2～ 2 .0 17h之间。 6 4、32、16、8、4、2 MIC硫酸粘杆菌素对大肠杆菌 CMCC4 4 10 3作用 2 h后 ,10 0 0倍稀释去除药物 ,硫酸粘杆菌素 PAE的平均值分别为 0 .5 17、2 .4 87、2 .0 37、1.72 7、1.4 6 7、1.0 5 7h,PAE与药物浓度呈正相关。

Risk factors and clinical responses of pneumonia patients with colistin-resistant Acinetobacter baumannii-calcoaceticus

BACKGROUND Nosocomial infections with carbapenem-resistant Acinetobacter baumanniicalcoaceticus complex(ABC)strains are great problem for intensive care units.ABC strains can develop resistance to all the antibiotics available.Carbapenem resistance is common and colistin resistance is rare in our country.Knowing the risk factors for colistin resistance is important since colistin seems to be the only remaining therapeutic option for the patients with pneumonia due to extensively drug resistant ABC for our country.AIM To investigate the comparison of clinical responses and outcomes between pneumonia patients with colistin-susceptible and-resistant Acinetobacter sp.Strains.METHODS During the study period,108 patients with pneumonia due to colistin-susceptible strains and 16 patients with colistin-resistant strains were included retrospectively.Continuous variables were compared with the Mann-Whitney U test,and categorical variables were compared using Pearson’s chi-square test or Fisher’s Exact chi-square test for two groups.A binary logistic regression model was developed to identify the potential independent factors associated with colistin resistance in patients with colistin-resistant strains.RESULTS High Acute Physiology and Chronic Health Evaluation II scores(OR=1.9,95%CI:1.4-2.7;P<0.001)and prior receipt of teicoplanin(OR=8.1,95%CI:1.0-63.3;P=0.045)were found to be independent risk factors for infection with colistin-resistant Acinetobacter sp.Different combinations of antibiotics including colistin,meropenem,ampicillin/sulbactam,amikacin and trimethoprim/sulfamethoxazole were used for the treatment of patients with colistin-resistant strains.Although the median duration of microbiological cure(P<0.001)was longer in the colistin-resistant group,clinical(P=0.703),laboratory(P=0.277),radiological(P=0.551),microbiological response(P=1.000)and infection related mortality rates(P=0.603)did not differ between the two groups.Among the patients with infections due to colistin-resistant strains,seven were treated with antibiotic combinations that included sulbactam.Clinical(6/7)and microbiological(5/7)response rates were quite high in these patients.CONCLUSION The optimal therapy regimen is unclear for colistin-resistant Acinetobacter sp.infections.Although combinations with sulbactam seems to be more effective in our study patients,data supporting the usefulness of combinations with sulbactam is very limited.

Baicalin inhibits colistin sulfate-induced apoptosis of PC12 cells

Baicalin, a type of flavonoid extracted from the dried root of Scutellaria baicalensis georgi, has been shown to effectively inhibit cell apoptosis. Therefore, we assumed that baicalin would suppress colistin sulfate-induced neuronal apoptosis. PC12 cells exposed to colistin sulfate （62.5-500 μg/mL） for 24 hours resulted in PCl2 cell apoptosis. In addition, caspase-3 activity, lactate dehydrogenase level and free radical content increased in a dose-dependent manner. Subsequently, PC12 cells were pretreated with baicalin （25, 50 and 100 pg/mL）, and exposed to 125 pg/mL colistin sulfate. Cell morphology markedly changed, and cell viability increased. Moreover, caspase-3 activity, lac- tate dehydrogenase level and free radical content decreased. Results indicated that baicalin inhib- ited colistin sulfate-induced PC12 cell apoptosis by suppressing free radical injury, and reducing caspase-3 activity and lactate dehydrogenase activity.

Studies on the Injectable Solution of Colistin Sulfate and Its Pharmacokinetics

The present studies were conducted to compose an injectable solution of colistin sulfate containing local anaesthesia, antioxidant and other additions. Results showed that the novel preparation was stable either to heat or to light. The term of validity of the preparation was 2 years at room temperature.The preparation containing 25. 0 mg ml-1 colistin sulfate showed no local tissue irritation, but the concentration of 50. 0 mg ml-1 colistin sulfate showed obvious local tissue irritation. Result of acute toxicity test showed that the LD50 of intramuscular injection in mice was 38. 72 mg kg-1, and oral LD50 was 431. 39 mg kg-1. The evidence of neurotoxicity was observed in mice in the acute toxicity test. A dose of 10.0 mg kg-1 b. w. or 15:0 mg kg-1b. w. was administered intramuscularly to piglet once daily for 5 days. No changes were detected in the piglet body except for the slight epithelial tissue's granular degenerations in the kidney and liver at the dose of the 10.0 mg kg-1. While at the dose of 15. 0 mg kg-1, the obvious neurotoxicity was observed at 4-5 days. The epithelial tissues in the kidney and liver showed moderate granular degenerations, especially in the tubuli renales cells. Blood cell's morphosis indexes were normal. With relation to liver's function, the indexes went beyond the normal scope. But with relation to kidney's function, the indexes showed mostly normal.When the preparation was separately administered into muscle(i. m.) in piglets with the dose of 2. 5 and 5. 0 mg kg-1 b. w, whose Cmax were 3.73±0. 28 and 6. 40±0.18 Abstract:g ml-1; Tmax were 32±1. 5 and 34±1.8min; t1/2β were 256±14 min and 264±29 min, respectively. t1/2βt was 251±13 min for the injection given into aural vein( i. v.) with the dose of 2.5 mg kg1-1 b. w.. Samples of the experimentally determined plasma concentration of colistin sulfate generated two-exponential model with first-order absorption. The mean absolute bioavail-ability coefficient of 2.5 and 5. 0 mg kg-1 b. w. (i. m.) were 98. 30 and 88. 54%, respectively. The high bio-availability and the long maintaining time of the valid blood-drug concentration showed that the injectable solution was suitable for i. m. in pigs, whose recommended dose was 2.5 mg kg-1 b. w. , twice daily.

A One-Health Sampling Strategy to Explore the Dissemination and Relationship Between Colistin Resistance in Human,Animal,and Environmental Sectors in Laos

This study was designed to investigate the molecular epidemiology of mobile colistin resistance(mcr)using a"One-Health"approach in Laos and to predict whether any dominant plasmid backbone and/or strain type influences the dissemination of mcr.We collected 673 samples from humans(rectal normal flora),poultry,and the environment(water,flies,birds,etc.)in Vientiane,Lao People’s Democratic Republic(Laos),from May to September 2018.A total of 238 Escherichia coli(E.coli)isolated from nonduplicative samples,consisting of 98 MCR-positive E.coli(MCRPEC)("mcr"denotes the gene encoding mobile colistin resistance,and"MCR"denotes the subsequent protein encoded by mcr)and 140 MCRnegative E.coli(MCRNEC),were characterized by phenotype and Illumina sequencing.A subset of MCRPEC was selected for Min ION sequencing,conjugation assay,plasmid stability,and growth kinetics in vitro.The prevalence of MCRPEC was found to be 14.6%(98/673),with the highest prevalence in human rectal swabs(45.9%(45/98),p<0.0001,odds ratio(OR):0.125,95% confidence interval(CI):0.077-0.202).The percentages of MCRPEC from other samples were 14.3%(2/14)in dog feces,12.0%(24/200)in flies,11.0%(11/100)in chicken meat,8.9%(8/90)in chicken cloacal,8.0%(4/50)in chicken caeca,and 7.5%(4/53)in wastewater.MCRPEC was significantly more resistant to co-amoxiclav,sulfamethoxazoletrimethoprim,levofloxacin,ciprofloxacin,and gentamicin than MCRNEC(p<0.05).Genomic analysis revealed the distribution of MCRPEC among diverse clonal types.The putative plasmid Inc types associated with mcr-1 were Inc X4,Inc HI2,Inc P1,Inc I2,and Inc FIA,and those associated with mcr-3 were Inc FII,Inc FIA,Inc FIB,Inc P1,and Inc R.Recovery of highly similar plasmids from both flies and other sampling sectors implied the role of flies in the dissemination of mcr-1.mcr-positive plasmids were shown to be conjugative,and a significantly high transfer rate into a hypervirulent clone ST1193 was observed.Plasmids containing mcr irrespective of Inc type were highly stable and invariably did not exert a fitness effect upon introduction into a new host.These findings signify the urgent need for a standard infection control program to radically decontaminate the source of resistance.

Linear Thanatin Is an Effective Antimicrobial Peptide against Colistin-Resistant <i>Escherichia coli in Vitro</i>

Colistin has been regarded as the last line antibiotic for treatment of infections caused by multidrug resistant gram-negative bacteria. Therefore, the increasing emergence of colistin resistance among gram-negative bacteria represents a serious problem. The objective of this study was to characterize the effectiveness of the chemically synthesized thanatin in linear form against colistin-resistant E. coli isolated from a pig farm in China. Agar diffusion assay and broth microdilution test were employed to analyze the susceptibility of colistin-sensitive E. coli (ATCC25922) and colistin-resistant E. coli (SHP45) to linear thanatin (L-thanatin). Combinatory effect of linear thanatin and colistin against E. coli was also determined by fractional inhibition concentration index (FICI) analysis. The results showed that L-thanatin at a concentration of 1 mg/ml produced larger inhibition zone on agar against ATCC25922 than SHP45. In the quantitative microdilution test, L-thanatin had the same MIC of 3.2 μg/ml for ATCC25922 and SHP45. Based on the FICI analysis, additive effect was obtained with 1.56 μg/ml of L-thanatin and 0.125 μg/ml of colistin for ATCC25922;but with 1.56 μg/ml of L-thanatin and 0.25 μg/ml of colistin or with 2 μg/ml of colistin and 0.39 μg/ml of L-thanatin for SHP45. These data proved that L-thanatin is an effective antimicrobial peptide against colistin-resistant E. coli.

Rapid one-step enzyme immunoassay and lateral flow immunochromatographic assay for colistin in animal feed and food

Background:Colistin(polymyxin E)is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention,treatment,and growth promotion.However,the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings,and its residue in animal-origin food may also pose serious health hazards to humans.Thus,it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin food.Results:A one-step enzyme-linked immunosorbent assay(ELISA)and a lateral flow immunochromatographic assay(LFIA)for colistin were developed based on a newly developed monoclonal antibody.The ELISA showed a 50%inhibition value(IC50)of 9.7 ng/m L with assay time less than 60 min,while the LFIA had a strip reader-based detection limit of 0.87 ng/m L in phosphate buffer with assay time less than 15 min.For reducing the non-specific adsorption of colistin onto sample vial,the components of sample extraction solution were optimized and proved to greatly improve the assay accuracy.The spiked recovery experiment showed that the recoveries of colistin from feed,milk and meat samples were in the range of 77.83%to 113.38%with coefficient of variations less than 13%by ELISA analysis and less than 18%by LFIA analysis,respectively.Furthermore,actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis.Conclusions:The developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.

An LC-MS/MS method for simultaneous analysis of the cystic fibrosis therapeutic drugs colistin,ivacaftor and ciprofloxacin

Inhaled antibiotics such as colistin and ciprofloxacin are increasingly used to treat bacterial lung infections in cystic fibrosis patients.In this study,we established and validated a new HPLC-MS/MS method that could simultaneously detect drug concentrations of ciprofloxacin,colistin and ivacaftor in rat plasma,human epithelial cell lysate,cell culture medium,and drug transport media.An aliquot of200 μL drug-containing rat plasma or cell culture medium was treated with 600 μL of extraction solution(acetonitrile containing 0.1% formic acid and 0.2% trifluoroacetic acid(TFA)).The addition of 0.2% TFA helped to break the drug-protein bonds.Moreover,the addition of 0.1% formic acid to the transport medium and cell lysate samples could significantly improve the response and reproducibility.After vortexing and centrifuging,the sample components were analyzed by HPLC-MS/MS.The multiple reaction monitoring mode was used to detect the following transitions:585.5-101.1(colistin A),578.5-101.1(colistin B),393.2-337.2(ivacaftor),332.2-314.2(ciprofloxacin),602.3-101.1(polymyxin B1 as internal standard(IS)) and 595.4-101.1(polymyxin B2 as IS).The running time of a single sample was only 6 min,making this a time-efficient method.Linear correlations were found for colistin A at 0.029-5.82 μg/m L,colistin B at 0.016-3.14 μg/m L,ivacaftor at 0.05-10.0 μg/m L,and ciprofloxacin at 0.043-8.58 μg/m L.Accuracy,precision,and stability of the method were within the acceptable range.This method would be highly useful for research on cytotoxicity,animal pharmacokinetics,and in vitro drug delivery.

Synergistic combination of colistin with imipenem, amikacineor ciprofloxacin against Acinetobacter baumannii andPseudomonas aeruginosa carbapenem-resistant isolated inAnnaba hospital Algeria

Objective:The aim of this study is to detect in vitro the synergetic activity of colistin in combination with imipenem,amikacin or ciprofloxacin,at sub-inhibitory concentrations,against carbapenems-resistant(CR)Acinetobacter baumannii and Pseudomonas aeruginosa strains isolated from various wards in Annaba teaching hospital in eastern Algeria.Materials and Methods:The minimal inhibitory concentrations(MIC)were determined by broth macrodilution(BMD).Carbapenemase encoding genes were screened using polymerase chain reaction(PCR).The activity of colistin in combination with second antibiotic was evaluated by the Checkerboard Technique.Results:39 CR P.aeruginosa and 21 CR A.baumanni strains where collected.The MIC values ranging from(0.25 to 4μg/ml)to colistin,≥16μg/ml for imipenem,≥4μg/ml to amikacin and≥8μg/ml ciprofloxacin.The PCR reveals the presence of the genes blaOXA23(n=12),blaOXA24(n=6),blaNDM1(n=3)in A.baumannii and blaVIM2(n=12)in P.aeruginosa.The combination of colistin with imipenem showed synergistic effect on 57.14%and 46.15%of A.baumannii and P.aeruginosa isolates,respectively.For colistin and amikacin,the synergistic effect is detected in 28.6%of A.baumannii and 30.8%of P.aeruginosa.While colistin and ciprofloxacin showed synergy on 14.29%and 15.38%of A.baumannii and P.aeruginosa isolates,respectively.Conclusion:CR A.baumannii and P.aeruginosa remain the most prevalent infection agents in patients from high-risk wards at Annaba Hospital.Colistin associated with imipenem or with amikacin at sub-inhibitory concentrations gives very encouraging results allowing better management of infections caused by this type of bacteria.

LC-MS/MS Method for Determination of Colistin in Human Plasma: Validation and Stability Studies

A simple and reliable high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the determination of colistin A and colistin B in human plasma was developed and validated. Clarithromycin was used as an internal standard (IS). Plasma extraction was performed using C-18 cartridges and methanol containing 0.1% formic acid. Analysis was performed using Atlantis dC18 (2.1 × 100 mm, 3 μm) column at room temperature and a mobile phase of 0.2% formic acid in acetonitrile and water (50:50, v:v), delivered at a flow rate of 0.2 ml/minute. Eluent was detected in the positive ion mode using electrospray ionization at the following transitions of mass to charge (m/z): colistin A, 585.6 → 101.4;colistin B, 578.7 → 101.3;and IS, 748.6 → 158.4. No interference by components of blank plasma or commonly used drugs was observed. The relationship between colistin A colistin B concentrations and their corresponding peak height ratios to the IS was linear over the range of 0.05 - 10 μg/ml. Inter-day coefficient of variation and bias were, respectively, ≤11.5% and −3.0 to 6.0 for colistin A and ≤9.9 and −4.7 to 3.0 for colistin B. Mean extraction recovery of colistin A, colistin B, and the IS were 97%, 94%, and 97%, respectively. The method was applied to assess the stability of colistin A and colistin B in processed samples (24 hr. at room temperature, 48 hours at −20°C) and unprocessed samples (24 hr. at room temperature, 8 weeks at −20°C) and after three cycles of freeze and thaw found to be ≥87%.

Colistin Use and Its Resistance in Kingdom of Saudi Arabia: A Narrative Review

Antibiotic resistance is steadily increasing all over the world and has become a major public health challenge. To this end, colistin, an old bactericidal antibiotic of polymyxins family, has been recently re-introduced as only available last-resort antibiotic arsenal for treatment of infections caused by multidrug resistant (MDR)-Gram-negative bacteria. However, the continual and extensive use of colistin has led to the emergence and rapid spreading of its bacterial resistance and non-susceptibility that is currently experiencing a critical healthcare issue with extensive global concern. Both transferable and intrinsic mechanisms of bacterial resistance to colistin have been documented in several countries and, therefore, comprehensive epidemiological data and reports are urgently needed to better understand the current status of this important antibiotic to properly optimize its clinical significance. In consistency, the present narrative review highlights both clinical use and reported bacterial resistance of colistin in the Kingdom of Saudi Arabia.

Synergistic Combination of Carbapenems and Colistin against P. aeruginosa and A. baumannii

Background: Intubated patients are particularly at risk of developing infections caused by these pathogens, specifically, P. aeruginosa and A. baumannii. In the past fifteen years, Carbapenems were known to be the drugs of choice for these bacteria. With the increase in the use and misuse of antibiotics, these bacteria became highly resistant, and almost all available antibiotics, including Carbapenems, became inefficient. Synergistic combination therapy may be a useful strategy in slowing as well as overcoming the emergence of resistance. The aim of this study was to evaluate the anti-bacterial activity on P. aeruginosa and A. baumannii of the combination of two antibiotics: Colistin and a Carbapenem (Meropenem or Imipenem). Methods: The antibacterial activity was assessed by determining the MIC. Then, the effect of combining the antibiotics was studied using the Checkerboard Technique described by White et al., 1996. The Fractional Inhibitory Concentration (FIC) for each strain was then calculated and classified as synergy, additive, indifference or antagonism. 11 strains of A. baumannii and 11 strains of P. aeruginosa were tested in the presence of Meropenem combined with Colistin or Imipenem combined with Colistin. Results: For the combination of Meropenem and Colistin, 6 strains of A. baumannii and 3 strains of P. aeruginosa showed synergy while 5 strains of A. baumannii and 7 strains of P. aeruginosa showed additive effect, only 1 strain of P. aeruginosa showed antagonism. For Imipenem and Colistin, only 1 strain of A. baumannii and 3 strains of Pseudomonas showed synergy while 8 strains of Acinetobacter and 8 strains of Pseudomonas showed additive effect. Conclusion: The “in vitro” combination Colistin-Carbapenem is associated with an improvement in MIC. In the majority of the cases, this improvement suggests a synergistic combination or an additive effect.

Mixed Micellar Electrokinetic Chromatographic Analysis of Colistin, Polypeptide Antibiotic, Using Laser-Induced Fluorescence Detection

The main goal of this work was to quantify the detection of colistin at low levels in urine samples through the practical application of mixed surfactant micellar electrokinetic chromatography–laser-induced fluorescence (MEKC-LIF) analysis method using its advantage of sensitivity and to examine direct injection of biological samples. Colistin (po- lymyxin E) has neither strong UV chromophore nor fluorophore. So, its assay for metabolism, pharmacokinetics studies for bioavailability and bioequivalence are difficult because of poor detectability. Therefore an enhanced UV or fluores-cence detection by chemical derivatization is required. MEKC-LIF method was proposed for colistin with a 488/520 nm argon-ion laser using a pre-CE derivatization with fluorescein isothiocyanate (FITC). Borate buffer was used as background buffer (BGB). The different parameters affecting the proposed derivatization reaction including concentration of the derivatizing reagent, reaction time and temperature were studied and optimized. The derivative was stable for up to 3 days. Different micelles (TX-100 and SDS) were examined as BGB additives separately but negative-charged mixed micelles (SDS/TX-100) were shown to be the best additive to BGB for the analysis of colistin particularly in human urine as they enhance both selectivity and sensitivity of the proposed method. BGB was used with pH 9.5, 10 kV, 8 s inj time, capillary length 75 cm × 75 μm ID (66 cm effective length), detection was LIF Ex 488 nm;Em 520 nm. The method was applied to colistin analysis in human urine and the recovery was > 98% (n = 5). LOD and LOQ in urine after pre-column derivatization using FITC were 100 and 250 ng/ml, respectively. Urine samples were analysed by direct injection without sample pre-treatment. The mechanism of enhancement of fluorescence of the derivative by surfactant was proposed.

<i>In Vitro</i>Activity of Colistin and Vancomycin or Azithromycin Combinations on Extensively Drug Resistant <i>Acinetobacter baumannii</i>Clinical Isolates

Background: Extensively drug resistant Acinetobacter baumannii (XDR-AB) presents an increasing challenge to health care in Egypt as they are among the most common bacteria isolated in hospital setting. Treatment of such infections usually involves the use of antimicrobial agents in combination. Various combinations have been proposed, with colistin serving as the backbone in many of them even for colistin resistant isolates. Aim: The study was conducted in order to test the in vitro combined effects of colistin and vancomycin or azithromycin against (XDR-AB) causing infections at Alexandria Main University Hospital in Egypt, in an attempt to detect the possibility of a beneficial combination therapy. Material/Methods: Thirty XDR-AB clinical isolates were included in the study. Antibiotic susceptibility testing was performed using automated Vitek 2 compact system and disc diffusion method. Colistin antibiotic disc diffusion test was compared with broth microdilution method. Organisms were also tested against colistin and vancomycin or azithromycin in combination using checkerboard synergy test and FICI (Fractional Inhibitory Concentration Index) was calculated. Synergy was defined as a FICI of ≤0.5. Results: On comparing the two methods used to detect susceptibility to colistin to broth microdilution for MIC (minimum inhibitory concentration) determination, as a reference method, the Vitek showed 100% categorical agreement (CA), on the other hand, the disc diffusion showed CA of 93% with very major errors. Synergy was detected for all isolates (100%) when combining colistin with vancomycin (FICI mean = 0.08). As for azithromycin, 21 strains had FICI range from 0.7 to 1.001, denoting indifference;the remaining 9 strains showed synergy with FICI range from 0.06 to 0.241. The mean colistin/azithromycin FICI was 0.71 for the 30 isolates. Conclusion: These findings suggest that regimens containing vancomycin may confer therapeutic benefit for infection due to XDR-AB;however, other methods (time-kill assay) should be used to confirm such synergy. Furthermore, the optimal combination treatment for serious XDR-AB infection should be addressed in a prospective clinical trial.

Wild-type MIC Distribution and Epidemiological Cut-off Value and Resistant Characteristics of Colistin Against Escherichia Coli from Chickens

The aim of the present study was to investigate minimum inhibitory concentration(MIC)distributions by broth microdilution(BMD)method and to determine the preliminary epidemiological cut-off value(ECV)of colistin by epidemiological cut-off(ECOFF)finder against E.coli from chickens in China.Anal swabs were collected from chicken farms in China.BMD method was used to measure MIC50 and MIC90 of colistin which were 2 and 4μg•mL^(-1),respectively.MIC frequency distributions for colistin were used to estimate preliminary ECV(8μg•mL^(-1)).High percentages of resistance to ampicillin(94.12%),nalidixic acid(94.12%),enrofloxacin(94.12%),tetracycline(94.12%),ciprofloxacin(88.24%),florfenicol(88.24%),neomycin(64.71%),gentamicin(58.82%),levofloxacin(58.82%),doxycycline(88.24%)and cefalexin(76.47%)were found.In addition,low percentages of resistance to amikacin(5.88%),spectinomycin(17.65%)and fosfomycin(41.18%)were noted.Notably,amoxicillin,sulfisoxazole and trimethoprim resulted in a 100%resistance generation efficacy rate.Prevalence of mcr-1 in E.coli(9/17)in chromosomal DNA was higher than mcr-4(2/17)gene,and mcr-1(5/17)was higher than mcr-4(3/17)in plasmid.

Characteristics of colistin-resistant Escherichia coli from pig farms in Central China

The emergence and dissemination of colistin resistance in Enterobacterioceae mediated by plasmid-borne mcr genes in recent years now pose a threat to public health.In this study,we isolated and characterized colistin-resistant and for mcr-positive£coli from pig farms in Central China.Between 2018 and 2019,594 samples were collected and recovered 445 E.coli isolates.Among them,33 with colistin resistance phenotypes and 37 that were positive for mcr genes were identified,including 34 positive for mcr-1,one positive for mcr-3,and two positive for both mcr-1 and mcr-3.An insertion of nine bases("CTGGATACG")into mcr-7 in four mcr-positive isolates led to gene dysfunction,and therefore did not confer the colistin resistance phenotype.Antimicrobial susceptibility testing revealed that 37 mcr-positive isolates showed severe drug resistance profiles,as 50% of them were resistant to 20 types of antibiotics.Multilocus sequence typing revealed a heterogeneous group of sequence types in mcr-positive isolates,among which ST10(5/37),ST156(5/37),and 5T617(4/37)were the predominant types.Plasmid conjugation assays showed that mcr-carrying plasmids of 25 mcr-positive isolates were conjugated with£coli recipient,with conjugation frequencies ranging from 1.7 × 10^(-6) to 4.1 × 10^(-3) per recipient.Conjugation of these mcr genes conferred a colistin resistance phenotype upon the recipient bacterium.PCR typing of plasmids harbored in the 25 transconjugants determined six types of plasmid replicons,including lncX4(14/25),FrepB(4/25),Incl2(3/25),lncHI2(2/25),FIB(1/25),and Inch(1/25).This study contributes to the current understanding of antibiotic resistance and molecular characteristics of colistin-resistant£coli in pig farms.

Identification of 2-aminothiazoyl piperidine derivatives as a new class of adjuvants potentiating the activity of colistin against Acinetobacter baumannii

The rapid prevalence of antibiotic resistance has led to a significant global health problem. Although colistin is the last resort antibiotic, it is limited by dose dependent toxicity. A critical approach to solve this problem is to use an antibiotic adjuvant, which is able to potentiate the activity of antibiotic and reduce the dosage of antibiotic. Herein, we reported a novel 2-aminothiazoyl piperidine adjuvant, which enhanced the activity of colistin against Acinetobacter baumannii(A. baumannii). Two pilot libraries of 40compounds were prepared and their adjuvant activities were evaluated. The most potential compound11j enabled to cause16-fold reduction in the minimum inhibitory concentration(MIC) of colistin at 8μg/m L. Besides, time-kill curves exhibited that compound 11j had significant adjuvant activity to kill the bacteria. The predicted ADMET analysis showed that 2-aminothiazoyl piperidine derivatives had good drug-likeness and acceptable physicochemical properties. Furthermore, membrane permeability experiments demonstrated that compound 11j was beneficial for colistin to destroy the outer membrane of bacteria. Also, the comparative molecular similarity indices analysis(Co MSIA) and the density functional theory(DFT) calculations were conducted. The results drawn from these analyses indicated that the novel scaffold provided helpful information for the finding of new adjuvant lead.

Low prevalence of colistin-resistant Escherichia coli from companion animals,China,2018-2021

China banned colistin as growth promoter for animals in the year of 2017.A decrease of colistin-resistant Escherichia coli(COREC)and mcr-1-positive Escherichia coli(MCRPEC)were observed in livestock(pigs and chickens)and humans after the ban policy.However,the prevalence of COREC among Chinese companion animals after the ban policy has not been investigated.Here,we recovered 771 E.coli isolates from the China Antimicrobial Resistance Surveil-lance Network for Pets(CARPet)surveillance system(19 provinces/municipalities)from 2018 to 2021.We identified 12 COREC from eight dogs and four cats,among which one feline and three canine isolates were MCRPEC.The prevalence of COREC and MCRPEC in pets from 2018-2021(1.1%-2.2%and 0.8%-1.1%)were lower than those from 2012-2016(7.1%-17.8%and 6.1%-14.3%).The phylogenetic analysis revealed that the four MCRPEC isolates displayed genetic diversity,while one canine isolate exhibited only 26 SNPs difference with one human MCRPEC isolate in the same city,suggesting the exchange of MCRPEC isolates between companion animals and humans.In three MCRPEC isolates,mcr-1 was located on an IncI2 plasmid,which exhibited 99.5%-99.9%nucleotide sequence identity with plasmid pHNSD133-MCR from E.coli of chicken origin.In the remaining MCRPEC,mcr-1 was chromosomally located flanked by intact ISApl1 elements forming a unit of ISApl1-mcr-1-pap2-ISApl1.Despite the low prevalence of COREC and MCRPEC observed in companion animals after the ban policy,the association of pet-derived MCRPEC and mcr-carrying plasmids with those from humans and farm animals suggest that annual surveillance of colistin resistance in bacteria of pet origin is essential.