scholarly journals A novel method for measuring phenotypic colistin resistance in Escherichia coli populations from chicken flocks

2020 ◽  
Author(s):  
Nhung Thi Nguyen ◽  
Nguyen Thi Phuong Yen ◽  
Nguyen Van Ky Thien ◽  
Nguyen Van Cuong ◽  
Bach Tuan Kiet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Production Cycle ◽  
Broth Microdilution ◽  
Colistin Resistance ◽  
E Coli ◽  
Microdilution Method ◽  
Faecal Samples ◽  
Broth Microdilution Method ◽  
Field Samples ◽  
Over Time

ABSTRACTColistin is extensively used in animal production in many low- and middle-income countries. There is a need to develop methodologies to benchmark and monitor changes in resistance in commensal bacterial populations in farms. We aimed to evaluate the performance of a broth microdilution method based on culturing a pooled Escherichia coli suspension (30-50 organisms) from each sample. In order to confirm the biological basis and sensitivity of the method, we prepared 16 standard suspensions containing variable ratios of colistin-susceptible and mcr-1 encoded colistin-resistant E. coli which were grown in 2mg/L colistin. The optical density (OD600nm) readings over time were used to generate a growth curve, and were adjusted to the values obtained in the absence of colistin. The median limit of detection of the method was 1 colistin-resistant in 104 susceptible colonies [1st - 3rd quartile, 1:102 – 1:105]. We applied this method to 108 pooled faecal samples from 36 chicken flocks in the Mekong Delta (Vietnam) over the production cycle. The correlation between this method and the prevalence of colistin resistance in individual colonies harvested from field samples, determined by the Minimum Inhibitory Concentration (MIC), was established. The overall prevalence of colistin resistance at sample and isolate level was 38.9% and 19.4%, respectively. Increased colistin resistance was associated with recent (2 weeks) use of colistin and other, non-colistin antimicrobials (OR=3.67 and OR=1.84, respectively). Our method is a sensitive and affordable approach to monitor changes in colistin resistance in pooled E. coli populations from faecal samples over time.IMPORTANCEColistin (polymyxin E) is an antimicrobial with poor solubility properties, and therefore broth microdilution is the only appropriate method for testing colistin resistance. However, estimating colistin resistance in commensal mixed Escherichia coli populations is laborious since it requires individual colony isolation, identification and susceptibility testing. We developed a growth-based microdilution method suitable for pooled faecal samples. We validated the method by comparing it with results from individual MIC testing of 909 E. coli isolates. We used the method to investigate phenotypic colistin resistance in 108 pooled faecal samples from 36 healthy chicken flocks, each sampled three times over the production cycle. A higher level of resistance was seen in flocks recently supplemented with colistin in drinking water, although the observed generated resistance was short-lived. Our method is affordable, and may potentially be integrated into surveillance systems aiming at estimating the prevalence of resistance at colony level in flocks/herds. Furthermore, it may also be adapted to other complex biological systems, such as farms and abattoirs.

scholarly journals A novel method for measuring phenotypic colistin resistance in Escherichia coli populations from chicken flocks

2020 ◽  
pp. AEM.02597-20
Author(s):  
Nhung Thi Nguyen ◽  
Nguyen Thi Phuong Yen ◽  
Nguyen Van Ky Thien ◽  
Nguyen Van Cuong ◽  
Bach Tuan Kiet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Production Cycle ◽  
Broth Microdilution ◽  
Colistin Resistance ◽  
E Coli ◽  
Microdilution Method ◽  
Faecal Samples ◽  
Broth Microdilution Method ◽  
Field Samples ◽  
Over Time

Colistin is extensively used in animal production in many low- and middle-income countries. There is a need to develop methodologies to benchmark and monitor changes in resistance among mixed commensal bacterial populations in farms. We aimed to evaluate the performance of a broth microdilution method based on culturing a pooled Escherichia coli suspension (30-50 organisms) obtained from each sample. To confirm the biological basis and sensitivity of the method, we cultured 16 combinations of one colistin-susceptible and one mcr-1 encoded colistin-resistant E. coli in the presence of 2mg/L colistin. Optical density (OD600nm) readings over time were used to generate a growth curve, and these values were adjusted to the values obtained in the absence of colistin (adjusted Area Under the Curve, AUCadj). The median limit of detection was 1 resistant in 104 susceptible colonies [1st - 3rd quartile, 102:1 –105:1]. We applied this method to 108 pooled faecal samples from 36 chicken flocks from the Mekong Delta (Vietnam), and determined the correlation between this method and the prevalence of colistin resistance in individual colonies harvested from field samples, determined by the Minimum Inhibitory Concentration. The overall prevalence of colistin resistance at sample and isolate level (estimated from the AUCadj) was 38.9% [95%CI, 29.8-48.8%] and 19.4% (SD± 26.3%), respectively. Increased colistin resistance was associated with recent (2 weeks) use of colistin (OR=3.67) and other, non-colistin antimicrobials (OR=1.84). Our method is a sensitive and affordable approach to monitor changes in colistin resistance in E. coli populations from faecal samples over time.IMPORTANCE Colistin (polymyxin E) is an antimicrobial with poor solubility in agar-based media, and therefore broth microdilution is the only available method for phenotypic resistance. However, estimating colistin resistance in mixed Escherichia coli populations is laborious since it requires individual colony isolation, identification and susceptibility testing. We developed a growth-based microdilution method suitable for pooled faecal samples. We validated the method by comparing it with individual MIC of 909 E. coli isolates; we then tested 108 pooled faecal samples from 36 healthy chicken flocks collected over their production cycle. A higher level of resistance was seen in flocks recently treated with colistin in water, although the observed generated resistance was short-lived. Our method is affordable, and may potentially be integrated into surveillance systems aiming at estimating the prevalence of resistance at colony level in flocks/herds. Furthermore, it may also be adapted to other complex biological systems, such as farms and abattoirs.

Tentative epidemiologic cut-off value and resistant characteristic detection of apramycin against Escherichia coli from chickens

2019 ◽  
Vol 366 (16) ◽  
Author(s):  
Erjie Tian ◽  
Ishfaq Muhammad ◽  
Wanjun Hu ◽  
Zhiyong Wu ◽  
Rui Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Multiple Drug Resistance ◽  
Aminoglycoside Antibiotic ◽  
Multiple Drug ◽  
Broth Microdilution ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Resistant Strains

ABSTRACT Escherichia coli are important foodborne zoonotic pathogens. Apramycin is a key aminoglycoside antibiotic used by veterinarians against E. coli. This study was conducted to establish the epidemiological cut-off value (ECV) and resistant characteristics of apramycin against E. coli. In this study, 1412 clinical isolates of E. coli from chickens in China were characterized. Minimum inhibitory concentrations (MICs) of apramycin were assessed by broth microdilution method. MIC50 and MIC90 for apramycin against E. coli (0.5–256 µg/mL) were 8 and 16 µg/mL, respectively. In this study, the tentative ECV was determined to be 16 µg/mL by the statistical method and 32 µg/mL by ECOFFinder software. Besides, the percentages of aac(3)-IV positive strains ascended with the increase of MIC values of apramycin, and the gene npmA was detected in strains with higher MICs. Sixteen apramycin highly resistant strains displayed multiple drug resistance (100%) to amoxicillin, ampicillin, gentamicin, doxycycline, tetracycline, trimethoprim and florfenicol, while most of them were susceptible to amikacin and spectinomycin. In summary, the tentative ECV of apramycin against E. coli was recommended to be 16 µg/mL.

scholarly journals A novel report of colistin-resistant Escherichia coli carrying mcr-1 gene from animal and human feacal samples in Nigeria

2018 ◽  
Vol 1 (1) ◽  
pp. 7-10 ◽  
Author(s):  
Olugbenga A. Olowe ◽  
Rita A. Olowe ◽  
Adeolu S. Oluremi ◽  
Olusolabomi J. Adefioye
Keyword(s):  
Escherichia Coli ◽  
Animal Husbandry ◽  
Multiple Drug ◽  
Colistin Resistance ◽  
Pcr Assay ◽  
Southwestern Nigeria ◽  
E Coli ◽  
Multiple Drug Resistant ◽  
Microbiological Methods ◽  
Faecal Samples

Background: The mobilized colistin resistance (m cr)-1 gene confers transferable colistin resistance. Reports of mcr-1-positive Escherichia coli (MCRPE) have attracted substantial attention. However, in Nigeria, there is no report of mcr-1 gene resistance. Since colistin is a last resort for multiple drug-resistant isolates, this study therefore report the prevalence of mcr-1 gene among E. coli isolated from human and animal sources. Methods: Out of a total of 280 samples collected from animal and hum an faecal samples from selected farms in Oyo and Osun States, Southwestern Nigeria between July 2015 and June 2016, 60 E. coli were identified using standard microbiological methods. The mcr-1 gene was detected in the isolates by conventional PCR assay. Results: The m cr-1 gene was low and not statistically significant (p≥0.05). It was detected in 5 (8.3%) of 60 E. coli isolates (4= animals; 1= human) Conclusion: This study is the first report of mcr -1 gene from E. coli from human and animal sources in Nigeria. This calls for urgent caution in the use of colistin in animal husbandry.

In vitro activity of antimicrobial agents with and without sulbactam, EDTA and sulbactam-EDTA on ESBLs-producing Escherichia coli isolated from healthy Tibetan yaks

2020 ◽  
Author(s):  
Baoguang Liu ◽  
Xiaoling Yuan ◽  
Yiheng Chen ◽  
Xiaoshen Li ◽  
Ming Bai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nasal Swab ◽  
Antimicrobial Agents ◽  
Synergistic Effects ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Third Generation Cephalosporins ◽  
Enhance Activity

Abstract Background The spread of ESBLs-producing bacteria has been strikingly rapid in many regions of the world and it causes therapeutic difficulties in everyday practice. The aims of this study were to investigate the prevalence and susceptibilities of ESBLs-producing Escherichia coli isolates from healthy Tibetan yaks in China, to evaluate the activity of drug combinations on ESBLs-producing E. coli isolates. Methods From July 2018 to August 2019, a total of 750 nasal swab samples were tested for the presence of E. coli and ESBLs-producing strains. The MICs of 11 antimicrobial agents alone and combinations with sulbactam, EDTA or sulbactam-EDTA against 240 ESBLs-producing E.coli strains were determined by the broth microdilution method. Results Overall, 59.87% (n = 449) of the samples were positive for E. coli, 240 (53.45%) of 449 E. coli isolates were confirmed to be ESBLs-producing. The addition of sulbactam to the third generation cephalosporins, amikacin and fosfomycin for all isolates resulted in low MICs, increasing the level of susceptibility from 0, 0 and 0% to 50 ~ 87.5, 4.2 and 100% respectively. The addition of EDTA to fluoroquinolones, doxycycline, florfenicol, amikacin and fosfomycin, showed improved activities and resulted in low MICs, increasing the level of susceptibility from 0, 0, 8.3, 0 and 0% to 4.2 ~ 29.2, 33.3, 33.3, 66.7 and 45.8%, respectively. All other antibacterials (except fluoroquinolones, doxycycline and florfenicol), when combined with sulbactam-EDTA, were found to be more active than combinations only with sulbactam or with EDTA against most of isolates, with lower MIC50s and MIC90s. Conclusion In conclusion, ESBLs-producing E. coli isolates were widespread in healthy Tibetan yaks in China. ESBLs-producing E. coli isolates exhibited varying degrees of multidrug resistance. This study these findings suggested that sulbactam can enhance activity of β-lactams and some non-β-lactams of antimicrobial agents and had a synergistic effects with EDTA in improving activities of some families of antimicrobials.

Rosemary and Tea Tree Essential Oils Exert Antibiofilm Activities In Vitro against Staphylococcus aureus and Escherichia coli

2020 ◽  
Vol 83 (7) ◽  
pp. 1261-1267
Author(s):  
TING LIU ◽  
JINGFAN WANG ◽  
XIAOMAN GONG ◽  
XIAOXIA WU ◽  
LIU LIU ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Essential Oil ◽  
Biofilm Formation ◽  
Gas Chromatography Mass Spectrometry ◽  
Tea Tree ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method

ABSTRACT The purpose of the present study was to determine the bioactive compounds in rosemary essential oil (REO) and tea tree essential oil (TEO) and to investigate their antibacterial and antibiofilm activities against Staphylococcus aureus and Escherichia coli in vitro. The MIC and MBC assays were performed to assess the antibacterial activity of these two EOs against S. aureus and E. coli with the broth microdilution method. A crystal violet assay was used to ascertain the effects of EOs on the biofilm formation of the test strains, and a tetrazolium bromide (MTT) assay was used to measure the level of inactivation of mature biofilms by EOs. Gas chromatography–mass spectrometry revealed 15 compounds in REO and 27 compounds in TEO, representing 97.78 and 98.13% of the total EO, respectively. Eucalyptol and α-pinene were found in high concentrations in REO, and the two major compounds in TEO were 4-terpineol and terpinolene. The MICs of REO for the two S. aureus and E. coli test strains were both 0.5 mg/mL, and the MICs of TEO for the two strains were both 0.25 mg/mL. Therefore, these EOs can significantly inhibit the formation of biofilms and induced morphological biofilm changes, as verified by scanning electron microscopy. Both EOs had destructive effects on the mature biofilm of the two test strains. TEO was more inhibitory than REO for biofilm formation by the two test strains. HIGHLIGHTS

scholarly journals Susceptibility to antibiotic combinations of Klebsiella pneumoniae and Acinetobacter baumannii strains isolated from COVID-19 patients

2021 ◽  
Vol 18 (4) ◽  
pp. 33-40
Author(s):  
D. V. Tapalski ◽  
E. V. Karpova
Keyword(s):  
Klebsiella Pneumoniae ◽  
Bactericidal Activity ◽  
Antibacterial Effect ◽  
Bactericidal Effect ◽  
Bacteriostatic Activity ◽  
Broth Microdilution ◽  
Colistin Resistance ◽  
Microdilution Method ◽  
Carbapenem Resistant ◽  
Broth Microdilution Method

Objective. To assess the susceptibility of K.pneumoniae and A.baumanii strains isolated from hospitalized COVID-19 patients to antibiotics and their combinations.Materials and methods. The minimum inhibitory concentrations (MICs) of meropenem and colistin were determined for 47 A.baumannii and 51K.pneumoniaestrains isolated from the hospitalized COVID-19 patients by the broth microdilution method. The susceptibility to 11 antibiotic combinations was assessed using the method of multiple combination bactericidal testing.Results. Colistin resistance was detected in 31.9 % of A.baumannii strains (MIC50 — 0.5 mg/l, MIC90 — 16 mg/l) and in 80.4 % of K.pneumoniaestrains (MIC50 — 16 mg/l, MIC90 — 256 mg/l). It has been shown that double antibiotic combinations with the inclusion of colistin exhibit bactericidal or bacteriostatic activity against 76.6–87.2 % of A.baumannii strains. Combinations with the addition of meropenem, colistin and macrolides exhibited bactericidal activity against 78.4–80.4 % of K.pneumoniae strains. Combinations of two carbapenems were not active, the combination of meropenem-colistin had a bactericidal effect only in 13.7 % of K.pneumoniae strains.Conclusion. Widespread colistin resistance was found in carbapenem-resistant K.pneumoniae and A.baumannii strains isolated from the hospitalized COVID-19 patients. The combinations of antibiotics that have a synergistic antibacterial effect in their pharmacokinetic/pharmacodynamic concentrations have been determined.

scholarly journals In Vitro Activities of Ceftazidime–Avibactam and Aztreonam–Avibactam at Different Inoculum Sizes of Extended-Spectrum β-Lactam-Resistant Enterobacterales Blood Isolates

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1492
Author(s):  
Moonsuk Bae ◽  
Taeeun Kim ◽  
Joung Ha Park ◽  
Seongman Bae ◽  
Heungsup Sung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
In Vitro Activity ◽  
Broth Microdilution ◽  
Microdilution Method ◽  
Extended Spectrum ◽  
Broth Microdilution Method ◽  
Tertiary Center ◽  
Inoculum Effect

β-lactam–avibactam combinations have been proposed as carbapenem-sparing therapies, but little data exist on their in vitro activities in infections with high bacterial inocula. We investigated the in vitro efficacies and the inoculum effects of ceftazidime–avibactam and aztreonam–avibactam against extended-spectrum β-lactam-resistant Enterobacterales blood isolates. A total of 228 non-repetitive extended-spectrum β-lactam-resistant Escherichia coli and Klebsiella pneumoniae blood isolates were prospectively collected in a tertiary center. In vitro susceptibilities to ceftazidime, aztreonam, meropenem, ceftazidime–avibactam, and aztreonam–avibactam were evaluated by broth microdilution method using standard and high inocula. An inoculum effect was defined as an eightfold or greater increase in MIC when tested with the high inoculum. Of the 228 isolates, 99% were susceptible to ceftazidime–avibactam and 99% had low aztreonam–avibactam MICs (≤8 mg/L). Ceftazidime–avibactam and aztreonam–avibactam exhibited good in vitro activities; MIC50/MIC90 values were 0.5/2 mg/L, 0.125/0.5 mg/L, and ≤0.03/0.25 mg/L, respectively, and aztreonam–avibactam was more active than ceftazidime–avibactam. The frequencies of the inoculum effect with ceftazidime–avibactam and aztreonam–avibactam were lower than with meropenem (14% vs. 38%, p < 0.001 and 30% vs. 38%, p = 0.03, respectively). The β-lactam-avibactam combinations could be useful as carbapenem-sparing strategies, and aztreonam–avibactam has the better in vitro activity but is more subject to the inoculum effect than ceftazidime–avibactam.

Discrepancy between colistin and polymyxin B susceptibility results among Escherichia coli and Klebsiella pneumoniae clinical isolates

2021 ◽  
Author(s):  
Serap Süzük Yıldız ◽  
Can Hüseyin Hekimoğlu ◽  
Zekiye Bakkaloğlu ◽  
Emine Alp
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Resistant Bacteria ◽  
Major Error ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method

AbstractThe selection of therapeutic agent to be used for the treatment of multidrug-resistant bacteria is a major concern. Polymyxin B use has been commenced in Turkey, although its clinical breakpoint is not listed in the EUCAST. This study aimed to determine the correlation between the MIC values of polymyxin B and colistin. A total of 505 isolates, including 122 isolates of Escherichia coli and 383 isolates of Klebsiella pneumoniae were included in the present study. All the isolates were assessed for colistin and polymyxin B using the broth microdilution method. The categorical agreement in the E. coli isolates was 98.4%, and the rate of very major error was 33.3%. The categorical agreement in the K. pneumoniae isolates was 99.5%, the rate of major error was 0.36%, and the rate of very major error was 0.98%. In the evaluation of the essential agreement, 1.6% error in E. coli and 2.3% error in K. pneumoniae were observed. It was concluded that polymyxin B should never be used in the treatment of the isolates reported as colistin-resistant, and if the MIC values are above 4 mg/L in E. coli and K. pneumoniae. Our results indicate importance of reporting both polymyxin B and colistin susceptibility results of clinical isolates.

scholarly journals The Anti-Staphylococcal Potential of Ethanolic Polish Propolis Extracts

Molecules ◽  
2019 ◽  
Vol 24 (9) ◽  
pp. 1732 ◽  
Author(s):  
Katarzyna Grecka ◽  
Piotr M. Kuś ◽  
Piotr Okińczyc ◽  
Randy W. Worobo ◽  
Justyna Walkusz ◽  
...  
Keyword(s):  
Hplc Analysis ◽  
Bactericidal Effect ◽  
Gram Negative Bacteria ◽  
Broth Microdilution ◽  
Inhibit Protein Synthesis ◽  
Gram Negative ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Ethanol Extracts

The principal objective of this study was to determine the anti-staphylococcal potential of ethanol extracts of propolis (EEPs). A total of 20 samples of propolis collected from apiaries located in different regions of Poland were used in the study. The two-fold broth microdilution method revealed some important differences in the antimicrobial activity of investigated EEPs. Up to the concentration of 4096 µg/mL no activity was observed against Gram-negative bacteria (E. coli and P. aeruginosa). Staphylococci exhibited much higher susceptibility. The highest efficiency observed for EEP12 and EEP20 (MIC values ranged between 32 and 256 µg/mL). However, the achievement of bactericidal effect usually required higher concentrations. In the case of clinical isolates of S. aureus MBC values for EEP12 and EEP20 ranged from 512 to 1024 µg/mL. The HPLC analysis revealed that these two products contained a higher concentration of flavonoids (flavonols, flavones, and flavanones) compared to other investigated EEPs. In checkerboard test, a synergistic anti-staphylococcal effect was observed for the action of EEP20 in combination with amikacin, kanamycin, gentamycin, tetracycline, and fusidic acid (all these antibiotics inhibit protein synthesis). Moreover, the investigated EEPs effectively eradicated staphylococcal biofilm. The obtained results clearly confirm the high anti-staphylococcal potential of propolis harvested in Polish apiaries.

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