Detection of Plasmid-Mediated Colistin Resistant mcr-1 Gene in Escherichia coli Isolated from Infected Chicken Livers in Nepal

Background: Plasmid-mediated resistance to the colistin in poultry is considered as an emerging problem worldwide. While poultry constitutes the major industry in Nepal, there is a paucity of evidence on colistin resistance in Escherichia coli isolates causing natural infections in poultry. This study aimed to explore the prevalence of plasmid-mediated colistin resistance gene, mcr-1 in E. coli isolated from liver samples of dead poultry suspected of E. coli infections. Methods: A total of two hundred and seventy liver samples (227 broilers and 43 layers) from dead poultry suspected of colibacillosis were collected from post-mortem in the Central Veterinary Laboratory (CVL), Kathmandu, between 1 February and 31 July 2019. The specimens were processed to isolate and identify E. coli; an antimicrobial susceptibility test (AST) using disk diffusion method was performed with 12 different antibiotics: Amikacin (30 µg), ampicillin (10 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), cefoxitin (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cotrimoxazole (25 µg), gentamicin (10 µg), imipenem (10 µg), levofloxacin (5 µg) and tetracycline (30 µg). Colistin resistance was determined by agar dilution method and colistin-resistant strains were further screened for plasmid-mediated mcr-1 gene, using conventional polymerase chain reaction (PCR). Results: Out of 270 liver samples, 53.3% (144/270) showed growth of E. coli. The highest number (54%; 109/202) of E. coli isolates was obtained in the liver samples from poultry birds (of both types) aged less than forty days. In AST, 95.1% (137/144) and 82.6% (119/144) of E. coli isolates were resistant against tetracycline and ciprofloxacin, respectively, while 13.2% (19/144) and 25.7% (37/144) isolates were resistant to cefoxitin and imipenem, respectively. In the same assay, 76.4% (110/144) E. coli isolates were multi-drug resistant (MDR). The phenotypic prevalence of colistin resistance was 28.5% (41/144). In the PCR assay, 43.9% (18/41) of colistin-resistant isolates were screened positive for plasmid-mediated mcr-1. Conclusion: The high prevalence of mcr-1 in colistin-resistant E. coli isolates in our study is a cause of concern for the probable coming emergence of colistin resistance in human pathogens, due to horizontal transfer of resistant genes from poultry to human isolates.

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Plasmid mediated colistin resistant mcr-1 and co-existence of OXA-48 among Escherichia coli from clinical and poultry isolates: first report from Nepal

Gut Pathogens ◽

10.1186/s13099-020-00382-5 ◽

2020 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Bijaya Muktan ◽

Upendra Thapa Shrestha ◽

Binod Dhungel ◽

Bagish Chandra Mishra ◽

Nabaraj Shrestha ◽

...

Keyword(s):

Escherichia Coli ◽

Resistance Gene ◽

Diffusion Method ◽

Dilution Method ◽

Agar Dilution Method ◽

Colistin Resistance ◽

Clinical Specimens ◽

E Coli ◽

Resistant Genes ◽

Rectal Swabs

Abstract Background Plasmid-mediated resistance to the last-resort drugs: carbapenems and colistin is an emerging public health threat. The studies on the prevalence and co-expression of resistant genes among livestock and human pathogens are rare in Nepal. This is the first study in Nepal exploring the prevalence and co-existence of colistin resistance gene, mcr-1 along with carbapenemase resistance gene, OXA-48 in Escherichia coli isolated from poultry and clinical specimens. Methods A total of 240 rectal swabs from chickens of five different poultry farms of Kathmandu valley and 705 mid-stream urine samples from human subjects attending Kantipur Hospital, Kathmandu were collected between August, 2018 and March, 2019. Rectal swabs and urine specimens were cultured. E. coli isolated from the specimens were screened for antimicrobial susceptibility testing (AST) using disk diffusion method’. Minimum inhibitory concentration (MIC) of colistin was determined by agar dilution method using 0.5 µg/ml to 32 µg/ml. The E. coli isolates were first screened for mcr-1 followed by screening for OXA-48 genes using conventional Polymerase chain reaction (PCR). Results Of the total samples analyzed, E. coli was isolated from 31.7% (76/240) of poultry and 7.9% (56/705) of clinical specimens. In AST, 80% (61/76) of E. coli from poultry and 79% (44/56) from clinical specimens were MDR. The phenotypic prevalence of colistin resistance in poultry specimens were 31.6% (24/76) and clinical specimens were 21.4% (12/56). In PCR assay, 27.6% (21/76) of poultry and 19.6% (11/56) of clinical isolates had colistin resistant mcr-1 gene. MICs value of E. coli isolates ranged from 4 to 32 (µg/ml) in both clinical and poultry isolates. Prevalence of co-existing carbapenem resistance gene, OXA-48, among colistin resistant mcr-1 positive isolates was 38% (8/21) in poultry specimens and 18.2% (2/11) in clinical specimens. Conclusions The high prevalence of colistin and carbapenem resistant genes, and their co-existence in plasmid DNA of E. coli isolates in this study suggests the possible spread to other animal, human and environmental pathogens. Molecular methods in addition to the conventional diagnostics in laboratories can help in early diagnosis, effective management and control of their potential transmission.

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Comparison of methods for the determination of antimicrobial resistance in Campylobacter spp. human and the food chain isolates

Veterinární Medicína ◽

10.17221/2030-vetmed ◽

2008 ◽

Vol 52 (No. 4) ◽

pp. 169-174

Author(s):

M. Holasova ◽

R. Karpiskova ◽

S. Karpiskova ◽

V. Babak ◽

J. Schlegelova

Keyword(s):

Nalidixic Acid ◽

Antimicrobial Agents ◽

Disk Diffusion ◽

Diffusion Method ◽

Dilution Method ◽

Agar Dilution Method ◽

Disk Diffusion Method ◽

Microdilution Method ◽

Study Results ◽

Agar Dilution

With a microdilution method, using the commercial diagnostic test Sensititre Susceptibility Plates for Campylobacter MIC (Trek Diagnostic Systems, Cleveland, OH, USA), disk diffusion and agar dilution method, resistance to six antimicrobial agents were examined in a reference strain Campylobacter jejuni ATCC 33560 and 73 thermo-tolerant isolates of Campylobacter spp. For the microdilution method and all tested antimicrobial agents, our determined values of microbiological breakpoints of resistant strains were suggested as the minimum inhibitory concentration (MICR) for ciprofloxacin ≥ 0.5, erythromycin ≥ 4, gentamicin ≥ 4, nalidixic acid ≥ 32 and tetracycline ≥ 4 μg/ml. On the basis of our study results, strains resistant to clindamycin were MICR ≥ 2 μg/ml for the dilution methods and a zone diameter R Ã¢ÂÂ¤ 16 mm for the disk diffusion method. Comparison of the results of the resistance examination, a microdilution method and disk diffusion method with the reference agar dilution method, showed that all compared methods yielded identical results with the exception of the resistance determination in erythromycin and nalidixic acid. The errors were mostly the result of the interpretation criteria for MICR of agar dilution method and different conditions of cultivation used. However, the compared methods, provide results comparable with the reference method having greater convenience of measurement.

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Inaccuracy of the Disk Diffusion Method Compared with the Agar Dilution Method for Susceptibility Testing of Campylobacter spp.

Journal of Clinical Microbiology ◽

10.1128/jcm.01090-11 ◽

2011 ◽

Vol 50 (1) ◽

pp. 52-56 ◽

Cited By ~ 31

Author(s):

Mirva Lehtopolku ◽

Pirkko Kotilainen ◽

Pauli Puukka ◽

Ulla-Maija Nakari ◽

Anja Siitonen ◽

...

Keyword(s):

Susceptibility Testing ◽

Disk Diffusion ◽

Diffusion Method ◽

Dilution Method ◽

Agar Dilution Method ◽

Disk Diffusion Method ◽

Agar Dilution ◽

Campylobacter Spp

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The study of reduced susceptibility of methicillin resistant Staphylococcus aureus to various antibiotics with special reference to glycopeptides in a tertiary care hospital in central India

International Journal of Community Medicine and Public Health ◽

10.18203/2394-6040.ijcmph20191132 ◽

2019 ◽

Vol 6 (4) ◽

pp. 1426

Author(s):

Swati S. Kale ◽

Ashwini Patil

Keyword(s):

Staphylococcus Aureus ◽

Tertiary Care ◽

Diffusion Method ◽

Dilution Method ◽

Agar Dilution Method ◽

Care Hospital ◽

Diagnostic Laboratory ◽

Disk Diffusion Method ◽

Methicillin Resistant ◽

Agar Dilution

Background: Staphylococcus aureus has emerged over the past several decades as a leading cause of hospital-associated and community acquired infections. Methicillin resistant S. aureus (MRSA), which are often resistant to several classes of antibiotics, is the most common cause of nosocomial infections and pose a great threat to the world. Vancomycin is regarded as the first-line drug for treatment of MRSA but resistance to this drug is being reported now a day.Methods: It was carried out for a period between January 2014 to June 2017 in the microbiology diagnostic laboratory. MRSA detection was performed by cefoxitin disk diffusion method. Screening for the vancomycin intermediate and the vancomycin resistant S. aureus (VISA and VRSA respectively) was carried out by using vancomycin screen. MIC (minimum inhibitory concentration) of vancomycin was tested by agar dilution method and E strip on all MRSA isolates.Results: A total of 287 S. aureus clinical isolates were included in the study. All MRSA were inoculated on vancomycin screen agar. Visible growth was present in 8 isolates. Five (3.73%) MRSA isolates with MIC of 4 were termed VISA (vancomycin intermediate S. aureus) by agar dilution method. Six isolates had the MIC of 4 and were termed as VISA.Conclusions: As disc diffusion method is not recommended by CLSI for S. aureus, vancomycin screen agar and MIC determination by either of the methods viz. agar dilution or E test can be used.

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ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACT FROM STEM BARK OF Cinnamomum sintoc

Jurnal Natural ◽

10.24815/jn.v0i0.8049 ◽

2017 ◽

Vol 17 (2) ◽

pp. 77

Author(s):

Nurdin Saidi ◽

Hira Helwati ◽

Lailatul Qhadariah Lubis ◽

Muhammad Bahi

Keyword(s):

Escherichia Coli ◽

Antimicrobial Activity ◽

Antibacterial Activity ◽

Methanol Extract ◽

Stem Bark ◽

Dilution Method ◽

Agar Dilution Method ◽

E Coli ◽

Paper Disk ◽

Agar Dilution

Antimicrobial activity of methanol extract from stem bark of Cinnamomum sintoc has been evaluated against Candida albicans, Staphylococcus aureus and Escherichia coli. The extraction of compound was carried out by maceration, then isolation by column chromatograph, which yielded five (5) subfractions (A-E). Activity against fungus C. albicans, S. aureus bacteria dan E. coli using agar dilution method in paper disk. Methanol extract was not potent against antifungal activity but shows antibacterial activity with medium category. Subfraction C showed that antibacterial activity against S. aureus and E. coli with weak category, but subfractions D and E did not show any activity.

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INVESTIGATIONS ON THE RESISTANCE OF COMMENSAL SWINE ESCHERICHIA COLI TO SOME AMINOGLYCOSIDES-AMINOCYCLITOLS

Archives of Veterinary Medicine ◽

10.46784/e-avm.v8i1.102 ◽

2016 ◽

Vol 8 (1) ◽

pp. 13-26

Author(s):

Valentina Urumova ◽

Mihni Lyutskanov ◽

Vladi Petrov

Keyword(s):

Escherichia Coli ◽

Antimicrobial Agents ◽

Dilution Method ◽

Agar Dilution Method ◽

Aminoglycoside Resistance ◽

E Coli ◽

Swine Farms ◽

Resistance Patterns ◽

Agar Dilution ◽

Aminoglycoside Resistance Genes

Th e aim of this study was to describe the prevalence of antibiotic resistance to some aminoglycosides, streptomycin, spectinomycin and gentamicinand three aminoglycoside- resistance genes in Escherichia coli isolated from feces and lagoon manure in six swine farms in Republic of Bulgaria. Atotal of 274 E. coli isolates from 270 fecal samples and twelve samples from lagoon manure were tested by disk diff usion method to determine resistance patterns to 11 antimicrobial agents. Aminoglycosides resistance also was determined by E-test, agar dilution method, PCR and qPCR. Th e highest resistance observed to streptomycin (70.0%) and spectinomycin (65.5%). Multi-resistance patterns in studied E. coli strains showed that the resistance to streptomycin/spectinomycin was most frequently seen together with resistance to ampicillin, tetracycline, and sulfonamides (39.6%). The E. coli isolates resistant to streptomycin, spectinomycin were examined for the presence of strA/strB, aadA1 genes, and resistant isolates to gentamicin were evaluated for the presence of the aacC1 gene. Th e most common gene determining resistance to aminoglycosides was aadA1 which was found in 54.0% of swine isolates and lagoon manure isolates followed by straA/strB genes (32.3%). Th e aacC1gene was not identifi ed in E. coli isolates resistant to gentamicin.

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Comparative Analysis of Gradient Diffusion and Disk Diffusion with Agar Dilution for Susceptibility Testing of Elizabethkingia anophelis

Antibiotics ◽

10.3390/antibiotics10040450 ◽

2021 ◽

Vol 10 (4) ◽

pp. 450

Author(s):

Chien-Tung Chiu ◽

Chung-Hsu Lai ◽

Yi-Han Huang ◽

Chih-Hui Yang ◽

Jiun-Nong Lin

Keyword(s):

Antimicrobial Agents ◽

Susceptibility Testing ◽

Disk Diffusion ◽

Diffusion Method ◽

Dilution Method ◽

Agar Dilution Method ◽

Disk Diffusion Method ◽

Gradient Diffusion ◽

Minimum Requirements ◽

Agar Dilution

Elizabethkingia anophelis has recently emerged as a cause of life-threatening infections. This study compared the results of antimicrobial susceptibility testing (AST) conducted for E. anophelis through different methods. E. anophelis isolates collected between January 2005 and June 2019 were examined for their susceptibility to 14 antimicrobial agents by using disk diffusion, gradient diffusion (Etest; (bioMérieux S.A., Marcy l’Etoile, France), and agar dilution methods. The agar dilution method was the reference assay. According to the agar dilution method, the isolates exhibited the highest susceptibility to minocycline (100%), doxycycline (97.6%), rifampin (95.2%), and levofloxacin (78.6%). A very major error rate of >1.5% was observed for nine antibiotics tested using the disk diffusion method. The overall categorical agreement rate between the disk diffusion and agar dilution methods was 74.8%, and ceftazidime, minocycline, levofloxacin, and rifampin met the minimum requirements for discrepancy and agreement rates. The Etest method tended to produce lower log2 minimum inhibitory concentrations for the antibiotics, except for trimethoprim–sulfamethoxazole and rifampin; the method resulted in very major errors for nine antibiotics. The overall essential and categorical agreement rates between the Etest and agar dilution methods were 67.3% and 76.1%, respectively. The Etest method demonstrated acceptable discrepancy and agreement rates for ceftazidime, minocycline, doxycycline, levofloxacin, and rifampin. AST results obtained through the disk diffusion and Etest methods for multiple antibiotics differed significantly from those obtained using the agar dilution method. These two assays should not be a routine alternative for AST for E. anophelis.

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Effects of selected Taiwanese endemic plants on anti-lipid peroxidation and antibacterial activities against clinically isolated extended-spectrum-β-lactamase producing Klebsiella pneumoniae and Escherichia coli

10.21203/rs.3.rs-168014/v1 ◽

2021 ◽

Author(s):

Po-Wei Tsai ◽

Pei-Chin Lin ◽

Ling-Ling Yang ◽

Ming Shun Wu

Keyword(s):

Escherichia Coli ◽

Lipid Peroxidation ◽

Medicinal Plants ◽

Antibacterial Activities ◽

Inhibitory Effect ◽

Dilution Method ◽

Agar Dilution Method ◽

Kidney Mitochondria ◽

E Coli ◽

Agar Dilution

Abstract It has been a challenge for many clinicians to treat a complicated extended-spectrum-β-lactamase (ESBL) producing Klebsiella Pneumoniae (Kp) and Escherichia coli (E. coli) infection due to widespread antibiotic abuse with renal damage as one of its common side effects. Therefore, this study aimed to assess the antibacterial activity of extracts from several Taiwanese folk medicinal plants against ESBL- Kp and E. coli. with renal protecting ability against lipid peroxidation (LPO) on mice kidney mitochondria. Preliminary antibacterial activities of ethanol extracts from twenty (20) Taiwanese folk medicinal plants were measured by agar-dilution method against standard ESBL strains of E. coli (ATCC 25922, ATCC 35218) and Kp (ATCC 23856, ATCC 700603). Rhus semialata var. roxburghiana DC. (RSR) exerted the most inhibitory effect and then further extracted with n-hexane, ethyl acetate, acetone, ethanol, and water, respectively. Each extract also evaluated against the four standard ATCC microorganisms. Their MIC50, MIC90, and time kill assay were adapted with detecting the maximum inhibitory activities and the antibacterial spectrum range of each extract was measured against twenty-four (24) kinds of microbes. Which were used including gram-positive, gram-negative bacteria and fungus by agar dilution method. Finally, renal protective ability was detected inhibitory effect of ferrous induced lipid peroxidation on mice mitochondria. Among 20 Taiwanese folk medicinal plants tested, Rhus semialata var. roxburghiana DC. (RSR) exhibited maximum inhibition against clinical ESBL-producing Kp and E. coli strains with acetone extracts showing MIC50/MIC90 values at 1000 µg/mL, the course of antimicrobial action was bacteriostatic and with inhibitions to all 24 kinds of microbial including Gram positive and negative bacteria and fungi. Furthermore, result of thiobarbituric acid reactive substances (TBARS) assay from this extract showed high lipid peroxidative (LPO) protective capability on mice kidney mitochondria (IC50: 29.29 ± 0.35µg/mL). RSR acetone extract, with its maximum activity against clinical isolated ESBL-producing Kp and E. coli, antimicrobial effect against other wide spectral range bacteria and relatively high LPO protective ability on mice kidney mitochondria, is a potential source, albeit further studies have yet to be conducted, to develop an antimicrobial drug against ESBL-Kp and E. coli.

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Molecular identification and antimicrobial resistance profiles of Campylobacter strains of poultry origin in India with special emphasis on fluoroquinolone resistance

Asian Journal of Medical and Biological Research ◽

10.3329/ajmbr.v1i1.25491 ◽

2015 ◽

Vol 1 (1) ◽

pp. 1-8

Author(s):

SM Lutful Kabir ◽

Masahiro Asakura ◽

Sachi Shiramaru ◽

Amit Pal ◽

Atsushi Hinenoya ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Quinolone Resistance ◽

Disk Diffusion ◽

Diffusion Method ◽

Dilution Method ◽

Agar Dilution Method ◽

Slight Variation ◽

Gyra Gene ◽

Disk Diffusion Method ◽

Agar Dilution

The current study aimed to identify the selected number of Campylobacter strains of poultry origin in India that were isolated in the Laboratory of International Prevention of Epidemics, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Japan to the species level with the aid of cdtA/B/C gene-based multiplex PCR assays as well as to investigate their antimicrobial resistance profiles. C. jejuni (4 strains) and C. coli (16 strains) were identified. The poultry isolates identified were subjected to susceptibility testing with the aid of disk diffusion method using 12 antimicrobial agents. Again, the resistant and intermediate isolates confirmed by the disk diffusion method were subjected to determination of minimum inhibitory concentration by agar dilution method. To elucidate the mechanism of quinolone resistance, a total number of 20 quinolone resistant strains were subjected to sequence determination and analysis of the gyrA gene in the quinolone-resistance determining region. The results of the disk diffusion method were consistent with the results of the agar dilution method with slight variation in case of ampicillin, levofloxacin, ofloxacin and fosfomycin. All C. jejuni and C. coli strains had the Thr 86 to Ile substitution in gyrA. Results of this study support the emergence of resistance of C. jejuni and C. coli strains of poultry origin in India to a variety of antimicrobials especially fluoroquinolones.Asian J. Med. Biol. Res. June 2015, 1(1): 1-8

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Susceptibility pattern of Escherichia coli from urinary tract infections to antibiotics and methanol extracts of Olax subscorpioidea and Sida corymbosa

Journal of Pharmacy & Bioresources ◽

10.4314/jpb.v17i1.12 ◽

2020 ◽

Vol 17 (1) ◽

pp. 75-80

Author(s):

Philip A. Idowu ◽

Babatunde M. Okanlawon ◽

Habeeb O. Salam

Keyword(s):

Escherichia Coli ◽

Urinary Tract ◽

Urinary Tract Infections ◽

Diffusion Method ◽

Dilution Method ◽

Agar Dilution Method ◽

Traditional Use ◽

E Coli ◽

Methanol Extracts ◽

Tract Infections

Antibiotic resistance in urinary tract infections (UTIs), of which Escherichia coli causes about 80% of cases is on increase, causing mortality, morbidity and increased health care costs. Clinical isolates of E. coli (13) from UTIs were tested for susceptibility to standard antibiotics and extracts of Olax subscorpioidea and Sida corymbosa. Methanol extracts of the plants were screened at 20, 40, 80 and 100 mg/ml against the isolates using agar-welldiffusion method while antibiogram was determined by Kirby-Bauer disc diffusion method. The minimum inhibitory concentrations (MICs) of the plants` extracts and two antibiotics were determined by agar dilution method. The isolates were mostly susceptible to ofloxacin and 100% resistance to augmentin. Extracts of the plants showed good and dose-dependent activities, even on the multidrug resistant E. coli isolates. The zones of inhibition of the extracts ranged 9-16 mm while the MICs ranged 0.5-10 mg/ml on the isolates. This study has shown that MDR E. coli in UTIs are still prevalent and that the roots of O. subscorpioidea and stem of S. corymbosa extracts have good antibacterial activities against the isolates. The results justified the traditional use of the plants to treat infections generally and the potential utilization in the treatment of UTIs. Keywords: Urinary tract infection; Antibiotics; Escherichia coli; Olax subscorpioidea; Sida corymbosa

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