Bacterial strains, S. aureus ATCC 25923, E. coli ATCC 25922, S. Enteritidis ATCC 13076, S. Typhimurium ATCC 13311, and S. Typhimurium UK-1 ATCC 68169, were inoculated in nutrient broth (Acumedia, Lansing, MI, USA, USA), and incubated at 37 °C for 24 h. Cultures reached a concentration of 109 CFU/mL. After the growth, the bacterial strains were serially diluted in a saline 10-fold solution up to 10−7, and dilution was plated in nutrient agar to confirm the total CFUs. A volume of 10 µL of each dilution was placed on the film’s square with and without bio-AgNP and stored in a sterile Petri dish at room temperature for 24 h. The initial cellular density in contact with films ranged from 106 CFU/cm2 to 1 CFU/cm2. After contact with bacteria, film samples were inoculated in nutrient broth and placed at 37 °C for 24 h. Bacterial growth was confirmed by samples plating in agar medium, E. coli in MacConkey Agar (Acumedia, Lansing, MI, USA, USA), S. aureus in Mannitol Salt Agar (Difco®, Tucker, GA, USA), and Salmonella sp. in SS Agar (Difco®, Tucker, GA, USA).
Macconkey agar
Manufactured by Acumedia
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About the product
MacConkey agar is a selective and differential culture medium used for the isolation and enumeration of Gram-negative bacteria, particularly members of the Enterobacteriaceae family. It contains bile salts and crystal violet to inhibit the growth of Gram-positive bacteria, while allowing the growth of Gram-negative bacteria.
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MacConkey Agar, originally produced by Acumedia, is now available under Neogen's branding. The product is actively listed on Neogen's official website, indicating its continued commercialization.
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22 protocols using «macconkey agar»
1
Antimicrobial Efficacy of Bio-AgNP Films
Corresponding organizations : Universidade Estadual de Londrina, Universidade Federal do ABC
2
Identification of Uropathogenic Bacteria
Urine samples were initially streaked on MacConkey agar (Acumedia, Michigan, US) and Cysteine Lactose Electrolyte Deficient (C.L.E.D.) agar (Hi Media laboratory Pvt Ltd, Mumbai, India) (MacConkey agar served as a selective as well as a differential culture medium. MacConkey agar was used for the isolation of Gram-negative uropathogens while C.L.E.D. agar was used for the differentiation of uropathogens based on color morphology. All media were prepared according to the manufacturer’s instructions. Urine samples were streaked on media using sterile cotton swabs. Media plates were incubated at 37 ºC overnight. Next day, plates were observed for growth. The colony of interest was picked from media containing selected pathogen using a sterile loop and mixed in 3% H2O2 on the slide for catalase test. Air bubbles and water were observed on a slide for a positive result (Habeeb et al. 2013 (link)). Triple sugar iron test is used for the characterization of various pathogens. This test is based on the pathogen’s ability for fermentation of sugars, H2S production and gas production. For TSI agar (BIOCHEM chemopharma, France), colony inoculation was performed by stabbing through the center of the medium to the bottom of the test tube and then streaking was also performed on the slant. Test tubes were incubated at 37 ºC for 18 h.
Corresponding organizations : Government College University, Faisalabad, National Institute for Biotechnology and Genetic Engineering, Pakistan Institute of Engineering and Applied Sciences
3
Isolation and Identification of E. coli from Wild Birds and Rodents
Sample collection: A total of 96 faecal samples from wild birds and 135 samples of rodents’ droppings were conveniently collected in 2017, as part of zoonotic disease surveillance programmes. The wild birds were collected by the Environmental Health Institute of National Environment Agency, where the wild birds’ carcasses were collected from urban areas and recreational parks. An approximate 1 g of wild bird faecal matter was collected after dissection of each wild bird. Samples of rodents’ droppings (rodent species were unidentified) were collected by Rodent Control Unit, of National Environment Agency Central Regional Office. These rodents’ droppings were found at bin chute, drain and kitchen areas.
Sample isolation was carried out according to the method as follows: 1–10 g of wild bird faecal or rodent droppings samples were incubated in 9 mL of Universal Pre-Enrichment Broth (Acumedia, Lansing, MI, USA) under aerobic conditions at 37 ± 1 °C for 16–18 h. A 10 μL loopful of enriched broth was streaked onto Eosine Methylene Blue agar (Acumedia, Lansing, MI, USA) and incubated under the same conditions. Single colonies were streaked on MacConkey agar (Acumedia, Lansing, MI, USA) and incubated under the same conditions for further isolation. The purified colonies were then streaked onto Tryptic Soy Agar (Acumedia, Lansing, MI, USA) and incubated under the same condition. E. coli confirmation was performed with an indole test. A pure colony from Tryptic Soy Agar was inoculated into Peptone water (Acumedia, Lansing, MI, USA) and incubated under the same conditions. Five drops (0.5 mL) of Remel™ Kovacs Indole Reagent (Thermo Scientific, Lenexa, KS, USA) was dispensed to the enriched Peptone water. A pink ring interfaced between Peptone water and indole reagent was observed for E. coli isolate. Next, randomly selected separate E. coli isolates (one colony per sample) were stored in Brain Heart Infusion broth with 15% glycerol until further usage.
DNA extraction of the presumptive E. coli isolates was carried out using DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany). Confirmation of E. coli isolates was performed by 16S ribosomal RNA polymerase chain reaction using forward primer 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer 1492r (5′-GGTTACCTTGTTACGACTT-3′). Polymerase chain reaction conditions are described as follows [11 ]: A 50 μL reaction mix consisted of 10 μL of 5x Phusion High-Fidelity Buffer (Thermo Scientific, Vilnius, Lithuania), 1 μL of dNTP mix (1st BASE, Seri Kembangan, Malaysia), 0.5 μL (10 µM) of each primer (Integrated DNA Technologies, Singapore), 0.5 μL of Phusion Hot Start II DNA Polymerase (Thermo Scientific, Vilnius, Lithuania), 5 μL of DNA template and 32.5 μL of molecular grade water was used. PCR was conducted using thermocycler (Applied Biosystems, Waltham, MA, USA) with conditions as follows: initial denaturation at 98 °C for 30 s, 35 cycles consisting of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s and extension at 72 °C for 30 s and a final extension at 72 °C for 10 min. The amplified fragments were visualised at 2% agarose gel. Those isolates with an expected band size of 1465 bp were sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA). Raw sequences were assembled using BioEdit version 7.2.6.1 software. Assembled reads were uploaded to nucleotide BLAST database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE = BlastSearch) (National Centre for Biotechnology Information, United States) for E. coli identification (>90%).
Sample isolation was carried out according to the method as follows: 1–10 g of wild bird faecal or rodent droppings samples were incubated in 9 mL of Universal Pre-Enrichment Broth (Acumedia, Lansing, MI, USA) under aerobic conditions at 37 ± 1 °C for 16–18 h. A 10 μL loopful of enriched broth was streaked onto Eosine Methylene Blue agar (Acumedia, Lansing, MI, USA) and incubated under the same conditions. Single colonies were streaked on MacConkey agar (Acumedia, Lansing, MI, USA) and incubated under the same conditions for further isolation. The purified colonies were then streaked onto Tryptic Soy Agar (Acumedia, Lansing, MI, USA) and incubated under the same condition. E. coli confirmation was performed with an indole test. A pure colony from Tryptic Soy Agar was inoculated into Peptone water (Acumedia, Lansing, MI, USA) and incubated under the same conditions. Five drops (0.5 mL) of Remel™ Kovacs Indole Reagent (Thermo Scientific, Lenexa, KS, USA) was dispensed to the enriched Peptone water. A pink ring interfaced between Peptone water and indole reagent was observed for E. coli isolate. Next, randomly selected separate E. coli isolates (one colony per sample) were stored in Brain Heart Infusion broth with 15% glycerol until further usage.
DNA extraction of the presumptive E. coli isolates was carried out using DNeasy Blood & Tissue Kits (Qiagen, Hilden, Germany). Confirmation of E. coli isolates was performed by 16S ribosomal RNA polymerase chain reaction using forward primer 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer 1492r (5′-GGTTACCTTGTTACGACTT-3′). Polymerase chain reaction conditions are described as follows [11 ]: A 50 μL reaction mix consisted of 10 μL of 5x Phusion High-Fidelity Buffer (Thermo Scientific, Vilnius, Lithuania), 1 μL of dNTP mix (1st BASE, Seri Kembangan, Malaysia), 0.5 μL (10 µM) of each primer (Integrated DNA Technologies, Singapore), 0.5 μL of Phusion Hot Start II DNA Polymerase (Thermo Scientific, Vilnius, Lithuania), 5 μL of DNA template and 32.5 μL of molecular grade water was used. PCR was conducted using thermocycler (Applied Biosystems, Waltham, MA, USA) with conditions as follows: initial denaturation at 98 °C for 30 s, 35 cycles consisting of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s and extension at 72 °C for 30 s and a final extension at 72 °C for 10 min. The amplified fragments were visualised at 2% agarose gel. Those isolates with an expected band size of 1465 bp were sequenced using BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA). Raw sequences were assembled using BioEdit version 7.2.6.1 software. Assembled reads were uploaded to nucleotide BLAST database (
Corresponding organizations : National Environment Agency, Nanyang Technological University
4
Isolation and Preservation of E. coli
The gastrointestinal tract, from proventriculus to cloaca, was removed from each European starling, placed into a sterile Whirl-Pak bag (Nasco, Fort Atkinson, WI), and homogenized for 120 sec at 230 rpm using a Stomacher 400 Circulator (Seward, Islandia, NY). The resulting homogenate was inoculated using a sterile cotton tipped applicator onto MacConkey agar (Acumedia, Lansing, MI) (MAC) supplemented with 2 μg/mL CTX (Calbiochem, EMD Millipore, Billerica, MA) and onto MAC supplemented with 1 μg/mL CIP (Enzo Life Sciences, Farmingdale, NY) and incubated at 37 °C for 24 hr. From each media, 1–2 presumptive E. coli colonies (occasionally lactose negative isolates were also collected) were subcultured on either MAC-CTX or MAC-CIP to yield purified isolates. Purified isolates were propagated overnight in Brain Heart Infusion Broth (BD, Franklin Lakes, NJ) at 37 °C with shaking at 200 rpm, then mixed 1:1 in 40% glycerol, and stored at −80 °C until further use.
Corresponding organizations : University of Wyoming, Food and Agriculture Organization of the United Nations, University of Guelph, Iowa State University, Colorado State University
5
Evaluation of Sanitization Treatments on Strawberries
Samples of strawberries were obtained as described in Section 2.1 , selected and washed with sterile distilled water. E. coli ATCC 11229 was obtained from the culture stock of the Microbiology Laboratory of the Federal University of Espírito Santo. The culture was maintained in 1 mL microtubes (Eppendorf®, São Paulo, Brazil) containing Brain Heart Infusion (BHI) agar (Difco®, Sparks, MD, USA) at −80 °C and activated by two consecutive BHI replications and incubation at 37 °C for 24 h until reaching populations of 108 and 109 CFU/mL.
To evaluate the effect of the treatments, inoculation of 100 g of strawberries was performed in previously sterilized plastic bags, and 200 mL of 0.1% peptone water was added to the inoculum (10 mL) to give a solution with approximately 106 CFU/mL. The plastic bag containing the inoculum and the sample was shaken gently for 5 min. The strawberries were kept in static contact with the cell suspension for 60 min at 24 ± 1 °C. Then, the cell suspension was drained, and the intentionally inoculated strawberries were subjected to sanitization treatments. The sanitization procedures were conducted as described inSection 2.1 . Samples to be treated with ultrasound were placed in previously sterilized plastic bags that were then placed inside the equipment. The purpose of this procedure was to prevent contamination of the equipment with the microorganism. After each treatment, 25 g of strawberries was transferred to sterile plastic bags with 225 mL of 0.1% peptone water and then homogenized for two minutes. Thereafter, appropriate dilutions were prepared and plated on MacConkey agar (Acumedia®, Indaiatuba, SP, Brazil). After plating and incubation for 48 h at 35 °C, colonies were counted.
To evaluate the effect of the treatments, inoculation of 100 g of strawberries was performed in previously sterilized plastic bags, and 200 mL of 0.1% peptone water was added to the inoculum (10 mL) to give a solution with approximately 106 CFU/mL. The plastic bag containing the inoculum and the sample was shaken gently for 5 min. The strawberries were kept in static contact with the cell suspension for 60 min at 24 ± 1 °C. Then, the cell suspension was drained, and the intentionally inoculated strawberries were subjected to sanitization treatments. The sanitization procedures were conducted as described in
Corresponding organizations : Universidade Federal do Espírito Santo, Universidade Vila Velha
Top 5 most cited protocols using «macconkey agar»
1
Salmonella typhimurium Isolation from Tissues
The isolation of S. typhimurium in tissues followed the reported method.21 (link) Briefly, tissues (100–200 mg) from internal organs were homogenized, and the supernatants (100 μL) were aseptically inoculated into 5 mL buffered peptone water and grown overnight. Aliquots were further grown in Rappaport–Vassiliades broth (Acumedia) for enrichment and incubated at 37°C for 24 hours. A loop full of the Rappaport–Vassiliades broth was spread on to MacConkey agar and Salmonella–Shigella selective agar (Acumedia). Typical colonies of Salmonella showed transparency of colonies with black centers on the Salmonella–Shigella agar after overnight culture.
Corresponding organizations : National Taipei University, National Chung Hsing University, China Medical University, National Taiwan University, Moscow Research and Clinical Center for Neuropsychiatry
2
Profiling Gut Microbial Diversity
Each fecal sample (1 g) was homogenized in a stomacher, with sterile peptone water (99 g). Subsequent tenfold serial dilutions were plated in triplicate on the following selective media to distinguish bacterial genera: Enterococcus spp.: KF Streptococcus agar (Acumedia) [19 (link)]; Lactobacillus spp.: MRS agar (Merck, Darmstadt, Deutschland, Germany) [20 ]; Bifidobacterium spp.: Bifidobacterium iodoacetate medium 25 (BIM-25) [21 (link)]; Enterobacteria: MacConkey agar (Acumedia) [22 (link)]; Clostridium spp.: RCA agar (Difco, Le Point de Claix, France) [23 (link)]. Plates for Enterococcus spp. and Enterobacteria were incubated at 37°C for 48 h. Plates for Lactobacillus spp., Clostridium spp. and Bifidobacterium spp were incubated anaerobically, in anaerobic jars with gas-generating kits, at 37°C for 48 h, 48 h and 72 h, respectively.
Corresponding organizations : Universidade Estadual Paulista (Unesp), Universidade de São Paulo
3
Antagonistic Effects of Lactobacillus on Food Pathogens
In order to test the direct antagonism of the strains under study against food pathogens (S. enterica var. Typhimurium ATTCC 14028, S. flexi ATTCC 11060 and E. coli ATCC 25922), a co-cultivation test was done. The method was adapted from Hütt, Shchepetova, Lõivukene, Kullisaar, and Mikelsaar (2006) (link). An overnight culture of each pathogen and the Lactobacillus strain were inoculated together (1% v/v) in 1 ml of BHI broth (Acumedia, Neogen, Lansing, MI, USA) and incubated for 16 h (37 C, aerobiosis). Control cultures were also prepared. Cell counts of the pathogen after incubation were done using MacConkey agar (Acumedia), incubated for 24 h (37 C, aerobiosis). The growth of the pathogen in BHI broth, inoculated alone or co-inoculated with a strain of lactobacilli was compared.
Corresponding organizations : Universidade Estadual de Santa Cruz, Universidade Federal de Minas Gerais, National University of the Littoral, Consejo Nacional de Investigaciones Científicas y Técnicas
4
Salmonella Prevalence in Acute Pediatric Diarrhea
This study comprised a total of 157 children (73 girls and 84 boys, age range 1-48 months, mean age 11.7 months, median 8.0 months) from low socioeconomic background (monthly income below U$300.00), who searched for assistance at Hospital Infantil João Paulo II, Belo Horizonte-MG, from March 2004 to March 2005. All of them presented acute diarrhea and no patient had history of hospitalization or antimicrobial therapy in the last 15 days prior to specimen collection. Clinical and epidemiological data were obtained through questionnaires filled in by the children's guardians.
Stool samples were individually transferred to two sterile leak proof wide mouth screw cap vials, one of them containing a transport medium of equal parts of glycerol and 0.033 M phosphate buffer, maintained in an ice bath, and processed within one hour.
For leukocytes search, fecal smears were stained with May-Grünwald-Giemsa and examined under bright field microscopy at 400× and 1,000×.
The specimens transported in buffered glycerol were streaked onto MacConkey Agar (Difco, Sparks, MD, USA) and SS Agar (Difco). They were also inoculated into Tetrathionate Broth (Acumedia, Baltimore, MD, USA) and, following incubation for about 18 h at 35ºC, subcultured on SS Agar. All agar cultures were incubated for up to 24 h at 35ºC. Afterwards, around five lactosenegative colonies from MacConkey Agar and five lactose-negative and five H 2 S-positive colonies from each SS Agar plate were picked and inoculated into Triple Sugar Iron Agar (Acumedia), Escola Paulista de Medicina (EPM) (36) and Citrate medium (Biobrás, Montes Claros-MG, Brazil). Whenever possible, morphologically different colonies were selected.
Following identification, Salmonella isolates were antigenically characterized by using somatic antisera directed against A, B, C1, C2, D, and E serogroups (Probac, São Paulo-SP, Brazil), according to manufacturer's instructions. Bacterium suspensions were boiled for 10 min before performing agglutination test when necessary. Subsequently, one S. enterica isolate obtained from each child was sent to Instituto Oswaldo Cruz (IOC) (Rio de Janeiro-RJ, Brazil) and serotyped by use of polyvalent and monovalent antisera against somatic and flagelar S. enterica antigens (19) .
Antimicrobial susceptibility profile of S. enterica strains were determined by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) guidelines (6) . Ampicillin (AMP), ceftriaxone (CRO), chloramphenicol (CLO), ciprofloxacin (CIP), nalidixic acid (NAL), and trimethoprim/sulfamethoxazole (SUT) (Cecon, São Paulo-SP, Brazil) were tested. Screening for extendedspectrum β-lactamases (ESBL) was performed by using ceftriaxone, ceftazidime, aztreonam, and cefotaxime (Cecon) (6) .
When strains isolated from the same patient showed divergent susceptibility profiles determined by disk diffusion technique, minimum inhibitory concentration was evaluated by agar dilution technique (2) . This was the case exclusively for chloramphenicol. Drug concentrations from 4 to 128 µg/ml were employed. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were included as controls for antimicrobial susceptibility testing.
In order to search for virulence determinants, S. enterica isolates were cultivated overnight on Tryptic Soy Agar (Difco) plates at 35ºC, bacterial cells were suspended in 500 μl sterile distilled water and centrifuged for 15 min at 6,000 g. Total deoxyribonucleic acid (DNA) was isolated by a phenol-chloroform method (12) and employed in amplification reactions targeting invA (5) and iroB (3) . Additionally, plasmidial DNA extracted by a protocol proposed by Birnboim & Doly (4) was used for detecting spvC (5) .
About 20 ng of bacterial DNA was used as template for amplification reactions. Details of the protocols are described in Amplicons were resolved in 8% polyacrylamide gels, stained with ethidium bromide, and visualized under UV light. Standard of 100 bp (Life Technologies, Gaithersburg, MD, USA) was used as molecular size marker. Positive, negative, and negative internal controls (sterile water) were included in each batch of reaction.
The results generated were analyzed by using the χ 2 test with Yates' correction or Fisher's exact test. The level of significance was set at 0.05.
Stool samples were individually transferred to two sterile leak proof wide mouth screw cap vials, one of them containing a transport medium of equal parts of glycerol and 0.033 M phosphate buffer, maintained in an ice bath, and processed within one hour.
For leukocytes search, fecal smears were stained with May-Grünwald-Giemsa and examined under bright field microscopy at 400× and 1,000×.
The specimens transported in buffered glycerol were streaked onto MacConkey Agar (Difco, Sparks, MD, USA) and SS Agar (Difco). They were also inoculated into Tetrathionate Broth (Acumedia, Baltimore, MD, USA) and, following incubation for about 18 h at 35ºC, subcultured on SS Agar. All agar cultures were incubated for up to 24 h at 35ºC. Afterwards, around five lactosenegative colonies from MacConkey Agar and five lactose-negative and five H 2 S-positive colonies from each SS Agar plate were picked and inoculated into Triple Sugar Iron Agar (Acumedia), Escola Paulista de Medicina (EPM) (36) and Citrate medium (Biobrás, Montes Claros-MG, Brazil). Whenever possible, morphologically different colonies were selected.
Following identification, Salmonella isolates were antigenically characterized by using somatic antisera directed against A, B, C1, C2, D, and E serogroups (Probac, São Paulo-SP, Brazil), according to manufacturer's instructions. Bacterium suspensions were boiled for 10 min before performing agglutination test when necessary. Subsequently, one S. enterica isolate obtained from each child was sent to Instituto Oswaldo Cruz (IOC) (Rio de Janeiro-RJ, Brazil) and serotyped by use of polyvalent and monovalent antisera against somatic and flagelar S. enterica antigens (19) .
Antimicrobial susceptibility profile of S. enterica strains were determined by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) guidelines (6) . Ampicillin (AMP), ceftriaxone (CRO), chloramphenicol (CLO), ciprofloxacin (CIP), nalidixic acid (NAL), and trimethoprim/sulfamethoxazole (SUT) (Cecon, São Paulo-SP, Brazil) were tested. Screening for extendedspectrum β-lactamases (ESBL) was performed by using ceftriaxone, ceftazidime, aztreonam, and cefotaxime (Cecon) (6) .
When strains isolated from the same patient showed divergent susceptibility profiles determined by disk diffusion technique, minimum inhibitory concentration was evaluated by agar dilution technique (2) . This was the case exclusively for chloramphenicol. Drug concentrations from 4 to 128 µg/ml were employed. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were included as controls for antimicrobial susceptibility testing.
In order to search for virulence determinants, S. enterica isolates were cultivated overnight on Tryptic Soy Agar (Difco) plates at 35ºC, bacterial cells were suspended in 500 μl sterile distilled water and centrifuged for 15 min at 6,000 g. Total deoxyribonucleic acid (DNA) was isolated by a phenol-chloroform method (12) and employed in amplification reactions targeting invA (5) and iroB (3) . Additionally, plasmidial DNA extracted by a protocol proposed by Birnboim & Doly (4) was used for detecting spvC (5) .
About 20 ng of bacterial DNA was used as template for amplification reactions. Details of the protocols are described in Amplicons were resolved in 8% polyacrylamide gels, stained with ethidium bromide, and visualized under UV light. Standard of 100 bp (Life Technologies, Gaithersburg, MD, USA) was used as molecular size marker. Positive, negative, and negative internal controls (sterile water) were included in each batch of reaction.
The results generated were analyzed by using the χ 2 test with Yates' correction or Fisher's exact test. The level of significance was set at 0.05.
5
Antibiotic Resistance Profiling of E. coli in Broiler Chickens
The experimental proposal was submitted and approved by the Ethics Committee on Animal Use of the Federal University of Paraiba (CEUA/UFPB). Two hundred fertile eggs were obtained from a commercial hatchery (Guaraves Alimentos Ltda, Guarabira, PB, Brazil), originated from Cobb500 breeders with 44 weeks of age. They were placed in egg incubators with standard temperature (37.7°C) and humidity (60%) conditions and automatic turning at each two hours. After hatch, a total of 168 chicks were weighed individually (mean weight 47.0 ± 0.5g) and used in a completely randomized design with two treatments and six repetitions, with 14 animals per repetition. The chicks were kept into boxes (50 x 50 x 50 cm). Each box was equipped with feeder and drinker and was covered with nylon to avoid contamination between boxes by vectors such as flies. Thermo-hygrometers (Oregon Scientific, Portland, EUA) were used to monitor temperature and relative humidity in the room.
A corn-soybean meal diet was formulated for the initial phase with the following levels: 22.2% crude protein, 2,950 kcal of metabolizable energy/kg of diet, 1.31% digestible lysine, 0.94% digestible methionine + cystine and 0.852% digestible threonine. Animals in the control group (CG) were administered 0.2 mL of Marek’s vaccine suspended in sterile saline solution subcutaneously, whereas the animals in the antimicrobial-administered group (AG) received 0.2 mL of Marek’s vaccine suspended in sterile ceftiofur solution (0.2 mg ceftiofur sodium).
Cloacal swabs were randomly collected from two animals per repetition before vaccination (day 0) and at 3, 5, 7, 9, 11 and 14 days post-hatching. After swab collection the animals were euthanized. The swabs were placed into Luria-Bertani broth (Himedia, India) supplemented with ceftiofur (2mg/L) and incubated at 37°C for 24 h. Then, a 20μL aliquot was spread onto MacConkey Agar (Acumedia, EUA) supplemented with ceftiofur (2mg/L) and incubated at 37°C for 24 h. Lactose fermenting colonies (four per plate) showing characteristics of E. coli were transferred to Eosin Methylene Blue Agar (EMB) (Himedia, India) and further confirmed as E. coli by the following biochemical tests: Triple Sugar Iron Agar (TSI) (Himedia, India), Lysine Iron Agar (LIA) (Himedia, India), Sulfide Indole Motility (SIM) (Acumedia, EUA), Simmons Citrate Agar (Oxoid, UK) and Urea Agar Base (Oxoid, UK).
The Clinical Laboratory Standards Institute (CLSI) disk diffusion method [7 ] was used to test E. coli- confirmed colonies for antimicrobial susceptibility to the following drugs: amoxicillin/clavulanate (Amx/Clv, 20/10 μg, Cecon, São Paulo, Brazil), aztreonam (ATM, 30 μg, Cecon) cefotaxime (CTX, 30 μg, Cecon), ceftazidime (CAZ, 30 μg, Cecon), ceftriaxone (CRO, 30 μg, Cecon), ciprofloxacin (CIP, 5 μg, Cecon), chloramphenicol (C, 30 μg, Cecon), gentamicin (GM, 10 μg, Cecon), sulfisoxazole / trimethoprim (SXT, 23.75 / 1.25 μg, Cecon) and tetracycline (Te, 30 μg, Cecon).
From each plate, all isolates showing different resistance patterns were taken for further confirmation, therefore, in some cases more than one isolate was recovered per sample (bird). We also determined the minimum inhibitory concentration (MIC) of ceftiofur (CTF) by the broth microdilution method [7 ] using 96-well microtiter plates containing final ceftiofur concentrations ranging from 0.5 μg/mL to 256 μg/mL. CLSI [7 ] criteria were used to interpret MIC results as susceptible (MIC ≤2 mg/L), intermediate (4 mg/L), or resistant (≥8 mg/L).
Phenotypic ESBL detection was carried out by double-disk synergy test using CTX, CAZ and CRO disks placed at a distance of 20 mm concentrically to the Amx/Clv disk [7 ]. E. coli isolates were also tested by PCR targeting the ESBL genes (blaCTX-M, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8 and blaSHV) as well as plasmid-mediated AmpC genes (blaACC, blaCMY-2, blaDHA, blaFOX, blaMOX and blaMIR), using primer sequences and conditions as previously described [8 (link)–11 (link)] and DNA extracted by phenol/chloroform/isoamyl-alcohol (25:24:1) as described by Fritsch et al. [12 ]. Afterwards, the DNA extracted from confirmed ESBL-producing E. coli was adjusted to 50 ng/ μL using a microvolume spectrometer (Colibri, Titertek Berthold, Germany) and Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) was used as genotyping method, as previously described [13 (link)]. Shortly, reactions were performed in 25 μL containing 1 pmol of primer, 200 mM of each dNTP, 3 mM of MgCl2, 100 ng of genomic DNA, and 1U of Taq DNA polymerase (Invitrogen, Brazil). Amplification was performed in a thermal cycler (TPersonal Thermocycler, Biometra, Germany). Products were analyzed by electrophoresis in 2% agarose gel (LGC Biotechnology, Brazil) stained with GelRed (Biotium, USA). The presence or absence of bands was analyzed visually under ultraviolet light. ERIC-PCR band patterns were scanned and analyzed using the Dice product moment correlation coefficient (2% tolerance) by BioNumerics software (Version 7.1, Applied Maths, Belgium). Clustering analysis was carried out by the unweighted pair group method with arithmetic averages (UPGMA). E. coli ATCC 25922 was used as internal control (outgroup). Details on the DNA extraction and PCR protocols that were used in this study are described in the Supplementary material (S1 Text and S1 Table ). The discriminatory power (D-value) was calculated as described by Hunter [14 (link)].
Fisher’s exact test at 5% probability was used to compare the overall frequency of phenotypic ESBL-E. coli isolates between control group (CG) and antimicrobial-administered group (AG). A Bayesian binomial logistic regression (BLR) approach with 8,000 repetitions was used to infer the probability of the ESBL occurrence between the treatment groups along the experimental period. Statistical analyses were performed in R environment [15 ] using brms package obtained from CRAN (https://cran.r-project.org/web/packages/brms/index.html )
A corn-soybean meal diet was formulated for the initial phase with the following levels: 22.2% crude protein, 2,950 kcal of metabolizable energy/kg of diet, 1.31% digestible lysine, 0.94% digestible methionine + cystine and 0.852% digestible threonine. Animals in the control group (CG) were administered 0.2 mL of Marek’s vaccine suspended in sterile saline solution subcutaneously, whereas the animals in the antimicrobial-administered group (AG) received 0.2 mL of Marek’s vaccine suspended in sterile ceftiofur solution (0.2 mg ceftiofur sodium).
Cloacal swabs were randomly collected from two animals per repetition before vaccination (day 0) and at 3, 5, 7, 9, 11 and 14 days post-hatching. After swab collection the animals were euthanized. The swabs were placed into Luria-Bertani broth (Himedia, India) supplemented with ceftiofur (2mg/L) and incubated at 37°C for 24 h. Then, a 20μL aliquot was spread onto MacConkey Agar (Acumedia, EUA) supplemented with ceftiofur (2mg/L) and incubated at 37°C for 24 h. Lactose fermenting colonies (four per plate) showing characteristics of E. coli were transferred to Eosin Methylene Blue Agar (EMB) (Himedia, India) and further confirmed as E. coli by the following biochemical tests: Triple Sugar Iron Agar (TSI) (Himedia, India), Lysine Iron Agar (LIA) (Himedia, India), Sulfide Indole Motility (SIM) (Acumedia, EUA), Simmons Citrate Agar (Oxoid, UK) and Urea Agar Base (Oxoid, UK).
The Clinical Laboratory Standards Institute (CLSI) disk diffusion method [7 ] was used to test E. coli- confirmed colonies for antimicrobial susceptibility to the following drugs: amoxicillin/clavulanate (Amx/Clv, 20/10 μg, Cecon, São Paulo, Brazil), aztreonam (ATM, 30 μg, Cecon) cefotaxime (CTX, 30 μg, Cecon), ceftazidime (CAZ, 30 μg, Cecon), ceftriaxone (CRO, 30 μg, Cecon), ciprofloxacin (CIP, 5 μg, Cecon), chloramphenicol (C, 30 μg, Cecon), gentamicin (GM, 10 μg, Cecon), sulfisoxazole / trimethoprim (SXT, 23.75 / 1.25 μg, Cecon) and tetracycline (Te, 30 μg, Cecon).
From each plate, all isolates showing different resistance patterns were taken for further confirmation, therefore, in some cases more than one isolate was recovered per sample (bird). We also determined the minimum inhibitory concentration (MIC) of ceftiofur (CTF) by the broth microdilution method [7 ] using 96-well microtiter plates containing final ceftiofur concentrations ranging from 0.5 μg/mL to 256 μg/mL. CLSI [7 ] criteria were used to interpret MIC results as susceptible (MIC ≤2 mg/L), intermediate (4 mg/L), or resistant (≥8 mg/L).
Phenotypic ESBL detection was carried out by double-disk synergy test using CTX, CAZ and CRO disks placed at a distance of 20 mm concentrically to the Amx/Clv disk [7 ]. E. coli isolates were also tested by PCR targeting the ESBL genes (blaCTX-M, blaCTX-M-1, blaCTX-M-2, blaCTX-M-8 and blaSHV) as well as plasmid-mediated AmpC genes (blaACC, blaCMY-2, blaDHA, blaFOX, blaMOX and blaMIR), using primer sequences and conditions as previously described [8 (link)–11 (link)] and DNA extracted by phenol/chloroform/isoamyl-alcohol (25:24:1) as described by Fritsch et al. [12 ]. Afterwards, the DNA extracted from confirmed ESBL-producing E. coli was adjusted to 50 ng/ μL using a microvolume spectrometer (Colibri, Titertek Berthold, Germany) and Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) was used as genotyping method, as previously described [13 (link)]. Shortly, reactions were performed in 25 μL containing 1 pmol of primer, 200 mM of each dNTP, 3 mM of MgCl2, 100 ng of genomic DNA, and 1U of Taq DNA polymerase (Invitrogen, Brazil). Amplification was performed in a thermal cycler (TPersonal Thermocycler, Biometra, Germany). Products were analyzed by electrophoresis in 2% agarose gel (LGC Biotechnology, Brazil) stained with GelRed (Biotium, USA). The presence or absence of bands was analyzed visually under ultraviolet light. ERIC-PCR band patterns were scanned and analyzed using the Dice product moment correlation coefficient (2% tolerance) by BioNumerics software (Version 7.1, Applied Maths, Belgium). Clustering analysis was carried out by the unweighted pair group method with arithmetic averages (UPGMA). E. coli ATCC 25922 was used as internal control (outgroup). Details on the DNA extraction and PCR protocols that were used in this study are described in the Supplementary material (
Fisher’s exact test at 5% probability was used to compare the overall frequency of phenotypic ESBL-E. coli isolates between control group (CG) and antimicrobial-administered group (AG). A Bayesian binomial logistic regression (BLR) approach with 8,000 repetitions was used to infer the probability of the ESBL occurrence between the treatment groups along the experimental period. Statistical analyses were performed in R environment [15 ] using brms package obtained from CRAN (
Corresponding organizations : Universidade Federal da Paraíba, The Ohio State University, Ohio Department of Health
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