Macconkey agar

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MacConkey agar is a selective and differential culture medium used for the isolation and identification of Gram-negative enteric bacteria, particularly members of the Enterobacteriaceae family. It inhibits the growth of Gram-positive bacteria while allowing the growth of Gram-negative bacteria.

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460 protocols using macconkey agar

Screening for ESBL-PE and MRSA in ICU

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Rectal and nasal swabs were collected within 48 hours of ICU admission for MDR organisms screening. Rectal swabs were screened for ESBL-PE, and MRSA colonization was screened from nasal swabs. Rectal swab specimens were cultured on selective and non-selective media.
The rectal swabs were inoculated on MacConkey agar (Oxoid, UK) supplemented with 2μg/ml of ceftazidime and MacConkey agar (Oxoid, UK) non supplemented by ceftazidime. Both plates were incubated aerobically at 37 °C for 18–24 hours. The plain MacConkey agar plate aimed to check if the swabs contained viable bacteria. Nasal swabs were cultured on sheep blood agar (Oxoid, UK) and incubated aerobically at 33 ⁰C for 18–24 hours for isolation S. aureus.

Manyahi J., Majigo M., Kibwana U., Kamori D, & Lyamuya E.F. (2022). Colonization of Extended-spectrum β-lactamase producing Enterobacterales and meticillin-resistant S. aureus in the intensive care unit at a tertiary hospital in Tanzania: Implications for Infection control and prevention. Infection Prevention in Practice, 4(2), 100212.

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Bacterial Isolation from Blood and Urine

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Blood samples: 1 ml of blood was collected with a sterile syringe and mixed with 20 ml of a Brain-Heart Infusion broth (Conda Pronadisa, Spain). The mixture was incubated at 37°C for 7 days, streaked onto the surface of blood agar and MacConkey agar (Oxoid, UK) and incubated at 37°C for 24 h (23 ). Urine samples were collected in sterile containers. A loopful of the urine samples were streaked onto the surface of blood agar and MacConkey agar (Oxoid, UK) and incubated at 37°C for 24 h. All isolates were identified according to morphological and biochemical tests (Gram stain, capsule stain, motility test, indole production test, urease production test, Methyl Red test, Voges-Proskauer test) (24 ) and confirmed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (Microflex, Bruker Daltonics, Bremen, Germany).

Khaertynov K.S., Anokhin V.A., Rizvanov A.A., Davidyuk Y.N., Semyenova D.R., Lubin S.A, & Skvortsova N.N. (2018). Virulence Factors and Antibiotic Resistance of Klebsiella pneumoniae Strains Isolated From Neonates With Sepsis. Frontiers in Medicine, 5, 225.

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Microbial Analysis of Meat and Milk

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Minced raw meat samples (25 g each) were cultured on nutrient agar, MacConkey agar, and UTI Chrome agar (Oxoid, Hampshire, UK). Milk samples (100 µL each) were serially diluted (10-fold) and cultured on nutrient agar, MacConkey agar, and UTI Chrome agar (Oxoid, Hampshire, UK), and then aerobically incubated overnight at 37 ºC. The isolates were preliminarily identified based on colony morphology, cultural characteristics, and Gram staining. The VITEK 2 compact instrument was employed to automatically identify isolates using GN cards (bioMérieux, Marcy-l’Étoile, France) with 64 different biochemical substrates.

Qamar M.U., A.a.t.i.k.a., Chughtai M.I., Ejaz H., Mazhari B.B., Maqbool U., Alanazi A., Alruwaili Y, & Junaid K. (2023). Antibiotic-Resistant Bacteria, Antimicrobial Resistance Genes, and Antibiotic Residue in Food from Animal Sources: One Health Food Safety Concern. Microorganisms, 11(1), 161.

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Comprehensive Microbial Identification and Susceptibility Testing

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Stool samples were routinely cultured on MacConkey agar, xylose lysine deoxycholate agar, and Hektoen agar (Oxoid Ltd, Basingstoke, UK). Stool samples were also inoculated into selenite F broth (Oxoid Ltd, Basingstoke, UK). Blood samples were collected in blood culture bottles and incubated in the BD BACTEC™ blood culture system (Becton, Dickinson and Company, Franklin Lakes, USA). A blood culture sample flagged as positive by the system was inoculated on blood agar, chocolate agar, and MacConkey agar. Identification of microbes from urine samples was performed by plating the sample onto MacConkey agar and cystine lactose electrolyte deficient (CLED) agar (Oxoid Ltd, Basingstoke, UK). Samples, other than stool or blood, e.g. urine, abdominal abscess, and surgical wound swabs, were cultivated as per standard operating procedures followed at the microbiology laboratory at KFHU.
Identification and antimicrobial susceptibility testing of all bacterial isolates was performed by the Vitek2 automated card system (bioMérieux Vitek Inc., Hazelwood, MO, USA). When required, an antimicrobial susceptibility test was performed manually using the disc diffusion method following the Clinical and Laboratory Standards Institute guidelines.²⁰
Salmonella serogrouping was performed using the BD Salmonella antisera (Becton, Dickinson and Company, Franklin Lakes, USA).

Aljindan R.Y, & Alkharsah K.R. (2020). Pattern of increased antimicrobial resistance of Salmonella isolates in the Eastern Province of KSA. Journal of Taibah University Medical Sciences, 15(1), 48-53.

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Isolation and Identification of Antibiotic-Resistant E. coli

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Thawing of samples was done only once, just before testing. All samples were first cultured in buffered peptone water for 18 ± 3 h at 37 °C. After pre-incubation, the samples were simultaneously tested for Salmonella spp. according to ISO 6579-1:2017-04 standard and E. coli isolation methods used in official AMR monitoring (652/2013/EC Commission Implementing Decision) [50 ]. For isolation of commensal, cephalosporin-, and carbapenem-resistant E. coli, each sample was simultaneously streaked on, respectively, MacConkey agar (Oxoid, Hampshire, UK), MacConkey agar supplemented with cefotaxime (1 mg/L, Oxoid, Hampshire, UK), chromID™ CARBA, and chromID™ OXA-48 agar (bioMérieux, Marcy l’Etoile, France). Additionally, samples were streaked on MacConkey supplemented with colistin (2 mg/L, Oxoid, Hampshire, UK) for detection of colistin-resistant E. coli. Presumed E. coli were identified with polymerase chain reaction (PCR) targeting the universal shock protein A gene (uspA) according to a previously described protocol [51 (link)].

Skarżyńska M., Zając M., Kamińska E., Bomba A., Żmudzki J., Jabłoński A, & Wasyl D. (2020). Salmonella and Antimicrobial Resistance in Wild Rodents—True or False Threat?. Pathogens, 9(9), 771.

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Isolation and Identification of E. coli

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For enrichment, samples in buffered peptone water were incubated at 37°C for 24 h in an incubator. A loopful from each of the overnight bacterial broth was then inoculated onto MacConkey agar (Oxoid, UK) and incubated at 37°C for 24 h. Pink-colored colonies on MacConkey agar were suspected as E. coli colonies. Smears from the suspected colonies were examined microscopically to detect Gram-negative rods using gram staining method. Colonies positive for Gram-negative rods were then streaked onto Eosin Methylene Blue (EMB) Agar (Oxoid, UK) and presumptively identified E. coli by observing characteristic green colonies with metallic sheen. The presumptive isolates were subcultured into tryptic soy broth (TSB) at 37°C for 24 h. Then, TSB cultures for each presumptive isolate were preserved by adding 15% glycerin and stored at −80°C for further use.

Gupta M.D., Sen A, & Das A. (2018). Occurrence of Escherichia coli carrying Shiga toxin-producing genes in buffaloes on smallholdings in Bangladesh. Veterinary World, 11(10), 1454-1458.

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Isolation and Identification of E. coli

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After collection, all samples were homogenised using stomacher and 1 mL of the suspension was transferred into 10 mL buffered peptone water using disposable pipette and the homogenates were incubated at 37°C for 16–18 h. After an overnight incubation in the preenrichment broth, a small amount of inoculum from BPW was transferred into MacConkey agar (Oxoid, UK) using a disposable pipette and streaked using a sterile wire loop. The inoculated plates samples were then incubated at 37°C for 24 h. After inoculation and isolating colonies from MacConkey agar, EMB (Oxoid, UK) was further used for screening. Greenish metallic sheen colonies on EMB were transferred to Nutrient Agar to nourish and maintain the colonies before conducting biochemical test for E. coli. Further biochemical tests and Gram's staining were conducted to identify E. coli phenotypically.

Aklilu E, & Raman K. (2020). MCR-1 Gene Encoded Colistin-Resistant Escherichia coli in Raw Chicken Meat and Bean Sprouts in Malaysia. International Journal of Microbiology, 2020, 8853582.

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Blood Culture Identification of S. Typhi

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For blood culture, 1–3 mL for children < 5 years of age and 5–10 mL for those 5–16 years of age was collected in a syringe, placed into Bactec media bottles (Becton-Dickinson, New Jersey, USA), incubated at 37°C in a computerised BACTEC 9,050 Blood Culture System (Becton-Dickinson), and subcultured after 24–48 hr onto blood, chocolate and MacConkey agar (Oxoid, Basingstoke, UK) plates. All isolates, whether from cases or carriers were cultured on selenite F (Oxoid) broth aerobically at 37°C overnight. Broth cultures were then subcultured on MacConkey agar and Salmonella-Shigella agar (Oxoid) and incubated at 37°C overnight. Blood and stool isolates were identified using a series of standard biochemical and serological tests as described previously Mbae et al., 2020 (link). Briefly, colonies of S. Typhi identified from biochemical and PCR testing were subjected to serological identification as S. Typhi using the slide agglutination technique and applying monovalent antisera (Murex Diagnostics, Dartford, UK). A drop of the antisera to be tested was mixed with a bacterial smear on a slide to observe for the presence or absence of agglutination in two minutes.

Kariuki S., Dyson Z.A., Mbae C., Ngetich R., Kavai S.M., Wairimu C., Anyona S., Gitau N., Onsare R.S., Ongandi B., Duchene S., Ali M., Clemens J.D., Holt K.E, & Dougan G. (2021). Multiple introductions of multidrug-resistant typhoid associated with acute infection and asymptomatic carriage, Kenya. eLife, 10, e67852.

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Stool Sample Analysis for Salmonella and Shigella

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Prior to subjecting the sample to culture media, physical analysis of the stool sample was performed in order to check whether the sample was diarrheal or not, presence of blood, pus and mucus. Children with diarrhea, blood, mucus were included. Diarrhea stool specimen was inoculated using sterile wire loop on MacConkey Agar (Oxoid, Ltd), and Xylose lysine- Deoxycholate agar (XLD) (Oxoid Ltd). The inoculated plates ware incubated at 37 °C for 24 h (SOPs) [12 ]. After 24 h incubation, the plates were checked for colony characteristics of Salmonella and Shigella species. Furthermore, colorless to yellow colonies on MacConkey Agar and pink to red colonies Xylose Lysine Deoxycholate (XLD) (Oxoid Ltd) [12 ] agar were typically considered as Non-lactose fomenters. In addition, the colonies considered as non-lactose fomenters were further identified by biochemical tests to confirm the identification to genus level of the pathogens Biochemical tests such as klingler iron agar (KIA), motility indole, lysine medium, simon’s citrate agar, and unease test were used [13 ].

Muse K., Urgessa K., Shume T., Tahir B, & Weldegebreal F. (2024). Prevalence, associated factors and antimicrobial susceptibility patterns of Salmonella and Shigella species among diarrheic under five children in Sultan Sheik Hassan Yabere referral Hospital, Jigjiga, Eastern Ethiopia. BMC Pediatrics, 24, 311.

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Detecting Carbapenem-Resistant Enterobacterales

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Primary processing was dependent on the specimen following standard laboratory procedures with cysteine lactose electrolyte deficient agar-CLED (Oxoid, Basingstoke, UK) for urine, 5% Sheep blood agar (SBA), chocolate agar, and MacConkey agar for positive automated blood culture vials, SBA, and MacConkey agar (Oxoid, Basingstoke, UK) for other specimen types. MacConkey and SBA were incubated in ambient air and Chocolate agar in 5% CO₂ for 18–24 h at 35–37 °C. The identification and antimicrobial susceptibility testing (AST) of suspected Enterobacterales bacterial colonies were conducted using the Phoenix system (BD Diagnostic Systems, Sparks, MD, US) and interpreted according to Clinical and Laboratory Standards Institute (CLSI) M100 30th Edition guidelines²⁹. The control strains E. coli ATCC 25922, and K. pneumoniae ATCC 700600 were used for quality control. At both IFAIN and Cepheid sites, Xpert^® Carba-R (Cepheid, Sunnyvale, CA, USA) was used for identification of five carbapenem resistance genes (bla_KPC, bla_VIM, bla_OXA-48, bla_IMP, and bla_NDM).

Medugu N., Tickler I.A., Duru C., Egah R., James A.O., Odili V., Hanga F., Olateju E.K., Jibir B., Ebruke B.E., Olanipekun G., Tenover F.C, & Obaro S.K. (2023). Phenotypic and molecular characterization of beta-lactam resistant Multidrug-resistant Enterobacterales isolated from patients attending six hospitals in Northern Nigeria. Scientific Reports, 13, 10306.

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