Macconkey s agar

The collected milk sample was kept in a sterile tube and transported to the laboratory within 4 h. Milk samples were cultured on nutrient agar, blood agar, mannitol salt agar, and MacConkey's agar (Oxoid, Hampshire, UK), and then the cultured plates were incubated for 24–48 h at 37°C. The pure colonies were first identified according to their Gram staining, morphological characteristics, hemolysis, and biochemical characteristics as described in [23 (link)]. Positive isolates of S. aureus were examined by multiplex PCR to determine their MRSA status [24 (link)]. Isolates of S. aureus were kept at −20°C in tryptic soy broth (TSB; Becton Dickinson, Wokingham, UK), containing 20% volume of glycerol.

The isolation procedures were conducted by scorching the surface of the organs with a hot spatula and then sterilizing loopfuls, and inoculating swabs onto Tryptone Soya Broth (TSB, OXOID, Hampshire, United Kingdom). The inoculated TSBs were then incubated aerobically for 24 h at 37 °C and streaked onto 5% sheep blood agar and MacConkey’s agar (OXOID, Hampshire, United Kingdom) for 24 h incubation period at 37 °C. Pure colonies were recognized morphologically using the Gram stain, Leishman's staining method, and biochemical assays (catalase, oxidase, nitrate, methyl red, Voges–Proskauer, sugar fermentation, indole, citrate, gelatin liquefaction and urease) (Carter 1984 ; Markey et al. 2013 ).

Samples were initially screened based on clinical history, symptoms, and postmortem examination. A total of one hundred (100) suspected samples were randomly selected. Samples were pre-enriched in buffered peptone water (Oxoid^®, Hampshire, UK) at a dilution ratio of 1:10 and were incubated overnight at 37 °C. A loopful of each culture was streaked on Eosin Methylene Blue (EMB) (Hi Media, India) agar and MacConkey’s agar (Oxoid^®, Hampshire, UK) and plates were incubated at 37 °C for 24 h. Characteristic metallic sheen colonies on EMB agar were selected and subsequently sub-cultured on nutrient agar (NA) (Oxoid^®, Hampshire, UK), and biochemical tests were performed for further confirmation, including triple sugar iron (TSI), motility indole urease, oxidase and catalase tests, and Gram staining. Single E. coli colonies were stored in Brain Heart Infusion broth (Oxoid^®, Hampshire, UK) in the presence of 15–20% glycerol and kept at −20 °C for further use.

A bacteriological examination was performed following Shukla and Mishra [28 ]. Under strict aseptic conditions, visceral organ samples (liver, heart, lung, and unabsorbed yolk sac) were collected from diseased and freshly deceased broiler chicks, as well as the yolk sac of dead in-shell chicks. All samples were pre-enriched in 9 mL buffer peptone water (PBW) (Oxoid Ltd, Hampshire, England) and incubated aerobically at 37 °C for 24 h. A loopful of inoculated broth was streaked onto the surface of Cetrimide and MacConkey’s agar (Oxoid Ltd, Hampshire, England) and incubated aerobically for 24–48 h at 37 °C. Suspected colonies appeared as large, transparent (non-lactose fermenter) on MacConkey’s agar, and yellowish green pigmentation with a fruity smell on Cetrimide agar were selected and purified on Trypticase Soya Agar (TSA) (Oxoid Ltd, Hampshire, England). Purified colonies were examined morphologically by gram’s staining and characterized biochemically using catalase, oxidase, indole, methyl red, voges-proskauer, citrate utilization, nitrate reduction, and glucose fermentation tests [29 ]. For the further identification, all suspected strains were preserved in 70% glycerol at − 20 °C.

P. aeruginosa clinical isolates and associated data (n = 160 data not shown, only new STs strains are described in this study) were collected from the Pakistan Institute of Medical Sciences (PIMS), Islamabad, Pakistan, in 2017. A 100 µL suspension of the samples was plated initially on blood agar (Oxoid) and MacConkey’s Agar (Oxoid, UK). The plates were incubated for 18–24 h at 37 °C. Morphologically distinct colonies were sub-streaked on Pseudomonas cetrimide agar (Oxoid) and incubated for 24 h at 37 °C. After confirmation by colony morphology, the isolates were identified using MALDI-TOF MS VITEK v2.3.3 (bioMérieux, Durham, NC, USA) [51 (link)].

The collected nasal swabs were incubated at 37 °C for 24 h in brain heart infusion (BHI) broth (Oxoid, Hampshire, UK). However, the lung specimens were homogenized using an automatic grinder instrument (Bertin Technologies, Dieppe, France). The tissue suspensions were centrifuged at 8000× g at 4 °C for 10 min and enriched in BHI broth overnight at 37 °C. A loopful from the enrichment broth was streaked onto various selective media, including MacConkey’s agar (Oxoid, Hampshire, UK), eosin methylene blue agar (Oxoid, Hampshire, UK), and HiCrome Klebsiella Selective Agar (Himedia, Mumbai, India). Suspected colonies were purified, Gram stained, and identified as Klebsiella spp. using biochemical tests including indole, methyl red, Voges–Proskauer, and citrate, as well as their characteristic reactions on triple sugar iron agar media [18 ].