Api 20e test

The bacteria that were used in the experiment were isolated from the inlet of the wastewater treatment plant in Katowice (Bz4) and the polluted river Leśnica in Wodzisław Śląski (Rz7) located in south Poland. The wastewater treatment plant in Katowice has a daily flow of approximately 43,000 m³ day⁻¹. According to Polish law (Dz. U. 2012 r. poz.145; Dz. U. Nr257, poz. 1545), Leśnica river is classified as a river with poor ecological status.
The bacteria strains were isolated using the spread plate method on nutrient agar (BTL), which was supplemented with 0.1 g l⁻¹ of brilliant green. After 72 h of incubation, a discolouration zone was observed around some bacterial colonies, which proved their decolourisation abilities. The colonies with the largest discoloured area, which was measured from the edge of the colony (11 and 18 mm for Bz4 and Rz7, respectively), were isolated and purified on nutrient agar (BTL) using the streak plate method. Selected strains were classified as Klebsiella planticola (Rz7) and Klebsiella sp. (Bz4) using the API 20E test (Biomerieux).

The P. mirabilis strains (KP; 5628) were isolated at the Department of Microbiology from the urine of patients of the Children’s Memorial Health Institute in Warsaw, Poland, who had been diagnosed with infectious urolithiasis. The P. mirabilis K8/MC strain was obtained from urinary stones and provided by the Provincial Specialist Hospital M. Pirogow in Lodz. The strains were identified using the API 20E test (Biomerieux, Marcy-I’Etoile, France) and cultured on TSB (tryptic soy broth, BTL, Warsaw, Poland) at 37 °C for 24 h.
Lactobacillus strains were isolated from the urinary tract of healthy people with the consent of the Committee for Bioethics of Scientific Research of the University of Lodz (4(I)/KBBN-UŁ/II/2020), and were deposited in the bacterial strain collection at the Department of Biology of Bacteria, University of Lodz. The method of isolation of these strains and their characteristics had been described previously [17 (link)]. Briefly, the strains were obtained from human urine of both men and women who had not been treated with antibiotics and probiotics in the last 3 months. They were identified by their morphology and mass spectrometry using a MALDI/TOF Microflex LT (Bruke, Billerica, MA, USA). Lactobacillus spp. were cultured on APT broth (BD Difco, Franklin Lakes, NJ, USA) and incubated in 5% CO₂ at 37 °C for 48 h.

All E. coli isolates (n = 730) investigated in this study were obtained from clinical specimens submitted to the Diagnostic Laboratory of the Division of Microbiology, Faculty of Veterinary Medicine at the Warsaw University of Life Sciences (Poland), from January 2007 to December 2013. Clinical samples were collected from dogs and cats with various types of infections (Table 1). E. coli isolates were cultured and identified using standard microbiological diagnostic techniques including the API 20 E test (bioMérieux, France). Based on the presence or absence of β-haemolysis on blood agar, the isolates were classified as haemolytic or nonhaemolytic, respectively. Based on clinical and microbiological diagnosis, all the E. coli isolates included in the study were recognized as the sole agent involved in the infection. In case of fecal samples, a parasitological examination was additionally performed to exclude parasitic disease. A reference strain, E. coli ATCC 25922, was used as a quality control in the susceptibility tests.

Blood cultures submitted to the Aga Khan University clinical microbiology laboratory (November 2016 to September 2017) grew S. Typhi that demonstrated high MICs against ceftriaxone and cefotaxime (>64 µg/ml). MICs were confirmed by two methods, Etest and Vitek 2 (bioMérieux). The identification of S. Typhi was confirmed by the API 20E test (bioMérieux) and agglutination with genus- and serotype-specific antisera (Salmonella poly antiserum A-I [Difco], Salmonella O antiserum [Difco], and Salmonella Vi antiserum [Difco]).

First, cecal content was removed and homogenized. Then, pools of six animals from the same experimental group were prepared: 5 pools from day-old-chicks (30 samples), 10 pools from animals in CFC at mid-period (60 samples), 10 from animals in IFC at mid-period (60 samples), 10 pools from animals in CFC at the end of the growing period (60 samples) and 10 pools from animals in IFC at the end of the growing period (60 samples). The pools’ content was cultured directly onto a Coliform Chromogenic agar (Scharlab, S.L., Barcelona, Spain) in duplicate, and agar plates were incubated at 37 ± 1 °C for 24 h. After incubation, suspected colonies were streaked onto a nutrient medium (Scharlab, S.L., Barcelona, Spain) and incubated at 37 ± 1 °C for 24 h. Then, API-20E test (Biomerieux, S.L., Barcelona, Spain) was performed to confirm E. coli.

A 14-year-old Lusitano horse with signs of abdominal pain was referred to a referral equine hospital in Lisbon in June 2016 and diagnosed with a small intestinal inguinal hernia. Laparotomy was performed, and the incarcerated portion of non-viable small intestine was resected under general anesthesia. The horse had never been subjected to antimicrobial therapy before and was treated with penicillin at a dose of 22 000 UI/kg for 7 days and gentamicin at a dose of 6,6 mg/kg for 5 days post-surgery. The patient developed a surgical site infection of the abdominal wall that was detected on day 4 post-surgery. A swab sample was collected from the site and further streaked in Blood and MacConkey agar. After a 24 h incubation at 37°C, pure bacterial cultures were obtained, posteriorly identified as K. pneumoniae using API 20 E test (Biomérieux, Marcy-Étoile, France).
The Laboratory of Microbiology of the Faculty of Pharmacy of the University of Porto provided the seven ST348 human isolates (Rodrigues et al., 2016 (link), 2017 (link) and unpublished data).