Cm0007

Isolation was conducted following standard procedures described by [36 ]. A loopful of semen used for count was cultured on blood (Oxoid: CM00558) and MacConkey agar (Oxoid: CM0007) at the same time and incubated at 37°C for 24 hours. Growth characteristics were recorded including haemolysis on blood agar and lactose fermentation on MacConkey. Primary tests including Gram's reaction, catalase, oxidase, oxidation-fermentation (O-F), motility, glucose, and growth on blood and MacConkey agar were conducted. The coagulase test was also conducted on Staphylococcus isolates.

The feces examined in this study originated from healthy donors and from salmonellosis patients in remission of two different clinical trials. The feces were collected and homogenized with tryptic soy broth plus glycerol before being stored at −80°C. For plating, an aliquot of the sample was scraped off the frozen stock and homogenized for 1 min at 25 Hz in PBS using a Qiagen TissueLyser. Serial dilutions were prepared in PBS and plated on MacConkey agar (Oxoid; CM0007) (with incubation overnight at 37°C under aerobic conditions). Morphologically different colonies were picked and streaked on MacConkey agar to ensure purity of the culture (with incubation overnight at 37°C under aerobic conditions). A single colony was picked to inoculate a 3-mL liquid LB culture (37°C overnight). The pellet of the culture was used for DNA extraction (Qiagen DNeasy blood and tissue kit).

Two milliliters of venous blood sample taken from patients was inoculated into 20 ml brain-heart infusion broth (BHI) (CM1135; Oxoid Ltd., England, UK). The culture bottles were incubated at 37 °C for 7 days and examined daily for evidence of bacterial growth, including turbidity and hemolysis. If bacterial growth was observed, a subculture was made after 24 h on blood agar (CM0271, Oxoid Ltd., England UK), Salmonella Shigella agar (SS) (CM0099, Oxoid Ltd., England, UK), and MacConkey agar (CM0007, Oxoid Ltd., England, UK).

The representative specimens (n = 280) were pre-enriched in 10 mL of tryptone soy broth (CM0129, Oxoid, Hampshire, UK) supplemented with 4 mg/L cefotaxime (Caisson C032-100G, Smithfield, UT, USA) and incubated at 37 °C for 24 h. Pre-enriched broth (100 µL) was streaked on MacConkey agar (Oxoid CM0007, Hampshire, UK) plates supplemented with 4 mg/L cefotaxime and incubated at 37 °C for 24 h [49 (link)]. Individual colonies selected from MacConkey agar were sub-cultured in tryptone soy broth to obtain a pure culture. A double disc synergy test (DDST) was used to confirm pure cultures as ESBL producers by following the protocol described in the M100 performance standards for antimicrobial susceptibility testing by the Clinical and Laboratory Standards Institute (CLSI) [50 ]. Briefly, the 0.5 McFarland standard equivalent test culture was swabbed onto the Mueller-Hinton (CM0337B, Oxoid, Hampshire, UK) agar plates and two antibiotic discs cefotaxime (CTX-30) and amoxicillin/clavulanic acid (AMC-30) were placed at a center-center distance of 20 mm and plates were incubated at 37 °C for 24 h. Expansion of the zone of inhibition of CTX-30 toward the AMC-30 disc was marked as a positive DDST. The species of ESBL bacteria were identified by using API-20E and 20NE strips (bioMérieux, Craponne, France) according to the manufacturer’s instructions.

Raw milk used for the definition of calibration curves experiments was collected aseptically from healthy cows. Conventional microbiological tests were performed according to NMC protocols [16 ], to confirm no bacterial growth. Briefly, a raw milk sample (10 μL) was plated on a Columbia agar plate and on MacConkey agar plate (CM0007, Oxoid, Hampshire, UK) and both were incubated at 37 °C for 48 h. The absence of growth on both plates was considered to be equivalent to the presence of no viable bacteria in the milk.
Each sample for biosensor testing had a 500 µL volume consisting of 2 µL of a suspension of functionalized NPs, 98 µL of PBST, and 400 µL of one of seven bacterial suspensions with pre-defined bacterial concentrations in PBS or sterile raw milk. The incubation of these samples was performed at RT for 3 h, under agitation.
All raw milk samples were submitted to a pre-treatment of 15 min at 60 °C in a dry bath incubator (Grant, model QBD2, Leicestershire, UK) and 15 min of continuous centrifugation in a vortex mixer (Labnet International Inc., Edison, NJ, USA) after adding bacteria and PBST. Only then, 2 µL of functionalized NPs suspension were added for final incubation step.

The presence of eight specific VAGs (papC, iucD, irp2, tsh, vat, astA, iss, and cva/cvi) was tested by a multiplex PCR [9 (link)]. The virulence-associated genes encode P-fimbria associated with adhesion [16 (link)], iron acquisition systems (iucD and irp2) [24 (link),25 (link)], and the temperature-sensitive autotransporter protein associated with high virulence [26 (link)]. The vat gene encodes a cytotoxic vacuolating autotransporter protein [27 (link)] the enteroaggregative heat-stable enterotoxin (astA) [28 (link)] a protein for increased serum survival (iss) [29 (link)] and the plasmid-borne genes cva/cvi which cause disruption of the membrane of sensitive cells [30 (link)]. E. coli isolated from MacConkey without any antimicrobials was used (Oxoid, CM0007). In brief, DNA was extracted by using a Maxwell^® RSC Instrument and Cultured Cells DNA kits (AS1620, Maxwell, Nacka, Sweden), as recommended by the manufacturer. PCR running conditions [9 (link)] were followed, except that agarose gels were run for 80 min at 75 V. A GeneRuler 100 bp plus DNA ladder (Thermo Scientific, SM0323, Vilnius, Lithuania) was used as a size marker on each gel.