Mcconkey agar

The swabs were placed in a sterile diluent (buffered peptone water; Oxoid) and then vortexed. From this initial dilution, two sequential 1/100 dilutions were prepared, and 100 microliters were plated onto Tryptone Soy Agar (Oxoid, TSA) and McConkey agar (Oxoid, McC). The agar plates were incubated at 37 °C for 24 and 48 h, after which the colony count per swab was determined.

After 8 h, the number of PAR5 cells present in the culture was estimated by making an appropriate decimal dilution of the bacteria in PBS and counting the bacterial colonies plated on McConkey agar (Oxoid) (Fig. 1a). The remaining volume of the PAR5 culture was centrifuged for 10 min at 500g. Supernatant was transferred to a fresh tube, filtered with a 0.22-µm Millipore membrane, and stored at 4 °C until needed. This solution was labeled as BCM-8 h (planktonic bacterial-conditioned medium). The pellet of PAR5-8 h bacterial cells was washed with 10 ml PBS (pH 7.4) and stored at 4 °C.

One μl of urine specimen was cultured on blood agar and McConkey agar (Oxoid). The presence of 10³ colony-forming units (CFUs) of a single uropathogen or more per milliliter was considered positive. Cultures were quantified from 10³, 10⁴ to >10⁵. Identification and antibiotic susceptibility testing was performed according to standard microbiological procedures.

Reference strains were from American Type Culture Collection (ATCC): S. aureus ATCC 25923 (Sa25923) and ATCC 6538 (Sa6538); Staphylococcus epidermidis ATCC 14990 (Se14990) and ATCC 12228 (Se12228); K. pneumoniae ATCC 700603 (Kp700603) and ATCC 13883 (Kp13883); P. aeruginosa ATCC 47085 (Pa47085), ATCC 9027 (Pa9027), and Pa14 (Pa14); Ralstonia mannitolilytica LMG 6866 (LMG6866) and BK931 (BK931). Bacteria were cultured aerobically at 37°C; culture media were blood agar, chocolate agar or McConkey agar (Oxoid, Hampshire, UK).

Milk samples were diluted at 0.1% peptone water (Oxoid Ltd, England) according to a 1-fold dilution pattern and incubated at 37°C for 24–48 h. Diluted samples were inoculated into McConkey agar (Oxoid, UK) and 5% sheep blood agar using the pour plate method and incubated at 37°C for 24–48 h. All suspected colonies were characterized and picked up for inoculation into buffered peptone water at 37°C for 24–48 h for streaking on specific culture media such as eosin methylene blue (Oxoid) for E. coli, Mannitol salt agar (Oxoid) for Staphylococcus spp., and modified Edwards medium (Oxoid) for Streptococcus spp. Isolates were identified by colony morphology, Gram staining, biochemical characterization, and molecular characterization using polymerase chain reaction with specific genes [20 (link)–22 ].

All patients included in this study presumably acquired OXA-48 producing Enterobacteriaceae during a large nosocomial outbreak in the Netherlands from 2009 to 2011 [13 ]. The bla_OXA-48 carrier status of colonised patients was determined every two months after hospital discharge, until six consecutive negative cultures with two months in-between cultures. There were no attempts for elimination of bla_OXA-48 colonisation. Screening swabs from rectum, throat, and possible infection sites were inoculated overnight in broth containing ertapenem (0.125 mg/L). The specimens were then tested by PCR for bla_OXA-48. Positive samples were inoculated on CRE (Oxoid Brilliance™ CRE Agar) and McConkey agar (Oxoid) and the presence of bla_OXA-48 was reconfirmed by PCR in every morphologically different isolate [13 ]. Data for the current study was collected until March 2013. Data collection was not designed for research purposes but for clinical care. Patient information was anonymised and de-identified prior to analysis. The Medical Ethics Review Committee of the Maasstad Ziekenhuis determined that this study was exempted from evaluation with regard to the Dutch Medical Research Involving Subjects Act. Details on microbiological methods, outbreak management, and impact of infection control measures can be found in Dautzenberg et al [13 ].