Mic test strip method

For the samples sent to the NRL, the serogroup was identified or confirmed by slide agglutination with commercial antisera (Thermo Scientific, Waltham, Massachusetts, US) or by multiplex PCR [15 (link)]. For the bacterial isolates, susceptibility to cefotaxime, ceftriaxone, ciprofloxacin, penicillin G and rifampicin was determined by the minimum inhibitory concentration (MIC) test strip method (Liofilchem, Roseto degli Abruzzi, Italy) on Mueller-Hinton agar (Thermo Scientific, Waltham, Massachusetts, US) supplemented with 5% of sheep blood. The breakpoints were those recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [16 ]. Chromosomal DNA was extracted using the QiAmp mini kit (Qiagen, Hilden, Germany) from an overnight culture or directly from the clinical sample, blood or cerebrospinal fluid (CSF). Multilocus sequence typing (MLST), PorA and FetA typing, MenB vaccine antigen variants and penA gene were identified using the PubMLST.org database (http://pubmlst.org/neisseria/). The genotypic formula comprised capsular group: porA (P1).VR1,VR2: fetA VR: ST(cc). The MenW/cc11 isolates were characterised for the allelic profile of six antigen-encoding genes (porA, porB, fetA, nadA, nhba and fHbp) suggested by Mustapha et al. as typical of the main MenW/cc11 sublineages [4 (link)].

MICs for beta-lactam antibiotics, including amoxicillin, cefotaxime, cefoxitin, meropenem, and penicillin G, were determined by the MIC test strip method (Liofilchem, Waltham, MA) according to the manufacturer’s instructions. Strains were grown overnight on tryptic soy agar with 5% sheep blood (Becton, Dickinson), and fresh colonies were collected with the BBL prompt inoculation system (Becton, Dickinson) and plated onto Mueller-Hinton agar (Remel Microbiology Products, Lenexa, KS) with the antibiotic test strips. MICs (in milligrams per liter) were read after 24 h of incubation independently by two technologists.