Cephalexin

The B.p strain 2016-CY-41 used in this study was recently isolated from a patient in China and was obtained from the National Institutes for Food and Drug Control (Beijing, China). The polymorphisms in the PT promoter (ptxP), PT subunit 1 (ptxA), pertactin (prn), fimbrial (fim)2 and fim3 were assessed by DNA sequencing. The genotype of 2016-CY-41 was ptxP1/ptxA1/prn1/fim2–1/fim3–1. For B.p infection experiments, bacteria were grown on Bordet-Gengou agar (B-G) plates (BG, Hopebio, CHN) containing 20% defibrinated sheep blood (Nanjinglezhen, CHN) for 48 to 72 h at 37 °C. Colonies from fresh B-G plates were resuspended in isotonic saline, diluted to a concentration of 10¹¹ CFU/mL using a turbidimetric method, and used within 2 h of preparation. For culture of nasopharyngeal wash (NPW) bacteria, Regan-Lowe plates prepared from Regan-Lowe charcoal agar base with 10% defibrinated sheep blood and 40 μg/mL cephalexin (Oxoid, US) were used.

We characterized five B. pertussis clinical isolates S1, S2, S3, S4 and S5 recovered from clinical samples collected from patients hospitalized or visited with clinical features such as pertussis visited or admitted in a pediatric hospital in the Pune region within India [65 (link)]. Nasopharyngeal swabs samples were received in Regan-Lowe transport medium (RTM) (Copan) under ambient conditions [26 (link)]. Detailed information of all clinical samples is included in Table 1. The swabs were streaked on Bordet-Gengou Agar (BGA, Difco) plates with defibrinated horse blood 15% (v/v) (in house) with cephalexin (40 µg/mL) (Oxoid, UK) to inhibit common Gram-positive nasopharyngeal flora in the sample [66 (link)]. The plates were incubated at 35 °C for 3–5 days. Suspected colonies were sub-cultured from mixed microbial growth observed on the same medium for 24–48 h. Suggestive B. pertussis colonies were confirmed using phenotypic and molecular identification techniques [26 (link),27 (link),65 (link)].

A modified form of the Kirby Bauer method was employed to evaluate the antibiotic susceptibility of bacteria isolated from urine specimens [20 (link),21 ]. The antibiotics tested included co-trimoxazole, ceftizoxime, chloramphenicol, cephalexin, tetracycline, ciprofloxacin, amikacin, ampicillin, sparfloxacin, ofloxacin, norfloxacin, and levofloxacin (Oxoid Ltd., Basingstoke, UK). The antibiotic susceptibility testing procedure that was employed is briefly described as follows. Pure culture of the test organism was emulsified in peptone water until the turbidity was comparable with that of 0.5% McFarland’s standard solution. A loopful of the suspension of the test organism was transferred onto a Mueller–Hinton agar plate, and a sterile cotton swab was then used to streak the entire surface of the agar. Sterile forceps were used to apply the antibiotic discs to the surface of the agar plate and incubated at 37 °C for 18–24 h. Zone diameters formed around the antibiotic discs were measured and classified as sensitive or resistant based on the Clinical Laboratory Standard Institute (CLSI) break point system [21 ].

A modified Kirby Bauer disk diffusion method was used to test each isolate for in vitro antimicrobial susceptibility based on the Clinical and Laboratory Standards Institute criteria [24 ]. In brief, standard inoculum adjusted to 0.5 McFarland standard turbidity was uniformly distributed over the surface of Mueller Hinton agar (Oxoid, Ltd., UK). Antimicrobial disks, including (Oxoid, Ltd., UK) cephalexin (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), erythromycin (15 μg), gentamycin (10 μg), penicillin (10 μg), tetracycline (30 μg), and cefoxitin (10 μg), were applied on Mueller Hinton agar plates using automatic disk dispenser. Following overnight incubation at 37°C, the zone of inhibition was measured and interpreted as sensitive, intermediate sensitive or resistant per the standard criteria [24 ]. Methicillin resistance was confirmed for all cefoxitin-resistant S. aureus using a PBP2a-agglutination test as described by Atay and Gulay [25 ]. An isolate was considered multidrug resistant (MDR) if it is resistant to at least one agent in three or more antimicrobials of structurally different categories [26 (link)].

Antimicrobial susceptibility testing was conducted on paired samples using the agar disc diffusion method following the recommendations of the Clinical and Laboratory Standards Institute (CLSI) [20 ].
To conduct this test, a standardized amount of S. aureus was inoculated (standard 0.5 by Mc Farland) in Mueller-Hinton agar medium (Scharlau Chemie S.A.) and then the sensi-disks were placed: oxacillin (OXA, 1 μg), cefoxitin (FOX, 30 μg), cephalexin (LEX, 30 μg), gentamicin (GEN, 10 μg), ciprofloxacin (CIP, 5 μg), erythromycin (ERY, 15 μg), clindamycin (CLI, 2 μg), trimethoprim/sulfamethoxazole (SXT 1.25/23.75 μg), tetracycline (TET, 30 μg), chloramphenicol (CHL, 30 μg), vancomycin (VAN, 30 μg), imipenem (IPM, 10 μg), and penicillin (PEN, 10 U) (Oxoid). To verify the action of the sensi-disks, during susceptibility analysis the ATCC 25923 strain of S. aureus was used as control.
Determination of MRSA isolates was performed according to the results obtained in the evaluation of oxacillin and cefoxitin and by detecting the mecA gene [21 (link), 22 (link)].

Escherichia coli isolates were examined for resistance to the following 17 antibiotics using the Kirby-Bauer disk diffusion method according to Clinical Laboratory Standards Institute guidelines [19 ]: amoxicillin/clavulanic acid (AMC, 20 μg/10 μg), ampicillin (AMP, 10 μg), cephalexin (30 μg), cefoxitin (CEF, 30 μg), ceftiofur (30 μg), aztreonam (30 μg), imipenem (10 μg), meropenem (10 μg), ciprofloxacin (CIP, 5 μg), norfloxacin (10 μg), nalidixic acid (30 μg), chloramphenicol (CHL, 30 μg), trimethoprim-sulfamethoxazole (SXT, 1.25 μg/23.75 μg), gentamicin (GEN, 10 μg), tobramycin (TOB, 30 μg), amikacin (AMK, 30 μg), and tetracycline (TET, 15 μg) (Oxoid). Escherichia coli isolates resistant to at least one antibiotic in three or more classes were considered multidrug-resistant [20 ].