Trimethoprim

Ascitic fluid samples of all patients were subjected to biochemical and cytological investigations at RMLH, New Delhi. The left over ascitic fluid was transferred to the AIIMS laboratory and processed for liquid culture and Xpert on the same day. Briefly, liquid culture for M. tuberculosis was performed from the sediment obtained after centrifugation of ascitic fluid in Middlebrook 7H9 broth enriched with oleic acid, albumin, dextrose, catalase (OADC) and an antibiotic cocktail; Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim and Azlocillin (PANTA) [Becton and Dickinson (BD), Franklin Lakes, New Jersey, United States] (Fig 1). The supernatant was stored at -80°C and used later for cfMTB-DNA extraction (Fig 1). Quantification of cfMTB-DNA was performed targeting the devR region of M. tuberculosis by qPCR.

Antimicrobial susceptibility testing was performed using the disk-diffusion method and the antibiotics ampicillin (AM), amoxicillin-clavulanic acid (AMC), cefotaxime (CTX), nalidixic acid (Na), ciprofloxacin (CIP), gentamicin (GM), kanamycin (K), streptomycin (S), sulfamethoxazole (Su), trimethoprim (TMP), tetracycline (T), and chloramphenicol (C) (Becton Dickinson, Heidelberg, Germany). Results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) performance standards (CLSI, 2016 ). For sulfamethoxazole, for which breakpoints are not listed separately from trimethoprim, an inhibition zone of ≤10 mm was interpreted as resistant. Isolates displaying resistance to three or more classes of antimicrobials (counting β-lactams as one class) were defined as multidrug-resistant (MDR). Synergistic effects between AMC and CTX were regarded as an indication of the presence of an ESBL producer (Kaur et al., 2013 (link)).

M. tuberculosis H37Rv and the clinical Beijing and LAM genotype families used in this study were grown in Middlebrook 7H9 (Becton-Dickenson, Franklin Lakes, NJ, USA) media supplemented with 0.2% glycerol (Sigma, Springfield, MO, USA), 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC, Becton-Dickenson) and 0.05% Tween 80 (Sigma, Missouri, USA) to mid-log phase (OD_600nm 0.5–0.8) at 37 °C without shaking. Five individual strains from the Beijing family and five from the LAM family were thawed from −80 °C freezer stocks, streaked on BBL™ Middlebrook 7H11 (Sigma, Springfield, MO, USA) media and incubated for 3 weeks at 37 °C. Single colonies were selected and sub-cultured for 7 days at 37 °C in 5 mL of Middlebrook 7H9 media supplemented with OADC and Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim and Azlocillin (PANTA, Becton-Dickenson, Franklin Lakes, NJ, USA) reconstituted in OADC, to ensure the strains were free of contaminants. Once the cultures reached mid-log phase (OD_600nm 0.5–0.8), the cells were used as a preculture to regrow the strains, again in Middlebrook 7H9 media supplemented with OADC and PANTA, to ensure any remaining contamination was killed. The mid-log liquid culture was then spread on BBL™ Middlebrook 7H11 plates supplemented with OADC to confirm that the Beijing and LAM strains were free of contamination.

To assess antibiotic resistance profiles of E. coli isolates, about every fifth of the enrichments prepared for the detection of Shiga toxin genes were selected (EE broth; 18–24 h, 37 °C). The enriched samples (one loopful: approx. 10 µl) were subcultured for 24 h at 37 °C on chromogenic RAPID’ E. coli 2 agar (Bio-Rad Laboratories, Reinach, Switzerland). One E. coli colony (violet to pink on RAPID’ E. coli 2 agar) from each of the 95 samples was selected and subjected to susceptibility testing against 12 antimicrobial agents by the disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) protocols and criteria [8 ]. The panel included ampicillin (AM, 10 µg), amoxicillin-clavulanic acid (AMC, 30 µg), chloramphenicol (C, 30 µg), cephalothin (CF, 30 µg), ciprofloxacin (CIP, 5 µg), cefotaxime (CTX, 30 µg), gentamicin (GM, 10 µg), kanamycin (K, 30 µg), nalidixic acid (NA, 30 µg), streptomycin (S, 10 µg), tetracycline (TE, 30 µg), and trimethoprim (TMP, 5 µg) (Becton–Dickinson).

Susceptibility testing was performed for the 192 isolates using the Kirby Bauer disk diffusion method and the antimicrobials nalidixic acid, ciprofloxacin, ampicillin, amoxicillin/clavulanic acid, cephalotin, cefotaxime, gentamicin, kanamycin, streptomycin, tetracycline, sulfamethoxazole, trimethoprim and chloramphenicol (Becton Dickinson, Heidelberg, Germany). Results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) [16 ]. Hereby, breakpoints for ciprofloxacin are ≥31 mm (susceptible), 21–30 mm (intermediate) and ≤20 mm (resistant).
Multidrug-resistance (MDR) was defined as resistance to conventional antityphoid drugs ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol (13).