Sulfamethoxazole

The method used for the isolation of spores from environmental samples was that described in OIE Terrestrial Manual 2012 [15 ], with some modifications. For culturing and isolation of B. anthracis the TSMP medium was used, consisting in the semi-selective Columbia blood agar added with trimethoprim (16 mg/lt), sulfamethoxazole (80 mg/lt), methanol (5 ml/lt) and polymyxin (300,000 units/lt). Based on our experience, TSMP has the same efficacy of PLET in isolating B. anthracis (data not shown). Briefly, to each 7.5 gram aliquot of soil sample were added 22.5 ml of deionized sterile water. After 30 minutes of washing by vortexing, the suspension was incubated at 64°C for 20 min to eliminate any vegetative forms of soil contaminants [16 (link)].From each sample, 10 ml of supernatant were collected and dilutions of 1:10 and 1:100 were made using normal saline solution.
Subsequently, 10 plates of TMSP were seeded with the undiluted suspension (100 μl/plate), 10 plates with the 1:10 dilution and 10 plates with the 1:100 dilution. After 24 and 48 hours of incubation at 37°C, each plate was examined for the presence of suspect colonies of B. anthracis and of contaminants. All colonies were counted. B. anthracis colonies were identified by Gram staining, colony morphology and anthrax-specific PCRs [17 (link)].

At 1, 14, or 28 dpi with either PBS or 10⁸ cfu of Kan^R, all mice were treated with trimethoprim and sulfamethoxazole in the drinking water daily for 10 days at concentrations of 54 and 270 µg/ml, respectively [46] (link). During this time, longitudinal urinalysis was continued weekly to confirm clearance of bacteriuria. Four weeks after the initiation of antibiotic therapy, mice from each test group (previously infected or naïve) were challenged with either PBS or 10⁷ cfu of UTI89 Spc^R. Longitudinal urinalysis was then performed as for the primary infection (except now triplicate plating on LB, LB/Kan25 and McConkey agar with 50 µg/ml spectinomycin (McC/Spc50) to identify mice with persistent bacteriuria and the responsible strain. Mice were sacrificed 4 weeks after challenge and tissue titers determined as above, triplicate plating on LB, LB/Kan25, and LB/Spc50.

The national public health laboratories within the Food‐ and Waterborne Diseases and Zoonoses (FWD) network has agreed on a panel of priority antimicrobials and optional antimicrobials to test for and report to ECDC (ECDC, 2016 , 2021 ). Compared with earlier recommendations, a second beta‐lactam (ceftazidime) and a carbapenem (meropenem) were added. For 2021, all MS reported results on meropenem and all but four for ceftazidime. Three last‐line antimicrobials – azithromycin, colistin and tigecycline – are also included in the priority list. For colistin, however, the methodology is complicated due to chemical properties of the substance and a joint EUCAST and Clinical and Laboratory Standards Institute (CLSI) subcommittee confirmed that broth microdilution is so far the only valid method for colistin susceptibility testing (CLSI and ECDC, 2016 ). Disk diffusion does not work because of poor diffusion of the large colistin molecule in the agar and tested gradient strips also underestimate colistin MIC values, again most likely due to poor diffusion in the agar (Matuschek et al., 2018 (link)). Therefore, only countries performing broth microdilution (or those predicting resistance from WGS) should report on colistin resistance. Nine MSs reported AST results for azithromycin, tigecycline and colistin for 2021.
Due to the problems in detecting low‐level fluoroquinolone resistance in Salmonella spp. using disk diffusion, nalidixic acid was, for a long time, used as a marker for fluoroquinolone resistance. After the discovery that plasmid‐mediated fluoroquinolone resistance is often not detected using nalidixic acid, EUCAST studied alternative disks and concluded that pefloxacin was an excellent surrogate marker (except for isolates having the aac(6′)‐Ib‐cr gene as the only resistance determinant) (Skov and Monnet, 2016 (link)). Since 2014, EUCAST has recommend this agent for screening of low‐level fluoroquinolone resistance in Salmonella with disk diffusion (EUCAST, 2014 ) and, since June 2016, this is also reflected in the EU protocol. In 2021, all countries reporting measured values for disk diffusion tested with pefloxacin instead of ciprofloxacin, except for Latvia where this information is unknown. Eleven countries reported the combination drug co‐trimoxazole (trimethoprim–sulfamethoxazole) in addition to, or instead of, testing the substances separately, partly because this combination is used for clinical treatment and partly because no EUCAST interpretive criterion exists for sulfamethoxazole for Salmonella.