trimethoprim obtained from clinical equine Staphylococcus aureus isolates using different methods

Journal Pre-proof Heterogeneity of antimicrobial susceptibility testing results for sulfamethoxazole/trimethoprim obtained from clinical equine Staphylococcus aureus isolates using different methods Anissa D. Scholtzek, Dennis Hanke, Inga Eichhorn, Birgit Walther, Antina Lubke-Becker, ¨ Engeline van Duijkeren, Robin K¨ock, Stefan Schwarz, Andrea T. Feßler

PII:

S0378-1135(19)31396-3

DOI:

https://doi.org/10.1016/j.vetmic.2020.108600

Reference:

VETMIC 108600

To appear in:

Veterinary Microbiology

Received Date:

29 November 2019

Revised Date:

21 January 2020

Accepted Date:

30 January 2020

Please cite this article as: Scholtzek AD, Hanke D, Eichhorn I, Walther B, Lubke-Becker ¨ A, van Duijkeren E, K¨ock R, Schwarz S, Feßler AT, Heterogeneity of antimicrobial susceptibility testing results for sulfamethoxazole/trimethoprim obtained from clinical equine Staphylococcus aureus isolates using different methods, Veterinary Microbiology (2020), doi: https://doi.org/10.1016/j.vetmic.2020.108600

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Heterogeneity of antimicrobial susceptibility testing results for sulfamethoxazole/trimethoprim obtained from clinical equine Staphylococcus aureus isolates using different methods

Short title:

SXT antimicrobial susceptibility testing

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Anissa D. Scholtzeka, Dennis Hankea, Inga Eichhorna, Birgit Waltherb, Antina Lübke-Beckera, Engeline van Duijkerenc, Robin Köckd,e, Stefan Schwarza, Andrea

Institute of Microbiology and Epizootics, Centre for Infection Medicine, Department of

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T. Feßlera*

Veterinary Medicine, Freie Universität Berlin, Berlin, Germany Advanced Light and Electron Microscopy (ZBS-4), Robert Koch Institute, Berlin, Germany

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National Institute for Public Health and the Environment (RIVM), Centre for Infectious

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Disease Control (CIb), Bilthoven, Netherlands

Institute of Medical Microbiology, University Hospital Münster, Münster, Germany

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Institute of Hygiene, DRK Kliniken Berlin, Berlin, Germany

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* Corresponding author: Tel.: +49-30-838-63074. Fax: +49-30-838-451851. E-mail address: [email protected]

Highlights: 

Five AST methods yielded various results for SXT among equine S. aureus isolates.

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All isolates harbored a trimethoprim resistance gene (dfrG or dfrS1).

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Three isolates had a folP mutation conferring sulfonamide resistance.

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All isolates were classified as hetero-resistant to SXT and SUL.

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Automated AST methods classified the isolates more easily as resistant.

Abstract

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Based on antimicrobial susceptibility testing (AST), correct classifications as susceptible, intermediate or resistant are challenging for some antimicrobial agent-bacterial species combinations. In this study, we investigated 19 equine Staphylococcus aureus isolates for their

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susceptibility to the combination sulfamethoxazole/trimethoprim (SXT) by using broth microdilution (BMD), agar disk diffusion (DD) and automated test systems. To elucidate the

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presence of the corresponding genetic resistance properties among the isolates, whole genome sequence analysis was performed and the genomes were screened for trimethoprim (TMP)

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resistance genes and mutations in the deduced FolP amino acid (aa) sequences, known to confer sulfonamide (SUL) resistance. To check for hetero-resistance, zone diameters in DD were

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screened after 18 and 42 h of incubation. All 19 isolates harboured one of the TMP resistance genes dfrG or dfrS1. Three isolates had the aa exchange in their FolP aa sequence (F17L), which

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has previously been described to result in SUL resistance. These isolates were classified as SXT-resistant by all methods. The remaining 16 isolates were classified as SXT-susceptible or

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-intermediate (BMD and/or DD) or SXT-resistant (mainly automated test systems). These isolates had no relevant aa variations in their FolP aa sequences. All 19 isolates showed slight growth within their SXT inhibition zone by DD, pointing towards hetero-resistance. Overall, automated test systems classified isolates lacking genetic resistance determinants more frequently as SXT-resistant than DD and BMD. Therefore, further studies are needed to define a reliable method for SXT susceptibility testing.

Key words:

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antimicrobial resistance, diagnostics, hetero-resistance, VITEK

Introduction Antimicrobial-resistant bacteria are a threat to both, human and veterinary medicine since

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treatment options might be limited (Levy and Marshall, 2004). Thus, identifying the most appropriate agent via antimicrobial susceptibility testing (AST) plays an important role to foster the prudent use of antibiotics (Morency-Potvin et al., 2017). The Clinical Laboratory Standard

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Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) offer a range of AST methods that provide either quantitative (broth microdilution

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methods, BMD) or qualitative (agar disk diffusion, DD) results (CLSI, 2018a, EUCAST, 2019). The quantitative methods yield minimal inhibitory concentrations (MICs), which can be

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categorized according to CLSI document VET08 (CLSI, 2018b) as susceptible (S), intermediate (I) (if available) or resistant (R). In all three cases, the classifications are based on approved

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clinical breakpoints. In general, the variation of a MIC value by ± one dilution step in a twofold dilution series is considered as an acceptable error that is inherent to the test system (CLSI,

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2018a). Some combinations of antimicrobial agent and bacterial species show trailing

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endpoints. In such cases, a growth reduction of ≥80% is considered as the MIC. Trailing endpoints in growth are commonly seen when staphylococci are tested for their susceptibility to the combination of sulfamethoxazole and trimethoprim (SXT) (CLSI, 2018a,b). The phenomenon of hetero-resistance describes the presence of subpopulations of an isolate showing different susceptibilities to an antimicrobial (El-Halfawy and Valvano, 2015). As hetero-resistance has been described in connection with SXT resistance, it considered when interpreting SXT susceptibility data.

needs to be

Besides DD or BMD (which are often performed manually), automated test systems are available that promise standardized quick results (Li et al., 2017). The automated test systems’ repeatedly detect turbidity and/or oxidation-reduction indicators in early states of bacterial growth in the presence of a certain antimicrobial agent, calculate the expected growth rate and therefrom derive the most probable MIC, which then can be categorized by the corresponding expert system software. Staphylococcus aureus is an important veterinary pathogen, which causes up to 22.8% of

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all equine wound infections in Germany. As 41.3% of these S. aureus isolates are methicillinresistant (MRSA) (Vincze et al., 2014), the treatment options are limited with respect to the licensed and available antimicrobials e.g. in Germany (https://www.vetidata.de). Based on AST

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data obtained (Cuny et al., 2016, Vincze et al., 2014), SXT may be a promising therapeutic option. Hence, the accuracy of phenotypic AST for SXT becomes increasingly important. In

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the present study, we comparatively investigated 19 equine S. aureus isolates for their

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susceptibility to SXT and the single compounds, sulfisoxazole, a sulfonamide (SUL), and trimethoprim (TMP), by various AST methods. In addition, whole genome sequences of the 19 isolates were searched for genes and mutations conferring resistance to the respective

Materials and methods

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2.

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antimicrobial agents.

2.1. Bacterial strains A total of 19 S. aureus isolates identified during routine diagnostics between 07/2015 and

06/2017 at the Institute of Microbiology and Epizootics of the Department of Veterinary Medicine, Freie University Berlin were included in this study. The isolates were initially selected because of their reduced susceptibility to oxacillin according to VITEK® 2 results

(bioMérieux, Nürtingen, Germany) (Scholtzek et al., 2019). The isolates originated from samples of a wound infection, a respiratory disease and 17 surgical site infections of altogether 17 individual horses treated at the Equine Clinic: Surgery and Radiology, Freie Universität Berlin. Further background information of the 19 equine isolates as well as their genomic relationships have been described previously (Scholtzek et al., 2019).

2.2. Antimicrobial susceptibility testing

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AST for SXT was carried out by VITEK® 2 and BD PhoenixTM (BD, Heidelberg, Germany) according to the manufacturers’ instructions. Additional AST was carried out by BMD according to CLSI standards (CLSI, 2018a), using sensititre™ microtitre plates (BMD1,

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Thermo Fisher, Wesel, Germany). These microtitre plates corresponded to those that were used in the German National Monitoring program GERM-Vet and contained SXT in the

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concentrations 0.015/0.29 – 32/608 mg/L in a two-fold dilution series. Moreover, SXT was also tested by BMD using the Merlin “micronaut-S anaerob” microtitre plates (BMD2, Merlin,

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Berlin, Germany). The micronaut microtitre plates contained SXT in the concentrations 0.0625/1.1875 – 8/152 mg/L, in two-fold dilution series, and were used since SXT was available

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in an appropriate test range on this panel. To test the susceptibility of the 19 isolates to sulfonamides and trimethoprim separately, additional susceptibility testing was performed for

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sulfisoxazole (Acros organics, Darmstadt, Germany) and trimethoprim (Sigma®, Darmstadt, Germany). For this, microtitre plates were set up according to CLSI standards (CLSI, 2018a)

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with a twofold dilution series for the agents. The tested ranges were 1 – 512 mg/L for sulfisoxazole (SUL) and 0.125 – 64 mg/L for trimethoprim (TMP) (BMD3). In all BMD approaches, S. aureus ATCC® 29213 served as quality control strain (CLSI, 2018b). Agar disk diffusion (DD) was performed for SXT (23.75µg + 1.25µg), TMP (5µg) (both BBLTM Sensi Discs, BD, Heidelberg, Germany) and SUL (300µg) (Oxoid, Munich, Germany) in accordance with the CLSI standards (CLSI, 2018b, 2019). For DD, S. aureus ATCC® 25923

served as quality control strain. All DD approaches were carried out in duplicates. Margins of zone diameters were inspected carefully after 18 h and after 42 h of incubation at 35°C ± 2°C to check for slight growth, which indicates hetero-resistance. To rule out slight growth because of high thymine-levels, Enterococcus faecalis ATCC® 29212 was tested side-by-side with the 19 test isolates following CLSI recommendations (CLSI, 2018a). Moreover, the universal recipient strain S. aureus RN4220, which is susceptible to virtually all antimicrobial agents, was investigated for its susceptibility to SXT, SUL and TMP

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by DD as well as for its susceptibility to SUL by BMD3. All results obtained by BMD and DD methods were evaluated using CLSI clinical breakpoints (CLSI 2018b, 2019).

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2.3. Whole genome sequence analysis

The whole genome sequences (WGSs) of the 19 equine S. aureus isolates have been

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determined previously (Scholtzek et al., 2019) and are available under BioProject accession no.

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PRJNA561466. Multilocus sequence types, derived from these whole genome sequences showed that the isolates were assigned to two different sequence types (STs), namely ST1 (n = 3) and ST1660 (n = 16) (Scholtzek et al., 2019). Further investigations for the presence of

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trimethoprim resistance genes within these sequences were performed by using ResFinder of the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/) with a

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threshold of 80 % and minimum length of 60 % or specific search for staphylococcal dfr genes

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in the whole genome sequences. In addition, the dihydropteroate synthase (DHPS) gene folP was investigated for mutations resulting in amino acid (aa) exchanges using Geneious v11.1.4. For this, we chose the well characterized, SXT-susceptible S. aureus strain RN4220 (GenBank accession no. NZ_AFGU01000103.1 with the protein sequence WP_000167924.1) for comparison.

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Statistical analysis

Non-parametric Mann-Whitney-U-test was performed using IBM®SPSS® Statistics Version 25, to compare the zone diameters for SUL of the isolates.

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Results and discussion

3.1. Antimicrobial susceptibility testing AST of the 19 S. aureus strains revealed different results regarding SXT (Table 1). Three isolates, which all belonged to ST1 and were closely related by cgMLST patterns (Scholtzek et

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al., 2019), were classified as SXT-resistant by all methods. The remaining16 isolates belonged to ST1660, were also closely related in cgMLST analysis (Scholtzek et al., 2019), but differed in their SXT results: BMD1, using the GERM-Vet microtitre plates, classified all 16 isolates as

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susceptible, while BMD2, using the micronaut microtitre plates, classified all but one isolate (IMT37083) as resistant. The first automated test system, VITEK® 2, classified two isolates

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(IMT37083 and IMT41452) as susceptible and the remaining 14 isolates as resistant, while BD

eleven as intermediate.

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PhoenixTM identified all isolates as resistant. DD classified five isolates as susceptible and

The three ST1 isolates that were classified as SXT-resistant by all methods were also

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classified as SUL-resistant by the single-compound testing via BMD3, using the self-made microtitre plates, but as SUL-susceptible by DD. The remaining 16 isolates, which belonged to

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ST1660, were tested as SUL-susceptible by BMD3 and DD. Interestingly, the zone diameters

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of the three ST1 isolates were significantly smaller (18 – 20 mm) than those of the remaining 16 ST1660 isolates (26 – 30 mm, Mann-Whitney-U-Test: U = 0.0; p = 0.005). All isolates were classified as resistant to TMP by both, BMD3 and DD (Table 1). The

quality control strains ATCC® 29213 (BMD) and ATCC® 25923 (DD) were always within the acceptable ranges (CLSI, 2018b). Method-dependent differences for SXT in staphylococci have already been described in previous studies (Coombs et al., 2019; Griffith et al., 2009). Reading of the results in BMD for

S. aureus and SXT poses some difficulties, since S. aureus commonly shows trailing endpoints. Thus, a growth reduction of ≥80% is considered as the MIC (CLSI, 2018a,b). As a consequence, determination of the endpoint can be subjective, even when well-trained personnel reads the microtitre plates. Similar difficulties occur in the interpretation of zone diameters in DD, where an appearance of ≤20% growth shall be ignored (CLSI, 2018b). Determining zone diameters in DD can be challenging, not only due to growth variation. The inhibition zone might not always appear exactly circular and even slightly variable thickness of

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the Mueller-Hinton agar can cause differences in the distribution of the antimicrobial agent, resulting in deviating zone diameters. To rule out slight growth due to excessive thymidine or thymine levels in the test medium, which may reverse the effects of SUL and TMP, E. faecalis

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ATCC® 29212 was tested as control. In our agar plates, the zone diameters for E. faecalis ATCC® 29212 were ≥ 20mm for SXT and the zones were clear and distinct, without any slight

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growth within the respective inhibition zones. This observation indicates acceptable thymidine

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and thymine levels (CLSI, 2018b). In the 19 tested isolates, there was slight growth in all inhibition zones of SXT and also in those of SUL, although to a lesser extent (Figure 1). In

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2017, hetero-resistance to SXT was described for the first time, in African S. aureus isolates from humans (Coelho et al., 2017). Hetero-resistance is commonly defined as a phenomenon, where different subpopulations of an isolate show different susceptibilities to an antimicrobial

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agent (El-Halfawy and Valvano, 2015). Coelho et al. (2017) tested S. aureus isolates regarding

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SXT, TMP and sulfamethoxazole via DD. They classified the isolates as fully susceptible when no colonies were within the inhibition zone and as hetero-resistant, when distinct colonies or slight growth occurred within the zone diameter. Using this definition, all 19 isolates in this study would be considered hetero-resistant. This might be a possible explanation for differing results, since there is no standardized method that detects hetero-resistance (El-Halfawy and Valvano, 2015). Interestingly, also S. aureus RN4220 had a zone of slight growth next to a clear inhibition zone, both within diameters classified as susceptible (28/34 mm for SXT, 26/34 mm

for SUL and 20/32 mm for TMP, Supplemental Figure 1). Thus, hetero-resistance cannot only occur when one subpopulation of an isolate is resistant and the other susceptible, but also when different subpopulations of an isolate exhibit different susceptibilities as seen for S. aureus RN4220 (El-Halfawy and Valvano, 2015; Falagas et al., 2008). To investigate the zone diameters further, we incubated the plates for another 24 h after initial reading of results after 18 h. There was no change in the zone diameter, but in the growth within the inhibition zones. In all isolates, growth in the inhibition zones of SUL and SXT intensified. In the 16 ST1660

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isolates not resistant to SXT, the growth within the SXT inhibition zone increased more distinctly than in the SUL inhibition zone (Figure 1). This finding points towards an overall greater SUL susceptibility of the ST1660 isolates compared to the ST1 isolates, which was also

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reflected by the significantly (p = 0.005) smaller SUL-associated zones of growth inhibition of the ST1 isolates.

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There are several studies implying that also in automated test systems, errors in the

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classification of clinical isolates regarding SXT can occur (Carroll et al., 2006; Coombs et al., 2019; Fahr et al., 2003; Huh et al., 2018). This effect might be associated with the phenomenon of the trailing endpoints, which are potentially not detected by the automated test systems. For

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SXT, the number of major errors (MEs, classified as resistant when reference is susceptible) seems to be higher than those of very major errors (VMEs, classified as susceptible when

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reference is resistant) in two studies (Carroll et al., 2006; Coombs et al., 2019). The percentages

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of MEs for SXT ranged from 1.5% up to 60% of tested S. aureus isolates (Carroll et al., 2006; Coombs et al., 2019). However, higher numbers of VMEs of about 6% to 9% (compared to MEs) were observed among the SXT susceptibility testing of staphylococci in two other studies (Fahr et al., 2003; Huh et al., 2018). MEs result in an unnecessary limitation of therapeutic options, as described by Coombs et al. (2019). Nonetheless, VMEs pose a greater threat to the population, since the application of the misclassified antimicrobial agent may result in treatment failure.

3.2. Genomic analyses All isolates harboured a trimethoprim resistance gene, either dfrG (ST1) or dfrS1 (ST1660), the latter also known as dfrA. This observation is in accordance with the results of phenotypic susceptibility testing, since both methods, BMD3 and DD, identified all isolates as resistant (Table 1). The trimethoprim-insensitive dihydrofolate reductase DfrS1 differs only by

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three aa variations (V31I, G43A, F98Y) from the trimethoprim-sensitive dihydrofolate reductase DfrC, which occurs regularly in Staphylococcus epidermidis (Dale et al., 1995). As the corresponding genes, the trimethoprim resistance gene dfrS1 and the regular dihydrofolate

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reductase gene dfrC, which does not represent a resistance gene, are most closely related, dfrS1 is not identifiable via ResFinder 3.2 and the gene was also not listed in the database (date last

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accessed 11.11.2019). Instead a detailed search of the WGSs of the respective isolates is necessary to identify this gene.

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To date, a gene conferring sulfonamide resistance in staphylococci has not yet been identified. Three mechanisms are suspected to mediate resistance to sulfonamides in

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staphylococci, including either (i) mutations or (ii) overexpression of the folP gene, encoding the targeted dihydropteroate synthase (DHPS), and (iii) an increased production of p-

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aminobenzoic acid (Schwarz et al., 2018). Since testing for susceptibility to SXT and SUL

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showed divergent results here, we decided to look for the genetic basis of SUL resistance. Therefore, we compared the WGSs of all isolates with the respective sequences of the well characterized, SXT-, SUL- and TMP-susceptible S. aureus RN4220. The analysis of the deduced amino acid (aa) sequence of the folP gene, encoding for a SUL-susceptible DHPS, showed that within the ST1660 isolates, all isolates had eight aa exchanges in their deduced FolP sequences (I30V, N31T, I37M, V58I, M64L, M101I, I117V, I126V; Figure 2). To our knowledge, none of these aa exchanges have been described to be associated with sulfonamide

resistance. The ST1 isolates only had two aa exchanges, namely F17L and A184V. F17L is one of three primary aa exchanges responsible for sulfonamide resistance in staphylococci (Griffith et al., 2018). This finding is in accordance with the results of BMD3. In the DD, the zone diameters regarding SUL were significantly smaller for the ST1 isolates than for the ST1660 isolates. According to CLSI (CLSI, 2019), a zone diameter of ≥17mm is classified as susceptible. The ST1 isolates had diameters of 18 – 20mm, which are very close to being classified as intermediate (13 – 16mm). Interestingly, Griffith et al. (2018) also discovered that

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primary mutations leading to SUL resistance also increased the susceptibility to trimethoprim. In the ST1 isolates, this effect could not be noted due to the presence of the trimethoprim

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resistance gene dfrG.

Implications for diagnostics

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Sulfonamides and trimethoprim alone each have a bacteriostatic effect. Both inhibit subsequent steps in the folate biosynthesis of the bacterial cell (Sköld, 2001). Regarding SXT,

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only the isolates resistant to both single agents (ST1) were classified consistently as SXTresistant. Overall, automated systems seemed to predict MICs which usually classified the

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isolates as SXT-resistant more frequently than BMD. Most automated systems detect growth repeatedly in the initial growth phase and extrapolate expected growth rates from those

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measurements. As mentioned above, the presence of trailing endpoints might lead to problems regarding the classification of the results. Since bacteriostatic agents only inhibit growth, but

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do not stop it completely, it seems possible, that the initial growth can point towards resistance, whereas the result after 18 h would be considered as susceptible. MEs and VMEs have been described occurring in automated test systems (Fahr et al., 2003;Carroll et al., 2006; Huh et al., 2018 Coombs et al., 2019). These findings indicate, that the difficulties regarding a reliable SXT susceptibility testing are not only present in the collection described in our study.

Therefore, further research on this topic is needed to evaluate the SXT susceptibility testing

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methods in correlation with the genetic resistance properties.

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Conclusions SXT is commonly used to treat staphylococcal infections in veterinary and human

medicine (Bowen et al., 2017; De Briyne et al., 2014). Therefore, a correct classification of clinical isolates as susceptible, intermediate (when available) or resistant is of major importance in the light of a prudent use of antimicrobial agents. Especially for SXT, using different AST methods can yield different results, making the choice of treatment more difficult. Previous studies revealed that when automated test systems are used, major errors occur in varying

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proportions (Carroll et al., 2006; Coombs et al., 2019; Fahr et al., 2003; Huh et al., 2018). PCR can help identifying trimethoprim resistance genes in staphylococci. However, since there is no known gene to confer sulfonamide resistance in staphylococci yet, a PCR approach does not fit

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regular needs in routine diagnostics. Therefore, further research is needed to define a reliable

Conflict of interest

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None to declare.

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method for SXT susceptibility testing.

Funding

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This research was funded by has been funded by the Federal Ministry of Education and Research (BMBF) under project numbers 01KI1727D and 01KI1727F as part of the Research Network Zoonotic Infectious Diseases.

Acknowledgements

The authors thank Andrea Schmidt, Angela Schellin, and Julian Brombach for excellent

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technical assistance and Beneditta Suwono for statistical advice.

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Figure 1. Agar disk diffusion results after 18 hours (A) and 42 hours (B), for a representative ST1 isolate (1) and a representative ST1660 isolate (2), showing a higher increase of

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growth within the inhibition zones of SXT over SUL

Figure 2. Comparison of the aa sequence of FolP. IMT39129 represents all ST1 isolates and

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ST39637 represents all ST1660 isolates. S. aureus RN4220 served as a reference (GenBank accession no. NZ_AFGU01000103.1 with the protein sequence WP_000167924.1). The dots indicate aa identity, while letters indicate aa exchanges.

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Table 1: Susceptibility testing results archived using different methods

®

SXT BMD1 (in mg/L)

BMD2 (in mg/L)

DD (in mm)

ST

VITEK 2 (in mg/L)

IMT39129 IMT39173 IMT39701

1 1 1

16/304 16/304 16/304

≥4/76 ≥4/76 ≥4/76

4/76 4/76 4/76

≥16/304 8/152 4/76

6 6 6

512 512 512

IMT39637 IMT37083 IMT37341 IMT37410 IMT37728 IMT39233 IMT39841 IMT40768 IMT40820 IMT40952 IMT41452 IMT41468 IMT43228 IMT43231 IMT43240 IMT41899

1660 1660 1660 1660 1660 1660 1660 1660 1660 1660 1660 1660 1660 1660 1660 1660

8/152 0.5/9.5 16/304 8/152 8/152 16/304 16/304 16/304 8/152 16/304 2/38 16/304 16/304 16/304 16/304 16/304

≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76 ≥4/76

1/19 0.12/2.38 0.5/9.5 0.5/9.5 0.5/9.5 1/19 1/19 0.5/9.5 1/19 0.5/9.5 0.5/9.5 1/19 1/19 1/19 1/19 0.5/9.5

8/152 0.25/4.75 8/152 8/152 4/76 4/76 4/76 8/152 4/76 4/76 8/152 4/76 8/152 ≥16/304 8/152 8/152

14 24 14 16 16 14 14 16 14 14 14 16 14 14 14 12

32 64 32 32 64 32 32 32 256 128 64 64 64 32 64 64

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Isolate-ID

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BD Phoenix (in mg/L)

TM

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ST = sequence type, SXT = sulfamethoxazole/trimethoprim, SUL = sulfisoxazole, TMP = trimethoprim, BMD = broth microdilution, DD = agar disk diffusion; Bold numbers indicate classification as resistant, italic numbers indicate classification as intermediate, for SXT the concentrations are given for the two compounds trimethoprim and sulfamethoxazole; BMD1 = sensititre™ microtitre plates (Thermo Fisher, Wesel, Germany); BMD2 = “micronaut-S anaerob” microtitre plates (Merlin, Berlin, Germany).; BMD3 = selfmade panels. The results were interpreted using to the CLSI breakpoints (CLSI 2018b, 2019).

BMD (in mg