Abstract
Objectives
Recently, reports on antimicrobial-resistant Bacteroides and Prevotella isolates have increased in the Netherlands. This urged the need for a surveillance study on the antimicrobial susceptibility profile of Bacteroides, Phocaeicola, Parabacteroides and Prevotella isolates consecutively isolated from human clinical specimens at eight different Dutch laboratories.
Methods
Each laboratory collected 20–25 Bacteroides (including Phocaeicola and Parabacteroides) and 10–15 Prevotella isolates for 3 months. At the national reference laboratory, the MICs of amoxicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem, metronidazole, clindamycin, tetracycline and moxifloxacin were determined using agar dilution. Isolates with a high MIC of metronidazole or a carbapenem, or harbouring cfiA, were subjected to WGS.
Results
Bacteroides thetaiotaomicron/faecis isolates had the highest MIC90 values, whereas Bacteroides fragilis had the lowest MIC90 values for amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem and moxifloxacin. The antimicrobial profiles of the different Prevotella species were similar, except for amoxicillin, for which the MIC50 ranged from 0.125 to 16 mg/L for Prevotella bivia and Prevotella buccae, respectively. Three isolates with high metronidazole MICs were sequenced, of which one Bacteroides thetaiotaomicron isolate harboured a plasmid-located nimE gene and a Prevotella melaninogenica isolate harboured a nimA gene chromosomally.
Five Bacteroides isolates harboured a cfiA gene and three had an IS element upstream, resulting in high MICs of carbapenems. The other two isolates harboured no IS element upstream of the cfiA gene and had low MICs of carbapenems.
Conclusions
Variations in resistance between species were observed. To combat emerging resistance in anaerobes, monitoring resistance and conducting surveillance are essential.
Introduction
Anaerobic bacteria are a major part of the human microbiota and important pathogens in human infections.1 They are believed to be susceptible to common antimicrobial agents used to treat polymicrobial infections with anaerobic bacteria, such as metronidazole and carbapenems. However, antimicrobial resistance among anaerobes is steadily increasing.2 Bacteroides and Prevotella isolates are important anaerobic clinical pathogens that can be cultured from a wide range of clinical samples.3 Resistance among Bacteroides spp. to important antimicrobial agents has been reported, including metronidazole and meropenem, and Bacteroides spp. can carry many antimicrobial resistance genes (ARGs).4 Taxonomic changes have been made among the species of the genus Bacteroides. In 2006, Bacteroides distasonis, Bacteroides merdae and Bacteroides goldsteinii were accommodated in the genus Parabacteroides.5 More recently, several other species, including Bacteroides vulgatus, Bacteroides dorei and Bacteroides massiliensis have been reclassified as Phocaeicola vulgatus, Phocaeicola dorei and Phocaeicola massiliensis.6
Prevotella spp. are often described as an important part of the human oral and urogenital microbiota. They can cause serious infections in the head/neck region, but are also isolated from other sites, including the abdomen.7,8 In recent years, resistance within this genus has increased, and metronidazole-resistant isolates have been described, as well as β-lactamase-producing isolates.9,10 Prevotella isolates can also carry multiple ARGs, but to a lesser extent compared with Bacteroides.11
In 2019, five MDR Bacteroides fragilis isolates harbouring several mobile genetic elements (MGEs) carrying ARGs were reported in five hospitals in the Netherlands.12 The finding of MDR B. fragilis isolates in different hospitals in the Netherlands is worrisome and underscores the need to perform an antibiotic susceptibility surveillance of Bacteroidales isolates (specifically Bacteroides, Parabacteroides, Phocaeicola and Prevotella) from hospitals located throughout the Netherlands. This prospective study describes the antimicrobial susceptibility profile of Bacteroides, Phocaeicola, Parabacteroides and Prevotella spp. isolated from clinical specimens in eight laboratories in the Netherlands in 2021.
Materials and methods
Collection of isolates
From February to July 2021, seven university hospitals and one regional laboratory providing microbiological services to regional hospitals and GPs, scattered across the Netherlands, consecutively collected 20–25 Bacteroides, Phocaeicola and Parabacteroides isolates and 10–15 Prevotella isolates during a maximum period of 3 months from a selection of clinically relevant materials using selective culture media provided by the national reference centre at the University Medical Center Groningen (UMCG), Groningen, the Netherlands. Sinus fluids, and pus/drain fluids from the abdominal region, thorax region, and head/neck region were cultured on Brucella laked blood agar supplemented with 80 mg/L kanamycin and 8 mg/L vancomycin (BBKV; Mediaproducts, Groningen, the Netherlands) and Bacteroides bile esculin agar (BBE; Mediaproducts, Groningen, the Netherlands) in addition to the standard isolation protocols of the laboratories. Plates were incubated at 37°C in an anaerobic atmosphere according to the standard protocol of the participating laboratories. After 48 h, plates were checked for bacterial growth. All morphologically different colonies were identified using MALDI-TOF MS, according to the standard protocol of the participating laboratories. Per patient, one isolate per species was included and stored in duplicate at −80°C using Microbank vials (Pro-Lab diagnostics, Birkenhead, UK). Positive blood culture bottles and other clinical materials were also included when containing Bacteroides, Phocaeicola, Parabacteroides or Prevotella spp. After the inclusion period, Microbank vials were sent by courier to the national reference centre. Upon arrival, all isolates were cultured on Brucella blood agar (BBA; Mediaproducts, Groningen, the Netherlands), supplemented with 10 mg/L haemin and 20 mg/L vitamin K1. Isolates were checked for purity and re-identified using MALDI-TOF MS (Bruker Microflex LT/SH, Bruker Daltonics, Bremen, Germany), with the version 11 database, including the MALDI Biotyper Subtyping Module, which was used to detect the cfiA gene in B. fragilis isolates.13 MALDI-TOF MS cannot differentiate between Bacteroides thetaiotaomicron and Bacteroides faecis; Bacteroides ovatus and Bacteroides xylanisolvens; and between P. vulgatus and P. dorei. Therefore, these species were combined and reported as B. thetaiotaomicron/faecis, B. ovatus/xylanisolvens and P. vulgatus/dorei complex, respectively.14
Antimicrobial susceptibility testing
Prior to antimicrobial susceptibility testing, isolates were cultured on BBA for 48 h at 37°C in an anaerobic atmosphere (80% N2, 10% H2, 10% CO2). Antimicrobial susceptibility testing for amoxicillin (Duchefa Farma BV, Haarlem, the Netherlands), amoxicillin/clavulanic acid (fixed ratio of 2:1) (Sigma–Aldrich, St. Louis, MO, USA), piperacillin/tazobactam (fixed concentration of 4 mg/L tazobactam; Fresenius Kabi, Bad Homburg vor den Höhe, Germany), meropenem (Fresenius Kabi), imipenem (Thermo Scientific, Waltham, MA, USA), metronidazole (B. Braun, Melsungen, Germany), moxifloxacin (Thermo Scientific) and tetracycline (Duchefa Farma BV) was performed using the agar dilution method, which is the recommended reference method for antimicrobial susceptibility testing of anaerobic organisms, using the EUCAST- and CLSI-recommended medium fastidious anaerobic agar supplemented with 5% defibrinated horse blood (FAA-HB; Mediaproducts) supplemented with the antimicrobial agent in increasing concentrations of a 2-fold serial dilution with a range of 0.008–256 mg/L.15 Of each bacterium, 2 µL of a 0.5 McFarland suspension was pipetted on the agar plates containing a dilution of the antibiotic tested at that moment and left to dry for about 10 min in an ambient atmosphere. Plates were incubated at 37°C for 48 h in an anaerobic atmosphere. Quality control was performed on every agar dilution plate using B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741, and for each antimicrobial agent, a control plate without antimicrobial agent was incubated under aerobic and anaerobic conditions. The MIC was the antibiotic concentration at which a significant reduction in growth was observed, as advised by the CLSI guidelines.15
The quality control strains for clindamycin resulted in a higher MIC value than the CLSI’s range.16 Due to these quality control issues, the clindamycin MIC was assessed using ETEST (bioMérieux, Marcy-l’ Étoile, France), according to the manufacturer’s recommendations. The MIC50 and MIC90 were determined per species when at least 10 isolates were tested for each antimicrobial agent. Species with fewer than 10 isolates were grouped as species (e.g. Bacteroides species). The use of different breakpoints between studies hampers the interpretation of differences in resistance between studies based on the percentage resistance. Therefore, the results of this study will be discussed using the MIC50 and MIC90. A complete overview of the percentage resistance using the different breakpoints is provided in Table S1 (available as Supplementary data at JAC Online). Furthermore, the results of the Phocaeicola isolates were joined with Bacteroides to facilitate comparison with other studies.
WGS
Isolates with MICs higher than 4, 8 and 4 mg/L to metronidazole, meropenem and imipenem, respectively, or carrying the cfiA gene, as determined by MALDI-TOF MS, were subjected to WGS. DNA extraction was performed using the DNeasy Ultraclean Microbial kit (MO BIO Laboratories, Carlsbad, CA, USA). Library preparation was performed using the Nextera XT v2 kit (Illumina, San Diego, CA, USA), followed by short-read sequencing on an Illumina MiSeq (Illumina) generating 250–300 bp paired-end reads using the MiSeq reagent kit v2 or v3, respectively.17 Using CLC Genomics workbench v12 (QIAGEN, Hilden, Germany), de novo assembly was performed.17 Analysis of WGS data, including detection of ARGs and MGEs, was performed as described previously.12 The identity of the isolates was confirmed by comparing the 16S rRNA gene with the NCBI database using BLASTn (https://blast.ncbi.nlm.nih.gov/).
Results
Clinical isolates
In total, 298 isolates were sent to the national reference centre, varying from 31 to 45 per participating centre. A total of 280 isolates were included in the study: 184 Bacteroides, Phocaeicola and Parabacteroides isolates and 96 Prevotella isolates. Eighteen isolates were not included in the study: six were identified as a species belonging to a different genus, 12 could not be resuscitated. Most isolates were cultured from abdominal samples (43%), followed by head/neck region samples (11%) and positive blood cultures (11%). An overview of isolates and associated clinical specimens is shown in Table 1.
Table 1.
Overview of clinical specimens
| Isolates | Blood culture | (Intra-)abdominala,b | Hepato-pancreato-biliary systema | Groin regiona | Head/neck regiona | Thorax regiona | Vascular prosthesisa | Orthopaedic materiala | Bone tissue | Urine | Not specifieda |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Bacteroides total (n = 139) | 22 | 71 | 8 | 12 | 2 | 1 | 2 | 2 | 1 | 0 | 18 |
| B. fragilis (n = 62) | 17 | 29 | 2 | 4 | 0 | 0 | 1 | 0 | 0 | 0 | 9 |
| B. ovatus/xylanisolvens (n = 25) | 0 | 16 | 4 | 3 | 0 | 0 | 0 | 1 | 0 | 0 | 1 |
| B. thetaiotaomicron/faecis (n = 31) | 4 | 11 | 2 | 3 | 2 | 1 | 1 | 1 | 0 | 0 | 6 |
| Bacteroides spp.c (n = 22) | 1 | 15 | 0 | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 2 |
| Phocaeicola total (n = 26) | 1 | 16 | 5 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| P. massiliensis (n = 1) | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| P. vulgatus/dorei (n = 25) | 1 | 15 | 5 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| Parabacteroides total (n = 19) | 2 | 15 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Parabacteroides distasonis (n = 14) | 2 | 11 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Parabacteroides spp.d (n = 5) | 0 | 4 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Prevotella total (n = 96) | 6 | 18 | 6 | 9 | 30 | 7 | 2 | 2 | 0 | 2 | 13 |
| Prevotella bivia (n = 13) | 0 | 5 | 0 | 2 | 1 | 3 | 0 | 1 | 0 | 0 | 1 |
| Prevotella buccae (n = 15) | 3 | 3 | 1 | 1 | 4 | 0 | 1 | 1 | 0 | 0 | 1 |
| Prevotella spp.e (n = 68) | 4 | 10 | 5 | 6 | 25 | 4 | 1 | 0 | 0 | 2 | 11 |
| Total all isolates (n = 280) | 32 | 120 | 21 | 23 | 32 | 8 | 4 | 4 | 1 | 2 | 33 |
aIncluding abscess/fluid/pus/tissue.
bIncluding ascites, perianal and peritoneal samples.
c Bacteroides spp. includes Bacteroides caccae (n = 6), Bacteroides cellulosilyticus (n = 2), Bacteroides clarus (n = 1), Bacteroides coagulans (n = 1), Bacteroides nordii (n = 1), Bacteroides salyersiae (n = 3), Bacteroides stercoris (n = 2), Bacteroides uniformis (n = 5).
d Parabacteroides spp. includes Parabacteroides goldsteinii (n = 1), Parabacteroides merdae (n = 4).
e Prevotella spp. includes Prevotella baroniae (n = 3), Prevotella bergensis (n = 3), Prevotella dentalis (n = 1), Prevotella denticola (n = 8), Prevotella disiens (n = 7), Prevotella histicola (n = 1), Prevotella intermedia (n = 6), Prevotella jejuni (n = 1), Prevotella loescheii (n = 1), Prevotella maculosa (n = 1), Prevotella melaninogenica (n = 7), Prevotella nanceiensis (n = 1), Prevotella nigrescens (n = 9), Prevotella oralis (n = 2), Prevotella oris (n = 8), Prevotella pallens (n = 3), Prevotella salivae (n = 3), Prevotella timonensis (n = 2), Prevotella veroralis (n = 1).
Antimicrobial susceptibility profile of Bacteroides, Phocaeicola and Parabacteroides isolates
Table 2 shows the differences in antimicrobial profile observed among the Bacteroides, Phocaeicola, Parabacteroides and Prevotella isolates per species. An overview of percentages resistance using EUCAST v11.0, EUCAST v13.1 and CLSI 2023 breakpoints is given in Table S1. For some isolates, susceptibility testing could not be performed due to issues in the growth and purity of the culture.
Table 2.
MIC50 and MIC90 (mg/L) of all isolates for the different antibiotics
| Isolates (n = 280) | Amoxicillin | Amoxicillin/clavulanic acid | Piperacillin/tazobactam | Meropenem | Imipenem | Metronidazole | Clindamycin | Tetracycline | Moxifloxacin | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | |
| Bacteroides total (138–139)a | 32 | >256 | 2 | 16 | 4 | 32 | 0.5 | 4 | 1 | 2 | 0.5 | 1 | 1.5 | >256 | 64 | 128 | 1 | 16 |
| B. fragilis (62) | 32 | 256 | 1 | 4 | 2 | 8 | 0.25 | 1 | 0.25 | 2 | 0.5 | 1 | 0.25 | >256 | 64 | 128 | 0.5 | 16 |
| B. thetaiotaomicron/faecis (31) | 64 | 256 | 2 | 32 | 16 | 32 | 0.5 | 4 | 1 | 4 | 0.5 | 1 | 4 | >256 | 32 | 128 | 2 | 16 |
| B. ovatus/xylanisolvens (25) | 64 | >256 | 2 | 16 | 8 | 32 | 0.5 | 4 | 1 | 4 | 0.5 | 1 | 3 | >256 | 64 | 128 | 2 | 16 |
| Bacteroides spp. (20–21)a,b | 32 | >256 | 2 | 4 | 4 | 8 | 0.25 | 1 | 1 | 1 | 0.5 | 0.75 | 1 | 4 | 32 | 64 | 2 | 4 |
| Phocaeicola total (25–26)a,c | 128 | >256 | 2 | 16 | 8 | 32 | 0.5 | 2 | 1 | 4 | 0.5 | 1 | 0.023 | 0.75 | 32 | 64 | 1 | 128 |
| P. vulgatus/dorei (24–25)a | 128 | >256 | 2 | 16 | 8 | 32 | 0.5 | 2 | 1 | 4 | 0.5 | 1 | 0.023 | 0.75 | 32 | 64 | 1 | 128 |
| Bacteroides and Phocaeicola total (163–165)a | 32 | >256 | 2 | 16 | 4 | 32 | 0.5 | 2 | 1 | 4 | 0.5 | 1 | 0.75 | >256 | 32 | 128 | 1 | 16 |
| Parabacteroides total (19)d | >256 | >256 | 8 | 32 | 4 | 16 | 1 | 2 | 2 | 2 | 1 | 1 | 1 | 3 | 64 | 64 | 0.5 | 1 |
| Parabacteroides distasonis (14) | >256 | >256 | 8 | 32 | 4 | 16 | 0.5 | 2 | 2 | 2 | 1 | 1 | 1.5 | 4 | 64 | 64 | 0.5 | 2 |
| Prevotella total (87–96)a | 4 | 128 | 0.5 | 4 | 2 | 4 | 0.125 | 0.25 | 0.125 | 0.5 | 0.5 | 1 | 0.016 | >256 | 1 | 64 | 1 | 4 |
| P. bivia (12–13)a | 16 | 64 | 2 | 4 | 1 | 4 | 0.125 | 0.125 | 0.125 | 0.125 | 1 | 1 | 0.023 | >256 | 1 | 64 | 2 | 4 |
| P. buccae (14–15)a | 0.125 | 256 | 0.25 | 8 | 2 | 8 | 0.125 | 0.5 | 0.25 | 0.5 | 0.5 | 1 | 0.016 | >256 | 0.5 | 32 | 0.5 | 1 |
| Prevotella spp. (60–68)a,e | 4 | 128 | 0.5 | 4 | 2 | 4 | 0.0625 | 0.25 | 0.125 | 0.25 | 0.25 | 1 | <0.016 | >256 | 1 | 64 | 0.5 | 2 |
aNot all strains were tested for all antibiotics.
b Bacteroides spp. includes Bacteroides caccae (n = 6), Bacteroides cellulosilyticus (n = 2), Bacteroides clarus (n = 1), Bacteroides coagulans (n = 1), Bacteroides nordii (n = 1), Bacteroides salyersiae (n = 3), Bacteroides stercoris (n = 2), Bacteroides uniformis (n = 5).
c Phocaeicola total includes Phocaeicola vulgatus/dorei (n = 25), Phocaeicola massiliensis (n = 1).
d Parabacteroides total includes Parabacteroides distasonis (n = 14), Parabacteroides goldsteinii (n = 1), Parabacteroides merdae (n = 4).
e Prevotella spp. includes Prevotella baroniae (n = 3), Prevotella bergensis (n = 3), Prevotella dentalis (n = 1), Prevotella denticola (n = 8), Prevotella disiens (n = 7), Prevotella histicola (n = 1), Prevotella intermedia (n = 6), Prevotella jejuni (n = 1), Prevotella loescheii (n = 1), Prevotella maculosa (n = 1), Prevotella melaninogenica (n = 7), Prevotella nanceiensis (n = 1), Prevotella nigrescens (n = 9), Prevotella oralis (n = 2), Prevotella oris (n = 8), Prevotella pallens (n = 3), Prevotella salivae (n = 3), Prevotella timonensis (n = 2), Prevotella veroralis (n = 1).
The MIC90 of amoxicillin/clavulanic acid was similar for B. thetaiotaomicron/faecis, B. ovatus/xylanisolvens, P. vulgatus/dorei and P. distasonis, with MIC90 values between 16 and 32 mg/L. However, B. fragilis and the Bacteroides ‘other species’ group had much lower MIC90 values of 4 mg/L. A similar tendency was seen for piperacillin/tazobactam. The MIC90 of meropenem and imipenem ranged from 1 to 4 mg/L. The highest MIC90, for both antibiotics, was 4 mg/L for B. thetaiotaomicron/faecis and B. ovatus/xylanisolvens isolates, and the imipenem MIC90 of P. vulgatus/dorei was 4 mg/L. Similar to amoxicillin/clavulanic acid and piperacillin/tazobactam, the meropenem and imipenem MIC90 of B. fragilis and the Bacteroides ‘other species’ group had the lowest values for both antibiotics. Four of the 62 B. fragilis isolates harboured the cfiA gene as determined by MALDI-TOF MS MBT Subtyping Module. Two isolates had high MICs of meropenem and imipenem, where one isolate had MICs of 256 and 128 mg/L, and the other had MICs of 16 and 8 mg/L, respectively. The other two isolates had lower MIC values; one isolate had MICs of 4 and 2 mg/L and the other had MICs of 0.5 and 0.25 m/L for meropenem and imipenem, respectively.
The MIC90 of metronidazole was similar for all species, ranging from 0.75 to 1 mg/L. The MIC90 of clindamycin was >256 mg/L for B. fragilis, B. thetaiotaomicron/faecis and B. ovatus/xylanisolvens. For all other Bacteroides, Phocaeicola and Parabacteroides species, the MIC90 was lower, ranging from 0.75 mg/L for P. vulgatus/dorei to 4 mg/L for P. distasonis. The MIC90 of tetracycline was mostly similar for all species, with MIC90 values between 64 and 128 mg/L. For moxifloxacin, Parabacteroides isolates had the lowest MIC90 value of 1 mg/L, and Phocaeicola isolates had the highest values of 128 mg/L.
Antimicrobial susceptibility profile of Prevotella isolates
Of 96 Prevotella isolates, most species were represented by <10 isolates, with only Prevotella bivia and Prevotella buccae represented by ≥10 isolates. Therefore, most Prevotella species were grouped, and only these two species could be compared with the average Prevotella data (Table 2). The MIC50 and MIC90 values for most antimicrobial agents were similar for P. bivia, P. buccae and the other species. The biggest difference was visible for amoxicillin, where the MIC50 was only 0.125 mg/L for P. bivia but 16 mg/L for P. buccae, with an average for all other species of 4 mg/L.
WGS of metronidazole-resistant isolates
The assembly output of the sequenced isolates is shown in Table S2. Three isolates with an MIC value of 8 mg/L for metronidazole (resistant according to EUCAST guidelines) were subjected to WGS, of which two harboured a nim gene encoding metronidazole resistance in their genome. The B. thetaiotaomicron isolate carried a nimE gene on a pBFS01_2 plasmid, and the Prevotella melaninogenica isolate harboured a nimA gene in its chromosome. The latter nim gene was not located on an MGE. The Prevotella nanceiensis isolate did not harbour a nim gene. Furthermore, the cfxA gene (n = 3), the tet(Q) gene (n = 1) and the mef(En2) gene (n = 1) were encountered in these isolates.
WGS of carbapenem-resistant isolates
A total of 12 isolates were subjected to WGS to detect carbapenem resistance determinants. Of six B. fragilis isolates, four harboured the cfiA gene. Two had either IS1186 or IS614 upstream and had high MICs of meropenem and imipenem (meropenem MIC: 16 and 256 mg/L; imipenem MIC: 8 and 128 mg/L, respectively). The other two cfiA-harbouring B. fragilis isolates did not have an IS element upstream and had low MICs of meropenem and imipenem (meropenem MIC: 4 and 0.5 mg/L; imipenem MIC: 2 and 0.25 mg/L, respectively). Two B. fragilis isolates sequenced due to high MICs of imipenem (MIC: 8 and 8 mg/L), but with low MICs of meropenem (MIC: 2 and 0.25 mg/L), did not harbour a cfiA gene. Of the other six sequenced isolates [i.e. B. thetaiotaomicron (n = 1), B. xylanisolvens (n = 1), Bacteroides spp. (n = 1) and P. vulgatus (n = 3)] the Bacteroides spp. isolate harboured the cfiA gene with an IS614 upstream. Other genes encountered in the 12 sequenced isolates were the cepA gene (n = 2), the cfxA gene (n = 2), the mef(A) gene (n = 1), erm(F) gene (n = 2), the mef(En2) gene (n = 1), the tet(X) gene (n = 1) and the tet(Q) gene (n = 4).
Discussion
This study presents the first national surveillance of antimicrobial resistance in Bacteroides, Phocaeicola, Parabacteroides and Prevotella within the Netherlands. Through a collaboration involving eight clinical laboratories across the country, we achieved a comprehensive understanding of the nationwide susceptibility patterns among clinical isolates of these genera. Thus far, the only comparable study on antimicrobial susceptibility patterns in the Netherlands was from the national reference laboratory in 2011–13.18 For Bacteroides and Phocaeicola isolates, an increase in the MIC90 of amoxicillin/clavulanic acid is observed, from an MIC90 of 1.5 mg/L in 2011–13 to 16 mg/L in this study. Among Prevotella isolates, an increase in MIC values was observed for amoxicillin/clavulanic acid, clindamycin and metronidazole. The MIC50 and MIC90 of amoxicillin/clavulanic acid went from 0.094 and 1 mg/L in 2011–13 to 0.5 and 4 mg/L in 2021. For clindamycin, the MIC50 was identical, at 0.016 mg/L; however, the MIC90 went from 32 mg/L in 2011–13 to >256 mg/L in 2021. For amoxicillin and metronidazole, similar values were found, with MIC90 values of 128 and 0.75 mg/L, respectively, in 2011–13, to 128 and 1 mg/L, respectively, in 2021.
Three isolates of different species (i.e. B. thetaiotaomicron, P. melaninogenica and P. nanceiensis) had a metronidazole MIC of 8 mg/L. Two of these isolates harboured a nim gene, which plays a role in metronidazole resistance.19 In the B. thetaiotaomicron isolate, the nimE gene was present on a pBFS01_2 plasmid, together with an ISBf6 insertion sequence element, which has been previously described by Sydenham et al.20 from an MDR B. fragilis isolate. This plasmid was also detected in 4/5 MDR B. fragilis isolates from the Netherlands and 2/23 cfiA-harbouring B. fragilis isolates from Hong Kong.12,21 The P. melaninogenica isolate resistant to metronidazole harboured a nimA gene in its chromosome. The nimA gene can be present on plasmids and in the chromosome. It is often associated with an IS1168 element, which was not detected in this isolate.19 The nimA gene has been observed in Bacteroides spp., but is less prevalent in Prevotella isolates.22 The third isolate resistant to metronidazole, P. nanceiensis, did not harbour any nim gene in its genome. Either this isolate harbours a not-yet-described nim gene or another resistance mechanism.23
WGS of isolates with high MICs of a carbapenem antibiotic, or harbouring the cfiA gene as determined by MALDI-TOF MS, showed that in five strains the cfiA gene was present, of which three were accompanied by an IS element. These IS elements were located upstream of the gene and were assigned to the IS1186 and IS614 family, which has been described previously by Soki et al.24 These IS elements have been reported to activate the cfiA gene, resulting in higher MIC values, as was seen in the carbapenem resistance in these three isolates. In this study, one Bacteroides non-fragilis isolate was found harbouring the cfiA gene with an IS614 upstream, with meropenem MICs of >256 mg/L and imipenem MICs of 64 mg/L. WGS results showed that an identical cfiA element was present in a B. fragilis isolate retrieved from the same clinical sample, indicating that horizontal gene transfer (HGT) occurred between the two isolates.25
Differences in antimicrobial susceptibility profiles can occur between different hospitals in the same country due to factors such as disparities in patient populations (e.g. patient origins), specialties of the associated university hospitals, and previous antibiotic treatments, among others. Therefore, surveillance of a single laboratory cannot fully represent the antimicrobial susceptibility patterns of a species across an entire country.26
With antimicrobial resistance rising among clinical Bacteroidales isolates, performing antimicrobial resistance testing is essential to guide appropriate treatment decisions.
This study represents the first national surveillance in the Netherlands involving isolates collected nationwide. By conducting regular surveillance of antimicrobial susceptibility profiles in clinically significant anaerobic bacteria, empirical treatments can be optimized, and trends in antimicrobial resistance can be closely monitored.
Supplementary Material
Contributor Information
K E Boiten, University Medical Center Groningen, Department of Medical Microbiology and Infection Prevention, University of Groningen, Groningen, The Netherlands.
D W Notermans, Centre for Infectious Disease Control (CIb), National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands; Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands.
R J Rentenaar, Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands.
J van Prehn, Department of Medical Microbiology, Leiden University Center for Infectious Diseases, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
L G M Bode, Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center (Erasmus MC), Rotterdam, The Netherlands.
I Maat, Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.
W van der Zwet, Department of Medical Microbiology, Infectious Diseases and Infection Prevention, Maastricht University Medical Center+, Maastricht, The Netherlands.
A Jansz, PAMM Laboratory of Medical Microbiology, Veldhoven, The Netherlands.
T J H Siebers, University Medical Center Groningen, Department of Medical Microbiology and Infection Prevention, University of Groningen, Groningen, The Netherlands; Department of Medical Microbiology, Certe, Groningen, The Netherlands.
J W A Rossen, University Medical Center Groningen, Department of Medical Microbiology and Infection Prevention, University of Groningen, Groningen, The Netherlands; Laboratory of Medical Microbiology and Infectious Diseases, and Isala Academy, Isala Hospital, Zwolle, The Netherlands; Department of Pathology, University of Utah School of Medicine, Salt Lake City, USA.
S C de Greeff, Centre for Infectious Disease Control (CIb), National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
A P A Hendrickx, Centre for Infectious Disease Control (CIb), National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
E J Kuijper, Centre for Infectious Disease Control (CIb), National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands; Department of Medical Microbiology, Leiden University Center for Infectious Diseases, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
A C M Veloo, University Medical Center Groningen, Department of Medical Microbiology and Infection Prevention, University of Groningen, Groningen, The Netherlands.
Funding
This work was supported by the Dutch National Institute for Public Health and the Environment (RIVM) (grant number V/150157/02/OT).
Transparency declarations
A.C.M.V. has received a grant from the Dutch National Institute for Public Health and the Environment (RIVM). All other authors: none to declare.
Author contributions
Study conceptualization: K.E.B., T.J.H.S., E.J.K., A.C.M.V.; study design: all authors; data collection: K.E.B., A.C.M.V.; data interpretation: K.E.B., A.C.M.V.; manuscript writing: K.E.B.; manuscript reviewing: all authors; project supervision: J.W.A.R., E.J.K., A.C.M.V.
Supplementary data
Tables S1 and S2 are available as Supplementary data at JAC Online.
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