554
Letters to the Editor / International Journal of Antimicrobial Agents 45 (2015) 550–561
Antibiotic susceptibility profiles of Prevotella species in The Netherlands Sir, Strict anaerobic bacteria comprise a significant part of the human microbiome at dermal, dental and mucosal surfaces and play an important role in health and disease. Prevotella spp. can be involved in head and neck infections, pleuropulmonary infections and abscess formation at different body sites. Antibiotic resistance is increasing not only among aerobic pathogens but also in anaerobic pathogens [1]. In anaerobes, production of -lactamases is the most common mechanism of -lactam resistance and is most frequently encountered in the Bacteroides fragilis group and the genus Prevotella, with significant interspecies differences [2]. It has been reported that 58% of Prevotella strains in Europe produce -lactamases [2], but studies on the antibiotic susceptibility profiles of anaerobes are rather limited and information to species level is scarce. Antibiotic treatment is often empirical and it is therefore important to conduct surveillance studies in which antibiotic susceptibility profiles of anaerobic bacteria are studied. In this study, the antibiotic susceptibility profiles of 113 Prevotella strains representing 18 species were established. Strains were isolated from a variety of human clinical specimens at the diagnostic laboratory of the Department of Clinical Microbiology at University Medical Center Groningen (Groningen, The Netherlands). Strains were revived from storage by subculturing on Brucella blood agar (Mediaproducts BV, Groningen, The Netherlands) supplemented with haemin (5 mg/L) and vitamin K1 (1 mg/L) and were incubated in an anaerobic atmosphere (80% N2 , 10% CO2 , 10% H2 ) at 37 ◦ C for 48 h. Strains were identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) as described previously [3]. MALDI-TOF MS measurements were performed using a microflexTM MALDI-TOF MS system and Biotyper 3.0 software (Bruker Daltonik GmbH, Bremen, Germany). Log scores ≥2 were considered reliable species identification. Minimum inhibitory concentrations (MICs) were determined for amoxicillin, amoxicillin/clavulanic acid (AMC), clindamycin, metronidazole, tetracycline, piperacillin/tazobactam (TZP), meropenem and cefoxitin using Etest strips (bioMérieux, Marcyl’Étoile, France) after 48 h of incubation. Bacteroides fragilis ATCC 25285 was used as a quality control strain for all tested antibiotics, except for amoxicillin for which Streptococcus pneumoniae ATCC 49619 was used. Breakpoints advised by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used for amoxicillin, AMC, clindamycin, metronidazole, TZP and meropenem (http://www.eucast.org). Clinical and Laboratory Standards Institute (CLSI) breakpoints were used for cefoxitin and
tetracycline. The -lactamase activity of Prevotella strains was determined using the BBLTM CefinaseTM disk (BBL Microbiology Systems, Cockeysville, MD) as recommended by the manufacturer. The antibiotic susceptibility profiles for the different species of Prevotella are shown in Table 1. Antimicrobial resistance was observed for amoxicillin (46%), clindamycin (10%), metronidazole (3%) and tetracycline (28%). All Prevotella strains were susceptible to AMC, TZP, meropenem and cefoxitin. Interspecies differences in antimicrobial resistance profiles were significant. For amoxicillin, the percentage of resistance varied from 0% (Prevotella intermedia, Prevotella corporis) to 80% (4/5 Prevotella baroniae). Significant interspecies variation in susceptibility was also observed for clindamycin (0–33%) and tetracycline (0–67%). Metronidazole resistance was encountered in three Prevotella bivia strains, with individual MICs of 8, 64 and >256 mg/L. Tetracycline resistance was not observed in Prevotella nigrescens and P. intermedia, but was present in all other species tested. In contrast, tetracycline resistance among Prevotella melaninogenica and P. bivia was 67% and 44%, respectively. The resistance rate for other individual Prevotella spp. to tetracycline was between 14% and 33%. -Lactamase production was detected in 58% (66/113) of the Prevotella strains. Interspecies differences in -lactamase production were observed and ranged from 0% (P. intermedia) to 100% (P. melaninogenica) (Table 1). This study shows that antibiotic resistance among Prevotella spp. from clinical specimens in The Netherlands is prevalent and differs between species. Multidrug-resistant (MDR) bacteria are usually defined as ‘acquired non-susceptibility to at least one agent in three or more antimicrobial categories’ [4]. This definition is applied for aerobic bacteria and may also be used for anaerobic bacteria. MDR Prevotella strains were reported by Shilnikova and Dmitrieva [5], who described two Prevotella isolates resistant to clindamycin, penicillin and metronidazole. In the present study, seven Prevotella strains with resistance to at least three antimicrobial categories were found, including five strains with resistance to amoxicillin, clindamycin or metronidazole, and tetracycline, and two strains with resistance to amoxicillin, clindamycin, metronidazole and tetracycline. No nim genes were detected in the three metronidazole-resistant strains. Four of the seven MDR strains were identified as P. bivia. To our knowledge, this is the first study to report on Prevotella strains showing resistance to four categories of antimicrobial agents. This emphasises the necessity of species identification of Prevotella strains and regular antibiotic susceptibility testing. Special attention should be paid to P. bivia since this a frequently encountered and most resistant species. Multidrug resistance within the genus Prevotella is extremely worrisome, especially in combination with resistance to metronidazole.
Letters to the Editor / International Journal of Antimicrobial Agents 45 (2015) 550–561
555
Table 1 Minimum inhibitory concentrations (MICs), percentage resistance to eight different antibiotics and percentage -lactamase-positive (%BL + ve) among Prevotella spp. clinical isolates. Strain
Amoxicillin
Prevotella spp. (n = 113) MIC range (mg/L) MIC50 (mg/L) MIC90 (mg/L) % resistant %BL + ve
256 1.5 >256 46 58
Prevotella bivia (n = 27) MIC range (mg/L) MIC50 (mg/L) MIC90 (mg/L) % resistant %BL + ve
0.032 to >256 3 >256 56 67
Prevotella buccae (n = 21) MIC range (mg/L) MIC50 (mg/L) MIC90 (mg/L) % resistant %BL + ve
0.032 to >256 0.094 >256 43 43
AMC
Clindamycin
Metronidazole
Meropenem
256 256 0.19 0.75 3
0.008–0.75 0.047 0.125 0
256 0.5 4 11
0.023–0.75 0.064 0.125 0
Letters to the Editor / International Journal of Antimicrobial Agents 45 (2015) 550–561
Antibiotic susceptibility profiles of Prevotella species in The Netherlands Sir, Strict anaerobic bacteria comprise a significant part of the human microbiome at dermal, dental and mucosal surfaces and play an important role in health and disease. Prevotella spp. can be involved in head and neck infections, pleuropulmonary infections and abscess formation at different body sites. Antibiotic resistance is increasing not only among aerobic pathogens but also in anaerobic pathogens [1]. In anaerobes, production of -lactamases is the most common mechanism of -lactam resistance and is most frequently encountered in the Bacteroides fragilis group and the genus Prevotella, with significant interspecies differences [2]. It has been reported that 58% of Prevotella strains in Europe produce -lactamases [2], but studies on the antibiotic susceptibility profiles of anaerobes are rather limited and information to species level is scarce. Antibiotic treatment is often empirical and it is therefore important to conduct surveillance studies in which antibiotic susceptibility profiles of anaerobic bacteria are studied. In this study, the antibiotic susceptibility profiles of 113 Prevotella strains representing 18 species were established. Strains were isolated from a variety of human clinical specimens at the diagnostic laboratory of the Department of Clinical Microbiology at University Medical Center Groningen (Groningen, The Netherlands). Strains were revived from storage by subculturing on Brucella blood agar (Mediaproducts BV, Groningen, The Netherlands) supplemented with haemin (5 mg/L) and vitamin K1 (1 mg/L) and were incubated in an anaerobic atmosphere (80% N2 , 10% CO2 , 10% H2 ) at 37 ◦ C for 48 h. Strains were identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) as described previously [3]. MALDI-TOF MS measurements were performed using a microflexTM MALDI-TOF MS system and Biotyper 3.0 software (Bruker Daltonik GmbH, Bremen, Germany). Log scores ≥2 were considered reliable species identification. Minimum inhibitory concentrations (MICs) were determined for amoxicillin, amoxicillin/clavulanic acid (AMC), clindamycin, metronidazole, tetracycline, piperacillin/tazobactam (TZP), meropenem and cefoxitin using Etest strips (bioMérieux, Marcyl’Étoile, France) after 48 h of incubation. Bacteroides fragilis ATCC 25285 was used as a quality control strain for all tested antibiotics, except for amoxicillin for which Streptococcus pneumoniae ATCC 49619 was used. Breakpoints advised by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used for amoxicillin, AMC, clindamycin, metronidazole, TZP and meropenem (http://www.eucast.org). Clinical and Laboratory Standards Institute (CLSI) breakpoints were used for cefoxitin and
tetracycline. The -lactamase activity of Prevotella strains was determined using the BBLTM CefinaseTM disk (BBL Microbiology Systems, Cockeysville, MD) as recommended by the manufacturer. The antibiotic susceptibility profiles for the different species of Prevotella are shown in Table 1. Antimicrobial resistance was observed for amoxicillin (46%), clindamycin (10%), metronidazole (3%) and tetracycline (28%). All Prevotella strains were susceptible to AMC, TZP, meropenem and cefoxitin. Interspecies differences in antimicrobial resistance profiles were significant. For amoxicillin, the percentage of resistance varied from 0% (Prevotella intermedia, Prevotella corporis) to 80% (4/5 Prevotella baroniae). Significant interspecies variation in susceptibility was also observed for clindamycin (0–33%) and tetracycline (0–67%). Metronidazole resistance was encountered in three Prevotella bivia strains, with individual MICs of 8, 64 and >256 mg/L. Tetracycline resistance was not observed in Prevotella nigrescens and P. intermedia, but was present in all other species tested. In contrast, tetracycline resistance among Prevotella melaninogenica and P. bivia was 67% and 44%, respectively. The resistance rate for other individual Prevotella spp. to tetracycline was between 14% and 33%. -Lactamase production was detected in 58% (66/113) of the Prevotella strains. Interspecies differences in -lactamase production were observed and ranged from 0% (P. intermedia) to 100% (P. melaninogenica) (Table 1). This study shows that antibiotic resistance among Prevotella spp. from clinical specimens in The Netherlands is prevalent and differs between species. Multidrug-resistant (MDR) bacteria are usually defined as ‘acquired non-susceptibility to at least one agent in three or more antimicrobial categories’ [4]. This definition is applied for aerobic bacteria and may also be used for anaerobic bacteria. MDR Prevotella strains were reported by Shilnikova and Dmitrieva [5], who described two Prevotella isolates resistant to clindamycin, penicillin and metronidazole. In the present study, seven Prevotella strains with resistance to at least three antimicrobial categories were found, including five strains with resistance to amoxicillin, clindamycin or metronidazole, and tetracycline, and two strains with resistance to amoxicillin, clindamycin, metronidazole and tetracycline. No nim genes were detected in the three metronidazole-resistant strains. Four of the seven MDR strains were identified as P. bivia. To our knowledge, this is the first study to report on Prevotella strains showing resistance to four categories of antimicrobial agents. This emphasises the necessity of species identification of Prevotella strains and regular antibiotic susceptibility testing. Special attention should be paid to P. bivia since this a frequently encountered and most resistant species. Multidrug resistance within the genus Prevotella is extremely worrisome, especially in combination with resistance to metronidazole.
Letters to the Editor / International Journal of Antimicrobial Agents 45 (2015) 550–561
555
Table 1 Minimum inhibitory concentrations (MICs), percentage resistance to eight different antibiotics and percentage -lactamase-positive (%BL + ve) among Prevotella spp. clinical isolates. Strain
Amoxicillin
Prevotella spp. (n = 113) MIC range (mg/L) MIC50 (mg/L) MIC90 (mg/L) % resistant %BL + ve
256 1.5 >256 46 58
Prevotella bivia (n = 27) MIC range (mg/L) MIC50 (mg/L) MIC90 (mg/L) % resistant %BL + ve
0.032 to >256 3 >256 56 67
Prevotella buccae (n = 21) MIC range (mg/L) MIC50 (mg/L) MIC90 (mg/L) % resistant %BL + ve
0.032 to >256 0.094 >256 43 43
AMC
Clindamycin
Metronidazole
Meropenem
256 256 0.19 0.75 3
0.008–0.75 0.047 0.125 0
256 0.5 4 11
0.023–0.75 0.064 0.125 0
