Prevotella melaninogenica, a Sentinel Species of Antibiotic Resistance in Cystic Fibrosis Respiratory Niche?

The importance and abundance of strict anaerobic bacteria in the respiratory microbiota of people with cystic fibrosis (PWCF) is now established through studies based on high-throughput sequencing or extended-culture methods. In CF respiratory niche, one of the most prevalent anaerobic genera is Prevotella, and particularly the species Prevotella melaninogenica. The objective of this study was to evaluate the antibiotic susceptibility of this anaerobic species. Fifty isolates of P. melaninogenica cultured from sputum of 50 PWCF have been included. Antibiotic susceptibility testing was performed using the agar diffusion method. All isolates were susceptible to the following antibiotics: amoxicillin/clavulanic acid, piperacillin/tazobactam, imipenem and metronidazole. A total of 96% of the isolates (48/50) were resistant to amoxicillin (indicating beta-lactamase production), 34% to clindamycin (17/50) and 24% to moxifloxacin (12/50). Moreover, 10% (5/50) were multidrug-resistant. A significant and positive correlation was found between clindamycin resistance and chronic azithromycin administration. This preliminary study on a predominant species of the lung “anaerobiome” shows high percentages of resistance, potentially exacerbated by the initiation of long-term antibiotic therapy in PWCF. The anaerobic resistome characterization, focusing on species rather than genera, is needed in the future to better prevent the emergence of resistance within lung microbiota.


Introduction
The importance and abundance of strict anaerobic bacteria in the respiratory microbiota of people with cystic fibrosis (PWCF) is now established. These recent data are based on high-throughput sequencing [1,2] and cultural techniques [3][4][5]. Anaerobes colony count has been evaluated between 1.10 4 to 9.10 7 colony forming units per milliliter in sputum culture [3]. Numerous anaerobic genera have been identified as part of the CF core lung microbiota such as Prevotella, Veillonella, Porphyromonas, Fusobacterium, or Peptostreptococcus [2,6,7]. However, these bacteria remain the unknowns of the lung, both in terms of diversity, resistance or impact on the pathophysiology of CF disease [8]. One of the most prevalent and abundant anaerobic genera described in CF lung is Prevotella, and particularly the species Prevotella melaninogenica [4,5,9]. Contradictory hypotheses on the potential role of Prevotella genus have been previously developed [8]. On the one hand, it has been associated with better lung function and less inflammation [2,[10][11][12]. On the other hand, Prevotella species are able to enhance other bacteria's pathogenicity in lung [13], to produce pro-inflammatory short chain fatty acids [14] and are described to be resistant to antibiotics [15]. Focusing on this last point, previous studies have reported resistance acquisition of Prevotella isolates, due to the production of enzyme (mainly betalactamase [13,16,17]) or the carrying of the resistance gene [18,19]. Moreover, in CF, an over-expression of antibiotic resistance may be observed due to several reasons: repeated or chronic use of antimicrobial treatment, administration of high doses of antibiotic therapy, or prescription of antibiotic molecules at sub-clinical doses for anti-inflammatory properties. As the issue of antibiotic resistance is crucial, even more in PWCF, antibiotic susceptibility monitoring of this main anaerobic genus is necessary.
The objective of this study was to evaluate the antibiotic resistance rate of Prevotella melaninogenica, the most prevalent anaerobic species of the bronchopulmonary microbiota of PWCF.

Patients Characteristics
Fifty PWCF were included in the study. The following sociodemographic and clinical parameters were recorded: age, sex, CFTR (CF transmembrane conductance regulator) gene mutation, lung function, diabetes, chronic antibiotics administration (azithromycin, aztreonam, colistin, and tobramycin), and antibiotic administration one month before the sample. The patients' lung involvement was assessed by the measurement of the maximum expiratory volume per second (FEV 1 ) by spirometry tests, which was defined by three stages: early (FEV 1 > 70%), intermediate (40 < FEV 1 < 70%) and advanced (FEV 1 < 40%) [20].

Statistical Analysis
Statistical analysis was performed using the Chi-square and Fisher tests. The alpha risk has been reported significant when it was less than 0.05.

Antibiotic Susceptibility Testing
Antibiotic susceptibilities of P. melaninogenica isolates are presented in Table 2. All isolates were susceptible to the following antibiotics: amoxicillin/clavulanic acid, piperacillin/ tazobactam, imipenem, and metronidazole. A total of 96% of P. melaninogenica isolates (48/50) were classified as beta-lactamase producers (MIC for amoxicillin > 0.5 mg/L), and 38% of these isolates (18/48) had a MIC for amoxicillin superior to 256 mg/L ( Figure 1). The MIC for amoxicillin for the two non-beta-lactamase producers isolates have been evaluated at 0.094 mg/L and inferior to 0.016 mg/L (Figure 1). A total of 34% (17/50) and 24% (12/50) of isolates were resistant to clindamycin and moxifloxacin, respectively. Ten percent of isolates (5/50) were categorized MDR, due to a combined resistance to amoxicillin, clindamycin and moxifloxacin.

Associations between Antibiotic Resistance and Antibiotic Administration
Amoxicillin, clindamycin and moxifloxacin resistance percentages were compared to antibiotic administration one month before the sample (oral or intravenous: yes or no) and chronic antibiotic administration (azithromycin, aztreonam, colistin or tobramycin: yes or no). A significantly positive correlation was found between clindamycin resistance and oral chronic azithromycin administration (p = 0.002, Chi-square test).

Discussion
The potential impact of strict anaerobic bacteria in the pathophysiology of CF disease and in the respiratory function of PWCF is still at the hypothesis stage. The important and now undeniable place of these bacteria within the CF respiratory microbiota led to the study of their antibiotic susceptibilities to better understand the issue of resistance. For the genus Prevotella, disparities in resistance rates have been observed among the different studies but all the results agree on an increase in antibiotic resistance from clinical isolates in CF, as well as in other diseases [15,23].
Concerning beta-lactams, resistance is mainly due to the production of beta-lactamases, which induce a loss of susceptibility to penicillin, and that of the first, second, and third (oral) generation cephalosporins [21]. Several beta-lactamase detection methods can be used, combining phenotypic (mostly performed in routine practice) and molecular (cfxA resistance genes detection approaches [18,19]). Indeed, the detection of cfxA genes in Prevotella isolates has been associated with high MICs of amoxicillin [19,24]. However it remains important to determine if the isolates are functionally resistant to amoxicillin [25]. In this study focusing on P. melaninogenica, a high percentage of isolates (96%) were categorized as beta-lactamase producers using phenotypic detection based on the MIC of amoxicillin determination. This result is slightly higher than previous results for this species (Table 3) which can be explained by three main reasons. Firstly, the other studies used the nitrocefin test for phenotypic detection of beta-lactamase. However the recommendation of EUCAST/CASFM 2019 [21] is to avoid nitrocefin, considered as a poor substrate which may underestimate beta-lactamase activity detection. Moreover interference with the isolates pigmentation (as P. melaninogenica) represents a limit to this test based on a colorimetric reaction [26]. As formerly demonstrated, the determination of the MIC of amoxicillin is a more reliable method of detection [27]. Secondly, this study focused only on P. melaninogenica species while others' studies are based on several Prevotella species, for which resistance rates may differ [26]. Thirdly, in this study, isolates have been obtained from sputum of PWCF, for which antibiotic selective pressure is higher due to chronic antibiotic therapy administration and may lead to diminution of antibiotic susceptibility [15]. Moreover, CF lung has been described as a potential reservoir of resistance genes (as oral sphere) because cfxA genes coding beta-lactamase could be transferred among bacteria by horizontal transfer gene mechanisms [8,28,29]. Fourthly, as demonstrated for other genera known to carry cfxA genes (Capnocytophaga, Bacteroides, . . . ), the expression of beta-lactamase may differ according to major substitutions in the cfxA gene sequence or to the influence of the genetic environment of cfxA [30]. This molecular part has not been evaluated in this study.
For the other beta-lactams, all of the isolates of the present study were susceptible to piperacillin/tazobactam, amoxicillin/clavulanic acid, and imipenem (Table 4). No resistance have been described to date for amoxicillin/clavulanic acid and imipenem, however resistance to piperacillin/tazobactam was previously reported in the literature for Prevotella spp. [3,31].
As beta-lactams, emerging resistance to macrolides and fluoroquinolone has been described for Prevotella spp. [42,43]. In our study, 24% of P. melaninogenica isolates were resistant to moxifloxacin, which was concordant with the results of Ulger Toprak et al. [26] (Table 4). Thirty-four percent of P. melaninogenica isolates were resistant to clindamycin, which was higher than previously described in non-CF individuals, as no resistance data are available for PWCF [18,26,32]. Chronic azithromycin administration, prescribed at sub-clinical doses for anti-inflammatory properties, was significantly correlated with clindamycin resistance (p < 0.05, Chi-square test), as previously highlighted in PWCF [16]. This result is consistent with the fact that one of the main factors leading to antibiotic resistance identified for anaerobic bacteria is long-term antibiotic use [43,44]. Sherrard et al. [15] showed that the administration of betalactam or tetracycline in the year prior to pulmonary sample collection in PWCF was associated with higher MICs of amoxicillin, ceftazidime and tetracycline for Prevotella spp. In the future, the worsening resistance of the Prevotella genus is a threat and should be monitored. Firstly, different studies highlighted the increase of MDR anaerobic isolates. Ulger Toprak et al. [26] have demonstrated that nearly 10% of Prevotella isolates were categorized MDR (antibiotics involved: ampicillin, clindamycin, moxifloxacin and tetracycline). In our study, a similar rate of MDR isolates was determined (five isolates of P. melaninogenica resistant to amoxicillin, moxifloxacin and clindamycin). Secondly, in addition to beta-lactamase activity, Prevotella species are able to produce extended-spectrum beta-lactamase (ESBLs), associated with treatment failure with cephalosporin antibiotics. In a study focused on Prevotella isolates isolated from respiratory samples of PWCF, 76% were found to produce ESBLs [17]. This resistance is rarely investigated in routine practice, as in our study, and may be underestimated. Finally, resistance to metronidazole, an anti-anaerobic molecule, has been reported for P. melaninogenica [18] and Prevotella spp. [31,42,[45][46][47] with a resistance rate under 10% (Table 4). Tunney et al. [3] have observed a higher resistance rate evaluated at 46% in Prevotella spp. isolates (including four species: P. corporis, P. disiens, P. melaninogenica and P. salivae). No metronidazole resistant isolates of P. melaninogenica were described in our study.
For all the antimicrobial categories, results obtained in our study compared with previous ones showed the importance of the species level when considering antibiotic resistance and the difficulty to compare antibiotic resistance from isolates of different species belonging to the same genus [26] (Table 4). The targeted study based on species is therefore the most informative. In routine practice, this approach requires equipment (e.g., anaerobic chamber, collecting device for anaerobic atmosphere preservation) for the detection and identification of strict anaerobic bacteria in CF respiratory samples as well as for the realization of antibiogram.

Conclusions
This preliminary study of 50 clinical isolates of a predominant species of the lung "anaerobiome" showed a high percentage of resistance and the presence of MDR isolates, potentially exacerbated by the initiation of long-term antibiotic therapy in PWCF. In the future, it will be important to continue to characterize the anaerobic resistome by focusing on species rather than genera in order to better prevent the emergence of resistance within pulmonary microbiota.