The Synergetic Effect of Sitaoxacin-Arbekacin Combination in the Mycobacteroides abscessus Complex

Mycobacteroides abscessus (M. abscessus) is the most commonly isolated rapidly growing mycobacteria (RGM) and is one of the most antibiotic-resistant RGM with rapid progression, therefore, treatment of M. abscessus is still challenging. The genus mycobacterium has long been known to have both rough and smooth colony phenotypes. Rough morphotypes generally are more virulent than smooth morphotypes. Recently, the ecacy of sitaoxacin, new uoroquinolone (FQ), containing regimens against M. abscessus complex have been reported. We here presented a new combination treatment with sitaoxacin that targeted rough morphotypes of M. abscessus, causing aggressive infections.


Conclusion
We demonstrated the synergistic effect of the sita oxacin-arbekacin combination against M. abscessus complex. Further, this combination regimen might be more effective against M. abscessus subsp. abscessus or rough morphotypes of M. abscessus complex.

Background
Nontuberculous mycobacteria (NTM) are environmental pathogens that can cause diverse types of infectious diseases in humans. NTM are classi ed into RGM where colony formation requires less than seven days and slowly growing mycobacteria (SGM) forming colonies at least seven days. M. abscessus is the most commonly isolated RGM and the third most common cause of respiratory NTM in the United States [1]. Pulmonary disease caused by M. abacessus mostly occur in the setting of structural lung conditions such as bronchiectasis, chronic obstructive pulmonary disease (COPD), and cystic brosis (CF). These infections are often incurable and associated with rapid lung function decline [2,3]. M. abscessus isolates are uniformly resistant to the standard antituberculous agents. New NTM treatment guidelines were published in 2020 [4]. The guidelines introduced new treatment options, including inhaled amikacin, tigecycline, and clofazimine [5][6][7]; however, the treatment bene ts were limited to negative conversion of sputum, and radical cure for M. abscessus pulmonary disease was obtained with limited disease and a combination of surgical resection of involved lung and chemotherapy [1]. Recently, the e cacy of sita oxacin, new uoroquinolone (FQ), containing regimens against M. abscessus complex have been reported [8,9]. FQs are recommended for use as second-choice drugs in treating TB, although they are only weakly e cacious against MAC infections [10][11][12]. Sita oxacin, with a chloro substituent at the C-8 position, is a newly developed oral quinoline, exhibiting good antimicrobial activity against extracellular and intramacrophage MAC compared to levo oxacin in vitro and in vivo [13][14][15][16]. These previous papers suggest that uoroquinolone combining regimens could have a potency for the effective treatment of M. abscessus complex.
Mycobacterium genus included Mycobacterium tuberculosis (TB), and some NTM have long been known to have both rough and smooth colony phenotypes [17,18]. These phenotypes are formed by the expression levels of glycopeptidolipids (GPLs). GPLs are produced by several NTMs, including RGMs (M. abscessus, M. chelonae, and M. smegmatis) [19][20][21] and Mycobacterium avium complex (MAC) members [22][23][24]. M. abscessus can spontaneously change between a smooth form, which expresses GPLs, and a rough form, lacking GPLs. The smooth form can form bio lms and colonize surfaces; conversely, the rough morphotypes cannot form bio lms but can multiply in macrophages and cause persistent infection [25]. Rough morphotypes are generally more virulent than smooth variants for isolates lacking GPLs enhanced releasing TNF-α from macrophage [25][26][27]. Conversely, to form bio lms, smooth variants were related to protecting from surrounding factors such as other bacteria, acidic pHs, hydrogen peroxide (H 2 O 2 ), and treatment with amikacin or azithromycin [28]. Here, we presented the new sita oxacin-arbekacin combination regimens, which are more effective on rough morphotypes, causing aggressive infections, and could be a potential treatment option against M. abscessus subsp. abscessus. Susceptibility to antimicrobial agents in combination with sita oxacin Methods of incubation time and susceptibility testing were used as a reference to our previous report [29]. The difference of MICs between both colony phenotypes was evaluated (Figure 1), and MICs of sita oxacin and amikacin in rough phenotypes were signi cantly lower than smooth phenotypes (p values of sita oxacin and amikacin were 0.0004 and 0.002, respectively). Therefore, we investigated the best combination partners of sita oxacin as the potential regimens for M. abscessus, especially rough morphotypes. The susceptibility to a combination of sita oxacin and antimicrobial agents was compared to that of the antimicrobial agents alone, categorized into each subspecies of M. abscessus complex (Figure 2). The MICs of three antimicrobial agents (amikacin, imipenem, and arbekacin) were measured with or without sita oxacin. Ten isolates (90.9%) of M. abscessus subsp. abscessus were susceptible to both sita oxacin and arbekacin in the combination administration; while, 11 isolates (50.0%) of M. abscessus subsp. massiliense were susceptible. The median MICs of sita oxacin and arbekacin were signi cantly lower in the combination administration (Table S2). Figure 2 also showed the colony phenotype and antimicrobial resistance gene status. We next evaluated the most synergistic combinations by using the fractional inhibitory concentration (FIC) index as described in previous paper ( Figure 3 and Table 2) [30]. Susceptibility was divided into two classes, synergy and additive as a synergistic effect and indifference and antagonism as an antagonistic effect. Synergistic effects were shown in 8 strains (23.5%) treated with sita oxacin-amikacin combination, 9

Association of clinical features with susceptibilities to the sita oxacin-arbekacin combination
We investigated whether susceptibility to the sita oxacin-arbekacin combination might associate with clinical or isolate status. The rough colony morphotypes revealed more synergistic effects than antagonistic effects (p = 0.008) ( Table 3). The other clinical parameters such as age, sex, smoking history, bronchiectasis lesion, and treatment history of antibiotics did not in uence the sita oxacinarbekacin combination.

Discussion
We demonstrated here the e cacy of the new sita oxacin-arbekacin combination regimen. The regimen revealed that both sita oxacin and arbekacin MICs reduction when the combination administration and signi cantly high synergistic effect against M. abscessus complex. The combination regimen showed a higher rate of susceptibility and synergistic effects against M. abscessus subsp. abscessus than M. abscessus subsp. Massiliense. These results might suggest that the combination therapy was more effective against M. abscessus subsp. abscessus, showing a high level of antimicrobial resistance. Furthermore, the combination revealed higher e cacy for the treatment of M. abscessus complex in rough morphotypes associated with aggressive infections.
In 2020, the new treatment guidelines for NTM pulmonary disease had been published [4], and new treatment strategies recommended changing the regimens based on macrolide resistance gene status. The systematic review described that treatment of macrolide containing regimens for M. abscessus subsp. massiliense led to a better culture conversion rate than M. abscessus subsp. abscessus [31]. However, the treatment options for macrolide resistance species of M. abscessus complex were lacking. We focused on a novel, highly effective FQ, sita oxacin, as a potential treatment option for the refractory M. abscessus infections.
Sita oxacin is approved in Japan, and it has been clinically used against most mycobacterial infections, mainly MAC, by its exceedingly low MIC level compared to other FQs. Eighteen patients with pulmonary MAC disease who received sita oxacin for at least 4 weeks for pulmonary MAC disease were evaluated; 10 patients (55.6%) showed improved radiological characteristics, and 8 patients (44.4%) showed negative sputum cultures at 6 months [32]. Thirty-one patients with refractory MAC-lung disease, who received sita oxacin-containing regimen for ≥ 4 weeks, were evaluated, 26% and 19% of patients showed symptomatic and radiological responses, respectively 12 months after receiving, and low sita oxacin MIC (≤ 1 µg/mL) were signi cant predictors of negative sputum culture conversion [33]. In a recent study, sita oxacin e cacy against M. abscessus has been reported. Bedaquiline-clofazimine-sita oxacin combination revealed a synergistic effect against 11 isolates of 70 M. abscessus subsp. abscessus (15.7%) [8]. In a Japanese retrospective study of 13 M. abscessus pulmonary disease, all 4 patients who received sita oxacin-containing regimens achieved negative sputum conversion after 1 year of treatment and improved radiological ndings [9]. Together with our data and previous reports, sita oxacin could be an effective antimicrobial combination partner against refractory M. abscessus complex.
M. abscessus exist in two distinct morphotypes, smooth and rough, that differ in their gross colony appearances when grown on solid media due to their differing amounts of cell wall GPLs. The smooth morphotype initially colonizes the airway mucosa, and had generally lower pathogenicity in this state. Subsequently switching from smooth morphotypes to rough morphotypes, aggressive pulmonary disease cause. Smooth morphotypes have an advantage in survival due to bio lm formation, leading to inhibit bacteria-induced apoptosis [34]. Conversely, rough morphotypes, without bio lms, induce the invasion ability mediated by apoptotic cell death [35,36]. Several clinical data have revealed increased pathogenicity from the rough morphotype. The rates of isolation of rough morphotype is higher in the CF patients with clinical symptoms [37], and case reports describe that the CF patients with rough morphotypes lead to dramatic declines of respiratory function and/or death [26,38]. Thus, the development of new treatment targeted rough morphotypes has become imperative. Our study revealed that sita oxacin-arbekacin combination had the higher synergy in rough morphotypes, the combination could be useful treatment for the patients who are isolated with rough morphotypes and/or whose disease have progressed. On the other hands, the combination had the less synergy in smooth morphotypes by bio lm activity. Interestingly, in 17 out of 19 smooth morphotypes (89.5%), sita oxacin susceptibility in the combination treatment improved as compared to alone. This data suggested that sita oxacin-arbekacin combination could be also partially effective as the treatment for smooth morphotypes of M. abscessus.

Conclusion
Our in vitro study demonstrated the synergistic effect of the sita oxacin-arbekacin combination against M. abscessus complex. Further, this combination regimen might be more effective against not only rough morphotype of M. abscessus, causing severe disease, but also M. abscessus subsp. abscessus, which is thought to reveal high resistance to antibiotics. Further studies are required to assess the clinical e cacy and adverse reactions of the sita oxacin-arbekacin combination; however, we expect that the combination could be a potential treatment option for the patients who are refractory to the standard combination therapy.  (Table 4) [39]. Sita oxasin breakpoints were treated as the same as moxi oxacin breakpoints, and arbekacin as the same as tobramycin. The effect of each agent combined with sita oxacin was evaluated using FIC index analysis [30].

Materials And Methods
Further methodological details are described in the Supplementary Materials and Methods.

Statistical analysis
Categorical variables were compared using the chi-square test or Fisher's exact test. The evaluation of changes in MIC was performed using the Wilcoxon signed-rank test. Differences were considered signi cant at p <0.05. When the chi-square test results were statistically signi cant, adjusted residuals were calculated to determine which particular associations were signi cant. Adjusted residuals were signi cant at p < 0.05 level if they were less than −1.96 or more than 1.96 and were signi cant at p < 0.01 level if they were less than −2.58 or more than 2.58. All statistical analyses were performed using the SPSS software program (version 20, IBM Japan, Japan). The study was approved by the Independent Ethics Committee at Juntendo University Hospital (approval no. 18-010 and 19-038) and adhered to the tenets of the Declaration of Helsinki.

Consent for publication
Not applicable.

Availability of data and materials
The datasets during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.