A 10-year single-center experience on Stenotrophomonas maltophilia resistotyping in Szeged, Hungary

Stenotrophomonas maltophilia is an aerobic, oxidase-negative and catalase-positive bacillus. S. maltophilia is a recognized opportunistic pathogen. Due to the advancements in invasive medical procedures, organ transplantation and chemotherapy of malignant illnesses, the relevance of this pathogen increased significantly. The therapy of S. maltophilia infections is challenging, as these bacteria show intrinsic resistance to multiple classes of antibiotics, the first-choice drug is sulfamethoxazole/trimethoprim. Our aim was to assess the epidemiology of S. maltophilia from various clinical samples and the characterization of resistance-levels and resistotyping of these samples over a long surveillance period. The study included S. maltophilia bacterial isolates from blood culture samples, respiratory samples and urine samples and the data for the samples, received between January 2008 until December 2017, a total of 817 S. maltophilia isolates were identified (respiratory samples n = 579, 70.9%, blood culture samples n = 175, 21.4% and urine samples n = 63, 7.7%). Levofloxacin and colistin-susceptibility rates were the highest (92.2%; n = 753), followed by tigecycline (90.5%, n = 739), the first-line agent sulfamethoxazole/trimethoprim (87.4%, n = 714), while phenotypic resistance rate was highest for amikacin (72.5% of isolates were resistant, n = 592). The clinical problem of sulfamethoxazole/trimethoprim-resistance is a complex issue, because there is no guideline available for the therapy of these infections.


INTRODUCTION
in ventilator machines and other equipments used for invasive procedures, in addition to water taps, humidifiers or mattress covers in hospital wards [7,8].
S. maltophilia (previously Xanthomonas maltophilia) is an aerobic, oxidase-negative and catalase-positive bacillus, which is the principal human pathogen of the genus, currently consisting of 16 different species [9]. In publications before the 1980's, S. maltophilia was reported as an infrequently isolated microorganism from clinical samples, mostly from hospital-acquired infections. Nevertheless, due to the advancements in invasive medical procedures, organ transplantation and chemotherapy of malignant illnesses, the relevance of this pathogen increased significantly since the 2000's (in correlation with the increased number of patients at risk to develop infections by bacteria with low virulence) [10]. S. maltophilia is a recognized opportunistic pathogen. The incidence of S. maltophilia infections in nosocomial settings is reported to be around 7-38 cases/ 10,000 discharges, and it is a frequent cause of outbreaks at intensive care units; in addition, increasing amount of reports highlight the role of these bacteria in community-acquired infections as well [11,12]. The main clinical manifestations of S. maltophilia infections are respiratory infections (i.e., tracheobronchitis) and bacteremia, however, infections from almost all anatomical regions have been described (e.g., meningitis, skin and soft tissue infections, genitourinary infections) [13,14]. The crude mortality rate for invasive S. maltophilia infections is quite high, especially if the patients receive inappropriate empiric therapy: 20-60% in case of bacteremia/sepsis and 20-70% in case of pneumonia [15,16]. The colonization of cystic fibrosis patients with S. maltophilia has also been extensively described, often leading to more frequent exacerbations and worse outcomes [17].
The therapy of S. maltophilia infections is challenging, as these bacteria show intrinsic resistance to multiple classes of antibiotics [9]. From a clinical perspective, resistance against b-lactam antibiotics (most notably, the carbapenem group) is a major concern; this is conferred by two zinc-dependent, chromosomally mediated b-lactamases (L1 and L2) [14,18]. In addition, a resistant phenotype may be expressed through a multitude of other mechanisms, e.g., lipopolysaccharidechanges or modifying enzymes for aminoglycosides, or through the over-expression of energy-dependent efflux pumps (e.g., SmeDEF, SmeVWX, SmeYZ), affecting susceptibility to several drugs [19]. Based on clinical experiences and current recommendations, the first-choice drug for the therapy of S. maltophilia infections is sulfamethoxazole/trimethoprim (or co-trimoxazole; 15 mg kg À1 day À1 ) [12,14]. Additionally, a recent meta-analysis has concluded that the use of levofloxacin in these infections is non-inferior to sulfamethoxazole/trimethoprim [20]. Nonetheless, in certain clinical situations (hypersensitivity to the drug, vulnerable patient population to fluoroquinolones) and in case of resistance to these agents, alternative drugs must be considered, usually in combination: these antibiotics include the tetracyclines (doxycycline, minocycline, and tigecycline), some remaining b-lactams with retained activity (ticarcillin/ clavulanate, ceftazidime), colistin, rifampin and chloramphenicol [9,12,14]. Resistance to the first-line agent sulfamethoxazole/trimethoprim is around 2-10% in Western Europe and in the US, however, resistance rates as high as 30-48% were reported from the Far East (China, Taiwan) [21]; resistance levels are generally higher in colonizer strains from cystic fibrosis patients (20-80%) [22]. Multidrug resistant (MDR) and extensively drug resistant (XDR) strains of S. maltophilia are concerning from both therapeutic and infection control perspectives, thus, the World Health Organization listed this pathogen as a "priority pathogen" for pharmaceutical companies to incentivize development of novel antibiotics [23].
Several surveillance studies have been published on the epidemiology of this pathogen, however, these epidemiological trends and resistance levels vary greatly in each hospital and geographical region; while the knowledge of local data is necessary to reflect on the regional/national situation and to allow for the appropriate choice of therapy [24]. In the present study, our aim was to assess the epidemiology of S. maltophilia from various clinical samples and the characterization of resistance-levels in these samples over a long surveillance period in a tertiary-care teaching hospital in Southern Hungary.

Clinical center
The present retrospective microbiological study was carried out at the Albert Szent-Gy€ orgyi Clinical Center, a tertiarycare teaching hospital in Szeged, Hungary. The study included S. maltophilia bacterial isolates from blood culture samples, respiratory samples and urine samples and the data for the samples from all outpatient Clinics and impatient departments, corresponding to the time period between January 2008 until December 2017. Bacterial isolates were considered separate if they were detected more than 14 days apart, or S. maltophilia isolates with different antibiotic susceptibilities were isolated [12]. Isolates collected for surveillance/infection control purposes from hospital environments were excluded from the analysis.

Sample processing and bacterial identification
Blood culture samples, respiratory samples and urine samples were processed in the Institute in accordance with international guidelines in routine bacteriology. Between 2008 and 2012, the BD Bactec (Beckton Dickinson, Franklin Lakes, NJ, USA) automated blood culture system was employed in the Institute, while from 2013 onwards, the BacT/ALERT 3D (bioM erieux, Marcy-l' Etoile, France) detection system was utilized. Blood culture bottles were incubated for 5 days (21 days, if endocarditis was suspected). Samples were cultured on blood agar, chocolate agar, eosinemethylene blue or UriSelect agar (in case of urine samples) plates (agar plates purchased from Bio-Rad, Berkeley, CA, USA). Culture plates were incubated at 37 8C for 24-48 h, aerobically. Between 2008 and 2012, phenotypic methods and VITEK 2 Compact ID/AST (bioM erieux, Marcy-l' Etoile, France) were used, while following 2012, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Bremen, Germany) was introduced to the diagnostic workflow of the laboratory. Sample preparation methods and the technical specifications for MALDI-TOF MS measurements were described elsewhere [25].

Statistical analysis
Descriptive statistical analysis was performed using Microsoft Excel 2013 (Redmond, WA, Microsoft Corp.). Additional statistical analyses were performed with SPSS software version 24 (IBM SPSS Statistics for Windows 24.0, IBM Corp. Armonk, NY, USA), using the c 2 -test and two-sample-test (isolation frequency and resistance trends). P values <0.05 were considered statistically significant.

Ethics
The study was deemed exempt from ethics review by the Institutional Review Board of the University of Szeged and informed consent was not required, as patient data was not collected and data anonymity was maintained.

RESULTS
Isolation frequency of S. maltophilia
The distribution of isolates into various resistotypes is shown in Table 1.; Type 0 represents fully-susceptible isolates (24.1%), Type I includes isolates resistant to amikacin or tigecycline only (65.4%), while Type II (1.8%) and Type III (4.0%) introduces resistance to sulfamethoxazole/trimethoprim and levofloxacin, respectively. Type IV (7.0%) represents resistance to three, while Type V (1.0%) represents resistance to four individual antibiotics. Type VI (2.2%) encompasses strains showing resistance to all tested agents. Based on EUCAST Expert Rules, isolates in Type IV and V categories also represent MDR S. maltophilia isolates, while isolates in the Type VI category should be considered XDR.

DISCUSSION
The aim of our present study was to characterize the resistance levels of S. maltophilia in a tertiary-care teaching hospital in the southern region of Hungary over a long surveillance period using phenotypic methods. S. maltophilia is an emerging, opportunistic pathogen with low levels of invasiveness, mainly affecting severely debilitated patients [29]. The following risk groups have been identified based on the literature: ICU patients or patient with a long hospital stay, extensive surgeries, immunosuppressive therapy or acquired immunosuppression (e.g., HIV-infection, severe neutropenia), mechanical ventilation, dialysis, patients with chronic illnesses (e.g., diabetes, respiratory disorders) or cancer, or a developmental abnormality [30]. Prevention of S. maltophilia acquisition and infection is very important from an infection control point-of-view, in addition to controlling antibiotic consumption for reducing the emergence of resistant strains [31]. Extensive use of carbapenems (both on a patient-level and institutional-level) has also been described as a potential risk factor for these infections (due to the selection pressure) [32]. The gastrointestinal tract, infected central venous catheters and the colonized/infected lungs were described as sources of infection, leading to invasive disease [33]. Due to its limited invasiveness, S. maltophilia must somehow bypass natural host defenses to cause illness; nonetheless, virulence factors, such as biofilm-formation (important for survival on abiotic surfaces), a positively charged cell surface and fimbriae are all considered important during the pathogenesis of these infections [34]. Previously it was hypothesized that S. maltophilia infections are characterized by the lack of an inflammatory response, however, this dogma has been recently challenged in a murine model, where it was shown that airway epithelial cells and macrophages react with an increased expression of IL-8 and TNF-a [35].
Empiric therapy of S. maltophilia infections is sulfamethoxazole/trimethoprim, combined with levofloxacin or ticarcillin/clavulanate (if available); the therapeutic protocol should be revised after the susceptibility results are available [1,2,12,14]. Resistance rates to sulfamethoxazole/trimethoprim (12.6%) was higher than the range of resistance in Western European countries (2-10%), although outlier countries with higher resistance (e.g., Spain: 25-27%, Turkey: 10-15%) have already been noted [36,37]. In contrast, the low level of levofloxacin resistance is an advantageous development, as it seems that there is no relevant difference in the clinical efficacy of these two drugs [20]. The relevance of the other three tested agents in clinical situations is harder to ascertain, as there are no evidence or clinical trials correlating their efficacy in the therapy of S. maltophilia infections [38]. In addition (as demonstrated in the Methods section), there are also contradictory information regarding susceptibility-testing method for these bacteria: based on EUCAST, disk diffusion is only available for sulfamethoxazole/trimethoprim, while CLSI offers disk diffusion testing breakpoints for levofloxacin and minocycline as well [12,14,39]. Some drugs, not even MIC breakpoints are available (thus, clinical microbiologists should not interpret them as susceptible or resistant for the treating physicians), as the pharmacokinetic/pharmacodynamic attributes, outcomes and antimicrobial efficacy of these antibiotics have not been characterized in relation with S. maltophilia infections [12,14,39]. The clinical problem of sulfamethoxazole/trimethoprim-resistance (mediated by the sul1-sul3 genes) is a complex issue. Because there is no guideline available for the therapy of these infections, clinicians often act upon national and/or institutional guidelines [40]. The development of guidelines would require reliable data from multiple clinical trials utilizing antibiotics other than sulfamethoxazole/trimethoprim, with clearlydefined case definitions and clinical endpoints; unfortunately, such data is currently not available [39,40].
The definition of resistotyping is the grouping of bacterial isolates by resistance patterns to a set of arbitrarily chosen antibiotics that are characteristic to specific strains by phenotypic methods; resistotyping is mainly used for epidemiological purposes [41]. Although data has been generated on the resistance-levels of S. maltophilia in other   [39]), resistotyping for this pathogen has not been previously described locally or in any other studies published previously. To highlight their importance, resistotypes may be correlated with clinical-therapeutic decisions: e.g., resistotypes 0, IA and IB are pan-susceptible, or resistant only to ancillary antibiotics, thus, the first-line drug (sulfamethoxazole/trimethoprim alone or in combination) may be used without difficulty, if the underlying conditions or the patient's medical history allows for it. Resistotypes IIA and IIB are resistant to sulfamethoxazole/trimethoprim, but susceptible to levofloxacin, which is presumably just as clinically-effective as the first-line drug; depending on the age of the patient, this drug may be clinically used alone or in combination (with ticarcillin/clavulanate, ceftazidime or rifampin). Resistotypes IIIA and IIIB are resistant to the tested fluoroquinolone drug, but susceptible to sulfamethoxazole/trimethoprim, corresponding to a similar therapeutic approach like resistotypes 0, IA and IB. Therapy of these infections becomes especially problematic starting from the IVA resistotype all the way onto resistotype VI, where, in addition to resistance against sulfamethoxazole/ trimethoprim and/or levofloxacin, the utility of possible secondary antibiotics is also narrowing: resistance to amikacin, tigecycline and colistin means that only very few antibiotics are left for therapy and for most of these agents, clinical evidence of efficacy is limited to case reports [42]. The relevance of amikacin is often questioned, as resistance may quickly develop due to membrane impermeability or alterations in the bacterial LPS, while colistin is considered as one of the last-resort agents, due to its nephrotoxic and neurotoxic adverse events and difficult dosing [43,44]. Several reports highlight the efficacy of tetracycline-derivatives, especially minocycline as a potential therapeutic alternative for resistant S. maltophilia infections, demonstrating high cure rates and advantageous outcomes. However, the adverse effect-profile of these drugs and the low serum concentrations achieved by tigecycline should also be taken into consideration [45]. Besides this, concerns have been raised that the frequent use of minocycline for the therapy of Acinetobacter calcoaceticus-baumannii complex may lead to the emergence of resistance in S. maltophilia [46].
The following limitations of the study should be noted: i) as the clinical data of the individual patients affected could not be accessed, the correlation between the symptoms and the isolation of S. maltophilia is unknown; thus, all true pathogens and colonizers were included in this study; ii) resistance of these isolates were characterized only phenotypically, the genetic nature of these resistance-determinants were not detected using molecular biological methods; iii) minocycline susceptibility-testing was not performed as this drug is not licensed or available in Hungary; iv) referral/ selection bias as the clinical center is a tertiary-care, specialized hospital.

CONCLUSIONS
S. maltophilia is an emerging opportunistic pathogen predominantly isolated from blood culture and respiratory tract samples, most often causing bacteremia, tracheobronchitis and soft tissue infections in hospitalized, immunocompromised patients. It is often difficult to distinguish between colonization and true infection, if the bacteria have been isolates from non-sterile body sites, however, the surveillance of colonizers is also relevant as in most cases, these microorganisms will initiate infections in susceptible hosts. The pharmacotherapy of S. maltophilia infections is limited by high-level intrinsic resistance, which is often worsened by acquired non-susceptibility. In our study, 87.4 and 90.5% of isolates were susceptible to sulfamethoxazole/trimethoprim and levofloxacin, respectively; in these cases, first-line agents are appropriate for use, bearing in mind the adverse events and contraindications associated with these drugs for specific patient groups. On the other hand, 8.0% were MDR and 2.2% was to be considered XDR strains. Thus, clinical management of infections, wherefor whatever reasonnone of the first-line agents are available for use, depends on the susceptibility of the pathogen to ancillary agents and the availability of these antibiotics on an institutional/regional/ national level. Additionally, more studies are needed to adequately assess the relevance of such antibiotics (i.e., colistin, minocycline, tigecycline, amikacin, rifampin, ticarcillin/clavulanate or ceftazidime) in the management of resistant S. maltophilia.
Funding sources: No financial support was received for this study.
Authors' contributions: M.G. and E.U. conceived and designed the study. E.U. was the senior microbiologist, performing bacterial isolation, identification and susceptibility-testing. M.G. performed data collection and analysis. M.G., E.U. wrote and revised the full paper. All authors have read and agreed to the published version of the manuscript.