Antibiotic resistance, biofilm formation, and biofilm-associated genes among Stenotrophomonas maltophilia clinical isolates

Objective The purpose of the present study was to investigate the antimicrobial susceptibility pattern, biofilm production, and the presence of biofilm genes among the S. maltophilia clinical isolates. A total of 85 clinical isolates of S. maltophilia were collected from patients referred to several hospitals. Susceptibility to antibiotics was investigated by disc diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). By the crystal violet staining method, the capability of biofilm formation was examined. The genes associated with biofilm production were investigated by the PCR-sequencing techniques. Results All isolates were resistant to doripenem, imipenem, and meropenem. Minocycline, trimethoprim/sulfamethoxazole and levofloxacin exhibited the highest susceptibility of 100%, 97.65%, and 95.29%, respectively. The results of crystal violet staining assay showed that all isolates (100%) form biofilm. Moreover, 24 (28.23%), 32 (37.65%), and 29 (34.12%) of isolates were categorized as weak, moderate, and strong biofilm producers, respectively. Biofilm genes including rpfF, spgM and rmlA had an overall prevalence of 89.41% (76/85), 100% (85/85) and 84.71% (72/85), respectively. Rational prescribing of antibiotics and implementation of infection control protocols are necessary to prevent further infection and development of antimicrobial resistance. Combination strategies based on the appropriate antibiotics along with anti-biofilm agents can also be selected to eliminate biofilm-associated infections.


Introduction
Stenotrophomonas maltophilia, previously known as Pseudomonas maltophilia or Xanthomonas maltophilia, has become nowadays a major opportunistic pathogen in hospitalized or immunocompromised patients worldwide [1]. This organism is the most prevalent nonfermenting Gram-negative bacilli in clinical laboratories after P. aeruginosa and Acinetobacter baumannii [2]. In addition, it is known to causes severe infections such as acute exacerbations of chronic obstructive pulmonary disease (COPD), pneumonia, bacteremia, sepsis, bone, and joint infections, eye infections, endocarditis, and meningitis [3,4]. S. maltophilia isolates show resistance to a variety of antibacterial agents, with various types of antimicrobial resistance mechanisms [5][6][7], leading to a great challenge for physicians and clinical microbiologists to manage related infections [8,9].
A prominent feature of S. maltophilia is its capability to adhere to abiotic surfaces, host tissues and biofilm formation [10,11]. S. maltophilia has been identified on the surfaces of biomaterials used in prosthetic devices, intravenous cannula, dental unit waterlines and nebulizers [12][13][14]. The biofilm-forming capacity of S. maltophilia has increasingly been accepted as an important virulence factor and is thought to play a significant role in the persistence of S. maltophilia infections in hospital settings [10,[15][16][17][18][19] The molecular mechanisms of biofilm formation in S. maltophilia is poorly investigated [18,19]. Several genes are associated with biofilm-formation. The spgM gene encodes a bifunctional enzyme with both phosphoglucomutase and phosphomannomutase activities that is involved in LPS biosynthesis, playing an important role in biofilm formation [18,20]. Mutation in spgM gene, may cause fewer LPS production and shorter O polysaccharide chains [21]. The rmlA gene encodes glucose-1-phosphate thymidyltransferas that is involved in LPS/EPS-coupled biosynthetic pathway It is reported that mutations in rmlA and rpfF genes resulted in reduced biofilm formation in S. maltophilia [4,19]. The rpfF gene, encoding the DSF (diffusible signal factor) synthase regulates the virulence expression, such as motility, extracellular proteases, LPS, and biofilm production. RpfF protein has some amino acid sequences similar to enoyl coenzyme A hydratases [19].
Considering the potential of biofilm in increasing antimicrobial resistance and subsequently, the increased rates of chronic infections caused by S. maltophilia, identification of the isolates with such factor will be benefit to better understand the pathogenesis of the organism. The aim of this survey was to investigate the pattern of antibiotic susceptibility, the ability of biofilm production, and the presence of biofilm-related genes in clinical S. maltophilia isolates.

Bacterial isolates and species identification
S. maltophilia isolates included in this study were originated from different clinical samples of patients admitted at selected hospitals in Tehran, Iran from January 2018 to January 2019. All of the isolates were identified by standard microbiological and biochemical methods, including catalase and oxidase tests, reactions in media, such as triple sugar iron agar, deoxyribonuclease test agar, and SIM (Merck company, Germany). S. maltophilia isolates were then confirmed by the PCR amplification of the 16S rRNA gene and sequencing ( Table 1). All isolates were stored in Luria-Bertani (LB) liquid medium (Merck company-Germany) containing 15% glycerol at − 80 °C. Escherichia coli ATCC 25,922 and S. maltophilia ATCC 13,637 were used as the quality control strains.

DNA preparation
S. maltophilia isolates were cultivated on Columbia agar medium (bioMérieux Italia S.p.A-Italy) supplemented with 5% sheep blood and incubated at 37 °C for 24 h. The DNA samples were extracted from the grown colonies of each isolate with high pure PCR Template Preparation Kit (Roche company-Germany). The total DNA concentration was evaluated by Nanodrop (WPA Biowave II Nanospectrophotometer-USA).

PCR-sequencing techniques
The presence of biofilm-encoding genes, including rpfF, spgM, and rmlA was investigated in S. maltophilia isolates by PCR technique using the specific primers (Table 1). PCRs were performed on a thermal cycler (Eppendorf, Master Cycler Gradient-Germany) in 25-μl reaction volumes with 1 μl (20 ng) of DNA template, 1 × PCR buffer, 12.5 μl of 2 × Master Mix (SinaClon-Iran), 3 mmol/L MgCl 2 , 0.4 mmol/L dNTPs, 9.5 μl of sterile distilled water, 1 μl of 10 pmol of each primer, and 0.08 IU of Taq DNA polymerase. PCR conditions were under the following: denaturation at 95 °C for 10 min, and then 36 cycles at 94 °C for 60 s, annealing at 52-60 °C, depending to the primers for each gene, for 60 s, and extension at 72 °C for 60 s, followed by a final extension at 72 °C for 5 min. PCR 16srRNA-R ACG GCA GCA CAG AAG AGC spgM-F ATA CCG GGG TGC GTT GAC spgM [18] spgM-R CAT CTG CAT GTG GAT CTC GT rpfF-F CAC GAC AGT ACA GGG GAC C rpfF [18] rpfF-R GGC AGG AAT GCG TTGG rmlA-F CGG AAA AGC AGA ACA TCG rmlA [3] rmlA-R GCA ACT TGG TTT CAA TCA CTT products were electrophoresed by 1.2-1.5% agarose gel, visualized by DNA Safe staining, and then photographed under UV light. The amplicons were purified using a PCR purification Kit (BioFact Co., South Korea), and then sequenced by an ABI PRISM 3700 sequencer (Applied Biosystems Inc., USA). The nucleotide sequences were analyzed using FinchTV software and comparisons were made using the NCBI BLAST program (http:// www. ncbi. nlm. nih. gov/ BLAST/).

Biofilm formation assay
Biofilm formation by S. maltophilia was evaluated using crystal violet staining method as previously described by Stepanović et al. [23]. All experiments were run in triplicate. An overnight culture of isolates was adjusted to the turbidity of a 1.0 McFarland standard. Suspensions were diluted at a ratio of 1:100 in 200 ml tryptic soy broth (TSB) (Merck, Darmstadt-Germany) containing 1% glucose and, were dispensed to the sterile flat-bottomed 96-well polystyrene microplates (JET Biofil, Guangzhou, China). After 24 h of incubation at 37 °C, microplates were washed three times with sterile phosphate-buffered saline (PBS, pH 7.3). Adherent biofilms were fixed for 1 h at 60 °C, stained by 200 µl Hucker modified crystal violet (Sigma Chemical Company-USA) for 5 min at room temperature and then rinsed with water and allowed to dry. Biofilm samples were destained with 200 ml 33% glacial acetic acid for 15 min. The optical density (OD) was measured at 492 nm using a microtiter plate reader (BioTek, Bad Friedrichshall, Germany). A cut-off value (ODc) was established. It is defined as three standard deviations (SD) above the mean OD of the negative control: ODc = average OD of negative control + (3 × SD of negative control). The isolates were categorized into four groups according to the following criteria: nonbiofilm producer (OD < ODc), weak-biofilm producer (ODc < OD < _2 × ODc), moderate-biofilm producer (2 × ODc < OD < 4 × ODc), and strong-biofilm producer (4 × ODc < OD).

Statistical analysis
The statistical analysis of data was performed with statistical software SPSS, 20.0 (SPSS Inc., Chicago, IL, USA). Chi-squared test was used to determine the association between categorical variables. A p-value ≤ 0.05 was considered statistically significant.

Patients and bacterial isolates
During one-year period, 85 S. maltophilia isolates were gathered from several health centers in Tehran, Iran. Among them, 49 isolates were from males and 36 isolates from females (male: female ratio = 1.36). Most of the S. maltophilia (90.03%) were isolated from the blood, while the rest (9.97%) were from the cough swabs. The range of patients' age was from 2 months to 85 years.

Discussion
S. maltophilia is increasingly identified as a cause of nosocomial infections, especially among immunosuppressed patients [24]. Treatment of infections caused by this pathogen is a problem for clinicians because of its resistance to a broad array of antimicrobial drugs [25]. In the present study, all isolates were resistant to carbapenems (p ≤ 0.001). Similarly, a previous study showed that resistance rates to imipenem and meropenem in S. maltophilia were 100% and 92.4%, respectively [26]. Moreover, our results showed a susceptibility rate of 28.24% to ceftazidime. A study by Farrell et al. showed that susceptibility of S. maltophilia against ceftazidime was 32.51% in Latin America, North America, Asian-Pacific, and Europe [26]. In contrast, Jamali et al. reported the susceptibility rate of 82% against this drug [27]. In our study, 100% and 95.29% of S. maltophilia were susceptible to minocycline and levofloxacin, respectively. Duan et al. showed also the susceptibility rates of 100% and 95.7% to minocycline and levofloxacin, respectively [28]. These findings indicate that such antibiotics serve as effective agents for treatment of S. maltophilia infections. On the other hand, the most effective antimicrobial agent used to treat S. maltophilia infections is SMX/TMP [29]. In a study by Jamali et al., 5% of isolates were resistant to SMX/TMP [30]. The susceptibility rates were reported as high as 95% in several studies conducted in most regions, including Latin America, North America, Europe [30][31][32]. In our study, 97.65% of isolates were found to be susceptible to SMX/TMP, indicating this antibiotic has increasingly become the last resort drug for the treatment of multi-resistant S. maltophilia infections. However, the highest rates of resistance have been reported in isolates obtained from patients in Asian countries, such as Malaysia, Korea, and Taiwan [33]. The present study generally reveals a low frequency of antibiotic resistance among the S. maltophilia. However, monitoring of the antibiotic resistance trends is nesserary either geographically or over time.
Biofilms have been recognized to be involved in many chronic and intractable infections [13,37]. The results of this study showed biofilm formation significantly correlated with ceftazidime and SMX/TMP resistance. Similarly, biofilm formation has been shown associated with resistance to different antibiotics, including ceftazidime, piperacillin/tazobactam, cefepime, ticacillin/clavulanic acid, aztreonam, and gentamicin [38]. more understanding of biofilm dynamics can lead to development of a effective treatment, and control, strategies for eradication of infections, improving patient care [39].
In the present study, we investigated the relationship between biofilm formation and the presence of its related genes rpfF, spgM, and rmlA. Sixty-three isolates of S. maltophilia strains had all genes studied, while only 81 strains carried the spgM. Overall, our results revealed that the presence of the spgM significantly promoted biofilm formation, in accordance to those obtained by Pompilio et al. [40]. In a study by Zhongliang Duan et al. the rates of spgM, rmlA, and rpfF biofilm genes were 100%, 83.7%, and 45.2%, respectively  [17]. Zhuo et al. indicated that biofilm formation was considerably associated with the presence of rpfF and spgM genes [18]. Moreover, the presence of either rpfF or spgM was significantly correlated to biofilm production, although the strongest biofilm was formed when both genes were present [15]. The presence of spgM, rpfF, and rmlA genes improved significantly the biofilm production in our isolates.
In conclusion, although the rate of resistance to multiple antibiotics among our S. maltophilia isolates was relatively low, cautious antimicrobial use and high standards of infection prevention and control are needed to prevent further development of resistant isolates. Additionally, combination strategies based on the proper antibiotics with anti-biofilm agents can be used to improve the treatment of biofilm-associated infections.

Limitations
A limitation of this study may be the lack of evaluation of expression levels of biofilm-associated genes by quantitative real-time PCR, an approach that may help to assess the role of each corresponding gene in biofilm production.