The Relationship between the Biofilm Genes and Antibiotic Resistance in Stenotrophomonas maltophilia

Objectives Today, Stenotrophomonas maltophilia (S. maltophilia) is a major opportunistic pathogen among hospitalized or immunocompromised patients. Antibiotic-resistant clinical isolates are increasing in several parts of the world. Various antibiotic-resistance and biofilm-forming genes are identified in this bacterium. Its capacity to form biofilms is an important virulence factor that may impact antibiotic-resistance patterns. In the current study, we evaluated the biofilm-formation capacity, antibiotic-resistance profile, and prevalence of biofilm-forming genes as well as antibiotic resistance genes among S. maltophilia isolates. Materials and Methods In this cross-sectional study, 94 clinical S. maltophilia isolates were recovered from four tertiary-care hospitals in Iran between 2021 and 2022. The presence of the selected antibiotic-resistance genes and biofilm-forming genes was examined by polymerase chain reaction (PCR). The ability of biofilm formation was examined by microtiter plate assay. The Kirby–Bauer disc diffusion method was used to evaluate the trimethoprim-sulfamethoxazole (TMP-SMX), levofloxacin, and minocycline resistance. Results S. maltophilia is mainly isolated from bloodstream infections. Notably, 98.93% of isolates were biofilm producers, of which 19.35%, 60.22%, and 20.43% produced strong, moderate, and weak biofilm, respectively. The frequency of biofilm genes was 100%, 97.88%, 96.80%, and 75.53% for spgM, rmlA, smf-1, and rpfF, respectively. Isolates with the genotype of smf-1+/rmlA+/spgM+/rpfF+ were mostly strong biofilm producers. Among the antibiotic-resistance genes, the Smqnr, L1, and sul1 had the highest prevalence (76.59%, 72.34%, and 64.89), respectively. Antimicrobial susceptibility evaluation showed 1.06%, 3.19%, and 6.3% resistance to minocycline, TMP-SMX, and levofloxacin. Conclusion The results of the current study demonstrated that S. maltophilia isolates differ in biofilm-forming ability. Moreover, smf-1, rmlA, and spgM genes were presented in all strong biofilm producers. Although the overall resistance rate to the evaluated antibiotics was high, there was no statistically significant relation between antibiotic resistance and the type of biofilm.


Introduction
Stenotrophomonas maltophilia (S. maltophilia), an opportunistic, Gram-negative pathogen, can cause a wide variety of infections, including pneumonia, bacteremia, sepsis, meningitis, endocarditis, urinary tract infections, skin and soft tissue infections, and endophthalmitis [1].One of the fundamental problems in the diagnosis of S. maltophilia is the high similarity with other nonfermenting Gram-negative bacteria, which has created many challenges in the diagnosis, treatment, and the prevalence of antibiotic resistance around the world.To overcome the problems associated with defnitive identifcation, molecular methods have been developed [2].It is mainly found in hospitalized patients, especially among the immunosuppressed and immunocompromised and those with medical implants, receiving broad-spectrum antibiotics, or sufering from cystic fbrosis [3].Various virulence factors could be involved in the pathogenesis of S. maltophilia, including protease StmPr1, exopolysaccharides, lipopolysaccharides, siderophores, and the ability to form the bioflm [4].Trough bioflm formation, these bacteria could grow on biotic and abiotic surfaces, acting as a source of various infections and associated with more than 60% of acquired nosocomial infections [5].Bioflms play a crucial role in the persistence of S. maltophiliahealthcare-associated infections, especially in patients with implanted medical devices and cystic fbrosis patients [6].Furthermore, bacterial bioflms increase the survival rate of bacteria under harsh conditions in the host imposed by the immune system or antibiotic therapies.Te occurrence of antibiotic resistance due to bioflm formation makes treatment options more difcult [7].It should be noted that S. maltophilia is usually resistant to several antibiotics because it confers various mechanisms of drug resistance, such as decreased permeability, production of beta-lactamase and carbapenemase enzymes, aminoglycoside-modifying enzymes, and MDR efux pumps [8,9].At least two chromosomally mediated inducible beta-lactamases, L1 and L2, are produced by S. maltophilia.An ampR-class A beta-lactamase module comprises the nearby ampR gene and gene L2, which encodes a class A beta-lactamase.L1 is a class B beta-lactamase with no nearby regulatory genes resembling ampR [10].Te presence of L1 contributes to hydrolyzing the carbapenems and resistance to clavulanic acid, cephalosporins, and penicillins.L2 confers resistance to cephalosporins, penicillins, and aztreonam.Smqnr causes intrinsic resistance to quinolones [11].Generally, this organism usually is susceptible to fuoroquinolones, minocycline, and ceftazidime [12].Several studies have reported that trimethoprimsulfamethoxazole (TMP-SMX) remains the drug of frst choice for treating S. maltophilia infections.However, resistance to this antibiotic has been attributed to the presence of sul1, sul2, and dfrA genes [13].Bioflms hinder the penetration of antibiotics; therefore, therapies may be faced with failure [14].Accordingly, investigating the genes involved in bioflm formation and their role in the degree of bioflms has great importance.It can be helpful in the identifcation of strong bioflm producer isolates and adopting treatment strategies.So, the purpose of the present study was to evaluate antibiotic-susceptibility patterns, bioflm-forming capacity, antibiotic resistance genes, bioflm formation-associated genes, and the relationship between these genes with the degree of bioflm-formation capacity in S. maltophilia isolates.

Sample Collection.
In this cross-sectional study, conducted from December 2021 to August 2022, 94 diferent clinical isolates of S. maltophilia were recovered from patients admitted to four tertiary-care hospitals in Iran (Tehran, Mashhad, Shiraz, and Qazvin).Tis study was approved by the Ethics Committee of Shahed University "IR.SHAHED.REC.1400.175."Te samples were identifed using conventional microbiological and biochemical methods, including catalase and oxidase tests, DNase, urease, and reactions in media such as triple sugar iron (TSI) agar (Merck, Germany), Simmons citrate agar (Merck, Germany), and sulfde indole motility (SIM) (Merck, Germany) [15].Stock cultures were stored in a tryptone soy broth (TSB) medium (Merck, Germany) containing 20% glycerol at −80 °C until analysis.Escherichia coli (E.coli) ATCC 25922 and S. maltophilia ATCC 13637 were quality control strains.

DNA Extraction and Molecular
Identifcation.All confrmed clinical isolates of S. maltophilia were cultivated on blood agar medium (Merck, Germany) and incubated at 37 °C overnight.DNA was then extracted from colonies using the DNall Plus Kit (ROJE Co., Iran) according to the manufacturer's protocol.Te total DNA concentration and purity were checked using the NanoDrop (WPA Biowave II Nanospectrophotometer, USA).
Finally, clinical isolates were confrmed based on the PCR amplifcation of the 23S rRNA gene and sequencing.PCR was conducted in a fnal volume of 25 μl containing three μl of template DNA, 12.5 μl of Master Mix (2X) (ROJE, Iran), 1 μl of each primer (10 pmol), and 7.5 μl of sterile distilled water.More details about the primers and PCR conditions are given in Table 1.

Molecular Detection of Bioflm Formation and Antibiotic-
Resistance Genes.Te presence of bioflm-formation genes, including smf-1, rmlA, spgM, and rpfF, along with the antibiotic-resistance genes, including L1, L2, sul1, sul2, sul3, dfrA13, and Smqnr, was assessed by PCR using the primers and conditions indicated in Table 1.Amplifcations were carried out in a thermocycler (Eppendorf, Master Cycler Gradient, Germany).PCR products were analyzed by electrophoresis on 1.5% agarose gel in TBE bufer, stained with DNA-safe stain (SinaClon-Iran), and then visualization under the UV transilluminator.
2.4.Bioflm-Formation Assay.Microtitre plate assay method was used to investigate bioflm-formation capacity (strong, moderate, or weak) in S. maltophilia isolates, as described previously [21].Briefy, an overnight culture of S. maltophilia in TSB with an optical density (OD) at OD600 � 0.1 (10 8 CFU/ml) was prepared; then, 200 μl of suspension was inoculated into a sterile 96-well fatbottomed polystyrene microplate (SPL, Korea).Negative control wells contained fresh TSB media, and all experiments were triplicate.Te microplates were incubated aerobically for 24 h at 37 °C.After incubation, wells were washed three times with 250 μl of the phosphate-bufered saline (PBS) (pH 7.2).Te adherent bioflms were fxed with 250 μl of methanol; after 15 min, the wells were discarded and dried at room temperature and stained for 15 min with 200 μl of 1% crystal violet.Each well was rinsed three times with PBS to remove extra dye and then dried.In the end, bioflm samples were resolubilized with 200 μl of acetic acid (33%) for 15 min, and the OD of each well was measured by 490 nm using a microplate reader (Elx808, BioTek, USA).

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International Journal of Microbiology

Patients and Samples.
Ninety-four clinical isolates of S. maltophilia were collected during the study period.Te results of tests for initial identifcations of clinical isolates are demonstrated in Figure 1.Moreover, the identity of all of the isolates was confrmed by amplifying the 23S rRNA gene by molecular PCR method; and in all of the clinical isolates, the PCR product was 520 base pairs long (Figure 2).Te isolates were recovered from 57 (60.64%) males and 37 (39.36%)females.Te patient's ages ranged from 7 months to 95 years.Isolate sources included blood (n � 86), urine (n � 3), tracheal secretion (n � 2), sputum (n � 2), and wound (n � 1) (Table 2).Te majority of isolates were collected from patients hospitalized in the ICU (n � 40), followed by surgery (n � 19), internal (n � 17), emergency ward (n � 12), pediatric ward [5], and coronary care unit (CCU) (n � 1).3).

Investigation of Antibiotic-Resistance Genes and Teir
Relation with the Degree of Bioflms.Te most and least genes present in S. maltophilia isolates were Smqnr (76.59%) and dfrA13 (5.32%), respectively (Table 4).Te sul3 gene was not observed in any of the clinical specimens.Te most antibiotic-resistance genes in strong and moderate bioflms strains were sul1 and dfrA13 genes, respectively (Table 4).Te gel electrophoresis image of PCR products is demonstrated in Figure 4.

Antibiotic-Susceptibility Profle. S. maltophilia isolates
showed the highest resistance to levofoxacin.Resistance to levofoxacin and TMP-SMX was 6.3% and 3.19%, respectively.Only one isolate was resistant to minocycline (Table 5).

Discussion
S. maltophilia is an opportunistic pathogen whose infectioncrude mortality rate is about 14% to 69% of patients [24].Te ability to produce bioflms on biotic and abiotic surfaces, including indwelling medical devices, leads to an increasing prevalence of drug resistance in these Gram-negative bacteria [25].Tis study demonstrates the genes involved in bioflm formation and antibiotic resistance and the correlation of these genes with the degree of bioflm-formation capacity in clinical isolates.Similar to the fndings of this study, Bostanghadiri et  International Journal of Microbiology and 1.06% of isolates were strong, moderate, weak, and none-bioflm producers, respectively.Consistently, in various studies that investigated bioflm-forming ability in S. maltophilia isolates, a signifcant variation was reported [5,16,29].Tese variations may be related to the presence of various bioflm-forming genes.In our research, we investigated the prevalence of bioflm-forming genes among S. maltophilia strains.We observed that the frequency of spgM, rmlA, smf-1, and rpfF genes was 100%, 96.80%, 97.88, and 75.53, respectively.Since the formation of bioflms involved in antimicrobial resistance as well as immune evasion, the average ability of bioflm formation of S. maltophilia isolates has been investigated by various studies, including strong bioflm producers with a frequency of 10% to 98.4%, moderate bioflm producers with the frequency of 21.3% to 46.6%, and weak-bioflm producers with a frequency of 16% to 36.6% [30].spgM encodes a bifunctional enzyme with both phosphoglucomutase and phosphomannomutase activities [5].Diferent reports revealed that the spgM gene could play an important role in  International Journal of Microbiology bioflm development, and its presence was signifcantly associated with producing strong bioflm in S. maltophilia isolates [16,29].et al. [31] and Duan et al. [32] reported that all S. maltophilia isolates harbor the spgM gene, similar to our study.Te rpfF gene plays a signifcant role in producing the signal molecule known as a difusible signal factor, a highly conserved quorum sensing signal in Gramnegative bacteria [33].Madi et al. [34] and Zhuo et al. [5] reported that the lowest frequency of bioflm-forming genes belonged to rpfF, similar to the current study.Tese results demonstrated that geographical variation does not afect the prevalence of this gene in S. maltophilia isolates.smf-1 involves adhesion to various surfaces and the initial stages of bioflm formation.In China and Egypt, the frequency of the smf-1 gene in S. maltophilia isolates was as high as 100% and 90%, respectively [28,32].Unlike our study, a low level    [35].Tis might be due to time of study.Te rmlA encodes a glucose-1-phosphate thymidyl transferase.Tese products are essential for the biosynthesis of lipopolysaccharide (LPS) O-antigen, which is involved in bioflm formation and twitching motility [36].In previous studies, the rmlA gene had a high frequency in S. maltophilia isolates [5,16,34].Our fndings demonstrated increased resistance to several antibiotics commonly used in S. maltophilia infections in Iran [11,31,37,38].Te antimicrobial susceptibility profle showed that 1.06%, 3.19%, and 6.3% of isolates were resistant to minocycline, TMP-SMX, and levofoxacin, respectively.Tese results emphasize the urgent need to address the inappropriate use and  International Journal of Microbiology prescription of antibiotics and implement robust control policies to limit the dissemination of resistant S. maltophilia strains and protect public Gajdács and Urbán reported that 12.1% of S. maltophilia isolates were TMP-SMXresistant, while 8.99% were resistant to levofoxacin [39].
Compared to our study, the resistance to TMP-SMX was almost four times higher, possibly due to the diference in the geographical area, type of antimicrobial susceptibility test, and the year of study.TMP-SMX is the primary antimicrobial drug of choice for treating S. maltophilia infections although, in recent years, increasing rates of resistance from 2.3% to 77% were reported [30].Based on a systematic review study, fuoroquinolones demonstrate comparable efects on the mortality of S. maltophilia infection compared with TMP-SMX, supporting the use of fuoroquinolones (mainly levofoxacin) in S. maltophilia infections [40].However, a high resistance level is also increasingly mentioned [26].Te increase in antimicrobial resistance continues to be a global health crisis [41].One of the mechanisms involved in resistance to various antibiotics in S. maltophilia isolates is the presence of antibioticresistance genes.Our research investigated the prevalence of antibiotic-resistance genes, including L1, L2, Smqnr, sul1, and sul2, among S. maltophilia strains in patients referred to diferent hospitals in Iran.Our fndings revealed that 64.89%, 32.97%, and 76.59 strains harbored the sul1, sul2, and Smqnr genes, respectively, in accordance with those obtained by Ebrahim-Saraie [42].Tis result indicated high prevalence of antibiotic-resistance genes in Iran, and this issue can be alarming for spread of resistant isolates in society.In a study conducted by Yinsai et al. in Tailand, only 6% and 2% of S. maltophilia strains were positive for the sul1 and sul2 genes, respectively, which contradict our results [43].Tese variations in prevalence could be attributed to diferences in population, time of study, clinical samples, and geographical factors.In the current study, the presence of dfrA13 was reported in clinical isolates of S. maltophilia for the frst time in Iran, and the frequency of this gene was 5.32%.Similar to other studies, sul3 was not detected in clinical isolates of S. maltophilia [44,45].Tis newest sulfonamide-resistance gene is usually located on plasmid and especially isolated in nonclinical specimens including water, soil, sewage loving animals, and animal farm [18].More clinical studies are required to fgure out better.Similarly, various studies revealed that most S. maltophilia isolates were recovered from blood and mainly isolated from males [11].Te advent of bloodstream infections caused by S. maltophilia and its associated complications has signifcantly increased in recent years, with the range of mortality rate as 30-51% [46].Tis rise can be attributed to the excessive and frequent consumption of available antibiotics.Notably, our study found that all resistant isolates were obtained from blood samples of patients with bacteremia in diferent regions of Iran.S. maltophilia was mainly isolated from the ICU, which is similar to the study by Ibn Saied et al. [47].Considering that most patients who are hospitalized in the ICU are immunocompromised, more attention should be given to the accurate identifcation and proper treatment of this opportunistic bacterium.Tis study has some limitations that can be mentioned as follows: First, almost all S. maltophilia isolates were bioflm producers; therefore, we could not compare various genes in clinical specimens with/without bioflm.Second, the other mentioned antibiotics based on ETEST according to CLSI guidelines were not reported in the current study; therefore, we could not demonstrate a comprehensive antimicrobial drug susceptibility profle in S. maltophilia isolates.Tird, we could not present the phylogenetic relationship of antibioticresistant and strong bioflm-producer isolates in the current study due to the lack of molecular typing.

Conclusion
Te ability to form bioflms in S. maltophilia makes antibiotics inefective and rapidly growing drug resistance of pathogenic bacteria.It is demonstrated that although the capacity to form bioflm in clinical isolates of S. maltophilia was highly conserved, there are multiple phenotypic variations among them.Moreover, the spgM gene was presented in bioflm-and nonbioflm-producing isolates.Furthermore, the most antibiotic-resistance gene in the two mentioned groups was Smqnr.

Figure 1 :
Figure 1: Conventional microbiological and biochemical methods for identifcation of S. maltophilia: (a) positive reaction for DNase.(b) Growth on blood agar medium after 24 hours of incubation at 37 °C.(c) 1: nonfermenting organisms in TSI medium; 2: motile in SIM medium; 3: growth on Simmons citrate agar.(d) Negative urease test.
2.6.Statistical Analysis.Data analysis was performed using SPSS version 25.0 (IBM Corp., Armonk, NY, USA).Te chisquare (χ 2 ) test was conducted to determine the association between the two categorical variables.P value ≤0.05 was considered statistically signifcant.

Table 2 :
Clinical sources of S. maltophilia and their prevalence in diferent bioflm degrees.
of the smf-1 gene was seen in the study conducted by Mohagheghzadeh et al.