Characterization of Phenotypic and Genotypic Diversity of Stenotrophomonas maltophilia Strains Isolated From Selected Hospitals in Iran

Abstract

<i>Stenotrophomonas maltophilia</i> is an environmental Gram-negative bacterium that has rapidly emerged as an important nosocomial pathogen in hospitalized patients. Treatment of <i>S. maltophilia</i> infections is difficult due to increasing resistance to multiple antibacterial agents. The purpose of this study was to determine the phenotypic and genotypic characterization of <i>S. maltophilia</i> isolates recovered from patients referred to several hospitals. A total of 164 clinical isolates of <i>S. maltophilia</i> were collected from hospitals in various regions in Iran between 2016 and 2017. Antibiotic susceptibility testing was performed by disc diffusion method and E-test assay according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The ability of biofilm formation was assessed with crystal violet staining and then, biofilm-associated genes were investigated by PCR-sequencing method. The presence of <i>L1</i> (a metallo-β-lactamase), <i>L2</i> (a clavulanic acid-sensitive cephalosporinase), <i>sul1</i> and <i>sul2</i> (resistance to Trimethoprim/Sulfamethoxazole), Sm<i>qnr</i> (intrinsic resistance to quinolones), and <i>dfrA</i> genes (dihydrofolate reductase enzyme that contributes to trimethoprim resistance) was also examined by PCR-sequencing. Relative gene expression of <i>smeDEF</i> efflux pump was assessed by real-time PCR. Genotyping was performed using the multi-locus sequencing typing (MLST) and repetitive extragenic palindromic-PCR (Rep-PCR). Isolates were resistant to imipenem (100%), meropenem (96%), doripenem (96%), and ceftazidime (36.58%). Notably, 5 (3.04%) isolates showed resistant to trimethoprim-sulfamethoxazole (TMP-SMX), an alarming trend of decreased susceptibility to TMP-SMX in Iran. Minocycline and levofloxacin exhibited the highest susceptibility of 91.46 and 99.39%, respectively. Using the crystal violet staining, 157 (95.73%) isolates had biofilm phenotype: 49 (29.87%), 63 (38.41%), and 45 (27.43%) isolates were categorized as strong-, moderate- and weak-biofilm producer while 7 isolates (4.26%) were identified a non-biofilm producer. Biofilm genes had an overall prevalence of 145 (88.41%), 137 (83.53%), and 164 (100%) of <i>rmlA</i>, <i>rpfF</i>, and <i>spgM</i>, respectively. <i>L1</i>, <i>L2</i>, <i>Smqnr</i>, <i>sul1</i>, and <i>sul2</i> resistance genes were detected in 145 (88.41%), 156 (96.12%), 103 (62.80%), 89 (54.26%), and 92 (56.09%) isolates, respectively. None of the <i>S. maltophilia</i> isolates were positive for <i>dfrA12</i>, <i>dfrA17</i>, and <i>dfrA27</i> genes. Gene expression analysis showed that <i>smeD</i> efflux system was overexpressed in two out of the five clinical isolates (40%) that showed resistance to TMP-SMX. Most of the isolates were genetically unrelated. Two new sequence types (ST139 and ST259) were determined. Our results showed that TMP-SMX was still an effective antibiotic against <i>S. maltophilia</i>. The findings of the current study revealed an increasing prevalence of antibiotic resistance and biofilm genes in clinical <i>S. maltophilia</i> isolates in Iran.