Summary

Introduction: Stenotrophomonas maltophilia, a non-fermentative bacterium, predominantly causes opportunistic infections in immunocompromised individuals and those receiving long-term, large-dose, broad spectrum antimicrobial agents. It is inherently resistant to most of the available antimicrobial agents. Trimethoprim-sulfamethoxazole (TMP/SMX), ceftazidime (CAZ), and levofloxacin (LVX) are the drugs of choice used for the infection treatment. The study aims to investigate the presence of plasmid-mediated quinolone resistance determinants of QnrA, QnrB, QnrC, QnrD, QnrS, and OqxAB genes in S. maltophilia isolates.
Materials and Methods: A total of 122 S. maltophilia isolates from various clinical specimens were tested in the study. The susceptibility of TMP/SMX and LVX was determined by disk diffusion method. The minimum inhibitory concentration value of CAZ was determined by the gradient diffusion test. Qnr (A, B, C, S, D) determinants and OqxAB were investigated using multiplex polymerase chain reaction. OqxA was investigated in the isolates and the presence of OqxB in OqxA-positive isolates.
Results: Five (4%) of the S. maltophilia isolates were resistant to TMP/SMX. Levofloxacin and CAZ resistances were determined as 6.5% and 56.5%, respectively. QnrS was detected in two of the isolates, and OqxA gene was found in three isolates. However, these isolates were not OqxB positive. One of these three positive isolates has been previously found to be QnrS-positive.
Conclusion: In our study, QnrS was detected in two and OqxA gene in three of the S. maltophilia isolates.

Introduction

Stenotrophomonas maltophilia, a non-fermentative Gram-negative bacterium, can cause nosocomial infections and is characterized by intrinsic resistance to several antibiotics^[1]. Several mechanisms contribute to its multidrug resistance (MDR), including plasmids, integrons, and transposons^[2].

Fluoroquinolones show a good activity against S. maltophilia. Nonetheless, in the last years, a considerable increase in the resistance to quinolones has been observed^{[3, 4]}. In general, two mechanisms, i.e., target modification (in the DNA gyrase and topoisomerase intravenous) and efflux by MDR pumps, contribute to the resistance to these antimicrobial agents^[5]. However, the outer membrane protein changes and the overexpression of efflux systems have been associated with an increased levels of quinolone resistance^[6].

The clinical importance of plasmid-mediated quinolone resistance is uncertain, although it is suggested to help stabilize or select for mutations in the quinolone resistance-determining region of DNA gyrase and topoisomerase, which confers high-level quinolone resistance^[7]. However, the emergence of plasmid-mediated  fluoroquinolone resistance has also been reported; different kind of mechanisms are determined:( 1) qnr, (2) aminoglycoside acetyltransferase aac(6’)-Ib-cr, (3) OqxAB and QepA^{[8, 9]}.The primary structures of QnrA, QnrB, and QnrS are similar: nine pentapeptide repeat units connected by a single glycine, followed by a cysteine, with variable number of units (22 in QnrS; 28 in QnrA; and 29 in QnrB, QnrC, and QnrD). Two plasmid-mediated quinolone transporters have been found: OqxAB and QepA^[10]. Qnr gene exists in several bacterial genera. It is found in the S. maltophilia chromosome as Qnr, which encoded a protein that contributes to the intrinsic resistance to quinolones^[11]. This gene could be plasmid-borne, resulting in high resistance to quinolones in wild-type and mutant bacteria. The first plasmid-mediated quinolone resistance determinant (later termed QnrA1) was first reported in 1998 from Klebsiella pneumoniae^[12-14]. The plasmid-mediated QnrA gene source has been identified in the Shewanella algae chromosome, while the possible source of plasmid-mediated QnrS determinants was identified in the Vibrio splendidus chromosome^{[15, 16]}.

This study aimed to investigate the presence of plasmid-mediated quinolone resistance determinants of QnrA, QnrB, QnrC, QnrD, QnrS, OqxA, and OqxB genes in S. maltophilia isolates.

Methods

A total of 122 S. maltophilia isolates were collected in January-December 2016, from various clinical specimens. Only one isolate per patient were tested in the study. Bacterial identification was made using standard algorithms (microscopy, culture characteristics, oxidase reaction), followed by an automated system using the Vitek MS (bioMerieux, France). The susceptibility of trimethoprim-sulfamethoxazole (TMP/SMX) and levofloxacin (LVX) were determined using the disk diffusion method, and the minimum inhibitory concentration (MIC) value of ceftazidime (CAZ) was determined using the gradient diffusion test. Levofloxacin and CAZ and TMP/SMX susceptibilities were interpreted according to the Clinical and Laboratory Standards Institute criteria and the European Committee on Antimicrobial Susceptibility Testing, respectively, established for S. maltophilia^{[17, 18]}. The isolates were stored at -60 °C until the study time.

DNA extraction was performed using a boiling technique for polymerase chain reaction (PCR).

Qnr (A, B, C, S, D) and OqxAB determinants were investigated by multiplex PCR using specific primers^[19-21]. OqxA and the presence of OqxB were investigated in the isolates and in OqxA-positive isolates. The primers used in the PCR assays were given in Table 1. QnrA-, QnrB-, QnrC-, QnrS-, and OqxAB-positive strains were used as a positive control and Escherichia coli ATCC 25928 as negative control in the PCR assay. The Ethical Committee of the Ondokuz Mayıs University, Faculty of Medicine, approved our study (decision number: B.30.2.ODM.0.20.08/471). The study followed the Declaration of Helsinki principles. Informed parental consent was not obtained due to the nature of the study-only the isolates were tested, and the patients’ electronic data were used without ID information.

Table 1: Sequence of primers

Statistical Analysis

All data were recorded in Excel spreadsheets, and categorical variables were expressed as numbers and percentages.

Results

The clinical sites of S. maltophilia infection were primarily the bloodstream (35.3%) and respiratory tract (33.6%) (Table 2).

Table 2: Distribution of the clinical specimens and QnrS- and OqxA-positive isolates

Out of 122 S. maltophilia isolates, 5 (4%) were resistant to TMP/SMX. Levofloxacin and CAZ resistances were determined as 6.5% and 56.5%, respectively.

QnrS was detected in two isolates (2 and 54). QnrA, QnrB, QnrC, and QnrD were not detected. The two QnrS-positive isolates were sequenced and confirmed and found that one was susceptible to LVX and the other was resistant to LVX. Both QnrS-positive isolates were susceptible to TMP/SMX and CAZ.

The presence of OqxA gene was found in three isolates (54, 145, 147) (Figure 1). One of these isolates has been previously found to be QnrS-positive (isolate 54). However, the presence of OqxB gene was not found. There was no positive isolates for OqxAB. One of the OqxA-positive isolate was resistant to LVX, and the other two were susceptible.

Figure 1: Polymerase chain reaction gel image made with OqxAB-positive strain (M, marker; 1, OqxAB-positive strain; bp:392 2; one of the clinical strains found to be OqxAB-positive and detected to be OqxAB-negative)

Discussion

S. maltophilia is accepted as a pathogen with gradually increasing importance recently. This could be caused by increasing number of immune-compromised patients, prolonged hospital stay and increasing use of wide spectrum antibiotics like carbapenems. S. maltophilia is a bacterium occurring in almost any aquatic or humid environment, including the drinking water supply^[22]. Also, it has emerged as an opportunistic pathogen affecting primarily the hospitalized and debilitated host. Nevertheless, the ability of S. maltophilia to colonize airway epithelia and plastic surfaces of indwelling medical devices made it a major nosocomial pathogen, especially in the ICU setting^[23].

Quinolones are widely used due to their broad spectrum. In a study, it has been stated that ciprofloxacin is the most commonly used antibacterial agent all over the world^[24].

Quinolones inhibit topoisomerization by forming drug-enzyme-DNA complexes. By creating a barrier in front of replication-competent RNA polymerase and DNA helicase, they cause events resulting in cell death^{[25, 26]}.

The quinolone resistance occurs through the three main mechanisms: (i) the changes in the chromosomal targets of the quinolones, (ii) reduced accumulation due to reduced membrane permeability and/or excessive working of the efflux pump systems, and (iii) plasmid-mediated resistance^[27].

The first plasmid-mediated quinolone was termed Qnr (standing for quinolone resistance). Currently, there are several named and described alleles: QnrA, QnrS, QnrB, QnrC, QnrD, QnrE, and QnrVC. The second, aac(6’)-Ib-cr, is a variant aminoglycoside acetyltransferase, capable of reducing ciprofloxacin activity. Finally, quinolone extrusion, a prevalent chromosomally encoded resistance mechanism, has also been found to be plasmid-borne and mediated by QepA or OqxAB^{[28, 29]}.

Qnr determinants isolated from many bacteria belong to Enterobacteriaceae family so far, although they are isolated most often from Klebsiella pneumoniae, Enterobacter spp., E. coli, and Salmonella enterica isolates^[28]. The Qnr determinants were also determined in Pseudomonas sp. isolates, with prevalence ranging 0.2-94%^{[10, 28]}. S. maltophilia spp. contains a chromosomally encoded Qnr gene, which confers low-level quinolone resistance upon its expression in a heterologous host. Plasmid-mediated quinolone resistance genes were investigated in S. maltophilia isolates in some studies. Qnr is identified in the genome of S. maltophilia strain R551-3. It codes for a 219 amino acid protein that shares about 60% amino acid identity with qnrB and has two domains of 5 and 28 pentapeptide repeats separated by a glycine^{[30, 31]}.

Furlan et al.^[32] investigated the antimicrobial resistance profile, the presence of mutations in the quinolone resistance-determining region and acquired resistance genes, and the different plasmid families in S. maltophilia isolated from Brazilian soils (150 varieties of agricultural soils). Study results showed the presence of QnrA and OqxAB genes (first time) and QnrB and QnrS genes (second time) in S. maltophilia. Ebrahim-Saraie et al.^[33] reported the occurrence of antibiotic resistance and resistance mechanisms among S. maltophilia clinical isolates (n=44) from Iranian patients. Qnr gene was found in 29 (65.9%) of the isolates.

In a study, the molecular epidemiology of S. maltophilia strains associated with an outbreak in the Children’s Hospital of México Federico Gómez was investigated. Twenty-one clinical S. maltophilia strains were recovered from cultures of blood and urine samples from 10 pediatric patients at the emergency department. Nine environmental S. maltophilia strains recovered from faucets in the same area were included. The presence of sul1, qnr, intl1, pilU, hlylII, and rmlA were investigated by multiplex PCR. Qnr genes were detected in 26/30 isolate (86.66%)^[34].

The pOLA52 plasmid, causing olaquindox (quinoxaline derivative used in agricultural feeds as growth promoter) antibiotic resistance, was identified from E. coli strains isolated from porcine embryos^[35]. OqxAB, a multidrug efflux pump of resistance-nodulation-division family and recently detected in E. coli isolates from humans, was shown as the resistance’s reason^[36]. It is also found in K. pneumoniae chromosome and causes olaquindox resistance according to different expression ratios^[21]. The plasmid pOLA52 was detected to cause an 8-16-fold increase in the MIC values of nalidixic acid and ciprofloxacin in E. coli strain without the AcrA gene^[37].

Among the 556 (1.8%) E. coli strains isolated between 1995 and 1998 in Denmark and Sweden, 10 have an MIC of olaquindox of 64 g/ml, of which 9 strains had oqxA gene detected in their isolates^[36]. Plasmid-mediated OqxAB was first detected in a human clinical E. coli isolate from South Korea^[21].

Fluoroquinolone resistance in S. maltophilia isolates occurred by multiple chromosomally encoded resistance determinants, including efflux pumps, plasmid-mediated quinolone resistance determinants, enzymes, and the quinolone resistance protein SmQnr^[38]. Levofloxacine resistance mechanisms in isolates tested in this study could be other mechanisms than plasmid-mediated. In our study, we determined two QnrS-positive isolates. The presence of OqxA gene was found in three isolates. However, OqxB was not detected. This was the first study that investigated the presence of OqxAB in S. maltophilia isolates. Our study’s limitation is that the number of isolates is limited because S. maltophilia was uncommon than the Gram-negative bacteria, Enterobacterales or P. aeruginosa.

Conclusion

S. maltophilia is an important opportunistic pathogen and resistant to many antibiotics because the patients’ early isolation prevented the clonal spread of S. maltophilia clinical isolates. In our study, QnrS was detected in two and OqxA gene in three S. maltophilia isolates. Monitoring S. maltophilia’s clinical distribution and antimicrobial resistance is of great significance for the bacterial infection’s clinical therapy.

Ethics

Ethics Committee Approval: The Ethical Committee of the Ondokuz Mayıs University, Faculty of Medicine, approved our study (decision number: B.30.2.ODM.0.20.08/471).

Informed Consent: Informed parental consent was not obtained due to the nature of the study.

Peer-review: Externally and internally peer-reviewed.

Authorship Contributions

Concept: Y.T.Ç., A.B., Design: Y.T.Ç., Data Collection or Processing: Y.T.Ç., İ.B., Analysis or Interpretation: Y.T.Ç., İ.B., A.B., Literature Search: İ.B., A.B., Writing: Y.T.Ç., İ.B.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.