Multiple antibiotics resistant among environmental isolates of Stenotrophomonas maltophilia

: In this study we assessed the functionality of integrons, melanin-like pigment and biofilm formation on multidrug resistance among environmental isolates of Stenotrophomonas maltophilia . Marked resistances were noted against aztreonam (60%), cefepime (68%), ceftazidime (77%), ciprofloxacin (72%), gentamicin (65%), meropenem (75%), piperacillin/tazobactam (65%) in S. maltophilia . Ticarcillin/clavulanic acid (66%) and trimethoprim/sulfamethoxazole (75%) were the active antibiotics against S. maltophilia. Class 1 intergron was significantly detected in 56.3% (54/96) of S. maltophilia strains. Integron-positive strains were significantly resistant to cefepime (69%), ceftazidime (78%), ciprofloxacin (74%), gentamicin (65%), and meropenem (72%). Gene cassettes arrays within integrons were identified as aminoglycoside resistance genes aacA4 , aadA2, aadB, aacC4 , and aacA6'-Ib ; β-lactams resistance genes bla IMP , bla OXA , and bla CARB ; chloramphenicol resistance genes cmlA and catB2 ; quaternary ammonium compound (QAC) resistance genes smr and qac ; and multi-gene cassettes: smr/aacA4 and bla IMP /aac6-II/aadA5. High-pigment-producing S. maltophilia strains revealed significant correlation with resistance to cefepime, ceftazidime, ciprofloxacin, levofloxacin and piperacillin/ tazobactam. Biofilm formation was not significant with resistance to ciprofloxacin, levofloxacin, meropenem, ticarcillin/clavulanic and trimethoprim /sulfamethoxazole. Our findings characterize the significant roles of integrons, melanin-like pigment and biofilm formation in the multidrug resistance of S. maltophilia. The range of antibiotics resistance genes and mobile genetic elements found suggests that the organism could potentially act as a reservoir of drug resistance determinants in environmental and clinical settings, which is an issue of public health concern.©

http://dx.doi.org/10.4314/jasem.v18 i2.16 Introduction: Stenotrophomonas maltophilia (formerly known as Pseudomonas maltophilia, Xanthomonas maltophilia) is a ubiquitous species from the gamma sub-division of Proteobacteria (Hayward et al., 2010). S. maltophilia is a Gram-negative opportunistic pathogen, and increasing incidence of nosocomial and community-acquired S. maltophilia infections is of particular concern for immunocompromised individuals, as this bacterial pathogen is associated with a significant fatality rate (Jumaa et al., 2006;Brooke, 2012). S. maltophilia is an environmental bacterium found in aqueous habitats, plant rhizospheres, animals, foods, contaminated medical care fluids, and water sources (Ryan et al., 2009). Infections of S. maltophilia can occur in a range of organs and tissues; the organism is commonly found in respiratory tract infections (Brooke, 2012).
S. maltophilia has emerged as one of the most frequently found bacteria in cystic fibrosis (CF) patients (Waters et al., 2007). S. maltophilia has been associated with infections of the eyes (Penland and Wilhelmus, 1996), urinary and respiratory tracts infections (Vartivarian et al., 1996). S. maltophilia possess endogenous β-lactamase production and low outer membrane permeabilit resulting to its resistant to many broad-spectrum of antibiotics including penicillins, carbapenems, and aminoglycosides (Gilligan and Whittier, 1999). The molecular mechanisms underlying pathogenicity of S. maltophilia are mainly unknown. Although its high capacity to adhere to various surfaces with biofilm formation or its intrinsic resistance to majority of commonly used antibiotics are factors that positively contribute to the infection process (Looney et al., 2009). S. maltophilia exhibits variety of mechanisms that singly or collectively contribute to its multidrug resistance (MDR) status. Intrinsic resistance includes inducible efflux pumps (Li and Nikaido, 2004;Liaw et al., 2010) and multiple β-lactamase expression (Avison et al., 2002) but not mutations in the quinolone resistance-determining region (Valdezate et al., 2005). In addition, S. maltophilia can acquire resistance through integrons, transposons, and plasmids (Barbolla et al., 2004;Liaw et al., 2010). Class 1 integrons have been characterized from S. maltophilia strains isolated in Argentina and Taiwan, which indicates that they contribute to trimethoprim /sulfamethoxazole (TMP/SMX) resistance through the sul1 gene carried as part of the 3′ end of the class 1 integron (Barbolla et al., 2004). Integrons are located on transposons or plasmids that facilitate the rapid spread of integrons to other strains and bacterial species (Jones et al., 1997). In this study we examine the functionality of integrons, melanin-like pigment and biofilm formation on multidrug resistance (MDR) among environmental isolates of S. maltophilia.

MATERIALS AND METHODS
Collection of samples: Samples were collected from different environmental settings in Benin City, Nigeria which includes:-abattoir (runoff water and effluent); hospital environment (wastewater outlets, pollutant dumpsite and reservoir water) and animal farms (feeds, water and effluent discharge).

Isolation of S. maltophilia isolates:
Stenotrophomonas maltophilia strains were isolated from environmental samples; samples were process and incubated overnight using Nutrient broth (Merck, South Africa). Aliquots of the broth cultures were spread on Mueller-Hinton agar (Merck, South Africa) and imipenem disks (Mast Diagnostics, Merseyside, United Kingdom) were applied on the bacterial lawn. Tiny colonies observed in the inhibition zone after 18 h of growth at 30 o C were carefully picked and purified (Bollet et al., 1995). All pure isolates were identified by phenotypic characteristics and standard biochemical reaction using API 20 NE test kit (bioMerieux, France). Isolate inoculation into API 20 NE strips was performed as recommended by the manufacturer (bioMerieux, France). After incubation for 48 h at 30 o C, results were read and analysed using the Analytical Profile Index (API) database (V4.1) with the apiweb TM identification software. Only confidence levels expressed as 'excellent identification' or 'very good identification' was considered in this study. All candidate isolates were confirmed by polymerase chain reaction with S. maltophilia-specific primers SM1f/SM4 as describe by Adamek et al. (2011).

Pigment bioassay
A melanin-like pigment bioassay was carried out as described by Wang et al. (2000). S. maltophilia were cultured in L-tyrosine-containing agar to facilitate the observation of brown pigment production. Pigment formation were recorded as 0, 1+, 2+ or 3+, with increased intensity and intensity equal to or greater than 2+ designated as high pigment expression.
Biofilm bioassay: The biofilm bioassay was carried out as described by Di Bonaventura et al. (2004). S. maltophilia were cultured overnight in Luria-Bertani medium, cultures were aseptically transferred to the wells of polystyrene microtitre plates. The plates were incubated at 37 o C for 10 h and subsequently stained with crystal violet.

DISCUSSION
The rapid spread of antibiotic resistance genes among bacterial isolates is an increasing problem in infectious disease control. In the study, we examined the roles of integrons, melanin and biofilm formation in relation to multidrug resistance in S. maltophilia. A significant correlation was established between the mechanisms studied and multidrug resistance. Studies have revealed that conserved DNA sequence, integron, may be carried on these episomal genetic structures (Stokes and Hall, 1989;Rowe-Magnus and Mazel, 1999). Integrons possess two conserved segments separated by a variable region that includes integrated cassettes, which often include antibiotic resistance genes (Recchia and Hall, 1995). Many resistance genes are present as gene cassettes within integrons, which may themselves be located on transmissible plasmids and transposons (Recchia and Hall, 1995).
Antimicrobial Surveillance Program isolates indicated that the newer fluoroquinolones demonstrated good efficacy; the most active were levofloxacin (6.5% resistance) and gatifloxacin (14.1%) (Sader and Jones, 2005). The resistance to levofloxacin was higher in our study (58% resistance) compare to previous the study of Sader and Jones (2005). A recent study encompassing data from Europe, Latin America and North America indicates that the level of resistance to TMP/SMX is 3.8%; however, previous studies indicate that the level is higher in Latin America than North America (Gales et al., 2001;Fedler et al., 2006). The current drug of choice for treating S. maltophilia infections is trimethoprim-sulphamethoxazole, but resistance is seen in S. maltophilia isolates due to a mobile determinant (Toleman, et al., 2007;Nicodemo and Paez, 2007). Other drugs with reasonable activity against S. maltophilia are minocycline and newer fluoroquinolones (Nicodemo and Paez, 2007). However, mutants resistant to these last resort drugs are readily selected in vitro. One mutation may be sufficient to cause resistance to these drugs, and worryingly, this mutation can be selected for in the presence of a front-line antimicrobial such as amikacin (Gould and Avison, 2006). Although surveillance studies are few, resistance to TMP/SMX appears to be emerging, and recent in vitro modeling studies have shown that combination therapies of TMP/SMX plus ciprofloxacin and TMP/SMX plus tobramycin exhibit a greater killing capacity then TMP/SMX alone (Zelenitsky et al., 2005;Al-Jasser, 2006 (2004). Such variation could be attributed to the different origins of the samples collected. The finding that the most common gene cassette carried by class 1 integron was comprised of aminoglycoside resistance determinants may account for the significant difference in susceptibility to gentamicin between the integron-positive and integron-negative isolates (strains). Integrons were significantly associated with resistance to certain antibiotics, including aminoglycosides (gentamicin), quinolones (levofloxacin and ciprofloxacin), and beta-lactam agents. This is not surprising, since many antibiotic resistance gene cassettes encoding resistance to a wide range of antibiotics have been reported previously (Sallen et al., 1995;Rowe-Magnus and Mazel, 1999).
Integrons capture genes as part of a genetic element known as a gene cassette. Most cassettes within integrons with known functions confer antibiotic or quaternary ammonium compound (QAC) resistance. Most of the integron arrays contained more than one resistance gene cassette, which can mediate resistance to multiple antibiotics. Increasing proportions of isolates containing class 1 integrons were detected in S. maltophilia (Barbolla et al., 2004;Toleman et al., 2007). Among the 17 SXT-resistant isolates, 15 (88%) carried class 1and 2 integrons of them possessed

ETINOSA O. IGBINOSA, FAITH E. OVIASOGIE
sul1genes. This report confirms the increased incidence of class 1 integron in SXT-resistant clinical isolates of S. maltophilia (Barbolla et al., 2004;Toleman et al., 2007). Toleman et al. (2006) indicates that resistance genes are linked to insertion sequence common region (ISCR) elements, which are DNA sequences found beyond and close to the 3' conserved sequences of class 1 integrons. These ISCR elements have been identified in numerous Gram-negative bacteria and a few Grampositive bacteria, and are responsible for the mobility and dissemination of many antibiotic resistance genes, including extended spectrum β-lactamase, carbapenemase genes, aminoglycoside, chloramphenicol, quinolone as well as trimethoprim resistance genes (Toleman et al., 2006). The use of quaternary ammonium compounds (QAC) in the natural environment has the potential to select for antibiotics resistance (Gaze et al., 2005). The frequent occurrence of the qac gene cassette within class 1integron in the present study implies that multidrug resistance may result from the introduction of biocides into hospital settings.
Melanin-like pigment has been shown to protect cells from environmental attack in bacteria and fungi (Coyne and al-Harthi, 1992;Gomez and Nosanchuk, 2003). In this study, it was revealed that pigment production in S. maltophilia was associated with antibiotic resistance. Our findings show that cell with and without pigment formation indicates that pigment-bearing cells were more resistant to antibiotics. The MAR phenotype and biofilm formation of S. maltophilia observed gives a clearer insight why the bacterium is persistent, and difficult to eradicate.
In conclusion this study revealed that all resistance determinants contributed to the multidrug resistant phenotype of S. maltophilia. Along with the presence of pigment production, biofilm formation and class 1 integron may possibly play important roles leading to multidrug resistance of S. maltophilia. The varieties of antibiotics resistance genes and mobile genetic elements found suggests that the organism could potentially act as a reservoir of drug resistance determinants in a environmental and clinical settings, which is an issue of public health concern.
Table 1: The relationship between antibiotic susceptibility profile and integrons in S. maltophilia