Characterization of Acquired Antimicrobial Resistance Genes in Environmental Stenotrophomonas maltophilia Isolates from Brazil

Abstract

Stenotrophomonas maltophilia is an opportunist pathogen that has intrinsic resistance to the majority of antibiotics and has a high ability to adapt in different environments; however, there are few reports of acquired resistance genes in S. maltophilia. The aim of this study was to investigate the antimicrobial resistance profile, the presence of mutations in the quinolone-resistance determining region, the presence of acquired resistance genes, and the different plasmid families in S. maltophilia isolated from Brazilian soils. A total of 16 isolates were obtained from a variety of agricultural soils with different cultures of Brazil and they were nonsusceptible to most of the antibiotics tested. No mutations were detected in the gyrA gene and only one (Ser-80-Ile) was detected in the parC gene. A diversity of acquired resistance genes was found, including the qnrA, qnrB, qnrS, oqxA, oqxB, bla_SHV, bla_CTX-M-Gp1, bla_PER, bla_OXA-1-like, bla_OXA-48-like, and sul1. All isolates presented ColE-like plasmids and only one presented IncL/M. These results show, for the first time, the presence of qnrA and oqxAB genes and the presence of qnrB and qnrS genes for the second time in the world in S. maltophilia.

Introduction

Stenotrophomonas maltophilia is a nonfermenting Gram-negative bacilli (NFGNB) widely distributed in the environment being reported in different sources, such as soil, plant roots, and water.¹ In the hospital environment, it is considered an opportunist pathogen associated with several types of infections, the respiratory infections being the most prevalent. Some characteristics contribute to the emergence of this pathogen as one of the main infective NFGNB in immunocompromised patients, such as the high capacity of adherence to different surfaces, biofilm formation, and intrinsic resistance to the great majority of antibiotics.^2,3

Trimethoprim–sulfamethoxazole, levofloxacin, and ceftazidime are the most commonly chosen antibiotics for the treatment of infections caused by S. maltophilia and the resistance to these antibiotics is associated with different molecular mechanisms, such as multidrug efflux pumps (RND, ABC, and MFS families), Qnr (Smqnr), mutations in the bacterial topoisomerase and gyrase encoding genes, lipopolysaccharide (SpgM), and intrinsic and acquired β-lactamases.^1,3,4 This study aimed to investigate the antimicrobial resistance profile, the presence of mutations in the quinolone-resistance determining region (QRDR), the presence of acquired resistance genes, and the different plasmid families in S. maltophilia isolated from Brazilian soils.

Materials and Methods

Obtaining isolates

A total of 150 soil samples were obtained from different cultures, cities, and states of Brazil between 2015 and 2017 (Table 1). The bacterial isolation was performed according to Mukherjee et al.,⁵ using MacConkey Agar (Oxoid, United Kingdom). Then, the isolates were stored at −80°C in brain heart infusion broth (Oxoid) with 15% glycerol.

Table 1.

Culture/Source, City, State, and Region of the Isolates Used in This Study

Isolate	Culture/source	City	State	Region
S359	Banana	Teresina	Piauí	Northeast
S365	Cashew	Jardinópolis	São Paulo	Southeast
S369	Landfill	Ribeirão Preto	São Paulo	Southeast
S370	Landfill	Ribeirão Preto	São Paulo	Southeast
S371	Not identified	Jardinópolis	São Paulo	Southeast
S377	Sunflower	Chapadão do Sul	Mato Grosso do Sul	Midwest
S487	Corn	Ribeirão Preto	São Paulo	Southeast
S488	Coffee	Tabatinga	São Paulo	Southeast
S489	Soy	Maringá	Paraná	South
S490	Sugarcane	Tambaú	São Paulo	Southeast
S491	Citrus	Água de Santa Bárbara	São Paulo	Southeast
S492	Peanut	Cafelândia	São Paulo	Southeast
S493	Strawberry	Atibaia	São Paulo	Southeast
S494	Coffee	Pedregulho	São Paulo	Southeast
S495	Soy	Jardinópolis	São Paulo	Southeast
S496	Sugarcane	Jardinópolis	São Paulo	Southeast

Identification of isolates

The isolates were first identified by MALDI-TOF VITEK^® MS (bioMérieux, Inc., Durham, NC) according to the manufacturer's recommendations and confirmed using the molecular identification. The genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN) and used for the sequencing of the 23S rRNA gene according to Hunt et al.⁶

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed according to CLSI⁷ using the disk diffusion method for trimethoprim + sulfamethoxazole (SXT) and minocycline. The minimum inhibitory concentration (MIC) method was used for levofloxacin and ceftazidime with a range from 1 to 256 μg/mL. Escherichia coli ATCC^® 25922 and Pseudomonas aeruginosa ATCC^® 27853 strains were used as control in these experiments.

Detection of mutations in the QRDR

Detection of mutations in QRDR of gyrA and parC genes was performed according to Jia et al.⁸ The alignment of gyrA and parC genes was performed using the S. maltophilia ATCC 13637 (GenBank accession number CP008838) using the Clustal Omega EMBL-EBI multiple sequence alignment.

Detection of acquired resistance genes and integrons

PCRs were performed for amplification of some plasmid-mediated quinolone resistance (PMQR) genes, such as qnrA, qnrB, and qnrS according to Cattoir et al.⁹; qepA according to Karczmarczyk et al.¹⁰; oqxA and oqxB according to Chen et al.¹¹ PCRs were performed for amplification of the genes bla_{CTX-M-Gp1-2-8-9}, bla_KPC, bla_CMY, bla_SHV, bla_GES, bla_PER, bla_VEB, bla_OXA-1-like, bla_OXA-48-like, bla_IMP, and bla_VIM according to Dallenne et al.¹²; bla_GIM and bla_SIM according to Ellington et al.,¹³ and bla_NDM according to Peirano et al.¹⁴ PCRs were performed for amplification of sul1, sul2, and sul3 genes according to Kerrn et al.,¹⁵ and Perreten and Boerlin.¹⁶ The classes 1, 2, and 3 integrons were researched using the primers described by Koeleman et al.¹⁷ and Ploy et al.¹⁸

Plasmid replicon typing

The plasmids were searched by PCR-based replicon typing using primers for 20 incompatibility groups, such as IncHI1, IncHI2, IncI1, IncFIA, IncFIB, IncFIC, IncFIIA, IncF_repB, IncL/M, IncP, IncA/C, IncW, IncX, IncN, IncT, IncK, IncB/O, IncY, IncU, and IncR according to Carattoli et al.¹⁹ and García-Fernández et al.²⁰ The ColE-like plasmids were also searched using primers described by García-Fernández et al.²⁰

Results

In this study, 50 different soil samples from four Brazilian regions were used for the isolation of 16 S. maltophilia. Fourteen isolates were obtained from a variety of agricultural soils with different cultures in several cities, states, and regions of Brazil and two of them were obtained from a landfill. The majority of these isolates (13) were obtained from soil samples from the southeast region and three other isolates were obtained from south, northeast, and midwestern regions (Table 1). The sequences obtained from the 23S rRNA gene were deposited in the GenBank (www.ncbi.nlm.nih.gov/Genbank) with accession numbers MH321509–MH321524.

Four antibiotics were tested using disk diffusion and MIC methods. Among the isolates, 15 (93.75%) were nonsusceptible (resistant or intermediate) to ceftazidime, 12 (75%) to levofloxacin, 8 (50%) to SXT, and 4 (25%) to minocycline. High MICs representing nonsusceptibility were observed for ceftazidime, having a range of 32 to >256 μg/mL, and for levofloxacin, with a range of 4–8 μg/mL. Two isolates (S365 and S490) were susceptible to levofloxacin and only one (S492) to ceftazidime (Table 2).

Table 2.

Resistance Profile, Mutation Points in Quinolone-Resistance Determining Region, Plasmid-Mediated Resistance Genes and Plasmid Families in Stenotrophomonas maltophilia Isolated from Soil

	Resistance profile
		MIC (μg/mL)		QRDR
Isolate	DD	LVX	CAZ	gyrA	parC	PMQR genes	β-Lactamases encoding genes	SRG + integron	Plasmid family
S359	SUT	8	>256	—	—	qnrA, qnrB, qnrS, oqxA, oqxB	—	intI1, sul1	ColE-like
S365	SUT	2	128	—	—	—	bla _SHV	intI1	ColE-like
S369	SUT	4	128	—	—	qnrS, oqxA, oqxB	—	intI1	ColE-like
S370	SUT	4	128	—	—	—	bla _SHV	intI1	ColE-like
S371	SUT	4	128	—	—	oqxB	bla _CTX-M-Gp1 , bla _SHV	intI1	ColE-like
S377	SUT	4	>256	—	S80I	qnrA, qnrB, qnrS, oqxA, oqxB	bla _CTX-M-Gp1, bla _OXA-48-like	intI1	ColE-like
S487	MIN	8	>256	—	—	—	bla _OXA-1-like	intI1	ColE-like
S488	SUT	4	>256	—	—	qnrA, qnrS	bla _OXA-1-like	intI1, sul1	ColE-like, IncL/M
S489	—	4	>256	—	—	—	bla_PER, bla_OXA-1-like	intI1	ColE-like
S490	—	2	32	—	—	—	bla _PER	intI1	ColE-like
S491	—	4	32	—	—	qnrA, qnrB, qnrS	bla_SHV, bla_PER, bla_OXA-48-like	intI1	ColE-like
S492	—	4	4	—	—	qnrA	—	intI1	ColE-like
S493	MIN	2	128	—	—	—	—	intI1	ColE-like
S494	SUT	2	128	—	—	—	bla_CTX-M-Gp1, bla_SHV	intI1, sul1	ColE-like
S495	MIN	4	>256	—	—	—	—	intI1	ColE-like
S496	MIN	4	>256	—	—	—	—	intI1	ColE-like

DD, disk diffusion; SUT, trimethoprim + sulfamethoxazole; MIN, minocycline; MIC, minimum inhibitory concentration; LVX, levofloxacin; CAZ, ceftazidime; S, serine; I, isoleucine; SRG, sulfonaminde resisistance genes; —, negative result; QRDR, quinolone-resistance determining region; PMQR, plasmid-mediated quinolone resistance.

All isolates were investigated for mutations in the QRDR. In gyrA gene, no mutations were found, and in parC gene, the substitution Ser-80-Ile (GenBank accession number MH332896) was found only in the isolate S377 (Table 2). Several acquired resistance genes were researched and a total of 39 amplicons for different genes were obtained. Among the PMQR genes, qnrS was the most prevalent, being detected in six isolates, followed by qnrA and oqxB in four isolates, and qnrB and oqxA in three isolates. Seven isolates did not present any of the PMQR genes researched (Table 2).

The β-lactamases encoding genes were found in 10 different isolates. The bla_SHV was detected in five isolates, followed by bla_CTX-M-Gp1, bla_PER, and bla_OXA-1-like in three isolates and bla_OXA-48-like in two isolates (Table 2). Among the sulfonamide resistance genes, sul1 was the only one found, being detected in three isolates (S359, S488, and S494). Class 1 integron (intI1) was detected in all isolates, however, classes 2 and 3 integrons (intI2 and intI3) were not detected (Table 2). Among the different plasmid families researched, the ColE-like plasmid was detected in all isolates and the IncL/M plasmid only in the S488 isolate (Table 2). The sequences obtained from the acquired resistance genes were deposited in the GenBank with accession numbers MH332885–MH332895.

Discussion

Nonsusceptibility to SXT, levofloxacin, and ceftazidime has increased, which causes concern, especially for SXT, considered the first-choice in the treatment of S. maltophilia infections.²¹ Chang et al.⁴ reported a decrease in the susceptibility over the years to ceftazidime and fluoroquinolones in worldwide SENTRY studies. These results corroborate those found in this study, since a low percentage of susceptibility to these antibiotics was found. Nonsusceptibility rates for SXT vary geographically, but are generally <10%. These data are different from those presented in this study, wherein 50% of environmental isolates showed nonsusceptibility to SXT.^4,22

The substitutions at the Ser-83 and Asp-87 codons of the gyrA gene and Ser-80 and Glu-84 of the parC gene are the main substitutions associated with nonsusceptibility to fluoroquinolones.^23,24 The substitution found in the parC gene has already been described in some studies; however, substitutions in the parC gene alone are not relevant for nonsusceptibility to fluoroquinolones.^24,25

The PMQR genes are frequently reported in clinical isolates and some reports of aquatic isolates have already occurred.^26,27 The first report of qnrB and qnrS in S. maltophilia was described by Rizek et al.²⁸ and Margaritis et al.,²⁹ respectively, and there are no reports of other qnr and oqxAB genes in this species. Some studies have investigated the PMQR genes in isolates nonsusceptible to levofloxacin; however, no gene was detected.^7,30 The majority of studies relate nonsusceptibility to fluoroquinolones with intrinsic and acquired efflux pumps.³¹ Our results show, for the first time, the presence of qnrA and oqxAB genes and the presence of qnrB and qnrS genes for the second time in the world in S. maltophilia.

Al Naiemi et al.³² reported bla_SHV and bla_CTX-M-1 for the first time in a clinical isolate of S. maltophilia in the Netherlands, and later, Lavigne et al.³³ and Maravić et al.³⁴ reported the presence of bla_CTX-M-15 in S. maltophilia isolates from different origins, such as clinical, mussels, and seawater. The bla_PER gene in an environmental isolate of S. maltophilia was identified for the first time in 2018, inside an IncA/C plasmid.³⁵ The bla_OXA-like gene has already been described in different bacterial genera from different sources; however, there are no reports of these genes in S. maltophilia since the majority of the studies report the intrinsic resistance to β-lactam by bla_L1 and/or bla_L2 genes.^36,37

The association of sul genes, including sul1, with the different integrons is associated with resistance to SXT.^34,38 Liaw et al.³⁹ reported that the presence of class 1 integron is associated with nonsusceptibility to multiple antibiotics in which different cassettes with several resistance genes have been described. Among these genes, dfrA and dfrB have already been described causing nonsusceptibility to SXT in S. maltophilia and these genes may be related to isolates that were nonsusceptible to SXT and did not present the sul genes.⁴⁰

In the isolates that present nonsusceptibility phenotype and did not present the genes studied, other mechanisms are associated.³ The plasmids belonging to the ColE-like and IncL/M families have been reported in different bacterial genera carrying several resistance genes, including the genes presented in this study.⁴¹ Ferreira et al.⁴² reported the presence of ColE-like plasmids carrying PMQR genes in isolates obtained from poultry in Brazil. The studies related to detection of acquired resistance genes associated with plasmid families are mainly concentrated in clinical isolates and among environmental bacteria, the most studied aquatic isolates, and few studies use bacterial isolates from soil.

Conclusion

S. maltophilia is intrinsically multidrug resistant to several antibiotics; however, some acquired mechanisms have also been described in this pathogen. The high occurrence of antibiotic and manure in agriculture selects bacteria resistant to the several antibiotics that can migrate to the hospital environment. Therefore, our results indicate that S. maltophilia isolated from soil harbor several acquired resistance genes for different antibiotics. Besides that, many isolates were nonsusceptible for almost indicated antibiotics, thus infections by environmental S. maltophilia with this resistance profile limit the therapeutic options, which is a great concern.

Footnotes

Acknowledgments

This work was supported by São Paulo Research Foundation—FAPESP (grant number 2015/18990-2). The authors thank J.D.D. Pitout (University of Calgary, Calgary, AB, Canada) for kindly providing the control strains used in this study.

Disclosure Statement

No competing financial interests exist.

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