Genomic information on Stenotrophomonas maltophilia ST264 isolated from a cystic fibrosis pediatric patient in Brazil

Abstract

Stenotrophomonas maltophilia is one of the Gram-negative bacilli most frequently found in the airways of cystic fibrosis patients. This opportunistic pathogen is intrinsically multidrug-resistant, and therefore, its treatment presents a challenge. The genetic characterization of S. maltophilia is largely unknown, especially from those strains that colonize/infect the airways of cystic fibrosis patients. This work reports the draft genome sequences of three S. maltophilia isolates recovered from the sputum of a cystic fibrosis pediatric patient in Southeast Brazil. Several resistance- and virulence-related genes were detected. Furthermore, one intact phage and one incomplete prophage region were also identified in all strains. Multilocus sequence typing showed that all strains belonged to a new sequence type (ST264). Interestingly, all S. maltophilia strains were genetically identical, showing persistence for at least 16 months. To our knowledge, this is the first report of S. maltophilia draft genome sequences obtained from a cystic fibrosis pediatric patient in Brazil.

Stenotrophomonas maltophilia is a Gram-negative bacillus that has emerged as an important opportunistic pathogen in cystic fibrosis (CF) patients. However, the consequences of its presence in this group of patients remain controversial [1]. A recent study associated this micro-organism with an acceleration in lung function decline, particularly in long-term colonized/infected patients [2]. S. maltophilia is intrinsically resistant to multiple antibiotic classes and has been described in 10 to 18% of CF patients. Knowledge about the mechanisms this pathogen uses to colonize and persist in the airways of CF patients is imperative to inform and improve the treatment [3, 4]. Genomic features are largely understudied; therefore, there is a need to further investigate the genomic characteristics of S. maltophilia strains that colonize/infect CF patients.

This study describes the draft genome sequences of three S. maltophilia strains recovered from the sputum of a single non-hospitalized CF pediatric patient attended at the Instituto Nacional da Saúde da Mulher, da Criança e do Adolescente Fernandes Figueira (IFF-FIOCRUZ), in the state of Rio de Janeiro (Southeast Brazil). Considering the period between 2012 and 2014, four S. maltophilia strains were obtained from the bacterial collection of the Departamento de Microbiologia, Imunologia e Parasitologia (DMIP) at Universidade do Estado do Rio de Janeiro (UERJ). These strains were identified with the numbers 15180 (isolated in September 2012), 16140, 16185 (isolated in March 2013), and 17307 (isolated in January 2014). Strain 16140 was inadequate and therefore was excluded from this study.

S. maltophilia strains were identified by conventional methods [5]. Disk diffusion tests, conducted and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) [6], showed that the strains were susceptible to levofloxacin, minocycline, and sulfamethoxazole/trimethoprim (SXT). The minimum inhibitory concentration (MIC) of three antibiotics—ciprofloxacin (CIP), ceftazidime (CAZ), and SXT—commonly used in the CF patients attended at IFF-FIOCRUZ, were determined by the microdilution method and breakpoints established for P. aeruginosa were used for interpreting CIP values [6]. All isolates were 128 mg/mL for CAZ, 4 to 16 mg/L for CIP, and 0.25 to 1 mg/L for SXT, showing susceptibility to SXT and resistance to CIP and CAZ.

The PureLink™ Genomic DNA Mini kit (Invitrogen, Carisbad, EUA) was used for the genomic DNA extraction according to the protocol provided by the manufacturer. Genomic libraries were constructed with the Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, USA). The DNA sequencing was performed for each strain with the 500 cycle MiSeq Reagent Kit v2 (Illumina Inc., San Diego, USA) on an Illumina MiSeq instrument (Illumina Inc., San Diego, USA). We have obtained 2.555.336, 2.608.756, and 2.776.152 paired-end sequence reads for strains 15180, 16185, and 17307, respectively. The quality score of the sequence reads of all strains was checked using FastQC v0.11.8 (available at http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were corrected and assembled de novo into scaffolds with the A5 assembly pipeline [7]. Raw read sequences were deposited in the sequence read archive (SRA) of the NCBI (available from https://submit.ncbi.nlm.nih.gov) with the following accession numbers: SRR9046642 (strain 15180), SRR10059485 (strain 16185), and SRR10059486 (strain 17307). In order to compare S. maltophilia DNA sequences, the average nucleotide identity (ANI) was calculated using the OrthoANI-usearch tool (available at https://www.ezbiocloud.net/tools/orthoaniu) [8]. All complete S. maltophilia genomes available at the time of this study in the NCBI database (n = 10) were aligned and compared. Among them, S. maltophilia NCTC10258 was the most genetically similar (98%) to our three S. maltophilia strains. The ANI analysis also revealed high identity (99.9%) among the 15180, 16185, and 17307 strains.

The Rapid Annotation Using Subsystems Technology (RAST) (available at http://rast.nmpdr.org) [9, 10] was used for genome annotation. The resulting estimated genome size ranged from 4.475.139 to 4.478.162 bp (Table 1).

Table 1.

Genomic features of S. maltophilia strains obtained from the sputum of a single cystic fibrosis pediatric patient

Genomic features	Strains
	15180	16185	17307
Genome size (bp)	4.475.139	4.478.162	4.477.335
GC Content (%)	66.6	66.6	66.6
Number of contigs (with PEGs)	54	50	46
Reads	2.555.336	2.608.756	2.776.152
Fold coverage	86x	87x	93x
N50 (bp)	165.645	166.323	195.024
L50 (bp)	10	10	8
CDS	4.170	4.172	4.163
RNAs	80	78	78
Number of subsystems	761	761	761

GC guanine and cytosine, bp base pairs, CDS coding sequences

Genes involved in antimicrobial resistance were investigated by Resfinder 3.0 tool (available at https://cge.cbs.dtu.dk/services/ResFinder/) [11] and Resistance Gene Identifier (RGI) (available at https://card.mcmster.ca) [12]. In order to refine these analyses, manual comparison with various annotated reference genomes were performed using the BLASTN tool (available at https://www.ncbi.nlm.nih.gov) [13]. Using these approaches, a gene repertoire associated with resistance to aminoglycosides (aac(6′)-Iz, aph (3′)-IIc), fluoroquinolones (smqnr, smqnR, smtcrA,), and beta lactams (bla_L1, ampR, bla_L2, ampN, ampG, ampC, ampD_I) was observed. In addition, several genes related to multidrug efflux pumps were identified (smeABC, smeT, smeDEF, smeIJK, tolCsm, pcm, smeRo, smeOP, smeRv, smeU, smeW, smeU2, smeXYZ, smeMN, smeGH, smeYZ, emrRsm, emrABCsm, sugE, emrE, smrA, macABCsm, fuaR, fuaABC, pmpM, norM).

Manual comparison with the BLASTN tool (available at https://www.ncbi.nlm.nih.gov) [13] was also used to identify virulence determinant genes. This study has also detected genes related to extracellular enzymes (stmPr1, stmPr2), adhesion (smf-1), polysaccharides (xanAB, rmlABCD, etfAB, ipsl, spgM), inner membrane protein (creBCD), and biofilm (rpfBFCG, fsnR, flhABROPNMLF, fliAKJIHGFE, ISPsyg).

A search for phage regions was performed using the Phage Search Tool (PHASTER) (available at http://phaster.ca) [14]. This approach detected one intact phage (PHAGE_Escher_vB_EcoM_ECO1230_10) and one incomplete prophage region (PHAGE_Xantho_CP2).

The Multilocus Sequence Typing (MLST) 1.8 tool, hosted by the Center for Genomic Epidemiology platform (CGE) (available at https://cge.cbs.dtu.dk/services/MLST/) [15] was used for molecular typing. This in silico MLST analysis resulted in the assignment of a new Sequence Type (ST), which was designated as ST264 (allelic profile: 21, 4, 97, 4, 25, 134, and 6 for atpD, gapA, guaA, mutM, nuoD, ppsA, and recA, respectively) after curation by the MLST website (https://pubmlst.org/smaltophilia/) [16]. This new ST described in this study was classified as a singleton.

This investigation presented whole-genome sequencing of three S. maltophilia isolates that belonged to the same clone, from strains that persisted in a single CF pediatric patient. Detailed genomic analysis will be provided a base for future studies about the role of S. maltophilia, particularly in CF patients colonized/infected in long-term infected patients.

These Whole Genome Shotgun projects have been deposited at the DNA Data Bank of the Japan/European Molecular Biology Laboratory/Genbank under the following accession numbers: PYHM00000000, PYHN00000000, and PYBT00000000. The versions described in this paper are versions PYHM02000000, PYHN02000000, and PYBT02000000.

Funding information

This study received funding support from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This study received funding support from FAPERJ- Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de 404 Janeiro – Grant E-26/110.742/2013.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.