Identification and Characterization of a Serious Multidrug Resistant Stenotrophomonas maltophilia Strain in China

An S. maltophilia strain named WJ66 was isolated from a patient; WJ66 showed resistance to more antibiotics than the other S. maltophilia strains. This bacteraemia is resistant to sulphonamides, or fluoroquinolones, while the representative strain of S. maltophilia, K279a, is sensitive to both. To explore drug resistance determinants of this strain, the draft genome sequence of WJ66 was determined and compared to other S. maltophilia sequences. Genome sequencing and genome-wide evolutionary analysis revealed that WJ66 was highly homologous with the strain K279a, but strain WJ66 contained additional antibiotic resistance genes. Further analysis confirmed that strain WJ66 contained an amino acid substitution (Q83L) in fluoroquinolone target GyrA and carried a class 1 integron, with an aadA2 gene in the resistance gene cassette. Homology analysis from the pathogen-host interaction database showed that strain WJ66 lacks raxST and raxA, which is consistent with K279a. Comparative genomic analyses revealed that subtle nucleotide differences contribute to various significant phenotypes in close genetic relationship strains.


Introduction
Stenotrophomonas maltophilia (S. maltophilia) is an aerobic Gram-negative bacillus ubiquitous in the natural and hospital environment [1].In addition, S. maltophilia is emerging as a global multidrug resistant (MDR) human opportunistic pathogen.In a clinical environment, S. maltophilia has been isolated from several sources, including the suction system tubing of dental chair units, contaminated endoscopes, and tap water, all of which present possible patient exposure sources.S. maltophilia contaminated central venous catheters and tap water faucets have been implicated in cutaneous and soft tissue infections in patients with neutropenia [2].
S. maltophilia is most commonly associated with respiratory and other serious infections, especially in immunocompromised patients [3].In polymicrobial infections, S. maltophilia was present in 45.8% of patients [4].S. maltophilia is being isolated with increasing frequency from patients with intensive care unit (ICU) associated infections [5].ICUacquired infections related to S. maltophilia were independently associated with increased morbidity and mortality [6,7].Specifically, early reports showed that this important nosocomial pathogen is associated with crude mortality rates ranging from 14 to 69% in patients with bacteraemia and the ICU mortality rate attributed to bacteraemia was 16.7% according to an 11 years review [4,8,9].
Since the first genome of S. maltophilia K279a was reported in 2008 [10]; bioprojects of 48 S. maltophilia strains can be found in NCBI.However, the majority of these strains are not available.
Here, we isolated a S. maltophilia strain from a human blood sample, named WJ66.This strain exhibited resistance to more antibiotics than the other S. maltophilia strains.Drug resistance test showed that S. maltophilia WJ66 is resistant to nearly all drugs, including sulphonamides and fluoroquinolones.We present the draft genome sequence of S. maltophilia WJ66 and conduct a comparison genomic and phenotypic analysis.Genome sequencing revealed that strain WJ66 contained additional antibiotic resistance genes and an amino acid substitution (Q83L) in fluoroquinolone target GyrA and carried a class 1 integron, with an aadA2 gene in the resistance gene cassette.This paper may lead to a better understanding of the correlation between pathogenic traits and specific genes or gene clusters of S. maltophilia.

Bacterial Strains and Drug
Resistance.Blood cultures and in vitro susceptibility testing confirmed the presence of a multidrug resistant S. maltophilia isolate, termed WJ66, in an infected patient.The patient was an elderly man in ICU with underlying diseases including type 2 diabetes, diabetic nephropathy, chronic renal insufficiency, uraemia, and a lung infection.Clinical tests showed that the isolates were resistant to nearly all drugs available for treatment, including sulphonamides and fluoroquinolones [8,11].Multilocus sequence typing (MLST) sequencing showed that S. maltophilia WJ66 belongs to ST31 (1, 4, 12, 6, 6, 22, and 7 for atpD, gapA, guaA, mutM, nuoD, ppsA, and recA).
We obtained S. maltophilia K279a from the ATCC and examined the drug resistant differences by Etest.Table 1 indicates that both K279a and WJ66 are resistant to imipenem, doxycycline, and tetracycline.K279a is sensitive to polymyxin, while WJ66 is moderately sensitive to polymyxin.Levofloxacin and trimethoprim-sulfamethoxazole are two of the few drugs that are generally efficacious against clinical S. maltophilia infections.K279a is sensitive to both these drugs, but WJ66 is resistant.The mechanisms of this resistance will be described in the following sections.Susceptibility results of control Escherichia coli strains (ATCC25922) and Pseudomonas aeruginosa (ATCC27853) are in line with CLSI standards.

Total Genome Overview of S. maltophilia WJ66.
The draft genomic sequence of S. maltophilia WJ66 was obtained by an Illumina Hiseq 2000.A total of 258 contigs, covering a total of 4,642,973 bp, were generated.The average length of a contig is 17,996 bp with a G + C content of 66.48%.The N50 contig   2).The genome sequence of WJ66 makes it possible for a comparative and functional genomic analysis of S. maltophilia isolates.

Phylogenetic Relationships among Sequenced S. maltophilia Strains
Using Whole Genome Comparisons.The S. maltophilia WJ66 genomic sequence was compared with 11 other S. maltophilia strains, whosegenomic sequence data are in the GenBank database.Figure 1 shows the phylogenetic relationships between different S. maltophilia isolate genomes.We took the Xanthomonas campestris pv.campestris (Xcc) stain 8004 as an outgroup construct for the phylogenetic tree.WJ66, K279A, EPM1, and Ab 55555 are on the same branch, indicating that they are highly homologous.Strain K279a is a blood sample isolate from the United Kingdom [10], strain WJ66 is also a blood sample isolate from China, strain Ab55555 is an American isolate, and strain EPM1 was found during the sequencing of two human-derived strains of Giardia duodenalis [13].The environmental isolate MF89, an oyster-associated bacteria, shows potential for crude oil hydrocarbon degradation [14] and is the most closely related to the above four isolates.In addition, JV3, D457, and AU12-09 are close relatives with each other.D457 is a clinical isolate [15] and AU12-09 is an intravascular catheters isolate [16].Homologous strains R551-3 and RA8 were isolated from the poplar Populus trichocarpa [17] and from sewage plant effluent [18], respectively.SKK35 is ulcer swab isolate [9].Overall, the S. maltophilia strains can be divided into two groups: the clinical isolates and the environmental isolates.The mutual affinity within groups is closer than the relationship between the groups.As the Xcc strain 8004 is the only non-S.maltophilia strain, the genomic differences between Xcc and S. maltophilia are the largest.

Comparing the Genomes of S. maltophilia WJ66 and
K279a.Because S. maltophilia strains WJ66 and K279a exist in the same isolated background and are closely related, more comparison need to be performed.Global alignments were performed by the MUMER software [19], and the results showed a large homology between WJ66 and K279a (Figure 2).

The Comprehensive Antibiotic Resistance Database (CARD)
. Within the CARD, 3411 genes are tagged for antibiotic molecule, biosynthesis, resistance, or inhibition of resistance, and 1859 genes were specifically annotated for antibiotic resistance.The Resistance Gene Identifier (RGI) version 2 provides an automated annotation of DNA sequences based upon the data available in the CARD, thus providing a prediction of antibiotic resistance genes [20].
Open reading frames (ORFs) obtained from the Gene-Marks annotation were subjected to BLAST analysis against the CARD.This approach highlighted the presence of 255 genes related to antibiotic resistance.The largest proportion of homology to antibiotic resistance genes was found to be efflux pump genes (approximately 41 homologous genes), the beta-lactamase gene (33 homologous genes), and quinolone resistance related genes (7 homologous genes).By comparing with the CARD, we found that WJ66 04174 and WJ66 01359 codes for the qepA gene, whose gene product is a quinolone efflux pump, are homologous with an antibiotic resistance gene in E. coli 1520 genome.The protein QnrB encoded by WJ66 00023 is a S. maltophilia inherent quinolone resistance gene.WJ66 04112 (gyrA), WJ66 03278 (gyrB), WJ66 01551 (parC), WJ66 03996 (parC), and WJ66 03111 (parE) were annotated as fluoroquinolone target genes, and mutation in these genes causes resistance to quinolones.GMS 00385 encodes dihydropteroate synthetase type 1 (sul1); downstream sul1 is GMS 00386 (WJ66 01310) coding an aadA2 aminoglycoside acetyltransferase; these two genes are homologous with that found in Salmonella enterica subsp.enterica serovar Typhimurium DT104.Advanced analyses found that sul1 and aadA2 are members of a class 1 integron and were not predicted by GeneMark analysis.Moreover, the two genes are 100% identical with latter strain Typhimurium DT104, indicating that they were probably transferred from another bacterium by mobile elements.

The Trimethoprim-Sulfamethoxazole Associated Integron.
In many Gram-negative bacteria, integrons carrying antibiotic resistance genes have been identified by mobile elements such as transposons and plasmids that facilitate the transfer of antibiotic resistance genes between different species.In the present study, the distribution of class 1 and 2 integrons was examined in a collection of clinical S. maltophilia isolates [21].
Integrons were initially described at the end of the 1980s [22].This family of elements appears formally distinct from other known mobile DNA elements such as transposons.Class 1 integrons play an important role in the emergence and spread of antimicrobial resistance determinants.Typically, class 1 integrons have three distinct genetic regions of which two are highly conserved, the 5  -conserved segment (5  -CS) and the 3  -conserved segment (3  -CS) flank the central variable region where the gene cassettes are located.The 5  -CS includes the integrase intI1 gene, the recombination site attI1, and the promoters Pc and P2 when present.The 3  -CS consists of the qacEΔ1 gene, which encodes an incomplete version of a protein that mediates the resistance to certain detergents, the sul1 gene, which encodes resistance to sulphonamides, and an open reading frame of unknown function orf5 [23].
Although most of class 1 integrons have this classic structure, an increasing number of class 1 integrons, related to trimethoprim-sulphamethoxazole resistance, with different structures have been reported [24,25].Although trimethoprim-sulfamethoxazole is considered the drug of choice for treating S. maltophilia infections, 25% of the isolates were resistant [21].
The WJ66 strain carries a detectable class 1 integron, whereas class 2 and class 3 integrons were not identified.In addition to the sul1 gene encoding a sulphonamide-resistant dihydrofolate synthase, the aadA2 gene, which encodes an aminoglycoside acetyltransferase giving resistance to aminoglycosides including amikacin, was identified as the only other gene in the resistance gene cassette (Figure 4).More details about the integron can be found in supplementary data 1 (see Supplementary Materials available online at http://dx.doi.org/10.1155/2015/580240).
Because class 1 integron has had a major role in the spread of antibiotic resistance and has led to worldwide difficulties in controlling bacterial infection, understanding the origin of these elements is important for the practical control of antibiotic resistance and for exploring the means by which bacterial lateral gene transfer can seriously impact, and be impacted by, human activities.

The Fluoroquinolone Levofloxacin Resistance Associated
Gene.Quinolones are synthetic antibacterial reagents with broad-spectrum activity.They inhibit the enzyme topoisomerase II, a DNA gyrase that is necessary for the replication of the microorganism.Topoisomerase II enzyme produces a negative DNA supercoil on DNA, which permits transcription or replication.By inhibiting this enzyme, DNA replication and transcription are blocked.
The earliest quinolone, nalidixic acid, was first used in a clinical setting in 1962.The targets of quinolone action are the essential bacterial enzymes DNA gyrase, as stated above, and DNA topoisomerase IV.Both are large, complex enzymes composed of 2 pairs of subunits.The subunits of DNA gyrase are GyrA, a 97-kDa protein encoded by the gyrA gene, and GyrB, a 90-kDa protein encoded by the gyrB gene.The subunits of topoisomerase IV are ParC (75 kDa) and ParE (70 kDa).
One of the mechanisms of quinolone resistance is the accumulation of mutations that alter the drug targets.In Gram-negative bacteria, DNA gyrase is more susceptible to inhibition by quinolones than is topoisomerase IV, whereas in Gram-positive bacteria, topoisomerase IV is usually the prime target and DNA gyrase is intrinsically less susceptible.Consequently, resistant mutations are found in gyrA in Gram-negative bacteria and in parC in Gram-positive bacteria [26].
The MIC of levofloxacin for K279a is 1 g/mL, the MIC of norfloxacin for D457 is 6 g/mL [27], and the MIC of levofloxacin for WJ66 is more than 32 g/mL, corresponding to susceptibility results: sensitive, moderately sensitive, and resistant, respectively.Sequence alignments show that the resistant WJ66 strain contained a point mutation (Q83L) in its GyrA (Figure 5).While no mutations in GyrB were identified, WJ66 contained mutations in ParC (S86I and E421D) and ParE (M227L and F519C).

Efflux Pumps in WJ66
Genome.Efflux as a mechanism for antibiotic resistance was first described in 1980 [28].In Gram-negative nosocomial pathogens, MDR is usually mediated by the overproduction of resistance-nodulation-division (RND) type efflux pumps.These pumps tend to have broad substrate profiles, including organic solvents, disinfectants, and antimicrobial drugs from a number of different classes.In the case of Gram-negative bacteria, efflux pumps consist of cytoplasmic, periplasmic, and outer membrane proteins (OMP) that associate to form multicomponent efflux systems [29].A periplasm spanning membrane-fusion protein (MFP) is usually specific to each RND efflux protein, and it is common to find the pair encoded as part of an operon.A third component, the outer membrane protein, can be encoded in the same operon, but there tends to be fewer OMPs than RND/MFP pairs in a cell, meaning that the OMPs are often promiscuous [10].The K279a sequence encodes nine RNDtype efflux pump genes that fall into the drug resistance type based on sequence homology.WJ66 also possessed these efflux pumps (Table 3).

The Pathogen-Host Interactions Database (PHI-base).
The PHI-base (http://www.phi-base.org/)contains expertly curated molecular and biological information on genes proven to affect the outcome of pathogen-host interactions.Information is also given on the target sites of some antiinfective molecules.
Little is known about pathogenesis of S. maltophilia and the genomic diversity exhibited by clinical isolates complicates the study of pathogenicity and virulence factors.A recent study about relative virulence in adult-zebrafish model of S. maltophilia infection revealed that abundance of the quorum-sensing factor Ax21 in four strains of S. maltophilia correlates with mortality [30].ORFs obtained from the GeneMarks annotation were subjected to BLAST analysis against the PHI-base, resulting in the identification of 252 homologous genes.Two genes, ax21 and avrXa21, were found to be homologous to Xanthomonas oryzae genes that are related to plant virulence.The ax21 and avrXa21 genes also have homologs in Metarhizium anisopliae and Saccharomyces cerevisiae.Seven genes were annotated as lethal to host (plant); the first two have homologs in Gibberella zeae, and the other five have homologs in Aspergillus fumigatus.Seven genes were annotated to be resistant to chemical reagents including disinfectants and chemotherapy.Seven genes were annotated with mixed outcomes.A majority of genes, approximately 26, were annotated as partial loss of Ax21 signalling in this opportunistic pathogen strain, these genes include raxB, raxC, raxH, raxR, raxH 2 /phoQ, and raxH 2 /phoP, which has a homolog in Xanthomon oryzae.Thirty-three genes were annotated as genes that do not affect pathogenicity, while 42 genes and 125 genes were annotated as genes that lead to pathogenic loss and virulence attenuation, respectively.While a majority of pathogenic genes were avirulent, foreign outbreaks are examples of pathogenicity and virulence still worthy of our attention.The rice XA21 receptor binds the AxYS22 peptide corresponding to the N-terminal region of Ax21, a type I-secreted protein that is highly conserved in all Xanthomonas species as well as in Xylella fastidiosa and S. maltophilia [31].
Ax21 is a 194-amino-acid protein and an activator of innate immunity mediated by the XA21 pattern recognition receptor [32].S. maltophilia displayed Ax21 (Smlt0378 in K279a) functions in the signal between cells.An Ax21 knockout leads to decreased virulence but a lack of raxST sulphated species modifications that do not affect the signal.These findings suggest that sulfation is not required for the regulatory action of Ax21 within S. maltophilia K279a [33].Ax21 associated genes in S. maltophilia WJ66 are shown in Table 4. Homology analysis showed that WJ66 lacks raxST and raxA, which is consistent with K279a.WJ66 03588 is homologous to the raxB gene, a 169-amino-acid protein that functions in Colicin V secretion and had 97.78% homology to an ABC transporter ATP-binding protein.EPM1 predicted that Smlt2001 from K279a is a putative 590-amino-acid ABC transporter ATP-binding protein that has high homology to PHI 1141 (719 amino acids).Ax21 secretion depends on the RaxB homologous protein Smlt2001.Because of the size difference, confirming WJ66 03588 functions as Smlt2001 will need further verification.Additionally, raxP is missing in WJ66, and raxQ has only 37.04% homology.

Conclusions
In summary, the genomic sequence of the resistant bacteraemia-associated S. maltophilia isolate WJ66 is highly homologous with K279a.Tiny differences in nucleic acid levels lead to different in vitro antibiotic susceptibility phenotypes.Comparative genomic analyses revealed a considerable diversity among S. maltophilia strains, providing new insights into the differences and similarities that may explain the diverse nature of these strains.Drug-resistant gene mutations, drug-resistant enzymes, and horizontal drug resistance gene transfer in bacteria contribute to drug resistance.We obtained useful information about the unexplored aspects of S. maltophilia.The work will improve our understanding of the correlation between pathogenic traits and specific genes or gene clusters of S. maltophilia.

Bacterial Strains, In Vitro Antimicrobial Sensitivity Assays,
and Genomic DNA Extraction.S. maltophilia WJ66 was originally isolated in a blood sample from an elder male patient undergoing chemotherapy.The patient has diabetic nephropathy, chronic renal insufficiency, uraemia, renal anaemia, lung infection, hypertension, cerebral infarction (old), urinary tract infection, and hyperuricemia and did not respond to therapy with piperacillin-tazobactam, ceftazidime, or imipenem.The bacterium was performed the antimicrobial susceptibility assay and the MLST sequencing as previously described [34].
Susceptibility assays for the different antibiotics were performed in Mueller Hinton (MH) broth, using the twofold dilution method in 96-well microporous plates.The results were recorded after 20 h incubation at 37 ∘ C [35].MICs were determined in MH medium by Etest (AB BIOMERIEUX, Arizona City, Sweden), according to the manufacturer's instructions.To ensure that the observed changes were consistent, all MICs were determined in three independent assays, using different bacterial cultures on different days.In most cases, there were no interassay differences in the MIC values.In a few cases, there were one dilution differences.For the latter, the assay was repeated one more time to further assure assay reliability.Control strains were Escherichia coli (ATCC25922) and Pseudomonas aeruginosa (ATCC27853).Genomic DNA was extracted using Invitrogen PureLink Genomic DNA Mini Kit according to the manufacturer's instructions.% identity 1 and % identity 2 values were results from the identity when subjected to BLAST analysis against PHI database and NR database, respectively.
4.2.Sequence Assembly, Prediction, and Annotation.After the DNA was prepared, a whole-genome shotgun (WGS) sequencing strategy and Illumina HiSeq 2000 sequencing technology with a paired-end protocol was used to determine the genome sequence of S. maltophilia WJ66.The low-quality reads were filtered, and the remaining reads were assembled into contigs with Velvet by the de novo assembly method.ORFs were predicted by the GeneMark analysis.The rRNA was predicted by using RNAmmer and the tRNA was identified with tRNA scan-SE.Then, the genomic sequence was annotated automatically using blast software for searching the SwissPort, UniPort, and the NR database; the cutoff of E-value was 1 − 5.

Nucleotide Sequence Accession Number.
This entire genome shotgun project has been deposited in the DDBJ/EMBL/ GenBank under the accession AZRF00000000.The version described in this paper is the first version, accession AZRF00000000.

Phylogenetic Analysis.
After a maximum likelihood phylogenetic analysis, we construct a neighbour-joining phylogenetic tree with one single copy gene family.Sequences from Xanthomonas campestris pv.campestris strain 8004 were used as an outgroup.

Genome Alignments of S. maltophilia WJ66 and K279a.
Annotated genome sequences and statistics of strain K279a from GenBank can be accessed through genome.Line figures depict the whole genome alignment results using MUMmer [19,36].The query genomic sequence was the strain WJ66 and the subject genome was K279a.The red and blue lines represent direct and reverse alignments, respectively.

Figure 1 :
Figure1: Evolutionary relationships among S. maltophilia strains.Neighbour-joining phylogram of S. maltophilia.Neighbour-joining phylogenetic tree was constructed based on a single copy gene family with the bootstrap set to 1000.Sequences from Xanthomonas campestris pv.campestris strain 8004 were used as an outgroup.The black spot indicates S. maltophilia WJ66.R551 and AU12 are short for R551-3 and AU12-09, respectively.

Figure 2 :
Figure 2: Genome alignment of S. maltophilia WJ66 and K279a.Line figures depict the results of whole genome alignment results using MUMmer.The query genomic sequence was the strain WJ66, and the subject genome was K279a.The red and blue lines represent direct reverse alignments, respectively.