Characterization of phage evolution and phage resistance in drug-resistant Stenotrophomonas maltophilia.

Phage therapy has become a viable antimicrobial treatment as an alternative to antibiotic treatment, with an increase in antibiotic resistance. Phage resistance is a major limitation in the therapeutic application of phages, and the lack of understanding of the dynamic changes between bacteria and phages constrains our response strategies to phage resistance. In this study, we investigated the changing trends of mutual resistance between Stenotrophomonas maltophilia (S. maltophilia) and its lytic phage, BUCT603. Our results revealed that S. maltophilia resisted phage infection through mutations in the cell membrane proteins, while the evolved phage re-infected the resistant strain primarily through mutations in structure-related proteins. Compared with the wild-type strain (SMA118), the evolved phage-resistant strain (R118-2) showed reduced virulence, weakened biofilm formation ability, and reduced resistance to aminoglycosides. In addition, the evolved phage BUCT603B1 in combination with kanamycin could inhibit the development of phage-resistant S. maltophilia in vitro and significantly improve the survival rate of S. maltophilia-infected mice. Altogether, these results suggest that in vitro characterization of bacteria-phage co-evolutionary relationships is a useful research tool to optimize phages for the treatment of drug-resistant bacterial infections.IMPORTANCEPhage therapy is a promising approach to treat infections caused by drug-resistant Stenotrophomonas maltophilia (S. maltophilia). However, the rapid development of phage resistance has hindered the therapeutic application of phages. In vitro evolutionary studies of bacteria-phage co-cultures can elucidate the mechanism of resistance development between phage and its host. In this study, we investigated the resistance trends between S. maltophilia and its phage and found that inhibition of phage adsorption is the primary strategy by which bacteria resist phage infection in vitro, while phages can re-infect bacterial cells by identifying other adsorption receptors. Although the final bacterial mutants were no longer infected by phages, they incurred a fitness cost that resulted in a significant reduction in virulence. In addition, the combination treatment with phage and aminoglycoside antibiotics could prevent the development of phage resistance in S. maltophilia in vitro. These findings contribute to increasing the understanding of the co-evolutionary relationships between phages and S. maltophilia.

The Potential of Phage Therapy against the Emerging Opportunistic Pathogen Stenotrophomonas maltophilia.

The isolation and characterization of bacteriophages for the treatment of infections caused by the multidrug resistant pathogen Stenotrophomonas maltophilia is imperative as nosocomial and community-acquired infections are rapidly increasing in prevalence. This increase is largely due to the numerous virulence factors and antimicrobial resistance genes encoded by this bacterium. Research on S. maltophilia phages to date has focused on the isolation and in vitro characterization of novel phages, often including genomic characterization, from the environment or by induction from bacterial strains. This review summarizes the clinical significance, virulence factors, and antimicrobial resistance mechanisms of S. maltophilia, as well as all phages isolated and characterized to date and strategies for their use. We further address the limited in vivo phage therapy studies conducted against this bacterium and discuss the future research needed to spearhead phages as an alternative treatment option against multidrug resistant S. maltophilia.

Characterization and Genomic Analysis of ɸSHP3, a New Transposable Bacteriophage Infecting Stenotrophomonas maltophilia.

This study describes a novel transposable bacteriophage, ɸSHP3, continuously released by Stenotrophomonas maltophilia strain c31. Morphological observation and genomic analysis revealed that ɸSHP3 is a siphovirus with a 37,611-bp genome that encodes 51 putative proteins. Genomic comparisons indicated that ɸSHP3 is a B3-like transposable phage. Its genome configuration is similar to that of Pseudomonas phage B3, except for the DNA modification module. Similar to B3-like phages, the putative transposase B of ɸSHP3 is a homolog of the type two secretion component ExeA, which is proposed to serve as a potential virulence factor. Moreover, most proteins of ɸSHP3 have homologs in transposable phages, but only ɸSHP3 carries an RdgC-like protein encoded by gene 3, which exhibits exonuclease activity in vitro Two genes and their promoters coding for ɸSHP3 regulatory proteins were identified and appear to control the lytic-lysogenic switch. One of the proteins represses one promoter activity and confers immunity to ɸSHP3 superinfection in vivo The short regulatory region, in addition to the canonical bacterial promoter sequences, displays one LexA and two CpxR recognition sequences. This suggests that LexA and the CpxR/CpxA two-component system might be involved in the control of the ɸSHP3 genetic switch.IMPORTANCES. maltophilia is an emerging global pathogenic bacterium that displays genetic diversity in both environmental and clinical strains. Transposable phages have long been known to improve the genetic diversity of bacterial strains by transposition. More than a dozen phages of S. maltophilia have been characterized. However, no transposable phage infecting S. maltophilia has been reported to date. Characterization of the first transposable phage, ɸSHP3, from S. maltophilia will contribute to our understanding of host-phage interactions and genetic diversity, especially the interchange of genetic materials among S. maltophilia.

Biological characteristics and genomic analysis of a Stenotrophomonas maltophilia phage vB_SmaS_BUCT548.

Stenotrophomonas maltophilia (hereinafter referred to as S. maltophilia) has developed into an important opportunistic pathogenic bacterium, which is prevalent in nosocomial and community infections, and has adverse effects on patients with a compromised immune system. Phage vB_SmaS_BUCT548 was isolated from sewage of Beijing 307 Hospital with S. maltophilia (strain No.824) as a host. Phage morphology was observed by transmission electron microscopy and its biological and genomic characteristics were determined. The electron microscope shows that the bacteriophage belonged to the Siphoviridae and MOI is 0.001. One-step growth curve shows that the incubation period is 30 min and the burst size is 134 PFU/Cell. The host range is relatively wide and it can lysis 11of 13 S. maltophilia strains. Next-Generation Sequencing (NGS) results show that the genome sequence is a dsDNA with 62354 bp length, and the GC content is 56.3% (GenBank: MN937349). One hundred and two online reading frames (ORFs) are obtained after RAST online annotation and the BlastN nucleic acid comparison shows that the phage had low homology with other phages in NCBI database. This study reports a novel S. maltophilia phage named vB_SmaS_BUCT548, which has a short incubation period, strong lytic ability, and a wide host range. The main characteristic of this bacteriophage is the novelty of the genomic sequence and the analysis of the other characteristics provides basic data for further exploring the interaction mechanism between the phage and the host.

Isolation and Characterization of the Novel Bacteriophage AXL3 against Stenotrophomonas maltophilia.

The rapid increase in the number of worldwide human infections caused by the extremely antibiotic resistant bacterial pathogen Stenotrophomonas maltophilia is cause for concern. An alternative treatment solution in the post-antibiotic era is phage therapy, the use of bacteriophages to selectively kill bacterial pathogens. In this study, the novel bacteriophage AXL3 (vB_SmaS-AXL_3) was isolated from soil and characterized. Host range analysis using a panel of 29 clinical S. maltophilia isolates shows successful infection of five isolates and electron microscopy indicates that AXL3 is a member of the Siphoviridae family. Complete genome sequencing and analysis reveals a 47.5 kb genome predicted to encode 65 proteins. Functionality testing suggests AXL3 is a virulent phage and results show that AXL3 uses the type IV pilus, a virulence factor on the cell surface, as its receptor across its host range. This research identifies a novel virulent phage and characterization suggests that AXL3 is a promising phage therapy candidate, with future research examining modification through genetic engineering to broaden its host range.

Novel Stenotrophomonas maltophilia temperate phage DLP4 is capable of lysogenic conversion.

BACKGROUND: Temperate bacteriophages are capable of lysogenic conversion of new bacterial hosts. This phenomenon is often ascribed to "moron" elements that are acquired horizontally and transcribed independently from the rest of the phage genes. Whereas some bacterial species exhibit relatively little prophage-dependent phenotypic changes, other bacterial species such as Stenotrophomonas maltophilia appear to commonly adopt prophage genetic contributions. RESULTS: The novel S. maltophilia bacteriophage DLP4 was isolated from soil using the highly antibiotic-resistant S. maltophilia strain D1585. Genome sequence analysis and functionality testing showed that DLP4 is a temperate phage capable of lysogenizing D1585. Two moron genes of interest, folA (BIT20_024) and ybiA (BIT20_065), were identified and investigated for their putative activities using complementation testing and phenotypic and transcriptomic changes between wild-type D1585 and the D1585::DLP4 lysogen. The gp24 / folA gene encodes dihydrofolate reductase (DHFR: FolA), an enzyme responsible for resistance to the antibiotic trimethoprim. I-TASSER analysis of DLP4 FolA predicted structural similarity to Bacillus anthracis DHFR and minimum inhibitory concentration experiments demonstrated that lysogenic conversion of D1585 by DLP4 provided the host cell with an increase in trimethoprim resistance. The gp65 / ybiA gene encodes N-glycosidase YbiA, which in E. coli BW25113 is required for its swarming motility phenotype. Expressing DLP4 ybiA in strain ybiA770(del)::kan restored its swarming motility activity to wildtype levels. Reverse transcription-PCR confirmed the expression of both of these genes during DLP4 lysogeny. CONCLUSIONS: S. maltophilia temperate phage DLP4 contributes to the antibiotic resistance exhibited by its lysogenized host strain. Genomic analyses can greatly assist in the identification of phage moron genes potentially involved in lysogenic conversion. Further research is required to fully understand the specific contributions temperate phage moron genes provide with respect to the antibiotic resistance and virulence of S. maltophilia host cells.

Identification and Characterization of Type IV Pili as the Cellular Receptor of Broad Host Range Stenotrophomonas maltophilia Bacteriophages DLP1 and DLP2.

Bacteriophages DLP1 and DLP2 are capable of infecting both Stenotrophomonas maltophilia and Pseudomonas aeruginosa strains, two highly antibiotic resistant bacterial pathogens, which is unusual for phages that typically exhibit extremely limited host range. To explain their unusual cross-order infectivity and differences in host range, we have identified the type IV pilus as the primary receptor for attachment. Screening of a P. aeruginosa PA01 mutant library, a host that is susceptible to DLP1 but not DLP2, identified DLP1-resistant mutants with disruptions in pilus structural and regulatory components. Subsequent complementation of the disrupted pilin subunit genes in PA01 restored DLP1 infection. Clean deletion of the major pilin subunit, pilA, in S. maltophilia strains D1585 and 280 prevented phage binding and lysis by both DLP1 and DLP2, and complementation restored infection by both. Transmission electron microscopy shows a clear interaction between DLP1 and pili of both D1585 and PA01. These results support the identity of the type IV pilus as the receptor for DLP1 and DLP2 infection across their broad host ranges. This research further characterizes DLP1 and DLP2 as potential “anti-virulence” phage therapy candidates for the treatment of multidrug resistant bacteria from multiple genera.

Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress.

Several bacterial species produce membrane vesicles (MVs) in response to antibiotic stress. However, the biogenesis and role of MVs in bacterial antibiotic resistance mechanisms have remained unclear. Here, we studied the effect of the fluoroquinolone ciprofloxacin on MV secretion by Stenotrophomonas maltophilia using a combination of electron microscopy and proteomic approaches. We found that in addition to the classical outer membrane vesicles (OMV), ciprofloxacin-stimulated cultures produced larger vesicles containing both outer and inner membranes termed outer-inner membrane vesicles (OIMV), and that such MVs are enriched with cytosolic proteins. Remarkably, OIMV were found to be decorated with filamentous structures identified as fimbriae. In addition, ciprofloxacin stress leads to the release of bacteriophages and phage tail-like particles. Prophage induction by ciprofloxacin has been linked to pathogenesis and horizontal gene transfer in several bacterial species. Together, our findings show that ciprofloxacin treatment of S. maltophilia leads to the secretion of a heterogeneous pool of MVs and the induction of prophages that are potentially involved in adverse side-effects during antibiotic treatment.

The isolation and characterization of Stenotrophomonas maltophilia T4-like bacteriophage DLP6.

Increasing isolation of the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia has caused alarm worldwide due to the limited treatment options available. A potential treatment option for fighting this bacterium is 'phage therapy', the clinical application of bacteriophages to selectively kill bacteria. Bacteriophage DLP6 (vB_SmoM-DLP6) was isolated from a soil sample using clinical isolate S. maltophilia strain D1571 as host. Host range analysis of phage DLP6 against 27 clinical S. maltophilia isolates shows successful infection and lysis in 13 of the 27 isolates tested. Transmission electron microscopy of DLP6 indicates that it is a member of the Myoviridae family. Complete genome sequencing and analysis of DLP6 reveals its richly recombined evolutionary history, featuring a core of both T4-like and cyanophage genes, which suggests that it is a member of the T4-superfamily. Unlike other T4-superfamily phages however, DLP6 features a transposase and ends with 229 bp direct terminal repeats. The isolation of this bacteriophage is an exciting discovery due to the divergent nature of DLP6 in relation to the T4-superfamily of phages.

The isolation and characterization of two Stenotrophomonas maltophilia bacteriophages capable of cross-taxonomic order infectivity.

BACKGROUND: A rapid worldwide increase in the number of human infections caused by the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia is prompting alarm. One potential treatment solution to the current antibiotic resistance dilemma is "phage therapy", the clinical application of bacteriophages to selectively kill bacteria. RESULTS: Towards that end, phages DLP1 and DLP2 (vB_SmaS-DLP_1 and vB_SmaS-DLP_2, respectively) were isolated against S. maltophilia strain D1585. Host range analysis for each phage was conducted using 27 clinical S. maltophilia isolates and 11 Pseudomonas aeruginosa strains. Both phages exhibit unusually broad host ranges capable of infecting bacteria across taxonomic orders. Transmission electron microscopy of the phage DLP1 and DLP2 morphology reveals that they belong to the Siphoviridae family of bacteriophages. Restriction fragment length polymorphism analysis and complete genome sequencing and analysis indicates that phages DLP1 and DLP2 are closely related but different phages, sharing 96.7 % identity over 97.2 % of their genomes. These two phages are also related to P. aeruginosa phages vB_Pae-Kakheti_25 (PA25), PA73, and vB_PaeS_SCH_Ab26 (Ab26) and more distantly related to Burkholderia cepacia complex phage KL1, which together make up a taxonomic sub-family. Phages DLP1 and DLP2 exhibited significant differences in host ranges and growth kinetics. CONCLUSIONS: The isolation and characterization of phages able to infect two completely different species of bacteria is an exciting discovery, as phages typically can only infect related bacterial species, and rarely infect bacteria across taxonomic families, let alone across taxonomic orders.

Genomic sequence of temperate phage Smp131 of Stenotrophomonas maltophilia that has similar prophages in xanthomonads.

BACKGROUND: Stenotrophomonas maltophilia is a ubiquitous Gram-negative bacterium previously named as Xanthomonas maltophilia. This organism is an important nosocomial pathogen associated with infections in immunocompromised patients. Clinical isolates of S. maltophilia are mostly resistant to multiple antibiotics and treatment of its infections is becoming problematic. Several virulent bacteriophages, but not temperate phage, of S. maltophilia have been characterized. RESULTS: In this study, a temperate myophage of S. maltophilia (Smp131) was isolated and characterized. Sequence analysis showed that its genome is 33,525-bp long with 47 open reading frames (ORFs). Its similarity to P2-like phages and prophages in S. maltophilia and several Xanthomonas pathovars includes genomic organization, arrangement of several operons, and possession of a slippery sequence T7G for translational frameshifting in tail assembly genes. Smp131 encodes a tyrosine family integrase that shares low degrees of similarity with those of other phages and a lysin belonging to family 19 chitinase that is observed in plants and some bacteria, although not in phages. tRNA are the preferred sites for host integration of Smp131 and the related phages: tRNA-Thr for Smp131 and prophage of S. maltophilia K279a; tRNA-Lys for prophages of X. campestris pv. campestris ATCC33913, X. oryzae pv. oryzae strains MAFF311018, and KACC10331; and tRNA-Asn for prophage of X. oryzae pv. oryzae PXO99A and remnant of X. axonopodis pv. citri 306. Regions flanking the prophages are varied highly in nucleotide sequence and rich in transposase genes, suggesting that frequent insertion/excision had occurred. CONCLUSIONS: Prevalence of closely related prophages in Stenotrophomonas and Xanthomonads may have contributed to the diversity of these closely related species owing to possible horizontal gene transfer mediated by the phages.

Genomic characterization and integrative properties of phiSMA6 and phiSMA7, two novel filamentous bacteriophages of Stenotrophomonas maltophilia.

Two novel filamentous phages, phiSMA6 and phiSMA7, were isolated from Stenotrophomonas maltophilia environmental strain Khak84. We identified and annotated 11 potential open reading frames in each phage. While the overall layout of the functional gene groups of both phages was similar to that of the known filamentous phages, they differed from them in their molecular structure. The genome of phiSMA6 is a mosaic that evolved by acquiring genes from at least three different filamentous S. maltophilia phages and one Xanthomonas campestris phage related to Cf1. In the phiSMA6 genome, a gene similar to the bacterial gene encoding the mating pair formation protein trbP was also found. We showed that phiSMA6 possesses lysogenic properties and upon induction produces high-titer lysates. The genome of phiSMA7 possesses a unique structure and was found to be closely related to a prophage present in the chromosome of the completely sequenced S. maltophilia clinical strain D457. We suggest that the other three filamentous phages of S. maltophilia described previously also have the capacity to integrate into the genome of their bacterial host.

Complete genome sequence of IME15, the first T7-like bacteriophage lytic to pan-antibiotic-resistant Stenotrophomonas maltophilia.

T7-like bacteriophages are a class of virulent bacteriophages which have a clearer genetic background and smaller genomes than other phages. In addition, it grows faster and is easier to culture than other phages. At present, the numbers of available T7-like bacteriophage genomes and Stenotrophomonas maltophilia genomes are small, and IME15 is the first T7-like virulent Stenotrophomonas phage whose sequence has been reported. It shows effective lysis of S. maltophilia. Here we announce its complete genome, and major findings from its annotation are described.

Complete Genome Sequence of IME13, a Stenotrophomonas maltophilia bacteriophage with large burst size and unique plaque polymorphism.

Stenotrophomonas maltophilia bacteriophage IME13 is a virulent phage with a large burst size, exceeding 3,000, much larger than that of any other stenotrophomonas phage reported before. It showed effective lysis of Stenotrophomonas maltophilia. Additionally, the phage IME13 developed at least three obviously different sizes of plaques when a single plaque was picked out and inoculated on a double-layer Luria broth agar plate with its host. Here we announce its complete genome and describe major findings from its annotation.

Isolation and characterization of a novel filamentous phage from Stenotrophomonas maltophilia.

In this study, a novel filamentous phage, φSHP1, of the environmental Stenotrophomonas maltophilia strain P2 was isolated and characterized. Electron microscopy showed that φSHP1 resembled members of the family Inoviridae and was about 2.1 µm long. The 6,867-nucleotide genome of φSHP1 was a circular single-stranded DNA and had a replication form designated pSH1. Ten putative open reading frames (ORFs) were found in the φSHP1 genome, and six predicted proteins showed similarity to proteins in databases. Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis of φSHP1 displayed one major structural polypeptide of approximately 4.0 kDa. N-terminal sequencing showed that it was the mature product of ORF5 and that its N-terminal 27 amino acid residues had been cleaved off from the predicted nascent protein. Finally, phylogenetic trees were constructed to analyze the phylogenetic relationship of φSHP1 to other known filamentous phages. φSHP1 appears to be the first reported Stenotrophomonas filamentous phage.

Stenotrophomonas maltophilia, un patógeno nosocomial emergente de importancia creciente / Stenotrophomonas maltophilia: an emergent nosocomial pathogen with an increasing significance

En las últimas décadas se ha estado aislando con frecuencia creciente Stenotrophomonas maltophilia de muestras clínicas, según reporta la literatura mundial. En la provincia de Villa Clara está constituyendo una causa cada vez más relevante de infección intrahospitalaria. El trabajo se propone destacar la importancia del conocimiento médico de este microorganismo, de sus características fundamentales y de las principales propuestas de fármacos para su tratamiento.

In the last decades, Stenotrophomonas maltophilia has been increasingly isolated from clinical samples according to world literature. In Villa Clara province, it is an ever-increasing important cause of intrahospital infection. The aim of this paper was to emphasize the significance of the medical knowledge of this microorganism, its fundamental characteristics, and the main drugs recommended of for its treatment.

Characterization of a novel T4-type Stenotrophomonas maltophilia virulent phage Smp14.

Stenotrophomonas maltophilia (Sm), with most of the isolates being resistant to multidrugs, is an opportunistic bacterium causing nosocomial infections. In this study, a novel virulent Sm phage, Smp14, was characterized. Electron microscopy showed that Smp14 resembled members of Myoviridae and adsorbed to poles of the host cells during infection. It lysed 37 of 87 clinical Sm isolates in spot test, displayed a latent period of ca. 20 min, and had a burst size of ca. 150. Its genome (estimated to be 160 kb by PFGE), containing m4C and two unknown modified bases other than m5C and m6A as identified by HPLC, resisted to digestion with many restriction endonucleases except MseI. These properties indicate that it is a novel Sm phage distinct from the previously reported phiSMA5 which has a genome of 250 kb digestible with various restriction enzymes. Sequencing of a 16 kb region revealed 12 ORFs encoding structural proteins sharing 15-45% identities with the homologues from T4-type phages. SDS-PAGE displayed 20 virion proteins, with the most abundant one being the 39 kDa major capsid protein (gp23), which had the N-terminal 52 amino acids removed. Phylogenetic analysis based on gp23 classified Smp14 into a novel single-membered T4-type subgroup.

Isolation and characterization of novel giant Stenotrophomonas maltophilia phage phiSMA5.

Stenotrophomonas maltophilia is one of the most prevalent opportunistic bacteria causing nosocomial infections. It has become problematic because most of the isolates are resistant to multiple antibiotics, and therefore, development of phage therapy has attracted strong attention. In this study, eight S. maltophilia phages were isolated from clinical samples including patient specimens, catheter-related devices, and wastewater. These phages can be divided into four distinct groups based on host range and digestibility of the phage DNAs with different restriction endonucleases. One of them, designated phiSMA5, was further characterized. Electron microscopy showed it resembled Myoviridae, with an isometric head (90 nm in diameter), a tail (90 nm long), a baseplate (25 nm wide), and short tail fibers. The phiSMA5 double-stranded DNA, refractory to digestion by most restriction enzymes, was tested and estimated to be 250 kb by pulsed-field gel electrophoresis. This genome size is second to that of the largest phage, phiKZ of Pseudomonas aeruginosa. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 25 virion proteins were visualized. N-terminal sequencing of four of them suggested that each of them might have had its N terminus cleaved off. Among the 87 S. maltophilia strains collected in this study, only 61 were susceptible to phiSMA5, indicating that more phages are needed toward a phage therapy strategy. Since literature search yielded no information about S. maltophilia phages, phiSMA5 appears to be the first reported.