New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci

Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin- and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin- and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin- and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.     

Comparative genomics and DNA array-based genotyping of pandemic Staphylococcus aureus strains encoding Panton-Valentine leukocidin

Within the last few years, methicillin-resistant Staphylococcus aureus (MRSA) strains encoding Panton-Valentine leukocidin (PVL) have emerged and spread worldwide. This epidemic can be attributed to a small number of distinct clones. The present study used a novel assay, based on multiplex linear DNA amplification and subsequent microarray hybridisation, to simultaneously detect all relevant exotoxins, antimicrobial resistance determinants and the allelic variants of agr. The genes of the staphylococcal exotoxin-like (set) locus were also included for typing purposes. This assay, together with multilocus sequence typing (MLST) and spa typing, was applied to 56 clinical isolates and reference strains representing all major pandemic PVL-MRSA lineages, as well as to phylogenetically-related strains and putative ancestors. Array hybridisation results allowed the assignment of isolates to clonal groups, which were in accordance with MLST and spa typing data. Ten distinct clonal groups of PVL-MRSA (ST1, ST5, ST8, ST22, ST30, ST59/359, ST80/583, ST88, ST93 and ST152), including 12 MLST types, were identified and analysed with regard to resistance determinants and genes coding for exotoxins. The array hybridisation data confirmed that pandemic PVL-positive strains originate from very diverse genetic backgrounds, and provided insights into the evolution of some lineages. The DNA microarray technique provides a valuable epidemiological tool for the detailed characterisation of clinical isolates and comparison of strains at a global level.     

Use of a DNA microarray for simultaneous detection of antibiotic resistance genes among staphylococcal clinical isolates

We developed a multiplex asymmetric PCR (MAPCR)-based DNA microarray assay for characterization of the clinically relevant antibiotic resistance genes leading to penicillin, methicillin, aminoglycoside, macrolide, lincosamide, and streptogramin B (MLS(B)) resistance in staphylococci. The DNA-based assay involves detection of specific conserved regions of the mecA, blaZ (methicillin and penicillin resistance), aac(6')-Ie-aph(2') (aminoglycoside resistance), ermA and ermC genes (MLS(B) resistance), and the msrA gene (macrolide and streptogramin B resistance). The microarray uses a variable sequence region of the 16S rRNA gene to broadly differentiate between Staphylococcus aureus and other coagulase-negative staphylococci (CoNS). The performance of the microarray was validated with a total of 178 clinically important S. aureus and 237 CoNS isolates, with correlations of 100% for S. aureus to CoNS discrimination and more than 90% for antibiotic resistance between the genotypic analysis determined by the microarray and the phenotype determined by standard methods of species identification and susceptibility testing. The major discrepant results were 17 mecA-positive CoNS and 60 aac(6')-Ie-aph(2')-positive CoNS isolates measured by microarray that were susceptible to the corresponding antibiotics based on disk diffusion assay. Overall, this microarray-based assay offers a simultaneous, fast (< or =5 h), and accurate identification of antibiotic resistance genes from a single colony, as well as species classification. Our extensive validation of the microarray suggests that it may be a useful tool to complement phenotypic susceptibility testing in clinical laboratories and to survey the spread of antibiotic resistance determinants in epidemiological studies.     

Putative link between Staphylococcus aureus bacteriophage serotype and community association

Methicillin-resistant Staphylococcus aureus (MRSA) from humans can be broadly separated into 3 groups: healthcare-associated (HA), community-associated (CA), and livestock-associated (LA) MRSA. Initially based on epidemiological features, division into these classes is becoming increasingly problematic. The sequencing of S. aureus genomes has highlighted variations in their accessory components, which likely account for differences in pathogenicity and epidemicity. In particular, temperate bacteriophages have been regarded as key players in bacterial pathogenesis. Bacteriophage-associated Panton-Valentine leukocidin genes (luk-PV) are regarded as epidemiological markers of the CA-MRSA due to their high prevalence in CA strains. This paper describes the development and application of a partial composite S. aureus virulence-associated gene microarray. Epidemic, pandemic, and sporadic lineages of UK HA and CA S. aureus were compared. Phage structural genes linked with CA isolates were identified and in silico analysis revealed these to be correlated with phage serogroup. CA strains predominantly carried a PVL-associated phage either of the A or Fb serogroup, whilst HA strains predominantly carried serogroup Fa or B phages. We speculate that carriage of a serogroup A/Fb PVL-associated phage rather than the luk-PV genes specifically is correlated with CA status.     

Multiplex PCR for the detection of genes encoding aminoglycoside modifying enzymes and methicillin resistance among Staphylococcus species

We developed multiplex polymerase chain reaction (PCR) to detect aac(6 ')/aph(2 "), aph(3 ')-IIIa, and ant(4 ')-Ia, the genes encoding the most clinically relevant amino-glycoside modifying enzymes (AME), and simultaneously, the methicillin resistant gene, mecA, in Staphylococcus species. Clinical isolates of 45 S. aureus and 47 coagulase negative staphylococci (CNS) from tertiary university hospitals were tested by conventional susceptibility testing, using the agar dilution method and by multiplex PCR. Of a total of 92 isolates, 61 isolates were found to be methicillin-resistant. Of these, 54 isolates (89%) were found to be harboring mecA. Seventy-five percent of the 92 isolates demonstrated resistance to at least one of the aminoglycosides tested. Moreover, resistance to aminoglycosides was closely associated with methicillin-resistance (p<0.05). The most prevalent AME gene was aac(6 ')/aph(2 ") which was found in 65% of the isolates, and ant(4 ')-Ia and aph(3 ')-IIIa were present in 41% and 9% of the isolates, respectively. The concordance between methicillin-resistance and the presence of mecA gene was 98% in S. aureus and 81% in CNS. The concordance between gentamicin resistance and the presence of aac(6 ')/aph(2 ") gene was 100% in S. aureus and 85% in CNS. The multiplex PCR method that we developed appears to be both a more rapid and reliable than conventional method.     

Use of a three-dimensional microarray system for detection of levofloxacin resistance and the mec A gene in Staphylococcus aureus

We evaluated a novel three-dimensional microarray (Pam Chip microarray) system to detect the presence of levofloxacin-related resistance mutations and the mec A gene. The results were compared to those obtained for 27 Staphylococcus aureus isolates by conventional DNA sequencing or PCR methods. Hybridization and fluorescence detection were performed using an FD 10 system designed for Pam Chip microarray under conditions optimized for each target/probe on the array. In dilution series analysis using multiplex PCR samples, the sensitivity of the microarray was about 10 times greater than that of conventional PCR methods. A high level of data reproducibility was also confirmed in those analyses. Various point mutations in quinolone resistance-determining regions detected by our system corresponded perfectly to the results obtained by conventional DNA sequencing. The results of the mec A gene detection using our system also corresponded to the PCR method; that is, signal/band was detected in all isolates of methicillin-resistant S. aureus, and no signal/band was detected in any isolate of methicillin-susceptible S. aureus. In conclusion, our novel three-dimensional microarray system provided rapid, specific, easy, and reproducible results for the simultaneous detection of levofloxacin resistance and the mec A gene in S. aureus.     

Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.     

Molecular epidemiology of invasive methicillin-susceptible Staphylococcus aureus strains circulating at a Swiss University Hospital

In contrast to methicillin-resistant Staphylococcus aureus, little is known of the distribution of spa types among methicillin-susceptible S. aureus (MSSA). We have analyzed 101 nonrepetitive invasive MSSA isolates from infected patients, consecutively isolated during 14 months between 2006 and 2007 at University Hospital Basel. They were genetically characterized according to S. aureus protein A (spa) types and important virulence-associated genes. Sixty-five different spa types corresponding to nine different spa clonal complexes were observed. Analysis of different virulence genes showed a frequency of 17% for toxic-shock syndrome toxin and 5% for exfoliative toxin D. In conclusion, spa typing revealed a great genetic diversity without predominant spa type, not providing evidence for clonal spreading.     

Use of Multiplex PCR To Detect Classical and Newly Described Pyrogenic Toxin Genes in Staphylococcal Isolates

Staphylococcus aureus may contain one or more genes that encode a variety of immunomodulatory pyrogenic toxins (PTs), including the staphylococcal enterotoxins and toxic shock syndrome toxin (TSST). The PTs interact with several cellular targets to produce disease, such as food poisoning and toxic shock syndrome. At present, nine serologically distinct enterotoxins and one immunoreactive form of TSST have been identified and characterized. As isolates of S. aureus are further assessed, it is anticipated that this number will increase. To facilitate screening, a multiplex PCR was designed to simultaneously determine which of these 10 currently known PT genes an individual S. aureus isolate possesses. We show here, using S. aureus isolates with characterized PT phenotypes, that this novel PCR technique reliably detects each of the known PTs in a single reaction.     

Multiplex PCR for Simultaneous Identification of Staphylococcus aureus and Detection of Methicillin and Mupirocin Resistance

In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the mecA (encoding high-level methicillin resistance), ileS-2 (encoding high-level mupirocin resistance), and femB (encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The femB PCR fragment allows the specific identification of S. aureus. Validation of the method was performed using 50 human isolates of methicillin-resistant S. aureus (MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.     

Reliability of two novel methods, Alamar and E test, for detection of methicillin-resistant Staphylococcus aureus.

E test strips (AB Biodisk, Culver City, Calif.) tested on 2% NaCl-supplemented Mueller-Hinton agar and Alamar panels (Alamar, Sacramento, Calif.) correctly characterized 127 of 127 methicillin-resistant Staphylococcus aureus isolates and 100 of 100 methicillin-susceptible S. aureus isolates. These overnight antimicrobial susceptibility test systems reliably detect this clinically important resistance.     

Identification and characterization of bacterial pathogens causing bloodstream infections by DNA microarray

Bloodstream infections are potentially life-threatening and require rapid identification and antibiotic susceptibility testing of the causative pathogen in order to facilitate specific antimicrobial therapy. We developed a prototype DNA microarray for the identification and characterization of three important bacteremia-causing species: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The array consisted of 120 species-specific gene probes 200 to 800 bp in length that were amplified from recombinant plasmids. These probes represented genes encoding housekeeping proteins, virulence factors, and antibiotic resistance determinants. Evaluation with 42 clinical isolates, 3 reference strains, and 13 positive blood cultures revealed that the DNA microarray was highly specific in identifying S. aureus, E. coli, and P. aeruginosa strains and in discriminating them from closely related gram-positive and gram-negative bacterial strains also known to be etiological agents of bacteremia. We found a nearly perfect correlation between phenotypic antibiotic resistance determined by conventional susceptibility testing and genotypic antibiotic resistance by hybridization to the S. aureus resistance gene probes mecA (oxacillin-methicillin resistance), aacA-aphD (gentamicin resistance), ermA (erythromycin resistance), and blaZ (penicillin resistance) and the E. coli resistance gene probes blaTEM-106 (penicillin resistance) and aacC2 (aminoglycoside resistance). Furthermore, antibiotic resistance and virulence gene probes permitted genotypic discrimination within a species. This novel DNA microarray demonstrates the feasibility of simultaneously identifying and characterizing bacteria in blood cultures without prior amplification of target DNA or preidentification of the pathogen.     

Rapid genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolates using miniaturised oligonucleotide arrays

This study evaluated a DNA oligonucleotide array that recognised 38 different Staphylococcus aureus targets, including all relevant resistance determinants and some toxins and species-specific controls. A new method for labelling sample DNA, based on a linear multiplex amplification that incorporated biotin-labelled dUTP into the amplicon, was established, and allowed detection of hybridisation of the amplicons to the array with an enzymic precipitation reaction. The whole assay was validated by hybridisations with a panel of reference strains and cloned specific PCR products of all targets. To evaluate performance under routine conditions, the assay was used to test 100 methicillin-resistant S. aureus (MRSA) isolates collected from a university hospital in Saxony, Germany. The results showed a high correlation with conventional susceptibility data. The ermA and ermC macrolide resistance genes were found in 40% and 32% of the isolates, respectively. The most prevalent aminoglycoside resistance gene was aphA3 (57% of the isolates), followed by aacA-aphD (29%) and aadD (29%); tet genes, mupR and dfrA were rare. There were no isolates with van genes or genes involved in resistance to quinupristin-dalfopristin. Enterotoxins were detected in 27% of the isolates. Genes encoding Panton-Valentine leukocidin, toxic shock syndrome toxin and exfoliative toxins were not found. The DNA array facilitated rapid and reliable detection of resistance determinants and toxins under conditions used in a routine laboratory and has the potential to be used for array-based high-throughput screening.     

Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy.     

Use of oligoarrays for characterization of community-onset methicillin-resistant Staphylococcus aureus

Until recently, methicillin-resistant Staphylococcus aureus (MRSA) was considered the prototype of a hospital-acquired bacterial pathogen. However, recent reports have shown that MRSA has now emerged in the community. Characterization of specific markers for distinguishing the origin of isolates could contribute to improved knowledge of MRSA epidemiology. The release of whole-genome sequences of hospital- and community-acquired S. aureus strains allowed the development of whole-genome content analysis techniques, including microarrays. We developed a microarray composed of 8,191 open reading frame-specific oligonucleotides covering >99% of the four sequenced S. aureus genomes (N315, Mu50, MW2, and COL) to evaluate gene contents of hospital- and community-onset S. aureus strains. In parallel, pulsed-field gel electrophoresis, variable number of tandem repeats, antibiogram, staphylococcal cassette chromosome-mec element typing, and presence of the Panton-Valentine leukocidin gene were evaluated in a collection of 15 clinical isolates. Clusters obtained with microarrays showed a high degree of similarity with those obtained by pulsed-field gel electrophoresis or variable number of tandem repeats. Clusters clearly segregated hospital-onset strains from community-onset strains. Moreover, the microarray approach allowed definition of novel marker genes and chromosomal regions specific for given groups of isolates, thus providing better discrimination and additional information compared to pulsed-field gel electrophoresis and variable number of tandem repeats. Finally, the comparative genome hybridization approach unraveled the occurrence of multiple horizontal transfer events leading to community-onset MRSA as well as the need for a specific genetic background in recipient strains for both the acquisition and the stability of the mec element.     

Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus

In this study we describe a multiplex PCR assay for the detection of nine clinically relevant antibiotic resistance genes of Staphylococcus aureus. Conditions were optimized to amplify fragments of mecA (encoding methicillin resistance), aacA-aphD (aminoglycoside resistance), tetK, tetM (tetracycline resistance), erm(A), erm(C) (macrolide-lincosamide-streptogramin B resistance), vat(A), vat(B), and vat(C) (streptogramin A resistance) simultaneously in one PCR amplification. An additional primer pair for the amplification of a fragment of the staphylococcal 16S rDNA was included as a positive control. The multiplex PCR assay was evaluated on 30 different S. aureus isolates, and the PCR results correlated with the phenotypic antibiotic resistance data obtained by the broth microdilution assay. The multiplex PCR assay offers a rapid, simple, and accurate identification of antibiotic resistance profiles and could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.     

Rapid identification of staphylococci from prosthetic joint infections using MALDI-TOF mass-spectrometry

Hospital-acquired infections associated with implanted medical devices are most commonly caused by staphylococci. Current methods of species identification are slow, costly, and sometimes unreliable. We evaluated the ability of a Bruker Daltonics Microflex MALDI-TOF/MS in conjunction with MALDI Biotyper software to identify 158 characterized staphylococcal isolates from prosthetic joint infections, including 36 Staphylococcus aureus, 100 Staphylococcus epidermidis, 10 Staphylococcus capitis, 8 Staphylococcus lugdunensis, 2 Staphylococcus warneri, and 2 Staphylococcus haemolyticus isolates using the extraction method recommended by Bruker Daltonics. The suggested species identification by the MALDI Biotyper software was correct for all isolates, indicating reliable differentiation between S. aureus and coagulase-negative staphylococci. Applying the recommended criteria of the MALDI Biotyper software all 158 isolates gave scores ≥2.0, implying secure genus and probable species identification for all isolates. 34/36 S. aureus, 36/100 S. epidermidis, 5/10 S. capitis, 6/8 S. lugdunensis, 2/2 S. haemolyticus, 0/2 S. warneri displayed scores ≥2.3 implying highly probable species identification. For S. epidermidis 25/100 additional isolates had a score close to 2.3. It appears that additional clinically relevant staphylococcal isolates in the data base might aid in identification at scores implying highly probable species identification. The ability of the MALDI Biotyper software to recognize clonally-related strains within a species group (i.e. sub-typing) was investigated, and showed great potential. In conclusion, the MALDI-TOF/MS MALDI Biotyper system provides a promising rapid and reliable method of identifying clinical isolates from prosthetic joint infections to the species level, and has potential for sub-typing.     

Increased vancomycin MICs for Staphylococcus aureus clinical isolates from a university hospital during a 5-year period

Staphylococcus aureus is one of the most commonly isolated organisms in nosocomial infections. While the prevalence of methicillin-resistant S. aureus (MRSA) continues to increase worldwide, there is concern about an increase in vancomycin MICs among S. aureus strains. The prevalence of MRSA and vancomycin MIC trends in S. aureus from patients in a university hospital were analyzed. Clinical Laboratory Standards Institute (CLSI, formerly NCCLS) reference broth microdilution MIC testing was performed on all clinically relevant S. aureus isolates from January 2000 through December 2004. A total of 6,003 S. aureus isolates were analyzed. No vancomycin-resistant S. aureus isolates were detected. One MRSA isolate had a vancomycin MIC of 8 mug/ml and was confirmed as vancomycin-intermediate S. aureus. Among the 6,002 remaining isolates, a shift in vancomycin MICs from 相似文献   

Simultaneous analysis of multiple staphylococcal enterotoxin genes by an oligonucleotide microarray assay

Staphylococcal enterotoxins (SEs) are a family of 17 major serological types of heat-stable enterotoxins that are one of the leading causes of gastroenteritis resulting from consumption of contaminated food. SEs are considered potential bioweapons. Many Staphylococcus aureus isolates contain multiple SEs. Because of the large number of SEs, serological typing and PCR typing are laborious and time-consuming. Furthermore, serological typing may not always be practical because of antigenic similarities among enterotoxins. We report on a microarray-based one-tube assay for the simultaneous detection and identification (genetic typing) of multiple enterotoxin (ent) genes. The proposed typing method is based on PCR amplification of the target region of the ent genes with degenerate primers, followed by characterization of the PCR products by microchip hybridization with oligonucleotide probes specific for each ent gene. We verified the performance of this method by using several other techniques, including PCR amplification with gene-specific primers, followed by gel electrophoresis or microarray hybridization, and sequencing of the enterotoxin genes. The assay was evaluated by analysis of previously characterized staphylococcal isolates containing 16 ent genes. The microarray assay revealed that some of these isolates contained additional previously undetected ent genes. The use of degenerate primers allows the simultaneous amplification and identification of as many as nine different ent genes in one S. aureus strain. The results of this study demonstrate the usefulness of the oligonucleotide microarray assay for the analysis of multitoxigenic strains, which are common among S. aureus strains, and for the analysis of microbial pathogens in general.     

Stenotrophomonas maltophilia strains from cystic fibrosis patients: genomic variability and molecular characterization of some virulence determinants

The genetic relatedness of 52 Stenotrophomonas maltophilia strains, collected from various environmental and clinical sources, including cystic fibrosis (CF) patients, as well as the presence and the expression of some virulence-associated genes were studied. Pulsed-field gel electrophoresis (PFGE) analysis identified 47 profiles and three clusters of isolates with an identical PFGE pattern considered to be indistinguishable strains. Restriction fragment length polymorphism of the gyrB gene grouped the 52 strains into nine different profiles. Most CF clinical isolates (29 out of 41) showed profile 1, while the analysis of the hypervariable regions of the 16S rRNA gene revealed five distinct allelic variations, with the majority of CF isolates (23 out of 41) belonging to sequence group 1. Furthermore, the strains were characterized for motility and expression of virulence-associated genes, including genes encoding type-1 fimbriae, proteases (StmPr1 and StmPr2) and esterase. All S. maltophilia strains exhibited a very broad range of swimming and twitching motility, while none showed swarming motility. A complete smf-1 gene was PCR-amplified only from clinically derived S. maltophilia strains. Finally, the virulence of representative S. maltophilia strains impaired in the expression of proteases and esterase activities was evaluated by infecting larvae of the wax moth Galleria mellonella. The results obtained strongly indicate that the major extracellular protease StmPr1 may be a relevant virulence factor of S. maltophilia.