METHODS AND MATERIALS FOR REDUCING BIOFILMS

This application is a continuation of U.S. application Ser. No. 12/664,574, filed Jun. 11, 2010, which is a U.S. National Stage application under 35 U.S.C. §371 of International Application No. PCT/US2008/066913, having a filing date of Jun. 13, 2008, which claims the benefit of U.S. Provisional Application Ser. No. 60/944,019, filed Jun. 14, 2007. The disclosures of the prior applications are incorporated by reference in their entirety.

1. Technical Field

This document relates to methods and materials involved in reducing biofilms. For example, this document provides enzymes (e.g., glycosyl hydrolases) and methods for using enzymes to reduce biofilms.

2. Background Information

Bacteria growing in biofilms are estimated to be involved in greater than 60 percent of all human bacterial infections. Biofilms are dynamic populations of bacteria in a surface-associated mode of growth that are covered in a protective, self-excreted extracellular polymeric substance (EPS) matrix. Biofilms provide protection against harsh environmental conditions, traditional antimicrobial therapies, and host immune defenses, thus making biofilm-associated infections difficult to treat. Biofilm-associated infections of indwelling medical devices (e.g., intravascular and urinary catheters, prosthetic heart valves, prosthetic joint implants, and hardware) represent a particularly important health problem, as removal and replacement of infected devices is often required.

The EPS matrix of a diverse number of biofilm-forming bacterial species can be composed of chains of polymeric β-1,6-linked N-acetyl-glucosamine (PNAG). Biofilm-forming strains of Staphylococcus aureus, S. epidermidis, Bordetella spp., Actinobacillus spp., and Escherichia coli are known to utilize PNAG as a major component of their EPS biofilm matrix (Cramton et al., Infect. Immun., 67:5427-5433 (1999); Kaplan et al., J. Bacteriol., 186:8213-8220 (2004); Mack et al., J. Bacteriol., 178:175-183 (1996); Parise et al., J. Bacteriol., 189:750-760 (2007); and Wang et al., J. Bacteriol., 186:2724-2734 (2004)). The N-acetyl-β-hexosaminidase, dispersin B, first purified from the Gram negative periodontal pathogen Actinobacillus actinomycetemcomitans (Kaplan et al., J. Bacteriol., 185:4693-8 (2003)), can cleave the β-1,6-linkages of PNAG in the biofilm matrices of Staphylococcus spp., Yersinia pestis, Actinobacillus spp., Bordetella spp., and E. coli (Itoh et al., J. Bacteriol., 187:382-387 (2005); Kaplan et al., J. Bacteriol., 185:4693-8 (2003); Kaplan et al., Antimicrob. Agents Chemother., 48:2633-2636 (2004); Kaplan et al., J. Bacteriol., 186:8213-8220 (2004); and Parise et al, J. Bacteriol., 189:750-760 (2007)).

This document provides methods and materials related to reducing biofilms. For example, this document provides enzymes (e.g., glycosyl hydrolases), nucleic acid molecules encoding enzymes, host cells containing nucleic acid encoding enzymes, and methods for using enzymes to reduce biofilms and infections associated with biofilms. Reducing biofilms and infections associated with biofilms can allow clinicians to treat patients effectively and can help reduce the incidence of infections in mammals (e.g., mammals containing or using a medical device that is susceptible to biofilms).

S. lugdunensis is an atypically virulent Gram positive human pathogen that is able to form biofilms (Frank et al., Antimicrob. Agents Chemother., 51:888-895 (2007)). As described herein, a polypeptide obtained from S. lugdunensis can have glycosyl hydrolase activity and can be used as a biofilm-releasing enzyme to treat or prevent a range of biofilm-associated bacterial infections.

In general, one aspect of this document features an isolated nucleic acid molecule that encodes a polypeptide having a length of at least 300 amino acid residues and at least about 95 percent identity to the amino acid sequence set forth in SEQ ID NO:2 over the length. The polypeptide can comprise a glycosyl hydrolase activity. The isolated nucleic acid molecule can comprise the nucleic acid sequence set forth in SEQ ID NO:1.

In another aspect, this document features an isolated nucleic acid molecule comprising at least 15 nucleotides in length, wherein the isolated nucleic acid molecule hybridizes under hybridization conditions to the sense or antisense strand of the sequence set forth in SEQ ID NO:1. The hybridization conditions can be highly stringent hybridization conditions. The isolated nucleic acid molecule can comprise at least 1000 nucleotides in length. The isolated nucleic acid molecule can comprise the sequence set forth in SEQ ID NO:1.

In another aspect, this document features a cell comprising an isolated nucleic acid molecule that (a) encodes a polypeptide having a length of at least 300 amino acid residues and at least about 95 percent identity to the amino acid sequence set forth in SEQ ID NO:2 over the length, or (b) comprises at least 15 nucleotides in length, wherein the isolated nucleic acid molecule hybridizes under hybridization conditions to the sense or antisense strand of the sequence set forth in SEQ ID NO:1. The cell can be a prokaryotic cell.

In another aspect, this document features a substantially pure polypeptide comprising an amino acid sequence having a length of at least 300 amino acid residues and at least about 95 percent identity to the amino acid sequence set forth in SEQ ID NO:2 over the length. The polypeptide can comprise a glycosyl hydrolase activity. The polypeptide can be encoded by a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO:l. The polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:2.

In another aspect, this document features a method for reducing biofilm present on a surface, wherein the method comprises contacting the surface with a polypeptide of claim 10 under conditions wherein the presence of the biofilm on the surface is reduced. The biofilm can comprise pathogenic bacteria. The surface can be a surface of a catheter. The polypeptide can comprise a glycosyl hydrolase activity. The polypeptide can be encoded by a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO:l. The polypeptide can comprise the amino acid sequence set forth in SEQ ID NO:2. The presence of the biofilm on the surface can be reduced to below the level of detection after the contacting step.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

This document provides isolated nucleic acid molecules, host cells that contain an isolated nucleic acid molecule, and substantially pure polypeptides. In addition, this document provides methods and materials for reducing biofilms, preventing biofilms, and treating infections associated with biofilms.

The term “nucleic acid” as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

The term “isolated” as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5′ end and one on the 3′ end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

The term “isolated” as used herein with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence.

It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.

The term “exogenous” as used herein with reference to nucleic acid and a particular cell refers to any nucleic acid that does not originate from that particular cell as found in nature. Thus, all non-naturally-occurring nucleic acid is considered to be exogenous to a cell once introduced into the cell. It is important to note that non-naturally-occurring nucleic acid can contain nucleic acid sequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a cell once introduced into the cell, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid.

Nucleic acid that is naturally-occurring can be exogenous to a particular cell. For example, an entire chromosome isolated from a cell of person X is an exogenous nucleic acid with respect to a cell of person Y once that chromosome is introduced into Y′s cell.

This document provides isolated nucleic acid molecules that contain a nucleic acid sequence having (1) a length, and (2) a percent identity to an identified nucleic acid sequence over that length. This document also provides isolated nucleic acid molecules that contain a nucleic acid sequence encoding a polypeptide that contains an amino acid sequence having (1) a length, and (2) a percent identity to an identified amino acid sequence over that length. Typically, the identified nucleic acid or amino acid sequence is a sequence referenced by a particular sequence identification number, and the nucleic acid or amino acid sequence being compared to the identified sequence is referred to as the target sequence. For example, an identified sequence can be the sequence set forth in SEQ ID NO:1 2, 5, or 6.

A length and percent identity over that length for any nucleic acid or amino acid sequence is determined as follows. First, a nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 Sequences (B12seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from the State University of New York—Old Westbury campus library as well as at Fish & Richardson's web site (world wide web at “fr.com/blast/”) or the U.S. government's National Center for Biotechnology Information web site (world wide web at “ncbi.nlm.nih.gov”). Instructions explaining how to use the B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to −1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\B12seq -ic:\seq1.txt -j c:\seq2.txt—p blastn -o c:\output.txt -q -1-r2. To compare two amino acid sequences, the options of Bl2seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences. Once aligned, a length is determined by counting the number of consecutive nucleotides or amino acid residues from the target sequence presented in alignment with sequence from the identified sequence starting with any matched position and ending with any other matched position. A matched position is any position where an identical nucleotide or amino acid residue is presented in both the target and identified sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acid residues. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides or amino acid residues are counted, not nucleotides or amino acid residues from the identified sequence.

The percent identity over a determined length is determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100. For example, if (1) a 1000 nucleotide target sequence is compared to the sequence set forth in SEQ ID NO:1, (2) the Bl2seq program presents 200 nucleotides from the target sequence aligned with a region of the sequence set forth in SEQ ID NO:1 where the first and last nucleotides of that 200 nucleotide region are matches, and (3) the number of matches over those 200 aligned nucleotides is 180, then the 1000 nucleotide target sequence contains a length of 200 and a percent identity over that length of 90 (i.e., 180÷200*100=90).

It will be appreciated that a single nucleic acid or amino acid target sequence that aligns with an identified sequence can have many different lengths with each length having its own percent identity. For example, a target sequence containing a 20 nucleotide region that aligns with an identified sequence as follows has many different lengths including those listed in Table 1.

It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It is also noted that the length value will always be an integer.

This document provides isolated nucleic acid molecules containing a nucleic acid sequence having a length and a percent identity to the sequence set forth in SEQ ID NO:1 or 5 over that length such that the length is at least about 500 nucleotides (e.g., at least about 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or more nucleotides) and the percent identity is at least about 80 percent (e.g., at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100 percent). For example, an isolated nucleic acid molecule provided herein can contain a length of about 1000 nucleotides (e.g., about 1035 nucleotides) with a 90, 95, 99, or 100 percent identify over that length to the sequence set forth in SEQ ID NO:l.

In some cases, an isolated nucleic acid molecule provided herein can contain the nucleotide sequence set forth in SEQ ID NO:1 or 5 without any nucleotide additions, deletions, or substitutions. In some cases, an isolated nucleic acid molecule provided herein can contain the nucleotide sequence set forth in SEQ ID NO:1 or 5 with one or more nucleotide additions, deletions, or substitutions (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 50, or 100 nucleotide additions, deletions, or substitutions).

This document also provides isolated nucleic acid molecules that are at least about 12 bases in length (e.g., at least about 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 100, 250, 500, 750, 800, 850, 900, 950, 1000, 1035, 1050, 1100, 1150, 1200, 1250, 1300, 1400, 1500, 2000, 3000, 4000, or 5000 bases in length) and hybridize, under hybridization conditions, to the sense or antisense strand of a nucleic acid having the sequence set forth in SEQ ID NO:1 or 5. The hybridization conditions can be moderately or highly stringent hybridization conditions. Such nucleic acid molecules can be molecules that do not hybridize to the sense or antisense strand of a nucleic acid having the sequence encoding the full amino acid sequence set forth in SEQ ID NO:3 or 4.

For the purpose of this invention, moderately stringent hybridization conditions mean the hybridization is performed at about 42° C. in a hybridization solution containing 25 mM KPO₄ (pH 7.4), 5×SSC, 5× Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5×10⁷ cpm/μg), while the washes are performed at about 50° C. with a wash solution containing 2×SSC and 0.1% sodium dodecyl sulfate.

Highly stringent hybridization conditions mean the hybridization is performed at about 42° C. in a hybridization solution containing 25 mM KPO₄ (pH 7.4), 5×SSC, 5× Denhart's solution, 50 μg/mL denatured, sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and 1-15 ng/mL probe (about 5×10⁷ cpm/μg), while the washes are performed at about 65° C. with a wash solution containing 0.2×SSC and 0.1% sodium dodecyl sulfate.

Isolated nucleic acid molecules provided herein can be obtained using any method including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, PCR can be used to obtain an isolated nucleic acid molecule containing a nucleic acid sequence sharing similarity to the sequences set forth in SEQ ID NO:1. PCR refers to a procedure or technique in which target nucleic acid is amplified in a manner similar to that described in U.S. Pat. No. 4,683,195, and subsequent modifications of the procedure described therein. Generally, sequence information from the ends of the region of interest or beyond are used to design oligonucleotide primers that are identical or similar in sequence to opposite strands of a potential template to be amplified. Using PCR, a nucleic acid sequence can be amplified from RNA or DNA. For example, a nucleic acid sequence can be isolated by PCR amplification from total cellular RNA, total genomic DNA, and cDNA as well as from bacteriophage sequences, plasmid sequences, viral sequences, and the like. When using RNA as a source of template, reverse transcriptase can be used to synthesize complimentary DNA strands.

Isolated nucleic acid molecules provided herein also can be obtained by mutagenesis. For example, an isolated nucleic acid containing a sequence set forth in SEQ ID NO:1 can be mutated using common molecular cloning techniques (e.g., site-directed mutagenesis). Possible mutations include, without limitation, deletions, insertions, and substitutions, as well as combinations of deletions, insertions, and substitutions.

In addition, nucleic acid and amino acid databases (e.g., GenBank) can be used to obtain an isolated nucleic acid molecule provided herein. For example, any nucleic acid sequence having some homology to a sequence set forth in SEQ ID NO:1 or 5, or any amino acid sequence having some homology to a sequence set forth in SEQ ID NO:2 or 6 can be used as a query to search GenBank®.

Further, nucleic acid hybridization techniques can be used to obtain an isolated nucleic acid molecule provided herein. Briefly, any nucleic acid molecule having some homology to a sequence set forth in SEQ ID NO:1 or 5 can be used as a probe to identify a similar nucleic acid by hybridization under conditions of moderate to high stringency. Once identified, the nucleic acid molecule then can be purified, sequenced, and analyzed to determine whether it is a nucleic acid molecule described herein.

Hybridization can be done by Southern or Northern analysis to identify a DNA or RNA sequence, respectively, that hybridizes to a probe. The probe can be labeled with a biotin, digoxygenin, an enzyme, or a radioisotope such as³²P. The DNA or RNA to be analyzed can be electrophoretically separated on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and hybridized with the probe using standard techniques well known in the art such as those described in sections 7.39-7.52 of Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring harbor Laboratory, Plainview, N.Y. Typically, a probe is at least about 20 nucleotides in length. For example, a probe corresponding to a 20 nucleotide sequence set forth in SEQ ID NO:1 can be used to identify an identical or similar nucleic acid. In addition, probes longer or shorter than 20 nucleotides can be used.

This document provides isolated nucleic acid molecules that contain the entire nucleic acid sequence set forth in SEQ ID NO:1 or 5. In addition, this document provides isolated nucleic acid molecules that contain a portion of the nucleic acid sequence set forth in SEQ ID NO:1 or 5. For example, this document provides an isolated nucleic acid molecule that contains a 600 nucleotide sequence identical to any 600 nucleotide sequence set forth in SEQ ID NO:1, including, without limitation, the sequence starting at nucleotide number 1 and ending at nucleotide number 600, the sequence starting at nucleotide number 2 and ending at nucleotide number 601, the sequence starting at nucleotide number 3 and ending at nucleotide number 602, and so forth. It will be appreciated that this document also provides isolated nucleic acid molecules that contain a nucleotide sequence that is greater than 600 nucleotides (e.g., 650, 700, 750, 800, 850, 900, 950, 1000, 1035, 1050, 1100, 1150, 1200, or more nucleotides) in length and identical to any portion of the sequence set forth in SEQ ID NO:1. For example, this document provides an isolated nucleic acid molecule that contains a 1000 nucleotide sequence identical to any 800 nucleotide sequence set forth in SEQ ID NO:1 including, without limitation, the sequence starting at nucleotide number 1 and ending at nucleotide number 1000, the sequence starting at nucleotide number 2 and ending at nucleotide number 1001, the sequence starting at nucleotide number 3 and ending at nucleotide number 1002, and so forth. Additional examples include, without limitation, isolated nucleic acid molecules that contain a nucleotide sequence that is 25 or more nucleotides (e.g., 50, 100, 150, 200, 250, 300, 350, or more nucleotides) in length and identical to any portion of the sequence set forth in SEQ ID NO:1.

In addition, this document provides isolated nucleic acid molecules that contain a variation of the nucleic acid sequence set forth in SEQ ID NO:1 or 5. For example, this document provides an isolated nucleic acid molecule containing a nucleic acid sequence set forth in SEQ ID NO:1 that contains a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e.g., single deletion together with multiple insertions). This document also provides isolated nucleic acid molecules that contain a variant of a portion of the nucleic acid sequence set forth in SEQ ID NO:1 or 5 as described herein.

This document provides isolated nucleic acid molecules that contain a nucleic acid sequence that encodes the entire amino acid sequence set forth in SEQ ID NO:2 or 6. In addition, this document provides isolated nucleic acid molecules that contain a nucleic acid sequence that encodes a portion of the amino acid sequence set forth in SEQ ID NO:2 or 6. For example, this document provides isolated nucleic acid molecules that contain a nucleic acid sequence that encodes a 25 amino acid sequence identical to any 25 amino acid sequence set forth in SEQ ID NO:2 including, without limitation, the sequence starting at amino acid residue number 1 and ending at amino acid residue number 25, the sequence starting at amino acid residue number 2 and ending at amino acid residue number 26, the sequence starting at amino acid residue number 3 and ending at amino acid residue number 27, and so forth. It will be appreciated that this document also provides isolated nucleic acid molecules that contain a nucleic acid sequence that encodes an amino acid sequence that is greater than 25 amino acid residues (e.g., 26, 30, 50, 100, 150, 200, 250, 300, 325, 344, or more amino acid residues) in length and identical to any portion of the sequence set forth in SEQ ID NO:2 or 6. For example, this document provides isolated nucleic acid molecules that contain a nucleic acid sequence that encodes a 150 amino acid sequence identical to any 150 amino acid sequence set forth in SEQ ID NO:2 including, without limitation, the sequence starting at amino acid residue number 1 and ending at amino acid residue number 150, the sequence starting at amino acid residue number 2 and ending at amino acid residue number 151, the sequence starting at amino acid residue number 3 and ending at amino acid residue number 152, and so forth. Additional examples include, without limitation, isolated nucleic acid molecules that contain a nucleic acid sequence that encodes an amino acid sequence that is 300 or more amino acid residues (e.g., 300, 310, 320, 330, 340, 344, or more amino acid residues) in length and identical to any portion of the sequence set forth in SEQ ID NO:2.

In addition, this document provides isolated nucleic acid molecules that contain a nucleic acid sequence that encodes an amino acid sequence having a variation of the amino acid sequence set forth in SEQ ID NO:2 or 6. For example, this document provides isolated nucleic acid molecules containing a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO:2 that contains a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e.g., single deletion together with multiple insertions). This document also provides isolated nucleic acid molecules containing a nucleic acid sequence encoding an amino acid sequence that contains a variant of a portion of the amino acid sequence set forth in SEQ ID NO:2 or 6 as described herein.

The isolated nucleic acid molecules provided herein can encode a polypeptide having glycosyl hydrolases activity. Any method can be use to determine whether or not a particular nucleic acid molecule encodes a polypeptide having glycosyl hydrolase activity. For example, cells transfected with a particular nucleic acid molecule can be used to determine whether the expressed polypeptide has glycosyl hydrolase activity.

This document provides substantially pure polypeptides. The term “substantially pure” as used herein with reference to a polypeptide means the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. Thus, a substantially pure polypeptide is any polypeptide that is removed from its natural environment and is at least 60 percent pure. A substantially pure polypeptide can be at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel.

A substantially pure polypeptide provided herein can have an amino acid sequence encoded by a nucleic acid molecule provided herein. In some cases, a substantially pure polypeptide provided herein can contain an amino acid sequence having a length and a percent identity to the sequence set forth in SEQ ID NO:2 or 6 over that length as determined herein provided the length is at least about 250 amino acid residues (e.g., at least about 260, 270, 280, 290, 300, 310, 320, 330, 340, 344, or more amino acid residues) and the percent identity is at least about 80 percent (e.g., at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100 percent). For example, a substantially pure polypeptide provided herein can have an amino acid sequence that contains a length of about 340 amino acid residues (e.g., about 344 amino acid residues) with a 90, 95, 99, or 100 percent identify over that length to the sequence set forth in SEQ ID NO:2 or 6.

Any method can be used to obtain a substantially pure polypeptide. For example, common polypeptide purification techniques such as affinity chromotography and HPLC as well as polypeptide synthesis techniques can be used. In addition, any material can be used as a source to obtain a substantially pure polypeptide. For example, transfected cell cultures or S. lugdunensis cultures can be used as a source material. In addition, tissue culture cells engineered to over-express a particular polypeptide of interest can be used to obtain substantially pure polypeptide. Further, a polypeptide can be engineered to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix. For example, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ tag (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini. Other fusions that could be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.

This document provides polypeptides that contain the entire amino acid sequence set forth in SEQ ID NO:2 or 6. In addition, this document provides polypeptides that contain a portion of the amino acid sequence set forth in SEQ ID NO:2 or 6. For example, this document provides polypeptides that contain a 50 amino acid sequence identical to any 50 amino acid sequence set forth in SEQ ID NO:2 including, without limitation, the sequence starting at amino acid residue number 1 and ending at amino acid residue number 50, the sequence starting at amino acid residue number 2 and ending at amino acid residue number 51, the sequence starting at amino acid residue number 3 and ending at amino acid residue number 52, and so forth. It will be appreciated that this document also provides polypeptides that contain an amino acid sequence that is greater than 50 amino acid residues (e.g., 75, 100, 150, 200, 250, 300, 325, 330, 335, 340, 344, or more amino acid residues) in length and identical to any portion of the sequence set forth in SEQ ID NO:2 or 6. For example, this document provides polypeptides that contain a 300 amino acid sequence identical to any 300 amino acid sequence set forth in SEQ ID NO:2 including, without limitation, the sequence starting at amino acid residue number 1 and ending at amino acid residue number 300, the sequence starting at amino acid residue number 2 and ending at amino acid residue number 301, the sequence starting at amino acid residue number 3 and ending at amino acid residue number 302, and so forth. Additional examples include, without limitation, polypeptides that contain an amino acid sequence that is 300 or more amino acid residues (e.g., 321, 322, 323, 324, 325, or more amino acid residues) in length and identical to any portion of the sequence set forth in SEQ ID NO:2.

In addition, this document provides polypeptides containing an amino acid sequence having a variation of the amino acid sequence set forth in SEQ ID NO:2 or 6. For example, this document provides polypeptides containing an amino acid sequence set forth in SEQ ID NO:2 that contains a single insertion, a single deletion, a single substitution, multiple insertions, multiple deletions, multiple substitutions, or any combination thereof (e.g., single deletion together with multiple insertions). This document also provides polypeptides containing an amino acid sequence that contains a variant of a portion of the amino acid sequence set forth in SEQ ID NO:2 or 6 as described herein.

The substantially pure polypeptides provided herein can have glycosyl hydrolase activity. Any method can be use to determine whether or not a particular polypeptide has glycosyl hydrolase activity. For example, cells expressing a particular polypeptide can be used to determine whether the polypeptide has glycosyl hydrolase activity.

This document also provides host cells such as any cell containing at least one isolated nucleic acid molecule described herein. Such cells can be prokaryotic cells (e.g., E. coli, S. aureus, or S. carnosus) or eukaryotic cells (e.g., insect cells, mammalian cells, or yeast cells). It is noted that cells containing an isolated nucleic acid molecule provided herein are not required to express a polypeptide. In addition, the isolated nucleic acid molecule can be integrated into the genome of the cell or maintained in an episomal state. Thus, host cells can be stably or transiently transfected with a construct containing an isolated nucleic acid molecule provided herein.

Host cells can contain an exogenous nucleic acid molecule that encodes a polypeptide having glycosyl hydrolase activity. Such host cells can express the encoded polypeptide such that the host cells produce a polypeptide having glycosyl hydrolase activity that can be purified.

Any methods can be used to introduce an isolated nucleic acid molecule into a cell. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods that can be used to introduce an isolated nucleic acid molecule into a cell.

The polypeptides provided herein (e.g., an S. lugdunensis glycosyl hydrolase) can be used to reduce biofilms, prevent biofilms, or treat infections associated with biofilms. The biofilm can be located on any surface including a surface of a foreign object at least partially located in a mammal's body. For example, a polypeptide provided herein such as a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 can be used as a catheter lock agent, wherein a solution containing the polypeptide can be distilled into the lumen of an intravascular catheter during periods of catheter disuse, in order to prevent biofilm formation in the catheter lumen. In some cases, a polypeptide provided herein can be used as an anti-biofilm coating on surfaces of catheters and other medical devices. In some cases, a polypeptide provided herein can be used as a therapeutic to detach pre-formed biofilms from a surface of a medical device. In some cases, a polypeptide provided herein can be used to diagnose S. lugdunensis infections.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Microorganisms used in this example are listed in Table 1. Bacteria stored at −70° C. were freshly streaked on sheep's blood agar or trypticase soy agar before each replicate experiment. For microtiter plate biofilm formation and detachment assays, immunodetection assays, and confocal microscopy assays (unless stated otherwise), isolated colonies from 16 to 24 hour-old plates were grown in trypticase soy broth (BD BBL, Franklin Lakes, N.J.) supplemented with 1% (w/v) sterile-filtered glucose (TSB_gluc1%) with shaking at 130 rpm for 22-24 hours. Unless otherwise noted, all experiments were incubated in ambient air at 37° C.

Biofilm formation was assayed using a microtiter plate assay as described elsewhere (Christensen et al., J. Clin. Microbiol., 22:996-1006 (1985)), with modifications as described elsewhere (Chaignon et al., Appl. Microbiol. Biotechnol., 75(1):125-132 (2007) and Frank and Patel, Diagn. Microbiol. Infect. Dis., 57:355-359 (2007)). Briefly, cultures were adjusted with TSB_gluac1% to match the turbidity of a 1.0 McFarland standard (˜1-2×10⁸ cfu/mL) and diluted 1:50 in TSB_gluc1% or TSB_gluc1% containing various concentrations of sodium chloride (1-5%, w/v) or ethanol (0.5-4%, v/v). For experiments examining the effect of glucose concentration on biofilm formation, bacteria were grown overnight, adjusted to 1.0 McFarland, and diluted in TSB containing the corresponding amount of glucose being tested (0.5-5%, w/v). 200 μL aliquots of each diluted culture were placed into four wells of 96-well microtiter plates (Nuclon Delta, Nalge Nunc International, Rochester, N.Y.), and incubated for 24 hours. Cell growth was measured by reading the OD_{600 nm} on a microplate reader (Multiskan, Thermo Electron, Waltham, Mass.). Culture media was discarded, and wells were washed twice by fully submerging plates in deionized water to remove non-adherent cells and allowed to air-dry overnight. Biofilms remaining in the wells were stained with 0.1% safranin for 1 minute, rinsed under running tap water to remove excess stain, and air-dried overnight. In order to ensure homogeneity among stained material in the wells, stained biofilms were resuspended in 200 μL of 30 percent glacial acetic acid, and the OD_{492 nm} was measured. Wells containing uninoculated media served as sterility controls and spectrophotometric blanks Each condition was assayed three times on separate days with similar results.

Ten mm diameter disks were cut from 0.020-inch thick non-reinforced medical grade silicone elastomer sheeting (Bentec Medical, Woodland, Calif.) with a skin biopsy punch and sterilized by autoclaving. An overnight culture of S. lugdunensis IDRL-2640 was adjusted to match the turbidity of a 1.0 McFarland standard (˜1×10⁸ cfu/mL) and diluted 1:50 in TSB_gluc1%. One mL aliquots of the diluted culture or sterile media were added to wells of a 24-well plate (Falcon; BD Biosciences, Franklin Lakes, N.J.). Disks were placed in the bottoms of wells with sterile forceps and incubated for 24 hours at 37° C. in 5% CO₂ to allow for biofilm formation. Following incubation, disks were removed from wells, soaked for 5 minutes in 1 mL of sterile phosphate-buffered saline (PBS) to remove planktonic bacteria, and stained with 1 mL 0.1% safranin for 1 minute. Excess stain was removed by repeatedly dipping each disk in sterile water.

To visualize biofilms by electron microscopy, disks were incubated as described above, soaked in 1 mL of sterile water for 5 minute, and placed into Trumps fixative (4% formaldehyde and 1% glutaraldehyde in phosphate buffer, pH 7.3). Following critical-point drying and gold-palladium sputter-coating, disks were imaged by cold field-emission SEM using a Hitachi S-4700 instrument (Hitachi Ltd., Tokyo, Japan).

Genomic DNA used for Southern blotting and restriction site PCR was prepared from S. lugdunensis isolates, S. aureus IDRL-2590, S. epidermidis IDRL-2873, and Escherichia coli ATCC 10798. S. aureus IDRL-2590 and S. epidermidis IDRL-2873, used as positive controls in Southern blots, are prosthetic joint infection isolates previously determined to be icaA positive by PCR (Frank et al., J. Clin. Microbiol., 42:4846-4849 (2004)). E. coli ATCC 10798 served as a negative control in Southern blots. A single colony of each organism was grown overnight in 200 mL TSB at 37° C. in 5% CO₂. Cells were pelleted, resuspended in 4 ml of 200 μg/mL lysostaphin (Sigma-Aldrich, Saint Louis, Mo.), and incubated at 37° C. for 30 minutes. An equal volume of DNA Stat-60 reagent (Tel-test, Inc., Friendswood, Tex.) was added, and cells were mixed by inversion and incubated for 20 minutes at room temperature to facilitate complete lysis prior to DNA extraction and precipitation as recommended by the manufacturer.

Low Stringency icaA and icaR Southern Blots

Genomic DNA (˜5 μg) was digested with restriction enzymes EcoRI (Roche Applied Science, Indianapolis, Ind.) or HaeIII (Invitrogen, Corp., Carlsbad, Calif.), as recommended by the manufacturer, separated by electrophoresis on a 1% agarose gel, and transferred to a nylon membrane by downward capillary transfer (Nytran SuperCharge Turboblotter Kit, Whatman, Inc., Florham Park, N.J.). Southern blotting and washes were performed under low stringency conditions with DIG EasyHyb hybridization solution and the DIG Wash and Block Buffer Set (Roche Applied Science) following the protocol described by the manufacturer for filter hybridization applications. DIG-labeled icaA probes were generated by PCR with the PCR DIG Probe Synthesis Kit (Roche Applied Science) from S. aureus IDRL-2590 and S. epidermidis IDRL-2873 DNA using primers KFicaAF and KFicaAR (Table 2). DIG-labeled icaR probes were generated from S. aureus and S. epidermidis DNA with primer pairs SAicaRF/SAicaRR and SEicaRF/SEicaRR, respectively. Bound probes were visualized on X-ray film with the chemiluminescent substrate CSPD (Roche Applied Science) following immunological detection with an alkaline phosphatase-labeled anti-DIG antibody (Roche Applied Science).

Primers KFicaAF and icaAR, which were designed from regions of high homology between the icaA genes of S. aureus and S. epidermidis and were reportedly used to amplify icaA from a S. lugdunensis isolate causing ventriculoperitoneal shunt infection (Sandoe et al., Clin. Microbiol. Infect., 7:385-387 (2001)), were used in standard PCR reactions with S. lugdunensis genomic DNA. PCR was performed using AmpliTaq Gold with Buffer I (Applied Biosystems, Foster City, Calif.) under low stringency annealing conditions (42° C.). The resulting PCR products were sequenced with the same primers. Restriction-site PCR (Sarkar et al., PCR Methods Appl., 2:318-322 (1993)), a primer walking strategy that couples outward facing primers of known sequence with a universal primer that recognizes a specific restriction enzyme recognition site, was used to acquire the sequence of the entire region encompassing the icaADBC locus from S. lugdunensis IDRL-2414 and IDRL-2664. Outward facing primers that annealed to the 5′ and 3′ regions of the initial S. lugdunensis icaA sequence were paired in standard PCR reactions with one of four restriction-site PCR primers, mRS-BamHI, mRS-EcoRI, mRS-Sau3AI, or mRS-TaqI (Table 2). One microliter of the first reaction was used as the template in a second PCR reaction containing a primer located internal to the first round primer and the restriction-site PCR primer used in the first round. Resulting products were analyzed by gel electrophoresis and sequenced with the second round specific primer. All sequence was verified bi-directionally. Table 2 lists primers used for restriction-site PCR or bi-directional sequence verification. Sequencing was performed on an ABI Prism 377 DNA sequencer with an ABI Prism Big Dye Terminator cycle sequencing ready reaction kit (Perkin-Elmer Applied Biosciences, Foster City, Calif.) at the Mayo Clinic DNA Sequencing core facility. Primers were synthesized by the Mayo Clinic DNA Synthesis core facility or Integrated DNA Technologies, Inc. (Coralville, Iowa).

S. lugdunensis ica Locus PCR Screen

Primer pairs KLF64F/KLF82R and KLF32F/KLF67R (Table 2) were used to PCR amplify 7.6 kb and 4.9 kb products, respectively, from the region spanning the S. lugdunensis icaADBC locus. PCR was performed with Platinum PCR SuperMix High Fidelity (Invitrogen) using template DNA prepared by alkaline wash, as described elsewhere (Frank et al., J. Clin. Microbiol., 42:4846-4849 (2004)). S. lugdunensis IDRL-2414, IDRL-2664, IDRL-5204, IDRL-5256, and IDRL-5258 PCR products were bi-directionally sequenced. Sequencing primers are listed in Table 2. Sequence alignments and comparisons were performed with Sequencher software (Gene Codes Corp., Ann Arbor, Mich.).

Immunoblot Detection of PNAG and other Polysaccharides in Static Phase or Biofilm S. lugdunensis Cells

The production of PNAG was assessed as previously elsewhere (Cramton et al., Infect. Immun., 67:5427-5433 (1999)), with some modifications. For static phase cells, bacteria were grown overnight in TSB_gluc1%, and equivalent amounts (1-2 mL) of cells, as determined by optical density, were harvested. For biofilm cells, biofilms were established as described above in 48 well microtiter plates (Nuclon Delta, Nalge Nunc International) containing 500 μL of culture. Biofilms from two wells per organism were scraped with a pipette tip, resuspended in the culture media, and pooled. Cell pellets were washed in sterile PBS, resuspended in 0.5 M EDTA, sonicated for 5 minutes at 40 kHz in a bath sonicator (Zenith Ultrasonics, Norwood, N.J.), boiled 5 minutes, and centrifuged. Supernatants were treated with 200 μg proteinase K for 30 minutes at 65° C., then 80° C. for 15 minutes to heat inactivate the enzyme. Extracts were spotted onto nitrocellulose, and blots were blocked 1 hour in 3% bovine serum albumin (BSA) in tris-buffered saline (TBS). Blots were probed overnight at 4° C. with a 1:5,000 dilution of goat anti-deacetylated PNAG antibody (Maira-Litran et al., Infect. Immun., 73:6752-6762 (2005)) in TBS-0.05% Tween-20 with 3% BSA. The anti-deacetylated PNAG antibody was obtained from Jerry Pier at Harvard Medical School. Blots were washed and probed with a 1:10,000 dilution of rabbit anti-goat horseradish peroxidase conjugate (Pierce, Rockford, Ill.) in TBS-0.05% Tween-20 with 3% skim milk for 1-4 hours at room temperature. Alternatively, blots were probed with wheat germ agglutinin horseradish peroxidase conjugate (Sigma Aldrich), as described elsewhere (Jefferson and Cerca, Bacterial-bacterial cell interactions in biofilms: detection of polysaccharide intercellular adhesins by blotting and confocal microscopy, p. 119-126. In S. P. Colgan (ed.), Methods Mol. Biol., vol. 341. (2006) Humana Press, Totowa). Bound probes were visualized on X-ray film with the chemiluminesence ECL kit (Amersham Biosciences, Pittsburgh, Pa.).

The biotinylated lectin screening kit-I (Vector Laboratories, Burlingame, Calif.) was used to screen blots for the presence of other polysaccharides released from static phase cells. Blots were blocked at room temperature for at least 1 hour in TBS-0.1% Tween-20 with or without 5% BSA, depending on optimized conditions for individual lectins, followed by 1 hour incubation at room temperature with 5 μg/mL lectin in TBS-0.1% Tween-20. Bound lectins were detected with the Vectastain Elite ABC kit (Vector Laboratories), as recommended by the manufacturer for the use of biotinylated lectins in Western blotting applications, and visualized on X-ray film with the ECL kit.

Scanned film images were adjusted with the brightness and contrast functions in Microsoft Office Picture Manager software.

40 mM sodium metaperiodate (NaIO₄, Sigma-Aldrich) in water, 40 μg/mL purified recombinant dispersin B (obtained from Kane Biotech Inc., Winnipeg, Manitoba, Canada) in sodium phosphate buffer (50 mM sodium phosphate (pH 5.8), 100 mM NaCl), 100 μg/mL proteinase K (Roche Applied Science) in 10 mM Tris-HCl pH 7.5, 10 U/mL trypsin (Promega Corp., Madison, Wis.) in 10 mM Tris-HCl pH 7.5, 100 μg/mL chymotrypsin (Sigma-Aldrich) in 10 mM Tris-HCl pH 7.5, and 100 μg/mL thermolysin (Sigma-Aldrich) in 10 mM Tris-HCl pH 7.5 were tested for their ability to detach pre-formed S. lugdunensis biofilms from polystyrene microtiter plate wells. For certain experiments, proteinase K was inactivated by boiling for 40 minutes.

Biofilms grown in TSB_gluc1% were formed in wells of microtiter plates and washed twice with deionized water, as described above for the microtiter plate biofilm formation assay. 100 μL of NaIO₄, enzyme, or suitable control was carefully added so as not to mechanically detach biofilms on the bottoms of wells. Plates were incubated at 37° C. for 2 hours, and contents of wells were discarded and washed twice with deionized water. Plates were air-dried overnight, stained with 0.1% safranin for 1 minute, and processed as described above to quantify the amount of stained biofilm remaining after treatment, relative to treatment with the control reagent. Four wells were measured for each treatment condition. Assays were repeated two or three times on separate days with similar results.

Biofilms were grown for microscopy in 4-well chambered coverglass (Lab-Tek II, Nalge Nunc International). Overnight cultures were adjusted and diluted 1:50 with TSB_gluc1% as described above for the microtiter plate biofilm formation assay. One mL aliquots were added to chamber wells and statically incubated for 20-24 hours. Media was removed from wells, biofilms were rinsed with 1 mL PBS, and stained for fluorescent CSLM. To visualize PNAG among biofilm cells, biofilms were incubated in the dark for 15 minutes with 1 mL PBS containing 0.09 mg/mL wheat germ agglutinin-Oregon Green 488 conjugate (Molecular Probes, Eugene, Oreg.) and 5 μg/mL FM 4-64 (Molecular Probes), a lipophilic styryl membrane dye used previously to image bacterial cell membranes (Sharp and Pogliano, Proc. Natl. Acad. Sci. U.S.A., 96:14553-14558 (1999)). Stains were removed and wells were rinsed with 2 mL PBS before imaging. Extracellular proteins among biofilm cells were visualized by incubating in the dark for 30 minutes with 1 mL undiluted SYPRO Ruby protein gel stain (Molecular Probes) containing 0.167 μM Syto-9 nucleic acid stain (Molecular Probes). Stains were removed before imaging.

Confocal images were acquired on an LSM510 equipped with an Axiovert 100M inverted microscope using a Plan-Apochromat 100×/1.4 NA oil immersion objective (Carl Zeiss, Inc., Thornwood, N.Y.). An argon laser was used to excite the fluorophores at the following wavelengths: 458 nm for SYPRO Ruby; 488 nm for Oregon Green (wheat germ agglutinin), FM 4-64, and Syto-9. Red fluorescence from SYPRO Ruby and FM 4-64 was detected with a LP 650 filter. Green fluorescence was detected from Oregon Green with a BP 505-550 filter and from Syto-9 with a BP 505-530 filter. Microscopy was performed on at least three different days. Images were prepared with the LSM510 software. Red/green fluorescence ratios to assess biofilm protein were calculated on SYPRO Ruby/Syto-9 images with KS 400 version 3.0 software (Carl Zeiss, Inc.). The fluorescence area (in pixel) was averaged for images taken in two areas per biofilm from two independent biofilms.

Data were analyzed with the Student's t-test using JMP 6.0.0 Software (SAS Institute, Inc., Cary, N.C).

Accession numbers for complete S. lugdunensis icaADBC operon sequences that are in GenBank®, National Center for Biotechnology Information, are: S. lugdunensis isolate IDRL-2414, EF546620; S. lugdunensis isolate IDRL-2664, EF546621; S. lugdunensis isolate IDRL-5204, EF546622; S. lugdunensis isolate IDRL-5256, EF546623; and S. lugdunensis isolate IDRL-5258, EF546624.

S. lugdunensis Clinical Isolates Form Biofilm

The biofilm antimicrobial susceptibility profiles and the effects that subinhibitory antibiotic concentrations elicit on biofilm formation of a collection of S. lugdunensis clinical isolates were determined (Frank et al., Antimicrob. Agents Chemother., 51:888-895 (2007)). A majority of the isolates were recovered from infections known to be associated with biofilm etiology, including prosthetic joint infection, endocarditis, and intravascular catheter infection (Table 1). To further characterize S. lugdunensis biofilm formation, this collection was evaluated using in vitro biofilm formation assays under growth conditions known to support staphylococcal biofilm formation, namely TSB supplemented with 1% glucose (Deighton et al., Methods Enzymol., 336:177-95 (2001) and Knobloch et al., Med. Microbiol. Immunol., 191:101-106 (2002)).

S. lugdunensis IDRL-2640, a folliculitis isolate, was incubated for 24 hours in TSB_gluc1% with a disk cut from silicone elastomer, a type of material used to manufacture intravascular catheters. Planktonic cells were removed by gentle washing, and the disk was stained with safranin to visualize adherent bacteria. The organism formed a strong confluent layer on the disk (FIG. 1A). SEM visualization of a duplicate disk revealed cells growing in clusters with the development of microcolonies across the disk surface (FIG. 1B). When viewed at higher magnification (FIG. 1C), cells appeared to be organized into a three-dimensional architecture held together by an extracellular polymeric substance. These properties are consistent with organisms growing as a biofilm.

The relative ability of each S. lugdunensis isolate to form biofilm was assessed using a polystyrene microtiter plate biofilm formation assay. All isolates were able adhere to polystyrene (FIG. 1D). Compared to the strong biofilm-forming strains S. aureus SA113 and S. epidermidis RP62A, the biofilm formation phenotype varied widely among the S. lugdunensis isolates. In particular, isolates IDRL-2664 and IDRL-5204 were poor biofilm formers, whereas isolates IDRL-2394, IDRL-5256, and IDRL-5258 formed robust biofilms. No correlation between the source of infection (Table 1) and the degree of biofilm formation existed for this set of staphylococci.

S. lugdunensis Biofilm Formation is Affected by Environmental Conditions

Changes in exogenous factors present in growth media that enrich or stress the growth environment, including glucose (Christensen et al., Infect. Immun., 37:318-26 (1982); Dobinsky et al., J. Bacteriol., 185:2879-2886 (2003); and Lim et al., J. Bacteriol., 186:722-729 (2004)), increasing osmolarity (Knobloch et al., J. Bacteriol., 183:2624-2633 (2001); Lim et al., J. Bacteriol., 186:722-729 (2004); and Moretro et al., Appl. Environ. Microbiol., 69:5648-5655 (2003)), and alcohols (Knobloch et al., J. Antimicrob. Chemother., 49:683-7 (2002) and Knobloch et al., Med. Microbiol. Immunol., 191:101-106 (2002)), influence S. aureus and S. epidermidis in vitro biofilm formation. The effect of increasing concentrations of glucose, sodium chloride, and ethanol on the collection of S. lugdunensis isolates was tested. Statistically measurable increases in biofilm formation in response to heightened glucose levels were observed for S. aureus SA113, S. epidermidis RP62A, and 80% (12/15) of the S. lugdunensis isolates (FIG. 2A). Three S. lugdunensis isolates, IDRL-856, IDRL-5254, and IDRL-5258, formed equivalent amounts of biofilm independent of the amount of glucose present in the environment.

Media enrichment with sodium chloride at concentrations up to 5% (w/v) has been shown to increase microtiter plate biofilm formation in S. aureus, S. epidermidis, and several other CNS. S. aureus SA113 and S. epidermidis RP62A biofilm levels were found to increase when grown in TSB_gluc1% with 1-2% or 1-3% sodium chloride, respectively (FIG. 2B). Likewise, 73% (11/15) of the S. lugdunensis isolates formed more biofilm in media containing 1 or 2% sodium chloride. In contrast, higher concentrations of salt substantially reduced S. lugdunensis adherence to microtiter wells (FIG. 2B). Essentially no measurable quantity of biofilm could be detected when S. lugdunensis isolates were incubated with 5% sodium chloride. Compared to baseline biofilm formation, S. epidermidis RP62A biofilm was not affected in 5% sodium chloride, whereas the level of S. aureus SA113 biofilm was significantly reduced, yet still discernable. Growth of all species tested was not affected by the increasing salt concentrations, as verified by measuring the optical density of the biomass in each well prior to the removal of planktonic cells. These results indicate that high sodium chloride concentrations can directly interfere with the process of S. lugdunensis biofilm formation.

Ethanol concentrations of up to 6% (v/v) are capable of stimulating biofilm formation by clinical S. epidermidis isolates (Knobloch et al., J. Antimicrob. Chemother., 49:683-7 (2002)). Positive biofilm-forming S. aureus and S. epidermidis reference strains both exhibited significant reductions in biofilm production with increasing ethanol concentrations (FIG. 2C). Most ethanol concentrations also reduced the levels of biofilm production of all S. lugdunensis isolates that formed biofilm at a baseline level of OD₄₉₂≧0.150 nm (FIG. 2C). Not only did 2% ethanol prevent the adherence of all S. lugdunensis that formed measurable baseline biofilms, but 4% ethanol was bactericidal for all S. lugdunensis isolates. S. lugdunensis IDRL-2554, IDRL-2640, and IDRL-5254 were the only isolates killed after incubation in 2% ethanol. These results indicate that S. lugdunensis clinical isolates are more susceptible to ethanol than are S. epidermidis clinical isolates, and that ethanol appears to be a negative regulator of S. lugdunensis biofilm formation.

Identification and Sequencing of icaADBC Homologues in S. lugdunensis

In order to determine whether the studied isolates lacked icaA, Southern blots were performed with S. aureus and S. epidermidis icaA probes under low stringency conditions. Hybridization signals were detected for 100% (n=7) of S. lugdunensis prosthetic joint infection isolates tested. This data suggests that the inability to detect icaA in S. lugdunensis by PCR might be due to mismatches between primers and the S. lugdunensis icaA sequence. Using the primer sequences reported elsewhere (Sandoe et al., Clin. Microbiol. Infect., 7:385-387 (2001)) under low stringency annealing conditions, a short region of an icaA homologue from S. lugdunensis was amplified and sequenced. This sequence was extended in both the 5′ and 3′ directions from S. lugdunensis IDRL-2414 and IDRL-2664 using restriction-site PCR, a primer-walking technique developed to acquire unknown sequence surrounding a region of known sequence (Sarkar et al., PCR Methods Appl., 2:318-322 (1993)).

A 7.6 kb region of the S. lugdunensis genome, which included open reading frames (ORFs) with high degrees of similarity (˜30-60% identical at the predicted amino acid level) to icaADBC from S. aureus, S. epidermidis, and S. caprae, were sequenced and annotated (FIG. 3). The predicted amino acid sequences for S. lugdunensis IcaA and IcaB are shorter than their homologues in other staphylococci, whereas IcaD and IcaC are longer than their counterparts. icaA overlaps icaD by 31 nucleotides, icaD overlaps icaB by 17 nucleotides, and icaB overlaps icaC by 23 nucleotides. An icaR homologue was not located upstream of icaA, as would be predicted from the conserved genomic organization of the ica loci in S. aureus, S. epidermidis, and S. caprae. Rather, a novel ORF that lacks homology with known staphylococcal sequences was found directly upstream of, and in the same orientation as, icaA. The ORF-icaADBC genes span a 4.75 kb region that includes a 251 nucleotide intergenic region separating the ORF and icaA. Open reading frames with high degrees of similarity to yycJ and yycl genes from S. aureus and S. epidermidis flanked the ORF-icaADBC region on the 5′ and 3′ sides, respectively. The finding that yycJ and yycl are separated by greater than 4.7 kb was surprising, as these two genes otherwise occur as part of the YycFG two-component system operon that is highly conserved among Gram positive bacteria.

Inability to Detect icaR in the S. lugdunensis Genome

The absence of icaR upstream of icaA indicated that it might be located elsewhere in the genome of S. lugdunensis. No hybridization signals were detected upon low stringency Southern blotting with probes generated from the full-length sequences of icaR from either S. aureus or S. epidermidis. Low annealing temperature PCR with the primers used to generate the icaR Southern probes from S. aureus and S. epidermidis was attempted; however, this resulted in non-specific annealing. These results strongly suggest that any icaR homologue would have very little similarity to known icaR sequences from other staphylococci.

A Novel Open Reading Frame with Predicted Glycosyl Hydrolase Activity

The 1,035 nucleotide ORF (FIG. 7) located upstream of the icaA start codon is predicted to encode a 344 amino acid polypeptide (FIG. 8) that is not similar to any currently known staphylococcal sequences as determined by nucleotide and protein BLAST searches SignalP 3.0 analysis of the sequence predicts that the hypothetical polypeptide contains an N-terminal signal sequence between amino acid residues 25 and 26 (Bendtsen et al., J. Mol. Biol., 340:783-795 (2004)). In particular, the ORF is located in the S. lugdunensis genome 251 nucleotides upstream of the start codon of the icaADBC locus, and 593 nucleotides downstream of the yycJ gene. The Conserved Domains Database at NCBI's web site was used to identify a putative conserved domain within the predicted ORF amino acid sequence that closely resembles the glycosyl hydrolase family 20 catalytic domain (Accession Number: pfam00728) and a group of N-acetyl-β-hexosaminidases (called Chb; Accession Number: COG3525.2), which are involved in carbohydrate transport and metabolism (Henrissat, Biochem. J., 280 (Pt 2):309-316 (1991)). The substrate binding pocket of family 20 glycosyl hydrolases are lined with several tryptophan residues that create a hydrophobic environment (Tews et al., Nat. Struct. Biol., 3:638-648 (1996)); such tryptophan residues are conserved in the predicted ORF amino acid sequence.

The most closely related translated polypeptide sequences to the translated ORF sequence, as identified in a TBLASTX 2.2.16 search, were the dispersin B (dspB) homologues from Actinobacillus pleuropneumoniae and Actinobacillus actinomycetemcomitans (Kaplan et al., J. Bacteriol., 185:4693-8 (2003) and Kaplan et al., J. Bacteriol., 186:8213-8220 (2004)). Dispersin B is an N-acetyl-β-hexosaminidase that cleaves the β-1,6-linkages in polymers of N-acetylglucosamine. A ClustalW (World Wide Web at “ebi.ac.uk/clustalw/”) comparison indicated that the ORF amino acid sequence is 26% identical to the dispersin B homologues from the two Actinobacillus species.

The ORF sequence was cloned, with and without the N-terminal signal sequence, into an E. coli overexpression vector (pET-30b). Each plasmid contained its respective ORF sequence (with or without the signal sequence) downstream of a T7 promoter, lac operator, and ribosome binding site, and upstream of sequence that will add a C-terminal thrombin cleavage site and hexa-histidine tag (to facilitate purification with nickel affinity chromatography) to the expressed protein. A brief attempt to induce expression and purify the encoded polypeptide in E. coli did not result in detectable polypeptide.

The 7.6 kb genomic region spanning from yycJ through yycI (FIG. 3) was amplified in all S. lugdunensis isolates except IDRL-2492 and IDRL-2639. A 4.9 kb product that encompasses the ORF-icaADBC region (FIG. 3) was amplified from the two remaining isolates, indicating that the ORF-icaADBC genes are intact in all isolates in our collection.

The following was performed to determine whether the variability in biofilm formation among S. lugdunensis isolates (FIG. 1D) could be explained by differences in the ORF-icaADBC primary sequence. In addition to the two isolates that were sequenced by restriction-site PCR (IDRL-2414 and IDRL-2664), 7.6 kb PCR products from IDRL-5204, IDRL-5256, and IDRL-5258 were sequenced. These isolates included a poor biofilm producer (IDRL-5204), intermediate biofilm producers (IDRL-2414, IDRL-2664), and strong biofilm producers (IDRL-5256, IDRL-5258) (FIG. 1D). The sequences of IDRL-2414, IDRL-2664, and IDRL-5204 were identical, but the sequences of the strongest biofilm formers were different at one or more locations. IDRL-5258 contained an R274Q change at amino acid 274 of IcaA. IDRL-5256 contained many single nucleotide changes throughout the sequenced region. Eleven variations were found in the yycJ-ORF intergenic region; one variation occurred in the ORF-icaA intergenic region; and four variations were located between icaC-yycI. In addition, the following silent (non-coding) and coding mutations were found in each of the coding regions: yycJ-6 silent; ORF-4 silent, S26A, E44D; icaA: 8 silent; icaD: 4 silent; icaB: 4 silent, H23Q; icaC: 3 silent; yycl-4 silent. Despite the correlation of sequence variations found in the most proficient biofilm forming isolates, it is not clear from these results whether the ORF-icaADBC locus primary sequence is a contributing factor in the relative ability of S. lugdunensis isolates to form biofilm.

Elucidation of the Role of PNAG in S. lugdunensis Biofilm Formation

The functional role of the S. lugdunensis icaADBC genes in biofilm formation was tested by assaying for PNAG in static phase or biofilm cells. Extracts from static phase cells were blotted on nitrocellulose membranes and probed with labeled wheat germ agglutinin (WGA). PNAG was strongly detected in S. epidermidis strains RP62A and CSF41498, and a PNAG over-expressing strain of S. carnosus TM300 that carries plasmid pCN27 (FIG. 4A). PNAG production by S. aureus strains RN4220 and SA113 was less abundant. A minimal signal was apparent in the PNAG-negative SA113 ica::tet isogenic knockout strain, most likely due to non-biofilm sources of N-acetylglucosamine. Unexpectedly, PNAG was not detected in any of the 15 S. lugdunensis isolate. PNAG was also not detected in the negative control strain S. carnosus TM300.

To confirm the lack of detection of PNAG from S. lugdunensis cells, immunoblotting experiments with static phase cells (data not shown) and biofilm cells (FIG. 4B) were performed using an anti-deacetylated PNAG antibody. Signal levels from all S. lugdunensis strains were equivalent to the non-PNAG producing S. aureus SAl 13 ica::tet and S. carnosus TM300 negative controls.

CSLM was used to visualize S. lugdunensis biofilms in comparison with biofilms formed by PNAG-producing S. epidermidis RP62A (FIG. 6A,B). Cells were stained with FM 4-64, a red lipophilic plasma membrane dye, and PNAG was stained with Oregon green conjugated wheat germ agglutinin. S. epidermidis RP62A formed a thick, multi-layered biofilm interspersed with large and abundant structures of PNAG (FIG. 6A). In contrast, under identical microscopy settings, S. lugdunensis IDRL-2640 formed a thick and dense biofilm void of detectable PNAG (FIG. 6B). Similar results were observed with S. lugdunensis IDRL-5258 biofilms. These images indicate that (1) S. lugdunensis isolates are able to form thick, multi-cellular biofilms, and (2) PNAG is not a recognizable component of S. lugdunensis in vitro biofilms.

In the absence of PNAG, alternative polysaccharides may be present in the matrix of S. lugdunensis biofilms. Six lectins—concanavalin A (binds glucose and mannose), DBA (binds N-acetylgalactosamine), SBA (binds galactose and N-acetylgalactosamine), PNA (binds galactose), RCA₁₂₀ (binds galactose and N-acetylgalactosamine), and UEA-1 (binds fucose)—were chosen to assay for other extracellular matrix polysaccharides in S. lugdunensis static phase cell extracts by immunoblotting. Concanavalin A bound to all staphylococcal strains tested, which was not unexpected based on its broad specificity for common sugars. SBA selectively bound to S. carnosus isolates, and RCA₁₂₀ selectively bound to S. epidermidis and the S. carnosus PNAG over-expressing strain. However, none of the lectins bound to the S. lugdunensis extracts, providing evidence that several other types of polysaccharides, in addition to PNAG, are not part of the S. lugdunensis biofilm matrix.

Detachment of S. lugdunensis biofilms with proteases but not carbohydrate-degrading Reagents

A chemical and enzymatic detachment approach was used to examine the composition of the biofilm matrix. Sodium metaperiodate and dispersin B degrade PNAG, thereby releasing PNAG-containing biofilms from their associated surfaces (Kaplan et al., J. Bacteriol., 185:4693-8 (2003); Kaplan et al., Antimicrob. Agents Chemother., 48:2633-2636 (2004); and Wang et al., J. Bacteriol., 186:2724-2734 (2004)). As expected, pre-formed biofilms of S. epidermidis RP62A and PNAG over-producing S. carnosus TM300+pCN27 that were treated with sodium metaperiodate (FIG. 5A) and dispersin B (FIG. 5B) were susceptible to detachment by both reagents (P-value≦0.001, Student's t-test). S. carnosus biofilms, which likely contain high amounts of PNAG without additional stabilizing factors, were essentially completely released from microtiter wells, compared to buffer-only treatment controls. In contrast, despite substantial detachment, much greater levels of S. epidermidis RP62A biofilms remained after treatment with either sodium metaperiodate or dispersin B. The biofilm matrix components of this strain are known to include extracellular teichoic acids and polypeptides, in addition to large amounts of PNAG, which may have assisted in protecting or stabilizing the biofilm from detachment. Confirmatory of the immunoblotting and microscopy data, S. lugdunensis biofilms resisted detachment by dispersin B (FIG. 5B). S. lugdunensis IDRL-2622 was the only isolate that exhibited statistically significant, yet minimal, detachment following incubation with dispersin B (P-value=0.045, Student's t-test). In addition, sodium metaperiodate had moderate to little effect on the release of 53% (8/15) S. lugdunensis biofilms (FIG. 5A). Seven S. lugdunensis biofilms (IDRL-2394, IDRL-2526, IDRL-2554, IDRL-2588, IDRL-2622, IDRL-2640, and IDRL-5258) demonstrated greater susceptibility to sodium metaperiodate (P-value≦0.047, Student's t-test), suggesting that the biochemical constituents in the extracellular matrix of S. lugdunensis biofilms may vary among isolates. The ability of several proteases with varying substrate specificities to detach biofilms of S. lugdunensis and the PNAG-containing control strains was tested. Proteinase K had no effect on the PNAG-positive control strains, while biofilm of all S. lugdunensis isolates that formed substantial biofilms were completely removed (FIG. 5C; P-value≦0.005, Student's t-test). In order to demonstrate that the overwhelming detachment of S. lugdunensis biofilms was due to the enzymatic activity of proteinase K, the experiment was repeated with heat-inactivated proteinase K, and the detachment effect was found to be abolished upon proteinase K denaturation. Concordant results were obtained upon treatment of biofilms with trypsin (FIG. 5D, P-value≦0.041, Student's t-test), except that a few S. lugdunensis isolates (IDRL-2414 and IDRL-2526) were noticeably more resistant to release by trypsin. Two other proteases, thermolysin and chymotrypsin, were also found to detach S. lugdunensis biofilms selectively, but not PNAG-containing biofilms formed by S. epidermidis RP62A or S. carnosus TM300+pCN27. These results provide evidence that polypeptides are important for S. lugdunensis in vitro biofilm formation on polystyrene when cells are grown in TSB_gluc1%.

Visualization of Extracellular Proteins in S. lugdunensis Biofilms by CSLM

Biofilms of S. epidermidis RP62A and S. lugdunensis IDRL-5258 were stained with the red fluorescent protein dye SYPRO Ruby in order to visualize extracellular polypeptides among biofilm cells, which were stained with the green nucleic acid stain Syto-9. Extracellular polypeptides were visible in biofilms of both S. epidermidis RP62A (FIG. 6C) and S. lugdunensis IDRL-5258 (FIG. 6D). S. lugdunensis biofilms appeared to contain more polypeptides than S. epidermidis biofilms. To more accurately assess the relative abundance of extracellular polypeptides per number of cells in biofilms formed by either organism, the ratio of polypeptide to cell fluorescence was calculated. The average polypeptide to cell fluorescence measured in S. lugdunensis IDRL-5258 biofilms was statistically higher than the same measurement in S. epidermidis RP62A biofilms (0.965, 0.285SD versus 0.385, 0.114SD, respectively; P-value<0.01, Student's t-test), supporting the hypothesis that polypeptides are a significant component of this organism's biofilm matrix.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.