INTRODUCTION

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Coagulase-negative staphylococci are commensal organisms of human skin flora but have become major etiological agents of nosocomial bacteremia and can colonize a variety of medical devices (18). These nosocomial infections are usually (>80%) caused by Staphylococcus epidermidis (18, 24). However, Staphylococcus saprophyticus is the second most frequently encountered agent of acute urinary tract infections (UTIs) after Escherichia coli (10, 11). S. saprophyticus is often isolated from the urine of young, sexually active female outpatients presenting with symptoms of acute UTI (1, 15, 25) indistinguishable from the symptoms of UTIs caused by Escherichia coli. These coagulase-negative staphylococci are rarely found as a cause of UTIs in hospitalized patients or as a contaminant of urine cultures (15) and are characterized by the low bacterial counts (less than 10⁵ CFU per ml) required to elicit a UTI (24). S. saprophyticus could be the cause of chronic bacterial prostatitis in men (4), and there is evidence that suggests that this staphylococcal species could be the etiological agent of sexually transmitted urethritis (9). The use of spermicide-coated condoms has now been associated with an increased risk of UTIs caused by S. saprophyticus (6).

The classical phenotypic identification of staphylococci by Kloos and Schleifer (19) remains the "gold standard" for reference laboratories, but it is too lengthy and cumbersome for routine use in hospital microbiology laboratories. S. saprophyticus is differentiated from other urinary coagulase-negative staphylococci (i.e., S. epidermidis) by its uniform resistance to novobiocin, aerobic growth requirements, urease production, and carbohydrate utilization (15). Several culture-based commercially available systems including API Staph strips and the RapiDEC system have been evaluated for the identification of S. saprophyticus (12, 26). However, these systems require at least 20 h for staphylococcal species identification and occasionally misidentify S. saprophyticus. A few DNA-based assays that target variable regions of the 16S rRNA gene of S. saprophyticus have been developed (7, 8). However, these assays have not been evaluated for direct detection of S. saprophyticus from clinical specimens.

Although S. saprophyticus is easy to cultivate, phenotypic analysis requires overnight growth of the microorganism. A rapid and sensitive DNA-based assay which is specific for S. saprophyticus and which is suitable for direct detection of the organism from urine specimens would allow a significant reduction in the time required for the diagnosis of S. saprophyticus infections. In this study, we present the development of an S. saprophyticus-specific DNA-based assay. An arbitrarily primed PCR (AP-PCR) protocol was used to find a prominent fingerprinting product of 380 bp shared by a panel of clinical strains of S. saprophyticus but not encountered in other closely related bacterial species. This DNA fragment was sequenced and used to design a pair of PCR primers suitable for the specific and ubiquitous detection of S. saprophyticus. This S. saprophyticus-specific PCR assay was adapted for direct detection of the organism from urine specimens. This assay will be combined in multiplex with other PCR assays currently under development in our laboratory to allow the concomitant detection of other bacteria frequently associated with UTIs.

	MATERIALS AND METHODS

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Bacterial strains. The bacterial isolates used in this study were selected from the culture collection of the Microbiology Laboratory of the Centre Hospitalier Universitaire de Québec (Pavillon Centre Hospitalier de l'Université Laval [CHUL], Ste-Foy, Québec, Canada). Three S. saprophyticus strains obtained from the American Type Culture Collection (ATCC; strains ATCC 15305, ATCC 35552, and ATCC 43867) were also used for this study. The strains were cultured on sheep blood agar or in brain heart infusion (BHI) medium. Bacterial cultures were stored frozen (80°C) in BHI broth containing 10% glycerol.

Clinical specimens. A total of five culture-negative urine specimens received at the Microbiology Laboratory of CHUL, all collected from different patients, were used in this study. Urine samples (stored at 4°C) were tested by PCR less than 48 h after reception at the laboratory.

DNA isolation. Genomic DNA was purified with the G NOME kit (Bio 101, Inc., Vista, Calif.) according to the manufacturer's instructions, except that the bacterial cells were initially resuspended in 250 µl of a lysis solution containing 200 µg of lysostaphin (Sigma Chemical Co., St. Louis, Mo.) per ml, 20 mM Tris, 2 mM EDTA, and 1.2% Triton X-100 and were incubated for 30 min at 37°C. Purified genomic DNA was diluted at a concentration of 1 ng/µl in TE buffer (10 mM Tris [pH 8.0], 1 mM EDTA).

AP-PCR amplification. Twenty 10-nucleotide primers (kit AD; Operon Technologies Inc., Alameda, Calif.) were used for AP-PCR to search for a specific amplicon shared exclusively by the species of interest, S. saprophyticus (5, 27, 28). Amplifications were performed directly from 1 µl (0.1 ng/µl) of purified genomic DNA from 5 S. saprophyticus strains and 27 other staphylococcal (non-S. saprophyticus) strains. The 25-µl AP-PCR mixture contained 50 µM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 1 of the 20 10-nucleotide primers at a concentration of 1.5 µM, 200 µM (each) the four deoxynucleoside triphosphates, and 0.5 U of Taq DNA polymerase (Promega Corp., Madison, Wis.) combined with the TaqStart antibody (Clontech Laboratories Inc., Palo Alto, Calif.). The TaqStart antibody, which is a neutralizing monoclonal antibody of Taq DNA polymerase, was added to all PCR mixtures to enhance the efficiency of the amplifications (17). The PCR mixtures were subjected to thermal cycling (3 min at 96°C and then 42 cycles of 1 min at 94°C for the denaturation step, 1 min at 31°C for the annealing step, and 2 min at 72°C for the extension step) with a PTC-200 thermal cycler (MJ Research Inc., Watertown, Mass.). A final extension step of 7 min at 72°C was performed to allow completion of amplicons. Random amplified polymorphic DNA (RAPD) fragment fingerprints were obtained by electrophoresis in 1.5% agarose gels containing 0.5 µg of ethidium bromide per ml in Tris-borate-EDTA buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at 4 V/cm for 90 min. The gels were visualized under 254-nm UV light. The sizes of the amplification products were estimated by comparison with a 50-bp-molecular-size standard ladder.

Conventional PCR amplification. Amplifications were performed either from 1 µl of a purified genomic DNA preparation or from a standardized bacterial suspension whose turbidity was adjusted to equal that of a 0.5 McFarland standard, which corresponds to approximately 1.5 × 10⁸ bacteria per ml. The 20-µl PCR mixture contained 0.4 µM (each) the two S. saprophyticus-specific primers 5'-TCA AAA AGT TTT CTA AAA AAT TTA C-3' (annealing positions 169 to 193) and 5'-ACG GGC GTC CAC AAA ATC AAT AGG A-3' (annealing positions 355 to 379), 200 µM (each) the four deoxyribonucleoside triphosphates (Pharmacia Biotech Inc., Baie d'Urfé, Québec, Canada), 10 µM Tris-HCl (pH 9.0), 50 µM KCl, 0.1% Triton X-100, 2.5 mM MgCl₂, 3.3 µg of bovine serum albumin (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada) per ml, 10 copies of linearized plasmid pSL1138, which served as a target for the internal control, and 0.5 U of Taq DNA polymerase (Promega Corp.) combined with the TaqStart antibody (Clontech Laboratories Inc.) (16, 22). An internal control was integrated into every PCR mixture (16). Use of this control allowed verification of the efficiency of the amplification and ensured that significant PCR inhibition was absent. The PCR mixtures were subjected to thermal cycling (3 min at 96°C and then 30 or 40 cycles of 1 s at 95°C for the denaturation step and 30 s at 55°C for the annealing-extension step with a PTC-200 thermal cycler). Analysis by agarose gel electrophoresis was performed as described previously (22).

Nucleotide sequence accession number. The nucleotide sequence of the S. saprophyticus-specific AP-PCR amplicon is available from GenBank as accession no. AF144088.

	RESULTS

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Isolation of an S. saprophyticus-specific DNA fragment by AP-PCR. The generation of RAPD fingerprints for 5 S. saprophyticus strains (including the 3 ATCC strains) and 29 other staphylococcal species with the 20 different AP-PCR primers (10-mer) allowed determination of which primer produced amplification patterns specific for the 5 S. saprophyticus strains. Primer OPAD-16 (5'-AACGGGCGTC-3') allowed isolation of a DNA fragment of 380 bp found in all RAPD patterns for the 5 S. saprophyticus strains tested but absent from the RAPD patterns for the other bacterial species tested (Fig. 1). Subsequently, we confirmed that this 380-bp amplification product was also absent from a wider array of bacterial species consisting of 19 other genetically related gram-positive species (Table 1). This S. saprophyticus-specific amplification product was gel purified and then cloned into the T/A cloning vector pCR 2.1. 

View larger version (67K): [in this window] [in a new window]   FIG. 1.   AP-PCR amplification with the OPAD-16 primer performed with 100 pg of purified genomic DNA from reference and clinical strains of S. saprophyticus, various staphylococcal species, and gram-positive bacteria genetically related to S. saprophyticus. The content of each lane is as follows: 2, S. saprophyticus ATCC 15305; 3, S. saprophyticus ATCC 33552; 4, S. saprophyticus ATCC 43867; 5, S. saprophyticus Cssa-18; 6, S. saprophyticus Ssa-165; 7, S. aureus ATCC 43300; 8, S. capitis subsp. capitis ATCC 27840; 9, S. epidermidis ATCC 14990; 10, S. haemolyticus ATCC 29970; 11, S. hominis ATCC 27844; 12, S. simulans ATCC 27848; 13, S. warneri ATCC 27836; 14, Bacillus subtilis ATCC 27370; 15, Enterococcus faecalis ATCC 29212; 16, Lactobacillus acidophilus ATCC 4356; 17, Listeria monocytogenes ATCC 15313; 18, Streptococcus pneumoniae ATCC 27336; 1 and 19, controls to which no DNA was added; M, 50-bp ladder (molecular size standard).

PCR assays. Specificity tests performed with the panel of gram-positive and gram-negative bacterial species (Table 1) with 30 and 40 cycles of amplification showed that the selected PCR primer pair amplified only DNA from S. saprophyticus strains. In order to ensure that the negative PCR results obtained with the bacterial species other than the target species were not attributable to PCR inhibitors or to the inadequacy of the PCR assay, all reactions included an internal control simultaneously amplified. This control was always efficiently amplified when no target DNA was present, thereby showing the absence of PCR inhibitors. It is important that the S. saprophyticus-specific PCR assay did not yield any specific amplification product with 27 staphylococcal species other than S. saprophyticus (Table 1). No false-positive results with the set of 49 gram-positive bacteria comprising 30 staphylococcal species and 19 other genetically related gram-positive species was observed, indicating that the targeted genomic sequences are unique to S. saprophyticus. Increasing the number of amplification cycles from 30 to 40 did not appear to affect the specificity of the S. saprophyticus-specific PCR assay because all staphylococcal species other than S. saprophyticus as well as closely related species (Table 1) could not be amplified by the 40-cycle PCR assay (data not shown).

	DISCUSSION

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S. saprophyticus is relatively easy to culture and identify by phenotypic methods. However, there is a need for the development of rapid and sensitive DNA-based assays which are suitable for the direct detection of S. saprophyticus from clinical specimens, especially urine specimens, to improve the rapidity and the accuracy of the diagnosis of S. saprophyticus infections. At present, resistance to novobiocin is still used in most laboratories to presumptively identify S. saprophyticus, but other coagulase-negative species, including S. epidermidis, are occasionally resistant to novobiocin. We have previously demonstrated the usefulness of nucleic acid amplification by PCR for detection and identification of S. aureus, S. epidermidis, and their clinically relevant antibiotic resistance genes (21-23). In the present study, we have developed a rapid PCR-based assay suitable for specific detection of S. saprophyticus in urine specimens. Initially, a set of 20 10-mer AP-PCR primers was tested with five different strains of S. saprophyticus in order to find a prominent shared amplicon. By this strategy, we were able to obtain such an amplicon of 380 bp that was consistently found in all S. saprophyticus strains tested but that was absent from other staphylococcal species and genetically related gram-positive bacteria. This S. saprophyticus-specific AP-PCR amplicon was sequenced and used to derive optimal PCR primers for the detection of S. saprophyticus. The S. saprophyticus-specific PCR assay developed in this study was specific because it did not amplify DNAs from a variety of gram-positive and gram-negative bacterial species including 26 staphylococcal species other than S. saprophyticus. Furthermore, this assay was shown to be 100% ubiquitous on the basis of testing of 60 S. saprophyticus clinical isolates from various patients, of which 92% were etiologic agents of UTIs. All 60 of these clinical isolates from CHUL as well as the three strains from ATCC were initially reconfirmed to be S. saprophyticus with the MicroScan Autoscan-4 system, thereby showing a perfect correlation with the identification obtained by the S. saprophyticus-specific PCR assay. Therefore, the S. saprophyticus genomic target of unknown coding potential selected for the PCR assay appears to be present in all S. saprophyticus strains and also appears to be well conserved in this species at the nucleotide level but either absent from or distinct in other closely related bacterial species including other staphylococcal species. The PCR assay, which was performed directly from standardized bacterial suspensions or urine specimens spiked with a known number of S. saprophyticus cells, was designed and optimized to be simple and performed in approximately an hour and a half.

Others have developed S. saprophyticus-specific PCR amplification assays targeting variable regions V3 and V6 of the 16S rRNA gene (7, 8). However, these assays have not been applied for direct detection of S. saprophyticus from clinical specimens. In our study, we have used a different approach to develop S. saprophyticus-specific DNA-based diagnostic tests. Our goal was to develop a simple and rapid PCR assay which is specific and ubiquitous for S. saprophyticus and which can be applied to detection directly from bacterial cultures or urine specimens in about 1 h. The 30-cycle PCR protocol showed sensitivity levels of about 100 copies of the S. saprophyticus genome. This sensitivity level is sufficient for culture confirmation assays from urine specimens or blood cultures. Increased levels of sensitivity of the PCR are required for detection of S. saprophyticus directly from urine specimens, in which the number of target cells can be much lower. The 40-cycle PCR protocol, which showed sensitivity levels of about six copies of the S. saprophyticus genome per PCR mixture or 300 to 1,400 CFU/ml of urine, appears to be suitable for that purpose. Such a high sensitivity level will be particularly critical in patients with UTIs with low S. saprophyticus cell counts (i.e., less than 10⁴ CFU/ml), found in approximately one-third of women with acute lower UTIs caused by S. saprophyticus (14, 20). Similarly, acute pyelonephritis caused by UTIs have been reported in association with low bacterial counts in voided urine (13).

The sensitivity assays performed with S. saprophyticus cells diluted in PBS or urine samples demonstrated the efficacy of the IDI DNA extraction kit for (i) control of the PCR inhibitors present in urine samples and (ii) lysis of S. saprophyticus cells. On the basis of the minimum number of CFU detected in PBS, this extraction kit allows a cell lysis which is about 1,300 times more efficient than that from the direct addition of diluted cells to the PCR mixture without pretreatment. Furthermore, the sensitivity levels achieved with spiked urine samples and cells diluted in PBS prepared with the IDI DNA extraction kit were both very similar, thereby suggesting that it eliminated PCR inhibition completely. For comparison, there was partial to complete PCR inhibition with spiked urine samples added directly to the PCR mixture without pretreatment.

Preliminary studies performed with the IDI DNA extraction kit, which requires about 10 min for urine sample preparation, indicate that it is also suitable for efficient recovery of DNA from gram-negative bacilli including E. coli, enterococci, and streptococci, which are also frequently encountered in urine specimens. However, this application needs to be confirmed in a clinical study to validate the procedure for the diagnosis of UTIs. We have previously developed PCR assays for the specific detection of other staphylococcal species as well as associated antibiotic resistance genes (21-23). The S. saprophyticus PCR assay reported in this study will be combined in multiplex with these PCR assays as well as with others which are under development, especially for the identification and detection from urine specimens of E. coli and other bacteria frequently associated with UTIs. A direct impact of such diagnostic tests is that they should allow the faster establishment of effective antibiotic therapy and a reduction of empirical treatments with broad-spectrum antibiotics which are associated with high costs and toxicity (2, 3). The consequent reduction of antibiotic use should reduce the emergence of resistance.