Copper Resistance Promotes Fitness of Methicillin-Resistant Staphylococcus aureus during Urinary Tract Infection.

Urinary tract infection (UTI) is one of the most common infectious conditions affecting people in the United States and around the world. Our knowledge of the host-pathogen interaction during UTI caused by Gram-positive bacterial uropathogens is limited compared to that for Gram-negative pathogens. Here, we investigated whether copper and the primary copper-containing protein, ceruloplasmin, are mobilized to urine during naturally occurring UTI caused by Gram-positive uropathogens in patients. Next, we probed the role of copper resistance in the fitness of methicillin-resistant Staphylococcus aureus (MRSA) during experimental UTI in a murine model. Our findings demonstrate that urinary copper and ceruloplasmin content are elevated during UTI caused by Enterococcus faecalis, S. aureus, S. epidermidis, and S. saprophyticus. MRSA strains successfully colonize the urinary tract of female CBA mice with selective induction of inflammation in the kidneys but not the bladder. MRSA mutants lacking CopL, a copper-binding cell surface lipoprotein, and the ACME genomic region containing copL, exhibit decreased fitness in the mouse urinary tract compared to parental strains. Copper sensitivity assays, cell-associated copper and iron content, and bioavailability of iron during copper stress demonstrate that homeostasis of copper and iron is interlinked in S. aureus. Importantly, relative fitness of the MRSA mutant lacking the ACME region is further decreased in mice that receive supplemental copper compared to the parental strain. In summary, copper is mobilized to the urinary tract during UTI caused by Gram-positive pathogens, and copper resistance is a fitness factor for MRSA during UTI. IMPORTANCE Urinary tract infection (UTI) is an extremely common infectious condition affecting people throughout the world. Increasing antibiotic resistance in pathogens causing UTI threatens our ability to continue to treat patients in the clinics. Better understanding of the host-pathogen interface is critical for development of novel interventional strategies. Here, we sought to elucidate the role of copper in host-Staphylococcus aureus interaction during UTI. Our results reveal that copper is mobilized to the urine as a host response in patients with UTI. Our findings from the murine model of UTI demonstrate that copper resistance is involved in the fitness of methicillin-resistant S. aureus (MRSA) during interaction with the host. We also establish a critical link between adaptation to copper stress and iron homeostasis in S. aureus.

Assessment of Silver Levels in a Closed-Incision Negative Pressure Therapy Dressing: In Vitro and In Vivo Study.

Objective: In recent years, reticulated open-cell foam-based closed-incision negative pressure therapy (ROCF-ciNPT) has shown effectiveness in management of various postoperative incisions. These dressings consist of a skin interface layer that absorbs fluid from the skin surface and reduces the potential for microbial colonization within the dressing by means of ionic silver. This study examines the ability of silver to reduce the bioburden within the dressing as well as the localized effect due to potential silver mobility. Approach: Ability of silver to reduce bioburden within the ROCF-ciNPT dressing was assessed using Staphylococcus aureus, Pseudomonas aeruginosa, and Candida spp. Furthermore, silver mobility was assessed using an in vitro skin model to study the zone of inhibition along with released silver quantification. Using a porcine model, diffusion of silver into blood and tissue was studied using emission spectrometry and histology. Results: Microbial growth in the ROCF-ciNPT dressing was significantly reduced (∼2.7-4.9 log reduction) compared to a silver-free negative control. No zone of inhibition was observed for microbial colonies for up to 7 days with minimal localized silver release (<5.5 ppm release). In vivo studies demonstrated no measurable concentration (<0.2 µg/g) of silver in the blood, urine, feces, kidney, and liver tissue biopsy. Innovation: This study provides an important insight into silver concentration and mobility within the ROCF-ciNPT dressing, given emerging concerns associated with potential silver cytotoxicity. Conclusion: These results indicate the concentration of silver (0.019% silver by weight) in the ROCF-ciNPT dressings has been adequate to reduce bioburden within the skin interface layer, while severely limiting the amount of silver leaching out.

Genotypic and phenotypic analysis of biofilm formation Staphylococcus epidermidis isolates from clinical specimens.

OBJECTIVES: Staphylococcus epidermidis is the primary causative agent of infections associated with indwelling biomaterials. Antibiotic susceptibility patterns, Biofilm formation capability, and screening of responsible genes in biofilm formation procedure in clinical isolates (icaA, icaB, icaC, icaD, sdrG, and atlE) were assigned as the main objectives in this study. The clinical samples were analyzed via standard biochemical assays for identifying different bacteria which were confirmed using the multiplex colony PCR method. Subsequently, biofilm-formation capability, antibiotic susceptibility testing, and the frequency of genes responsible for biofilm formation in the confirmed strains were checked. RESULTS: Out of 183 clinical specimens 54 S. epidermidis isolates were detected by targeting a housekeeping gene (sesc) taking advantage of the PCR procedure. All of the strains were Biofilm forming producers. The in vitro biofilm formation assays determined that 45 (83.33%), 5 (9.26%), 4 (7.41%) were strong, moderate, and weak biofilm former strains respectively. Among the isolated strains, the specific frequencies of the biofilm-forming genes were specified to be (98%) for sdrG, (84%) for atlE, (80%) for icaC, and (70%) for icaD. Cefamandole and Amikacin are the most effective antibiotics in isolated strains. All strains were ascertained to be methicillin and amoxicillin/clavulanic acid resistant.

Changing antibiotic resistance profile of Staphylococcus aureus isolated from HIV patients (2012-2017) in Southern India.

PURPOSE: Emergence of multidrug-resistant and methicillin-resistant Staphylococcus aureus (MRSA) infections in HIV patients limit the treatment options and challenge the clinical management of infections. The periodic monitoring of S. aureus infections and its drug resistance profile in HIV patients are of paramount importance in clinical management. MATERIALS AND METHODS: A total of 7204 clinical specimens from HIV patients from 2012 to 2017 were processed for the isolation of S. aureus strains using conventional culture techniques and cultures were identified using standard biochemical test. Antibiotic susceptibility of S. aureus strains was tested by Kirby-Bauer disk diffusion method. RESULTS: A total of 380 (5.3%) S. aureus strains were isolated from HIV patients in the study period. High percentage of S. aureus strains were isolates from urine (69.5%) specimen and 58.4% of S. aureus infections were noted among hospitalized patients. Antibiotic susceptibility profile reveals S. aureus was highly resistant to penicillin (95.2%) followed by cephalexin (84.6%). Methicillin resistance was highly observed in the year 2017 (86%) and the rate of MRSA steadily increasing from 51.8% in 2012 to 86% in 2017. Significant increase of S. aureus infections (35%; p<0.001) and MRSA (76%; p=0.0007) were observed in the year 2016. CONCLUSIONS: This study reports the increasing trends of S. aureus infections and MRSA among HIV patients from Southern India. Multidrug-resistance profile of S. aureus could complicate the selection of proper antibiotic regimens and time cure of HIV patients.

Ultrasensitive and selective detection of Staphylococcus aureus using a novel IgY-based colorimetric platform.

To develop a specific method for the detection of S. aureus, chicken anti-protein A IgY was adopted for specifically capturing S. aureus, depending on the specific recognition of staphylococcal protein A (SPA) by chicken anti-protein A IgY, which can eliminate the interference from protein G-producing Streptococcus. HRP labeled IgG, Fc region of which has a high affinity towards SPA, was paired with IgY for the colorimeter analysis of the system. By optimizing the system, a super-low detection limit of 11 CFU of S. aureus in 100 µL PBS without enrichment, with a linear range from 5.0 × 102 CFU mL-1 to 5.0 × 104 CFU mL-1 was obtained. The entire assay was accomplished in less than 90 min and no cross-reactivity with the other tested bacterial species was observed. Moreover, the developed assay has been applied for the detection of S. aureus in three different types of real samples (sodium chloride injection, apple juice and human urine) with satisfactory results. To the best of our knowledge, it is the first time to report using chicken anti-protein A IgY and any IgG to detect S. aureus based on the dual-recognition mode of SPA. The novel method opened up a way for monitoring S. aureus in food samples with high sensitivity, specificity and simple operation.

Clinical pharmacokinetic and pharmacodynamic analysis of daptomycin and the necessity of high-dose regimen in Japanese adult patients.

The objective of this study was to explore the optimal dosage regimen of daptomycin and to determine the necessity and validity of a high-dose regimen from the perspectives of PK/PD parameters using Monte Carlo Simulation (MCS) and therapeutic drug monitoring (TDM) in a Japanese clinical setting. The volume of distribution (0.13 ± 0.012 L/kg) in this study was greater than that in healthy volunteers reported in Japan. The range of half-lives was between 8.9 and 34.9 h, which were gradually prolonged as creatinine clearance decreased. In MCS, the cumulative fractions of response (CFR) of the peak/MIC â‰§ 60 and the AUC/MIC â‰§ 666 at the 6 mg/kg q 24 h were 72.0% and 78.8% but at the 10 mg/kg q 24 h, the CFRs improved to both 99%. In TDM with 6 mg/kg q 24 h regimen, the patients who reached the peak and AUC target were 40% (2 out of 5 patients), respectively. The intraindividual variability in daptomycin PK may indicate the necessity of TDM and high-dose regimen, such as over 8 mg/kg, may be needed to ensure the effectiveness especially on Japanese patients with normal renal function.

Acute Proximal Myopathy in a Young Male-A Case of Infectious Myositis.

Background and objectives: Acute proximal muscle weakness has a broad differential. Infectious myositis is difficult to differentiate clinically from inflammatory myopathy, often causing a delayed diagnosis. Infectious myositis should be thought of as a differential for proximal muscle pain and weakness in the right context. Case Presentation: A 40-year-old male with diabetes presented with exquisite pain and weakness of proximal extremities. He denied trauma, recent travel, new medications, or substance use. He denied prior rheumatologic, thyroid, or musculoskeletal disorders. The urine culture revealed staphylococcal infection with negative blood cultures. Rheumatologic and endocrine workups were negative. Random muscle biopsy was negative for inflammatory infiltrate. MRI of thighs and arms showed innumerable foci of nodular and ring enhancement in the proximal muscle groups. The patient noted improvement after about 10 days of antibiotics with complete resolution at 2 months. Discussion and Conclusion: Bacterial myositis is most often due to Staphylococcus aureus (70%) and affects a single muscle. Multifocal abscesses are rare and strongly suggest transient bacteremia. Our patient most likely had transient initiating staphylococcal bacteremia leading to diffuse myositis and hematogenous urinary tract infection (UTI). A delay in treatment can be life-threatening.

Monic acid A: a biomarker in clinical intra-nasal mupirocin medication for MRSA decolonisation.

CONTEXT: Mupirocin (BactrobanR) is widely prescribed for intra-nasal decolonisation of MRSA for in-patients awaiting surgery or self-medicated for out-patients although adherence for the latter is not monitored. Non-adherence is a widespread pharmaceutical problem but could encourage selection of antibiotic resistance. Mupirocin is only a topical antibiotic because it decomposes in stomach acidity to monic acid A, but this has not previously been exploited as a biomarker for clinical intra-nasal medication. MATERIALS AND METHODS: Urine from three catheterised patients in two London hospitals during and after mupirocin medication, was passed through Waters Oasis cartridges to isolate organic acids. Sensitive LC-MS-MS analysis for monic acid A in methanolic eluate has been developed to identify ∼10 pg. RESULTS: Monic acid A was quantified in all samples from one patient, translating into 6-46 ng from 12 mg mupirocin, assuming 1 L daily urine output. However, no urinary monic acid A was detected for two other patients. DISCUSSION AND CONCLUSIONS: Consistent occurrence of monic acid A in urine of one mupirocin patient shows for the first time that antibiotic distribution across nasal mucous membranes had generally been maintained during medication. In contrast, consistent absence in the two other patients requires wider study in hospital.

Evaluation of MALDI-TOF VITEK®MS and VITEK® 2 system for the identification of Staphylococcus saprophyticus.

AIM: To compare two identification methods for coagulase-negative staphylococci (CoNS) isolated from patients with urinary tract infections, VITEK® 2 and MALDI-TOF VITEK®MS, with genotypic identification by internal transcribed spacer PCR (ITS-PCR). RESULTS: A total of 217 CoNS isolates were studied. Agreement of the VITEK® 2 system with ITS-PCR was 84.8%, with 98% sensitivity and 100% specificity. Thirty-one of the 33 strains incorrectly identified by VITEK® 2 belonged to the species Staphylococcus saprophyticus. MALDI-TOF VITEK®MS showed an excellent correlation with ITS-PCR since it correctly identified all CoNS isolates. CONCLUSION: MALDI-TOF VITEK®MS is more accurate than the automated VITEK® 2 system in identifying CoNS isolated from urinary tract infections to species level, particularly urinary isolates of S. saprophyticus.

What about Urinary Tract Infections and its Antibiotic Resistance Bacteria in Ilam, Iran?

BACKGROUND: Because of the unknown situation of antibiotic resistance pattern in the main hospital in Ilam, Iran, we aimed to evaluate the antibiotic resistance pattern of uropathogenic bacteria obtained from referred patients to Imam Khomaini Hospital, Ilam, Iran. So, 114 bacteria were collected during 9-month period and evaluated for their antibiotic resistance patterns. RESULTS: Our results demonstrated that Escherichia coli as the dominant responsible for urinary tract infection. Our results demonstrated that 61.4 % (n = 70) of isolates were positive for E.coli, while lowest prevalence was observed for Staphylococcus aureus and Acinetobacter baumannii. The results also showed that 6.4% (n = 7) were metallo beta lactamase (MBL) producers. Our findings showed only 4 gram positive bacteria were obtained from patients with urinary tract infections including one methicillin resistant S. aureus (MRSA) and 2 vancomycin resistant Enterococcus faecalis (VRE). CONCLUSION: In conclusion, we strongly recommended to perform a perfect study among all hospitals in Iran to evaluate the situation of antibiotic resistance and make a real panel to control this issue.

Antimicrobial resistance in urine and skin isolates in Timor-Leste.

OBJECTIVES: High rates of antimicrobial resistance (AMR) are seen throughout Southeast Asia. However, limited AMR data exist for Timor-Leste, which is situated on the south-eastern portion of the Malay Archipelago. The purpose of this study was to identify AMR in bacteria isolated from urine and skin swabs from patients in Dili, the capital of Timor-Leste. METHODS: Urine and skin swabs were collected from symptomatic patients in Timor-Leste and were processed for bacterial culture. Isolates were processed in Australia using a VITEK®2 system for bacterial identification and to determine antimicrobial susceptibility according to Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: A total of 154 urine isolates and 57 skin isolates were analysed. Of the Enterobacteriaceae, 35% were resistant to ceftriaxone with an extended-spectrum ß-lactamase (ESBL)-producing phenotype. Carbapenem resistance was not observed in any of the Gram-negative isolates. Of the Staphylococcus aureus isolates, 11% were of the community-associated methicillin-resistant S. aureus (CA-MRSA) phenotype. CONCLUSIONS: A moderately high proportion of Gram-negative urine isolates in Timor-Leste demonstrate phenotypic ESBL production, and a relatively low proportion of S. aureus isolates were methicillin-resistant. Improved understanding of AMR rates in Timor-Leste can help guide antimicrobial prescribing and inform antimicrobial stewardship strategies.

Fluorescent turn-on sensing of bacterial lipopolysaccharide in artificial urine sample with sensitivity down to nanomolar by tetraphenylethylene based aggregation induced emission molecule.

A tetraphenylethylene based aggregation induced emission (AIE) probe, TPEPyE, bearing a positively charged pyridinium pendant was designed and synthesized. The positively charged TPEPyE can efficiently bind to the negatively charged lipopolysaccharide (LPS) through electrostatic interactions between the two oppositely charged species. As a result, upon the addition of LPS into the PBS solution of TPEPyE, this probe aggregated immediately onto the surface of LPS and resulted over 22-fold of fluorescence enhancement. TPEPyE exhibited good selectivity and high sensitivity toward LPS in PBS buffer solution and the detection limit was calculated to be 370 pM (3.7ng/mL). More notably, TPEPyE also retained good sensitivity and selectivity in artificial urine system (with much higher ionic strength) with the detection limit down to nanomolar. Moreover, this probe can also make a distinction between gram-positive bacteria Staphylococcus aureus (S. aureus) and gram-negative bacteria Escherichia coli (E. coli), making it a promising sensor for clinical monitoring of urinary tract infections.

Staphylococcus saprophyticus bacteremia after ESWL in an immunocompetent woman.

Staphylococcus saprophyticus is a well-known cause of uncomplicated urinary tract infections, especially in young and sexually active women. Presence in blood cultures is rare and often attributed to contamination. When bacteremia is significant, it occurs mostly in patients with hematologic malignancies and is predominantly catheter-related. However, we describe a case of significant bacteremia with S. saprophyticus associated with urinary tract infection after extracorporeal shock wave lithotripsy of an ureterolithiasis in an otherwise healthy patient.

Agarose-based microfluidic device for point-of-care concentration and detection of pathogen.

Preconcentration of pathogens from patient samples represents a great challenge in point-of-care (POC) diagnostics. Here, a low-cost, rapid, and portable agarose-based microfluidic device was developed to concentrate biological fluid from micro- to picoliter volume. The microfluidic concentrator consisted of a glass slide simply covered by an agarose layer with a binary tree-shaped microchannel, in which pathogens could be concentrated at the end of the microchannel due to the capillary effect and the strong water permeability of the agarose gel. The fluorescent Escherichia coli strain OP50 was used to demonstrate the capacity of the agarose-based device. Results showed that 90% recovery efficiency could be achieved with a million-fold volume reduction from 400 µL to 400 pL. For concentration of 1 × 10(3) cells mL(-1) bacteria, approximately ten million-fold enrichment in cell density was realized with volume reduction from 100 µL to 1.6 pL. Urine and blood plasma samples were further tested to validate the developed method. In conjugation with fluorescence immunoassay, we successfully applied the method to the concentration and detection of infectious Staphylococcus aureus in clinics. The agarose-based microfluidic concentrator provided an efficient approach for POC detection of pathogens.

Antimicrobial susceptibility of methicillin-resistant Staphylococcus pseudintermedius isolated from veterinary clinical cases in the UK.

Staphylococcus pseudintermedius is a leading aetiologic agent of pyoderma and other body tissue infections in dogs and cats. In recent years, an increased prevalence of methicillin-resistant S. pseudintermedius (MRSP) has been reported. Isolation of MRSP in serious infections poses a major therapeutic challenge as strains are often resistant to all forms of systemic antibiotic used to treat S. pseudintermedius -related infections. This study investigates the occurrence of MRSP from a total of 7183 clinical samples submitted to the authors' laboratories over a 15-month period. Identification was based on standard microbiological identification methods, and by S. pseudintermedius-specific nuc polymerase chain reaction (PCR). Methicillin resistance was confirmed by PBP2a latex agglutination and mecA PCR. Susceptibility against non-beta-lactam antibiotics was carried out using a disc-diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. In addition, susceptibility to pradofloxacin--a new veterinary fluoroquinolone--was also investigated. SCCmec types were determined by multiplex PCR. Staphylococcus pseudintermedius was isolated from 391 (5%) samples and 20 were confirmed as MRSP from cases of pyoderma, otitis, wound infections, urinary tract infection and mastitis in dogs only. All 20 isolates were resistant to clindamycin and sulphamethoxazole/trimethoprim. Nineteen were resistant to chloramphenicol, enrofloxacin, gentamicin, marbofloxacin and pradofloxacin; additionally, seven isolates were resistant to tetracycline. Fifteen isolates carried SCCmec type II-III, four isolates had type V and one harboured type IV. To date, only a few scientific papers on clinical MRSP strains isolated from the UK have been published, thus the results from this study would provide additional baseline data for further investigations.

Etiology of acute pyelonephritis: species composition and populational levels of urine microflora.

Acute urinary tract infection is considered to be a growing burden in both medical and socio-economic terms. The exact etiologic structure of acute pyelonephritis pathogens, their role and interference is unclear. Thus, we aimed on revealing the species composition and populational levels of microorganisms causing acute inflammation of kidney and perirenal tissues. The study included 145 patients with acute pyelonephritis (mean age 34.15±6.79); 67 (47.2%) male and 78 (53.8%) female. Microbiota was defined in three different portions (samples) of urine taken during on urination. Species were considered as dominant when constancy index was 50.0% or higher, additional - from 25.0% to 50.0% and random species if value less than 25.0%. Received data expectedly correlates with other studies. Conditionally pathogenic Enterobacteriaceae play major role in etiology of acute pyelonephritis. Our study provides further evidence of higher etiologic significance of conditionally pathogenic flora (normal E. coli) over of the traditional pathogenic microorganisms like enteropathogenic Escherichia and Staphylococcus aureus.

[Staphylococcus saprophyticus isolated from urine culture in outpatients: epidemiology and antimicrobial susceptibility (Label Bio Elbeuf study - November 2007-July 2009)]. / Staphylococcus saprophyticus isolés d'examens cytobactériologiques urinaires en ville : épidémiologie et sensibilité aux antibiotiques (étude Label Bio Elbeuf - novembre 2007-juillet 2009).

OBJECTIVE: The aim of the study was to determine the distribution and the antibiotic susceptibility of Staphylococcus saprophyticus patterns isolated from urine culture in outpatients (population: 57,000, Elbeuf, Normandie, France). DESIGN: Prospective study from November 2007 to October 2009 in collaboration with three private medical laboratories. Determination of susceptibility to oxacillin by disk diffusion (cefoxitin, and moxalactam), automated method (Vitek BioMérieux 2) and mecA PCR's detection. Determination of the minimum inhibitory concentration by microbroth dilution for other antibiotics. RESULTS: Five thousand and fifty-one bacterial strains isolated, 91 strains of S. saprophyticus (1.8%), 89 in women (2.25%) and two in men (0.18%). S. saprophyticus represented 10.3% and 14.5% of isolates (women respectively aged between 11 and 30; 16 and 20 years); S. saprophyticus is isolated less frequently in winter. mecA PCR detection was positive for two strains. All strains tested were susceptible to ciprofloxacin and furans. Only one strain is resistant to cotrimoxazole. CONCLUSIONS: S. saprophyticus is found mostly in women between 11 to 30 years. Cotrimoxazole (after susceptibility testing) is efficient in case of S. saprophyticus's cystitis. Furans (probabilistic treatment) have to be reevaluated because of the potential for serious adverse effects.

The relative importance of Staphylococcus saprophyticus as a urinary tract pathogen: distribution of bacteria among urinary samples analysed during 1 year at a major Swedish laboratory.

To determine the distribution of urinary tract pathogens with focus on Staphylococcus saprophyticus and analyse the seasonality, antibiotic susceptibility, and gender and age distributions in a large Swedish cohort. S. saprophyticus is considered an important causative agent of urinary tract infection (UTI) in young women, and some earlier studies have reported up to approximately 40% of UTIs in this patient group being caused by S. saprophyticus. We hypothesized that this may be true only in very specific outpatient settings. During the year 2010, 113,720 urine samples were sent for culture to the Karolinska University Hospital, from both clinics in the hospital and from primary care units. Patient age, gender and month of sampling were analysed for S. saprophyticus, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Species data were obtained for 42,633 (37%) of the urine samples. The most common pathogens were E. coli (57.0%), Enterococcus faecalis (6.5%), K. pneumoniae (5.9%), group B streptococci (5.7%), P. mirabilis (3.0%) and S. saprophyticus (1.8%). The majority of subjects with S. saprophyticus were women 15-29 years of age (63.8%). In this age group, S. saprophyticus constituted 12.5% of all urinary tract pathogens. S. saprophyticus is a common urinary tract pathogen in young women, but its relative importance is low compared with E. coli even in this patient group. For women in other ages and for men, growth of S. saprophyticus is a quite uncommon finding.

Development of PCR primers and a DNA macroarray for the simultaneous detection of major Staphylococcus species using groESL gene.

Staphylococcus spp., including S. aureus, S. intermedius, S. hyicus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. xylosus, and S. carnosus, are major bacterial species associated with food poisoning, and human and veterinary clinics. Traditional methods for the identification of these staphylococci are time-consuming, laborious, or inaccurate. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and polymerase chain reaction (PCR) primers for the detection of the aforementioned Staphylococcus species. These primers were proved to be specific for the detection of their corresponding target strains. Furthermore, by using a consensus primer pair, we were able to co-amplify the intergenic region of groES-groEL for these staphylococci. Followed by a chromogenic macroarray system with the specific probes on the plastic chips, these staphylococci in milk products or clinical samples could be simultaneously detected. When the system was used for the inspection of milk or urine samples containing N × 10° target cells per milliliter of the sample, all these staphylococcal species could be identified after an 8-h pre-enrichment step. This system also allowed the adequate diagnosis of bacteremia, since N × 10° target cells per milliliter of the blood samples could be detected after a 12-h pre-enrichment. Compared to the multiplex PCR method, this approach has the additional advantage that it allowed the discrimination of more bacterial strains-even some bacterial strains that may generate PCR products with the same molecular sizes.