Staphylococcus aureus identification: thermonuclease agar for direct testing of blood isolates and a new slide agglutination test.

Simulated blood cultures were used to evaluate Thermonuclease agar (Remel) for distinguishing Staphylococcus aureus from coagulase-negative staphylococci (CNS) without subculture to agar media, and a new slide agglutination test (Staphylochrome; Innovative Diagnostic Systems) was evaluated for its ability to distinguish S. aureus from CNS after growth on blood agar. A total of 125 S. aureus and 124 CNS isolates were tested by each method. Reference identification methods included tube coagulase, thermonuclease detection from solid media, and biochemical characterization. Direct thermonuclease testing with simulated blood cultures correctly identified all 249 isolates. Staphylochrome correctly identified 121 of 125 S. aureus and all CNS isolates. S. aureus was reliably distinguished from CNS by both tests evaluated in this study.     

Prosthetic joint infection due to Staphylococcus lugdunensis

Staphylococcus lugdunensis, a coagulase-negative staphylococcus, is being increasingly recognized as the cause of serious infections. We report 2 cases of total knee arthroplasty infection caused by S lugdunensis. S lugdunensis frequently produces a clumping factor that can result in a positive slide (short) coagulase test result. If the microbiology laboratory does not use the tube coagulase (long) test to confirm the slide coagulase test result, the organism may be misidentified as Staphylococcus aureus. S lugdunensis is more virulent than other coagulase-negative staphylococci and in many clinical situations behaves like S aureus, further increasing the confusion. However, S lugdunensis differs from S aureus in that it is susceptible to most antibiotics. This fact may alert the microbiology laboratory or the clinician that the isolate is likely not S aureus and prompt further testing of a specific isolate. Accurate identification of S lugdunensis isolates facilitates studies to define the epidemiology and pathogenesis of prosthetic joint infection due to S lugdunensis and delineates optimal medical and surgical therapies.     

Genotypic versus phenotypic characterization, with respect to susceptibility and identification, of 17 clinical isolates of Staphylococcus lugdunensis

OBJECTIVES: To compare different methods for the identification and determination of susceptibility to penicillin and methicillin of Staphylococcus lugdunensis. METHODS: Seventeen clinical isolates of S. lugdunensis (identified by PCR amplification and sequencing of the rpoB gene) were studied using the ATB32-Staph, Crystal, Vitek 2 and Wider commercial systems. The clumping factor test and the tube coagulase test were also performed. Beta-lactamase production was studied by chromogenic methods. Methicillin resistance was phenotypically studied by the MRSA slide latex agglutination test, growth in MRSA agar, and the Vitek 2 and Wider systems (based on oxacillin MIC), and genotypically studied by detection of the mecA gene by PCR. RESULTS: The clumping factor test was negative in 35.3% of strains. All isolates were correctly identified to species level by the ATB32-Staph system. Species misidentification rates were 5.9%, 23.5% and 29.4% with the Crystal, the Vitek 2 and the Wider systems, respectively, mostly as Staphylococcus haemolyticus. Beta-lactamase was present in 11.8% of strains. Whereas 76.5% and 47.1% of strains exhibited oxacillin resistance (MIC range 0.5-2 mg/L) by the Vitek 2 system and the Wider system, respectively, none of the strains was positive in the MRSA slide latex agglutination test or grew in MRSA agar. All strains lacked the mecA gene. CONCLUSIONS: The clumping factor test and some commercial systems may misidentify S. lugdunensis. Oxacillin resistance detected by commercial systems is not indicative of the presence of the mecA gene. These facts, together with beta-lactamase production, may preclude adequate treatment of infections by this virulent coagulase-negative Staphylococcus.     

Rapid MRSA detection by a latex kit.

Methicillin resistant strains of Staphylococcus aureus (MRSA) are implicated in serious infections and nosocomial outbreaks, and show resistance to a wide range of antibiotics, thus limiting the treatment options. Therefore, rapid detection is clinically crucial for both treatment and infection control measures. This study assessed the performance of a rapid latex agglutination kit marketed to detect MRSA clinical isolates (MRSA-Screen test Denka Seiken Co Ltd, Tokyo, Japan) based on detecting a specific penicillin binding protein 2a (PBP2a) in comparison to the NCCLS oxacillin salt agar screen plate, the 1 microg oxacillin disk diffusion test, and the oxacillin MIC by E-test. Testing was carried out on 133 isolates consisting of 99 MRSA and 34 methicillin sensitive strains of S. aureus (MSSA). Concordant results were observed between the latex kit and all the other tests for the 99 MRSA isolates. Only 1 of the 34 MSSA isolates gave a positive agglutination reaction in the latex kit. The kit sensitivity and specificity were determined to be 100% and 97%, respectively. This reliable performance indicates that the MRSA-Screen latex test is very useful test for the rapid detection of MRSA isolates in the clinical microbiology laboratory.     

Rapid, direct identification of Staphylococcus aureus and Streptococcus pneumoniae from blood cultures using commercial immunologic kits and modified conventional tests.

To develop safe and rapid methods for identification of Staphylococcus aureus and Streptococcus pneumoniae directly from positive blood culture bottles (BCB) (BACTEC, Johnston Laboratories), several commercial biochemical and immunological tests as well as modified conventional tests were evaluated. Preliminary studies demonstrated that both S. aureus and St. pneumoniae could be identified directly using only a small aliquot (100 microliters) of the blood culture broth obtained via vent without need for centrifugation or other separation steps. A simple tube coagulase exhibited 98% sensitivity and 100% specificity for 32 S. aureus isolates and 157 blood cultures positive for coagulase-negative staphylococci when read at 2 hr. All systems employed for direct identification of St. pneumoniae exhibited excellent sensitivity and specificity using aliquots from blood culture broths, but Pneumoslide (BBL Microbiology Systems, Cockeysville, MD) was easiest to perform and interpret. The results of this study show that S. aureus and St. pneumoniae can be identified directly from blood culture broth aliquots using rapid methods that eliminate the need for centrifugation or use of needles and syringes.     

Evaluation of a latex agglutination assay for rapid detection of oxacillin resistant Staphylococcus aureus

Oxacillin resistance in Staphylococcus aureus is mediated by the mecA gene, resulting in production of penicillin-binding protein 2a (PBP2a), which is not present in the oxacillin susceptible strains. We evaluated the ability of a 30-min latex agglutination (LA) test (Seiken, Tokyo, Japan) to detect production of PBP2a in 315 clinical isolates of S. aureus. The LA results were compared with results of susceptibility testing using the Vitek GPS-SV test card. The latex test was positive for all 206 isolates determined to be methicillin resistant by Vitek (sensitivity 100%), the latex test was negative for 108 of 109 isolates determined to be oxacillin susceptible by Vitek, and the latex test was positive for 1 isolate determined to be susceptible by Vitek (specificity 99.1%). The discrepant isolate was negative for the mecA gene by polymerase chain reaction (PCR). The LA test is a rapid and reliable method for detecting oxacillin resistant S. aureus.     

Gold nanoparticle-based immunochromatographic test for identification of Staphylococcus aureus from clinical specimens

BACKGROUND: Staphylococcus aureus is one of the most important human pathogens, causing both nosocomial and community-acquired infections. Therefore, a method for rapidly detecting for S. aureus would be useful. We describe an analytical system of immunochromatographic assay based on gold nanoparticles developed for the detection of S. aureus in patient specimen. METHODS: The assay was in the sandwich format, using anti-protein A IgG with 2 distinct specificities. One anti-protein A IgG was immobilized in a defined detection zone on a porous nitrocellulose membrane, while the other anti-protein A IgG was conjugated with gold nanoparticles. The mixture was then passed along the porous membrane by capillary action past the anti-protein A IgG in the detection zone, binding the particles that to which surface protein A was already bound to their surface, yielding a red color. RESULTS: The sensitivity and specificity in the immunochromatographic test were 100% and 94.7-100% for 130 S. aureus strains and 36 non-S. aureus strains, respectively. The results were comparable to the conventional coagulase test and latex agglutination test of different bacteria. CONCLUSION: This method may be useful for analyzing S. aureus in patient specimen. It was quick, easy to perform, and with a long shelf life at room temperature.     

Virulent gentamicin-induced small colony variants of Staphylococcus aureus.

Stable nonhemolytic small colony variants were isolated in pure culture from nine of 30 Staphylococcus aureus clinical strains after incubation of log10 7.0 cfu for 48 hr in MH broth containing 1.0 microgram/ml gentamicin. The variants resembled Staphylococcus epidermidis on blood agar, but they were positive for tube coagulase and thermostable nuclease at 24 hr and fermented mannitol slowly. The infectivity and virulence of four variants were compared to four parent S. aureus and three S. epidermidis strains in a rabbit model of endocarditis. Log10 5.0 cfu of the variant S. aureus, parent S. aureus, or S. epidermidis strains were injected intravenously into rabbits with intracardiac catheters. Quantitative culture of vegetations demonstrated endocardial infection in 47 of 49 (96%) animals injected with S. aureus variants, 44 of 44 injected with S. aureus parent strains, and four of 21 (19%) S. epidermidis-injected animals. The mortality rate in untreated animals within 4 days was five of 49 (10%) for variant S. aureus, 33 of 44 (75%) for parent S. aureus, and 0 of 21 for S. epidermidis. Small colony variants of S. aureus may be mistaken for S. epidermidis, but the variants are significantly more infective than S. epidermidis and are more likely to cause endocarditis. Gentamicin-induced S. aureus small colony variants are as infective but less virulent than their parent S. aureus strains.     

Microscopic examination of stools and a latex slide agglutination test for the rapid identification of bacterial enteric infections in Khmer children

Eighty-five stools collected from 50 children with diarrhea at an evacuation site on the Thai-Kampuchean border were (1) examined microscopically for fecal leukocytes, (2) tested after 24 hr enrichment in brain/heart infusion broth by a latex slide agglutination test for detection of Salmonella and Shigella, and (3) examined with microbiological techniques to identify bacterial, viral, and parasitic pathogens. If the 65 specimens in which one or no pathogens are considered, 6 or more fecal leukocytes/hpf were found on microscopic examination of stools in both children infected with Shigella spp., the one child infected with Salmonella spp., and three of eight children infected with Campylobacter spp. Less than or equal to 5 leukocytes/hpf were found in 70% (7/10) of children infected with rotavirus, 100% (2/2) infected with Cryptosporidium, 100% (2/2) infected with Giardia, 89% (8/9) infected with enterotoxigenic Escherichia coli, and 77% (24/31) with diarrhea in whom no etiologic agent was identified. The Salmonella slide latex test had a sensitivity of 50%, a specificity of 92%, and a positive predictive value of 12%. The Shigella slide latex test had a sensitivity of 0%, a specificity of 95%, and a positive predictive value of 0%. Forty-five percent of the latex slide agglutination tests from enrichment cultures were nonspecific. Microscopic examination of diarrheal stools for fecal leukocytes, though nonspecific, appears to be the best way to differentiate Shigella spp. from rotavirus and parasitic infections. Examining stools for fecal leukocytes was less helpful in differentiating Shigella from other bacterial infections.     

A comparison of the rapid thermonuclease test and the lysostaphin susceptibility test in the presumptive identification of Staphylococcus aureus from positive Bactec blood cultures

We compared the ability of a rapid 2-hour thermonuclease test and a Gram-stained lysostaphin susceptibility test to presumptively identify Staphylococcus aureus from 72 blood cultures. The thermonuclease test identified 25 of 27 S. aureus; there were no false positives. The predictive values of positive and negative thermonuclease tests were 100% and 95.7%, respectively. The lysostaphin test correctly identified 24 of 27 S. aureus; however, there were eight false positives. The predictive values of positive and negative lysostaphin tests were 75% and 95.2%, respectively. Lysostaphin MICs were greater than or equal to 1.6 micrograms/ml for only 28 of 41 coagulase negative staphylococci strains. We now routinely apply the thermonuclease test in the clinical laboratory. Of the first 304 blood cultures tested in this setting, the thermonuclease test correctly identified 63 of 68 S. aureus. There have been no false positives. The thermonuclease test is superior to the lysostaphin test and accurately identifies S. aureus in blood cultures within 2 hours of the first positive reading.     

fbl gene as a species‐specific target for Staphylococcus lugdunensis identification

Staphylococcus lugdunensis is an unusually virulent coagulase‐negative species, associated with severe infections. The present report describes the development of a single‐step, species‐specific PCR protocol for S. lugdunensis identification. fbl gene, encoding a fibrinogen‐binding adhesin, was exploited and assessed as a suitable nucleic acid target. The gene was detected in all 17 S. lugdunensis isolates examined, while no amplification product was obtained from 98 isolates representing 11 staphylococcal and 17 nonstaphylococcal species. Forty‐seven percent of the S. lugdunensis strains produced a positive slide coagulase reaction, which is consistent with varying levels of Fbl protein expression within the species. J. Clin. Lab. Anal. 24:119–122, 2010. © 2010 Wiley‐Liss, Inc.     

Evaluation of different methods for detecting methicillin (oxacillin) resistance in Staphylococcus aureus

OBJECTIVES: To evaluate the performance of oxacillin, cefazolin, cefoxitin, cefotaxime and imipenem discs; Etest for oxacillin; microdilution; agar screening plates with 2 and 6 mg/L of oxacillin; and PBP2' agglutination for detection of methicillin-resistant Staphylococcus aureus (MRSA). METHODS: A total of 102 clinical S. aureus isolates, including 51 MRSA isolates, tested by PCR for the presence or absence of the mecA gene (gold standard method), isolated from different patients and at different times, were tested with: oxacillin (1 microg), cefazolin, cefoxitin, cefotaxime and imipenem (all 30 microg) discs; Etest for oxacillin; microdilution with oxacillin; agar screening tests (ORSAB medium) with 2 mg/L or 6 mg/L of oxacillin; and PBP2' agglutination with two different kits for detection of MRSA strains. RESULTS: The cefoxitin disc, ORSAB medium and PBP2' detection all showed 100% sensitivity. The cefoxitin, cefazolin and imipenem discs, Etest for oxacillin, microdilution and agar screening method with 6 mg/L at 24 h showed the highest specificity (100%), although variable degrees of sensitivity. The cefoxitin disc, which showed negative and positive predictive values of 100% and 98%, respectively was the best method for detecting MRSA isolates. CONCLUSIONS: In the absence of availability of molecular biology techniques, the cefoxitin disc was the best predictor of methicillin resistance in S. aureus from among the techniques tested.     

A rapid test to detect the most clinically significant Staphylococcus species

A new two-hour test system (RAPIDEC staph) to detect the main staphylococci was evaluated. Of 124 reference strains tested, 83 represented species most often found in clinical specimens, the remaining 41 strains representing five species primarily of animal origin. RAPIDEC staph detected all Staph, aureus, Staph. epidermidis and seven of eight Staph. saprophyticus strains. Of the animal species, all Staphylococcus intermedius strains were assigned to the correct category, the remainder were misidentified as Staph. epidermidis. With 121 catalase-positive Gram-positive cocci isolated from urines the test system correctly detected all Staph. aureus strains, 39 of 41 Staph. epidermidis, and 34 of 36 Staph. saprophyticus. Of 25 atypical Staph. aureus strains which were either slide or tube coagulase negative the test system correctly detected all 17 slide coagulase negative strains but failed to detect eight which were tube coagulase negative. There were no false positives. RAPIDEC staph is a rapid, accurate system for the detection of the three main clinically important species of staphylococci.     

安徽省铜陵地区2009年细菌耐药性监测

目的 了解安徽省铜陵地区临床分离菌株耐药状况.方法 2009年1～12月铜陵地区临床分离菌1 977株用KirbyBauer法进行药敏试验.结果 1977株细菌中革兰阳性菌430株,占21.8%;革兰阴性菌1 547株,占78.2%;MRSA和MRCNS分别占金葡菌和CNS的21.5%和61.6%;MRSA和MRCNS对庆大霉素、环丙沙星、克林霉索和红霉素等均高度耐药,对利福平和氯霉素的耐药率均较低;未见耐万古霉素和替考拉宁葡萄球菌;粪肠球菌对青霉素、氨苄西林、呋喃妥因、磷霉素和氯霉素的耐药率较低;屎肠球菌对磷霉素和氯霉素耐药率较低,未见耐万占霉素和替考拉宁肠球菌.大肠埃希菌和克雷伯菌属中产ESBLs株分别占53.4%和44.4%,产ESBLs株除对亚胺培南和美罗培南均无耐药外,对其他19种抗菌药物的耐药率均较非产ESBLs株高;不发酵糖菌对常用抗菌药物的耐药率较往年上升.结论 革兰阳性菌对糖肽类抗生素无耐药;肠杆菌科细菌对亚胺培南、美罗培南、头孢哌酮-舒巴坦、哌拉西林-他唑巴坦和阿米卡星等耐药率低;不发酵糖菌对常用抗菌药物的耐药率较往年上升.     

First report of a left ventricular assist device infection caused by Staphylococcus schleiferi subspecies coagulans: a coagulase-positive organism

Staphylococcus schleiferi subspecies coagulans is often misclassified as S. aureus given the production of a pseudocoagulase turning the tube coagulase test positive. It is therefore infrequently reported in the literature. We describe the first case of S. schleiferi subspecies coagulans left ventricular assist device infection in a patient awaiting heart transplantation.     

Occurrence and reproducibility of the "skip" phenomenon in bactericidal testing of Staphylococcus aureus

A "skip" phenomenon, in which subcultures to determine bactericidal endpoints show discordant results, can make bactericidal testing of Staphylococcus aureus difficult to interpret. Either a single or several consecutive concentrations may be skipped, with insignificant growth from these concentrations, but heavy growth from higher antimicrobic concentrations. Replicate macrodilution minimum bactericidal concentration testing of cephalothin against S. aureus was investigated to determine the frequency and reproducibility of the skip phenomenon in this test. In preliminary testing, the skip phenomenon occurred at 24 hr in 11 of the 30 tests performed on ten S. aureus strains. Incubation of tubes for 48 hr before subculture markedly reduced the number of tests containing a skip tube (four of 30). The skip phenomenon was not reproducible either within concurrent replicate testing or on repeat testing of the same isolate on subsequent days. The ten strains were retested in replicative trials using the macrodilution test comparing Mueller-Hinton broth (MHB) and MHB supplemented with Tween 80. The number of skips occurring in the supplemented broth test was the same (two of 22) at 24 hr as nonsupplemented broth. For reproducible results, macrodilution minimum bactericidal testing requires performance of the test in duplicate, with repeat subculture at 48 hr if the skip phenomenon occurs in both trials at the 24-hr subculture. Killing curves performed on these isolates did not appear to demonstrate any relationship between the skip phenomenon and the paradoxical effect described by Eagle.     

胶东地区耐甲氧西林金黄色葡萄球菌耐药性的研究

目的了解胶东地区耐甲氧西林金黄色葡萄球菌(MRSA)的耐药情况。方法采用VITEK2compact微生物分析仪进行细菌鉴定和药物敏感试验;头孢西丁Kirby-Bauer法和青霉素结合蛋白2a胶乳凝集(PBP2a)试验对分离出的金黄色葡萄球菌进行MRSA确认;酸测定法和D试验测定MRSA产β-内酰胺酶和红霉素诱导克林霉素耐药实验。结果头孢西丁纸片扩散法与PBP2a胶乳凝集法对MRSA检出率无统计学上差别(P>O.05);331株MR-SA产β内酰胺酶率和克林霉素诱导试验阳性率分别为91.14%和16.62%;全部菌株对万古霉素、替加环素和利奈唑胺敏感,但对红霉素、克林霉素耐药率高达82.19%和77.34%。结论 MRSA耐药机制复杂,呈现多重耐药,应根据药敏结果选用抗生素。     

Detection of methicillin resistance in staphylococci by using a DNA probe.

A DNA probe derived from the PBP 2a gene of the methicillin-resistant Staphylococcus aureus COL was compared with phenotypic microbiologic tests for its ability to identify methicillin-resistant and -susceptible staphylococci. Lysates were applied to nitrocellulose with a dot blot apparatus. Isolates tested were both S. aureus and coagulase-negative staphylococci that had been recovered from a variety of geographic and clinical sources. When compared with a spread plate phenotypic test, the DNA probe gave sensitivity, specificity, and predictive values for both positive and negative tests of 100% for 204 S. aureus isolates (103 positive, 101 negative) and 99, 95, 99, and 95%, respectively, for 249 coagulase-negative staphylococci (210 positive, 39 negative). The probe was more sensitive than broth microdilution and more specific than agar dilution in identifying methicillin-resistant and -susceptible coagulase-negative staphylococci; all tests were equally accurate in identifying the methicillin susceptibility of S. aureus. DNA probe analysis for determining the methicillin susceptibility of staphylococci was rapid, easily interpretable, and equally accurate with radioactive and nonradioactive probes, and it gave results equivalent to the most sensitive microbiologic test for all staphylococcus species studied.     

Evaluation of a semiquantitative C-reactive protein latex slide test as compared to quantitative turbidimetric measurement in hospital laboratory practice

Serum C-reactive protein (CRP) concentrations of 708 patients with suspected bacterial disease were estimated with a semiquantitative CRP latex slide test using cut-off levels of 20 and 40 mg/l and with quantitative turbidimetric technique. Latex slide tests gave 11.8% (80/708) discordant results. About 7% of tests gave clear disconcordant results (5% of those were positive and 2% negative tests). We conclude that CRP latex slide test is not useful in emergency laboratory in hospital material with a high incidence of bacterial infections.     

Detection of mecA-mediated resistance using reference and commercial testing methods in a collection of Staphylococcus aureus expressing borderline oxacillin MICs

Phenotypic methods for detecting mecA-mediated resistance in Staphylococcus aureus include both oxacillin and cefoxitin susceptibility tests; many laboratories perform multiple tests. Conflicting oxacillin and cefoxitin susceptibility results are most likely to occur for isolates that either have reduced susceptibility to oxacillin by a non-mecA-mediated mechanism or are mecA positive but are very heteroresistant. To understand the performance of oxacillin and cefoxitin tests for such isolates, we tested 135 S. aureus isolates using either cefoxitin or oxacillin and compared the results with mecA polymerase chain reaction. These strains either expressed borderline oxacillin MICs (1-4 microg/mL) and had undetermined mecA status or were mecA positive but were not detected by oxacillin broth microdilution (BMD) or disk diffusion (DD) in original testing. For 24-h readings, performance of cefoxitin tests (sensitivity/specificity) were DD (99/100), Etest using < or =6 microg/mL as susceptible (99/98), and Phoenix MIC using < or =4 microg/mL as susceptible (98/100). Using 6 microg/mL of cefoxitin as a screen test in both BMD and agar dilution also worked well (98/98-100). Sensitivity/specificity of oxacillin methods were oxacillin agar screen (BBL: 80/86; Remel, Lenexa, KS: 85/50), DD (91/59), BMD (85/88), MicroScan (89/96), VITEK Legacy (82/93), VITEK 2 (91/73), and Phoenix, (67/96). These results suggest that a cefoxitin test can be used alone to predict mecA-mediated resistance in S. aureus.