No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Comparison of coagulase, deoxyribonuclease (DNase), and heat-stable nuclease tests for identification of Staphylococcus aureus
Abstract
One thousand and thirty-five clinical isolates of the genus staphylococcus were used to compare the coagulase test with the deoxyribonuclease (DNase) and heat-stable nuclease tests as methods for identifying Staphylococcus aureus. Conflicting results were obtained with 65 isolates when the coagulase test was compared with the DNase test but with only one isolate when the coagulase test was compared with the heat-stable nuclease test. The heat-stable nuclease test produced reliable results after four hours' incubation and was considered a satisfactory substitute for the coagulase test in the clinical laboratory.
... In a recent edition of the Journal of Clinical Microbiology, Qian et al. described the sensitivity and specificity of the direct tube coagulase (DTC) test for the presumptive identification of Staphylococcus aureus directly from positive blood culture bottles from which Gram stains were consistent with staphylococci (7). The reported sensitivities of 34% at 2 h of incubation and 65% at 4 h of incubation (7) are considerably lower than those previously described and suggest an unacceptably high rate of false negatives (1,4,5,6,8,9,11). The authors draw the conclusion that despite its low sensitivity the DTC test is clinically valuable, particularly in experienced hands, because of its low cost and simplicity (7). ...
... However, Qian et al. neither evaluated nor commented on the use of the thermostable DNase test for the presumptive identification of S. aureus directly from positive blood culture bottles, as described by Madison and Baselski (4) and others (1,6,8,9,10). In these studies, the thermostable DNase test has been reported to be sensitive (85% to 100%) and specific (93 to 100%), to have a high degree of correlation with the DTC test (92.7 to 100%), to be relatively simple to perform, and to have low material cost (1,4,6,8,9,10). ...
... However, Qian et al. neither evaluated nor commented on the use of the thermostable DNase test for the presumptive identification of S. aureus directly from positive blood culture bottles, as described by Madison and Baselski (4) and others (1,6,8,9,10). In these studies, the thermostable DNase test has been reported to be sensitive (85% to 100%) and specific (93 to 100%), to have a high degree of correlation with the DTC test (92.7 to 100%), to be relatively simple to perform, and to have low material cost (1,4,6,8,9,10). In our laboratory, the material cost per test is 79¢, compared to 22 ¢ for the DTC test. ...
... human nucleases can be distinguished based on their nuclease activity profile [26], and both bacterial nuclease activity [27] and nuclease patterns [28] have already been used and proposed, respectively, to identify and discriminate between bacterial species. The use of bacterial nuclease activity blueprints as novel diagnostic biomarkers is particularly promising, and novel approaches are expanding its potential for the successful identification and characterization of clinically relevant bacterial pathogens. ...
... However, to overcome the specificity issues posed by the production of thermolabile extracellular nucleases by micrococci and coagulase-negative Staphylococci, such as Staphylococcus epidermidis [100], a derivative of the DNase test, known as the thermonuclease (TNase) test, that exploits the thermostability of MN was developed. TNase is not only more specific than the DNase test, but its accuracy has been shown to match that of the tube coagulase test (TCT) for the identification of S. aureus in food and clinical isolates of Gram-positive cocci [27,[101][102][103]. Moreover, it represents a simple, rapid (~ 2,5 hours) and inexpensive methodology for the detection at very low concentrations (5-10 ng/g -approx. ...
Introduction:
In the increasingly challenging field of clinical microbiology, diagnosis is a cornerstone whose accuracy and timing are crucial for the successful management, therapy, and outcome of infectious diseases. Currently employed biomarkers of infectious diseases define the scope and limitations of diagnostic techniques. As such, expanding the biomarker catalog is crucial to address unmet needs and bring about novel diagnostic functionalities and applications.
Areas covered:
This review describes the extracellular nucleases of 15 relevant bacterial pathogens and discusses the potential use of nuclease activity as a diagnostic biomarker. Articles were searched for in PubMed using terms: "nuclease", "bacteria", "nuclease activity" or "biomarker". For overview sections, original and review articles between 2000 and 2019 were searched for using terms: "infections", "diagnosis", "bacterial", "burden", "challenges". Informative articles were selected.
Expert opinion:
Using the catalytic activity of nucleases offers new possibilities compared to established biomarkers. Nucleic acid activatable reporters in combination with different transduction platforms and delivery methods can be used to detect disease-associated nuclease activity patterns in vitro and in vivo for prognostic and diagnostic applications. Even when these patterns are not obvious or of unknown etiology, screening platforms could be used to identify new disease reporters.
... Assays that identify S. aureus in blood cultures by measuring heat-denaturation resistant nuclease activity in positive blood cultures have been described over 50 years ago [11]. While various reports have since documented the value of this approach, nuclease activity in these studies was only measured after the bacteria were cultured [15][16][17]. ...
... Current microbiological laboratory methods for diagnosing SAB depend on an initial blood culturing step that takes a day or longer (e.g., see time-to-positivity values in S1 and S2 Tables). S. aureus is typically identified from agar-plated bacterial colonies (these are grown as 18-24 hour subcultures from positive blood culture bottles) with biochemical methods (e.g., measurement of coagulase activity [17]) that have been used for decades. Methods developed more recently for S. aureus identification include PCR-and mass spectrometry-based approaches that eliminate subculture on plates, but not broth-based blood culture. ...
S. aureus bacteremia (SAB) is a common condition with high rates of morbidity and mortality. Current methods used to diagnose SAB take at least a day, and often longer. Patients with suspected bacteremia must therefore be empirically treated, often unnecessarily, while assay results are pending. In this proof-of-concept study, we describe an inexpensive assay that detects SAB via the detection of micrococcal nuclease (an enzyme secreted by S. aureus) in patient plasma samples in less than three hours. In total, 17 patient plasma samples from culture-confirmed S. aureus bacteremic individuals were tested. 16 of these yielded greater nuclease assay signals than samples from uninfected controls or individuals with non-S. aureus bacteremia. These results suggest that a nuclease-detecting assay may enable the rapid and inexpensive diagnosis of SAB, which is expected to substantially reduce the mortality and morbidity that result from this condition.
... Work of several investigators has shown considerable disagreement over how well the DNase test predicts a clinical isolate's identity as S. aureus. Negative DNase results were seen with no more than 2% of the tube coagulase positive organisms (DNase "false-negatives") tested by several groups of investigators, 1,7,8,16 however, their incidences of tube coagulase negative organisms found to be DNase positive (DNase "falsepositives") showed considerable variation ranging from 3.5%' and 4.3% 16 to 13.5% 8 and 18.2%. 7 Two groups 1,8 tested Micrococcaceae and two groups 7,16 tested only members of the genus Staphylococcus, but high and low DNase false-positive rates were observed in both categories of test organisms. ...
... Negative DNase results were seen with no more than 2% of the tube coagulase positive organisms (DNase "false-negatives") tested by several groups of investigators, 1,7,8,16 however, their incidences of tube coagulase negative organisms found to be DNase positive (DNase "falsepositives") showed considerable variation ranging from 3.5%' and 4.3% 16 to 13.5% 8 and 18.2%. 7 Two groups 1,8 tested Micrococcaceae and two groups 7,16 tested only members of the genus Staphylococcus, but high and low DNase false-positive rates were observed in both categories of test organisms. Zarzour and Belle 16 used a methyl green indicator system which has been found to give more positive reactions 8 than the 1 N HC1 indicator system used by the other three groups, yet they had a low rate of false-positives. ...
Simultaneous testing for clumping factor, coagulase, deoxyribonuclease, and thermonuclease was performed on 189 clinical isolates of gram-positive cocci with strong catalase activity to determine the suitability of the thermonuclease test as a routine procedure for the identification of Staphylococcus aureus. Positive reactions to all four tests were exhibited by 72 of the strains while 88 of the isolates gave uniformly negative results. Although discrepancies were found between the reactions of 29 organisms, differences were found between the reactions of 29 organisms, differences between tube coagulase ant thermonuclease results were rare. Greater than 90% of positive reactions for both tube coagulase and thermonuclease tests were detected within a four-hour incubation period. The thermonuclease test was found to be simple, reliable, inexpensive and rapid. This test gave easily interpretable reactions within an eight-hour workday, even when only one or two isolated colonies were used for testing. The thermonuclease test is well suited for use as a primary clinical laboratory procedure for the identification of Staphylococcus aureus.
... MSSA and MRSA strains were included in this study if all of the following four criteria had been fulfilled: (i) typical morphology of colonies (Bannerman, 2003) on 5 % Columbia sheep blood agar, (ii) Gram stain showing Grampositive cocci in clumps, (iii) at least one positive reaction in the tube coagulase test evaluated after 2, 4 and 24 h (bioMérieux) or detection of the coagulase gene encoding free coagulase by genotyping using PCR and (iv) detection of the fibrinogen receptor gene encoding the clumping factor by PCR. As it is known that there are DNase-negative (Menzies, 1977) or catalase-negative (Tu & Palutke, 1976) S. aureus strains, and some S. aureus strains fail to grow on mannitol salt agar (Kampf et al., 1997), we did not exclude strains with these characteristics. ...
... aureus strains' in the CNS group. As DNase-positive CNS strains are documented (Menzies, 1977), as well as growth of CNS on mannitol salt agar (Bannerman, 2003), we did not exclude strains with these characteristics. ...
Most routine laboratory detection of Staphylococcus aureus isolates is based on rapid agglutination test systems. Failure of agglutination assays to identify meticillin-resistant S. aureus strains (MRSA) has been demonstrated. The aim of this study was to evaluate six commercially available agglutination tests for the detection of meticillin-sensitive S. aureus (MSSA) and mecA-positive MRSA strains. The Dry Spot Staphytect Plus test (Oxoid), the Pastorex Staph Plus test (Bio-Rad), the Slidex Staph-Kit and Slidex Staph Plus test (bioMérieux), the Staphaurex Plus test (Remel) and the Staphylase Test (Oxoid) were used. As determined by pulsed field gel electrophoresis, 52 distinct MRSA strains from five countries, 83 MSSA strains and 150 coagulase-negative staphylococci were included. Species identification and determination of susceptibility patterns were performed using colony morphology, Gram stain, catalase testing, tube coagulase testing, DNase testing, mannitol fermentation, susceptibility testing towards oxacillin by Etest, coagulase gene PCR, fibrinogen receptor gene PCR and PCR of the mecA gene. Sensitivity of the agglutination tests ranged from 82.7 to 100.0 % for MRSA strains and 92.8 to 100.0 % for MSSA strains, respectively. Specificity of the test systems ranged from 91.3 to 99.1 %. None of the six agglutination assays produced correct reactions for all staphylococci tested. Only the Dry Spot Staphytect Plus test correctly identified all 52 MRSA strains. For the other tests kits, sensitivity of MRSA detection was lower than for MSSA isolates. Depending upon the local MRSA prevalence and the parameter of interest (sensitivity or specificity), these test systems may be useful for routine diagnostic purposes.
... Coagulase and DNAse Production: It was detected by the method using ethylenediaminetetraacetic acid (EDTA) treated coagulase plasma by the formation of a clot after 1, 2, 4 or 24 h recorded as positive. During the investigation, 0-1 % DNA (BDH) was added to this medium to enhance the detection of DNase 32 . ...
Marine bacteria have the potentiality to produce diverse
bioactive molecules such as pigment. Therefore, it needs to exploit and
identifying a novel type of pigment from marine bacteria for Industrial
applications. This study aimed to investigate the marine bacterial pigment
against antioxidant and anti-cancer properties; the marine bacteria producing
pigment were isolated from water samples collected at the coastal of
Marakkanam (TN), India. The isolates were screened out based on the
growth characteristics and performance of different media and the strain
designated as MB4, which was taken as further studies. The strain MB4
characterized by SEM analysis showed that coccoid cell morphology,
nonsporulating, Gram-positive with yellow pigmentation and positive for
MR-VP, catalase, lipase, acetoin production, and hemolysis. The cells were
able to tolerate 10 percent NaCl concentration and ability to grown pH 9.
The MB4 strain was shown a higher wave-number (1395.77) cm-1 against
Raman Intensity to identify pigment production. The methanolic extracted
pigment was produced at a maximum peak at 260 nm. The yellow-pigmented
crude extract checked for anti-cancer properties using the colon cancer cell
line (HCT15), the cell viability has been reduced after treatment of the extract
(25-500 μg ml-1) and also exhibits IC50 value of 255.58 ± 43.51 mg ml-1
antioxidant DPPH radical scavenging activity. Due to their yellow pigment
productions which have antioxidant activity and anti-cancer properties, this
could be a novel pigment-producing strain for biomedical and industrial
applications.
... A control was executed without the addition of extracts or standards. Percentage scavenging and IC 50 value, which is the concentration of the sample required to scavenge 50% of the free radicals, was calculated [26]. ...
Elaeocarpus ganitrus is commonly known as Rudraksha exhibit a wide range of pharmacological activities which include anti-inflammatory, analgesic, and antimicrobial properties. Medicinal plants traditionally used against bovine mastitis are therapeutically active against bacterial pathogens. This study was aimed to evaluate the antimicrobial potential of E. ganitrus plant seed extracts against biofilm-forming Staphylococcus spp. E. ganitrus plant seed extracts were phytochemically screened for Alkaloids, Flavonoids, glycosides, and Saponins and checked for in vitro antibacterial activity at different concentrations against different Staphylococcus spp by agar well diffusion method. Silver nanoparticles (AgNPs) synthesized from the E. ganitrus were characterized by UV spectroscopy, Fourier Infrared Spectrometry (FTIR), Scanning Electron Microscopy (SEM), and Energy Dispersive X-ray analysis (EDAX). E. ganitrus extract showed positive results for flavonoids, tannins, reducing sugars and steroids and the absence of glycosides and alkaloids. UV spectroscopy, SEM, EDAX, and FTIR confirm the presence of AgNPs. The Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of methanol extract and AgNPs against fifteen Methicillin-Resistant Staphylococcus aureus (MRSS) samples were determined and found to significantly inhibit the activity against the tested bacterial strains. The Minimum Inhibitory effect was observed when the microbes were treated with the AgNPs extract (p-value-0.0001) when compared to the plant seed extract. E. ganitrus and AgNPs extracts were found to be potential against MRSS causing bovine mastitis infection and support the possible use of these phototherapeutic agents in the clinical management of the disease with low cost and fewer side effects.
... After culture onto blood agar medium, suspected colonies were identified using conventional biochemical tests and thermonuclease (nuc) gene amplification. Subsequently, the isolates were transferred into the trypticase soy broth medium containing 30% glycerol and kept at −20°C for further process (Chesneau and El Solh, 1992;Kateete et al., 2010;Menzies, 1977). ...
Background
Virulent strains of Staphylococcus aureus (S. aureus) express a series of virulence factors which cause severe infections such as skin and soft tissue infections which can be life-threatening. Additionally, extensive antibiotic resistance among nosocomial pathogens has left limited choices for their eradication. Our objective was investigation of antibiotic resistance and virulence determinants of S. aureus from skin infections.
Materials and methods
Two-hundred non-duplicate S. aureus isolates were collected from skin infections. Antibiotic susceptibility profile was evaluated using disc diffusion and methicillin resistance was confirmed. Biofilm formation was assessed by microtiter tissue plate assay. Determinants of virulence factors including hlA-α, tsst-1, pvl, PSM's, eta and etb genes and also the mupirocin resistance mup A gene and class I integron were detected by PCR. MLST was performed for isolates containing the pvl gene.
Results
The age range of patients included 12–76 years (mean ± SD = 56.34 ± 5.43). MRSA (70/200) were isolated significantly higher among ages>50 years (p < .001) but not significantly different between both genders (p = .112). Prior antibiotic consumption and hospitalization were significantly associated with MRSA (81.42%) and MDR (84.28%) isolation. The existence of int1 gene (44.5%) was significantly higher in multidrug-resistant (MDR) isolates (p < .001). Thirty-six (18%) MDR isolates carried the mupA gene. The existence of mupA gene was significantly associated with prior hospitalization (n = 33, 91.66%, p < .0001) and antibiotic consumption (n = 30, 83.33%, p < .001). Predominant virulence determinant included PSMα (61.5%), followed by tsst-1 (17.5%), hla-α (9.5%), pvl (2.5%), eta (2.5%) and etb (1%). The rate of strong biofilm producers (totally 25%) was not significantly different between MRSA and MSSA. The existence of PSM-α gene was significantly higher among strong biofilm producers compared to biofilm non-producers (p = .002). PFGE exhibited no genetic relation among strains.
Conclusion
Virulence factors of S. aureus from skin infections contributed in severity of infection and biofilm formation. MDR phenotype was more common among older patients with history of hospitalization and prior antibiotic consumption. Mupirocin resistance has emerged among MDR and MRSA isolates. Hence suitable control strategies must be performed to inhibit the spread of these strains in healthcare and community settings.
... Se empezó con la identificación mediante la observación de las características propias de la colonia (presencia y tipo de hemolisis, color y textura). Después se procedió a realizar las siguientes 3 pruebas bioquímicas: Prueba de coagulasa en tubo (Sperber y Tatini., 1975), prueba de sal manitol (Cervantes- García et al., 2014) y prueba de DNasa (Menzies, 1977). ...
The genetic variability of S. aureus strains isolated from some cases of bovine mastitis was determined. 335 cows from 27 stables were sampled in 10 municipalities in the state of Jalisco. S. aureus strains were identified from milk samples of each mammary gland of each cow, which were grown in blood agar and based on the characteristics of the culture, biochemical tests, and finally their molecular confirmation by PCR. The genetic variation in the strains was identified d by pulsed field electrophoresis technique. The images of the gels were analyzed using the Bionumerics® software. 2.26% of clinical mastitis and 40.45% of subclinical mastitis were diagnosed with the California test. A frequency of appearance of S. aureus of 9.8% of the total sampled glands was recorded. A genetic variation of 14.9% was observed. The 32 strains analyzed were grouped into pulsotypes with 95% or more of genetic similarity, resulting in 12 pulsotypes. It is concluded that there is great diversity in the genetic variability of S. aureus strains from different stables in the state of Jalisco, and that there is a great genetic similarity of strains within the stable
... Extracellular DNase activity can enhance the prevalence of pathogenic bacteria and increase the incidence of diseases. Menzies (1977), Gündoğan et al (2006) and Kateete et al (2010), have reported DNase activity levels of 98%, 94.5% and 75%, respectively, for S. aureus. These results agree with the DNase activity determined for S. aureus in the present study. ...
Aim: Mastitis is one of the most common diseases of dairy cattle and causes significant economic losses. Staphylococcus aureus produces many virulence factors that facilitate the adhesion and penetration of damaged tissues, and thereby, cause subclinical mastitis. This study was aimed at investigating the phenotypic and genotypic characteristics and antimicrobial susceptibility of S. aureus.. Materials and Methods: A total of 241 S. aureus strains isolated from bovine mastitis cases were tested phenotypically (catalase, coagulase, haemolysis, DNase, mannitol fermentation and biofilm formation) and genotypically (by the polymerase chain reaction (PCR) technique). Antimicrobial susceptibility was tested using 15 different antibiotics. Results: While the isolates showed different levels of haemolytic activity (β 47%, α 42%, γ 10% and δ 1%), only the β-haemolytic strains produced a positive CAMP-like reaction. Although all isolates were able to grow on MSA containing 7.5% NaCl, mannitol fermentation activity was observed in 80.5% of the isolates. The nuc gene was detected in all isolates, but only 84.2% of the isolates showed DNase activity. The Congo red agar method can be used to detect the biofilm forming capability of isolates, but the crystal violet staining method gives more reliable results. The sec gene was the most common en-terotoxin genes (84%). Three isolates harboured the mecA gene, but were sensitive to methicillin. Conclusion: Phenotypic variations among isolates result in the misclassification of S. aureus strains and require the use of molecular methods. The rapid and accurate molecular typing of S. aureus can aid in both determining the prevalence of this infectious microorganism and preventing epidemic infections.
... The plasma of different species i.e. rabbit, bovine, pig, human etc were used in coagulase reaction for the identification of S. aureus. It was noted that the mixture of pig and rabbit plasma improve the reliability as compared than either plasma 17 . Human plasma shows more sensitive than sheep plasma for the tube coagulase test (91% and 81%, respectively) but both had low specificity (11% and 7%, respectively). ...
Objective: To characterize different uropathogens and determine their antibiotic susceptibility pattern.
Material and Methods: In the present study, 300 patients with clinical symptoms were investigated from tertiary care
hospitals of Khyber Pakhtunkhwa from January 2015 to December 2015. Clean catch midstream urine of patients were
collected, leukocyte count was determined and were cultured. The recovered bacteria were identified using different
biochemical tests. Susceptibility to antimicrobial agents was determined using disc diffusion method of Kirby-Bauer.
Results: From a total of 300 samples, 28.6% were positive for both bacterial culture and pus cells while 9.6% and
15.6% samples were having only pus cells and bacterial culture respectively. Microorganisms recovered and identified
were E. coli (73%) followed by Citrobacter freundii (9%) Staphylococcus aureus (4%), Pseudomonas aeruginosa (4%),
Morganella morganii (3%), Proteus, Streptococcus spp. and Candida albicans (1% each), while 4% were identified as
normal flora of the urinary tract. The recovered microorganisms were mostly sensitive to tazocin and amikacin while
they showed resistance against norfloxacin and tobramycin.
Conclusion: E. coli was the most common uropathogen. There is a strong association between pus cells and UTI. The
susceptibility pattern of different uropathogens show increasing resistance to commonly used antimicrobial agents.
Key Words: Urinary tract infection, pus cells, uropathogens.
... Besides plasma coagulation and beta haemolysis, the presence of thermonuclease activity is often used as an additional marker for the identification of CoPS, particularly S. aureus (Menzies, 1977;Baird, 1996;Boerlin et al., 2003). All these tests have previously been used to identify atypical variants of S. aureus strains (Kloos and Schleifer, 1975;Heltberg and Bruun, 1984;Fox and Gay, 1993;Laevens et al., 1996;Matthews et al., 1997;Garbacz et al., 2002;Malinowski et al., 2009). ...
Avian leukosis virus (ALV) is an economically important poultry pathogen and its infection may result in low
productivity and mortality in chickens. Based on the viral envelope, glycoprotein antigenic structure, host range
and mutual interference among different strains, ALVs can be classified as 10 subgroups, A–J. Subgroups A–E and
J exist in chickens. ALV-A, B and J are the most common exogenous subgroups that cause chicken tumours,
whereas tumours caused by subgroups C, D appear to be rare. Subgroup E is an endogenous leukosis virus, which
has low or no pathogenicity to chickens directly, but studies have shown that chickens infected with ALV-E remained
viraemic with exogenous virus for longer time and developed neoplasm at a much higher frequency than did the
control chickens not infected with ALV-E. Liver samples (35 flocks) were collected from commercial broiler birds
which showed liver abnormalities like hepatomegaly, paleness, necrotic spots and haemorrhages. The liver samples
were processed and DNA was extracted and polymerase chain reaction PCR amplification of 466 bp glycoprotein
gene of ALV subgroup A–E was amplified. Out of 35 flocks, 27 flocks were found positive for ALV and sequence
Sequence analysis revealed that 94–96% homology with envelope gene of ALV. Phylogenetic analysis showed that
it belonged to ALV subgroup E. Eradication of ALV from primary breeding stocks is the most effective means to
control ALV infection.
... Some human strains of CNST also elaborate an extracellular DNAse, but these enzymes are heat labile (ie, they are inactivated by boiling for 15 minutes). 38 A variety of methods have been developed for detecting thermonuclease production by staphylococci. 38 4547 ' 48 One of the most convenient is the method of Barry et al, 47 in which a colony of staphylococcus is transferred to 0.5 mL brainheart infusion broth and incubated for two to six hours at 35 °C. ...
... The plasma of different species i.e. rabbit, bovine, pig, human etc were used in coagulase reaction for the identification of S. aureus. It was noted that the mixture of pig and rabbit plasma improve the reliability as compared than either plasma 17 . Human plasma shows more sensitive than sheep plasma for the tube coagulase test (91% and 81%, respectively) but both had low specificity (11% and 7%, respectively). ...
Objective: To evaluate performance (sensitivity & specificity) of a range of phenotypic tests which are currently used in tertiary care hospitals for the indentification of S aureus and to identify an optimal phenotypic test that is reliagle and cost effective and can be used with confidence for confirmation of S. aureus. Material and Methods: The present study was conducted at clinical microbiology laboratory department of Pathology of Northwest General Hospital and Research Centre, Peshawar from January 2012 to September 2013. The study group consisted of 300 samples were collected from different clinical sources i.e. patient’s blood, body fluids, pus swabs, wound swabs, urine and sputum. During sampling safety methods were adopted and data were collect regarding age, sex, types of specimens and present health condition were also recorded. Results: All clinical samples evaluated with slide coagulase (SCT), mannitol salt fermented test (MSA) and DNase test the result were 95%, 87% and 86%, respectively. Combination of these tests with different sera used in SCT their sensitivity results increased considerably. The human plasma when used in SCT test the positive results were 95%, but when used in combination with MSA test and DNase test then sensitivity increased to 98% and 96%, respectively. Similarly, while using horse plasma, 90% were slide coagulase positive and combination of SCT with mannitol test and DNase test depicted 94% and 91% positive results respectively. In similar manner, when cow plasma was used, slide Coagulase test showed 85% sensitivity and in combination with mannitol fermentation test and DNase test depicted 87% and 86% sensitivity, respectively. Conclusion: It is concluded that there is no single phenotypic test that can give 100% reliable identification of S. aureus and we have to use combination of tests for maximum reliable results. The combination of slide coagulase test with mannitol fermentation test provide the maximum reliable results while using human plasma followed by using horse and cow plasma.
... Among such recognisable exoproducts are DNAase, fibrinolysin, proteinase and lipase-esterase. Menzies (1977) examined 728 strains of coagulase-positive staphylococci and found that heatstable DNAase was produced by all of them whereas in a collection of 307 coagulase-negative staphylococci only one produced heat-stable DNAase. Nevertheless 56 out of 307 (1 8.2%) of the coagulase-negative staphylococci exhibited some DNAase activity. ...
... The plasma of different species i.e. rabbit, bovine, pig, human etc were used in coagulase reaction for the identification of S. aureus. It was noted that the mixture of pig and rabbit plasma improve the reliability as compared than either plasma 17 . Human plasma shows more sensitive than sheep plasma for the tube coagulase test (91% and 81%, respectively) but both had low specificity (11% and 7%, respectively). ...
... An aliquot of the blood culture broth was removed, and 5 l was inoculated directly into the BD Max StaphSR sample buffer tube (SBT), mixed by vortexing for 15 s, and then loaded onto the BD Max system for automated extraction, PCR amplification, and analysis. The results of molecular testing were compared with the gold standard of subculture onto solid medium (blood, chocolate, trehalose, DNase [10][11][12], mannitol salt, and Brilliance MRSA 2 [Oxoid, Hampshire, United Kingdom] agars) with 18 to 24 h of incubation at 35°C, species identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) (Bruker, Karlsruhe, Germany), and antimicrobial susceptibility using the PMIC-84 panel on the BD Phoenix II automated microbiology system. In addition, rapid rabbit plasma coagulase (Bio-Rad Laboratories, Hercules, CA) testing was performed directly on the blood culture broth sediment (12,13). ...
The BD MAX™ Staph SR assay is an automated qualitative
in vitro
diagnostic test for the direct detection and differentiation of methicillin-susceptible
S. aureus
(MSSA) and methicillin-resistant
S. aureus
(MRSA). 460 specimens were tested and the results compared with standard culture-based identification. MRSA was detected in 48 samples (sensitivity 100%; PPV 100%). MSSA was detected in 112 samples (sensitivity 99.1%; PPV 100%) and 299 samples containing coagulase-negative staphylococcus and non-staphylococcal species were negative by the BD MAX™ Staph SR assay (specificity 100%; NPV 99.7-100%).
... There is considerable interest in the use of TNase for detection of staphylococcal growth and the likely presence of enterotoxins in foods (13,14,25). The TNase assay, which takes 4 h, was also considered a satisfactory substitute for the coagulase test, which may take as long as 24 h in the clinical laboratory (18,22). ...
The activities of coagulase and thermostable nuclease (TNase) and the production of protein A were studied in 338 bacterial strains. These included 213 isolates of Staphylococcus aureus to determine which characteristic was most specific for the identification of S. aureus. The evaluation of different protocols for interpretation of coagulase results was also undertaken. Protein A was analyzed by a sandwich enzyme-linked immunosorbent assay using microtiter plates coated with anti-protein A antibodies. Coagulase activities were determined according to the criteria recommended by Association of Official Analytical Chemists (AOAC; any degree of clot formation is a positive reaction), American Public Health Association (APHA; coagulase activities ≥ 3+ are positive reactions), and the Bacteriological Analytical Manual (BAM; only 4+ reaction is positive). It was found that the AOAC protocol, which had a test sensitivity of 97.7% and a specificity of 95.1% and could be completed within six hours, was more practical than the methods used by APHA and BAM. Compared with coagu1ase and TNase, protein A was a better marker of S. aureus; a high sensitivity (100%) and specificity (96.8%) were obtained by using protein A for the identification of S. aureus.
... 13 The thermostable nuclease test, which detects the presence of deoxyribonucleases, provides high sensitivity and specificity but is not widely used in the clinical setting because of time and manipulative constraints. 10 Towards this end, promising results have been reported for the coagglutination procedures employing either sheep red blood cells (SRBCs) or latex particles variously sensitized to react with specific cell components of S. aureus, such as protein A and clumping factor. 5,9 Although protein A content varies considerably in strains of S. aureus, both specificity and sensitivity of more than 98% have been reported for approaches using SRBCs sensitized with anti-SRBC IgG to detect protein A by reaction with the F c portion of IgG. 6 Similarly, although clumping factor reactivity varies considerably among S. aureus strains, sensitivities and specificities of more than 98% have been reported for procedures using SRBCs sensitized with fibrinogen to detect clumping factor. 2 Comparable results also have been described for the use of latex particles sensitized with both IgG and fibrinogen by plasma coating so as to detect both protein A and clumping factor. ...
Three commercial coagglutination tests--Sero-STAT, Accu-Staph, and Staphyloslide--were performed in parallel with slide coagulase, tube coagulase, and thermostable nuclease tests on 100 methicillin-susceptible Staphylococcus aureus (MSS) strains, 100 methicillin-resistant S. aureus (MRS) strains, and 100 non-S. aureus staphylococcal strains (NSA). All three coagglutination tests showed sensitivities of 100% for MSS strains. For MRS strains, sensitivities were, respectively, 99%, 100%, and 99%. False-positive reactions were, respectively, 10%, 2%, and 2%. A marked difference in slide coagulase test sensitivity was found for MSS strains (79%) and MRS strains (14%). These findings suggest that the coagglutination tests may be less sensitive for detecting MRS strains than for detecting MSS strains and that these properties may be related to clumping factor reactivity. The high false-positive rate for Sero-STAT and even the 2% false-positive rate for Accu-Staph and Staphyloslide make clinical usefulness at this time somewhat problematic and debatable. In view of these findings, the authors prefer to retain the tube coagulase test and thermostable nuclease test for differentiation of S. aureus from non-S. aureus strains in their laboratory.
... When suitable reagents are used and appropriate test logistics are adhered to, the tube coagulase test has been found to be an acceptably accurate, technically nondemanding method for identifying S. aureus (1). Even under optimum conditions, however, rare strains of Staphylococcus other than S. aureus have been shown to possess tube coagulase activity (8,9,11,12). Furthermore, a small number of clinical isolates of S. aureus have been found to lack tube coagulase activity (5, 7). ...
286 clinical isolates of staphylococci and 19 Micrococcus spp. were tested in a new latex agglutinating test to detect bound coagulase and protein A simultaneously. Coagulase-positive strains of staphylococci (n = 119) were all found to be latex-positive. Negative latex agglutination test results were obtained with 154 out of 167 coagulase-negative strains, the other 13 (7.8%) strains gave positive latex tests. Furthermore 3 out of 19 Micrococcus strains yielded positive agglutinating results. According to the results presented a negative latex test allows a rapid exclusion of S. aureus. A positive latex test requires the determination of further typical characteristics to differentiate among the staphylococcal isolates.
Ältere Menschen sind gegenüber invasiven Infektionen und Sepsis besonders vulnerabel mit ungünstiger Prognose. Staphylococcus aureus und Haemophilus influenzae können beide invasive Infektionen verursachen. Oft geht eine asymptomatische Besiedelung einer Infektion voraus und ist ein Risikofaktor für eine invasive Infektion. Daher wurde eine bizentrische Querschnittstudie in den Regionen Aachen und Würzburg durchgeführt, um die Prävalenz von H. influenzae, S. aureus und MRSA (Methicillin resistenter S. aureus) bei asymptomatischen Senioren zu bestimmen, wie auch Risikofaktoren für eine Besiedelung. Von Oktober 2012 bis Mai 2013 wurden 677 Erwachsenen im Alter von 65 Jahren oder älter eingeschlossen, die zu Hause oder in Seniorenheimen lebten. Die Prävalenz von H. influenzae bei älteren Menschen war mit einer Trägerrate von nur 1,9% ([95% CI: 1,0 - 3,3%]; 13/677) sehr niedrig. Trägerisolate waren überwiegend nicht typisierbare H. influenzae, zeigten eine hohe clonale Diversität und waren alle Ampicillin-sensibel. Die Prävalenz von S. aureus war mit 28,5% ([95% CI: 25,1 - 32,1%]; 193/677) hoch, wie für die deutsche Allgemeinbevölkerung bekannt, während MRSA bei weniger als 1% der Teilnehmer gefunden wurde (0,7% [95% CI: 0,2 - 1,7%]; 5/677). Die Prävalenz von H. influenzae, S. aureus und MRSA unterschied sich nicht signifikant zwischen selbständig zu Hause lebenden Senioren und Pflegeheimbewohnern. Ältere, selbständig lebende Menschen mit höherem Bildungsniveau hatten signifikant höhere Kolonisierungsraten mit S. aureus (adjusted OR: 1,905 [95% CI: 1,248 - 2,908]; p = 0,003). Bei Pflegeheimbewohnern war eine Kolonisierung signifikant mit Verheiratet sein assoziiert (adjusted OR: 3,367 [95% CI: 1,502 - 7,546]; p = 0,003). Diese Ergebnisse unterstreichen die Bedeutung von sozio-demographischen Faktoren für eine Kolonisierung mit S. aureus und schließen eine Lücke bei epidemiologischen Daten zu H. influenzae.
Introduction: Staphylococcus aureus bacteremia causes significant morbidity and mortality, mainly by methicillin-resistant S. aureus (MRSA). Currently, vancomycin is the main choice for the treatment of infections by MRSA. Broth microdilution (BMD) remains the gold standard for measuring vancomycin MIC. However, most clinical laboratories employ practical methods in the routines, but these methods may not determine accurate vancomycin MIC values. Objectives: This study aimed to evaluate the accuracy of VITEK®2, Phoenix® and Etest® methods against BMD. Materials and Methods: A total of 78 strains (27 methicillin-sensitive S. aureus and 51 MRSA) were isolated from bloodstream infections. The vancomycin MIC was determined following CLSI and the manufacturers' recommendations. We also performed SCCmec typing, in order to identify their vancomycin MIC ratio values. Results: Most of all isolates showed values of MIC = 1 μg/mL by BMD and Phoenix®, while Etest® and VITEK® 2 determined the majority with MIC = 1.5 and 0.5 μg/mL, respectively. Thus, Etest® and VITEK® 2 tended to overestimate and underestimate, respectively, the MIC values. Three MRSA isolates that were vancomycin susceptible by the BMD were vancomycin-intermediate by Etest®. The SCCmec II (39%) and IV (51%) were the most frequent, and there was no relationship between the type of SCCmec and the MIC values. Conclusions: The results showed that vancomycin MICs vary according to the test method. It is essential that clinicians consider the differences in MIC results determined by different methods, since the MIC value is generally the parameter used by clinicians to select the appropriate therapy.
Background
Virulent strains of Staphylococcus aureus (S. aureus) express a series of virulence factors which cause severe infections such as skin and soft tissue infections which can be life-threatening. Additionally, extensive antibiotic resistance among nosocomial pathogens has left limited choices for their eradication. Our objective was investigation of antibiotic resistance and virulence determinants of S. aureus from skin infections.
Materials and methods
Two-hundred non-duplicate S. aureus isolates were collected from skin infections. Antibiotic susceptibility profile was evaluated using disc diffusion and methicillin resistance was confirmed. Biofilm formation was assessed by microtiter tissue plate assay. Determinants of virulence factors including hlA-α, tsst-1, pvl, PSM's, eta and etb genes and also the mupirocin resistance mup A gene and class I integron were detected by PCR. MLST was performed for isolates containing the pvl gene.
Results
The age range of patients included 12–76 years (mean ± SD = 56.34 ± 5.43). MRSA (70/200) were isolated significantly higher among ages>50 years (p < .001) but not significantly different between both genders (p = .112). Prior antibiotic consumption and hospitalization were significantly associated with MRSA (81.42%) and MDR (84.28%) isolation. The existence of int1 gene (44.5%) was significantly higher in multidrug-resistant (MDR) isolates (p < .001). Thirty-six (18%) MDR isolates carried the mupA gene. The existence of mupA gene was significantly associated with prior hospitalization (n = 33, 91.66%, p < .0001) and antibiotic consumption (n = 30, 83.33%, p < .001). Predominant virulence determinant included PSMα (61.5%), followed by tsst-1 (17.5%), hla-α (9.5%), pvl (2.5%), eta (2.5%) and etb (1%). The rate of strong biofilm producers (totally 25%) was not significantly different between MRSA and MSSA. The existence of PSM-α gene was significantly higher among strong biofilm producers compared to biofilm non-producers (p = .002). PFGE exhibited no genetic relation among strains.
Conclusion
Virulence factors of S. aureus from skin infections contributed in severity of infection and biofilm formation. MDR phenotype was more common among older patients with history of hospitalization and prior antibiotic consumption. Mupirocin resistance has emerged among MDR and MRSA isolates. Hence suitable control strategies must be performed to inhibit the spread of these strains in healthcare and community settings.
Multidrug-resistant Staphylococcus aureus, including MRSA (Methicillin-Resistant) and VRSA (Vancomycin-resistant), causes serious healthcare-associated infections, even sepsis and death. Here, we identified six novel cathelicidins (CATHPb1-6) from Python bivittatu, and CATHPb1 displayed the best in vitro pharmacological and toxicological profile. We further show that CATHPb1 exhibited evident protection in mice MRSA/VRSA infection models, either given 24h before or 4h after infection. The protection was all effective through different administration routes, but was blocked by in vivo depletion of monocyte/macrophages or neutrophils. CATHPb1 can rapidly and massively modulate macrophages/monocytes and neutrophils trafficking to the infection site, and potentiate their bactericidal functions. Meanwhile, CATHPb1 remarkably augmented neutrophil-mediated bacteria killing by facilitating neutrophil extracellular traps (NETs) formation and preventing its degradation. Acting through MAPKs and NF-κB pathways, CATHPb1 selectively enhanced the levels of chemokines while reducing the production of pro-inflammatory cytokines without undesirable toxicities. The much improved serum half-life and stabilities confer CATHPb1 an excellent prospect to become a novel therapeutic agent against multidrug-resistant staphylococcal infections.
Staphylococcus aureus is a nosocomial pathogen that resides in the soft tissues causing many diseases. The current study was conducted to determine the prevalence of Methicillin Resistant S. aureus (MRSA) in ear discharge and pus of patients and antibacterial activity of crude methanolic extract (Cr. MeOH Ext.) and various fractions of M. Africana and V. agnus castus against clinical isolates of MRSA. A total of 40 samples were collected from ear, nose and throat (ENT) outpatient department and wards of Khyber Teaching Hospital (KTH), Peshawar. Out of 40 samples, 36 (90%) samples showed growth on Mannitol Salt Agar (MSA) media out of which 9(25%) were MRSA and the remaining 27(75%) were methicillin susceptible S. aureus (MSSA). A good antibacterial activity was observed for the Cr. MeOH Ext. (76.1%) and ethyl acetate (EtOAc) fraction of V. agnus castus against S11 (71.4%). The n-hexane fraction also showed good antibacterial effect (70%) against S26. The chloroform (CHCl3), butanol (BuOH) and aqueous fractions of M. africana showed good antibacterial activity against S11 (71.4%), S32 (70%) and S26 (75%), respectively. The above results revealed that the selected plants can be further utilized for isolation of the active ingredients as the crude extracts were found good for inhibition of MRSA.
The variation in coagulase enzyme production and clotting blood plasma by methicillin resistant (MRSA) and sensitive (MSSA) strains of Staphylococcus aureus was studied after growing them in various culture media (brain heart infusion broth, tryptone phosphate broth and peptone broth) and analyzing with different blood plasma (rabbit, sheep, pig and human). High number of test MRSA and MSSA isolates from clinical specimens and carrier samples had maximum coagualse activity when grown in brain heart infusion broth. Similarly, more than 90% of the test isolates could clot human plasma preferably. Determination of coagulase production by S. aureus may be a rapid and cost effective method of identifying these pathogens from clinical specimens and carrier samples.
Decades ago, neutrophil granulocytes have been recognized as professional phagocytes. In their granules they store a massive array of antimicrobial enzymes and peptides which they can release either to the outside or into the phagosome, where phagocytosed microorganisms are quickly killed. Some years ago a different antimicrobial function of neutrophils was discovered: once stimulated, neutrophils can undergo a cell death program that induces massive structural changes and finally leads to the formation of Neutrophil Extracellular Traps (NETs), which can bind and kill microorganisms outside the cell. In this review, the current knowledge about antimicrobial properties of NETs is summarized and microbial strategies to escape NETs are discussed.
RESUME Objectif : Le coût élevé du plasma lyophilisé de lapin utilisé dans le cadre de l'identification des souches de Staphylococcus aureus constitue une des raisons d'inadéquation entre les dépenses et les recettes des laboratoires d'analyses biomédicales. Pour contourner cette difficulté, les techniciens de laboratoire ont recours à des méthodes d'identification moins coûteuses. La présente étude a eu pour objectif de déterminer dans un but économique le plus petit volume de plasma de lapin capable de révéler la présence de la staphylocoagulase libre. Méthodologie et résultat : Pour ce faire, 25 souches de Staphylococcus aureus isolées de différents échantillons biologiques et identifiées sur la base de la présence de la staphylocoagulase libre ont été utilisées. Ces souches ont été soumises à la recherche de la staphylocoagulase libre avec des volumes décroissants de 0,4 ml ; 0,3ml ; 0,2ml et 0,1ml de plasma lyophilisé de lapin. Toutes les souches bactériennes ont montré un résultat positif en 24 heures avec 0,2 ml comme plus petit volume. Le coagulum formé présente la même consistance que celui obtenu avec le volume de 0,5 ml indiqué par le protocole normal. Conclusion et application de résultats: Au total, le volume de plasma de lapin peut-être donc réduit de 0,5 à 0,2 ml sans aucun impact sur la qualité du résultat. Dans le contexte de pays moins avancé comme le Bénin, cela est un premier jet en vue de l'efficience des méthodes de diagnostic au laboratoire d'analyses biomédicales. Mots-clés : staphylocoagulase libre, plasma de lapin, efficience. ABSTRACT Objective: The high cost of lyophilized rabbit's plasma, which is used for the identification of Staphylococcus aureus, is one of the reasons of inadequacy between income and expenses of biomedical analysis laboratories. To circumvent this difficulty, laboratory technicians resort to less expensive methods of identification. This work was done to determine the lowest volume of rabbit's plasma that can reveal the presence of free staphylocoagulase.
369 staphylococcal strains isolated from clinical material were examined for tubeagglutination with sensitized sheep red cells in a standarized assay to study its reliability for routine identification of S. aureus. Colonies isolated from blood agar plates correlated in 99.5% with the (optimized) coagulase reaction. The test is easily to perform and results can be read after 2 hours, whereas the reference methods coagulase, hyaluronidase and deoxyribonuclease took as much as 24 h. The reliability of these tests is discussed.ZusammenfassungAn 369 aus klinischem Einsendematerial isolierten Staphylokokkenstämmen wurde ein standardisierter Protein-A-Hämagglutinationstest (PAHT) auf seine Verwendbarkeit für die routinemäßige Erfassung von S. aureus-Spezies überprüft. Dabei sollte insbesondere untersucht werden, ob der Test auch ohne Zwischenschaltung optimierender Kulturmedien eine gute Korrelation mit den konventionellen Reaktionen Plasmakoagulase, Hyaluronidase und Desoxyribonuclease zeigt.Nach Anzüchtung der zu untersuchenden Stämme auf einer Blutagarplatte stimmte der PAHT in 99,5% mit der (optimierten) Koagulasereaktion überein. Der PAHT hat gegenüber den oben genannten Methoden den Vorteil der schnelleren Ablesbarkeit und eignet sich gut für das Mikrotiterplatten-Verfahren. Die Verläßlichkeit der konventionellen Methoden wird diskutiert.
Objective: To report the isolation and characterization of four coagulase-negative methicillin-resistant Staphylococcus aureus isolated from patients admitted to the ICU of a Kuwait hospital by bacteriological and molecular methods. Methods: The isolates were characterized by cultural characteristics, Gram stain, catalase, coagulase, DNase and biochemical tests and typed by a combination of antibiogram and pulsed-field gel electrophoresis to evaluate their relatedness. Results: These isolates were gram-positive cocci in clusters, catalase, DNase and slide coagulase-positive but tube coagulase-negative. They gave negative results for ornithine decarboxylase and pyrrolidonyl-arylamidase tests, which indicated that these were not Staphylococcus lugdunensis or Staphylococcus schleiferi, and were identified as S. aureus by API Staph. They had identical antibiograms and pulsed-field gel electrophoresis patterns which suggested that they had a common origin. Conclusion: The results highlight the need to complement the coagulase tests with other tests such as DNase and biochemical tests to correctly identify S. aureus. Coagulase-negative S. aureus appears to be an increasing problem that clinical laboratories should be aware of. They are as virulent as those producing coagulase and can colonize, cause infections and spread among patients.
Bacteriological analysis of milk samples from quarters of a dairy cow suffering from subclinical mastitis yielded two isolates of Staphylococcus aureus which gave a negative reaction in the standard coagulase test. Both isolates were also clumping factor and thermonuclease negative, and gave a negative reaction in the Staphaurex® test. The isolates were identified by using commercial biochemical systems, and by PCR analysis of different staphylococcal cell surface protein and exoprotein genes. Further molecular identification of the isolates, which included sequencing of the 16S rRNA gene and RT-PCR of coagulase (coa), clumping-factor (clfA) and thermonuclease (nuc) genes, was consistent with the diagnosis phenotypically 'coagulase-negative variant of Staph. aureus'. The fact that coagulase-negative Staph. aureus variants can occur in the context of intramammary infections in cattle may result in the incorrect diagnosis 'coagulase-negative staphylococci (CNS)' in routine mastitis diagnostic, at least in rare cases. To fully ensure correct species diagnosis, sequencing of the 16S rRNA gene and amplification of specific genes such as coa is necessary in these cases.
As key players in the host innate immune response, neutrophils are recruited to sites of infection and constitute the first line of defense. They employ three strategies to eliminate invading microbes: microbial uptake, the secretion of antimicrobials, and the recently described release of Neutrophil Extracellular Traps (NETs). Composed of decondensed chromatin and antimicrobial proteins, NETs bind and kill a variety of microbes including bacteria, fungi, and parasites. In addition to using a repertoire of known antimicrobials, NETs incorporate histones into the antimicrobial arsenal. Furthermore, NETs may contribute to microbial containment by forming a physical barrier and a scaffold, to enhance antimicrobial synergy while minimizing damage to host tissues. Their role in innate immunity is only now being uncovered.
Strains isolated from pigeons belonging to the coagulase-positive species Staphylococcus intermedius, coagulase-negative Staph. hyicus subsp. chromogenes strains from cattle and pigs, and Staph. aureus strains from poultry, gave weakly positive reactions in DNase plate culture tests and heat-resistant DNase tests. Staph. aureus and Staph. intermedius strains from other sources and coagulase-negative and coagulase-positive Staph. hyicus subsp. hyicus strains reacted strongly in these tests. A standardized plate culture test procedure is proposed and the use of DNase tests in the identification of staphylococci isolated from animals is discussed.
The speciation of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant diagnostic problem when rapid identification methods such as slide agglutination tests, are used, because of the high proportion of false-negative reactions. 150 perfectly identified MRSA strains were tested on 5 commonly used agglutination reagents ("Bacto staph latex test", "Monostaph", "Pastorex staph", "Staphaurex", and "Staphyslide test") in comparison with a new micromethod ("RAPIDEC staph") which detects a type of staphylocoagulase within 2 hours by a fluorescence test. The "RAPIDEC staph" reagent enabled identification of all the MRSA while the agglutination tests gave poorer results: "Monostaph" correctly identified 64.6% of strains, "Staphyslide", 59.3%, "Bacto staph latex test", 44.6%, "Pastorex staph", 38.6% and "Staphaurex", 28.6%. These results show that agglutination slide tests are not reliable enough for the identification of MRSA which are more and more encountered in hospital wards. The authors recommend not to use slide agglutination methods. They suggest the tube test for coagulase which is the reference technique, although it is time-consuming and not well standardized. The results of this evaluation encourage the use of the "RAPIDEC staph" reagent since it is an easy-to-use, reliable technique for the rapid identification of Staphylococcus aureus.
The detection of thermonuclease by the Oxford strain and eight clinical isolates of Staphylococcus aureus in a variety of bacteriological broths with and without added blood was examined using a toluidine blue-DNA-agar plate method. In Isosensitest, brain-heart infusion, tryptic soy, nutrient and gas-liquid chromatography broths (all of which do not contain liquoid) thermonuclease detection was uncomplicated. In Bactec broths (containing liquoid) detectable thermonuclease activity was greatly reduced in the absence of blood. The addition of 10% blood to the Bactec broths restored the activity. Liquoid was shown to be responsible for the inhibition of thermonuclease activity, and its effect could be neutralised by the addition of blood, albumin, or haemoglobin. In specimens containing no blood, or insufficient blood to neutralise the liquoid in culture broths, more has to be added to prevent false negative reporting of S aureus. This can be done after growth at the time of thermonuclease testing. Clinical consequences of delayed identification of S aureus in routine blood cultures may be serious. The application of the thermonuclease test to blood culture broths is both fast and specific.
A rapid latex agglutination test, Staphaurex, was tested for its ability to identify Staphylococcus aureus using 72 reference strains and 785 clinical isolates of the family Micrococcaceae. All reference strains of Staphylococcus aureus were Staphaurex-positive. Non-Staphylococcus aureus reference strains were negative. Using clinical strains, the results of the Staphaurex test were compared with the results of other tests commonly used to identify Staphylococcus aureus. A total of 393 clinical isolates were classified as Staphylococcus aureus. The Staphaurex, slide coagulase, tube coagulase/human plasma and tube coagulase/rabbit plasma tests correctly identified 98%, 93.6%, 93.6% and 97.5% of the Staphylococcus aureus strains, respectively. The performance of the Staphaurex test, in terms of sensitivity and specificity, was significantly better than the slide coagulase test. It was as sensitive and almost as specific as the tube coagulase rabbit test and more sensitive than the tube coagulase human test.
Four rapid latex agglutination assays, StaphAurex (Wellcome Diagnostics, Research Triangle Park, N.C.), Bacto Staph (Difco Laboratories, Detroit, Mich.), SeroSTAT (Scott Laboratories, Inc., Fiskeville, R.I.), Veri-Staph (Zeus Technologies, Raritan, N.J.), and two hemagglutination tests, Staphyloslide (BBL Microbiology Systems, Cockeysville, Md.) and Hemastaph (Remel, Lenexa, Kans.), were compared with the conventional slide coagulase, tube coagulase (TC), and thermonuclease (TNase) tests for the identification of Staphylococcus aureus. A total of 118 clinical isolates of S. aureus (52 methicillin resistant), 50 S. epidermidis, 5 S. capitis, 2 S. hominis, 3 S. simulans, 6 S. saprophyticus, and 2 S. warneri were tested. The slide coagulase, TC and TNase tests detected 115 (97.5%), 117 (99.2%), and 118 (100%) of the S. aureus isolates, respectively. All showed 100% specificity. The StaphAurex, Veri-Staph, Staphyloslide, Hemastaph, SeroSTAT, and Bacto Staph assays correctly identified 117 (99.2%), 117 (99.2%), 116 (98.3%), 110 (93.2%), 108 (91.5%), and 107 (90.7%) of the S. aureus isolates, respectively. For methicillin-resistant S. aureus isolates, StaphAurex, Veri-Staph, Staphyloslide, Hemastaph, SeroSTAT, and Bacto Staph showed 1 (2%), 1 (2%), 2 (4%), 7 (13.5%), 7 (13.5%), and 8 (15.4%) false-negative results, respectively. All the commercial agglutination assays demonstrated false-positive results with strains of S. capitis, S. saprophyticus and S. warneri. The overall accuracy of the commercial agglutination assays compared with TC and TNase ranged from 90.7 to 99.2%. We recommend that negative reactions with the rapid commercial test kits for methicillin-resistant Staphylococcus isolates be confirmed with the TC or TNase test.
Several commercial systems are available to distinguish between Staphylococcus aureus and the coagulase-negative species of the Micrococcaceae family. Four latex agglutination systems (Accu-Staph, SeroSTAT, Staphaurex, and Staphylatex) and two hemagglutination systems (Hemastaph and Staphyloslide) were compared for their performance in the rapid identification of 232 isolates of staphylococci, including 114 of methicillin-resistant S. aureus. Accu-Staph, Staphaurex, and Staphyloslide correctly identified 100% of the methicillin-resistant S. aureus isolates; Hemastaph and Staphylatex, 99.1%; and SeroSTAT, 94.7%. Most reactions were easy to interpret, although 15% of the SeroSTAT reactions were weak. Autoagglutination occurred only with isolates of coagulase-negative staphylococci. False-positive reactions were rare and occurred only with systems which did not detect autoagglutination. Five of these six systems appear to be adequate for the rapid identification of S. aureus, including methicillin-resistant isolates.
A rapid, in situ thermonuclease test that identifies colonies of Staphylococcus aureus among staphylococci isolated from swimming pool water by membrane filtration recovery on various selective and differential media is described.
A latex agglutination test (SeroSTAT Staph; Scott Laboratories, Fiskeville, R.I.) and two hemagglutination tests (Staphyloslide; BBL Microbiology Systems, Cockeysville, Md.; and Hemastaph; Remel, Lenexa, Kans.) were compared with the slide coagulase (SC) and tube coagulase (TC) tests at room temperature (22 to 25 degrees C) and at 37 degrees C for the rapid identification of Staphylococcus aureus. A total of 380 clinical strains of staphylococci were tested. The TC test performed at room temperature yielded the largest number of TC-positive results (n = 239), and based on this observation 239 organisms were classified as S. aureus and 141 were classified as non-S. aureus. The SC, TC (37 degrees C), SeroSTAT Staph, Staphyloslide, and Hemastaph tests correctly identified 210 (87.9%), 221 (92.5%), 238 (99.6%), 239 (100%), and 236 (98.7%) of the S. aureus isolates, respectively. Of the S. aureus isolates that were TC positive at room temperature 68% required 24 h of incubation before coagulase production was detected. There was one false-negative SeroSTAT Staph result and one false-negative Hemastaph result. The Staphyloslide test yielded two noninterpretable results (both organisms were later confirmed as non-S. aureus), whereas there were six noninterpretable results recorded with the Hemastaph test (four organisms were classified as non-S. aureus, and two were classified as S. aureus). The SeroSTAT Staph, Staphyloslide, and Hemastaph tests were all more sensitive than the conventional SC and TC (37 degrees C) tests and were considerably more rapid than the TC test at either temperature.
The detection of thermonuclease activity in 86 blood culture samples containing gram-positive cocci showed 100% correlation with the subsequent identification of the isolate as Staphylococcus aureus by the coagulase test. No positive thermonuclease results were found with 66 samples containing coagulase-negative staphylococci and 56 samples containing other gram-positive organisms. The thermonuclease test provides a rapid, reliable method to identify S. aureus in blood cultures.
Two coagulase-variant forms of Staphylococcus aureus were isolated from blood cultures of a patient with infective endocarditis. The coagulase-positive isolate was hemolytic, whereas the coagulase-negative isolate was nonhemolytic. All other properties examined were identical in both strains. Since coagulase-negative S. aureus strains have been isolated from clinical specimens, laboratories should consider using a combination of other biological properties along with coagulase production for the identification of S. aureus.
A total of 57 bacterial strains were isolated from tooth surfaces, oral mucosa, skin of the upper lip, and rectum of 3 persons. Identification of the strains indicated that each type of surface had a characteristic microflora. Aggregation of bacterial suspensions, induced by salivary agglutinins, was measured spectrophotometrically as the decrease in optical density (OD) by time. The aggregation curves for the oral strains followed a sigmoid pattern. The aggregation rates varied between individuals and strains. Most fecal strains showed an aggregation pattern which differed from that of the oral strains and was characterized by only a small, initial decrease in OD. The few strains, mainly Propionibacterium strains, collected from the skin of the upper lip did not aggregate. It appears from the data that several mechanisms are involved in the retention of bacteria to surfaces.
THE INFLUENCE of seasonal and weather variations on recovery of Staphylococcus aureus in patients having boil infections was investigated. Recovery of the organ ism from patients was found to be higher during the dry season when there was acute shortage of water and little or no rainfall. The results showed that as the rainy season begins, prevalence of coagulas-e positive Staphylococci from the boil infections reduced con siderably. The difference in recovery rate of the causa tive agent of boil was Statistically significant (P<0.001) between the dry and the rainy seasons. Therefore lower relative humidity coupled with higher temperature and shortage of water are the most important environmen tal factors predisposing patients to Staphylococci boil infections.
A rapid method utilizing Kirby-Bauer susceptibility plates was developed to determine bacterial tolerance to antibiotic bactericidal activity. After completion of initial antibiotic disk susceptibility testing, the disks containing cephalothin, cefazolin, nafcillin, oxacillin, and methicillin were removed and replaced with disks containing a potent beta-lactamase. The plates were reincubated for 18-24 hours and examined for regrowth of organisms within the original zone of inhibition. For 15 of 16 patients who had serious Staphylococcus aureus infections, the method correlated with clinical outcome of antibiotic chemotherapy. Broth dilution tests for bactericidal activity only correlated with clinical response for 11 of 16 patients. One hundred consecutive clinical S. aureus isolates tested with the new method demonstrated tolerance in 27% of strains to cephalothin, 15% to cefazolin, 1% to oxacillin, and 2% of nafcillin.
A latex slide agglutination test detecting clumping factor and protein A simultaneously is recommended for rapid and reliable routine identification of Staphylococcus aureus. Strains (836) of staphylococci isolated from clinical specimens were examined, all S. aureus strains identified by conventional methods were correctly differentiated by the latex test, and no false-positive results occurred with other staphylococci. The reagent is easy to prepare since plasma is the coating material.
A new, commercially available latex agglutination test (SeroSTAT Staph; Scott Laboratories, Inc., Fiskeville, R.I.) was compared with the tube coagulase and slide coagulase tests as means for identifying Staphylococcus aureus. Of 160 clinical isolates of S. aureus, 159 (99.4%) yielded positive results with the latex agglutination test. Negative latex agglutination test results were obtained with 266 of 267 clinical isolates of Micrococcus spp. and staphylococcal species other than S. aureus (99.6%). The latex agglutination test was found to be a rapid, technically nondemanding method for identifying S. aureus. It was as accurate as the tube coagulase test and more accurate than the slide coagulase test.
In an era characterized by increasing emphasis on minimizing laboratory costs, reliable and cost-effective methods for rapidly identifying bacteria and fungi directly from blood cultures have a great deal of appeal to clinical microbiologists. A variety of methods have been evaluated and found to be useful under certain conditions, although none of the methods has been standardized and questions remain as to whether their use improves patient care or reduces hospital costs. Even if these methods do not improve patient care or reduce hospital costs, their use and expense could be justified if they improve laboratory work flow or decrease laboratory costs or both. Several issues remain unresolved, one of which is whether the use of rapid identification methods with a continuous-monitoring blood culture system might allow for a clinically important decrease in the time required to identify blood culture isolates. Another issue is whether subsequent isolation by culture is necessary for microorganisms with predictable antimicrobial susceptibility patterns. These and other issues need to be studied further before the exact clinical usefulness of rapid methods will be known. At this time, no commercial product has been cleared or approved by the Food and Drug Administration (FDA) for the direct detection or identification of both of pathogenic microorganisms from blood culture bottles (Sharon Hansen, PhD, personal communication, 1993). Consequently, laboratory directors should exercise caution in the use of commercial or other products for direct blood culture testing, because manufacturers assume no liability for products that are used for purposes other than that for which they have been approved. In addition, such use of commercial products may be in violation of the rules set forth in the Clinical Laboratory Improvement Act of 1988. Furthermore, as discussed previously, the clinical performance characteristics of many products typically have not been determined, and, therefore, test reference ranges, sensitivity, specificity, and positive and negative predictive values have not been established. Other issues, such as the effect of different blood culture media and additives, also have not been studied adequately, nor are specific controls defined. Therefore, laboratory staff who would like to use commercial products to test blood cultures directly must themselves establish the performance characteristics of the product (keeping in mind the issue of liability) or, preferably, persuade manufacturers to sponsor large-scale controlled clinical trials both to establish performance characteristics and to obtain FDA clearance or approval for such usage of the product.(ABSTRACT TRUNCATED AT 400 WORDS)
Based on the metachromatic property of Toluidine Blue O, three, convenient agar-diffusion methods have been developed that enable detection of the nuclease of Staphylococcus aureus at concentrations as low as 0.005 mug/ml in agar and broth cultures. The interactions of agar and deoxyribonucleic acid with Toluidine Blue O are discussed.
Some strains of Staphylococcus epidermidis and Micrococcus sp. produce nucleases. However, thermal stability was shown to be unique to the nucleases of S. aureus. In addition, two micromethods for susceptibility testing to lysostaphin were more precise and convenient than anaerobic glucose fermentation in distinguishing between the genera Staphylococcus and Micrococcus.
A total of 91 enterotoxigenic strains of Staphylococcus auerus isolated from foods and tested for production of coagulase and thermostable nuclease and the ability to ferment glucose and mannitol showed, with the exception of four strains, a complete correlation among these properties. A similar correlation was observed with 103 cultures of S. aureus isolated from clinical material. In all instances, the coagulase reactions were sufficiently strong to be scored at either the 3+ or 4+ levels. Presumptive staphylococcal cultures isolated during routine examination of foods and yielding 2+ coagulase reactions or lower were invariably negative for thermostable nuclease production. It is suggested that the thermostable nuclease test be performed on cultures with doubtful coagulase reactions before classifying them as S. aureus.
Use of the agar plate test for the enzyme deoxyribonucleate 3'-nucleotidohydrolase (deoxyribonuclease) can result in frequent misdiagnosis of Staphylococcus epidermidis as S. aureus.
SUMMARY A study has been made of 607 cultures of Gram-positive and catalase- positive cocci received from workers and collections in different parts of the world. These cultures were examined for a wide range of morphological and physiological characters and representative cultures were further studied to determine the chemical constituents of the organisms. Five hundred and sixty-four of the cultures received were aerobic members of the Micrococcaceae, and of these 96% were classified in the author's groups and subgroups; a further subgroup was, however, introduced to accommodate the not previously studied pink-pigmented micrococci. It appears that the Gram-positive and catalase-positive cocci are best separated intq,the genus Staphylococcus and the genus Micrococcus on the ability of members of Staphylococcus to grow and produce acid from glucose anaero- bically; six subgroups of staphylococci were recognized and eight of micrococci. The relationship of named species, pups and subgroups to the author's classification was examined ; several species and groups of micro- cocci had been incorrectly classified. Thus, M. denitrijcaas, M. halodenitri- $cans and M. radiodurans possess characters which suggest that they should be reclassified with the Gram-negative genera and that Abd-El- Malek and Gibson's group IIIB should be classified with the Gram-positive microbacteria.