Improvement of lysine effect on aldehyde-ruthenium red preservation and staining of the staphylococcal glycocalyx

1995 ◽  
Vol 53 ◽  
pp. 1072-1073
Author(s):  
Theresa A. Fassel ◽  
Paul E. Mozdziak ◽  
James R. Sanger ◽  
Charles E. Edmiston
Keyword(s):  
Staphylococcus Epidermidis ◽  
Frozen Storage ◽  
Ruthenium Red ◽  
Blood Agar ◽  
Sodium Cacodylate ◽  
Coagulase Negative Staphylococci ◽  
Gram Positive ◽  
Cationic Reagent ◽  
Negative Mutant

The cationic reagent ruthenium red (RR) has been used to enhance ultrastructural preservation and staining of the bacterial glycocalyx. Lysine was needed in prefixation to preserve the polysaccharide glycocalyx material for the gram-positive, coagulase-negative staphylococci species. The inclusion of lysine in the prefixative of glutaraldehyde (GA)-RR enhanced observation of elaborate and extensive glycocalyx material. However, prefixation in 75 mM lysine in GA-RR is restricted to 20 minutes because gelling or solidification frequently occurs at longer intervals resulting in loss of sample. To improve the utility of this approach, the effect of the Karnovsky ratio of 2% paraformaldehyde to 2.5% GA was investigated.Cells of gram-positive, coagulase-negative staphylococci species, Staphylococcus epidermidis RP62, a polysaccharide producer, and Staphylococcus epidermidis M187-SN3, a polysaccharide negative mutant, were recovered from frozen storage and grown on blood agar plates. After 24 hours, cells were transferred to trypticase soy broth for 18 hours, and incubated at 35°C. Half of the cells were prefixed in 75 mM L-lysine, 0.075% RR, 2.5% GA in 0.1 M sodium cacodylate pH 7.2 for 20 minutes.

Lysine effect on ruthenium red and alcian blue preservation of the staphylococci glycocalyx

1993 ◽  
Vol 51 ◽  
pp. 374-375
Author(s):  
Theresa A. Fassel ◽  
James R. Sanger ◽  
Charles E. Edmiston
Keyword(s):  
Staphylococcus Aureus ◽  
Alcian Blue ◽  
Staphylococcus Epidermidis ◽  
Latent Infection ◽  
Frozen Storage ◽  
Ruthenium Red ◽  
Coagulase Negative Staphylococci ◽  
En Bloc ◽  
Initial Adhesion ◽  
Staphylococcus Hominis

The gram-positive coagulase-negative staphylococci are opportunistic organisms important in latent infection associated with prosthetic biomaterials. Their highly charged polysaccharide glycocalyx, or slime, aids in proliferation of bacteria on surfaces after an initial adhesion event, possibly at the time of surgical insertion. The cationic reagents, ruthenium red (RR) and alcian blue (AB), in en bloc procedures have aided visualization of the polysaccharide glycocalyx material in TEM or SEM applications. Further, enhancement of RR preservation or staining of slime by inclusion of lysine in a prefixation stage has been observed. The effect of lysine with and without RR and AB on preservation/staining of the staphylococci glycocalyx is investigated further in this study.Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis RP62 (RP62) and Staphylococcus hominis SP2 (SP2) were cultured in trypticase soy broth for 18 hrs at 35°C, following recovery from frozen storage and plating on blood agar plates for 24 hrs. Cells with 75mM lysine in the prefixation were incubated for 20 minutes with 2.5% glutaraldehyde (GA), 2.5% GA and 0.075% RR, or 2.5% GA and 0.075% AB, respectively.

scholarly journals Contaminant bacteria in traditional-packed honey

2007 ◽  
Vol 19 (1) ◽  
Author(s):  
Hening Tjaturina Pramesti ◽  
Karlina Hardjawinata ◽  
Putra Qadri Fath
Keyword(s):  
Staphylococcus Epidermidis ◽  
Honey Sample ◽  
Coagulase Negative Staphylococci ◽  
Gram Positive ◽  
Bacterial Colony ◽  
Colony Forming Units ◽  
Morphology Characteristics ◽  
Gram Positive Cocci ◽  
Opportunist Pathogen ◽  
Colony Counter

Honey may be contaminated by microorganisms during its harvesting, processing, and packaging. Honey selected for clinical purposes must safe, sterile, and contain antimicrobial activity, so it must be evaluated using laboratory testing. The aim of this descriptive laboratory study was to isolate and identify the bacterial contaminant in the traditional-packed honey dealing with the use of honey for medical purposes. the colony forming units of honey sample cultured on blood agar were counted using Stuart bacterial colony counter. The suspected bacterial colonies were isolated and identified based on cultural morphology characteristics. The isolates of suspected bacterial colonies were stained according to Gram and Klein method and then were examined by the biochemical reaction. The results showed that there were two contaminant bacteria. Gram-positive cocci which were presumptively identified as coagulase-negative Staphylococci and gram-positive rods which were presumptively identified as Bacillus subtilis. In conclusion, the contaminant bacteria were regarded as low pathogen bacteria. The subtilin enzyme of B subtilis may cause an allergic reaction and coagulase-negative Staphylococci, Staphylococcus epidermidis is also an opportunist pathogen. Inevitably, for medical purposes, traditional-packed honey must be well filtered, water content above 18%, and standardized sterilization without loss of an antibacterial activity or change in properties.

scholarly journals Comparative In Vitro Activities of Ciprofloxacin, Gemifloxacin, Grepafloxacin, Moxifloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, and Other Antimicrobial Agents against Bloodstream Isolates of Gram-Positive Cocci

2000 ◽  
Vol 44 (3) ◽  
pp. 802-805 ◽  
Author(s):  
Dwight Hardy ◽  
Daniel Amsterdam ◽  
Lionel A. Mandell ◽  
Coleman Rotstein
Keyword(s):  
Staphylococcus Epidermidis ◽  
Antimicrobial Agents ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Coagulase Negative Staphylococci ◽  
Gram Positive ◽  
Gram Positive Cocci

ABSTRACT The in vitro activity of gemifloxacin against 316 bloodstream isolates of staphylococci, pneumococci, and enterococci was compared with the activities of six fluoroquinolones and three other antimicrobial agents. Of the antimicrobial agents tested, gemifloxacin was the most potent against penicillin-intermediate and -resistant pneumococci, methicillin-susceptible and -resistantStaphylococcus epidermidis isolates, and coagulase-negative staphylococci.

scholarly journals In Vitro activity of a Novel Benzoquinolizine Antibiotic, Levonadifloxacin (WCK 771) against Blood Stream Gram-Positive Isolates from a Tertiary Care Hospital

2020 ◽  
Vol 12 (03) ◽  
pp. 230-232
Author(s):  
Dhruv Mamtora ◽  
Sanjith Saseedharan ◽  
Ritika Rampal ◽  
Prashant Joshi ◽  
Pallavi Bhalekar ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Tertiary Care Hospital ◽  
Tertiary Care ◽  
Blood Stream ◽  
Tolerability Profile ◽  
Care Hospital ◽  
Coagulase Negative Staphylococci ◽  
Gram Positive ◽  
Methicillin Resistant

Abstract Background Blood stream infections (BSIs) due to Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are associated with high mortality ranging from 10 to 60%. The current anti-MRSA agents have limitations with regards to safety and tolerability profile which limits their prolonged usage. Levonadifloxacin and its oral prodrug alalevonadifloxacin, a novel benzoquinolizine antibiotic, have recently been approved for acute bacterial skin and skin structure infections including diabetic foot infections and concurrent bacteremia in India. Methods The present study assessed the potency of levonadifloxacin, a novel benzoquinolizine antibiotic, against Gram-positive blood stream clinical isolates (n = 31) collected from January to June 2019 at a tertiary care hospital in Mumbai, India. The susceptibility of isolates to antibacterial agents was defined following the Clinical and Laboratory Standard Institute interpretive criteria (M100 E29). Results High prevalence of MRSA (62.5%), quinolone-resistant Staphylococcus aureus (QRSA) (87.5%), and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) (82.35%) were observed among bacteremic isolates. Levonadifloxacin demonstrated potent activity against MRSA, QRSA, and MR-CoNS strains with significantly lower minimum inhibitory concentration MIC50/90 values of 0.5/1 mg/L as compared with levofloxacin (8/32 mg/L) and moxifloxacin (2/8 mg/L). Conclusion Potent bactericidal activity coupled with low MICs support usage of levonadifloxacin for the management of BSIs caused by multidrug resistant Gram-positive bacteria.

scholarly journals Prevalence of Antimicrobial-Resistant Pathogens in Canadian Hospitals: Results of the Canadian Ward Surveillance Study (CANWARD 2008)

2010 ◽  
Vol 54 (11) ◽  
pp. 4684-4693 ◽  
Author(s):  
George G. Zhanel ◽  
Melanie DeCorby ◽  
Heather Adam ◽  
Michael R. Mulvey ◽  
Melissa McCracken ◽  
...  
Keyword(s):  
Staphylococcus Epidermidis ◽  
Susceptibility Testing ◽  
Multidrug Resistant ◽  
Surveillance Study ◽  
Bacterial Isolates ◽  
Coagulase Negative Staphylococci ◽  
Clinical Specimens ◽  
E Coli ◽  
Polymyxin E ◽  
Resistance Rates

ABSTRACT A total of 5,282 bacterial isolates obtained between 1 January and 31 December 31 2008, inclusive, from patients in 10 hospitals across Canada as part of the Canadian Ward Surveillance Study (CANWARD 2008) underwent susceptibility testing. The 10 most common organisms, representing 78.8% of all clinical specimens, were as follows: Escherichia coli (21.4%), methicillin-susceptible Staphylococcus aureus (MSSA; 13.9%), Streptococcus pneumoniae (10.3%), Pseudomonas aeruginosa (7.1%), Klebsiella pneumoniae (6.0%), coagulase-negative staphylococci/Staphylococcus epidermidis (5.4%), methicillin-resistant S. aureus (MRSA; 5.1%), Haemophilus influenzae (4.1%), Enterococcus spp. (3.3%), Enterobacter cloacae (2.2%). MRSA comprised 27.0% (272/1,007) of all S. aureus isolates (genotypically, 68.8% of MRSA were health care associated [HA-MRSA] and 27.6% were community associated [CA-MRSA]). Extended-spectrum β-lactamase (ESBL)-producing E. coli occurred in 4.9% of E. coli isolates. The CTX-M type was the predominant ESBL, with CTX-M-15 the most prevalent genotype. MRSA demonstrated no resistance to ceftobiprole, daptomycin, linezolid, telavancin, tigecycline, or vancomycin (0.4% intermediate intermediate resistance). E. coli demonstrated no resistance to ertapenem, meropenem, or tigecycline. Resistance rates with P. aeruginosa were as follows: colistin (polymyxin E), 0.8%; amikacin, 3.5%; cefepime, 7.2%; gentamicin, 12.3%; fluoroquinolones, 19.0 to 24.1%; meropenem, 5.6%; piperacillin-tazobactam, 8.0%. A multidrug-resistant (MDR) phenotype occurred frequently in P. aeruginosa (5.9%) but uncommonly in E. coli (1.2%) and K. pneumoniae (0.9%). In conclusion, E. coli, S. aureus (MSSA and MRSA), P. aeruginosa, S. pneumoniae, K. pneumoniae, H. influenzae, and Enterococcus spp. are the most common isolates recovered from clinical specimens in Canadian hospitals. The prevalence of MRSA was 27.0% (of which genotypically 27.6% were CA-MRSA), while ESBL-producing E. coli occurred in 4.9% of isolates. An MDR phenotype was common in P. aeruginosa.

scholarly journals Case Study: Misdiagnosis of Nonhemolytic Staphylococcus aureus Isolates from Cases of Bovine Mastitis as Coagulase-Negative Staphylococci

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 252
Author(s):  
Valerie E. Ryman ◽  
Felicia M. Kautz ◽  
Steve C. Nickerson
Keyword(s):  
Staphylococcus Aureus ◽  
Bovine Mastitis ◽  
Dairy Farm ◽  
Blood Agar ◽  
Coagulase Negative Staphylococci ◽  
Mannitol Salt Agar ◽  
Lactating Cows ◽  
Presumptive Identification ◽  
Microbiological Techniques

Staphylococcus aureus is one of the most concerning mastitis-causing pathogens in dairy cattle. Using basic microbiological techniques, S. aureus is typically identified by colony characteristics and hemolysis on blood agar where isolates without hemolysis are typically considered to be coagulase-negative staphylococci (CNS) isolates. Herein, we present a decade-long case study where suspected S. aureus isolates from one Georgia dairy farm were further tested to confirm presumptive identification. Presumptive identification of bacterial growth from 222 mammary secretions from bred Holstein heifers and lactating cows was conducted at the time of collection. Presumptive identification of S. aureus on blood agar was based on observation of colony morphology, color, and presence or absence of a broad zone of incomplete hemolysis and a smaller zone of complete hemolysis at 48 h. Those without hemolysis were presumptively characterized as CNS. All isolates were further plated on mannitol salt agar and a coagulase test was performed. A positive for both of these tests together was deemed to be S. aureus. A selection of isolates was tested using API® Staph to biochemically confirm S. aureus identification. Data showed that 63.96% of isolates presumed to be CNS isolates were identified as S. aureus, 9.46% of isolates presumed to be CNS isolates were identified as coagulase-positive staphylococci (CPS) species (but not S. aureus), and 26.58% of samples that were presumed to be CNS isolates were identified correctly.

Impaired Cytokine Responses to Live Staphylococcus epidermidis in Preterm Infants Precede Gram-positive, Late-onset Sepsis

2020 ◽  
Author(s):  
Tobias Strunk ◽  
Julie Hibbert ◽  
Dorota Doherty ◽  
Elizabeth Nathan ◽  
Karen Simmer ◽  
...  
Keyword(s):  
Preterm Infants ◽  
Staphylococcus Epidermidis ◽  
Late Onset ◽  
Gram Positive ◽  
Cytokine Responses ◽  
Inflammatory Protein ◽  
Late Onset Sepsis ◽  
Immunological Mechanisms ◽  
Factor Α

Abstract Background Late-onset sepsis (LOS) with Staphylococcus epidermidis is common in preterm infants, but the immunological mechanisms underlying heightened susceptibility are poorly understood. Our aim is to characterize the ontogeny of cytokine responses to live S. epidermidis in preterm infants with and without subsequent Gram-positive LOS. Methods We conducted a prospective, observational cohort study of preterm infants (<30 weeks gestational age [GA]) with blood sampling on Days 1, 7, 14, 21, and 28 of life. Cytokine responses in peripheral whole blood stimulated with live S. epidermidis were analyzed by 11-plex immunoassay. Results Of 129 infants (mean GA, 26.2 weeks; mean birth weight, 887g), 23 (17.8%) had confirmed LOS with Gram-positive organisms and 15 (11.6%) had clinical sepsis, with median onsets at 13 and 15 days, respectively. Blood cytokine responses to an in vitro S. epidermidis challenge were similar between infected and uninfected infants on Day 1, but diverged thereafter. Infants with subsequent LOS displayed broadly reduced S. epidermidis–induced responses from Day 7 onwards, compared to those who did not develop LOS. This pattern was observed with chemokines (interleukin [IL]-8, monocyte chemotactic protein–1, and macrophage inflammatory protein–1α), pro-inflammatory cytokines (IL-1, IL-6, and tumor necrosis factor–α) and the regulatory cytokine IL-10. Conclusions Cytokine responses to a live S. epidermidis challenge are impaired in infants with LOS and precede the onset of clinical illness. Quantifying pathogen-specific cytokine responses at Day 7 may identify those high-risk preterm infants at the greatest risk of LOS, and prospective replication is warranted.

scholarly journals Finding of coagulase negative staphylococci in the herd with an increased number of somatic cells in milk and their antimicrobial susceptibility

2013 ◽  
Vol 67 (3-4) ◽  
pp. 175-185
Author(s):  
Vera Katic ◽  
Natasa Rajic-Savic
Keyword(s):  
Clavulanic Acid ◽  
Somatic Cells ◽  
Inhibition Zone ◽  
Subclinical Mastitis ◽  
Blood Agar ◽  
Penicillin G ◽  
Coagulase Negative Staphylococci ◽  
Milk Samples ◽  
Causative Agents ◽  
Amoxicillin Clavulanic Acid

Coagulase-negative staphylococci (CNS) are generally considered to be opportunistic pathogens. Controlling CNS mastitis is difficult because the epidemiology is not clear, and the CNS group consists of about 40 different Staphylococcus species. Therefore, the aim of this study was to determine the prevalence of coagulasenegative staphylococci in milk of the cows with subclinical mastitis, as well as to determine different CNS species isolated from quarter milk samples for their susceptibility to antimicrobials used commonly for mastitis therapy. On the farm where there was found an increase of somatic cells in bulk milk, 112 dairy cows were examined by mastitis test. From 52 udder quarters where mastitis test showed an increase of somatic cells, milk samples were taken for bacteriological examination. For isolating the causes of mastitis there was used blood agar. Identification of the causative agents of mastitis was carried out on the basis of colony appearance on blood agar and their physiological characteristics. Coagulasepositive staphylococci sensitivity which cause mastitis was tested by Kirby Bauer method. For susceptibility testing there were used commercially produced discs containing: 10 IU penicillin, amoxicillin/clavulanic acid (20 +10 ?g), cloxacillin 25 ?g, 30 ?g amoxicillin, cephalexin 30 ?g, ceftiofur 30 ?g, 15 ?g lincomycin, gentamicin and tetracycline 30 ?g. The sensitivity of microorganisms was evaluated on the basis of inhibition zone diameter recommended by the manufacturer and was labeled as sensitive (S) moderately sensitive (I) or resistant (R). Coagulase-negative staphylococci were isolated from 61.53% of samples from cows with subclinical mastitis, making them the most common cause of subclinical mastitis. The highest resistance of coagulase-negative staphylococci was found to penicillin G (58.33% of isolates). Full sensitivity of coagulase-negative staphylococci was found to amoxicillin / clavulanic acid (100% of isolates), a good sensitivity to ceftiofur (83.33% of isolates), cefalexin (70.83% of isolates) and ceftriaxone (41.66% of isolates).

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