Sheep Blood Agar Plates Research Articles

In vitro efficacy of a povidone–iodine 0.4% and dexamethasone 0.1% suspension against ocular pathogens

To assess the efficacy of a povidone-iodine 0.4%-dexamethasone 0.1% suspension against bacterial, fungal, and Acanthamoeba clinical isolates. Bascom Palmer Eye Institute, McKnight Research Building, Miami, Florida, USA. Experimental study. One hundred milliliters of 10(4) colony-forming units/mL of ocular isolates of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Serratia marcescens, Candida albicans, Fusarium solani, and Acanthamoeba castellanii were inoculated into 100 μL of a povidone-iodine 0.4%-dexamethasone 0.1% suspension in a 96-well microtiter plate incubated at room temperature. Organism viability was assessed at 15, 30, and 60 seconds by removing 10 μL aliquots and streaking onto a 5.0% sheep blood agar plate (fungi and bacteria) and agar-agar (Acanthamoeba) using a 0.001 calibrated loop. The plates were then incubated at 35 °C and monitored for up to 7 days. Isolates were inoculated into 200 μL of trypticase soy broth as controls. The number of colonies was counted and compared with controls to determine the kill rate. A 99.9% kill was observed for MRSA, P aeruginosa, S marcescens, and C albicans after 15 seconds of exposure and for F solani after 60 seconds. Acanthamoeba castellanii cyst viability was not inhibited by exposure to the povidone-iodine and dexamethasone suspension. Organism growth was achieved on all control broth. Povidone-iodine 0.4%-dexamethasone 0.1% suspension killed all bacterial and candida isolates within 15 seconds of exposure. It killed Fusarium isolates at 60 seconds but was ineffective against Acanthamoeba cysts. Drs. Pelletier and Miller have no financial or proprietary interest in any material or method mentioned. Additional disclosures are found in the footnotes.

In vitro Production of Clostridium difficile Spores for Use in the Efficacy Evaluation of Disinfectants: A Precollaborative Investigation

Clostridium difficile is a strict anaerobic spore-forming bacterium, and an increasingly common nosocomial pathogen. The U.S. Environmental Protection Agency (EPA) is responsible for the registration of disinfectants, including products designed to treat environmental surfaces contaminated with spores of C. difficile. Product efficacy data are required for registration; however, there is a lack of methodology for generating high-quality spore suspensions for evaluating product performance. As such, a study was carried out to select a suitable C. difficile strain and to develop a stand-alone method to prepare a spore suspension that meets specific criteria necessary for quantitative testing of disinfectants. The criteria are: (1) a spore titer of > 8 log10/mL, (2) > or = 90% spores to vegetative cells, and (3) resistance of spores (determined by viability) to 2.5 M hydrochloric acid (HCl). Several strains of C. difficile (toxigenic and nontoxigenic) were grown on various media (solid and liquid) for varying lengths of time to determine the best combination of incubation conditions and media to optimize spore production and quality. Once the spore production procedure was optimized, a toxigenic strain of C. difficile [American Type Culture Collection (ATCC) 43598] was selected for use in trials to verify repeatability from one production run to the next. The spore suspension was initiated by spreading vegetative cells of C. difficile (ATCC 43598) on CDC anaerobic 5% sheep blood agar plates and incubating for 7-10 days at 36 +/- 1 degrees C under anaerobic conditions. Spores were harvested when > or = 90% of the cells converted to spores as determined by observation using phase-contrast microscopy. The spores were washed three times with saline-Tween-80, resuspended in cold deionized water, heated to 70 degrees C for 10 min, evaluated microscopically for quality, and enumerated on cycloserine-cefoxitin-fructose agar containing horse blood and taurocholate. The spore suspension was used to inoculate brushed stainless steel carriers (1 cm in diameter) with and without a soil load in accordance with the Standard Quantitative Carrier Disk Test Method (ASTM E-2197-02) to determine carrier load. Once it was determined that > 6 log10 spores/carrier could be recovered, spores were evaluated for resistance to HCI. The sporulation method presented in this report is simple and repeatable and results in spore suspension of high titer (> 8 log10/mL) and quality (> or = 90% spores to vegetative cells) that met acid resistance criteria (spores were resistant to 2.5 M HCI for 10 min). In addition, recovery from brushed stainless steel carriers with and without soil load was > 6 log10 spores/carrier. A 6 log10 performance standard was set forth in the EPA's interim guidance for generating data to support a label claim for effectiveness against C. difficile spores on hard, nonporous surfaces. This precollaborative investigation successfully demonstrated the use of a methodology for in vitro production of C. difficile spores (ATCC 43598) necessary for conducting efficacy tests. A proposal will be submitted to the AOAC INTERNATIONAL Methods Committee on Antimicrobial Efficacy Testing for a collaborative study; see Appendix.

A case of wound dual infection with Pasteurella dagmatis and Pasteurella Canis resulting from a dog bite - limitations of Vitek-2 system in exact identification of Pasteurella species

BackgroundPasteurella species, widely known as indigenous orgganisms in the oral and gastrointestinal floras of many wild and domestic animals, are important pathogens in both animals and humans. Human infections due to Pasteurella species are in most cases associated with infected injuries following animal bites. We encountered a rare case of dual infections caused by different two Pasteurella species occurred in a previously healthy 25-year-old female sustaining injury by a dog-bite.MethodologyExudates from the open wound of her dog-bite site, together with the saliva of the dog were submitted for bacteriological examination. Predominantly appearing grayish-white smooth colonies with almost the same colonial properties but slightly different glistening grown on chocolate and sheep blood agar plates were characterized morphologically by Gram's stain, biochemically by automated instrument using Vitek 2 system using GN cards together with commercially available kit system, ID-Test HN-20 rapid panels, and genetically by sequencing the 16S rRNA genes of the organism using a Taq DyeDeoxy Terminator Cycle Sequencing and a model 3100 DNA sequencer instrument.ResultsThe causative isolates from the dog-bite site were finally identified as P. canis and P. dagmatis from the findings of the morphological, cultural, and biochemical properties together with the comparative sequences of the 16S rRNA genes. Both the isolates were highly susceptible to many antibiotics and the patient was successfully treated with the administration of so-called the first generation cephalosporin, cefazolin followed by so-called the third generation cephalosporin, cefcapene pivoxil. The isolate from the dog was subsequently identified as P. canis, the same species as the isolate from the patient.ConclusionsTo the best of our knowledge, this was the second report of a dual infection with Pasteurella species consisting of P. dagmatis and P. canis resulting from a dog-bite, followed by the first report of dual infections due to P. dagmatis and P. multocida in 1988. Our isolate finally identified as P. dagmatis was misidentified as P. pneumotripica by means of the Vitek 2 system. The species name "P. dagmatis" was not included in the database of the system. It is also important for routine clinical microbiology laboratories to know the limitation of the automated Vitek 2 system for the accurate identification of Pasteurella species especially P. dagmatis. It should be emphasized that there still exists much room for improvement in Vitek 2 system. Significant improvement of Vitek 2 system especially in the identification of Pasteurella species is urgently desired.

Streptococcus pyogenes Ser/Thr Kinase-regulated Cell Wall Hydrolase Is a Cell Division Plane-recognizing and Chain-forming Virulence Factor

Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.

Expression pattern and apoptosis-inducing activity to murine macrophages and hepatocytes of Leptospira interrogans Sph2 hemolysin

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira. Key words: Leptospira interrogans; sph2 gene/recombinant expression/transient expression; Sphingomyelinase hemolysin; Hemolytic activity; Cell apoptosis

Immune Response and Pasteurella Resistance in Rabbits Fed Diets Containing Various Amounts of Black Cumin Seeds

Problem statement: The consumption of black cumin (Nigella sativa) seed has immunomodulatory and anti-bacterial activity, but in rabbits this had not yet been tested. Approach: In the present studies, rabbits were fed diets without or with black cumin seed and antibody production, phagocytotic activity, hypersensitivity and resistance against Pasteurellosis were assessed. Results: Feeding black cumin seed significantly increased serum concentrations of antibodies in response to intramusculary injected serum bovine albumin. Blood derived from rabbits fed the diets containing either 15 or 20% black cumin seed significantly reduced the growth of Staphylococcus aureus on sheep-blood agar plates. Skin thickness as index of hypersensitivity towards tuberculin was significantly reduced at 48 h after intradermal injection of the agent. Ingestion of black cumin seed significantly extended survival time after intraperitoneal administration of Pasteurella multocida. Conclusion: The feeding of black cumin seed to rabbits stimulated their immune system, but did not enhance inflammation.

Analysis of the sporicidal activity of chlorine dioxide disinfectant against Bacillus anthracis (Sterne strain)

Routine surface decontamination is an essential hospital and laboratory procedure, but the list of effective, noncorrosive disinfectants that kill spores is limited. We investigated the sporicidal potential of an aqueous chlorine dioxide solution and encountered some unanticipated problems. Quantitative bacteriological culture methods were used to determine the log(10) reduction of Bacillus anthracis (Sterne strain) spores following 3min exposure to various concentrations of aqueous chlorine dioxide solutions at room temperature in sealed tubes, as well as spraying onto plastic and stainless steel surfaces in a biological safety cabinet. Serial 10-fold dilutions of the treated spores were then plated on 5% sheep blood agar plates, and the survivor colonies were enumerated. Disinfection of spore suspensions with aqueous chlorine dioxide solution in sealed microfuge tubes was highly effective, reducing the viable spore counts by 8log(10) in only 3min. By contrast, the process of spraying or spreading the disinfectant onto surfaces resulted in only a 1log(10) kill because the chlorine dioxide gas was rapidly vaporised from the solutions. Full potency of the sprayed aqueous chlorine dioxide solution was restored by preparing the chlorine dioxide solution in 5% bleach (0.3% sodium hypochlorite). The volatility of chlorine dioxide can cause treatment failures that constitute a serious hazard for unsuspecting users. Supplementation of the chlorine dioxide solution with 5% bleach (0.3% sodium hypochlorite) restored full potency and increased stability for one week.

Correlates of vaginal colonization with group B streptococci among pregnant women

Background:A study of genital colonization by group B streptococcus (GBS) was conducted in pregnant women in their third trimester, which is a known risk factor of morbidity and mortality among newborns.Aims:The present study was undertaken to study the prevalence and the correlates of vaginal colonization by GBS among pregnant women.Setting and Design:This observational cross-sectional study was conducted during September 2002 to March 2004 on 524 pregnant women.Materials and Methods:Three high vaginal swabs were obtained from all the pregnant women admitted at term and in preterm labor. Two swabs were used for aerobic culture and the third one for gram staining. The first set of swabs was cultured on 5% Sheep blood agar plates. The second set of swabs were inoculated into Todd–Hewitt broth and then subcultured in 5% Sheep blood agar plates. The main outcome measures were the presence of GBS infection in comparison to the age group, gravida, gestational age, premature rupture of membrane (PROM), preterm labor and association with febrile spells of the present pregnancy.Results:The culture positivity rate of GBS was 4.77% and coexistent organisms isolated were Candida species (36%), Staphylococcus aureus (8%) and Enterococcus species (8%). Culture positivity in the age group of 18–25 years was 5.71%, of which 5.74% were in their first pregnancy. The correlation between age group and gravida with GBS culture positivity was statistically insignificant. The culture positivity in <36 weeks of gestational age was 6.93%. This relation was statistically significant. Twenty-eight percent developed PROM. Sixty-four percent of culture positives had preterm labor.Conclusion:GBS infection among pregnant women was significantly correlated with the gestational age, PROM and preterm labor. In pregnancy GBS colonization causes asymptomatic bacteriuria or UTI. It is a well known cause of puerperal infections with amnionitis, endometritis and sepsis being the most commonly reported infections.[5]

Functional analysis of Leptospira interrogans sphingomyelinase hemolysin genes and their transcriptional level alterations in the infected host cells

Objective To determine the hemolytic activity of products of sphingomyelinase hemolysin encoding genes of Leptospira interrogaas, and the transcriptional level alterations in the infected host cells. Methods By using genomic DNAs of pathogenic L. interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai and serogroup Pomona serovar Pomona strain Luo, and non-pathogenic L. biflexa serogroup Sama-ranga serovar Patoc strain Patoc Ⅰ as templates, PCRs were performed to amplify entire sph1-sph4 genes. The amplified products were sequenced after T-A cloning. Prokaryotic expression systems of sph1-sph4 genes were re-spectively constructed, and the expressions of target recombinant proteins rSph1-rSph4 were examined by SDS-PAGE. Ni-NTA affinity chromatographic column was used to extract the expressed rSph1-rSph4. Hemolytic ac-tivities of rSph1-rSph4 on sheep blood agar plate were identified. Transcription alterations of sphl-sph4 genes in L. interrogans strain Lai after infected J774A. 1 cells were measured by real-time fluorescence quantitative RT-PCR. Results From genomic DNAs of both L. interrogans strain Lai and Luo, but not from that of L. biflexa strain Patoc Ⅰ , the target fragments of sph1-sph4 genes could be amplified. All the cloned sph1-sph4 genes had 100% nucleotide sequence identities compared to the corresponding reported sequences. The constructed pro-karyotic expression systems were able to efficiently express the target recombinant proteins rSph1-rSph4, respec-tively. All the rSph1-rSph4 had hemolytic activities, and among the four products rSph2 displayed the strongest hemolytic activity. After L. interrogaas strain Lai infecting J774A. 1 cells, the transcriptional levels of sph1-sph4 genes were remarkably up-regulated, especially for mRNA levels of sph2 and sph4 genes. Conclusion sph1- sph4 genes exist only in pathogenic L. interrogans species, and their products have hemolytic activity. The up-regulation of sph1-sph4 gene transcriptional levels in L. interrogans strain Lai after infected cells implies that the sphingomyelinase hemolysins may play important roles in the process of L. interrogans infection in hosts. Key words: Leptospira interrogaas; Sphingomyelinase hemolysin; Recombinant expression; Hemolytic activity; Transcriptional level

Characterization of a Hemolysin Gene ytjA from Bacillus subtilis

Bacillus subtilis is popularly used as a probiotic in many fields. Although recent research has found that some secondary metabolites of B. subtilis could cause hemolysis, the hemolytic mechanism in B. subtilis is still unclear. In this paper, the hemolysis-associated gene ytjA was cloned and expressed in Escherichia coli, and the hemolytic activity of the expressed soluble protein was indicated by the presence of clear hemolytic zones on sheep blood agar plates. In addition, reverse transcriptase-polymerase chain reaction analysis confirmed that the ytjA gene was transcribed in B. subtilis. These results suggest that ytjA is one of the hemolysin genes responsible for hemolysis in B. subtilis.

Differential Time to Positivity and Quantitative Cultures for Noninvasive Diagnosis of Catheter-Related Blood Stream Infection in Children

Accurate diagnosis of catheter-related blood stream infection (CRBSI) is necessary to make a decision about removal of the catheter. Differential time to positivity (DTP) and the ratio of quantitative cultures (RQC) between central and peripheral blood cultures have not been evaluated against a strict standard in children, namely catheter tip culture. Our aim is to compare DTP and RQC in the diagnosis of catheter tip-confirmed catheter-related infection in children. Prospective study performed in 2 large hospitals in Santiago, Chile. Children with clinically suspected CRBSI had 2 peripheral and central vein blood samples obtained for automated culture in Bact/Alert and for quantitative cultures in 5% sheep blood agar plate. The catheter tip was cultured. Sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratios (LR), and accuracy of DTP and RQC were compared against catheter tip-confirmed CRBSI. During a 3-year period, 344 clinically suspected CRBSIs were diagnosed in children of which 124 episodes met study criteria. Catheter tip culture-confirmed CRBSI in 25 (20%) of 124 episodes. A total of 34 microorganisms were cultured from 25 CRBSI; 8 of 25 (32%) episodes were polymicrobial. Staphylococcus aureus followed by coagulase-negative Staphylococcus were the most common microorganisms. For CRBSI, DTP and RQC reached a sensitivity of 75% versus 24% (P < 0.001), specificity of 86 versus 94%, positive predictive value of 58% versus 50%, negative predictive value of 93% versus 82%, LR of 5.48 versus 4.50, and accuracy of 0.84 versus 0.79. In children, DTP was better than RQC for diagnosis of catheter tip-confirmed CRBSI.

Lack of Evidence for “ Acinetobacter septicus ” as a Species Different from Acinetobacter ursingii ?

In their recent paper, Kilic et al. (2) reported on a hospital outbreak of bloodstream infections caused by a non-glucose-acidifying Acinetobacter strain. The authors showed that this strain (AK001) was phenotypically most similar to Acinetobacter ursingii but had two properties atypical of this species: i.e., the ability to hemolyse sheep blood and the inability to grow on citrate. Additional taxonomic methods to clarify the taxonomic status of AK001 included 16S rRNA and rpoB gene sequence analysis and DNA-DNA reassociation. Although the results of the sequence analyses supported the close relationship of the strain to A. ursingii, the value of DNA-DNA reassociation between AK001 and the A. ursingii type strain was found to be 64.7 to 68.7%, which is slightly less than 70%, the recommended threshold for species delineation (6). The authors concluded that the strain represents a new species, for which the name “Acinetobacter septicus” was proposed. As, in our opinion, the data provided by Kilic et al. (2) do not convincingly substantiate the proposal of a novel species, we analyzed strain AK001 using methods that had previously been used to resolve Acinetobacter taxonomy (3). AK001 was obtained by the Culture Collection, University of Goteborg (CCUG) from Y.-W. Tang and was designated CCUG 56015. The strain was shown to have properties consistent with the original description of A. ursingii, except for the inability to utilize citrate (4). It is of note, however, that there are other citrate-negative A. ursingii strains in our current collection, which indicates the intraspecies variation of this property. Importantly, the hemolytic activity of AK001 reported by Kilic et al. (2) could not be confirmed, although sheep blood agar plates obtained from different suppliers were used at different cultivation temperatures and with several control hemolytic Acinetobacter strains. In the study by Kilic et al. (2), the 16S rRNA gene sequences of AK001 were 99.5% similar to that of the A. ursingii type strain, which is the value that can be found in strains within one species (4). To obtain more comprehensive information on the rpoB-based position of AK001 within the genus Acinetobacter, we determined the rpoB sequence of AK001 and compared it to that of eight A. ursingii strains and all hitherto-described species. The similarity values between AK001 (GenBank no. {type:entrez-nucleotide,attrs:{text:EU742582,term_id:190688713,term_text:EU742582}}EU742582) and the A. ursingii strains ({type:entrez-nucleotide,attrs:{text:EU477105,term_id:526150767,term_text:EU477105}}EU477105 and {type:entrez-nucleotide-range,attrs:{text:EU742575 to EU742581,start_term:EU742575,end_term:EU742581,start_term_id:190688699,end_term_id:190688711}}EU742575 to EU742581) ranged from 96.9% to 98.0%, which partially overlaps with the range of intraspecies values found for Acinetobacter beijerinckii (3). In contrast, the rpoB similarity values between AK001 and the other species were between 78.8% and 84.5%. The proposal of “A. septicus” was based mainly on the results of DNA-DNA reassociation. However, DNA-DNA reassociation values close to the proposed value of 70% have to be treated with caution as incongruent results for the same strains can be obtained in different laboratories and/or by different methods, which may have taxonomic implications (5). Concerning also other limitations of DNA-DNA reassociation (1), no conclusions based on these intermediate values should be made without further evidence of genetic isolation between strains. To address this problem, the relatedness of AK001 to hitherto-described species was investigated by AFLP (Keygene NV) analysis, a whole-genome fingerprinting method. Previous studies using this method have shown that strains of the same (genomic) species cluster together at a ≥50% similarity level (4). AFLP analysis showed that AK001 and 25 reference A. ursingii strains were linked in a distinct cluster at a 59% similarity level, while AK001 was separated from other species at 34% similarity, which strongly supports the assignment of AK001 to A. ursingii. Based on the above arguments, strain AK2001 is considered as belonging to A. ursingii and not representing a novel species.

Urinary Tract Infection Caused by CapnophilicEscherichia coli

Urinary Tract Infection Caused by Capnophilic<i>Escherichia coli</i>

Nocardia asteroides peritoneal dialysis-related peritonitis: a case of successful treatment and return to peritoneal dialysis

A 66-year-old male from rural Manukau, New Zealand, presented to hospital with 2 weeks of generalized abdominal pain and cloudy PD effluent. He had been on PD for 3 years, with only one previous episode of PD-related peritonitis secondary to Streptococcus oralis a year prior. Other significant co-morbidity included well-controlled Type 2 diabetes mellitus on diet therapy, hypertension, mild chronic obstructive airways disease and quiescent gout. On initial examination, the patient was afebrile with blood pressure of 140/70mmHg. Abdominal examination showed generalized tenderness and normal bowel sounds. The PD catheter exit site was normal. Microscopy of dialysate showed 1600 leucocytes, 86% of which were neutrophils, and no organisms. After initial investigations, antibiotic treatment was commenced for PD-related peritonitis with empirical intraperitoneal cephazolin and gentamicin. His dialysate sample was processed in our microbiology laboratory; this involved inoculation of 10 mL into both BacT/ALERT FA aerobic and FN anaerobic blood culture bottles (bioMerieux, Marcy-l’Etoile, France), with the deposit from 10 mL of centrifuged sample inoculated onto two sheep blood agar plates, incubated in 5% CO2 and anaerobically, and one chocolate agar plate incubated in 5% CO2. After 5 days incubation, the BacT/ALERT FA aerobic bottle was reported as growing a fungus. Since one of the enrichment broths had become positive, the primary agar plates were held longer than the usual 5 days, but remained culture negative after 21 days. The patient was therefore treated as fungal peritonitis with a change in his antibiotic regimen to fluconazole 200 mg orally on alternate days and removal of his PD

Concentração inibitória mínima (CIM) de oito antimicrobianos frente isolados de Streptococcus suis

Avaliou-se a concentração inibitória mínima (CIM) de 75 isolados de Streptococcus suis frente a oito antimicrobianos freqüentemente utilizados no controle da infecção por esse microrganismo. A CIM foi realizada em placas previamente preparadas com ágar sangue, contendo concentrações variando de 0,25 a 256 µg/ml dos seguintes antimicrobianos: amoxicilina, ampicilina, penicilina, ceftiofur sódico, florfenicol, lincomicina, penicilina, sulfametoxazol-trimetoprim e tetraciclina. A amoxicilina nas concentrações de 1 e 2 µg/ml, o florfenicol a 1 µg/ml e o sulfametoxazol-trimetoprim nas concentrações de 2 e 8 µg/ml, foram os antimicrobianos frente aos quais os microrganismos apresentaram menor resistência. Em contraste, a ampicilina, tetraciclina, ceftiofur sódico e lincomicina, foram menos efetivos, apresentando CIM de (64 e 128 µg/ml), (64 e 128 µg/ml), (128 e 256 µg/ml) e (&gt;;256 µg/ml), respectivamente. Os resultados deste estudo demonstram que a amoxicilina e o florfenicol são os antibióticos de escolha para o tratamento de infecções pelo S. suis em suínos.

Highly Efficient Classification and Identification of Human Pathogenic Bacteria by MALDI-TOF MS

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10(4) cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future.

Antimicrobial Effect of Triclosan and Triclosan with Gantrez on Five Common Endodontic Pathogens

Microbial control of the root canal system is one of the key objectives of root canal therapy. Triclosan is a widely accepted broad spectrum antimicrobial agent proven to be effective against many gram-positive and gram-negative bacteria. Triclosan acts by blocking bacterial fatty acid biosynthesis. The addition of Gantrez copolymer has been shown to enhance the antimicrobial activity of triclosan. The purpose of this study was to determine the minimum inhibitory and bactericidal concentrations of triclosan and triclosan with Gantrez against Prevotella intermedia, Fusobacterium nucleatum, Actinomyces naeslundii, Porphyromonas gingivalis, and Enterococcus faecalis. The minimum inhibitory concentration (MIC) of both test solutions was determined for each of the 5 microorganisms by using microtiter serial dilutions. Samples were streaked on 5% sheep blood agar plates and placed in an anaerobic incubator to determine the minimum bactericidal concentration (MBC). The MBC of triclosan ranged from 12-94 microg/mL. The MBC of triclosan with Gantrez ranged from <0.3-10.4 microg/mL. The addition of Gantrez enhanced the bactericidal activity of triclosan. Both triclosan and triclosan with Gantrez demonstrated bactericidal activity against the 5 specific endodontic pathogens.

Clinical Validation of the Molecular BD GeneOhm StaphSR Assay for Direct Detection of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in Positive Blood Cultures

The rapid detection of Staphylococcus aureus bacteremia and a swift determination of methicillin susceptibility has serious clinical implications affecting patient mortality. This study evaluated the StaphSR assay (BD GeneOhm, San Diego, CA), a real-time PCR assay, for the identification and differentiation of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) from 300 positive blood cultures. The BD GeneOhm StaphSR assay was performed and interpreted according to the manufacturer's recommendations. Positive blood cultures (containing predominantly gram-positive cocci in clusters) were subcultured on 5% sheep blood agar plates. After 18 to 24 h of incubation, isolates morphologically consistent with S. aureus were presumptively identified by latex agglutination (Staphaurex Plus; Remel, Lenexa, KS). Susceptibility testing was initially performed with the Phoenix automated microbiology system (BD Diagnostics, Sparks, MD). Additional susceptibility testing of samples with discrepant results was done using BBL oxacillin screen agar (BD Diagnostics, Sparks, MD), oxacillin and cefoxitin Etests (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar, an immunoassay for penicillin binding protein 2' (Denka Seiken Co., Tokyo, Japan), and mecA PCR. The sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm StaphSR assay for MSSA detection were 98.9, 96.7, 93.6, and 99.5%, respectively. For the detection of MRSA, the BD GeneOhm StaphSR assay was 100% sensitive and 98.4% specific; positive and negative predictive values for MRSA detection were 92.6 and 100%, respectively. Inhibition was seen with only one sample, and the issue was resolved upon retesting. The BD GeneOhm StaphSR assay appears to be a valuable diagnostic tool for quickly differentiating bacteremia caused by MSSA and MRSA from that caused by other gram-positive cocci.

Acetobacter cibinongensisBacteremia in Human

<i>Acetobacter cibinongensis</i>Bacteremia in Human

The Effectiveness of Current Sterility Techniques in Natural Orifice Transluminal Endoscopic Surgery (NOTES)

Background: NOTES represents a potentially less invasive alternative to conventional laparoscopy. However, breach of the gut wall raises the specter of intra-peritoneal infection. Various anti-microbial techniques have been employed in animal survival studies—including sterile saline rinse, intravenous and topical antibiotics, and Betadine—despite a paucity of data regarding their general effectiveness. Aim: To assess the effectiveness of existing sterilization techniques for NOTES by quantifying and speciating colony-forming units (CFU's) before and after treatment in ex vivo porcine organs. Method: Stomachs and distal colons were harvested en bloc from six fasted adult female white pigs following euthanasia. Four of these animals received 1g of Cefazolin intravenously prior to sacrifice. After the organs were filleted open in sterile fashion, they were further sub-divided into 2.0 cm × 2.0 cm full-thickness square samples. Each tissue sample was then assigned to one of five treatment arms: (1) sterile 0.9% saline lavage, (2) Betadine lavage, (3) lavage with a Cefazolin/Metronidazole suspension, (4) 4% Chlorhexidine lavage, or (5) no treatment. Following 10 minutes of contact time with the treatment solution, the mucosal surface of each tissue sample was swabbed with a sterile cotton applicator and inoculated in 5 ml of normal saline for 1 hour, followed by serial dilutions. 100 mL aliquots of each dilution were plated onto Sheep's blood agar plates and incubated in aerobic conditions at 24 degrees Celsius. Colony-forming units (CFU's) were quantified after seven days, and representative samples were sent to a commercial veterinary laboratory for speciation. Results: Initial bacterial density was estimated to be 1.0 × 106 CFU's/cm2 for stomach and 1.2 × 106 CFU's/cm2 for colon. The predominant organisms were Escherichia coli in stomach and both Escherichia coli and Enterococcus sp in colon. Sterile normal saline lavage caused a 1-log reduction to 2.3 × 105 CFU's/cm2. Antibiotic lavage caused a 2-log reduction to 6 × 104 CFU's/cm2. Betadine lavage a 5-log reduction to 4.1 × 101 CFU's/cm2. Finally, Chlorhexidine lavage also caused a 5-log reduction to 6.0 × 101 CFU's/cm2. The use of intravenous antibiotics alone diminished bacterial load to 10 CFU's/cm2. The combined use of intravenous antibiotics and Betadine or Chlorhexidine decreased counts to zero. Data was analyzed using ANOVA 2-way analysis method and found to be statistically significant (P = 0.001). Conclusions: The use of intravenous antibiotics in addition to topical Betadine or Chlorhexidine effectively eradicated bacteria in both gastric and colonic mucosa in this porcine model.