Comparison of Inoculation with the InoqulA and WASP Automated Systems with Manual Inoculation.

The quality of sample inoculation is critical for achieving an optimal yield of discrete colonies in both monomicrobial and polymicrobial samples to perform identification and antibiotic susceptibility testing. Consequently, we compared the performance between the InoqulA (BD Kiestra), the WASP (Copan), and manual inoculation methods. Defined mono- and polymicrobial samples of 4 bacterial species and cloudy urine specimens were inoculated on chromogenic agar by the InoqulA, the WASP, and manual methods. Images taken with ImagA (BD Kiestra) were analyzed with the VisionLab version 3.43 image analysis software to assess the quality of growth and to prevent subjective interpretation of the data. A 3- to 10-fold higher yield of discrete colonies was observed following automated inoculation with both the InoqulA and WASP systems than that with manual inoculation. The difference in performance between automated and manual inoculation was mainly observed at concentrations of >10(6) bacteria/ml. Inoculation with the InoqulA system allowed us to obtain significantly more discrete colonies than the WASP system at concentrations of >10(7) bacteria/ml. However, the level of difference observed was bacterial species dependent. Discrete colonies of bacteria present in 100- to 1,000-fold lower concentrations than the most concentrated populations in defined polymicrobial samples were not reproducibly recovered, even with the automated systems. The analysis of cloudy urine specimens showed that InoqulA inoculation provided a statistically significantly higher number of discrete colonies than that with WASP and manual inoculation. Consequently, the automated InoqulA inoculation greatly decreased the requirement for bacterial subculture and thus resulted in a significant reduction in the time to results, laboratory workload, and laboratory costs

Factors associated with positive urine cultures in cats with subcutaneous ureteral bypass system implantation

Objectives The aims of this study were to report the postoperative incidence of subcutaneous ureteral bypass (SUB)-associated bacteriuria and risk factors in a large population of UK cats, to identify the commonly implicated isolates in these cases and to report associations of positive postoperative urine cultures with device occlusion or a need for further surgery. Methods Electronic clinical records were reviewed to identify cats with ureteral obstruction that underwent unilateral or bilateral SUB implantation between September 2011 and September 2019. In total, 118 client-owned cats were included in the study population. Information recorded included signalment, history, surgical and biochemical factors, urinalysis and culture results. Multivariable logistic regression was performed to identify variables associated with a positive postoperative culture. Results In total, 10 cats (8.5%) had a positive postoperative culture within 1 month postsurgery and 28 cats (23.7%) within 1 year postsurgery. Cats with a positive preoperative culture were significantly more likely to have a positive culture within 6 months postoperatively (odds ratio [OR] 4.09, 95% confidence interval [CI] 1.18–14.18; P = 0.026). Of the 14 cats with a positive preoperative culture, six (42.9%) returned a positive culture within 1 year postoperatively, and in four cases (66.7%) the same isolate was identified. Cats with a higher end-anaesthetic rectal temperature were significantly less likely to return a positive culture within 3 months (OR 0.398, 95% CI 0.205–0.772; P = 0.006) postsurgery. Cats culturing positive for Escherichia coli at any time point (OR 4.542, 95% CI 1.485–13.89; P = 0.008) were significantly more likely to have their implant removed or replaced. Conclusions and relevance Perioperative hypothermia and preoperative positive culture were independent predictors of a postoperative positive culture and this should be taken into consideration when managing these cases. Positive postoperative culture rates were higher than have previously been reported

Propolis: a potential natural product to fight Candida species infections

Aim: To evaluate the effect of propolis against Candida species planktonic cells and its counterpart's biofilms. Materials & methods: The MIC values, time-kill curves and filamentation form inhibition were determined in Candida planktonic cells. The effect of propolis on Candida biofilms was assessed through quantification of CFUs. Results: MIC values, ranging from 220 to 880 µg/ml, demonstrated higher efficiency on C. albicans and C. parapsilosis than on C. tropicalis cells. In addition, propolis was able to prevent Candida species biofilm's formation and eradicate their mature biofilms, coupled with a significant reduction on C. tropicalis and C. albicans filamentation. Conclusion: Propolis is an inhibitor of Candida virulence factors and represents an innovative alternative to fight candidiasis.The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (Cnpq) and Fundação Araucária for the financial support received. Flávia Tobaldini-Valerio acknowledges the financial support of CAPES – Proc. 9469/14-1. The authors also thank FCT for the Strategic Project of the UID/BIO/04469/2013 unit, FCT and European Union funds (FEDER/COMPETE) for the project RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed

Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes

Background Staphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health. Although vancomycin-resistant Staphylococcus aureus (VRSA) is currently not as widespread as methicillin-resistant S. aureus (MRSA), genome evolution of MRSA into VRSA, including strains engineered within the same patient under anti-staphylococcal therapy, may build up to future public health concern. To further complicate diagnosis, infection control and anti-microbial chemotherapy, non-sterile sites such as the nares and the skin could contain both S. aureus and coagulase-negative staphylococci (CoNS), either of which could harbour mecA the gene driving staphylococcal methicillin-resistance and required for MRSA-VRSA evolution. Results A new heptaplex PCR assay has been developed which simultaneously detects seven markers for: i) eubacteria (16S rRNA), ii) Staphylococcus genus (tuf), iii) Staphylococcus aureus (spa), iv) CoNS (cns), v) Panton-Valentine leukocidin (pvl), vi) methicillin resistance (mecA), and vii) vancomycin resistance (vanA). Following successful validation using 255 reference bacterial strains, applicability to analyse clinical samples was evaluated by direct amplification in spiked blood cultures (n = 89) which returned 100 % specificity, negative and positive predictive values. The new assay has LoD of 1.0x103 CFU/mL for the 16S rRNA marker and 1.0x104 CFU/mL for six other markers and completes cycling in less than one hour. Conclusion The speed, sensitivity (100 %), NPV (100 %) and PPV (100 %) suggest the new heptaplex PCR assay could be easily integrated into a routine diagnostic microbiology workflow. Detection of the cns marker allows for unique identification of CoNS in mono-microbial and in poly-microbial samples containing mixtures of CoNS and S. aureus without recourse to the conventional elimination approach which is ambiguous. In addition to the SA-CoNS differential diagnostic essence of the new assay, inclusion of vanA primers will allow microbiology laboratories to stay ahead of the emerging MRSA-VRSA evolution. To the best of our knowledge, the new heptaplex PCR assay is the most multiplexed among similar PCR-based assays for simultaneous detection of STAAR

Detecting imipenem resistance in Acinetobacter baumannii by automated systems (BD Phoenix, Microscan WalkAway, Vitek 2); high error rates with Microscan WalkAway

<p>Abstract</p> <p>Background</p> <p>Increasing reports of carbapenem resistant <it>Acinetobacter baumannii </it>infections are of serious concern. Reliable susceptibility testing results remains a critical issue for the clinical outcome. Automated systems are increasingly used for species identification and susceptibility testing. This study was organized to evaluate the accuracies of three widely used automated susceptibility testing methods for testing the imipenem susceptibilities of <it>A. baumannii </it>isolates, by comparing to the validated test methods.</p> <p>Methods</p> <p>Selected 112 clinical isolates of <it>A. baumanii </it>collected between January 2003 and May 2006 were tested to confirm imipenem susceptibility results. Strains were tested against imipenem by the reference broth microdilution (BMD), disk diffusion (DD), Etest, BD Phoenix, MicroScan WalkAway and Vitek 2 automated systems. Data were analysed by comparing the results from each test method to those produced by the reference BMD test.</p> <p>Results</p> <p>MicroScan performed true identification of all <it>A. baumannii </it>strains while Vitek 2 unidentified one strain, Phoenix unidentified two strains and misidentified two strains. Eighty seven of the strains (78%) were resistant to imipenem by BMD. Etest, Vitek 2 and BD Phoenix produced acceptable error rates when tested against imipenem. Etest showed the best performance with only two minor errors (1.8%). Vitek 2 produced eight minor errors(7.2%). BD Phoenix produced three major errors (2.8%). DD produced two very major errors (1.8%) (slightly higher (0.3%) than the acceptable limit) and three major errors (2.7%). MicroScan showed the worst performance in susceptibility testing with unacceptable error rates; 28 very major (25%) and 50 minor errors (44.6%).</p> <p>Conclusion</p> <p>Reporting errors for <it>A. baumannii </it>against imipenem do exist in susceptibility testing systems. We suggest clinical laboratories using MicroScan system for routine use should consider using a second, independent antimicrobial susceptibility testing method to validate imipenem susceptibility. Etest, whereever available, may be used as an easy method to confirm imipenem susceptibility.</p

Infusions and decoctions of Castanea sativa flowers as effective antitumor and antimicrobial matrices

Chestnut trees are one of the most important crops in the north-eastern part of Portugal, representing millions of euros of yearly income. There are many ancestral claims of the health benefits of the consumption of chestnut flowers in infusions that remain unproven. In this manuscript, the antitumor and antimicrobial potential of chestnut flowers from two cultivars, Judia and Longal, extracted through infusions and decoctions are reported. In terms of antitumor activity, the most sensitive cell lines were HepG2 and HCT15 with the cultivar Judia showing higher activity for HCT15 and Longal for HepG2, regardless of the extraction methods. Regarding the antibacterial activity of the extracts, decoctions proved to be more effective with lower minimum inhibition concentrations, while infusions were better in terms of antifungal activity. The good overall antimicrobial activity could justify the inclusion of the flowers in food chain processing to act as a natural antimicrobial. Furthermore, the results corroborate some of the ancestral claims of the consumption of these flowers. (C) 2014 Elsevier B.V. All rights reserved.The authors are grateful to PRODER Project No 46577 - PlantLact and to the Foundation for Science and Technology (FCT, Portugal) for financial support to the research center CIMO (Pest-OE/AGR/UI0690/2011) and Ricardo Calhelha's grant (SFRH/BPD/68344/2010), and to the Serbian Ministry of Education and Science for the Science and Technological Development Grant No. 173032

Evaluation of the best method to assess antibiotic potentiation by phytochemicals against staphylococcus aureus

The increasing occurrence of bacterial resistance to antibiotics has now reached a critical level. Finding antibiotic coadjuvants capable to inhibit the bacterial resistance mechanisms would be a valuable mid-term solution, until new classes of antibiotics are discovered. Selected plant alkaloids were combined with 5 antibiotics against 10 Staphylococcus aureus strains, including strains expressing distinct efflux pumps and methicillin-resistant S. aureus strains. The efficacy of each combination was assessed using the microdilution checkerboard, time-kill, Etest, and disc diffusion methods. The cytotoxicity of the alkaloids was evaluated in a mouse fibroblast cell line. Potentiation was obtained in 6% of all 190 combinations, especially with the combination of: ciprofloxacin with reserpine (RES), pyrrolidine (PYR), and quinine (QUIN); tetracycline with RES; and erythromycin with PYR. The highest cytotoxicity values were found for QUIN (half maximal inhibitory concentration [IC50] = 25 ± 2.2 mg/L) and theophylline (IC50 = 100 ± 4.7 mg/L).The authors are very grateful to Professor Simon Gibbons (Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, UCL School of Pharmacy, London) for providing some of the bacterial strains. This work was supported by Operational Programme for Competitiveness Factors - COMPETE and by FCT Portuguese Foundation for Science and Technology through Projects Bioresist - PTDC/EBB-EBI/105085/2008; Phytodisinfectants - PTDC/DTP-SAP/1078/2012 and the PhD grants awarded to Ana Abreu (SFRH/BD/84393/2012) and Anabela Borges (SFRH/BD/63398/2009)

Spread of a highly mucoid Streptococcus pyogenes emm3/ST15 clone

<p>Abstract</p> <p>Background</p> <p>Hyaluronic acid capsule plays a key role in <it>Streptococcus pyogenes </it>virulence. Circulation of mucoid or highly encapsulated strains has been related to rheumatic fever epidemics and invasive disease in several countries. In 2009, an outbreak of mucoid <it>S. pyogenes </it>isolates was detected in northern Spain. The aim of the study was to describe clinical and molecular characteristics of mucoid strains causing this outbreak and to compare them with a sample of non-mucoid <it>S. pyogenes </it>isolates obtained during the same period of time.</p> <p>Methods</p> <p>All <it>S. pyogenes </it>isolates with a mucoid colony morphology (n = 132), 10% of non-mucoid (n = 144) and all invasive <it>S. pyogenes </it>isolates (n = 7) obtained in 2009 were included. Characterization was performed by T-agglutination, <it>emm </it>typing, pulsed field gel electrophoresis and multilocus sequence typing.</p> <p>Results</p> <p>One clone characterized as <it>emm</it>3.1/ST15 comprised 98.5% (n = 130) of all mucoid isolates. Subjects of all ages were affected. Main clinical manifestations were pharyngitis and scarlet fever, but this clone also caused invasive disease: two cases of streptococcal toxic shock syndrome, one arthritis, and one celullitis with a fatal outcome. Mucoid isolates were more prone to cause invasive disease than non-mucoid isolates (p = 0.001).</p> <p>Conclusions</p> <p>Although no acute rheumatic fever cases were detected, the most worrisome characteristics of this clone were the success for causing invasive disease and the merge of two virulent features: the serotype, <it>emm</it>3, and capsule hyper-production, expressed as a mucoid morphology.</p