A new method for normalized interpretation of antimicrobial resistance from disk test results for comparative purposes.

Objective To evaluate a calibration method for disk diffusion antibiotic susceptibility tests, using zone diameter values generated in the individual laboratory as the internal calibrator for combinations of antibiotic and bacterial species. Methods The high-zone side of zone histogram distributions was first analyzed by moving averages to determine the peak position of the susceptible population. The accumulated percentages of isolates for the high zone diameter values were calculated and converted into probit values. The normal distribution of the ideal population of susceptible strains was then determined by using the least-squares method for probit values against zone diameters, and the ideal population was thereby defined, including mean and standard deviation. Zone diameter values were obtained from laboratories at the Karolinska Hospital (KS) and Växjö Hospital (VX), and from two laboratories (LabA, LabB) in Argentina. The method relies on well standardized disk tests, but is independent of differences in MIC limits and zone breakpoints, and does not require the use of reference strains. Resistance was tentatively set at below 3 SD from the calculated, ideal mean zone diameter of the susceptible population. Results The method, called normalized interpretation of antimicrobial resistance, was tested on results from the KS and VX clinical microbiology laboratories, using the disk diffusion method for antimicrobial susceptibility tests, and for two bacterial species, Staphylococcus aureus and Escherichia coli. In total, 114 217 test results were included for the clinical isolates, and 3582 test results for control strains. The methodology at KS and VX followed the standard of the Swedish Reference Group for Antibiotics (SRGA). Zone diameter histograms for control strains were first analyzed to validate the procedure, and a comparison of actual means with the calculated means showed a correlation coefficient of r = 0.998. Results for clinical isolates at the two laboratories showed an excellent agreement for 54 of 57 combinations of antibiotic and bacterial species between normalized interpretations and the interpretations given by the laboratories. There were difficulties with E. coli and mecillinam, and S. aureus and tetracycline and rifampicin. The method was also tested on results from two laboratories using the NCCLS standard, and preliminary results showed very good agreement with quality-controlled laboratory interpretations. Conclusions The normalized resistance interpretation offers a new approach to comparative surveillance studies whereby the inhibition zone diameter results from disk tests in clinical laboratories can be used for calibration of the test

Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10

AbstractThe MIC wild-type (WT) distribution for Mycobacterium tuberculosis in BACTEC 960 MGIT is not defined, which may result in poor reproducibility for drug susceptibility testing (DST), as several DST methods with different breakpoints are in use. In a comparison between MGIT and Middlebrook 7H10 medium of seven first- and second-line drugs, including 133 MIC determinations of 15 WT isolates, we found an agreement of 91.7% within ± one MIC dilution step. The results confirm the agreement in MIC testing between 7H10 and MGIT and indicate that breakpoints could be harmonized in order to avoid misclassification

Invasive disease caused by Haemophilus influenzae in Sweden 1997–2009; evidence of increasing incidence and clinical burden of non‐type b strains

Introduction of a conjugated vaccine against encapsulated Haemophilus influenzae type b (Hib) has led to a dramatic reduction of invasive Hib disease. However, an increasing incidence of invasive disease by H. influenzae non‐type b has recently been reported. Non‐type b strains have been suggested to be opportunists in an invasive context, but information on clinical consequences and related medical conditions is scarce. In this retrospective study, all H. influenzae isolates ( n  =   410) from blood and cerebrospinal fluid in three metropolitan Swedish regions between 1997 and 2009 from a population of approximately 3 million individuals were identified. All available isolates were serotyped by PCR ( n  =   250). We observed a statistically significant increase in the incidence of invasive H. influenzae disease, ascribed to non‐typeable H. influenzae (NTHi) and encapsulated strains type f (Hif) in mainly individuals >60 years of age. The medical reports from a subset of 136 cases of invasive Haemophilus disease revealed that 48% of invasive NTHi cases and 59% of invasive Hif cases, respectively, met the criteria of severe sepsis or septic shock according to the ACCP/SCCM classification of sepsis grading. One‐fifth of invasive NTHi cases and more than one‐third of invasive Hif cases were admitted to intensive care units. Only 37% of patients with invasive non‐type b disease had evidence of immunocompromise, of which conditions related to impaired humoral immunity was the most common. The clinical burden of invasive non‐type b H. influenzae disease, measured as days of hospitalization/100 000 individuals at risk and year, increased significantly throughout the study period.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87052/1/j.1469-0691.2010.03417.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/87052/2/CLM_3417_sm_FigS1.pd

Impact of inter-lab variation on the estimation of epidemiological cut-off values for disc diffusion susceptibility test data for Aeromonas salmonicida

Peer-reviewed. Released with a Creative Commons Attribution Non-Commercial No Derivatives LicenseTwo laboratories investigated the susceptibility of 106 Aeromonas salmonicida strains (from Denmark, France, Ireland, Norway and Scotland) to erythromycin, gentamicin, oxytetracycline and oxolinic acid using the disc diffusion protocols (M42-A) published by the Clinical and Laboratory Standards Institute. In studies of susceptibility to florfenicol an additional 15 Canadian strains were included. Comparison of the data generated by the two laboratories demonstrated that for each disc both detected a similar pattern of distribution but that there was a significant numerical difference in the zone sizes they recorded. Analysis of the extent of this lateral shift between the data generated in two laboratories indicated that the application of a single laboratory-independent epidemiological cut-off value for each disc could result in disagreement between the laboratories as to whether a strain should be classified as wild-type or non wild-type. Normalised resistance interpretation was employed to generate epidemiological cut-off values from the data obtained by each laboratory. The use of these laboratory-specific cut-off values resulted in both laboratories achieving complete agreement as to the classification of all strains to all agents

A Simple & Convenient Solid Phase Synthesis of Bacterial Origin Octapeptide Sequence, Glu-Asp-Gly-Asn-Lys-Pro-Gly-Lys-OH

The repeating octapeptide sequence, Glu-Asp-Gly-Asn-Lys-Pro-Gly-Lys-OH derived from the glycoprotein found in Staphylococcus aureus cell wall is assembled by simple solid phase peptide synthesis methodology using a base labile linker

Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins

Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies

Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG1, the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 1011 affibody molecules. Four rounds of selection using three different mouse IgG1 mAbs as alternating targets resulted in the identification of binders with broad mIgG1 recognition and dissociation constants (KD) in the low nanomolar to low micromolar range. For one of the binders, denoted Zmab25, competition in binding to full length mIgG1 by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG1 Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG1 had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG1 nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Zmab25. This selective binding profile towards murine IgG1 was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant