Species-driven interpretation guidelines in case of a single-sampling strategy for blood culture.

The purpose of this paper is to define guidelines to interpret positive blood cultures (BCs) to distinguish bloodstream infection (BSI) from contamination in BCs drawn with a single venipuncture. During a 2-year period, each positive BC set (comprising six bottles from a single venipuncture) was prospectively categorised by clinicians, bacteriologists and hospital epidemiologists as BSI or contamination. For each case, the number of positive bottles per set, results from Gram staining and microorganism identification were analysed in order to define interpretation guidelines. We analysed 940 positive BC sets. The BSI rate in monomicrobial BC sets was positively correlated with the number of positive bottles. The positive predictive value was 88% with one and 100% with ≥2 positive bottles for Escherichia coli; 100% for Staphylococcus aureus, Pseudomonas and Candida spp., regardless of the number of positive bottles; 3.5%, 61.1%, 78.9% and 100% for coagulase-negative staphylococci (CoNS) with one, two, three and ≥4 positive bottles, respectively. Using a single-sampling strategy, interpretation guidelines for monomicrobial positive BCs are based on the number of positive bottles per set, results from Gram staining and microorganism identification: ≥4 positive bottles (≥2 with Gram-negative bacilli) always led to a diagnosis of BSI. The CoNS BSI rate positively correlates with the number of positive bottles.

16S rRNA sequencing in routine bacterial identification: a 30-month experiment.

Accurate identification of bacterial isolates is an essential task in clinical microbiology. Phenotypic methods are time-consuming and either fail to identify some bacteria such as Gram-positive rods entirely or at least fail to do so in some clinical situations. 16S rDNA sequencing is a recent method of identification which offers a useful alternative. In this study, we investigate the usefulness of this method for identifying a range of bacteria in a clinical laboratory under routine conditions. Over a period of 30 months, 683 isolates were obtained from clinical specimens, sequenced and analysed. For 568 of these isolates (83.1%), the sequence provided species level identification. For 108 isolates (15.8%), the identification was limited to the genus level, and for 7 isolates (1%), the sequence remained unidentifiable by 16S rDNA sequence analysis. For the isolates identified only to the genus level, the 16S rDNA approach failed to identify bacteria to the taxonomic level for 3 reasons: failure to differentiate between species in 72 isolates (66%), the lack of any closely related sequence in the database for 15 isolates (13.8%) and the presence of more than 1% of undetermined position in the sequence for 13 isolates (12%).

[From prevalence to predictive values: considerations on the antimicrobial susceptibility testing dealing with change of susceptibility rates]. / De la prévalence aux valeurs prédictives: l'antibiogramme face à l'évolution de la résistance aux antibiotiques.

The aim of the study was to quantify the influence of the variation of the susceptibility prevalence (frequencies of susceptible, intermediate and resistant isolates) on the predictive value of antimicrobial susceptibility testing reports performed with disk diffusion method, under several scenarios of distinct susceptibility prevalence. Prevalence variation effect was assessed through a modeling approach that enabled to take into account the technical variability and to control prevalence through scenarios. Results show how the prevalence impacts on the disk diffusion performance with level of discrepancies varying with prevalence. The phenomenon may be quantitatively noteworthy for some antimicrobial agents considering the prevalence recorded with time or location, leading to lowered performances. For instance, with amoxicillin+clavulanic acid, predictive values of susceptible and resistant reports varied respectively from 70 to 96 and from 33 to 97 %. For a 18-mm diameter, the probability the isolate is truly susceptible varied from 16 to 73 % according to the prevalence tested. Characteristic of the prevalence effect are described and consequences on zone diameter breakpoint policy raised. They include to (i) reevaluate disk diffusion breakpoint consistency when the prevalence impact is noteworthy and (ii) estimate the consequences of an international harmonized breakpoint policy on prediction quality and appropriate patient management.

A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model.

Advances in DNA sequencing and the increasing number of sequences available in databases have greatly enhanced the bacterial identification process. Several species within the genus Mycobacterium cause serious human and animal diseases. In order to assess their relative positions in the evolutionary process, four gene fragments, from the 16S rRNA (564 bp), hsp65 (420 bp), rpoB (396 bp) and sod (408 bp) genes, were sequenced from 97 strains, including all available type strains of the genus Mycobacterium. The results demonstrate that, in this case, the concatenation of different genes allows significant increases in the power of discrimination and the robustness of the phylogenetic tree. The sequential and/or combined use of sequences of several genes makes it possible to refine the phylogenetic approach and provides a molecular basis for accurate species identification.

BIBI, a bioinformatics bacterial identification tool.

BIBI was designed to automate DNA sequence analysis for bacterial identification in the clinical field. BIBI relies on the use of BLAST and CLUSTAL W programs applied to different subsets of sequences extracted from GenBank. These sequences are filtered and stored in a new database, which is adapted to bacterial identification.

Modeling the lag time of Listeria monocytogenes from viable count enumeration and optical density data.

The following two factors significantly influence estimates of the maximum specific growth rate ( micro (max)) and the lag-phase duration (lambda): (i) the technique used to monitor bacterial growth and (ii) the model fitted to estimate parameters. In this study, nine strains of Listeria monocytogenes were monitored simultaneously by optical density (OD) analysis and by viable count enumeration (VCE) analysis. Four usual growth models were fitted to our data, and estimates of growth parameters were compared from one model to another and from one monitoring technique to another. Our results show that growth parameter estimates depended on the model used to fit data, whereas there were no systematic variations in the estimates of micro (max) and lambda when the estimates were based on OD data instead of VCE data. By studying the evolution of OD and VCE simultaneously, we found that while log OD/VCE remained constant for some of our experiments, a visible linear increase occurred during the lag phase for other experiments. We developed a global model that fits both OD and VCE data. This model enabled us to detect for some of our strains an increase in OD during the lag phase. If not taken into account, this phenomenon may lead to an underestimate of lambda.

A mathematical model describing the thermal virus inactivation.

A new mathematical model is proposed to describe the inactivation of viruses at different temperatures. This model takes into account the exponential decrease of the viral titer with time, the inactivation rate being an exponential function of the temperature. A one-step non-linear regression was used to fit oral poliovirus vaccine (OPV) experimental data. In one of the applications of the model, we illustrate the use of our model to compare the accelerated degradation test of OPV new formulations to standard OPV. Such a model is both simple and convenient to use. It should be a useful tool in optimizing formulations for live viral vaccines.

Fitness and competitive growth advantage of new gentamicin-susceptible MRSA clones spreading in French hospitals.

Since 1991, new epidemic methicillin-resistant Staphylococcus aureus (MRSA) strains characterized by the unexpected reappearance of heterogeneous phenotypic expression of resistance to methicillin and by susceptibility to gentamicin and various other antibiotics (GS-MRSA) have been reported in France. GS-MRSA strains have progressively replaced MRSA clones expressing homogeneous resistance to methicillin and resistance to gentamicin (GR-MRSA). In this study, we investigated the physiological characteristics of these new clones. In particular, we evaluated and compared the maximal growth rate and the deduced generation times (related to fitness of strains) of the major French epidemic MRSA clones. The population studied consisted of 79 isolates including (i) GR-MRSA that comprised six different types on the basis of PFGE; (ii) GS-MRSA the majority of which clustered into two PFGE types, A1 (usually resistant to erythromycin) and B (usually susceptible to erythromycin); (iii) methicillin-susceptible S. aureus (MSSA). GS-MRSA-A1 and MSSA strains were shown to have a significant fitness benefit (about 20%) with shorter generation times (theta = 23.7 +/- 0.1 and 22.9 +/- 0.05 min, respectively) than GR-MRSA and GS-MRSA-B strains (theta = 30.3 +/- 0.2 and 32.5 +/- 0.5 min, respectively). These data suggest that a link exists between genetic patterns, resistance profiles and physiological properties. In vitro competitive experiments indicated that GS-MRSA- A1 strains were able to rapidly outgrow GR-MRSA strains. The growth advantage observed should be taken into account in understanding the spread of some new clones of MRSA.

Characterization of unexpected growth of Escherichia coli O157:H7 by modeling.

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.

A model describing the relationship between regrowth lag time and mild temperature increase for Listeria monocytogenes.

In order to comply with the consumer demand for ready-to-eat and look 'fresh' products, mild heat treatment will be used more and more in the agrofood industry. Nonetheless there is no tool to define the most appropriate mild heat treatment. In order to build this tool, it is necessary to study and describe the response of a bacterial population to a mild increase in temperature, from the dynamic point of view. The response to a mild increase in temperature, defined by stress duration and temperature, consisted in a mortality phase followed by the lag time of the survivors and their exponential growth. The effect of the mild increase in temperature on the mortality phase was described in a previous paper (Bréand et al., Int. J. Food Microbiol., in press). The effect of the stress duration on the lag was presented in a previous paper (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). In particular, the biphasic relationship between the lag and the stress duration was observed and modelled with a four parameter nonlinear model: the primary model (Bréand et al., Int. J. Food Microbiol. 38 (1997) 157-167). The study presented in this paper deals with the effect of the stress temperature on the biphasic relationship between the lag time and the stress duration. The secondary models describing the effect of the stress temperature on this biphasic relationship, were empirically built from our experimental data concerning Listeria monocytogenes. This work pointed out that the higher the stress temperature, the narrower the range of stress duration for which the lag time increased. Since the primary and the secondary models of the lag time were available, the global model describing the effect of the mild increase duration and temperature directly on the lag was fitted. This model allowed an improvement of the parameter estimator precision. The potential contribution in mild heat treatment optimization of this global model and the one built for the mortality phase (Bréand et al., Int. J. Food Microbiol., in press) is discussed.

Model of the influence of time and mild temperature on Listeria monocytogenes nonlinear survival curves.

Heat treatment has long been regarded as one of the most widely used and most effective means of destroying pathogens in food. Up to now the linear relationship between the death rate and the temperature has been used when choosing the best heat treatment to apply. However, the information given by this linear relationship is no longer sufficient when nonlinear survival curves are observed. Consequently, the agri-food industry needs a tool to choose the best mild heat treatment to apply in the case of nonlinear survival curves. This study deals with the temperature-induced death of Listeria monocytogenes CIP 7831 in the stationary phase of growth. Eleven temperatures were tested. With the proposed primary and secondary models good fits of our data were obtained. A model describing both the effect of the duration of treatment and the temperature on the logarithm of the number of survivors was then built. A clear increase in the precision of the estimation of the parameters was obtained with this model. Moreover, with this model a new graphical strategy to choose a mild heat increase regarding a maximal survivor number has been proposed.

Homogeneous two-site immunometric assay kinetics as a theoretical tool for data analysis.

The easily accessible kinetics of a new homogeneous two-site fluorometric immunoassay for prolactin was studied, in order to determine its usefulness for assay data reduction and optimization. The combined use of a simple descriptive model fitted to experimental data and a mechanistic model to simulate the kinetics revealed that (i) the kinetics curve presented an early inflexion point. Its time of occurrence was constant as long as the antigen concentration was below the smallest antibody concentration and decreased to zero for higher concentrations. It may therefore be used as an indicator of hooked samples. (ii) The kinetics steepest slope was correlated with antigen concentration. Its use as a dose-response curve variable would allow higher concentrations to be assayed than with the classical end-point dose-response curve. The results suggest that control and exploitation of kinetic parameters could help to improve the rapidity, analytical range, and reliability of homogeneous two-site immunometric assays.

Simple relationship between acid dissociation constant and minimal pH for microbial growth in laboratory medium.

A simple relationship was observed in growth medium, between the dissociation constant (via the pKa) of the acid used to control pH and the minimum pH at which Salmonellae and Escherichia coli initiate growth. From this new relationship, a simple method was proposed to predict the minimum growth pH for a given strain and different acid types. This method, illustrated on Listeria monocytogenes, would merely require the knowledge of two minimum pH values, one for a strong acid (e.g. hydrochloric acid) and one for a weak acid (e.g. acetic or propionic acid). From these two values, it seems possible to estimate for a given growth medium, the minimum pH value for any other acid within the defined pKa range.

A descriptive model for the kinetics of a homogeneous fluorometric immunoassay.

A descriptive mathematical model was chosen to fit the antigen-antibody association kinetics of a new homogeneous immunometric assay for prolactin, involving time-resolved fluorescence detection (TRACE technology, Time Resolved Amplified Cryptate Emission). We paid special attention to the methodology and criteria applied, to yield a convenient and statistically valid model, designed to allow potential exploitation of kinetic information in the data processing of the assay. We compared specific parameterizations of an hyperbolic model, the Gompertz, and the monomolecular models on the basis of morphological considerations, a statistical analysis of fit, and an assessment of the parameters estimation quality, over a wide range of antigen concentrations. The monomolecular model gave the best fit, and the most precise and stable estimation of its parameters. The study of parameter properties confirmed this choice.

Reappraisal of the effect of temperature on the growth kinetics of Aeromonas salmonicida.

The effect of temperature (1-34 degrees C) on the maximum specific growth rate of Aeromonas salmonicida could not be described by the classical growth models; for some strains, two optimal temperatures at 23 degrees C and 30 degrees C were observed, as well as an unexpected increase in the pseudolag time above 27 degrees C. This could be explained by the presence of two subsets, notably S-layer+ and S-layer- sub-populations. The A- cells had higher growth parameters (Topt and mu opt) than the A+ cells and were selected by subcultures above 30 degrees C. Yet the relative proportion of A+ cells did not explain all the variation of mu max versus temperature, and the growth kinetics of an Aer. salmonicida isolate remained unpredictable.

A model describing the relationship between lag time and mild temperature increase duration.

When a bacterial population undergoes an unfavourable increase in temperature for a given duration, called stress duration, a death phase followed by a lag and a growth phase are observed. The lag phase is actually of great interest in regard to foodstuff safety in choosing a suitable protocol for the detection of microorganisms which have undergone a mild heat treatment. The extension of lag time with the severity of the increase in temperature has been highlighted by previous papers. Our experimental results concerning Listeria monocytogenes and Escherichia coli revealed that a two phase relationship between lag time and stress duration is observed for a specific increase in temperature. The first phase consists of an increase in lag time up to a peak; the second one consists of a decrease from this peak to a steady threshold. The mathematical model presented, describing the relationship between lag time and stress duration was empirically built from our experimental data concerning L. monocytogenes and E. coli. The fit evaluation carried out led us to consider this model as a good description of the relationship studied. The potential contribution of our model in heat treatment optimization is discussed.

Application of a modified disc diffusion technique to antimicrobial susceptibility testing of Vibrio anguillarum and Aeromonas salmonicida clinical isolates.

Two techniques for antimicrobial susceptibility testing of Vibrio anguillarum and Aeromonas salmonicida strains were compared. The first method was the reference test that determines Minimal Inhibitory Concentrations (MIC); the second was a modified diffusion test that measures the Inhibitory Concentrations in Diffusion (ICD) by carrying out the diffusion test with five discs of differing contents. ICD measurement was not applicable for the susceptibility testing of oxytetracycline and sulfadimethoxine. On the other hand, a good correlation between the MICs and the ICDs was observed for oxolinic acid, sarafloxacin, chloramphenicol and trimethoprim. Moreover, the ICD values were close to those obtained for the MIC values. A. salmonicida resistant strains were detected by ICD determination. Thus, ICD could be used instead of MIC for oxolinic acid, sarafloxacin, trimethoprim and chloramphenicol susceptibility testings. The ICD technique is easy to carry out and is not dependent on the growth characteristics of bacteria.

An economic approach to the MIC.

The determination of the Inhibitory Concentration in Diffusion (ICD) is proposed as an alternative to the agar dilution Minimal Inhibitory Concentration (MIC) that is time-consuming and cumbersome for routine use. Based on the technique of the disk diffusion test, it consists in calculating a continuous variable, the ICD, corresponding to the antibiotic concentration in the agar at the edge of the inhibition zone. Six antibiotics were tested (ampicillin, cefotaxime, erythromycin, gentamicin, nalidixic acid and rifampicin) each against 17 to 51 strains of enterobacteria, Staphylococcus aureus and Enterococcus spp. and six other antibiotics (cefsulodin, ceftazidime, imipenem, piperacillin, ticarcillin and tobramycin), against 13 to 25 strains of Pseudomonas aeruginosa. A total of 284 antibiotic-strain combinations were tested. Three different antibiotic charges were obtained for each antibiotic by cutting commercial disks in two and four equal pieces. The ICD was calculated for each strain from the size of inhibition zones around a full disk, a half and a quarter of a disk. Concurrently, the MIC was performed, using a conventional agar dilution method. There was a good correlation between the two methods and reproducibility for the ICD proved to be correct. This reliable technique is very efficient both in terms of laboratory time and cost of materials and could be proposed for widespread use in clinical laboratories.