The influence of heteroresistance on minimum inhibitory concentration (MIC), investigated using weak-acid stress in food spoilage yeasts

Populations of microbial cells may resist environmental stress by maintaining a high population-median resistance (IC50) or, potentially, a high variability in resistance between individual cells (heteroresistance); where heteroresistance would allow certain cells to resist high stress, provided the population was sufficiently large to include resistant cells. This study sets out to test the hypothesis that both IC50 and heteroresistance may contribute to conventional minimal-inhibitory-concentration (MIC) determinations, using the example of spoilage-yeast resistance to the preservative sorbic acid. Across a panel of 26 diverse yeast species, both heteroresistance and particularly IC50 were positively correlated with predicted MIC. A focused panel of 29 different isolates of a particular spoilage yeast was also examined (isolates previously recorded as Zygosaccharomyces bailii, but genome resequencing revealing that several were in fact hybrid species, Z. parabailii and Z. pseudobailii). Applying a novel high-throughput assay for heteroresistance, it was found that IC50 but not heteroresistance was positively correlated with predicted MIC when considered across all isolates of this panel, but the heteroresistance-MIC interaction differed for the individual Zygosaccharomyces subspecies. Z. pseudobailii exhibited higher heteroresistance than Z. parabailii whereas the reverse was true for IC50, suggesting possible alternative strategies for achieving high MIC between subspecies. This work highlights the limitations of conventional MIC measurements due to the effect of heteroresistance in certain organisms, as the measured resistance can vary markedly with population (inoculum) size.Importance Food spoilage by fungi is a leading cause of food waste, with specialised food spoilage yeasts capable of growth at preservative concentrations above the legal limit, in part due to heteroresistance allowing small subpopulations of cells to exhibit extreme preservative resistance. Whereas heteroresistance has been characterised in numerous ecological contexts, measuring this phenotype systematically and assessing its importance are not encompassed by conventional assay methods. The development here of a high-throughput method for measuring heteroresistance, amenable to automation, addresses this issue and has enabled characterisation of the contribution that heteroresistance may make to conventional MIC measurements. We used the example of sorbic acid heteroresistance in spoilage yeasts like Zygosaccharomyces spp, but the approach is relevant to other fungi and other inhibitors, including antifungals. The work shows how median resistance, heteroresistance and inoculum size should all be considered when selecting appropriate inhibitor doses in real-world antimicrobial applications such as food preservation

Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group.

One hundred representative strains of Bacillus cereus were selected from a total collection of 372 B. cereus strains using two typing methods (RAPD and FT-IR) to investigate if emetic toxin-producing hazardous B. cereus strains possess characteristic growth and heat resistance profiles. The strains were classified into three groups: emetic toxin (cereulide)-producing strains (n=17), strains connected to diarrheal foodborne outbreaks (n=40) and food-environment strains (n=43), these latter not producing the emetic toxin. Our study revealed a shift in growth limits towards higher temperatures for the emetic strains, regardless of their origin. None of the emetic toxin-producing strains were able to grow below 10 degrees Celsius. In contrast, 11% (9 food-environment strains) out of the 83 non-emetic toxin-producing strains were able to grow at 4 degrees Celsius and 49% at 7 degrees Celsius (28 diarrheal and 13 food-environment strains). non-emetic toxin-producing strains. All emetic toxin-producing strains were able to grow at 48 degrees Celsius, but only 39% (16 diarrheal and 16 food-environment strains) of the non-emetic toxin-producing strains grew at this temperature. Spores from the emetic toxin-producing strains showed, on average, a higher heat resistance at 90 degrees Celsius and a lower germination, particularly at 7 degrees Celsius, than spores from the other strains. No difference between the three groups in their growth kinetics at 24 degrees Celsius, 37 degrees Celsius, and pH 5.0, 7.0, and 8.0 was observed. Our survey shows that emetic toxin-producing strains of B. cereus have distinct characteristics, which could have important implication for the risk assessment of the emetic type of B. cereus caused food poisoning. For instance, emetic strains still represent a special risk in heat-processed foods or preheated foods that are kept warm (in restaurants and cafeterias), but should not pose a risk in refrigerated foods

Phenotypic Prediction: Linking in vitro Virulence to the Genomics of 59 Salmonella enterica Strains

The increased availability of whole-genome-sequencing techniques generates a wealth of DNA data on numerous organisms, including foodborne pathogens such as Salmonella. However, how these data can be used to improve microbial risk assessment and understanding of Salmonella epidemiology remains a challenge. The aim of this study was to assess variability in in vitro virulence and genetic characteristics between and within different serovars. The phenotypic behavior of 59 strains of 32 different Salmonella enterica serovars from animal, human and food origin was assessed in an in vitro gastro-intestinal tract (GIT) system and they were analyzed for the presence of 233 putative virulence genes as markers for phenotypic prediction. The probability of in vitro infection, P(inf), defined as the fraction of infectious cells passing from inoculation to host cell invasion at the last stage of the GIT system, was interpreted as the in vitro virulence. Results showed that the (average) P(inf) of Salmonella serovars ranged from 5.3E-05 (S. Kedougou) to 5.2E-01 (S. Typhimurium). In general, a higher P(inf) on serovar level corresponded to higher reported human incidence from epidemiological reporting data. Of the 233 virulence genes investigated, only 101 showed variability in presence/absence among the strains. In vitro P(inf) was found to be positively associated with the presence of specific plasmid related virulence genes (mig-5, pef, rck, and spv). However, not all serovars with a relatively high P(inf), > 1E-02, could be linked with these specific genes. Moreover, some outbreak related strains (S. Heidelberg and S. Thompson) did not reveal this association with P(inf). No clear association with in vitro virulence P(inf) was identified when grouping serovars with the same virulence gene profile (virulence plasmid, Typhoid toxin, peg operon and stk operon). This study shows that the in vitro P(inf) variation among individual strains from the same serovar is larger than that found between serovars. Therefore, ranking P(inf) of S. enterica on serovar level alone, or in combination with a serovar specific virulence gene profile, cannot be recommended. The attribution of single biological phenomena to individual strains or serovars is not sufficient to improve the hazard characterization for S. enterica. Future microbial risk assessments, including virulence gene profiles, require a systematic approach linked to epidemiological studies rather than revealing differences in characteristics on serovar level alone

A quantitative approach towards a better understanding of the dynamics of Salmonella spp. in a pork slaughter-line.

Pork contributes significantly to the public health disease burden caused by Salmonella infections. During the slaughter process pig carcasses can become contaminated with Salmonella. Contamination at the slaughter-line is initiated by pigs carrying Salmonella on their skin or in their faeces. Another contamination route could be resident flora present on the slaughter equipment. To unravel the contribution of these two potential sources of Salmonella a quantitative study was conducted. Process equipment (belly openers and carcass splitters), faeces and carcasses (skin and cutting surfaces) along the slaughter-line were sampled at 11 sampling days spanning a period of 4 months. Most samples taken directly after killing were positive for Salmonella. On 96.6% of the skin samples Salmonella was identified, whereas a lower number of animals tested positive in their rectum (62.5%). The prevalence of Salmonella clearly declined on the carcasses at the re-work station, either on the cut section or on the skin of the carcass or both (35.9%). Throughout the sampling period of the slaughter-line the total number of Salmonella per animal was almost 2log lower at the re-work station in comparison to directly after slaughter. Seven different serovars were identified during the study with S. Derby (41%) and S. Typhimurium (29%) as the most prominent types. A recurring S. Rissen contamination of one of the carcass splitters indicated the presence of an endemic 'house flora' in the slaughterhouse studied. On many instances several serotypes per individual sample were found. The enumeration of Salmonella and the genotyping data gave unique insight in the dynamics of transmission of this pathogen in a slaughter-line. The data of the presented study support the hypothesis that resident flora on slaughter equipment was a relevant source for contamination of pork

A theory on the vertical dispersal of splash-borne pathogen units influenced by arable crop characteristics

An analytical mechanistic model was proposed to study the vertical spread of splash-borne spores in arable crop canopies. Three crop types were considered, with different LAI distributions. The influences of crop characteristics and rain properties on vertical spread were investigated. The LAI affected the amount of rain being intercepted by the canopy and the vertical displacement of splashed spores. Splash dispersal was concentrated in the upper canopy layers in a crop having LAI constant or increasing with height. Splash probabilities were greatest and most spores were intercepted in the layers just beneath the upper layers in a crop having LAI decreasing with height

Validation of EN ISO 6579-1 - Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 1 detection of Salmonella spp.

The European and International Standard method for the detection of Salmonella spp. in samples from the primary production stage, EN ISO 6579:2002/Amd.1:2007, was validated by an interlaboratory study in the frame of Mandate M/381, ordered by the European Commission and accepted by the European Standardisation Organisation (CEN). In addition to this study, results from two interlaboratory studies organised earlier by the European Union Reference Laboratory (EURL) for Salmonella were used for determination of the performance characteristics. Parallel to the performance evaluation for the Mandate, the revision of EN ISO 6579:2002 started. Part of this revision was the incorporation of the standardised method for detection of Salmonella in samples from the primary production stage (EN ISO 6579:2002/Amd.1:2007) and its performance characteristics in the new part 1 of EN ISO 6579. The 2002 version of EN ISO 6579 already contained performance characteristics for the detection of Salmonella in food samples, but LOD50 values (contamination level at which 50% of the samples are found positive) were not yet included. To be in line with the performance characteristics determined for detection of Salmonella spp. in samples from the primary production stage, LOD50 values for detection of Salmonella in food samples were calculated from the raw data of the validation studies performed in 2000. In this paper, the performance characteristics of EN ISO 6579-1:2017 are determined based not only on the results of the interlaboratory study carried out in 2013 under the Mandate, but also on several other interlaboratory studies. These performance characteristics consist of specificity, sensitivity and LOD50

Critical comparison of statistical methods for quantifying variability and uncertainty of microbial responses from experimental data

Variability and uncertainty are important factors for quantitative microbiological risk assessment (QMRA). In this context, variability refers to inherent sources of variation, whereas uncertainty refers to imprecise knowledge or lack of it. In this work we compare three statistical methods to estimate variability in the kinetic parameters of microbial populations: mixed-effect models, multilevel Bayesian models, and a simplified algebraic method previously suggested. We use two case studies that analyse the influence of three levels of variability: (1) between-strain variability (different strains of the same species), (2) within-strain variability (biologically independent reproductions of the same strain) and, at the most nested level, (3) experimental variability (species independent technical lab variability resulting in uncertainty about the population characteristic of interest) on the growth and inactivation of Listeria monocytogenes. We demonstrate that the algebraic method, although relatively easy to use, overestimates the contribution of between-strain and within-strain variability due to the propagation of experimental variability in the nested experimental design. The magnitude of the bias is proportional to the variance of the lower levels and inversely proportional to the number of repetitions. This bias was very relevant in the case study related to growth, whereas for the case study on inactivation the resulting insights in variability were practically independent of the method used. The mixed-effects model and the multilevel Bayesian models calculate unbiased estimates for all levels of variability in all the cases tested. Consequently, we recommend using the algebraic method for initial screenings due to its simplicity. However, to obtain parameter estimates for QMRA, the more complex methods should generally be used to obtain unbiased estimates

Comparative Exposure Assessment of ESBL-Producing Escherichia coli through Meat Consumption.

The presence of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC (pAmpC) producing Escherichia coli (EEC) in food animals, especially broilers, has become a major public health concern. The aim of the present study was to quantify the EEC exposure of humans in The Netherlands through the consumption of meat from different food animals. Calculations were done with a simplified Quantitative Microbiological Risk Assessment (QMRA) model. The model took the effect of pre-retail processing, storage at the consumers home and preparation in the kitchen (cross-contamination and heating) on EEC numbers on/in the raw meat products into account. The contribution of beef products (78%) to the total EEC exposure of the Dutch population through the consumption of meat was much higher than for chicken (18%), pork (4.5%), veal (0.1%) and lamb (0%). After slaughter, chicken meat accounted for 97% of total EEC load on meat, but chicken meat experienced a relatively large effect of heating during food preparation. Exposure via consumption of filet americain (a minced beef product consumed raw) was predicted to be highest (61% of total EEC exposure), followed by chicken fillet (13%). It was estimated that only 18% of EEC exposure occurred via cross-contamination during preparation in the kitchen, which was the only route by which EEC survived for surface-contaminated products. Sensitivity analysis showed that model output is not sensitive for most parameters. However, EEC concentration on meat other than chicken meat was an important data gap. In conclusion, the model assessed that consumption of beef products led to a higher exposure to EEC than chicken products, although the prevalence of EEC on raw chicken meat was much higher than on beef. The (relative) risk of this exposure for public health is yet unknown given the lack of a modelling framework and of exposure studies for other potential transmission routes