Lüppo Ellerbroek

PCR inhibitors – occurrence, properties and removal

The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of sample types. However, the method is prone to inhibiting substances, which may be present in the analysed sample and which may affect the sensitivity of the assay or even lead to false‐negative results. The PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action. Some of them are predominantly found in specific types of samples thus necessitating matrix‐specific protocols for preparation of nucleic acids before PCR. A variety of protocols have been developed to remove the PCR inhibitors. This review focuses on the general properties of PCR inhibitors and their occurrence in specific matrices. Strategies for their removal from the sample and for quality control by assessing their influence on the individual PCR test are presented and discussed.

Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method

A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project (http://www.pcr.dk). Each laboratory analysed by the PCR a set of independent obtained presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole broiler carcass-rinse resulted in a detection limit of less than 5 cells per 25 g meat or 100 ml broiler rinse. A total of 435 samples from four countries, including pig carcass swabs (n = 285), whole broiler carcass-rinse (n = 25), various raw meat (n = 33), and environmental samples (n = 92) were investigated. The interlaboratory diagnostic accuracy, i.e. diagnostic specificity and sensitivity, was shown to be 97.5%. The co-amplification of an internal amplification control indicated possible inhibitory substances derived from the sample. This work can contribute to the quality assurance of PCR-based diagnostic methods and is currently proposed as international standard document.

Species distribution and antibiotic resistance patterns of enterococci isolated from food of animal origin in Germany

Presently, enterococci take the third place of bacterial pathogens associated with nosocomial infections, after staphylococci and Escherichia coli. Especially, the resistances of enterococci to several available antibiotics are threatening. We attempted to determine which species of enterococci could be found in food of animal origin and their significance according to their antibiotic resistances for human beings. From November 2000 to May 2002 we investigated 155 samples of food of animal origin bought in retail outlets in Germany: 27 samples of sausages, 19 of ham, 83 of minced meat, 26 of cheese. From these food samples we isolated 416 enterococcal strains. The most frequent species was Enterococcus faecalis (299 strains); furthermore, we found Enterococcus faecium (54 strains), Enterococcus durans together with Enterococcus hirae (24 strains), Enterococcus casseliflavus (22 strains), Enterococcus avium (9 strains) and Enterococcus gallinarum (8 strains). We focused on the resistance patterns of 118 selected E. faecium and E. faecalis strains to 13 antimicrobial active agents (ampicillin, amoxicillin/clavulanic acid, avilamycin, chloramphenicol, enrofloxacin, erythromycin, flavomycin, gentamicin, penicillin, quinupristin/dalfopristin, teicoplanin, tetracycline and vancomycin). From the clinical point of view, the situation of antibiotic resistance to the examined antimicrobial agents seemed to be favourable. The investigated strains were sensitive to ampicillin and amoxicillin/clavulanic acid. These antibiotics are, in combination with an aminoglycoside, for example gentamicin, agents of choice for the treatment of enterococcal infections in human medicine. Only one E. faecium strain was resistant to penicillin, while all strains were sensitive to the glycopeptide antibiotics, vancomycin and teicoplanin. Resistances found against the antibiotics, tetracycline, quinupristin/dalfopristin and erythromycin, are causes for concern.

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle

BackgroundCampylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans.ResultsHere, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified.ConclusionsThe genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments.

Analysis of risk factors for Campylobacter species infection in broiler flocks

We have sampled 146 German broiler flocks at slaughter from May 2004 to April 2005 to determine the prevalence of Campylobacter spp. and to investigate risk factors for the presence of Campylobacter spp. at flock level. Cecal samples were tested in accordance to ISO 10727, and potential risk factors were analyzed using farm- and flock-specific information obtained from questionnaires. Of the flocks tested, 44% were Campylobacter-positive, and most were infected with Campylobacter jejuni. Higher Campylobacter prevalence was found during the months of May to October (52%). Using farm- and flock-specific information obtained from questionnaires, we identified 3 risk factors for Campylobacter colonization. Campylobacter prevalence was significantly higher in flocks from free-range and organic farms, in flocks with a size up to 15,000 birds and with more than 25,000 birds, and in flocks using nipple drinkers with trays. We found no evidence of an effect of slaughter age, time interval between successive flocks, hygiene measures, number of broiler houses on a farm, partial slaughter, source of water supply, and number of farm employees on the Campylobacter infection rate.

Application of a Swab Sampling Method for the Detection of Norovirus and Rotavirus on Artificially Contaminated Food and Environmental Surfaces

Noroviruses and rotaviruses are the leading causes of non-bacterial gastroenteritis in humans worldwide. Virus-contaminated food and surfaces represent an important risk to public health. However, established detection methods for the viruses in food products are laborious and time-consuming. Here, we describe a detailed swabbing protocol combined with real-time RT-PCR for norovirus and rotavirus detection on artificially contaminated food and environmental surfaces. Recovery rates between 2 and 78% for norovirus and between 8 and 42% for rotavirus were determined for contaminated food surfaces of apple, pepper, cooked ham and salami. From contaminated environmental surfaces (stainless steel, ceramic plate, polyethylene, wood), recovery rates between 26 and 52% (norovirus) and between 10 and 58% (rotavirus) were determined. The results demonstrate the suitability of the swab sample method for virus detection on food and environmental surfaces. Compared to other methods, it is easy to perform and significantly time-saving, predestining it for routine testing.

Detection of hepatitis E virus RNA in raw sausages and liver sausages from retail in Germany using an optimized method

Hepatitis E virus (HEV) is a pathogen of increasing importance, which can be zoonotically transmitted from domestic pigs, wild boar, and deer to humans. Foodborne transmission by consumption of raw and undercooked liver, meat, or sausages prepared from infected animals has been documented. The aim of this study was to investigate the distribution of HEV in different types of sausages sold in Germany. As no standardized methods for HEV detection in food exist, several techniques of sample homogenization, virus concentration and nucleic acid extraction followed by real-time RT-PCR were compared using artificially contaminated sausages. A method using TRI Reagent® Solution showed the best efficacy of matrix disruption and a treatment with chloroform followed by a silica-based RNA extraction method resulted in the highest HEV detection rates. The detection limit of the method was 2.9 × 10(3) and 5.3 × 10(4) genome equivalents per 5 g raw sausage and 2 g liver sausage, respectively. Application of the method to raw and liver sausages from retail in Germany resulted in the HEV genome detection in 14 out of 70 (20%) raw sausages and in 11 out of 50 (22%) liver sausages. The detected HEV sequences showed a high diversity and belonged to different subtypes of HEV genotype 3. The results indicate a broad distribution of HEV-RNA in meat products sold in Germany; however, the infectivity of the detected virus remains to be assessed in future.

Comparison of two extraction methods for viruses in food and application in a norovirus gastroenteritis outbreak

Noroviruses are important causes of gastroenteritis; however, due to a lack of sensitive detection methods, the distinct role of contaminated food in norovirus outbreaks remains unclear. Two published virus extraction procedures combined with real-time RT-PCR for the detection of norovirus from food inoculated experimentally were compared. The elution-precipitation method was most efficient in all food matrices tested showing detection limits of 20 RT-PCRU for lettuce and ham, and 200 RT-PCRU for raspberries. The average recovery rates were 23%, 7% and 24% for lettuce, raspberries and ham, respectively. The ultrafiltration method yielded detection limits of 200 RT-PCRU for lettuce and ham, and 2000 RT-PCRU for raspberries; recovery rates were 9%, 7%, 3%, respectively. Subsequently, food items implicated in a norovirus outbreak were examined by the elution-precipitation method. Virus recovery rates determined by using MS2 phage ranged from 1 to 20% depending on the food matrix. However, norovirus could not be detected in the food items examined. This negative result may be explained by a low virus titer and heterogeneous virus distribution, or by random selection of food samples that contained no norovirus. Both, proper sampling and virus extraction from foods may be improved further to identify vehicles of infection.

Campylobacter spp. in Broiler Flocks at Farm Level and the Potential for Cross-Contamination During Slaughter

Screening of broiler flocks for their Campylobacter carriage on farm level and consequently the spread of Campylobacter spp. during slaughtering can help to identify hygiene control points. Therefore, between December 2001 and August 2002 in total 51 broiler flocks from three farms of different geographical regions in Germany were analysed for thermophilic Campylobacter. Campylobacter spp. were isolated from 45% of the broiler flocks examined. Subsequently, 1101 samples were taken from 22 flocks during different stages of processing. Samples were collected from: transport crates before and after cleaning/disinfection, evisceration, post‐scalded and post‐chilled carcasses and endproducts. Additionally, 45 selected Campylobacter isolates of droppings were genotyped by pulsed‐field gel electrophoresis (PFGE). Campylobacter carriage of flocks showed seasonal variation, with the highest contamination rate during the period of June to August. No evidence was found for a horizontal transmission from one broiler flock to the next via a persistent house‐contamination. In each positive flock, one to three different genotypes were found. One or two clones dominated isolations obtained from the farm level. The fact that in different flocks indistinguishable isolates of clonal origin were detected during the same rearing period suggested a transmission between the broiler flocks or an intermittent common external source. In one case, isolates of clonal origin were detected in various farms during different rearing periods. Sampling during processing confirmed that the entrance of a positive flock resulted in contamination of the abattoir environment. Campylobacter spp. were isolated from all sampling stages along the processing line, with a percentage of 91.1–100 of isolates at different stages of slaughtering.

Limits of control--crucial parameters for a reliable quantification of viable campylobacter by real-time PCR.

The unsuitability of the “CFU” parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR.