Relation between broiler and human Campylobacter jejuni strains isolated in Belgium from 2011 to 2013.

AIMS: This study inquires the relationship between Campylobacter jejuni isolated from broiler meat carcasses (n = 97) and human clinical samples (n = 72) in Belgium, from 2011 to 2013. METHODS AND RESULTS: The evaluation of the relation was based on the characteristics determined using multilocus sequence typing (MLST) alone and combined with flagellin gene A restriction fragment length polymorphism (flaA-RFLP) typing, antibiotic microbiological resistance profiling (AMRp), lipooligosaccharide class typing or virulence gene profiling (Vp). Clusters containing both human and broiler meat strains were more common when MLST was used alone, followed by MLST/flaA-RFLP and then by MLST/AMRp. More logical chronologically relations broiler-human were obtained for MLST/flaA-RFLP, then for MLST, and finally for MLST/AMRp: i.e. the isolates would first be detected in the broiler meat and at the same time or later in humans. CONCLUSIONS: In several cases, the C. jejuni strains isolated from the consumed broiler meat and from the campylobacteriosis case had the same profile, according to the used typing methods. The circulating Campylobacter strains appear to have remained the same from 2011 till 2013 in Belgium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study corroborates previously published data from Belgium that suggest a strong correlation between C. jejuni strains isolated from broiler meat and from campylobacteriosis patients.

Effect of exposure to stress conditions on propidium monoazide (PMA)-qPCR based Campylobacter enumeration in broiler carcass rinses.

Campylobacter quantification by qPCR is unable to distinguish viable vs. dead cells in contrast to the culture-based ISO 10272-2 reference method. Propidium monoazide (PMA) has been used to overcome this disadvantage. A Campylobacter PMA-qPCR enumeration method was evaluated for its consistency and compared to the culture-based enumeration for both artificially and natural contaminated broiler carcass rinses. The PMA effect was further evaluated on stressed cells. Five conditions, commonly encountered during the slaughter process and storage (acid, heat, cold, oxidation and freezing), were inflicted to the broiler carcass rinses artificially contaminated with Campylobacter jejuni or Campylobacter coli. A better correlation between the reference method and the qPCR enumeration was obtained when PMA was used. The two cultured-based methods used showed a significant CFU reduction for heat, cold and acid stresses although the PMA-qPCR enumeration showed that viable bacteria were underestimated. Freezing showed the highest reduction effect, while the reduction extend was also overestimated by the microbiological enumeration procedure. Exposure to a mild oxidative stress was the only stress condition applied at temperatures permitting adaptation of Campylobacter and did not lead to either reduction in CFU nor in the PMA-qPCR signal.

Indications for worldwide increased norovirus activity associated with emergence of a new variant of genotype II.4, late 2012.

Globally, surveillance systems showed an increasein norovirus activity in late 2012. Molecular datashared through the NoroNet network suggest thatthis increase is related to the emergence of a newnorovirus genotype II.4 variant, termed Sydney 2012.Healthcare institutions are advised to be prepared fora severe norovirus season.

Microbiological and molecular investigation of an increase of human listeriosis in Belgium, 2006-2007.

In Belgium, the majority of cases of listeriosis are sporadic cases. In this study we present evidence for an episode of listeriosis: a time-linked cluster of cases that occurred in 2006 and 2007, and the identification of identical strains. The episode involved 11 patients, infected with Listeria monocytogenes of serovar 4b. The source of infection was not detected.

Whole-genome sequencing of Listeria monocytogenes serotype 4b isolated from ready-to-eat lentil salad in Algiers, Algeria.

Listeria monocytogenes is a Gram-positive food-borne pathogen causing a serious threat for public health. Here we announce the whole genome sequence (3 011 693 bp) of Listeria monocytogenes serotype 4b, isolated from ready-to-eat lentil salad in Algiers and belonging to sequence type 2, lineage I and clonal complex 2.

Whole-Genome Sequencing of Six Strains of Salmonella enterica Isolated from Imported Meat in Algeria.

Nontyphoidal Salmonella (NTS) is one of the main causes of foodborne disease worldwide. In this report, we announce the first whole-genome sequencing of six strains of Salmonella enterica isolated from imported meat in Algeria. The genome sizes ranged from 4,601,209 to 4,958,962 bp. Antimicrobial resistance (AMR) genes, plasmids, and virulence factors were detected.

Whole-Genome Sequencing of Multidrug-Resistant Escherichia coli Strains Harboring the mcr-1 Gene, Isolated from Seawater of the Algiers Coast in Algeria.

Colistin resistance has emerged worldwide and is threatening the treatment efficacy of multiresistant Escherichia coli strains in humans and animals. Here, we communicate the whole-genome sequencing (WGS) of two colistin-resistant E. coli strains, M49 and M78, with genomes sizes of 4,947,168 and 5,178,716 bp, respectively, isolated from seawaters of the Algiers coast.

Quantification of Campylobacter spp. in chicken carcass rinse by real-time PCR.

AIMS: In this study, a real-time quantitative polymerase chain reaction (PCR) method was examined for its ability to quantify Campylobacter spp. in chicken carcass rinses and compared with bacteriological culturing. METHODS AND RESULTS: The linearity of the real-time PCR quantification protocol was assessed on pure DNA. The amplification efficiency was 100% and the square regression coefficient (R(2)) was 0.998. Quantification was linear over at least 7 log units. Using a crude cell lysate gave the highest sensitivity and the detection limit of the method was 3.3 log CFU per carcass. The statistical correlation between the bacteriological enumeration and the real-time quantitative (Q)-PCR determined using chicken carcasses sampled at the end of the slaughter line was 0.733. The difference in detection levels was probably because of the detection of stressed, dead or viable but not culturable cells by Q-PCR. CONCLUSION: The real-time Q-PCR method described in this study is a powerful tool for determining the number of Campylobacter cells on carcasses. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time Q-PCR method is available to quantify the Campylobacter contamination at the slaughterhouse level and could be used to evaluate primary production.

Quantification, distribution and diversity of ESBL/AmpC-producing Escherichia coli on freshly slaughtered pig carcasses.

This study quantified cefotaxime-resistant E. coli (CREC) on nine different carcass areas of 104 freshly slaughtered pig carcasses. In 49% [95% confidence interval (95% CI): 29-69%] of the carcasses CREC could be isolated and enumerated (using Tryptone Bile Agar with X-Glucuronide supplemented with 1 mg/L cefotaxime). Proportions of positive samples varied between carcass areas from 1% [95% CI: 0-10%] (loin) to 23% [95% CI: 10-44%] (head). Maximum concentrations on positive samples ranged between -0.6 log10 CFU/cm2 (loin, elbow before evisceration) and 1.7 log10 CFU/cm2 (head). The head was significantly more frequently contaminated than the loin (p = 0.027) and ham (3% [95% CI: 1-15%]). The foreleg was significantly more frequently contaminated (20% [95% CI: 13-30%]) than the ham. Combination disk diffusion assays revealed that 81% of the CREC isolates were extended-spectrum beta-lactamases (ESBL) producers, 13% were AmpC cephalosporinases (AmpC) producers and 2% ESBL and AmpC co-producers. Genotyping denoted blaCTX-M-gr1 (63%) and blaTEM (40%) as most present antibiotic resistance genes. Multiple gene combinations in one isolate and multiple combinations of genotypes and phenotypes among isolates of one sample were observed. These quantitative data can be used for intervention strategies to lower human exposure to CREC.

Mechanisms of erythromycin resistance of Campylobacter spp. isolated from food, animals and humans.

Macrolides are regarded as drugs of choice for treatment of human campylobacteriosis. The use of antimicrobials for this purpose as well as in food animal production could result in macrolide resistance in Campylobacter species. Campylobacter isolates exhibit two different phenotypes with regard to erythromycin resistance: high-level resistance (HLR) and low-level resistance (LLR). Thirty-six food/animal and human isolates of Campylobacter jejuni and C. coli were examined for their mechanisms of resistance to erythromycin. The data presented here confirm the previous findings that the A2075G mutation in the 23S rRNA gene is the most frequently reported mechanism of high-level erythromycin resistance in Campylobacter isolates. The efflux pump inhibitor PAbetaN increased susceptibility to erythromycin for at least 16-32-fold in all examined HLR isolates, suggesting that the efflux mechanism acts in synergy with the 23S rRNA mutation to confer high-level erythromycin resistance. This was also confirmed in the isolates with sequence variation in the efflux pump cmeB gene. Additionally, the PAbetaN restored the susceptibility of LLR strains to the level of minimal inhibitory concentrations (MICs) of the susceptible strains and also reduced the MICs of the susceptible C. jejuni and C. coli isolates. The data suggest that active efflux contributes to the intrinsic resistance to erythromycin in Campylobacter and also contribute to high-level resistance.

High abundance and diversity of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faeces and tonsils of pigs at slaughter.

This cross-sectional study investigates the abundance of cefotaxime-resistant Escherichia coli (CREC) in the faeces and tonsils of 96 pigs during slaughter. Moreover, different isolates from a selected number of pigs were tested to study the diversity of blaESBL genes within E. coli isolates from one pig. Cefotaxime-resistant bacteria (based on enumeration results on MacConkey agar supplemented with 1mg/L cefotaxime) were found in the faeces of 77 pigs (80%; 95% CI: 70-87%) and the tonsils of 91 pigs (95%; 95% CI: 88%-98%). Cefotaxime-resistant E. coli (based on enumeration results on Tryptone Bile X-glucuronide agar supplemented with 1mg/L cefotaxime) were detected in 72 faecal samples (75%; 95% CI: 64-83%) and 45 tonsil samples (47%; 95% CI: 35-59%), in numbers up to 5.5 and 5.6log10 CFU/g, respectively. On average, around 1/10,000 E. coli in both faeces and tonsils were cefotaxime-resistant, though large variations were observed between pigs. Within one sample, CREC isolates with up to five different combinations of ESBL genes were observed. In three out of 16 faecal samples and six out of 14 tonsil samples, only one ESBL gene profile was found. The high numbers of CREC that are occasionally found in the faeces and tonsils of pigs during slaughter may represent an important source of contamination of carcasses and subsequently pork.

Comparative performance of isolation methods using Preston broth, Bolton broth and their modifications for the detection of Campylobacter spp. from naturally contaminated fresh and frozen raw poultry meat.

The performance of different isolation methods was evaluated for the detection of Campylobacter from naturally contaminated raw poultry meat. Therefore, fresh and frozen poultry meat samples were analysed using the standard procedure (ISO 10272-1:2006), enrichment in Preston broth, and enrichment in modified Bolton broth (supplemented with (i) potassium clavulanate (C-BB), (ii) triclosan (T-BB), (iii) polymyxin B (P-BB)). The enrichment cultures were streaked onto both modified charcoal cefoperazone deoxycholate agar (mCCDA) and RAPID'Campylobacter agar (RCA). Moreover, direct plating on mCCDA and RCA was performed to quantify Campylobacter. In total, 33 out of 59 fresh retail meat samples (55.9%) were Campylobacter positive. For both fresh and frozen poultry meat samples, enrichment in Bolton broth (ISO 10272-1:2006) resulted in a higher number of positive samples than enrichment in Preston broth. Supplementation of Bolton broth with potassium clavulanate (C-BB) and triclosan (T-BB) enhanced the Campylobacter recovery from fresh poultry meat compared to non-supplemented Bolton broth, although the use of C-BB was less applicable than T-BB for Campylobacter recovery from frozen samples. Additionally, the use of RCA resulted in a higher isolation rate compared to mCCDA. The present study demonstrates the impact of culture medium on the recovery of Campylobacter from fresh and frozen naturally contaminated poultry meat samples and can support laboratories in choosing the most appropriate culturing method to detect Campylobacter.

Genetic resistance towards Salmonella infection in pigs.

A PCR-RFLP of intron 1 and intron 2 of the Nramp1 gene described as a potential candidate gene in controlling pig resistance to Salmonella infections was optimised and an association between the Salmonella status and the polymorphisms was investigated. For intron 1 new primers were designed and the amplicon of 622 bp was restricted with Hinf I to give fragments of 280 bp, 220 bp and 100 bp for allele A and 76 bp, 100 bp, 144 bp, and 280 bp for allele B. For intron 2 a new polymorphism was detected (a deletion) with fragments of 210 bp and 199 bp after amplification respectively for allele A and B. The frequency of allele B and A was 0.93 and 0.90 on a total of 180 and 205 pigs for the polymorphism in intron 1 and intron 2 respectively in a mixed population of hybrid and purebred pigs. Similar allele frequencies of allele B and A of 0.90 and 0.91 respectively in intron 1 and intron 2 were found in a sample of 392 and 391 slaughter pigs. In the latter population, no association between the Nramp1 genotypes and the colon Salmonella status or the ELISA status could be demonstrated.

Limited genetic diversity and gene expression differences between egg- and non-egg-related Salmonella Enteritidis strains.

Salmonella Enteritidis strains of egg- and non-egg-related origin were characterized molecularly to find markers correlated with the egg-contaminating property of Salmonella Enteritidis. Isolates were examined by random amplified polymorphic DNA (RAPD), plasmid profiling and phage typing. Furthermore, the presence of 30 virulence genes was tested by PCR. In genetic fingerprinting and gene content, only small differences between the strains were found and no correlation was observed with the origin (egg-related versus non-egg-related). A major RADP group was present in both egg- and non-egg-related strains, but other smaller RAPD groups were present as well in both categories of strains. Phage types PT4 and PT21 were predominant. Differential mRNA expression levels of fimA and agfA under conditions of growth simulating the conditions during egg formation were determined by real-time RT-PCR. Although differences in fimA and agfA expression levels were observed between the strains, these could not be correlated with the origin of the strains (egg-related versus non-egg-related). The highest expression levels of agfA and fimA were only found in two non-egg-related strains, which seemed to be correlated with the presence of a 93 kb plasmid instead of the 60 kb virulence plasmid. Our results seem to indicate only a limited role for at least type I fimbriae (encoded by fim operon) in egg contamination by Salmonella Enteritidis.

Reported foodborne outbreaks due to noroviruses in Belgium: the link between food and patient investigations in an international context.

The Belgian data for foodborne norovirus (NoV) outbreaks became available for the first time with the introduction of an extraction and detection protocol for NoV in the National Reference Laboratory for foodborne outbreaks in September 2006. In 2007, 10 NoV foodborne outbreaks were reported affecting 392 persons in Belgium. NoV became the most detected agent in foodborne outbreaks followed by Salmonella (eight foodborne outbreaks). The major implicated foods were sandwiches (4/10), where food handlers reported a history of gastroenteritis in two outbreaks. A food handler was implicated in the limited number of Belgian NoV outbreaks which is in accord with internationally recorded data. Forty foodborne and waterborne outbreak events due to NoV, epidemiological and/or laboratory confirmed, from 2000 to 2007 revealed that in 42.5% of the cases the food handler was responsible for the outbreak, followed by water (27.5%), bivalve shellfish (17.5%) and raspberries (10.0%).

Characterization of virulence properties of Listeria monocytogenes serotype 4b strains of different origins.

In this study, the virulence heterogeneity of Listeria monocytogenes serotype 4b strains of different origins was analysed on different levels. On one hand, the survival of L. monocytogenes strains in synthetic gastric fluid was studied. On the other hand, the pathogenic potential of strains with different inlB expression levels was analysed in an A/J mouse model for gastrointestinal listeriosis. Differences in survival capacity in gastric fluid and in in vivo virulence potential were observed between the tested strains. No clear correlation between the origin and the obtained data could be made. However, these results confirm the existence of heterogeneity in virulence potential of L. monocytogenes serotype 4b strains.

Identification of lactobacilli isolated from the cloaca and vagina of laying hens and characterization for potential use as probiotics to control Salmonella Enteritidis.

AIMS: To select Lactobacillus strains from laying hens for potential use as probiotic to control Salmonella Enteritidis infection. METHODS AND RESULTS: One hundred and eighty-six lactobacilli were isolated from the cloaca and vagina of laying hens, and identified at the species level by a polyphasic taxonomic approach. All isolates belonged to the Lactobacillus acidophilus, Lactobacillus reuteri or Lactobacillus salivarius phylogenetic groups, with the L. reuteri group being the most predominant group. Based on genetic diversity, about 50 representative strains were selected and tested for in vitro properties that could be predictive for probiotic activity in laying hens. Salmonella inhibition was shown to be species dependent, and correlated to some extent with the production of lactic acid. A selection of strains was evaluated in a S. Enteritidis challenge experiment. Two strains, L. reuteri R-17485 and Lactobacillus johnsonii R-17504 significantly decreased the colonization of chicks by S. Enteritidis in caeca, liver and spleen. CONCLUSIONS: Lactobacilli isolated from laying hens were observed to inhibit Salmonella growth in vitro, most probably through production of lactic acid, and to decrease in vivo the S. Enteritidis colonization of chicks. SIGNIFICANCE AND IMPACT OF THE STUDY: The data demonstrate that Lactobacillus isolates from laying hens may have probiotic potential in reducing S. Enteritidis infection.

Prevalence of Salmonella, Campylobacter and VTEC on pig farms.

Four different pig farms were sampled for the prevalence of Salmonella, Campylobacter and verotoxigenic E. coli (VTEC). Pigs of different age groups and pigs of the same age were tested by taking rectal swabs. The farm environment was tested by examining overshoes, and the feed and drinking water in the pens. From a total of 215 rectal samples of individual pigs, 15 rectal samples taken from animals at the same farm were positive for Salmonella. The Salmonella status of the pigs at this farm differed from one age group to another. S. Typhimurium was isolated from all the positive rectal samples and S. Typhimurium and S. Schwarzengrund were isolated from the environment. On two other farms Salmonella was only present in the environment with S. London and S. Typhimurium as serotypes. The presence of Campylobacter was tested in 150 rectal swabs, 51 of these, spread over the four farms, turned out to be positive. At all four pig farms Campylobacter was isolated from the environment as well. All the strains were identified as Campylobacter coli by a species-specific PCR. To determine if pigs are a reservoir of VTEC a total of 289 samples were screened for the presence of VTEC and 54 strains were isolated that each carried one virulence gene. Thirty-one strains carried the vt2e variant of the vt2 gene wich causes the endema disease in young pigs, four strains harboured the hlyA gene and 19 the eaeA gene.

Salmonella on pig carcasses: positive pigs and cross contamination in the slaughterhouse.

AIMS: The purpose of this study was to investigate the prevalence of Salmonella in pigs at the moment of slaughter and in the slaughterhouse environment. METHODS AND RESULTS: In total, five different commercial slaughterhouses were sampled during eight slaughterhouse visits. Carcass swabs, colon content and mesenteric lymph nodes were taken to reflect the animal status and from the slaughterhouse environmental samples were taken. Salmonella was isolated from 37% of the carcass samples as a mean value. High variations were noticed between different slaughterhouses (between 0 and 70%) and sampling days in the same abattoir (between 3 and 52%). A correlation was found between the carcass contamination and the status of the delivered animals (P=0.01675). Cross contamination was estimated to account for 29% of the positive carcasses. The slaughterhouse environment was highly contaminated; before starting the slaughtering activities 25% of the samples were positive on average. The most prevalent serotypes isolated at the slaughterhouse environment and from the colon content were S. Typhimurium, S. Livingstone and S. Derby. On carcasses S. Typhimurium was predominately isolated (71%). The biggest variability of serotypes was found in the mesenteric lymph nodes. Serologically 56.3% of the pigs were found positive for Salmonella using a cut-off level of the optical density percentage higher than 10 (O.D.% > or = 10). While on individual pig level the correlation between the bacteriological and serological data was poor, because of recent Salmonella infections, a better correlation was found at the herd level on the moment of slaughtering. CONCLUSION: A high degree of carcass contamination is noticed after slaughtering. This contamination resulted from the delivery of Salmonella-positive pigs and cross-contamination from the slaughterhouse environment. SIGNIFICANCE AND IMPACT OF THE STUDY: In pigs, Salmonella carriage is high, but it is obvious that slaughterhouse hygiene is a determinative factor for managing carcass contamination.