Characteristics of Listeria Monocytogenes Strains Persisting in a Meat Processing Facility over a 4-Year Period.

Listeria monocytogenes can persist in food production facilities, resulting in serious threats to consumers due to the high mortality associated with listeriosis, especially in the very young, old and pregnant. We subtyped 124 strains of L. monocytogenes isolated from a meat processing facility in Switzerland by serotyping, multi locus sequence typing (MLST) typing and whole genome sequencing. We then analyzed their ability to form biofilms and their resistance to the disinfectants benzalkonium chloride (BC) and peracetic acid (PAA). The genotyping results of the strains showed that several clonal populations of L. monocytogenes belonging to CC9, CC204 and CC121 had persisted in this meat processing facility for at least four years. All of the strains showed biofilm forming capacity comparable to a known high biofilm forming strain. Known efflux pumps for BC were present in CC204, CC9 (brcABC) and CC121 (qacH) strains, while strains from other CC showed very low minimal inhibitory concentrations (MICs) for BC. For PAA, minimal bactericidal concentrations of 1.2⁻1.6% for 20 min and minimal inhibitory concentrations between 0.1 and 0.2% were observed. These values were close to or above the recommended concentration for use (0.5⁻1%), suggesting that PAA might be ineffective at controlling L. monocytogenes in this and potentially other meat processing facilities.

Comparative Genomic Characterization of the Highly Persistent and Potentially Virulent Cronobacter sakazakii ST83, CC65 Strain H322 and Other ST83 Strains.

Cronobacter (C.) sakazakii is an opportunistic pathogen and has been associated with serious infections with high mortality rates predominantly in pre-term, low-birth weight and/or immune compromised neonates and infants. Infections have been epidemiologically linked to consumption of intrinsically and extrinsically contaminated lots of reconstituted powdered infant formula (PIF), thus contamination of such products is a challenging task for the PIF producing industry. We present the draft genome of C. sakazakii H322, a highly persistent sequence type (ST) 83, clonal complex (CC) 65, serotype O:7 strain obtained from a batch of non-released contaminated PIF product. The presence of this strain in the production environment was traced back more than 4 years. Whole genome sequencing (WGS) of this strain together with four more ST83 strains (PIF production environment-associated) confirmed a high degree of sequence homology among four of the five strains. Phylogenetic analysis using microarray (MA) and WGS data showed that the ST83 strains were highly phylogenetically related and MA showed that between 5 and 38 genes differed from one another in these strains. All strains possessed the pESA3-like virulence plasmid and one strain possessed a pESA2-like plasmid. In addition, a pCS1-like plasmid was also found. In order to assess the potential in vivo pathogenicity of the ST83 strains, each strain was subjected to infection studies using the recently developed zebrafish embryo model. Our results showed a high (90-100%) zebrafish mortality rate for all of these strains, suggesting a high risk for infections and illness in neonates potentially exposed to PIF contaminated with ST83 C. sakazakii strains. In summary, virulent ST83, CC65, serotype CsakO:7 strains, though rarely found intrinsically in PIF, can persist within a PIF manufacturing facility for years and potentially pose significant quality assurance challenges to the PIF manufacturing industry.

Hepatic toxicity of dronedarone in mice: role of mitochondrial ß-oxidation.

Dronedarone is an amiodarone-like antiarrhythmic drug associated with severe liver injury. Since dronedarone inhibits mitochondrial respiration and ß-oxidation in vitro, mitochondrial toxicity may also explain dronedarone-associated hepatotoxicity in vivo. We therefore studied hepatotoxicity of dronedarone (200mg/kg/day for 2 weeks or 400mg/kg/day for 1 week by intragastric gavage) in heterozygous juvenile visceral steatosis (jvs(+/-)) and wild-type mice. Jvs(+/-) mice have reduced carnitine stores and are sensitive for mitochondrial ß-oxidation inhibitors. Treatment with dronedarone 200mg/kg/day had no effect on body weight, serum transaminases and bilirubin, and hepatic mitochondrial function in both wild-type and jvs(+/-) mice. In contrast, dronedarone 400mg/kg/day was associated with a 10-15% drop in body weight, and a 3-5-fold increase in transaminases and bilirubin in wild-type mice and, more accentuated, in jvs(+/-) mice. In vivo metabolism of intraperitoneal (14)C-palmitate was impaired in wild-type, and, more accentuated, in jvs(+/-) mice treated with 400mg/kg/day dronedarone compared to vehicle-treated mice. Impaired ß-oxidation was also found in isolated mitochondria ex vivo. A likely explanation for these findings was a reduced activity of carnitine palmitoyltransferase 1a in liver mitochondria from dronedarone-treated mice. In contrast, dronedarone did not affect the activity of the respiratory chain ex vivo. We conclude that dronedarone inhibits mitochondrial ß-oxidation in and ex vivo, but not the respiratory chain. Jvs(+/-) mice are slightly more sensitive for the effect of dronedarone on mitochondrial ß-oxidation than wild-type mice. The results suggest that inhibition of mitochondrial ß-oxidation is an important mechanism of hepatotoxicity associated with dronedarone.

Effects of diazepam on gene expression and link to physiological effects in different life stages in zebrafish Danio rerio.

We applied zebrafish whole genome microarrays to identify molecular effects of diazepam, a neuropharmaceutical encountered in wastewater-contaminated environments, and to elucidate its neurotoxic mode of action. Behavioral studies were performed to analyze for correlations between altered gene expression with effects on the organism level. Male zebrafish and zebrafish eleuthero-embryos were exposed for 14 d or up to 3 d after hatching, respectively, to nominal levels of 273 ng/L and 273 µg/L (determined water concentrations in the adult experiment 235 ng/L and 291 µg/L). Among the 51 and 103 altered transcripts at both concentrations, respectively, the expression of genes involved in the circadian rhythm in adult zebrafish and eleuthero-embryos were of particular significance, as revealed both by microarrays and quantitative PCR. The swimming behavior of eleuthero-embryos was significantly altered at 273 µg/L. The study leads to the conclusion that diazepam-induced alterations of genes involved in circadian rhythm are paralleled by effects in neurobehavior at high, but not at low diazepam concentrations that may occur in polluted environments.