Assessing the value of bacteriological examination as a diagnostic tool in relation to meat inspection in cattle.

In Denmark, lesions indicating acute generalised infection identified at meat inspection will result in total condemnation. An official bacteriological examination (BU) protocol can be used for slaughtered animals with lesions such as endocarditis and endophlebitis as an assisting diagnostic tool to confirm whether the condition is local or generalised. If local, the carcass can be approved after condemnation of the relevant organs. The BU involves cultivating samples from the spleen and muscle. The aim of this study was to assess the value of the BU protocol. The study was conducted from February to May 2019 at a Danish cattle abattoir. Three groups of slaughtered cattle were included: 24 cases consisting of cattle with endocarditis and endophlebitis, 25 control animals consisting of cattle fully approved at inspection and 16 animals condemned at inspection due to endocarditis and endophlebitis with complications. Samples were taken from the heart, liver, kidney, lung, spleen and muscles of each animal. The BU protocol was used for cultivation. Different types of colonies were identified using MALDI-TOF-MS analysis. One or more samples with bacterial growth were found in all condemned animals - in 16 out of the 24 case animals and in two out of 25 control animals. In all three groups, Trueperella pyogenes was the most frequently isolated bacterium (60%) followed by Fusobacterium necrophorum (10%). For the case animals, the organ most commonly found with bacterial growth was the liver (46%), followed by the lung (38%) and the kidney (38%), while 96% of the muscle samples were negative. For the condemned group, bacterial growth was found in 75% of the spleen samples, 56% of liver and lung samples, and 50% of the muscle samples. A statistical analysis of the samples from cases and controls showed strong pair-wise associations for the presence of bacteria between organs, but no pair-wise associations between presence of bacteria in the muscle and any of the organs. Hence, if bacteria are found e.g. in the liver, they are likely to be found in other organs, but not in the muscle. In total, 20 of the 24 case animals were fully or partly approved in accordance with the current rules for judgement. It was concluded that the BU protocol using spleen and muscle samples would be suitable as a diagnostic tool for the judgement of slaughtered animals in cases where there is doubt about the stage of the lesions observed.

Chitin Attenuates Expression of Listeria monocytogenes Virulence Genes in vitro.

External signals are crucial for bacteria to sense their immediate environment and fine-tune gene expression accordingly. The foodborne pathogen Listeria monocytogenes senses a range of environmental cues in order to activate or deactivate the virulence-inducing transcriptional factor PrfA during transition between infectious and saprophytic lifecycles. Chitin is an abundant biopolymer formed from linked ß-(1-4)-N-acetyl-D-glucosamine residues associated with fungi, the exoskeleton of insects and often incorporated into foods as a thickener or stabilizer. L. monocytogenes evolved to hydrolyse chitin, presumably, to facilitate nutrient acquisition from competitive environments such as soil where the polymer is abundant. Since mammals do not produce chitin, we reasoned that the polymer could serve as an environmental signal contributing to repression of L. monocytogenes PrfA-dependent expression. This study shows a significant downregulation of the core PrfA-regulon during virulence-inducing conditions in vitro in the presence of chitin. Our data suggest this phenomenon occurs through a mechanism that differs from PTS-transport of oligosaccharides generated from either degradation or chitinase-mediated hydrolysis of the polymer. Importantly, an indication that chitin can repress virulence expression of a constitutively active PrfA∗ mutant is shown, possibly mediated via a post-translational modification inhibiting PrfA∗ activity. To our knowledge, this is the first time that chitin is reported as a molecule with anti-virulence properties against a pathogenic bacterium. Thus, our findings identify chitin as a signal which may downregulate the virulence potential of the pathogen and may provide an alternative approach toward reducing disease risk.

The association between meat inspection codes, footpad lesions and thinning of broiler flocks in the Danish broiler production.

The foundation of the condemnation practices in Post-Mortem Inspection (PMI) of poultry should be based on up-to-date scientific evidence about the cause of infection and hence whether the lesions observed are of food safety, animal health or welfare concerns. This study aimed to investigate the association between meat inspection codes, footpad lesions, and thinning of flocks in Danish broiler production. The data set was based on the delivery of chicken flocks to one of the two larger chicken slaughterhouses in Denmark, representing 71 farms, 174 houses, and 4,068 flocks over three years from January 2016 to December 2018. Post-mortem condemnation data of slaughtered chickens recorded and stored in the Danish Quality Assurance System (KIK) database was used in the study. Five potentially causal models were developed to investigate whether there was an association between dermatitis, arthritis, systemic infection, emaciation, mortality and possible explaining factors` (footpad lesion, age at slaughter, scratches, ascites, systemic infection and thinning of the flock). These five ecological logistic regression models were analyzed with the three levels: farm, house, and flock. Data from a total number of 126,137,002 (N) slaughtered chickens recorded in KIK databases were used for modeling and analyses. The prevalence of condemned carcasses was 1.1 % (n = 1,420,812). Overall, 12 individual reasons for condemnation of carcasses were recorded. The most frequently observed reason for condemnation was skin disease (scratches and dermatitis) with a prevalence of 0.5 %. Prevalence of ascites was 0.2 %, discoloration 0.09 %, emaciation 0.09 %, hepatitis 0.09 % and arthritis 0.07 %. In the first model, dermatitis was shown to be positively associated with age at slaughter with an OR = 1.04 (CI95 %: 1.02-1.05), while arthritis was considered an intervening variable. Moreover, there was a small protective effect of thinning of the flock for first and second delivery. There was a positive association between arthritis and age at the time of slaughter with an OR = 1.13 (CI95 %: 1.12-1.15). Systemic infections were associated with scratches with an OR = 24.5 (CI95 %: 16.6-36.3) and footpad lesions with an OR = 1.007 (CI95 %: 1.006-1.008). Further modelling of emaciation and mortality was not considered because of unbalanced groups in the data probably caused by the fact that some condemnation codes were rare. We observed that the most common causal factors of condemnation in the systemic infection models were scratches and footpad lesion, therefore preventing and controlling such lesions could reduce losses. Specific management and environmental etiological factors of the main infections causing condemnation in Danish broilers should be determined.

Molecular Characteristics and Zoonotic Potential of Salmonella Weltevreden From Cultured Shrimp and Tilapia in Vietnam and China.

Salmonella Weltevreden is increasingly reported from aquatic environments, seafood, and patients in several Southeast Asian countries. Using genome-wide analysis, we characterized S. Weltevreden isolated from cultured shrimp and tilapia from Vietnam and China to study their genetic characteristics and relatedness to clinical isolates of S. Weltevreden ST-365. The phylogenetic analysis revealed up to 312 single-nucleotide polymorphism (SNP) difference between tilapia isolates, whereas isolates from shrimp were genetically more closely related. Epidemiologically unrelated isolates from Vietnam were closely related to isolates from China, e.g., 20 SNPs differences between strains 28V and 75C. In comparison with strains from other parts of the world, our environmental isolates predominantly clustered within the continental South Asia lineage, constituted mostly of strains from human stool with as low as seven SNPs difference, e.g., 30V versus Cont_ERR495254. All sequenced isolates were MLST type ST-365 and contained the major virulence-related genes encoded by the Salmonella Pathogenicity Islands 1-5. Ten of the isolates harbored the IncFII(S) plasmid similar to the virulence genes-mediated plasmid pSPCV of S. Paratyphi C, and one isolate had the IncQ1 plasmid on the same contig with strA/B, sul2, and tetA resistance genes similar to the IncQ1 type, pNUC of S. Typhimurium. A pangenomic analysis yielded 7891 genes including a core genome of 4892 genes, with a closely related accessory genome content between clinical and environmental isolates (Benjamini p > 0.05). In a search for differences that could explain the higher prevalence of S. Weltevreden in aquatic samples, genomes were compared with those of other Salmonella enterica serovars. S. Weltevreden revealed specific regions harboring glpX (Fructose-1;6-bisphosphatase; class II), rfbC (dTDP-4-dehydrorhamnose 3;5-epimerase), and cmtB (PTS Mannitol-specific cryptic phosphotransferase enzyme IIA component) involved in carbohydrate biosynthesis pathways. Our study builds grounds for future experiments to determine genes or pathways that are essential when S. Weltevreden are in aquatic environments and microbial interactions providing survival advantages to S. Weltevreden in such environments.

Ciprofloxacin-induced persister-cells in Campylobacter jejuni.

Campylobacter jejuni is a major bacterial foodborne-pathogen. Ciprofloxacin is an important antibiotic for the treatment of C. jejuni, albeit high rates of fluoroquinolone resistance have limited its usefulness. Persister-cells are transiently antibiotic-tolerant fractions of bacterial populations and their occurrence has been associated with recalcitrant and persistent bacterial infections. Here, time-kill assays with ciprofloxacin (200×MIC, 25 µg ml-1) were performed in C. jejuni strains 81-176 and RM1221 and persister-cells were found. The frequency of survivors after 8 h of ciprofloxacin exposure was approx. 10-3 for both strains, while after 22 h the frequency was between 10-5-10-7, depending on the strain and growth-phase. Interestingly, the stationary-phase cultures did not display more persister-cells compared to exponential-phase cultures, in contrast to what has been observed in other bacterial species. Persister-cells after ampicillin exposure (100×MIC, 200 µg ml-1) were not detected, implying that persister-cell formation in C. jejuni is antibiotic-specific. In attempts to identify the mechanism of ciprofloxacin persister-cell formation, stringent or SOS responses were not found to play major roles. Overall, this study reports ciprofloxacin persister-cells in C. jejuni and challenges the notion of persister-cells as plainly dormant non-growing cells.

Chitinase Expression in Listeria monocytogenes Is Influenced by lmo0327, Which Encodes an Internalin-Like Protein.

The chitinolytic system of Listeria monocytogenes thus far comprises two chitinases, ChiA and ChiB, and a lytic polysaccharide monooxygenase, Lmo2467. The role of the system in the bacterium appears to be pleiotropic, as besides mediating the hydrolysis of chitin, the second most ubiquitous carbohydrate in nature, the chitinases have been deemed important for the colonization of unicellular molds, as well as mammalian hosts. To identify additional components of the chitinolytic system, we screened a transposon mutant library for mutants exhibiting impaired chitin hydrolysis. The screening yielded a mutant with a transposon insertion in a locus corresponding to lmo0327 of the EGD-e strain. lmo0327 encodes a large (1,349 amino acids [aa]) cell wall-associated protein that has been proposed to possess murein hydrolase activity. The single inactivation of lmo0327, as well as of lmo0325 that codes for a putative transcriptional regulator functionally related to lmo0327, led to an almost complete abolishment of chitinolytic activity. The effect could be traced at the transcriptional level, as both chiA and chiB transcripts were dramatically decreased in the lmo0327 mutant. In accordance with that, we could barely detect ChiA and ChiB in the culture supernatants of the mutant strain. Our results provide new information regarding the function of the lmo0325-lmo0327 locus in L. monocytogenes and link it to the expression of chitinolytic activity.IMPORTANCE Many bacteria from terrestrial and marine environments express chitinase activities enabling them to utilize chitin as the sole source of carbon and nitrogen. Interestingly, several bacterial chitinases may also be involved in host pathogenesis. For example, in the important foodborne pathogen Listeria monocytogenes, the chitinases ChiA and ChiB and the lytic polysaccharide monooxygenase Lmo2467 are implicated in chitin assimilation but also act as virulence factors during the infection of mammalian hosts. Therefore, it is important to identify their regulators and induction cues to understand how the different roles of the chitinolytic system are controlled and mediated. Here, we provide evidence for the importance of lmo0327 and lmo0325, encoding a putative internalin/autolysin and a putative transcriptional activator, respectively, in the efficient expression of chitinase activity in L. monocytogenes and thereby provide new information regarding the function of the lmo0325-lmo0327 locus.

Antigen Uptake during Different Life Stages of Zebrafish (Danio rerio) Using a GFP-Tagged Yersinia ruckeri.

Immersion-vaccines (bacterins) are routinely used for aquacultured rainbow trout to protect against Yersinia ruckeri (Yr). During immersion vaccination, rainbow trout take up and process the antigens, which induce protection. The zebrafish was used as a model organism to study uptake mechanisms and subsequent antigen transport in fish. A genetically modified Yr was developed to constitutively express green fluorescent protein (GFP) and was used for bacterin production. Larval, juvenile and adult transparent zebrafish (tra:nac mutant) received a bath in the bacterin for up to 30 minutes. Samples were taken after 1 min, 15 min, 30 min, 2 h, 12 h and 24 h. At each sampling point fish were used for live imaging of the uptake using a fluorescence stereomicroscope and for immunohistochemistry (IHC). In adult fish, the bacterin could be traced within 30 min in scale pockets, skin, oesophagus, intestine and fins. Within two hours post bath (pb) Yr-antigens were visible in the spleen and at 24 h in liver and kidney. Bacteria were associated with the gills, but uptake at this location was limited. Antigens were rarely detected in the blood and never in the nares. In juvenile fish uptake of the bacterin was seen in the intestine 30 min pb and in the nares 2 hpb but never in scale pockets. Antigens were detected in the spleen 12 hpb. Zebrafish larvae exhibited major Yr uptake only in the mid-intestine enterocytes 24 hpb. The different life stages of zebrafish varied with regard to uptake locations, however the gut was consistently a major uptake site. Zebrafish and rainbow trout tend to have similar uptake mechanisms following immersion or bath vaccination, which points towards zebrafish as a suitable model organism for this aquacultured species.

Identification and Antimicrobial Resistance of Bacteria Isolated from Probiotic Products Used in Shrimp Culture.

Probiotics are increasingly used in aquaculture to control diseases and improve feed digestion and pond water quality; however, little is known about the antimicrobial resistance properties of such probiotic bacteria and to what extent they may contribute to the development of bacterial resistance in aquaculture ponds. Concerns have been raised that the declared information on probiotic product labels are incorrect and information on bacterial composition are often missing. We therefore evaluated seven probiotics commonly used in Vietnamese shrimp culture for their bacterial species content, phenotypic antimicrobial resistance and associated transferable resistance genes. The bacterial species was established by 16S rRNA sequence analysis of 125 representative bacterial isolates. MIC testing was done for a range of antimicrobials and whole genome sequencing of six multiple antimicrobial resistant Bacillus spp. used to identify resistance genes and genetic elements associated with horizontal gene transfer. Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus spp. were identified. Although our culture-based isolation and identification may have missed a few bacterial species present in the tested products this would represent minor bias, but future studies may apply culture independent identification methods like pyro sequencing. Only 6/60 isolates were resistant to more than four antimicrobials and whole genome sequencing showed that they contained macrolide (ermD), tetracycline (tetL), phenicol (fexA) and trimethoprim (dfrD, dfrG and dfrK) resistance genes, but not known structures associated with horizontal gene transfer. Probiotic bacterial strains used in Vietnamese shrimp culture seem to contribute with very limited types and numbers of resistance genes compared to the naturally occurring bacterial species in aquaculture environments. Approval procedures of probiotic products must be strengthened through scientific-based efficacy trials and product labels should allow identification of individual bacterial strains and inform the farmer on specific purpose, dosage and correct application measures.

Clonal Occurrence of Salmonella Weltevreden in Cultured Shrimp in the Mekong Delta, Vietnam.

This study investigated the occurrence, serovar and antimicrobial resistance of Salmonella spp. in shrimp samples from intensive and extensive farms located in three different provinces in the Mekong Delta, Vietnam. Shrimp from 11 of the 48 farms all contained S. Weltevreden, except for one farm yielding S. Agona, with no difference in Salmonella occurrence between the two production systems. Pulsed field gel electrophoresis (PFGE) of S. Weltevreden showed closely related XbaI pulse types, suggesting a clonal relationship despite the farms and shrimp samples being epidemiologically unrelated. S. Weltevreden was susceptible to most antimicrobials tested, with a few strains being resistant to florfenicol, chloramphenicol, sulfamethoxazole or trimethoprim. Future studies of the ecology of S. Weltevreden should establish if this serovar may survive better and even multiply in warm-water shrimp farm environments compared to other Salmonella serovars.

Assessment of human health risk associated with pyaemia in Danish finisher pigs when conducting visual-only inspection of the lungs.

The most important lesion to be overlooked when performing visual-only inspection of the lungs is embolic pneumonia. The aim of the present study was to assess the additional human health risk represented by overlooking cases of pyaemia represented by embolic pneumonia in finisher pigs, when conducting visual-only compared to palpation of the lungs, as is the traditional meat inspection procedure. An examination of bacteria isolated from 19 finisher pigs identified with embolic pneumonia at traditional meat inspection was undertaken. From each pig samples were taken from various organs (lungs, spleen, heart, liver and kidney), from the carpal joints (A. carpi) and flexor muscle (M. flexor digitorum superficialis) on the right foreleg. These data were included in a risk assessment following OIE guidelines. Bacteria were isolated from 78 out of 127 tissue and swap samples taken (61% positive samples). Staphylococcus aureus (N=37) was the most frequently isolated bacterium. The predominant site of S. aureus was the lung. S. aureus was detected although less frequently in low numbers in some organs (<100CFU/sample) and muscle samples (<10CFU/sample). Only one MRSA isolate was found. Staphylococcus warneri (N=24) was the second most commonly found bacterium. There was no predominant site and the number of S. warneri was less than 50CFU per sample. The risk of a food-borne intoxication from S. aureus in relation to pyaemia in pigs was considered very low due to the low quantitative numbers of S. aureus in muscle tissue samples. Implementing visual-only inspection will reduce the exposure of S. aureus due to less cross-contamination and handling of the plucks by the meat inspectors. The human health risk associated with S. warneri was considered very low, due to the limited zoonotic potential of this bacterium. In conclusion, the additional human health risk in relation to possibly overlooking pyaemia in Danish finisher pigs was considered negligible when conducting visual-only compared to traditional meat inspection.

Chitinase expression in Listeria monocytogenes is positively regulated by the Agr system.

The food-borne pathogen Listeria monocytogenes encodes two chitinases, ChiA and ChiB, which allow the bacterium to hydrolyze chitin, the second most abundant polysaccharide in nature. Intriguingly, despite the absence of chitin in human and mammalian hosts, both of the chitinases have been deemed important for infection, through a mechanism that, at least in the case of ChiA, involves modulation of host immune responses. In this study, we show that the expression of the two chitinases is subject to regulation by the listerial agr system, a homologue of the agr quorum-sensing system of Staphylococcus aureus, that has so far been implicated in virulence and biofilm formation. We demonstrate that in addition to these roles, the listerial agr system is required for efficient chitin hydrolysis, as deletion of agrD, encoding the putative precursor of the agr autoinducer, dramatically decreased chitinolytic activity on agar plates. Agr was specifically induced in response to chitin addition in stationary phase and agrD was found to regulate the amount of chiA, but not chiB, transcripts. Although the transcript levels of chiB did not depend on agrD, the extracellular protein levels of both chitinases were reduced in the ΔagrD mutant. The regulatory effect of agr on chiA is potentially mediated through the small RNA LhrA, which we show here to be negatively regulated by agr. LhrA is in turn known to repress chiA translation by binding to the chiA transcript and interfering with ribosome recruitment. Our results highlight a previously unrecognized role of the agr system and suggest that autoinducer-based regulation of chitinolytic systems may be more commonplace than previously thought.

Staphylococcus aureus but not Listeria monocytogenes adapt to triclosan and adaptation correlates with increased fabI expression and agr deficiency.

BACKGROUND: The ability of pathogens to adapt to the widely used biocide, triclosan, varies substantially. The purpose of the study was to examine bacterial adaptation over an extended period of time to low increments of triclosan concentrations. Focus was two human pathogens, S. aureus and L. monocytogenes that previously have displayed inherent high and low adaptability, respectively. RESULTS: Three strains of L. monocytogenes and two strains of S. aureus including the community-acquired USA300 were exposed to increasing, sub-lethal concentrations of triclosan in triclosan-containing agar gradients. Following 25 days of exposure on agar plates to sub-lethal concentrations of triclosan with a twofold concentration increase every second day, minimum inhibitory concentration (MIC) for S. aureus increased from 0.125 (8325-4) and 0.0625 (USA 300) mg/L to 4 mg/L. The MIC of all three L. monocytogenes strains was initially 4 mg/L and remained unaltered by the exposure. The adapted S. aureus isolates retained normal colony size but displayed increased expression of fabI encoding an essential enzyme in bacterial fatty acid synthesis. Also, they displayed decreased or no expression of the virulence associated agrC of the agr quorum sensing system. While most adapted strains of USA300 carried mutations in fabI, none of the adapted strains of 8325-4 did. CONCLUSIONS: Adaptability to triclosan varies substantially between Gram positive human pathogens. S. aureus displayed an intrinsically lower MIC for triclosan compared to L. monocytogenes but was easily adapted leading to the same MIC as L. monocytogenes. Even though all adapted S. aureus strains over-expressed fabI and eliminated expression of the agr quorum sensing system, adaptation in USA300 involved fabI mutations whereas this was not the case for 8325-4. Thus, adaptation to triclosan by S. aureus appears to involve multiple genetic pathways.

Recently introduced qacA/B genes in Staphylococcus epidermidis do not increase chlorhexidine MIC/MBC.

OBJECTIVES: Chlorhexidine is used as a disinfectant to prevent surgical infections. Recently, studies have indicated that chlorhexidine usage has selected methicillin-resistant Staphylococcus aureus strains that are tolerant to chlorhexidine and that this may be related to the presence of the qacA/B-encoded efflux pumps. Here, we evaluated if high-level exposure to chlorhexidine selects for tolerant colonizing Staphylococcus epidermidis and we addressed the consequences of long-term exposure to chlorhexidine. METHODS: Chlorhexidine susceptibility and carriage of qacA/B was determined for colonizing S. epidermidis isolated from scrub nurses heavily exposed to chlorhexidine and were compared with isolates from non-users of chlorhexidine hand rubs. S. epidermidis blood isolates from the 1960s, before the wider introduction of chlorhexidine to the market, were also tested and compared with recently collected S. epidermidis blood isolates. RESULTS: There was no correlation between the use of chlorhexidine in scrub nurses and the presence of qacA/B genes in S. epidermidis isolates or increased MICs/MBCs of chlorhexidine for S. epidermidis isolates. While 55% of current blood isolates harboured the qacA/B genes, none of the 33 historical S. epidermidis isolates did, although their MICs and MBCs of chlorhexidine were comparable to those for current isolates. CONCLUSIONS: Chlorhexidine used as a hand rub does not select for S. epidermidis isolates with increased MICs or MBCs of chlorhexidine. However, the absence of qacA/B genes in S. epidermidis isolates obtained in the 1960s suggests that long-term use of biocides like chlorhexidine or related compounds may select for the presence of qacA/B genes.

Initial adhesion of Listeria monocytogenes to fine polished stainless steel under flow conditions is determined by prior growth conditions.

Listeria monocytogenes is a food-borne pathogen known to persist in food production environments, where it is able to attach and form biofilms, potentially contaminating food products ready for consumption. In this study the first step in the establishment of L. monocytogenes in a food-processing environment was examined, namely the initial adhesion to stainless steel under specific dynamic flow conditions. It was found that the intrinsic ability of L. monocytogenes to adhere to solid surfaces under flow conditions is dependent on nutrient availability. The addition of L-leucine to the growth medium altered the fatty acid composition of the L. monocytogenes cells and increased adhesion. The growth conditions resulting in the highest adhesion (growth medium with added glucose) had cells with the highest electron donating and lowest electron accepting properties, whereas growth conditions resulting in lowest adhesion (growth medium with added mannose) had cells with the lowest electron donating properties and highest electron accepting properties. The highest and lowest adhesion conditions correlated with differences in expression of cell surface protein of L. monocytogenes and among these the autolysin amidase (Ami). This study implies that food composition influences the adhesion of L. monocytogenes to solid surfaces during dynamic flow conditions.

Staphylococcus epidermidis isolated in 1965 are more susceptible to triclosan than current isolates.

Since its introduction to the market in the 1970s, the synthetic biocide triclosan has had widespread use in household and medical products. Although decreased triclosan susceptibility has been observed for several bacterial species, when exposed under laboratory settings, no in vivo studies have associated triclosan use with decreased triclosan susceptibility or cross-resistance to antibiotics. One major challenge of such studies is the lack of strains that with certainty have not been exposed to triclosan. Here we have overcome this challenge by comparing current isolates of the human opportunistic pathogen Staphylococcus epidermidis with isolates collected in the 1960s prior to introduction of triclosan to the market. Of 64 current S. epidermidis isolates 12.5% were found to have tolerance towards triclosan defined as MIC≥0.25 mg/l compared to none of 34 isolates obtained in the 1960s. When passaged in the laboratory in the presence of triclosan, old and current susceptible isolates could be adapted to the same triclosan MIC level as found in current tolerant isolates. DNA sequence analysis revealed that laboratory-adapted strains carried mutations in fabI encoding the enoyl-acyl carrier protein reductase isoform, FabI, that is the target of triclosan, and the expression of fabI was also increased. However, the majority of the tolerant current isolates carried no mutations in fabI or the putative promoter region. Thus, this study indicates that the widespread use of triclosan has resulted in the occurrence of S. epidermidis with tolerance towards triclosan and that the adaptation involves FabI as well as other factors. We suggest increased caution in the general application of triclosan as triclosan has not shown efficacy in reducing infections and is toxic to aquatic organisms.

Bacterial flora and antimicrobial resistance in raw frozen cultured seafood imported to Denmark.

Intensified aquaculture includes the use of antimicrobials for disease control. In contrast to the situation in livestock, Escherichia coli and enterococci are not part of the normal gastrointestinal flora of fish and shrimp and therefore not suitable indicators of antimicrobial resistance in seafood. In this study, the diversity and phenotypic characteristics of the bacterial flora in raw frozen cultured and wild-caught shrimp and fish were evaluated to identify potential indicators of antimicrobial resistance. The bacterial flora cultured on various agar media at different temperatures yielded total viable counts of 4.0 × 10(4) to 3.0 × 10(5) CFU g(-1). Bacterial diversity was indicated by 16S rRNA sequence analysis of 84 isolates representing different colony types; 24 genera and 51 species were identified. Pseudomonas spp. (23% of isolates), Psychrobacter spp. (17%), Serratia spp. (13%), Exiguobacterium spp. (7%), Staphylococcus spp. (6%), and Micrococcus spp. (6%) dominated. Disk susceptibility testing of 39 bacterial isolates to 11 antimicrobials revealed resistance to ampicillin, amoxicillin-clavulanic acid, erythromycin, and third generation cephalosporins. Resistance to third generation cephalosporins was found in Pseudomonas, a genus naturally resistant to most ß-lactam antibiotics, and in Staphylococcus hominis. Half of the isolates were susceptible to all antimicrobials tested. Results indicate that identification of a single bacterial resistance indicator naturally present in seafood at point of harvest is unlikely. The bacterial flora found likely represents a processing rather than a raw fish flora because of repeated exposure of raw material to water during processing. Methods and appropriate indicators, such as quantitative PCR of resistance genes, are needed to determine how antimicrobials used in aquaculture affect resistance of bacteria in retailed products.

Modeling the growth of Listeria monocytogenes in soft blue-white cheese.

The aim of this study was to develop a predictive model simulating growth over time of the pathogenic bacterium Listeria monocytogenes in a soft blue-white cheese. The physicochemical properties in a matrix such as cheese are essential controlling factors influencing the growth of L. monocytogenes. We developed a predictive tertiary model of the bacterial growth of L. monocytogenes as a function of temperature, pH, NaCl, and lactic acid. We measured the variations over time of the physicochemical properties in the cheese. Our predictive model was developed based on broth data produced in previous studies. New growth data sets were produced to independently calibrate and validate the developed model. A characteristic of this tertiary model is that it handles dynamic growth conditions described in time series of temperature, pH, NaCl, and lactic acid. Supplying the model with realistic production and retail conditions showed that the number of L. monocytogenes cells increases 3 to 3.5 log within the shelf life of the cheese.

A small RNA controls expression of the chitinase ChiA in Listeria monocytogenes.

In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for efficient binding to its target. In the present study, we sought to gain more insight into the functional role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment, and we provide evidence that both LhrA and Hfq act to down-regulate the expression of lmo0302 and chiA. Furthermore, in vitro binding experiments show that Hfq stimulates the base pairing of LhrA to chiA mRNA. Finally, we demonstrate that LhrA has a negative effect on the chitinolytic activity of L. monocytogenes. In marked contrast to this, we found that Hfq has a stimulating effect on the chitinolytic activity, suggesting that Hfq plays multiple roles in the complex regulatory pathways controlling the chitinases of L. monocytogenes.

Listeria monocytogenes efficiently invades Caco-2 cells after low-temperature storage in broth and on deli meat.

The objective of this study was to investigate how various growth conditions influence the virulence of Listeria monocytogenes monitored by its ability to invade the epithelial cell lines Caco-2 and INT-407. The growth conditions examined were modified atmosphere-packaged deli meat and brain heart infusion broth (BHI) with and without salt. Five strains of L. monocytogenes were selected to investigate their invasiveness and all strains invaded Caco-2 cells at higher levels than INT-407 cells. Further, the clinical strains (3443 and 3734) were more invasive (p < 0.05) than the strains isolated from meat and food-processing environments (3008, 3126, and 4140) after grown in BHI at 30 degrees C. This attenuation could not be ascribed to a defective Internalin A as all strains encoded an intact inlA gene. To determine the influence of food products on virulence, the ability of L. monocytogenes to invade Caco-2 cells was compared after growth on a fermented sausage and on cured cooked ham to that of bacteria grown in BHI broth supplemented with salt. Samples were stored under chilling conditions for up to 4 weeks. The results showed no difference (p > 0.05) in invasiveness after 7 days at 10 degrees C in BHI broth or on sausage, whereas a slight increase (p < 0.05) was observed after incubation on ham for 2 and 4 weeks compared to that in BHI broth. Most importantly, our results show that L. monocytogenes efficiently invade Caco-2 cells even after 4 weeks of storage at chilled temperature. This is highly relevant for safety assessment of this organism in food as these conditions reflect storage of ready-to-eat food products in domestic refrigerators.

Poor invasion of trophoblastic cells but normal plaque formation in fibroblastic cells despite actA deletion in a group of Listeria monocytogenes strains persisting in some food processing environments.

We determined mammalian cell invasion and virulence gene (inlA, inlB, and actA) sequences of Listeria monocytogenes strains belonging to a molecular subtype (RAPD 9) that often persists in Danish fish-processing plants. These strains invaded human placental trophoblasts less efficiently than other L. monocytogenes strains, including clinical strains, and they carry a premature stop codon in inlA. Eight of 15 strains, including the RAPD 9 and maternofetal strains, had a 105-nucleotide deletion in actA that did not affect cell-to-cell spread in mouse fibroblasts. The RAPD 9 strains may still be regarded as of low virulence with respect to human listeriosis.