Biofilm formation and desiccation survival of Listeria monocytogenes with microbiota on mushroom processing surfaces and the effect of cleaning and disinfection.

Microbial multispecies communities consisting of background microbiota and Listeria monocytogenes could be established on materials used in food processing environments. The presence, abundance and diversity of the strains within these microbial multispecies communities may be affected by mutual interactions and differences in resistance towards regular cleaning and disinfection (C&D) procedures. Therefore, this study aimed to characterize the growth and diversity of a L. monocytogenes strain cocktail (n = 6) during biofilm formation on polyvinyl chloride (PVC) and stainless steel (SS) without and with the presence of a diverse set of background microbiota (n = 18). L. monocytogenes and background microbiota strains were isolated from mushroom processing environments and experiments were conducted in simulated mushroom processing environmental conditions using mushroom extract as growth medium and ambient temperature (20 °C) as culturing temperature. The L. monocytogenes strains applied during monospecies biofilm incubation formed biofilms on both PVC and SS coupons, and four cycles of C&D treatment were applied with a chlorinated alkaline cleaning agent and a disinfection agent based on peracetic acid and hydrogen peroxide. After each C&D treatment, the coupons were re-incubated for two days during an incubation period for 8 days in total, and C&D resulted in effective removal of biofilms from SS (reduction of 4.5 log CFU/cm2 or less, resulting in counts below detection limit of 1.5 log CFU/cm2 after every C&D treatment), while C&D treatments on biofilms formed on PVC resulted in limited reductions (reductions between 1.2 and 2.4 log CFU/cm2, which equals a reduction of 93.7 % and 99.6 %, respectively). Incubation of the L. monocytogenes strains with the microbiota during multispecies biofilm incubation led to the establishment of L. monocytogenes in the biofilm after 48 h incubation with corresponding high L. monocytogenes strain diversity in the multispecies biofilm on SS and PVC. C&D treatments removed L. monocytogenes from multispecies biofilm communities on SS (reduction of 3.5 log CFU/cm2 or less, resulting in counts below detection limit of 1.5 log CFU/cm2 after every C&D treatment), with varying dominance of microbiota species during different C&D cycles. However, C&D treatments of multispecies biofilm on PVC resulted in lower reductions of L. monocytogenes (between 0.2 and 2.4 log CFU/cm2) compared to single species biofilm, and subsequent regrowth of L. monocytogenes and stable dominance of Enterobacteriaceae and Pseudomonas. In addition, planktonic cultures of L. monocytogenes were deposited and desiccated on dry surfaces without and with the presence of planktonic background microbiota cultures. The observed decline of desiccated cell counts over time was faster on SS compared to PVC. However, the application of C&D resulted in counts below the detection limit of 1.7 log CFU/coupon on both surfaces (reduction of 5.9 log CFU/coupon or less). This study shows that L. monocytogenes is able to form single and multispecies biofilms on PVC with high strain diversity following C&D treatments. This highlights the need to apply more stringent C&D regime treatments for especially PVC and similar surfaces to efficiently remove biofilm cells from food processing surfaces.

Variability in growth and biofilm formation of Listeria monocytogenes in Agaricus bisporus mushroom products.

Foods and food production environments can be contaminated with Listeria monocytogenes and may support growth of this foodborne pathogen. This study aims to characterize the growth and biofilm formation of sixteen L. monocytogenes strains, isolated from mushroom production and processing environments, in filter-sterilized mushroom medium. Strain performance was compared to twelve L. monocytogenes strains isolated from other sources including food and human isolates. All twenty-eight L. monocytogenes strains showed rather similar growth performance at 20 °C in mushroom medium, and also significant biofilm formation was observed for all strains. HPLC analysis revealed the presence of mannitol, trehalose, glucose, fructose and glycerol, that were all metabolized by L. monocytogenes, except mannitol, in line with the inability of L. monocytogenes to metabolize this carbohydrate. Additionally, the growing behavior of L. monocytogenes was tested on whole, sliced and smashed mushroom products to quantify performance in the presence of product-associated microbiota. A significant increase of L. monocytogenes was observed with higher increase of counts when the mushroom products were more damaged, even with the presence of high background microbiota counts. This study demonstrated that L. monocytogenes grows well in mushroom products, even when the background microbiota is high, highlighting the importance to control (re)contamination of mushrooms.

Growth performance of Listeria monocytogenes and background microbiota from mushroom processing environments.

Interaction between Listeria monocytogenes and resident background microbiota may occur in food processing environments and may influence the survival of this pathogen in a factory environment. Therefore the aim of this study was to characterize the growth performance of microbiota isolated from the processing environments of frozen sliced mushrooms, and to investigate the competitive performance of L. monocytogenes when co-cultured with accompanying environmental microbiota. Acinetobacter, Enterobacteriaceae, Lactococcus and Pseudomonas were the most prominent background microbiota isolated from the processing environment of frozen sliced mushrooms. All individual microbiota strains were able to grow and form biofilm in filter-sterilized mushroom medium, with the mannitol-consumers Raoultella and Ewingella as top performers, reaching up to 9.6 and 9.8 log CFU/mL after 48 h incubation at room temperature. When L. monocytogenes mushroom isolates were co-cultured with the microbiota strains, L. monocytogenes counts ranged from 7.6 to 8.9 log CFU/mL after 24 h of incubation, while counts of the microbiota strains ranged from 5.5 to 9.0 log CFU/mL. Prolonged incubation up to 48 h resulted in further increase of L. monocytogenes counts when co-cultured with non-acidifying species Pseudomonas and Acinetobacter reaching 9.1 to 9.2 log CFU/mL, while a decrease of L. monocytogenes counts reaching 5.8 to 7.7 log CFU/mL was observed in co-culture with Enterobacteriaceae and acidifying Lactococcus representatives. In addition, L. monocytogenes grew also in spent mushroom media of the microbiota strains, except in acidified spent media of Lactococcus strains. These results highlight the competitive ability of L. monocytogenes during co-incubation with microbiota in fresh and in spent mushroom medium, indicative of its invasion and persistence capacity in food processing factory environments.

Calling for a systems approach in microbiome research and innovation.

Microbiomes are all around us in natural and cultivated ecosystems, for example, soils, plants, animals and our own body. Microbiomes are essential players of biotechnological applications, and their functions drive human, animal, plant and environmental health. The rapidly developing microbiome research landscape was studied by a global mapping excercise and bibliometric analysis. Although microbiome research is performed in many different science fields, using similar concepts within and across fields, microbiomes are mostly investigated one ecosystem at-a-time. In order to fully understand microbiome impacts and leverage microbial functions, research needs to adopt a systems approach connecting microbiomes and research initiatives in divergent fields to create understanding on how microbiomes can be modulated for desirable functions as a basis of sustainable, circular bioeconomy.

Genomic characteristics of Listeria monocytogenes isolated during mushroom (Agaricus bisporus) production and processing.

Listeria monocytogenes is a foodborne pathogen ubiquitously found in nature and which has been isolated from food and food processing environments. This study aimed to characterize L. monocytogenes strains isolated from the production and processing environments of frozen sliced mushrooms (Agaricus bisporus). An analysis was executed along the mushroom processing chain including one mushroom grower and two mushroom processing factories. A total of 153 L. monocytogenes strains were isolated, which could be grouped in three PCR serogroups, namely, serogroup 1/2a-3a (39.2%), serogroup 1/2b-3b-7 (34.0%) and serogroup 4b-4d-4e (26.8%). A selection of 44 L. monocytogenes strains isolated from the processing environment after cleaning and disinfection (C&D) and from frozen sliced mushrooms was genotyped by whole genome sequencing (WGS), because these strains pose a potential risk for product contamination after C&D and for human consumption. Multilocus sequence typing (MLST) revealed 11 clonal complexes (CCs), with strains belonging to CC1, CC4, CC37 and CC87 being detected in both processing factories. Comparative WGS analysis of the 44 strains showed the presence of Listeria pathogenicity island 1 (LIPI-1) with a disrupted version of actA in all CC1, CC4, CC5, CC59 strains, and all but one CC224 strains. Notably, both inlA and inlB were detected as full-length loci in every strain, except for inlA in a CC6 strain that harbored a three amino acid deletion. LIPI-3 was detected in all CC1, CC4, CC6 and CC224 strains, while LIPI-4 was detected in all CC4 and CC87 strains. In addition, antibiotic susceptibility tests showed susceptibility towards fourteen antibiotics tested. The bcrABC operon was found in one CC5 strain, that showed a higher tolerance towards benzalkonium chloride than any other strain tested with confluent growth till 12.5 µg/ml for the CC5 strain compared to 2.5 µg/ml for the other strains. This study highlights that the ecology of L. monocytogenes in the frozen sliced mushroom production chain is highly diverse, and shows the importance of hygienic measures to control L. monocytogenes along the frozen sliced mushroom production chain.

Geographic and temporal variations in population dynamics of Ixodes ricinus and associated Borrelia infections in The Netherlands.

In a countrywide investigation of the ecological factors that contribute to Lyme borreliosis risk, a longitudinal study on population dynamics of the sheep tick Ixodes ricinus and their infections with Borrelia burgdorferi sensu lato (s.l.) was undertaken at 24 sites in The Netherlands from July 2006 to December 2007. Study sites were mature forests, dune vegetations, or new forests on land reclaimed from the sea. Ticks were sampled monthly and nymphal ticks were investigated for the presence of Borrelia spp. I. ricinus was the only tick species found. Ticks were found in all sites, but with significant spatial and temporal variations in density between sites. Peak densities were found in July and August, with lowest tick numbers collected in December and January. In some sites, questing activities of I. ricinus nymphs and adults were observed in the winter months. Mean monthly Borrelia infections in nymphs varied from 0% to 29.0% (range: 0%-60%), and several sites had significantly higher mean nymphal Borrelia infections than others. Four genospecies of Borrelia burgdorferi s.l. were found, with B. afzelii being dominant at most sites. Borrelia infection rates in nymphal ticks collected in July, September, and November 2006 were significantly higher (23.7%, p<0.01) than those in the corresponding months of 2007 (9.9%). The diversity in Borrelia genospecies between sites was significantly different (p<0.001). Habitat structure (tree cover) was an effective discriminant parameter in the determination of Borrelia infection risk, as measured by the proportion of nymphal ticks infected with B. burgdorferi s.l. Thickness of the litter layer and moss cover were positively related to nymphal and adult tick densities. The study shows that Borrelia-infected ticks are present in many forest and dune areas in The Netherlands and suggests that in such biotopes, which are used for a wide variety of recreational activities, the infection risk is high.

Properties of bacterial endophytes and their proposed role in plant growth.

Bacterial endophytes live inside plants for at least part of their life cycle. Studies of the interaction of endophytes with their host plants and their function within their hosts are important to address the ecological relevance of endophytes. The modulation of ethylene levels in plants by bacterially produced 1-aminocyclopropane-1-carboxylate deaminase is a key trait that enables interference with the physiology of the host plant. Endophytes with this capacity might profit from association with the plant, because colonization is enhanced. In turn, host plants benefit by stress reduction and increased root growth. This mechanism leads to the concept of 'competent' endophytes, defined as endophytes that are equipped with genes important for maintenance of plant-endophyte associations. The ecological role of these endophytes and their relevance for plant growth are discussed here.

PCR detection of oxytetracycline resistance genes otr(A) and otr(B) in tetracycline-resistant streptomycete isolates from diverse habitats.

A range of European habitats was screened by PCR for detection of the oxytetracycline resistance genes otr(A) and otr(B), found in the oxytetracycline-producing strain Streptomyces rimosus. Primers were developed to detect these otr genes in tetracycline-resistant (Tc(R)) streptomycete isolates from environmental samples. Samples were obtained from bulk and rhizosphere soil, manure, activated sludge and seawater. The majority of Tc(R) streptomycetes originated from bulk and rhizosphere soil. Fewer Tc(R) streptomycetes were isolated from manure and seawater and none from sewage. By PCR, three out of 217 isolates were shown to contain the otr(A) gene and 13 out of 217 the otr(B) gene. Surprisingly, these genes were detected in taxonomic groups not known as tetracycline-producing strains. The majority of the otr gene-carrying strains was assigned to S. exfoliatus or S. rochei and originated from all habitats from which Tc(R) streptomycetes were obtained. Our results indicated that the occurrence of otr(A) and otr(B) genes in natural environments was limited and that otr(B), in comparison to otr(A), seemed to be more common.

The Low-Temperature-Induced Viable-But-Nonculturable State Affects the Virulence of Ralstonia solanacearum Biovar 2.

ABSTRACT The physiology and virulence of Ralstonia solanacearum biovar 2 strain 1609, kept in water at 4 and 20 degrees C, were studied. At 20 degrees C, total cell and plate count (colony forming units; CFU) numbers were similar, between log 5.03 and log 5.55 CFU, and log 5.03 and log 5.51 cells per ml, at days 0 and 132, respectively. However, CFU in the cultures kept at 4 degrees C dropped from log 6.78 CFU/ml at day 0 to below detection after 84 days. The presence of catalase in the agar resulted in higher CFU, and at day 84, log 1.95 CFU/ml still was detectable. No colonies were observed at day 125. The presence of viable-but-nonculturable (VBNC) cells in the 4 degrees C cultures was confirmed using SYTO9 viability staining. Viable cell numbers were log 1.77 higher than CFU on plates with catalase. At day 84 and after 125 days, log 3.70 viable cells per ml still were present. Shifts in subpopulations differing in viability were found by flow cytometric sorting of 4 degrees C-treated cells stained with SYTO9 (healthy) and propidium iodide (PI; compromised). The SYTO9-stained cell fractions dropped from 99 to 39%, and the PI-stained fractions increased from 0.7 to 33.3% between days 0 and 125. At 20 degrees C, the SYTO9-stained fraction remained stable at 99% until day 132. SYTO9-stained cells sorted from 4 degrees C cultures at day 100 were injected into tomato plants. Upon incubation for 30 days, these plants did not show wilting. However, more than log 4.19 CFU and log 8.17 cells were recovered from these plants. Cells from colonies isolated from the nonwilted plants did not regain their virulence as demonstrated by subsequent injection into several new sets of tomato plants. Cells from 4 degrees C cultures injected at day 125 were not able to cause wilting of, or proliferate in, tomato plants. The threat posed by VBNC R. solanacearum cells upon incubation at 4 degrees C was thus ephemeral because cells lost their capacity to cause disease after 125 days.

The complete nucleotide sequence and environmental distribution of the cryptic, conjugative, broad-host-range plasmid pIPO2 isolated from bacteria of the wheat rhizosphere.

Plasmid pIPO2 is a cryptic, conjugative, broad-host-range plasmid isolated from the wheat rhizosphere. It efficiently self-transfers between alpha, beta and gamma Proteobacteria and has a mobilizing/retromobilizing capacity for IncQ plasmids. The complete nucleotide sequence of pIPO2 is presented on the basis of its mini-Tn5::luxABtet-tagged derivative, pIPO2T. The pIPO2 sequence is 39815 bp long and contains at least 43 complete ORFs. Apart from a suite of ORFs with unknown function, all of the genes carried on pIPO2 are predicted to be involved in plasmid replication, maintenance and conjugative transfer. The overall organization of these genes is different from previously described plasmids, but is similar to the genetic organization seen in pSB102, a conjugative plasmid recently isolated from the bacterial community of the alfalfa rhizosphere. The putative conjugative transfer region of pIPO2 covers 23 kb and contains the genes required for DNA processing (Dtr) and mating pair formation (Mpf). The organization of these transfer genes in pIPO2 is highly similar to the genetic organization seen in the environmental plasmid pSB102 and in pXF51 from the plant pathogen Xylella fastidiosa. Plasmids pSB102 and pXF51 have recently been proposed to form a new family of environmental broad-host-range plasmids. Here it is suggested that pIPO2 is a new member of this family. The proposed Mpf system of pIPO2 shares high amino acid sequence similarity with equivalent VirB proteins from the type IV secretion system of Brucella spp. Sequence information was used to design primers specific for the detection of pIPO2. Environmental DNA from a range of diverse habitats was screened by PCR with these primers. Consistently positive signals for the presence of pIPO2 were obtained from a range of soil-related habitats, including the rhizospheres of young wheat plants, of field-grown oats and of grass (all gramineous plants), as well as from the rhizosphere of tomato plants. These data add to the growing evidence that plasmids carry advantageous genes with as yet undefined functions in plant-associated communities.

Prevalence of streptomycin-resistance genes in bacterial populations in European habitats.

The prevalence of selected streptomycin (Sm)-resistance genes, i.e. aph (3''), aph (6)-1d, aph (6)-1c, ant (3'') and ant (6), was assessed in a range of pristine as well as polluted European habitats. These habitats included bulk and rhizosphere soils, manure from farm animals, activated sludge from wastewater treatment plants and seawater. The methods employed included assessments of the prevalence of the genes in habitat-extracted DNA by PCR, followed by hybridisation with specific probes, Sm-resistant culturable bacteria and exogenous isolation of plasmids carrying Sm-resistance determinants. The direct DNA-based analysis showed that aph (6)-1d genes were most prevalent in the habitats examined. The presence of the other four Sm-modifying genes was demonstrated in 58% of the tested habitats. A small fraction of the bacterial isolates (8%) did not possess any of the selected Sm-modifying genes. These isolates were primarily obtained from activated sludge and manure. The presence of Sm-modifying genes in the isolates often coincided with the presence of IncP plasmids. Exogenous isolation demonstrated the presence of plasmids of 40-200 kb in size harbouring Sm-resistance genes from all the environments tested. Most plasmids were shown to carry the ant (3'') gene, often in combination with other Sm-resistance genes, such as aph (3'') and aph (6)-1d. The most commonly found Sm-modifying gene on mobile genetic elements was ant (3''). Multiple Sm-resistance genes on the same genetic elements appeared to be the rule rather than the exception. It is concluded that Sm-resistance genes are widespread in the environmental habitats studied and often occur on mobile genetic elements and ant (3'') was most often encountered.

A polyphasic approach for studying the interaction between Ralstonia solanacearum and potential control agents in the tomato phytosphere.

Ralstonia solanacearum biovar 2, the causative agent of brown rot in potato, has been responsible for large crop losses in Northwest Europe during the last decade. Knowledge on the ecological behaviour of R. solanacearum and its antagonists is required to develop sound procedures for its control and eradication in infested fields.A polyphasic approach was used to study the invasion of plants by a selected R. solanacearum biovar 2 strain, denoted 1609, either or not in combination with the antagonistic strains Pseudomonas corrugata IDV1 and P. fluorescens UA5-40. Thus, this study combined plating (spread and drop plate methods), reporter gene technology (gfp mutants) and serological (imunofluorescence colony staining [IFC]) and molecular techniques (fluorescent in situ hybridization [FISH], PCR with R. solanacearum specific primers and PCR-DGGE on plant DNA extracts). The behaviour of R. solanacearum 1609 and the two control strains was studied in bulk and (tomato) rhizosphere soil and the rhizoplane and stems of tomato plants. The results showed that an interaction between the pathogen and the control strains at the root surface was likely. In particular, R. solanacearum 1609 CFU numbers were significantly reduced on tomato roots treated with P. corrugata IDV1(chr:gfp1) cells as compared to those on untreated roots. Concomitant with the presence of P. corrugata IDV1(chr:gfp1), plant invasion by the pathogen was hampered, but not abolished.PCR-DGGE analyses of the tomato rhizoplane supported the evidence for antagonistic activity against the pathogen; as only weak R. solanacearum 1609 specific bands were detected in profiles derived from mixed systems versus strong bands in profiles from systems containing only the pathogen. Using FISH, a difference in root colonization was demonstrated between the pathogen and one of the two antagonists, i.e. P. corrugata IDV1(chr:gfp1); R. solanacearum strain 1609 was clearly detected in the vascular cylinder of tomato plants, whereas strain IDV1 was absent.R. solanacearum 1609 cells were also detected in stems of plants that had developed in soils treated with this strain, even in cases in which disease symptoms were absent, indicating the occurrence of symptomless infection. In contrast, strain 1609 cells were not found in stems of several plants treated with either one of the two antagonists. The polyphasic analysis is valuable for testing antagonistic strains for approval as biocontrol agents in agricultural practice.