Characterization of foamed plastic litter on Danish reference beaches - Pollution assessment and multivariate exploratory analysis.

Foamed litter comprise a significant amount of the pollution at beaches globally. This group represents a variety of foamed items and fragments originating from different applications and sources. Although foamed plastic contributes importantly to the marine environmental pollution, there is generally limited knowledge of the composition of this litter pool. The aim of this study was to characterize item types and polymer materials of foamed litter from six Danish reference beaches during the period 2018-2021. The foamed litter were classified into ten categories, including identifiable items, as well as fragments of foamed PS, or pieces of other foamed polymers of rigid or flexible sponges. Foamed PS (42%) and PUR (49%) were identified as the dominant polymers by FTIR analysis. Multivariate exploratory analysis was performed to investigate PUR foam, and specific spectra features for rigid and flexible foam were demonstrated. Furthermore, we assessed different correlation methods for identification of PUR foams.

A nationwide assessment of plastic pollution in the Danish realm using citizen science.

Plastic pollution is considered one of today's major environmental problems. Current land-based monitoring programs typically rely on beach litter data and seldom include plastic pollution further inland. We initiated a citizen science project known as the Mass Experiment inviting schools throughout The Danish Realm (Denmark, Greenland and the Faeroe Islands) to collect litter samples of and document plastic pollution in 8 different nature types. In total approximately 57,000 students (6-19 years) collected 374,082 plastic items in 94 out of 98 Danish municipalities over three weeks during fall 2019. The Mass Experiment was the first scientific survey of plastic litter to cover an entire country. Here we show how citizen science, conducted by students, can be used to fill important knowledge gaps in plastic pollution research, increase public awareness, establish large scale clean-up activities and subsequently provide information to political decision-makers aiming for a more sustainable future.

Airborne MRSA and Total Staphylococcus aureus as Associated With Particles of Different Sizes on Pig Farms.

Airborne methicillin-resistant Staphylococcus aureus (MRSA) have previously been found on pig farms, which may lead to nasal deposition of MRSA in humans via inhalation. The anterior nares are the main niche for S. aureus, and S. aureus can cause, e.g. wound infection and pneumonia. The aim of this study was to acquire knowledge about the potential deposition of airborne MRSA, specifically, and of total S. aureus (including both methicillin-sensitive S. aureus and MRSA, in the following called S. aureus) in the different parts of the airways during occupancy on pig farms. Measurements of airborne MRSA and S. aureus were performed on four pig farms using a six and a three-stage sampler during different work tasks, such as high-pressure cleaning and everyday inspection. MRSA were quantified using MRSA-selective agar, and S. aureus were quantified using Staphylococcus selective agar. The identity of the bacteria were confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The geometric mean (GM) concentrations of MRSA and S. aureus were 447 cfu/m3 air and 1.8 × 103 cfu/m3 air, respectively. The highest concentrations of MRSA and S. aureus were found among pigs in a weaner stable and during high-pressure cleaning of an empty stable, respectively. The lowest concentrations of MRSA and S. aureus were found in a stable with sick pigs and in feed-storages, respectively. Most MRSA and S. aureus were associated with particles between 7 and 12 µm. On average, the particle size fractions potentially depositing in the upper airways constituted 70%, in the primary and secondary bronchi 22%, and in the terminal bronchi and alveoli 8% of the inhalable MRSA and S. aureus concentration. Across the sampled areas, the geometric mean diameter (Dg) of particles with MRSA and S. aureus were 7.2 and 6.4 µm, respectively, and no significant difference was found between these Dgs. The Dg of the airborne particles with the studied bacterium was significantly associated with the different locations on the farms. The largest Dgs were found in the air samples from the aisles and on the fence to the pens, while the smallest Dgs were found in samples from the pens among the pigs and in samples taken at greater distances from the pigs: in the hallway, feed-storage, and entry room. In conclusion, airborne MRSA and S. aureus were found in sample fractions potentially depositing in all six parts of the airways. However, the majority was found to potentially deposit in the upper airways. The concentration of airborne MRSA and S. aureus and MRSA, as well as the fraction potentially depositing in the different parts of the airways, depended on the specific work task being performed and the location on the farm.

Survival of LA-MRSA in Dust from Swine Farms.

Dust is suspected to be an important factor in transmission of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) between pigs and pig farmers and their families. The aim of this study was to determine the rate of decay for Staphylococcus aureus and LA-MRSA in dust from swine farms. Electrostatic dust fall collectors (EDCs) were used for passive sampling of settling airborne dust in 11 stable sections from six swine farms. Extraction, plating, identification, and enumeration of cultivable S. aureus and LA-MRSA from the EDCs were performed after storage for 0-30 days postsampling. The survival of S. aureus was measured in 196 dust samples from all farms, and data were used to estimate the decay constant λ according to a model for exponential decay: N(t) = N0 × e-λt. The number of S. aureus colonies was up to 600-fold higher than the number of LA-MRSA colonies on MRSA selective agar. The data showed a good fit to the model (λ = 0.13, r2 = 0.86) even with a large difference in initial concentrations of S. aureus between stables. The loads of S. aureus and LA-MRSA in the dust were significantly reduced by storage time, and the half-life was 5 days for both S. aureus and LA-MRSA. In dust samples with high initial concentrations, LA-MRSA and S. aureus could still be cultivated 30 days after sampling. On all farms MRSA isolates belonged to the clonal complex (CC) 398, and at one farm some isolates also belonged to CC30. A screening for other Staphylococcus species in the farm dust revealed 13 different species numerically dominated by Staphylococcus equorum. Based on the exponential decay model, S. equorum had a half-life of 4 days. In conclusion, the presence of MRSA in airborne dust from five of six farms indicates that dust might be an important vehicle for transmission of LA-MRSA. LA-MRSA and S. aureus was found to survive well in farm dust with half-lives of 5 days, and dependent on the initial concentration they could be found in farm dust for weeks. The 99.9% die-off rate was 66 days for LA-MRSA. Thus, farm dust can pose an exposure risk for humans in the farm environment, but also when transported to other environments. On the other hand, the risk will decrease by time. These results provide important knowledge to diminish spread from farm environments to other environments on, e.g., tools or clothing, and in relation to cleaning of emptied LA-MRSA-positive stables.

No apparent transmission of livestock-associated methicillin-resistant Staphylococcus aureus CC398 in a survey of staff at a regional Danish hospital.

Background: In recent years, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) multi locus sequence type CC398 has spread widely in the livestock production in Europe. The rates of LA-MRSA in hospitals have been found to be largely determined by contact to and density of livestock in the area. Methods: This is a cross sectional study of the prevalence of LA-MRSA among hospital staff in a Danish hospital situated in a livestock production region. We analysed nasal swabs, air and dust samples for the presence of MRSA using PCR and mass spectrometry. Results: Of 1745 employees, 545 (31%) contributed nasal swabs. MRSA was not detected in any participant, nor was it detected in air or dust at the hospital or in houses of employees living on farms. Four percent of the participants had contact to pigs either directly or through household members. LA-MRSA was detected in two of 26 samples from animal sheds, both of them from pig farms. The participation rate was relatively low, but participants were representative for the source population with regards to animal contact and job titles. Conclusions: The study suggests a low point prevalence of LA-MRSA carriage in Danish hospital staff even in regions where livestock production is dense. Should more studies confirm our findings we see no need for additional hospital precautions towards LA-MRSA in Denmark at the moment. We think that our data might reduce potential stigmatization of hospital workers with contact to LA-MRSA positive farms at their work places and in their communities.

Transmission of Methicillin-Resistant Staphylococcus aureus to Human Volunteers Visiting a Swine Farm.

Transmission of methicillin-resistant Staphylococcus aureus (MRSA) from animals to humans is of great concern due to the implications for human health and the health care system. The objective was to investigate the frequency and duration of MRSA carriage in human volunteers after a short-term exposure in a swine farm. The experimental study included 34 human volunteers staying 1 h in a MRSA-positive swine farm in four trials. In two of the trials, the influence of farm work involving pig contact was studied using a crossover design. The quantities of MRSA in nasal swabs, throat swabs, and air samples were measured at different time points and analyzed in relation to relevant covariates. This investigation showed that, overall, 94% of the volunteers acquired MRSA during the farm visit. Two hours after the volunteers left the stable, the nasal MRSA count had declined to unquantifiable levels in 95% of the samples. After 48 h, 94% of the volunteers were MRSA-negative. Nasal MRSA carriage was positively correlated to personal exposure to airborne MRSA and farm work involving pig contact and negatively correlated to smoking. No association was observed between MRSA carriage and face touching behavior, nasal methicillin-susceptible Staphylococcus aureus (MSSA) carriage, age, or gender. The increase in human MRSA carriage among the volunteers with pig contact seems to be dependent on the increased concentration of airborne MRSA of the surrounding air and not directly on physical contact with pigs. MRSA was not detected in any of the throat samples.IMPORTANCE The experimental approach made it possible to elucidate the contributions of airborne MRSA levels and farm work to nasal MRSA carriage in a swine farm. Short-term exposure to airborne MRSA poses a substantial risk for farm visitors to become nasal carriers, but the carriage is typically cleared within hours to a few days. The risk for short-term visitors to cause secondary transmissions of MRSA is most likely negligible due to the observed decline to unquantifiable levels in 95% of the nasal samples after only 2 h. The MRSA load in the nose was highly correlated to the amount of MRSA in the air and interventions to reduce the level of airborne MRSA or the use of face masks might consequently reduce nasal contamination.

Establishment of Bacterial Herbicide Degraders in a Rapid Sand Filter for Bioremediation of Phenoxypropionate-Polluted Groundwater.

In this study, we investigated the establishment of natural bacterial degraders in a sand filter treating groundwater contaminated with the phenoxypropionate herbicides (RS)-2-(4-chloro-2-methylphenoxy)propanoic acid (MCPP) and (RS)-2-(2,4-dichlorophenoxy)propanoic acid (DCPP) and the associated impurity/catabolite 4-chlorophenoxypropanoic acid (4-CPP). A pilot facility was set up in a contaminated landfill site. Anaerobic groundwater was pumped up and passed through an aeration basin and subsequently through a rapid sand filter, which is characterized by a short residence time of the water in the filter. For 3 months, the degradation of DCPP, MCPP, and 4-CPP in the sand filter increased to 15 to 30% of the inlet concentration. A significant selection for natural bacterial herbicide degraders also occurred in the sand filter. Using a most-probable-number (MPN) method, we found a steady increase in the number of culturable phenoxypropionate degraders, reaching approximately 5 × 10(5) degraders per g sand by the end of the study. Using a quantitative PCR targeting the two phenoxypropionate degradation genes, rdpA and sdpA, encoding stereospecific dioxygenases, a parallel increase was observed, but with the gene copy numbers being about 2 to 3 log units higher than the MPN. In general, the sdpA gene was more abundant than the rdpA gene, and the establishment of a significant population of bacteria harboring sdpA occurred faster than the establishment of an rdpA gene-carrying population. The identities of the specific herbicide degraders in the sand filter were assessed by Illumina MiSeq sequencing of 16S rRNA genes from sand filter samples and from selected MPN plate wells. We propose a list of potential degrader bacteria involved in herbicide degradation, including representatives belonging to the Comamonadaceae and Sphingomonadales.

Degradation of trace concentrations of the persistent groundwater pollutant 2,6-dichlorobenzamide (BAM) in bioaugmented rapid sand filters.

Groundwater is an important drinking water resource. Yet, this resource is threatened by pollution from chemicals, such as pesticides and their degradation products. To investigate the potential for remediation of groundwater polluted by trace concentrations of the pesticide residue 2,6-dichlorobenzamide (BAM), we established a pilot waterworks including two sand filters. The waterworks treated groundwater polluted with 0.2 µg/L BAM at flow conditions typical for rapid sand filters. Bioaugmentation of the sand filter with a specific BAM-degrading bacterium (Aminobacter sp. MSH1) resulted in significant BAM degradation to concentrations below the legal threshold level (0.1 µg/L), and this without adverse effects on other sand filter processes such as ammonium and iron oxidation. However, efficient degradation for more than 2-3 weeks was difficult to maintain due to loss of MSH1-bacteria, especially during backwashing. By limiting backwash procedures, the period of degradation was prolonged, but bacteria (and hence degradation activity) were still lost with time. Protozoa were observed to grow in the filters to a density that contributed significantly to the general loss of bacteria from the filters. Additionally, the concentration of easily assimilable organic carbon (AOC) in the remediated water may have been too low to sustain a sufficient population of degrader bacteria in the filter. This study shows that scaling up is not trivial and shortcomings in transferring degradation rates obtained in batch experiments to a rapid sand filter system are discussed. Further optimization is necessary to obtain and control more temporally stable systems for water purification. However, for the first time outside the laboratory and at realistic conditions a potential for the biodegradation of recalcitrant micropollutants in bioaugmented rapid sand filters is shown.

Pesticide Side Effects in an Agricultural Soil Ecosystem as Measured by amoA Expression Quantification and Bacterial Diversity Changes.

BACKGROUND AND METHODS: Assessing the effects of pesticide hazards on microbiological processes in the soil is currently based on analyses that provide limited insight into the ongoing processes. This study proposes a more comprehensive approach. The side effects of pesticides may appear as changes in the expression of specific microbial genes or as changes in diversity. To assess the impact of pesticides on gene expression, we focused on the amoA gene, which is involved in ammonia oxidation. We prepared soil microcosms and exposed them to dazomet, mancozeb or no pesticide. We hypothesized that the amount of amoA transcript decreases upon pesticide application, and to test this hypothesis, we used reverse-transcription qPCR. We also hypothesized that bacterial diversity is affected by pesticides. This hypothesis was investigated via 454 sequencing and diversity analysis of the 16S ribosomal RNA and RNA genes, representing the active and total soil bacterial communities, respectively. RESULTS AND CONCLUSION: Treatment with dazomet reduced both the bacterial and archaeal amoA transcript numbers by more than two log units and produced long-term effects for more than 28 days. Mancozeb also inhibited the numbers of amoA transcripts, but only transiently. The bacterial and archaeal amoA transcripts were both sensitive bioindicators of pesticide side effects. Additionally, the numbers of bacterial amoA transcripts correlated with nitrate production in N-amended microcosms. Dazomet reduced the total bacterial numbers by one log unit, but the population size was restored after twelve days. The diversity of the active soil bacteria also seemed to be re-established after twelve days. However, the total bacterial diversity as reflected in the 16S ribosomal RNA gene sequences was largely dominated by Firmicutes and Proteobacteria at day twelve, likely reflecting a halt in the growth of early opportunists and the re-establishment of a more diverse population. We observed no effects of mancozeb on diversity.

High-resolution melt analysis for rapid comparison of bacterial community compositions.

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.

Bactericidal antibiotics do not appear to cause oxidative stress in Listeria monocytogenes.

Oxidative stress can be an important contributor to the lethal effect of bactericidal antibiotics in some bacteria, such as Escherichia coli and Staphylococcus aureus. Thus, despite the different target-specific actions of bactericidal antibiotics, they have a common mechanism leading to bacterial self-destruction by internal production of hydroxyl radicals. The purpose of the present study was to determine if a similar mechanism is involved in antibiotic killing of the infectious human pathogen, Listeria monocytogenes. We treated wild-type L. monocytogenes and oxidative stress mutants (Δsod and Δfri) with three different bactericidal antibiotics and found no difference in killing kinetics. In contrast, wild-type E. coli and an oxidative stress mutant (ΔsodA ΔsodB) differed significantly in their sensitivity to bactericidal antibiotics. We conclude that bactericidal antibiotics did not appear to cause oxidative stress in L. monocytogenes and propose that this is caused by its noncyclic tricarboxylic acid (TCA) pathway. Hence, in this noncyclic metabolism, there is a decoupling between the antibiotic-mediated cellular requirement for NADH and the induction of TCA enzyme activity, which is believed to mediate the oxidative stress reaction.

Characterization of a small erythromycin resistance plasmid pLFE1 from the food-isolate Lactobacillus plantarum M345.

This paper reports the complete 4031 bp nucleotide sequence of the small erythromycin resistance plasmid pLFE1 isolated from the raw-milk cheese isolate Lactobacillus plantarum M345. Analysis of the sequence revealed the coding regions for the erythromycin resistance determinant Erm(B). A replication initiation protein RepB was identified belonging to the RepB proteins of the pMV158 family of rolling-circle replicating plasmids. The transcriptional repressor protein CopG and a small counter transcribed RNA, two elements typically involved in replication control within this family were also found. A putative replication initiation site including a single-strand origin (sso) -like region succeeded by a characteristic pMV158 family double-strand origin (dso) was located upstream of the replication region. An open reading frame following a typical origin of transfer (oriT) site and coding for a putative truncated mobilization (Mob) protein with a size of 83 aa was detected. The product of the putative mob gene showed large similarity to the N-terminal region of the pMV158 family of Pre/Mob proteins, but was much smaller than other proteins of this family. We therefore suggest that the Mob function in pLFE1 is supplied in trans from another plasmid present in L. plantarum M345.Filter-mating experiments showed that pLFE1 has a broad host-range with transconjugants obtained from Lactobacillus rhamnosus, Lactococcus lactis, Listeria innocua, the opportunistic pathogen Enterococcus faecalis and the pathogen Listeria monocytogenes.

A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species.

Optimal conditions and a standardized method for conjugation between two model lactococcal strains, Lactococcus lactis SH4174 (pAMbeta1-containing, erythromycin resistant donor) and L. lactis Bu2-60 (plasmid-free, erythromycin sensitive recipient), were developed and tested in a inter-laboratory experiments involving five laboratories from different countries. The ultimate goal of the study was to assess the microbial potential of antibiotic resistance transfer among Lactic Acid Bacteria (LAB). The influence of culture age (various OD values) and ratios of donor and recipient cultures as well as filter, solid and liquid mating techniques, were examined in order to optimize the conjugation protocol. In the result of these studies, we concluded that the donor-to-recipient ratio appear to be important; the most efficient technique for conjugation was filter mating and the optimal conditions for gene transfer were observed when late logarithmic cultures of both donor and recipient were used. Comparison of conjugal transfer frequencies between five partner laboratories showed that results are sufficiently intra-laboratory repeatable and inter-laboratory comparable. This is the first study of this kind, in which a standardized protocol of conjugal mating for testing antibiotic resistance dissemination among LAB was established and validated.

Selective pressure affects transfer and establishment of a Lactobacillus plantarum resistance plasmid in the gastrointestinal environment.

OBJECTIVES AND METHODS: A Lactobacillus plantarum strain recently isolated from French raw-milk cheese was tested for its ability to transfer a small plasmid pLFE1 harbouring the erythromycin resistance gene erm(B) to Enterococcus faecalis. Mating was studied in vitro and in different gastrointestinal environments using gnotobiotic rats as a simple in vivo model and streptomycin-treated mice as a more complex model. Transfer and establishment of transconjugants in the intestine were investigated with and without selective pressure. RESULTS: Compared with the relatively low transfer frequency of approximately 5.7 x 10(-8) transconjugants/recipient obtained in vitro by filter mating, a surprisingly high number of transconjugants (10(-4) transconjugants/recipient) was observed in gnotobiotic rats even without antibiotic treatment. When erythromycin was administered, a transfer rate of approximately 100% was observed, i.e. the recipient population turned completely into transconjugants (3 x 10(9) cfu/g faeces). Additionally, the time to reach a stable transconjugant population level was much faster in the erythromycin-treated gnotobiotic rats (1 day) than in the untreated animals (4-5 days). Transconjugants persisted in the gut in relatively stable numbers at least 12 days after termination of antibiotic treatment. In the streptomycin-treated mice, no transfer was observed either with or without erythromycin treatment. CONCLUSIONS: The overall results imply that the gastrointestinal tract may comprise a more favourable environment for antibiotic resistance transfer than conditions provided in vitro. However, the indigenous gut microbiota severely restricts transfer, thus minimizing the number of detectable transfer events. Treatment with erythromycin strongly favoured transfer and establishment of pLFE1.