Improvements of weaned pigs barn hygiene to reduce the spread of antimicrobial resistance.

The spread of antimicrobial resistance (AMR) in animal husbandry is usually attributed to the use of antibiotics and poor hygiene and biosecurity. We therefore conducted experimental trials to improve hygiene management in weaned pig houses and assessed the impact on the spread. For each of the two groups examined, the experimental group (EG) and the control group (CG), three replicate batches of piglets from the same pig breeder, kept in pre-cleaned flat decks, were analyzed. In the flat decks of the experimental groups, the hygiene conditions (cleaning, disinfection, dust removal and fly control) were improved, while regular hygiene measures were carried out in the control groups. The occurrence and spread of AMR were determined in Escherichia coli (E. coli; resistance indicator) using cultivation-dependent (CFU) and -independent (qPCR) methods as well as whole genome sequencing of isolates in samples of various origins, including feces, flies, feed, dust and swabs. Surprisingly, there were no significant differences (p > 0.05) in the prevalence of resistant E. coli between the flat decks managed with conventional techniques and those managed with improved techniques. Selective cultivation delivered ampicillin- and sulfonamide-resistant E. coli proportions of up to 100% and 1.2%, respectively. While 0.5% E. coli resistant to cefotaxime and no ciprofloxacin resistance were detected. There was a significant difference (p < 0.01) in the abundance of the blaTEM-1 gene in fecal samples between EG and CG groups. The colonization of piglets with resistant pathogens before arrival, the movement of flies in the barn and the treatment of bacterial infections with antibiotics obscured the effects of hygiene improvement. Biocide tolerance tests showed no development of resistance to the farm regular disinfectant. Managing hygiene alone was insufficient for reducing antimicrobial resistances in piglet rearing. We conclude that the complex factors contributing to the presence and distribution of AMR in piglet barns underscore the necessity for a comprehensive management strategy.

Selection of aquatic microbiota exposed to the herbicides flufenacet and metazachlor.

Herbicides are important, ubiquitous environmental contaminants, but little is known about their interaction with bacterial aquatic communities. Here, we sampled a protected natural freshwater habitat and characterised its microbiome in interaction with herbicides. We evolved the freshwater microbiomes in a microcosm assay of exposure (28 days) to flufenacet and metazachlor at environmental concentrations of 0.5, 5 and 50 µg L-1 . Inhibitory effects of herbicides were exemplarily assessed in cultured bacteria from the same pond (Pseudomonas alcaligenes, Paenibacillus amylolyticus and Microbacterium hominis). Findings were compared to long-term concentrations as provided by local authorities. Here, environmental concentrations reached up to 11 µg L-1 (flufenacet) and 76 µg L-1 (metazachlor). Bacteria were inhibited at minimum inhibitory concentrations far above these values; however, concentrations of 50 µg L-1 of flufenacet resulted in measurable growth impairment. While most herbicide-exposed microcosm assays did not differ from controls, Acidobacteria were selected at high environmental concentrations of herbicides. Alpha-diversity (e.g., taxonomic richness on phylum level) was reduced when aquatic microbiomes were exposed to 50 µg metazachlor or flufenacet. One environmental strain of P. alcaligenes showed resistance to high concentrations of flufenacet (50 g L-1 ). In total, this study reveals that ecologic imbalance due to herbicide use significantly impacts aquatic microbiomes.

Tracking Antimicrobial Resistant E. coli from Pigs on Farm to Pork at Slaughter.

Antimicrobial-resistant bacteria might be transferred via the foodchain. However, that risk is rarely tracked along different production steps, e.g., from pigs at farm to meat. To close that gap, we performed a prospective study in four conventional and two organic farms from the moment pigs entered the farm until meat sampling at slaughter. Antimicrobial use was recorded (0 to 11 agents). Antimicrobial susceptibility (AMS) against 26 antibiotics, including critically important substances, was tested by microdilution, and tetA-tetB-sulI-sulII-strA-strB-bla-CTXM-qacEΔ1 were included in PCR-genotyping. From 244 meat samples of 122 pigs, 54 samples (22.1%) from 45 animals were positive for E. coli (n = 198). MICs above the breakpoint/ECOFF occurred for all antibiotics except meropenem. One isolate from organic farming was markedly resistant against beta-lactams including fourth-generation cefalosporines. AMS patterns differed remarkably between isolates from one piece of meat, varying from monoresistance to 16-fold multiresistance. Amplicon-typing revealed high similarity between isolates at slaughter and on farm. Prior pig lots andeven the farmer might serve as reservoirs for E. coli isolated from meat at slaughter. However, AMS phenotyping and genotyping indicate that antimicrobial resistance in E. coli is highly dynamic, impairing reliable prediction of health risks from findings along the production chain.

Spread of antimicrobial resistance genes via pig manure from organic and conventional farms in the presence or absence of antibiotic use.

AIMS: Antibiotic-resistant bacteria affect human and animal health. Hence, their environmental spread represents a potential hazard for mankind. Livestock farming is suspected to be a key factor for spreading antibiotic resistance; consumers expect organic farming to imply less environmental health risk. This study aimed to assess the role of manure from organic and conventional farms for spreading antimicrobial resistance (AMR) genes. METHODS AND RESULTS: AMR-genes-namely tet(A), tet(B), tet(M), sul2 and qacE/qacEΔ1 (potentially associated with multiresistance) were quantified by qPCR. Antimicrobial use during the study period was qualitatively assessed from official records in a binary mode (yes/no). Median concentrations were between 6.44 log copy-equivalents/g for tet(A) and 7.85 for tet(M) in organic liquid manure, and between 7.48 for tet(A) and 8.3 for sul2 in organic farmyard manure. In conventional manure, median concentrations were 6.67 log copy-equivalents/g for sul2, 6.89 for tet(A), 6.77 for tet(B) and 8.36 for tet(M). Integron-associated qac-genes reached median concentrations of 7.06 log copy-equivalents/g in organic liquid manure, 7.13 in conventional manure and 8.18 in organic farmyard manure. The use of tetracyclines or sulfonamides increased concentrations of tet(A) and tet(M), or of sul2, respectively. Comparing farms that did not apply tetracyclines during the study, the relative abundance of tet(A) and tet(M) was still higher for conventional piggeries than for organic ones. CONCLUSIONS: Relative abundances of AMR genes were higher in conventional farms, compared to organic ones. Antibiotic use was linked to the relative abundance of AMR-genes. However, due to the bacterial load, absolute concentrations of AMR-genes were comparable between fertilizers of organic and conventional farms. SIGNIFICANCE AND IMPACT OF STUDY: To our knowledge, this is the first absolute quantification of AMR-genes in manure from organic farms. Our study underlines the importance of long-term reduction in the use of antimicrobial agents in order to minimize antibiotic resistance.

Phenotypic and Genotypic Characterization of C. perfringens Isolates from Dairy Cows with a Pathological Puerperium.

Clostridium perfringens (C. perfringens) forms part of the intestinal microbiome, but is also a known pathogen in histotoxic infections. The significance of the pathogen as a cause of uterine infections in cattle has been little studied so far. Here, we analyzed the association between a pathological puerperium in cattle and the detection of C. perfringens in a prospective longitudinal study. Clostridium perfringens were only found in vaginal and uterine samples of diseased cattle, and were absent in healthy controls. Isolates (n = 21) were tested for the production of major toxins (alpha-, beta-, epsilon-toxin) by ELISA and for the potential of production of major (alpha-, beta-, iota-toxin) and minor toxins (beta2 toxin) by PCR. Furthermore, antimicrobial susceptibility was also tested phenotypically by microdilution. Despite the frequent use of tetracycline treatment in cows suffering from puerperal disorders, no isolate showed phenotypic tetracycline resistance. Most isolates did not release major amounts of toxin. The strict association of C. perfringens with puerperal disease, together with the absence of major toxins might hint towards a major role of other or unknown clostridial virulence factors in uterine disease.

Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples.

The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony-forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α- and ß-diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.

Effects of the herbicides metazachlor and flufenacet on phytoplankton communities - A microcosm assay.

Agrochemicals are the main pollutants in freshwater ecosystems. Metazachlor and flufenacet are two common herbicides applied in fall (i.e., August-October) to agricultural fields in Northern Germany. High concentrations of these herbicides are often found in adjacent aquatic ecosystems. Phytoplankton are one of the highly susceptible non-targeted aquatic organismal groups for herbicides and effects on phytoplankton may initiate a chain of consequences in meta communities through trophic interactions. Few studies have focused on responses of the phytoplankton community for metazachlor and, no studies have focused on flufenacet. We studied the effects of metazachlor and flufenacet on the phytoplankton community by conducting a microcosm experiment exposing natural fall phytoplankton communities to environmentally realistic concentrations as 0 (control), 0.5, 5 and 50 µg L-1 of metazachlor and flufenacet treatments over a 4-week period. We measured changes in density, composition (i.e., in phyla and species level), taxonomic diversity indices, and functional features of phytoplankton communities as a response to herbicides. A reduction in the density of Chlorophyta species (e.g., Koliella longiseta, Selenastrum bibraianum) and Cyanobacteria species (e.g., Merismopedia tenuissima and Aphanocapsa elegans) was observed in herbicide treatments compared to controls. The phytoplankton community shifted towards a high density of species from Bacillariophyta (e.g., Nitzschia fonticola and Cyclotella meneghiniana), Miozoa (i.e., Peridinium willei), and Euglenozoa (i.e., Trachelomonas volvocina) in herbicide treatments compared to controls. Metazachlor and flufenacet showed significant negative effects on taxonomic diversity indices (e.g., species richness, the Shannon-Wiener index) and functional features (e.g., functional dispersion and redundancy) of the phytoplankton communities, with increasing herbicide concentrations. Our study provides insights into direct, selective, and irrecoverable effects of metazachlor and flufenacet on phytoplankton communities in the short-term. The comprehensive understanding of these effects of environmentally realistic herbicide concentrations on aquatic biota is essential for a sustainable management of aquatic ecosystems in agricultural areas.

Mycobacterium avium subsp. paratuberculosis Proteome Changes Profoundly in Milk.

Mycobacterium avium subspecies paratuberculosis (MAP) are detectable viable in milk and other dairy products. The molecular mechanisms allowing the adaptation of MAP in these products are still poorly understood. To obtain information about respective adaptation of MAP in milk, we differentially analyzed the proteomes of MAP cultivated for 48 h in either milk at 37 °C or 4 °C or Middlebrook 7H9 broth as a control. From a total of 2197 MAP proteins identified, 242 proteins were at least fivefold higher in abundance in milk. MAP responded to the nutritional shortage in milk with upregulation of 32% of proteins with function in metabolism and 17% in fatty acid metabolism/synthesis. Additionally, MAP upregulated clusters of 19% proteins with roles in stress responses and immune evasion, 19% in transcription/translation, and 13% in bacterial cell wall synthesis. Dut, MmpL4_1, and RecA were only detected in MAP incubated in milk, pointing to very important roles of these proteins for MAP coping with a stressful environment. Dut is essential and plays an exclusive role for growth, MmpL4_1 for virulence through secretion of specific lipids, and RecA for SOS response of mycobacteria. Further, 35 candidates with stable expression in all conditions were detected, which could serve as targets for detection. Data are available via ProteomeXchange with identifier PXD027444.

First Molecular Characterization of Siphoviridae-Like Bacteriophages Infecting Staphylococcus hyicus in a Case of Exudative Epidermitis.

Exudative epidermitis (EE), also known as greasy pig disease, is one of the most frequent skin diseases affecting piglets. Zoonotic infections in human occur. EE is primarily caused by virulent strains of Staphylococcus (S.) hyicus. Generally, antibiotic treatment of this pathogen is prone to decreasing success, due to the incremental development of multiple resistances of bacteria against antibiotics. Once approved, bacteriophages might offer interesting alternatives for environmental sanitation or individualized treatment, subject to the absence of virulence and antimicrobial resistance genes. However, genetic characterization of bacteriophages for S. hyicus has, so far, been missing. Therefore, we investigated a piglet raising farm with a stock problem due to EE. We isolated eleven phages from the environment and wash water of piglets diagnosed with the causative agent of EE, i.e., S. hyicus. The phages were morphologically characterized by electron microscopy, where they appeared Siphoviridae-like. The genomes of two phages were sequenced on a MiSeq instrument (Illumina), resulting in the identification of a new virulent phage, PITT-1 (PMBT8), and a temperate phage, PITT-5 (PMBT9). Sequencing of three host bacteria (S. hyicus) from one single farm revealed the presence of two different strains with genes coding for two different exfoliative toxin genes, i.e., exhA (2 strains) and exhC (1 strain). The exhC-positive S. hyicus strain was only weakly lysed by most lytic phages. The occurrence of different virulent S. hyicus strains in the same outbreak limits the prospects for successful phage treatment and argues for the simultaneous use of multiple and different phages attacking the same host.

Peripheral blood bovine lymphocytes and MAP show distinctly different proteome changes and immune pathways in host-pathogen interaction.

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogen causing paratuberculosis in cattle and small ruminants. During the long asymptomatic subclinical stage, high numbers of MAP are excreted and can be transmitted to food for human consumption, where they survive many of the standard techniques of food decontamination. Whether MAP is a human pathogen is currently under debate. The aim of this study was a better understanding of the host-pathogen response by analyzing the interaction of peripheral blood lymphocytes (PBL) from cattle with MAP in their exoproteomes/secretomes to gain more information about the pathogenic mechanisms of MAP. Because in other mycobacterial infections, the immune phenotype correlates with susceptibility, we additionally tested the interaction of MAP with recently detected cattle with a different immune capacity referred as immune deviant (ID) cows. In PBL, different biological pathways were enhanced in response to MAP dependent on the immune phenotype of the host. PBL of control cows activated members of cell activation and chemotaxis of leukocytes pathway as well as IL-12 mediated signaling. In contrast, in ID cows CNOT1 was detected as highly abundant protein, pointing to a different immune response, which could be favorable for MAP. Additionally, MAP exoproteomes differed in either GroEL1 or DnaK abundance, depending on the interacting host immune response. These finding point to an interdependent, tightly regulated response of the bovine immune system to MAP and vise versa.

Short communication: Selection of extended-spectrum ß-lactamase-producing Escherichia coli in dairy calves associated with antibiotic dry cow therapy-A cohort study.

Antimicrobial residues in milk have been discussed as a possible selector for Enterobacteriaceae that produce extended-spectrum ß-lactamases (ESBL) in dairy herds. Such residues are found in waste milk after antibiotic treatment of mastitis, but antibiotic dry cow therapy might also lead to antibiotic residues in colostrum and in milk during early lactation. While it is known that feeding of waste milk selects ESBL bacteria in calves, this was not investigated for colostrum yet, which is supposed to contain much lower antibiotic concentrations than waste milk. In this observational prospective case study on 2 farms, we hypothesized that blanket dry cow treatment with ß-lactams would have more selective (here: increasing) effects on ESBL concentrations than selective (here: individually chosen) antibiotic dry cow therapy. Thus, we compared concentrations of ESBL-producing Enterobacteriaceae in feces of calves (n = 50) at 2 dairy farms with different management of antibiotic dry cow therapy. Considerably higher concentrations of ESBL-producing Escherichia coli were observed in blanket antibiotic dry cow therapy on d 3 of the calf's life (7.6 vs. 5.3 log cfu/g of calf feces). Both farms used narrow-spectrum penicillin combined with aminoglycosides for drying off, and the majority of ESBL isolates (93%) were co-resistant to aminoglycosides. No waste milk was fed to calves and no calf was treated with ß-lactam antibiotics or aminoglycosides during the first 3 d of life, thus differences were most likely associated with different frequency of antibiotic dry cow therapy on farms (19 of 25 mother cows on farm A, 9 of 25 on farm B). Even though the presumable selection effect of antibiotics used for drying off decreased within the next 3 wk, this result further emphasizes the need for the reduction and prudent use of antibiotic dry cow therapy on farms.

Antimicrobial resistance of Enterobacter cloacae complex isolates from the surface of muskmelons.

The increasing antimicrobial resistance (AMR) among pathogenic and opportunistic pathogenic microorganisms is one of the main global public health problems. The consumption of food contaminated with such bacteria (ARB), especially of raw products, might result in the direct acquisition of ARB and in a spread of resistant bacteria along the food chain. The aim of the study was to characterize the antimicrobial susceptibility of potentially extended spectrum ß-lactamase (ESBL) producing or AmpC resistant Enterobacteriaceae isolated from the surface of 147 muskmelons from wholesale and retail. A phenotypic analysis was carried out by using minimum inhibitory concentration (MIC) test strips for ESBL detection and MIC susceptibility plates against 14 antimicrobials. Furthermore, ESBL genes, sul-genes and plasmid-mediated AmpC resistance were analyzed by real-time PCR. Additionally, a further insight in the AmpC resistance of isolates of the Enterobacter cloacae complex (ECC) was obtained by analyzing the sequence of the ampC regulatory region (n = 15). A total of 73 potentially resistant Enterobacteriaceae were isolated from 56 muskmelons. Of these, 15 isolates of the ECC were suspicious for ESBL/AmpC resistance, and eleven thereof were positive for the AmpC family EBC. Phenotypic analysis showed diminished susceptibility against "critically" and "highly important" antimicrobials, according to the WHO classification. Furthermore, divergence in the ampC regulatory region was detected between the 15 isolates. These findings highlight the important role that raw produce might play in the transmission of antimicrobial resistances along the food chain.

A Listeria monocytogenes ST2 clone lacking chitinase ChiB from an outbreak of non-invasive gastroenteritis.

An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.

Unraveling the Role of Vegetables in Spreading Antimicrobial-Resistant Bacteria: A Need for Quantitative Risk Assessment.

In recent years, vegetables gain consumer attraction due to their reputation of being healthy in combination with low energy density. However, since fresh produce is often eaten raw, it may also be a source for foodborne illness. The presence of antibiotic-resistant bacteria might pose a particular risk to the consumer. Therefore, this review aims to present the current state of knowledge concerning the exposure of humans to antibiotic-resistant bacteria via food of plant origin for quantitative risk assessment purposes. The review provides a critical overview of available information on hazard identification and characterization, exposure assessment, and risk prevention with special respect to potential sources of contamination and infection chains. Several comprehensive studies are accessible regarding major antimicrobial-resistant foodborne pathogens (e.g., Salmonella spp., Listeria spp., Bacillus cereus, Campylobacter spp., Escherichia coli) and other bacteria (e.g., further Enterobacteriaceae, Pseudomonas spp., Gram-positive cocci). These studies revealed vegetables to be a potential-although rare-vector for extended-spectrum beta-lactamase-producing Enterobacteriaceae, mcr1-positive E. coli, colistin- and carbapenem-resistant Pseudomonas aeruginosa, linezolid-resistant enterococci and staphylococci, and vancomycin-resistant enterococci. Even if this provides first clues for assessing the risk related to vegetable-borne antimicrobial-resistant bacteria, the literature research reveals important knowledge gaps affecting almost every part of risk assessment and management. Especially, the need for (comparable) quantitative data as well as data on possible contamination sources other than irrigation water, organic fertilizer, and soil becomes obvious. Most crucially, dose-response studies would be needed to convert a theoretical "risk" (e.g., related to antimicrobial-resistant commensals and opportunistic pathogens) into a quantitative risk estimate.

Probiotic Bacillus cereus Strains, a Potential Risk for Public Health in China.

Bacillus cereus is an important cause of foodborne infectious disease and food poisoning. However, B. cereus has also been used as a probiotic in human medicine and livestock production, with low standards of safety assessment. In this study, we evaluated the safety of 15 commercial probiotic B. cereus preparations from China in terms of mislabeling, toxin production, and transferable antimicrobial resistance. Most preparations were incorrectly labeled, as they contained additional bacterial species; one product did not contain viable B. cereus at all. In total, 18 B. cereus group strains-specifically B. cereus and Bacillus thuringiensis-were isolated. Enterotoxin genes nhe, hbl, and cytK1, as well as the ces-gene were assessed by PCR. Enterotoxin production and cytotoxicity were confirmed by ELISA and cell culture assays, respectively. All isolated B. cereus group strains produced the enterotoxin Nhe; 15 strains additionally produced Hbl. Antimicrobial resistance was assessed by microdilution; resistance genes were detected by PCR and further characterized by sequencing, transformation and conjugation assays. Nearly half of the strains harbored the antimicrobial resistance gene tet(45). In one strain, tet(45) was situated on a mobile genetic element-encoding a site-specific recombination mechanism-and was transferable to Staphylococcus aureus and Bacillus subtilis by electro-transformation. In view of the wide and uncontrolled use of these products, stricter regulations for safety assessment, including determination of virulence factors and transferable antimicrobial resistance genes, are urgently needed.

Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA.

Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed.

Reconstruction of the original mycoflora in pelleted feed by PCR-SSCP and qPCR.

Ground feeds for pigs were investigated for fungal contamination before and after pelleting (subsamples in total n = 24) by cultural and molecular biological methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the resulting acrylamide gel, more than 85% of DNA bands of ground feeds were preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was equivalent to 4.77-5.69 log10  CFU g(-1) , calculated by qPCR using a standard curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in average ± 0.07 log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51 log10  CFU g(-1) culturable fungi, while there was < 2.83 log10  CFU g(-1) detected in pelleted feeds. This result shows that, while the process of pelleting reduced the amount of living fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the described methodology was able to reconstruct the fungal microbiota in feeds and reflected a considerable fungal contamination of raw materials such as grains.

Insusceptibility to disinfectants in bacteria from animals, food and humans-is there a link to antimicrobial resistance?

Enterococcus faecalis (n = 834) and Enterococcus faecium (n = 135) from blood and feces of hospitalized humans, from feces of outpatients and livestock and from food were screened for their susceptibility to a quaternary ammonium compound (didecyldimethylammoniumchloride, DDAC) and to 28 antibiotics by micro-/macrodilution. The maximum DDAC-MIC in our field study was 3.5 mg/l, but after adaptation in the laboratory, MIC values of 21.9 mg/l were observed. Strains for which DDAC had MICs > 1.4 mg/l ("non-wildtype," in total: 46 of 969 isolates/4.7%) were most often found in milk and dairy products (14.6%), while their prevalence in livestock was generally low (0-4%). Of human isolates, 2.9-6.8% had a "non-wildtype" phenotype. An association between reduced susceptibility to DDAC, high-level-aminoglycoside resistance and aminopenicillin resistance was seen in E. faecium (p < 0.05). No indications for a common source of non-wildtype strains were found by RAPD-PCR; however, several non-wildtype E. faecalis shared the same variant of the emeA-gene. In addition, bacteria (n = 42) of different genera were isolated from formic acid based boot bath disinfectant (20 ml of 55% formic acid/l). The MICs of this disinfectant exceeded the wildtype MICs up to 20-fold (staphylococci), but were still one to three orders of magnitude below the used concentration of the disinfectant (i. e., 1.1% formic acid). In conclusion, the bacterial susceptibility to disinfectants still seems to be high. Thus, the proper use of disinfectants in livestock surroundings along with a good hygiene praxis should still be highly encouraged. Hints to a link between antibiotic resistance and reduced susceptibility for disinfectants-as seen for E. faecium-should be substantiated in further studies and might be an additional reason to confine the use of antibiotics.

Presence of the vancomycin resistance gene cluster vanC1, vanXYc, and vanT in Enterococcus casseliflavus.

The three chromosomally located clustered genes vanC1, vanXYc, and vanT confer intrinsic resistance to vancomycin and are used for species identification of Enterococcus gallinarum. In this study, 28 strains belonging to the E. gallinarum/casseliflavus group isolated from cloacal swabs from laying hens were screened for the presence of vanC1. As confirmed by species-specific multiplex PCR, 11 vanC1-positive strains were identified as E. gallinarum. Surprisingly, one yellow pigmented strain, verified as E. casseliflavus by species-specific multiplex PCR, was also vanC1 positive; vanXYc and vanT were additionally detectable in this strain. To our knowledge, this is the first report of vanC1, vanXYc, and vanT in E. casseliflavus. The minimum inhibitory concentration of vancomycin was 4 mg/L. Real-time reverse transcription-PCR revealed that none of the clustered genes was expressed in this strain. Even if the genes seem not to be active, there is a certain risk that they will be transferred to other bacteria where they might be functionally expressed. Therefore, it may be advisable to expand the search for vanC1, vanXYc, and vanT from E. gallinarum to other (enterococcal) species. This study confirms that enterococci live up to their name as being reservoir bacteria and should therefore always be closely monitored.