[Health risks from crop irrigation with treated wastewater containing antibiotic residues, resistance genes, and resistant microorganisms]. / Gesundheitsrisiken durch die Bewässerung von Nutzpflanzen mit aufbereitetem Abwasser, das Antibiotikarückstände, Resistenzgene und resistente Mikroorganismen enthält.

This review describes the effects and potential health risks of resistant microorganisms, resistance genes, and residues of drugs and biocides that occur when re-using wastewater for crop irrigation. It focusses on specific aspects of these contaminants and their interactions, but does not provide a general risk assessment of the microbial load when using reclaimed water.Antimicrobial residues, antimicrobial resistant microorganisms, and resistance genes are frequently detected in treated wastewater. They have effects on the soil and plant-associated microbiota (total associated microorganisms) and can be taken up by plants. An interaction of residues with microorganisms is mainly expected before using the water for irrigation. However, it may also occur as a combined effect on the plant microbiome and all the abundant resistance genes (resistome). Special concerns are raised as plants are frequently consumed raw, that is, without processing that might reduce the bacterial load. Washing fruits and vegetables only has minor effects on the plant microbiome. On the other hand, cutting and other processes may support growth of microorganisms. Therefore, after such process steps, cooling of the foods is required.Further progress has to be made in the treatment of wastewater that will be used for crop irrigation with respect to removing micropollutants and microorganisms to minimize the risk of an increased exposure of consumers to transferable resistance genes and resistant bacteria.

Pseudomonas rustica sp. nov., isolated from bulk tank raw milk at a German dairy farm.

Here we present the description of a novel Pseudomonas species, designated Pseudomonas rustica sp. nov., which was isolated from raw milk samples obtained from Germany. Results of initial 16S rRNA gene sequence analysis assigned the strain into the genus Pseudomonas and showed Pseudomonas helmanticensis, Pseudomonas neuropathica and Pseudomonas atagonensis to be its closest relatives. Further studies including sequence analysis of the rpoB gene, multi-gene phylogenetic tree reconstruction, whole-genome sequence comparisons, cellular fatty acid analysis and chemotaxonomic characterization showed a clear separation from the known Pseudomonas species. Isolate MBT-4T was closely related to Pseudomonas helmanticensis, 'Pseudomonas crudilactis' and Pseudomonas neuropathica with average nucleotide identities based on blast values of 88.8, 88.8 and 88.6%, respectively. Therefore, the strain can be classified into the Pseudomonas koreensis subgroup of the Pseudomonas fluorescens group. The G+C content of strain MBT-4T was 58.9 mol%. The strain was catalase- and oxidase-positive, while the ß-galactosidase reaction was negative. Growth occurred between 4 and 30 °C and at pH values from pH 6.0 to 8.0. In conclusion, strain MBT-4T belongs to a novel species, for which the name Pseudomonas rustica sp. nov. is proposed. The type strain is MBT-4T (=DSM 112348T=LMG 32241T) and strain MBT-17 is also a representative of this species.

Taxonomic Evaluation of the Heyndrickxia (Basonym Bacillus) sporothermodurans Group (H. sporothermodurans, H. vini, H. oleronia) Based on Whole Genome Sequences.

The genetic heterogeneity of Heyndrickxia sporothermodurans (formerly Bacillussporothermodurans) was evaluated using whole genome sequencing. The genomes of 29 previously identified Heyndrickxiasporothermodurans and two Heyndrickxia vini strains isolated from ultra-high-temperature (UHT)-treated milk were sequenced by short-read (Illumina) sequencing. After sequence analysis, the two H. vini strains could be reclassified as H. sporothermodurans. In addition, the genomes of the H.sporothermodurans type strain (DSM 10599T) and the closest phylogenetic neighbors Heyndrickxiaoleronia (DSM 9356T) and Heyndrickxia vini (JCM 19841T) were also sequenced using both long (MinION) and short-read (Illumina) sequencing. By hybrid sequence assembly, the genome of the H. sporothermodurans type strain was enlarged by 15% relative to the short-read assembly. This noticeable increase was probably due to numerous mobile elements in the genome that are presumptively related to spore heat tolerance. Phylogenetic studies based on 16S rDNA gene sequence, core genome, single-nucleotide polymorphisms and ANI/dDDH, showed that H. vini is highly related to H. sporothermodurans. When examining the genome sequences of all H.sporothermodurans strains from this study, together with 4 H. sporothermodurans genomes available in the GenBank database, the majority of the 36 strains examined occurred in a clonal lineage with less than 100 SNPs. These data substantiate previous reports on the existence and spread of a genetically highly homogenous and heat resistant spore clone, i.e., the HRS-clone.

Polyphasic study of antibiotic-resistant enterobacteria isolated from fresh produce in Germany and description of Enterobacter vonholyi sp. nov. isolated from marjoram and Enterobacter dykesii sp. nov. isolated from mung bean sprout.

Forty-two antibiotic-resistant enterobacteria strains were isolated from fresh produce obtained from the northern German retail market. A polyphasic characterization based on both phenotypic and genotypic methods was used to identify predominant strains as Citrobacter (C.) gillenii, C. portucalensis, Enterobacter (En.) ludwigii, Escherichia (E.) coli and Klebsiella (K.) pneumoniae. 38.1% of the enterobacteria strains were resistant to tetracycline, while 23.8% and 9.5% of strains were resistant to streptomycin and chloramphenicol, respectively. A high percentage of Klebsiella (100%), Enterobacter (57.1%) and Citrobacter (42.9%) strains were also resistant to ampicillin, with some strains showing multiple resistances. For unequivocal species identification, the genomes of thirty strains were sequenced. Multilocus sequence analysis, average nucleotide identity and digital DNA-DNA hybridization showed that Enterobacter strains E1 and E13 were clearly clustered apart from Enterobacter species type strains below the species delineation cutoff values. Thus, strains E1T (=DSM 111347T, LMG 31875T) represents a novel species proposed as Enterobacter dykesii sp. nov., while strain E13T (=DSM 110788T, LMG 31764T) represent a novel species proposed as Enterobacter vonholyi sp. nov. Strains often possessed different serine ß-lactamase genes, tet(A) and tet(D) tetracycline resistance genes and other acquired antibiotic resistance genes. Typical plasmid replicon types were determined. This study thus accurately identified the enterobacteria from fresh produce as species belonging to the genera Citrobacter, Enterobacter, Escherichia and Klebsiella, but also showed that these can carry potentially transferable antibiotic resistance genes and may thus contribute to the spread of these via the food route.

Complete Genome Sequence of a Shiga Toxin-Producing Escherichia coli O26:H11 Strain (Sequence Type 21) and Two Draft Genome Sequences of Listeria monocytogenes Strains (Clonal Complex 1 [CC1] and CC59) Isolated from Fresh Produce in Germany.

The complete genome sequence of a Shiga toxin-producing Escherichia coli (STEC) O26:H11 strain, MBT-5 (sequence type 21 [ST21], stx 1a, stx 2a, eae, ehxA), and two draft genome sequences of Listeria monocytogenes strains MBT-6 and MBT-7 belonging to the virulent sequence types 1 (ST1, clonal complex 1 [CC1]) and 59 (ST59, CC59), respectively, were determined. The strains were isolated in 2015 from ready-to-eat mixed greens in Germany.

Draft Genome Sequences of 12 Shiga Toxin-Producing Escherichia coli Strains Isolated from Dairy Cattle in Portugal.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen transmitted from animals to humans through contaminated food. Cattle are the main reservoir of STEC, but their genetic diversity is still poorly characterized, especially regarding strains isolated in Portugal. We therefore present the draft genomic sequences of 12 STEC strains isolated from cattle in the north of Portugal.

Fast and Easy Phage-Tagging and Live/Dead Analysis for the Rapid Monitoring of Bacteriophage Infection.

Use of bacteriophages, which are viruses that kill bacteria, for biocontrol of pathogens and antimicrobial resistant bacteria has become increasingly important in recent years. As traditional culture-based methods are laborious and time-consuming, practicable use of bacteriophages will hinge on development of rapid and high throughput methods to analyze, characterize and screen large bacteriophage libraries. We thus established a novel method to fluorescently tag bacteriophages for virus screening and interaction studies, without the need for complicated and laborious purification procedures or genetic engineering of viruses to express fluorescent proteins. Bacteriophage PMBT14 was tagged using DNA dye Syto 13. Simply by using a membrane filter, tagged bacteriophages can be separated from non-sequestered excess dye rapidly, effortlessly, and cheaply. The procedure takes less than 30 min and makes use of simple laboratory consumables that are already commonly used for bacteriophage preparations. As proof of concept, we present here flow cytometric methods to analyze bacteriophage binding, infection and killing that are very accessible for high throughput analysis. We show that the resulting fluorescently tagged bacteriophage can be used to specifically stain its host bacterium Pseudomonas fluorescens DSM 50090. Individual fluorescent bacteriophages, their binding to and initial infection of bacteria could also be observed using confocal microscopy. The infection process was halted by the metabolic inhibitor sodium azide, suggesting a requirement of host metabolic processes for penetration by PMBT14. Flow cytometric live/dead assays was used as a complementary method to determine bacteriophage infection of its host. We made preliminary efforts to adapt the tagging method to two other bacteriophages and discuss potential pitfalls and solutions in the use of tagged phages. Fluorescent phage tagging has previously been demonstrated to facilitate analysis of bacteriophage-host interactions. The method adopted in this study makes it fast, easy as well as cost effective.

Genome analysis of the temperate bacteriophage PMBT6 residing in the genome of Bifidobacterium thermophilum MBT94004.

The Siphoviridae phage PMBT6 was identified by transmission electron microscopy in the supernatant of Bifidobacterium thermophilum MBT94004 bioreactor fermentation culture, where it occurred at a moderately high titer. Genome analysis of the bacterial DNA confirmed the presence of this prophage within the genome of the lysogenic host. Under laboratory conditions, the prophage could not be induced by mitomycin C, ultraviolet C irradiation or hydrogen peroxide, suggesting that the prophage was released by spontaneous induction under (yet unknown) bioreactor conditions. Genome sequencing of the virion resulted in a linear, double-stranded DNA molecule of 36,561 bp with a mol% G + C content of 61.7 and 61 predicted open reading frames with low similarity to other Bifidobacterium spp. genomes, confirming that PMBT6 represents a novel temperate phage for this genus.

Antibiotics resistance and toxin profiles of Bacillus cereus-group isolates from fresh vegetables from German retail markets.

BACKGROUND: This study aimed to evaluate the safety of raw vegetable products present on the German market regarding toxin-producing Bacillus cereus sensu lato (s.l.) group bacteria. RESULTS: A total of 147 B. cereus s.l. group strains isolated from cucumbers, carrots, herbs, salad leaves and ready-to-eat mixed salad leaves were analyzed. Their toxinogenic potential was assessed by multiplex PCR targeting the hemolysin BL (hbl) component D (hblD), non-hemolytic enterotoxin (nhe) component A (nheA), cytotoxin K-2 (cytK-2) and the cereulide (ces) toxin genes. In addition, a serological test was used to detect Hbl and Nhe toxins. On the basis of PCR and serological results, none of the strains were positive for the cereulide protein/genes, while 91.2, 83.0 and 37.4% were positive for the Hbl, Nhe and CytK toxins or their genes, respectively. Numerous strains produced multiple toxins. Generally, strains showed resistance against the ß-lactam antibiotics such as penicillin G and cefotaxim (100%), as well as amoxicillin/clavulanic acid combination and ampicillin (99.3%). Most strains were susceptible to ciprofloxacin (99.3%), chloramphenicol (98.6%), amikacin (98.0%), imipenem (93.9%), erythromycin (91.8%), gentamicin (88.4%), tetracycline (76.2%) and trimethoprim/sulfamethoxazole combination (52.4%). The genomes of eight selected strains were sequenced. The toxin gene profiles detected by PCR and serological test mostly agreed with those from whole-genome sequence data. CONCLUSIONS: Our study showed that B. cereus s.l. strains encoding toxin genes occur in products sold on the German market and that these may pose a health risk to the consumer if present at elevated levels. Furthermore, a small percentage of these strains harbor antibiotic resistance genes. The presence of these bacteria in fresh produce should, therefore, be monitored to guarantee their safety.

Pseudomonas kielensis sp. nov. and Pseudomonas baltica sp. nov., isolated from raw milk in Germany.

In this study, nine Gram-negative, motile and rod-shaped bacteria were isolated during a Germany-wide investigation of raw milk microbiota. The strains could be differentiated from their closest relatives by phenotypic and chemotaxonomic characterization and average nucleotide identity (ANIb) values calculated from draft genome assemblies. Strains MBT-1T, MBT-8, MBT-9, MBT-10, MBT-11 and MBT-12 were related to the Pseudomonas chlororaphis subgroup. Isolates MBT-2T, MBT-13 and MBT-14 were closely related to Pseudomonas rhizosphaerae DSM 16299T with an ANIb of 88.2 % and a genome-to-genome distance result of 36.0 %. The G+C content of the DNA of strains MBT-1T and MBT-2T was 60.84 and 62.48 mol%, respectively. The major fatty acids were C16 : 1 ω7c (summed feature 3), C16 : 0 and C18 : 1 ω7c (summed feature 8). The strains were catalase-positive, while production of urease, ß-galactosidase and indole were negative. Growth occurred at 4-30 °C and at pH values of pH 6.0-8.0. Based on these results, we conclude that the strains belong to two novel species, for which the names Pseudomonas kielensis sp. nov. and Pseudomonas baltica sp. nov. are proposed. The type strains are MBT-1T (=DSM 111668 T= LMG 31954T) and MBT-2T (=DSM 111761 T=LMG 31955T).

Diversity and Antibiotic Susceptibility of Acinetobacter Strains From Milk Powder Produced in Germany.

Forty-seven Acinetobacter spp. isolates from milk powder obtained from a powdered milk producer in Germany were investigated for their antibiotic resistance susceptibilities, in order to assess whether strains from food harbor multiple antibiotic resistances and whether the food route is important for dissemination of resistance genes. The strains were identified by 16S rRNA and rpoB gene sequencing, as well as by whole genome sequencing of selected isolates and their in silico DNA-DNA hybridization (DDH). Furthermore, they were genotyped by rep-PCR together with reference strains of pan-European groups I, II, and III strains of Acinetobacter baumannii. Of the 47 strains, 42 were identified as A. baumannii, 4 as Acinetobacter Pittii, and 1 as Acinetobacter calcoaceticus based on 16S rRNA gene sequencing. In silico DDH with the genome sequence data of selected strains and rpoB gene sequencing data suggested that the five non-A. baumannii strains all belonged to A. pittii, suggesting that the rpoB gene is more reliable than the 16S rRNA gene for species level identification in this genus. Rep-PCR genotyping of the A. baumannii strains showed that these could be grouped into four groups, and that some strains clustered together with reference strains of pan-European clinical group II and III strains. All strains in this study were intrinsically resistant toward chloramphenicol and oxacillin, but susceptible toward tetracycline, tobramycin, erythromycin, and ciprofloxacin. For cefotaxime, 43 strains (91.5%) were intermediate and 3 strains (6.4%) resistant, while 3 (6.4%) and 21 (44.7%) strains exhibited resistance to cefepime and streptomycin, respectively. Forty-six (97.9%) strains were susceptible to amikacin and ampicillin-sulbactam. Therefore, the strains in this study were generally not resistant to the clinically relevant antibiotics, especially tobramycin, ciprofloxacin, cefepime, and meropenem, suggesting that the food route probably poses only a low risk for multidrug resistant Acinetobacter strains or resistance genes.

Draft Genome Sequence of the Intimin-Positive Enteropathogenic Escherichia albertii Strain MBT-EA1, Isolated from Lettuce.

The genome of the intimin (eae)-harboring Escherichia albertii strain MBT-EA1, isolated from lettuce in Germany, was sequenced. Sequence analysis showed the assembled draft genome size to be 4,560,948 bp, containing a predicted total of 4,414 protein-encoding genes, 11 rRNAs, and 82 tRNAs. Furthermore, three plasmid sequences were found.

Persistence and reduction of Shiga toxin-producing Escherichia coli serotype O26:H11 in different types of raw fermented sausages.

Fermented sausages have been identified as source of several outbreaks of Shiga toxin-producing Escherichia coli (STEC). Illnesses linked to non-O157 STEC serotypes appear to be on the rise worldwide, and serogroup O26 is the second most reported in Europe after O157. However, data on the behavior of serogroup O26 in food are rare, so that the aim of this study was to investigate the survival of STEC O26:H11 in different types of fermented sausages ("Teewurst", fast-ripened and long-fermented salami). Challenge studies were performed with an inoculation cocktail which consisted of three STEC O26:H11 strains isolated from human, cattle and food sources. In the short-ripened spreadable sausage type "Teewurst" STEC counts decreased by only 0.5 log10 within 28days. In contrast, STEC reductions from 2.2 to 2.6 log10 units were observed in the different salami products, while the most pronounced decrease of 1.0 log10 unit within one day was detected in fast-ripened sausages with glucono delta-lactone (GdL). Moreover, numbers of the food-associated E. coli O26:H11 strain were significantly higher (p<0.001) than those of the human and cattle STEC O26:H11 strains in all types of fermented sausages. Approximately 60% of all STEC isolates from GdL salami shared the genotypic virulence profile of the food-associated E. coli O26:H11 strain. In summary, hurdles of acidification and drying during salami ripening resulted in reductions of STEC O26:H11 counts. However, our results also indicate that STEC O26:H11 can persist in the environment of "Teewurst" and might therefore pose a risk to public health.

Presence of Human Pathogens in Produce from Retail Markets in Northern Germany.

Two hundred fresh produce samples (cucumber, carrots, herbs, leaf lettuce, and ready-to-eat mixed salad leaves) were obtained from retail in northern Germany in 2015. These were investigated for microbial contamination and the presence of foodborne pathogens, including Listeria monocytogenes, Salmonella serovars, presumptive Bacillus (B.) cereus, and Shiga toxin-producing Escherichia coli using culture-dependent (enrichment, plating on selective media) and -independent (real-time polymerase chain reaction [PCR]) techniques. Overall, our results showed that the fresh produce samples generally showed high mean aerobic mesophilic bacterial counts of between 7 and 8 log10 cfu/g. However, there was also a considerable variation in total aerobic bacterial counts between different product samples. Although real-time PCR signals for pathogenic E. coli were detected in 14.0% of total samples analyzed, only one (0.5%) Shiga toxin-producing E. coli isolate of serotype O26:H11 was recovered from mixed salad leaves and contained stx1, stx2, and eae genes. Two L. monocytogenes isolates (1% of total samples) were recovered from packaged mixed salad leaves and belonged to PCR serogroups IIb and IVb, respectively. One Salmonella isolate (0.5%) was recovered after selective enrichment also from mixed salad leaves and it was identified as Salmonella Szentes serotype 16:k:1,2. Overall the incidence of foodborne pathogens on the northern German retail market in 2015 was very low.

Genome Sequence of Klebsiella pneumoniae Bacteriophage PMBT1 Isolated from Raw Sewage.

A bacteriophage virulent for extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae strain 182 was isolated from sewage. The double-stranded DNA (dsDNA) genome showed high similarity to the genomes of other Klebsiella pneumoniae phages. It comprises 175,206 bp with a mol% G+C content of 41.9 and contains 276 putative open reading frames (ORFs) and one tRNA.

Draft Genome Sequence of Lactobacillus plantarum BFE 5092 Isolated from Maasai Fermented Milk.

The draft genome of Lactobacillus plantarum BFE 5092 isolated from the Maasai traditional fermented milk product kule naoto was sequenced, and sequence analysis showed the assembled genome size to be 3,285,094 bp, containing a predicted total of 3,111 protein-encoding genes, 17 rRNAs, and 70 tRNAs.