Epidemiological cut-off values for non-O1/ non-O139 Vibrio cholerae disc diffusion data generated by standardised methods.

This work generates the data needed to set epidemiological cut-off values for disc-diffusion zone measurements of Vibrio cholerae. The susceptibility of 147 European isolates of non-O1/non-O139 V. cholerae to 19 antibiotics was established using a standardised disc diffusion method which specified incubation of Mueller Hinton agar plates at 35°C. Epidemiological cut-off values were calculated by analysis of the zone size data with the statistically based normalised resistance interpretation method. Cut-off values for 17 agents were calculated by analysis of the aggregated data from all 4 laboratories participating in this study. The cut-off values calculated were ≥18 mm for amoxicillin/clavulanate, ≥18 mm for amikacin, ≥19 mm for ampicillin, ≥27 mm for cefepime, ≥31 mm for cefotaxime, ≥24 mm for ceftazidime, ≥24 mm for chloramphenicol, ≥31 mm for ciprofloxacin, ≥16 mm for erythromycin, ≥ 27 mm for florfenicol, ≥16 mm for gentamicin, ≥23 mm for imipenem, ≥25 mm for meropenem, ≥29 mm for nalidixic acid, ≥28 mm for norfloxacin, ≥13 mm for streptomycin and ≥23 mm for tetracycline. For the other 2 agents the data from 1 laboratory was excluded from the censored aggregation because the data from that laboratory was considered excessively imprecise. The cut-off values for these 2 agents calculated for the aggregation of the data from 3 laboratories were ≥23 mm for trimethoprim and ≥24 mm for trimethoprim/sulfamethoxazole. These zone size data will be submitted to the Clinical Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) for their consideration in setting international consensus epidemiological cut-off values for non O1/non-O139 V. cholerae.

[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.

Characterization of qnrB-carrying plasmids from ESBL- and non-ESBL-producing Escherichia coli.

BACKGROUND: Escherichia coli carrying clinically important antimicrobial resistances [i.e., against extended-spectrum-beta-lactamases (ESBL)] are of high concern for human health and are increasingly detected worldwide. Worryingly, they are often identified as multidrug-resistant (MDR) isolates, frequently including resistances against quinolones/fluoroquinolones. RESULTS: Here, the occurrence and genetic basis of the fluoroquinolone resistance enhancing determinant qnrB in ESBL-/non-ESBL-producing E. coli was investigated. Overall, 33 qnrB-carrying isolates out of the annual German antimicrobial resistance (AMR) monitoring on commensal E. coli (incl. ESBL-/AmpC-producing E. coli) recovered from food and livestock between 2013 and 2018 were analysed in detail. Whole-genome sequencing, bioinformatics analyses and transferability evaluation was conducted to characterise the prevailing qnrB-associated plasmids. Furthermore, predominant qnrB-carrying plasmid-types were subjected to in silico genome reconstruction analysis. In general, the qnrB-carrying E. coli were found to be highly heterogenic in their multilocus sequence types (STs) and their phenotypic resistance profiles. Most of them appeared to be MDR and exhibited resistances against up to ten antimicrobials of different classes. With respect to qnrB-carrying plasmids, we found qnrB19 located on small Col440I plasmids to be most widespread among ESBL-producing E. coli from German livestock and food. This Col440I plasmid-type was found to be highly conserved by exhibiting qnrB19, a pspF operon and different genes of unassigned function. Furthermore, we detected plasmids of the incompatibility groups IncN and IncH as carriers of qnrB. All qnrB-carrying plasmids also exhibited virulence factors and various insertion sequences (IS). The majority of the qnrB-carrying plasmids were determined to be self-transmissible, indicating their possible contribution to the spread of resistances against (fluoro)quinolones and other antimicrobials. CONCLUSION: In this study, a diversity of different plasmid types carrying qnrB alone or in combination with other resistance determinants (i.e., beta-lactamase genes) were found. The spread of these plasmids, especially those carrying antimicrobial resistance genes against highest priority critically important antimicrobial agents, is highly unfavourable and can pose a threat for public health. Therefore, the dissemination pathways and evolution of these plasmids need to be further monitored.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Host Range, Morphology and Sequence Analysis of Ten Temperate Phages Isolated from Pathogenic Yersinia enterocolitica Strains.

Yersinia enterocolitica is a heterogeneous species comprising highly pathogenic, weakly pathogenic and non-pathogenic strains. Previous data suggest that gene exchange may occur in Yersinia. Only scarce information exists about temperate phages of Y. enterocolitica, even though many prophage sequences are present in this species. We have examined 102 pathogenic Y. enterocolitica strains for the presence of inducible prophages by mitomycin C treatment. Ten phages were isolated from nine strains belonging to the bio (B)/serotypes (O) B2/O:5,27, B2/O:9 and 1B/O:8. All phages are myoviruses showing lytic activity only at room temperature. Whole-genome sequencing of the phage genomes revealed that they belong to three groups, which, however, are not closely related to known phages. Group 1 is composed of five phages (type phage: vB_YenM_06.16.1) with genome sizes of 43.8 to 44.9 kb, whereas the four group 2 phages (type phage: vB_YenM_06.16.2) possess smaller genomes of 29.5 to 33.2 kb. Group 3 contains only one phage (vB_YenM_42.18) whose genome has a size of 36.5 kb, which is moderately similar to group 2. The host range of the phages differed significantly. While group 1 phages almost exclusively lysed strains of B2/O:5,27, phages of group 2 and 3 were additionally able to lyse B4/O:3, and some of them even B2/O:9 and 1B/O:8 strains.

ESKAPE Bacteria and Extended-Spectrum-ß-Lactamase-Producing Escherichia coli Isolated from Wastewater and Process Water from German Poultry Slaughterhouses.

The wastewater of livestock slaughterhouses is considered a source of antimicrobial-resistant bacteria with clinical relevance and may thus be important for their dissemination into the environment. To get an overview of their occurrence and characteristics, we investigated process water (n = 50) from delivery and unclean areas as well as wastewater (n = 32) from the in-house wastewater treatment plants (WWTPs) of two German poultry slaughterhouses (slaughterhouses S1 and S2). The samples were screened for ESKAPE bacteria (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Escherichia coli Their antimicrobial resistance phenotypes and the presence of extended-spectrum-ß-lactamase (ESBL), carbapenemase, and mobilizable colistin resistance genes were determined. Selected ESKAPE bacteria were epidemiologically classified using different molecular typing techniques. At least one of the target species was detected in 87.5% (n = 28/32) of the wastewater samples and 86.0% (n = 43/50) of the process water samples. The vast majority of the recovered isolates (94.9%, n = 448/472) was represented by E. coli (39.4%), the A. calcoaceticus-A. baumannii (ACB) complex (32.4%), S. aureus (12.3%), and K. pneumoniae (10.8%), which were widely distributed in the delivery and unclean areas of the individual slaughterhouses, including their wastewater effluents. Enterobacter spp., Enterococcus spp., and P. aeruginosa were less abundant and made up 5.1% of the isolates. Phenotypic and genotypic analyses revealed that the recovered isolates exhibited diverse resistance phenotypes and ß-lactamase genes. In conclusion, wastewater effluents from the investigated poultry slaughterhouses exhibited clinically relevant bacteria (E. coli, methicillin-resistant S. aureus, K. pneumoniae, and species of the ACB and Enterobacter cloacae complexes) that contribute to the dissemination of clinically relevant resistances (i.e., blaCTX-M or blaSHV and mcr-1) in the environment.IMPORTANCE Bacteria from livestock may be opportunistic pathogens and carriers of clinically relevant resistance genes, as many antimicrobials are used in both veterinary and human medicine. They may be released into the environment from wastewater treatment plants (WWTPs), which are influenced by wastewater from slaughterhouses, thereby endangering public health. Moreover, process water that accumulates during the slaughtering of poultry is an important reservoir for livestock-associated multidrug-resistant bacteria and may serve as a vector of transmission to occupationally exposed slaughterhouse employees. Mitigation solutions aimed at the reduction of the bacterial discharge into the production water circuit as well as interventions against their further transmission and dissemination need to be elaborated. Furthermore, the efficacy of in-house WWTPs needs to be questioned. Reliable data on the occurrence and diversity of clinically relevant bacteria within the slaughtering production chain and in the WWTP effluents in Germany will help to assess their impact on public and environmental health.

Yersinia pseudotuberculosis Prevalence and Diversity in Wild Boars in Northeast Germany.

In this study, the prevalence of Yersinia pseudotuberculosis in wild boars in northeast Germany was determined. For that purpose, the tonsils of 503 wild boars were sampled. The presence of Y. pseudotuberculosis was studied by diagnostic PCR. Positive samples were analyzed by cultural detection using a modified cold enrichment protocol. Ten Y. pseudotuberculosis isolates were obtained, which were characterized by biotyping, molecular serotyping, and multilocus sequence typing (MLST). In addition, whole-genome sequences and the antimicrobial susceptibility of the isolates were analyzed. Yersinia pseudotuberculosis was isolated from male and female animals, most of which were younger than 1 year. A prevalence of 2% (10/503) was determined by cultural detection, while 6.4% (32/503) of the animals were positive by PCR. The isolates belonged to the biotypes 1 and 2 and serotypes O:1a (n = 7), O:1b (n = 2), and O:4a (n = 1). MLST analysis revealed three sequence types, ST9, ST23, and ST42. Except one isolate, all isolates revealed a strong resistance to colistin. The relationship of the isolates was studied by whole-genome sequencing demonstrating that they belonged to four clades, exhibiting five different pulsed-field gel electrophoresis (PFGE) restriction patterns and a diverse composition of virulence genes. Six isolates harbored the virulence plasmid pYV. Besides two isolates, all isolates contained ail and inv genes and a complete or incomplete high-pathogenicity island (HPI). None of them possessed a gene for the superantigen YPM. The study shows that various Y. pseudotuberculosis strains exist in wild boars in northeast Germany, which may pose a risk to humans.IMPORTANCEYersinia pseudotuberculosis is a foodborne pathogen whose occurrence is poorly understood. One reason for this situation is the difficulty in isolating the species. The methods developed for the isolation of Yersinia enterocolitica are not well suited for Y. pseudotuberculosis We therefore designed a protocol which enabled the isolation of Y. pseudotuberculosis from a relatively high proportion of PCR-positive wild boar tonsils. The study indicates that wild boars in northeast Germany may carry a variety of Y. pseudotuberculosis strains, which differ in terms of their pathogenic potential and other properties. Since wild boars are widely distributed in German forests and even populate cities such as Berlin, they may transmit yersiniae to other animals and crop plants and may thus cause human infections through the consumption of contaminated food. Therefore, the prevalence of Y. pseudotuberculosis should be determined also in other animals and regions to learn more about the natural reservoir of this species.

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes.

Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

Molecular Tracing to Find Source of Protracted Invasive Listeriosis Outbreak, Southern Germany, 2012-2016.

We investigated 543 Listeria monocytogenes isolates from food having a temporal and spatial distribution compatible with that of the invasive listeriosis outbreak occurring 2012-2016 in southern Germany. Using forensic microbiology, we identified several products from 1 manufacturer contaminated with the outbreak genotype. Continuous molecular surveillance of food isolates could prevent such outbreaks.

Differential detection of pathogenic Yersinia spp. by fluorescence in situ hybridization.

Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis are pathogens of major medical importance, which are responsible for a considerable number of infections every year. The detection of these species still relies on cultural methods, which are slow, labour intensive and often hampered by the presence of high amounts of accompanying flora. In this study, fluorescence in situ hybridization (FISH) was used to develop a fast, sensitive and reliable alternative to detect viable bacteria in food. For this purpose, highly specific probes targeting the 16S and 23S ribosomal RNA were employed to differentially detect each of the three species. In order to enable the differentiation of single nucleotide polymorphisms (SNPs), suitable competitor oligonucleotides and locked nucleic acids (LNAs) were used. Starved cells still showed a strong signal and a direct viable count (DVC) approach combined with FISH optimized live/dead discrimination. Sensitivity of the FISH test was high and even a single cell per gram of spiked minced pork meat could be detected within a day, demonstrating the applicability to identify foodborne hazards at an early stage. In conclusion, the established FISH tests proved to be promising tools to compensate existing drawbacks of the conventional cultural detection of these important zoonotic agents.

Rapid characterisation of Klebsiella oxytoca isolates from contaminated liquid hand soap using mass spectrometry, FTIR and Raman spectroscopy.

Microbiological monitoring of consumer products and the efficiency of early warning systems and outbreak investigations depend on the rapid identification and strain characterisation of pathogens posing risks to the health and safety of consumers. This study evaluates the potential of three rapid analytical techniques for identification and subtyping of bacterial isolates obtained from a liquid hand soap product, which has been recalled and reported through the EU RAPEX system due to its severe bacterial contamination. Ten isolates recovered from two bottles of the product were identified as Klebsiella oxytoca and subtyped using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF MS), near-infrared Fourier transform (NIR FT) Raman spectroscopy and Fourier transform infrared (FTIR) spectroscopy. Comparison of the classification results obtained by these phenotype-based techniques with outcomes of the DNA-based methods pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis of whole-genome sequencing (WGS) data revealed a high level of concordance. In conclusion, a set of analytical techniques might be useful for rapid, reliable and cost-effective microbial typing to ensure safe consumer products and allow source tracking.

Rapid screening for antibiotic resistance elements on the RNA transcript, protein and enzymatic activity level.

BACKGROUND: The emerging threat posed by antibiotic resistance has affected public health systems all over the world. Surveillance of resistant bacteria in clinical settings and identifying them in mixed cultures is of paramount importance and can contribute to the control of their spreading. Culture-independent monitoring approaches are highly desirable, since they yield results much faster than traditional susceptibility testing. However, many rapid molecular methods like PCR only detect the sole presence of a potential resistance gene, do not provide information regarding efficient transcription, expression and functionality and, in addition, cannot assign resistance genes to species level in mixed cultures. METHODS: By using plasmid-encoded TEM ß-lactamase mediated ampicillin resistances as a proof of principle system, we (1) developed a fluorescence in situ hybridization-test (FISH) capable to detect the respective mRNAs, (2) implemented an immunofluorescence test to identify the corresponding proteins and (3) compared these two microscopic tests with an established colorimetric nitrocefin assay to assess the enzymatic activity. RESULTS: All three methods proved to be suitable for the testing of antibiotic resistance, but only FISH and immunofluorescence were able to differentiate between susceptible and resistant bacteria on the single cell level and can be combined with simultaneous species identification. CONCLUSIONS: Fluorescence in situ hybridization and immunofluorescence tests are promising techniques in susceptibility testing since they bridge the gap between the slow, but accurate and sound cultural methods and molecular detection methods like PCR with much less functional relevance.

FISHing for bacteria in food--a promising tool for the reliable detection of pathogenic bacteria?

Foodborne pathogens cause millions of infections every year and are responsible for considerable economic losses worldwide. The current gold standard for the detection of bacterial pathogens in food is still the conventional cultivation following standardized and generally accepted protocols. However, these methods are time-consuming and do not provide fast information about food contaminations and thus are limited in their ability to protect consumers in time from potential microbial hazards. Fluorescence in situ hybridization (FISH) represents a rapid and highly specific technique for whole-cell detection. This review aims to summarize the current data on FISH-testing for the detection of pathogenic bacteria in different food matrices and to evaluate its suitability for the implementation in routine testing. In this context, the use of FISH in different matrices and their pretreatment will be presented, the sensitivity and specificity of FISH tests will be considered and the need for automation shall be discussed as well as the use of technological improvements to overcome current hurdles for a broad application in monitoring food safety. In addition, the overall economical feasibility will be assessed in a rough calculation of costs, and strengths and weaknesses of FISH are considered in comparison with traditional and well-established detection methods.

A suggested classification for two groups of Campylobacter myoviruses.

Most Campylobacter bacteriophages isolated to date have long contractile tails and belong to the family Myoviridae. Based on their morphology, genome size and endonuclease restriction profile, Campylobacter phages were originally divided into three groups. The recent genome sequencing of seven virulent campylophages reveal further details of the relationships between these phages at the genome organization level. This article details the morphological and genomic features among the campylophages, investigates their taxonomic position, and proposes the creation of two new genera, the "Cp220likevirus" and "Cp8unalikevirus" within a proposed subfamily, the "Eucampyvirinae"

American crocodiles (Crocodylus acutus: Reptilia: Crocodilidae) visiting the facilities of a freshwater aquaculture of the Northern Pacific region, Costa Rica, carry tetracycline-resistant Escherichia coli.

Apex predators are exposed to antimicrobial compounds and resistant microbes, which accumulate at different trophic levels of the related ecosystems. The study aimed to characterize the presence and the antimicrobial resistance patterns of fecal Escherichia coli isolated from cloacal swab samples obtained from wild-living American crocodiles (Crocodylus acutus) (n = 53). Sampling was conducted within the distinctive context of a freshwater-intensive aquaculture farm in Costa Rica, where incoming crocodiles are temporarily held in captivity before release. Phenotypic antimicrobial susceptibility profiles were determined in all isolates, while resistant isolates were subjected to whole-genome sequencing and bioinformatics analyses. In total, 24 samples contained tetracycline-resistant E. coli (45.3%). Isolates carried either tet(A), tet(B), or tet(C) genes. Furthermore, genes conferring resistance to ß-lactams, aminoglycosides, fosfomycin, sulfonamides, phenicol, quinolones, trimethoprim, and colistin were detected in single isolates, with seven of them carrying these genes on plasmids. Genome sequencing further revealed that sequence types, prevalence of antibiotic resistance carriage, and antibiotic resistance profiles differed between the individuals liberated within the next 24 h after their capture in the ponds and those liberated from enclosures after longer abodes. The overall presence of tetracycline-resistant E. coli, coupled with potential interactions with various anthropogenic factors before arriving at the facilities, hinders clear conclusions on the sources of antimicrobial resistance for the studied individuals. These aspects hold significant implications for both the aquaculture farm's biosecurity and the planning of environmental monitoring programs using such specimens. Considering human-crocodile conflicts from the One Health perspective, the occurrence of antimicrobial resistance underscores the importance of systematical surveillance of antibiotic resistance development in American crocodiles.

Spread of a distinct Stx2-encoding phage prototype among Escherichia coli O104:H4 strains from outbreaks in Germany, Norway, and Georgia.

Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the world's largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.

Impact and Diversity of ESBL-Producing Klebsiella pneumoniae Recovered from Raw Chicken Meat Samples in Türkiye.

The interrelationship between human, animal and environmental sectors leads to the spread of antibiotic resistance due to selective pressures, evolutionary traits and genomic evolution. In particular, the frequent use of antibiotics in livestock inevitably influences the emergence of specific resistance determinants in human strains, associated with reduced treatment options in clinical therapy. In this study, ESBL-producing Klebsiella pneumoniae strains isolated from chicken meat samples were evaluated for public health implications in Türkiye. Whole-genome sequencing was used for genetic dissection and phylogenetic comparison of their genomes. The isolates were assigned to four MLST types (ST147, ST37, ST2747 and ST219); two of them were found to represent the ST147 clone associated with severe human infections worldwide. In addition to cephalosporins, high resistance levels to quinolones/fluoroquinolones were identified phenotypically, caused by acquired resistance genes and chromosomal point variations. One isolate was also found to carry the qacE∆1 efflux transporter gene, which confers tolerance to quaternary ammonium compounds. The detection of virulence genes (i.e., that coding for enterobactin) associated with the pathogenicity of K. pneumoniae suggests a public health impact. Thus, comprehensive information on the occurrence and impact of K. pneumoniae from livestock is needed to derive appropriate management strategies for consumer protection. In this study, it was shown that poultry meat serves as a reservoir of clinically emerging multidrug-resistant high-risk clones.

The Novel Yersinia enterocolitica Telomere Phage vB_YenS_P840 Is Closely Related to PY54, but Reveals Some Striking Differences.

Telomere phages are a small group of temperate phages, whose prophages replicate as a linear plasmid with covalently closed ends. They have been isolated from some Enterobacteriaceae and from bacterial species living in aquatic environments. Phage PY54 was the first Yersinia (Y.) enterocolitica telomere phage isolated from a nonpathogenic O:5 strain, but recently a second telomeric Yersinia phage (vB_YenS_P840) was isolated from a tonsil of a wild boar in Germany. Both PY54 and vB_YenS_P840 (P840) have a siphoviridal morphology and a similar genome organization including the primary immunity region immB and telomere resolution site telRL. However, whereas PY54 only possesses one prophage repressor for the lysogenic cycle, vB_YenS_P840 encodes two. The telRL region of this phage was shown to be processed by the PY54 protelomerase under in vivo conditions, but unlike with PY54, a flanking inverted repeat was not required for processing. A further substantial difference between the phages is their host specificity. While PY54 infects Y. enterocolitica strains belonging to the serotypes O:5 and O:5,27, vB_YenS_P840 exclusively lyses O:3 strains. As the tail fiber and tail fiber assembly proteins of the phages differ significantly, we introduced the corresponding genes of vB_YenS_P840 by transposon mutagenesis into the PY54 genome and isolated several mutants that were able to infect both serotypes, O:5,27 and O:3.

Free-floating extracellular DNA (exDNA) in different wastewaters: Status quo on exDNA-associated antimicrobial resistance genes.

Wastewater treatment plants (WWTPs) have been reported as major anthropogenic reservoirs for the spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) into the environment, worldwide. While most studies mainly focus on the intracellular DNA (iDNA), extracellular DNA (exDNA) accounting for a significant proportion of the total DNA in wastewater, was usually neglected. Following the One Health approach, this study focuses on wastewaters of municipal, clinical, and livestock origins (n = 45) that undergo different treatment processes (i.e., conventional activated sludge, ultrafiltration, and ozonation). Water samples were analysed for 12 ARGs as indicators of the different compartments associated with iDNA and exDNA by quantitative real-time PCR (qPCR). Taxonomic profiling of exDNA-fractions, obtained using nucleic acid adsorption particles, was conducted by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Notified exDNA concentrations varied between on-site WWTPs and treatment stages, and ranged from 314.0 ± 70.2 ng/mL in untreated livestock wastewater down to 0.7 ± 0.1 ng/mL in effluents after ultrafiltration. In general, influents exhibited higher concentrations compared to effluents, while wastewater treated by advanced treatment processes (i.e., ultrafiltration and ozonation) showed the lowest exDNA concentrations. Despite the lower concentrations, free-floating exDNA accounted for up to 80.0 ± 5.8% of the total DNA in effluents. Target ARGs were more common in the iDNA (100%, n = 45/45), compared to the exDNA-fractions (51.1%, n = 23/45), whereas exDNA-ARGs were mostly detected in clinical and slaughterhouse wastewaters as well as in the municipal influents. Compared to the iDNA-ARGs, the concentrations of exDNA-ARGs were in general lower. Nevertheless, significant higher concentrations for exDNA-associated genes were measured in clinical wastewaters for blaNDM (4.07 ± 0.15 log gene copies (GC)/L) and blaVIM-2 (6.0 ± 0.2 log GC/L). Overall, our results suggest that depending on the origin of wastewater and its treatment methods, exDNA represents an important reservoir for ARGs, particularly in clinical wastewater.

Characterization of Temperate LPS-Binding Bordetella avium Phages That Lack Superinfection Immunity.

Bordetella avium causes a highly infectious upper respiratory tract disease in turkeys and other poultry with high economic losses. Considering the antimicrobial resistance crisis, bacteriophages (phages) may be an alternative approach for treating bacterial infections such as bordetellosis. Here, we describe seven B. avium phages, isolated from drinking water and feces from chicken and turkey farms. They showed strong bacteriolytic activity with a broad host range and used lipopolysaccharides (LPS) as a host receptor for their adsorption. All phages are myoviruses based on their structure observed by transmission electron microscopy. Genome sequence analyses revealed genome assembly sizes ranging from 39,087 to 43,144 bp. Their permutated genomes were organized colinearly, with a conserved module order, and were packed according to a predicted headful packing strategy. Notably, they contained genes encoding putative markers of lysogeny, indicative of temperate phages, despite their lytic phenotype. Further investigation revealed that the phages could indeed undergo a lysogenic life cycle with varying frequency. However, the lysogenic bacteria were still susceptible to superinfection with the same phages. This lack of stable superinfection immunity after lysogenization appears to be a characteristic feature of B. avium phages, which is favorable in terms of a potential therapeutic use of phages for the treatment of avian bordetellosis. IMPORTANCE To maintain the effectiveness of antibiotics over the long term, alternatives to treat infectious diseases are urgently needed. Therefore, phages have recently come back into focus as they can specifically infect and lyse bacteria and are naturally occurring. However, there is little information on phages that can infect pathogenic bacteria from animals, such as the causative agent of bordetellosis of poultry, B. avium. Therefore, in this study, B. avium phages were isolated and comprehensively characterized, including whole-genome analysis. Although phenotypically the phages were thought to undergo a lytic cycle, we demonstrated that they undergo a lysogenic phase, but that infection does not confer stable host superinfection immunity. These findings provide important information that could be relevant for potential biocontrol of avian bordetellosis by using phage therapy.