Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

The Lytic Siphophage vB_StyS-LmqsSP1 Reduces the Number of Salmonella enterica Serovar Typhimurium Isolates on Chicken Skin.

Phage-based biocontrol of bacteria is considered a natural approach to combat foodborne pathogens. Salmonella spp. are notifiable and highly prevalent pathogens that cause foodborne diseases worldwide. In this study, six bacteriophages were isolated and further characterized that infect food-derived Salmonella isolates from different meat sources. The siphovirus VB_StyS-LmqsSP1, which was isolated from a cow's nasal swab, was further subjected to in-depth characterization. Phage-host interaction investigations in liquid medium showed that vB_StyS-LmqsSP1 can suppress the growth of Salmonella species isolates at 37°C for 10 h and significantly reduce the bacterial titer at 4°C. A reduction of 1.4 to 3 log units was observed in investigations with two food-derived Salmonella isolates and one reference strain under cooling conditions using multiplicities of infection (MOIs) of 104 and 105. Phage application on chicken skin resulted in a reduction of about 2 log units in the tested Salmonella isolates from the first 3 h throughout a 1-week experiment at cooling temperature and with an MOI of 105. The one-step growth curve analysis using vB_StyS-LmqsSP1 demonstrated a 60-min latent period and a burst size of 50 to 61 PFU/infected cell for all tested hosts. Furthermore, the genome of the phage was determined to be free from genes causing undesired effects. Based on the phenotypic and genotypic properties, LmqsSP1 was assigned as a promising candidate for biocontrol of Salmonella enterica serovar Typhimurium in food. IMPORTANCE Salmonella enterica is one of the major global causes of foodborne enteritis in humans. The use of chemical sanitizers for reducing bacterial pathogens in the food chain can result in the spread of bacterial resistance. Targeted and clean-label intervention strategies can reduce Salmonella contamination in food. The significance of our research demonstrates the suitability of a bacteriophage (vB_StyS-LmqsSP1) for biocontrol of Salmonella enterica serovar Typhimurium on poultry due to its lytic efficacy under conditions prevalent in food production environments.

Properties of Two Broad Host Range Phages of Yersinia enterocolitica Isolated from Wild Animals.

Yersinia (Y.) enterocolitica and Y. pseudotuberculosis are important zoonotic agents which can infect both humans and animals. To combat these pathogens, the application of strictly lytic phages may be a promising tool. Since only few Yersinia phages have been described yet, some of which demonstrated a high specificity for certain serotypes, we isolated two phages from game animals and characterized them in terms of their morphology, host specificity, lytic activity on two bio-/serotypes and genome composition. The T7-related podovirus vB_YenP_Rambo and the myovirus vB_YenM_P281, which is very similar to a previously described phage PY100, showed a broad host range. Together, they lysed all the 62 tested pathogenic Y. enterocolitica strains belonging to the most important bio-/serotypes in Europe. A cocktail containing these two phages strongly reduced cultures of a bio-/serotype B4/O:3 and a B2/O:9 strain, even at very low MOIs (multiplicity of infection) and different temperatures, though, lysis of bio-/serotype B2/O:9 by vB_YenM_P281 and also by the related phage PY100 only occurred at 37 °C. Both phages were additionally able to lyse various Y. pseudotuberculosis strains at 28 °C and 37 °C, but only when the growth medium was supplemented with calcium and magnesium cations.

Characterization of mcr-5-Harboring Salmonella enterica subsp. enterica Serovar Typhimurium Isolates from Animal and Food Origin in Germany.

We characterized eight mcr-5-positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany. Five plasmid types were identified harboring mcr-5 on Tn6452 or putative mobile insertion cassettes. The mobility of mcr-5 was confirmed by integration of Tn6452 into the bacterial chromosomes of two strains and the detection of conjugative mcr-5 plasmids. The association with mobile genetic elements might further enhance mcr-5 distribution.

Carbapenemase VCC-1-Producing Vibrio cholerae in Coastal Waters of Germany.

During antimicrobial drug resistance testing for Vibrio spp. from coastal waters of Germany, we identified 4 nontoxigenic, carbapenem-resistant V. cholerae isolates. We used whole-genome sequencing to identify the carbapenemase gene blaVCC-1. In addition, a molecular survey showed that more blaVCC-1-harboring isolates are present in coastal waters of Germany.

Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B.

OBJECTIVES: Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L. METHODS: A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing. RESULTS: In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011-13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed. CONCLUSIONS: Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health.

Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes).

Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).

Campylobacter group II phage CP21 is the prototype of a new subgroup revealing a distinct modular genome organization and host specificity.

BACKGROUND: The application of phages is a promising tool to reduce the number of Campylobacter along the food chain. Besides the efficacy against a broad range of strains, phages have to be safe in terms of their genomes. Thus far, no genes with pathogenic potential (e.g., genes encoding virulence factors) have been detected in Campylobacter phages. However, preliminary studies suggested that the genomes of group II phages may be diverse and prone to genomic rearrangements. RESULTS: We determined and analysed the genomic sequence (182,761 bp) of group II phage CP21 that is closely related to the already characterized group II phages CP220 and CPt10. The genomes of these phages are comprised of four modules separated by very similar repeat regions, some of which harbouring open reading frames (ORFs). Though, the arrangement of the modules and the location of some ORFs on the genomes are different in CP21 and in CP220/CPt10. In this work, a PCR system was established to study the modular genome organization of other group II phages demonstrating that they belong to different subgroups of the CP220-like virus genus, the prototypes of which are CP21 and CP220. The subgroups revealed different restriction patterns and, interestingly enough, also distinct host specificities, tail fiber proteins and tRNA genes. We additionally analysed the genome of group II phage vB_CcoM-IBB_35 (IBB_35) for which to date only five individual contigs could be determined. We show that the contigs represent modules linked by long repeat regions enclosing some yet not identified ORFs (e.g., for a head completion protein). The data suggest that IBB_35 is a member of the CP220 subgroup. CONCLUSION: Campylobacter group II phages are diverse regarding their genome organization. Since all hitherto characterized group II phages contain numerous genes for transposases and homing endonucleases as well as similar repeat regions, it cannot be excluded that these phages are genetically unstable. To answer this question, further experiments and sequencing of more group II phages should be performed.

Biological and genomic characterization of three psychrophilic Y. enterocolitica phages.

Yersinia (Y.) enterocolitica is an important foodborne pathogenic species that is mainly transmitted by the consumption of contaminated meat, particularly pork. To combat the bacteria along the food chain, the application of strictly lytic phages may be a promising tool. As the temperatures in the gut of animals and during food processing can differ significantly, a phage cocktail intended to be used for applications should comprise phages that are active at various temperatures. In this study, we isolated and characterized three phages with a myoviridal morphology (vB_YenM_P8, vB_YenM_P744 and vB_YenM_P778), which lysed the most important Y. enterocolitica serotypes O:3, O:9 and O:5,27 at a low multiplicity of infection (MOI) and at low temperatures down to 6°C. While vB_YenM_P8 is a member of the T4 family, vB_YenM_P744 and vB_YenM_P778 are novel phages that do not show relationship to known phages. The three phages were mixed in a cocktail with the already described phages vB_YenM_P281 and vB_YenP_Rambo. The cocktail revealed a strong lytic activity and lysed a mixture of Y. enterocolitica serotypes at room temperature (RT) within few hours with a reduction of up to 4.8 log10 units. Moreover, at even lower temperatures the mixture was significantly reduced after incubation overnight. The strongest reductions were determined at 6°C (4.0 log10 units) suggesting that the cocktail can lyse the psychrophilic Y. enterocolitica also during food processing. To determine possible phage resistance, 100 colonies that survived the infection by the phages were isolated and analysed regarding their serotype and phage susceptibility. Most isolates belonged to serotype O:9, but all of them were still sensitive to at least one phage of the cocktail.

Draft genome sequence of Klebsiella pneumoniae KLEB-33: a convergent biofilm hyperforming multiresistant strain belonging to the emerging ST16 lineage harboring multiple hypervirulence genes.

The emergence of convergent Klebsiella pneumoniae strains showing multiresistance, characteristic of nosocomial pathotypes and hypervirulent traits typical of community-acquired isolates, makes them important models for studying K. pneumoniae pathogenesis. Here, we describe the convergent, multidrug-resistant KLEB-33 strain harboring several hypervirulence genes and make its genome available to the scientific community.

The complete genome sequence of bacteriophage CP21 reveals modular shuffling in Campylobacter group II phages.

Campylobacter group II phages described so far share a high degree of sequence similarity. We report the 182,833-bp genomic sequence of the closely related group II phage CP21 and show that it has a completely different genomic organization. As in other group II phages, the CP21 genome is composed of large modules separated by long DNA repeat regions which obviously trigger recombination and modular shuffling.

Shiga toxin-producing Escherichia coli strains from cattle as a source of the Stx2a bacteriophages present in enteroaggregative Escherichia coli O104:H4 strains.

Enteroaggregative, Shiga toxin-producing E. coli (EAEC-STEC) O104:H4 strains are emerging pathogens causing life threatening diseases in humans. EAEC-STEC O104:H4 strains isolated between 2001 and 2011 were found to harbor a distinct type of Shiga toxin 2a- (Stx2a) encoding prophage. This phage type shows only <65% genetic similarity to so far described viable Stx phages due to differences in the modules for DNA replication, metabolism, regulation and host specificity. Stx production in EAEC is rarely observed and the source of the Stx2a phage in the EAEC-STEC O104:H4 strains is not known. We identified two DNA segments derived from orf15 and the cI gene of the O104:H4 Stx2a phage P13374 that are characteristic for Stx2a prophages present in EAEC-STEC O104:H4 strains. By PCR, these sequences were detected in 14 (5.8%) of 241 Stx2-positive STEC from animals and food. Infectious Stx2a phages could be isolated from four bovine STEC strains. These were found highly similar to P13374 for orf15, cI and stx2a sequences, the chromosomal integration site (wrbA), for phage DNA restriction profiles, virion morphology and superinfection immunity. Stx2a phages of the four bovine STEC strains formed lysogens on the E. coli K-12 strain C600. Phage P13374 from an EAEC-STEC O104:H4 outbreak strain and one of the bovine STEC phages (P13803) lysogenized the Stx-negative EAEC O104:H4 strain CB14647 by integrating in the wrbA gene of CB14647 and converted it into a Stx2a producer. Our findings provide experimental evidence that EAEC-STEC O104:H4 strains have evolved by uptake of Stx2a phages from the bovine reservoir.

Isolation and characterization of phages with lytic activity against methicillin-resistant Staphylococcus aureus strains belonging to clonal complex 398.

Some years ago, MRSA clonal complex (CC) 398 emerged, which spread extensively in livestock animals. People in contact with food production animals are at high risk of colonization. A reduction of MRSA CC398 in livestock might be achieved by application of virulent phages. However, there have not yet been any reports published on phages lysing MRSA CC398 strains. In this study, three virulent phages (PSa1, PSa2 and PSa3) with lytic activity against MRSA CC398 strains were isolated from German pig farms. Morphologically, the phages are members of the family Podoviridae, and they exhibited an identical host range. They lysed 52 (60 %) out of 86 tested MRSA CC398 strains representing 18 different spa types. While the PSa1 and PSa3 genomes have a similar size of approximately 17.5 kb, the PSa2 genome is somewhat larger (ca. 18.5 kb). Southern hybridization revealed strong DNA homologies between the phages, which was confirmed by sequence analysis of cloned restriction fragments and PCR products. Moreover, the whole PSa3 genomic sequence (17,602 bp) showed a close relationship to 44AHJD-like phages, which are not known to contain virulence-associated genes. To assess whether these phages might be candidates for applications, in vitro experiments were carried out in which the number of MRSA CC398 cells could be reduced by up to four log10 units. The phages were stable at a wide range of temperatures and pH values.

Genomic Evidence of mcr-1.26 IncX4 Plasmid Transmission between Poultry and Humans.

Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (mcr). mcr-1.26 is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, mcr-1.26-carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of mcr-1.26 exclusively on IncX4 plasmids. mcr-1.26 was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS6-like element. Based on the genetic diversity of E. coli isolates, transmission of the mcr-1.26 resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn2 transposon on the mcr-1.26 IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described mcr-1.26-carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function. IMPORTANCE Evolutionary events causing the emergence of new resistances/variants are usually rare and challenging to predict. Conversely, common transmission events of widespread resistance determinants are quantifiable and predictable. One such example is the transmissible plasmid-mediated colistin resistance. The main determinant, mcr-1, has been notified in 2016 but has successfully established itself in multiple plasmid backbones in diverse bacterial species across all One Health sectors. So far, 34 variants of mcr-1 are described, of which some can be used for epidemiological tracing-back analysis to identify the origin and transmission dynamics of these genes. Here, we report the presence of the rare mcr-1.26 gene in E. coli isolated from poultry since 2014. Based on the temporal occurrence and high similarity of the plasmids between poultry and human isolates, our study provides first indications for poultry husbandry as the primary source of mcr-1.26 and its transmission between different niches.

Phage Therapy in Germany-Update 2023.

Bacteriophage therapy holds promise in addressing the antibiotic-resistance crisis, globally and in Germany. Here, we provide an overview of the current situation (2023) of applied phage therapy and supporting research in Germany. The authors, an interdisciplinary group working on patient-focused bacteriophage research, addressed phage production, phage banks, susceptibility testing, clinical application, ongoing translational research, the regulatory situation, and the network structure in Germany. They identified critical shortcomings including the lack of clinical trials, a paucity of appropriate regulation and a shortage of phages for clinical use. Phage therapy is currently being applied to a limited number of patients as individual treatment trials. There is presently only one site in Germany for large-scale good-manufacturing-practice (GMP) phage production, and one clinic carrying out permission-free production of medicinal products. Several phage banks exist, but due to varying institutional policies, exchange among them is limited. The number of phage research projects has remarkably increased in recent years, some of which are part of structured networks. There is a demand for the expansion of production capacities with defined quality standards, a structured registry of all treated patients and clear therapeutic guidelines. Furthermore, the medical field is still poorly informed about phage therapy. The current status of non-approval, however, may also be regarded as advantageous, as insufficiently restricted use of phage therapy without adequate scientific evidence for effectiveness and safety must be prevented. In close coordination with the regulatory authorities, it seems sensible to first allow some centers to treat patients following the Belgian model. There is an urgent need for targeted networking and funding, particularly of translational research, to help advance the clinical application of phages.

Campylobacter jejuni group III phage CP81 contains many T4-like genes without belonging to the T4-type phage group: implications for the evolution of T4 phages.

CP81 is a virulent Campylobacter group III phage whose linear genome comprises 132,454 bp. At the nucleotide level, CP81 differs from other phages. However, a number of its structural and replication/recombination proteins revealed a relationship to the group II Campylobacter phages CP220/CPt10 and to T4-type phages. Unlike the T4-related phages, the CP81 genome does not contain conserved replication and virion modules. Instead, the respective genes are scattered throughout the phage genome. Moreover, most genes for metabolic enzymes of CP220/CPt10 are lacking in CP81. On the other hand, the CP81 genome contains nine similar genes for homing endonucleases which may be involved in the attrition of the conserved gene order for the virion core genes of T4-type phages. The phage apparently possesses an unusual modification of C or G bases. Efficient cleavage of its DNA was only achieved with restriction enzymes recognizing pure A/T sites. Uncommonly, phenol extraction leads to a significant loss of CP81 DNA from the aqueous layer, a property not yet described for other phages belonging to the T4 superfamily.

Shiga toxin-producing Escherichia coli O104:H4: a new challenge for microbiology.

In 2011, Germany experienced the largest outbreak with a Shiga toxin-producing Escherichia coli (STEC) strain ever recorded. A series of environmental and trace-back and trace-forward investigations linked sprout consumption with the disease, but fecal-oral transmission was also documented. The genome sequences of the pathogen revealed a clonal outbreak with enteroaggregative E. coli (EAEC). Some EAEC virulence factors are carried on the virulence plasmid pAA. From an unknown source, the epidemic strains acquired a lambdoid prophage carrying the gene for the Shiga toxin. The resulting strains therefore possess two different mobile elements, a phage and a plasmid, contributing essential virulence genes. Shiga toxin is released by decaying bacteria in the gut, migrates through the intestinal barrier, and is transported via the blood to target organs, like the kidney. In a mouse model, probiotic bifidobacteria interfered with transport of the toxin through the gut mucosa. Researchers explored bacteriophages, bacteriocins, and low-molecular-weight inhibitors against STEC. Randomized controlled clinical trials of enterohemorrhagic E. coli (EHEC)-associated hemolytic uremic syndrome (HUS) patients found none of the interventions superior to supportive therapy alone. Antibodies against one subtype of Shiga toxin protected pigs against fatal neurological infection, while treatment with a toxin receptor decoy showed no effect in a clinical trial. Likewise, a monoclonal antibody directed against a complement protein led to mixed results. Plasma exchange and IgG immunoadsoprtion ameliorated the condition in small uncontrolled trials. The epidemic O104:H4 strains were resistant to all penicillins and cephalosporins but susceptible to carbapenems, which were recommended for treatment.

Global colistin use: a review of the emergence of resistant Enterobacterales and the impact on their genetic basis.

The dramatic global rise of MDR and XDR Enterobacterales in human medicine forced clinicians to the reintroduction of colistin as last-resort drug. Meanwhile, colistin is used in the veterinary medicine since its discovery, leading to a steadily increasing prevalence of resistant isolates in the livestock and meat-based food sector. Consequently, transmission of resistant isolates from animals to humans, acquisition via food and exposure to colistin in the clinic are reasons for the increased prevalence of colistin-resistant Enterobacterales in humans in the last decades. Initially, resistance mechanisms were caused by mutations in chromosomal genes. However, since the discovery in 2015, the focus has shifted exclusively to mobile colistin resistances (mcr). This review will advance the understanding of chromosomal-mediated resistance mechanisms in Enterobacterales. We provide an overview about genes involved in colistin resistance and the current global situation of colistin-resistant Enterobacterales. A comparison of the global colistin use in veterinary and human medicine highlights the effort to reduce colistin sales in veterinary medicine under the One Health approach. In contrast, it uncovers the alarming rise in colistin consumption in human medicine due to the emergence of MDR Enterobacterales, which might be an important driver for the increasing emergence of chromosome-mediated colistin resistance.