Rare Plasmid-Mediated AmpC Beta-Lactamase DHA-1 Located on Easy Mobilized IS26-Related Genetic Element Detected in Escherichia coli from Livestock and Food in Germany.

AmpC beta-lactamases cause resistance to third-generation cephalosporins, including beta-lactamase inhibitors. In Escherichia coli from the German food production chain, the majority of AmpC beta-lactamase activity can be attributed to plasmid-mediated CMY-2 or overproduction of chromosomal AmpC beta-lactamase, but occasionally other enzymes like DHA-1 are involved. This study investigated the prevalence of the AmpC beta-lactamase DHA-1 in ESBL/AmpC-producing E. coli (n = 4706) collected between 2016 and 2021 as part of a German antimicrobial resistance monitoring program along the food chain. Eight isolates (prevalence < 0.2%) were detected and further characterized by PFGE, transformation and conjugation experiments as well as short-read and long-read sequencing. All eight strains harbored blaDHA-1 together with qnrB4, sul1 and mph(A) resistance genes on an IS26 composite transposon on self-transferable IncFII or IncFIA/FIB/II plasmids. During laboratory experiments, activation of the translocatable unit of IS26-bound structures was observed. This was shown by the variability of plasmid sizes in original isolates, transconjugants or transferred plasmids, and correspondingly, duplications of resistance fragments were found in long-read sequencing. This activation could be artificial due to laboratory handling or naturally occurring. Nevertheless, DHA-1 is a rare AmpC beta-lactamase in livestock and food in Germany, and its dissemination will be monitored in the future.

Genetic Characterization of Carbapenem-Resistant Klebsiella spp. from Municipal and Slaughterhouse Wastewater.

Currently, human and veterinary medicine are threatened worldwide by an increasing resistance to carbapenems, particularly present in opportunistic Enterobacterales pathogens (e.g., Klebsiella spp.). However, there is a lack of comprehensive and comparable data on their occurrence in wastewater, as well as on the phenotypic and genotypic characteristics for various countries including Germany. Thus, this study aims to characterize carbapenem-resistant Klebsiella spp. isolated from municipal wastewater treatment plants (mWWTPs) and their receiving water bodies, as well as from wastewater and process waters from poultry and pig slaughterhouses. After isolation using selective media and determination of carbapenem (i.e., ertapenem) resistance using broth microdilution to apply epidemiological breakpoints, the selected isolates (n = 30) were subjected to WGS. The vast majority of the isolates (80.0%) originated from the mWWTPs and their receiving water bodies. In addition to ertapenem, Klebsiella spp. isolates exhibited resistance to meropenem (40.0%) and imipenem (16.7%), as well as to piperacillin-tazobactam (50.0%) and ceftolozan-tazobactam (50.0%). A high diversity of antibiotic-resistance genes (n = 68), in particular those encoding ß-lactamases, was revealed. However, with the exception of blaGES-5-like, no acquired carbapenemase-resistance genes were detected. Virulence factors such as siderophores (e.g., enterobactin) and fimbriae type 1 were present in almost all isolates. A wide genetic diversity was indicated by assigning 66.7% of the isolates to 12 different sequence types (STs), including clinically relevant ones (e.g., ST16, ST252, ST219, ST268, ST307, ST789, ST873, and ST2459). Our study provides information on the occurrence of carbapenem-resistant, ESBL-producing Klebsiella spp., which is of clinical importance in wastewater and surface water in Germany. These findings indicate their possible dissemination in the environment and the potential risk of colonization and/or infection of humans, livestock and wildlife associated with exposure to contaminated water sources.

Slaughterhouse wastewater as a reservoir for extended-spectrum ß-lactamase (ESBL)-producing, and colistin-resistant Klebsiella spp. and their impact in a "One Health" perspective.

Klebsiella spp. are ubiquitous bacteria capable of colonizing humans and animals, and sometimes leading to severe infections in both. Due to their high adaptability against environmental/synthetic conditions as well as their potential in aquiring antimicrobial/metal/biocide resistance determinants, Klebsiella spp. are recognized as an emerging threat to public health, worldwide. Currently, scarce information on the impact of livestock for the spread of pathogenic Klebsiella spp. is available. Thus, the phenotypic and genotypic properties of extended-spectrum ß-lactamase-producing, and colistin-resistant Klebsiella strains (n = 185) from process- and wastewater of two poultry and pig slaughterhouses as well as their receiving municipal wastewater treatment plants (mWWTPs) were studied to determine the diversity of isolates that might be introduced into the food-production chain or that are released into the environment by surviving the wastewater treatment. Selectively-isolated Klebsiella spp. from slaughterhouses including effluents and receiving waterbodies of mWWTPs were assigned to various lineages, including high-risk clones involved in human outbreaks, and exhibited highly heterogeneous antibiotic-resistance patterns. While isolates originating from poultry slaughterhouses showed the highest rate of colistin resistance (32.4%, 23/71), carbapenem-resistant Klebsiella spp. were only detected in mWWTP samples (n = 76). The highest diversity of resistance genes (n = 77) was detected in Klebsiella spp. from mWWTPs, followed by isolates from pig (n = 56) and poultry slaughterhouses (n = 52). Interestingly, no carbapenemase-encoding genes were detected and mobile colistin resistance genes were only obeserved in isolates from poultry and pig slaughterhouses. Our study provides in-depth information into the clonality of livestock-associated Klebsiella spp. and their determinants involved in antimicrobial resistance and virulence development. On the basis of their pathogenic potential and clinical importance there is a potential risk of colonization and/or infection of wildlife, livestock and humans exposed to contaminated food and/or surface waters.

Phenotypic and Genotypic Properties of Fluoroquinolone-Resistant, qnr-Carrying Escherichia coli Isolated from the German Food Chain in 2017.

Fluoroquinolones are the highest priority, critically important antimicrobial agents. Resistance development can occur via different mechanisms, with plasmid-mediated quinolone resistance (PMQR) being prevalent in the livestock and food area. Especially, qnr genes, commonly located on mobile genetic elements, are major drivers for the spread of resistance determinants against fluoroquinolones. We investigated the prevalence and characteristics of qnr-positive Escherichia (E.) coli obtained from different monitoring programs in Germany in 2017. Furthermore, we aimed to evaluate commonalities of qnr-carrying plasmids in E. coli. We found qnr to be broadly spread over different livestock and food matrices, and to be present in various sequence types. The qnr-positive isolates were predominantly detected within selectively isolated ESBL (extended spectrum beta-lactamase)-producing E. coli, leading to a frequent association with other resistance genes, especially cephalosporin determinants. Furthermore, we found that qnr correlates with the presence of genes involved in resistance development against quaternary ammonium compounds (qac). The detection of additional point mutations in many isolates within the chromosomal QRDR region led to even higher MIC values against fluoroquinolones for the investigated E. coli. All of these attributes should be carefully taken into account in the risk assessment of qnr-carrying E. coli from livestock and food.

Clinically Relevant Escherichiacoli Isolates from Process Waters and Wastewater of Poultry and Pig Slaughterhouses in Germany.

Escherichia coli is frequently associated with multiple antimicrobial resistances and a major cause of bacterial extraintestinal infections in livestock and humans. However, data on the epidemiology of (i) multidrug-resistant (MDR) and (ii) extraintestinal pathogenic E. coli (ExPEC) in poultry and pig slaughterhouses in Germany is currently lacking. Selected E. coli isolates (n = 71) with phenotypic resistance to cephalosporins from two poultry and two pig slaughterhouses expressing high MDR rates (combined resistance to piperacillin, cefotaxime and/or ceftazidime, and ciprofloxacin) of 51.4% and 58.3%, respectively, were analyzed by whole-genome sequencing. They constituted a reservoir for 53 different antimicrobial resistance determinants and were assigned various sequence types, including high-risk clones involved in human infections worldwide. An ExPEC pathotype was detected in 17.1% and 5.6% of the isolates from poultry and pig slaughterhouses, respectively. Worryingly, they were recovered from scalding water and eviscerators, indicating an increased risk for cross-contaminations. Uropathogenic E. coli (UPEC) were detected in the effluent of an in-house wastewater treatment plant (WWTP) of a poultry slaughterhouse, facilitating their further dissemination into surface waters. Our study provides important information on the molecular characteristics of (i) MDR, as well as (ii) ExPEC and UPEC regarding their clonal structure, antimicrobial resistance and virulence factors. Based on their clinical importance and pathogenic potential, the risk of slaughterhouse employees' exposure cannot be ruled out. Through cross-contamination, these MDR E. coli pathotypes may be introduced into the food chain. Moreover, inadequate wastewater treatment may contribute to the dissemination of UPEC into surface waters, as shown for other WWTPs.

Draft Genome Sequences of Pseudomonas sp. Isolates Recovered from Ghanaian Fish Food Samples in 2018.

The genus Pseudomonas represents a broad diversity of opportunistic and pathogenic species that are able to colonize a wide range of ecological niches. Here, we report on draft genome sequences of 35 Pseudomonas sp. isolates that were recovered from small processed Ghanaian fishes offered at food markets in 2018.

Draft Genome Sequences of Acinetobacter baumannii Isolates Recovered from Sewage Water from a Poultry Slaughterhouse in Germany.

Acinetobacter baumannii is an important human pathogen usually associated with severe hospital-acquired infections. Here, we announce the draft genome sequences of two livestock-associated isolates recovered from sewage water from a poultry slaughterhouse in Germany. Short-read whole-genome sequencing was conducted to determine the genetic basis of their antimicrobial resistance phenotype.

Diversity of CTX-M-1-producing E. coli from German food samples and genetic diversity of the blaCTX-M-1 region on IncI1 ST3 plasmids.

Antimicrobial resistance to cephalosporins is commonly mediated by extended-spectrum ß-lactamases (ESBL) or plasmidic AmpC ß-lactamases (pAmpC). In livestock blaCTX-M-1 is the most frequently detected ESBL-encoding gene. As transmission to consumers through contaminated food is often proposed, this study characterized ESBL/pAmpC-producing E. coli collected from food samples. Therefore, samples from food products of animal origin and vegetables were screened for phenotypically resistant E. coli by selective cultivation. The ESBL genotype was confirmed for 404 isolates with the majority of them (n = 212) harboring the blaCTX-M-1 gene. PFGE and MLST analyses as well as plasmid characterization were carried out for 89 isolates, selected under epidemiological aspects. In addition, 44 isolates were investigated by whole genome sequencing and/or sequencing of their plasmids on an Illumina Miseq platform. MLST and PFGE indicated a diverse population of CTX-M-1-producing E. coli in German food samples with no spread of single clonal lineages. The majority of the isolates harbored the blaCTX-M-1 gene on IncI1 plasmids. Frequently, the gene was associated with the ISEcp1 element and located on a ∼100 kb IncI1 plasmid depicting the plasmid multilocus sequence type (ST) 3. The blaCTX-M-1 gene and its flanking sequences were located within the shufflon of the type IV pilus region in diverse orientations. In conclusion, dissemination of the CTX-M-1 ß-lactamase within food samples of animal origin is driven by the transmission of a ∼100 kb large IncI1 ST3 plasmid. Apart from conjugal transfer of IncI1 ST3 plasmids the transmission of the blaCTX-M-1 gene might be further promoted through mobilization due to its location within a recombination hot-spot of IncI1 plasmids.

Complete Draft Genome Sequence of an Extended-Spectrum ß-Lactamase-Producing Citrobacter freundii Strain Recovered from the Intestine of a House Sparrow (Passer domesticus) in Germany, 2017.

Here, we announce the genome of an extended-spectrum ß-lactamase-producing Citrobacter freundii strain isolated from the cecum of a house sparrow that was found dead in Berlin-Lichtenberg, Germany, in 2017. This isolate exhibits increased MICs for several antimicrobials and a comprehensive set of acquired resistance determinants potentially involved in horizontal gene transfer.

CTX-M-15-Producing E. coli Isolates from Food Products in Germany Are Mainly Associated with an IncF-Type Plasmid and Belong to Two Predominant Clonal E. coli Lineages.

Extended-spectrum beta-lactamases (ESBL) mediating resistance to 3rd generation cephalosporins are a major public health issue. As food may be a vehicle in the spread of ESLB-producing bacteria, a study on the occurrence of cephalosporin-resistantu Escherichia coli in food was initiated. A total of 404 ESBL-producing isolates were obtained from animal-derived food samples (e.g., poultry products, pork, beef and raw milk) between 2011 and 2013. As CTX-M-15 is the most abundant enzyme in ESBL-producing E. coli causing human infections, this study focusses on E. coli isolates from food samples harboring the blaCTX-M-15 gene. The blaCTX-M-15 gene was detected in 5.2% (n = 21) of all isolates. Molecular analyses revealed a phylogenetic group A ST167 clone that was repeatedly isolated from raw milk and beef samples over a period of 6 months. The analyses indicate that spread of CTX-M-15-producing E. coli in German food samples were associated with a multireplicon IncF (FIA FIB FII) plasmid and additional antimicrobial resistance genes such as aac(6)-Ib-cr, blaOXA-1, catB3, different tet-variants as well as a class 1 integron with an aadA5/dfrA17 gene cassette. In addition, four phylogenetic group A ST410 isolates were detected. Three of them carried a chromosomal copy of the blaCTX-M-15 gene and a single isolate with the gene on a 90 kb IncF plasmid. The blaCTX-M-15 gene was always associated with the ISEcp1 element. In conclusion, CTX-M-15-producing E. coli were detected in German food samples. Among isolates of different matrices, two prominent clonal lineages, namely A-ST167 and A-ST410, were identified. These lineages may be important for the foodborne dissemination of CTX-M-15-producing E. coli in Germany. Interestingly, these clonal lineages were reported to be widely distributed and especially prevalent in isolates from humans and livestock. Transmission of CTX-M-15-harboring isolates from food-producing animals to food appears probable, as isolates obtained from livestock and food samples within the same time period exhibit comparable characteristics as compared to isolates detected from human. However, the routes and direction of transmission need further investigation.

Spread and persistence of VIM-1 Carbapenemase-producing Enterobacteriaceae in three German swine farms in 2011 and 2012.

The occurrence of carbapenemase-producing Enterobacteriaceae in livestock is considered as a threat for public health. In Germany, VIM-1-producing Escherichia (E.) coli sequence type (ST) 88 and Salmonella Infantis isolates harbouring blaVIM-1IncHI2 plasmids have been isolated from swine and poultry farms. A retrospective study was performed to determine if there was a broader distribution of VIM-1-positive isolates in any of the carbapenemase-positive swine farms. Selective incubation (carbapenem-containing broth) of 249 conserved cultures collected in three farms (2011-2012), allowed the detection of 40 blaVIM-1-positive isolates. Apart from the already known non-motile Salmonella Infantis isolate R25 (farm S1) and R27 (S2), a third isolate was recovered from farm S3. For E. coli, additional to isolates R29 and R178 (S2), 35 new isolates were identified in the same farm during all the sampling periods (three dates, 2011) and in samples from different animals, farm environment, manure and flies. The newly identified E. coli and Salmonella isolates showed similar genetic and phenotypic characteristics (XbaI-PFGE profiles, antimicrobial resistance patterns, plasmid content, phylogroups, antigenic formula) to those in the previously described strains, suggesting microevolution within the clonal lines within one fattening period. The study shows that persistence of carbapenemase-producing clonal lines in livestock farms is possible, and underlines the need for harmonised monitoring and surveillance studies to follow up the occurrence of such bacteria in European livestock.