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 β-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

Binding Specificities of the Telomere Phage ϕKO2 Prophage Repressor CB and Lytic Repressor Cro

Temperate bacteriophages possess a genetic switch which regulates the lytic and lysogenic cycle. The genomes of the temperate telomere phages N15, PY54, and ϕKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor (cI or cB), the lytic repressor (cro) and a putative antiterminator (q). The roles of these products are thought to be similar to those of the lambda proteins CI (CI prophage repressor), Cro (Cro repressor), and Q (antiterminator Q), respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ϕKO2 are reminiscent of lambda-like phages. We determined binding sites of the ϕKO2 prophage repressor CB and lytic repressor Cro on the ϕKO2 genome in detail by electrophoretic mobility shift assay (EMSA) studies. Unexpectedly, ϕKO2 CB and Cro revealed different binding specificities. CB was bound to three OR operators in the intergenic region between cB and cro, two OL operators between cB and the replication gene repA and even to operators of N15. Cro bound exclusively to the 16 bp operator site OR3 upstream of the ϕKO2 prophage repressor gene. The ϕKO2 genes cB and cro are regulated by several strong promoters overlapping with the OR operators. The data suggest that Cro represses cB transcription but not its own synthesis, as already reported for PY54 Cro. Thus, not only PY54, but also phage ϕKO2 possesses a genetic switch that diverges significantly from the switch of lambda-like phages

Genome-Based Analysis of Klebsiella spp. Isolates from Animals and Food Products in Germany, 2013–2017

The increase in infections with multidrug-resistant and virulent Klebsiella pneumoniae (K. pneumoniae) strains poses a serious threat to public health. However, environmental reservoirs and routes of transmission for Klebsiella spp. that cause infections in humans and in livestock animals are not well understood. In this study, we aimed to analyze the distribution of antibiotic resistance genes and important virulence determinants (ybt, clb, iro, iuc, rmpA/A2) among 94 Klebsiella spp. isolates from different animal and food sources isolated between 2013 and 2017 in Germany. Antibiotic susceptibility testing was performed, and the genomes were sequenced by Illumina and Nanopore technology. Genetic relationships were assessed by conducting core genome multilocus sequence typing (cgMLST). Kleborate was used to predict resistance and virulence genes; Kaptive was used to derive the capsule types. The results revealed that 72 isolates (76.6%) belonged to the K. pneumoniae sensu lato complex. Within this complex, 44 known sequence types (STs), 18 new STs, and 38 capsule types were identified. Extended-spectrum beta-lactamase (ESBL) genes were detected in 16 isolates (17.0%) and colistin resistance in one (1.1%) K. pneumoniae isolate. Virulence genes were found in 22 K. pneumoniae isolates. Overall, nine (9.6%) and 18 (19.1%) isolates possessed the genes ybt and iuc, respectively. Notably, aerobactin (iuc lineage 3) was only detected in K. pneumoniae isolates from domestic pigs and wild boars. This study provides a snapshot of the genetic diversity of Klebsiella spp. in animals and food products in Germany. The siderophore aerobactin was found to be more prevalent in K. pneumoniae strains isolated from pigs than other sources. Further investigations are needed to evaluate if pigs constitute a reservoir for iuc lineage 3.Peer Reviewe

High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (OR3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (OR3) upstream of the prophage repressor gene. The OR3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages

Genetic and Phenotypic Virulence Potential of Non-O1/Non-O139 Vibrio cholerae Isolated from German Retail Seafood

Non-O1 and non-O139 Vibrio cholerae (NOVC) can cause gastrointestinal infections in humans. Contaminated food, especially seafood, is an important source of human infections. In this study, the virulence potential of 63 NOVC strains isolated from retail seafood were characterized at the genotypic and phenotypic levels. Although no strain encoded the cholera toxin (CTX) and the toxin-coregulated pilus (TCP), several virulence factors, including the HlyA hemolysin, the cholix toxin ChxA, the heat-stable enterotoxin Stn, and genes coding for the type 3 and type 6 secretion systems, were detected. All strains showed hemolytic activity against human and sheep erythrocytes: 90% (n = 57) formed a strong biofilm, 52% (n = 33) were highly motile at 37 °C, and only 8% (n = 5) and 14% (n = 9) could resist ≥60% and ≥40% human serum, respectively. Biofilm formation and toxin regulation genes were also detected. cgMLST analysis demonstrated that NOVC strains from seafood cluster with clinical NOVC strains. Antimicrobial susceptibility testing (AST) results in the identification of five strains that developed non-wildtype phenotypes (medium and resistant) against the substances of the classes of beta-lactams (including penicillin, carbapenem, and cephalosporin), polymyxins, and sulphonamides. The phenotypic resistance pattern could be partially attributed to the acquired resistance determinants identified via in silico analysis. Our results showed differences in the virulence potential of the analyzed NOVC isolated from retail seafood products, which may be considered for further pathogenicity evaluation and the risk assessment of NOVC isolates in future seafood monitoring

Identification of a blaVIM-1-Carrying IncA/C2 Multiresistance Plasmid in an Escherichia coli Isolate Recovered from the German Food Chain

Within the German national monitoring of zoonotic agents, antimicrobial resistance determination also targets carbapenemase-producing (CP) Escherichia coli by selective isolation from food and livestock. In this monitoring in 2019, the CP E. coli 19-AB01133 was recovered from pork shoulder. The isolate was assigned to the phylogenetic group B1 and exhibited the multi-locus sequence-type ST5869. Molecular investigations, including whole genome sequencing, of 19-AB01133 revealed that the isolate carried the resistance genes blaVIM-1, blaSHV-5 and blaCMY-13 on a self-transmissible IncA/C2 plasmid. The plasmid was closely related to the previously described VIM-1-encoding plasmid S15FP06257_p from E. coli of pork origin in Belgium. Our results indicate an occasional spread of the blaVIM-1 gene in Enterobacteriaceae of the European pig population. Moreover, the blaVIM-1 located on an IncA/C2 plasmid supports the presumption of a new, probably human source of carbapenemase-producing Enterobacteriaceae (CPE) entering the livestock and food chain sector

Genetic but No Phenotypic Associations between Biocide Tolerance and Antibiotic Resistance in Escherichia coli from German Broiler Fattening Farms

Biocides are frequently applied as disinfectants in animal husbandry to prevent the transmission of drug-resistant bacteria and to control zoonotic diseases. Concerns have been raised, that their use may contribute to the selection and persistence of antimicrobial-resistant bacteria. Especially, extended-spectrum β-lactamase- and AmpC β-lactamase-producing Escherichia coli have become a global health threat. In our study, 29 ESBL-/AmpC-producing and 64 NON-ESBL-/AmpC-producing E.coli isolates from three German broiler fattening farms collected in 2016 following regular cleaning and disinfection were phylogenetically characterized by whole genome sequencing, analyzed for phylogenetic distribution of virulence-associated genes, and screened for determinants of and associations between biocide tolerance and antibiotic resistance. Of the 30 known and two unknown sequence types detected, ST117 and ST297 were the most common genotypes. These STs are recognized worldwide as pandemic lineages causing disease in humans and poultry. Virulence determinants associated with extraintestinal pathogenic E.coli showed variable phylogenetic distribution patterns. Isolates with reduced biocide susceptibility were rarely found on the tested farms. Nine isolates displayed elevated MICs and/or MBCs of formaldehyde, chlorocresol, peroxyacetic acid, or benzalkonium chloride. Antibiotic resistance to ampicillin, trimethoprim, and sulfamethoxazole was most prevalent. The majority of ESBL-/AmpC-producing isolates carried blaCTX-M (55%) or blaCMY-2 (24%) genes. Phenotypic biocide tolerance and antibiotic resistance were not interlinked. However, biocide and metal resistance determinants were found on mobile genetic elements together with antibiotic resistance genes raising concerns that biocides used in the food industry may lead to selection pressure for strains carrying acquired resistance determinants to different antimicrobials

Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli

Antimicrobial resistance (AMR) is a major threat to public health worldwide. Currently, AMR typing changes from phenotypic testing to whole-genome sequence (WGS)-based detection of resistance determinants for a better understanding of the isolate diversity and elements involved in gene transmission (e.g., plasmids, bacteriophages, transposons). However, the use of WGS data in monitoring purposes requires suitable techniques, standardized parameters and approved guidelines for reliable AMR gene detection and prediction of their association with mobile genetic elements (plasmids). In this study, different sequencing and assembly strategies were tested for their suitability in AMR monitoring in Escherichia coli in the routines of the German National Reference Laboratory for Antimicrobial Resistances. To assess the outcomes of the different approaches, results from in silico predictions were compared with conventional phenotypic- and genotypic-typing data. With the focus on (fluoro)quinolone-resistant E.coli, five qnrS-positive isolates with multiple extrachromosomal elements were subjected to WGS with NextSeq (Illumina), PacBio (Pacific BioSciences) and ONT (Oxford Nanopore) for in depth characterization of the qnrS1-carrying plasmids. Raw reads from short- and long-read sequencing were assembled individually by Unicycler or Flye or a combination of both (hybrid assembly). The generated contigs were subjected to bioinformatics analysis. Based on the generated data, assembly of long-read sequences are error prone and can yield in a loss of small plasmid genomes. In contrast, short-read sequencing was shown to be insufficient for the prediction of a linkage of AMR genes (e.g., qnrS1) to specific plasmid sequences. Furthermore, short-read sequencing failed to detect certain duplications and was unsuitable for genome finishing. Overall, the hybrid assembly led to the most comprehensive typing results, especially in predicting associations of AMR genes and mobile genetic elements. Thus, the use of different sequencing technologies and hybrid assemblies currently represents the best approach for reliable AMR typing and risk assessment

Genetic characterization of a multidrug-resistant Salmonella enterica serovar Agona isolated from a dietary supplement in Germany

Salmonella enterica subsp. enterica serovar Agona has a history of causing food-borne outbreaks and any emergence of multidrug-resistant (MDR) isolates in novel food products is of concern. Particularly, in food products frequently consumed without sufficient heating prior to consumption. Here, we report about the MDR isolate, 18-SA00377, which had been isolated from a dietary supplement in Germany in 2018 and submitted to the German National Reference Laboratory for Salmonella. WGS-based comparative genetic analyses were conducted to find a potential reservoir of the isolate itself or mobile genetic elements associated with MDR. As a phylogenetic analysis did not yield any closely related S. Agona isolates, either globally or from Germany, a detailed analysis of the largest plasmid (295,499 bp) was performed as it is the main carrier of resistances. A combined approach of long-read and short-read sequencing enabled the assembly of the isolate’s chromosome and its four plasmids. Their characterization revealed the presence of 23 different antibiotic resistance genes (ARGs), conferring resistance to 12 different antibiotic drug classes, as well as genes conferring resistance to six different heavy metals. The largest plasmid, pSE18-SA00377-1, belongs to the IncHI2 plasmid family and carries 16 ARGs, that are organized as two distinct clusters, with each ARG associated with putative composite transposons. Through a two-pronged approach, highly similar plasmids to pSE18-SA00377-1 were identified in the NCBI database and a search for Salmonella isolates with a highly similar ARG resistance profile was conducted. Mapping and structural comparisons between pSE18-SA00377-1 and these plasmids and Salmonella isolates showed that both the plasmid backbone and identical or similar ARG clusters can be found not only in Salmonella isolates, originating mostly from a wide variety of livestock, but also in a diverse range of bacterial genera of varying geographical origins and isolation sources. Thus, it can be speculated that the host range of pSE18-SA00377-1 is not restricted to Salmonella and its spread already occurred in different bacterial populations. Overall, this hints at a complex history for pSE18-SA00377-1 and highlights the importance of surveilling multidrug-resistant S. enterica isolates, especially in novel food items that are not yet heavily regulated

Occurrence and Genomic Characterization of Clone ST1193 Clonotype 14-64 in Uncomplicated Urinary Tract Infections Caused by Escherichia coli in Spain

We conducted a prospective, multicenter, specific pilot study on uncomplicated urinary tract infections (uUTI). One-hundred non-duplicated uropathogenic Escherichia coli (UPEC) from uUTI occurred in 2020 in women attending 15 primary care centers of a single health region of northern Spain were characterized using a clonal diagnosis approach. Among the high genetic diversity showed by 59 different phylogroup-clonotype combinations, 11 clones accounted for 46% of the isolates: B2-ST73 (CH24-30); B2-ST73 (CH24-103); B2-ST131 (CH40-30); B2-ST141 (CH52-5); B2-ST372 (CH103-9); B2-ST404 (CH14-27); B2-ST404 (CH14-807); B2-ST1193 (CH14-64); D-ST69 (CH35-27); D-ST349 (CH36-54), and F-ST59 (CH32-41). The screening of the UPEC status found that 69% of isolates carried ≥ 3 of chuA, fyuA, vat, and yfcV genes. Multidrug resistance to at least one antibiotic of ≥ 3 antimicrobial categories were exhibited by 30% of the isolates, with the highest rates of resistance against ampicillin/amoxicillin (48%), trimethoprim (35%), norfloxacin (28%), amoxicillin-clavulanic acid (26%), and trimethoprim-sulfamethoxazole (24%). None extended-spectrum beta-lactamase/carbapenemase producer was recovered. According to our results, fosfomycin and nitrofurantoin should be considered as empirical treatment of choice for uUTI by E. coli (resistance rates 4% and 2%, respectively). We uncover the high prevalence of the pandemic fluoroquinolone-resistant ST1193 clone (6%) in uUTI, which represents the first report in Spain in this pathology. The genomic analysis showed similar key traits than those ST1193 clones disseminated worldwide. Through the SNP comparison based on the core genome, the Spanish ST1193 clustered with isolates retrieved from the Enterobase, showing high genomic similarity than the global ST1193 described in the United States, Canada and Australia. IMPORTANCE Analyzing the clonal structure and antimicrobial resistance of E. coli isolates implicated in uncomplicated urinary tract infections, one of the most frequent visits managed in primary health care, is of interest for clinicians to detect changes in the dynamics of emerging uropathogenic clones associated with the spread of fluoroquinolone resistance. It can also provide consensus concerning optimal control and antibiotic prescribingThis study was supported by the projects and funds PID2019-104439RB-C21/AEI/10.13039/501100011033 from the Agencia Estatal de Investigación (AEI, Spain), co-funded by the European Regional Development Fund of the European Union: A Way to Make Europe (ERDF); FIS PI17-00728 (Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain), co-funded by ERDF; GRUPIN IDI/2022/000033 by the Regional Ministry of Science of Asturias (IDI/2022/000033). ED431C 2021/11 from the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and ERDF. I.G-M. and V.G. acknowledge the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia for their post-doctoral grants (Grant Number ED481B-2021-006 and ED481-B2018/018, respectively). The research stay of I.G-M at the Hospital Universitario Central de Asturias was funded by a grant from the Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC). L.L. acknowledges the Ministry of Education of Spain for her predoctoral grant FPU19/01127.S