7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

Professor

Characterization of Klebsiella species from slaughterhouses in Edo State, Nigeria: Insights from phenotypic and whole-genome analysis

Epidemiology and antimicrobial resistance of zoonotic pathogens 

BackgroundSlaughterhouses are known reservoirs for pathogenic bacteria, and elicit food safety apprehensions. Klebsiella species, recognized opportunistic pathogens, may occur on meat surfaces and require a thorough understanding of their slaughterhouse characteristics for accurate assessment of associated transmission and human health risks. This study investigated phenotypic and genomic features of Klebsiella isolates, shedding light on antimicrobial resistance (AMR) and potential virulence factors, offering valuable insights for public health considerations.MethodologyBefore and after disinfection, surface samples from slaughterhouse underwent Klebsiella species isolation and identification via MALDI-TOF. Antimicrobial and biocide susceptibilities were ascertained for relevant substances, evaluating both minimum inhibitory concentration (MIC) and additionally minimum bactericidal concentration for biocides. Isolates with high biocide MICs were subjected to efflux pump inhibition testing. Further, Klebsiella underwent screening for biofilm formation capabilities. Whole genome sequencing (WGS) was employed to explore genetic diversity and potential traits contributing to persistence.ResultsThe majority of recovered isolates was identified as K. pneumoniae (n=105). Additional species included K. aerogenes (n=6), K. quasipneumoniae (n=7), and K. variicola (n=13). Biocide susceptibility varied, with two isolates showing triclosan resistance compared to in-use concentrations. Efflux pump inhibition resulted in increased susceptibility to octenidine dihydrochloride and didecyldimethylammonium chloride. Out of the total isolates, 31 K. pneumoniae (23.7%) exhibited multidrug resistance. Two K. pneumoniae isolates were extended-spectrum β-lactamase producers. Biofilm formation was prevalent, with stronger biofilm forming ability post-disinfection. WGS revealed a broad genetic diversity, encompassing AMR, virulence, biocide, and biofilm genes.ConclusionsOur findings highlight various public health relevant characteristics of Klebsiella isolates from slaughterhouses, emphasizing diverse AMR profiles and genetic traits.

Oral Presentation

Cefiderocol resistance on the rise: Emergence of resistant Enterobacterales in the One Health context

Enterobacterales, e.g., Escherichia coli (EC) and Klebsiella pneumoniae (KP), stand out for their ability to evolve and acquire resistance to last-line antibiotics such as carbapenems, contributing to difficult-to-treat infections. Cefiderocol (FDC) was developed to combat such multidrug-resistant (MDR) strains by using a mechanism that bypasses common resistance mechanisms. Unfortunately, as with most new drugs, resistance to FDC has been reported with increasing frequency, always associated with hospitalization and antibiotic therapy. However, little is known about the prevalence of FDC-resistant EC and KP beyond the hospital walls. Our studies aimed to fill this gap by investigating the presence of these pathogens in a One Health context.We sampled surface water from different sites in a Western European (Germany) and a Sub-Saharan African (Ghana) setting. Bacteria were initially selected for their MDR phenotype, whole genome sequenced and subsequently tested for FDC resistance. In addition, strains from various human, animal, and environmental sources from our strain collection were systematically tested for FDC resistance.We collected a total of 116 MDR EC and 28 MDR KP. EC strains with phenotypic resistance to FDC were present in both sets of water samples. Interestingly, typical mutations described in the literature as resistance markers (e.g., ompC) were not confirmed by genomic analysis. Knock-in and knock-out studies are ongoing. In addition, we identified FDC-resistant, convergent KP strains of sequence type 307 in our strain collection that were isolated during a clonal outbreak in Germany that preceded the clinical use of FDC. Of note, the underlying mutation type in the cirA gene has not yet been described in this context.Our research has revealed the emergence of FDC-resistant strains in surface waters in Germany and Ghana. More importantly, FDC resistance is not a recent phenomenon and may have predated the clinical use. These findings highlight the need for comprehensive One Health studies that integrate genomic and epidemiologic data to effectivly monitor and manage antimicrobial resistance at the human-animal-environmental interface.

Low zoonotic potential but multi-drug resistance in Streptococcus suis isolates from healthy and diseased birds

Streptococcus suis is a respiratory commensal of pigs, with some lineages causing serious swine disease and zoonotic disease in humans. In recent years, S. suis has also been isolated from cats, dogs, cattle, sheep, wild boars, and different bird species, including chicken. It is generally assumed that, as in humans, these infections are due to &quot;spillovers&quot; from pigs, but no genomic investigation of respective isolates has yet been performed. This project aimed at investigating the zoonotic potential of these isolates by employing bioinformatics analyses with a special emphasis on host adaptation, virulence, and antimicrobial resistance.Over the last six years, we collected and whole-genome sequenced S. suis samples from birds in the veterinary diagnostics department of the Freie Universität Berlin. Most of these birds were diseased, and S. suis was isolated among other pathogens. We combined these samples with published genomes of Vietnamese healthy chicken isolates and compared them to S. suis lineages isolated from pigs, wild boars, and human. In addition, to examine potential survival advantages of bird isolates in birds, we performed growth experiments in chicken vs. pig blood.All the bird isolates clustered phylogenetically within a group of largely commensal isolates of pigs, and distinct from the lineages responsible for most zoonoses, lacking most known virulence genes. Using genome-wide association studies, we detected several unique mobile genetic elements specific to bird isolates, suggestive of S. suis adaptation to birds. Concordantly, S. suis bird isolates showed a better survival in chicken than in porcine blood, although this effect was not significant. Furthermore, bird isolates had a larger genome size than closely related pig isolates. Finally, our analyses revealed that bird isolates were often multi-drug resistant.In summary, our results imply that S. suis could be persisting in bird populations independently of pigs, at least for short periods of time, but that birds are unlikely to be a source of zoonotic infection in humans. Our study also suggests a potential role for antibiotic resistance in host switching.

Single nucleotide polymorphisms (SNPs) in dacB/ampD alter population plasticity of gene amplification based heteroresistance

Introduction Heteroresistance (HR) describes the ability of a subpopulation to grow in the presence of inhibitory antibiotic concentrations. We found HR to ceftazidime (CAZ) in a clinical Enterobacter cloacae complex (ECC) strain (IMT49658). Material &amp; Methods We performed extensive phenotypic (population analysis profiles, stability analysis of resistance, ScanLag) and molecular microbiological techniques (qRT-PCR, whole genome sequencing, raw read analysis) in order to show the plasticity and mechanism of HR in this ECC strain. We re-investigated the genome and phenotype of IMT 49658 after long-term evolution in 32 µg/ml CAZ and after exposure to 128 µg/ml CAZ in plates.Goals By revealing mechanism and evolution of HR to conventional resistance, we aim for a better detection and treatment of bacterial heteroresistant infections. Results WGS detected a plasmidal gene amplification with β-lactamase ampC blaDHA-1. qRT-PCR showed a high genomic copy number of blaDHA‑1 in resistant subpopulations, decreasing when they reverted to susceptibility. Gene amplifications varied in single cells of one colony (raw read analysis). Resistant subpopulations showed heterogeneous lag times in ScanLag. After evolving ECC for 21 days in CAZ, we discovered a SNP in dacB and ampD, encoding for a stop codon. These mutants displayed low amplification levels but resistance in disk diffusion and homogenous lag times. The dacB mutant was more frequent than the ampD mutant on plates with 128 µg/ml CAZ.Summary Long-term evolution in antibiotic niches drives the emergence of new resistant mutants, balancing the fitness costs of e.g., gene amplifications. Comprehension of the transition from HR to resistance is inevitable for successful treatment of bacterial infections.

Phage regulation and persister cell formation upon ciprofloxacin treatment via the toxin TisB in Salmonella Typhimurium

IntroductionPersisters are drug-tolerant cells that survive bactericidal concentrations of antibiotics, therefore playing an important role in clinical therapy failure [1]. Mechanisms influencing bacterial survival during treatment with antibiotics include the activity of bacterial toxins, induction of prophages, and activation of stress responses [2, 3].Material/methodWe examined the effects of a tisB toxin deletion mutant in Salmonella Typhimurium on the ATP concentrations during treatment with four-fold the MIC of ciprofloxacin. Furthermore, we also examined the TisB-dependent induction of the prophages and their impact on persister cell formation comparing a phage-free variant of the wild type strain in a ∆tisB background.ResultsDeletion of tisB significantly increased ATP concentration and SOS response when exposed to ciprofloxacin, with a reduction in the formation of persistent cells. Interestingly, a 90-fold reduction in bacterial survival was observed four hours after challenging the phage-carrying ∆tisB strain with ciprofloxacin. In contrast, deletion of all prophages in the tisB background slowed initial killing and had a less significant impact on drug persistence.DiscussionThe SOS response is controlled by RecA, which requires ATP for its activity. We suggest that TisB inhibits ATP formation, and interferes with DNA repair responses. This significantly reduces the SOS response-dependent prophage induction and thus increases persister cell formation.Brauner, A., et al., Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat Rev Microbiol, 2016. 14(5): p. 320-30.Dorr, T., M. Vulic, and K. Lewis, Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS Biol, 2010. 8(2): p. e1000317.Braetz, S., et al., Prophage Gifsy-1 Induction in Salmonella enterica Serovar Typhimurium Reduces Persister Cell Formation after Ciprofloxacin Exposure. Microbiol Spectr, 2023: p. e0187423.

Phylogenomic analyses identifies dog-human sharing of genetically closely related Escherichia coli, including carbapenemase OXA-244 producing and colistin resistant strains

Introduction: In Nigeria, a large proportion of dogs are free roaming or semi-restricted; a practice that brings them in close contact with potentially virulent and antibiotic resistant bacteria in the community. We investigated the role of dogs as community reservoir for dissemination of Escherichia coli resistant to "high priority critically important antimicrobials" in Makurdi, North central Nigeria.Methods: We subjected a total of 137 isolates to whole genome sequencing and uploaded the reads unto the EnteroBase server which facilitates genome assembly, core and whole genome based phylogenomic analyses. Genome assemblies were queried for resistance determinants using AMRFinder while phenotypic expression of resistance was assessed based on the antibiotic minimum inhibitory concentration (MIC).Results: Hierarchical clustering analyses of core genome multilocus sequence types (cgMLST) revealed a genetically diverse population of 34 clonal complexes (CC) predominated by CC10. We identified the blaOXA244 cabarpenemase gene in two isolates and the plasmid mediated colistin resistance gene variants: mcr-1.22, mcr-10.1, with the former located on an IncX4 plasmid. Multiple strains harbored extended spectrum β-lactamases (ESBL) genes predominantly blaCTX-M-15 (n=50) and blaCTX-M-55 (n=6). Isolates expressed cefotaxime and colistin resistance and a meropenem MIC of 0.125 µg/ml, respectively. Comparative phylogenomic analyses revealed close genetic relationship at HC5 (pairwise genetic distance of ≤ 5 cgMLST allelic difference) between canine isolates and human strains recovered from the same geographic location in Nigeria, suggestive of potential human-dog exchange of strains. Additional closely related genomes were detected among sequence data publicly available from EnteroBase.Conclusion: Our findings show that dogs in developing countries are an important reservoir for potential community dissemination of colistin resistant, ESBL and carbapenemase producing E. coli, reechoing concerns for zoonotic and bidirectional spread.

Oral Session
Epidemiology and Antimicrobial Resistance of Zoonotic Pathogens

Appointment

Chair

Topic

Programme

Oral SessionEpidemiology and Antimicrobial Resistance of Zoonotic Pathogens

Appointment

Chair

Topic

Programme

Oral Session
Epidemiology and Antimicrobial Resistance of Zoonotic Pathogens