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- ItemConsensusPrime—A Bioinformatic Pipeline for Ideal Consensus Primer Design(Basel : MDPI, 2022) Collatz, Maximilian; Braun, Sascha D.; Monecke, Stefan; Ehricht, RalfBackground: High-quality oligonucleotides for molecular amplification and detection procedures of diverse target sequences depend on sequence homology. Processing input sequences and identifying homogeneous regions in alignments can be carried out by hand only if they are small and contain sequences of high similarity. Finding the best regions for large and inhomogeneous alignments needs to be automated. Results: The ConsensusPrime pipeline was developed to sort out redundant and technical interfering data in multiple sequence alignments and detect the most homologous regions from multiple sequences. It automates the prediction of optimal consensus primers for molecular analytical and sequence-based procedures/assays. Conclusion: ConsensusPrime is a fast and easy-to-use pipeline for predicting optimal consensus primers that is executable on local systems without depending on external resources and web services. An implementation in a Docker image ensures platform-independent executability and installability despite the combination of multiple programs. The source code and installation instructions are publicly available on GitHub.
- ItemMolecular characterisation of extended-spectrum ß-lactamase producing Escherichia coli in wild birds and cattle, Ibadan, Nigeria(London : BioMed Central, 2021) Fashae, Kayode; Engelmann, Ines; Monecke, Stefan; Braun, Sascha D.; Ehricht, RalfBackground: Antimicrobial resistance (AMR) is an increasing global health concern reducing options for therapy of infections and also for perioperative prophylaxis. Many Enterobacteriaceae cannot be treated anymore with third generation cephalosporins (3GC) due to the production of certain 3GC hydrolysing enzymes (extended spectrum beta-lactamases, ESBLs). The role of animals as carriers and vectors of multi-resistant bacteria in different geographical regions is poorly understood. Therefore, we investigated the occurrence and molecular characteristics of ESBL-producing Escherichia coli (E. coli) in wild birds and slaughtered cattle in Ibadan, Nigeria. Cattle faecal samples (n = 250) and wild bird pooled faecal samples (cattle egrets, Bubulcus ibis, n = 28; white-faced whistling duck, Dendrocygna viduata, n = 24) were collected and cultured on cefotaxime-eosin methylene blue agar. Antimicrobial susceptibility was determined by agar diffusion assays and all 3GC resistant isolates were genotypically characterised for AMR genes, virulence associated genes (VAGs) and serotypes using DNA microarray-based assays. Results: All 3GC resistant isolates were E. coli: cattle (n = 53), egrets (n = 87) and whistling duck (n = 4); cultured from 32/250 (12.8%), 26/28 (92.9%), 2/24(8.3%), cattle, egrets and whistling duck faecal samples, respectively. blaCTX-M gene family was prevalent; blaCTX-M15 (83.3%) predominated over blaCTX-M9 (11.8%). All were susceptible to carbapenems. The majority of isolates were resistant to at least one of the other tested antimicrobials; multidrug resistance was highest in the isolates recovered from egrets. The isolates harboured diverse repositories of other AMR genes (including strB and sul2), integrons (predominantly class 1) and VAGs. The isolates recovered from egrets harboured more AMR genes; eight were unique to these isolates including tetG, gepA, and floR. The prevalent VAGs included hemL and iss; while 14 (including sepA) were unique to certain animal isolates. E. coli serotypes O9:H9, O9:H30 and O9:H4 predominated. An identical phenotypic microarray profile was detected in three isolates from egrets and cattle, indicative of a clonal relationship amongst these isolates. Conclusion: Wild birds and cattle harbour diverse ESBL-producing E. coli populations with potential of inter-species dissemination and virulence. Recommended guidelines to balance public health and habitat conservation should be implemented with continuous surveillance. © 2021, The Author(s).
- ItemWhat the Phage: a scalable workflow for the identification and analysis of phage sequences(Oxford : Oxford University Press, 2022) Marquet, Mike; Hölzer, Martin; Pletz, Mathias W; Viehweger, Adrian; Makarewicz, Oliwia; Ehricht, Ralf; Brandt, ChristianPhages are among the most abundant and diverse biological entities on earth. Phage prediction from sequence data is a crucial first step to understanding their impact on the environment. A variety of bacteriophage prediction tools have been developed over the years. They differ in algorithmic approach, results, and ease of use. We, therefore, developed "What the Phage"(WtP), an easy-to-use and parallel multitool approach for phage prediction combined with an annotation and classification downstream strategy, thus supporting the user's decision-making process by summarizing the results of the different prediction tools in charts and tables. WtP is reproducible and scales to thousands of datasets through a workflow manager (Nextflow). WtP is freely available under a GPL-3.0 license (https://github.com/replikation/What_the_Phage).
- ItemThe First Report of mcr-1-Carrying Escherichia coli Originating from Animals in Serbia(Basel : MDPI, 2021) Mišić, Dušan; Kiskaroly, Ferenc; Szostak, Michael P.; Cabal, Adriana; Ruppitsch, Werner; Bernreiter-Hofer, Tanja; Milovanovic, Viktoria; Feßler, Andrea T.; Allerberger, Franz; Spergser, Joachim; Müller, Elke; Schwarz, Stefan; Braun, Sascha D.; Monecke, Stefan; Ehricht, Ralf; Korus, Maciej; Benković, Damir; Korzeniowska, Malgorzata; Loncaric, IgorThe aim of this study was continuous monitoring of the presence of mcr-1 to mcr-5 genes in Enterobacterales isolated from cattle, pigs, and domestic poultry at intensive breeding facilities in Northern Vojvodina, Serbia, from 1 January 1 to 1 October 2020. Out of 2167 examined samples, mcr-1 was observed in five E. coli isolates originating from healthy turkeys. Four isolates belonged to the phylogenetic group B1, and one isolate to the phylogenetic group A. Detected E. coli serogenotypes (somatic O and flagellar H antigens) were O8:H25 and O29:H25. Core-genome multi-locus sequence typing (cgMLST) revealed three ST58 isolates clustering together in Clonal Complex (CC) 155 and two singletons of ST641-CC86 and ST410-CC23, respectively. Clonotyping revealed CH4-32 (n = 3), CH6-53 (n = 1) and CH4-24 (n = 1). In all isolates, the mcr-1 gene was located on a large IncX4 replicon type plasmid. Eight virulence-associated genes (VAGs) typical of avian pathogenic E. coli (APEC) (fyuA, fimH, hlyF, iss, ompT, sitA, traT, iroN) were detected in four isolates. These isolates were investigated for susceptibility to four biocides and revealed MIC values of 0.125% for glutardialdehyde, of 0.00003-0.00006% for chlorohexidine, of 4-6% for isopropanol and of 0.001-0.002% for benzalkonium chloride. All obtained MIC values of the tested biocides were comparable to the reference strain, with no indication of possible resistance. This is the first report of mcr-1.1-carrying E. coli from Serbia. Although only samples from turkeys were mcr-positive in this study, continuous monitoring of livestock samples is advised to prevent a spill-over from animals to humans.
- ItemPhenotypic and Molecular Detection of Biofilm Formation in Staphylococcus aureus Isolated from Different Sources in Algeria(Basel : MDPI, 2020) Achek, Rachid; Hotzel, Helmut; Nabi, Ibrahim; Kechida, Souad; Mami, Djamila; Didouh, Nassima; Tomaso, Herbert; Neubauer, Heinrich; Ehricht, Ralf; Monecke, Stefan; El-Adawy, HosnyStaphylococcus aureus is an opportunistic bacterium causing a wide variety of diseases. Biofilm formation of Staphylococcus aureus is of primary public and animal health concern. The purposes of the present study were to investigate the ability of Staphylococcus aureus isolated from animals, humans, and food samples to form biofilms and to screen for the presence of biofilmassociated and regulatory genes. In total, 55 Staphylococcus aureus isolated from sheep mastitis cases (n = 28), humans (n = 19), and from food matrices (n = 8) were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The ability of Staphylococcus aureus for slime production and biofilm formation was determined quantitatively. A DNA microarray examination was performed to detect adhesion genes (icaACD and biofilmassociated protein gene (bap)), genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), regulatory genes (accessory gene regulator (agr) and staphylococcal accessory regulator (sarA)), and the staphylococcal cassette chromosome mec elements (SCCmec). Out of 55 Staphylococcus aureus isolates, 39 (71.0%) and 23 (41.8%) were producing slime and biofilm, respectively. All Staphylococcus aureus strains isolated from food showed biofilm formation ability. 52.6% of the Staphylococcus aureus strains isolated from sheep with mastitis, and 17.9% of isolates from humans, were able to form a biofilm. Microarray analysis typed the Staphylococcus aureus into 15 clonal complexes. Among all Staphylococcus aureus isolates, four of the human isolates (21.1%) harbored the mecA gene (SCCmec type IV) typed into 2 clonal complexes (CC22-MRSA-IV and CC80-MRSA-IV) and were considered as methicillin-resistant, while two of them were slime-producing. None of the isolates from sheep with mastitis harbored the cna gene which is associated with biofilm production. The fnbB gene was found in 100%, 60% and 40% of biofilm-producing Staphylococcus aureus isolated from food, humans, and sheep with mastitis, respectively. Three agr groups were present and agr group III was predominant with 43.6%, followed by agr group I (38.2%), and agr group II (18.2%). This study revealed the capacity of Staphylococcus aureus isolates to form biofilms and highlighted the genetic background displayed by Staphylococcus aureus isolates from different sources in Algeria. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemExploring the evolution and epidemiology of European CC1-MRSA-IV: tracking a multidrug-resistant community-associated meticillin-resistant Staphylococcus aureus clone(London : Soc., 2021) Earls, Megan R.; Steinig, Eike J.; Monecke, Stefan; Samaniego Castruita, José A.; Simbeck, Alexandra; Schneider-Brachert, Wulf; Vremerǎ, Teodora; Dorneanu, Olivia S.; Loncaric, Igor; Bes, Michèle; Lacoma, Alicia; Prat Aymerich, Cristina; Wernery, Ulrich; Armengol-Porta, Marc; Blomfeldt, Anita; Duchene, Sebastian; Bartels, Mette D.; Ehricht, Ralf; Coleman, David C.This study investigated the evolution and epidemiology of the community-associated and multidrug-resistant Staphylococcus aureus clone European CC1-MRSA-IV. Whole-genome sequences were obtained for 194 European CC1-MRSA-IV isolates (189 of human and 5 of animal origin) from 12 countries, and 10 meticillin-susceptible precursors (from North-Eastern Romania; all of human origin) of the clone. Phylogenetic analysis was performed using a maximum-likelihood approach, a time-measured phylogeny was reconstructed using Bayesian analysis, and in silico microarray genotyping was performed to identify resistance, virulence-associated and SCCmec (staphylococcal cassette chromosome mec) genes. Isolates were typically sequence type 1 (190/204) and spa type t127 (183/204). Bayesian analysis indicated that European CC1-MRSA-IV emerged in approximately 1995 before undergoing rapid expansion in the late 1990s and 2000s, while spreading throughout Europe and into the Middle East. Phylogenetic analysis revealed an unstructured meticillin-resistant S. aureus (MRSA) population, lacking significant geographical or temporal clusters. The MRSA were genotypically multidrug-resistant, consistently encoded seh, and intermittently (34/194) encoded an undisrupted hlb gene with concomitant absence of the lysogenic phage-encoded genes sak and scn. All MRSA also harboured a characteristic ~5350 nt insertion in SCCmec adjacent to orfX. Detailed demographic data from Denmark showed that there, the clone is typically (25/35) found in the community, and often (10/35) among individuals with links to South-Eastern Europe. This study elucidated the evolution and epidemiology of European CC1-MRSA-IV, which emerged from a meticillin-susceptible lineage prevalent in North-Eastern Romania before disseminating rapidly throughout Europe.
- ItemMolecular investigations on a chimeric strain of Staphylococcus aureus sequence type 80(San Francisco, California, US : PLOS, 2020) Gawlik, Darius; Ruppelt-Lorz, Antje; Müller, Elke; Reißig, Annett; Hotzel, Helmut; Braun, Sascha D.; Söderquist, Bo; Ziegler-Cordts, Albrecht; Stein, Claudia; Pletz, Mathias W.; Ehricht, Ralf; Monecke, StefanA PVL-positive, methicillin-susceptible Staphylococcus aureus was cultured from pus from cervical lymphadenitis of a patient of East-African origin. Microarray hybridisation assigned the isolate to clonal complex (CC) 80 but revealed unusual features, including the presence of the ORF-CM14 enterotoxin homologue and of an ACME-III element as well as the absence of etD and edinB. The isolate was subjected to both, Illumina and Nanopore sequencing allowing characterisation of deviating regions within the strain´s genome. Atypical features of this strain were attributable to the presence of two genomic regions that originated from other S. aureus lineages and that comprised, respectively, 3% and 1.4% of the genome. One deviating region extended from walJ to sirB. It comprised ORF-CM14 and the ACME-III element. A homologous but larger fragment was also found in an atypical S. aureus CC1/ST567 strain whose lineage might have served as donor of this genomic region. This region itself is a chimera comprising fragments from CC1 as well as fragments of unknown origin. The other deviating region comprised the region from htsB to ecfA2, i.e., another 3% of the genome. It was very similar to CC1 sequences. Either this suggests an incorporation of CC1 DNA into the study strain, or alternatively a recombination event affecting “canonical” CC80. Thus, the study strain bears witness of several recombination events affecting supposedly core genomic genes. Although the exact mechanism is not yet clear, such chimerism seems to be an additional pathway in the evolution of S. aureus. This could facilitate also a transmission of virulence and resistance factors and therefore offer an additional evolutionary advantage.
- ItemUse of meat juice and blood serum with a miniaturised protein microarray assay to develop a multi-parameter IgG screening test with high sample throughput potential for slaughtering pigs(London : BioMed Central, 2020) Loreck, Katharina; Mitrenga, Sylvia; Heinze, Regina; Ehricht, Ralf; Engemann, Claudia; Lueken, Caroline; Ploetz, Madeleine; Greiner, Matthias; Meemken, DianaBackground: Serological screening of pig herds at the abattoir is considered a potential tool to improve meat inspection procedures and herd health management. Therefore, we previously reported the feasibility of a miniaturised protein microarray as a new serological IgG screening test for zoonotic agents and production diseases in pigs. The present study investigates whether the protein microarray-based assay is applicable for high sample throughput using either blood serum or meat juice. Material and methods: Microarrays with 12 different antigens were produced by Abbott (formerly Alere Technologies GmbH) Jena, Germany in a previously offered 'ArrayTube' platform and in an 'ArrayStrip' platform for large-scale use. A test protocol for the use of meat juice on both microarray platforms was developed. Agreement between serum and meat juice was analysed with 88 paired samples from three German abattoirs. Serum was diluted 1:50 and meat juice 1:2. ELISA results for all tested antigens from a preceding study were used as reference test to perform Receiver Operating Characteristic analysis for both test specimens on both microarray platforms. Results: High area under curve values (AUC > 0.7) were calculated for the analysis of T. gondii (0.87), Y. enterocolitica (0.97), Mycoplasma hyopneumoniae (0.84) and Actinobacillus pleuropneumoniae (0.71) with serum as the test specimen and for T. gondii (0.99), Y. enterocolitica (0.94), PRRSV (0.88), A. pleuropneumoniae (0.78) and Salmonella spp. (0.72) with meat juice as the test specimen on the ArrayStrip platform. Cohens kappa values of 0.92 for T. gondii and 0.82 for Y. enterocolitica were obtained for the comparison between serum and meat juice. When applying the new method in two further laboratories, kappa values between 0.63 and 0.94 were achieved between the laboratories for these two pathogens. Conclusion: Further development of a miniaturised pig-specific IgG protein microarray assay showed that meat juice can be used on microarray platforms. Two out of twelve tested antigens (T. gondii, Y. enterocolitica) showed high test accuracy on the ArrayTube and the ArrayStrip platform with both sample materials. © 2020 The Author(s).
- ItemCharacterization of antibiotic and biocide resistance genes and virulence factors of staphylococcus species associated with bovine mastitis in Rwanda(Basel : MDPI AG, 2020) Antók, Fruzsina Irén; Mayrhofer, Rosa; Marbach, Helene; Masengesho, Jean Claude; Keinprecht, Helga; Nyirimbuga, Vedaste; Fischer, Otto; Lepuschitz, Sarah; Ruppitsch, Werner; Ehling-Schulz, Monika; Feßler, Andrea T.; Schwarz, Stefan; Monecke, Stefan; Ehricht, Ralf; Grunert, Tom; Spergser, Joachim; Loncaric, IgorThe present study was conducted from July to August 2018 on milk samples taken at dairy farms in the Northern Province and Kigali District of Rwanda in order to identify Staphylococcus spp. associated with bovine intramammary infection. A total of 161 staphylococcal isolates originating from quarter milk samples of 112 crossbred dairy cattle were included in the study. Antimicrobial susceptibility testing was performed and isolates were examined for the presence of various resistance genes. Staphylococcus aureus isolates were also analyzed for the presence of virulence factors, genotyped by spa typing and further phenotypically subtyped for capsule expression using Fourier Transform Infrared (FTIR) spectroscopy. Selected S. aureus were characterized using DNA microarray technology, multi-locus sequence typing (MLST) and whole-genome sequencing. All mecA-positive staphylococci were further genotyped using dru typing. In total, 14 different staphylococcal species were detected, with S. aureus being most prevalent (26.7%), followed by S. xylosus (22.4%) and S. haemolyticus (14.9%). A high number of isolates was resistant to penicillin and tetracycline. Various antimicrobial and biocide resistance genes were detected. Among S. aureus, the Panton–Valentine leukocidin (PVL) genes, as well as bovine leukocidin (LukM/LukF-P83) genes, were detected in two and three isolates, respectively, of which two also carried the toxic shock syndrome toxin gene tsst-1 bovine variant. t1236 was the predominant spa type. FTIR-based capsule serotyping revealed a high prevalence of non-encapsulated S. aureus isolates (89.5%). The majority of the selected S. aureus isolates belonged to clonal complex (CC) 97 which was determined using DNA microarray based assignment. Three new MLST sequence types were detected. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemCharacterisation of Methicillin-Resistant Staphylococcus aureus from Alexandria, Egypt(Basel : MDPI, 2023) Monecke, Stefan; Bedewy, Amira K.; Müller, Elke; Braun, Sascha D.; Diezel, Celia; Elsheredy, Amel; Kader, Ola; Reinicke, Martin; Ghazal, Abeer; Rezk, Shahinda; Ehricht, RalfThe present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt’s second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+fus] (n = 6), CC1-MRSA-[V+fus+tir+ccrA/B-1] (PVL+) (n = 5) as well as CC1-MRSA-[V+fus+tir+ccrA/B-1] and CC1153-MRSA-[V+fus] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCCmec V or VI elements that included the fusidic acid resistance gene fusC. The SCCmec [V+fus+tir+ccrA/B-1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ, aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCCmec elements comprising mecA and fusC. This is an unsettling trend, but more MRSA typing data from Egypt are required.