Sybille Schöneberger

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The detailed reconstruction of the plasmid sequences revealed that in all outbreak-associated isolates bla NDM—1 was located on similar composite transposons, which were also very similar to Tn previously described for Acinetobacter baumannii.

In contrast to Tn , these structures were flanked by IS 26 elements, which could facilitate horizontal gene transfer.

Moreover, the identical plasmid was found to be shared by E. Our results highlight the importance of detailed genome-based analyses for complex nosocomial outbreaks, allowing the identification of causal genetic determinants and providing insights into potential mechanisms involved in the dissemination of antibiotic resistances between different bacterial species.

The rapid spread of antimicrobial resistance AMR in nosocomial pathogens poses an urgent threat to patients and public health worldwide.

In particular, the increasing incidence of multidrug-resistant MDR Gram-negative bacteria that are resistant to fluoroquinolones, third-generation cephalosporins, and carbapenems is of major concern, as these MDR pathogens dramatically limit therapeutic options Pfeifer et al.

For this reason, bacterial pathogens such as Acinetobacter baumanii , Pseudomonas aeruginosa , and various Enterobacterales species have been identified by the World Health Organization as first priority for research and development of new antibiotics Tacconelli and Magrini, Due to the rapid spread and worldwide dissemination of ESBL-producing Enterobacterales and other third-generation cephalosporin-resistant Gram-negative bacteria, carbapenems and other antibiotics of last resort have been increasingly used since the early s Hawkey and Livermore, This enzyme hydrolyzes penicillins, third-generation cephalosporins and carbapenems but not the monobactam aztreonam Nordmann et al.

These plasmids usually co-harbor large collections of genetic AMR determinants, thereby mediating a MDR phenotype Pfeifer et al.

Since its first description, NDM-1 has been recorded worldwide in a great diversity of clinical Enterobacterales species and has often been associated with a travel history to endemic regions like India, Pakistan, and the Balkan states Nordmann et al.

The potential of bla NDM—1 to rapidly disseminate between related species of Enterobacterales is facilitated by the high genetic mobility of bla NDM—1 and an association of this resistance determinant with promiscuous plasmids that have a broad host range Nordmann et al.

As a result of effective horizontal gene transfer HGT , identical mobile genetic elements MGEs occur in different bacteria.

However, identical MGEs shared by different bacteria are not necessarily proof of HGT, as they may originate from different sources.

The reliable reconstruction of transmission chains is therefore essential to the analysis of multi-species outbreaks, although often difficult and laborious Pirs et al.

Outbreaks of NDMproducing Enterobacterales have been reported, as described for the United Kingdom Fairley et al. In Europe and more particular in Germany, the prevalence of invasive Enterobacterales with resistance to carbapenems is still low, and NDMproducing isolates have only been observed occasionally Becker et al.

However, data based on the voluntary submission of MDR isolates to the National Reference Centre for Multidrug-resistant Gram-negative Bacteria NRC in Bochum, Germany, indicate an increasing number of carbapenemase-producing isolates in Germany over the past years Robert Koch-Institute, From to , the number of NDMproducing Enterobacterales isolates received from hospitals across Germany rose from 40 to Focusing on all carbapenemase-positive Enterobacterales , a total of isolates were registered in , including Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae.

In the present study, molecular typing methods and whole genome sequencing were combined to trace the prolonged nosocomial outbreak of different NDMproducing Enterobacterales species back to clonal spread and cross-species exchange of plasmids and possibly transposons.

In a German hospital with 18 wards and approx. Two of the first three isolates were obtained from patients with travel history.

A clinical case definition based on the successful molecular detection of NDM-1 in Enterobacterales was adopted to track a possible spread of these pathogens.

Analyses were performed and interpreted according to EUCAST 1. In case of ambiguous results, carbapenemase detection was conducted using a commercial cassette PCR system considering genes encoding for KPC, NDM, VIM, IMP-1, and OXA Isolates were sent to the NRC to confirm the diagnosis and carbapenemase type.

Finally, a total of 51 carbapenemase-positive Enterobacterales isolates collected between July and October were submitted to the Robert Koch-Institute RKI for detailed molecular analyses and strain typing Supplementary Table 1.

At the RKI, antimicrobial susceptibility testing was repeated by the use of broth microdilution BMD according DIN and EUCAST guidelines v9.

Bacterial strain typing was performed by Xba I-macrorestriction followed by PFGE. Fragment patterns were interpreted according to the criteria of Tenover et al.

PCR-based replicon typing PBRT was performed using a commercial kit PBRT KIT, Diatheva, Italy. Plasmid linearization and separation were performed by S1 nuclease restriction of whole genomic DNA donor strains and transconjugants combined with PFGE as previously described Barton et al.

Brain Heart Infusion BHI broth was used for the cultivation of bacteria. ONT sequencing was carried out at the RKI, while SMRT sequencing was conducted by GATC Biotech Konstanz, Germany.

Quality of raw sequence data was checked using FastQC v0. Additionally, Kraken v0. For reference-based read alignments, raw reads were trimmed using Trimmomatic v0.

Illumina-only read sets were de novo assembled using SPAdes v2. The following cgMLST templates were used for E.

For Citrobacter freundii and E. For this purpose, C. Minimum spanning trees were inferred by ignoring pairwise missing values. Since no stable task templates are available for Morganella morganii , phylogenetic relationships were reconstructed on the level of single nucleotide polymorphisms SNPs.

A reference sequence was selected by using refRank Becker et al. Subsequently, trimmed paired-end reads were aligned to Morganella morganii subsp.

Alignment of consensus sequences were reduced to variant positions using SNPFilter v3. De novo assembled contigs were used for both resistance gene and plasmid replicon type identification.

For the detection of acquired AMR genes, the ResFinder tool v3. In silico replicon typing was performed with the PlasmidFinder v2. For the reconstruction of outbreak related MGEs, different strategies have been used Figure 1.

Briefly, de novo assembled contigs were screened for bla NMD—1 , bla KPC—2 , and replication sequences using BLAST Camacho et al.

In order to identify defective assemblies, trimmed Illumina reads were aligned to the selected contigs Geneious v Claudio Dietrich.

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Department of Psychiatry and Psychoherapy, Semmelweis University, Budapest. Praveenlal Kuttichira. Pushpagiri Institue of Medical Sciences, Thiruvalla, Kerala, Dept.

Dena Sadeghi Bahmani. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran. Kermanshah University of Medical Sciences, Substance Abuse Prevention Research Center and Sleep Disorders Research Center.

Evan Matthews. Mark Weiser. Andrea Boscutti. Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy.

Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy. University of Modena and Reggio Emilia, Clinical and Experimental Medicine PhD Program, University of Modena and Reggio Emilia, Modena, Italy.

Child and Adolescent Neuropsychiatry Unit, Maternal-Child Integrated Care Department, Integrated University Hospital of Verona, Italy.

Section of Psychiatry, Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Italy. Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy.

Department of Pathophysiology and Transplantation, Psychiatric Clinic, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy. Akihiro Takamiya.

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Sybille Schöneberger
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