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Sybille SchГ¶neberger One of the most demanding challenges in infection control is the worldwide dissemination of multidrug-resistant (MDR) bacteria in clinical settings. Especially the increasing prevalence of carbapenemase producing Gram-negative pathogens poses an urgent threat to public health, as these enzymes confer resistance to almost all β-lactam antibiotics including carbapenems. Baza genealogiczna potomków Sejmu Wielkiego (Czteroletniego) pod redakcją Marka Jerzego Minakowskiego. Sejm Czteroletni czyli Wielki to parlament Polski i Litwy obradujący To help identify the effects and factors influencing the impact of the COVID pandemic on physical and mental well-being, please click on the link below, and answer the survey.

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Nahezu alle. 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.

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Irrua, Edo State, Nigeria. Afzal Javed. Chairman Pakistan Psychiatric Research Centre, Fountain House, Lahore-Pakistan.

Dean, Faculty of Psychiatry, College of Physicians and Surgeons Pakistan. Yasser Abu Jamei. The different analyses tasks are shown in italics bright colored boxes.

The respective input and output reads, contigs, draft and final sequences is highlighted by boxes with strong colors.

If a method could not successfully applied, alternative methods were used indicated by red arrows. For a detailed description of the reconstruction process, see Supplementary Data Sheet 1.

Primers were obtained from Integrated DNA Technologies Leuven, Belgium Supplementary Table 2. In July , a carbapenem-resistant C. The hospital includes 18 hospital wards that cover almost all medical disciplines.

These were collected from 38 patients and three environmental sources rinse water samples across seven different wards I-VII between July and October Most patients were primarily hospitalized at the hematologic oncology ward I Supplementary Table 1.

Containment and finally cessation of transmission was achieved by extensive staff training and the introduction of substantial organizational, structural and infection control measures.

Figure 2. Emergence of NDMproducing Enterobacterales between July and October in a German hospital. The bla NDM—1 gene was detected in 35 isolates from 29 patients and two environmental sources Figure 2 , Supplementary Table 1.

NDMpositive isolates were identified as C. Among these, seven isolates additionally encoded KPC Moreover, we identified 16 bla NDM—1 negative but bla KPC—2 positive isolates from 13 patients and one environmental source, that revealed K.

Resistance to colistin was only measured in intrinsically resistant isolates M. Conjugation assays combined with S1-nuclease PFGE, PBRT and hybridization experiments showed an association of bla NDM—1 with plasmids of different sizes and Inc types Supplementary Table 1.

The 19 E. The three clusters comprised nine ST isolates, seven ST isolates, and two ST isolates, respectively, which was consistent with PFGE results Supplementary Table 1.

Within each cluster, isolates did not show more than eight allele differences in the cgMLST Figure 3A. Three different clusters and one singleton have also been detected for the 22 K.

Less than eleven different alleles were detected between K. For three C. Likewise, both ST E. Since a cgMLST scheme was not yet available for M.

By this, a maximal difference of six SNPs was found data not shown. Figure 3. Genetically related clusters of NDMpositive Enterobacterales based on cgMLST.

For E. The number of different alleles between isolates is shown next to black lines. Please note: the distances displayed are not proportional to the number of differing targets.

Isolate names are shown within color-coded circles representing MLST-based sequence types. To assess whether bla NDM—1 associated MGEs in the different Enterobacterales isolates were genetically related or not, WGS data were used to reconstruct the involved genetic elements.

For this purpose, data from short- and long-read sequencing were utilized in combination with results of conjugation assays, S1-PFGE-based plasmid size determination and Southern hybridization, thus allowing the complete reconstruction of eleven bla NDM—1 carrying plasmids present in 34 isolates Table 1 and Supplementary Image 1.

Only for one isolate of this study C. A detailed description of the sequence reconstruction processes is given in Supplementary Data Sheet 1.

They were not considered to be involved in local transmission events and therefore not addressed in further details.

Figure 4. Genetic organizations of bla NDM—1 associated Tn composite transposons. A Structure of the genuine Tn composite transposon originally described by Bontron et al.

B Organization of Tn like structures in plasmids potentially involved in horizontal transmission. The complete reconstruction of pCFT8 failed Tn-type 8.

C bla NDM—1 associated structures from isolates of patients with travel history demonstrating a different Tn structure. ORFs and respective orientations are represented by arrows.

These regions were located upstream the antibiotic resistance island ARI-A that mainly affected genes coding for conjugative transfer proteins Supplementary Image 1.

Within ARI-A, we found the bla NDM—1 gene located on a previously described Tn transposon structure Bontron et al. This structure, however, was consistently flanked by two IS 26 elements which were integrated into the adjacent IS Aba elements of Tn Figure 4.

As a consequence, the genuine composite transposon structure of Tn is turned into an IS 26 -dependent composite transposon structure.

In the E. This observation was confirmed by hybridization results data not shown. The second E. The K. This plasmid pKPC-2 was found in all NDMpositive ST39 K.

In accordance to results from conjugation and hybridization experiments, bla NDM—1 has also been found to be located on the IncN plasmid pECT6 in several isolates Table 1.

In pECT6, the above described IS 26 flanked composite transposon has been integrated upstream of Hsp20 and a putative transposase Supplementary Image 1.

Interestingly, pECT6 could be detected in both E. A minor structural variation of pECT6 named pECT7 was present in four E. This variant is defined by an insertion of Tn2 in the Tn like transposon structure of pECT6, thereby forming a new transposon and plasmid structure Tn-type 7 Figure 4.

Since carbapenems belong to the most important last resort antibiotics for the treatment of infections caused by MDR Gram-negative bacteria, the emergence of bacteria producing carbapenem hydrolysing enzymes is of particular interest.

KPC-2 producing strains, especially K. Hospital-associated infections caused by NDMproducing Enterobacterales have recently been described in Spain, England, and Italy ECDC, ; Fairley et al.

Colonization and infections with carbapenem-resistant Enterobacterales and A. Considering a substantial underreporting of the number of NDM-1 cases, we could still assume and extrapolate that during the time of the present study —, bla NDM—1 was a rare carbapenemase type in Germany.

Therefore, the spatial and temporal clustering of NDMpositive isolates in a single hospital — as described in this study — strongly pointed to possible transmissions across different bacterial species.

To verify this, we complemented and combined results from traditional typing methods such as PFGE and MLST with WGS data from NGS and ultra-long sequencing technologies.

This combination made it possible to track and resolve different potential transmission routes Figure 5. Figure 5. The illustrated plasmids are horizontally aligned to date of isolation of the respective strain.

Plasmid- and Tn-types are represented by color-coded circles and fillings, respectively see legend on right side. Upper numbers bold within plasmid rings highlight Tn-types, while the numbers below show the plasmid size [kb].

The reconstructed transposon structures are schematically illustrated beneath the corresponding plasmids. Directed dotted lines indicate possible vertical black and horizontal red transmission routes, while gray-shaded lines hypothesize possible transfers of the respective bla NDM—1 carrying transposon.

At the bottom, the Tn like structure is shown in detail Tn-type 3. Two bla NDM—1 carrying plasmids belong to isolates collected from patients with a travel history and, thus not linked to the outbreak indicated by airplane symbols.

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