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Objectives To characterize the genetic environment of metallo-β-lactamases (MBL) in carbapenem-resistant clinical Acinetobacter pittii isolates. Methods Seventeen carbapenem-resistant A. pittii isolates harbouring an MBL were collected between 2010 and 2015 in Germany. Antimicrobial susceptibility testing was performed using agar dilution. Presence of MBLs was confirmed by PCR and their genetic location determined by S1-pulsed-field gel electrophoresis followed by Southern blot hybridization. Whole-genome sequencing was performed using the Miseq and MinION platforms. Isolates were typed using an ad hoc core genome MLST scheme. Conjugation into A. baumannii was tested by broth mating. Results In 10 isolates the MBL was plasmid-encoded and in seven isolates chromosomally encoded. bla GIM-1 and bla VIM-2 were plasmid-encoded, bla VIM-4 was chromosomally encoded, while bla NDM-1 was chromosomally encoded in four and plasmid-encoded in three isolates. Seven of ten plasmids were conjugative into A. baumannii. Although most isolates were unrelated, the backbones of the MBL-encoding plasmid showed >99% similarity and only differed in the MBL-encoding area. bla NDM-1-harbouring plasmids were highly similar to other plasmids from Acinetobacter isolates worldwide while the bla VIM-2- and bla GIM-1-encoding plasmids have not been described. Conclusions These data show the existence of a promiscuous plasmid circulating in A. pittii isolates in Germany that differs only in the MBL-encoding region. Its plasmid backbone has been found globally among multiple Acinetobacter spp. These data should raise awareness of an epidemic conjugative plasmid that has independently acquired MBLs. We should also consider that future comparative plasmid analysis will look beyond solely the resistome and include the mobile elements carrying the resistance genes. [ABSTRACT FROM AUTHOR]

Objectives: To examine the impact on carbapenem resistance of mutations in Escherichia coli PBP2 detected in clinical isolates showing increased MICs of imipenem, but not of meropenem.Methods: The mutations in the PBP2-encoding gene mrdA were introduced into E. coli DH5α using the helper plasmid pTKRED. β-Lactam MICs were determined by broth microdilution and interpreted according to EUCAST. Mutants were screened for secondary mutations by WGS. To detect a possible fitness cost related to these mutations, the generation times of the mutants and E. coli DH5α were measured and their cell morphology was examined.Results: The 10 mutation patterns introduced into mrdA increased the MICs of imipenem and doripenem in all cases and of meropenem and mecillinam in some cases, but had no effect on ertapenem resistance. While no significant alteration of the generation time of the mutants could be detected, several mutants showed increased transverse diameters and thus altered cell morphology.Conclusions: Here we describe mutation patterns in PBP2 that contribute to increased MICs of carbapenems for clinical isolates of E. coli. We show that different mutations affect carbapenem MICs differently and that the mutations do not impact generation time, although some mutations affect cell morphology. [ABSTRACT FROM AUTHOR]

Objectives: To identify and characterize a novel MBL gene conferring carbapenem resistance to an isolate of Enterobacter cloacae from Austria.Methods: The novel MBL gene was heterologously expressed in Escherichia coli TOP10 to conduct comparative MIC studies and biochemical assays. Furthermore, WGS was performed using Illumina MiSeq and Oxford Nanopore MinION instruments to analyse the genetic environment of the novel MBL gene.Results: The novel MBL showed highest sequence homology to a predicted MBL precursor from the marine bacterium Rheinheimera pacifica and hence belongs to Ambler subgroup B3. The comparative MIC studies and biochemical assays showed activity of the novel enzyme against penicillins, cephalosporins and carbapenems, but not against aztreonam. It was named Linz MBL (LMB-1). The blaLMB-1 gene was shown to be located on a 108 kb plasmid of Inc type IncFIB(K). Of note, a gene adjacent to blaLMB-1 coded for a glycerophosphoryl diester phosphodiesterase that was also previously detected in R. pacifica.Conclusions: Homologies of the MBL gene itself and another gene located on the same plasmid to genes detected in marine bacterial species strongly suggest that this novel MBL was transferred to E. cloacae from a marine bacterium. This underlines the importance of natural reservoirs supplying hitherto unknown resistance genes to clinically relevant bacterial species and the importance of ongoing surveillance and research. [ABSTRACT FROM AUTHOR]

Objectives: Characterization of Proteus mirabilis isolates harbouring bla OXA-58 with emphasis on the genetic environment of this resistance determinant.Methods: Strains of P. mirabilis ( n  =   37) isolated from different patients were tested for the presence of bla OXA-58 . The genetic context of bla OXA-58 was determined by WGS of two strains and Sanger sequencing. Clonality of the strains was assessed by PFGE. Susceptibility testing was performed by microdilution according to EUCAST.Results: Four strains isolated in different geographical regions of Germany were positive for bla OXA-58 , and WGS showed that this resistance gene was harboured on a plasmid. Sanger sequencing confirmed the presence of two nearly identical plasmids, 6219 and 6208 bp in size, in all four strains. Upstream of bla OXA-58 an IS Aba 3-like transposase gene was located. The P. mirabilis strains were not clonally related according to PFGE. MICs of meropenem for three of the strains were only just above the EUCAST breakpoint and the Carba NP test was positive for only two of the strains.Conclusions: To our knowledge, this is the first description of bla OXA-58 in the species P. mirabilis . The resistance gene is harboured by almost identical plasmids in strains not clonally related and from different geographical regions. Apart from an IS Aba 3-like transposase gene upstream of bla OXA-58 the genetic context is different from bla OXA-58 harboured on plasmids in the genus Acinetobacter . With MICs of meropenem well below the EUCAST breakpoint or only just above it and equivocal or false negative results from the Carba NP test, bla OXA-58 can be easily overlooked in P. mirabilis . [ABSTRACT FROM AUTHOR]

Objectives: To characterize a novel subclass B1 metallo-β-lactamase (MBL) found in an MDR Pseudomonas aeruginosa clinical isolate.Methods: The isolate P. aeruginosa NRZ-03096 was recovered in 2012 from an anal swab from a patient hospitalized in Northern Germany and showed high MICs of carbapenems. MBL production was analysed by several phenotypic tests. Genetic characterization of the novel bla gene and MLST was performed by WGS. The novel bla gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization to determine the kinetic parameters K m and k cat .Results: P. aeruginosa NRZ-03096 was resistant to all tested β-lactams and showed an MBL phenotype. Shotgun cloning experiments yielded a clone producing a novel subclass B1 enzyme with only 74.3% identity to the next nearest relative, KHM-1. The novel MBL was named HMB-1 (for Hamburg MBL). Analysis of WGS data showed that the bla HMB-1 gene was chromosomally located as part of a Tn 3 family transposon that was named Tn 6345 . Expression of bla HMB-1 in E. coli TOP10 led to increased resistance to β-lactams. Determination of K m and k cat revealed that HMB-1 had different hydrolytic characteristics compared with KHM-1, with lower hydrolytic rates for cephalosporins and a higher rate for imipenem.Conclusions: The identification of HMB-1 further underlines the ongoing spread and diversification of carbapenemases in Gram-negative human pathogens and especially in P. aeruginosa . [ABSTRACT FROM AUTHOR]

Objectives: The objective of this study was to characterize a novel IMP-type metallo-β-lactamase (MBL) found in an MDR clinical isolate of Pseudomonas aeruginosa. Methods: The P. aeruginosa isolate NRZ-00156 was recovered from an inguinal swab from a patient hospitalized in Western Germany and showed high MICs of carbapenems. MBL production was analysed by Etest for MBLs, an EDTA combined disc test and an EDTA bioassay. Typing of the isolate was performed by MLST. Genetic characterization of the new blaIMP genewas performed by sequencing the PCR products. A phylogenetic treewas constructed. The novel blaIMP gene was expressed in Escherichia coli TOP10 and the enzyme was subjected to biochemical characterization. Results: The P. aeruginosa isolate NRZ-00156 expressed the ST235 allelic profile and was resistant to all the b-lactams tested except aztreonam. The isolate was positive for MBL production and harboured a new IMP allele, blaIMP-31, located on a disrupted class I integron [also carrying the blaOXA-35, aac(6')-Ib, aac(3)-Ic and aphA15 genes]. Its closest relative was IMP-35, with 96.7% amino acid identity. Expression of blaIMP-31 demonstrated that E. coli TOP10 producing IMP-31 had elevated resistance to all the β-lactams tested except aztreonam. Kinetic data were obtained for both IMP-31 and IMP-1. In comparison with IMP-1, IMP-31 showed weaker hydrolytic activity against all the β-lactams tested, which resulted from lower kcat values. Conclusions: The characterization of the new IMP-type gene blaIMP-31 froman ST235 P. aeruginosa isolate indicates an ongoing spread of highly divergent IMP-type carbapenemases in clinical P. aeruginosa strains and highlights the continuous need for the prevention of nosocomial infections caused by MDR Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Objectives Antibiotic resistance among Escherichia coli is globally an increasing problem in public healthcare. Understanding the spread of plasmid-mediated ESBL genes is of great importance in elucidating their molecular epidemiology. However, differentiation of subtypes and alleles is frequently hampered by the lack of comprehensive diagnostic tools. We therefore developed a novel universal blaSHV, blaTEM and blaCTX-M subtyping assay based on PCR and Sanger sequencing that results in large amplicons of >700 bp, enabling differentiation of bla alleles as precisely as possible. Methods The assay was established using 10 reference strains with known bla genotypes that represent all examined primer groups and 101 uncharacterized ESBL-producing E. coli of clinical and livestock-associated origins from different German regions. All isolates were tested in parallel with established blaSHV, blaTEM and blaCTX-M subtyping assays for the respective β-lactamases and their alleles. Results The novel assay yielded equal (n = 92) or better (n = 47) subtyping results compared with established subtyping methods and reliably detected all expected enzymes in the reference strains. Overall, the occurring enzymes could be differentiated into groups representing one (n = 9), two (n = 5) or three (n = 4) highly similar alleles. Moreover, ESBL and non-ESBL allelic variants of blaSHV and blaTEM occurring in the same isolate were distinguished reliably. Conclusions We established a highly discriminatory assay for the subtyping of clinically important ESBL genes that can easily be used in epidemiological analyses. [ABSTRACT FROM PUBLISHER]

Objectives To characterize the mechanisms involved in the reduced carbapenem susceptibility of five Acinetobacter pittii strains isolated from different regions of Germany. Methods The strains were analysed by susceptibility testing, phenotypic tests for metallo-β-lactamase production, sequencing of the integron structure and strain typing by PFGE, as well as multilocus sequence typing (MLST) and plasmid analysis by S1 restriction and hybridization. Results Despite GIM-1 production, the MICs of imipenem were only 4 mg/L for four strains and some methods of phenotypic MBL detection failed. According to PFGE and MLST, the strains belonged to four different clones, but blaGIM-1 was present in identical integron structures in all strains and carried on plasmids of ∼60 kb. Conclusions For the first time, GIM-1 has been demonstrated in A. pittii. This resistance mechanism has previously been reported only in Enterobacteriaceae and Pseudomonas aeruginosa. As GIM-1 was found in strains with diverse clonal backgrounds, but encoded on plasmids of a similar size, further spread among Acinetobacter spp. seems possible. The detection of GIM-1 production might be challenging in some strains due to the low MICs of carbapenems. [ABSTRACT FROM PUBLISHER]

Objectives To characterize the mechanisms involved in reduced susceptibility to carbapenems in two Enterobacter cloacae clinical isolates. Methods Two E. cloacae isolates recovered from different regions in Germany and showing reduced susceptibility to carbapenems were analysed. Susceptibility testing, conjugation, transformation assays, plasmid analysis, sequencing and molecular typing using rep-PCR were performed. Results The two clinical isolates carried the blaGIM-1 gene and showed resistance to ertapenem, with variable MIC values of imipenem and meropenem. The isolates were clonally unrelated. The blaGIM-1 gene was located on self-transferable and non-typeable plasmids. Both isolates harboured distinct plasmids and integron structures containing the blaGIM-1 gene cassette. Interestingly, one of the two plasmids was able to replicate in Pseudomonas aeruginosa, demonstrating its broad host range. Conclusions This is the first identification in E. cloacae of the blaGIM-1 gene, which is responsible for reduced susceptibility to carbapenems. We showed that this gene, previously identified in P. aeruginosa, was located in a different genetic background in E. cloacae. The blaGIM-1 gene might spread quite efficiently in Enterobacteriaceae and P. aeruginosa, as it is difficult to detect and in addition is located on conjugative plasmids. [ABSTRACT FROM PUBLISHER]

Objectives To analyse the mechanisms responsible for carbapenem resistance in one Acinetobacter baumannii isolate recovered from a patient transferred to Germany from an Egyptian hospital. Methods PCR and sequencing were used to search for β-lactamase and 16S RNA methylase genes. Multilocus sequence typing was used to determine the sequence type (ST) of the isolate. Results Sequencing of the PCR product obtained using primers for blaNDM-1 revealed a variant of NDM-1 that had a C to G substitution at position 82 resulting in an amino acid substitution of proline to alanine at position 28. This variant was designated NDM-2. Genes encoding extended-spectrum β-lactamases or 16S RNA methylase were not detected. The strain lacked detectable plasmids and blaNDM-2 was not transferred by conjugation. MLST showed that the isolate belonged to a new ST, ST103. Conclusions This work further underlines the spread of NDM carbapenemases in A. baumannii, and the spread of the corresponding gene in the Middle East. It also describes the first variant of NDM-1. [ABSTRACT FROM PUBLISHER]