Search

Little information is available on the local epidemiology of mobile genetic elements such as plasmids harboring acquired beta-lactamase genes in Western African Ghana. In the present study, we screened for plasmids in three Escherichia coli and four Klebsiella pneumoniae isolates expressing extended spectrum beta-lactamases (ESBL) mediated by the blaCTX-M-15 gene from chronically infected wounds of Ghanaian patients. Bacterial isolates were subjected to combined short-read and long-read sequencing to obtain the sequences of their respective plasmids. In the blaCTX-M-15-gene-carrying plasmids of the four ESBL-positive K. pneumoniae isolates, IncFIB/IncFII (n = 3) and FIA (n = 1) sequences were detected, while in the blaCTX-M-15-gene-carrying plasmids of the three ESBL-positive E. coli isolates, IncFIA/IncFIB (n = 2) and IncFIB (n = 1) sequences were found. The three IncFIB/IncFII sequence-containing plasmids were almost identical to a K. pneumoniae plasmid reported from France. They belonged to the clonal lineages ST17, ST36 and ST39 of K. pneumoniae, suggesting transversal spread of this obviously evolutionary successful plasmid in Ghana. Other resistance gene-encoding plasmids observed in the assessed Enterobacterales harbored IncFIA/IncR and IncFII sequences. International spread was confirmed by the high genetic similarity to resistance-mediating plasmids published from Asia, Australia, Europe and Northern America, including a blaCTX-M-15-gene-carrying plasmid isolated from a wild bird in Germany. In conclusion, the study contributed to the scarcely available information on the epidemiology of third-generation cephalosporine resistance-mediating plasmids in Ghana. Furthermore, the global spread of resistance-mediating plasmids provided hints on the evolutionary success of individual resistance-harboring plasmids by transversal spread among K. pneumoniae lineages in Ghana. [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]

The distribution of carbapenemase genes in Escherichia coli strains isolated between September 2009 and May 2013 in Germany was investigated. Out of 192 isolates with carbapenemase production OXA-48 was found in 44.8%, VIM-1 in 18.8%, NDM-1 in 11.5% and KPC-2 in 6.8%. Patients with VIM-1 producing E. coli ( n = 36) differed from patients with OXA-48 by an older age, less frequent mention of travel history and an increased proportion of clinical over screening specimens. These data might indicate that introduction from abroad is of minor importance for VIM-1 producing E. coli compared to other carbapenemases. Multilocus sequence typing revealed that E. coli with VIM-1 were mostly multiclonal, emphasizing the role of horizontal gene transfer in its spread. Susceptibility testing of VIM-1 producing E. coli demonstrated aztreonam susceptibility in 55.6%. Among non-β-lactams susceptibility rates of >90% were observed for amikacin, tigecycline, colistin, fosfomycin and nitrofurantoin. [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 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]

We studied the presence of extended spectrum beta lactamases (ESBLs) in 44 clinical isolates of Escherichia coli collected from out-patients in two university teaching hospitals in South-Eastern Nigeria. Species identification was performed by standard microbiology methods and re-confirmed by MALDI-TOF technology. Phenotypic characterization of ESBL enzymes was done by double disc synergy test and presence of ESBL genes was determined by specific PCR followed by sequencing. Transfer of plasmid DNA was carried out by transformation using E. coli DH5 as recipient strain. Phenotypic characterization identified all isolates to be ESBL positive. 77% of strains were from urine, 13.6% from vaginal swabs and 9.0% from wound swabs. 63.6% were from female patients, 68% were from outpatients and 95.5% from patients younger than 30 years. All ESBL producers were positive in a PCR for blaCTX-M-1 cluster, in exemplary strains blaCTX-M-15 was found by sequencing. In all strains ISEcp1 was found upstream and ORF477 downstream of blaCTX-M. PCR for blaTEM and blaOXA-1 was positive in 93.1% of strains, whereas blaSHV was not detected, aac(6')-Ib-cr was found in 97.7% of strains. RAPD analysis revealed seven different clonal groups named A through G with the majority of the strains (65.9%) belonging to clone A. Transfer of an ESBL plasmid with co-resistance to gentamicin, kanamycin, tobramycin, doxycycline and trimethropim-sulfamethoxazolewas successful in 19 (43.2%) strains. This study showed a high rate of CTX-M-1 cluster - ESBLs in South-Eastern Nigeria and further confirms the worldwide spread of CTX-M ESBL in clinical isolates. [ABSTRACT FROM AUTHOR]

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]

During the time period from April 2012 to May 2013, 13 newborns (1 - 4 weeks old) and one child in a paediatric hospital ward in Germany were colonized with an extended spectrum betalactamase (ESBL) producing Klebsiella oxytoca (CTX-M-15). A microbiological source-tracking analysis, including human and environmental samples, was carried out to identify the source and transmission pathways of the K. oxytoca clone. In addition, different hygienic intervention methods were evaluated. Isolates of the K. oxytoca strains were detected in the detergent drawer and on the rubber door seal of a domestic washer-extractor machine, used in the same ward to wash laundry of the newborns as well as in two sinks. These strains were typed using PFGE and multi-locus sequence typing (MLST). The environmental findings were compared with those of the human strains and the isolates detected on the clothing. The results showed that the strains were identical using both techniques, (ST201 and pulse-field-gel-electrophoresis [PFGE] type-00531, a clone specific to this hospital and not previously isolated in Germany), emphasizing the washing machine as a reservoir and fomite for the transmission of these multidrug-resistant bacteria (MDR). After the washing machine had been taken out of use, no further colonizations were detected over a following 4-year period. [ABSTRACT FROM AUTHOR]

Recently Gram-negative bacteria co-producing multiple carbapenemases have emerged in different parts of the world. We report the first isolation of an Escherichia coli strain co-producing the carbapenemases NDM-1 and OXA-232. [ABSTRACT FROM AUTHOR]