the emergence and spread of carbapenemase - producing enterobacteriaceae is an increasing problem of global dimensions .
originally , metallo--lactamases ( mbls ) were associated with resistance in gram - negative non - fermenters , but they have become increasingly important regarding carbapenem resistance in enterobacteriaceae .
mbls confer resistance to almost all -lactam antibiotics and are not inactivated by -lactamase inhibitors , hence limiting treatment options in the individual patient and presenting a major challenge for infection control within the hospital setting 1 . to date , the occurrence of carbapenem - resistant enterobacteriaceae in germany is still a rare event ; however , outbreaks involving kpc and vim-1 carrying klebsiella pneumoniae isolates have been described 2,3 . in the year 2012 , vim-1 was the most prevalent mbl detected in enterobacteriaceae in germany , followed by ndm-1 and gim-1 2 .
imp type mbls were first identified in pseudomonas aeruginosa in japan 4 and have since been reported predominantly from asia 5 . with the exception of italy ,
imp - type enzymes have rarely been reported from other countries in europe 1,5,6 . here , we report the isolation of citrobacter freundii harbouring mbl imp-8 from three patients between march 2012 and march 2013 .
all three patients had underlying haematological conditions ( acute myeloid leukaemia n  =  2 and myelodysplastic syndrome
they were screened from rectal swabs for colonization with multidrug resistant gram - negative bacteria on a weekly routine schedule .
identification of the isolates was performed using matrix - assisted laser desorption / ionization time of flight mass spectrometry ( maldi tof - ms ) ( axima assurance , biomrieux sa , marcy letoile , france ; saramis database version 4.09 ) and the vitek 2 identification system ( biomrieux sa ) .
antimicrobial susceptibility testing was initially performed with the vitek 2 system ( biomrieux sa ) and confirmed by etest ( biomrieux sa ) .
results were interpreted according to the european committee on antimicrobial susceptibility testing ( eucast ) guidelines ( http://www.eucast.org/fileadmin/src/media/pdfs/eucast_files/breakpoint_tables/breakpoint_table_v_3.1.pdf ) .
the detection of extended - spectrum -lactamase genes blactx - m , blatem , blashv , 7 and carbapenemase genes blaoxa-48 
 8 , blakpc 
 9 , blandm 
 10 , blaimp and blavim 
 11 was performed as described previously .
sequencing of the imp genes was performed using class 1 integron primer 3cs ( 5-aag cag act tga cct ga-3 ) in combination with primer imp - a , and 5cs ( 5-ggc atc caa gca gca ag-3 ) in combination with primer imp - b 11 .
sequence identification was determined by comparison with the sequences available in genbank ( http://www.ncbi.nih.gov/blast ) and with the reference sequences of the lahey database ( http://www.lahey.org/studies ) .
alignment was performed using bioedit version 7.1.11 ( ibis biosciences , carlsbad , ca , usa ) .
genomic fingerprinting of the isolates was done by the enterobacterial repetitive intergenic consensus ( eric ) method using the eric2 primer as described previously 12 .
plasmids were extracted using the qiagen large construct kit ( qiagen , hilden , germany ) and digested using ecori and bamhi to allow for size estimation of the plasmids .
briefly , dna was blotted onto positively charged nylon membranes ( roche biochemicals , basel , switzerland ) in denaturation solution ( 3  m nacl , 0.4  m naoh ) by capillary transfer .
high stringency hybridization was performed in accordance with the instructions given by the manufacturer of the digoxigenin labelling and detection kit ( roche biochemicals ) .
digoxigenin - labelled dna probes were generated with a digoxigenin - labelling pcr kit as described in the manufacturer s instructions ( roche biochemicals ) using the oligonucleotides imp - a and imp - b .
l for the isolate of patients two and three , whereas meropenem mic for patient one was intermediate with an mic of 8 
molecular detection of esbl genes blactx - m , blatem , blashv and carbapenemase genes blaoxa-48 , blakpc , blandm and blavim was negative in all three isolates .
the imp pcr gave a positive result and subsequent determination of the nucleotide sequence revealed an mbl of the imp-8 type .
the sequence was compared with the reference sequence ( genbank accession number af322577 ) 13 .
the blaimp-8 gene detected in the three isolates harboured a non - coding point mutation at position 18 ( tc ) as shown in fig.1 .
additionally , an imp-8 carrying plasmid of approximately 28  500  bp could be isolated from all c.  freundii strains .
genomic fingerprinting by the eric method revealed indistinguishable pcr patterns in all isolates ( data not shown ) .
characteristics of imp-8 carrying citrobacter freundii isolates from rectal swabs of three hospitalized patients in germanya i , intermediate ; r , resistant ; s , susceptible ; an , amikacin ; atm , aztreonam ; caz , ceftazidime ; cip , ciprofloxacin ; col , colistin ; ctx , cefotaxime ; cxm , cefuroxime ; ert , ertapenem ; mem , meropenem ; tig , tigecycline ; tzp , piperacillin - tazobactam . interpretation according to the eucast clinical breakpoints ( http://www.eucast.org/fileadmin/src/media/pdfs/eucast_files/breakpoint_tables/breakpoint_table_v_3.1.pdf ) . sequence of the blaimp-8 gene of three citrobacter freundii strains isolated from hospitalized patients in germany . compared with the sequence of the imp-8 reference strain ( genbank accession number af322577 13 ) ,
a non - coding point mutation at position 18 was identified in the imp-8 from the three citrobacter freundii strains ( grey shaded ) .
imp-8 is very uncommon in europe , only once reported from portugal in a pseudomonas mendocina strain 6,14 . in contrast , imp-8 is frequently encountered in asia , especially in taiwan , where imp-8-producing enterobacteriaceae are involved in serious infections .
. reported on a case series of 37 patients with bloodstream infections caused by a large variety of imp-8-producing enterobacterial species including escherichia coli , k.  pneumoniae , enterobacter cloacae and c.  freundii 
 15 .
furthermore , aggravating the issue , phenotypic screening for imp-8-positive enterobacteriaceae is extremely difficult because of the lack of distinctive phenotypes .
investigation of 95 imp-8-positive enterobacteriaceae revealed susceptibility to ertapenem in 21% and to meropenem in 45% , whereas phenotypic combined disk tests using edta and phenylboronic acid were positive in only 40% of the isolates 16 .
these observations from taiwan suggest that imp-8 is capable of spreading between enterobacterial species , causing serious problems in terms of infection control measures and limiting therapeutic options in critically ill patients .
the role of faecal carriage of mbl - producing enterobacteriaceae remains unclear and has not been investigated in a large - scale epidemiological study .
faecal carriage of one c.  freundii vim-1 has been reported from spain in an outpatient , who was also colonized with two different vim-1-carrying k.  pneumoniae isolates 17 . in an italian hospital
, eight vim-1-carrying c.  freundii strains were isolated from rectal swab samples during active screening following the detection of a k.  pneumoniae carbapenemase ( kpc)-positive patient 18 .
none of our three patients became infected by the imp-8 c.  freundii strains , but nevertheless it is alarming that imp-8 mbl circulates in the gut flora of patients at high risk of nosocomial infections .
even more worrisome is the fact , that the imp-8 gene is located on a plasmid , which might facilitate the transfer of the resistance gene within enterobacterial species .
our findings emphasize the importance of establishing screening schemes and laboratory diagnostic algorithms to ensure the implementation of efficient infection control measures and therapeutic strategies , not only in high - prevalence countries , but also in countries with a low incidence of mbl producing enterobacteriaceae .
 metallo--lactamases ( mbls ) in enterobacteriaceae are an increasing problem worldwide . 
 this report describes the isolation of citrobacter freundii carrying imp-8 mbl from three patients during the period from march 2012 until march 2013 in germany . the blaimp-8 enzyme is predominantly found in asia , where imp-8 has spread to various enterobacterial species causing serious infections . to our best knowledge , this is the first report of blaimp-8 habouring enterobacteriaceae in europe . 
Introduction
Laboratory Analysis
Results
Conclusion
Conflict of Interest