Imported PER-1 producing Pseudomonas aeruginosa, PER-1 producing Acinetobacter baumanii and VIM-2-producing Pseudomonas aeruginosa strains in Hungary

Introduction Pseudomonas aeruginosa and Acinetobacter baumanii are important nosocomial pathogens with wide intrinsic resistance. However, due to the dissemination of the acquired resistance mechanisms, such as extended-spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) production, multidrug resistant strains have been isolated more often. Case presentation We report a case of a Hungarian tourist, who was initially hospitalized in Egypt and later transferred to Hungary. On the day of admission PER-1-producing P. aeruginosa, PER-1 producing A. baumannii, SHV-5-producing Klebsiella pneumoniae and VIM-2-producing P. aeruginosa isolates were subcultured from the patient's samples in Hungary. Comparing the pulsed-field gel electrophoresis (PFGE) patterns of the P. aeruginosa strains from the patient to the P. aeruginosa strains occurring in this hospital, we can state that the PER-1-producing P. aeruginosa and VIM-2-producing P. aeruginosa had external origin. Conclusion This is the first report of PER-1-producing P. aeruginosa,and PER-1-producing A. baumanii strains in Hungary. This case highlights the importance of spreading of the beta-lactamase-mediated resistance mechanisms between countries and continents, showing the importance of careful screening and the isolation of patients arriving from a different country.


Introduction
Pseudomonas aeruginosa and Acinetobacter baumanii are very important nosocomial pathogens mainly in intensive care units, being responsible for various types of infections with more and more limited therapeutic options [1].
The P. aeruginosa have significant intrinsic resistance to antibiotics [2]. Therefore, the antipseudomonal beta-lactams such as ticarcillin, piperacillin, ceftazidime, cefepime, aztreonam, and the carbapenems have an important therapeutic value. Three mechanisms of betalactam resistance are predominant: production of betalactamases, loss of outer membrane proteins and up-regulation of efflux pumps. Most strains of P. aeruginosa which are resistant to third-generation cephalosporins produce a chromosomally mediated molecular class C beta-lactamase, the AmpC enzyme [2]. However, acquired beta-lactamases encoded by mobile genetic elements are important resistance mechanism in P. aeruginosa and in Acinetobacter spp. as well. Acquired resistance to betalactams can lead to therapeutic failure, especially when it is associated with resistance to other classes of drugs, such as aminoglycosides and fluoroquinolones. Among these enzymes acquired, PER (Pseudomonas extended resistance), a class A extended-spectrum beta-lactamase (ESBL), occurring less frequently has clinical importance by conferring resistance to oxyimino beta-lactams [3]. Poor outcome as a result of infection caused by PER-1 producers has been reported [4]. The also acquired metallo betalactamases (MBLs) of molecular class B can cause resistance to all beta-lactams except monobactam. The genes of MBLs are usually part of class I integron together with gene cassettes encoding resistance genes. Several types of MBL enzymes have been identified in P. aeruginosa among which the VIM-type enzymes appear to be the most prevalent [5].
Here we report the first detection of PER-producing P. aeruginosa, A. baumannii isolates and VIM-producing P. aeruginosa in Hungary from a patient, who was hospitalized in Egypt and transferred to Hungary. This work illustrates the dissemination of bacteria carrying PER-type ESBL and VIM-type MBL enzymes.

Case presentation
In April, 2006 a 53-year-old Hungarian tourist was involved in a severe terror-attack in Egypt. He was initially hospitalized because of his burn, mechanical injuries and sepsis syndrome in Egypt. Five days later he was transferred in comatose, hypoxic and hypothermic condition to the Burn Unit of State Health Center, Budapest, Hungary. On the day of admission, bacterial cultures taken from burn wound. Based on the different colony morphology and antibiotic susceptibility patterns three different P. aeruginosa strains -an ESBL-producing P. aeruginosa (PA1), an imipenem-resistant P. aeruginosa (PA2), an MBL-producing P. aeruignosa (PA3), were observed and furthermore ESBL-producing Klebsiella pneumoniae (ESBL-KP), methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis (EF) were isolated. Next day ESBL-producing A. baumanii (ESBL-AB) and PA2 were cultured from the patient's canul. During his hospitalization from his nose PA1, ESBL-AB, ESBL-KP, MRSA, from his throat PA1, ESBL-AB, ESBL-KP, from his trachea PA3, ESBL-AB, ESBL.KP,-MRSA and from his wound PA1, PA3, ESBL-AB, ESBL-KP, MRSA and EF were isolated. Blood cultures were taken nine occasions and PA1, ESBL-KP, MRSA and EF were subcultured. The patient received adequate supportive treatment and empirically cefepime, subsequently meropenem and vancomycin were administered intravenously at high dosage. The aminoglycosides were synergy resistant. On the 8 th day of the hospitalization in Hungary the patient died.
The isolates were identified by VITEK 2 (BioMérieux, Marcy l'Etoile, France). On a routine antibiogram synergy was observed between amoxycillin/clavulanic acid and cefepime or ceftazidime disks (Oxoid, Basingstoke, Hampshire, United Kingdom) in K. pneumoniae and in A. baumanii strains but no synergy was observed in P. aeruginosa strains. The minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined by Etest (AB Biodisk, Solna, Sweden) and by using the interpretative criteria of the Clinical and Laboratory Standards Institute [6]. Pulsed-field gel electrophoresis (PFGE) of the SpeI-digested (New England Biolab, Beverly, MA) genomic DNA of P. aeruginosa was performed and analyzed as described previously [7]. Isoelectric focusing (IEF) was performed, as described previously [8]. In the PCR assays primers specific to bla TEM [9], bla SHV [9], bla PER [10], bla OXAs [11], bla IMP , bla VIM and bla classI genes [12] were used. The nucleotide sequences were determined using the ABI 3100 Genetic Analyzers (Applied Biosystems, Foster City, CA). Sequences were analyzed using NCBI BLAST search.
Three different antibiotic susceptibility patterns were observed in P. aeruginosa strains isolated from the patient on the day of admission ( The PCR and sequencing results are summarized in Table  2. TEM-1 genes were identified in A. baumanii and K. pneumoniae. OXA genes were recognized in PA1 and PA3 strains. PER-1 genes were found in PA1 and A. baumanii isolates and SHV-5 was found in K. pneumoniae. Only the PA3 isolate harbored the VIM-2 gene. The OXA-10 and VIM-2 genes were located on the same class I integrons, as the sequencing result of class I integron showed. According to IEF test results, all the isolates contained βlactamases. Isoelectric focusing confirmed the expression of PER-1 (pI 5.  Table 2).
The three P. aeruginosa isolates, PA1, PA2 and PA3, showed three different PFGE patterns (data not presented) suggesting that they belonged to different clones ( Table 2). Comparing these PFGE patterns to six other P. aeruginosa strains occurring in this Hungarian hospital during the same period, no similarity was found suggesting the external origin of PA1, PA2 and PA3.

Discussion
Based on the antibiotic susceptibilities, IEF and the sequencing result we can state that the PA1 strain and the A. baumanii strain produced PER-1 ESBL enzyme. The A. baumanii strain also produced TEM-1 and the PA1 strain OXA-10 broad-spectrum β-lactamase. The PA2 strain produced only chromosomal AmpC cephalosporinase as suggesting that loss of OprD was responsible for the imipenem resistance. The PA3 strain produced the OXA-1 broad-spectrum β-lactamase and the VIM-2 MBL enzyme. The K. pneumoniae strain produced SHV-5 and TEM-1 enzyme.
According to the type of ESBL enzyme produced by different bacteria species SHV-TEM-and CTX-M type ESBL-producing Klebsiella spp. and Escherichia coli strains have been isolated in Hungary so far [13,14] -while in Egypt the ESBL phenotype has been observed in K. pneumoniae and CTX-M-type ESBL in E. coli strains [15,16]. The PER-1 beta-lactamase has been considered to be significant only in Turkey for years [17]. However, the PER-1 beta-lactamase found mainly in P. aeruginosa has been detected in many countries such as Turkey, France, Belgium, Spain, Italy, Poland, Romania, Japan and South Korea until now [10,[18][19][20][21][22][23][24][25]. The PER-1 production in Acinetobacter spp. was observed more often in Turkey and in Korea [4,17,26,27]. According to our knowledge this is the first report of PER-1-producing P. aeruginosa and PER-1-producing A. baumanii in Hungary and since that other strains of PER-1 producing P. aeruginosa have been isolated [28].
Several types of MBL enzymes -IMP-type, VIM-type, SPM-1, GIM-1, SIM-1 -have been identified in P. aeruginosa [5]. The VIM enzymes are the most common in Europe  [29]. The VIM-2 enzyme detected in this case has been previously found in several species worldwide and it seems to be the most prevalent allelic form [30][31][32][33][34][35]. In Hungary VIM-producing P. aeruginosa strains have been isolated but in Egypt MBL-producing strains have not been reported until now [36,37].
Furthermore the result of the PFGE analysis of the PA1, PA2 and PA3 confirms, that the three P. aeruginosa strains -the PER-1 producing P. aeruginosa (PA1), the OprD-loss P. aeruginosa (PA2) and VIM-2-producing P. aeruginosa (PA3) strains -had external origin and could be transferred from Egypt to Hungary. The PER-1 producing P. aeruginosa and A. baumanii strains disappeared from the hospital, no more infections have been detected with these strains since then.
The emergence and subsequently spread of PER-producing and MBL-producing strains are alarming. Supposing, that these resistance mechanisms might also exist in other countries the dissemination of PER-enzyme could be more prevalent over the world, particularly due to the unsolved problem of routine screening for ESBL-production in P. aeruginosa strains. The CLSI recommendation for screening the ESBL-production in different bacteria species is uncomplete, exist just for Klebsiella spp. and Escherichia coli strains [6].
In conclusion, this work confirms the emergence of PER-1-producing P. aeruginosa and A. baumannii isolate, VIM-2 producing P. aeruginosa strains in Hungary. Furthermore it illustrates the possibility of the inter-country and the inter-continent spread of the beta-lactamase-mediated resistance mechanisms. Our study features the intercontinental spread of antimicrobial resistance, showing the importance of careful screening and the isolation patients arriving from a different country.