Emergence of Klebsiella pneumoniae with an AmpC(DHA-1) and blaSHV-11 in a Belgian hospital. Timothy Vanwynsberghe1, Katia Verhamme2, Marijke Raymaekers3, Reinoud Cartuyvels3, An Boel1, Hans De Beenhouwer1  1Clinical Microbiology, OLV Hospital, Aalst; 2Infection control and epidemiology, OLV Hospital, Aalst; 3Clinical Microbiology, Virga-Jesse Hospital, Hasselt, Belgium P671 Objectives Results (2) Results (3) From August 2006 to February 2007 we observed 28 unrelated clinical isolates of Klebsiella pneumoniae expressing an unusual antibiotic susceptibility pattern. Characterisation of these strains was performed in combination with an epidemiological investigation. These 28 strains of Klebsiella pneumoniae shared the same phenotype (Table 1, Figure 1). cefoxitin-resistant (Ø = 6mm). Amoxicillin-clavulanic acid (AMC) induced resistance for cefotaxime and aztreonam. Scattered colonies in the inhibition zones of ceftazidime, ceftriaxone, aztreonam, cefotaxime, and AMC, but not for cefepime. The cefepime inhibition zone showed a small enhancement (phantom zone) near AMC. PCR: presence of the blaSHV-11 gene, which is not classified as an ESBL-producing gene. presence of a DHA-1-type AmpC β-lactamase. PFGE: 3 types (Figure 3). All strains are ‘closely’ or ‘possibly related’ to the outbreak according to the Tenover criteria2. Methods Routine antibiotic susceptibility testing is performed by Phoenix® (BD). Isolates flagged as “possible extended-spectrum β-lactamases (ESBL) positive” were investigated with our modified double-disk synergy test. Modified double-disk synergy test: double disk testing according to Jarlier with amoxicillin-clavulanic acid, ceftazidime, ceftriaxone, supplemented with aztreonam cefepime and cefotaxime. An extra cefoxitin disk was added. AmpC disk test: presented by Black et al1, was performed in order to verify the presence of an AmpC β-lactamase Polymerase chain reaction (PCR): The clinical isolates were examined for the presence of blaTEM, blaTEM-24, blaSHV and blaCTX-M, using consensus primers for the different genes, and for the presence of an AmpC β-lactamase. DNA macrorestriction and PFGE analysis: DNA macrorestriction using the XBA1 restriction enzyme (Genepath group 6 reagent kits, Bio-Rad, Hercules, USA) was performed according to the manufacturer’s instructions. PFGE restriction patterns were obtained on a GenePath system (Bio-Rad, Hercules, USA) using following electrophoresis conditions: 6 V/cm for 17h with pulse times from 5.3 to 49.9s (non-linear ramp). The PFGE patterns were analysed and clustered into dendrograms with the Fingerprinting II software (Bio-Rad, Hercules, USA). Interpretation was done using the criteria of Tenover et al2. 2 1 4 5 3 Figure 1. Phenotype of one of the isolates on our modified double-disk synergy test (1: ceftazidime, 2: ceftriaxone, 3: aztreonam, 4: amoxicillin-clavulanic acid, 5: cefotaxime, 6: cefepime, and 7: cefoxitin). 7 6 Possible causes of cefoxitin-resistance: 1. porin loss 2. AmpC β-lactamase production 3. carbapenemase production. Figure 3. PFGE analysis. Strains of Klebsiella pneumoniae containing SHV-11 together with an AmpC β-lactamase are very rare and have been described mainly in Taiwan3. The literature reveals that the prevalence of AmpC β-lactamases in K. pneumoniae is rising and strains producing these enzymes have been reported from every continent, but particularly from East-Asia4.  In these strains the AmpC disk test was positive pointing to the presence of an AmpC β-lactamase (Figure 2). Conclusions 1 1 Figure 2. AmpC disk test: indentation towards cefoxitin points to the presence of an AmpC β-lactamase. The test strain is applied on a paper disk containing TRIS-EDTA and is placed near a cefoxitin (FOX) disk. (1: isolate nr 67224, 2: negative control) A Klebsiella pneumoniae harbouring DHA-1 AmpC β-lactamase together with blaSHV-11 is now being reported for the first time in an outbreak in Europe5. In order to screen for AmpC β-lactamases, applying a cefoxitin disk on a double-disk synergy test is useful. Infection with this K. pneumoniae was related with stay in the ICU and/or a history of urological conditions/interventions. 2 Epidemiology: 1) A retrospective analysis of unique clinical isolates of K. pneumoniae with the same antibiotic-profile, performed in our hospital: 8/264 in 2004, 9/287 in 2005, 10/130 in the first semester of 2006. Unfortunately, none of these isolates were available for further testing. Results (1) References   AMC ATM CAZ CRO CTX FEP FOX Ref.nr. MIC Ø 67224 >16 11 a <=2 23 a 20 a 26 / 23ab 29 c 6 Acknowledgement: We are grateful to Professor G.A. Jacoby for typing the AmpC β-lactamase by PCR. Black JA, Moland ES, Thomson KS. AmpC Disk Test for Detection of Plasmid-Mediated AmpC β-Lactamases in Enterobacteriaceae Lacking Chromosomal AmpC β-Lactamases. J Clin Microbiol 2005; 43: 3110-3. Tenover FC., Arbeit RD, Mickelsen PA, Murray BE, Persing DH, Swaminathan B. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: 2233-2239. Yan JJ, Ko WC, Yung JC, Chuang CL, Wu JJ. Emergence of Klebsiella pneumoniae isolates producing inducible DHA-1 β-lactamase in a university hospital in Taiwan. J Clin Microbiol 2002; 40: 3121-6. Song W, Kim JS, Kim HS, Yong D, Jeong SH, Park MJ, et al. Increasing trend in the prevalence of plasmid-mediated AmpC β-lactamases in Enterobacteriaceae lacking chromosomal AmpC gene at a Korean university hospital from 2002 to 2004. Diagn Microbiol Infect Dis 2006; 55: 219-24. Vanwynsberghe T, Verhamme K, Raymaekers M, Cartuyvels R, Boel A, De Beenhouwer H. Outbreak of Klebsiella pneumoniae strain harbouring an AmpC (DHA-1) and a blaSHV-11 in a Belgian hospital, August-December 2006. Euro Surveill. 2007 Feb 1; 12(2): E070201.3. 2) 28 isolates from: respiratory samples, urine, abdominal fluid, blood cultures. 3) 28 Patients: Ratio of men to women was 3:1 Review of the medical charts: infection related with stay in intensive care unit history of urological conditions and/or interventions Table 1. The characteristics of the first strain discovered. MIC in µg/ml, inhibition zone (Ø) in mm. AMC: amoxicillin-clavulanic acid, ATM: aztreonam, CAZ: ceftazidime, CRO: ceftriaxone, CTX: cefotaxime, FEP: cefepime, FOX: cefoxitin. a:scattered colonies in the inhibition zone, b: flattening of the inhibition zone towards AMC, c: phantom zone present towards AMC.