Extended-spectrum β-lactamases (ESBL) producing bacteria have been increasingly reported in both hospital and community patients. Production of ESBLs is the major mechanism of resistance to oxymino-cephalosporins and aztreonam in Gram-negative bacteria 1,2. Recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M β-lactamases) has been reported. Over 80 CTX-M enzymes have been described so far, which can be grouped into five main subgroups according to amino acid sequence identity (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25) 3 . In some countries, CTX-M β-lactamases are the most prevalent types of ESBLs, for instance in Russia 4, Greece 5 , Spain 6 , Switzerland 7, Japan 8, Taiwan 9, China 10 and Argentina 11 . These enzymes have been identified in countries near Croatia such is Italy 12, Hungary13 and Austria14

The aim of this study was to determine the prevalence and the types of CTX-M β lactamases produced by Klebsiella pneumoniae clinical isolates collected from October 2006 to January 2007 from both community- and hospital –based isolates were included (Figure 1.). 128 ESBL isolates were subjected to further analysis: screening with double disc diffusion test and confirmed by ESBL E test 15 

Ana Budimir

Branka Bedenić

Helena Četković

Ivana Mareković

Smilja Kalenić

Zrinka Bošnjak

English

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947Z. Fiolić et al.: Survey of Klebsiella Pneumoniae Producing CTX-M, Coll. Antropol. 39 (2015) 4: 947–951The aim of this study was to determine the prevalence and the types of CTX-M b lactamases produced by Kleb-siella pneumoniae clinical isolates collected from October 2006 to January 2007 from both community- and hospital – based isolates were included (Figure 1.). 128 ESBL iso-lates were subjected to further analysis: screening with double disc diffusion test and confirmed by ESBL E test15.Methods and ResultsPercentage of ESBL producing isolates was 56 and 44 from the hospitals and community, respectively. CTX-M was detected in 33 (25.8%) isolates. Seventy two strains origi-nated from hospitals and 56 strains from community. Com-munity isolates were obtained from outpatients deparment and according to medical records did not stay in hospitals within previous year. Duplicate isolates were excluded. Only single isolates (one isolate per patient) were analyzed.IntroductionExtended-spectrum b-lactamases (ESBL) producing bacteria have been increasingly reported in both hospital and community patients. Production of ESBLs is the ma-jor mechanism of resistance to oxymino-cephalosporins and aztreonam in Gram-negative bacteria1,2. Recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M b-lactamases) has been reported. Over 80 CTX-M enzymes have been described so far, which can be grouped into five main subgroups according to amino acid sequence identity (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25)3. In some countries, CTX-M b-lactamases are the most prevalent types of ESBLs, for instance in Russia4, Greece5, Spain6, Switzerland7, Japan8, Taiwan9, China10 and Argentina11. These enzymes have been identified in countries near Croatia such is Italy12, Hungary13 and Austria14.Coll. Antropol. 39 (2015) 4: 947–951Short communicationNationwide Survey of Klebsiella Pneumoniae Strains Producing CTX-M Extended-spectrum b-lactamases in CroatiaZlatko Fiolić1, Zrinka Bošnjak2, Branka Bedenić2, Ana Budimir2, Ivana Mareković2, Helena Cetkovic3, Smilja Kalenić21 University Hospital Centre Zagreb, Department of Surgery, Kispaticeva 12, Zagreb 10 000, Croatia2 University Hospital Centre Zagreb, Department of Clinical and Molecular Microbiology, Kispaticeva 12, Zagreb 10 000, Croatia3 »Rudjer Boskovic« Institute, Division of Molecular Biology, Bijenicka 54, Zagreb 10 000 CroatiaA B S T R A C TExtended-spectrum b-lactamases (ESBL) producing bacteria have been increasingly reported in both hospital and community patients. Production of ESBLs is the major mechanism of resistance to oxymino-cephalosporins and aztreonam in Gram-negative bacteria1,2. Recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M b-lactamases) has been reported. Over 80 CTX-M enzymes have been described so far, which can be grouped into five main subgroups according to amino acid sequence identity (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25)3. In some countries, CTX-M b-lactamases are the most prevalent types of ESBLs, for instance in Russia4, Greece5, Spain6, Switzer-land7, Japan8, Taiwan9, China10 and Argentina11. These enzymes have been identified in countries near Croatia such is Italy12, Hungary13 and Austria14. The aim of this study was to determine the prevalence and the types of CTX-M b lac-tamases produced by Klebsiella pneumoniae clinical isolates collected from October 2006 to January 2007 from both community- and hospital-based isolates were included (Figure 1.). 128 ESBL isolates were subjected to further analysis: screening with double disc diffusion test and confirmed by ESBL E test15.Key words: Klebsiella pneumoniae, CTX-M, surveyReceived for publication August 9, 2013948Z. Fiolić et al.: Survey of Klebsiella Pneumoniae Producing CTX-M, Coll. Antropol. 39 (2015) 4: 947–951After determination of minimal inhibitory concentra-tion (MIC), a phenotype consistent with production of CTX-M-type b-lactamase was defined by a cefotaxime MIC ≥ 8-fold higher than ceftazidime MIC, with the MICs of both agents reduced ≥ 8-fold in the presence of 4mg/L clavulanic acid. Strains producing CTX-M of group 1 b-lactamase demonstrated high-level resistance to ceftazi-dime with uniform susceptibility to carbapenems17,18. El-evated MICs of ceftazidime in our strains is consistent with the production of CTX-M-15 b-lactamase.The rates of resistance to non-b-lactam agents, including tetracy-cline, amikacin, ciprofloxacin and cotrimoxazole for CTX-M producing K.pneumoniae from hospitals were 85.7%, 29.4%, 25.3% and 72.1%, from community 87.3%, 25.0%, 27.1% and 75.2% respectively. There was no resistance observed to carbapenems. Based on the conjugation assay as describ ed previously19, seven bla genes could be trans-ferred to transconjugants with the frequency of transfer (10–4–10–6) to recipient strain E.coli, suggesting that they were plasmid mediated. E. coli A15R- resistant to rifam-picin was used as recipient in conjugation experiments.Resistance to chloramphenicol, tetracycline and cotri-moxazole were cotransferred alongside with CTX resis-tance from five, two and two isolates, respectively.Only two isolates cotransfer tetracycline and co-tri-moxazole resistance to E.coli recipient. In other strains, these resistance determinants were not located on trans-ferable plasmids.Isolates with a CTX-M phenotype were screened for bla CTX-M alleles by multiplex PCR with primers MA-1 (5’-SCS-ATG-TGC-AGY-ACC-AGT-AA-3’) and MA-2 (5’-CGC-CRA-TAT-GRT-TGG-TGG-TG-3’). PCR was performed under following conditions: 94° for 3 min, the 35 cycles consisting of 94°C for 30 s, 55°C for 30 s, and 72°C for 45 s each, followed by a final extension at 72°C for 5 min. All 33 isolates were confirmed to produce group 1 CTX-M en-zyme. More hospital isolates were positive [19 out of 33 (57.6%)] compared to community [14 out of 33 (42.4%)].Twenty eight isolates gave a 400 bp PCR product appar-ently linking an IS26 element with blaCTX-M which previ-ously reported in the UK18. The blaCTX-M genes from repre-sentative strains were sequenced using forward (MA-1) and reverse (MA-2) primers and primers for group – 1 CTX-M beta-lactamases in order to amplify the whole open – reading frame of blaCTX-M gene.Sequencing of PCR products of representative strains belonging to major clusters revealed the presence of CTX-M-15-b-lactamases.Isolation of chromosomal DNA was performed as de-scribed by Kaufman et al.20. Bacterial genomic DNA was compared by using PFGE of Xba I- digested fragments as described previously21. The PFGE patterns were compared following the criteria of Tenover21 for bacterial strain typ-ing and analyzed by computer software GelComparII (Ap-plied Maths, Belgium). An optimization of 0.50% and posi-tion tolerance of 3.00% was applied during the comparison of PFGE fingerprinting patterns. Three of the 33 isolates could not be typed using PFGE, due to either difficulties CTX-M positive isolates were more prevalent in hospi-tals (57.6%) than in community (42.4%).The gender ratio of hospital patients was [29.5%:70.5% (female: male)]. The CTX-M positive isolates from hospi-tals were found mostly in urine samples (47.4 %) but also from respiratory tract and wounds. A majority of patients were over 60 years old. Sixty patients were older than 60 years. Twenty one strain (16%) was obtained from respira-tory tract and twelve (9%) from wound swabs. Urinary catheter was not significant risk factor for colonization with ESBL producing organism in our study. Previous antibiotic therapy was found to be significant risk factor for infection or colonization with ESBL producer. Hi square test was used to determine the statistical signifi-cance of the risk factors.Most patients have been hospitalized in urology units or intensive care units. All of CTX-M-15 producers were isolated from urine from elderly men and woman (> 60 years). The gender ratio of community patients was [63.3%:36.7% (female: male)]. The CTX-M positive com-munity isolates originated mostly from urine (93.8%) which is in concordance with previous reports16.Fig. 1. Map of Croatia showing 12 hospitals and 13 Institutes of Public HealthCHC Zagreb-1, GH Sv.Duh Zagreb-2, CH Dubrava Zagreb-3, IPH Zagreb-4, IPH Varaždin-5, IPH Čakovec-6, GH Koprivnica-7, IPH Bjelovar-8, IPH Virovitica-9, GH Pakrac-10,  GH N. Gradiška-11, GH Požega-12, IPH Sl.Brod-13,  GH Našice-14, GH Vukovar-15, IPH Osijek-16, IPH Zabok-17, GH Zabok-18, IPH Pula-19, IPH Gospić-20, IPH Zadar-21,  GH Split-22, IPH Split-23, IPH-Dubrovnik-24 and GH Ogulin-25.CHC-Clinical Hospital Center, GH-General Hospital and IPH-Institute of Public HealthCircles indicate participating laboratory, whereas squares indicate laboratory in which a detected CTX-M positive strains.949Z. Fiolić et al.: Survey of Klebsiella Pneumoniae Producing CTX-M, Coll. Antropol. 39 (2015) 4: 947–951with lyses of the bacterial cell wall or the digestion of the DNA. Typing of the CTX-M producing strains by PFGE revealed 14 similarity clusters (I to XIV) three of which, I, III and V, were classified as major similarity clusters, which have shown specific geographical distribution (Fig-ure 2). Similarity cluster I harboured 5 of the 33 (15.2%) isolates, similarity cluster III harbored 8 of the 33 (24.2%) isolates; two isolates produced identical XbaI PFGE pat-terns, whereas similarity cluster V harboured 3 of the 33 (9.1%) isolates and they all originated from urine. Cluster VII, X and XII harboured two isolates each. Most isolates from both community and hospitals belonged to same large clusters. The remaining isolates showed unique, un-related PFGE profiles and were unlikely to be considered as outbreaks strains.Strains 13, 106, 26, 83 are single isolates which showed distinct PFGE patterns and were not related to other strains.Although several studies addressed the issue of emer-gence of CTX-M – producing Klebsiella pneumoniae world-Fig. 2. Dendrogram of the 33 CTX-M producing Klebsiella pneumoniae.Dendrogram of the 33 CTX-M producing Klebsiella pneumoniae.The seven columns on the right represent isolate code, centre, inpatient/out-patient, clinical ward, PFGE type, clinical specimen and date of collection.950Z. Fiolić et al.: Survey of Klebsiella Pneumoniae Producing CTX-M, Coll. Antropol. 39 (2015) 4: 947–951wide, multicenter epidemiological survey has not been carried out in Croatia until now. It has only been reported that resistance to b-lactam antibiotics is increasing among clinical isolates from some hospitals, expressing the need for further investigation. In the previous report from our country, we have shown presence of group 1 CTX-M pro-ducing isolates in E.coli 22.DiscussionThe present study is first national survey from twelve Croatian hospitals and from the community and offers an insight in to the current prevalence and molecular types of CTX-M- producing K.pneumoniae in both the hospital and community setting. The previous studies have shown CTX-M-3 b-lactamase to be the most prevalent in Croa-tia21. Since blaCTX-M-15 coding region differs from bla CTX-M-3 by only a single amino acid substitution (Asp240 → Gly) it can be hypothesized that bla CTX-M-15 evolved from bla CTX-M-3 by point mutation in the coding region of the gene.A number of risk factors have been identified as linked to the acquisition of community-acquired infection involv-ing CTX-M-positive isolates. These are previous hospital-ization or antibiotic therapy within the previous 3 month, old age (>60 years), male gender and urinary catheteriza-tion4,23,24. We have found two of above-mentioned risk fac-tors in our community patients: old age (>60 years) and male gender, but did not find prior hospital contact, anti-biotic therapy nor presence of urinary and vascular cath-eter in this group of patients.Our data suggest that CTX-M producers are wide-spread in at least three regions of Croatia, possibly as a consequence of the dissemination of major clones between hospitals and community, between regions, and the hori-zontal transfer of plasmids or mobile elements. The pres-ence of insertion sequences most likely facilitated the spread of bla CTX-M genes and enhanced the expression of the genes. The fact that isolates from different hospitals were clonally related points out that there was epidemic spread of related clones between hospitals in the same region. Our data suggest that these clones can spread from the community to the hospital and other way around. Their incidence may be rising with potential risk of trans-fer to other bacterial species, like E.coli, especially in com-munity. This demonstrates the need to monitor both hos-pitalized and community patients for further emergence of transferable resistance to extended-spectrum cephalo-sporins.In conclusion, this study highlights the need to estab-lish an antimicrobial resistance surveillance network for K. pneumoniae and to further monitor the trends and new resistance mechanisms in the hospitals and the commu-nity. The factors responsible for the selection and dissem-ination of the plasmids encoding CTX-M type enzymes and clonal dissemination of strains have to be identified, controlled and prevented to avoid outbreaks.AcknowledgementsThe authors are thankful to Professor Neil Woodford from Health Protection Agency, London, UK for providing the control strains for groups of CTX-M –b-lactamases.We wish to thank Dubravko Sijak and Stjepan Katic for their excellent technical assistance.This research was supported by Research Grant of Ministry of Science, Education and Sport of Republic of Croatia No: 108-1080114-0017.Competing interests: None declared.Ethical approval: Issued by Ethics Committee Univer-sity of Zagreb Medical School, No: 04-1058-2006.R E F E R E N C E S1. BRADFORD PA, Clin Microbiol Rev 14 (2001) 933.- 2. JACOBY GA, MUNOZ-PRICE LS, N Engl J Med 352 (2005) 380. — 3. http://www.lahey.org./Studies/other.asp/table1. — 4. 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EISNER A, FAGAN EJ, FEIERL G, KESSLER HH, MARTH E, LIVERMORE DM, WOODFORD N, Antimicrob Agents Chemother 50 (2006)785. — 15. CLINICAL AND LABORATORY STANDARDS INSTITUTE/NCCLS. Perfomance stan-dards for antimicrobial susceptibility testing. In: 17th informational supple-ment. CLSI/NCCLS document M100-S17(Eds) (Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, 2007). — 16. KIRASTIN P, APISARNTHANARAK A, SAIFON P, LAESRIPA C, KITPHATI R, MUNDY LM, Diagn. Microbiol. Infect. Dis. 58 (2007) 349. — 17. BONNET R, Antimicrob Agents Chemother 48(2004)1. — 18. WOODFORD N, WARD ME, KAUFMANN ME, TURTON J, FAGAN EJ, JAMES D, JOHNSON AP, PIKE R, WARNER M, CHEASTY T, PEARSON A, HARRY S, LEACH JB, LOUGHREY A, LOWES JA, WARREN RE, LIVERMORE DM, J Antimicrob Chemother 54 (2004) 735. — 19. ELWELL LP, FALKOW S, R plasmids and the detection of plasmid-specified genes. In: LORIAN V (Eds) Antibiotics in Laboratory Medicine (1986). — 20. KAUFMANN ME, Pulsed-Field Gel Electrophoresis. In: WOODFORD N, JOHNSON A (Eds) Molecular bacteriology. Protocols and clinical applications (1st ed Humana Press Inc. Totowa, New York, 1998). — 21. TENOVER FC, ARBEIT RD, GOERING RV, MICKELSEN PA, MURRAY BE, PERSING DH, SWAM-INATHAN B, J Clin Microbiol 33 (1995) 2233. — 22. TONKIĆ M, BEDENIĆ B, GOIĆ-BARIŠIĆ I, KATIĆ S, KALONA S, KAUFMANN ME, WOODFORD N, PUNDA-POLIĆ V, Journal of Chemother 19 (2007) 97. — 23. COLODNER R, ROCK W, CHAZAN B, KELLER N, GUY N, SAKRAN W, RAZR, Eur J Clin Microbiol Infect Dis 23 (2004) 163. — 24. BORER A, GILAD J, MENASCHE G, PELED N, RIESENBERG K, SCH-LAEFFER F, Med Sci Monit 8 (2002) CR44.951Z. Fiolić et al.: Survey of Klebsiella Pneumoniae Producing CTX-M, Coll. Antropol. 39 (2015) 4: 947–951Z. Bošnjak e-mail: zbosnjak@kbc-zagreb.hrNACIONALNO ISTRAŽIVANJE SOJEVA BAKTERIJE KLEBSIELLA PNEUMONIAE KOJI PROIZVODE CTX-M PROŠIRENI-SPEKTAR B-LAKTAMAZA U HRVATSKOJS A Ž E T A KProšireni spektar beta-laktamaza (ESBL) kojr proizvode bakterije se povišeno pojavljuje kod bolničkih pacijenta I pacijenata u zajednici. Proizvodnja ESBL-a je glavni mehanizam otpornosti na oxymino-cefalosporine i aztreoname kod gram-negativnih bakterija1,2. Nedavno je zabilježena nova obitelj ESBL-a s dominantnim djelovanjem protiv cefotaksima (CTX-M beta-laktamaze). Više od 80 CTX-M enzima je do sada opisano, koji se mogu grupirati u pet glavnih podsku-pina prema istovjetnosti aminokiselinskog slijeda (CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 i CTX-M-25)3. U nekim zemljama, CTX-M beta-laktamaze su najzastupljenije vrste ESBL-a, primjerice u Rusiji4, Grčkoj5, Španjolskoj6, Švicarskoj7, Japanu8, Tajvanu9, Kini10 i Argentini11. Ovi enzimi su identificirani u zemljama u blizini Hrvatske poput Italije12, Madžarske13 i Austrije14. Cilj ovog istraživanja bio je utvrditi učestalost i vrste CTX-M beta laktamaza koje proizvode klinički izolati bakterjie Klebsiella pneumoniae prikupljeni od listopada 2006. do siječnja 2007. godine od oba u zajednici i bolnica-based izolata su bili uključeni (slika 1.). 128 ESBL izolati bili podvrgnuti daljnjoj analizi: screening s dvostrukim disk difuzije testa i potvrđuje ESBL E test15.

Nationwide Survey of Klebsiella Pneumoniae Strains Producing CTX-M Extended-spectrum b-lactamases in Croatia

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