High diversity of extended-spectrum b-lactamases among clinical isolates of Enterobacteriaceae from Portugal

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1 Journal of Antimicrobial Chemotherapy Advance Access published October 3, 2007 Journal of Antimicrobial Chemotherapy doi: /jac/dkm381 High diversity of extended-spectrum b-lactamases among clinical isolates of Enterobacteriaceae from Portugal Elisabete Machado 1,2,3, Teresa M. Coque 2, Rafael Cantón 2,Ângela Novais 2, João Carlos Sousa 3, Fernando Baquero 2 and Luísa Peixe 1 * on behalf of The Portuguese Resistance Study Group 1 REQUIMTE, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; 2 Servicio de Microbiología, Hospital Universitario Rámon y Cajal, IMSALUD, Madrid, Spain; 3 Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Porto, Portugal Received 20 February 2007; returned 8 May 2007; revised 6 September 2007; accepted 10 September 2007 Objectives: To investigate the occurrence and the diversity of Ambler class A ESBLs among Enterobacteriaceae from different Portuguese clinical settings over a 2 year period ( ). Methods: One hundred and nine extended-spectrum b-lactamase (ESBL)-producing Enterobacteriaceae isolates from five geographically distant health institutions in Portugal were studied. ESBLs were characterized by isoelectric focussing, PCR and further sequencing. Antibiotic susceptibility testing, transfer of resistance genes and clonal diversity were determined by standard procedures. Plasmid relatedness was established by comparison of random amplified polymorphic DNA (RAPD) patterns. Results: ESBLs were identified as TEM (46%), SHV (30%), CTX-M (22%) and GES (2%) types; TEM-24, TEM-52, SHV-12 and CTX-M-15 enzymes being the most frequently found. Inter-hospital dissemination of epidemic strains harbouring the most prevalent ESBLs was detected, including the TEM-24-producing Enterobacter aerogenes European epidemic clone. Conjugative transfer of ESBLs was achieved for 67% of isolates and epidemic plasmids containing specific bla genes were detected (bla CTX-M-15 and bla TEM-24 ). We describe two new ESBLs, SHV-90 (A187T, G238S and E240K) and SHV-91 (P20S and E240K), and a new TEM-type enzyme conferring a phenotype resembling that of a complex mutant TEM b-lactamase, designated as TEM-154 (M69L and R164S). The broad-spectrum b-lactamases SHV-26, SHV-36 and TEM-110 were first observed in our country. Conclusions: We describe a complex ESBL epidemiology in Portugal, including widespread dissemination of known strains and plasmids coding for TEM-24 and CTX-M-15 enzymes as observed in other European countries. Keywords: TEM, SHV, CTX-M-15, clonal dissemination, epidemic plasmids, ESBLs epidemiology Introduction Extended-spectrum b-lactamases (ESBLs) have increasingly been reported worldwide since their first description in 1983, with significant geographical differences in the epidemiology and prevalence of different types. 1 The epidemiology of ESBLs is very complex and involves dissemination of epidemic strains and plasmids, as well as recombinatorial exchange between different hot spots often located in ESBL plasmids (as inti1, 3 0 CS and Tn402-tni module on class 1 integrons, or res site and mer operon in Tn21 derivatives, or a variety of insertion sequences). 2 5 Portugal is one of the European countries with the highest rates of ESBLs, as reported in work conducted by Bouchillon et al. 6 However, available studies on Portuguese ESBL epidemiology at the molecular level only describe the emergence of particular types, specific clonal outbreaks or occurrence of ESBLs in particular hospitals for short periods of time. 3,7,8 To better understand the recent and rapid spread of ESBL-producing organisms in our country, 3,7 we investigated the occurrence and... *Correspondence address. Laboratório de Microbiologia, Faculdade de Farmácia da Universidade do Porto, Rua Aníbal Cunha 164, Porto 4050, Portugal Tel: þ ; Fax: þ ; lpeixe@ff.up.pt... Page 1 of 5 # The Author Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 Machado et al. diversity of Ambler class A ESBLs among Enterobacteriaceae from different Portuguese clinical settings over a 2 year period. Materials and methods This study was designed to achieve ESBL detection and identification at the molecular level rather than ESBL prevalence among Enterobacteriaceae. Two hundred and eighty Enterobacteriaceae isolates exhibiting decreased susceptibility to expanded-spectrum cephalosporins from patients attending three hospitals (n ¼ 169) and two laboratories (n ¼ 111) in the North and Centre of Portugal were collected between October 2002 and November Isolates from the same individual showing identical susceptibility profiles and isolated during the same hospitalization week were not included. Isolates were recovered from urine (n ¼ 72), respiratory samples (n ¼ 25), blood (n ¼ 18), sputum (n ¼ 4) and other well-defined clinical sources. All these isolates were sent to our centre for further studies. Bacterial identification and preliminary susceptibility testing were performed by using the automated WIDER (Fco. Soria Melguizo, Madrid, Spain) or VITEK (biomérieux, Marcy l Étoile, France) systems. Susceptibility testing for non-b-lactam antibiotics was determined by the standard CLSI disc diffusion method. ESBL production was confirmed by the standard double disc synergy test on Mueller Hinton agar plates with and without cloxacillin (250 mg/l) (Sigma-Aldrich, St Quentin-Fallavier, France) and characterization of ESBLs was performed by isoelectric focusing, PCR for bla genes and further sequencing. 3,4 Conjugation experiments were performed as described previously. 4 Clonal relationships among isolates were established by PFGE, using SmaI (Proteus mirabilis) orxbai (all remaining species) as restriction enzymes (Amersham, Life Sciences, Uppsala, Sweden or Takara Bio Inc., Shiga, Japan) and the following electrophoresis conditions: 2 10 s for 30 h, 148C, 6 V/cm 2. 3 Representative isolates of the epidemic TEM-24-producing Enterobacter aerogenes clone recovered from French, Belgium and Spanish hospitals were included for clonal comparations. 1 Plasmid DNA was obtained by the alkaline lysis method for size determination or by using a QIAgen Plasmid Midi kit (QIAGEN, Hilden, Germany) for fingerprinting analysis. 4 Plasmid relatedness was established by comparison of restriction patterns obtained after DNA digestion with EcoRI or PstI, as described previously. 4 Results and discussion The isolates were identified as 133 Escherichia coli, 67 Klebsiella pneumoniae, 17 Serratia marcescens, 16 E. aerogenes, 16 P. mirabilis, 13Klebsiella oxytoca, 7 Enterobacter cloacae, 6 Citrobacter freundii, 3 Morganella morganii, 1 Providencia alcalifaciens and 1 Providencia stuartii. Presence of ESBLs was confirmed in 39% of the isolates resistant to expanded-spectrum cephalosporins (n ¼ 109: 37 E. coli, 46 K. pneumoniae, 7 K. oxytoca, 10 E. aerogenes, 3 E. cloacae and 6 P. mirabilis). ESBLs belonged to TEM (46%, 50/109), SHV (30%, 33/109), CTX-M (22%, 24/109) and GES (2%, 2/109) families and corresponded to 16 distinct types: TEM (TEM-10, -12, -24, -52, -116, -154), SHV (SHV-2, -5, -12, -55, -90, -91), CTX-M (CTX-M-1, -14, -15) and GES-1. A new TEM variant, TEM-154 (containing mutations M69L and R164S and conferring a phenotype resembling that of a complex mutant TEM b-lactamase 9 )(pi¼ 5.3), and two new SHV ESBL variants, SHV-90 (containing mutations A187T, G238S and E240K) ( pi ¼ 8.2) and SHV-91 (containing mutations P20S and E240K) ( pi ¼ 7.6), were first detected during this work. Other enzymes such as TEM-12, SHV-2 and the broad-spectrum b-lactamases TEM-110, SHV-26 or SHV-36 found in two P. mirabilis, six K. pneumoniae (also producing SHV-5) and one K. pneumoniae, respectively, were first observed in our country. The majority of the ESBL-producing isolates carried multiple b-lactamases (bla ESBL and bla OXA-1, bla TEM-1, bla TEM-26 or bla SHV-1 ). K. pneumoniae was the species most commonly found as an ESBL producer (42%, 46/109), although ESBL-producing E. coli was also detected frequently (34%, 37/109). TEM-type ESBLs were observed in all the bacterial species analysed; SHV types were only found among K. pneumoniae (n ¼ 25), K. oxytoca (n ¼ 6) and E. cloacae (n ¼ 2); CTX-M types were only detected among E. coli (n ¼ 22) and K. pneumoniae (n ¼ 2); and GES-1 was identified in K. pneumoniae (n ¼ 1) and E. coli (n ¼ 1). Isolates producing TEM, SHV and CTX-M ESBLs were mostly non-susceptible (intermediate or resistant) to streptomycin (88% versus 97% versus 88%, respectively), spectinomycin (96% versus 73% versus 83%), tobramycin (78% versus 91% versus 88%), kanamycin (78% versus 88% versus 92%) and tetracycline (76% versus 85% versus 92%). TEM- and SHV-producing isolates also showed non-susceptibility patterns to netilmicin (78% versus 79%), sulphonamides (88% versus 97%), trimethoprim (84% versus 94%) and chloramphenicol (74% versus 91%). Resistance rates greatly varied among TEM-, SHV- and CTX-M-producing isolates for ciprofloxacin (64% versus 48% versus 96%, respectively) and gentamicin (18% versus 91% versus 88%, respectively). Non-susceptibility to amikacin was lower than that observed for other aminoglycosides tested among all ESBL types (42% versus 27% versus 33%, respectively). Clonal and/or plasmid dissemination were frequently associated with the most prevalent ESBL types observed (TEM-24, CTX-M-15, SHV-12 and TEM-52). Inter-hospital dissemination of strains of TEM-24-producing E. aerogenes and K. pneumoniae, or CTX-M-15-producing E. coli was detected (Table 1). The E. aerogenes strain recovered from two Portuguese hospitals was clonally related to the epidemic European clone widely disseminated throughout France, Belgium and Spain, 1 and the E. coli producing CTX-M-15 recovered from both hospital and community settings was related to an epidemic CTX-M-15 E. coli clone recently detected. 5 It is of interest to highlight that K. pneumoniae isolates showing an identical PFGE-type (PFGE-type K) contained genes coding for ESBLs differing only in one or two amino acids: SHV-12, SHV-5 or SHV-90 [A187K variation (SHV-5/SHV-90), Q35L variation (SHV-5/SHV-12) and Q35L and A187T variation (SHV-12/SHV-90)], suggesting intra-clonal/intra-plasmid evolution of ESBLs, as described in other studies. 2,5 Transferability of bla genes was observed in 67% of the ESBL-producing isolates: 86% of TEM-producing isolates, 61% of SHV-producing isolates and 42% of CTX-M-producing isolates, whereas GES-producing isolates were not able to transfer. Two epidemic plasmids of 85 or 120 kb carrying bla CTX-M-15, bla OXA-1 and aac(6 0 )-Ib-cr were isolated from E. coli (n ¼ 4) and K. pneumoniae (n ¼ 1) strains from different hospitals, 3 and Page 2 of 5

3 ESBLs in Enterobacteriaceae from Portugal Table 1. Characterization of ESBL-producing Enterobacteriaceae from different Portuguese healthcentres ( ) ESBL Species (no. of isolates/no. of transconjugants) Isolation date (mm/yy) PFGE types found in different hospitals a Non-b-lactam resistance CIH NH CH AL phenotype b,c,d Conjugative plasmids found among different strains (kb) TEM-type (n ¼ 50) TEM-10 E. cloacae (1/1) Feb/03 A (n ¼ 1) Gm, Tb, Sp, Te, Nt, Km TEM-12 K. pneumoniae (1/1) Feb/03 B (n ¼ 1) Sm, Sp, Na TEM-24 E. coli (4/4) Mar/03 Oct/03 A (n ¼ 1), B(n ¼ 2) (Gm), (Tb), (Ak), Sm, (Sp), C(n ¼ 1) (Cp), Na, (Su), Tp, (Te), (Cm), Nt, K. pneumoniae (13/11) Apr/03 Apr/04 A (n ¼ 2), F(n ¼ 1) A(n ¼ 10) (Tb), (Ak), (Sm), (Sp), (Cp), (Na), (Su), (Tp), (Te), (Cm), (Nt), pt24-a (180) pt24-a (180) K. oxytoca (1/1) Mar/04 A (n ¼ 1) Tb, Sp, Su, Tp, Nt, Km ND P. mirabilis (6/6) Feb/03 Jul/04 A (n ¼ 6) (Tb), (Ak), (Sm), Sp, Cp, Na, pt24-a (180) Su, Tp, (Te), Cm, (Nt), E. aerogenes (8/8) Nov/02 Oct/03 A (n ¼ 3) A (n ¼ 5) (Tb), (Ak), (Sm), (Sp), Cp, Na, pt24-a (180) Su, (Tp), (Te), Cm, (Nt), TEM-52 E. coli (9/9) Feb/03 Aug/04 D (n ¼ 2), E(n ¼ 1), F(n ¼ 1), G(n ¼ 1), H(n ¼ 1), I(n ¼ 1), ND (n ¼ 2) (Gm), (Sm), (Sp), (Cp), (Na), (Su), (Tp), (Te), (Cm), K. pneumoniae (1/1) Aug/03 C (n ¼ 1) Tb, Sm, Sp, Cp, Na, Su, Tp, Cm, Nt, Km TEM-116 K. pneumoniae (3/1) Oct/02 Jan/03 D (n ¼ 1) B (n ¼ 1) E(n ¼ 1) (Gm), (Tb), Sm, Sp, Cp, Na, (Su), Tp, (Te), (Cm), (Nt), E. aerogenes (2/0) Oct/03 May/04 A (n ¼ 1) A (n ¼ 1) Gm, Tb, (Sm), Sp, Cp, Na, (Te), (Cm), Nt, Km TEM-154 E. coli (1/0) Apr/04 J (n ¼ 1) Gm, Tb, Sm, Sp, Cp, Na, Su, Tp, Te, Cm, Nt, Km SHV-type (n ¼ 33) SHV-2 K. pneumoniae (2/1) Jan/03 Mar/03 I (n¼2) (Gm), (Tb), (Sm), Sp, Cp, Na, Su, (Tp), (Te), (Cm), (Nt), SHV-5 K. pneumoniae (6/6) Oct/03 Nov/04 K (n ¼ 6) (Gm), (Tb), (Ak), (Sm), (Sp), Cm, Nt, Continued Page 3 of 5

4 Machado et al. Table 1. Continued ESBL Species (no. of isolates/no. of transconjugants) Isolation date (mm/yy) PFGE types found in different hospitals a Non-b-lactam resistance CIH NH CH AL phenotype b,c,d Conjugative plasmids found among different strains (kb) K. oxytoca (1/1) Apr/03 ND (n ¼ 1) Su, Tp, Te SHV-12 K. pneumoniae (6/5) Apr/03 Aug/04 G (n ¼ 1), H(n ¼ 1), J(n ¼ 1), K(n ¼ 1), L(n ¼ 1), M(n ¼ 1) (Gm), Tb, (Ak), (Sm), (Sp), (Cm), (Nt), K. oxytoca (5/0) Jan/03 Aug/04 B (n ¼ 4) C (n ¼ 1) Gm, Tb, (Ak), Sm, (Sp), Cp, Na, Su, Tp, (Te), Cm, (Nt), E. cloacae (2/2) Jan/03 B (n ¼ 2) (Gm), (Tb), Ak, Sm, Sp, Na, (Su), Tp, Te, Cm, Nt, Km SHV-55 K. pneumoniae (4/0) Jan/03 Jul/03 N (n ¼ 4) Gm, (Tb), Sm, Sp, Su, (Tp), Te, Cm, (Nt), SHV-90 K. pneumoniae (6/5) Nov/03 Aug/04 K (n ¼ 6) (Gm), Tb, (Ak), (Sm), (Sp), (Cm), Nt, SHV-91 K. pneumoniae (1/0) Jul/03 I (n ¼ 1) Tb, Sm, Sp, Nt, Km variable size ND CTX-M-type (n ¼ 24) CTX-M-1 E. coli (1/1) Sep/03 ND (n ¼ 1) Sm, Sp, Su, Tp, Te CTX-M-14 E. coli (4/4) Aug/03 Jun/04 K (n ¼ 4) (Gm), (Tb), Sm, Sp, Cp, Na, (Su), (Tp), (Te), Km CTX-M-15 E. coli (17/4) Jan/03 Oct/04 M (n ¼ 7), M(n ¼ 7) M (n ¼ 1) (Gm), Tb, (Ak), (Sm), (Sp), Cp, N(n ¼ 1), Na, (Su), (Te), (Cm), (Nt), O(n ¼ 1) K. pneumoniae (2/1) Oct/03 Sep/04 O (n ¼ 2) (Gm), (Tb), Sm, Sp, Cp, Na, Su, Tp, (Te), (Cm), GES-type (n ¼ 2) E. coli (1/0) Jan/04 L (n ¼ 1) Tb, Ak, Sm, Sp, Su, Tp, Te, Nt, Km K. pneumoniae (1/0) Sep/04 P (n ¼ 1) Gm, Tb, Sm, Sp, Cp, Na, Su, Tp, Te, Cm, Nt, Km pc15-b (120, n ¼ 1) pc15-a (85, n ¼ 3) pc15-a (85, n ¼ 1) a CIH, Hospital de São Teotónio, located in Viseu, in the Centre Interior region of Portugal; NH, Hospital Geral de Santo António, located in Oporto, in the North region of Portugal; CH, Hospitais Universitários de Coimbra, located in the Centre region of Portugal; AL, Ambulatory Laboratories, located in the North region of Portugal. b Gm, gentamicin; Tb, tobramycin; Ak, amikacin; Sm, streptomycin; Sp, spectinomycin; Nt, netilmicin; Km, kanamycin; Su, sulphonamides; Tp, trimethoprim; Te, tetracycline; Cm, chloramphenicol; Cp, ciprofloxacin; Na, nalidixic acid. c Transfer of a resistance phenotype is indicated by underlining. d Variable presence or transferability of resistance phenotype is indicated by parentheses. Page 4 of 5

5 ESBLs in Enterobacteriaceae from Portugal one plasmid of 180 kb containing bla TEM-24b was recovered from E. coli, K. pneumoniae, E. aerogenes and P. mirabilis (Table 1). All three plasmids shared RFLP profiles identical to those recently recovered in Spain, 5,10 reflecting international dissemination of these elements. All TEM-52-producing isolates showed plasmid bands of 100 kb (Table 1). In summary, we described a complex ESBL epidemiology in Portugal including dissemination of strains and plasmids coding for TEM-24 and CTX-M-15 enzymes, probably imported from other European countries. We also detected ESBLs already spread in Europe, intra-plasmid evolution of bla genes resulting in new ESBL variants and the frequent recovery of CTX-M variants in Portugal. 3,7 The present epidemiological information anticipates a shift in ESBL distribution as has happened in other regions and will be useful in the design of interventions to control the dissemination of ESBL-producing organisms and/or ESBLs in our country. Acknowledgements Contributing members of the Portuguese Resistance Study Group are: Helena Ramos, Hospital Geral de Santo António, Porto, Portugal; Graça Ribeiro and Clementina Vital, Hospitais Universitários de Coimbra, Coimbra, Portugal; João Fernandes Ribeiro and Francisco Freitas, Hospital de São Teotónio; and Sónia Magalhães, LabFafe, Fafe, Portugal. Funding E. M. was supported by a fellowship from Fundação para a Ciência e Tecnologia de Portugal (SFRH/BD/11304/2002). This work was partially supported by research grants from the Ministry of Science and Technology of Spain (SAF ), and from the European Commission (LSHM-CT ). Transparency declarations R. C. received grants from Wyeth and consultancies from Novartis. Other authors: none to declare. References 1. Cantón R, Novais A, Valverde A et al. Prevalence and spread of extended-spectrum b-lactamase-producing Enterobacteriaceae in Europe. Clin Microbiol Infect 2007; in press. 2. Baraniak A, Fiett J, Mrowka A et al. Evolution of TEM-type extended-spectrum b-lactamases in clinical Enterobacteriaceae strains in Poland. Antimicrob Agents Chemother 2005; 49: Machado E, Coque TM, Cantón Ret al. Dissemination in Portugal of CTX-M-15-, OXA-1-, and TEM-1-producing Enterobacteriaceae strains containing the aac(6 0 )-Ib-cr gene, which encodes an aminoglycoside- and fluoroquinolone-modifying enzyme. Antimicrob Agents Chemother 2006; 50: Novais A, Cantón R, Valverde A et al. Dissemination and persistence of bla CTX-M-9 are linked to class 1 integrons containing CR1 associated with defective transposon derivatives from Tn402 located in early antibiotic resistance plasmids of IncHI2, IncP1-a, and IncF1 groups. Antimicrob Agents Chemother 2006; 50: Novais A, Cantón R, Moreira R et al. Emergence and dissemination of Enterobacteriaceae isolates producing CTX-M-1-like enzymes in Spain are associated with IncFII (CTX-M-15) and broad-host-range (CTX-M-1, -3, -32) plasmids. Antimicrob Agents Chemother 2007; 51: Bouchillon SK, Johnson BM, Hoban DJ et al. Determining incidence of extended-spectrum b-lactamases producing Enterobacteriaceae, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus in 38 centres from 17 countries: the PEARLS study Int J Antimicrob Agents 2004; 24: Mendonça N, Leitão J, Manageiro V et al. Spread of extendedspectrum b-lactamase CTX-M-producing Escherichia coli clinical isolates in community and nosocomial environments in Portugal. Antimicrob Agents Chemother 2007; 51: Brízio A, Vasco S, Gonçalves AR et al. Survey of extendedspectrum b-lactamases in Escherichia coli isolates from a Portuguese hospital and characterisation of a novel class 1 integron (In60A) carrying the bla CTX-M-9 gene. Int J Antimicrob Agents 2006; 28: Robin F, Delmas J, Schweitzer C et al. Evolution of TEM-type enzymes: biochemical and genetic characterization of two new complex mutant TEM enzymes, TEM-151 and TEM-152, from a single patient. Antimicrob Agents Chemother 2007; 51: Novais A, Cantón R, MachadoEet al. International dissemination of extended-spectrum b-lactamase TEM-24 among Enterobacteriaceae species is caused by spread of both epidemic IncA/C2 plasmid and strains. Clin Microbiol Infect 2007; 13 Suppl 1: Abstract P1988. Page 5 of 5