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Emergence of SHV-12 extended spectrum beta-lactamase among clinical isolates of Enterobacter cloacae in Tunisia
Microbial Pathogenesis 53 (2012) 64e65
Contents lists available at SciVerse ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Emergence of SHV-12 extended spectrum beta-lactamase among clinical isolates of Enterobacter cloacae in Tunisia Hella Lahlaoui a, *, Ben Haj Khalifa Anis b, Kheder Mohamed b, Ben Moussa Mohamed a a b
Laboratoire de Microbiologie, Hôpital Militaire de Tunis, 1089 Monfleury, Tunisia Laboratoire de Microbiologie, Hôpital Tahar Sfar de Mahdia, 5100 Mahdia, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2011 Received in revised form 10 April 2012 Accepted 13 April 2012 Available online 21 April 2012
A collection of seven multidrug-resistant clinical isolates of Enterobacter cloacae with reduced susceptibility to ceftazidime and cefepime recovered from 2009 to 2010 at the University Hospital of Mahdia, Tunisia, was analysed. PCR analysis and sequencing demonstrated that all study isolates harbored SHV12 b-lactamase that was transferred by conjugation. Characterization of the regions surrounding the blaSHV-12 showed that this gene was flanked by two IS26 elements. pulsed-field gel electrophoresis (PFGE) revealed differents profiles indicating that the study isolates were not clonally related. Diffusion of E. cloacae producing SHV-12 ESBL in our hospital is the consequence of disseminations of plasmids harboring the SHV-12 gene. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: E. cloacae SHV-12 BLSE Tunisia
Resistance to cephalosporins, due to the production of extended-spectrum b-lactamases (ESBLs), is an ever increasing problem and is a cause of serious concern worldwide [1]. The majority of these enzymes are derived by amino acid substitutions from the narrow spectrum precursors, TEM-1,2 and SHV-15 [2]. SHV-type ESBLs, encoded by blaSHV genes, confer resistance to a broad spectrum of b-lactam antimicrobial agents and are of significant therapeutic concern for infections caused by many species of gram-negative bacteria [3,4]. At present there are more than 140 varieties of SHV b-lactamases, the majority of which confer the extended-spectrum b-lactamase (ESBL) phenotype (www.lahey.org/Studies). This enzyme has been detected in many species of the family Enterobacteriaceae, including Enterobacter spp which is a leading cause of ventilator-associated pneumonia, bloodstream infections, and urinary tract infections in hospitalized patients [5]. Here we report the emergence of blaSHV-12 gene in clinical isolate of Enterobacter cloacae from Tunisia and investigate the ESBL expressing strain for their molecular and clinical characteristics. A total of nine isolates of E. cloacae were collected between October 2009 and April 2010 from different patients hospitalized at the University Hospital of a Tunisian coastal city. All isolates were
* Corresponding author. Tel.: þ216 50778984. E-mail address: [email protected] (H. Lahlaoui). 0882-4010/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2012.04.003
recovered from various pathological specimens and were identified by the API 20E system (bioMérieux) (Table 1). ESBL detection was performed by the double disk diffusion test and revealed synergies between amoxicilineclavulanic acid and oxyiminocephalosporins (cefotaxime, ceftazidime) containing disks for all isolates. Seven isolates showed a clearly higher level of resistance to ceftazidime and cefpime were submitted to further analysis (Table 1). Minimum inhibitory concentrations (MICs) were determined by a microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [6] and showed similar susceptibility profiles, characterized by elevated MICs (256 mg/ml) of amoxicillin, ceftazidime and cefepime but remained susceptible to imipenem (Table 1). Screening for ESBL gene was performed by PCR using the primers for blaTEM, blaCTX-M and blaSHV [7,8]. An 875-pb fragment of SHV-type gene was amplified which, after sequencing and comparing them against the NCBI nucleotide databases, was identified as an SHV-12- gene b-lactamase for all the isolates. This SHV-12 b-lactamase is characterized by three point mutations in the SHV-1 precursor (Gln35, Ser238, and Lys240), according to Ambler nomenclature [9]. PCR mapping of the genetic environment surrounding the blaSHV-12 gene with primers specific for insertion sequence IS26 (primers IS26-1 and IS26-2) [10] combined with forward and reverse primers for blaSHV gene showed that the all resistant strains contained the same IS26 elements, which were detected both upstream and downstream of blaSHV-12. This study
H. Lahlaoui et al. / Microbial Pathogenesis 53 (2012) 64e65
65
Table 1 Characteristics of extended-spectrum b-lactamase-producing Enterobacter clocace isolates. Isolate
WK1 WK2 WK3 WK4 WK5 WK6 WK7
Source
Sang Urine Placenta Urine Placenta Urine Urine
Ward
Paediatrics Gynaecology Gynaecology Paediatrics Gynaecology Gynaecology Paediatrics
Isolation
17/10/2009 09/11/2009 28/12/2009 29/12/2009 09/01/2010 06/04/2010 17/04/2010
Sex
Male Female Female Female Female Female Male
MIC (mg/ ml) AMX
AMC
CTX
CAZ
FOX
CEF
AZT
IMP
256 256 256 256 256 256 256
2 2 2 2 4 2 2
128 64 64 64 128 64 64
256 256 256 256 256 256 256
64 64 64 64 64 64 64
256 256 256 256 256 256 256
32 64 32 32 64 64 64
1 1 1 1 2 1 1
Transconjugation
PFGE clone
ESBL
þ þ þ þ þ þ þ
1 2 3 3 4 5 3
SHV-12 SHV-12 SHV-12 SHV-12 SHV-12 SHV-12 SHV-12
MIC, minimum inhibitory concentration; AMX, amoxicillin; AMC, amoxicillin-clavulanic; CTX, cefotaxime; CAZ, ceftazidime; FOX, cefoxitin; CEF, cefepime; AZT,aztreonam IPM, imipenem; þ, Positive result.
suggests that the dissemination of SHV-12 among different E. cloace strains may be mediated by the mobile element. Conjugation experiments for the blaSHV-12 gene were carried out between the donors and the azide-resistant recipient strain E. coli J53 and selected on MH agar plates supplemented with ceftazidime (2 mg/L) and sodium azide (100 mg/L) [11]. Transconjugants were obtained in all E. coli strains that yielded a production of ESBLs by the double-disc synergy test; the presence of SHV-12 was confirmed by sequence analysis of the PCR product. In all, the experiments indicated that the SHV-12 was located on over 100 kb single conjugative plasmid. The genetic relationship between the isolates was evaluated by pulsed-field gel electrophoresis (PFGE) as previously described [12], using 50 U of XbaI restriction enzyme for 4 h at 37 C and separated by electrophoresis in a 1% agarose gel. PFGE analysis revealed five distinct profiles as shown in Table 1. This study constitutes the first report of a hospital outbreak due to an SHV-12-producing E. cloacae. This blaSHV-12 gene was found to be plasmid-located and associated to the insertion sequence, IS26. SHV-12 ESBL was first identified in 1997 in Switzerland [13] and later reported from various country including Taiwan, Korea, and Italy [14e16]. In Tunisia, the SHV-12 was described for the first time in Klebsiella pneumoniae strains from a Tunisian hospital on 2004 [17] and is identified with SHV-2-a the most prevalent extendedspectrum SHV enzymes among Enterobacteriacae. In fact, the genes surrounding blaSHV- 12 in this study is identical to the genes surrounding blaSHV- 2a. This suggests that blaSHV-12 identified in this study has evolved from blaSHV-2a as supposed by the evolutionary approach [18]. SHV-2a and SHV-12 differ from SHV-1, SHV-2 and SHV-5, respectively, by one amino acid change from leucine to glutamine at position 35, which is far from the active site and known not to alter the isoelectric point. Further, the IS26 element is not detected in SHV-1, SHV-2, SHV-3, SHV-4 or SHV-5. These results reinforce the hypothesis that SHV-12 may have evolved directly from SHV-2-a, not from SHV-2 to 5 [10]. In conclusion, The identification of the E. cloacae ESBL as an SHV-12 b-lactamase confirms the widespread diffusion in Tunisia of this enzyme, which was one of the most common variants found in most hospitals and in several different species of Enterobacteriaceae. However, we propose that careful attention be given to the transfer of these genes, especially with respect to the acquisition of multidrug resistance.
Acknowledgment This work was funded by grants from the Tunisian Ministry of Higher Education, Scientific Research and Technology.
References [1] Al Naiemi N, Zwart B, Rijnsburger MC, Roosendaal R, Debets-Ossenkopp YJ, Mulder JA, et al. Extended-spectrum-beta-lactamase production in a Salmonella enterica serotype Typhi strain from the Philippines. J Clin Microbiol 2008;46:2794e5. [2] Kasap M, Fashae K, Torol S, Kolayli F, Budak F, Vahaboglu H. Characterization of ESBL (SHV-12) producing clinical isolate of Enterobacter aerogenes from a tertiary care hospital in Nigeria. Ann Clin Microbiol Antimicrob 2010;9:1. [3] al Naiemi N, Schipper K, Duim B, Bart A. Application of minimal sequence quality values prevents misidentification of the blaSHV type in single bacterial isolates carrying different SHV extended-spectrum b-lactamase genes. J Clin Microbiol 2006;44:1896e8. [4] Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14:933e51. [5] Szabó D, Bonomo RA, Silveira F, Pasculle AW, Baxter C, Linden PK, et al. SHV-type extended-spectrum beta-lactamase production is associated with reduced cefepime susceptibility in Enterobacter cloacae. J Clin Microbiol 2005;43:5058e64. [6] Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; 20th informational supple-ment M100eS20. Wayne, PA: Clinical and Laboratory Standards Institute; 2010. [7] Sharma J, Sharma M, Ray P. Detection of TEM & SHV genes in Escherichia coli & Klebsiella pneumoniae isolates in a tertiary care hospital from India. Indian J Med Res 2010;132:332e6. [8] Lartigue MF, Zinsius C, Wenger A, Bille J, Poirel L, Nordmann P. Extendedspectrum {beta}-lactamases of the CTX-M type now in Switzerland. Antimicrob Agents Chemother 2007;51:2855e60. [9] Ambler RP, Coulson AFW, Frère JM, Ghuysen JM, Joris B, Forsman M, et al. A standard numbering scheme for the class A b-lactamases. Biochem J 1991; 276:269e72. [10] Kim J, Shin HS, Seol SY, Cho DT. Relationship between bla SHV-12 and bla SHV-2a in Korea. J Antimicrob Chemother 2002;49:261e7. [11] Ryoo NH, Kim EC, Hong SG, Park YJ, Lee K, Bae IK, et al. Dissemination of SHV12 and CTX-M-type extended-spectrum beta-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES3 in Korea. J Antimicrob Chemother 2005;56:698e702. [12] Biendo M, Manoliu C, Laurans G, Castelain S, Canarelli B, Thomas D, et al. Molecular typing and characterization of extended-spectrum TEM, SHV and CTX-M beta-lactamases in clinical isolates of Enterobacter cloacae. Res Microbiol 2008;159:590e4. [13] Nüesch-Inderbinen MT, Kayser FH, Hachler H. Survey and molecular genetics of SHV b-lactamases in Enterobacteriaceae in Switzer-land: two novel enzymes, SHV-11 and SHV-12. Antimicrob Agents Chemother 1997;41:943e9. [14] Kim J, Kwon Y, Pai H, Kim JW, Cho DT. Survey of Klebsiella pneumoniae strains producing extended-spectrum b-lactamases: prevalence of SHV-12 and SHV2a in Korea. J Clin Microbiol 1998;36:1446e9. [15] Perilli M, Dell’Amico E, Segatore B, de Massis MR, Bianchi C, Luzzaro F, et al. Molecular characterization of extended-spectrum b-lactamases produced by nosocomial isolates of Enterobacteriaceae from an Italian nationwide survey. J Clin Microbiol 2002;40:611e4. [16] Yan JJ, Wu SM, Tsai SH, Wu JJ, Su IJ. Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum b-lactamases and identification of a novel AmpC enzyme (CMY-8) in Southern Taiwan. Antimicrob Agents Chemother 2000;44:1438e42. [17] Ben-Hamouda T, Foulon T, Ben-Mahrez K. Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended-spectrum beta-lactamaseproducing Klebsiella pneumoniae in a Tunisian neonatal ward. Microb Drug Resist 2004;10:132e8. [18] Ford PJ, Avison MB. Evolutionary mapping of the SHV beta-lactamase and evidence for two separate IS26-dependent blaSHV mobilization events from the Klebsiella pneumoniae chromosome. J Antimicrob Chemother 2004;54:69e75.
Contents lists available at SciVerse ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Emergence of SHV-12 extended spectrum beta-lactamase among clinical isolates of Enterobacter cloacae in Tunisia Hella Lahlaoui a, *, Ben Haj Khalifa Anis b, Kheder Mohamed b, Ben Moussa Mohamed a a b
Laboratoire de Microbiologie, Hôpital Militaire de Tunis, 1089 Monfleury, Tunisia Laboratoire de Microbiologie, Hôpital Tahar Sfar de Mahdia, 5100 Mahdia, Tunisia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 December 2011 Received in revised form 10 April 2012 Accepted 13 April 2012 Available online 21 April 2012
A collection of seven multidrug-resistant clinical isolates of Enterobacter cloacae with reduced susceptibility to ceftazidime and cefepime recovered from 2009 to 2010 at the University Hospital of Mahdia, Tunisia, was analysed. PCR analysis and sequencing demonstrated that all study isolates harbored SHV12 b-lactamase that was transferred by conjugation. Characterization of the regions surrounding the blaSHV-12 showed that this gene was flanked by two IS26 elements. pulsed-field gel electrophoresis (PFGE) revealed differents profiles indicating that the study isolates were not clonally related. Diffusion of E. cloacae producing SHV-12 ESBL in our hospital is the consequence of disseminations of plasmids harboring the SHV-12 gene. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: E. cloacae SHV-12 BLSE Tunisia
Resistance to cephalosporins, due to the production of extended-spectrum b-lactamases (ESBLs), is an ever increasing problem and is a cause of serious concern worldwide [1]. The majority of these enzymes are derived by amino acid substitutions from the narrow spectrum precursors, TEM-1,2 and SHV-15 [2]. SHV-type ESBLs, encoded by blaSHV genes, confer resistance to a broad spectrum of b-lactam antimicrobial agents and are of significant therapeutic concern for infections caused by many species of gram-negative bacteria [3,4]. At present there are more than 140 varieties of SHV b-lactamases, the majority of which confer the extended-spectrum b-lactamase (ESBL) phenotype (www.lahey.org/Studies). This enzyme has been detected in many species of the family Enterobacteriaceae, including Enterobacter spp which is a leading cause of ventilator-associated pneumonia, bloodstream infections, and urinary tract infections in hospitalized patients [5]. Here we report the emergence of blaSHV-12 gene in clinical isolate of Enterobacter cloacae from Tunisia and investigate the ESBL expressing strain for their molecular and clinical characteristics. A total of nine isolates of E. cloacae were collected between October 2009 and April 2010 from different patients hospitalized at the University Hospital of a Tunisian coastal city. All isolates were
* Corresponding author. Tel.: þ216 50778984. E-mail address: [email protected] (H. Lahlaoui). 0882-4010/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2012.04.003
recovered from various pathological specimens and were identified by the API 20E system (bioMérieux) (Table 1). ESBL detection was performed by the double disk diffusion test and revealed synergies between amoxicilineclavulanic acid and oxyiminocephalosporins (cefotaxime, ceftazidime) containing disks for all isolates. Seven isolates showed a clearly higher level of resistance to ceftazidime and cefpime were submitted to further analysis (Table 1). Minimum inhibitory concentrations (MICs) were determined by a microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [6] and showed similar susceptibility profiles, characterized by elevated MICs (256 mg/ml) of amoxicillin, ceftazidime and cefepime but remained susceptible to imipenem (Table 1). Screening for ESBL gene was performed by PCR using the primers for blaTEM, blaCTX-M and blaSHV [7,8]. An 875-pb fragment of SHV-type gene was amplified which, after sequencing and comparing them against the NCBI nucleotide databases, was identified as an SHV-12- gene b-lactamase for all the isolates. This SHV-12 b-lactamase is characterized by three point mutations in the SHV-1 precursor (Gln35, Ser238, and Lys240), according to Ambler nomenclature [9]. PCR mapping of the genetic environment surrounding the blaSHV-12 gene with primers specific for insertion sequence IS26 (primers IS26-1 and IS26-2) [10] combined with forward and reverse primers for blaSHV gene showed that the all resistant strains contained the same IS26 elements, which were detected both upstream and downstream of blaSHV-12. This study
H. Lahlaoui et al. / Microbial Pathogenesis 53 (2012) 64e65
65
Table 1 Characteristics of extended-spectrum b-lactamase-producing Enterobacter clocace isolates. Isolate
WK1 WK2 WK3 WK4 WK5 WK6 WK7
Source
Sang Urine Placenta Urine Placenta Urine Urine
Ward
Paediatrics Gynaecology Gynaecology Paediatrics Gynaecology Gynaecology Paediatrics
Isolation
17/10/2009 09/11/2009 28/12/2009 29/12/2009 09/01/2010 06/04/2010 17/04/2010
Sex
Male Female Female Female Female Female Male
MIC (mg/ ml) AMX
AMC
CTX
CAZ
FOX
CEF
AZT
IMP
256 256 256 256 256 256 256
2 2 2 2 4 2 2
128 64 64 64 128 64 64
256 256 256 256 256 256 256
64 64 64 64 64 64 64
256 256 256 256 256 256 256
32 64 32 32 64 64 64
1 1 1 1 2 1 1
Transconjugation
PFGE clone
ESBL
þ þ þ þ þ þ þ
1 2 3 3 4 5 3
SHV-12 SHV-12 SHV-12 SHV-12 SHV-12 SHV-12 SHV-12
MIC, minimum inhibitory concentration; AMX, amoxicillin; AMC, amoxicillin-clavulanic; CTX, cefotaxime; CAZ, ceftazidime; FOX, cefoxitin; CEF, cefepime; AZT,aztreonam IPM, imipenem; þ, Positive result.
suggests that the dissemination of SHV-12 among different E. cloace strains may be mediated by the mobile element. Conjugation experiments for the blaSHV-12 gene were carried out between the donors and the azide-resistant recipient strain E. coli J53 and selected on MH agar plates supplemented with ceftazidime (2 mg/L) and sodium azide (100 mg/L) [11]. Transconjugants were obtained in all E. coli strains that yielded a production of ESBLs by the double-disc synergy test; the presence of SHV-12 was confirmed by sequence analysis of the PCR product. In all, the experiments indicated that the SHV-12 was located on over 100 kb single conjugative plasmid. The genetic relationship between the isolates was evaluated by pulsed-field gel electrophoresis (PFGE) as previously described [12], using 50 U of XbaI restriction enzyme for 4 h at 37 C and separated by electrophoresis in a 1% agarose gel. PFGE analysis revealed five distinct profiles as shown in Table 1. This study constitutes the first report of a hospital outbreak due to an SHV-12-producing E. cloacae. This blaSHV-12 gene was found to be plasmid-located and associated to the insertion sequence, IS26. SHV-12 ESBL was first identified in 1997 in Switzerland [13] and later reported from various country including Taiwan, Korea, and Italy [14e16]. In Tunisia, the SHV-12 was described for the first time in Klebsiella pneumoniae strains from a Tunisian hospital on 2004 [17] and is identified with SHV-2-a the most prevalent extendedspectrum SHV enzymes among Enterobacteriacae. In fact, the genes surrounding blaSHV- 12 in this study is identical to the genes surrounding blaSHV- 2a. This suggests that blaSHV-12 identified in this study has evolved from blaSHV-2a as supposed by the evolutionary approach [18]. SHV-2a and SHV-12 differ from SHV-1, SHV-2 and SHV-5, respectively, by one amino acid change from leucine to glutamine at position 35, which is far from the active site and known not to alter the isoelectric point. Further, the IS26 element is not detected in SHV-1, SHV-2, SHV-3, SHV-4 or SHV-5. These results reinforce the hypothesis that SHV-12 may have evolved directly from SHV-2-a, not from SHV-2 to 5 [10]. In conclusion, The identification of the E. cloacae ESBL as an SHV-12 b-lactamase confirms the widespread diffusion in Tunisia of this enzyme, which was one of the most common variants found in most hospitals and in several different species of Enterobacteriaceae. However, we propose that careful attention be given to the transfer of these genes, especially with respect to the acquisition of multidrug resistance.
Acknowledgment This work was funded by grants from the Tunisian Ministry of Higher Education, Scientific Research and Technology.
References [1] Al Naiemi N, Zwart B, Rijnsburger MC, Roosendaal R, Debets-Ossenkopp YJ, Mulder JA, et al. Extended-spectrum-beta-lactamase production in a Salmonella enterica serotype Typhi strain from the Philippines. J Clin Microbiol 2008;46:2794e5. [2] Kasap M, Fashae K, Torol S, Kolayli F, Budak F, Vahaboglu H. Characterization of ESBL (SHV-12) producing clinical isolate of Enterobacter aerogenes from a tertiary care hospital in Nigeria. Ann Clin Microbiol Antimicrob 2010;9:1. [3] al Naiemi N, Schipper K, Duim B, Bart A. Application of minimal sequence quality values prevents misidentification of the blaSHV type in single bacterial isolates carrying different SHV extended-spectrum b-lactamase genes. J Clin Microbiol 2006;44:1896e8. [4] Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14:933e51. [5] Szabó D, Bonomo RA, Silveira F, Pasculle AW, Baxter C, Linden PK, et al. SHV-type extended-spectrum beta-lactamase production is associated with reduced cefepime susceptibility in Enterobacter cloacae. J Clin Microbiol 2005;43:5058e64. [6] Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; 20th informational supple-ment M100eS20. Wayne, PA: Clinical and Laboratory Standards Institute; 2010. [7] Sharma J, Sharma M, Ray P. Detection of TEM & SHV genes in Escherichia coli & Klebsiella pneumoniae isolates in a tertiary care hospital from India. Indian J Med Res 2010;132:332e6. [8] Lartigue MF, Zinsius C, Wenger A, Bille J, Poirel L, Nordmann P. Extendedspectrum {beta}-lactamases of the CTX-M type now in Switzerland. Antimicrob Agents Chemother 2007;51:2855e60. [9] Ambler RP, Coulson AFW, Frère JM, Ghuysen JM, Joris B, Forsman M, et al. A standard numbering scheme for the class A b-lactamases. Biochem J 1991; 276:269e72. [10] Kim J, Shin HS, Seol SY, Cho DT. Relationship between bla SHV-12 and bla SHV-2a in Korea. J Antimicrob Chemother 2002;49:261e7. [11] Ryoo NH, Kim EC, Hong SG, Park YJ, Lee K, Bae IK, et al. Dissemination of SHV12 and CTX-M-type extended-spectrum beta-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES3 in Korea. J Antimicrob Chemother 2005;56:698e702. [12] Biendo M, Manoliu C, Laurans G, Castelain S, Canarelli B, Thomas D, et al. Molecular typing and characterization of extended-spectrum TEM, SHV and CTX-M beta-lactamases in clinical isolates of Enterobacter cloacae. Res Microbiol 2008;159:590e4. [13] Nüesch-Inderbinen MT, Kayser FH, Hachler H. Survey and molecular genetics of SHV b-lactamases in Enterobacteriaceae in Switzer-land: two novel enzymes, SHV-11 and SHV-12. Antimicrob Agents Chemother 1997;41:943e9. [14] Kim J, Kwon Y, Pai H, Kim JW, Cho DT. Survey of Klebsiella pneumoniae strains producing extended-spectrum b-lactamases: prevalence of SHV-12 and SHV2a in Korea. J Clin Microbiol 1998;36:1446e9. [15] Perilli M, Dell’Amico E, Segatore B, de Massis MR, Bianchi C, Luzzaro F, et al. Molecular characterization of extended-spectrum b-lactamases produced by nosocomial isolates of Enterobacteriaceae from an Italian nationwide survey. J Clin Microbiol 2002;40:611e4. [16] Yan JJ, Wu SM, Tsai SH, Wu JJ, Su IJ. Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum b-lactamases and identification of a novel AmpC enzyme (CMY-8) in Southern Taiwan. Antimicrob Agents Chemother 2000;44:1438e42. [17] Ben-Hamouda T, Foulon T, Ben-Mahrez K. Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended-spectrum beta-lactamaseproducing Klebsiella pneumoniae in a Tunisian neonatal ward. Microb Drug Resist 2004;10:132e8. [18] Ford PJ, Avison MB. Evolutionary mapping of the SHV beta-lactamase and evidence for two separate IS26-dependent blaSHV mobilization events from the Klebsiella pneumoniae chromosome. J Antimicrob Chemother 2004;54:69e75.