|Year : 2016 | Volume
| Issue : 2 | Page : 40-45
Molecular characterization of extended spectrum β-lactamases, ampccephalosporinases and carbapenemases in klebsiellapneumoniae causing bacteremia at charles nicolle Hospital of Tunisia
Elaa Maamar1, Samia Hammami2, Sana Ferjani1, Zaineb Hamzaoui1, Asma Jlizi3, M Saidani4, A Slim4, I Boutiba-Ben Boubaker4
1 University of Tunis El Manar, Faculty of Medicine of Tunis -LR99ES09 Research Laboratory «Antimicrobial resistance», 1007 Tunis-, Tunisia
2 University of Tunis El Manar, Faculty of Medicine of Tunis -LR99ES09 Research Laboratory «Antimicrobial resistance», 1007 Tunis; Charles Nicolle Hospital, Laboratory of Microbiology,1006-Tunis; University of Gafsa, Faculty of Sciences of Gafsa, Tunisia
3 Rabta University Hospital, Laboratory of Microbiology,1007-Tunis-, Tunisia
4 University of Tunis El Manar, Faculty of Medicine of Tunis -LR99ES09 Research Laboratory «Antimicrobial resistance», 1007 Tunis; Charles Nicolle Hospital, Laboratory of Microbiology, 1006-Tunis, Tunisia
|Date of Web Publication||6-Jul-2017|
University of Tunis El Manar, Faculty of Medicine of Tunis -LR99ES09 Research Laboratory «Antimicrobial resistance», 1007 Tunis
Source of Support: None, Conflict of Interest: None
Purpose of the Study: This study was conducted to detect and characterize the genes encoding extended spectrum β-lactamases and associated β-lactamases (carbapenemases and Ambler Class C β-lactamases).
Patients and Methods: In 2011, out of the 65 non-duplicative Klebsiellapneumoniae collected from blood culture at Charles Nicolle hospital of Tunisia, 36 were resistant to 3rd generation cephalosporin.
Results: All strains showed a double disk synergy test positive. They were mainly isolated in intensive care unit (31%). They were frequently resistant to most antibiotics tested, except colistin and tigecyclin. Five isolates (13%) showed reduced susceptibility to carbapenems. blaCTX-M-15 was harbored by 35 strains and blaSHV-12 by one. blaCTX-M-15 were associated with blaTEM-1 (n=21), blaOXA-48 and blaCMY-2 (n=1) and blaOXA-48and blaTEM-1 (n=4). The conjugation wassuccessfulfor4/5 strains (3 harboring blaCTX-M-15 and one blaSHV-12). The plasmids carrying the blaCTX-M-15 were assigned to IncN or IncL/M only for 2 strains. The remaining blaCTX-M-15-carrying plasmid was negative for all of the replicons tested as well as the blaSHV-12-carrying plasmid.
Conclusion: Our results confirm the spread of CTX-M-15 in our institution. To our knowledge, this is the first report of K. pneumoniae coproducing CTX-M-15, CMY-2 and OXA-48. The implementation of preventive measures against the spread of these multiresistant bacteria is needed.
Keywords: ESBL, AmpCcephalosporinase, Oxa-48, Klebsiellapneumoniae, Bacteremia
|How to cite this article:|
Maamar E, Hammami S, Ferjani S, Hamzaoui Z, Jlizi A, Saidani M, Slim A, Boubaker I B. Molecular characterization of extended spectrum β-lactamases, ampccephalosporinases and carbapenemases in klebsiellapneumoniae causing bacteremia at charles nicolle Hospital of Tunisia. Acta Med Int 2016;3:40-5
|How to cite this URL:|
Maamar E, Hammami S, Ferjani S, Hamzaoui Z, Jlizi A, Saidani M, Slim A, Boubaker I B. Molecular characterization of extended spectrum β-lactamases, ampccephalosporinases and carbapenemases in klebsiellapneumoniae causing bacteremia at charles nicolle Hospital of Tunisia. Acta Med Int [serial online] 2016 [cited 2019 Mar 21];3:40-5. Available from: http://www.actamedicainternational.com/text.asp?2016/3/2/40/209796
| Introduction|| |
Since their description in the mid-1980s, extended spectrum β-lactamase (ESBL)-producing organisms have become recognized as a worldwide problem. Although ESBLs have been detected in a wide variety of Gram-negative bacteria, Klebsiellapneumoniae has been found to be the most common species to produce ESBLs. Because ESBL- producing organisms are frequently multidrug resistant, therapeutic options for these infections are severely limited and treatment can be challenging. Also, there have been many reports of outbreaks caused by these organisms, and it has been demonstrated that ESBL production by infecting organisms adversely affects the clinical outcome with a significant morbidity and mortality.,,,
In Tunisia, epidemiological data of the spread of ESBL-producing K. pneumoniaestrains showed a rapid diffusion since their first description in 1984. In fact, their prevalence varies from 27.9% in 1999 to 51.5% in 2007 with a high incidence 87.5% in Mongi Slim Hospital followed by 19°/Ω in Children's Hospital.
This study was conducted to detect and characterize ESBLs and associated β-lactamases (carbapenemases and Ambler Class C β-lactamases (AmpC)) produced by K. pneumoniae resistant to 3rd generation cephalosporin (3rd GC) causing bacteremia at Charles Nicolle Hospital, during 2011.
| Materials and Methods|| |
Among the 65 non-duplicate K. pneumoniae isolated from blood cultures of 65 patients hospitalized at Charles Nicolle Hospital of Tunis from January to December 2011, 36 (55.3%) were resistant to 3rd GC. They were collected mainly from intensive care unit (31%), neonatology (26%) and surgery (23%) [Table 1].
|Table 1: Characteristics of extended spectrum β-lactamases K. pneumoniae producers isolated from blood cultures in 2011 at Charles Nicolle hospital|
Click here to view
Antibiotic Susceptibility Testing
Antimicrobial susceptibility testing was determined by the disk-diffusion method on Mueller-Hinton (MH) agar plates (Bio-Rad, Marnes-la-Coquette, France) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. ESBL production was detected by the double disk synergy test (DDST) with or without cloxacillin as described previously. Isolates with reduced susceptibility to carbapenems: imipenem (diameter zone ≤ 22 mm) and/or ertapenem (diameter zone ≤ 18 mm) were subjected to the modified Hodge screening test (MHT) for diffusible carbapenemase detection as previously described. The minimal inhibition concentrations (MICs) of ceftazidime, ceftazidime-clavulanic acid, cefotaxime, cefoxitine and cefepime were determined by agar dilution technique. For imipenem, ertapenem and meropenem, MICs were determined by E-test (Bio-Mérieux SA, Marcy l'étoile, France). Results were interpreted according to the CLSI guidelines.
Detection of β-lactamase-encoding Genes
K. pneumoniae isolates were screened by PCR for the following β-lactamases encoding genes: blaCTX-M phylogenetic lineage groups 1, 2 and 9,,, blaTEM, blaSHV, blaCIT' blaACC blaEBC blaMOX blaFOX, blaDHA, blaLAT, blaACT, blaMIR, blaOXA-48 and blaKpC as described previously. The PCR products were purified with a Purification Kit (BioMatik) and sequenced on an ABI PRISM 310 DNA Sequencer (Applied Biosystems). The nucleotide sequences were analyzed with the BLAST program (http://www.ncbi.nlm. nih.gov/BLAST/).
β-Lactam Resistance Transfer Assays
Conjugation assays were performed on 5 isolates selected on the basis of their susceptibility to rifampin. It was carried out in brain heart broth (Bio-Rad), with E. coli J53-2 as the recipient. Transconjugants were selected on Mueller Hinton agar plates containing rifampicin (500 mg/L) and ticarcillin (125 mg/L). If not successful at the first attempt, mating experiments were repeated up to 3 times. Transconjugant's were submitted to bacterial identification with the API-20E system (Bio-Mérieux SA, Marcy l'étoile, France), antibiotic susceptibility testing and PCR amplification of the bla genes mentioned above.
Incompatibility groups (Inc) were determined by PCR- based replicon typing method as previously described.
| Results|| |
Antibiotic Susceptibility Tests
All isolates showed a positive DDST. They showed high level of resistance to cefotaxime (MIC50= 512 μg/mL), ceftazidime (MIC50=128 μg/mL) and cefepóme (MIC50=128 μg/mL). MICs of ceftazidime decreased when clavulanic acid is added. However, 1 strain showed high level of resistance to cefoxitin (MIC=>512 μg/mL) [Table 1]. Five strains were resistant to ertapenem and showed a positive MHT. Three of them were resistant to imipinem and/or meropenem [Table 1].
All isolates were resistant to most antibiotics tested except colistin and tigecylin.They were frequently resistant to tobramycin (n=35), gentamicin (n=28), tetracycline (n=19), trimethoprim–sulfamethoxazole (n=30) and fluoroquinolones (n=26).
β-Lactamases Gene Characterization
The results of PCR and sequence analysis are summarized in [Table 1]. The blaSHV1 gene was detected in all K. pneumoniae strains. ESBL genes were detected in all strains, blaCTX-M-15 in 35 strains and blaSHV-12 in one. Moreover, 28 isolates harbored blaTEM-1. Ertapenem resistant strains carried blaOXA-48. The blaCMY-2 genewas detected in one strain that also coproduce 3β-lactamases genes (blaCTX-M-15, blaOXA-48 and blaTEM-1).
Plasmid Replicon Type Determination
For 2 strains, the plasmids carrying blaCTX-M-15 were assigned to the IncN and IncL/M replicon type. However, the remaining blaCTX-M-15-carrying plasmid was negative for all of the replicons tested, as well as the blaSHV-12-carrying plasmid [Table 1].
Theconjugationwassuccessfulfor4/5 strains (3 harboring blaCTX-M-15 and one blaSHV-12). We also observed co-transfer, following conjugation, of resistance to other antibiotics, such as i.e. gentamicin (n=1), tobramycin (n=2), netilmicin (n=1), minocycline (n=4) and chloramphenicol (n=2).
Production of ESBLs was detected in all transconjugants by the DDST. The transfer of ESBL genes was confirmed in all transconjugants; blaCTX-M-15 and blaSHV-12 were detected in 3 and 1 transconjugants, respectively. Only one of the 4 transconjugants was typeable for the incompatibility groups (IncL/M) [Table 2].
|Table 2: Resistance profils and β-lactamases genes of donor strains and their transconjugants|
Click here to view
| Discussion|| |
ESBL producing K. pneumoniae is one of the most important pathogens responsible for life threatening infections.,, In the present study, frequency of ESBL producing K. pneumoniae causing bacteremia is 47% which is lower than 87.5% reported in a Tunisian pediatric intensive care unit. A study conducted in India reported 56% ESBL K. pneumoniae causing septicemiaamongneonates.
In most European countries, Latin America, and East Asia, CTX-M variants have displaced TEM and SHV enzymesas the predominant β-lactamases produced by Gram-negative bacteria such as K. pneumoniae. Our work shows that CTX-M-15 is the most common ESBL. CTX-M-15, first described in India in 1999, has spread over the world, and seems to be the most common ESBL type., In Tunisia, dissemination of CTX-M-15 has been reported in K. pneumoniae, E. coli and Enterobacter cloacae strains.,,, blaCTX-M-15 gene is generally found on large conjugative plasmids and is located downstream of an ISEcpl insertion sequence which explains its remarkable transmission success. The large use of 3rdGC in clinical practice has significantly contributed to their selection.
Although SHV-12 was found in one isolate of our collection, previous Tunisian studies showed that this enzyme was associated with K. pneumoniaeinvolved in nosocomial outbreaks.,,,, Worldwide, the distribution of SHV- ESBLs showed that SHV-2, SHV-4 and SHV-5 enzymes are the most prevalent in the United States and Europe.
The ESBL-producing organisms additionally exhibited co-resistance against multiple antimicrobials from other classes, such as tetracycline, aminoglycosides and fluoroquinolones. This complexity in antimicrobials resistance combinations limits suitable drug of choice for antimicrobial therapy, leaving carbapenems the last options for treatment in some cases. However, the emergence of plasmidiccarbapenemases producers clearly compounds potential treatment options. Carbapenem resistance was found in 5 of our strains. All harbored OXA-48. This class D carbapenemasewas first identified in K. pneumoniae from Turkey in 2003 and then extensively spread in other countries and species. In Tunisia, recent studies reported the spread of OXA-48 producing Enterobacteriaceae.,
blaCMY-2 is the most frequently encountered plasmid encoded AmpC-type β-lactamase gene found in E. coli worldwide.,, the first AmpC type β-lactamase to be identified in Tunisia was in 1996 in a P. mirabilie isolate, and was characterized as a CMY-4 enzyme. In Tunisia, CMY-2 has already been detected in food samples and fecal flora of healthy chickens and pets but never in clinical isolates. In our study, blaCMY-2 was detected in one strain, which co-produced 3 plasmidicβ-lactamases (CTX-M-15, OXA-48 and TEM-1). The coexistence of several enzymes in the same strain is noteworthy and has been previously described in K. pneumoniae in Tunisia (VIM-4, CTX-M-15 and CMY-4).
Identification and classification of plasmids harbouring β-lactamases is helpful to analyze their distribution in nature and their relationship to host cells and to discover their evolutionry origins. In our study, incompatibility groups of blaCTX-M-15 carrying plasmids were determined only in 2 strains (IncN and IncL/M). However, other studies reported that some blaCTX-M-15 and blaSHV-12-carrying plasmids were negative for all of the replicons tested,, as well as described in our study. Further research is necessary to provide more information about our plasmids.
In conclusion, our study reports a widespread diffusion of CTX-M-15 with the emergence of OXA-48 in K. pneumoniae in Tunisia which seriously limits therapeutic options in cases of clinical infections with these bacteria. In addition, coexistence of ESBLs production with cephalosporinase or carbapenemase is a serious public health problem and requires continuous surveillance, monitoring and revision of the antibiotic use policies.
| Acknowledgments|| |
This work was funded by Grants from the Tunisian Ministry of Higher Education, Scientific Research and Technology.
All authors disclose no commercial associations that might create a conflict of interest in connection with this study.
| References|| |
Bradford PA. Extended-spectrum beta-lactamases in the 21st
century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 2001;14:933–51.
Elhani DL, Bakir MA, Passet V, Arlet G, Brisse S et al. Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae
strains in a university hospital in Tunis, Tunisia, 1999-2005. Clin Microbiol Infect. 2010;16:157–64.
Ben-Hamouda T, Foulon T and Ben-Mahrez K. Involvement of SHV-12 and SHV-2a encoding plasmids in outbreaks of extended- spectrum beta-lactamase-producing Klebsiella pneumoniae
in a Tunisian neonatal ward. Microb Drug Resist. 2004;10: 132–8.
Ben Jaballah N, Bouziri A, Mnif K, Hamdi A, Khaldi A, and W. Kchaou. Epidemiology of hospital-acquired bloodstream infections in a Tunisian pediatric intensive care unit: a 2-year prospective study. Am J Infect Control. 2007;35:613–8.
Mamlouk K, Boutiba-Ben Boubaker I, Gautier V, Vimont S, Picard B, Ben Redjeb S et al. Emergence and outbreaks of CTX-M beta- lactamase-producing Escherichia coli
and Klebsiella pneumoniae
strains in a Tunisian hospital. J Clin Microbiol. 2006;44:4049–56.
Paterson DL, Hujer KM., Hujer AM, et al. Extended-spectrum beta-lactamases in Klebsiella pneumoniae
bloodstream isolates from seven countries: dominance and widespread prevalence of SHV and CTX-M-type beta-lactamases. Antimicrob Agents Chemother. 2003; 47: 3554–60.
Boutiba-Ben Boubaker I, Ghozzi R, Ben Abdallah H, Mamlouk K, Kamoun A and Ben Redjeb S. Evolution of acquired resistance to third-generation cephalosporins in Enterobacteriaceae
in a Tunisian hospital 1993-2001. Clin Microbiol Infect. 2004; 10:665–7.
Ben Redjeb S and Boutiba Ben-Boubaker I. L'antibiorésistance en Tunisie: données 2004-2007.Tunis, LART-LR99ES09. 2008, p67.
Clinical and Laboratory Standards Institue. performance Standards for Antimicrobial Susceptibility Testing, 16 th informational supplement. 2006;CLSI document no. M100–S16.
Jarlier V, Nicolas MH, Fournier G and Philippon A. Extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis. 1988;10:867–78.
Lee K, Chong Y, Shin HB, Kim YA, Yong D and. Yum JH. Modified Hodge and EDTA-disk synergy tests to screen metallo-beta- lactamase-producing strains of Pseudomonas
and Acinetobacter species
. Clin Microbiol Infect. 2001;7:88–91.
Arlet G, Rouveau M and Philippon A. Substitution of alanine for aspartate at position 179 in the SHV-6 extended-spectrum beta- lactamase. FEMS Microbiol Lett. 1997; 152:163–167.
Eckert C, Gautier V, Saladin-Allard M, et al. Dissemination of CTX- M-type beta-lactamases among clinical isolates of Enterobacteriaceae
in Paris, France. Antimicrob Agents Chemother. 2004;48:1249–55.
Saladin M, Cao VT, Lambert T, et al. Diversity of CTX-M beta- lactamases and their promoter regions from Enterobacteriaceae
isolated in three Parisian hospitals. FEMS Microbiol Lett. 2002; 209: 161–168.
Perez-Perez FJ and Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol. 2002; 40: 2153–62.
Poirel L, Heritier C, Tolun V and Nordmann P. Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae
. Antimicrob Agents Chemother. 2004; 48:15–22.
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL and Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods. 2005; 63: 219–228.
Jain A and Mondal R. Prevalence & antimicrobial resistance pattern of extended spectrum beta-lactamase producing Klebsiella spp
isolated from cases of neonatal septicaemia. Indian J Med Res. 2007; 125: 89–94.
Oteo J, Cuevas O, Lopez-Rodriguez I, et al. Emergence of CTX-M- 15-producing Klebsiella pneumoniae
of multilocus sequence types 1, 11, 14, 17, 20, 35 and 36 as pathogens and colonizers in newborns and adults. J Antimicrob Chemother. 2009; 64: 524–8.
Hawkey PM. Prevalence and clonality of extended-spectrum beta- lactamases in Asia. Clin Microbiol Infect. 2008;14:159–65.
Karim A, Poirel L, Nagarajan S and Nordmann P. Plasmid- mediated extended-spectrum beta-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiol Lett. 2001;201:237–41.
Coque TM, Novais A, Carattoli A, et al. Dissemination of clonally related Escherichia coli
strains expressing extended-spectrum beta- lactamase CTX-M-15. Emerg Infect Dis. 2008;14:195–200.
Damjanova I, Toth A, Paszti J, et al. Expansion and countrywide dissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type beta-lactamase-producing Klebsiella pneumoniae
epidemic clones in Hungary in 2005--the new 'MRSAs'. J Antimicrob Chemother. 2008; 62: 978–85.
Hammami S, Boutiba-Ben Boubaker I, Saidani M, et al. Characterization and molecular epidemiology of extended spectrum beta-lactamase producing Enterobacter cloacae
isolated from a Tunisian hospital. Microb Drug Resist 2012; 18: 59–65.
Lavollay M, Mamlouk K, Frank T, et al. Clonal dissemination of a CTX-M-15 beta-lactamase-producing Escherichia coli
strain in the Paris area, Tunis, and Bangui. Antimicrob Agents Chemother 2006; 50: 2433–8.
Cao V, Lambert T, Nhu DQ, et al. Distribution of extended- spectrum beta-lactamases in clinical isolates of Enterobacteriaceae
in Vietnam. Antimicrob Agents Chemother. 2002; 46: 3739– 43.
Bonnet R. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother. 2004; 48: 1–14.
Mnif B, Harhour H, Jdidi J, et al. Molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli
in Tunisia and characterization of their virulence factors and plasmid addiction systems. BMC Microbiol. 2013; 13: 147.
Rejiba S, Mercuri PS, Power P and Kechrid A. Emergence and dominance of CTX-M-15 extended spectrum beta-lactamase among Escherichia coli
isolates from children. Microb Drug Resist. 2011;17:135–40.
Jacoby GA and Han P. Detection of extended-spectrum beta- lactamases in clinical isolates of Klebsiella
pneumoniae and Escherichia coli
. J Clin Microbiol. 1996;34: 908–11.
Nordmann P, Naas T and Poirel L. Global spread of Carbapenemase- producing Enterobacteriaceae
. Emerg Infect Dis. 2011;17:1791–98.
Ktari S, Mnif B, Louati F, et al. Spread of Klebsiella pneumoniae
isolates producing OXA-48 beta-lactamase in a Tunisian university hospital. J Antimicrob Chemother. 2011;66:1644–6.
Saidani M, Hammami S, Kammoun A, Slim A, and Boutiba- Ben Boubaker I. Emergence of carbapenem-resistant OXA-48 carbapenemase-producing Enterobacteriaceae
in Tunisia. J Med Microbiol. 2012;61:1746–1749.
Ben Slama K, Jouini A, Ben Sallem R, et al. Prevalence of broad- spectrum cephalosporin-resistant Escherichia coli
isolates in food samples in Tunisia, and characterization of integrons and antimicrobial resistance mechanisms implicated. Inter J Food Microbio. 2010;137:281–6.
Carattoli A, Lovari S, Franco A, Cordaro G, Di Matteo P and. Battisti A. Extended-spectrum beta-lactamases in Escherichia coli
isolated from dogs and cats in Rome, Italy, from 2001 to 2003. Antimicrobial Agents Chemother. 2005;49:833–5.
Pitout, J. D., and K. B. Laupland. Extended-spectrum beta- lactamase-producing Enterobacteriaceae:
an emerging public-health concern. Lancet Infect Dis. 2008;8: 159–66.
Verdet C, Arlet G, Ben Redjeb S, Ben Hassen A, Lagrange PH and Philippon A. Characterisation of CMY-4, an AmpC-type plasmid- mediated beta-lactamase in a Tunisian clinical isolate of Proteus mirabilis
. FEMS Microbiol Lett. 1998;169:235–40.
Sallem RB, Gharsa H, Slama KB, et al. First detection of CTX-M-1, CMY-2, and QnrB19 resistance mechanisms in fecal Escherichia coli
isolates from healthy pets in Tunisia. Vector Borne Zoonotic Dis. 2013;13:98–102.
Ktari S, Arlet G, Mnif B, et al. Emergence of multidrug-resistant Klebsiella pneumoniae
isolates producing VIM-4 metallo-beta- lactamase, CTX-M-15 extended-spectrum beta-lactamase, and CMY-4 AmpC beta-lactamase in a Tunisian university hospital. Antimicrob Agents Chemother. 2006;50: 4198–201.
Carattoli, A. Plasmids in Gram negatives: molecular typing of resistance plasmids. Int J Med Microbiol. 2011; 301:654–8.
[Table 1], [Table 2]