|Year : 2011 | Volume
| Issue : 3 | Page : 258-261
First detection of TEM-116 extended-spectrum β-lactamase in a Providencia stuartii isolate from a Tunisian hospital
H Lahlaoui1, S Dahmen2, MB Moussa1, B Omrane3
1 laboratory of microbiology, Department of biology, Military Hospital of Tunis, Tunis, Tunisia
2 Infections with Multiresistant Bacteria to Antibiotics (UR/29/04), Department of Microbiology, University Hospital of Sahloul, Sousse, Tunisia
3 Laboratory of Biochemistry and Biotechnology, Department of biology, Faculty of Sciences of Tunis, Campus Universitaire, Tunis, Tunisia
|Date of Submission||03-Jan-2011|
|Date of Acceptance||01-Jul-2011|
|Date of Web Publication||17-Aug-2011|
laboratory of microbiology, Department of biology, Military Hospital of Tunis, Tunis
Source of Support: Tunisian Ministry of Higher Education, Scientifi c
Research and Technology, Conflict of Interest: None
Purpose: To study the resistance to third-generation cephalosporins in Providencia stuartii strain isolated from hospitalized patient in Tunisia and to identify the responsible genes Materials and Methods: This strain was analysed by PCR and sequencing to identify the genes responsible for the β-lactamase resistance phenotypes. The transferability of the phenotypes was tested by conjugation to Escherichia coli J53. The isoelectric point was determinate by isoelectrofocalisation. Results: This resistance was carried by a 60 kb plasmid that encoded a β-lactamase with a pI of 5.4. This β-lactamase revealed identity with the blaTEM-1 gene encoding the TEM-1 β-lactamase, except for a replacement of the Val residue at position 84 by Ile, and the Ala residue at position 184 by Val. These two mutations were encountered in TEM-116 β-lactamase. Conclusion: This study demonstrates the ﬁrst description of TEM-116 in the P. stuartii species in the world and the ﬁrst one in a Tunisian hospital.
Keywords: Extended spectrum β-lactamases, Providencia stuartii, TEM-116, tunisia
|How to cite this article:|
Lahlaoui H, Dahmen S, Moussa M B, Omrane B. First detection of TEM-116 extended-spectrum β-lactamase in a Providencia stuartii isolate from a Tunisian hospital. Indian J Med Microbiol 2011;29:258-61
|How to cite this URL:|
Lahlaoui H, Dahmen S, Moussa M B, Omrane B. First detection of TEM-116 extended-spectrum β-lactamase in a Providencia stuartii isolate from a Tunisian hospital. Indian J Med Microbiol [serial online] 2011 [cited 2019 Sep 19];29:258-61. Available from: http://www.ijmm.org/text.asp?2011/29/3/258/83909
| ~ Introduction|| |
Among members of the family Enterobacteriaceae, extended spectrum β-lactamase (ESBL) production is the most important mechanism of resistance to β-lactam antimicrobial agents, and there are an increasing number of reports of the clinical failure of these drugs, especially in high-risk wards, such as intensive care units. , Typically, ESBLs hydrolyze third generation cephalosporins and aztreonam, but not carbapenems, and are inhibited by clavulanic acid and tazobactam.  These enzymes are mostly commonly classified according to two general schemes: The ambler molecular classification and the Busch-Jacoby-Medeiros functional system. ,
The most common ESBLs identified in the past were those of the TEM and SHV types, which evolved via mutation of earlier penicillinases. The TEM-type ESBLs were considered, until recently, to be the most frequent ESBLs.  Well over 100 TEM-type β-lactamases have been described, of which the majority are ESBLs. Their isoelectric points range from 5.2 to 6.5. All are TEM-1 or TEM-2 derivatives. The first plasmid-mediated β-lactamase in Gram-negatives, TEM-1, was described in the early 1960s.  The TEM-1 enzyme was originally found in a single strain of Escherichia More Details coli isolate from a patient in Athens, Greece, named Temoneira, hence the designation TEM.  Being plasmid and transposon mediated has facilitated the spread of TEM-1 to other species of bacteria. Within a few years after its first isolation, the TEM-1 β-lactamase spread worldwide and is now found in many different species of members of family Enterobacteriaceae such as Enterobacter aerogenes, Morganella morganii, Proteus mirabilis, Proteus rettgeri, and Providencia stuartii.
In this study, we describe the derivate of TEM-1 with an ESBL activity, TEM-116, which was detected in clinical isolate of P. stuartii from Hospital in Tunisia.
| ~ Materials and Methods|| |
Bacterial isolate. One ESBL-producing P.stuartii HL21 was isolated from hospitalized patient in Tunisia, on Febuary 2009. The source of this isolate was rectal swab from a 79-year-old patient hospitalized in intensive care unit (ICU) after a digestive hemorrhage . It was identified as P. stuartii either by automated (GNI+; Vitek) and by manual biochemical analyses using the Api 20E system.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed using the disk diffusion method on Mueller-Hinton agar plates with β-lactam and non-β-lactam antibiotic-containing disks according to the Guideliness Clinical Laboratory Standards Institute. Minimal inhibitory concentrations (MIC) of selected antimicrobial agents were determined by a microdilution technique.  The double-disk synergy test was used to detect the ESBL production as previously described. 
Culture was grown overnight at 37°C in Trypto-Caseine Soy broth (TCS). Bacterial suspension was disrupted by sonication in UP 400 S five times for 45 s each time. Crude extract was centrifuged at 11200 RCF for 15 min at 4°C. The supernatant was stored in aliquots at -20°C and was used for the determination of isoelectric points and β-lactamase activities.
The supernatant of the sonicate was subjected to isoelectric focusing on ampholine polyacrylamide gel with a pH range of 3-10 at a voltage range of 100-300.  Extracts from TEM-1 (Escherichia coli R111), TEM-2 (E. coli RP4), TEM-3 (E. coli D660) and SHV-1 (E. coli P453) producing strains were used as standards for pIs of 5.4, 5.6, 6.3 and 7.6, respectively. β-lactamases activities were revealed by iodemetric method using benzylpenicillin to 1 mM and cefotaxime (3 mM) as substrates in phosphate buffer (25 mM; pH 7). 
Analysis of plasmids and transfer of resistance
The plasmid DNA was extracted by an alkaline lysis method modified from the method described by Birnboim and Doly.  The plasmid size was estimated after digestion with various endonucleases and agarose gel electrophoresis.
Transfer of resistance phenotypes by conjugation was performed with E. coli J53 Azide R as the recipient strain. Transconjugants were selected on Mueller-Hinton agar supplemented with 100 mg/l sodium azide to inhibit the growth of the donor strain, and with 2.5 mg/l ceftazidime to inhibit the growth of the recipient strain. The resulting transconjugants were puriﬁed and identiﬁed with API 20 E.
Polymerase chain reaction ampliﬁcation of bla TEM genes
Total DNA extraction was carried out for all samples using the heat-shock technique.  Detection of TEM encoding gene was performed with previously described oligonucleotides and reaction conditions.  The DNA ampliﬁcation programs consisted of an initial denaturing at 94°C for 10 min, followed by 35 cycles of 1min at 94°C, 1 min at 59°C and 1 min at 72°C. A final extension was performed at 72°C for 10 min. PCR products were analyzed by agarose gel electrophoresis.
Sequencing and analysis of bla TEM
Product of PCR reaction was purified with the PCR purification kit and sequenced. DNA sequences were determined in both strands with an automated sequencer ABI 3100 sequencer. The nucleotide and deduced protein sequences were analysed with software available over the Internet at the National Center for Biotechnology Information.
| ~ Results|| |
β-Lactam susceptibility profile and associated resistance . P. stuartii HL21 has a significant degree of multiresistance as shown by the noticeable resistance level observed with various antibiotics [Table 1]. This strain was resistant to ticarcillin, cefotaxime, ceftazidime, cefoxitin, aztreonam (MIC 512 μg/ml) and cefpirome (MIC 128 μg/ml). The strain was also resistant to chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline and streptomycin, while it remained susceptible to imipenem. The transconjugants showed a signiﬁcant degree of resistance with ticarcillin , ceftazidime, cefotaxime and aztreonam [Table 1].
|Table 1: MICs of various antimicrobial agents obtained for the clinical isolate P. stuartii HL21, transconjugants and the E. coli J53 recipients|
Click here to view
The disk diffusion method showed synergy between ceftazidime, cefotaxime, aztreonam, ceftriaxone, and amoxicillin-clavulanic acid against the strain and its transformants, suggesting the presence of a class A extended-spectrum β-lactamase.
Plasmid profiles and transfer of resistance. Characterization of the resistance plasmid by agarose gel electrophoretic analysis of the DNA extracted from P. stuartii HL21 or E. coli transconjugants revealed the presence of a single plasmid. The plasmid size was estimated to be 60 kb by agarose gel electrophoresis.
Identification of β-lactamases. IEF revealed production of β-lactamases with pI values of 5.4 and 7.6 by P. stuartii HL21. The β-lactamases with a pI of 5.4 were transferred to E. coli J53. Thus, the pI 7.6 band probably represented the chromosomal SHV-1 β-lactamase of the P. stuartii strain. 
Characterization of the extended spectrum β-lactamase
P. stuartii HL21 and and its transformant E. coli DH5α /p HL21 gave positive PCR-based amplifications with specific primers of the blaTEM gene. Sequencing identified that it was identical to TEM-1 gene, except for two-point mutations. These mutations consisted of a replacement of the valine residue at position 84 by Isoleucine (Val84Ile) and that of Alanine at position 184 by Valine (Ala184Val). These two mutations were encountered in TEM-116 β-lactamase [Table 2]. 
| ~ Discussion|| |
The contribution of TEM-derived encoded ESBLs in the resistance mechanism to β-lactam-stable antibiotics has been reported worldwide.,, This ESBLs were considered, until recently, to be the most frequent. Nevertheless, over 180 TEM-type β-lactamases have been described, of which the majority are ESBLs.
In this study, we describe the derivate of TEM-1 with an ESBL activity, TEM-116, which was detected in clinical isolate of P. stuartii from Hospital in Tunisia. It was identical to TEM-1 gene, except for two-point mutations. These mutations consisted of a replacement of the valine residue at position 84 by Isoleucine (Val84Ile) the Alanine at position 184 by Valine (Ala184Val). These two mutations were encountered in TEM-116 β-lactamase.  These mutations, not previously detected in other TEM-type variants might be involved in the high affinity of the TEM-116 enzyme for ceftazidime, cefotaxime and the other extended-spectrum β-lactams.
The amino acid replacements at position 84 and 184 have not been observed in other TEM-type β-lactamases identified before TEM-116 but the amino acid replacements in these positions was observed in TEM-157 (GenBank DQ909059) and TEM-162 (GenBank EF468463) enzymes discovered after the apparition of TEM-116.
TEM-116 was first and most prevalently identified in Korea,  than in Spain,  and Uruguay in Klebsiella pneumonia and E. coli isolates associated, sometimes, with β-lactamases like: TEM-1, PER-1 and SHV-12.
Across the globe, TEM-1 β-lactamase exists at high frequencies in antibiotic-resistant pathogens.  While TEM-1 has a spectrum that is limited to penicillins and early cephalosporins, it has given rise to more than 180 descendent alleles, such as TEM-116. These alleles confer a resistance to most modern β-lactam antibiotics.
This is the first report of a TEM-116 extended spectrum β-lactamase in Providencia stuartii isolate. Therefore, these results highlight the remarkable adaptability of P. stuartii to selective antibiotic pressure.
| ~ Acknowledgments|| |
This work was funded by grants from the Tunisian Ministry of Higher Education, Scientific Research and Technology.
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[Table 1], [Table 2]
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