Distribution of bla TEM,bla SHV,bla CTX-M Genes Among Clinical Isolates of Klebsiella pneumoniae at Labbafinejad Hospital,Tehran,Iran

Abstract

Extended-spectrum β-lactamase (ESBL)-producing isolates of Klebsiella pneumoniae have been increasingly recognized in the hospital settings in Iran as well as throughout the world. The aim of this study was to detect and determine the genes encoding the ESBLs including bla_TEM, bla_SHV, and bla_CTX-M groups among the K. pneumoniae isolates at Labbafinejad Hospital by polymerase chain reaction (PCR) and characterize them by direct sequencing of PCR products. Eighty-nine isolates were isolated from patients at different wards during March 2008–March 2009. They were identified as K. pneumoniae using biochemical tests. Susceptibility of isolates to 17 different antimicrobial agents was determined using agar disk diffusion method. The phenotypic confirmatory test was used to screen the isolates for production of ESBLs. To amplify the bla_SHV the template DNA was extracted by boiling method. Plasmid DNA was extracted using minipreparation kit and used as template in PCR for detection of bla_TEM and bla_CTX-M. The selected PCR products were sequenced and analyzed. All 89 strains were susceptible to imipenem. The rates of resistance to different antibiotics were in the following order: aztronam (79.7%), cefexime (67.4%), cefpodoxime (66.2%), cefotaxime (65.1%), ceftazidime (61.7%). The phenotypic confirmatory test detected 62 isolates (69.7%) as ESBL-producing K. pneumoniae. The prevalence of genes encoding ESBLs were as follows: bla_TEM 54% (n = 48), bla_SHV 67.4% (n = 60), bla_CTX-M-I 46.51% (n = 40), and bla_CTX-M-III 29% (n = 25). The bla_CTX-M-II and bla_CTX-M-IV were not detected. All bla_TEM types were characterized as bla_TEM-1 and all bla_CTX-M-I were identified as bla_CTX-M-15. The SHV types were characterized as SHV-5, SHV-11, and SHV-12. The rate of ESBL at Labbafinejad Hospital was 25% increase in a 4-year study that ended in March 2009. It appears that bla_TEM-1, bla_SHV-5, bla_SHV-11, bla_SHV-12, and bla_CTX-M-15 are the dominant ESBLs among the resistant strains of K. pneumoniae in Iran.

Introduction

Extended-spectrum β-lactamases (ESBLs) were first described in Klebsiella pneumoniae and Serratia marcescens isolates in 1983 in Europe and subsequently found among K. pneumoniae and Esherichia coli isolates from the United States in 1989.¹¹ The ESBLs are enzymes that can be produced by bacteria, making them resistant to cephalosporins, for example, cefuroxime, cefotaxime, and ceftazidime, which are the most widely used antibiotics in many hospitals.¹⁴ More than 340 β-lactamases have been identified. Gram-negative betalactamases are divided into four classes designated as Classes A–D.²⁰ Class A enzymes include bla_TEM and bla_SHV families. SHV may be more frequently found in clinical isolates than any other type of ESBLs.²³ TEM-1 was first reported in 1965 from an E. coli isolate from a patient in Athens, Greece, named Temoneira (hence the designation TEM).²³ The TEM-type ESBLs are derivatives of TEM-1 and TEM-2. In 1989, a clinical isolate of E. coli that produced a non-TEM, non-SHV ESBL was reported. The enzyme was designated CTX-M-1, indicating its preferential hydrolytic activity against cefotaxime.¹² Although spread of CTX-M–producing isolates appeared to be limited to specific areas in the world during the 1990s, this situation has changed during the past decade. Recent epidemiological studies of ESBL-producing bacteria demonstrated a dramatic increase in the prevalence of CTX-M enzymes globally.^5,26 Over 40 different types of CTX-M have been found in different Enterobacteriaceae species.²² They are divided into five subgroups, namely, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25, according to the similarity of their amino acid sequences. According to the National Committee for Clinical Laboratory Standards' guidelines,⁷ antibiotics can be used for screening ESBLs in K. pneumoniae. Subsequently, a confirmatory test was done by adding clavulanic acid according to the manufacturer's recommendation. The aims of this study were to determine the prevalence of ESBL-producing isolates of K. pneumoniae at Labbafinejad Teaching Hospital and to detect and characterize the bla_TEM, bla_SHV, and bla_CTX-M genes by polymerase chain reaction (PCR) and DNA sequencing.

Materials and Methods

Bacterial strains

Eighty-nine isolates from hospitalized patients were identified as K. pneumoniae, as described previously.¹⁰ These isolates were cultured from the clinical specimens including urine (n = 70), trachea (n = 6), wounds (n = 5), blood (n = 3), sputum (n = 2), and others (n = 3).

E. coli ATCC 35218 (bla_TEM-1), K. pneumoniae ATCC 700603 (bla_SHV-18), and K. pneumoniae K.P 7881 containing bla_CTX-M type (kindly presented by Professor Nordmann) were used as positive controls for PCR reactions. K. pneumoniae ATCC 700603 was used as positive control, and E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as negative controls in antibiotic susceptibility testing.

Antimicrobial susceptibility testing

Isolates were plated on Mueller–Hinton agar and their susceptibilities to 17 different antibiotics were tested by disk diffusion method according to the Clinical and Laboratory Standard Institute's guidelines.¹²

ESBL screening

The strains were phenotypically screened using ceftazidime, +/− clavulanic acid and cefotaxime, +/− clavulanic acid.

DNA preparation

Bacteria were grown on nutrient agar at 37°C overnight. The plasmid DNA was used for detection of bla_TEM and bla_CTX-M groups. Plasmid DNA was extracted using Mini prep K0502 kit (Fermentas, Vilnius, Lithuania). To extract the chromosomal DNA, a loopful of bacterial colonies was harvested from a blood agar plate and suspended in 0.5 ml of sterile water. The suspension was heated at 95°C for 10 min. After centrifugation at 5,000 rpm, the sediment was frozen at −20°C for 20 min and then recentrifuged at 10,000 rpm. The supernatant containing DNA was used as the source of template for detection of bla_SHV.²⁸

PCR amplification

The primers for bla_TEM and bla_SHV genes were designed by downloading some sequences of both TEM and SHV variants available from the GenBank Database (K. pneumoniae bla_SHV-18 Accession Number AF132290 and E. coli bla_TEM-1 Accession Number AM886293). These variants were aligned using ClustalW2 software (clustal.ebi.ac.uk). Primers were designed from the consensus sequence and analyzed for hair-pin loop and primer dimer formation using Gene Runner software (Hasting software, Hastings Inc., Hudson, NY). The primers described by Pitout et al.²⁴ were used to amplify family- or group-specific bla_CTX-M genes. These groups were designated bla_CTX-M-I, bla_CTX-M-II, bla_CTX-M-III, and bla_CTX-M-IV. The bla_TEM, bla_SHV, and bla_CTX-M (including bla_CTX-M-I, bla_CTX-M-II, bla_CTX-M-III, bla_CTX-M-IV) were amplified and the amplicons from selected strains were sequenced. The primers, the PCR conditions, and sizes of the PCR products are shown in Table 1. Amplification was performed in a 25 μl volume containing 2.5 μl of 10 × PCR reaction buffer, 2 μl MgCl₂ (25 mM), 0.5 μl dNTPs (10 mM), 0.5 μl (each) primers (10 pmol/ml) with 0.2 μl (500 U/ml) Taq DNA polymerase (Fermentas). Five microliters of the template DNA was added to the reaction mixture. PCR amplifications were carried out on a thermal cycler (Eppendorf, Hamburg, Germany). The cycling conditions for amplification were as follows: for bla_SHV, initial denaturation at 95°C for 2 min and 30 cycles of 1 min at 95°C, 30 s at 56°C, and 30 s at 72°C, followed by 5 min at 72°C; for bla_TEM gene, initial denaturation of 2 min at 95°C and 30 cycles of 30 s at 95°C, 30 s at 58°C, and 1 min at 72°C, followed by 5 min at 72°C; for bla_CTX-M-I, bla_CTX-M-II, and bla_CTX-M-III, initial denaturation at 95°C for 2 min; and for bla_CTX-M-IV, initial denaturation at 95°C for 2 min and 30 cycles of 1 min at 95°C, 45 s at 62°C, and 45 s at 72°C, followed by 5 min at 72°C. The resulting PCR products were analyzed by electrophoresis with 1.5% agarose gels.

Table 1.

Primers Used for Amplification

Target	Primer	Sequence	Product size (bp)	Annealing temperature (°C)
bla _CTX-M-I	CTXM1-F3	GAC GAT GTC ACT GGC TGA GC	499	55
	CTXM1-R2	AGC CG C CGA CGC TAA TAC A
bla _CTX-M-II	TOHO1-2F	GCG ACC TGG TTA ACT ACA ATC C	351	55
	TOHO1-1R	CGG TAG TAT TGC CCT TAA GCC
bla _CTX-M-III	CTXM825F	CGC TTT GCC ATG TGC AGC ACC	307	55
	CTXM825R	GCT CAG TAC GAT CGA GCC
bla _CTX-M-IV	CTXM914F	GCT GGA GAA AAG CAG CGG AG	474	62
	CTXM914R	GTA AGC TGA CGC AAC GTC TG
bla _SHV	SHVF	TCAGCGAAAAACACCTTG	472	56
	SHVR	TCCCGCAGATAAATCACC
bla _TEM	TEMF	CTTCCTGTTTTTGCTCACC	632	58
	TEMR	AGCAATAAACCAGCCAGC

Sequencing

Based on the drug susceptibility patterns and the wards of the study hospital, amplicons from 34 isolates were selected and sequenced for bla_SHV (n = 4), bla_TEM (n = 17), and bla_CTX-M-I (n = 13). The sequences were analyzed using the BLAST program (www.ncbi.nlm.nih.gov/BLAST).

Results

Antimicrobial susceptibility testing

All isolates were susceptible to imipenem. With a resistant rate of 79.7%, aztronam and amoxiclave were the least effective antibiotics against isolates of K. pneumoniae in this study. The rates of resistance to other antibiotics were in the following order: cefexime (67.4%), cefpodoxime (66.2%), cefotaxime (65.1%), ceftazidime (61.7%), ceftriaxone (55%), cefepime (49.4%,), ciprofloxacin (42.7%), gentamicin (34.8%), nitruforantoin (22.5%), amikacin (16.9%), and piperacillin/tazobactam (14.6%).

ESBL screening

The phenotypic confirmatory test (PCT) detected 62 isolates (69.7%) as ESBL producers. The resistance patterns of these isolates are shown in Table 2.

Table 2.

Antimicrobial Susceptibilities of the Extended-spectrum β-lactamase–Producing Klebsiella pneumoniae Isolates

Antibiotic	Sensitive, n (%)	Intermediate, n (%)	Resistance, n (%)	Total, n (%)
Aztreonam	0 (0)	3 (4.8)	59 (95.2)	62 (100)
Amoxiclauv	1 (1.6)	1 (1.6)	60 (96.8)	62 (100)
Cefotaxime	2 (3.2)	4 (6.5)	56 (90.3)	62 (100)
Cefpodoxime	3 (4.8)	1 (1.6)	58 (93.5)	62 (100)
Ceftriaxone	7 (11.3)	8 (12.9)	47 (75.8)	62 (100)
Cefexime	4 (6.5)	0 (0)	58 (93.5)	62 (100)
Ceftazidime	5 (8)	4 (6.5)	53 (85.5)	62 (100)
Cotrimaxasol	13 (21)	2 (3.2)	47 (75.8)	62 (100)
Kanamicin	10 (16.1)	2 (3.2)	50 (80.6)	62 (100)
Tobramicin	12 (19.4)	0 (0)	50 (80.6)	62 (100)
Cefepime	14 (22.4)	4 (6.5)	44 (70.9)	62 (100)
Nitrofurantoin	28 (42.5)	18 (29)	16 (25.8)	62 (100)
Piperacillin/tazobactam	26 (41.9)	24 (38.7)	12 (19.4)	62 (100)
Ciprofloxacin	25 (40.4)	5 (8)	32 (51.6)	62 (100)
Gentamicin	28 (45.1)	4 (6.5)	30 (48.4)	62 (100)
Amikacin	38 (61.3)	10 (16.1)	14 (22.6)	62 (100)
Imipenem	62 (100)	0 (0)	0 (0)	62 (100)

PCR amplification

Amplification of DNA from 89 isolates showed the prevalence of bla_TEM, bla_SHV, bla_CTX-M-I, and bla_CTX-M-III as 54%, 67.4%, 46.5%, and 29%, respectively. The bla_CTX-M-II and bla_CTX-M-IV were not detected in this study. The sizes of amplicons for bla_SHV, bla_TEM, bla_CTX-M-I, and bla_CTX-M-III were 472, 636, 499, and 307 bp, respectively. To identify the group of the bla_CTX-M family, 13 samples of bla_CTX-M-I–positive isolates were sequenced. The sequencing results showed that they belong to bla_CTX-M-15. It is also shown that all of the bla_CTX-M-III isolates are bla_CTX-M-8.

All PCR products obtained for bla_TEM in this study were characterized as bla_TEM-1 after sequencing. Unlike bla_TEM, sequences of amplicons from bla_SHV were characterized as bla_SHV-12, bla_SHV-5, and bla_SHV-11. The sequences obtained for bla_TEM, bla_SHV5, and bla_CTX-M were submitted to GenBank under GQ470427 through GQ470460 accession numbers.

Discussion

ESBL-producing Enterobacteriaceae are now an increasing problem worldwide. The emergence and progressive spread of these bacteria seem to be caused mainly by extensive use of broad-spectrum β-lactamase in empiric therapy and rapid plasmid-mediated distribution of resistance genes between bacterial species.

More than 75% of the studies have addressed ESBL-producing infections with K. pneumoniae.²³ Using PCT protocol, our findings showed that 62 isolates were identified as ESBL producers. The number of isolates detected as ESBL producers by cefotaxime, +/− clavulanic (n = 60) exceeded those detected by ceftazidime, +/− clavulanic acid (n = 57).

The most prevalent genes detected in this study were bla_SHV (n = 60, 67.4%), followed by bla_TEM (n = 48, 54%), bla_CTX-M-I (n = 34, 38.2%), and bla_CTX-M-III (n = 24, 27%). Simultaneous existence of multiple genes encoding ESBLs was detected among the isolates and these genes include bla_TEM, _SHV, _CTX-M (n = 15), bla_TEM, _CTX-M (n = 21), bla_SHV, _CTX-M (n = 22), and bla_TEM, _SHV (n = 30). Four isolates (4.5%) of phenotypically confirmed ESBL isolates do not have any ESBL-encoding genes (bla_TEM, _SHV, _CTX-M). These isolates probably produce other ESBL enzymes. Moreover, of 60 isolates that were positive for bla_SHV, 17 were negative in PCT. This is not surprising because our sequencing detected type1 bla_SHV. This is one of the reasons for negativity of some isolates in PCT while they were positive in PCR assay. Almost all non–ESBL-producing K. pneumonia isolates have chromosomally mediated SHV-1 β-lactamases.⁴ Similarly, of 47 isolates that were positive for bla_TEM, 12 were not detected as ESBL producing by PCT in this study. The sequences obtained for bla_TEM corresponded to TEM-1 type. It should be noted that TEM-1 is not ESBL but is widespread in other species too.²³ In contrast to bla_TEM and bla_SHV, consistent results were obtained for bla_CTX-M because all isolates containing this type of genes were positive in PCT. Moreover, the sequencing demonstrated that bla_CTX-M-I and bla_CTX-M-III encode CTX-M-15 and CTX-M-8, respectively. This is the first report on the existence of bla_CTX-M-15 and bla_CTX-M-8 in Iran. These results are compliant with the previous studies in other countries such as Europe. Recently, clonal outbreaks caused by CTX-M-15 K. pneumoniae have been reported in Scandinavia.¹⁷ A recent dramatic increase in ESBL-producing organisms is being observed both in hospitals and in the community, mainly caused by the CTX-M-15 enzyme.¹⁶ This enzyme has now become the most prevalent enzyme in hospitals and in the community.^16,29 In France, the prevalence of ESBL production in Enterobacteriaceae reported in CTX-M variants belonged primarily to the CTX-M-I (85%) and CTX-M-9 (11.3%).³ TEM-24, CTX-M-15, CTX-M-32, and SHV-12 are frequently detected in both Spain and Portugal.^13,18 In Bulgaria, hospital outbreaks caused by CTX-M-3, CTX-M-15, and SHV-12 are described, often with an involvement of S. marcescens, in addition to K. pneumoniae.¹⁹ In Hungary, a recent eruptive and extensive spread of highly ciprofloxacin-resistant CTX-M-15 K. pneumoniae epidemic clones has been detected.⁹ In Turkey, CTX-M-15 is widely distributed^15,30 and epidemic strains of K. pneumoniae isolates producing the carbapenemase OXA-48 and the ESBLs SHV-12 or CTX-M-15 have emerged.⁶ The prevalence of bla_SHV, bla_TEM, and bla_CTX-M among isolates of K. pneumonia in Saudi Arabia has been reported as 97.3%, 84%, and 34%, respectively.¹ Characterization of CTX-M variants in that country demonstrated that they belonged to CTX-M-I (60%) and CTX-M-9 (40%). In Pakistan, the prevalence of ESBL-producing K. pneumonia was very high (70%).²⁷ In the United Arab Emirates, 36% of K. pneumonia isolates have been reported as positive for ESBL production.² In Lebanon, the prevalence of ESBL production in Enterobacteriaceae was reported in 72% strains and the majority of the strains (83%) expressed CTX-M-15 (83%) and SHV-5a (18%).²¹

It appears that the annual rate of infection with ESBL-producing strain of K. pneumoniae has increased at Labbafinejad hospital since 2005. The prevalence of these strains at Tehran hospitals has been reported as 45.5% at that time.¹⁰ Because of agreement between the results obtained from both PCT and PCR test for bla_CTX-M (100%), the CTX-M seems to be the most active enzyme against the new cephalosporins. Control of endemic ESBL producers is difficult and may only be possible after significant nursing and medical reorganization at a substantial financial cost.^8,25 Therefore, control of the initial outbreak of ESBL-producing organisms in a hospital or specialized unit of a hospital is of critical importance.

Footnotes

Acknowledgment

This project was jointly supported by the Tehran University of Medical Science (for Bacteriology and PCR experiments) and the Research Center for Infectious Diseases and Tropical Medicine of Shahid Beheshti University of Medical Sciences (for sequencing part of the project).

Disclosure Statement

No competing financial interests exist.

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