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TEM & SHV genes in extended spectrum [beta]-lactamase producing Klebsiella species & their antimicrobial resistance pattern.

Background & objectives: Extended spectrum [beta]-lactamases (ESBLs) are often plasmid mediated derived from mutations in the classic TEM and SHV genes by one or more amino acid substitution around the active site. Detection of TEM and SHV genes by molecular methods in ESBL producing bacteria and their pattern of antimicrobial resistance can provide useful information about its epidemiology and risk factors associated with these infections. We investigated the presence of TEM and SHV genes in ESBL producing Klebsiella spp. and their antimicrobial resistance pattern in cases of neonatal septicaemia in a tertiary care hospital.

Methods: A total of 130 clinical isolates of Klebsiella spp. isolated from septicaemic neonates of a neonatal intensive care unit (NICU) from a tertiary care hospital in north India, were screened for ESBL production by combined disk diffusion method. PCR was used to detect TEM and SHV genes in ESBL positive isolates. Isoelectric points of ESBL enzymes from a few isolates (n = 6) were noted for typing of ESBL by isoelectric focusing.

Results: Of the 64 ESBL producing Klebsiella spp. isolates, 17 (26.5%) had both TEM and SHV genes, 31 (48.4%) had TEM alone and 13 (20.3%) had SHV gene alone. Three (4.6%) ESBL positive isolates were negative for both TEM and SHV. Isolates with both TEM and SHV genes were highly resistant to antibiotics used. Degree of resistance for 3rd generation cephalosporins was also high in these isolates. Six randomly selected isolates were subjected to isoelectric focussing. Results of isoelectric focussing were comparable with PCR.

Interpretation & conclusions: Presence of TEM gene in ESBL producing Klebsiella spp. was more common than SHV gene. Frequency of antibiotic resistance was high in isolates having both TEM and SHV genes.

Key words ESBL-Klebsiella spp.-neonatal septicaemia-SHV-TEM

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Extended spectrum [beta]-lactamase (ESBL) producing Klebsiella spp. has been frequently implicated in outbreaks in paediatric intensive care units (PICUs) and neonatal intensive care units (NICUs) (1,2). ESBLs are often plasmid mediated and derived from mutations in the classic TEM (Temoneira) and SHV (Sulphydryl variable) genes by one or more amino acid substitution around the active site (3). TEM-1 [beta]-lactamase producing bacteria are spread worldwide and it is now the most commonly encountered mechanism of resistance to the [beta]-1actam group of drugs in Gram-negative bacilli (4). Over 100 variations and point mutations in TEM gene were reported by DNA sequencing (5). These mutations are the most responsible factor for resistance to beta lactams in these isolates (6). [The SHV family of [beta]-lactamases has been derived from Klebsiella spp. SHV-1 is universally found in K. pneumonia (7), evolved as a chromosomal gene in Klebsiella spp. and was later incorporated into a plasmid, which has spread to other enterobacterial species (6)]. A total of 40 types of SHV type ESBL enzymes are already reported (5).

There are various reports (8,9) on the prevalence of ESBL producing Klebsiella spp. from India. A recent study by Lal et al (10) describes occurrence of genetic variants in K. pneumoniae from clinical samples of various origin and reported that isolates having both TEM and SHV genes were more common than TEM and SHV alone. However, published information on genetic variability of ESBL producing Klebsiella spp. from India is limited.

We studied the presence of TEM and SHV genes in ESBL producing Klebsiella spp. isolated from septicaemic neonates admitted to a neonatal intensive care unit (NICU) of a tertiary care hospital in north India and their antibiotic resistance pattern.

Material & Methods

A total of 4497 blood samples, consecutively collected from suspected cases of neonatal septicaemia during a period of three years (December 15, 2003-December 14, 2006) admitted to the NICU of King George's Medical University, Lucknow, were referred to the Department of Microbiology for blood culture. Samples from older children/adults and from neonates with other clinical diagnosis were excluded. The study protocol was approved by the institutional ethical committee.

All the samples were processed by standard methods. Blood (1-2 ml) was collected into 10 ml of brain heart infusion broth (Hi-media laboratories, India) with 0.05 per cent sodium polyanethol sulphonate. The broth was incubated at 37[degrees]C, overnight. A blind subculture on McConkey agar plate and blood agar plate (Hi-media laboratories, India) was done. Any sign of growth was followed by subculture and identified by Gram staining. Gram-negative rods were identified by relevant biochemical test (11) i.e., motility test, MR/VP and sugar fermentation test. All isolates, which were confirmed as Klebsiella spp., were used for further analysis.

Antimicrobial susceptibility testing: Antimicrobial susceptibility testing was determined by Kirby-Bauer's (12) disc diffusion method as per Clinical Laboratory Standards Institute-2006 (CLSI) recommendations (13). A whole range of antibiotics (concentration of antibacterial in [micro]g) was included to study the resistance. Antimicrobial discs used were ampicillin (10), amoxycillin/clavulanic acid (10/20), piperacillin (100), piperacillin/tazobactem (100), ticarcillin (75), ticarcillin/clavulanic acid (75/10), cefixime (5), cefuroxime (30), cefpodoxime (10), cephotaxime (30), ceftazidime (30), aztreonam (30), netilmycin (30), amikacin (30), gentamycin (30), chlorophenicol (30), co-trimoxazol (30), tetracycline (30), imipenam (10) and meropenam (10). All the antibiotics were procured from Hi-media laboratories, Mumbai (India). Isolates showing inhibition zones [less than or equal to] 27 mm for cephotaxime and [less than or equal to] 22 mm for ceflazidime were screened as potential ESBL producers.

Minimum inhibitory concentration (MIC) testing: MIC was determined by agar dilution methods for ceftazidime (0.25-128 [micro]g/ml), cephotaxime (0.25-128 [micro]g/ml) and cefpodoxime (0.25-128 [micro]g/ml) using series of dilution according to CLSI-2006 guidelines (13), as described earlier (14). Confirmation of ESBL production was done (15) as described earlier (14).

Isoelectric focussing: The isoelectric focusing was performed by harvesting 20 h brain infusion broth (Hi-media laboratories, India) culture by centrifugation. Pellet was resuspended in 1 ml of phosphate buffer (0.05 M: PH 7). The enzymes were released by two cycles of freezing (-70[degrees]C) and thawing at room temperature and subjected to 10 pulses of sonication (full power, 0.5 seconds per pulse) for 5 min in a sonicator (Heat systems, Ultrasonics W 380, USA) in ice cold water. Isoelectric focusing was done in an ampholine gel (Ph 3.0-Ph 10) (16).

Detection of [bla.sub.TEM] and [bla.sub.SHV] gene: Genomic DNA was extracted from well grown cultures of Klebsiella spp. by the method described by Jain et al (17). Detection of [bla.sub.TEM] and [bla.sub.SHV] genes from total bacterial DNA was performed with a subset of primers (18,19) that amplified 867 bp [bla.sub.TEM] and 930 bp [bla.sub.SHV], coding region in Klebsiella spp. genome. The primers (Banglore Gennei, India) used were-:

For [bla.sub.TEM]

5'-ATAAAATTC TTGAAGACGAAA-3'

5'-GACAGT TACCAATGCTTAATCA-3'

For [bla.sub.SHV]

5'-GGGTTATTCTTATTTGTCGC-3'

5'-TTAGCGTTGCCAGTGCTC-3 '

Reactions were performed in a DNA thermal cycler (Techne, UK) in 20[micro]l mixtures containing 1.5 U Taq polymerase (Genome, India) and 1 x buffer consisting of 10 mM Tris HCl (pH-8.3), 1.5 mM Mg[Cl.sub.2], 50 mM KCl, 0.01 [micro]g of gelatin, each deoxynucleoside triphosphate at a concentration of 200[micro]M and each oligonucleotides primer at a concentration of 50 picomoles/[micro]l. PCR profile was an initial denturation at 94[degrees]C for 5 min followed by 35 cycles of 94[degrees]C for one min, 52[degrees]C for one min and 72[degrees]C for one min and a final extension cycle at 72[degrees]C for 10 min. PCR products were analyzed by electrophoresis with 1.5 per cent agarose gel. After staining with ethidium bromide the gel was photographed on an ultraviolet light transilluminator by gel documentation system (Sigma, USA).

Quality control for ESBL detection, isoelectric focussing and PCR: K. pneumoniae ATCC700603 (CMC, Vellore) (ESBL positive) was used as quality control for ESBL test and PCR. On disk diffusion testing the zone diameter ranges (in mm) for K. penumoniae ATCC700603 were as follows; cefpodoxime 9-16, ceftazidime 10-18, aztreonam 9-17 and cefotaxime 17-25. In disc diffusion phenotypic testing, K. pneumoniae ATCC700603 shows [less than or equal to] 5 mm increase in ceftazidime/clavulanic acid zone diameter. Preparations from standard strain known to harbour TEM-1, SHV-1 and SHV-5 were used as standards for isoelectric focussing. An ESBL negative strain (CMC, Vellore) was also used along with positive samples for isoelectric focussing and PCR.

Statistical analysis: Comparison of proportion was done by using chi square test, and 95 per cent confidence interval was applied where difference was statically significant for antibiotic resistance among groups using STATA version 9.2 (Texas, USA) software.

[FIGURE 1 OMITTED]

Results & Discussion

Of the 130 clinical isolates of Klebsiella spp., 64 were ESBL positive. All these were subjected to antimicrobial susceptibility testing and those with TEM and SHV gene showed high resistance (Table I).

Genotypic characterization by PCR of all ESBL positive isolates (n=64) of Klebsiella spp. revealed that 867 bp and 930 bp amplified products were seen in isolates with [bla.sub.TEM] and [bla.sub.SHV] genes respectively (Figs 1, 2). Forty eight isolates (75.0%) contained [bla.sub.TEM] gene, while 30 isolates (46.8%) contained [bla.sub.SHV] gene, 17 (26.5%) had both [bla.sub.TEM] and [bla.sub.SHV] genes and 31 (48.4%) had TEM gene alone. Thirteen (20.3%) had SHV gene alone. Three isolates (4.6%) did not show either TEM or SHV gene.

Six randomly selected isolates (2 each from TEM, SHV and TEM+SHV positive groups) were subjected to isoelectric focussing. Results showed that TEM positive isolates produced beta lactamases consistent with pI (isoelectric point) of 5.4 and 5.6. SHV positive isolates were categorized with pI of 7.6 and 8.2, representing the presence of SHV type beta lactamases. Both TEM +SHV positive isolates produced beta lactamase with pI of 5.6, and 8.0 signifying the presence of TEM and SHV both type ESBLs.

Frequency of antibiotic resistance was high in those isolates of Klebsiella spp., which had TEM and SHV both genes but they were statistically significant for antibiotic amoxicillin/clavulanic acid only (Table I). These isolates also had elevated MIC for cephotaxime and ceftazidime (Tables II, III).

[FIGURE 2 OMITTED]

Patients having infections caused by ESBL producing organism are at an increased risk of treatment failure with broad-spectrum [beta]-1actam antibiotics. Therefore, it is recommended that any organism confirmed for ESBL production be reported as resistant to all broad-spectrum [beta]-1actam antibiotic, regardless of the susceptibility test result (13). While some ESBL producing strains have overt resistance to broad-spectrum [beta]-1actam antibiotics, many will not be phenotypically resistant. Therefore, it is important for clinical microbiology laboratory to be aware of isolates that may show increased MICs of oxyimino-cephalosporins even though these may not be reported as resistant, as this might suggest the presence of ESBLs (19).

The phenotypic test only presumptively identifies the presence of ESBL. The task of identifying the specific ESBL present in clinical isolates is more complicated. In the early days, determination of the isoelectric points was sufficient to identify the ESBL. However, with >100 TEM type [beta]-lactamases, many of which present identical isoelectric points, determination of ESBL by isoelectric points is no longer possible (20). A similar situation is seen in the SHV, CTXM (cefotaxime MICs in the resistant range >64 [micro]g/ml) and OXA (oxaciliin-hydrolyzing) families of ESBLs. The easiest and most common molecular method used to detect the presence of a [beta]-lactamase belonging to a family of enzymes is PCR with oligonucleotide primers that are specific for beta-lactamase gene. These primers are usually chosen to anneal to regions where various point mutations are not known to occur. However, PCR will not discriminate among different variants of TEM or SHV and sequencing of open reading frames (ORF) are required to specify it (10).

It is interesting that specific ESBLs appear to be unique to certain country or region. TEM-10 has been responsible for several unrelated outbreaks in the United States (20) and recently reported from in Europe with the same frequency (22). Similarly TEM-3 is common in France, but has not been reported from United States (23). A study from Korea revealed that the SHV [beta]-lactamase is the most common ESBLs found in Korea (24). SHV [beta]-lactamase (especially SHV-5 [beta]-lactamase), is commonly encountered and reported worldwide (14,24). In India, reported frequency of ESBL producing Klebsiella spp. is between 6-87 per cent (2,25,26). Though the prevalence of Klebsiella spp. has been recognized and reported from various parts of the country, there is limited data about its genotypes.

Degree of resistance against third-generation cephalosporins can be highly variable among different ESBL enzymes (27) and phenotypic methods cannot differentiate ESBL types. Molecular methods like isoelectric focussing, PCR and DNA sequencing need to be used for detection and typing of different ESBL enzymes. In conclusion, we report the presence of TEM and SHV genes in ESBL producing Klebsiella spp. isolates from our neonatal septicaemia patients. Drug resistance was high in isolates having both TEM and SHV genes.

Acknowledgment

Authors acknowledge Dr M.P. Singh, Scientist, Industrial Toxicological Research Center, Lucknow, for his support during isoelectric focussing and the Indian Council of Medical Research, New Delhi for partial funding of this project.

Received July 3, 2007

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(27.) Knothe H, Shah P, Krcmery V, Antal M, Mitsuhashi S. Transferable resistance to cephotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection 1983; 11 : 315-7.

Amita Jain & Rajesh Mondal

Department of Microbiology, C.S.M. Medical University, Lucknow, India

Reprint requests: Dr Amita Jain, Professor, Post Graduate Department of Microbiology, C.S.M. Medical University

Lucknow 226 003, India

e-mail: amita602002@yahoo.com
Table I. Comparison of antibiotic resistance patterns of ESBL producing
Klebsiella spp. (n=64) on the basis of genotypes & resistance patterns
of non ESBLs (n=66)

Resistant (%) ESBL negative TEM positive
 (n=66) (n=31)

Ampicillin 42.2 96.9
Ticarcillin 24.2 96.9
Piperacillin 27.2 93.9
Amoxicillin/clavulanic * 13.6 12.1 (22, 58)
Ticarcillin/clavulanic 7.5 48.4
Piperacillin/tazobactem 18.1 6.1
Aztreonam 21.2 93.9
Imipenam 00 00
Meropenam 00 00
Ceftazidime 15.1 93.9
Cephotaxime 15.1 93.9
Ceufuroxime 7.5 66.6
Cefixime 12.1 66.6
Cefpodoxime 13.6 96.9
Amikacin 12.1 30.3
Gentamicin 36.3 58.15
Netilmicin 51.5 81.8
Tetracycline 54.5 84.4
Ciprofloxacin 15.1 45.4
Chloramphenicol 27.2 69.6
Co-trimoxazole 45.4 63.6

Resistant (%) SHV positive TEM + SHV
 (n=13) positive (n=17)

Ampicillin 100 100
Ticarcillin 100 100
Piperacillin 100 100
Amoxicillin/clavulanic * 94.0 (64, 100) 100
Ticarcillin/clavulanic 94.0 100
Piperacillin/tazobactem 23.5 85.7
Aztreonam 100 100
Imipenam 00 00
Meropenam 5.8 00
Ceftazidime 100 100
Cephotaxime 100 100
Ceufuroxime 100 100
Cefixime 100 100
Cefpodoxime 100 100
Amikacin 81.6 94.8
Gentamicin 94.2 100
Netilmicin 94.2 100
Tetracycline 100 100
Ciprofloxacin 76.4 85.7
Chloramphenicol 88.2 100
Co-trimoxazole 100 100

Resistant (%) TEM + SHV
 negative (n=3)

Ampicillin 100
Ticarcillin 100
Piperacillin 100
Amoxicillin/clavulanic * 66.6
Ticarcillin/clavulanic 33.3
Piperacillin/tazobactem 66.6
Aztreonam 100
Imipenam 100
Meropenam 100
Ceftazidime 66.6
Cephotaxime 100
Ceufuroxime 100
Cefixime 100
Cefpodoxime 100
Amikacin 66.6
Gentamicin 100
Netilmicin 100
Tetracycline 100
Ciprofloxacin 100
Chloramphenicol 100
Co-trimoxazole 100

* 95 per cent confidence interval is shown in parenthesis where
difference was statistically significant for antibiotic resistance
among groups

Table II. MIC of cephotaxime in relation with genotypes of ESBL
producing Klebsiella spp.

MIC TEM (n=31) SHV (N=13) TEM & SHV TEM & SHV
([micro]g/ml) n (%) n (%) (n=17) negative
 n (%) (n=3) n (%)

<2 2(6.4) 00 00 00
2 6(19.3) 1(7.6) 00 00
4 2(6.4) 00 00 00
8 4(12.8) 2(15.3) 1(5.8) 00
16 3(9.6) 1(7.6) 2(11.6) 00
32 2(6.4) 1(7.6) 1(5.8) 00
64 3(9.6) 1(7.6) 4(23.23) 2(66.6)
128 1(3.2) 2(15.3) 1(5.8) 00
>128 8(25.8) 5(38.4) 8(47.0) 1(33.3)

Table III. MIC of ceftazidime in relation with genotypes of ESBL
producing Klebsiella spp.

MIC TEM (n=31) SHV (N=13) TEM & SHV TEM & SHV
([micro]g/ml) n (%) n (%) (n=17) negative
 n (%) (n=3) n (%)

>2 1(3.2) 00 00 00
2 8(25.8) 4(30.7) 1(5.8) 00
4 1(3.2) 00 00 00
8 4(12.8) 2(15.3) 1(5.8) 00
16 2(6.4) 00 2(11.6) 1(33.3)
32 5(16.1) 00 1(5.8) 00
64 4(12.8) 1(7.6) 3(17.6) 1(33.3)
128 1(3.2) 2(15.3) 00 1(33.3)
>128 5(16.1) 4(30.7) 9(52.9) 00
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Title Annotation:Temoneira; Sulphydryl variable
Author:Jain, Amita; Mondal, Rajesh
Publication:Indian Journal of Medical Research
Article Type:Report
Geographic Code:9INDI
Date:Dec 1, 2008
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