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MICROBIOLOGY SECTION - ORIGINAL ARTICLE Table of Contents   
Year : 2008  |  Volume : 51  |  Issue : 1  |  Page : 130-136
The prevalence of extended-spectrum β-lactamase in environmental isolates of Enterobacter


1 Department of PG Studies and Research in Biological Sciences, RD University, Jabalpur, MP, India
2 Department of Microbiology, Regional Medical Research Center for Tribals (Indian Council of Medical Research), Jabalpur, MP, India

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   Abstract 

The incidence of extended-spectrum β-lactamase (ESBL)-producing strains and multidrug-resistant strains of Enterobacter spp. isolated from the 1312 km long river Narmada was investigated. Out of the 57 isolates of Enterobacter, 73.68% were found to be ESBL producers including the isolates of E. taylorae and isolates of E. agglomerans, which have been characterized for the first time. All the isolates were found susceptible to the antibiotic imipenem. AmpC gene was found in all the Enterobacter strains tested. AmpC β-lactamase-producing bacterial pathogens may cause major therapeutic failure if not detected and reported in time. It was seen that these enzymes are mainly chromosomally mediated along with several non-AmpC β-lactamase.

Keywords: Drug resistance, Enterobacter, extended-spectrum β-lactamase, polymerase chain reaction

How to cite this article:
Sharma A, Dour P, Singh TN. The prevalence of extended-spectrum β-lactamase in environmental isolates of Enterobacter. Indian J Pathol Microbiol 2008;51:130-6

How to cite this URL:
Sharma A, Dour P, Singh TN. The prevalence of extended-spectrum β-lactamase in environmental isolates of Enterobacter. Indian J Pathol Microbiol [serial online] 2008 [cited 2019 Aug 25];51:130-6. Available from: http://www.ijpmonline.org/text.asp?2008/51/1/130/40426



   Introduction Top


Since the first report in 1983, extended-spectrum β-lactamases (ESBLs) have contributed to the dramatic increase in resistance to β-lactam agents among the members of family Enterobacteriaceae. [1] The resistant strains gain their resistance by producing ESBLs, which are class A enzymes. ESBLs are the derivatives of common β-lactamases (TEM and SHV) that have undergone one or more amino acid substitutions near the active site of the enzyme, thus increasing their affinity and the hydrolytic activity against the third-generation cephalosporins and monobactams. [2] Extensive use of newer-generation cephalosporin has been the strong factor for the evolution of newer β-lactamases such as ESBLs. ESBLs are encoded by transferable conjugative plasmids, which often code resistance determinants to other antimicrobial agents such as aminoglycosides. These conjugative plasmids are responsible for the dissemination of resistance to other members of gram-negative bacteria in the community. In recent years, ESBLs have also become more and more prevalent among species with an inducible AmpC-type β-lactamase such as Enterobacter spp., Citrobacter freundii and Serratia marcescens .

Enterobacter spp. is the third most prevalent bacterium exhibiting resistance to the first- and second-generation cephalosporins and aminopenicillins, through a mechanism mediated by the production of β-lactamases. Due to the extensive spread of multidrug-resistant ESBL-producing strains, there has been renewed interest in Enterobacter infections. Moreover, there are no published data revealing the evidence of ESBL-producing strains in river water in India. There are no published data on the β-lactamases of E. taylorae and E. agglomerans spp. that were investigated in the present study.

The present study was therefore conducted with an objective to examine the incidence of ESBL-producing strains, multidrug-resistant strains and detection of bla SHV , bla TEM and Amp C genes of Enterobacter species recovered from the 1312 km long river Narmada.


   Materials and Methods Top


Bacterial isolates

The bacterial isolates under study were identified and confirmed up to the species level on the basis of morphological, cultural, and biochemical [3] characteristics with the help of Bergey's Manual of Systematic Bacteriology [4] and Probabilistic Identification of Bacteria (PIB) computer kit. [5] The isolates were further analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA).

Susceptibility testing

The sensitivity of the isolates of the third-generation cephalosporins, viz., ceftriaxone, cefodoxime, ceftazidime, cefotaxime and aztreonam each 30 痢/disc, and to the other antibiotics was determined by the disc diffusion method. [6] The results were interpreted as per the guidelines of the Clinical Laboratory Standard Institute (CLSI).  Escherichia More Details coli (NCIB 9482) - Genetic stock, classical recipient in conjugation, nonlysogenic, F Thr Leu Lac Thi Str (R) T1 (R) T2 (R) T6 (R) {3 37C} strain was used for quality control, obtained from MTCC, Chandigarh.

ESBL detection

The ESBL detection was based on the double disc synergy test (DDST). In DDST, synergy was determined between a disc of cefotaxime (30 痢) and a disc of cefotaxime + clavulanic acid (30 痢 + 10 痢) which were placed 20 mm apart on a lawn of overnight culture suspension of isolates, which was adjusted to the McFarland 0.5 on Muller Hinton Agar. Disc containing clavulanic acid was prepared by applying 10 無 of a 1000 痢/mL clavulanic acid stock solution to each disc. If the zone size around the cefotaxime + clavulanic acid was >5 mm in comparison to the third generation cephalosporin disc alone, indicates the production of lactamase by Enterobacter strains. This increase occurred because the β-lactamases produced by the isolates were inactivated by clavulanic acid.

Detection of bla TEM , bla SHV , and AmpC gene

Three primer sets were used to amplify an internal region of the bla TEM, bla SHV, and Amp C genes [Table - 1]. Heat treatment (98蚓, 10 minutes) was used to prepare the template DNA from the control and environmental isolates. PCR amplification was performed in 25 無 reaction mixtures containing 0.2 mM of each dNTP, 14 mM Tris HCl (pH 8.4), 35 mM KCl, 1.5 mM MgCl 2 , 1.5 然 of SHV primers, 1 然 of TEM primers and 0.5 然 of AmpC primers, 1 U of Taq DNA polymerase and 2 無 of Template DNA. A negative control and a positive control containing a DNA mixture of three control strains (SHV-1, TEM-2, and AmpC β-lactamases producers) were included. DNA was amplified with the gradient thermalcycler (Eppendrof, Germany) using the following cycling parameters: 32 cycles, with one cycle consisting of denaturation at 94蚓 for 30 seconds, annealing at 54蚓 and extension at 72蚓 for 1 minute. Each PCR program was preceded by denaturation step of 94蚓 for 5 minutes and a final step of 72蚓 for 10 minutes. The PCR products were analyzed on a 2% agarose gel with 1x Tris-borate EDTA buffer. The gel was stained with ethidium bromide (concentration, 0.5 痢/mL) and analyzed under UV light.


   Results Top


A total of 57 Enterobacter isolates, identified with the help of Bergey's Manual of Systematic Bacteriology and Probabilistic Identification of Bacteria (PIB) computer kit, were assigned BGCC numbers. Among 57 isolates of Enterobacter , 22 were identified as E. cloacae , 11 isolates were identified as E. taylorae , 9 were identified as E. aerogens , 4 were identified as E. sakazakii , 6 were identified as E. agglomerans , 4 were identified as E. gergoviae , and 1 was identified as E. asburiae . A detailed overview of the Enterobacter strains tested is presented in [Table - 2].

The 16S rRNA gene (16S rDNA) was enzymatically amplified for a total of 57 strains belonging to seven described genomic species of the genus Enterobacter , and the amplicon was restricted independently with a total of four different restriction enzymes. They were amplified by Eco RI [Figure - 1], Hind III [Figure - 2] Hae III [Figure - 3] and Alu I [Figure - 4]. All the four enzymes produced three to five fragments on digestion.

Out of the 57 isolates of Enterobacter , 42 (73.68%) were found to be ESBL producers. Among these 42 ESBL producers, 23 (40.35%) were found to be ESBL producers with derepressed mutants, while the remaining 19 (33.33%) were plain ESBL producers. Seven (12.28%) isolates of Enterobacter were only derepressed, and 2 (9.09%) isolates were AmpC producers. Six (10.52%) isolates of Enterobacter did not show any type of β-lactamase pattern [Table - 3].

The resistance patterns of different Enterobacter strains were remarkably similar, particularly towards the third-generation cephalosporins. However, they were completely susceptible towards imipenem. These strains E. cloacae showed resistance towards at least four antibiotics. Nine isolates (81.81%) of E. taylorae showed resistance towards ceftriaxone, cefpodoxime, cefepime, ceftazidime, cefuroxime, cefotaxime, ceftazidime + clavulanic acid, cefoxitin, aztreonam and chloramphenicol antibiotics. Similarly E. sakazakii (100%) and E. gergoviae (75%) showed resistance towards different antibiotics. However, it was observed that the resistance towards the third-generation cephalosporins gradually increased in majority of the Enterobacter strains.

A multiplex PCR was designed to detect bla TEM , bla SHV , and Amp C genes in Enterobacter strains. PCR amplification products for Amp C were obtained for all the Enterobacter strains tested. [Table - 4] shows the β-lactamase genes bla TEM and bla SHV in the Enterobacter strains.


   Discussion Top


Traditionally, identification of pathogenic bacteria has been based on pathogenic characteristics and mainly involves analysis of different metabolic properties. Recently, molecular analysis of phylogenetic markers has been recognized as a very useful tool for the identification of bacterial genera, species, and subspecies. Among these markers, 16S rRNA is particularly useful because these molecules are present in every living cell and their function is highly conserved. In the present study, ARDRA with a probe complementary to the 16S rRNA was performed. Repeated ARDRA establishes the similarity between the different Enterobacter isolates.

Within the family of Enterobacteriaceae, natural resistance to majority of the β-lactam antibiotics is attributed predominantly to the action of chromosomally expressed AmpC or class A β-lactamases. With few exceptions, each species within the Enterobacteriaceae is thought to express its own β-lactamase. Chromosomal Amp C expression is generally reflected by resistance or decreased susceptibilities to amoxicillin, the same susceptibilities to aminopenicillins in the presence of clavulanic acid as to amoxicillin alone, natural resistance to narrow-spectrum cephalosporins, and natural carboxypenicillin sensitivity. [7] In contrast, naturally occurring class A-producing bacteria are generally resistant to amino and carboxypenicillins but not to cephalosporins and aminopenicillins in the presence of β-lactamase inhibitors. Reports of a previous study revealed that the strains of Enterobacter are naturally resistant to amoxicillin, amoxicillin/clavulanate, cefoxitin, and cefaclor but naturally sensitive to ticarcillin.

As evident from this study, resistance to cefotaxime would suggest either a derepressed AmpC or an ESBL producer. Combination with clavulanic acid bringing it back to the completely susceptible level would indicate an ESBL alone. If there is an improvement with clavulanic acid but not to the completely susceptible range, it would suggest either a derepressed AmpC + ESBL or could also suggest the presence of an ESBL with several other (non-AmpC) β-lactamases. The current work indicated ESBL-producing Enterobacter spp. often produces multiple β-lactamases. [8]

If a strain is susceptible to cefepime and resistant to cefotaxime and cefoxitin, the AmpC is the likely player. If it is cefotaxime resistant, cefoxitin sensitive and cefepime resistant or sensitive, then probably it is an ESBL producer. High-level expression of Amp C prevents recognition of ESBL producer. This is common in E. cloacae and E. aerogenes that produce chromosomally encoded AmpC β-lactamases. High-level AmpC production has minimal effect on activity of cefepime, making it more reliable for ESBL detection in presence of AmpC. [8],[9]

According to Sykes and Matthou, [10] it appears that all bacteria produce at least one chromosomally mediated β-lactamase and that these enzymes are specific for genus, species, and subspecies. 82.57% of our strains, produced one of the inducible chromosomal β-lactamases. This percentage coincides with the results of an international collaborative study in which 78% of all Enterobacter strains were found to be resistant to cefoxitin.

It is obvious that the natural resistance of 82% of all Enterobacter strain is due to these enzymes since all these strains produce one of the β-lactamases. [11] Cefoxitin is the most effective inducer of all cephalosporins, [12] and the same enzyme produces cefoxitin resistance in E. coli after the transfer of E. cloacae β-lactamase gene into this species.

ESBL-mediated resistance to third-generation cephalosporins was found in 47.36% environmental isolates in this study. This prevalence rate is similar to other reports from India and abroad. During the past decade, ESBL-producing K. pneumonia has emerged as one of the multidrug-resistant bacteria. [13] The incidence of ESBL-producing Enterobacter isolates in the United States has been reported to be 5%. [14] In France and England, 14% to 16% ESBL producers among clinical isolates of Enterobacter have been reported. [15],[16] In a previous study in central India, 76.5% of Enterobacter isolates resistant to third-generation cephalosporin antibiotics were found to produce ESBLs by Double-Disc Synergy Test. However, the percentage of the third-generation cephalosporins may be much higher because the conventional disc diffusion criteria used in routine laboratory detection underestimate the incidence of these isolates.

In addition to resistance to the third-generation cephalosporins, 27.85% of the isolates showed resistance to cefepime, 53.57% to cefoxitin, 34.42% to aztreonam, and 32.71% to chloramphenicol. In this study, resistance to the third-generation cephalosporins was found to coexist with resistance to other antibiotics. Since all the isolates showed multidrug resistance, the therapeutic strategies to control infection due to Enterobacter spp. have to be carefully formulated. Since all the isolates were sensitive to imipenem, it might serve as the drug of choice for the treatment of infections.

The resistance to ceftazidime in these Enterobacter isolates was higher than in those studied by Brun-Buisson [17] on clinical samples, and the presence of ESBL activity was partly confirmed by the inhibition of cephalosporin by clavulanic acid and to a lesser extent by a sulbactam. Clavulanic acid is known to be more active than sulbactam.

Enterobacter spp. has been an important source of transferable antibiotic resistance. ESBL production is coded by genes that are predominantly located on large conjugative plasmid of 80 kb to 160 kb in size. These plasmids are easily transmitted among different members of Enterobacteriaceae, and accumulation of resistance genes by them results in evolution of multidrug-resistant strains. For this reason, ESBL-producing isolates are resistant to a variety of classes of antibiotics. Moreover, the emergence of these multiple resistant Enterobacter strains is unfortunately accompanied by a relatively high stability of the plasmid-encoding ESBLs. [18] Third-generation cephalosporins' resistance is often mediated by TEM- and SHV-type β-lactamases in Enterobacteriaceae.

E. taylorae and E. agglomerans have been reported for the first time as β-lactamases-positive species. It was concluded that these strains of Enterobacter are likely to express chromosomal AmpC β-lactamases. The present study elaborates the knowledge about the production of β-lactamases by these Enterobacter species that are likely to be human pathogens which are present in riverine environment.

 
   References Top

1.Subha A, Nanthan A. ESBL mediated resistance to 3 rd generation cephalosporins among K. pneumonia in Chennai. Indian J Med Microbiol 2002;20:92-5.  Back to cited text no. 1    
2.Rodriquez B, Hamill RJ, Houstan EE, Georghiou PR, Clarridge JE, Rodriquez RL, et al . Detection of β-lactamases in nosocomial gram-negative clinical isolates. Indian J Med Microbiol 2004;22:247-50.  Back to cited text no. 2    
3.Mac Faddin JF. Biochemical tests for identification of medical bacteria. 2 nd ed. Williams and Wilkins: London/Baltimore; 1980.  Back to cited text no. 3    
4.Holt J, Krieg N, Sneath P, Staley J, Williams S. Bergey's manual of determinative bacteriology, 9 th ed. 1994. p. 175-87.  Back to cited text no. 4    
5.Bryant TN. Probabilistic identification of bacteria, PIB computer kit, Medical Statistics and Computing University Southampton: SO94 XYUK; 2003.  Back to cited text no. 5    
6.Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1996;45:493-6.  Back to cited text no. 6    
7.Livermore DM. β-lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995;8:557-84.  Back to cited text no. 7    
8.Rice LB, Bonomo RA. β-lactamases: Which ones are clinically important? Drug Resistance Update 2003;3:178-89.  Back to cited text no. 8    
9.Rottman M, Benzerara Y, Bercot H, Bizet B, Philippon C, Arlet A. Chromosomal AmpC genes in Enterobacter species other than Enterobacter cloacae and ancestral association of the ACT-1 plasmid-encoded cephalosporinase to E. asburiae. FEMS Microbiol Lett 2002;210:87-92.  Back to cited text no. 9    
10.Sykes RB, Matthew M. The β-lactamases of gram-negative bacteria and their role in resistance to β-lactam antibiotics. J Antimicrob Chemother 1976;2:115-57.  Back to cited text no. 10    
11.Sudha P, Yu Y, Sader HS, Jones RN, Chen M. Susceptibility testing accuracy of a CTX-M-type extended-spectrum β-lactamase organism-producing population of enterobacteriaceae: Intermethod analysis for 9#x3b2 lactams. Diagnost Microbiol Infect Dis 2006;53:131-41.  Back to cited text no. 11    
12.Sanders CC, Sanders WE Jr, Goering RV. In vitro antagonism of β-lactam antibiotics by cefoxitin. Antimicrob Agents Chemother 1982;21:968-75.  Back to cited text no. 12    
13.Minami S, Yotsuji A, Inoue M, Mitsuhashi S. Induction of β-lactamases by various β-lactam antibiotics in Enterobacter cloacae. Antimicrob Agents Chemother 1980;18:382-5.  Back to cited text no. 13    
14.Lucet JC, Decre D, Fochelle A, Guillou ML, Pernet M, Deblangy C. Control of a prolonged outbreak of extended spectrum-β-lactamase producing Enterobacteriaceae in a University Hospital. Clin Infect Dis 1999;29:1411-8.  Back to cited text no. 14    
15.Jacoby GA, Sutton L. Properties of plasmids responsible for production of extended spectrum β-lactamases. Antimicrob Agents Chemother 1991;35:164-9.  Back to cited text no. 15    
16.Sirot JN, Chardon MH. Susceptibility of Enterobacteriaceae lactam agents and fluoroquinolones: A three year survey in France. Clin Microbiol Infect 2002;8:207-13.  Back to cited text no. 16    
17.Brun BC, Legrand P, Phillippon A, Montravers F, Ansquer M, Dural J. Transferable enzymatic resistance to third generation cephalosporins during nosocomial outbreak of multi-resistance Klebsiella pneumonia. Antimicrob Agents Chemother 1987;2:302-6.  Back to cited text no. 17    
18.Velasco C, Romero L, Martinez JM, Bano JR, Pascual A. Analysis of plasmid encoding ESBLs from Eschirichia coli isolated from non-hospitalized patients in Seville. Int J Antimicrob Agents 2007;29:89-92.  Back to cited text no. 18    

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Correspondence Address:
Anjana Sharma
Bacteriology Laboratory, Department of PG Studies and Research in Biological Sciences, RD University, Jabalpur - 482 001, MP
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.40426

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    Figures

  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4]
 
 
    Tables

  [Table - 1], [Table - 2], [Table - 3], [Table - 4]

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