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Characterization of Escherichia coli and Klebsiella pneumoniae isolates for [bla.sub.ESBLs], [bla.sub.ampC], and [bla.sub.NDM-1] from a north Indian tertiary hospital: a preliminary time trend study.


The development of resistance to antimicrobial agents among Gram-negative pathogenic organisms has been progressive and inexorable. Of the few new drugs available, many have already become targets for bacterial resistance mechanisms. Pathogen of particular concern are the extended-spectrum [beta]-lactamase (ESBL)-producing ones. ESBLs are plasmid-mediated [beta]-lactamases which have an ability to hydrolyze [beta]-lactamase antibiotics containing an oxyimino group (e.g. ceftazidime, ceftriaxone, cefotaxime, or aztreonam). They have been most commonly found in Klebsiella pneumoniae, but are increasingly reported in Escherichia coli, Proteus mirabilis, and other members of Enterobacteriaceae.

ESBL enzymes initially come up through point mutations in the genes encoding the classic TEM and SHV [beta]-lactamase, resulting in one or more amino acids substitution near the active site of enzyme thus increasing their affinity and hydrolytic activity against third generation cephalosporins and monobactams, and hence an array of oxyimino-[beta]-lactam hydrolyzing enzymes with wider spectrum of activity has been generated. AmpC beta-lactamases are bacterial enzyme that confer resistance to oxyimino- and 7 [alpha]-methoxy cephalosporins and contribute to an appreciable resistance in the clinical isolates (1). In due course, various surveillance studies in different parts of the world have shown the predominance of TEM or SHV ESBLs, but in recent years, a new trend has been emerging i.e. the rapidly growing involvement of acquired AmpC beta-lactamase and CTX-M enzymes (2-4).

The genes encoding ESBLs and AmpC enzymes are located on plasmids which mediates their distribution. Recently, their mobilization through various insertion sequences and transposons have also been reported. The growing resistance rate in different microbial population can be attributed to these genetic vehicles which not only mediate their transfer in similar species but also results in inter-species relocation. Antibiotic resistance to a large extent is determined by acquisition of mobile genetic elements (MGEs). Such bundling of resistance genes on MGEs may lead to even faster acquisition of resistance genes. ESBL-producing organisms are also often found resistant to other non-beta-lactam antibiotics; it may be because of the presence of genes encoding such resistance mechanism on the same mobile genetic elements, viz. plasmids, integrons and transposons, along with genes for ESBLs (5).

Since the ESBL-producing organisms are posing major threat for clinical therapeutics, it is mandatory to identify the prevalence of such strains in hospitals and illustrate their epidemiology in order to control the spread of these strains by determining suitable preventive measures and treatment policies. Therefore, the present study was designed to characterize E. coli and K. pneumoniae isolates genotypically and also to compare the prevalence of resistance genes over a period of time.


Sample collection

A total of 125 isolates (109 E. coli and 16 K. pneumoniae) that were previously characterized for the presence of [bla.sub.ampC] were looked for the co-existence of [bla.sub.CTX-M], [bla.sub.TEM] and [bla.sub.SHV] (6). These isolates were categorized in two groups based on the period of collection of samples. Group I comprises of 91 isolates that were collected over a period of one year in 2009 and 34 isolates were included in group II, which were collected in the year 2010. Moreover, a subset of 54 isolates (37 E. coli and 17 K. pneumoniae) was collected after a gap of two year (in 2012) in order to compare the prevalence of [bla.sub.ampC], and [bla.sub.ESBLs] in the respective years. The isolates of third group were obtained from multiple sources including 37 from pus, 12 from urine, 2 from catheter tip, and one each from blood, drain, and peritoneal dialysis fluid.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed by the Kirby-Bauer method and interpreted as per CLSI (formerly NCCLS) guidelines (7). E. coli ATCC 25922 was used as a control strain.

Detection of [bla.sub.ESBLs], [bla.sub.ampC], and [bla.sub.NDM-1]

Crude genomic DNA was extracted from the isolates by heat lysis method. Briefly 2-3 bacterial colonies were suspended in 50 [micro]L of molecular grade water and the cells were lysed by heating at 95[degrees]C for 5 min. and then immediately cooled to 4[degrees]C. The DNA was then screened for the presence of Class A ESBLs, more specifically, [bla.sub.CTXM], [bla.sub.TEM] and [bla.sub.SHV] by the PCR using the primers that span the universal region of CTX-M, TEM and SHV genes, as described previously (4). Presence of [bla.sub.NDM-1] was established by PCR using primer that specifically target NDM gene (8), whereas [bla.sub.ampC] was detected by the protocol of Feria et al. (2002) with some modifications (9).

Detection of mobilizing genetic elements (MGEs)

The study isolates were screened for various MGEs including insertion sequences (ISEcp1, IS26 and ISCR1) and Sul-1-type class1 integrons. Details of primers and size of amplified product are shown in Table 1.

Randomly amplified polymorphic DNA (RAPD) typing

RAPD-PCR typing of the [bla.sub.NDM-1]-harboring isolates was done as described previously (10) to determine whether any specific clone is circulating in the hospital environment. Typing results were analyzed by using gel documentation system (BioRad) and clustering was done by Quantity One Software.


Antimicrobial resistance pattern

Among Enterobacterial isolates collected in the year 2012, 50% (27/54) were found to be imipenem resistant. Moreover, resistance to aminoglycosides is still low as compared to cephalosporins and fluoroquinolones. Detailed antibiotic resistance patterns of the study isolates are shown in Table 2.

Occurrence of various beta-lactamase genes

In the isolates collected in the year 2012, the maximum prevalence among ESBLs was noted to be that of [bla.sub.CTX-M] (75.9%) followed by [bla.sub.TEM] (61.1%) and then [bla.sub.SHV] (44.4%) (Figure 1). Whereas, [bla.sub.ampC] was found in 33.3% (18/54) isolates only (Figure 2). All the 18 AmpC-harboring isolates except one, were found to possess any of the Class A ESBL (CTX-M or TEM or SHV) gene or combination of more than one gene was also noticed. This clearly indicates their co-carriage on single plasmid or they are transferring through mobile genetic elements like insertion sequences and integrons. Surprisingly, we have noticed occurrence of [bla.sub.NDM-1] in 27.8% (15/54) isolates that were collected in the year 2012 (Figure 3). Whereas, none of the isolates were found to harbor [bla.sub.NDM-1] in the previous year's collection.

Analysis of [bla.sub.NDM-1]-harboring isolates

As we observed that out of 15 NDM-1 positive isolates, only five showed the presence of Sul-1-type integrons indicating that probably in our study isolates, NDM-1 (along with combination of other [bla.sub.ESBLs]) is mobilizing through insertion sequences and integrons is playing less role in their mobilization as compared to those of insertion sequences. Moreover, 12/15 NDM-harboring Enterobacterial isolates showed the presence of ISCR7 indicating its probability as major mobilizing genetic element responsible for mobilization of this particular [beta]-lactamase gene. Detailed genetic organization is shown in Table 3. Amplification pattern of various MGEs is shown in Figure 4.

RAPD analysis

Out of 15 NDM-1-harboring isolates, two (one each of E. coli and K. pneumoniae) were found untypable. However, diversity in bacterial population is still maintained and no particular clone was noticed among the study isolates. RAPD profile of [bla.sub.NDM-1]-harboring E. coli and K. pneumoniae is shown in Figure 5.

Comparison of the Enterobacterial isolates for the presence of various bla genes in 2009, 2010 and 2012

When we compare the occurrence of AmpC and ESBLs in 2009, 2010 and 2012, we observed a slight decrease in [bla.sub.ampC] prevalence in the first two years (80.2% in 2009 to 76.5% in 2010) but after a gap of one year, a drastic decrease was noticed (33.3% in 2012). Among ESBLs, [bla.sub.CTX-M] prevalence was found to be 78.0% in 2009, increasing slightly to 88.2% in 2010, and dropping slightly to 75.9% in 2012. Occurrence of [bla.sub.TEM] and [bla.sub.SHV] showed more or less similar pattern, with the occurrence of [bla.sub.TEM] almost identical in 2010 and 2012.

Prevalence of [bla.sub.TEM] was noted to be 37.4% in 2009, increased to 64.7% in 2010 and then decreased slightly to 61.1% in 2012. Moreover, the occurrence of [bla.sub.SHV] was noticed to be 38.5% in 2009, it then increased to 76.5% in 2010 but a significant decrease was noticed in 2012 with a 44.4% prevalence rate. Occurrence of [bla.sub.NDM-1] was noticed only in the isolates collected in 2012. Comparative trends can be seen in Figure 6.


Emerging bacterial resistance is the most serious problem being faced in the clinical practice today. The term ESBLs was originally applied to describe the TEM and SHV variants that can hydrolyze oxyiminocephalosporins (11). The extended-spectrum of activity can be defined in terms of hydrolysis of oxyiminocephalosporins or aztreonam at >10% of the activity of hydrolyzing benzylpenicillins. Generally they confer resistance to 1st, 2nd and 3rd generation cephalosporins along with monobactams, but are sensitive to cephamycins and carbapenems (12). Most of ESBls-coding genes are plasmid-borne but can be located on transposons and integrons (13) that facilitate their mobilization. CTX-M type of ESBL was reported in 1989 and is now considered as one of the most dominant type of ESBL in many countries. AmpC beta-lactamases are clinically important cephalosporinases, confer resistance to cephalothin, cefazolin, cefoxitin, most penicillins and [beta]-lactamase-[beta]-lactamase inhibitor combinations.

Reports on the prevalence of ESBLs in India have been recorded since the 1990s (14). Jemima and Verghese reported a prevalence of [bla.sub.CTX-M-1] in 15.8% isolates from the southern part of India (15). In the southern part of India, prevalence of ESBLs was reported to be 40% (16). Moreover, Anandan et al. reported 80% Klebsiella spp. and 63.6% E. coli as ESBL-producers in pediatric patients with septicemia (17). Recently, in a study performed in Rajasthan by Dalela, 73.5% E. coli and 58.1% Klebsiella pneumoniae isolates were reported as ESBL producers (18).

However, we isolated 78.0% CTX-M-producers in 2009, slightly increased to 88.2% in 2010, and then in 2012 the occurrence of [bla.sub.CTX-M] was very similar to that in 2009. Moreover, TEM-producers were found to be 37.4% in 2009, increased to 64.7% in 2010 and 61.1% in 2012. Prevalence of [bla.sub.SHV] was noticed to be 38.5% in 2009, 76.5% in 2010 and 44.4% in 2012.

Manoharan et al. reported 36.5% cefoxitin-resistant isolates as AmpC-producers in a study conducted on isolates taken from five Indian medical centers (19). However, in our study isolates, occurrence of [bla.sub.ampC] was found to be 80.2% in the year 2009, decreased to 76.5% in 2010 and a further decrease was observed in the 2012 with a prevalence of 33.3%.

Resistance to carbapenem is often mediated by production of MBL, a class B-type beta-lactamases that requires bivalent metal ion, usually Zn for their activity. NDM-1 share only 32.4% amino acid sequence homology with the closely related MBLs (VIM-1/VIM-2), and transpose hastily to other organisms via rolling circle mechanism facilitated by ISCR1 (20). NDM has been originally identified in a K. pneumoniae isolate from a Swedish patient of Indian origin, has now been reported in isolates worldwide among Gram-negative bacterial species (2023). A study from Pakistan reported occurrence of [bla.sub.NDM-1] in 27.1% of inpatients and 13.8% of outpatients (24). We also noticed occurrence of [bla.sub.NDM-1] in almost similar fraction (27.8%) of our Enterobacterial isolates.

In addition to Enterobacteriaceae, [bla.sub.NDM-1] has also been reported in non-fermentative bacteria like Acinetobacter spp. (21). NDM-1 [beta]-lactamases has also been reported in Hong Kong and Taiwan in E. coli and K. pneumoniae isolates (25,26). Co-carriage of [bla.sub.NDM-1] and [bla.sub.ESBLs], more specifically [bla.sub.CTX-M-15] and [bla.sub.CTX-M-14] has also been reported (23, 27). Recently, a novel variant of NDM (NDM-8) has been reported by Tada et al. in a multidrug-resistant E. coli isolate (28). Furthermore, 1.0% of Enterobacterial isolates from Indian hospitals were reported to harbour [bla.sub.NDM-1] (29). Kumarasamy et al. reported prevalence of NDM-1 in 1.2% enterobacterial isolates from Chennai and 13.1% isolates from Haryana (30). Shweta et al. have recently reported occurrence of [bla.sub.NDM-1] in 3 (out of 74) Acinetobacter baumanii isolates from a tertiary care hospital of Northern India (31). They also reported co-production of EBC, DHA, and CIT AmpC families in all the three NDM-harboring isolates. It has been reported that [bla.sub.NDM] is closely associated with ISCR1 and ISCR16 and is supposed to get mobilized through these insertion sequences. However some studies showed its mobilization through ISAba125 sequences (31). We also observed association of [bla.sub.NDM-1] with ISCR1 rather than Sul-1-type class 1 integrons in our bacterial population; however, it is in contrast to the report of Farzana et al. who reported that all NDM-1-harbouring isolates (8/31) were associated with class 1 integrons (32).

The free exchange of genetic material across the bacterial world ensures the survival of infectious agents. It is a well known fact that the resistance determinants were present in bacterial population before the therapeutic use of antibiotics; the current widespread prevalence is certainly due to selection pressure generated by human activity. Furthermore, conjugational transfer of antibiotic resistance genes across bacterial species and genera has amplified the problem of antibiotic resistance among pathogenic organisms. It has also been reported that [bla.sub.NDM-1] harboring plasmid, also possess an array of co-resistance determinants including various [beta]-lactamase genes, genes responsible for quinolone resistance etc. (33).

Regular surveillance of antimicrobial drug resistance is highly recommended. In the present study the change in such a trend, i.e. an initial increase followed by a decrease could have resulted from (i) inconsistency in the sample collection time, (ii) inconsistent sample size, and (iii) may be due to implementation of a strict antibiotic policy at our institution following a few preceding publications in this regard. As we noticed wide provincial differences, so it seems necessary to take into account the local epidemiology; more specifically at the level of country, particular region, and even particular hospitals. It further helps in making decisions about empirical therapy. Moreover, proper application of surveillance is essential to reduce current drug resistance rate in hospitals as well as in communities. Surveillance of carbapenemase-producing organisms seems to be essential for proper implementation of infection control strategies and also for selection of appropriate antimicrobial therapy. Moreover, infection control measures will be key factor in minimizing spread of NDM-1-harboring plasmids in hospital settings.


This work was partially sponsored by Dr. D.S. Kothari Post Doctoral Fellowship [F.4-2/2006(BSR)/13-773/2012(BSR)] by the University Grants Commission (sanctioned to F. Sobia). We are thankful to Prof. Daniel Jonas, Department of Environmental Health Sciences, Frieberg, Germany for kindly providing the control strains for [bla.sub.CTX-M-15] (D3), [bla.sub.TEM] (D2), [bla.sub.SHV] (D1), and [bla.sub.ampC] ([bla.sub.CIT-] harboring D1).


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Author information

Farrukh Sobia, MSc PhD, Post Doctoral Fellow [1]

Moin Uddin, MSc PhD, Associate Professor [1]

Mohammad Shahid, MBBS MD PhD FNZIMLS, Associate Professor [2,3]

Haris M Khan, MBBS MD, Professor [2]

[1] Department of Biochemistry, JawaharLal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh-202002, Uttar Pradesh, India

[2] Section of Antimicrobial Resistance Research and Molecular Biology, Department of Microbiology, JawaharLal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh-202002, Uttar Pradesh, India

[3] Department of Microbiology, Immunology and Infectious diseases, College of Medicine and Medical Sciences, Arabian Gulf University, Manama, Bahrain

The authors declare no conflicts of interest.

Author for correspondence: Dr Farrukh Sobia, Dr. D. S. Kothari Postdoctoral Fellow, Department of Biochemistry, J. N. Medical College & Hospital, Aligarh Muslim University, Aligarh-202002, U.P., India. Email:

Table 1. Oligonucleotides used in the present study.

Target gene         Primer Sequence      Amplified   Reference

[bla.sub.ampC]    P1F: 5'-CCC CGC TTA     634 bp         9
                   TAG AGC AAC AA-3'

                  P1R: 5'-TCA ATG GTC
                   GAC TTC ACA CC-3'

[bla.sub.CTX-M]   P2F: 5'-ATG TGC AGY      593bp         4
                   ACC AGT AAR GT 3'

                  P2R: 5'-TGG GTR AAR
                   TAR GTS ACC AGA 3'
                    where Y, Wobble
                    (C+T); R, Wobble
                  (A+G) and S, Wobble

[bla.sub.TEM]     P3F: 5'-KAC AAT AAC      936bp         4
                   CCT GRT AAA TGC-3'

                  P3R: 5'-AGT ATA TAT
                   GAG TAA ACT TGG 3'
                    where K, Wobble
                  (G+T) and R, Wobble

[bla.sub.SHV]     P4F: 5'-TTT ATC GGC      930bp         4
                   CYT CAC TCA AGG-3'

                  P4R: 5'-GCT GCG GGC
                  CGG ATA ACG-3' where
                    Y, Wobble (C+T)

[bla.sub.NDM-1]   P5F: 5'-CAG CGC AGC      800bp         8
                       TTG TCG-3'

                  P5R: 5'-TCG CGA AGC
                       TGA GCA-3'

ISEcp1            P6F: 5'-AAA AAT GAT     1100 bp       10
                   TGA AAG GTG GT-3'

                  P6R: 5'-CAG CGC TTT
                   TGC CGT CTA AG-3'

IS26               P7F:5'-GCG GTA AAT    variable       10
                   CGT GGA GTG AT-3'

                  P7R: 5'-ATT CGG CAA
                   GTT TTT GCT GT-3'

ISCR1             P8F: 5'-CTC ACG CCC     600 bp         4
                   TGG CAA GGT TT-3'

                  P8R: 5'-CTT TTG CCC
                    TAG CTG CGG T-3'

Sul1-type         P9F: 5'-CTT CGA TGA     420 bp         4
class 1            GAG CCG GCG GC-3'
                  P9R: 5'-GCA AGG CGG
                   AAA CCC GCG CC-3'

Table 2. Antibiotic resistance rates in E. coli and K.
pneumoniae isolates.

Antimicrobial agents        % Resistance (n=54)
(disc potency)

Cefixime (5[micro]g)            92.59 (50)
Ceftriaxone (30[micro]g)        98.15 (53)
Cefotaxime (30[micro]g)         90.74 (49)
Cefepime (30[micro]g)           72.22 (39)
Gatifloxacin (5[micro]g)        92.59 (50)
Ofloxacin (5[micro]g)           96.30 (52)
Ciprofloxacin (5[micro]g)       94.44 (51)
Gentamicin (10[micro]g)         51.85 (28)
Amikacin (10[micro]g)           48.15 (26)
Imipenem (10[micro]g)           50.00 (27)

Figures in parentheses show number of resistant isolates.

Table 3. Association of NDM-1 harboring Enterobacterial isolates
with various mobilizing genetic elements.

Combination of               Mobilizing genetic elements
bla genes in
[bla.sub.NDM-1]-       Insertion                Integron
harboring              sequences
isolates (n=15)

[bla.sub.CTX-M] +      IS26 + IS       Sul1-type class 1 integron
[bla.sub.ampC] +       Ecp1 + IS
[bla.sub.TEM] +        CR1 (2/4)
(4/15)                IS Ecp1 + IS     Sul1-type class 1 integron
                       CR1 (1/4)

                       IS26 + IS                   --
                       Ecp1 + IS
                       CR1 (1/4)

[bla.sub.CTX-M] +      IS Ecp1 +       Sul1-type class 1 integron
[bla.sub.ampC] +      IS CR1 (3/3)
[bla.sub.TEM] +
[bla.sub.SHV] +

[bla.sub.CTX-M] +      IS26 + IS       Sul1-type class 1 integron
[bla.sub.TEM] +        Ecp1 (1/3)
[bla.sub.SHV] +
[bla.sub.NDM-1]     IS26 + IS Ecp1 +               --
(3/15)                IS CR1 (1/3)

                     IS Ecp1 (1/3)                 --

[bla.sub.CTX-M] +   IS26 + IS Ecp1 +               --
[bla.sub.TEM] +       IS CR1 (2/2)

[bla.sub.CTX-M] +      IS Ecp1 +       Sul1-type class 1 integron
[bla.sub.ampC] +      IS CR1 (1/2)
(2/15)                 IS26 + IS                   --
                       CR1 (1/2)

[bla.sub.TEM] +            --                      --
[bla.sub.SHV] +
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Author:Sobia, Farrukh; Uddin, Moin; Shahid, Mohammad; Khan, Haris M.
Publication:New Zealand Journal of Medical Laboratory Science
Article Type:Report
Date:Aug 1, 2014
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