Screening of Ocular Enterobacteriaceae Isolates for Presence of Chromosomal blaNDM-1 and ESBL Genes: A 2-Year Study at a Tertiary Eye Care Center

Abstract

Purpose.: Since, to our knowledge, there are no reports on the prevalence of the blaNDM-1 gene among ocular isolates of Enterobacteriaceae, and only limited information on the prevalence of extended spectrum beta-lactamases (ESBLs) among ocular bacterial isolates are available, our study was undertaken.

Methods.: A prospective study was done on 74 Enterobacteriaceae isolates from patients presenting with clinical suspicion of bacterial ocular infections during a period from January 2010–December 2011. All isolates were subjected to detection of ESBLs by double disc synergy and screened for the presence of CTX-M –I, II, III, and IV groups, and OXA, TEM, SHV, blaNDM-1 genes by PCR.

Results.: Of 74 ocular Enterobacteriaceae isolates 57 (77%) were ESBL producers tested by the double disc diffusion test. PCR-based DNA sequencing of these 57 ocular isolates showed the presence of CTX-M-15 (14.0%), blaOXA-1 (5.2%), blaSHV-1 (8.7%), and blaTEM-1 (7.0%) types. The blaNDM-1 was absent among these ocular isolates. The most widely disseminated ESBL gene among ocular isolates was CTX-M-15. Phenotypic and genotypic results showed 100% correlation.

Conclusions.: To our knowledge, this is the first extensive study performed to genotype ESBL-producing ocular Enterobacteriaceae isolates. The isolation of ESBL-producing Enterobacteriaceae organisms predominantly from conjunctival specimens indicates community-acquired infections/colonization by these bacteria in the conjunctiva of the patients, and cases are not related to hospital-acquired infections because of the short stay of ophthalmic patients in the hospitals. A shift in the resistance rates of ceftazidime from 37.5% to 79.7% over the years proves the increase in drug resistance among ocular clinical isolates.

Introduction

The eye, an organ of the human body, is impermeable to almost all external infectious agents, ¹ though the ocular surface invariably is exposed to a wide array of microorganisms. ² Microbial infection of the eye is via external sources or through intraocular invasion of micro-organisms that are disseminated from the bloodstream ³ or due to a breach in the ocular barriers. ⁴ Alteration of the normal flora contributes to varied ocular diseases, including blepharitis, conjunctivitis, canaliculitis, dacryocystitis, orbital cellulitis, keratitis, and finally infectious endophthalmitis, including panophthalmitis and so forth. ^1,5–7 The most common microorganisms causing ocular infections include Staphylococcus aureus, coagulase-negative staphylococci, Genus Streptococcus, Corynebacterium, Bacillus, Nocardia, Pseudomonas aeruginosa, Enterobacteriaceae, nonfermentors, and others. ^4,8–10  

Despite the protection by the components of tear along with blinking action of the eyelids, the resident bacteria of the conjunctival sac or environmental bacteria can establish infection, resulting in the need for antibiotic intervention to treat the disease. Knowledge of the antimicrobial resistance, especially to commonly used antibiotics, is very important. Hence, potentially sight-threatening infections can emerge if appropriate antibiotic therapy is not instituted rapidly. The factors contributing to development of drug resistance among ocular isolates include overuse of antibiotics for systemic infection as well as overuse of antibiotics in the eye. ^6,11 As a result, periodic susceptibility testing along with molecular biologic techniques for the detection of emerging antibiotic resistance among clinical isolates of bacteria is warranted to understand the prevalence of drug resistance to higher levels of drugs, and also to ensure the availability of broad-spectrum antimicrobials. 

Extended-spectrum β-lactamase (ESBL) enzymes are capable of hydrolyzing and inactivating a wide variety of beta lactams, including third generation cephalosporins, penicillins, and aztreonam. All of these β-lactamase enzymes are found commonly in the Enterobacteriaceae family. ¹² Infections due to ESBL-producing bacteria present a major therapeutic dilemma, since the choice of antibiotics is restricted. ¹³  

Prevalence of carbapenemase production among clinical isolates of Enterobacteriaceae has increased over the past decade. ¹⁴ Beta-lactams have been used widely as the mainstay of treatment for severe infections due to these bacteria, with carbapenems often representing the last line of defense. ¹⁵ Recently, the New Delhi metallo-beta-lactamase (NDM-1), a novel type of metallo-beta-lactamase (MBL) conferring resistance to almost all beta-lactam antibiotics, including carbapenems that are used in treatment of antibiotic-resistant bacterial infections, has emerged and, therefore, has alarmed the world. ^16,17 This worldwide spread of Enterobacteriaceae-carrying blaNDM-1, thus, has become a significant threat to human health due to its extensive drug resistance, leaving few or no therapeutic options and, therefore, becoming a major public health problem throughout the world, particularly in India. ¹⁸ Many reports on the existence and dissemination of blaNDM-1 have arisen at a rapid rate throughout the world and also from India. ^19–21  

So far, blaNDM-1 has not been screened among the ocular pathogens. The Enterobacteriaceae group of microorganisms is isolated at significantly higher rates among ocular pathogens. The major antibacterial drugs used in the treatment of ophthalmic bacterial infections include beta-lactam antibiotics, such as cefazolin and ceftazidime. Therefore, we aimed to explore ophthalmic clinical isolates belonging to the family of Enterobacteriaceae in quest of the existence of ESBLs and blaNDM-1 at a tertiary eye care center in South India. 

Materials and Methods

A prospective analysis was done on 74 bacteria belonging to Enterobacteriaceae isolated from patients with clinical suspicion of bacterial ocular diseases, such as conjunctivitis, canaliculitis, keratitis, dacryocystitis, endophthalmitis, and panophthalmitis, along with screening of donor corneal rim (DCR) and swab specimens from multiorgan donors (MOD) during the 2-year period from January 2010–December 2011, from a total of 7598 specimens, including extraocular and intraocular, received for microbial culture at L & T Microbiology Research Centre, Kamalnayan Bajaj Research Centre, Sankara Nethralaya, a tertiary eye care center at Chennai, India. The ocular clinical isolates were screened for ESBL production and presence of the blaNDM-1 gene by molecular methods. Table 1 shows the detailed distribution of Enterobacteriaceae among the ocular specimens. Ocular specimens were processed for isolation of pathogenic bacteria by standard conventional bacteriologic methods and the bacteria isolated were identified to species level using standard biochemical tests. ²² A positive culture was defined as growth of the microorganism in two or more media or confluent growth at the site of inoculation, and these were included for the study. Study protocol was approved by the institutional ethics sub-committee (IRB). The study was conducted according to the guidelines set forth in the Declaration of Helsinki. 

Table 1. 

 

Distribution of Ocular Enterobacteriaceae Isolates Included in the Present Study

Enterobacteriaceae Isolates	N of Isolates (%)	Intraocular Specimens* (n = 20)	Extraocular Specimens† (n = 34)	Other Specimens‡ (n = 20)
K. pneumoniae	16 (21.3%)	8	5	3
K. oxytoca	14 (18.9%)	2	6	6
E. coli	10 (13.5%)	0	7	3
E. aerogenes	9 (12.1%)	2	4	3
C. freundii	8 (10.8%)	2	4	2
P. mirabilis	5 (6.7%)	2	2	1
C. koseri	4 (5.4%)	3	1	0
S. marcescens	3 (4.0%)	0	3	0
P. rettgeri	1 (1.3%)	1	0	0
M. morganii	1 (1.3%)	0	1	0
E. cloacae	1 (1.3%)	0	1	0

*  Intraocular specimens include aqueous humor (n = 1), vitreous humor (n = 4), eviscerated material (n = 6), intraocular lens (n = 2), and silicon rod (n = 3).

†  Extraocular specimens include orbital biopsy (n = 2), canalicular pus (n = 2), contact lens (n = 7), corneal scraping (n = 7), corneal button (n = 1), conjunctival swab (n = 14), scleral tissue (n = 2), and lid abscesses (n = 3).

‡  Other specimens include DCR (n = 14) and (n = 6).

Screening for Production of ESBLs by Phenotypic Methods: Double Disc Synergy Test (DDST)

Requirements for DDST.

We used Müller-Hinton agar, Müller-Hinton broth, bacterial isolates to be tested (in 0.5 McFarland's standard concentration), quality control strains Escherichia coli ATCC 35218 (positive control) and Escherichia coli ATCC 25922 (negative control), antibiotic discs ceftazidime (30 μg) versus ceftazidime/clavulanic acid (30/10 μg), and cefotaxime (30 μg) versus cefotaxime/clavulanic acid (30/10 μg). 

Procedure for Performing the DDST.

We selected 3 to 5 well-isolated colonies of Enterobacteriaceae from an agar plate culture. The isolated colonies were picked with the edge of a sterile loop and inoculated into a tube containing 4 to 5 mL of Müller-Hinton broth. The broth culture was incubated at 37°C until it achieved or exceeded the turbidity of the 0.5 McFarland standards (usually 2–6 hours). Turbidity was adjusted to 0.5 McFarland standard. Optimally, within 15 minutes after adjusting the turbidity, a sterile cotton swab was dipped into the adjusted suspension, rotated several times and pressed firmly on the inside wall of the tube above the fluid level to remove excess inoculum from the swab. The dried surface of a Müller-Hinton agar plate was swab streaked over the entire sterile agar surface. This procedure was repeated by streaking two more times, rotating the plate approximately 60° each time to obtain a uniform lawn culture throughout the surface of the agar. The lid was left ajar for 3 to 5 minutes, but no more than 15 minutes, to allow any excess surface moisture to be absorbed before applying drug-impregnated disks. Disks containing the ceftazidime were placed 15 mm apart (edge-to-edge) from a ceftazidime/clavulanic acid disc containing 10 μg of the latter compound. Similarly, cefotaxime (30 μg) and cefotaxime/clavulanic acid (30/10 μg) antibiotics were placed parallel to the previous discs. Inoculated plates were incubated at 37°C for 18 to 24 hours, after which the plates were read. 

Reading Plates and Interpreting Results.

Any enhancement of the zone of inhibition between a beta-lactam disk and that containing the beta-lactamase inhibitor was indicative of the presence of an ESBL. ²³  

Uniplex PCR for Detection of ESBL Genes

All 57 clinical isolates of Enterobacteriaceae were subjected for uniplex PCR amplification, targeting CTX-M genes, ²⁴ TEM, ²⁵ OXA, ²⁶ SHV, ²⁷ and chromosomal-mediated blaNDM-1 genes, which was done using the primers and protocol as we described previously. ²⁸ Non-ESBL E. coli ATCC 25922, and ESBL-producing Klebsiella pneumoniae ATCC 700603 and E. coli ATCC 35218 strains were used as controls. 

Results

We included in our study a total of 74 Enterobacteriaceae isolated from various ocular specimens. The highest rate of Enterobacteriaceae was isolated from the male population (n = 44, 59.5%) compared to the female population (n = 30, 40.5%). The mean age was 40.1 years for the male group (range 5 months to 78 years) and 52.5 years for the female group (range 12–75 years). Of the 74 clinical isolates, the maximum number was isolated from DCR (16.2%) and conjunctival swab (16.2%) specimens. 

Among the 74 Enterobacteriaceae isolates, 34 (52.7%) specimens had Enterobacteriaceae isolated as a pure culture and the remaining 23 (31.1%) were isolated along with groups of bacterial strains other than Enterobacteriaceae, 10 (13.5%) strains were isolated along with fungi, and 2 (2.7%) strains were isolated along with other groups of bacteria and fungus. The data on the polymicrobial distribution of these isolates along with other bacteria and fungi are not provided. 

Phenotypic Results of ESBL Detection in Enterobacteriaceae Isolates

Of the total 74 ocular clinical isolates of Enterobacteriaceae tested for the production of ESBLs by the double disc diffusion test, 77% (57/74) exhibited ESBL positivity, of which 3.2% (3/57) were Citrobacter freundii, 14.0% (8/57) C. koseri, 12.2% (7/57) E. aerogenes , 17.5% (8/57) E. coli , 21.0% (12/57) K. pneumoniae , 17.5% (10/57) K. oxytoca, 10.5% (6/57) Proteus mirabilis , and 5.2% (3/57) Serratia marcescens , respectively. Results of screening tests for ESBLs are shown in Table 2. Among the 57 Enterobacteriaceae isolates that were positive for ESBL production, the highest percent ESBL production was exhibited by K. pneumoniae (21.0%), followed by K. oxytoca (17.5%). Ocular clinical isolates of Enterobacteriaceae showed overall 70.0% positivity for ESBL production. The rate of ESBL isolations from intraocular specimens was 30% (predominant specimen was eviscerated material) and in case of extraocular groups of ESBL specimens the isolation rate was 41.1% (predominantly from conjunctival swabs specimens). The rate of the isolation of ESBL-producing Enterobacteriaceae isolates recovered from DCR, MOD swab, silicon rod implants, or intraocular lenses (IOL) was 31.5% (n = 18/57), whereas the isolation rates from the rest of the specimens were 68.5% (n = 39/57). The result of DDST of a K. pneumoniae strain isolated from an eviscerated material along with positive and negative control strains is shown in the Figure. Details on the number of Enterobacteriaceae isolates recovered from various ocular disease conditions are shown in Table 2. 

Figure. 

 

Result of DDST of a K. pneumoniae strain isolated from an eviscerated material along with positive and negative control strains. CE, cefotaxime (30 g); CA, ceftazidime (30 g); CEC, cefotaxime + clavulanic acid (30/10 g); CAC, ceftazidime + clavulanic acid (30/10 g).

Table 2. 

 

Details on Number of Enterobacteriaceae Isolates Recovered from Various Ocular Disease Conditions

Organism (N = 74)	Conjunctivitis	Canalicularitis	Keratitis	Trauma	Post– PK	Post Suture	Vitrectomy	DCR	MOD	Endophthalmitis			Total N of Isolates Recovered
										Endogenous	Post– operative	Pan– ophthalmitis
C. freundii	–	–	3	–	1	–	1	–	1	–	1	1	8
C. koseri	–	–	1	–	–	–	1	1	1	–	–	–	4
E. aerogenes	1	–	1	1	1	–	–	–	1	–	2	2	9
E. clocae	–	–	–	1	–	–	–	–	–	–	–	–	1
E. coli	5	–	–	2	–	–	–	1	1	–	1	–	10
K. pneumoniae	3	–	1	2	–	–	2	4	2	–	3	1	18
K. oxytoca	3	1	–	1	1	2	–	3	–	2	1	–	14
Morganella morganii	–	–	1	–	–	–	–	–	–	–	–	–	1
P. mirabilis	1	1	–	1	1	–	–	1	–	–	–	–	5
Providencia rettgeri	–	–	1	–	–	–	–	–	–	–	–	–	1
S. marcescens	1	–	2	–	–	–	–	–	–	–	–	–	3
Total specimens (N = 74)	14 (18.9%)	2 (2.7%)	11 (14.9%)	8 (10.8%)	3 (4.0%)	2 (2.7%)	4 (5.4%)	10 (13.5%)	6 (8.1%)	2 (2.7%)	8 (10.8%)	4 (5.4%)	74

Genotypic Results of ESBL Detection in Enterobacteriaceae Isolates

All 57 ESBL-producing isolates were subjected to ESBL detection by the molecular method. Among the 57 isolates, single ESBL genes were present in 20 (35.0%), of which blaCTX-M (n = 8, 14.0%) was the most common ESBL gene detected, followed by blaSHV (n = 5, 8.7%), blaTEM (n = 4, 7.0%), and blaOXA (n = 3, 5.2%). Among the 22 (38.5%) isolates harboring two ESBL genes, blaTEM + blaCTX-M was the most common combination (n = 10, 17.5%) followed by blaSHV + blaTEM (n = 6, 10.5%), blaOXA + blaTEM (n = 3, 5.2%), and blaOXA + blaSHV (n = 3, 5.2%). Nine isolates carried three ESBL genes, and the predominant combination was blaCTX-M + blaTEM + blaOXA (n = 4, 7.0%), followed by blaCTX-M + blaOXA + blaSHV (n = 2, 3.5%), and the remaining 3 (1.7%) combinations were blaCTX-M + blaSHV + blaTEM, blaCTX-M + blaTEM + blaSHV, and blaOXA+ blaTEM + blaSHV. Six (10.5%) isolates had all 4 gene combinations, that is blaCTX-M + blaOXA + blaSHV + blaTEM. Distribution ESBLs in Enterobacteriaceae isolates according to genotypic combinations are shown in Table 2. Table 3 shows the organism-wise distribution of ESBL producers. Other CTX-M genes (II, III, and IV) and NDM-1 were not detected in any of the isolates. The blaNDM-1 gene was absent among all the ESBL producers. Phenotypic and genotypic methods of ESBL detection showed a 100% correlation (Table 4). 

Table 3. 

 

Enterobacteriaceae Isolates Distributed according to the Clinical Source and the Results of ESBL Screening Test

Ocular Specimens	ESBL Screening Test Results
	N of Isolates Tested	ESBL Positive (% Positivity)	ESBL Negative (% Negativity)
A. Intraocular specimens	16	12 (75.0)	4 (25.0)
Aqueous humor	1	1 (100.0)	0
Vitreous humor	4	2 (50.0)	2 (50.0)
Eviscerated material	6	5 (83.3)	1 (16.7)
Intraocular lens	2	1 (50.0)	1 (50.0)
Silicon rod	3	3 (100.0)	0
B. Extraocular specimens	38	31 (81.5)	7 (18.5)
Orbital biopsy	2	2 (100.0)	0
Canalicular pus	2	2 (100.0)	0
Contact lens	7	5 (71.4)	2 (28.6)
Corneal scraping	7	6 (85.7)	1 (14.3)
Corneal button	1	1 (100.0)	0
Conjunctival swab	14	12 (85.7)	2 (14.3)
Scleral tissue	2	0	2 (100.0)
Lid abscesses	3	3 (100.0)	0
C. Other specimens	20	14 (70.0)	6 (30.0)
DCR	14	12 (85.7)	2 (14.3)
MOD	6	2 (33.3)	4 (66.7)
Totals	74	57 (77.0)	17 (23.0)

Table 4. 

 

ESBL Genotypes Detected among 57 Ocular Enterobacteriaceae Strains that Were Tested Positive for ESBL Production

Positive by PCR for ESBL Genes	ESBL Producer (N = 57)
	K. pneumoniae (n = 12, 21.0%)	K. oxytoca (n = 10, 17.5%)	E. coli (n = 8, 14.0%)	E. aerogenes (n = 7, 12.2%)	C. freundii (n = 3, 5.2%)	P. mirabilis (n = 6, 10.5%)	C. koseri (n = 8, 14.0%)	S. marscensens (n = 3, 5.2%)	Total n = 57 (%)
A. Single ESBL gene positivity
blaCTX-M only	2	2	1	2	–	–	1	–	8 (14.0%)
blaOXA only	–	–	3	–	–	–	–	–	3 (5.2%)
blaSHV only	4	1	–	–	–	–	–	–	5 (8.7%)
blaTEM only	1	–	–	–	1	–	2	–	4 (7.0%)
B. Two genes positivity
blaCTX-M + blaTEM	4	3	–	1	–	1	–	1	10 (17.5%)
blaOXA + blaTEM		1	–	1	–	–	1	–	3 (5.2%)
blaSHV + blaTEM	1	1	1	1	–	1	1	1	6 (10.5%)
blaOXA + blaSHV	–	–	–	–	–	1	1	1	3 (5.2%)
C. Three ESBL genes positivity
blaCTX-M + blaSHV	–	1	–	–	–	–	–	–	1 (1.7%)
blaCTX-M + blaTEM + blaOXA	–	–	–	1	2	–	1	–	4 (7.0%)
blaCTX-M + blaTEM + blaSHV	–	–	–	–	–	–	1	–	1 (1.7%)
blaCTX-M + blaOXA + blaSHV	–	1	–	–	1	–	–	–	2 (3.5%)
blaOXA + blaTEM + blaSHV	–	–		1	–	–	–	–	1 (1.7%)
D. Four ESBL genes positivity
blaCTX-M + blaOXA+ blaSHV + blaTEM	–	–	3	–	–	3	–	–	6 (10.5%)

Sensitivity of uniplex PCR's ranged from 10 to 50 ng DNA of ESBL-producing positive controls. The primers were highly specific, and no amplification was observed with fungal, viral, and human DNA. 

DNA Sequencing

PCR products were subjected to DNA sequencing with forward primer and reverse primer of the corresponding genes. Nucleotide sequence results were analyzed by BLAST (available online at www.ncbi.nlm.nih.gov/blast). The BLAST analysis showed that all genotypically CTX-M–I positive isolates were of the CTX-M 15 (blaCTX-M-15) beta-lactamase type, while the OXA gene belongs to class blaOXA-1, SHV gene to class blaSHV-1, and TEM gene to blaTEM-1 type. 

Nucleotide Sequence Accession Number

The nucleotide sequence data reported here will appear in the GenBank nucleotide sequence database under accession numbers JN019833-JN019840 (8 strains), JN043372 (1 strain), and JN043375-JN043380 (6 strains). 

Discussion

Our study clearly showed a high prevalence of ESBL-producing Enterobacteriaceae isolates among ocular clinical isolates. Most of these patients were not staying in the hospital, which indicated that ESBL genes in these isolates could be community-acquired whereas the blaNDM-1 gene was not detected in our study, indicating absence of the same in the community-acquired infections. Many studies on existence of the blaNDM-1 gene among Enterobacteriaceae isolated from patients who underwent transplantation, chronic disease, and accidents have been reported by Liang et al. ²⁹  

In our study, large numbers of Enterobacteriaceae were isolated from the conjunctival ocular surface followed by the DCR isolates. Predominant Enterobacteriaceae tested were isolated from patients suspected of having conjunctivitis (18.9%) followed by keratitis (14.9%). In the conjunctivitis cases, the primary pathogens isolated were E. coli followed by K. pneumoniae , whereas in cases of keratitis the predominant pathogen was C. freundii . 

Jones et al. recorded the incidence of ESBL positivity to be 60% to 68% in India, from strains isolated from major hospitals. ³⁰ However, the prevalence studies on ESBL-producing ocular isolates from India are very few. Bharathi et al. reported the incidence of ESBL among the ocular population to be 7%. ⁹ In our study, the rate of ESBL positivity was 77% by the double disc diffusion test. In our previous study conducted on DCR and MOD isolates (13), ²⁸ ESBL production was 58.3%. K. oxytoca was isolated from intraocular specimens of two patients with endogenous endophthalmitis whose urine and blood cultures also showed growth of the same organism. This confirms the endogenous spread of K. oxytoca in both cases, and the isolates were tested negative for ESBLs and the blaNDM-1 gene. 

To prevent external contamination, all donor corneas were procured by whole globe enucleation, followed by corneoscleral rim excision at the host eye bank with aseptic techniques. Under aseptic precautions, they were stored in McCarey and Kaufman medium containing gentamicin (100 μg/mL). At the time of surgery, the cornea was trephined for transplantation, and the remaining rim of corneoscleral tissue was transferred with M-K medium and sent to the clinical microbiology laboratory for bacterial culture within 15 minutes after performing penetrating keratoplasty. Enterobacteriaceae thus isolated from these donor corneal rims were tested for ESBLs. 

A total of 14 isolates (n = 14/20, 70%) from MOD and DCR specimens was positive for ESBL among Enterobacteriaceae tested. The organisms isolated among these MOD and DCR specimens were of nosocomial origin, since all patients would have had a reasonable period of stay in the hospital before the donation of organs. Similarly, isolation of ESBL-producing Enterobacteriaceae in case of silicon rods and intraocular lenses also was nosocomial as the patients gained infection predominantly during surgery despite all standard aseptic precautions, such as use of povidone-iodine, installation of antibiotics, sterilized instruments, and IOLs. 

ESBL-positive isolates showed increased susceptibility to gatifloxacin (74.4%), moxifloxacin (69.5%), and ofloxacin (68.9%). A higher degree of resistance was exhibited towards ceftazidime (79.7%) followed by the aminoglycoside group, which includes gentamicin (56.7%) and tobramycin (55.4%, data not shown). In a study conducted at our hospital by Madhavan et al. in 1999, 62.5% of sensitivity to ceftazidime was reported among gram-negative endophthalmitis isolates. ³¹ The shift in the resistance rates of ceftazidime (a commonly-used ocular antibacterial agent) from 37.5% to 79.7% proves the increasing spread of drug resistance among the ocular population during this intervening period. 

The most common type of ESBL enzyme encountered in our study is the CTX-M type; more specifically CTX-M-15. Studies conducted by Johann et al. ²⁴ and Ensor et al. ³² showed CTX-M-15 to be the most widely disseminated ESBL enzymes. Our present study reaffirms these findings. Six (10.5%) isolates had the 4-gene combinations, that is blaCTX-M + blaOXA + blaSHV + blaTEM were detected in 3 E. coli and 3 P. mirabilis isolates. These 6 isolates were from DCR and conjunctival swabs, indicating the prevalence of the drug resistance. Further studies may be needed to determine the reason for these findings. The blaNDM-1 gene was absent among Enterobacteriaceae isolates, whereas our previous study, done at our institution during the period of January 2011 to June 2011, ²⁸ demonstrated the existence of the blaNDM-1 gene in a strain of Acetinobacter baumannii, which was isolated from a swab collected from the conjunctiva of a multiorgan donor who met with a road traffic accident and was on a ventilator. ²⁹ Literature findings also confirm the existence of the blaNDM-1 gene in A. baumannii strains and in road traffic accident cases. ²⁸  

Although there are many reports on the presence of blaNDM-1 genes among clinical isolates in India, still to our knowledge only a single report stating the absence of blaNDM-1 genes among fecal flora exists. ²¹ Our study also showed absence of blaNDM-1 genes among ocular specimens. This demonstrates that at this time point, blaNDM-1 genes are not carried among the organisms isolated from extraocular specimens that are highly exposed to community-acquired infections and also from intraocular specimens, making them free from increased drug resistance and, thus, making the treatment not a hard task. However, the high rates of ESBL-producing isolates among ocular strains is an important finding. 

Cefazolin and cefotaxime are being used routinely in the treatment of ocular infections. Considering the increasing spread of such ESBL genes in the community, all patients with a culture-positive report should carry a note indicating whether the isolate is an ESBL producer or not. This will help clinicians to avoid use of penicillins, aztreonam, and all cephalosporins (except cephamycins), irrespective of their in vitro susceptibility. 

To our knowledge, this is the first extensive study to screen for predominant drug resistance-encoding ESBL genes among the ocular isolates. We demonstrated the common ESBL genotypes present among Enterobacteriaceae isolated in our hospital setup. Ours is a first study performed for screening blaNDM-1 genes on the ocular isolates of Enterobacteriaceae. The overall rate of nosocomial infections was 31.5% of hospitalized patients, while the remaining 68.5% were community–acquired. These important findings indicated the existence of community-associated strains of ESBL-producing Enterobacteriaceae and a remarkable percentage of such strain do exist that are hospital acquired. 

Acknowledgments

Vaidehi Tiru, Department of Microbiology, Sundaram Medical Foundation, Chennai, provided literature support. 

Footnotes

 Supported by Vision Research Foundation, Sankara Nethralaya, Chennai, India. The authors alone are responsible for the content and writing of the paper.

Footnotes

 Disclosure: M. Sowmiya, None; J. Malathi, None; H.N. Madhavan, None