COMPARATIVE STUDY OF CONVENTIONAL MEDIA AND LOW NUTRIENT MEDIA FOR THE  MICROBIOLOGICAL DIAGNOSIS OF ENDOPHTHALMITIS.

  1. Dr Asima Banu, M.D. Microbiology

Department of Microbiology

Bangalore Medical College and Research Institute

Bangalore – 560002

 

  1. Dr Nagaraj E.R, M.D. Microbiology

Professor of Microbiology

Sri Siddharatha Medical College

Tumkur – 572107

 

  1. Dr Meera Meundi, M.D. Microbiology

Professor & HOD of Microbiology

KVG Medical College

Sullia

 

  1. Dr Sriprakash K.S, M.S Ophthalmology

Professor of Ophthalmology

Director, MINTO Regional Institute of Ophthalmology

Bangalore – 560002

 

 

ABSTRACT:

 

BACKGROUND: Endophthalmitis is an ocular emergency and bacteria are the commonest etiological agents of infectious endophthalmitis. Any delay in treatment will result in serious complication like complete loss of vision.

OBJECTIVE: This study was undertaken with the aim of isolating bacteria causing endophthalmitis and studying their antibiogram which is very crucial for management; and to evaluate the use of a low nutrient agar for isolation of the etiological agent.

METHODS: 60 clinically diagnosed cases of endophthalmitis were studied during the period of three years. Aerobic culture and sensitivity was done on conventional media and low nutrient media (R2A agar). Antimicrobial sensitivity testing was performed.

RESULTS: Comparative evaluation of R2A agar versus conventional enriched media used for culture showed positive culture of 89% (42) cases in R2A agar against the 79% (37) cases in conventional media used. Sensitivity of the isolates against the antimicrobial agents exactly correlated with those used in ophthalmic infections. All the gram positive isolates (100%) were sensitive to vancomycin and the entire gram negative isolates (100%) sensitive to Ceftadizime.

 CONCLUSIONS: R2A agar yielded 10% higher growth when compared to other conventional media used. At the same time, conventional media are indispensable as few organisms failed to grow on R2A agar. It can be concluded that R2A and conventional medium can be used supplementary to each other.

 

Key words: Endophthalmitis, infectious endophthalmitis, low nutrient media

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

INTRODUCTION:

 

Bacteria are the commonest etiological agents of infectious endophthalmitis and clinical and experimental studies have firmly established that delay in therapy will result in poor visual outcome, especially in severe cases [1]. Culture of intraocular specimens, is considered the Gold standard in the diagnosis of endophthalimitis. Nevertheless, even under the most appropriate care traditional microbiological methods are difficult and yield positive results in only 25-60% of the clinically diagnosed typical cases of endophthalimitis[2]. Conventionally, media rich in proteinaceous substrates (blood, . Consequently, there is a need for a culture medium which allows recovery of slow-growing pathogenic bacteria.

 

In this present study we used a low nutrient media in comparison with the conventional media with an objective of evaluating its use in increasing the culture positivity in endophthalmitis cases that in turn will favour clinical outcomes. To the best of our knowledge, this is the first study of its kind done in the world and has not been reported previously.

 

 

 

 

 

 

 

 

MATERIALS AND METHODS:

 

This prospective study of 3 years was conducted in the Department of Microbiology of a tertiary care referral hospital. A total of 60 cases of clinically diagnosed endophthalimitis who presented themselves to Ophthalmology department were included in the study. Institutional ethical clearance was obtained and written, informed consent of the patient was taken according to prescribed guidelines. Controls could not be put up because an invasive procedure is involved for sample collection.

 A total of 80 specimens were obtained from post traumatic, post operative and endogenous endophthalmitis samples. Either the vitreous (93%, 81/87) or anterior chamber (7%, 6/87) samples collected by pars plana vitrectomy, vitreous tap or biopsy by standard techniques and were transported immediately to the lab[6,7] , processed by Gram’s stain and inoculation onto the following conventional liquid-and solid-phase media and low nutrient media[8,9,10,11] .

  1. 5% sheep blood agar, incubated at 370 C aerobically.
  2. Bacto R2A agar (from DIFCO) incubated at 300 C aerobically.
  3. Chocolate agar, incubated at 370 C in the presence of 5-10% of CO2.
  4. Brain heart infusion broth 5-10 ml, incubated at 370 C aerobically.
  5. Anaerobic culture was done using Robertson’s cooked meat media and incubated at 370 C for 2 weeks.

The culture on blood agar, chocolate agar, R2A and Brain heart infusion broth were incubated for 48 hours and if there was no growth, these media were incubated for 8 more days to allow the growth of slow growing or fastidious organisms. If the BHI broth showed turbidity, it was then subcultured onto blood agar, chocolate agar and MacConkey’s agar and the organisms were identified by standard techniques. If the broth media was still clear at the end of 10 days, a terminal blind subculture on blood agar and chocolate agar was performed before considering it negative . All the cultures were subjected to gram stain and later the isolated bacteria were identified by standard methods [12].

A Kirby-Bauer disk diffusion test was performed to determine antibiotic susceptibility [13]. All the isolates were tested for antibiotic sensitivity on Muller Hinton agar by Kirby Bauer disc diffusion technique using standard methods. The following discs were used for sensitivity.

 

GRAM POSITIVE     ORGANISMS GRAM NEGATIVE      ORGANISMS
amikacin 30 mg amikacin 30 mg
cephalexin 30 mg ceftazidime 10 mg
ciprofloxcin 01 mg ciprofloxacin 01 mg
tobramycin 10 mg tobramycin 10 mg
ampicillin 10 mg ticarcillin          75 mg
vancomycin   30 mg cephalexin  30 mg 

All the strains of Staphylococcus aureus that were resistant to ampicillin and cephalosporins were tested for methicillin resistance on Mueller Hinton agar with 4% NaCl using oxacillin (1mg) discs. The plates were incubated at 300 C and reading taken after 24 hours. The outcome measures included isolates identified and antibiotic sensitivity of the specimens.

 

The Statistical software namely SAS 9.2, SPSS 15.0, Stata 10.1, MedCalc 9.0.1 ,Systat 12.0 and R environment ver.2.11.1 were used for the analysis of the data and Microsoft word and Excel have been used to generate graphs, tables etc[14,15,16].

 

Diagnostic values based on Accuracy                                     Significant figures

0.9-1.0   Excellent test                             Suggestive significance (P value: 0.05<P<0.10)

0.8-0.9   Good test                                   Moderately significant ( P value:0.01<P £ 0.05)

0.7-0.8   Fair test                                     Strongly significant   (P value: P£0.01) 

0.6-0.7   Poor test

0.5-0.6   Fail

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

RESULTS:

 

Out of the 60 patients, culture was positive in 47 cases. The culture was positive in 89% (42/47) cases on R2A agar whereas on the conventional medium 79% (37/47) cases were positive. No anaerobes were isolated among these cases.

Among the post-operative cases, culture was positive in 16 (89%) cases on conventional media and 18 (100%) on R2A agar. In the post-traumatic cases, 20 (71%) cases were culture positive on conventional media whereas R2A agar yielded positive growth in 23 (82%) cases. In the case of endogenous endophthalmitis, the isolate grew both on conventional medium and R2A agar.

A total of 56 isolates were obtained from 47 culture positive cases. Single isolates were obtained in 38(81%) of the 47 culture positives, while 9 (19%) of the cases yielded two isolates. Among the 56 isolates, 49(87.5%) were Gram positive and 7(12.5%) were Gram negative. The commonest organism isolated was Bacillus cereus (21.42%). The second most common organism was Staphylococcus aureus (17.85%) followed by Staphylococcus epidermidis and Pseudomonas aeruginosa accounting for 12.5% cases each. The other etiological agents were Streptococcus pneumoniae and Corynebacterium species (10.71%) each, Bacillus species (5.35%), Streptococcus pyogenes, Streptococcus mitis, Corynebacterium jeikum, Nocardia asteroides and Brevibacterium species accounting for 1.79% each. No anaerobes were isolated.

12 (21.43%) organisms grew only on R2A agar and did not grow on conventional media. These isolates were 4 (7%) each of Bacillus cereus and Staphylococcus aureus, Corynebacterium species 2 (3.5%), Nocardia asteroides and Bacillus species 1 (1.75%) each. 6 (11%) isolates that grew in the conventional medium, failed to grow on R2A agar. These were Streptococcus pneumoniae 2 (3.6%) followed by Bacillus cereus, Streptococcus pyogenes, Brevibacterium species and Staphylococcus aureus (1.8%) each.

 

Statistical analysis (Table: 4) showed that the Findings of R2A are significantly associated with Conventional Media with p<0.001 with a sensitivity of 86.49% and a specificity of 56.52%. The positive and negative predictive values were 76.19% and 72.22 respectively.  The accuracy of the test is 75%.

 

Antibiogram showed that among the gram positive bacteria tested 48 of 49 (98%) were sensitive to amikacin, 40 of 49 (82%) to ciprofloxacin, 28 of 49 (57%) to cephalexin, 49 of 49 (100%) to vancomycin, 11 of 49 to ampicillin (22%), 26 of 49 (46%) to tobramycin.

Among the gram negative bacteria, 1 of 7 (14%) were sensitive to ampicillin, 6 of 7 (86%) to amikacin, 4 of 7 (57%) to ciprofloxacin, 5 of 7 (71%) to cephalexin, 7 of 7 (100%) to ceftazidime, 5 of 7 (71%) to ticarcillin, 4 of 7 (57%) to tobramycin.

 

 

 

 

 

 

 

 

DISCUSSION:

 

There are no microbiological studies on endophthalmitis using low nutrient media for isolation of pathogenic organisms. In the present study, an attempt was made to determine the various bacteria responsible for endophthalmitis, their antibiotic sensitivity and to compare the efficacy of the use of a low nutrient agar with that of conventional culture methods used for endophthalmitis.

Vitreous is a low molecular weight substance and contains approximately 99% water. Other macromolecular constituents are Type II collagen, Hyaluronic acid and Glycoprotein. Low molecular weight constituents include water-90%, Sodium, Potassium, Bicarbonate, Glucose  and  Amino acids [6].

 

In the present study, we found the conventional media that were used gave positive results in 78.7% cases (37 of 47 cases) and R2A agar gave positivity in 89.36% cases (42 of 47 cases). A total of 50 (89%) isolates were isolated by using R2A agar from the 42 culture positive cases. 12 isolates (21.43%) that did not grow on the conventional media used were isolated on R2A agar. This could be because the conventional media  like blood agar, chocolate agar and BHI broth rich in proteinaceous substrates (typically blood, meat and yeast extract) are used to culture material from infected eyes and it is assumed that pathogens will proliferate under optimal conditions. However, some bacteria exist in clinical environments very different from these highly nourished laboratory conditions [3] and may even reside within the eye as biofilms [4, 5]. These micro-environments may sustain bacteria adapted to more stringent conditions that will not necessarily be cultured by standard methods

 

. As the bacteria in the vitreous are adapted to low nutrient conditions, similar simulated media like R2A with low nutrient ingredients is used to culture these adapted organisms.

We found that pathogens were cultured consistently on R2A agar as well as being grown on conventional media. Except for 6 isolates that did not grow on R2A, all the other organisms were isolated. These isolates were from acute post traumatic cases with history of 24 hours. Some of the organisms that did not grow are fastidious organisms and in others the cases presented acutely, (i.e., within 12 hrs. of onset of inflammation). Thus, the organisms would have failed to grow on R2A agar. All the organisms that grew on R2A agar were from cases that presented late (5 days to 1 month) after onset of inflammation. All the isolates that grew on conventional medium, presented acutely (with 48 hrs. up to 4 days) of onset of inflammation.

 

In the present study, we used R2A agar plates that require no special preparation, only needing incubation at 300 C which is the room temperature in most areas and showed less contamination when incubated for longer periods than the conventional media. Even in testing centres with dedicated microbiological facilities, the culture positivity rate in various studies for endophthalmitis is low. In this context, the use of a low nutrient media may provide an increased rate of positive cultures where conventional culture is difficult. An increase culture positivity is achievable using low nutrient R2A agar as a supplementary medium along with conventional enriched medium.

 

Various studies showing the culture positivity rates using different techniques, like Ravi V et al [17] used Conventional and Bact /alert and had positivity of 40.4%, Anand et al[18] using only conventional culture got a positivity of 44.5% and Therese et al[19] used PCR and conventional culture and got a positivity of 75.8%. In the present study, using conventional culture and low nutrient R2A agar, we got a positivity of 78%. This shows very clearly that using routine media and R2A agar has increased the isolation rates as compared to even Bact/alert system and PCR.

The treatment of choice in cases of established bacterial endophthalmitis is intravitreal injections of antibiotics which include vancomycin for all gram positive bacteria and an aminoglycoside, amikacin or ceftazidine that show broad spectrum activity against gram negative bacteria [20]. Regarding antibiotic susceptibility patterns, vancomycin resistant gram-positive bacteria were not encountered in this study but a high degree of resistance was encountered in ampicillin (79%) and tobramycin (49%)  among all the isolates. Previously reported series of high sensitivity of intraocular isolates to amikacin, ciprofloxacin, cephalexin and ceftazidine was found in our study also where 86% of the total isolates were sensitive to amikacin, 79% to ciprofloxacin and 61% to cephalexin and 100% to ceftazidime. Hence, while determining choice of antibiotics for intravitreal infections, a high degree of invitro resistance of isolates to certain aminoglycosides like tobramycin should be considered.

R2A agar yielded 10% higher growth when compared to other conventional media used. At the same time, conventional media are indispensable as few organisms failed to grow on R2A agar. It can be concluded that R2A should be seen as complimentary to conventional media for isolation of organisms causing endophthalmitis and not as an alternative to them.

 

 

 

REFERENCES:

  1. Forster, R.K. Experimental Post-operative endophthalimitis. Trans Am Ophthalmol Societ 1990: 505-559.
  2. Sharma, S. Ocular Infections:Research in India. Indian J Med Microbiol2010; 28(2): 91-4
  3. Laws EA, Jones D, Karl DM. Method for assessing heterogeneity in turnover rates within microbial communities. Appl Environ Microbiol 1986; 52: 866-874.
  4. Elder MJ, Stapleton F, Evans E, Dart JKG. Biofilm-related infections in ophthalmology. Eye 1995; 9: 102-109.
  5. Morlet N, McLaughlin-Borlace L, Dart J. Bacterial biofilm on explanted intraocular lenses (IOLs). Invest Ophthalmol Vis Sci 1996; 37: S758.
  6. Madhavan HN, Anand A.R., Lily Therese K. Infectious endophthalmitis. Indian J Med Microbiol 1999; 17( 3 ):108-115.
  7. Chang, Guyer, Green, Shields, Yannuzzi. Retina-Vitreous-Macula. Vol 2. WB Saunders Co.; 1999. Diagnostic Vitrectomy. Glenn J. Jaffe; 1487-1501
  8. Derek Y. Kunimoto, Taraprasad Das, Savitri Sharma, Subhadra Jalali, Ajit B. Majji, Usha Gopinathan et al. Microbiologic spectrum and susceptibility of isolates. Part 1, Postoperative Endophthalmitis. American Journal of Ophthalmology 1999; 128:240-241.
  9. Taraprasad Das, Mangat R Dogra, Lingam Gopal, Subhadra Jalali, Atul Kumar et al. Post – surgical endophthalmitis diagnosis and treatment. Indian Journal of Ophthalmology 1995; 43:103-116.

10.  Collee J.G., Miles R.S. and Watt B. Tests for identification of bacteria. In: Mackie and McCartney, Practical Medical Microbiology (Vol. 2) 14th edition Collee J.G. Fraser A.G., Mrmion B.P. and Simon .A. Edenberg; Churchill, 1996:131-151.

11.  Khasravi AD, Mehdinejad M, Heidari M. Bacteriological findings in patients with ocular infections and antibiotic susceptibility pattern of isolated pathogens. Singapore Med J 2007;48:741-3

12.  Thomas A. Ciuella, Allen D. Beck, Trexler M Topping Ann Sullivan Baker, Blebitis, early Endophthalmiis and late endophthalmitis after glaucoma – filtering surgery. Ophthalmology 1997; 104: 986-994.

13.  Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; seventeenth informational supplement. Performance Standards for Antimicrobial Disk Susceptibility Tests: CLSI document M100-S17. M2-A9. Approved Standard. (19th ed) 2007;Vol 1

  1. Bernard Rosner (2000), Fundamentals of Biostatistics, 5th Edition, Duxbury, page 80-240
  2. Sunder Rao P S S , Richard J : An Introduction to Biostatistics, A manual for students in health sciences , New Delhi: Prentice hall of India. 86-160
  3. Griner PF, Mayewski RJ, Mushlin AI, Greenland P (1981) Selection and  interpretation of diagnostic tests and procedures. Annals of Internal Medicine, 94, 555-600.

17.  Ravi V., Sharma S., Lakshmi V., Ata-ur-rasheed M.D., Athmanathan S., Jalali S et al . Evaluation of bact / alert microbial detection system for the diagnosis of infectious endophthalmitis. Indian J Med Microbiol 2000; 18(1):15-20.

18.   Anand A. R, Therese K.L., Madhavan HN. Spectrum of Aetiological Agents of Post-operative Endophthalmitis and antibiotic susceptibility of Bacterial isolates. Ophthalmology 2000; 48:123-128.

19.   Therese K.L., Anand A.R., Madhavan H.N. PCR in the diagnosis of Bacterial Endophthalmitis.  Br J Ophthalmol 1998; 82:1078-1082.

20.   Chalita MR, Hofling-Lima AL, Paranhos A, Jr, Schor P, Belfort R.Jr. Shifting trends in in-vitro antibiotic susceptibilities for common ocular isolates during a period of 15 years. Am J Ophthalmol. 2004; 137:43–51.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

CHARTS & TABLES:

 

Table 1: showING comparative analysis of culture positivity on conventional media and R2A agar.

 

Total Cases

Culture Positive

Conventional

R2A

60

47 (78%)

37 (61.61%)

42 (70%)

 

 

 

Table 2: showING the positive cases with respect to diagnosis AND culture medium used

 

Type of Endophthalmitis

Total no. of cases

Total positive

Percentage

Culture Positive

On conventional media

on R2A

Post-operative

20(63.5%)

18

90%

16 (89%)

18 (100%)

Post-traumatic

38 (65.5%)

28

74%

20 (71%)

23 (82%)

Endogenous

02 (03%)

01

50%

01 (100%)

01 (100%)

Total

60 (100%)

47

 

37 (79%)

42 (89%)

 

 

 

 

Table 3: shows the total number of organisms isolated in the      present study.

 

Sl.

No.

Organism

Nos. of isolates

Percentage

1

Bacillus cereus

12

21.42%

2

Staphyclococcus aureus

10

17.85%

3

Staphylococcus epidermidis

07

12.50%

4

Pseudomonas aeruginosa

07

12.50%

5

Strep pneumonia

06

10.71%

6

Coryne bacterium species

06

10.71%

7

Bacillus subtilis

03

5.36%

8

Strep pyogenes

01

1.79%

9

Streptococcus mitis

01

1.79%

10

Corynebacterium jeikum

01

1.79%

11

Nocardia asteroids

01

1.79%

12

Brevibacterium species

01

1.79%

 

Total

56

100%

 

 

 

 

  

TABLE 4: CORRELATION OF CONVENTIONAL MEDIA AND R2A

 

R2A

Conventional Media

Yes

No

Yes

32(86.5%)

10(43.5%)

No

5(13.5%)

13(56.5%)

Total

37(100.0%)

23(100.0%)

Inference

Findings of R2A are significantly associated with Conventional Media with p<0.001**

Sensitivity %

86.49

Specificity %

56.52

PPV %

76.19

NPV %

72.22

Accuracy %

75.00

 

PPV: Positive Predictive value               

NPV: Negative predictive value

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5:  Sensitivity pattern of isolates (Percentage sensitive)

 

 

Organisms

Total Ampicillin Amkacin Ciproflox Cephalexin Ceftazidime Vancomycin Ticarcillin Tobramycin

Pseudomonas aeruginosa

7 114.28% 685.7% 457.1% 571.4% 7100% - 585.7% 457.1%

Bacillus Cereus

12 18.3% 1191.6% 1083.5% 541.6% - 12100% - 666.6%

Staph aureus

MSSA

 6  0%  6

100%

 4

66.6%

 4

66.6%

-  6

100%

 -  0%

MRSA

4 0% 233.3% 233.3% 0% - 4100% - 0%

Staph epidermidis

7 114.28% 7100% 571.4% 685.7% - 7100% - 685.7%

Strep pneumonia

6 583.3% 6100% 6100% 6100% - 6100% - 6100%

Coryne bacteria species

6 116.6% 6100% 583.3% 350% - 6100% - 116.6%

Bacillus species.

3 0% 266.6% 3100% 0% - 3100% - 3100%

Corynebacterium  jeikum

1 0% 0% 1100% 1100% - 1100% - 1100%

Streptococcus mitis

1 1100% 1100% 10% 1100% - 1100% - 1100%

Streptococcus pyogenes

1 1100% 1100% 1100% 1100% - 1100% - 1100%

Brevibacteriium species

1 1100% 1100% 1100% 1100% - 1100% - 1100%

Nocardia asteroids

1 0% 1100% 1100% 0% - 1100% - 0%
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