Year : 1972 | Volume
: 20 | Issue : 4 | Page : 164--170
Study of the normal conjunctival flora (bacterial and fungal) and its relations to external ocular infections
PN Srinivasa Rao1, K.N.A Rao2,
1 Dept. of Ophthalmology, Kasturba Medical College, Manipal, India
2 Dept. of Microbiology, Kasturba Medical College, Manipal, India
P N Srinivasa Rao
Dept. of Ophthalmology, Kasturba Medical College, Manipal
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Srinivasa Rao P N, Rao K. Study of the normal conjunctival flora (bacterial and fungal) and its relations to external ocular infections.Indian J Ophthalmol 1972;20:164-170
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Srinivasa Rao P N, Rao K. Study of the normal conjunctival flora (bacterial and fungal) and its relations to external ocular infections. Indian J Ophthalmol [serial online] 1972 [cited 2023 Apr 1 ];20:164-170
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This report is a study of the normal conjunctival flora and its relation, if any to external ocular infections, in a group of patients, that attended the ophthalmic department of the Kasturba General Hospital, Manipal from December 1966 to January 1968.
Manipal is located (altitude of 100 meters) on the West Coast, and has an annual rain fall of more than 120 inches with the maximum precipitation from June to September. The weather is "hot" throughout the year with an average of about 30°C. During the South West monsoon season there is a luxuriant and ubiquitous growth of fungi which becomes a real problem during this season. These factors made us feel that a study of the conjunctival flora during a period for a year was worth while.
Material and Methods
115 patients with no clinical evidence of extra ocular infections, otherwise healthy were selected. Some of these patients sought help for refractive errors and the others had come for some intra-ocular operations such as cataract. 58 cases of corneal ulcers were also studied.
Two sterile swabs, were used to collect materials. The swabs were thoroughly rubbed over the lower fornix of the eye from lateral to the medial side, sealed in sterile test tubes and sent to the laboratory soon after collection. In the case of corneal ulcers the conjunctival sacs were well washed in sterile saline and scrapings made from the deeper parts of the ulcers and also at their edge. These were sent to the laboratory for both bacteriological and mycological cultures, as was done with the swabs.
On receipt of the swabs or the scraped material in the laboratory, one swab was seeded on blood agar and then a smear was made. With the other a culture on Sabouraud's agar, a smear, as also the wet preparation were made.
These were routinely examined by a Lactophenol blue preparation, by gram's staining and giemsa stain for any evidence of fungi or bacteria.
The inoculated blood agar and Sabouraud's plates were incubated at 37° and room temperature respectively. The blood agar was rejected after 72 hrs. and the Sabouraud's after 3 weeks, if no growth was seen.
The growths on blood agar were identified by the usual methods up to the genus level, a species level where it was possible by the usual tests like gram's staining, motility, coagulase test if staphylococcus was isolated, or by other suitable tests like biochemical tests as warranted by the isolate for confirmation of the identification.
As a routine, antibiotic sensitivity test of bacterial isolates were done by the disc diffusion methods.
The fungi were identified by their morphology after the sub-cultures were made on corn-meal agar, and by slide culture as well to study the characteristic clamydo-spores etc, as indicated. Biochemical tests were also set up to identify the species. In a few cases of doubtful identification culture specimen were sent for confirmation of the identification.
Results and Observations
The organisms isolated from the 115 normal patients are given in a tabular form [Table 1] and [Table 2].
From the above table, it is obvious that the incidence of bacteria in the normal conjunctiva, increases with age. In contrast, the fungal incidence remains more or less the same.
Incidence in the sexes:
The conjunctival bacterial flora is more or less the same in either sex, ((22.5% males 18.2% females) but there is predominance of fungal incidence in the males. (28.2% males 18.2%females) This may be attributed to the outdoor work in this preeminantly agricultural area in which males take part more than females.
Relation to weather:
In this part of the country there is no marked seasonal changes. For all practical purposes, the year can be considered as one long season of summer, with relatively high humidity. November to March is slightly cooler and drier (28°C). April to October, the temperature is higher (31°C) and the atmosphere is saturated with humidity, on account of full precipitation of the South-West monsoon (120 inches).
In the hot season there is predominance of bacterial flora.
In a previous work (SRINIVASA RAO et al, 1967), it was the impression that fungal ulcers are perhaps more common in relatively dry and cool period (December to February). Such crowding of cases can perhaps, as was pointed out, be attributed to the harvest season. It is during the process of separating the paddy from the crop (thrashing) that foreign bodies (mostly husk) enter the eye, causing corneal abrasion and subsequent infection.
Out of the above, 13 cases, each showing fungal and bacterial infection in this series underwent intraocular surgery. All these 26 cases responded well to the usual regime of pre-operative medication, i.e. 7 days of topical broad spectrum antibiotic application, ,and 3 days of systemic antibiotic treatment after operation. There was no complication like endophthalmitis or iridocyclitis which can be attributed to the fungal or bacterial infection.
DUKE-ELDER quotes 13 authors, who have found a considerable proportion of normal conjunctival sacs free from bacteria as also varying from 72% (PUTTANNA 1958-1959) to as low as 17% (KHORAZO AND THOMPSON. There are other authors who have denied this and are of opinion that practically every conjunctiva exhibits some flora. DUKEELDER however concludes, that at birth the conjunctival sac is normally sterile, but soon it tends to get contaminated by organisms, either saprophytic or parasitic in character. Considering its exposure to the atmosphere, and its continuity with the skin, it is surprising it even remains sterile.
AGARWAL AND KHOSLA  in their series found that in the urban population only 23% of conjunctival sacs were sterile, as compared to 30% in rural population. This differs from the findings of HAMMAKE  where more positive results are from the rural areas. AGARWAL et alt explain this difference as due to the indiscriminate use of corticosteroid and antibiotic eye, ointment by the urban people.
ROHATAG1  in his series of 50 normal conjunctiva found bacteria in all of them Staphylococcus Albus Coagulase Negative in 16 out of 50 cases, and B. Xerosis in 13 out of 50.
The fact that the sac may be sterile and that organisms in it so frequently assume Innocuous characteristics is due in part to its low temperature, in part to the action of the lysozyme of the tears and, in part to the mechanical action of blinking and the slincing effect of the lacrimal secretion. Lysozyme probably plays an important part not only in depleting the total bacterial population but also in determining its character. Thus its action is insignificant in inhibiting such organisms as C. XEROSIS, MORAXELLA AND H. AEGYPTICUS, while its decreased concentration in conjunctivitis may explain the profusion and relative virulence of other organisms in diseased conditions (DE VINCENTIS).
KHORAZA AND THOMPSON, as quoted by GIVNER  made cultural studies of the normal conjunctiva in 1122 patients. They found staphylococci were present in 64 per cent of them, diphtheroids in 36 per cent, alpha haemolytic strepto-cocci in 3-4 per cent, streptococcus pyogenes in 1.2 per cent, pneumococci 2.6 per cent, sarcinae 1 per cent. More than one organisms were present in 25 per cent, and no organism was present in 17 per cent.
Surprisingly few investigations have been done on the mycotic flora of the healthy conjunctiva, but those that are available indicate that quite a proportion of apparently normal conjunctiva are contaminated in this way. Thus in Central Europe FAZAKAS , found in 24-257 (of cases; MITSUI AND HANABUSA  found 18.5% in Japan. HAMMEKE AND ELLIS  found Fungi in 10.3% of the normal eyes of the adults in ARKANSAS 5 % of children and 1.0% of new born infants. HINLEY and SMITH (England) found positive cultures for fungus in 27.9 per cent of normal conjunctiva. The most common are blastomycosis particularly Penicillium.
It would seem that most of these are air-born and it is interesting that their occurrence depends greatly on the occupation, since many of them live amidst vegetation (plants and vegetable material) they are found most commonly in the rural population. FAZAKAS  for example as quoted by DUKE-ELDER isolated fungi from the conjunctival sac, the lids and the lacrimal passages in 83% of harvesters and threshers. BALAKRISHNAN  opines that in primary keratomycosis, infection is carried from the soil to the cornea due to minor injuries.
WILLIAMSON, et al  from Great Britain isolated fungi from 2.9% of the healthy conjunctival sac examined, the incidence being higher in older subjects. Most species isolated were non-pathogenic transchient aerial contaminants, but some potential pathogens were found.
Out of a total of 58 cases of corneal ulcer scrapings, only in 14 bacterial growth was seen, as given in the table below:
Incidence of bacteria in the corneal ulcer scrapings is more or less the same as that of normal conjunctiva. Even the pattern seems to be more or less the same as that of normal conjunctiva. This rather low incidence of bacteria from the corneal ulcer scrapings may be due to the use of some antibiotic prior to hospitalisation. It is difficult to establish the cause and effect relationship. Repeated cultures were not taken nor an attempt was made to identify the organism in living cells. Besides, the scrapings from the floor of the ulcer were not examined for inclusion bodies.
On the other hand the incidence of fungus from the corneal ulcer scrapings is slightly higher. Here again, it is difficult to establish cause and effect relationship. By and large, no attempts were made for repeated culture. It may be that the presence of dead tissue simply favours the growth of saprophytic fungi, thereby accounting for higher incidence. Alternatively some of the cases are perhaps due to actual invasion of the tissue by the fungus. There is evidence to show that even non-pathogenic species under certain conditions assume a pathogenic character and proliferate on, and penetrate into tissues producing necrosis (PUTTANNA, 1969). This is specially seen in patients with low resistance as in debilitated individuals, diabetis, leukaemia and cancer. Prolonged use of corticosteroids and antibiotics are contributing factors. This is due to the fact that the normal balance that exists between bacteria and fungi have been disturbed and the opportunistic fungi gain the upper hand. Any minor trauma of the cornea favours such invasion. The pattern of fungus isolated from corneal ulcer scrapings appears to be the same, as that obtained in the normal conjunctival sac.
All the cases of corneal ulcers had the following treatment. The ulcer was scraped, the base of the ulcer was cauterized and suitable broad spectrum antibiotic applied, and sometimes supplemented by systemic antibiotic. After 48 hours, when the results of culture and sensitivity tests were known, the antibiotic was changed if necessary. Out of 58 cases, in 11 cases the treatment was considered as failures - i.e., the cases went for perforation. The study of these failure cases are interesting:
Out of 11 cases, 5 were sterile bacteriologically and mycologically. In 2 of these cases repeated cultures were done, both for bacteria and fungus. The ulcers progressed in spite of treatment. We are not able to account for these. The tests for inclusion bodies were not done.In 3 out of 4 cases, B. Pyocyaneus was identified, the ulcerations went on to perforation in spite of treatment. Inference is B. Pyocyaneus must be considered as the most dangerous organism as far as eye is concerned.One case was one of perforated corneal ulcer going on to Panophthalmitis. B. Coli were grown by cultural method and also a scanty growth of Aspergillus Flavus. Only 2 colonies of Aspergillus Flavus were grown and so the impression is that it was only a contaminant.In the remaining 2 cases, Fungus probably is the etiological agent.
In another case the examination at the time of commencement of treatment showed haemolytic streptococci culturally and no fungus. The case improving well. After about 1 month there was sudden recrudescence. Direct examination at the time showed budding yeast like cells by direct examination and Candida Albicans and coagulase negative staphylococci by cultural methods. The treatment was a failure and the impression is the recrudescence of the disease was due to Candida Albicans.
In one other case, colonies of mucor were found and no bacteria were identified. The ulcer may be attributed to fungal infection, since the treatment was a fuilure and mucor was not grown from the normal conjunctival sac.
115 normal conjunctival sacs were investigated, 24 (20.9%) of them showed some bacteria or other, and 28 of them fungus (24.3%. In general, they appear to be innocuous. 58 cases of corneal ulcers were investigated for bacteria or fungus. Only 14 (24.1%) of them showed some bacteria, this rather low incidence of bacteria was perhaps attributable to the prior use of antibiotic. 21 (36.2%) of them showed some fungus or other. It is difficult to establish the cause and effect relationship. In at least 2 of these cases, fungus proved to be the causative agent.
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