Contents lists available at ScienceDirect

Experimental Eye Research

journal homepage: www.elsevier.com/locate/yexer

Polybacterial community analysis in human conjunctiva through 16S rRNA

gene libraries
KrishnanNair Geetha Deepthia, Rajagopalaboopathi Jayasudhaa, Rameshan Nair Girisha,
Palanisamy Manikandanb,1, Rammohan Ramb, Venkatapathy Narendranb,
Solai Ramatchandirane Prabagarana,∗
a
Department of Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India
b
Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore, Tamil Nadu, India

A R T I C LE I N FO A B S T R A C T

Keywords: The conjunctival sac of healthy human harbours a variety of microorganisms. When the eye is compromised, an
16S rRNA gene library occasional inadvertent spread happens to the adjacent tissue, resulting in bacterial ocular infections.
Conjunctival swab Microbiological investigation of the conjunctival swab is one of the broadly used modality to study the aetio-
Cataract logical agent of conjunctiva. However, most of the time such methods yield unsatisfactory results. Hence, the
Culture independent identification
present study intends to identify the bacterial community in human conjunctiva of pre-operative subjects
Amplified ribosomal DNA restriction analysis
through 16S rRNA gene libraries. Out of 45 samples collected from preoperative patients undergoing cataract
surgery, 36 libraries were constructed with bacterial nested-PCR-positive samples. The representative clones
with unique restriction pattern were generated through Amplified Ribosomal DNA Restriction Analysis (ARDRA)
which were sequenced for phylogenetic affiliation. A total of 211 representative clones were obtained which
were distributed in phyla Actinobacteria, Firmicutes, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria,
Bacteroidetes, and Deinococcus–Thermus. Findings revealed the presence of polybacterial community, especially
in some cases even though no bacterium or a single bacterium alone was identified through cultivable method.
Remarkably, we identified 17 species which have never been reported in any ocular infections. The sequencing
data reported 6 unidentified bacteria suggesting the possibility of novel organisms in the sample.
Since, polybacterial community has been identified consisting of both gram positive and gram negative
bacteria, a broad spectrum antibiotic therapy is advisable to the patients who are undergoing cataract surgery.
Consolidated effort would significantly improve a clear understanding of the nature of microbial community in
the human conjunctiva which will promote administration of appropriate antibiotic regimen and also help in the
development of oligonucleotide probes to screen the predominant pathogens for early predisposition.

1. Introduction
The conjunctiva is a transparent lining of mucous membrane that
covers the posterior aspect of the eyelid together with the anterior
portion of the eyeball. Conjunctival epithelium has a unique defense to
protect against the ocular infection even if it is highly exposed to an
extensive array of microorganisms. Though infection of the bacteria is
usually restricted to ocular surface, occasional accidental spread takes
place in adjacent tissues like conjunctiva, cornea, inner eye, orbit and in
extreme cases even to the brain. Earlier reports claim that the healthy
conjunctiva hosts abundant bacterial community which encompasses
resident opportunistic pathogens and transient species (Dong et al.,
2011).
Occasionally host defense responses in the conjunctiva are prone to
attack by these organisms in those patients who have either undergone
intraocular surgery or penetrating trauma (Creuzot-Garcher et al.,
2016; Rishi et al., 2016). In such cases, the organisms residing on the
ocular surface are able to enter into the eye by direct inoculation with
subsequent proliferation causing a range of post-operative infections.
Therefore, an intense pre-operative examination of the microbiota of
ocular surface is imperative to reduce the risk thereby improving the
visual outcomes (Movahedan and Djalilian, 2012).
To date, conventional bacterial culture technique is the widely used
diagnostic modality to assess microbiota of the ocular surface (Zhang

∗
Corresponding author. Department of Biotechnology, Bharathiar University, Coimbatore, 641 046, Tamil Nadu, India.
E-mail address: prabagaran@buc.edu.in (S.R. Prabagaran).
1
Present Address: College of Applied Medical Sciences, Majmaah University, Majmaah 11952, Kingdom of Saudi Arabia.

https://doi.org/10.1016/j.exer.2018.05.011
Received 31 January 2018; Received in revised form 18 April 2018; Accepted 10 May 2018
K.G. Deepthi et al.
et al., 2017). However, these conventional methods are often inefficient
as they are laborious and time-consuming. Besides owing to limited
sample volume, they are unreliable for cultivation in media requiring
fastidious growth conditions (Schabereiter-Gurtner et al., 2001;
Graham et al., 2007). In such cases hampering appropriate treatment
will lead to prodigious visual consequences.
Hence, with the advent of molecular approaches such as polymerase
chain reaction (PCR) and DNA sequencing, rapid identification of
bacteria is possible. Unlike cultivable method, 16S rRNA gene PCR can
identify pathogenic bacteria even with small sample volume at high
sensitivity and more accuracy (Carroll et al., 2000). This helps to reduce
the time necessary for a confirmatory laboratory report to exemplify the
microorganisms involved in rapidly developing serious infections.
Usually, the detection of clinically relevant bacteria is possible by
the amplification of 16S rRNA gene which codes for the small subunit of
ribosomal RNA. It targets regions with highly conserved sequences that
are common among all previously studied bacteria, exploring the in-
terspersed highly variable or divergent sequences that can phylogen-
etically affiliate with the taxonomic species.
However, pertaining to polybacterial communities, direct sequen-
cing of 16S rRNA PCR products is not possible as they contain diverse
bacteria. Generally such heterogeneous population invites exclusive
cloning or demanding next-generation sequencing technologies for
better understanding (Jayasudha et al., 2014). To accomplish this, the
present study focuses on identification of polybacterial community in
human conjunctiva through 16S rRNA gene libraries employing Am-
plified Ribosomal DNA Restriction Analysis (ARDRA), DNA sequencing
and phylogenetic analysis.
2. Materials and methods
2.1. Conjunctival samples
Samples were collected from 45 pre-operative patients waiting to
undergo cataract surgery in a tertiary care eye hospital, Coimbatore,
India from September 2014 to June 2016. This study was conducted in
accordance with the tenets of the Declaration of Helsinki. As per the
advice of Institutional Review Board, a written informed consent was
obtained from the patients prior to sample collection. Under aseptic
conditions, sampling was performed in duplicate from the lower con-
junctival sac of the cataract eye using sterilized cotton swabs. Swabs
were collected in duplicate for either microbiological identification of
cultivable organism for analysis immediately or stored in saline at
−20 °C for culture independent investigation. Controls were main-
tained with sterile cotton swabs, in which no sampling was performed,
which were analyzed along with the conjunctival swab samples.
2.2. Microbiological assessment
All conjunctival swab samples were immediately processed for
culturing of aerobic and facultative bacteria, besides fungi. The samples
were seeded on to blood agar (tryptic soy agar with 5% sheep blood),
chocolate agar, potato dextrose agar, brain-heart infusion broth and
thioglycollate broth. Blood agar and chocolate agar plates were in-
cubated aerobically with 5% carbon dioxide at 37 °C for 24–48 h.
Thioglycollate and brain-heart infusion broth were incubated aero-
bically at 37 °C for 24–48 h. Potato dextrose agar plates were incubated
for a maximum of 3 weeks at 27 °C. Positive cultures from the plates
were identified by using Analytical Profile Index strip Method (Moore
et al., 2017). Eventually, if no growth was observed after incubation,
then the sample was considered as culture negative.
2.3. DNA extraction
From the stored conjunctival swab samples, bacterial genomic DNA
was extracted directly using QIAamp DNA Mini Kit (Qiagen, Hilden,
North Rhine-Westphalia, Germany) following the manufacturer's re-
commendations. The eluted DNA was stored under −20 °C, for further
analysis.
2.4. PCR amplification of 16S rRNA gene
Through nested PCR, 16S rRNA gene was amplified from the ex-
tracted DNA by using universal eubacterial primers (Integrated DNA
Technologies, Coralville, lowa, USA) considering the low copy number
of source DNA template. In first round PCR, 20 μL of reactions were
made containing 10 X PCR buffer, 1.5 mM MgCl2, 400 μM dNTPs
(Sigma-Aldrich, Vienna, Austria), 0.5 μM of forward primer (5′-GAGT
TTGATCCTGGCTCAG-3′) and reverse primer (5′-ACGGCTACCTTGTTA
CGACTT-3′) (Shivaji et al., 2000), 0.5 U Taq DNA polymerase (Sigma-
Aldrich, Vienna, Austria) and 50 ng of template DNA or an equal vo-
lume of sterile water (negative control). The PCR steps essentially
contained an initial denaturation at 94 °C for 5 min subsequently fol-
lowed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 48 °C
for 1 min, extension at 72 °C for 2 min and a final extension for
10 min at 72 °C. The second round nested PCR was carried out as same
in the first round PCR using 2 μL of first amplicon final product as
template along with 0.5 μM of forward inner primer (5′-TCCTACGGG
AGGCAGCAG-3′) and reverse inner primer (5′-GGCGGTGTGTACAAGG
CCC-3) (Albano et al., 2008; Neupert et al., 2008). The amplicons were
analyzed along with 100 bp DNA ladder as a molecular marker by
electrophoresis in 0.8% agarose gel containing ethidium bromide
(0.5 μg/ml) which were documented using gel documentation system
(Syngene International Ltd, Bangalore, India).
Nested PCR being highly sensitive, all the experiments were carried
out in a sterile workstation, using reagents prepared with UV treated
sterile nuclease free water (HiMedia Laboratories Pvt. Ltd, India) to
avoid cross contamination.
2.5. Clone libraries
Amplified 16S rRNA gene product was eluted from agarose gel using
elution kit (Bio Basic Inc, Canada). The extracted amplicon containing
mixed population were ligated into the pJET1.2/blunt cloning vector
(Thermo Scientific, Waltham, Massachusetts, United States) according
to manufacturer's protocol. The Ligation mixture was transformed into
Escherichia coli DH5α competent cells through calcium chloride medi-
ated transformation (Sambrook and Russell, 2006a). The recombinant
clones were propagated on Luria Bertani (LB) agar plate containing
100 μg/ml ampicillin. The plasmids were extracted using standard al-
kaline lysis method (Sambrook and Russell, 2006b) and inserts were
amplified with vector specific primers pJET 1.2 F (5′-CGACTCACTAT
AGGGAGAGCGGC-3′) and pJET 1.2 R (5′-AAGAACATCGATTTTCCAT
GGCAG-3′) using the same PCR steps described above except annealing
at 60 °C for 30 s.
2.6. ARDRA and 16S rRNA gene sequencing
Amplified clone inserts were digested using two restriction enzymes
HaeIII and TaqI for 4 h at 37 °C and 65 °C respectively. The digested
products were electrophoresed in a 2% agarose gel with Ethidium
bromide at 100 V for 2 h. The presence or absence of restriction di-
gestion pattern in each library was scored manually as ‘1’ or ‘0’ re-
spectively. All binary data was analyzed with NTSYSpc version 2.1
(Exeter software, Setauket, New York, USA); subsequently, based on
banding pattern, a dendrogram was constructed using UPGMA clus-
tering algorithm. The 16S rRNA gene of representative isolate from
each cluster after grouping were selected and subjected to Sanger se-
quencing (SciGenom, Cochin, Kerala, India) using primers pJET 1.2 F
and pJET 1.2 R. The resultant sequencing reads were assembled with
Lasergene Seqman Pro software (DNASTAR Inc., Madison, Wisconsin,
USA) and verified manually. Potential chimera sequences were
K.G. Deepthi et al.
screened and removed using UCHIME algorithm.
2.7. Phylogenetic analysis
The 16S rRNA gene sequences were compared for phylogenetic
neighbors in NCBI nucleotide sequence database using BLAST algo-
rithm and EzTaxon server (Kim et al., 2012); subsequently, reference
strains of all species of closely related genera were retrieved from these
databases and aligned using BioEdit software through multiple se-
quence alignment. The obtained alignments were used to determine the
phylogenetic relationship among 16S rRNA gene sequence of re-
combinant clones and reference strains using neighbor-joining, max-
imum likelihood and maximum parsimony algorithms of MEGA soft-
ware version 7 (Kumar et al., 2016) with 1000 bootstrap replications.
2.8. Statistical analysis of clone libraries
To estimate the bacterial composition among libraries, Principal
components analysis (PCA) was performed using PAST software pro-
gram (Hammer, 2001).
2.9. Nucleotide sequence accession numbers
The entire 16S rRNA gene sequences determined in this study were
deposited in the GenBank database and assigned the accession numbers
MG696907 to MG697113.
3. Results
Conjunctival swab samples from 45 patients who were scheduled to
undergo cataract surgery were examined for the incidence of bacteria
through routine microbiological culture and 16S rRNA gene libraries.
Among the total samples, 9 were observed negative in both culture and
PCR, which were not included eventually for further analysis. Of the
remaining 36 samples, though 12 alone were culture positive, all of
them were PCR positive through nested PCR. Thus, 24 samples were
claimed to be significant as they were detected positive only through
16S rRNA gene amplification. These results illustrate the sensitivity of
nested PCR assay which can detect organisms even at very low titre
compared to culture method.
3.1. Culture identification
In culture method, only 27% (12 samples) showed positive results
with single bacterial infection; which include Coagulase negative
Staphylococci (n = 6), Gram negative bacilli (n = 4) and α haemolytic
Streptococci (n = 2). All culture negative samples with no growth were
discarded only after 48 h of incubation.
3.2. Culture independent identification
Among 45 conjunctival swab samples, 36 were positive through
nested PCR, which were further investigated through 16S rRNA gene
libraries. Unique restriction pattern of recombinant clones were pooled
from each library that were scrutinized by ARDRA. Thus the total 211
unique patterns from the 36 libraries yielded 616 clones for DNA se-
quencing. Out of 36 libraries, 33 (91%) showed multiple restriction
patterns indicating the predominance of polybacterial flora, which
comprise 2 restriction patterns (3 libraries) or more up to 11 (30 li-
braries). Exceptionally, a library (CON12) had the highest diverse re-
striction pattern with 18 groups depicting the significance of our study
which was reported as culture negative in cultivable method. All the
211 clones with unique restriction patterns were subjected to 16S rRNA
gene sequencing which enabled the identification of different bacterial
communities (Table 1). Comparative sequence analyses were performed
using EzTaxon-e database based on sequence homology with known
bacteria which ranged between 93% and 100%.
Though 211 clones were sequenced, four (CON8-5, CON13-20,
CON12-3 and CON12-10) were identified with putative chimera due to
which they were dropped for further analysis. From the rest 207 clones,
6 clones (3%) showed ≤97% sequence identity remaining unclassified
suggesting that they could be some novel bacterial species. Yet the
remaining 164 clones (79%) showed ≥ 99% sequence homology to
known species, while 37 clones showed sequence homology between
97% and 99% providing identity up to genus level.
Overall, Fig. 1 shows distribution of 201 clones into phyla Actino-
bacteria, γ - Proteobacteria, Firmicutes, α-Proteobacteria, β-Proteobacteria,
Bacteroidetes, and Deinococcus–Thermus. Altogether, clone libraries re-
vealed the presence of 62 distinct species, 41 distinct genera and 34
distinct families (Fig. 2). Of the 41 identified genera, species affiliation
could not be determined for 9 genera viz., Apibacter sp, Nakamurella sp,
Reyranella sp, Brachybacterium sp, Thermus sp, Snodgrasella sp, Anaero-
coccus sp, Mesorhizobium sp and Dechloromonas sp. The analysis un-
veiled the presence of 118 g positive and 83 g negative representative
clones. At genus level, about 52% (104 clones) were grouped into 5
different genera and 48% (97 clones) belonged to remaining 36 genera.
About 61%of these genera (25 genera) were established with individual
libraries. The most often detected genera were Corynebacterium
(n = 30), Staphylococcus (n = 26) and Cutibacterium (n = 23), followed
by Escherichia (n = 13) and Acinetobacter (n = 12) However, profusion
of ubiquitous genera in each library varied depending on patients.
Among 36 libraries, 50% (18 libraries) contained both Gram posi-
tive and Gram negative bacteria, while the rest 50% had biased with
either Gram positive (7 Libraries) or Gram negative (11 libraries)
bacteria. Due to this fact, phyolgenetic lineages were separately ana-
lyzed for gram positive and gram negative clones using unambiguous
sequences (Fig. 3a and Fig. 3b). Those clones showing less than 600
base pair were not included for the construction of phylogenetic tree.
Phylogenetic analysis facilitates the affiliation of those genera which
were identified up to species level.
Out of 36 libraries, monobacterial infection was found in 3 libraries
with Paracoccus marcusii (CON9), Neisseria sp. (CON33) and Neisseria
flava (CON34). The highest bacterial diversity was found in CON2 with
11 different bacteria. Another 2 libraries (CON12 and CON22) con-
tained 9 different bacteria (Table 1). Predominance of genus Cor-
ynebacterium was found in 15 libraries. This was followed by Staphy-
lococcus and Cutibacterium which were distributed in 14 and 11 libraries
respectively. The other most frequently disseminated genera were Es-
cherichia (8 libraries), Paracoccus (7 libraries) and Bacillus (6 libraries).
Few libraries exhibited considerable frequency by a single bacterium
with different restriction patterns and varied sequence homology,
which could have been due to strain variations with in species. Con-
spicuously, CON14, CON17, CON25 and CON27 showed diverse pat-
terns occupied with a single bacterium, whereas other libraries like
CON15 and CON18 have more than one species with unreliable se-
quence homology and ARDRA pattern.
Principal component analysis (PCA) was performed to identify the
bacterial composition among 36 libraries which were evaluated based
on the relative abundance of bacterial phyla. The results clearly de-
monstrated the diversity and dominance of bacterial communities
among all libraries (Fig. 4). It elucidated 69.95% of total variance
among the individual library by first two components, where PC1 and
PC2 contributed 43.19% and 26.76% of total variance, respectively.
4. Discussion
This study demonstrated the microbial diversity in human con-
junctiva of pre-operative cataract patients, which is indispensable for
the timely prevention of intra ocular infection through prologue of
these organisms in to the anterior chamber. Our results explore diverse
bacterial composition present in conjunctival swab samples through
16S rRNA gene library. Detection of bacteria through conventional
K.G. Deepthi et al.

Table 1
Polybacterial community identified in the conjunctival swab samples through culture method and 16S rRNA gene libraries.
Case Library name Culture result No. of ARDRA pattern Clone name and accession Closest phylogenetic affiliation Sequence homology
clones number

P1 CON1 Coagulase negative 21 8 CON1-1 MG696907 Streptomyces heliomycin 99.57%

staphylococci CON1-2 MG696908 Corynebacterium lipophiloflavum 99.28%
CON1-6 MG696909 Snodgrasella sp. 97.74%
CON1-7 MG696910 Corynebacterium lipophiloflavum 99.10%
CON1-9 MG696911 Streptomyces caelestis 99.45%
CON1-10 MG696912 Escherichia coli 99.53%
CON1-13 MG696913 Staphylococcus simulans 99.57%
CON1-21 MG696914 Streptomyces heliomycini 99.91%
P2 CON2 Gram negative bacilli 13 11 CON2-1 MG696915 Bacilus sp. 98.07%
CON2-2 MG696916 Bacillus megaterium 99.09%
CON2-3 MG696917 Kocuria rhizophila 99.01%
CON2-4 MG696918 Paracoccus sanguinis 99.50%
CON2-5 MG696919 Micrococcus sp. 98.78%
CON2-6 MG696920 Tsukamurella carboxydivorans 99.53%
CON2-8 MG696921 Escherichia coli 99.87%
CON2-9 MG696922 Unidentified bacteria 95.55%
CON2-10 MG696923 Brevundimonas vesicularis 99.76%
CON2-11 MG696924 Corynebacterium macginleyi 99.62%
CON2-12 MG696925 Corynebacterium sp. 98.66%
P3 CON3 Culture negative 10 5 CON3-1 MG696926 Dechloromonas sp. 98.03%
CON3-2 MG696927 Ralstonia mannitolilytica 100%
CON3-6 MG696928 Cutibacterium sp. 98.60%
CON3-7 MG696929 Acinetobacter schindleri 99.39%
CON3-8 MG696930 Unidentified bacteria 96.77%
P4 CON4 Coagulase negative 16 4 CON4-1 MG696931 Enhydrobacter aerosaccus 100%
staphylococci CON4-2 MG696932 Enhydrobacter aerosaccus 99.08%
CON4-3 MG696933 Staphylococcus sp. 97.69%
CON4-7 MG696934 Bacillus infantis 99.81%
P5 CON5 Culture negative 19 8 CON5-1 MG696935 Corynebacterium propinquum 99.71%
CON5-2 MG696936 Corynebacterium propinquum 99.84%
CON5-3 MG696937 Corynebacterium macginleyi 99.48%
CON5-6 MG696938 Corynebacterium sp. 97.98%
CON5-7 MG696939 Dolosigranulum pigrum 99.68%
CON5-13 MG696940 Dolosigranulum pigrum 99.81%
CON5-14 MG696941 Corynebacterium sp. 97.89%
CON5-18 MG696942 Dolosigranulum pigrum 99.65%
P6 CON6 Culture negative 16 8 CON6-1 MG696943 Corynebacterium oculi 99%
CON6-3 MG696944 Corynebacterium accolens 99.02%
CON6-4 MG696945 Micrococcus aloeverae 99.78%
CON6-6 MG696946 Escherichia coli 99.85%
CON6-10 MG696947 Bradyrhizobium japonicum 99.33%
CON6-12 MG696948 Corynebacterium oculi 99.78%
CON6-14 MG696949 Unidentified bacteria 96.14%
CON6-16 MG696950 Corynebacterium sp. 98.70%
P7 CON7 Culture negative 16 6 CON7-1 MG696951 Staphylococcus hominis 99.72%
CON7-3 MG696952 Corynebacterium mucifaciens 99.40%
CON7-5 MG696953 Paracoccus sp. 97.73%
CON7-9 MG696954 Corynebacterium propinquum 99.81%
CON7-11 MG696955 Escherichia coli 99.81%
CON7-13 MG696956 Paucimonas lemoignei 99.21%
P8 CON8 Culture negative 18 5 CON8-1 MG696957 Enhydrobacter sp. 98.93%
CON8-2 MG696958 Oceanobacillus profundus 99.04%
CON8-4 MG696959 Thermus sp. 98.54%
CON8-15 MG696960 Ralstonia pickettii 99.53%
CON8-17 MG696961 Corynebacterium macginleyi 99.62%
P9 CON9 Culture negative 9 1 CON9-1 MG696962 Paracoccus marcusii 100%
P10 CON10 Gram negative bacilli 17 4 CON10-5 MG696963 Corynebacterium macginleyi 100%
CON10-10 MG696964 Streptococcus vestibularis 99.09%
CON10-13 MG696965 Corynebacterium macginleyi 99.90%
CON10-17 MG696966 Streptococcus thermophilus 99.76%
P11 CON11 Culture negative 14 5 CON11-1 MG696967 Aquabacterium parvum 99.22%
CON11-2 MG696968 Cutibacterium acnes 99.88%
CON11-4 MG696969 Mesorhizobium sp. 97.35%
CON11-8 MG696970 Cutibacterium acnes 99.65%
CON11-11 MG696971 Reyranella sp. 98.34%
(continued on next page)
K.G. Deepthi et al.

Table 1 (continued)

Case Library name Culture result No. of ARDRA pattern Clone name and accession Closest phylogenetic affiliation Sequence homology
clones number

P12 CON12 Culture negative 27 16 CON12-1 MG696972 Escherichia sp. 98.94%

CON12-2 MG696973 Cutibacterium acnes 99.52%
CON12-5 MG696974 Staphylococcus epidermidis 99.91%
CON12-7 MG696975 Escherichia coli 99.81%
CON12-8 MG696976 Cutibacterium acnes 99.81%
CON12-9 MG696977 Escherichia coli 100%
CON12-12 MG696978 Cutibacterium acnes 99.72%
CON12-14 MG696979 Unidentified bacteria 93.92%
CON12-15 MG696980 Escherichia sp. 98.94%
CON12-16 MG696981 Pseudomonas aeruginosa 99.71%
CON12-21 MG696982 Cutibacterium acnes 99.38%
CON12-22 MG696983 Brachybacterium sp. 98.38%
CON12-23 MG696984 Nesterenkonia massiliensis 99.81%
CON12-24 MG696985 Cutibacterium acnes 99.62%
CON12-25 MG696986 Kocuria rhizophila 99.34%
CON12-26 MG696987 Micrococcus aloeverae 99.81%
P13 CON13 Culture negative 26 5 CON13-1 MG696988 Cutibacterium acnes 99.78%
CON13-3 MG696989 Micrococcus aloeverae 99.52%
CON13-6 MG696990 Bacillus halotolerans 99.89%
CON13-15 MG696991 Micrococcus aloeverae 99.48%
CON13-22 MG696992 Kocuria rhizophila 100%
P14 CON14 Culture negative 21 7 CON14-1 MG696993 Rhodobacter johrii 100%
CON14-5 MG696994 Rhodobacter johrii 99.90%
CON14-7 MG696995 Rhodobacter johrii 99.90%
CON14-18 MG696996 Rhodobacter johrii 99.90%
CON14-19 MG696997 Rhodobacter johrii 100%
CON14-20 MG696998 Rhodobacter johrii 100%
CON14-21 MG696999 Rhodobacter johrii 99.80%
P15 CON15 Culture negative 24 10 CON15-2 MG697000 Paracoccus sphaerophysae 99.71%
CON15-3 MG697001 Paracoccus sphaerophysae 99.64%
CON15-5 MG697002 Cutibacterium acnes 99.67%
CON15-7 MG697003 Staphylococcus hominis 99.81%
CON15-9 MG697004 Staphylococcus hominis 99.22%
CON15-10 MG697005 Staphylococcus hominis 99.34%
CON15-15 MG697006 Staphylococcus hominis 99.53%
CON15-20 MG697007 Cutibacterium acnes 99.53%
CON15-21 MG697008 Corynebacterium 99.69%
tuberculostearicum
CON15-23 MG697009 Streptococcus infantis 99.74%
P16 CON16 Culture negative 16 2 CON16-3 MG697010 Staphylococcus epidermidis 99.73%
CON16-15 MG697011 Corynebacterium macginleyi 99.47%
P17 CON17 Culture negative 26 5 CON17-1 MG697012 Pseudomonas stutzeri 99.78%
CON17-6 MG697013 Pseudomonas stutzeri 99.89%
CON17-13 MG697014 Pseudomonas stutzeri 99.89%
CON17-19 MG697015 Pseudomonas stutzeri 99.78%
CON17-22 MG697016 Pseudomonas stutzeri 99.34%
P18 CON18 Gram negative bacilli 32 8 CON18-2 MG697017 Acinetobacter schindleri 99.07%
CON18-10 MG697018 Acinetobacter variabilis 100%
CON18-11 MG697019 Acinetobacter variabilis 99.81%
CON18-18 MG697020 Acinetobacter radioresistens 99.78%
CON18-21 MG697021 Acinetobacter variabilis 99.72%
CON18-22 MG697022 Escherichia coli 100%
CON18-28 MG697023 Acinetobacter radioresistens 99.16%
CON18-32 MG697024 Escherichia coli 99.91%
P19 CON19 Culture negative 22 8 CON19-1 MG697025 Corynebacterium macginleyi 99.75%
CON19-2 MG697026 Enhydrobacter aerosaccus 99.91%
CON19-4 MG697027 Neisseria sp. 98.92%
CON19-6 MG697028 Corynebacterium macginleyi 99.71%
CON19-10 MG697029 Staphylococcus cohnii 100%
CON19-13 MG697030 Corynebacterium macginleyi 99.68%
CON19-17 MG697031 Neisseria flava 99.35%
CON19-20 MG697032 Corynebacterium macginleyi 99.75%
P20 CON20 α haemolytic Streptococci 17 8 CON20-1 MG697033 Corynebacterium freiburgense 100%
CON20-3 MG697034 Bacillus sp. 98.60%
CON20-5 MG697035 Corynebacterium jeikeium 99.61%
CON20-6 MG697036 Staphylococcus pasteuri 99.88%
CON20-7 MG697037 Staphylococcus epidermidis 99.62%
CON20-8 MG697038 Unidentified bacteria 94.59%
CON20-11 MG697039 Nakamurella sp. 98.44%
CON20-16 MG697040 Streptococcus oralis 99.69%
(continued on next page)
K.G. Deepthi et al.

Table 1 (continued)

Case Library name Culture result No. of ARDRA pattern Clone name and accession Closest phylogenetic affiliation Sequence homology
clones number

P21 CON21 Culture negative 20 7 CON21-1 MG697041 Kocuria rhizophila 99.74%

CON21-3 MG697042 Cutibacterium acnes 99.88%
CON21-4 MG697043 Actinomyces oris 99.89%
CON21-5 MG697044 Corynebacterium sp. 98.59%
CON21-6 MG697045 Ralstonia pickettii 99.53%
CON21-12 MG697046 Moraxella osloensis 99.25%
CON21-16 MG697047 Bacillus halotolerans 100%
P22 CON22 Culture negative 22 12 CON22-1 MG697048 Micrococcus aloeverae 99.81
CON22-2 MG697049 Cutibacterium acnes 99.71%
CON22-4 MG697050 Micrococcus aloeverae 99.91%
CON22-6 MG697051 Unidentified bacteria 93.79%
CON22-8 MG697052 Corynebacterium matruchotti 99.41%
CON22-9 MG697053 Ralstonia sp. 98.44%
CON22-11 MG697054 Corynebacterium appendicis 100%
CON22-15 MG697055 Micrococcus lylae 99.60%
CON22-16 MG697056 Cutibacterium acnes 99.43%
CON22-17 MG697057 Paracoccus sp. 98.53%
CON22-18 MG697058 Cutibacterium acnes 99.52%
CON22-19 MG697059 Moraxella osloensis 99.79%
P23 CON23 Culture negative 24 3 CON23-1 MG697060 Cutibacterium acnes 99.79%
CON23-10 MG697061 Corynebacterium 99.67%
tuberculostearicum
CON23-20 MG697062 Staphylococcus hominis 99.53
P24 CON24 Culture negative 17 4 CON24-7 MG697063 Prevotella intermedia 99.47%
CON24-10 MG697064 Staphylococcus epidermidis 99.83%
CON24-14 MG697065 Cutibacterium acnes 99.72%
CON24-15 MG697066 Cutibacterium acnes 99.55%
P25 CON25 Culture negative 6 2 CON25-1 MG697067 Luteimonas terrae 99.91%
CON25-3 MG697068 Luteimonas terrae 99.86%
P26 CON26 Culture negative 26 8 CON26-2 MG697069 Cutibacterium acnes 99.81%
CON26-4 MG697070 Haemophilus aegyptius 99.15%
CON26-6 MG697071 Staphylococcus epidermidis 99.72%
CON26-7 MG697072 Haemophilus aegyptius 100%
CON26-17 MG697073 Cutibacterium acnes 99.43%
CON26-18 MG697074 Haemophilus sp. 98.96%
CON26-19 MG697075 Cutibacterium sp. 98.98%
CON26-23 MG697076 Staphylococcus epidermidis 99.30%
P27 CON27 Coagulase negative 17 3 CON27-1 MG697077 Staphylococcus epidermidis 99.91%
staphylococci CON27-2 MG697078 Staphylococcus epidermidis 99.62%
CON27-14 MG697079 Staphylococcus epidermidis 99.82%
P28 CON28 Culture negative 16 3 CON28-2 MG697080 Acinetobacter variabilis 99.72%
CON28-11 MG697081 Acinetobacter radioresistens 99.85%
CON28-14 MG697082 Escherichia coli 99.72%
P29 CON29 Culture negative 8 2 CON29-1 MG697083 Paracoccus marcusii 100%
CON29-4 MG697084 Paracoccus sp. 98.37%
P30 CON30 Gram negative bacilli 13 5 CON30-1 MG697085 Herbaspirillum aquaticum 99.84%
CON30-4 MG697086 Herbaspirillum aquaticum 99.88%
CON30-7 MG697087 Acinetobacter lwoffii 100%
CON30-9 MG697088 Acinetobacter lwoffii 100%
CON30-11 MG697089 Acinetobacter lwoffii 100%
P31 CON31 Coagulase negative 28 9 CON31-1 MG697090 Staphylococcus hominis 99.43%
staphylococci CON31-3 MG697091 Staphylococcus sp. 98.67%
CON31-6 MG697092 Cutibacterium acnes 99.29%
CON31-10 MG697093 Staphylococcus epidermidis 99.15%
CON31-12 MG697094 Anearococcus sp. 97.97%
CON31-20 MG697095 Cutibacterium acnes 99.62%
CON31-24 MG697096 Pseudomonas stutzeri 99.61%
CON31-27 MG697097 Citrobacter freundii 99.22%
CON31-28 MG697098 Anaerococcu sp. 97.48%
P32 CON32 Coagulase negative 6 4 CON32-2 MG697099 Bacillus sp. 98.87%
staphylococci CON32-4 MG697100 Staphylococcus sp. 97.99%
CON32-5 MG697101 Paracoccus marcusii 100%
CON32-6 MG697102 Bacillus sp. 98.27%
P33 CON33 Culture negative 5 2 CON33-2 MG697103 Neisseria sp. 98.96%
P34 CON34 Culture negative 1 CON34-1 MG697104 Neisseria flava 99.81%
P35 CON35 Coagulase negative 12 6 CON35-1 MG697105 Staphylococcus epidermidis 99.12%
staphylococci CON35-2 MG697106 Staphylococcus epidermidis 99.23%
CON35-3 MG697107 Staphylococcus epidermidis 99.13%
CON35-4 MG697108 Ochrobactrum intermedium 99.82%
CON35-7 MG697109 Pseudomonas geniculata 99.62%
CON35-9 MG697110 Pseudomonas geniculata 99.62%
P36 CON36 α haemolytic Streptococci 10 3 CON36-1 MG697111 Escherichia coli 99.34%
CON36-3 MG697112 Apibacter sp. 98.47%
CON36-10 MG697113 Escherichia coli 99.34%
K.G. Deepthi et al.

18

16

14

12

No. of clones
others
10
Fermicutes
8 γ Proteobacteria
6 β Proteobacteria
4 α Proteobacteria

2 Ac nobacteria

CON26
CON27

CON29
CON1
CON2

CON4
CON5
CON6
CON7
CON8
CON9
CON10
CON11
CON12
CON13
CON14
CON15
CON16
CON17
CON18
CON19
CON20
CON21
CON22
CON23
CON24
CON25

CON28

CON30
CON31
CON32
CON33
CON34
CON35
CON36
CON3

Library

Fig. 1. Distributions of different phylotypes from 36 clone libraries of conjunctival swab samples.

Fig. 2. Family level distribution of phylogenetically identified 207 clones. Maximum axis value is 30 and each internal axis tick for every 5 numbers.

culture methods endowed with a highly contradictory outlook that only
27% (12 samples) of eyes showed positive results. Such findings are in
accordance with the previous reports confirming the predominance of
Coagulase negative Staphylococci in conjunctival samples (Schabereiter-
Gurtner et al., 2001). This clearly demonstrated the limitation of tra-
ditional culture techniques, which require sufficient sample availability
and certain bacteria which are non cultivable or require fastidious
growth conditions (Aoki et al., 2013).
Polymerase Chain reaction (PCR) usually has enhanced diagnostic
precision which is capable of addressing such cases. In addition, nested
PCR would detect low bacterial load in the test sample yielding suffi-
cient amplicon to get detected in agarose gel (Prabagaran et al., 2017),
which intensified our study yielding 80% of the conjunctival swab
samples (36 Samples) as PCR positive. The remaining 20% (9 samples)
K.G. Deepthi et al.

Fig. 3a. Phylogenetic trees using neighbor-joining method based on partial 16S rRNA gene sequences from Gram positive bacteria of conjunctival swab samples.
Aquifex pyrophilus was used as an out group. The scale bar represents 0.02 substitutions per alignment position in the tree. Bootstrap values (expressed as percentage
of 1000 replications) greater than 50% are given at nodes.
K.G. Deepthi et al.

Fig. 3b. Phylogenetic trees using neighbor-joining method based on partial 16S rRNA gene sequences from Gram negative bacteria of conjunctival swab samples.
Methanothermococcus thermolithotrophicus was used as an out group. The scale bar represents 0.05 substitutions per alignment position in the tree. Bootstrap values
(expressed as percentage of 1000 replications) greater than 50% are given at nodes.
K.G. Deepthi et al.
Fig. 4. Principal Components Analysis (PCA) analysis of bacterial communities using the 16S rRNA gene sequences among 36 libraries of conjunctival swab samples.
All sequences within each library that had sequence identities of ≥97% were classified to phylum level based on BLAST identities.
showed negative results for both cultivable method and PCR. In several
cases, prior antibiotic treatment or PCR inhibitors are responsible for
such negative observations (Pandit et al., 2005). To exclude false po-
sitive result, two sterile cotton swabs were processed as negative con-
trol along with each batch of samples.
Inter individual comparison showed the incidence of diverse bac-
terial population including previously reported ocular bacteria such as
Corynebacterium (14%), Staphylococcus (13%), Propionibacterium (11%),
Acinetobacter (6%), Pseudomonas (4%), Bacillus (4%), Streptococcus
(2%), Ralstonia (2%), Kocuria (2%), Anaerococcus (1%), Aquabacterium
(0.5%), Bradyrhizobium (0.5%) and Brevundimonas (0.5%) (Graham
et al., 2007; Dong et al., 2011) (Fig. 2). Though, most of them were
ocular resident opportunistic pathogens, they are the one responsible
for various ocular diseases (Aoki et al., 2013). Notable, among them
Staphylococcus, Streptococcus, Bacillus, Propionibacterium, Pseudomonas
etc are causing post cataract bacterial endophthalmitis (Speaker et al.,
1991). The augmented probability of such genera in conjunctival swab
samples reflects the necessity of raising consciousness on these organ-
isms.
Despite this, our study found various bacteria that were not reported
previously in any sort of ocular infections. CON31 comprised of
Citrobacter freundii is an opportunistic pathogen reported in environ-
ment as well as intestine of animals causing blood, respiratory and
urinary infections (Edwards et al., 2015). Similarly Ochrobactrum in-
termedium in CON35 was associated with blood, urinary, bladder, liver
infection and infective endocarditis (Aujoulat et al., 2014). Acineto-
bacter schindleri is yet another potential pathogen taxonomically char-
acterized in 2001 found in CON18 library (Nemec et al., 2001).
In contrast, a new species Corynebacterium oculi which was initially
derived from human ocular specimens was found in CON6 (Bernard
et al., 2016). According to previous studies, Tsukamurella hongkongensis
and Tsukamurella sinensis were reported as novel species from patients
who have experienced keratitis, catheter-related bacteraemia and con-
junctivitis. Our study confined the presence of Tsukamurella carbox-
ydivorans (CON2-6) which was not previously reported in any other
clinical specimen rather than soil habitat (Park et al., 2009).
Indeed, some of the newly reported species which had never been
accounted before in any studies apart from taxonomic investigation
were also detected in our conjunctival swab samples (Table 1). CON12
is one of the well assorted samples which consisted of Nesterenkonia
massiliensis, reported from fecal flora of a HIV patient in France
(Edouard et al., 2014). Funke et al., 2009 isolated Corynebacterium
freiburgense for the first time from a lesion obtained through dog bite
which we witnessed in CON20. Paracoccus sanguinis from blood of
hospitalized patients in New York State (McGinnis et al., 2015) and
Corynebacterium appendicis acquired from abdominal swab associated
with appendicitis (Yassin et al., 2002) were observed in CON2-4 and
CON22-11 libraries respectively. CON18 and CON28 contained a range
of Acinetobacter variabilis strains reported from a variety of human and
animal specimens (Krizova et al., 2015). Interestingly while all the
above mentioned organisms were taxonomically explored by various
scientists from different countries during the last few decades, none of
them were previously reported from India. This suggests that numerous
enigmatic bacteria which were not previously identified in India in-
habited the human conjunctiva.
To the best of our knowledge, Paracoccus sphaerophysae (Deng et al.,
2011), Snodgrassella (Asraf, 2016), Micrococcus aloeverae (Prakash et al.,
2014), Luteimonas terrae (Ngo and Yin, 2016), Apibacter (Kwong and
Moran, 2016), Oceanobacillus profundus (Kim et al., 2007), Paucimonas
lemoignei (Jendrossek, 2001) and Bradyrhizobium japonicum (Jordan,
1982) were never detected previously in any of the clinical specimens.
Remarkably, most of them were exogenous microbial species, sug-
gesting the possibility of their entry through direct exposure. Conse-
quently, the bacterial composition among individual may vary de-
pending on anthropogenic effect, host immunological or biochemical
factors or normal ecological variations (Fierer et al., 2007).
We were unable to identify a few clones like CON2-9, CON3-8,
CON12-14, CON20-8, and CON22-6 which have no sequence similarity
to any of the previously described bacterial species. Such unidentified
bacteria could be novel which needs further characterization through
improved cultivation techniques. Among them we were able to affiliate
a few based on phylogenetic tree (Fig. 3a and b). Particularly, CON2-9
and CON3-8 were showing virtual similarity to Sediminibacterium sal-
moneum, while CON12-14 and CON22-6 affiliated to Snodgrasella alvi
K.G. Deepthi et al.
respectively; CON20-8 comprises Deinococcus navajonensis as their clo-
sest relative with 94.59% sequence identity which was reported in
2005. (Srinivasan et al., 2017). These taxonomic affiliations through
phylogenetic exploration are unexpected but valuable because it reveals
the incidence of enormous bacterial assortment in conjunctiva.
Significance of the study is that, most of the culture negative sam-
ples exhibit diverse bacterial community including a few unidentified
bacteria. Notably CON12 library with 27 clones exhibited 18 diverse
ARDRA patterns of varied sequence homology betrayed 9 different
bacteria from single patient. Similarly CON22 was with 12 diverse
ARDRA patterns and 9 different bacteria. Both the libraries had
Propionibacterium acnes represented predominantly in several ARDRA
patterns is an opportunistic pathogen that play a significant role in
corneal ulcer and postoperative endophthalmitis (Perry and Lambert,
2011). Reports on Propionibacterium acnes unveil its assorted sub-
species, signifying the possibility of different strains among particular
species with diverged ARDRA pattern (McDowell et al., 2016). Such
diverse paradigm is impossible to identify solely by microbiological
culture identification.
PCA plot established the visualization of various species represented
by clone libraries distributed within each cluster. Since the library
consist of diverse species range, the grouping of clones in different
clusters is based on variability in the percentage of diverse bacterial
phyla. In the plot, most libraries clustered together showing the dis-
tribution of common bacterial phylum among those libraries. While
CON19 and CON21 overlapped together with Actinobacteria, β –
Proteobacteria, γ – Proteobacteria and Firmicutes in almost equal ratio.
Predominance of phylum Actinobacteria was observed in CON12 and
CON22 in almost equal number which clustered separately far from
other phyla. Similarly, CON17 and CON18 were placed apart from the
cluster due to the occurrence of γ – Proteobacteria alone in the particular
libraries. All the remaining libraries were dispersed in the plot, de-
monstrating the quantum of bacterial diversity among those libraries.
Altogether, bacterial composition identified in this study highlights
the importance of understanding bacterial communities in conjunctiva.
Clone libraries of 16S rRNA sequencing exemplified complex and di-
verse bacterial community than understood through culture method,
where pathogenic bacteria contributed 80% of the total bacteria. In
contrast to the previous reports, our study found more or less even
distribution of both Gram positive and Gram negative bacteria in ocular
surface signifying the equal contribution of both in eye infections.
Significantly the investigation showed inimitable results even in culture
negative cases in conjunction with previous studies. This demonstrates
the ability of molecular approach through 16S rRNA gene libraries
which can detect even difficult-to-culture bacterial community up to
species level. Also the virulence of bacteria is found diverged in com-
bination with community variation alerting on the spectrum of bacteria
which will aid to evade post operative complications eventually. Thus
the study signifies the necessity for a broad spectrum antibiotic therapy
that needs to be envisaged to the patients. Studies in this line would
essence the development of a battery of oligonucleotide probes that can
be used to screen the predominant pathogens that threat vision.
Overall, this piece of work will aid ophthalmologists to address those
patients becoming a victim of post surgical complications.
Funding sources
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Conflicts of interest
No conflicting relationship exists for any author.
Acknowledgments
The author K. G. Deepthi wishes to acknowledge University Grant
Commission, New Delhi for her BSR fellowship (UGC/BSR/No.F.25.1/
2014-15/7-25/2007). The authors are grateful to UGC (Govt of India)
for support provided to establish infrastructure in the Department of
Biotechnology grant vide UGC/SAP/No.F.3-20/2013. The authors ac-
knowledge Prof Doraiswamy N. and Prof Themina from Presidency
College, Chennai for proof reading the manuscript.
References
Albano, H., Henriques, I., Correia, A., Hogg, T., Teixeira, P., 2008. Characterization of
microbial population of ‘Alheira’(a traditional Portuguese fermented sausage) by
PCR-DGGE and traditional cultural microbiological methods. J. Appl. Microbiol. 105,
2187–2194.
Aoki, R., Fukuda, K., Ogawa, M., Ikeno, T., Kondo, H., Tawara, A., Taniguchi, H., 2013.
Identification of causative pathogens in eyes with bacterial conjunctivitis by bacterial
cell count and microbiota analysis. Ophthalmology 120, 668–676.
Asraf, S.A., 2016. Gut microbiome of honey bee -An industrially relevant pollinator.
IIOAB J. 7, 21.
Aujoulat, F., Romano-Bertrand, S., Masnou, A., Marchandin, H., Jumas-Bilak, E., 2014.
Niches, population structure and genome reduction in Ochrobactrum intermedium:
clues to technology-driven emergence of pathogens. PLoS One 9, e83376.
Bernard, K.A., Pacheco, A.L., Loomer, C., Burdz, T., Wiebe, D., Huynh, C., Kaplen, B.,
Olson, A.B., Cnockaert, M., Eguchi, H., Kuwahara, T., 2016. Corynebacterium lowii sp.
nov. and Corynebacterium oculi sp. nov., derived from human clinical disease and an
emended description of Corynebacterium mastitidis. Int. J. Syst. Evol. Microbiol. 66,
2803–2812.
Carroll, N.M., Jaeger, E.E., Choudhury, S., Dunlop, A.A., Matheson, M.M., Adamson, P.,
Okhravi, N., Lightman, S., 2000. Detection of and discrimination between gram-po-
sitive and gram-negative bacteria in intraocular samples by using nested PCR. J. Clin.
Microbiol. 38, 1753–1757.
Creuzot-Garcher, C., Benzenine, E., Mariet, A.S., de Lazzer, A., Chiquet, C., Bron, A.M.,
Quantin, C., 2016. Incidence of acute postoperative endophthalmitis after cataract
surgery: a nationwide study in France from 2005 to 2014. Ophthalmology 123,
1414–1420.
Deng, Z.S., Zhao, L.F., Xu, L., Kong, Z.Y., Zhao, P., Qin, W., Chang, J.L., Wei, G.H., 2011.
Paracoccus sphaerophysae sp. nov., a siderophore-producing, endophytic bacterium
isolated from root nodules of Sphaerophysa salsula. Int. J. Syst. Evol. Microbiol. 61,
665–669.
Dong, Q., Brulc, J.M., Iovieno, A., Bates, B., Garoutte, A., Miller, D., 2011. Diversity of
bacteria at healthy human conjunctiva. Invest. Ophthalmol. Vis. Sci. 52, 5408–5413.
Edouard, S., Sankar, S., Dangui, N.P., Lagier, J.C., Michelle, C., Raoult, D., Fournier, P.E.,
2014. Genome sequence and description of Nesterenkonia massiliensis sp. nov. Strain
NP1 T. In: Stand Genomic Sci 9. pp. 866.
Edwards, G.B., Luna, A.J., Hernandez, A.C., Everett, G.F., 2015. Complete genome se-
quence of Citrobacter freundii myophage Moon. Genome Announc. 3 e01427–14.
Fierer, N., Breitbart, M., Nulton, J., Salamon, P., Lozupone, C., Jones, R., Robeson, M.,
Edwards, R.A., Felts, B., Rayhawk, S., Knight, R., 2007. Metagenomic and small-
subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and
viruses in soil. Appl. Environ. Microbiol. 73, 7059–7066.
Funke, G., Frodl, R., Bernard, K.A., Englert, R., 2009. Corynebacterium freiburgense sp.
nov., isolated from a wound obtained from a dog bite. Int. J. Syst. Evol. Microbiol. 59,
2054–2057.
Graham, J.E., Moore, J.E., Xu, J., Goodall, E., Dooley, J., Dartt, D., Downes, S., Moore, T.,
2007. Ocular pathogen or commensal: a PCR-based study of surface bacterial flora in
normal and dry eyes. Invest. Ophthalmol. Vis. Sci. 48, 5616–5623.
Hammer, O., 2001. PAST: paleontological statistics software package for education and
data analysis. Palaeontol. Electron. 4, 9.
Jayasudha, R., Narendran, V., Manikandan, P., Prabagaran, S.R., 2014. Identification of
polybacterial communities in patients with postoperative, posttraumatic, and en-
dogenous endophthalmitis through 16S rRNA gene libraries. J. Clin. Microbiol. 52,
1459–1466.
Jendrossek, D., 2001. Transfer of [Pseudomonas] lemoignei, a Gram-negative rod with
restricted catabolic capacity, to Paucimonas gen. nov. with one species, Paucimonas
lemoignei comb. nov. Int. J. Syst. Evol. Microbiol. 51, 905–908.
Jordan, D.C., 1982. Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium
gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int.
J. Syst. Evol. Microbiol. 32, 136–139.
Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H.,
Yi, H., Won, S., 2012. Introducing EzTaxon-e: a prokaryotic 16S rRNA Gene sequence
database with phylotypes that represent uncultured species. Int. J. Syst. Evol.
Microbiol. 62, 716–721.
Kim, Y.G., Choi, D.H., Hyun, S., Cho, B.C., 2007. Oceanobacillus profundus sp. nov., iso-
lated from a deep-sea sediment core. Int. J. Syst. Evol. Microbiol. 57, 409–413.
Krizova, L., McGinnis, J., Maixnerova, M., Nemec, M., Poirel, L., Mingle, L., Sedo, O.,
Wolfgang, W., Nemec, A., 2015. Acinetobacter variabilis sp. nov. (Formerly, DNA
group 15 sensu Tjernberg & Ursing), isolated from humans and animals. Int. J. Syst.
Evol. Microbiol. 65, 857–863.
Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics
analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874.
K.G. Deepthi et al.
Kwong, W.K., Moran, N.A., 2016. Apibacter adventoris gen. nov., sp. nov., a member of the
phylum Bacteroidetes isolated from honey bees. Int. J. Syst. Evol. Microbiol. 66,
1323–1329.
McDowell, A., Barnard, E., Liu, J., Li, H., Patrick, S., 2016. Proposal to reclassify
Propionibacterium acnes type I as Propionibacterium acnes subsp. acnes subsp. nov. and
Propionibacterium acnes type II as Propionibacterium acnes subsp. defendens subsp.
nov. Int. J. Syst. Evol. Microbiol. 66, 5358–5365.
McGinnis, J.M., Cole, J.A., Dickinson, M.C., Mingle, L.A., Lapierre, P., Musser, K.A.,
Wolfgang, W.J., 2015. Paracoccus sanguinis sp. nov., isolated from clinical specimens
of New York State patients. Int. J. Syst. Evol. Microbiol. 65, 1877–1882.
Moore, S.G., Ericsson, A.C., Poock, S.E., Melendez, P., Lucy, M.C., 2017. Hot topic: 16S
rRNA gene sequencing reveals the microbiome of the virgin and pregnant bovine
uterus. J. Dairy Sci. 100, 4953–4960.
Movahedan, A., Djalilian, A.R., 2012. Cataract surgery in the face of ocular surface dis-
ease. Curr. Opin. Ophthalmol. 23, 68–72.
Nemec, A., De Baere, T., Tjernberg, I., Vaneechoutte, M., Van Der Reijden, T.J.,
Dijkshoorn, L., 2001. Acinetobacter ursingii sp. nov. and Acinetobacter schindleri sp.
nov., isolated from human clinical specimens. Int. J. Syst. Evol. Microbiol. 51,
1891–1899.
Neupert, J., Karcher, D., Bock, R., 2008. Design of simple synthetic RNA thermometers for
temperature-controlled gene expression in Escherichia coli. Nucleic Acids Res. 36,
e124. http://dx.doi.org/10.1093/nar/gkn545.
Ngo, H.T., Yin, C.S., 2016. Luteimonas terrae sp. nov., isolated from rhizosphere soil of
Radix ophiopogonis. Int. J. Syst. Evol. Microbiol. 66, 1920–1925.
Pandit, L., Kumar, S., Karunasagar, I., Karunasagar, I., 2005. Diagnosis of partially treated
culture-negative bacterial meningitis using 16S rRNA universal primers and restric-
tion endonuclease digestion. J. Med. Microbiol. 54, 539–542.
Park, S.W., Kim, S.M., Park, S.T., Kim, Y.M., 2009. Tsukamurella carboxydivorans sp.
nov., a carbon monoxide-oxidizing actinomycete. Int. J. Syst. Evol. Microbiol. 59,
1541–1544.
Perry, A., Lambert, P., 2011. Propionibacterium acnes: infection beyond the skin. Expert
Rev. Anti. Infect. Ther. 9, 1149–1156.
Prabagaran, S.R., Kalaiselvi, V., Chandramouleeswaran, N., Deepthi, K.G., Brahmadathan,
K.N., Mani, M., 2017. Molecular diagnosis of Salmonella typhi and its virulence in
suspected typhoid blood samples through nested multiplex PCR. J. Microbiol. Meth.
139, 150–154.
Prakash, O., Nimonkar, Y., Munot, H., Sharma, A., Vemuluri, V.R., Chavadar, M.S.,
Shouche, Y.S., 2014. Description of Micrococcus aloeverae sp. nov., an endophytic
actinobacterium isolated from Aloe vera. Int. J. Syst. Evol. Microbiol. 64, 3427–3433.
Rishi, E., Rishi, P., Koundanya, V.V., Sahu, C., Roy, R., Bhende, P.S., 2016. Post-traumatic
endophthalmitis in 143 eyes of children and adolescents from India. Eye 30,
615–620.
Sambrook, J., Russell, D.W., 2006a. Preparation and transformation of competent E. coli
using calcium chloride. In: Cold Spring Harbor Protocols, pdb. prot 3932.
Sambrook, J., Russell, D.W., 2006b. Preparation of plasmid DNA by alkaline lysis with
SDS: minipreparation. In: Cold Spring Harbor Protocols, pdb.prot4084.
Schabereiter-Gurtner, C., Maca, S., Rölleke, S., Nigl, K., Lukas, J., Hirschl, A., Lubitz, W.,
Barisani-Asenbauer, T., 2001. 16S rDNA-based identification of bacteria from con-
junctival swabs by PCR and DGGE fingerprinting. Invest. Ophthalmol. Vis. Sci. 42,
1164–1171.
Shivaji, S., Vijaya Bhanu, N., Aggarwal, R.K., 2000. Identification of Yersinia pestis as the
causative organism of plague in India as determined by 16S rDNA sequencing and
RAPD-based genomic fingerprinting. FEMS Microbiol. Lett. 189, 247–252.
Speaker, M.G., Milch, F.A., Shah, M.K., Eisner, W., Kreiswirth, B.N., 1991. Role of ex-
ternal bacterial flora in the pathogenesis of acute postoperative endophthalmitis.
Ophthalmology 98, 639–650.
Srinivasan, S., Lim, S., Lim, J.H., Jung, H.Y., Kim, M.K., 2017. Deinococcus rubrus sp. nov.,
a bacterium isolated from Antarctic Coastal sea water. Int. J. Microbiol. Biotechnol.
27, 535–541.
Yassin, A.F., Steiner, U., Ludwig, W., 2002. Corynebacterium appendicis sp. nov. Int. J. Syst.
Evol. Microbiol. 52, 1165–1169.
Zhang, S.D., He, J.N., Niu, T.T., Chan, C.Y., Ren, C.Y., Liu, S.S., Qu, Y., Chong, K.L., Wang,
H.L., Tao, J., Pang, C.P., 2017. Bacteriological profile of ocular surface flora in
meibomian gland dysfunction. Ocul. Surf. 15, 242–247.