Research ArticleBacterial Diversity in Feline Conjunctiva Based on16S rRNA Gene Sequence Analysis: A Pilot Study
Katarzyna PBoneczka-Janeczko,1 Jacek Bania,2 Karolina Bierowiec,1
Maciej KieBbowicz,3 and ZdzisBaw KieBbowicz3
1Department of Epizootiology with Clinic for Birds and Exotic Animals, Faculty of Veterinary Medicine, Wrocaw University ofEnvironmental and Life Sciences, Norwida 31, 50-356 Wrocaw, Poland2Department of Food Hygiene and Consumer Health Protection, Faculty of Veterinary Medicine, Wrocaw University ofEnvironmental and Life Sciences, Norwida 31, 50-356 Wrocaw, Poland3Department andClinic of Veterinary Surgery, Faculty of VeterinaryMedicine,WrocawUniversity of Environmental and Life Sciences,Norwida 31, 50-356 Wrocaw, Poland
Correspondence should be addressed to Zdzisaw Kiebowicz; firstname.lastname@example.org
Received 30 August 2017; Revised 25 October 2017; Accepted 6 November 2017; Published 27 November 2017
Academic Editor: Daniele Corsaro
Copyright 2017 Katarzyna Poneczka-Janeczko et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.
Changes in the microbial populations in the conjunctival sacs of animals have traditionally been evaluated using conventionalmicrobiology techniques. The goal of this study was to examine the suitability of a methodology which may reveal a previouslyunknown microbiome inhabiting feline conjunctival membranes. In the present study, we determined the microbial diversity infeline conjunctivas based on 16S rRNA gene sequence analysis. Five taxa not described earlier in veterinary ophthalmology (i.e.,Staphylococcus caprae, Staphylococcus succinus, Propionibacterium acnes, Psychrobacter faecalis, and Bacillus subtilis) were identifiedin feline conjunctivas with a high similarity (99-100%). The study demonstrates that the feline conjunctival sacs are inhabitedby much more rich and diverse microbial communities than previously thought using culture-based methods. From the clinicalperspective, this could suggest that other laboratory procedures (e.g., extended incubation time in the case of Actinobacteria,formerly order Actinomycetales) or a new tool like culture-independent approaches (next-generation DNA sequencing) shouldbe taken into account.
Conjunctivitis, keratoconjunctivitis, and corneal sequestra-tion are common clinical problems in cats. Based on researchover the last few decades, characteristics of the bacterial florain feline conjunctival sacs show a similar composition, andthe occurrence of particular species of bacteria varies byfrequency of their isolation. However, Gelatt described thefeline conjunctival and corneal surface as being generallycolonized to a lower degree than in other domestic species .Among bacteria isolated from the conjunctiva, staphylococciare the most representative group. The presence of S. epider-midis, S. pseudintermedius, S. aureus, S. albus, S. haemolyticus,S. simulans, S. auricularis, S. saprophyticus, and S. felis hasbeen observed in the conjunctiva by many scientists .
A second group of frequently isolated microorganisms arehemolytic and nonhemolytic streptococci (i.e., S. viridans). Previous studies based on the microbiological identifi-cation of bacteria or the sequencing of amplicons generatedfrom microbial DNA have led to the identification of severalgenera in feline conjunctivas, such as Enterococcus spp.,Pseudomonas spp. (P. aeruginosa), Proteus spp., Pasteurellaspp., Bacillus spp., and Micrococcus spp. [3, 5]. Mycoplasma(M. felis, M. canadense, M. cynos, M. gateae, M. lipophilum,andM. hyopharyngis) have also been considered conjunctivalcommensals, which in some circumstances may be involvedin conjunctival pathology .Chlamydophila felis has beenidentified as an indisputable pathogen of feline conjunctiva.ThisGram-negative bacteriumhas already been isolated froma number of feline conjunctivitis cases [10, 11]. There is also
HindawiBioMed Research InternationalVolume 2017, Article ID 3710404, 5 pageshttps://doi.org/10.1155/2017/3710404
2 BioMed Research International
evidence that other Chlamydia-related microorganisms likeChlamydophila pneumoniae and Neochlamydia hartmannel-laemay be associated with conjunctiva [12, 13]. Investigatingconjunctival infections in cats with lepromatous lesions, Fyfeet al.  identified Mycobacterium spp. to have occurred.Based on a phylogenetic analysis, a novel species in theMycobacterium simiae-related group was identified . Onthe other hand, Fox et al.  described Salmonella-associatedconjunctivitis in cats.
Most of the previous research has investigated felineocular microflora using a classical microbiology approachinvolving the culture and further characterization of isolates.The aim of the present study was to examine the suitabilityof the methodology which may disclose microbial diversitywithin feline conjunctivas of healthy cats and animals withconjunctivitis symptoms, using partial sequencing of the 16SrRNA gene. To the best of our knowledge, it is a frontierresearch in the field of veterinary ophthalmology and apreliminary study linked to our next project concerning next-generation sequencing (NGS).
2. Materials and Methods
Conjunctival swabs obtained from three clinically healthycats with no ocular disorders and from three cats withconjunctivitis symptoms were included in the study. Basedupon our own clinical experience with chronic conjunctivitisin cats and for the purpose of the study, sick animalscomply with criteria such as manifestation of conjunctivi-tis (ocular discharge, chemosis, and conjunctival edema)lasting about six months and insufficient response to thestandard ophthalmological treatment (history of the treat-ment with ophthalmic ointments and eye drops). Sick catswere also tested by PCR and RT-PCR to determine thepresence ofChlamydia felis, feline herpesvirus-1 (FHV-1), andMycoplasma felis infections, according to published protocolsby Chalker et al. , Marsilio et al. , and Helps et al., whereby specificDNAwas not detected. Additionally, anophthalmic examination was performed on each cat; eyelashand cartilage abnormalities and incorrect positioning of theeyelids were ruled out. Irregularities of the drainage systemwere eliminated with a 1% fluorescein test and by irrigationvia a 26G catheter.
Swabs were taken prior to any other ocular examinationand transferred to the bacteriological laboratory at theDepartment of Epizootiology, Faculty of VeterinaryMedicine, Wrocaw. Material from the swabs was suspendedin 500 L of 0.9% NaCl by vortexing for 2 minutes. DNA wasextracted frommaterial released from the ocular swabs usingthe QIAamp UltraSens Virus Kit (Qiagen, Syngen Biotech,Wrocaw, Poland), in accordance with the manufacturersinstructions. The quantity of DNA was measured usingNanoDrop 2000. Amplification of the conserved regionwithin eubacterial 16S rRNA gene was performed withprimers 16S-27f AGAGTTTGATCMTGGCTCAG and16S-907r CCGTCAATTCMTTTRAGTTT, yielding an880 bp product. Next, nested PCR using 16S-27f primerand 16S-519r GWATTACCGCGGCKGCTG was performed,
Figure 1: Diversity of bacterial genera identified within felineconjunctivas, based on a 16S rRNA gene sequence analysis.
yielding a product of 492 bp (all primers were fromhttp://www.ridom.de/rdna/). PCR was performed underthe following conditions: initial denaturation at 94C for3min, followed by 35 cycles of 94C for 30 s, 54C for45 s, and 72C for 30 s. The 492 bp PCR products wereligated into the pJET1.2/blunt cloning vector (ThermoScientific). Laboratory E. coli NovaBlue strain (Novagen)was transformed with a ligation product using heatshock, and the cells were plated onto agar containingampicillin. Positive clones with amplicon-containingvectors were PCR-amplified using pJET1.2 sequencingprimers and sequenced. The sequences obtained from bothstrands of the PCR product were analyzed using BioEditsoftware (http://www.mbio.ncsu.edu/BioEdit/bioedit.html)and taxon identification was conducted using thehttps://blast.ncbi.nlm.nih.gov platform. Taxa were identifiedat a species level when the similarity of their 16S rRNAsequence and those from the GenBank database were >99%.Sequence similarity between 97 and 99% was the criterionfor identification of the taxon at the genus level.
A total of 48 sequence reads were obtained in the study;only the 30 high-quality sequence reads were used in furtheranalysis of the diversity of bacterial flora in the feline con-junctiva. Eight genera were identified among the sequencesfrom clinically healthy and diseased animals (Figure 1).Taking into consideration the maximal 16S rRNA distancescores < 1%, the following species were recognized: Bacillussubtilis, Psychrobacter faecalis, Psychrobacter pulmonis, Pro-pionibacterium acnes, Staphylococcus caprae, Staphylococcuscapitis, Staphylococcus succinus, Streptococcus infantarius, andStreptococcus lutetiensis. The low similarity in microfloracomposition at the genus level was observed between dis-eased and healthy conjunctivas (Table 1).