Identification of Novel Sequence Types among Staphylococcus haemolyticus Isolated from Variety of Infections in India

The aim of this study was to determine sequence types of 34 S. haemolyticus strains isolated from a variety of infections between 2013 and 2016 in India by MLST. The MEGA5.2 software was used to align and compare the nucleotide sequences. The advanced cluster analysis was performed to define the clonal complexes. MLST analysis showed 24 new sequence types (ST) among S. haemolyticus isolates, irrespective of sources and place of isolation. The finding of this study allowed to set up an MLST database on the PubMLST.org website using BIGSdb software and made available at http://pubmlst.org/shaemolyticus/. The data of this study thus suggest that MLST can be used to study population structure and diversity among S. haemolyticus isolates.


Introduction
Staphylococcus haemolyticus, one of the coagulase-negative staphylococci (CoNS), is an opportunistic pathogen that ranks second after S. epidermidis in the frequency of isolation from blood cultures [1]. S. haemolyticus is notoriously known to exhibit multidrug resistance and form biofilm [2][3][4][5][6][7][8]. Multilocus sequence typing (MLST), a highly discriminatory and reproducible DNA-based typing technique has been utilized to analyze the bacterial isolates. The genetic analysis reported, at least, seventeen sequence types (STs) among S. haemolyticus strains, however, there is no MLST database in the public domain [3]. Therefore, we analyzed strains of S. haemolyticus isolated from a variety of infections collected from different parts of India and set up an MLST database at http://pubmlst.org/shaemolyticus/.

Ethics statement
The study was approved by Institutional Review Board (IRB) of LV Prasad Eye Institute (LEC/ 08/110/2009), and by Institute Ethics Sub-Committee (IESC) of All India Institute of Medical Sciences, New Delhi (IESC/T-34/2013), and the data were analyzed anonymously and reported.

Bacterial isolates
A total of 34 S. haemolyticus isolates were included in this study. Seventeen isolates were from infected eyes, seven were from the healthy conjunctivae, six were from blood, and two isolates each were from pus and sputum, respectively. The isolates were identified by using Vitek 2 Compact System or Staph ID32 strips (bioMerieux, Marcy I'Etoile, France), 16S rDNA sequencing and amplification of S. haemolyticus specific nuc gene [9]. The strains were isolated from clinical samples sent to Microbiology Laboratories of LV Prasad Eye Institute, Bhubaneswar and Hyderabad, India, All India Institute of Medical Sciences, New Delhi, India, and Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, respectively. The strains from asymptomatic healthy conjunctivae were isolated only after clinical examination by optometry and ophthalmology faculty that ruled out any ocular surface infection.

Multilocus sequence typing and phylogenetic analysis
MLST of S. haemolyticus was performed using PCR product obtained with seven housekeeping genes [3]. Amplified product of seven housekeeping genes viz. arc, SH1200, hemH, leuB, SH1431, cfxE, and RiboseABC were purified (ExoSAP; Affymetrix, Cleveland, USA). Both the strands were sequenced using an ABI sequencer model 3500 (Life Technologies, Marsiling, Singapore) at the sequencing facility of the Institute of Life Sciences (Bhubaneswar, India). The nucleotide sequences were aligned using MEGA5.2 software and were manually compared with already reported alleles and STs. Sequencing was performed in biological duplicates to confirm the presence of novel alleles. New alleles were proposed and designated in comparison with the previously reported alleles [3]. A unique allele number was assigned to each sequence even if they differed at a single nucleotide site in a sequential manner and no weighting was applied to reflect the number of nucleotide differences between alleles. Subsequently, the novelty of new alleles was validated by Dr. Jorunn Pauline Cavanagh, Department of Pediatrics, University Hospital of North Norway (Personal communication).
The advanced cluster analysis was performed to define the clonal complexes (CC) by using Bionumerics software, version 7.1 (Applied Maths, Belgium). A minimum spanning tree (MST) was constructed using the MLST data and partitions were created to form clusters. The similarity in at least six alleles grouped isolates of S. haemolyticus in one CC. The central ST of each partition was used to designate a CC.

MLST database for S. haemolyticus
The MLST database for S. haemolyticus was successfully set up at the website PubMLST.org using the BIGSdb software, and made available at http://pubmlst.org/shaemolyticus/ to assign STs or designate new STs among S. haemolyticus isolates [10].

. Allelic profiles of sequence types (STs) of S. haemolyticus strains isolated from a variety of infections and also included previously reported STs (ST1-ST17
ST1-ST17: STs and profiles were reported previously by Cavanagh et al. [3] doi:10.1371/journal.pone.0166193.t001
The data of this study thus suggest that MLST scheme can be widely used to study population structure and diversity among S. haemolyticus isolates. The developed MLST database could select STs and assign new STs among S. haemolyticus after adding new sequence information to the database.