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Genome sequences of Salisediminibacterium haloalkalitolerans 10nlg, Bacillus lonarensis 25nlg, Bacillus caseinilyticus SP, Pelagirhabdus alkalitolerans S5, Salibacterium halotolerans S7 and Salipaludibacillus aurantiacus S9 six novel, recently described compatible solute producing bacteria.

The strain Salisediminibacterium haloalkalitolerans 10nlg was recently described as a novel species of the genus Salisedimini bacterium. The cells are Gram-stain-positive, rod shaped, non-motile, orange pigmented and non-endospore forming. The species showed catalase positive and oxidase negative activities (Sultanpuram et al., 2015a). The two novel Bacillus species described Bacillus lonarensis 25[nlg.sup.T] and Bacillus caseinilyticus [SP.sup.T] were Gram-stain-positive, terminal endospore forming, motile rods (Sultanpuram et al., 2015b; Sultanpuram et al., 2015c). Strains Pelagirhabdus alkalitolerans S5, Salibacterium halotolerans S7 and Salipaludibacillus aurantiacus S9 were three novel recently described members representing novel genus from the family Bacillaceae. These strains were Gram-stain-positive, rod shaped, non-motile and non-endospore forming, except Salipaludibacillus aurantiacus S9 (Sultanpuram et al., 2016; Sultanpuram et al., 2015d; Sultanpuram and Mothe 2016). Here we report the genome sequencing of these six strains.

MATERIAL AND METHODS

Library construction, genome sequencing and data analysis

Sequencing of the type strains was part of Genomic Encyclopedia of Type Strains, Phase III: the (KMG-III) project (Whitman et al., 2015). Project information is deposited in the Genomes on Line Database (GOLD) (Reddy et al., 2015) and the high quality draft genome sequence is deposited in GenBank and in the Integrated Microbial Genomes database (IMG) (Markowitz et al., 2014). Draft sequencing, initial gap closure and annotation were performed by the DOE Joint Genome Institute (JGI) using state-of-the-art sequencing technology (Mavromatis et al., 2012).

An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2500-1TB. Illumina paired-end reads of average 250-bp length and the clean data were assembled using Velvet (version 1.1.04)(Zerbino and Birney 2008), simulated paired end reads were created from Velvet contigs using wgsim and simulated read pairs were reassembled using Allpaths-LG (version r42328) (MacCallum et al., 2009). Protein-coding genes were identified using Prodigal (Hyatt et al., 2010), as part of the DOE-JGI genome annotation pipeline (Huntemann et al., 2015). Additional gene prediction analysis and manual functional annotation were performed within the Integrated Microbial Genomes (IMG) platform, which provides tools for analyzing and reviewing the structural and functional annotations of genomes in a comparative context (Markowitz et al., 2014). Genome annotation procedures are detailed in Markowitz et al. and references therein. Briefly, the predicted CDSs were translated and used to search the NCBI nonredundant database, UNIProt, TIGRFam, Pfam, KEGG, COG and InterPro databases. Transfer RNA genes were identified using the tRNAScan-SE tool and other non-coding RNAs were found using INFERNAL. Ribosomal RNA genes were predicted using hmmsearch against the custom models generated for each type of rRNA.

RESULTS AND DISCUSSIONS

Genome features

The Table 1 describes the classification, general features and genome sequencing projects information for the strains according to the MIGS recommendations (Field et al., 2008). The assembled draft genome of Salisediminibacterium haloalkalitolerans 10nlg contained 2,93,7914 bps with 47.13% G + C content. Further, the genome analysis predicted 2,282 proteins coding sequences (CDS) of which 80.35% were assigned to recognized functional genes. The genome analysis revealed 57 tRNA and 13 rRNA genes. The genome of Bacillus lonarensis 25[nlg.sup.T] contained 2,58,2209 bps with 43.26% G + C content. Further, the genome analysis predicted 2,581 proteins coding sequences (CDS) of which 96.34% were assigned to recognized functional genes with 67 tRNA and 16 rRNA genes. The assembled genome of Bacillus caseinilyticus [SP.sup.T] contained 5,87,2627 bps with 44.85% GC content. Further, the genome analysis predicted 5,199 proteins coding sequences (CDS) of which 97.41% were assigned to recognized functional genes. The genome analysis revealed 101 tRNA and 12 rRNA genes. The genome of Pelagirhabdus alkalitolerans S5 contained 2,52,7601 bps with 88.99% DNA coding bases and 37.08% GC content. The genome revealed 2,033 protein coding sequences which were about 81.61% of the total genome. Around 15.01% CDS present weren't assigned to any of the known functions. Further, it revealed 56 tRNA and 13 rRNA genes. Strain Salibacterium halotolerans S7 showed 3, 61, 4063 bps with 47.65% G + C mol%. Around 75.89% of the identified CDS were protein sequences with known functions and about 807 protein coding genes had some unknown functions. Sixty one tRNA and 13 rRNA genes were revealed in the analysis of strain Salibacterium halotolerans S7 genome. Salipaludibacillus aurantiacus S9 draft genome revealed 4,43,5185 bps genome size with 42.42% G + C mol%. The protein functions of 3349 CDS were known, whereas, 883 CDS protein functions were unknown. Further, it revealed 73 tRNA and 16 rRNA genes.

The genomes of all the above described six strains harboured a cluster of genes coding for compatible solute production, thus explaining their osmotolerance. These genes from strain Salisediminibacterium haloalkalitolerans 10nlg include, L- ectoine synthase (130 aa; locus tag SAMN05444126_10550; accession number F0GV01000005.1) and choline dehydrogenase (561 aa, locug tag SAMN05444126_10967; accession number F0GV01000009.1). The strain, Bacillus lonarensis 25[nlg.sup.T] contains ectoine synthase (127 aa; locus tag SAMN05421737_10167; accession number FMYM01000001), glycerol-3-phosphate dehydrogenase (NAD(P)+ (339 aa; locus tag SAMN05421737_105223; accession number FMYM01000005.1), 1-acyl-sn-glycerol-3-phosphate acyltransferase (192 aa; locus tag SAMN05421737_105218; accession number FMYM01000005.1), choline dehyrogenase 402 aa; locus tag SAMN05421737_10199; accession number FMYM01000001.1).The strain, Bacillus caseinilyticus [SP.sup.T] contains L-ectoine synthase (128 aa; locus tag SAMN05421736_104185; accession number FNPI01000004.1), glycerol-3-phosphate dehydrogenase (NAD(P) + (338 aa; locus tag SAMN05421736_10427; accession number FNPI01000004.1), glycerol-1-phosphate dehydrogenase (351 aa; locus tag SAMN05421736_10511; accession number FNPI01000005.1), glycerol-1phosphate dehydrogenase (561 aa; locus tag SAMN05421736_10785; accession number FNPI01000007.1), 1-acyl-sn-glycerol-3-phosphate acyltransferase (196 aa; locus tag SAMN05421736_10420; accession number FNPI01000004.1), choline dehyrogenase (569 aa; locus tag SAMN05421736_101228; accession number FNPI01000001.1). Pelagirhabdus alkalitolerans S5 contains ectoine synthase (129 aa; locus tag SAMN05421734_10588; accession number FMYI01000005.1), glycerol-3-phosphate dehydrogenase (NAD(P)+ (347 aa; locus tag SAMN05421734_102431; accession number FMYI01000002.1), 1-acylsn-glycerol-3-phosphate acyltransferase (243 aa; locus tag SAMN05421734_102368; accession number FMYI01000002.1/ 193 aa; locus tag SAMN05518683_10435; accession number FMYI01000002.1/ 237 aa; locus tag SAMN05518683_10446; accession number FMYI01000004.1). Whereas, strain Salibacterium halotolerans S7 codes for L- ectoine synthase (128 aa; locus tag SAMN05518683_10726; accession number FOXD01000007.1), ectoine hydroxylase (314 aa; locus tag SAMN05518683_10644; accession number FOXD01000006.1) and choline dehydrogenase (569 aa; locus tag SAMN05518683_10452; accession number FOXD01000004.1 / 265 aa; locus tag SAMN05518683_10464; accession number FOXD01000004.1 / 402 aa; locus tag SAMN05518683_12711; accession number FOXD01000027.1). Salipaludibacillus aurantiacus S9 codes for L- ectoine synthase (131 aa; locus tag SAMN05518684_11244; accession number FOGT01000012.1), ectoine hydroxylase (310 aa; locus tag SAMN05518684_10667; accession number FOGT01000006.1), choline dehydrogenase (563 aa; locus tag SAMN05518684_106165; accession number FOGT01000006.1) and betaine aldehydyde dehydrogenase (490 aa; locus tag SAMN05518684_106162; accession number FOGT01000006.1). These draft genome sequences will further help in understanding the genetic potential of the above described six strains belonging to Firmicutes for osmotolerance, especially in relation to ectoine and hydroxyectoine production which are very commercially viable products.

Nucleotide sequence accession numbers

The draft sequence of Salisedimini bacterium haloalkalitolerans 10nlg, Bacillus lonarensis 25[nlg.sup.T], Bacillus caseinilyticus [SP.sup.T], Pelagirhabdus alkalitolerans S5, Salibacterium halotolerans S7 and Salipaludibacillus aurantiacus S9 obtained in this Whole Genome Shotgun project has been deposited at GenBank under the accession numbers FOGV00000000, FMYM00000000, FNPI00000000, FOGV00000000, FOXD00000000 and FOGT00000000, respectively. The Genomes on Line Database (GOLD) IDs of the strains Salisediminibacterium haloalkalitolerans 10nlg, Bacillus lonarensis 25[nlg.sup.T], Bacillus caseinilyticus [SP.sup.T], Pelagirhabdus alkalitolerans S5, Salibacterium halotolerans S7 and Salipaludibacillus aurantiacus S9 are PRJEB16955, PRJEB15470, PRJEB16536, PRJEB15473, PRJEB17326 and PRJEB16964, respectively.

ACKNOWLEDGMENTS

We are thankful to Prof. William Whitman for proposing the genomic sequencing of these organisms under KMG-III. These organisms were isolated and described in Department of Science & Technology, Government of India, FTSYS project grants (SB/FT/LS-115/2012-13 and SB/FT/LS320/2012-13, respectively).

REFERENCES

[1.] Field D, Garrity G, Gray T, Morrison N, Selengut J, Strek P, Tatusova T, Thomson N, et al., The minimum information about a genomic sequence (MIGS) specification. Nat. Biotechnol., 2008; 26: 541-547.

[2.] Huntemann M, Ivanova NN, Mavromatis K, Tripp HJ, Paez-Espino D, Palaniappan K, et al., The standard operating procedure of the DOEJGI Microbial Genome Annotation Pipeline (MGAP v.4). Stand. GenomicSci., 2015; 10:86.

[3.] Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ., Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinfo., 2010; 11:119.

[4.] MacCallum I, Przybylski D, Gnerre S, Burton J, Shlyakhter I, Gnirke A, et al, ALLPATHS 2: small genomes assembled accurately and with high continuity from short paired reads. Genome Biol, 2009; 10:R103.

[5.] Markowitz VM, Chen IM, Palaniappan K, Chu K, Szeto E, Pillay M, et al., IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res., 2014; 42(Database issue):D560-7.

[6.] Mavromatis K, Land M, Brettin TS, Quest DJ, Copeland A, Clum A, et al., The fast changing landscape of sequencing technologies and their impact on microbial assemblies and annotation. PLoS One, 2012; 7:e48837.

[7.] Reddy TBK, Thomas A, Stamatis D, Bertsch J, Isbandi M, Jansson J, et al., The Genomes OnLine Database (GOLD) v.5: a metadata management system based on a four level (meta) genome project classification. Nucleic Acids Res, 2015; 43:D1099.

[8.] Sultanpuram, VR, Mothe, T, Mohammed, F., Salisediminibacterium haloalkalitolerans sp. nov., isolated from a soda lake, emended description of Bacillus locisalis as Salisediminibacterium locisalis comb. nov and emened description of genus Salisediminibacterium. Arch. Microbiol., 2015a; 197: 553-560.

[9.] Sultanpuram, VR, Mothe, T, Mohammed, F., Chintalapati, S, Chintalapati, VR., Bacillus lonarensis sp. nov., a haloalkalophilic bacterium isolated from a soda lake. Arch. Microbiol., 2015b; 197: 27-34.

[10.] Sultanpuram, VR, Mothe, T, Mohammed, F., Bacillus casienilyticus sp. nov., a bacterium isolated from a soda lake. Int. J. Sys. Evolu. Microbiol, 2015c; 65 (8): 2441-2446.

[11.] Sultanpuram, VR, Mothe, T, Chintalapati, S, Chintalapati, VR., Salibacterium halotolerans gen. nov., sp. nov., a novel bacterium isolated from a salt pan, reclassification of Bacillus qingdaonensis as Salibacterum qingdaonense comb. nov. and Bacillus halochares as Salibacterium halochares comb. nov. Int. J. Syst. Evolu. Microbiol., 2015d; 65: 4270-4275.

[12.] Sultanpuram, VR, Mothe, T., Salipaludibacillus aurantiacus gen. nov., sp. nov., a novel alkali tolerant bacterium, reclassification of Bacillus agaradharens as Salipaludibacillus agaradharens comb. nov. and Bacillus neizhouensis as Salipaludibacillus neizhouensis comb. nov. Int. J. Syst. Evolu. Microbiol., 2016; 66: 2747-2753.

[13.] Sultanpuram, VR, Mothe, T, Chintalapati, S, Chintalapati, VR., Pelagirhabdus alkalitolerans gen. nov., sp. nov., a novel alkali and thermo tolerant bacterium isolated from Pingaleshwar beach, India and reclassification of Amphibacillus fermentum as Pelagirhabdus fermentum com. nov. Int. J. Syst. Evolu. Microbiol., 2016; 66: 84-90.

[14.] Whitman WB, Woyke T, Klenk HP, Zhou Y, Lilburn TG, Beck BJ, et al., Genomic encyclopedia of bacterial and archaeal type strains, phase III: the genomes of soil and plant associated and newly described type strains. Stand. Genomic Sci., 2015; 10: 26.

[15.] Zerbino DR, Birney E, Velvet: algorithms for de novo short read assembly using de bruijn graphs. Genome Res., 2008; 18:821-829.

Vishnuvardhan Reddy Sultanpuram * and Thirumala Mothe

Microbial Ecology Laboratory, Department of Applied Biosciences, Mahatma Gandhi University, Anneparthy, Yellareddygudem (PO), Nalgonda--508 254, Telangana, India.

http://dx.doi.org/ 10.22207/JPAM.11.2.26

(Received: 03 February 2017; accepted: 30 April 2017)

* To whom all correspondence should be addressed.

E-mail: svr.micro@gmail.com
Table 1. Classification, general features and genome sequencing
projects information for the strains according to the MIGS
recommendations (Field et al., 2008)

MIGS ID    Property              Domain Bacteria
           Current
           Classification

                                 Phylum Firmicutes
                                 Class Bacilli
                                 Order Bacillales
                                 Family Bacillaceae
                                 Genus
                                 Salisediminibacterium
                                 Species
                                 haloalkalitolerans
                                 Strain lOnlg
           Gram stain            Positive
           Cell shape            Rod
           Motility              Non-motile
           Sporulation           Non-endospore former

           Temperature           20-50[degrees]C
           range Optimum Temp.   37[degrees]C
           Carbon                Varied
           source
           Energy                Heterotrophic
           source
MIGS-6     Habitat               Soda lake
MIGS-6.3   Salinity              NaCl
MIGS-22    Oxygen                Aerobic/Facultative
                                 anaerobe

MIGS-15    Biotic relationship   Free living
MIGS-14    Pathogenicity         None
MIGS-4     Geographic            Lonar soda lake,
           location              India

MIGS-5     Sample collection     05/08/2013
           time
MIGS-4.3   Depth                 1 m
MIGS-4.4   Altitude              Not recorded
MIGS-31    Finishing             Permanent draft
           quality
MIGS-28    Libraries used        Shotgun
MIGS-29    Sequencing            Illumina HiSeq
           platforms             25 00-1TB
MIGS-30    Assemblers            Newbler v.2.8
MIGS-32    Gene calling method   Glimmer 3.02
           NCBI project ED       335132
           NCBI Bio-             PRJNA335132
           Project
           Accession
           Project               Environmental
           relevance             and
                                 biotechnological

MIGS ID    Property              Domain Bacteria
           Current
           Classification

                                 Phylum Firmicutes
                                 Class Bacilli
                                 Order Bacillales
                                 Family Bacillaceae
                                 Genus
                                 Bacillus
                                 Species lonarensis

                                 Strain 2 5 nig
           Gram stain            Positive
           Cell shape            Rod
           Motility              Motile
           Sporulation           Terminal
                                 endospore
                                 former
           Temperature           20-50[degrees]C
           range Optimum Temp.   35-37[degrees]C
           Carbon                Varied
           source
           Energy                Heterotrophic
           source
MIGS-6     Habitat               Soda lake
MIGS-6.3   Salinity              NaCl
MIGS-22    Oxygen                Aerobic/
                                 Facultative
                                 anaerobe
MIGS-15    Biotic relationship   Free living
MIGS-14    Pathogenicity         None
MIGS-4     Geographic            Lonar soda
           location              lake, India

MIGS-5     Sample collection     05/08/2013
           time
MIGS-4.3   Depth                 1 m
MIGS-4.4   Altitude              Not recorded
MIGS-31    Finishing             Permanent
           quality               draft
MIGS-28    Libraries used        Shotgun
MIGS-29    Sequencing            Illumina
           platforms             HiSeq 2500-1TB
MIGS-30    Assemblers            Newbler v.2.8
MIGS-32    Gene calling method   Glimmer 3.02
           NCBI project ED       329819
           NCBI Bio-             PRJNA329819
           Project
           Accession
           Project               Environmental
           relevance             and
                                 biotechnological

MIGS ID    Property              Domain Bacteria
           Current
           Classification

                                 Phylum Firmicutes
                                 Class Bacilli
                                 Order Bacillales
                                 Family Bacillaceae
                                 Genus
                                 Bacillus
                                 Species
                                 caseinilyticus
                                 Strain SP
           Gram stain            Positive
           Cell shape            Rod
           Motility              Motile
           Sporulation           Terminal
                                 endospore
                                 former
           Temperature           15-60[degrees]C
           range Optimum Temp.   37[degrees]C
           Carbon                Varied
           source
           Energy                Heterotrophic
           source
MIGS-6     Habitat               Soda lake
MIGS-6.3   Salinity              NaCl
MIGS-22    Oxygen                Aerobic/
                                 Facultative
                                 anaerobe
MIGS-15    Biotic relationship   Free living
MIGS-14    Pathogenicity         None
MIGS-4     Geographic            Lonar
           location              soda lake,
                                 India
MIGS-5     Sample collection     05/08/2013
           time
MIGS-4.3   Depth                 1 m
MIGS-4.4   Altitude              Not recorded
MIGS-31    Finishing             Permanent
           quality               draft
MIGS-28    Libraries used        Shotgun
MIGS-29    Sequencing            Illumina
           platforms             HiSeq 2500-1TB
MIGS-30    Assemblers            Newbler v.2.8
MIGS-32    Gene calling method   Glimmer 3.02
           NCBI project ED       329820
           NCBI Bio-             PRJNA329820
           Project
           Accession
           Project               Environmental
           relevance             and
                                 biotechnological

MIGS ID    Property              Domain Bacteria
           Current
           Classification

                                 Phylum Firmicutes
                                 Class Bacilli
                                 Order Bacillales
                                 Family Bacillaceae
                                 Genus
                                 Pelagirhabdus
                                 Species
                                 alkalitolerans
                                 Strain S5
           Gram stain            Positive
           Cell shape            Rod
           Motility              Non-motile
           Sporulation           Non-
                                 Endospore-
                                 former
           Temperature           20-55[degrees]C
           range Optimum Temp.   37[degrees]C
           Carbon                Varied
           source
           Energy                Heterotrophic
           source
MIGS-6     Habitat               Marine
MIGS-6.3   Salinity              NaCl
MIGS-22    Oxygen                Aerobic/
                                 Facultative
                                 anaerobe
MIGS-15    Biotic relationship   Free living
MIGS-14    Pathogenicity         None
MIGS-4     Geographic            Pingaleshwar
           location              beach,
                                 India
MIGS-5     Sample collection     12/06/2015
           time
MIGS-4.3   Depth                 1 m
MIGS-4.4   Altitude              Not recorded
MIGS-31    Finishing             Permanent
           quality               draft
MIGS-28    Libraries used        Shotgun
MIGS-29    Sequencing            Illumina
           platforms             HiSeq 2500-1TB
MIGS-30    Assemblers            Newbler v.2.8
MIGS-32    Gene calling method   Glimmer 3.02
           NCBI project ED       343906
           NCBI Bio-             PRJNA330706
           Project
           Accession
           Project               Environmental
           relevance             and
                                 biotechnological

MIGS ID    Property              Domain Bacteria
           Current
           Classification

                                 Phylum Firmicutes
                                 Class Bacilli
                                 Order Bacillales
                                 Family Bacillaceae
                                 Genus
                                 Salibacterium
                                 Species
                                 halotolerans
                                 Strain S7
           Gram stain            Positive
           Cell shape            Rod
           Motility              Non-Motile
           Sporulation           Non-
                                 Endospore-
                                 former
           Temperature           25-45[degrees]C
           range Optimum Temp.   37[degrees]C
           Carbon                Varied
           source
           Energy                Heterotrophic
           source
MIGS-6     Habitat               Marine salt pan
MIGS-6.3   Salinity              NaCl
MIGS-22    Oxygen                Aerobic/
                                 Facultative
                                 anaerobe
MIGS-15    Biotic relationship   Free living
MIGS-14    Pathogenicity         None
MIGS-4     Geographic            Khavda,
           location              India

MIGS-5     Sample collection     18/09/2014
           time
MIGS-4.3   Depth                 1 m
MIGS-4.4   Altitude              Not recorded
MIGS-31    Finishing             Permanent
           quality               draft
MIGS-28    Libraries used        Shotgun
MIGS-29    Sequencing            Illumina
           platforms             HiSeq 2500-1TB
MIGS-30    Assemblers            Newbler v.2.8
MIGS-32    Gene calling method   Glimmer 3.02
           NCBI project ED       335130
           NCBI Bio-             PRJNA335130
           Project
           Accession
           Project               Environmental
           relevance             and
                                 biotechnological

MIGS ID    Property              Domain Bacteria
           Current
           Classification

                                 Phylum Firmicutes
                                 Class Bacilli
                                 Order Bacillales
                                 Family Bacillaceae
                                 Genus
                                 Salipalu dib aci Hus
                                 Species
                                 aurantiacus
                                 Strain S9
           Gram stain            Positive
           Cell shape            Rod
           Motility              Non-motile
           Sporulation           Endospore-
                                 former

           Temperature           20-45[degrees]C
           range Optimum Temp.   37[degrees]C
           Carbon                Varied
           source
           Energy                Heterotrophic
           source
MIGS-6     Habitat               Marine salt marsh
MIGS-6.3   Salinity              NaCl
MIGS-22    Oxygen                Aerobic/
                                 Facultative
                                 anaerobe
MIGS-15    Biotic relationship   Free living
MIGS-14    Pathogenicity         None
MIGS-4     Geographic            Narayan
           location              sarovar,
                                 India
MIGS-5     Sample collection     21/09/2014
           time
MIGS-4.3   Depth                 1 m
MIGS-4.4   Altitude              Not recorded
MIGS-31    Finishing             Permanent
           quality               draft
MIGS-28    Libraries used        Shotgun
MIGS-29    Sequencing            Illumina
           platforms             HiSeq 2500-1TB
MIGS-30    Assemblers            Newbler v.2.8
MIGS-32    Gene calling method   Glimmer 3.02
           NCBI project ED       335131
           NCBI Bio-             PRJNA335131
           Project
           Accession
           Project               Environmental
           relevance             and
                                 biotechnological
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Author:Sultanpuram, Vishnuvardhan Reddy; Mothe, Thirumala
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Geographic Code:9INDI
Date:Jun 1, 2017
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