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Annals of Clinical and Medical Microbiology

Respiratory and Gut Microbiota of Children with Cystic Fibrosis: A Pilot Study

Original Research | Open Access Volume 5 | Issue 1 |

  • 1. Postgraduate Program in Child and Adolescent Health, Federal University of Paraná (UFPR), Curitiba, Brazil. Gen. Carneiro Street, 181, Curitiba-PR, Brazil, Zip code 80060-900
  • 2. Department of Pediatrics, Federal University of Paraná (UFPR), Curitiba-PR, Brazil. Padre Camargo Street, 453, Curitiba-PR, Brazil, Zip code 80030-110.
  • 3. Department of Basic Pathology, Sector of Biological Sciences, Federal University of Paraná (UFPR). Coronel Francisco H. dos Santos Avenue, 100, Curitiba-PR, Brazil, Zip code 81531-980.
  • 4. Laboratory of Bacteriology, Hospital of Clinics, Federal University of Paraná (UFPR). Padre Camargo Street, 280 Curitiba-PR, Brazil, Zip code 80060-240.
  • 5. Instituto de Pesquisa Pelé Pequeno Príncipe, Silva Jardim Avenue, 1632, Curitiba – PR, Brazil, Zip code 80250-060.
  • 6. Neoprospecta Microbiome Technologies, Sapiens Parque, Florianópolis, SC, 88056- 000, Brazil, Zip code 88054-700.
  • 7. Federal University of Paraná (UFPR), Curitiba-PR, Brazil. Curitiba, Brazil. Gen. Carneiro Street, 181, Curitiba-PR, Brazil, Zip code 80060-900
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Corresponding Authors
Jannaina Ferreira de Melo Vasco, Padre Camargo Street, 453 - Alto da Glória, Curitiba-PR, Brazil, Zip code 80030-110 Tel.: +55 41 32086500
Abstract

Differences in the clinical presentation of cystic fibrosis (CF) may be due to microbiota components and their relationship with the host’s immune system. In this pilot study, we aimed to investigate the composition of the respiratory and gut microbiota of a cohort of clinically stable children with CF, homozygous for the p.Phe508del mutation. Oropharyngeal swabs and stool samples were obtained from these children attending the CF referral clinics at the Hospital of Clinics, Federal University Paraná (CHC – UFPR). Oropharyngeal and gut microbiota were assessed by V3-V4 sequencing of the 16S ribosomal RNA, and bioinformatics analyses were performed using a proprietary pipeline. We identified a total of 456 bacterial taxa belonging to 164 genera, of which 65 (39.6 %) were common to both the respiratory and gastrointestinal tracts. Taxa from eight genera dominated more than 75 % of the microbial composition of both the niches. Among these dominant taxa, only Prevotella spp. were common to both the sites. Overall, the respiratory and gut microbiota were homogeneous among all the patients. Longitudinal studies targeting a larger cohort are important for an improved understanding of how the composition of bacterial communities is related to changes in the clinical status of CF.

Keywords

•    Cystic fibrosis
•    Microbiota
•    Respiratory microbiota
•    Gut microbiota
•    16S rRNA sequencing

Citation

Vasco JFM, Riedi CA, Marconi C, Nogueira KS, Rodrigues LS, et al. (2021) Respiratory and Gut Microbiota of Children with Cystic Fibrosis: A Pilot Study. Ann Clin Med Microbiol 5(2): 1028.

ABBREVIATIONS

CF: cystic fibrosis; CFTR: cystic fibrosis transmembrane conductance regulator; EL-1: fecal elastase-1; URT; upper respiratory tract

INTRODUCTION

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene affects the respiratory and gut microbiota, even in the absence of antibiotic exposure [1–3]. Cystic fibrosis (CF) is associated with changes in the host´s microbiota, leading to lower diversity and reduced richness of bacterial communities [4–7]. Studies have highlighted that dysbiosis in individuals with CF may be related to physiological factors such as increased mucus secretion, altered dietary intake, medications such as pancreatic enzymes [4], increasing age, the use of antibiotics, decreased lung function, and disease progression [5– 7]. It has been hypothesized that differences in the clinical presentation of CF in infants and young children with the same CFTR mutation may be explained by the distinct microbiota composition and its interactions with host immunity, including the gut-lung axis [8, 9]. It is well acknowledged that the intestinal microbial colonization profile is a determinant of the microbiota of the respiratory tract. This is because both sites share several common genera, and intestinal colonization by certain species often precedes their detection in the respiratory tract [8, 10]. Longitudinal studies have identified consistent trends in the temporal dynamics of the respiratory microbiota according to the age of the host. In patients with CF aged less than two years, the respiratory tract microbiota is dominated by Streptococcus spp., Prevotella spp., Veillonella spp., Rothia spp., and Actinomyces spp. These species are progressively replaced by CF-associated pathogens, such as Pseudomonas spp., Burkholderia spp., Stenotrophomonas spp., and Staphylococcus spp., which further dominate this bacterial community in patients aged 6 years and above [6,11]. Gut microbiota in patients with CF is significantly different from that in non-CF patients, with a substantial decrease in bacterial richness and diversity from infancy to late adolescence [12–14]. In these niches, a greater abundance of taxa from the Firmicutes phylum (e.g., Staphylococcus spp., Streptococcus spp., Clostridioides difficile, and Veillonella dispar), was observed concomitantly with decreased abundance of Bacteroidetes (e.g., Bacteroides spp.,

Bifidobacterium adolescentis, Eubacterium spp., Ruminococcus spp., and Faecalibacterium prausnitzii) [15,16]. Culture-independent studies have demonstrated that although the gut and respiratory microbiota show differences in composition, there is some concordance of the gut and respiratory microbiota in infants with CF over time [15, 17]. Thus, there is evidence of a relationship and crosstalk between the intestine and the lung (the gut-lung axis) [18, 19]. To better understand the mechanistic basis of the relationship between the host´s microbiota and the clinical presentation of CF, it is essential to characterize the microbiota of both respiratory and gastrointestinal tracts of CF. Additionally; these results could allow the identification of bacterial predictors of clinical status, including pulmonary disease in CF [20, 21]. In order to establish methods for sampling and data analysis in a larger cohort study, we conducted a pilot cross-sectional study in which we aimed to characterize the gut and respiratory microbiota of clinically stable children with CF who were homozygous for the p.Phe508del mutation.

MATERIALS AND METHODS

Patients and sampling

Ten clinically stable children with CF were enrolled for the study. Homozygosity for p.Phe508del was determined during neonatal screening and CF was confirmed by the sweat chloride test and genetic testing (Table 1).

Table 1: Overview of the clinical characteristics of the 10 study participants.

Participant ID Sex Age (years) Pancreatic insufficiency, treatment given Levels of EL-1 (µ/g) PA DNase Vitamin supplement (ADEK) GI findings
          Culturea     Steatorrhea
CF 1 Female 6.4 Yes, Creon < 5,50 Negative Yes Yes Yes
CF 2 Male 2 Yes, Creon < 5,50 Negative No Yes Yes
CF 3 Male 6.3 Yes, Creon < 5,50 Negative No Yes Yes
CF 4 Female 6.2 Yes, Creon < 5,50 Negative Yes Yes Yes
CF 5 Male 4 Yes, Creon < 5,50 Negative Yes Yes Yes
CF 6 Female 3 Yes, Creon < 5,50 Negative Yes Yes Yes
CF 7 Female 4.8 Yes, Creon < 5,50 Negative Yes Yes Yes
CF 8 Female 2 Yes, Creon < 5,50 Negative Yes Yes Yes
CF 9 Male 5.9 Yes, Creon < 5,50 Negative Yes Yes Yes
CF 10 Female 12.6 Yes, Creon < 5,50 Negative Yes Yes Yes
Abbreviations: CF; cystic fibrosis; EL-1; fecal elastase-1; PA; Pseudomonas aeruginosa; GI; Gastrointestinal awithin the last month.

All participants showed pancreatic insufficiency and underwent pancreatic enzyme replacement therapy (e.g., Creon). The status of exocrine pancreatic function was defined based on steatorrhea and the levels of fecal elastase-1 (EL- 1). The values of the fecal elastase assay and enzyme linked immunosorbent assay stool test (BIOSERV Diagnostics, Rostock, Germany) for pancreatic elastase 1 were considered normal in the range of 200–500 mg/g of feces, borderline in the range of 100–200 mg/g feces, and were considered to have pancreatic insufficiency if less than 100 mg/g of feces. None of the participants had a culture positive for Pseudomonas aeruginosa or presented pulmonary exacerbation four weeks prior to sampling, and none of them were treated with antimicrobial drugs two weeks prior to sampling. The study protocol was approved by the Human Research Ethics Committee of Complexo Hospital de Clínicas - Federal University Paraná (CHC–UFPR), Curitiba-PR, Brazil (CEP-HC/UFPR ref no: 1.948.265). Parents of all the patients provided written informed consent for participation in the study. Stool and oropharyngeal swabs were collected from each participant using sterile flocked swabs (Copan Diagnostics, Murrieta, CA, USA) during regular scheduled visits to the CF outpatient clinic at CHC-UFPR. Each swab was placed in a separate microtube containing 1 mL of stabilization solution (NeoSampleZ, Neoprospecta, Brazil) and homogenized by microtube inversion. The samples were stored at 18–27 °C until shipment to the Neoprospecta laboratory (Florianopolis, SC, Brazil) within 30 days of sample collection. Microbiota analysis DNA was extracted from the preserved respiratory and fecal samples using the DNeasy PowerSoil Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Amplification of the 16S ribosomal RNA V3/V4 region was carried out using the 341F 5′- CCTACGGGRSGCAGCAG-3′ and 806R 5′-GGACTACHVGGGTWTCTAAT-3′ primer sets, according to a previously described method [22, 23]. The libraries were prepared using a double polymerase chain reaction (PCR) protocol containing TruSeq structure adapter (Illumina, USA), allowing a second PCR with indexing sequences, as previously described [24]. The final PCR reactions were cleaned using 132 AMPureXP beads (Beckman Coulter, USA), and samples were pooled in the sequencing libraries for quantification. The library pools were adjusted to a final concentration of 11.5 pM and sequenced using a MiSeq system (Illumina Inc., San Diego, CA), using the V2 kit (2 × 250 cycles) [25]. Raw read sequences were converted into FASTQ files, quality-filtered (Phred quality score < 20), and trimmed to remove primers. A final 283-bp read was thus obtained using the Sentinel pipeline (Neoprospecta Microbiome, Florianopolis, SC, Brazil). Identical reads (99 % similarity) were grouped into clusters. Clusters present in less than five samples were removed, since such structures are often related to chimeric sequences [26]. Operational taxonomic units (OTUs) were selected using the BLASTN v.2.6.0+ tool [27] with the 16S ribosomal gene database created by Neoprospecta Microbiome. For taxonomic assignments, only sequences with one of the following conditions were used in subsequent analyses: high bit score, low e-value, or taxonomies with greater representation. A total of 1,056,505 reads (average of 42,010 reads per sample) were retrieved by sequencing analyses. Finally, a bacterial profile for each sample was created based on the relative abundance of the bacterial genera. Relative abundance was calculated after normalization using the median values of all the reads retrieved from each sample. Graphs of the proportion of taxa in each patient and all the samples; Venn diagrams were constructed using the OTU table normalized by the median and Venn Diagram packages in R [28,29]. To determine the relative abundance of bacterial taxa at each site, the numbers of reads of an individual taxon were divided by the sum of all taxa reads from the sample in each participant. reads (average of 42,010 reads per sample)

RESULTS AND DISCUSSION

We identified 456 bacterial taxa representing 164 genera, of which 65 (39.6 %) were shared between the gastrointestinal and respiratory tracts Venn diagram (Figure 1).

Figure 1 Distribution of the genera identified in the sequencing analyses, based on the assessment of the two body sites (gastrointestinal and respiratory tracts).

Figure 1: Distribution of the genera identified in the sequencing analyses, based on the assessment of the two body sites (gastrointestinal and respiratory tracts).

Gut microbiota showed greater richness (130 genera) than respiratory microbiota (99 genera). The gut microbial community comprised of nine genera: Bacteroides, Blautia, Anaerostipes, Lachnoclostridium, Tyzzerella, Veillonella, Streptococcus, Clostridium, and Bifidobacterium. As shown in (Figure 2),

Figure 2 Distribution of the most abundant bacterial taxa, at the genus level, of the microbiomes in the intestinal and respiratory tracts. (A) and (B) – Stacked bar plots displaying the relative abundance (y-axis) of the eight most abundant taxa observed in the (A) Intestinal tracts and (B) respiratory tracts of the study population; the x-axis in each Graph corresponds to the 10 study participants. (C) And (D) – Pie chart of the eight most abundant genera across all the (C) intestinal tract samples and (D) respiratory tract samples

Figure 2: Distribution of the most abundant bacterial taxa, at the genus level, of the microbiomes in the intestinal and respiratory tracts. (A) and (B) – Stacked bar plots displaying the relative abundance (y-axis) of the eight most abundant taxa observed in the (A) Intestinal tracts and (B) respiratory tracts of the study population; the x-axis in each Graph corresponds to the 10 study participants. (C) And (D) – Pie chart of the eight most abundant genera across all the (C) intestinal tract samples and (D) respiratory tract samples

eight genera represented more than 75 % of the taxa found in either the gut or the respiratory niches. Among the most abundant genera, only Prevotella spp. was common to both the sites. Gut microbiota analysis revealed that the genus Bacteroides from the phylum Bacteroidetes showed the highest relative abundance in all the patients, except patient 10, who showed Prevotella spp. as the most abundant genus (Figure 2). The initial years of life are critical for the development of the immune system, and bacterial species play a critical role in this process [30]. Progressively, the relatively simple neonatal intestinal microbiota matures and increases the complexity of the microbiota, with enrichment in Firmicutes and Bacteroidetes. However, at the genus level, intestinal microbiota are significantly different in children and adults with CF [31]. External factors, such as antibiotics, seem to alter the bacterial composition in children below two years of age, and are associated with a decrease in the relative abundance of Bacteroides spp. [14]. Age is certainly considered as the main predictor of fecal bacterial composition and is positively associated with an increase in the relative abundance of the genus Blautia (phylum Firmicutes) [10, 32]. All the fecal samples showed a high abundance of this genus. The respiratory “core” microbial community was composed of five genera: Fusobacterium, Rothia, Streptococcus, Veillonella, and Haemophilus. The anaerobic Prevotella genera were found in the respiratory tracts of nine children and were the predominant taxon in three of them. The abundance of Prevotella spp. in the airways of patients with CF is unclear, and in this study, all patients were stable. Further characterization of the isolates will aid in determining the contribution of Prevotella spp. to airway disease associated with CF. Considering the respiratory “core” genera, Streptococcus spp. was the most abundant genus in the three samples. The composition of a healthy lung microbiota is determined by the balance of microbial immigration from the upper respiratory tract (URT) and its elimination capacity. In healthy children, although the lung microbiota is distinct from the URT microbiota, it is dominated by species that are also present in the nasopharynx and oropharynx, including Moraxella spp., Haemophilus spp., Staphylococcus spp., and Streptococcus spp. [33]. The adult lung microbiota seems to be dominated by the phyla Firmicutes and Bacteroidetes, including the genera Streptococcus spp., Veillonella spp., and Prevotella spp. [34]. Veillonella spp. have been previously associated with positive interactions with Streptococcus spp., Moraxella catarrhalis, and Haemophilus influenzae [35, 36] Except Moraxella spp., all other genera were detected in the respiratory samples. Although none of the study participants presented symptoms of respiratory exacerbation or a positive culture for P. aeruginosa, four of them (40 %) showed the presence of Pseudomonas spp. in the oropharyngeal microbiota. It is known that the genus Pseudomonas is enriched in the airways of clinically stable children with CF when compared to non-CF children [37, 38, and 39]. A previous study showed that decrease in the relative abundance of Parabacteroides spp. in the gut microbiota precedes chronic colonization by Pseudomonas aeruginosa [14,40]. Patient 01 showed less diversity in the respiratory microbiota compared to the other patients, with Staphylococcus aureus being the most abundant species. Although lower microbial diversity is associated with lower quality of respiratory microbiota, and S. aureus is an opportunistic pathogen in CF, interactions between species and with the host interfere in these dynamics [33]. Available data support the hypothesis that S. aureus alters its virulence to a commensal state when exposed to Corynebacterium species [41, 42, 43], genera of which were also detected in the respiratory sample of patient 01. Diversity and richness measurements are important for understanding the bacterial community structure and dynamics. Here, were identified 64 important genera in differential abundance between the gut and respiratory microbiota: Rothia, Bifidobacterium, Actinomyces, Atopobium, Collinsella, Slackia, Fusobacterium, Leptotrichia, Parasutterella, Sutterella, Lautropia, Achromobacter, Neisseria, Kingella, Eikenella, Escherichia, Enterobacter, Salmonella, Haemophilus, Terrahaemophilus, Aggregatibacter, Peptostreptococcus, Intestinibacter, Terrisporobacter, Peptoclostridium, Clostridium, Eubacterium, Mogibacterium, Peptoniphilus, Parvimonas, Mycoplasma, Granulicatella, Abiotrophia, Enterococcus, Staphylococcus, Streptococcus, Erysipelatoclostridium, Coprobacillus, Solobacterium, Veillonella, Dialister, Megasphaera, Tyzzerella, Roseburia, Coprococcus, Blautia, Lachnoclostridium, Catonella, Stomatobaculum, Lachnoanaerobaculum, Fusicatenibacter, Oribacterium, and Anaerostipes. These genera have increased abundance in the respiratory tract and reduced or null abundance in the intestinal tract, or vice versa, as exemplified in Figure 3.

Figure 3 Top 25 genera identified with differential abundance in the groups.

Figure 3: Top 25 genera identified with differential abundance in the groups.

Although clinical evidence of the impact of gut microbiota on lung infections and disease is not fully understood, studies have shown that lung diseases are associated with disorders of the gut microbiome [44, 45]. Studies indicate that alterations in gut microbial diversity and abundance have been linked to immune response, inflammation, and lung disease development. Reduced diversity of intestinal bacteria was previously associated with CF [37, 46], and gut colonization patterns were considered as determinants in the development of the respiratory tract microbiota in children, related to airway exacerbations [10]. A significant overrepresentation of Streptococcus spp. in the intestinal microbiota was also noted in the current study. Children with intestinal inflammation seem to be prone to CF and provide a basis for the important role of the gut-lung axis in the evolution of this systemic disease [38]. Schippa et al. [47] found that homozygosity of p.Phe508del is more closely associated with a distinct profile of the gut microbiota than other CFTR allelic variants, with an increased abundance of potentially pathogenic species such as Escherichia coli and Eubacterium biform. The bacterial genus Escherichia was detected in all the fecal samples and Eubacterium was detected in 70 % of the top 10 genera of the gut microbiota (Figure 2).

CONCLUSION

Despite the limited sample size of this pilot study, we successfully analyzed the microbial community of children with highly homogeneous CF without pulmonary exacerbation. The microbiota composition of the study population was fairly homogeneous among the patients, and the identified taxa corresponded to trends consistent with studies for the same age group in the CF patient homozygote DelF508. Current data provide information regarding the colonization of well acknowledged respiratory and intestinal pathogens in this population, warranting further longitudinal studies based on larger cohorts. These future studies will allow a better

understanding of how mutations in CF may affect the airway and intestinal microenvironment, which could lead to non-optimal microbial colonization. These studies will provide important information for the potential use of microbiota components as prognostic tools for determining the course of CF, response to treatment, and/or prevention of lung disease.

ACKNOWLEDGEMENTS

We gratefully acknowledge the participation of the patients and their families in this study. We would like to thank Wellington Pine Omori, at Neoprospecta® Microbiome Technologies, for aiding with the bioinformatics analyses and Elessandra S. Bitencourt, a nurse at the Federal University of Paraná, for aiding with sample collection. We would like to thank Editage (www. editage.com) for English language editing.

DECLARATIONS OF INTEREST FUNDING

This work was supported by Vertex® Pharmaceuticals Inc (Tracking Number: IIS-2017- 106050) for conducting the research and collecting and analyzing of data.

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Vasco JFM, Riedi CA, Marconi C, Nogueira KS, Rodrigues LS, et al. (2021) Respiratory and Gut Microbiota of Children with Cystic Fibrosis: A Pilot Study. Ann Clin Med Microbiol 5(2): 1028.

Received : 23 Aug 2021
Accepted : 21 Sep 2021
Published : 23 Sep 2021
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JSM Arthritis
ISSN : 2475-9155
Launched : 2016
JSM Head and Neck Cancer-Cases and Reviews
ISSN : 2573-1610
Launched : 2016
JSM General Surgery Cases and Images
ISSN : 2573-1564
Launched : 2016
JSM Anatomy and Physiology
ISSN : 2573-1262
Launched : 2016
JSM Dental Surgery
ISSN : 2573-1548
Launched : 2016
Annals of Emergency Surgery
ISSN : 2573-1017
Launched : 2016
Annals of Mens Health and Wellness
ISSN : 2641-7707
Launched : 2017
Journal of Preventive Medicine and Health Care
ISSN : 2576-0084
Launched : 2018
Journal of Chronic Diseases and Management
ISSN : 2573-1300
Launched : 2016
Annals of Vaccines and Immunization
ISSN : 2378-9379
Launched : 2014
JSM Heart Surgery Cases and Images
ISSN : 2578-3157
Launched : 2016
Annals of Reproductive Medicine and Treatment
ISSN : 2573-1092
Launched : 2016
JSM Brain Science
ISSN : 2573-1289
Launched : 2016
JSM Biomarkers
ISSN : 2578-3815
Launched : 2014
JSM Biology
ISSN : 2475-9392
Launched : 2016
Archives of Stem Cell and Research
ISSN : 2578-3580
Launched : 2014
JSM Pediatric Surgery
ISSN : 2578-3149
Launched : 2017
Journal of Memory Disorder and Rehabilitation
ISSN : 2578-319X
Launched : 2016
JSM Tropical Medicine and Research
ISSN : 2578-3165
Launched : 2016
JSM Head and Face Medicine
ISSN : 2578-3793
Launched : 2016
JSM Cardiothoracic Surgery
ISSN : 2573-1297
Launched : 2016
JSM Bone and Joint Diseases
ISSN : 2578-3351
Launched : 2017
JSM Bioavailability and Bioequivalence
ISSN : 2641-7812
Launched : 2017
JSM Atherosclerosis
ISSN : 2573-1270
Launched : 2016
Journal of Genitourinary Disorders
ISSN : 2641-7790
Launched : 2017
Journal of Fractures and Sprains
ISSN : 2578-3831
Launched : 2016
Journal of Autism and Epilepsy
ISSN : 2641-7774
Launched : 2016
Annals of Marine Biology and Research
ISSN : 2573-105X
Launched : 2014
JSM Health Education & Primary Health Care
ISSN : 2578-3777
Launched : 2016
JSM Communication Disorders
ISSN : 2578-3807
Launched : 2016
Annals of Musculoskeletal Disorders
ISSN : 2578-3599
Launched : 2016
Annals of Virology and Research
ISSN : 2573-1122
Launched : 2014
JSM Renal Medicine
ISSN : 2573-1637
Launched : 2016
Journal of Muscle Health
ISSN : 2578-3823
Launched : 2016
JSM Genetics and Genomics
ISSN : 2334-1823
Launched : 2013
JSM Anxiety and Depression
ISSN : 2475-9139
Launched : 2016
Clinical Journal of Heart Diseases
ISSN : 2641-7766
Launched : 2016
Annals of Medicinal Chemistry and Research
ISSN : 2378-9336
Launched : 2014
JSM Pain and Management
ISSN : 2578-3378
Launched : 2016
JSM Women's Health
ISSN : 2578-3696
Launched : 2016
Clinical Research in HIV or AIDS
ISSN : 2374-0094
Launched : 2013
Journal of Endocrinology, Diabetes and Obesity
ISSN : 2333-6692
Launched : 2013
Journal of Substance Abuse and Alcoholism
ISSN : 2373-9363
Launched : 2013
JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
Journal of Drug Design and Research
ISSN : 2379-089X
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Annals of Food Processing and Preservation
ISSN : 2573-1033
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
Clinical Research in Infectious Diseases
ISSN : 2379-0636
Launched : 2013
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
JSM Cell and Developmental Biology
ISSN : 2379-061X
Launched : 2013
Annals of Aquaculture and Research
ISSN : 2379-0881
Launched : 2014
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
Annals of Forensic Research and Analysis
ISSN : 2378-9476
Launched : 2014
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
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