Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described. The identification and characterization of virulence factors is of upmost relevance to discover new targets for the development of diagnostic methods, therapeutic drugs, and vaccines . HoweverMycoplasma hyopneumoniae and the commensal Mycoplasma flocculare [M. hyopneumoniae adheres to the host respiratory epithelium and causes the porcine enzootic pneumonia (PEP). M. flocculare also adheres to porcine respiratory epithelium and can be isolated from normal and pneumonic lungs. This species is usually regarded as non-pathogenic [M. hyopneumoniae [M. hyopneumoniae, there are some strains that vary in their virulence levels, or even are avirulent, such as M. hyopneumoniae J, which has reduced adhesion capacity to porcine cilia [M. hyopneumoniae pathogenic and non-pathogenic strains  revealed no extensive genomic differences [M. hyopneumoniae and M. flocculare [M. hyopneumoniae and M. flocculare include the absence, in M. flocculare, of the glpO gene, related to M. hyopneumoniae hydrogen peroxide generation and cytotoxicity [M. flocculare predicted surface proteins are shared with M. hyopneumoniae [M. hyopneumoniae strains, and between M. hyopneumoniae and M. flocculare so far do not fully explain their differential phenotypes of virulence/pathogenicity.Among the mycoplasmas that coinhabit the swine respiratory tract, there are two interesting species for comparative studies: the pathogenic occulare . M. hyopthogenic ,4, althoeumoniae . Despitene cilia . Compariocculare , which socculare . The diftoxicity ,12, and toxicity . Howevereumoniae , and theM. hyopneumoniae strains or between M. hyopneumoniae and M. flocculare. However, previous comparative transcriptomic studies between M. hyopneumoniae and M. flocculare [M. hyopneumoniae pathogenic (7448 and 7422) and non-pathogenic (J) strains [M. hyopneumoniae and M. flocculare secreted proteins revealed several virulence-related differences between these mycoplasma species [M. hyopneumoniae secretome included several virulence-related proteins, like adhesins, transporters, nucleases and uncharacterized proteins bearing virulence-related functional domains, not found in the M. flocculare, secretome. Overall, these previous studies indicate the necessity of further and more comprehensive comparative proteomic studies, to deeply investigate possible pathogenicity or virulence-related differences at the protein level.Differential expression of ortholog genes may also contribute to differences in pathogenicity or virulence level between occulare  failed t strains . Moreove species . This stM. hyopneumoniae strains 7448 (pathogenic) and J (non-pathogenic), and M. flocculare were compared by a mass spectrometry (MS)-based approach to identify differences in protein abundance associated with pathogenicity or virulence. Mycoplasma cells were fractioned into cytoplasmic- and surface-enriched protein fractions and their protein contents were analyzed by high-resolution and high-sensitivity MS. Several significant differences among M. hyopneumoniae strains and M. flocculare proteomes were depicted, and their biological significance for mycoplasma-host interactions, for virulence and PEP determination are discussed.Here, the whole cell proteomes of M. hyopneumoniae 7448, M. hyopneumoniae J, and M. flocculare, respectively. Detailed peptide and protein identification data are presented in Supplementary Tables 1 and 2, respectively. Around 70% (242) of the detected proteins were shared between M. hyopneumoniae 7448, J, and M. flocculare  for the proteins and peptides of all samples validated all MS/MS results.LC-MS/MS analyzes of proteins from soluble and insoluble fractions identified overall totals of 344 out of 695 (~ 50%), 343 out of 672 (~ 51%), and 315 out of 581(~ 54%) protein species from occulare , SupplemM. hyopneumoniae 7448, M. hyopneumoniae J, and M. flocculare samples, respectively. Most (188) of the identified proteins in this fraction type (considering ortholog ones) were shared between the two M. hyopneumoniae strains, and M. flocculare  proteins identified in these fractions are shared among all analyzed mycoplasmas  and J , and for M. flocculare  in comparison to those identified in the corresponding soluble extracts (~ 25%). Conversely, most proteins identified in the soluble fractions of M. hyopneumoniae 7448 , M. hyopneumoniae J  and M. flocculare  were predicted as cytoplasmic proteins. Overall, these preliminary results showed qualitative differences between extracts that demonstrate the enrichment of surface proteins in insoluble extracts of M. hyopneumoniae (7448 and J) and M. flocculare.The identified protein repertoires from soluble and insoluble protein extracts were compared to confirm the enrichment of the insoluble fraction with surface proteins. M. hyopneumoniae 7448, 125 out of 210 proteins (59%) were differentially abundant between these fractions, while 93 out of 200 (46%) and 89 out of 150 (59%) were differentially abundant for M. hyopneumoniae J, and for M. flocculare, respectively (Supplementary Table 4). Among overrepresented proteins, there was an evident enrichment of surface-predicted proteins in insoluble fractions for all analyzed mycoplasmas, while cytoplasmic proteins were more abundant in soluble fractions  and surface-enriched protein extracts (SuPE), respectively.M. hyopneumoniae 7448 was qualitatively and quantitatively analyzed and compared to those from M. hyopneumoniae J and M. flocculare. Qualitative comparisons were based on presence/absence of detected ortholog proteins, while quantitative comparisons were performed between ortholog proteins shared between M. hyopneumoniae 7448 and J, and between M. hyopneumoniae 7448 and M. flocculare.The whole-cell proteome (including proteins detected in both CyPE and SuPE) of M. hyopneumoniae 7448 samples in comparison to M. hyopneumoniae J and M. flocculare, respectively  were underrepresented in M. hyopneumoniae 7448 samples in comparison to the J strain.Quantitative analyzes were performed with shared proteins between samples based on emPAI values (Supplementary Table 5). M. hyopneumoniae 7448 to M. flocculare found 79 proteins differentially abundant. Twenty CyPE and 44 SuPE proteins were overrepresented in M. hyopneumoniae 7448 samples, with differences in abundance ranging from 1.7 to 63-fold. Nine CyPE and 6 SuPE proteins were underrepresented in M. hyopneumoniae 7448 samples in this comparison. Among these differentially abundant proteins from M. hyopneumoniae 7448 and M. flocculare, only 9 presented significant abundance differences in both CyPE and SuPE. Of these proteins seven were overrepresented in both protein extracts of M. hyopneumoniae 7448. The other 2 represent cases of differential enrichment between the subcellular fractions in these two species. An aminopeptidase was overrepresented in the CyPE and underrepresented in the SuPE, in M. hyopneumoniae 7448, and vice-versa, in M. flocculare. Conversely, an uncharacterized protein (MHP7448_0356), was underrepresented in the CyPE and overrepresented in the SuPE, in M. hyopneumoniae 7448, and vice-versa, in M. flocculare.Quantitative comparisons between M. hyopneumoniae 7448 and J strains, between M. hyopneumoniae 7448 and M. flocculare regarding whole-cell proteomes. These differences can be associated with the differential pathogenic and non-pathogenic natures of these mycoplasmas and may point out some potential PEP determinants as described in the next sections.Overall, these results showed important qualitative and quantitative differences between M. hyopneumoniae 7448 and its non-pathogenic counterparts were assumed to be potential PEP determinants. This assumption was validated by the fact that, among these differential proteins, there were many virulence-related proteins previously described in the literature, like adhesins, proteases, redox balancing protein, and membrane transporters. The observed qualitative differences are graphically represented in Differential proteins between M. hyopneumoniae 7448 (MHP7448_0173), M. hyopneumoniae J (MHJ_0169) and, M. flocculare (MFC_0210). The XAA-PRO aminopeptidase was exclusively detected in the CyPE in M. flocculare, while in both M. hyopneumoniae strains it was detected in both CyPE and SuPE. The neutrophil activating factor, which is involved in oxidative stress, was exclusively detected in M. hyopneumoniae 7448 CyPE samples (MHP7448_0457). Most of the detected membrane transporters protein species were found only in SuPE or in both CyPE and SuPE samples from all analyzed mycoplasmas. As expected, those membrane transporters shared between CyPE and SuPE samples were mostly enriched in the SuPE samples (see Supplementary Table 4).Qualitative comparisons revealed that most of the differential proteins were detected in both CyPE and SuPE samples for all analyzed mycoplasmas (see Supplementary Table 2). However, some proteins were exclusively detected in only one subcellular fraction, as follows. Methionine aminopeptidase was exclusively found in the CyPE from M. hyopneumoniae 7448 adhesin repertoire in comparison to those of the non-pathogenic counterparts. In comparison to M. hyopneumoniae J, the P97-like (MHP7448_0272) and MgpA-like (MHP7448_0005) adhesins were overrepresented in M. hyopneumoniae 7448 CyPE. On the other hand, the P95 (MHP7448_0099) and P102-copy 1 (MHP7448_0199) adhesins, were underrepresented in M. hyopneumoniae 7448 SuPE. Comparisons between M. hyopneumoniae 7448 and M. flocculare adhesins, showed that the P65 adhesin (MHP7448_0656) was overrepresented in M. hyopneumoniae 7448 in both CyPE and SuPE. Interestingly, in M. flocculare, the P95 (MFC_00492) and P60-like (MFC_01236) adhesins, in CyPE, and the P97 copy-2 adhesin (MFC_00472), in SuPE, were overrepresented in comparison to M. hyopneumoniae 7448.Considering the proteins detected in both CyPE and SuPE, several quantitative differences were observed involving the M. hyopneumoniae strains and M. flocculare samples, several quantitative differences were observed, which are suggestive of differential mechanisms for regulation of protein abundance and subcellular localization. Comparing proteases found in both M. hyopneumoniae 7448 and J SuPE, the oligoendopeptidase F and leucyl aminopeptidase were overrepresented in the pathogenic mycoplasma . In comparison to M. flocculare, 6 proteases were overrepresented in M. hyopneumoniae 7448 samples, as follows. An aminopeptidase (MHP7448_0129) and the methionine aminopeptidase (MHP7448_0173) were more abundant in M. hyopneumoniae 7448 CyPE. The oligoendopeptidase F (MHP7448_0521), the leucyl aminopeptidase (MHP7448_0464), the ATP-dependent protease binding protein (MHP7448_0101), and the lon protease (MHP7448_0524) were more abundant in M. hyopneumoniae 7448 SuPE. Interestingly, the MHP7448_0129 aminopeptidase, overrepresented in M. hyopneumoniae 7448 CyPE, was differentially enriched in M. flocculare, being ~ 15 times more abundant in SuPE.Proteases are often involved in the virulence of several pathogens, including pathogenic mycoplasmas. In the protease repertoires identified in M. hyopneumoniae neutrophil activating factor (MHP7448_0457) was detected only in M. hyopneumoniae 7448 CyPE. Regarding proteins differentially abundant, a thiol peroxidase (MHP7448_0096) and a thioredoxin (MHP7448_0384)  were overrepresented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J. In comparison to M. flocculare, a NADH oxidase (MHP7448_0082) and a thioredoxin reductase (MHP7448_0098) were overrepresented in the pathogenic mycoplasma.Redox balancing proteins can be also associated with virulence of several pathogens, including mycoplasmas, and some of them were differentially represented in the performed proteomic analyzes. M. hyopneumoniae and M. flocculare genomes. Around 47% (25) of membrane transporters species detected by LC-MS/MS were shared among the three analyzed proteomes. Among these membrane transporters, 6 (3 from CyPE and 3 from SuPE) and 3 (from SuPE) proteins were overrepresented and underrepresented, respectively, in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J. In comparison to M. flocculare, 7 (3 from CyPE and 4 from SuPE) and 1 (from CyPE) membrane transporters were overrepresented and underrepresented, respectively, in M. hyopneumoniae 7448.Membrane transport proteins, such as ABC transporters, permeases and PTS system proteins, correspond to ~ 12% of the proteins encoded by M. hyopneumoniae 7448 also presents several potential virulence-related enzymes that were differentially represented in comparison to M. hyopneumoniae J, and M. flocculare. In comparison to M. hyopneumoniae J, a phosphopentomutase (MHP7448_0161), and a ribonuclease (MHP7448_0037) were 3–4 times more abundant in M. hyopneumoniae 7448 CyPE. Comparing to M. flocculare, a lipase-esterase (MHP7448_0133), a ribonuclease (MHP7448_0037), two glycolytic enzymes, namely lactate dehydrogenase , and pyruvate dehydrogenase , a chaperone DnaJ (MHP7448_0068), and three translation elongation factors  were from 2.6 to ~ 63 times more abundant in M. hyopneumoniae 7448.Besides the canonical virulence-related proteins described above, M. hyopneumoniae 7448 proteome in comparison to the samples of non-pathogenic mycoplasmas. The potential of these proteins as PEP determinants deserves further investigation.Overall, these results showed important qualitative and quantitative differences in virulence-related proteins that might be PEP determinants. Importantly, along with these previously described virulence-related proteins, at least 47 other proteins were overrepresented in M. hyopneumoniae J and M. flocculare were also qualitative and quantitatively analyzed and compared between each other. Among the proteins detected in M. hyopneumoniae J and M. flocculare samples, 26 and 41 proteins were exclusively detected, respectively . The differences in abundance of both CyPE and SuPE proteins ranged from ~ 1.6 to ~ 19-times fold. Regarding CyPE differentially abundant proteins, 4 and 17 were overrepresented in M. hyopneumoniae J and M. flocculare, respectively. On the other hand, 63 and 5 SuPE proteins were overrepresented in M. hyopneumoniae J and M. flocculare, respectively. All 4 M. hyopneumoniae J overrepresented CyPE proteins were also overrepresented in SuPE samples. Interestingly, two M. hyopneumoniae J proteins, an ABC transporter (MHJ_0450) and an arginine-tRNA ligase (MHJ_0012), were differentially enriched, once they were underrepresented in CyPE and overrepresented in SuPE.The whole cell proteomes of vely see . Among tM. hyopneumoniae J and M. flocculare did not provide evidence of common features that could be clearly associated with the lack of virulence of these related bacteria. However, the observed qualitative and quantitative differences point out to physiological differences between them that deserve further investigation.Overall, the comparisons between the proteomes of M. hyopneumoniae 7448 proteins (Supplementary Table 6A), 292 M. hyopneumoniae J proteins (Supplementary Table 6B), and 286 M. flocculare proteins (Supplementary Table 6C) were categorized according to GO terms into “biological process” (BP), “molecular function” (MF), and “cellular component”(CC) categories. No annotations were retrieved for 52, 51, and 29 proteins of M. hyopneumoniae 7448, M. hyopneumoniae J, and M. flocculare, respectively. Several functional BP, CC, and MF subcategories were commonly overrepresented in all mycoplasma samples. On the other hand, some functional subcategories were exclusively found as overrepresented in each of the analyzed samples, as follows. “Cellular macromolecule metabolic process”, “phosphorus metabolic process” and “ribose phosphate metabolic process” (BP subcategories); and “nucleic acid binding”, “oxidoreductase activity”, and “translation factor activity, RNA binding” (MF subcategories) were enriched only in M. hyopneumoniae 7448. For M. hyopneumoniae J. only the MF subcategories “hydrolase activity”, “nucleoside-triphosphatase activity”, and “pyrophosphatase activity” (MF) were exclusively enriched. Finally, some subcategories involved in nucleotide metabolism, as “pyridine nucleotide metabolic process”, “nucleobase-containing compound biosynthetic process”, “aromatic compound biosynthetic process” (BP subcategories), and other nucleotide-metabolism related MF subcategories were exclusively enriched in M. flocculare.GO functional enrichment analyzes were performed for the whole cell protein sets of all mycoplasma samples to provide clues on functional differences between strains and species. Totals of 292 M. hyopneumoniae 7448 and J strains and M. flocculare present overall metabolic similarities, as expected. However, some interesting differences were highlighted among them, pointing out specific functional distinctions with possible impact for their proliferation, and survival capacities in the natural host.The performed GO functional analyzes showed that M. hyopneumoniae 7448 and J, and M. flocculare, respectively, establishing interesting subsets of mycoplasma uncharacterized proteins (Supplementary Table 3), which deserve further attention. The Venn diagram in M. hyopneumoniae 7448 and J, and M. flocculare uncharacterized proteins. Cell fraction analyzes revealed that ~ 44% of the uncharacterized protein species were found in both CyPE and SuPE  and J . For M. flocculare, only 24% of uncharacterized proteins were detected in SuPE.Our proteomic data provided experimental validation for 70, 73 and 69 genes previously regarded as hypothetical for and SuPE ). A largIn silico functional predictions were performed in order to provide clues on the functional roles of the detected uncharacterized proteins. A total of 41 different domains from the Pfam database were found distributed among 52 out of the total of 109 different uncharacterized proteins species detected in the analyzed samples (Supplementary Table 7). The “N-6 DNA methylase” domain was exclusively found in the M. hyopneumoniae 7448 set of uncharacterized proteins, while the DUF1410, ‘DUF4231ʹ and “tRNA synthetases class II” domains were exclusively found in the M. hyopneumoniae J set. Finally, domains related to replication initiation, peptidase and recombinase functions were found exclusively in the M. flocculare set.M. hyopneumoniae 7448 and J, and/or between M. hyopneumoniae 7448 and M. flocculare, 14 virulence-related domains were identified, including domains of potential peptidases, lipases, nucleases, permeases, thioredoxins and chaperones, were found distributed among 18 protein species. Among these proteins, only 4 were overrepresented in M. hyopneumoniae 7448, namely MHP7448_0431, bearing a “phosphatidylethanolamine-binding” domain; MHP7448_0064, bearing a “AAA domain”; MHP7448_0522, bearing a “GDSL-like lipase/acylhydrolase” domain; and MHP7448_0148, bearing a ‘Hsp33ʹ domain. While MHP7448_0431 and MHP7448_0064 were overrepresented in comparison to M. hyopneumoniae J, MHP7448_0431, MHP7448_0522 and MHP7448_0148 were overrepresented in comparison to M. flocculare.Among the uncharacterized proteins shared between M. hyopneumoniae strains , and M. flocculare, a non-pathogenic related species. For the first time, subcellular fractions of M. hyopneumoniae and M. flocculare were comparatively assessed using high-sensitivity high-resolution mass spectrometry. Qualitative and quantitative differences between the pathogenic M. hyopneumoniae 7448 and its non-pathogenic were found, involving potential PEP determinants, such as adhesins, proteases, and proteins related to redox balancing or membrane trafficking.Bacterial pathogenicity and virulence are multifactorial features that can be better assessed in comparative studies at the protein level, as protein abundance is the result of transcriptional regulation, post-translational processing and/or protein degradation. In this study, we compared the protein repertoires of cytoplasmic and surface-enriched protein fractions, comprehending the whole-cell proteomes, from the pathogenic and non-pathogenic In silico subcellular localization prediction associated with the quantitative proteomics of CyPE and SuPE, confirmed their enrichment with cytoplasmic and surface proteins, respectively, for all analyzed mycoplasma samples. A previous M. hyopneumoniae 7448 surface protein survey carried out by our group identified only 34 surface-predicted proteins detected using a biotin cell surface labeling approach [M. hyopneumoniae 7448 SuPE (38% of the predicted surfaceome).Cell fractioning procedures are useful to reduce proteome complexity, allowing the enrichment of low-abundance proteins. They improve the efficiency of MS-based protein identification and allow the association of different sets of proteins to specific cell compartments . The carapproach . Our fraet al. (2009). In comparison to the former data, proteome coverage was improved 28% (from 22% to 50%) for M. hyopneumoniae 7448, and 27% (from 24% to 51%) for M. hyopneumoniae J. The remaining ~ 50% of predicted proteins not covered by our proteomic data may not have been detected due to their low abundance or lack of expression in culture conditions.The cell fractioning approach combined with a high-resolution and sensitivity LC-MS/MS provided a high proteome coverage for all three mycoplasmas analyzed. The LC-MS/MS approach sensitivity was evidenced by comparing our data to those published by Pinto M. hyopneumoniae 7448 shared ~ 70% of the detected proteins with M. hyopneumoniae J and M. flocculare. Despite these high similarities between the sets of proteins detected for M. hyopneumoniae strains and M. flocculare, many qualitative and quantitative differences were detected, several of them likely associated with pathogenicity/PEP determination. Within the sets of proteins differentially represented in M. hyopneumoniae 7448 in comparison to the non-pathogenic samples, there are representatives of several classes of proteins and/or functions that may be potential PEP determinants, such as adhesins, proteases, oxidative stress-related proteins, and membrane transporters, among others.M. hyopneumoniae 7448 and J, and M. flocculare are quite similar, containing few qualitative differences between the adhesin repertoires of M. hyopneumoniae and M. flocculare [M. flocculare orthologs for one P97 paralog , and one P102 paralog , and some rearrangements in M. hyopneumoniae genomic regions containing adhesin genes in comparison to M. flocculare. Despite these differences, the overall high qualitative similarity between the M. hyopneumoniae and M. flocculare adhesin sets was confirmed at proteomic level by the data described here. However, our data also pointed out some interesting quantitative differences, as three adhesins  were more abundant in M. hyopneumoniae 7448 than in the non-pathogenic mycoplasmas, which may be associated with the higher adherence capacity of pathogenic M. hyopneumoniae. Conversely, four adhesins were more abundant in the non-pathogenic mycoplasmas. P95 and P97, for example, were more abundant in M. flocculare than in M. hyopneumoniae 7448. However, the M. flocculare orthologs are quite divergent (only ~ 55% of sequence identity to the M. hyopneumoniae orthologs), which may imply different adhesion properties. Moreover, M. flocculare has only one copy of P97 (MFC_00472), while M. hyopneumoniae has two, and, in this case, the overrepresentation of the single M. flocculare P97 may be resultant of a compensating mechanism.Genomic comparative analyzes demonstrated that the sets of adhesin-encoding genes from occulare . The onlM. hyopneumoniae and M. flocculare proteins, including adhesins. This suggests that these proteins are more fragmented in the cell surface than in in the cytoplasm, when they are expected to be mostly unprocessed. Previous studies have showed that adhesins are targets of post-translational proteolytic events [M. hyopneumoniae strains [Additionally, we observed higher peptide coverages in CyPE in comparison to those in SuPE, for c events –24 which strains . Along wM. hyopneumoniae proteome. Most of the proteases found in the whole cell proteomes of M. hyopneumoniae strains and M. flocculare were detected in both CyPE and SuPE. Interestingly, most of the overrepresented proteases of M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare were detected in SuPE. These differences in protease abundance between subcellular fractions and between pathogenic and non-pathogenic mycoplasmas could be resultant of differential enzyme activity or regulation for their targeting to preferential substrates in cell surface. With that, specific proteolytic activities could be targeted, for example, to the processing of surface adhesins.As mediators of post-translational proteolytic events and other important cell processes, proteases play an important role to shape the M. hyopneumoniae had their activities experimentally assessed [M. hyopneumoniae leucyl aminopeptidase has been associated with plasminogen, heparin and foreign DNA binding and is localized on mycoplasma cell surface [M. hyopneumoniae 7448 SuPE. Moreover, oligoendopeptidase F and XAA-PRO aminopeptidase were previously associated with host kallikrein-kinin system, participating in inflammatory processes [M. hyopneumoniae 7448, along with previous functional studies, indicate the involvement of these enzymes with important pathogenicity-related mechanisms from adhesion to host immunomodulation.Some of the proteases overrepresented in assessed –27. Inte surface , which cMycoplasma pneumoniae and Mycoplasma mycoides subsp. mycoides [M. hyopneumoniae were able to produce hydrogen peroxide from glycerol metabolism, but that the non-pathogenic strain J and M. flocculare were not [M. hyopneumoniae uptakes and metabolizes glycerol, while M. flocculare does not, failing to produce cytotoxic levels of hydrogen peroxide, which can be explained by the absence, in the M. flocculare genome, of the glpO gene, related to glycerol metabolism and hydrogen peroxide production [Endogenous production of hydrogen peroxide through glycerol metabolism is essential for cytotoxicity of pathogenic mycoplasmas, as mycoides ,29. In lwere not . M. hyopoduction .M. hyopneumoniae 7448 CyPE. In Helicobacter pylori, this protein was previously related to neutrophil activation by the production of reactive oxygen species (ROS) [M. hyopneumoniae 7448 than in M. hyopneumoniae J and M. flocculare. These results agreed with the functional enrichment analyzes, which demonstrated that the “oxidoreductase activity” subcategory, including all detected proteins related to redox balancing, was exclusively enriched in M. hyopneumoniae 7448. These differentially abundant proteins can be considered potential PEP determinants, due to their importance for bacterial survival in the context of endogenous (mycoplasma) and exogenous (host) ROS production [M. flocculare, its inability to produce endogenous hydrogen peroxide may be associated with its commensal nature, being less harmful to the host.Among proteins involved with oxidoreduction processes, a neutrophil activating factor was exclusively detected in es (ROS) . Moreoveoduction –33. For M. hyopneumoniae and M. flocculare genomes have ~ 80 membrane transporters coding genes each, including genes coding for ABC transporters, permeases and PTS systems. In the LC-MS/MS analyzes, ~ 68% of the sets of membrane transporters species identified in M. hyopneumoniae and M. flocculare predicted proteomes were detected, with a partial (~ 50%) overlapping. Moreover, abundance differences were found between membrane transporters orthologs shared by M. hyopneumoniae 7448 and M. hyopneumoniae J, or by M. hyopneumoniae 7448 and M. flocculare. Overall, these evident qualitative and quantitative differences among the sets of membrane transporters of M. hyopneumoniae 7448, M. hyopneumoniae J, and M. flocculare are suggestive of substantial differences in transporting activities/capabilities and may also contribute to their differential virulence/pathogenicity.Membrane transporters have been described as virulence-related proteins, as they may be associated with multidrug resistance, metal ions uptake, and cell attachment , which aM. hyopneumoniae 7448 and its non-pathogenic counterparts analyzed here. Functional enrichment analyzes showed some important metabolic subcategories specifically enriched in the M. hyopneumoniae 7448 whole cell proteome. The “phosphorous metabolic process” subcategory, which includes several glycolytic enzymes and kinases, and the “RNA binding” subcategory, which includes ribosomal proteins, translational elongation factors and aminoacyl tRNA ligases, were exclusively enriched in this pathogenic mycoplasma. In agreement to the functional enrichment analyzes, several proteins with canonical functions in metabolic pathways were overrepresented in M. hyopneumoniae 7448 protein repertoire, including the glycolytic enzymes LDH and pyruvate dehydrogenase, the pentose pathway enzyme phosphopentomutase, and translation-related proteins. Overall, the exclusive enrichment of all these metabolic functions suggests a higher metabolic capacity for the pathogenic M. hyopneumoniae strain, which may favor its proliferation and survival, contributing to the colonization, and infection of the porcine respiratory tract.Many proteins not classically related to virulence were also differential represented between M. hyopneumoniae 7448 proteins not usually regarded as virulence factors, such as proteins involved in pentose phosphate pathway, DNA replication, and translation may have also alternative (moonlighting) functions of relevance for pathogenicity [Glycolytic enzymes and other differential genicity –37. For genicity ,39, whilgenicity –42.M. hyopneumoniae strains and M. flocculare codes for hypothetical proteins. For pathogenic species, such set of hypothetical proteins is of particular interest, once it represents a potential reservoir of unknown virulence factors. For M. pneumoniae, many novel virulence factors were predicted upon in silico analyzes of hypothetical proteins [M. hyopneumoniae and M. flocculare coding DNA sequences (CDSs) whose putative products have been annotated as “hypothetical proteins” had their proteins products experimentally detected by LC-MS/MS. This allowed to confirm these CDSs as functional genes, and to change the status of their products to that of “uncharacterized proteins”. Among the detected uncharacterized proteins, several functional domains were predicted, including virulence-related ones, and most of them were conserved among the orthologs. More importantly, abundance differences between M. hyopneumoniae 7448 and its assessed non-pathogenic counterparts were observed for some of the uncharacterized proteins bearing functional domains, including virulence-related ones. Future analyzes of these and other uncharacterized proteins along with the characterization of their functional domains will be important steps towards the elucidation of their functions in M. hyopneumoniae biology and their possible roles as novel virulence factors.Around 37% of the sequenced genomes of proteins . In our M. hyopneumoniae strains and M. flocculare, and an extended list of tens of candidates to pathogenicity determinants, beyond those classically described. Several protein classes with potential virulence-related functions were identified as overrepresented in the M. hyopneumoniae 7448 pathogenic strain, including adhesins, proteases, oxidative stress proteins, membrane transporters, and proteins with moonlighting functions, along with many so far uncharacterized proteins. Based on our proteomics results, the pathogenic nature of M. hyopneumoniae may be explained, at least in part, by the overrepresentation of several virulence-related proteins. These overrepresented proteins are involved in a wide range of biological processes, including adhesin processing and cell adhesion regulation, detoxification, overall metabolism regulation, and host-pathogen cell trafficking, among others. Although no specific commensalism determinants were found, the underrepresentation of several virulence-related proteins encoded by the non-pathogenic mycoplasmas may be a key point to explain their commensal natures.Our results provided a comprehensive profiling of the whole cell proteomes of two M. hyopneumoniae strains and M. flocculare repertoires deserve future studies to elucidate mechanisms related to pathogenicity or commensalism, respectively. Of particular interest will be proteins with unknown function or with possible moonlighting functions overrepresented in the pathogenic M. hyopneumoniae 7448 strain. Moreover, the identification and characterization of M. hyopneumoniae virulence factors is of upmost relevance to discover new targets for the development of novel diagnostic methods, therapeutic drugs, and preventive vaccines against PEP.Several of the identified proteins in M. hyopneumoniae pathogenic strain 7448 was isolated from an infected swine from Lindóia do Sul  [M. hyopneumoniae non-pathogenic strain J , and the non-pathogenic M. flocculare  were acquired from American Type Culture Collection by the Empresa Brasileira de Pesquisa Agropecuária-Centro Nacional de Pesquisa de Suínos e Aves . For soluble and insoluble protein extracts, respectively, all bacteria were cultivated in 50 mL and 100 mL of Friis medium [s medium , at 37°Cg, 15 min, 4°C), and washed three-times with PBS (pH 7.4). Cells were resuspended and lysed by sonication at 25 Hz in an ice bath by five 30 s cycles with 1 min intervals between pulses. The lysates were centrifuged at 10,000 x g, for 20 min, at 4°C and the supernatant (soluble fraction) was recovered for proteomics analyzes. The pellet (insoluble fraction) was resuspended in RapiGest SF Surfactant . Soluble and insoluble protein extracts were quantified using the microBCA Protein Assay Kit  using a NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific). Three protein extracts were independently produced to provide the three biological replicates for each sample.For protein extraction, cultured mycoplasma cells were pelleted by centrifugation  at 37°C for 1 h, and alkylated with 10 μg of iodoacetamide  in the dark, at room temperature. Protein samples were diluted to a final 1 M urea concentration, and further digested with 1 μg of trypsin . For insoluble fraction analysis, samples resuspended in RapiGest SF were reduced with DTT (Bio-Rad) at 60°C for 30 min to a final concentration of 5 mM and alkylated with iodoacetamide (Bio-Rad) 15 mM  at room temperature for 30 min in the dark. Proteins were then digested overnight with 0.5 μg of trypsin (Promega) at 37°C, and RapiGest SF was removed as recommended by the manufacturer (Waters). Resulting soluble and insoluble fractions peptides were desalted in HLB cartridges , and eluted with 50% acetonitrile/0.1% TFA. Peptides were then lyophilized using a Concentrator Plus (Eppendorf), prior to MS analyzes.Processed peptide samples were analyzed for protein identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described ,46. BrieM. hyopneumoniae 7448 and J strains, and M. flocculare. These local databases were derived from the fully sequenced genomes from M. hyopneumoniae 7448 , M. hyopneumoniae J , and M. flocculare , and included all deduced amino acid sequences  from the corresponding genomes annotation , available at NCBI (https://www.ncbi.nlm.nih.gov/protein/) and Uniprot (http://www.uniprot.org/). The MASCOT search parameters for protein identification included a fragment ion mass tolerance of 0.5 Da, peptide ion tolerance of 7 ppm, and three missed cleavages of trypsin. Carbamidomethylation of cysteine was specified as a fixed modification, whereas the oxidation of methionine, acetylation of lysine and N-terminal ends of proteins, and phosphorylation of tyrosine and serine/threonine were specified as variable modifications [The MS/MS raw data were processed using msConvert version 3 (ProteoWizard) , and theScaffold software version 4.8.1  was used to validate the peptide and protein identifications. The peptide identifications were accepted if they could be established at greater than 99.0% probability as assigned by the Peptide Prophet algorithm . The proM. hyopneumoniae 7448 and J, and M. flocculare, ortholog sequences were determined using OrthoFinder [-6.In order to allow comparisons among protein repertoires from hoFinder . OrtholoM. hyopneumoniae 7448 and J strains, and M. flocculare were analyzed in silico to predict their subcellular localization, being classified as surface or cytoplasmic proteins. Membrane proteins were initially predicted based on positive predictions as lipoproteins, using LipoP 1.0 [Proteins identified in soluble and insoluble fractions of ipoP 1.0 , and PREipoP 1.0 . Non-lipipoP 1.0 , PhobiusipoP 1.0 , HMMTOP ipoP 1.0 , CW-PREDipoP 1.0 , and HMMipoP 1.0 . Non-traipoP 1.0 , iLoc-GpipoP 1.0 , and CELipoP 1.0 .Proteins not predicted as membrane proteins were then classified as secreted or cytoplasmic. Secreted proteins were predicted based on the presence of signal peptide or on non-classical secretion prediction. Signal peptide predictions were made using SignalP 4.1 , PhobiusM. hyopneumoniae 7448 and J, and M. flocculare insoluble fractions were compared to those of the corresponding soluble fractions. For that, differentially represented proteins, exclusively detected or more abundant in the insoluble protein fraction in comparison to the soluble extract of the same species or strain, were analyzed based on subcellular localization predictions. Protein abundance was measured based on normalized spectral abundance factor (NSAF) values [t-test, with the Benjamini-Hochberg FDR multiple-testing correction. A p-value < 0.05 was considered statistically significant. Proteins with differential abundances between surface-enriched and soluble protein extracts were represented in heat-maps using the Heatmapper web server (http://www.heatmapper.ca) using the Z-score calculation of NSAF values.To confirm the enrichment of surface proteins in the insoluble fractions, the LC-MS/MS datasets of proteins identified in the ) values  and quanM. hyopneumoniae 7448 and M. hyopneumoniae J; (ii) M. hyopneumoniae 7448 and M. flocculare; and (iii) M. hyopneumoniae J and M. flocculare, the analyzes were based on the exponentially modified protein abundance index (emPAI) values [M. hyopneumoniae 7448 vs. M. hyopneumoniae J; M. hyopneumoniae 7448 vs. M. flocculare; or M. hyopneumoniae J vs. M. flocculare) comparisons. The emPAI values were statistically compared using Student’s t-test using Prism GraphPad Software version 6 . Fold-changes (FC) were calculated for each pair of ortholog proteins. Proteins with a p-value < 0.05 and a FC > 1.5 were considered differentially abundant by both statistical and FC parameters.For quantitative comparisons between ortholog proteins shared between (i) ) values . EmPAI vIn silico functional analyzes of M. hyopneumoniae and M. flocculare proteins identified by LC-MS/MS were based on gene ontology (GO). Mycoplasma identified proteins were submitted to hierarchical GO overrepresentation tests using the Cytoscape 2.6.3 26 plugin BiNGO 2.3 [M. hyopneumoniae 7448 and J GO annotation files were acquired from Uniprot (http://www.uniprot.org/). M. flocculare GO annotations were acquired using BLAST2GO version 3.0 [−3). The ontology files were retrieved from the GO database (http://www.geneontology.org/). Both annotation and ontology files were edited in-house as BiNGO input files. The hypergeometric overrepresentation tests were performed at a 0.05 level of significance, with the Benjamini-Hochberg FDR multiple-testing correction. Uncharacterized proteins were further analyzed in order to predict functional domains using the Pfam software version 29.0 (http://pfam.xfam.org/) [iNGO 2.3 . Custom sion 3.0 . For thaam.org/) .


Hearing loss is an important public health issue, since it has a very negative impact on peoples’ lives, irrespective of the age at which it develops. However, globally there is a noticeable lack of epidemiological data for health outcomes for people who are deaf and hard of hearing. In Greece, people with hearing disabilities are systematically not included in health policy and planning processes, despite there being a marked tendency for global efforts aimed at improving their quality of life.The sample consisted of 140 adults with hearing loss (86 d/Deaf and 54 hard of hearing) and 97 normal hearing as the control group. We run data collection from April to June 2015, using the Greek version of the 36-Item Short Form Health Survey (SF-36v2). Socio-demographic and characteristics about non-medical determinants of health .were also collected and were analysed as possible determinants. Data analysis included bivariate and multivariate analyses such as linear regression models.Multivariate analyses identified that in all the SF-36v2 dimensions, the scores among deaf people were lower than those with normal hearing. Determinants included the hearing loss degree, educational level, body mass index, levels of physical activity, and alcohol consumption levels, while the variable “number of family members per household” was inversely associated with physical health summary scale score.Improving knowledge of the health-related determinants that affect quality of life for the population with hearing loss is an important step in designing targeted services and interventions. In light of these findings, a special effort must be made to ensure the wellbeing of this population. Hearing impairment, deafness or hearing loss is a partial or total inability to hear: it may be ranked as mild, moderate, moderately severe, severe or profound. Impairment may result from genetic causes, complications at birth, certain infectious diseases, chronic ear infections, the use of particular drugs, exposure to excessive noise and ageing . AlthougDespite the seriousness of this public health issue, there is a noticeable lack of epidemiological data for health outcomes for people who are deaf and hard of hearing . HoweverIn Greece, people with hearing disabilities are systematically excluded from health policy and planning processes, despite there being a marked tendency for global efforts aimed at improving their quality of life . FurtherIdentifying determinants of the health-related quality of life of deaf and hard of hearing people is of great importance so as their health status can be improved. This study aims to fill a gap in the literature, by investigating the epidemiological profile of deaf and hard of hearing, so thatthe futurepolicy on their healthcare needs would be evidence-based. The objectives of this study included an assessment of the health-related quality of life of deaf and hard of hearing adults (aged 18–65), and the investigation of the factors that affect it.deaf when referring to the audiological condition of not hearing, and the uppercase Deaf when referring to a particular group of deaf people who share a language – (GSL) – and a culture. The members of this group have adopted the GSL in their daily life, they use it as primarily for their communication and hold a set of beliefs about themselves and their connection to wider society. We distinguish them from, for example, those who find themselves losing their hearing because of illness, trauma or age; although those people share the condition of not hearing, they do not have access to the knowledge, beliefs and practices that make up the culture of Deaf people. Additionally, the distinction between the definition of “deaf” and “hard of hearing” is often given under different criteria, as it has strong ties with the individuals’ ideological positions about education, social inclusion and/or rehabilitation of people with hearing loss [A cross-sectional study was conducted, and the study population consisted of 140 young and middle-aged adults (18–65 years) with hearing loss (86 d/Deaf and 54 hard of hearing) and 97 hearing participants. There are several groups included within the broad “deaf and hard of hearing” category, as the population with hearing loss consists of subgroups with distinctly different cultural and communicational characteristics, which need to be separately examined. Factors that must be considered with Deaf/HH sub-populations include a) degree of hearing loss, b) age of onset of loss , c) preferred language, and d) psychological issues . Followiing loss .Deaf (upper case D) typically belong to the Deaf Community and use GSL as a primary language [Deaf – Persons referred to as language .deaf (lower case d), that do not consider themselves members of the Deaf Community, although they may be severely or profoundly deaf, strive to identify themselves with hearing people and regard their hearing loss solely according to biomedical model of health, using non-signing communication [deaf – Persons referred to as nication .Hard of hearing that communicate in terms of auditory-verbal approach  .Hard of hearing who communicate via lip-reading  .Hard of hearing who communicate using GSL as a preferred method of communication .In this study, the participants with hearing loss were categorised by combination of cultural self-identification, and their preferred method of communication, in order to self-identify themselves in one of the following five categories; a classification that fits best with the population with hearing loss in Greek culture.Regarding the sampling method, there is no accurate census of deaf and hard of hearing in Greece since members of the subcategories of this population are extremely difficult to be identified, except from the members of the first subcategory who are registered in deaf clubs- Thus, the probability or random sampling was not feasible and therefore a convenience sampling technique was used. In regards to the deaf and hard of hearing participants that communicate via GSL, the recruitment was done by visiting the 5 Deaf Clubs-members of the Hellenic Federation of the Deaf which are located in Attica . In addition, the Hellenic Federation of the Deaf embedded a link of an online survey in their website. This helped the wide distribution of the questionnaire and to reach the selected population, as deaf and hard of hearing individuals in Greece use this website to get local news and information, even if they do not communicate via sign-language. We also had placed a link on social networks, to provide also quick access to participants with similar demographics that had normal hearing, according to their answers in self-reported hearing difficulties.The questionnaire consisted of three sections. The first section explained the purpose of the questionnaire and provided a contact name and address for any enquiries regarding the study. The second and third section consisted of the 36-item Short Form Health Survey (SF-36v2), and questions on standard socio-demographic data such as gender, age, marital status, number of family members per household, complete educational status, work status, income and non-medical determinants of health . The SF-36 Health Survey Version 2, for the measurement of the perceived health-related quality of life, is a widely used questionnaire. The SF-36v2 Health Survey health domain scales are: Physical Functioning, Role-Physical, Bodily Pain, General Health, Vitality, Social Functioning, Role-Emotional, Mental Health. Higher scores across each subscale indicate greater HRQoL. The psychometrically-based physical component summary and mental component summary scores are also provided. The SF-36 has been translated and its psychometric properties tested for a Greek population by Pappa et al. .o3699/2008. The 6 pilot subjects were representative of the overall Deaf signers that were enrolled into this project, as they represent them unanimously on a regular basis in the Community, and they have a deep understanding of their educational and other needs. Face validity of the questionnaire was very good and no significant corrections were made.A pilot study with 6 deaf adults was carried out, in order to check the comprehensibility of the questionnaire, as for the Sign Language users the Greek language is being perceived as a second language, and it is being taught to the Schools of the Deaf along with the GSL, which is their first language. In addition, they are obligated to learn only the written form of the Greek language and not the oral form according to the Greek Law NThe data collection was done in person, so that the questions were presented in GSL from the first author (TD) when it was necessary, in order to improve accessibility and minimise the language barrier for Deaf, as they completed a lengthy written questionnaire in a second language. The completion of the questionnaire by the deaf and hard of hearing non-users of GSL and by the hearing participants was done in person, following a convenience sampling technique.All participants were informed about the aim and procedures of this study  and gave their consent. Personal data of the participants were not registered at any stage of the study. Participation in this study was voluntary and anonymous. The previous engagement of the first author in the Deaf community, as she has a National Certification in GSL and participates in their community activities, reinforced the feelings of trust and comfort of the participants, leading to a high response rate (91%). The response rate for the hearing participants was 93%.Categorical variables are presented as absolute (n) and relative (%) frequencies, while quantitative variables are presented as mean  or median (range). The Kolmogorov-Smirnov test and normal plots were used to test the normality of quantitative variable distribution.Firstly, we performed bivariate analyses between socio-demographic and non-medical characteristics and scores on SF-32v2 scales. Bivariate associations between categorical variables were assessed with chi-square test, while between categorical and ordinal variables with chi-square trend test. Student’s t-test and analysis of variance were used for the association between categorical variables and a continuous one that followed normal distribution. Also, we used Kruskal-Wallis test to assess the relation between categorical variables and a continuous one that did not follow normal distribution. We used Pearson’s and Spearman’s correlation coefficient to find out the correlation between continuous variables that followed and did not follow normal distribution respectively.p < 0.2) in bivariate analyses, then multivariate linear regression analysis was applied, using the backward stepwise linear regression model in order to eliminate confounding. For multivariate linear regression models, coefficients’ beta values, 95% confidence intervals and p values are presented.Scores on SF-32v2 scales were the dependent variables. In case that > 2 independent variables were associated with scores on SF-32v2 scales (n ≤ 10).‘Work status’ was not used as an independent variable due to the small number of participants in several categories . Therefore, the classification was limited to the three bigger categories (deaf/ hard of hearing/hearing) and it was not possible to consider the exact language preferences of the participants, but only their cultural self-identification. That means that the “deaf category” in the statistical analysis finally included those that self-identified themselves as severely or profoundly deaf and used to communicate via GSL or non-signing communication (category No1 and No2), while the “hard of hearing category” included those that self-identified themselves as having mild, moderate or moderately severe hearing loss and used to communicate in terms of auditory-verbal approach or via lip-reading (category No3 and No4).The two-tailed significance level was set ≤0.05. Data were analyzed using IBM SPSS 21.0 .n = 79) the GSL was the preferred method of communication, while for the 8.1% (n = 7) was not. Among the 54 hard of hearing, the 20.4% (n = 11) preferred to communicate via the oral method, the 50% (n = 27) via the lip-reading technique and the 29.6% (n = 16) via the GSL.Socio-demographic characteristics are presented in Table p = 0.02). There was a higher proportion of overweight (55.8%) and obese people (20.9%) among deaf and hard of hearing adults (55.6% and 18.5% accordingly), while almost half of the hearing participants (44.3%) had a normal BMI (p = 0.004) . Besides, the median number of years of smoking was higher for hard of hearing and deaf compared to the hearing participants (p = 0.01).Concerning tobacco consumption, the median number of cigarettes smoked per day was higher for hard of hearing and deaf compared to the hearing participants . The median number of units of alcohol per week was higher for deaf compared to hard of hearing and hearing participants (p < 0.001).Cronbach’s coefficient alpha across the domains of SF-36v2 ranging from 0.91 to 0.93, indicating high internal consistency reliability.p < 0.2) in bivariate analyses, then multivariate linear regression analysis was applied, using the backward stepwise linear regression model in order to eliminate confounding.The bivariate associations between all independent variables and scores on SF-32v2 scales are presented in Table Multivariate linear regression analyses revealed a statistically significant relation between hearing loss and lower SF-36v2 scores across subscales, even after controlling for confounders. If the confidence interval did not include the value of zero effect, it was assumed that there was a statistically significant result Table . ConcernRespondents with a higher degree of hearing loss had lower scores in all SF-36 sections, excluding the General Health score, while decreased body mass index (BMI) was associated with increased scores in all SF-36 sections, excluding the Bodily Pain score.Higher educational level was associated with increased scores in Physical Functioning, Role Physical, Bodily Pain, General Health, Social Functioning and Physical Health Component Summary scores and increased physical activity was associated with increased scores in Physical Functioning, General Health, Role Emotional and Physical Health Component Summary scores.Finally, decreased alcohol consumption was associated with increased scores in all the SF-36v2 dimensions except social functioning and decreased number of family members per household was associated with increased Physical Health Component Summary score.Previous studies have revealed that disabling hearing loss, which refers to hearing loss greater than 41 dB in the better hearing ear in adults, is associated with poorer physical health –23 and lDecreased numbers of household individuals, and particularly individuals living alone, has been associated with lower mental health levels, which is associated with depression among adults with hearing loss , 34. HowThe findings of our study showed that increased education was associated with increased scores in Physical Functioning, Role Physical, Bodily Pain, General Health, Social Functioning and Physical Health Component Summary and are in compliance with previous studies , 36, 38.Concerning the variable tobacco consumption, in this study the 55.8% of deaf were current tobacco consumers, while in the Health Interview Survey  the rateAlso, we found that decreased alcohol consumption was associated with increased scores in all the SF-36v2 dimensions except social functioning. The higher number of alcohol units per week has been associated also in a previous study  with lowhigher than what is considered as a healthy weight, has been associated also in previous studies with lower level of health-related quality of life, in both physical and mental health domains for population with hearing loss [Respectively, we found that 55.8% and 20.9% of deaf were overweight and obese, while the percentages in hearing people were 38.1% and 15.5%. In the Health Interview Survey , percenting loss , 23, 34.Also, in our study, increased physical activity was associated with increased scores in physical functioning, general health, role emotional and physical health component summary. In previous studies the lower physical activity levels have also been associated with population with hearing loss , 20.There are several limitations when conducting a study among deaf and hard of hearing participants. However, the previous engagement of the first author (TD) with the Deaf community, allowed many of these methodological issues being eliminated as much as possible.n ≤ 10). As a result, the classification was limited to the three bigger categories (deaf/ hard of hearing/hearing) and it was not possible to take into consideration the language preferences of the participants, but only their cultural self-identification. This should be considered by future studies aiming to assess the health-related quality of life in a larger sample of population with hearing loss, as e.g. the Deaf community struggles with a number of socioeconomic-based health disparities, many of which are not directly associated with the degree of hearing loss.The main limitation of our study is that we did not use an additional specific questionnaire for hearing loss – for example the Hearing Handicap Inventory for Adults (HHIA) – to identify problems that hearing loss may cause to participants, as there have been expressed doubts in literature concerning differential performance of SF-36 items in healthy adults with and without functional limitations . HoweverValidating and standardizing a conceptual model  of healtAs hearing loss is an important global health concern, health-related quality of life among deaf and hard of hearing adults must be estimated as a factor of great importance. In Greece, people with hearing disabilities are systematically excluded in health policy and planning processes, despite there is a marked tendency for global efforts aimed at improving their quality of life.Identifying determinants of the health- related quality of life of deaf and hard of hearing people is decisive, so as their health condition can be improved. The findings of this study suggest that hearing loss is capable of contributing to HRQoL deficits in Greek deaf and hard of hearing adults, as their health is poorer than that of the general population, with probable underdiagnosis and undertreatment of chronic conditions, putting them at risk of preventable ill health.Health information is not available in sign language for Deaf people and it may affect their skills in taking decisions in everyday life concerning health promotion. That is being reflected in several determinants . Programs that raise health knowledge in Deaf communities could contribute to improve their skills in take decisions in everyday life concerning the promotion of a healthy lifestyle. This could help in tackling health inequalities for people with hearing loss and in improving their quality of life.Future research should involve a more extensive population with hearing loss, in order to produce health statistics of deaf and hard of hearing populations, so as they can be included in the priorities for improving health and chronic disease prevention programs and stop therefore this health condition from being a “silent epidemic” .


The intravitreal dexamethasone implant Ozurdex is indicated for the treatment of macular edema due to diabetes and branch retinal vein occlusion. While the most common ocular side effects are elevated intraocular pressure and cataract formation, rare complications related to the injection have been reported. We present a case with extramacular retinal hole after Ozurdex injection. They reported that these values were lower than the 0.1-0.2 N necessary for a foreign body to damage the retina according to previous studies. In this case, which they referred to as the “magic bullet”, the authors believed that no mechanism other than direct contact by the implant could have created the retinal hole and suggested that this complication may be attributable to the retina becoming more susceptible to trauma in chronic retinal disease.Extramacular retinal hole following Ozurdex injection, as seen in our case, has only been reported previously by Christensen et al.4 reported in an experimental study that the Ozurdex implant exited the applicator at a speed of 0.8 m/s and decelerated progressively, and that its deceleration was more rapid in the vitreous compared to water. They concluded that the retinal impact energy calculated in their analyses did not reach the previously reported levels necessary to reach the retina. In addition, the authors followed the patient without treatment and reported that the hole was stable. For our patient, however, we preferred to treat with laser photocoagulation because the hole appeared to be large and causing traction.One point to consider here is the relationship between the speed at which the trigger is pushed and the velocity with which the implant is released. Meyer et al.In our case, the momentary hypotony observed immediately before pushing the trigger during injection may have shortened the distance between the entry site and retina, thus allowing the implant to cause direct damage to the retina. Therefore, we believe that patients who exhibit globe softening during implantation require special care, and that at the very least, the clinician should attempt to aim the implant toward the extramacular area.


Sensory deprivation, such as hearing loss, has been demonstrated to change the intrinsic functional connectivity (FC) of the brain, as measured with resting-state functional magnetic resonance imaging (rs-fMRI). Patients with sloping sensorineural hearing loss (SNHL) are a unique population among the hearing impaired, as they have all been exposed to some auditory input throughout their lifespan and all use spoken language.Twenty patients with SNHL and 21 control subjects participated in a rs-fMRI study. Whole-brain seed-driven FC maps were obtained, with audiological scores of patients, including hearing loss severity and speech performance, used as covariates.Most profound differences in FC were found between patients with prelingual  vs. postlingual onset  of SNHL. An early onset was related to enhancement in long-range network connections, including the default-mode network, the dorsal-attention network and the fronto-parietal network, as well as in local sensory networks, the visual and the sensorimotor. A number of multisensory brain regions in frontal and parietal cortices, as well as the cerebellum, were also more internally connected. We interpret these effects as top-down mechanisms serving optimization of multisensory experience in SNHL with a prelingual onset. At the same time, POST patients showed enhanced FC between the salience network and multisensory parietal areas, as well as with the hippocampus, when they were compared to those with PRE hearing loss. Signal in several cortex regions subserving visual processing was also more intra-correlated in POST vs. PRE patients. This outcome might point to more attention resources directed to multisensory as well as memory experience. Finally, audiological scores correlated with FC in several sensory and high-order brain regions in all patients.The results show that a sloping hearing loss is related to altered resting-state brain organization. Effects were shown in attention and cognitive control networks, as well as visual and sensorimotor regions. Specifically, we found that even in a partial hearing deficit (affecting only some of the hearing frequency ranges), the age at the onset affects the brain function differently, pointing to the role of sensitive periods in brain development. The brain develops the capability of a complex form based on its genetic makeup, that is modified by its sensory experience . The strOne MRI technique, which may serve as a window into experience-related brain development and plasticity, particularly, is resting state functional magnetic resonance imaging (rs-fMRI). It measures low-frequency spontaneous fluctuations of blood oxygen level-dependent (BOLD) signals across the whole brain. This effect was first described by Several canonical resting state networks (RSN) can be reproducibly identified using a variety of analysis approaches . These iSensory deprivation resulting from blindness or deafness provides an excellent opportunity to study experience-dependent brain plasticity . SimilarNevertheless, only scarce literature exists on brain resting brain network changes in patients with moderate to severe hearing loss, as is the case in the current study . The finAlthough some convergence can be seen in the published findings, suggesting the involvement of additional cognitive resources as well as possible enhanced collaboration between sensory regions indicating compensatory plasticity in hearing loss, the outcomes of the existing studies are hardly comparable. Besides the fact that diverse patient populations have been studied  and they were mostly very limited in size, the above-mentioned experiments selected only specific networks for the analysis  and involved fewer women (N = 3) than the POST group (N = 8) . All subjects were right-handed. The control group included 12 women and 9 men, aged 31.8 ± 7.2 years and was matched for education levels . All subjects in the control group had normal hearing (<25 dB for 0.125–8 kHz) and no tinnitus. Please see further details of participants in The study was approved by the Medical Ethics Committee of the Institute of Physiology and Pathology of Hearing (IPPH) in Kajetany/Warsaw, Poland. Participants gave their written informed consent prior to the study and received no monetary reward for their participation. Twenty patients  were recruited from the Institute of Physiology and Pathology of Hearing. Ten patients had higher (university degree), eight medium (high-school) and two basic (primary school) education. All patients had idiopathic sloping sensorineural hearing loss .A comprehensive audiometric evaluation was performed for each subject, both patients and the normal hearing individuals (NH) controls. The average pure tone audiometry for the whole group of patients for frequency ranges from 500 Hz to 2000 Hz (PTA) was 56.3 ± 24.3 dB for the left ear and 59.8 ± 24.4 dB for the right ear  = 2000 [ms]; time of echo (TE) = 30 [ms]; flip angle (FA) = 90°; voxel size = 3 × 3 × 3 mm; imaging matrix = 64 × 64; no of slices = 41; time of acquisition (TA) = 8.08 min; 250 data points. Participants were instructed to relax during scanning with their eyes closed and not to think of anything in particular. A structural T1 MR sequence had the following parameters: TR = 1900 [ms]; TE = 2.26 [ms]; time of inversion (TI) = 900 [ms]; flip angle (FA) = 9°; field of view (FOV) = 28.8 × 27.0 [cm]; matrix = 320 × 300; voxel size = 0.9 × 0.9 × 0.9 [mm]; pixel bandwidth = 200 Hz/pix; no of slices = 208; TA = 5:11 min.1. Preprocessing of the functional data included slice timing correction, motion correction, scrubbing, linear detrending, band-pass filtering (0.01 Hz < f < 0.1 Hz), co-registration to individual T1 structural scans, spatial normalization to MNI space, and spatial smoothing (6 mm Gaussian kernel). Each subject’s structural scan was segmented into gray matter, white matter, and cerebrospinal fluid (CSF) tissue classes using the unified segmentation approach implemented in SPM12. In addition, the Artifact Detection Tool2 was used to measure motion artifacts in all individuals. Linear regression of confounding effects was applied including: mean signal from white matter and CSF, motion parameters obtained in the realignment step, volumes that showed movement exceeding 0.5mm from the scrubbing step  and 10 first scans (effect of rest). It was found that the mean frame-wise displacement (±SD) was: 0.112 ± 0.038 mm in the NH and 0.128 ± 0.056 mm in patients with SNHL . Seven individual volumes required scrubbing (both groups together).A standard preprocessing pipeline was applied in CONN (Connectivity Toolbox) which uses functions from Statistical Parametric Mapping 12 (SPM12) software3. The first-level whole-brain seed-driven FC maps were obtained by estimating temporal correlations of BOLD signal in seeds corresponding to nodes of canonical RSN. In addition, we used nodes corresponding to the Heschl gyri obtained from the new default atlas (132 ROIs) combining the FSL Harvard-Oxford atlas cortical and subcortical areas and the AAL atlas cerebellar areas. The analysis was performed separately for each node . Since the hearing loss severity and speech understanding scores were comparable for both ears in all participants, averaged binaural PTA and WRS were used for the correlation analysis.For the patient group, to measure bivariate correlations between the connectivity strength and patients’ audiological scores, including WRS and PTA, seed-to-voxel correlation coefficients (Pearson’s R correlation coefficients) were computed and converted to normally distributed p < 0.001 uncorrected and then the cluster-size FWE-corrected level of p < 0.05 and with the Holm–Bonferroni method at p < 0.05 to account for the effect of comparisons between multiple networks and regions  for each RS network and to examine connectivity differences between groups, i.e., between the normal hearing subjects and patients, as well as between patient groups with prelingual onset of SNHL and postlingual onset of SNHL. In addition, we performed a comparison analysis between patients with tinnitus and without tinnitus, as well as with and without hearing aids. This was also done due to the fact that the proportion of patients with and without tinnitus/hearing aids was different in the PRE and the POST subgroup. For all these comparisons mean binaural PTA and WRS values were used as covariates. SPMs were generated for each outcome and thresholded at the voxel level at  regions .The outcomes of psychological questionnaires  were analyzed with SPSS ver. 20 software.anxiety as state 32 ± 7.7 and anxiety as trait 32.4 ± 7.4; NH group: anxiety as trait 28.1 ± 5.5 and anxiety as trait 29.5 ± 6.2) (p > 0.05). In both groups the symptoms indicated non-existent or only mild depressive and anxiety symptoms.The normal hearing and the patient group were not different in terms of their scores in BDI  and STAI (Patients: p < 0.05 and Holm–Bonferroni level at p < 0.05 to account for multiple comparisons of FCs). All functional connections that were found correlated with PTA and WRS values at FWEc < 0.05 but failed to exceed the Holm–Bonferroni p < 0.05 threshold are presented in Hearing severity  was found positively correlated with FC between the visual medial seed and bilateral occipital poles, as well as the left frontal eye field and the precuneus. At the same time, negative correlations  between the superior node of the sensorimotor network and the occipital fusiform gyrus, as well as the right superior temporal gyrus of the language network and the DMN prefrontal hub to postcentral gyrus left and precentral gyrus right, respectively.The only statistically significant correlation that was found for binaural WRS values in patients was a positive one between the left Heschl gyrus and bilateral lingual/intracalcarine cortex.p < 0.05 and Holm–Bonferroni corrected at p < 0.05). The reverse comparison showed decreased FC in the normal hearing group between bilateral inferior parietal sulci, and bilateral precentral gyri. With this stringent statistical approach there were no other functional connections that showed significant between-group differences  and those who acquired their HL later in life . We found differences in both directions, but mostly increased functional couplings in the patient group with early auditory deficits. These results are very interesting as they may reflect the neuronal effects of the onset of a partial hearing impairment vs. cross-hemisphere), with the effect present more often in the right brain hemisphere. We suggest in this patient population a mechanism of top-down modulation by the FPN axis. This mechanism serves to obtain optimal integration of inputs, including aspects of speech, coming from multiple sensory modalities.We found in patients with prelingual SNHL increased functional couplings both in long-ranging brain networks, such as the DMN, the FPN and the DAN, as well as in local sensory networks, i.e., the visual areas and the sensorimotor areas. We saw that most of the FC alterations involved ipsilateral connections  and AG  in inferior parietal cortex, and the inferior temporal gyrus (ITG). The ITG is, among other functions, involved in recognizing visual patterns, also those with linguistic features  auditory deprivation enhanced intrinsic FC within the visual network features . These ffeatures . In the We suggest that the demonstrated enhanced collaboration between the multisensory and “unisensory” brain areas in this specific hearing impaired population reflects, (a) processing of vibrotactile and spatial aspects of acoustic signal, such as people’s gestures or music-induced vibration, (b) experience with lip-reading and face feature analysis, (c) enhanced sensory-motor-visual integration . The latIn the PRE patients we also saw bilateral frontal poles having increased FC with dorsolateral prefrontal cortex. The latter is one of the main hubs of the SN, which serves environment monitoring and information integration. Frontal pole, due to its dense structural connectivity with many cortical and subcortical brain regions, has been proposed to participate in the highest level of unification of information coming from all sensory systems, as well as in processes such as attention and memory, decision making, novelty detection and multiple-task coordination . We alsoWe suggest the following underpinnings of all the described effects; In patients with a prelingual onset of SNHL, the incoming auditory input, including speech, is degraded from the very early stages of life and thus requires increased effort and attention to understand . At the In addition, the cerebellum was more intrinsically interconnected in the PRE patient population. The cerebellum is now recognized to play a major role in sensorimotor and cognitive processes, such as phonological fluency, semantic word association and even metalinguistic skills . There eIn sum we suggest that the control mechanisms described above, involving the FPN axis and maybe the cerebellum as well, serve to optimize integration of multisensory  aspects of the incoming stimulation, with the degraded auditory input or regardless of it (we saw no changes in the auditory network).Patients with acquired hearing deficits, in turn, revealed more functional couplings between various visual subregions and the frontal node of the DAN  – the frontal eye field, as well as in the SN . In this subpopulation the effects were relatively balanced in terms of the left and right hemisphere as well as ipsi- vs. contra-lateral changes in FC. These outcomes suggest that some specific functional changes in neuronal processing might accompany postlingual partial hearing deficits, which are slightly distinct to those that might characterize patients with early hearing loss onset.The role of the frontal eye field (FEF), which is on the border with the motor-related precentral gyrus, is control of visual attention and eye movement. Interestingly, it has been shown that FEF is densely and reciprocally structurally connected with the visual cortex . WhetherBesides the enhanced FEF-visual connections we also saw increased functional couplings between lateral parts of the visual network and insula, in patients with postlingual onset of SNHL. The lateral occipital cortex is a mid-level visual cortex area subserving object recognition. Interestingly, a recent study showed increased engagement of LOC in a visual verbal rhyming task in cochlear implant candidates with postlingual profound deafness . In addiThe tested postlingual subpopulation of patients also demonstrated increased FC between another Silence Network region, the multisensory and attention-related SMG, with the hippocampus. As cortico-hippocampal interaction was demonstrated to be implicated in successful memory formation  this finIn sum, we can speculate on the following compensatory mechanism occurring in late onset SNHL. The Silence Network (including the insula and the SMG) that was found to be more strongly internally coupled in patients with postlingual SNHL, integrates multiple internal and external stimuli . An audiIn the whole patient population, after we applied a very stringent statistical threshold, we found that increased severity of hearing loss correlated with increased FC strength between the left medial visual cortex and the left occipital pole. Both these regions can be considered the early visual cortex. We therefore hypothesize that subjects with more severe deafness (higher PTA) have to rely on visual information more when recognizing speech signal, as for example during lip-reading, recognizing facial expressions or gestures. Coordinates of regions that showed increased FC were almost the same as those found more connected in the patient subgroup with prelingual SNHL . This might suggest that although the PTA values in both patient populations were not statistically different, the fact that the PTA was slightly lower in the early-onset group (66 ± 21 dB in PRE vs. 58 ± 18 in POST) might have had some effect on RSN.N = 20), further investigation is essential.Another connection that was positively correlated with PTA was between FEF and precuneus, which might again imply an increased reliance on/attention to the incoming visual input in patients with partial hearing loss. At the same time, with better hearing  FC was enhanced between sensorimotor brain areas  and multisensory brain regions, including STG, PFC and higher visual cortex. This again suggests that collaboration of senses might serve as compensation for the degradation of the incoming auditory input. In addition, we saw that with increasing speech recognition scores FC between the early auditory cortex (HG) and the early visual cortex  was also enhanced. This finding of enhanced connections between early sensory cortices is indeed intriguing, as we also found that this coupling was stronger in the normal hearing population as compared to the patient group as a whole (see below). For more in-depth interpretation of these finding, due to the relatively small sample size of patients . We did not hypothesize that we would see any effects in the auditory system of the tested patients as they never experienced a total auditory deprivation. experienced joint exposure. However, we speculate that in case of normal hearing what we see is the effect of the very often of early sensory cortices to auditory and visual input, both not degraded. This experience might enhance functional coupling between early sensory brain regions. In the case of patients, especially those with early developed auditory deficits, such an experience is never the case. This does not exclude, however, enhanced collaboration between higher sensory and association regions in the hearing impaired. In fact, with only partial hearing deficit the early auditory cortex is never fully deprived of auditory input.In the current study it was especially important for us to take a whole-brain approach to the analysis of RSN. At the same time, in all but few recent studies involving patients with hearing deficits , the RSNN = 9) also suffered from tinnitus. We therefore cannot exclude the possibility that the presented outcomes are related to the presence of tinnitus. Indeed, we did find FC differences between patients with and without the experience of tinnitus. None of these differences overlapped with those found for comparisons between patients with early and late onset of SNHL. Studying the effect of tinnitus was not the aim of the current experiment and so we did not collect any further measures of tinnitus in individual subjects, such as tinnitus laterality, duration, severity and bothersomeness. Meanwhile, increasingly more rs-fMRI studies indicate that these parameters significantly change the way tinnitus affects brain FC (We acknowledge that an estimated 30–40% of patients with SNHL also have tinnitus which can affect FC, as it was demonstrated in a number of studies . Some stbrain FC . Such anFinally, the relatively small sample size in the current experiment should be mentioned as a study limitation. It is definitely the case that individual variability of FC can be high and thus can significantly influence group results if the studied groups is small. In addition, half of the study participants wore one or two hearing aids, with the other half not using any kind of amplification (which can affect FC, as shown in Taken together, the results of our study indicate that both a general loss of function and compensatory plasticity likely coexist between various networks in the auditory-deprived brain. Whether or not sensory deprivation caused by SNHL might be solely attributed to disruptions in connectivity patterns is, nevertheless, yet to be determined. However, earlier partial hearing deficits definitely seem to induce increase in FC particularly, such as by the additional involvement of subregions of the visual and sensorimotor systems. In sum we propose a mechanism of top-down modulation of perceptual processing by the frontal, parietal and cerebral brain regions that supports optimal integration of incoming multisensory information. Nevertheless, further rs-MRI studies are needed to address the effects of partial hearing loss of varying severity and age at onset, on intrinsic functional networks in the brain.The datasets generated for this study are available on request to the corresponding author.This study was approved by the Bioethical Committee of the Institute of Physiology and Pathology of Hearing.TW designed the fMRI paradigm, analyzed the fMRI data, and prepared the manuscript. KC designed the study, performed the fMRI study and psychological assessments, and prepared the manuscript. AP analyzed the fMRI data and prepared the manuscript. EW recruited the patients and performed the medical examinations. BB and HS consulted the study design and preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This single-center follow-up reveals the medical and technical reliability of a transcutaneous bone-conduction implant for hearing rehabilitation because complication and revision rates were low. The majority of patients still used the device at the end of the observation period. Implantation resulted in favorable hearing outcomes in comparison to that of unaided conditions. Cautious patient selection mainly regarding co-morbidities, the history of chronic otologic diseases and proper surgical technique seems to be crucial in reducing complications.Bone-conduction implants are a standard therapeutic option for patients with conductive, unilateral, or mixed hearing loss who either do not tolerate conventional hearing aids or can benefit from surgery. The aim of this study was to evaluate long-term medical and technical outcomes, and audiological results with the Bonebridge transcutaneous bone-conduction implant. This retrospective study included all patients implanted with a bone-conduction hearing implant at a tertiary medical referral center between March 2012 and October 2018. Medical and technical outcomes included the mean length of implant usage, medical and technical complications , explantations and revisions, coupling approaches, implant failure rate, implant survival and the implant loss for added follow-up years. Auditory results were measured by functional hearing gain and the Freiburger monosyllabic test at 65 dB sound pressure level. Sixty-four patients were included in the study; five of these were implanted bilaterally (69 devices). Five unilaterally implanted patients were lost to follow-up. The mean follow-up was 27.1 months (range: 0.2 months–6.3 years). The mean implant usage was 25.9 months (range: 0.2 months–6.3 years). Fifty-seven implants (89.1%) were in use at the end of the follow-up period. Complications occurred in six ears (9.4%). Five implants (7.8%) were explanted without reimplantation. Device failure occurred in one implant (1.6%), which was possibly caused by recurrent head trauma. The rate of implant loss due to technical device failure (damage to device) was 1 per 72 follow-up years. The mean improvement on the Freiburger monosyllabic test (52.1%,  Bone-conduction implants (BCI) are a standard therapeutic option for patients with conductive, unilateral, or mixed hearing loss who either do not tolerate conventional hearing aids or can benefit from surgery. Over the last few years, implants have also been extended to single-sided deafness (SSD), by routing the signal through the skull from the contralateral deaf to the normal-hearing ear .The Bonebridge (Bb)  active transcutaneous BCI was developed to circumvent typical postoperative complications found with percutaneous systems, such as high infection rates, fixture losses, the need for revision surgery and skin dampening effects ,3,4.As noted by Sprinzl et al. , the Bb Various authors have assessed hearing rehabilitation outcomes with the Bb. However, these studies mostly reported on audiological outcomes in a small number of patients. The largest cohorts included 38 , 28 7]  7] and 2The aim of the current study was to analyze all consecutive patients implanted with the Bb device in a tertiary referral medical center over a 6-year follow-up period. Long-term medical and technical outcomes including the mean length of implant usage (in months), medical and technical complications (including skin and wound infections), lack of benefit and technical failure were analyzed. Furthermore, revisions, reimplantations, explantations, implant survival and implant loss for added up follow-up years were assessed. Postoperative audiological benefit was also evaluated.This was a retrospective chart analysis of all patients implanted with the Bb at the Department of Otorhinolaryngology, Head and Neck Surgery at the Medical University of Vienna, between March 2012 and October 2018. Some patients that had been implanted with the Bb in our department and initially reported on in previous studies . Data on long-term medical and technical outcomes, including the mean length of implant usage (in months), medical and technical complications , revisions, reimplantations, explantations, implant survival and implant loss for added up follow-up years were retrieved using patient medical charts.Audiometric tests were performed in a sound-proofed room with routinely calibrated audiometers used in the clinical routine. Pure-tone audiograms and speech tests were performed prior to surgery and with the same settings postoperatively. The functional hearing gain (FHG) was calculated by comparing unaided free-field thresholds to thresholds with the Bonebridge activated. The contralateral ear was covered in all cases using earmuffs . Audiological testing was performed preoperatively, followed by postoperative measurements taken on a monthly basis for the first 6 months. For our analysis, the last available measurement was used. Testing included the sound-field (SF) pure-tone PTA4 thresholds . Functional gain (FG) was calculated as a difference between unaided postoperative and Bb-aided PTA4 thresholds for patients with atresia and combined hearing loss. Due to inadequacy, FG was not calculated for patients with SSD. One expert noted that traditional FG measurement was not applicable in patients with combined hearing loss implanted with bone-conduction implants and recommended calculating the effective gain in these cases . By defiThe statistical analysis was performed with the Statistical Program of Social Sciences . The statistical significance (Alpha) was set at 0.05, two-tailed. Comparison of complication rates between the two FMT coupling groups was performed using the Chi-Square test. In order to compare unaided and b-aided SF PTA4 thresholds and word recognition scores, a paired t-test was utilized. Distribution was tested by the Mann–Whitney U Test and the Shapiro–Wilk Test. The Kaplan–Meier survival curve was used to analyze implant survival. Descriptive analysis was performed to determine the mean and standard deviation (SD).The approval for this study was obtained from the ethics committee of the Medical University of Vienna .n = 69 Bb devices). Patient demographics including types of hearing loss are presented in A total of 64 patients were implanted with the Bb; five of these patients were implanted bilaterally , with an FG of 26.5 dB SPL (± 3.1 dB SD) in patients with atresia and combined hearing loss (see p = 0.0001 each). The effective gain in patients with combined hearing loss was −11.0 dB.The average SF PTA4 threshold improved significantly from 65.4 to 38.9 dB (loss see . Frequenp = 0.0001), from 13.3 % to 65.4 % (an improvement of 52.1 %). This rate of improvement was significant (p = 0.0001) for all three types of hearing loss . In all of these cases, an infection of the radical cavity resulted in skin dehiscence over the implant. Therefore, the implants had to be removed in all three patients. One patient suffered from comorbidities such as diabetes and obesity. Another patient who had a history of five recurrent cholesteatomas initially had a wound dehiscence and then complained about persisting pain over the implant which was caused by a radical cavity infection, resulting in explantation. Wound infection and chronic inflammation of the subcutis led to explantation in one patient who also had a past medical history of a modified radical cavity. Complications occurred statistically significantly more often in patients where the Bb was placed in a radical cavity  at 1 kHz, with a threshold of 50 dB SPL BC. This patient had to be explanted due to a complete lack of benefit. So far, only a few studies with a relatively small number of patients have assessed the surgical and technical outcomes with the Bb. One short-term analysis of six patients during a 6-month observation period reported no major complications . BaumgarTypically, percutaneous bone-anchored hearing aids have shown high rates of postoperative complications. One study on 41 BAHA (percutaneous bone-conduction implant) patients reported a skin complication rate of 29% . AnotherThe implant loss for added up follow-up years is an outcome parameter which indicates the number of devices lost due to technical complications ,20. GiveThe mean overall FG in patients with atresia and combined hearing loss was 26.5 dB, which is slightly lower than results found in the literature . Our results correspond to those reported by other authors [The mean overall preoperative and postoperative word recognition score on the Freiburger monosyllabic test for all patients at 65 dB SPL was 13.3% versus 65.4%, with a statistically significant mean improvement of 52.1% ( authors ,22,23.The present data confirm that the Bonebridge provides safe and effective long-term hearing rehabilitation. In conclusion, the Bb has proven to be a safe system as shown by very moderate complication and revision rates. The device failure rate was very low and caused by external trauma. The efficacy of the implant was confirmed by the audiological evaluation. Meticulous patient selection, mainly with regard to co-morbidities and proper surgical technique, seems to be a crucial factor in reducing complications. Furthermore, individual anatomy, especially in children, and a history of chronic otologic disease should be taken into account when planning a Bb implantation for hearing rehabilitation.