Bartonella spp. are zoonotic bacteria with small mammals as main reservoirs. Bartonella spp. prevalence in small mammals from Myanmar and Sri Lanka are yet unknown. (2) Methods: Small mammals were snap trapped in Sri Lanka and Myanmar in urban surroundings. Spleens-derived DNA was screened for Bartonella spp. using conventional PCR based on three target genes. Positive samples were sequenced. (3) Results: 994 small mammals were collected comprising 6 species: Bandicota bengalensis, Bandicota indica, Rattus exulans, Rattus rattus, Mus booduga, and Suncus murinus. In Myanmar, the Bartonella prevalence in Bandicoot rats (68.47%) was higher than in Rattus rattus (41.67%), Rattus exulans (21.33%), and Suncus murinus (3.64%). Furthermore the prevalence in Myanmar  was twice as high as in Sri Lanka . In Sri Lanka, Bartonella spp. occurred almost exclusively in R. rattus. In Myanmar, Bartonella kosoyi was mainly detected (56%), followed by Bartonella sp. KM2529 (15%), Bartonella sp. SE-Bart D (12%) and Bartonella henselae (1%). In Sri Lanka, B. phoceensis (60%) and Bartonella sp. KM2581 (33%) were predominant. (4) Conclusions: Bartonella spp. were detected in all investigated small mammal species from Myanmar and Sri Lanka for the first time. Bartonella kosoyi and B. henselae are zoonotic. As these small mammals originated from urban settlements, human bartonellosis seems likely to occur.(1) Background:  Bartonella spp. are vector-borne and commonly transmitted via blood-sucking arthropods. Rodents are known to be the main reservoir of different Bartonella spp. with fleas the main vector within and between rodent populations [Bartonella elizabethae, Bartonella grahamii, B. henselae, Bartonella tribocorum, and Bartonella vinsonii subsp. arupensis are described in association with rodent reservoirs [The genus Bartonella includes over 40 species and subspecies  of gram-ulations ,3. Amongservoirs ,4,5.Bartonella spp. infections are associated with various human diseases such as cat-scratch disease (Bartonella henselae), trench fever (Bartonella quintana), and Oroya fever (Bartonella bacilliformis). Cases of bartonellosis associated with rodents are described from all over the world with clinical symptoms such as fever, muscle and joint pain, neuroretinitis, and endocarditis [B. henselae in human patients from Laos and Taiwan [Bartonella tamiae and other Bartonella spp. were found in febrile patients from Thailand [carditis ,7. Regard Taiwan ,9. FurthThailand . Until n2 and population size is 54 million people [2 [Nonetheless, rodent-associated zoonotic Bartonellae can pose a risk in countries such as Myanmar and Sri Lanka where close contact between humans and rodents is common ,11. Myann people  of whichn people ,14. Aboueople [2 . As of 2eople [2 .The genera Bandicota, Rattus and Mus include synanthropic pest rodent species that live in close contact with humans in those countries . In otheBartonella spp. prevalence in fleas associated with cats and rodents in an area along the Thai-Myanmar border. Six out of 90 fleas were tested positive for Bartonella DNA and a newly described Bartonella species was detected from one flea collected from a Rattus surifer [Bartonella spp. infections in dogs with positive individuals showing even multiple infections [In Myanmar, a previous study  detected surifer . In Sri fections .Bartonella spp. to assess the risk for human health and companion animals.To date, no study of Bartonella prevalence in small mammals as possible reservoir hosts has been conducted in Myanmar and Sri Lanka. Because of the close contact between rodents and humans in various parts of these countries, it is important to study rodent-associated Thus, the main aim of this study was to detect small mammal species living close to human settlements in Myanmar and Sri Lanka and to evaluate the Bartonella prevalence in those small mammal species for the first time. Another purpose was to identify the Bartonella species in these samples by molecular techniques to evaluate their zoonotic potential.Small mammals were trapped at five sites in Myanmar and Sri Lanka in the years 2018 and 2019. Traps were set in villages at structures used for rice storage and rice processing in Myanmar and Sri Lanka .In Myanmar, study sites were located in granaries or mills in the rural delta and coastal zone, where the main agricultural production is located. Dayēbo  and Pike Kye We  are rural areas mainly characterized by rice production . At the Small mammal trapping in Sri Lanka was conducted in Pasyala (Gampaha District) i , Pasyala (Gampaha District) ii , Kahapathwala  i  and Kahapathwala  ii  that are characterized by an increase in housing units indicating population growth in these areas. Sinhapura (Polonnaruwa District) i  is characterized by rural agriculture with 39.1% employment in this sector ,33. TherSmall mammals were obtained and killed by snap trapping as part of regular pest rodent control operations. In the countries where the work was conducted, snap trapping is not regarded as animal experimentation. Hence, no animal ethics permits were required. In Myanmar, small mammals were trapped from June to December in 2018 and from May to December in 2019. In each site, 10 snap traps  and 5 local-made bamboo traps were baited with unhulled rice and set opportunistically in and around 10 small-holder rice storage facilities for 450 trap nights per site and year . In Sri Lanka, small mammals were captured in two trapping sessions in June–December 2018 and two trapping sessions in March–August 2019 (for details see ). In eaccytochrome b gene [Bartonella spp.Trapped small mammals were identified to species level based on morphology  supplemee b gene . Standar® 24 tissue homogenizer  was used for mechanical disruption with 5500 rpm for 2 × 15 s with a 10 s break between both runs.Before processing the spleen samples for DNA extraction, the ethanol was washed out with phosphate-buffered-saline  by transferring in PBS and incubating for four hours and drying afterwards. Then, all samples were homogenized with 0.6 g sterile ceramic beads  four times the sample’s weight with PBS (pH 7.2). The PrecellysAfterwards the DNA was extracted using the QIAamp DNA Mini Kit  according to the manufacturer’s instructions. The DNA samples were measured qualitatively and quantitatively with the NanoDrop ND-1000 (PeqLab Biotechnologie GmbH). Samples with a DNA concentration >40 ng/μL were diluted with the kit’s elution buffer to receive a DNA concentration of 20–40 ng/μL for each sample.Bartonella spp. was detected in DNA samples by conventional polymerase chain reaction (PCR) targeting the NADH dehydrogenase subunit (nuoG) with an amplicon size of 346 base pairs (bp). Additionally, all samples were further analyzed in two PCRs targeting the gltA gene (378 bp) and a fragment of the 16S-23S rRNA ITS region (453–780 bp) [The presence of –780 bp) ,37,38,39nuoG was detected with previously published PCR protocols and the genus specific primers nuoGF (5′-GGCGTGATTGTTCTCGTTA-3′) and nuoGR (5′-CACGACCACGGCTATCAAT-3′) [Bartonella spp. DNA based on the gltA gene the genus specific primers BhCS.781p (5′-GGGGACCAGCTCATGGTGG-3′) and BhCS.1137n (5′-AATGCAAAAAGAACAGTAAACA-3′) were used. The amplification consisted of 45 cycles of denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s and elongation at 72 °C for 1 min [The amplicon of the CAAT-3′) . For theor 1 min ,38. The https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 25 February 2021)) [PCR products were prepared with DNA Gel Loading Dye (6×)  for gel electrophoresis in 2% agarose. The results were visualized by UV light using the UVP GelSolo streamlined gel documentation . Afterwards 125 positive samples were selected for sequencing to cover all small mammal species detected at each location in Myanmar and Sri Lanka in both seasons of each collection year. The samples were purified for sequencing using the NucleoSpin Gel and PCR clean-up kit  as recommended by the manufacturer. Further, samples were sequenced commercially with forward and reverse primers of the 16S-23S rRNA ITS region . The sequences were trimmed using Bionumerics v.7.6.1.  and compared to available data in GenBank with BLASTn (y 2021)) . ObtaineBartonella spp. in small mammals were determined by the Clopper and Pearson method [Confidence intervals (95% CI) for the prevalence of n method  with setP (probability) < 0.05. Only samples tested positive for all three target genes were considered positive for Bartonella spp.The independence of compared prevalence rates concerning small mammal species, age, sex, trapping year, season, and in Myanmar additionally regarding the habitat, was tested using Pearson’s Chi-squared test. Tests with two variables were considered to be significant if Bandicota bengalensis ; B. indica ; Rattus exulans ; R. rattus ; Suncus murinus   and 166 at granaries . In Sri Lanka, 499 small mammals were captured representing six species: B. bengalensis (n2019 = 1); B. indica ; Mus booduga (n2018 = 4); R. exulans ; R. rattus ; S. murinus   . Of thes19 = 10) . All smaBartonella spp. DNA by conventional PCR targeting all three target genes altogether .Overall 248 out of 994 samples  tested positive for together . Altoget2 = 41.273, df = 1, P < 0.0001). In contrast, Bartonella prevalence in Sri Lanka was similar between years (10–28%) and sites (10–28%)  .Bandicota than in Rattus spp. and S. murinus . The prevalence of Bartonella in the genus Rattus was significantly higher compared to S. murinus  in Myanmar. Likewise, in Sri Lanka the prevalence in R. rattus was higher than in all other small mammal species which were caught by lower numbers .In Myanmar, prevalence was higher in Bartonella spp. in Myanmar was detected between males and females , between the years 2018 and 2019  and the rainy and dry season . In Myanmar, the prevalence of Bartonella in small mammals caught in granaries was significantly higher than in small mammals caught in mills . However, due to the unequal distribution of small mammal species in granaries and mills, these results may be regarded as slightly distorting. The prevalence of adult small mammals was significantly higher compared to the lower prevalence of sub-adult small mammals . There was no significant difference in Sri Lanka for the Bartonella prevalence between adults and sub-adults , males and females , the years 2018 and 2019 , and the seasons  [No significant difference in the prevalence of  0.9443)   and R. rattus  and at the sites Dayēbo  and Kadoke Phayargyi . In Sri Lanka, 11 out of 80 positively tested samples  showed double infections. Here, co-infections were detected only in one rodent species, R. rattus, caught at the sites Pasyala and Kahapathwala.Several Bartonella co-infections were detected in small mammals from Myanmar and Sri Lanka. In Myanmar 30 out of 168 positively tested small mammals  with double infections were detected by PCR targeting the ITS region. Double infections in Myanmar were most common in Bartonella-positive samples based on the ITS region (53%) and 11 different Bartonella strains were detected . Bartonella sp. KM2529  and Bartonella sp. SE-Bart-D  were detected in small mammals from Myanmar as well. The most frequently identified Bartonella species in Sri Lanka was B. phoceensis  followed by Bartonella spp. KM2581 . The detected Bartonella spp. were evenly distributed and no effect of small mammal species, age, season and habitat was apparent. However, B. kosoyi. was exclusively detected in B. indica from Myanmar.Sequence analyses were performed for 125 detected . In MyanBartonella spp., (Accession Number: EF407566) and (Acc. No.: EF213776) were detected in one R. exulans caught in Kan Nyi Naung, Myanmar. Two R. norvegicus from China were earlier positive for Bartonella sp. RN25BJ, respectively, Bartonella sp. RN28BJ. Furthermore, 10 samples showed <97% identity with sequences in GenBank, but 98–100% within group-similarity and can be assembled in groups with >98% agreement  in Sri Lanka which are most similar to Bartonella sp. SE-Bart-D. Three samples from S. murinus caught in 2019 at two different sites in Myanmar form Group 3 and show high similarity to an uncultured Bartonella clone 5199  are in agreement with the previously mentioned studies [Especially in Southeast Asia rodent-associated infectious diseases are emerging . The Baral hosts . Severalal hosts ), Laos , China al hosts ,21, and al hosts ). The Ba studies .R. rattus was introduced via ship trading to this island [Xenopsylla cheopis) [Bartonella spp. prevalence in rodents at different islands. An explanation for this distribution was the varying ecological condition affecting the flea populations as essential vectors for the infection of rodents [Bartonella spp.; however, the interaction between host and vector has not been clarified, yet [The prevalence of Bartonella in small mammals from Sri Lanka was significantly lower than in Myanmar. In Sri Lanka, the cosmopolitan species s island . A possicheopis) . Moreove rodents . Variousied, yet . The misied, yet .Bartonella spp. prevalence among small mammal species in Myanmar. Bandicoot rats had the highest (63%) and S. murinus the lowest prevalence (4%). In a recent study in Nepal, a low prevalence of Bartonella spp. was detected in B. bengalensis (26.3%) and high prevalence in S. murinus (64.1%) while R. rattus (43.3%) was intermediate. In Bangladesh both B. bengalensis (63.2%) and S. murinus (42.9%) showed higher prevalence than R. rattus [S. murinus might be due to a lack of host-specific vectors in Sri Lanka in our study. However, information about the ectoparasite fauna of S. murinus is lacking. Additionally, low Bartonella prevalence was detected in other insectivores (Sorex araneus) in England [S. araneus showed that some blood-sucking arthropods harboring rare Bartonella spp. may feed and transmit these Bartonella spp. exclusively to shrews. Furthermore, S. araneus was found to be less affected by ectoparasites compared to endemic rodents [S. murinus may be related to an adapted ectoparasite fauna in Bangladesh but not in Myanmar or Sri Lanka. In Sri Lanka, the predominant rodent species R. rattus showed the highest prevalence (18%)—apart from the single B. bengalensis, which also tested positive. The prevalence for R. rattus and the high prevalence of B. bengalensis in Myanmar are in line with R. rattus in Asia .Bartonella spp. in small mammals from urban regions [Bartonella spp., which can infect several mammals [Bartonella spp. in Sri Lanka was detected concerning the different sites. All small mammals were caught in small-holder rice storage facilities in Sri Lanka and thus conditions were not as contrasting as at the sites in Myanmar.In Myanmar, the prevalence was higher in urban sites (Pike Kye We and Dayēbo) compared to the three rural sites. In line with our study, other authors reported higher prevalence of  regions ,59,60. ABartonella spp. These bacteria can persist in the reservoir host without harming them drastically [Sigmodon hispidus). The decrease of prevalence may be due to the development of immunity with age [Bartonella spp. was detected in B. bengalensis and B. indica and in Sri Lanka especially in R. rattus. Paziewska et al. [Myodes glareolus overcomes Bartonella infections within 1–2 months, however the length of time it takes to overcome the infection in Apodemus flavicollis is not definitely known. Furthermore, reinfections in M. glareolus were detected less frequently than in A. flavicollis [Bandicota spp. (more often collected in Myanmar) longer than in Rattus spp. (more often collected in Sri Lanka), which would explain the contrasting results in both countries.In Myanmar, the prevalence in adult small mammals showed significantly higher prevalence than in subadults, possibly due to the life cycle of stically . In contstically  reportedwith age . No diffa et al.  showed tvicollis . Thus, BIn the present study, there was no effect of the seasons. Morway et al. showed that Bartonella prevalence rates were influenced by climatic conditions . MoreoveBartonella spp. strains for a long time and that single strains replace each other. This infection strategy may have an impact on the probability of double infections in small mammals [Bartonella spp. infections with more than one species and related Bartonella strains [B. bengalensis, R. rattus, and R. exulans in Myanmar and exclusively in R. rattus in Sri Lanka at all sites. High co-infection rates (56%) with two or more genotypes, including recombinant variants, were detected in gerbils and fleas from Israel [Bartonella spp. than their hosts [Kosoy et al.  showed t mammals . However strains . This dim Israel . In Taiwir hosts . In Soutir hosts ,71,72.B. kosoyi, first described in 2019, was the predominant species for the samples from Myanmar. Analysis confirmed a phylogenetic association of B. kosoyi with the zoonotic B. grahamii, B. tribocorum, and B. elizabethae [Bartonella elizabethae and B. elizabethae-like species show host-specificity for rodents of the genus Rattus but can infect other small mammals like Bandicoot rats and S. murinus as well [B. kosoyi mainly in Bandicoot rats . Before the Tel Aviv strain was named B. kosoyi sp. nov., this strain had been detected in Bandicoot rats and R. rattus from Bangladesh [B. kosoyi.Our sequence analysis of Bartonella-positive samples identified 11 different Bartonella strains. Moreover,  as well . We detengladesh  and Nepangladesh . Furtherngladesh . TherefoBartonella sp. SE-Bart-D isolated from the Oriental rat flea (X. cheopis) in Egypt. Bartonella sp. SE-Bart-D shared 85–90% similarity with B. tribocorum, B. grahamii, and B. elizabethae, which are all known as zoonotic agents [Some of the sequencing results from Myanmar were highly similar (99.81–100%) to c agents .Bartonella phoceensis was the predominant result for all sequenced samples. Bartonella phoceensis was first detected along with Bartonella rattimassiliensis in France in 2004 [B. phoceensis was detected in small mammals from Asia. In Japan and Thailand B. phoceensis was detected in R. rattus [S. murinus and Rattus tanezumi [B. phoceensis was associated, with a similarity of 94%, with uncultured Bartonella species found in Rattus norvegicus from Taiwan [B. phoceensis is not recognized as a zoonotic agent and no case of human illness has been described.In Sri Lanka,  in 2004 . Since t. rattus ,77 and itanezumi . Moreovem Taiwan . Until nBartonella henselae was detected in one S. murinus from Myanmar in the current study. The organism with the highest similarity B. henselae Q5BJ-CW was first isolated from a dog in China. Bartonella henselae is known to be mostly transmitted by wild and domestic cats to humans [B. henselae, is associated with fever and regional lymphadenopathy [B. henselae in Italy and New Zealand. Therefore, rodents can be a source of infection due to close contact to humans [Bartonella spp. can be an underestimated threat to public health. Although the number of zoonotic Bartonella spp. has emerged, bartonellosis in humans is reported very rarely [o humans . Cat screnopathy . Though o humans ,80. Due y rarely .Bartonella spp. were detected for the first time in small mammals from Myanmar (34%) and Sri Lanka (16%) in this study. The high prevalence is in line with previously reported results from other countries in Southeast Asia. The reasons for the lower prevalence in Sri Lanka may be missing vectors or different ecological conditions. All examined small mammal species were Bartonella-positive in Myanmar and Sri Lanka. Eleven different Bartonella strains were detected of which three are considered potentially zoonotic. Thus, small mammals may serve as reservoir hosts for zoonotic Bartonella spp. in these countries. In Myanmar and Sri Lanka, contact between humans and rodents is frequent in rice storage and processing facilities. With an increasing degree of urbanization and thus more frequent contact between humans and rodents, these rodent-associated pathogens may pose a higher risk for human health.


Methylmalonic acidemia is an organic acid metabolism disorder that usually has nonspecific clinical manifestations.A 3-month-old female infant was admitted to the hospital for developmental retardation. Her prenatal and birth history was unremarkable. After admission, she developed dyspnea and severe anemia and was subsequently transferred to the intensive care unit. Magnetic resonance imaging of her brain showed a Dandy-Walker malformation, and metabolic screening indicated methylmalonic acidemia. Thus, she was diagnosed with methylmalonic acidemia and Dandy-Walker malformation. The patient underwent treatment including acidosis correction, blood transfusion, antibiotics, mechanical ventilation and heat preservation. Unfortunately, her condition progressively worsened and she died of metabolic crisis.Dandy-Walker malformation may be a clinical manifestation of methylmalonic acidemia. Additionally, the co-existence of methylmalonic acidemia and Dandy-Walker malformation may be an uncharacterized syndrome which needs to be studied further. Methylmalonic acidemia is one of the most common organic acid metabolism disorders. Deficiency in the enzyme methylmalonyl-CoA mutase or adenosyl-cobalamin synthesis results in inborn errors of metabolism. The clinical manifestations of patients with methylmalonic acidemia are usually nonspecific. Patients with severe methylmalonic acidemia are prone to lethargy, vomiting, respiratory distress, hypothermia, severe ketoacidosis, and hyperammonemia . This co12/L, and a hemoglobin level of 33 g/L. After a blood transfusion, her hemoglobin gradually increased to a normal level. Blood gas analysis revealed an elevated lactic acid level of 14.62 mmol/L, a decreased pH of 6.84, partial pressure of carbon dioxide of 74.70 mmHg, partial pressure of oxygen of 21.50 mmHg, and potassium level of 6.3 mmol/L. Her C-reactive protein level was 18.40 mg/L. A chest radiograph showed a patchy dense shadow in both lungs. She was also positive for cytomegalovirus immunoglobulin M antibody. Magnetic resonance imaging of her brain showed cerebellar vermis hypoplasia, enlargement of the fourth ventricle, and supratentorial hydrocephalus (Fig. A 3-month-old female infant was admitted to the hospital for developmental retardation. Her prenatal and birth history was unremarkable. She grew slowly and had a below average height and weight. She could not laugh and raise her head. After admission, she developed dyspnea and severe anemia and was subsequently transferred to the intensive care unit. Auscultation of both lungs indicated some rales. Her lowest body temperature was 34.7℃, and she developed an irregular respiratory rhythm. Since her condition was progressively worsening, she was treated with invasive mechanical ventilation. Routine blood examination showed normal leukocyte and platelet counts, an erythrocyte count of 1.04 × 10Here, we report a case of methylmalonic acidemia in a patient with a Dandy-Walker malformation. To the best of our knowledge, this brain imaging finding is very rare in patients with methylmalonic acidemia. Most cases of Dandy-Walker malformations are reported in infants below the age of 1 year , althougMethylmalonic acidemia is a severe and even life-threatening metabolic disease. Methylmalonic acid is produced during the metabolism of some odd-chain fatty acids and amino acids. Methylmalonic acidemia is caused by defects in methylmalonyl-CoA mutase or its coenzyme . The incOur patient was positive for cytomegalovirus immunoglobulin M antibody. We are unsure whether the cytomegalovirus infection was congenital due to the lack of cytomegalovirus testing during the neonatal period. The patient had never received anti-cytomegalovirus treatment. Congenital cytomegalovirus infection usually leads to neurologic conditions in children . PrenataOur case highlights a rare association between methylmalonic acidemia and Dandy-Walker malformation. The co-existence of Dandy-Walker malformation and methylmalonic acidemia may be a yet uncharacterized syndrome. It is possible that Dandy-Walker malformation is just a special manifestation of methylmalonic acidemia or alternately was caused by a congenital infection in our patient with methylmalonic acidemia. Regardless, this association merits further study.


Chlamydia spp. was assessed in diseased and healthy eyes of domestic sheep and wild ruminants that share mountain habitats in northern Spain. The presence of Chlamydia spp. was tested by real-time PCR in 1786 conjunctival swabs collected from both eyes of 893 animals from mountain habitats in northern Spain, and chlamydial species were identified in the positive samples by ArrayTube microarray methods. Chlamydial DNA was detected in 0.6% (CI95% 0.2–1.3) of the Pyrenean chamois (Rupicapra pyrenaica) and 1.4% (CI95% <0.01–8.1) of the sheep (Ovis aries) sampled, with Chlamydia pecorum the only chlamydial species identified. No association of C. pecorum with ocular disease or co-infection with Mycoplasma conjunctivae was found. Further studies on the pathogenesis of infectious keratoconjunctivitis are needed to better understand the ecology of C. pecorum and its possible role as a ruminant pathogen at the wildlife–livestock interface.Infections by Chlamydiae are associated with ocular disease in humans and animals. In this study, the presence and diversity of  Chlamydiaceae family consists of the single genus Chlamydia, which comprises 13 species: Chlamydia (C.) trachomatis, C. pneumoniae, C. psittaci, C. abortus, C. felis, C. pecorum, C. suis, C. gallinacea, C. caviae, C. avium, C. serpentis, C. poikilothermis and C. muridarum [Chlamydiaceae can establish prolonged persistent infections, which are typically asymptomatic, but can also cause disease affecting the eyes, the genital tract, the joints or the respiratory tract, and occasionally cause systemic disease [C. abortus, C. psittaci, C. pecorum and C. pneumoniae are the species most commonly detected in association with disease [Chlamydiae are obligate intracellular gram-negative bacteria affecting both humans and animals. According to the current taxonomy, the  disease . Chlamyd disease .Chlamydia pecorum is one of the most common species detected in domestic ruminants, associated with reduced weight gain, milk production and conception rates, and therefore having an economic impact on livestock production [C. pecorum infection in ruminants include conjunctivitis, infectious arthritis and spontaneous abortion, as well as encephalomyelitis in cattle [Chlamydia pecorum has also been detected in wildlife, such as deer [Phascolarctos cinereus) [Chlamydia pecorum is a major pathogen in koalas [C. pecorum in wild hosts is mostly unknown.oduction ,6. Clinin cattle ,8. Chlam as deer ,10, ungu as deer ,12, koalinereus)  and birdinereus) . Althougn koalas , the patChlamydia spp. have occasionally been reported as the main cause of infectious keratoconjunctivitis (IKC) outbreak in bighorn sheep (Ovis canadensis) [C. pecorum has been involved in an outbreak of ocular disease in reindeer [Mycoplasma conjunctivae [Chlamydia spp. has pathogenic synergism in co-infection with M. conjunctivae or can cause sporadic ocular disease in wild ruminants. Ocular co-occurrence of Chlamydia spp. and M. conjunctivae has been reported in chamois, sheep and goats [adensis) , and C. reindeer . The cliunctivae , yet it nd goats ,18, and nd goats .Chlamydiaceae in the eyes of wild and domestic ungulates from shared mountain habitats in northern Spain, and to explore the association of Chlamydia spp. occurrence with ocular clinical signs, either alone or in co-infection with M. conjunctivae.The aim of this study is to assess the frequency and diversity of Chlamydiaceae was detected in seven swab samples from five of the 893 animals tested . Chlamydiaceae DNA was detected bilaterally in the eyes of two chamois. By species, the overall sample prevalence was 0.6% (CI95%: 0.2–1.5) in chamois and 1.4% (CI95%: <0.01–8.1) in sheep. No statistical association was observed between the detection of Chlamydia spp. and the animal species. Chlamydiaceae were only detected in the eyes of ruminants from the Eastern Pyrenees, but not from the Cantabrian Mountains (Chlamydiaceae was detected were 0.1% (CI95%: <0.01–0.9) in Alt Pallars National Game Reserve (PAP), 0.1% (CI95%: <0.01–0.9) in Cadí National Game Reserve (PCD) and 0.3% (CI95%: <0.01–1.1) in Freser-Setcases National Game Reserve (PFS). In sheep, Chlamydiaceae was only detected in conjunctival swabs from one flock from PAP, with a sample prevalence of 1.4% (CI95%: <0.01–8.1). For both chamois and sheep, the frequency of Chlamydiaceae detection was not statistically different between study areas.ountains . The samC. pecorum was identified by the ArrayMate microarray assay in six of the qPCR-positive samples, including bilateral ocular detections in two chamois and unilateral detections in one chamois and one sheep. The identification of chlamydial species was unsuccessful in the remaining qPCR-positive sample from a chamois. No other Chlamydiaceae were detected in this study.Chlamydiaceae-positive ruminants were also positive for M. conjunctivae or had signs of ocular disease. The detection of M. conjunctivae was correlated with the presence of ocular signs, which has been previously published [Among the sampled ruminants, 63 animals had ocular clinical signs that ranged from mild ocular discharge to corneal perforation . Howeverublished .Chlamydiaceae has been associated with ocular disease in wild and domestic animals, yet there is scarce knowledge about the frequency and diversity of chlamydial infections in the eyes of ruminants, and its association with ocular clinical signs. In this cross-sectional study, we found a low frequency of C. pecorum in asymptomatic domestic sheep and Pyrenean chamois among the ruminant communities from northern Spain. However, Chlamydiaceae may be present in anatomical sites other than the eyes, including the urogenital tract, rectum, joints, brain, lungs or spleen [Chlamydiaceae infections.r spleen ,8,21, whChlamydiaceae in Alpine ibex 1.2% (Capra ibex) and Alpine chamois 2.5% (Rupicapra rupicapra) from the Swiss European Alps [Chlamydiaceae detection [Our results are similar to those reported for the ocular detection of ean Alps . Howeverean Alps ,22. Methetection , furtherChlamydia pecorum, identified in both asymptomatic sheep and chamois in this study, has been previously detected in domestic ruminants presenting syndromes that include ocular disease and polyarthritis [C. pecorum in the eyes of wildlife inhabiting alpine ecosystems without association with clinical signs, including Alpine ibex [C. pecorum suggest that certain strains may be more pathogenic than others [Chlamydia spp. does not seem to play a major role in outbreaks of ocular-exclusive syndromes [Cervidae worldwide [rthritis . Similarine ibex  and Alpiine ibex . Althougn others . Accordin others . Howeveryndromes ,20, but orldwide ,16.Chlamydia spp. to cause ocular disease may also be affected by co-infections with other pathogens. Microbiological cultures from diseased eyes often yield different bacterial isolates, including M. conjunctivae, Moraxella spp., Pseudomonas spp. or Staphylococcus spp. [Chlamydiaceae and M. conjunctivae were detected in this study, other studies reported C. pecorum, C. abortus and C. psittaci co-infection with Mycoplasma species in IKC cases from domestic sheep [Chlamydia spp. and M. conjunctivae ocular co-occurrence in a severe IKC outbreak of Pyrenean chamois [M. conjunctivae alone is a sufficient cause for IKC in Caprinae [Chlamydia spp. in these cases of co-occurrence remains unclear. Experimental or longitudinal studies may provide more meaningful insights into the synergism between Chlamydia spp. and Mycoplasma spp. for the onset and progression of IKC.The potential of cus spp. . Althougic sheep ,27, and ic sheep , suggest chamois . HoweverCaprinae , and theC. pecorum in different domestic and wild hosts from alpine ecosystems suggests that transmission between different animal species may occur. However, previous studies reported a great variability between C. pecorum strains in co-grazing wildlife and livestock [C. pecorum strains circulating in chamois and sheep would help to better understand the possible epidemiological links between sympatric hosts from the Pyrenees.The detection of ivestock , suggestC. pecorum in the eyes of domestic sheep and Pyrenean chamois from the Eastern Pyrenees, with no relation to ocular disease or evidence of co-infection with M. conjunctivae. Chlamydiaceae probably did not participate significantly in the pathogenesis of sporadic ocular disease or in the epidemiology of infectious keratoconjunctivitis in the studied mountain habitats from northern Spain. However, further studies are needed to better understand the ecology and pathogenic potential of chlamydial species in wild ruminant hosts and their interface with livestock.In conclusion, we detected low-frequency This study was performed from 2009 to 2015 in two areas from the Cantabrian Mountains (NW Spain) and six different areas from the Eastern Pyrenees (NE Spain). The study areas in the Cantabrian Mountains were the National Game Reserves of Mampodre  and Riaño . In the Eastern Pyrenees, the study areas were PAP , Boumort National Game Reserve , PCD , Cerdanya-Alt Urgell National Game Reserve , PFS  and Vall d’Aran .(Rupicapra pyrenaica), sheep (Ovis aries), roe deer (Capreolus capreolus), red deer (Cervus elaphus), mouflon (Ovis aries musimon), fallow deer (Dama dama) and Iberian ibex (Capra pyrenaica). In total, 1786 conjunctival swabs were collected from 893 wild and domestic ruminants by introducing sterile dry cotton swabs to each eye separately. The wildlife sampled were hunted animals sampled during the regular hunting season, and included Pyrenean chamois cador Pathogen Kit , following the manufacturer’s instructions.Conjunctival swabs were thawed, cut and mixed during one minute with 0.5 mL of lysis buffer  in sterile tubes. The lysates were obtained by incubating the tubes at 60 °C for 60 min after adding 0.024 mL of proteinase K. Finally, proteinase K was inactivated at 97 °C for 15 min . DNA wasChlamydiaceae DNA was searched with a SYBR green-based qPCR assay using the primers Chuni-1F (5′-GGG CTA GAC ACG TGA AAC CTA-3′) and Chuni-2R (5′-CCA TGC TTC AAC CTG GTC ATA A-3′) and following previously reported cycling conditions. Briefly, each reaction consisted of 2.5 μL of DNA sample, 12.5 μL of SYBRGreen PCR Master Mix 2x , 400 nM of each forward and reverse primer and nuclease-free water to a total volume of 25 μL. C. psittaci DNA was used as positive control [ control .Chlamydiaceae real-time PCR Chlamydiaceae targeting the 23S rRNA gene using the primers Ch23S-F (5′-CTG AAA CCA GTA GCT TAT AAG CGG T-3′), Ch23S-R (5′- ACC TCG CCG TTT AAC TTA ACT CC-3′) and probe Ch23S-p (FAM-CTCATCATGCAAAAGGCACGCCG-TAMRA) [The samples positive for the SYBR green-based PCR were sent to the University of Zurich, Switzerland, for confirmation by a family-specific G-TAMRA) . ChlamydG-TAMRA) .M. conjunctivae in the eyes of the animals included in this study was previously published [The molecular detection of ublished ,34, and Chlamydiaceae detection according to the area and the animal species of study, Pearson’s Chi-squared test (χ2) was implemented using the R statistical software [p < 0.05.To assess the differences over software . Statist


Talaromyces (Penicillium) marneffei. Similar to other major causes of systemic mycoses, the extent of disease and outcomes are the results of complex interactions between this opportunistic human pathogen and a host’s immune response. This review will highlight the current knowledge regarding the dynamic interaction between T. marneffei and mammalian hosts, particularly highlighting important aspects of virulence factors, intracellular lifestyle and the mechanisms of immune defense as well as the strategies of the pathogen for manipulating and evading host immune cells.Talaromycosis (Penicilliosis) is an opportunistic mycosis caused by the thermally dimorphic fungus  Talaromyces species, Talaromyces (Penicillium) marneffei is the only thermally dimorphic species known to be pathogenic to mammals, including humans. T. marneffei is a primary lung pathogen that disseminates to other internal organs by lymphatic or hematogenous mechanisms [T. marneffei grows as a saprophytic mold in the environment, but undergoes phase transition to a pathogenic yeast-like cell at mammalian physiologic temperatures. Dr. Gabriel Segretain is credited with identifying the fungus and he named it in honor of Dr. Hubert Marneffe, the director of the Pasteur Institute of Indochina [Among the hundreds of chanisms . It causndochina . Dr. Segndochina . Seventendochina .T. marneffei infection in an HIV-infected patient of Southeast Asia was reported in 1989 from Bangkok, Thailand, and this coincided with the beginning of the HIV epidemic in the region [T. marneffei was described in a dog in Brazil co-infected with canine distemper virus (CDV), which represents the first description of talaromycosis in a dog and also extends the geographical distribution of T. marneffei outside the known endemic area [The first case reports from an endemic area were published in 1984, describing five patients in northern Thailand . In 1985e region . More remic area .T. marneffei was first isolated from bamboo rats (Rhizomys sinensis) as well as from other species of bamboo rat distributed in an endemic area of T. marneffei. The bamboo rat is recognized as an important natural reservoir host of T. marneffei [T. marneffei results in the establishment of infection in the wild bamboo rat [T. marneffei [In other mammals, arneffei ,9,10. Upmboo rat . Signifiarneffei .Histoplasma, T. marneffei can survive and replicate inside the phagosomal compartment of macrophages [T. marneffei is classified as a facultative intracellular pathogen as it is found inside macrophages and tissue histiocytes in talaromycosis patients [The mechanisms for the pathogenesis of talaromycosis are not definitively established. However, fungal morphogenesis appears to be a crucial virulence factor in the establishment of infection . Evidencrophages ,19,20,21patients ,22.T. marneffei and the host, particularly in the context of virulence factors and mechanisms of host immune defenses.The ability to transition from an environmental mold to a yeast form and resist to killing by host phagocyte is recognized as an important virulence mechanism of dimorphic pathogenic fungi  as the sT. marneffei require to (I) adhere to and colonize host tissue, (II) multiply, (III) escape or destroy host defense system and (IV) damage the host tissue [T. marneffei conidia entry is similar to that resulting in infections caused by another dimorphic fungus [T. marneffei conidia to pneumocytes and human extracellular matrix proteins (ECM) have been reported [T. marneffei conidia adhere to host ECMs, fibronectin and laminin, via an N-acetylneuraminic acid  dependent process [T. marneffei animal models.To establish the infection, pathogenic fungi including t tissue . The resc fungus . The rolreported . Moreove process ,28. Coni process . These ET. marneffei. Conidia are phagocytized by resident alveolar macrophage as well as polymorphonuclear neutrophils (PMNs) [T. marneffei are able to subvert the natural killing mechanisms of macrophages to survive and replicate inside the phagosome, persisting though lysosome fusion, and finally, escaping into the cytoplasmic environment [After conidial attachment, phagocytic cells are the first line of defense against s (PMNs) ,31. In ms (PMNs) ,32  can readily spread in a Trojan horse manner within these host cells throughout the host resulting in systemic infection [T. marneffei can also become latent with subsequent reactivation, which is clearly evidenced by patients developing talaromycosis years after spending time in an endemic area [T. marneffei crossing the human blood–brain barrier is still lacking [After establishing infection inside alveolar phagocytes, nfection ,34. The nfection . Insteadmic area ,36,37,38mic area  or in thmic area ,41,42. Tmic area . Centralmic area ,44. Amonmic area ,45. HoweAspergillus spp., Candida spp., and Cryptococcus spp. produce diverse virulence factors. For example, Cryptococcus spp. secretes hydrolytic enzymes and exotoxins as well as form a unique polysaccharide capsule [T. marneffei. For dimorphic fungi, a virulence factor is functionally defined as a factor that affects the survival and growth of the organism in its mammalian host, but is not required for parasitic phase growth in vitro [T. marneffei involves many virulent factors that provide fungal survival and persistence inside the host. Thermal tolerance for survival at temperatures at and above 37 °C is the main prerequisite for dissemination to other host internal organs [T. marneffei virulence determinants [Unlike various pathogenic bacteria that produced highly potent exotoxins to destroy host tissue, dimorphic pathogenic fungi do not directly produce exotoxin to facilitate the invasion of host tissue . Other m capsule ,47. Lessin vitro . Indeed,l organs . Currentl organs . A wide l organs . Severalrminants ,52,53,54Coccidioides, Paracoccidioides, Histoplasma, Blastomyces, Sporothrix, Emmonsia, Emergomyces, and T. marneffei, dimorphic switching from saprophytic mycelium to parasitic phase  has been linked to virulence [T. marneffei have been performed. The studies found that optimal temperature for mold growth ranged from 17 to 28 °C. The temperature capable of supporting yeast transition began at 32 °C, but morphogenesis optimally occurred at 37 °C. Also, 40 °C and above inhibits fungal growth. There is a broad range of pH (4.0–10.1) for supporting optimal growth at 37 °C. In addition, NaCl concentrations higher than 4% abrogated hyphal to yeast conversion [Indeed, in dimorphic fungal pathogens such as irulence ,56. Tranirulence ,57. In vnversion .T. marneffei [T. marneffei conidia germinate to produce hyphae at 25 to 28 °C. The conidia swell isotropically within the first 6 hr and undergo polarized growth to germinate in the short filament. At 37 °C, the conidia also swell isotropically prior to initiating polarized growth for germination, but the germlings subsequently generate hyphal-like structures that are often highly branched and these develop arthroconidia within 48 h. Eventually, the arthroconidia are liberated, then transform into uninucleate yeast cell arthroconidia that divided by fission [abaA gene is a master regulator of asexual development in filamentous fungi, especially in the aspect of conidiophore development [T. marneffei abaA deletion mutant displayed aberrant mold to yeast conversion and arthroconidial filamentations at 25 °C. Likewise, at 37 °C the yeast cell failed to switch correctly from multinucleate filamentous to uninucleate yeast cells and multiple nuclei were observed within either arthroconidium or yeast cells [Several molecular genetic studies have focused on the genetic element influencing the dimorphic switching of arneffei ,52. Unde fission . The abaelopment . Bornemast cells .T. marneffei infected macrophages contain the unicellular yeast cells which proliferate within these innate immune cells. Within phagocytes, T. marneffei conidia directly convert to fission yeast cells in a process called “conidia to unicellular yeast morphogenesis” [slnA and drkA genes are essential for dimorphism inside host macrophages at 37 °C. Within 24 h post infection, many fission yeast cells of T. marneffei wild type can be observed within conidia infected macrophages. In contrast, infection with slnA mutant revealed predominately ungerminated conidia whereas macrophages infected with drkA mutant contained mostly septate germlings and a few yeast cells [slnA and drkA genes influence germination inside macrophage environments.From antigenic expression studies, dimorphism inside in vitro host macrophages at 37 °C is different from that achieved in artificial culture medium alone . T. marngenesis” . Therefost cells . These fT. marneffei genes in yeast transition. The yeast cells produced at 37 °C by rasA or cflA mutants exhibit unusual morphologies that appeared swollen, branched, and missharpened [pakA and pakB that works downstream of RhoGTPase are also involved in conidial germination and correct yeast morphogenesis at 37 °C. The pakA mutant remains predominately ungerminated conidia in infected macrophage [pakA and pakB strongly affects the production of yeast cells inside macrophages in vivo, but has no effect in vitro at 37 °C [There are additional roles for other known harpened ,64. The crophage . Howeverat 37 °C . Recentlat 37 °C . Altogetat 37 °C .T. marneffei, this fungus is likely to have evolved several mechanisms of survival under oxidative stress (or respiratory burst) within the macrophage. Oxidative stress is one of the native defenses produced by phagocytes to kill several intracellular parasitic microorganisms. The phagocytes play a crucial role in eliminating fungal pathogens by producing reactive oxygen or nitrogen species, including superoxide radical anion (O2−), hydrogen peroxide (H2O2), hydroxyl radicals (HO•) and nitric oxide (NO) [T. marneffei. When the fungus produces acid phosphatase, it concomitantly might lead to a decrease in host cell intracellular pH and inhibit phagocyte respiratory burst, resulting in enhanced fungal survival [T. marneffei in a macrophage cell line infection model showed that 4-aminoquinoline changes macrophage intracellular pH and fungal growth is inhibited [Regarding the intracellular lifestyle of ide (NO) . The reaide (NO) . The temsurvival . A subsenhibited .T. marneffei produces potent enzymes that specifically detoxify ROS molecules inside host macrophage environments. For example, catalase-peroxidase is a powerful reducing agent that metabolizes H2O2 to water and oxygen. This enzyme is an established virulence factor in Mycobacterium tuberculosis and Aspergillus fumigatus [CpeA) in T. marneffei is associated with the upregulated expression of CpeA transcript both in yeast phase and during macrophage infection [CpeA controls fungal tolerance to H2O2 but not to a heat stress response. H2O2 treatments induced high expression of this gene in both mold and yeast phase. It is therefore proposed that the CpeA of T. marneffei is utilized to protect conidia and yeast cells from oxidative stress in the host macrophage environment [2−) into either ordinary molecular oxygen (O2) or hydrogen peroxide (H2O2). T. marneffei has been shown to survive and replicate as yeast inside the macrophage phagosome. Thirach et al. (2007) investigated the fungal SOD encoding gene (sodA). In T. marneffei, the putative SodA peptide consists of 154 amino acids with shared identity to fungal Cu, Zn- SOD. The results suggest that sodA might play a role in stress response and in the adaptation of T. marneffei inside the macrophage [umigatus ,72. The nfection . Pongpomironment . In addicrophage .T. marneffei yeast cells must adapt to the stress of glucose starvation while also attempting to exploit alternate carbon sources inside the host-cell environment, since glucose is either limited or absent in the phagosomal environment. Macrophage phagosomes contain a complex variety of alternate carbon sources, including amino acids, carboxylic acids and fatty acids [T. marneffei assimilates these alternate carbon sources for energy production in order to survive in this internal macrophage environment [T. marneffei infection, the glyoxylate cycle is induced and acuE and acuD genes are involved in this process. The T. marneffei acuD gene generates isocitrate lyase, the pivotal enzyme in glyoxylate cycle, and gene activity is regulated during the pathogenic yeast phase as acuD was highly expressed after macrophage internalization [acuE encodes malate synthase, and T. marneffei acuE is controlled in a manner similar to acuD as that responds to both temperature and acetate induction [T. marneffei in the human host and the fungus acquires this nutrient using several strategies to uptake host cell iron [T. marneffei [T. marneffei increased their susceptibility to killing by THP-1 human macrophages [T. marneffei to assimilate alternative nourishment under glucose restricted conditions.ty acids . T. marnironment . During lization . acuE ennduction . Likely ell iron . The stuarneffei . Subsequarneffei . The attrophages . These dT. marneffei is closely linked with growth at higher temperatures, heat shock proteins (HSPs) are proposed as potential virulence factors. HSPs are a group of chaperoning intracellular proteins produced by eukaryotic cells in response to stressful conditions and they are generally upregulated upon infection to prevent misfolding of damaged proteins [T. marneffei HSP70 was first isolated and identified by Kummasook et al. (2007). The results showed that the hsp70 transcription was upregulated during the mycelium to yeast transition. Upregulation was also observed when mycelial or yeast cells were subjected to a heat stress environment at 39 °C. T. marneffei HSP70 may play an important role to prevent the yeast proteins from damage during temperature increase [Since the pathogenic phase of proteins . T. marnincrease . The smaincrease .H. capsulatum, P. brasiliensis, C. neoformans, A. fumigatus and Sporothrix schenckii [C. neoformans cells are more negatively charged on the cell wall, and this phenomenon interferes with the phagocytotic mechanism [T. marneffei in vitro and during infection and found that melanins were generated during infection. In particular, patients with talaromycosis had melanized yeast cells in their skin lesion. Additionally, sera from T. marneffei inoculated mice contained IgM and IgG antibodies to fungal melanin. Given the well documented effects of melanin in other pathogenic fungi, T. marneffei melanin and melanin-like pigments may have a role in the virulence of this pathogenic microorganism [alb1) was characterized. These gene clusters were associated with regulating dihydroxynaphthalene (DHN) melanin biosynthesis [T. marneffei alb1 knockdown mutants showed the defects of melanin production and increased the susceptibility to H2O2. Moreover, the survival rate of BALB/c mice after infection with alb1 knockdown mutant was higher than for mice infected with the wild-type strain of T. marneffei [Melanin is a dark brown or black pigment of high molecular weight generated by oxidative polymerization of phenolic or indolic compounds. Melanins are produced by a remarkable variety of organisms such as bacteria, fungi, plants and animals. Melanins are involved in virulence of many pathogenic fungi including chenckii ,87. Melachenckii . For exaechanism . Youngchorganism . Subsequynthesis . Conidiaarneffei ,92.A. fumigatus and C. neoformans. In C. neoformans, this enzyme promotes the pathogenicity of C. neoformans by catalyzing the formation of melanin precursors. There multiple laccases in T. marneffei and the roles of these laccases in virulence and pathogenesis have been characterized. Significantly, a quadruple deletion mutant of T. marneffei laccase encoding genes  was more susceptible to an oxidative stressor, a cell wall stressor and antifungal agents, including itraconazole, fluconazole and clotrimazole [T. marneffei was also shown to be more susceptible to killing by THP-1 human macrophages compared to infections with wild-type T. marneffei. Moreover, the mutant T. marneffei stimulated a significantly higher production of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 by the THP-1 cells. Altogether, these results demonstrate that T. marneffei laccases are involved in resistance to the host immune response [Alongside being involved in melanin generation, p-diphenol dioxygen oxidoreductases or laccases are multi-copper containing oxidoreductase that catalyzes the oxidation of organic and inorganic substances including phenol containing amino acid, methoxy phenol and aromatics amine, with the concomitant four-electron reduction of oxygen to water. The essential properties of fungal laccases have been investigated and were shown to broadly influence fungal development, control phenotype and morphogenesis, detoxify toxins, control pathogenesis in pathogenic fungi and regulate stress response adaptation . Laccaserimazole . Subsequresponse .T. marneffei genes involved in transcriptional regulation and cellular signal transduction cascade have been studied. The rfxA gene is related to nuclear division and binary fission (cytokinesis), and interference with rfxA expression caused the defects in cellular division in both conidiation and yeast transition [T. marneffei YakA and rttA genes involved in controlling cellular morphogenesis, cell wall integrity and heat stress response have been investigated. YakA plays a crucial role in cell wall integrity and heat stress response at 39 °C. The rttA is associated with conidial germination and oxidative stress response. A Galleria mellonella infection model at 37 °C demonstrated that rttA mutant fails to generate the fission yeast forms in G. mellonella, whereas the fission yeasts were found in larvae infected with wild type or complemented strain [T. marneffei sakA and atfA genes affect the viability of T. marneffei conidia under nitrosative and oxidative stresses including adaptation to heat stress at 39–42 °C. Deletion of these genes resulted in the decreased survival rate of conidia inside mouse (J774) and human (THP-1) macrophage cell lines [T. marneffei was examined. Deletion of the MCD gene increased the susceptibility to killing by RAW264.7 mouse macrophage and reduced the mutant strain’s virulence in BALB/c mice [ansition . Furtherd strain ,98. T. mll lines ,100. RecB/c mice .T. marneffei yeast cells are taken up by RAW 264.7 murine macrophage cells and this process increased the expression of surface CD80, CD86, and MHC class II. Moreover, incubation of T. marneffei EV with macrophages stimulated the expression levels of ROS, NO, and several inflammatory factors including IL-1β, IL-6, IL-10, and TNF-α. The level of secreted inflammatory factors showed a significant decrease when EVs were destroyed by protease. The proteomic analysis by LC–MS/MS demonstrated T. marneffei EV contained highly immunogenic proteins and some bioactive components including heat shock protein, Mp1p mannoprotein, and peroxidase enzyme [T. marneffei potential virulence factor was summarized in Extracellular vesicles (EVs) are double-layer lipid membrane structures that contain various bioactive components that are released by cells into the extracellular environment. In addition to being released from mammalian cells, bacteria, and diverse other cells, the production and secretion of EVs plays an important role in pathogenic fungal infections ,103. Rece enzyme . The oveT. marneffei, the host depends on both innate and acquired immune responses to eradicate the microorganism and combat infection. The mononuclear phagocytes such as macrophage and dendritic cells (DCs) have major roles in the activation of cellular pathways and the production of several cytokines in both pro-inflammatory cytokine  and anti- inflammatory cytokine (IL-10). Recently, an investigation of human genetic polymorphisms was undertaken to predict susceptibility against T. marneffei infection. The approach focused on single nucleotide polymorphisms (SNPs) in Toll-like receptor (TLR) genes of Han Chinese with AIDS demonstrated that SNPs within the TLR2, TLR4, and TLR9 genes may contribute to increasing the susceptibility and severity to T. marneffei [After encountering arneffei . Moreovearneffei .T. marneffei infection and immunocompromised status resulting from various factors of acquired immunity impairment strongly suggests that host innate immunity must play a crucial role in controlling T. marneffei infection when CD4 T cells are dysfunctional or disabled. Accordingly, the phagocytic cells of the innate immune system including monocytes [T. marneffei inside the host.The association between onocytes , macrophonocytes ,33, PMNsonocytes ,34,107, onocytes  are invoT. marneffei and the host. In addition to human macrophages, the responses of pulmonary macrophages from rabbits and mice to T. marneffei conidia have been reported [T. marneffei conidia to monocytes/macrophages was demonstrated by Srinoulprasert et al. (2009) who found that engagement of the fungus could be significantly inhibited by monoclonal antibodies against pattern recognition receptors (PRRs), including a Mannose receptor, TLR1, TLR2, TLR4, TLR6, CD14, CD11a, and CD18. Additionally, monocytes co-cultured with T. marneffei conidia had increased expression of surface CD40 and CD86 molecules as well as higher TNF-α and IL-1β production [As mentioned above, macrophages have a central role in the interaction between reported ,110. Theoduction .T. marneffei conidia and yeast cells. A disparity in the susceptibility of T. marneffei conidia and yeast cells to the fungicidal activity of macrophages has been observed in vitro [T. marneffei, and fungicidal activity is mediated by nitric oxide (NO) [T. marneffei in vitro. These observations indicate that IFN-γ is a potent inducer for killing T. marneffei inside macrophages via a NO-dependent process [Interestingly, macrophages respond differently to in vitro , with yeide (NO) . Indeed,ide (NO) . Kudeken process .T. marneffei. Using heat-killed yeast, monocyte-derived macrophages readily internalized yeast even in the absence of opzonization and the major receptor(s) recognizing T. marneffei included a glycoprotein with N-acetyl-beta-D-glucosaminyl groups. Although T. marneffei stimulates the oxidative burst (respiratory burst) of macrophage regardless of whether opsonins are present, TNF-α production is upregulated only in the presence of opsonins. Thus, the ability of un-opsonized T. marneffei to parasitize macrophages without stimulating the production of TNF-α might be a critical step for the survival of this intracellular fungus [TNF-α plays an important role in host defense against r fungus .T. marneffei. Wright’s-stained peripheral blood smears from patients with AIDS-associated talaromycosis have revealed large numbers of fission yeast cells inside PMNs [2O2 and superoxide anion, mediated by enzyme myeloperoxidase, while the second is due to the action of anti-microbial enzymes exocytosed from their granules, such as lysozyme, lactoferrin, acid phosphatase, β-glucuronidase and elastase [T. marneffei yeast cells. Granulocyte-macrophage-colony-stimulating factor (GM-CSF) stimulated PMNs demonstrate strong killing activity against T. marneffei yeast forms, but not in conidia. It is speculated that T. marneffei yeast cell killing is executed through exocytosis of PMNs granular enzyme with direct (close) contact between fungus and PMNs. Moreover, the killing mechanism of GM-CSF stimulated PMNs on T. marneffei was not mediated by a superoxide dependent mechanism, because SOD failed to inhibit the fungicidal activity of GM-CSF-stimulated PMNs [In addition to macrophages, PMNs have fungicidal activity against ide PMNs . Likewiside PMNs . In geneelastase . In vitrted PMNs ,107.T. marneffei can be observed growing inside PMN whereas yeast forms were predominantly growing within macrophage. This observation implies that the intracellular milieu of those phagocytes may be a determinant of T. marneffei morphogenesis in vivo, which can override the influence of temperature. Moreover, this result may also explain the reason why macrophages are the preferred residence for infection and replication of T. marneffei as macrophage can serve as a protective shield for the yeast cells to evade PMN mediated destruction by the strong myeloperoxidase activity delivered in PMN granules [Recently, the relationship between macrophage and PMN in the pathogenesis of talaromycosis was explored in an ectothermal zebra fish model. In zebra fish, despite the lower incubation temperature (33 °C), the phase transition from conidia to yeast occurs. Additionally, filamentous forms of granules ,115.T. marneffei [T. marneffei and other pathogenic fungi have developed several strategies to escape host macrophage killing. For example, the attenuation of M1 and/or induction of M2 macrophages polarization are major immune escape tactics [H1 signaling. On the other hand, M2 macrophages are involved in anti-inflammatory responses and tissue repair mediated by TH2 signaling and are associated with an immunological response to parasitic infections or allergic asthma [Candida albicans, the fungus achieves immune escape via inducing macrophage M2 polarization [Cryptococcus neoformans, the fungus drives monocytes to affiliate an M2 macrophage polarization, which is permissive to fungal proliferation and spreading, in a disseminated model of cryptococcosis [T. marneffei stimulated macrophages induce M2 response, mediated by IL-10, in both a BALB/c mouse alveolar macrophage model [T. marneffei infected THP-1 human macrophages by disturbances CD86 (B7-2) expression [T. marneffei may escape macrophage killing to proliferate inside macrophage by inducing M2-like polarization [T. marneffei infected human macrophage have been investigated. Chen et al. (2014) demonstrated that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are essential for TNF-α production, whereas p38 mitogen activated protein kinase is essential for IL-10 production. These findings suggest that ERK1/2 might be essential for the initiation of proinflammatory responses to T. marneffei. In contrast, the p38 pathway activation may attenuate host immune response and promote the intracellular survival of T. marneffei [T. marneffei include the down regulation of the proinflammatory cytokine IL-6 produced by bronchial epithelial cells [Although macrophages are considered the key primary effector cells in host resistance against arneffei ,33. Howe tactics . Macroph tactics . For thi tactics . M1 macrc asthma . In Candrization . In Crypcoccosis . T. marnge model  and a PBge model . Moreovepression . The sturization . The cytarneffei . Other ial cells  and throal cells ,128.T. marneffei and host DCs. Nakamura et al. (2008) demonstrated that TLR2 and dectin-1 are essential in recognizing T. marneffei for the activation of bone marrow-derived DCs (BMDCs) [T. marneffei yeast cells were found to increase regulatory T cell (Treg) expansion and restrict TH17 cell responses by increased the production level of CD80, CD86, IL-6, IL-10 and TGF-β levels in the culture supernatant of T. marneffei-stimulated BMDCs [T. marneffei enhance HIV-1 trans-infection of primary CD4 T cells [DCs are considered the most powerful of the immune phagocytes. In fungal infection, DCs are extremely efficient at processing and presenting fungal antigen to CD4 T cells or alternatively through CD8 T cells  . Unfortu (BMDCs) . In a reed BMDCs . In the  T cells .T. marneffei infection and evolve over the course of disease [T. marneffei conidia or yeast cells are often reported [T. marneffei yeast cells, which is absent when conidia are used. From in vivo studies, NLRP3 deficient mice have higher fungal loads and increased mortality rates than wild-type mice after systemic T. marneffei infection [T. marneffei in patients who suffer from substantial loss of CD4 T cells or other functional impairment of acquired immunity as a result of HIV manifestation. Notably, patients with co-infections of T. marneffei and HIV with the most robust proinflammatory cytokine response or “cytokine-storm” had the worst outcomes, underscoring the need for a balanced immunological response [Although cytokine responses are very complex in  disease , a signireported ,106,112.nfection . This stnfection . The sturesponse .T. marneffei. However, in vivo model studies have determined that T lymphocytes play a protective role against T. marneffei infection. Indeed, there is a faster progression of T. marneffei infection in athymic nude mice compared to immunologically intact mice [T. marneffei to infected athymic nude has a profound effect on disease progression as it results in decreased numbers of viable T. marneffei cells in the lungs, livers and spleens of the animals receiving adoptive T cell transfer compared to non-treated infected athymic nude mice. These data show that T cell mediated immunity has a central role in host resistance to T. marneffei [Although depletions of CD4 T lymphocytes are closely associated with an increased risk for talaromycosis, there are relatively few studies specifically investigating the role of T-cell (CD4 and CD8)-mediated immune responses to act mice . Notablyarneffei .T. marneffei cells are occasionally identified in the peripheral blood by light microscopy of blood smears [T. marneffei. Notably, high titers of anti-IFN–γ neutralizing autoantibodies in AOID are associated with disseminated non-tuberculous mycobacteria (NTM), histoplasmosis, cryptococcosis, melioidosis, non-typhoidal salmonellosis, and varicella zoster virus infections as well as talaromycosis, which exemplifies the role of IFN –γ in regulating a host’s ability to combat diverse intracellular pathogen [T. marneffei, and their spleens and livers showed abundant yeast cells and an absence of granuloma formation. Moreover, TH1-polarization of cytokines with increases in IFN- γ and IL-12 was observed in the spleen [H1 immune response plays a crucial role in host defense to other intracellular pathogenic microorganism such as in infections with mycobacteria [H. capsulatum and C. neoformans [In talaromycosis patients co-infected with HIV, the histopathologic features depend on the degree of immunological suppression . At lowed smears ,137. In pathogen ,139,140.e spleen . These fbacteria  and introformans ,143.H1, another subset of CD4 T cells is the “TH17 and regulatory T cell (Treg)” that play an important role in the dynamic immune balance  of protective immunity and autoimmunity as well as immunopathogenesis [H. capsulatum infection, increases of TH17 cytokines and reductions in the number of Treg are associated with rapid fungal clearance in CCR5 deficient animals [H1/TH17 protective immunity and prevents fatal disease outcomes from the fungus [T. marneffei infection is not well-defined. Tang et al. 2020 demonstrated that after recognizing T. marneffei yeast cells, murine BMDCs increased Treg expansion by upregulating Foxp3 expression and restricting TH17 cell responses by downregulating RORγt expression. This phenomenon promotes immunological tolerance and thereby may be harmful to the host defense against T. marneffei infection [H1 defects including problem in IFN- γ/IL-12 production, TH17 defect, STAT1 mutation, STAT3 mutation, and CD40 ligand deficiency) are documented to be associated with talaromycosis and therefore are linked to increased susceptibility to T. marneffei infection [In addition to Togenesis ,145. In  animals . Moreovee fungus . Unfortunfection . OverallT. marneffei infection is unclear and, as no major investigations into this aspect of host immunity have been carried out to date, antibody mediated protection has yet to be effectively defined. However, the protective effect of antibodies against cell wall mannoprotein has been reported. Wong et al. (2002) demonstrated the production of murine specific IgM and IgG antibody responses to a highly immunogenic secreted cell wall mannoprotein, Mp1p, using both recombinant protein and DNA immunization strategies [Due to the immunocompromised status of the majority of individuals with talaromycosis, it is expected that there will be considerable defects of the humoral (antibody mediated) immune responses . Furtherrategies . NeverthT. marneffei has been obtained as a result of serological assays for clinical laboratory diagnosis [T. marneffei proteins failed to induce antibody production in immunocompromised patients [Indeed, the majority of data regarding humoral mediated immunity to iagnosis , in whiciagnosis ,153,154.iagnosis ,155. Morpatients ,156.There is, however, an important link between humoral and cellular immunity that is pertinent to talaromycosis, as protective antibodies are required for robust CD4 T cell-mediated isotype switching and affinity maturation . In the T. marneffei and decipher the processes that the fungus utilizes to modify the capacity of the host to combat infection. We highlight that the complexity underlying T. marneffei’s ability to establish infection from morphogenesis to intracellular survival. T. marneffei is remarkable in its capacity to adapt, survive and manipulate host responses, particularly phagocytic immune cells and especially macrophages. These complex strategies developed by T. marneffei are presented in As reviewed in this paper, researchers in several countries are working to characterize the putative virulence factors of T. marneffei, there are numerous questions that need to be addressed to robustly understand the biology and pathogenesis of T. marneffei [T. marneffei, especially in the context of pathway to survive intracellularly in infected cells (Trojan horse model) [T. marneffei infection, such as DCs, natural killer cells (NK cells) as well as other innate-like T lymphocytes, are rich areas for exploration. Likewise, the role of other CD4 T cells subset  T cells) are urgently needed areas for investigation. Studies on these research areas as well as rigorous explorations of antibodies-based therapy, such as the passive administration of therapeutic monoclonal antibodies [T. marneffei, or the utilization of anti-CD19 chimeric antigen receptor-modified T cell (CAR-T-cell) therapies [Although more than six decades have passed since Dr. Segretain discovered arneffei . For exae model)  and acqutibodies , prophylherapies  will be


OBJECTIVES/GOALS: Intravenous immunoglobulin (IVIG) is used for infection prevention in pediatric B-cell acute lymphoblastic leukemia (B-ALL), but evidence for this is lacking. We describe the prevalence of hypogammaglobulinemia in pediatric B-ALL, predictors of IVIG use and its efficacy for infection prevention. METHODS/STUDY POPULATION: We will conduct a retrospective review of children age 1-21 years with B-ALL treated at Aflac Cancer and Blood Disorders Center from 2010 to 2017. The cohort was identified through the cancer registry. Demographics, disease factors, laboratory values, medications and infection outcomes were linked between the electronic medical record and an institutional database. Outcomes of interest include emergency department (ED) visits, hospitalization days, and episodes of infection. Descriptive statistics will be performed. Outcomes will be compared between IVIG recipients and non-recipients. Univariate and multivariate logistic regression models will assess predictors of IVIG administration. RESULTS/ANTICIPATED RESULTS: We identified 443 patients with B-ALL during the study period who met inclusion criteria. Exclusion criteria included receipt of IVIG or hematopoietic stem cell transplant prior to diagnosis. The average age at diagnosis is 6.5 years (standard deviation 4.8 years); 52.6% are male; 61.6% are white; 61.0% are standard risk per National Cancer Institute criteria. Among eligible patients, 137 (31.1%) received IVIG. We hypothesize that IVIG initiation is associated with hypogammaglobulinemia and history of severe infection. We also anticipate that frequency of emergency department visits, hospitalization days, and episodes of infection will decrease after IVIG initiation. DISCUSSION/SIGNIFICANCE OF IMPACT: The immunological profile of children with B-ALL and factors influencing their susceptibility to infection are still incompletely understood. The benefits of IVIG are unknown. This study will provide evidence for IVIG prophylaxis recommendations in pediatric leukemia patients.


Cutibacterium acnes (previously known as Propionibacterium acnes) is frequently found on lipid-rich parts of the human skin. While C. acnes is most known for its role in the development and progression of the skin disease acne, it is also involved in many other types of infections, often involving implanted medical devices. C. acnes readily forms biofilms in vitro and there is growing evidence that biofilm formation by this Gram-positive, facultative anaerobic micro-organism plays an important role in vivo and is also involved in treatment failure. In this brief review we present an overview on what is known about C. acnes biofilms (including their role in pathogenesis and reduced susceptibility to antibiotics), discuss model systems that can be used to study these biofilms in vitro and in vivo and give an overview of interspecies interactions occurring in polymicrobial communities containing C. acnes. It was successfully cultivated in 1897 and formally named Bacillus acnes in 1900. Due to its morphological similarity to members of the genus Corynebacterium, it was reclassified as Corynebacterium acnes in 1923 [Propionibacterium, as Propionibacterium acnes [Propionibacterium spp. were traditionally subdivided in ‘classic’ and ‘cutaneous propionibacteria’. The ‘classic propionibacteria’ contain species isolated from dairy products such as Propionibacterium freudenreichii, while the ‘cutaneous propionibacteria’ comprise P. acnes, Propionibacterium avidum and Propionibacterium granulosum, all isolated from human skin [Cutibacterium was proposed for the cutaneous species, resulting in the reclassification of P. acnes as Cutibacterium acnes [C. acnes isolates and understand C. acnes phylogeny and clonal distribution. A first method was based on the sequences of the tly and the recA genes and allowed further subdivision of serotypes I and II into phylotypes IA1, IA2, IB, IC and II, and, moreover, revealed the additional phylotype III [C. acnes subsp. acnes, C. acnes subsp. defendens, and C. acnes subsp. elongatum for phylotypes I, II and III, respectively [. Subsequently, two multilocus sequence typing (MLST) schemes have been described for C. acnes. The first is based on the partial sequencing of nine housekeeping genes and was developed in 2010 [C. acnes strains is based on ribotyping. This method is based on sequence analysis of the gene coding for the 16S rRNA [1 and IA2. However, the scheme is not in full agreement with phylotyping, as for example type IB and IC share RT1 and RT5, while RT1 is also shared between type IA1 and IB.Already in 1972 two distinct groups were recognized within this species, based on differences in cell wall composition . Serologtype III . These dectively . Subsequ in 2010 . A secon in 2010 ,13. More in 2010 . An alte16S rRNA . The res2C. acnes can metabolize lipids produced by the human sebaceous glands, thrives in lipid-rich environments [6 colony forming units (CFU) per cm2 [2 CFU per cm2 - are found on dryer areas, such as skin of the lower extremities [C. acnes colonization of the follicle [C. acnes thrives in the lipid-rich and anaerobic environment. C. acnes colonization and the resulting activation of the immunocompetent keratinocytes and sebocytes play essential roles in acne pathogenesis. Triglycerides present in the sebum are hydrolyzed by bacterial lipases and the resulting free fatty acids have comedogenic properties and can act as damage associated molecular patterns, while the released glycerol acts as a nutrient source for C. acnes [C. acnes include co-hemolytic Christie–Atkins–Munch-Peterson (CAMP) factors and porphyrins that will propagate acne pathogenesis through the generation of pro-inflammatory cytokines in human keratinocytes and sebocytes. This includes IL-1β, which is produced as a consequence of activation of the NLRP3 inflammasome [C. acnes alone is not an explanation for the occurrence of acne, as the organism is present in both healthy and affected hair follicles and it seems equally unlikely that variation in relative abundance of C. acnes can explain the differences observed [C. acnes groups with disease pathology. C. acnes strains belonging to phylotype IA1 are more often found in acneic skin, while higher phylotypic diversity as well as an enrichment of phylotype II strains can typically be found on healthy skin. In addition, recent research has demonstrated that C. acnes strains belonging to phylotype III dominate in progressive macular hypomelanosis [. An ‘acne index’ was assigned to each RT by calculating its prevalence in acne patients. As RTs 4, 5, 8, and 10 are significantly enriched on the skin of acne patients, strains belonging to these RTs are characterized by a high acne index. In contrast, RTs 6 and 16 are strongly associated with healthy skin and therefore strains belonging to these RTs possess a low acne index [tad and sag genes  in RT4 and RT5, potentially providing a link between the acne index of a C. acnes strains and its virulence properties.ronments  and as a per cm2 . In contremities . Acne isfollicle ,25. Excefollicle . In thesC. acnes . Other fammasome ,28. Due ammasome . The preobserved ,24. Howeelanosis ,31. An ‘ne index ,32. Deta3C. acnes was first described in 1999, when it was shown that C. acnes forms biofilms on prosthetic hips [C. acnes biofilm formation on various biomaterials was subsequently confirmed in a wide range of other studies . In 200ic cells . Upregulic cells  as well lm model  were subC. acnes biofilm matrix has been explored in several in vitro studies. Biofilms of a C. acnes skin isolate grown in cell culture flasks contained polysaccharides, proteins and extracellular DNA (eDNA), with the polysaccharides containing α-mannopyranosyl and α-glucopyranosyl residues [C. acnes strains recovered from contaminated cardiac pacemaker devices and formed in 96-well plates was found to contain eDNA, proteins, and poly-N-acetyl glucosamine [C. acnes RT5 strain grown in vitro on cellulose acetate filters were found to contain polysaccharides (62.6%), proteins (9.6%), eDNA (4.0%) and other compounds  [C. acnes cell wall and contained N-acetylgalactosamine, N-acetylmannosamine, 2-acetamido-2-deoxy-galactose and 2,3-di-acetamido-2,3-dideoxy-mannuronic acid residues, but no evidence for the presence of poly-N-acetyl glucosamine was obtained. These data suggest that overall biofilm composition is similar to what is observed in other bacterial biofilms [C. acnes strains, suggesting that both eDNA and proteins are important for adhesion to abiotic surfaces, although sensitivity to DNase I was more strain-dependent [The composition of the residues . The matcosamine . Finallycursors) . In the biofilms  but can ependent .4C. acnes biofilm-like structures in acne skin biopsies; such biofilms were more frequently observed in follicles of acne patients than in those of healthy controls [in vivo C. acnes biofilms showed different morphologies, with some attaching to the follicle wall and/or the hair shaft, while others occurred in the lumen; interestingly these different colonization patterns could be observed in the same hair follicle [C. acnes biofilms were also observed in atherosclerotic carotid artery specimens where they are often part of a multispecies biofilm [Jahns, Alexeyev and co-workers ,47,48 decontrols . These i biofilm .C. acnes in human disease comes from implant-associated infections [C. acnes from these infections and there is now convincing evidence that C. acnes biofilms are involved in infections related to the use of prosthetic joints, other orthopedic devices, cerebrovascular devices, breast implants, and cardiovascular devices. In all orthopedic infections, evidence of a biofilm can be found if the fluid from the sonicated implant is investigated [C. acnes [C. acnes from frozen stocks grows within 2–3 days in a research laboratory but requires up to 14 day to grow from orthopedic infections samples strongly suggests they are present in these samples as biofilms [C. acnes is the dominant pathogen found after shoulder arthroplasty [Cutibacterium avidum dominates after hip arthroplasty [Cutibacterium spp. is about 85%, but worse if only a debridement is performed and the implant is retained [Cutibacterium spp. relapses, radical surgery and a prolonged antibiotic treatment over 6 weeks led to the best outcomes and avoided relapse of infection [C. acnes causes several cardiovascular device-related infections, such as prosthetic valve endocarditis, and pacemaker and cardiac implantable cardioverter-defibrillator infections. Infections can be divided into local infections (pocket infections) or device-related bloodstream infections, including device-related endocarditis [C. acnes. Endocarditis caused by C. acnes has been associated with both native and prosthetic valves but more often develops on valve prostheses, most commonly on the aortic valve [Cutibacterium spp. infection between 1995 and 2016 revealed 51 episodes of prosthetic valve endocarditis of which 63% underwent surgery, suggesting a mature biofilm infection that could not be treated with antibiotics alone [C. acnes biofilms in endocarditis has been confirmed with fluorescent in situ hybridization, which allows confirmation of biofilm-like structures within the histological context and rules out contamination [Staphylococcus aureus and Escherichia coli [C. acnes are rare, they occur. In 2010, Uckay et al. reported 29 patients with spondylodiscitis caused by C. acnes who presented with back pain [C. acnes are typical microorganisms that are identified next to S. aureus or Gram-negative pathogens [C. acnes in these cases seems to be an important virulence factor since most of the implant-associated infections requires removal of the implant to cure the infections. For example, Köder et al. found that treatment with biofilm-active antibiotics was associated with better treatment outcome and less postoperative pain intensity although this finding was not specific for C. acnes spine infection [C. acnes can play a role [C. acnes biofilms have been visualized in intervertebral discs of patients undergoing microdiscectomy [However, most evidence for a role of fections . The impstigated . Sonicatstigated . For exaC. acnes ,61. One C. acnes ,63. HoweC. acnes ,65. The biofilms . Among proplasty  while Curoplasty . Treatmeretained . This obnfection . This iscarditis . Diagnosic valve . A Swedics alone . The premination . Spondylhia coli ; while iack pain . In patiathogens ,76. The nfection . Also iny a role , and C. scectomy  and in sscectomy .5C. acnes has emerged worldwide [C. acnes strains are currently not only found in acne patients treated with antibiotics, but also in other types of infections, including infections related to the use of various medical devices.Treatment with topical antibiotics  is often prescribed in severe cases of acne, and it is thus not a surprise that resistance in orldwide . While torldwide . As coulC. acnes. For example, substantially higher concentrations of cefamandole, ciprofloxacin, and vancomycin are required for inhibition and eradication of in vitro grown C. acnes biofilms  compared to planktonic bacteria [C. acnes biofilms grown in microtiter plates were substantially less sensitive to killing by a range of anti-microbial products used for the treatment of acne, including 0.5% minocycline, 1% clindamycin, 0.5% erythromycin, 0.3% doxycycline, 0.5% oxytetracycline and 2.5–5% benzoyl peroxide [C. acnes biofilms formed on titanium disks required prolonged exposure to penicillin (7 days), linezolid or linezolid + rifampicin (14 days), and prevention of relapse (i.e. full eradication) required a 14 day treatment of penicillin or linezolid plus rifampicin, but could not be achieved by treatment with linezolid alone [C. acnes from glass beads required considerably higher antibiotic concentrations than those needed for killing planktonic cells for rifampicin (4-fold higher), daptomycin (16-fold higher), vancomycin (64-fold higher) and levofloxacin (256-fold higher), while for the β-lactam antibiotics penicillin G and ceftriaxon the difference was smaller (2-fold higher for both) [in vitro grown C. acnes biofilms, this is not the case for ciprofloxacin and clindamycin, and a four day treatment with the latter antibiotics at a concentration that is 50 times higher than the minimal inhibitory concentration showed no effect [in vitro studies using surface-attached biofilms, they clearly indicate that biofilm formation has the potential to contribute to the reduced antimicrobial susceptibility observed for C. acnes in vivo.It is well-known that microbial biofilm formation contributes to the failure of antimicrobial therapy  and failbacteria . Likewisperoxide . Partialid alone . Eradicaor both) . Finallyo effect . While t66.1staphylococci. Large-scale studies on the microbiome of acneic follicles have not yet been performed, but initial data point to a dominance of Cutibacterium spp., Staphylococcus spp. and Malassezia spp. (previously known as Pityrosporum spp.) [The human skin is home to a large number of different bacteria and the skin microbiome plays an important role in controlling colonization by pathogens and in modulating the cutaneous immune system ,89. Whilum spp.) .C. acnes inhibit biofilm formation by S. epidermidis and S.aureus (the latter to a lesser extent), but not of Pseudomonas aeruginosa or Bacillus subtilis [C. acnes fermentation products inhibit the growth of both S. aureus and S. epidermidis [C. acnes, reduces colonization of skin hair follicles by Staphylococcus species [C. acnes, while being an important player in the development of acne, also has a beneficial effect on the host, by limiting growth of potential pathogens on the skin.Microorganisms colonizing the same skin area influence each other through competition for the limited amount of nutrients on the skin and through the production of antimicrobial compounds . For exasubtilis . In addidermidis . Finally species . These dS. epidermidis towards C. acnes has also been described. This could be due to the fermentation of glycerol by S. epidermidis leading to the production of succinic acid or other short chain fatty acids that inhibit C. acnes [C. acnes biofilms may act as a ‘sanctuary’ for S. aureus, protecting it from harsh conditions during prolonged co-culture [S. aureus and C. acnes in polymicrobial communities are definitely not always passive, as it has been shown that S. aureus-induced haemolysis and cell lysis were increased when S. aureus was grown in the presence of C. acnes and that this is due to CAMP factors produced by C. acnes [C. acnes induces S. aureus aggregation and plasma-independent biofilm development on an abiotic surface; this biofilm promoting activity depends on sarA, a known biofilm regulator in S. aureus [C. acnes and S. aureus could be co-modulated by human natriuretic peptides [C. acnes responds, as it earlier had been shown that physiologically relevant levels of norepinephrine induce biofilm dispersion and stimulate expression of genes coding for various C. acnes virulence factors  [It should however be noted that these interactions are not a one way street and antagonistic activity of C. acnes  and/or tC. acnes . In addi-culture . These iC. acnes . In addi. aureus . Recent peptides . The late lyase) .6.2C. acnes can form multispecies biofilms with the dimorphic fungus Candida albicans [S. aureus and P. aeruginosa, in these multispecies biofilms C. acnes adheres both to yeast cells and hyphae and the presence of C. acnes in these in vitro biofilms significantly reduced the susceptibility of Candida albicans to the antifungal agent micafungin [Candida albicans enhanced early C. acnes biofilm formation in the presence of oxygen (but not in anaerobic conditions) [C. acnes and fungi  also appear to be important [C. acnes and Malassezia restricta were observed in a pre-clinical cell-culture based dandruff model [C. acnes and fungi in these polymicrobial communities is unknown.albicans ,103. Intcafungin . Also thmportant  and mixeff model . Current77.1in vitro and in vivo biofilm models is available [C. acnes biofilms is derived from studies in which biofilms are formed on abiotic surfaces (cell culture flask or microtiter plate) under conditions that bear little relevance for the in vivo situation. Recently a dynamic (flow-cell based) model was described as well [C. acnes biofilm biology can be obtained in these models, it is important to realize that such biofilms are different from in vivo biofilms [in vitro models in which biofilm formation on various biomaterials (including stainless steel and titanium) can be studied, have also been described [While a wide range of vailable ,107, mos as well . Althougbiofilms . To bettescribed ,35,110.in vitro study of C. acnes biofilms in the context of acne in more in vivo like conditions, a model using artificial sebum  was developed. In this model C. acnes biofilm formation can be studied, as well as the production of virulence factors like lipases, proteases, and CAMP factors [In order to allow the  factors  Fig. 1)in vitro 7.2C. acnes (biofilms) and the human host are also available. As acne is an exclusively human disease, these models require the use of human cell lines [Several cell-culture based models suitable to study the interaction between ll lines ,112.in vitro keratinocytes are primary normal human epidermal keratinocytes (NHEK), directly dissociated from parental tissue and subsequently grown as monolayers. Although these cells retain the features of the original tissue, the main disadvantage of primary cells is that they will reach senescence after approx. 15–20 passages, limiting the duration and scope of experiments. In contrast, immortalized cell lines can be cultured for an extended time and their genotype and phenotype remains stable over time. These immortalized cell lines can be established after spontaneous mutations or induced by introducing viral oncogenes that affect the cell cycle [The skin is the first line of defense against external influences and is comprised of immunocompetent cells including keratinocytes, which account for over 95% of all the cells in the epidermis . The mosll cycle ,114. An ll cycle . The HaCll cycle ). Other ll cycle .C. acnes [C. acnes  in a pre-clinical dandruff model [C. acnes [A more complex, tissue-like model is the reconstituted/reconstructed human epidermis (RHE), which requires NHEKs to obtain optimal tissue morphology i.e. a stratified epithelium. In this model, the NHEKs are cultured on a collagen matrix placed at an air-liquid interface, producing 8–12 layers of epidermis, that can be used to study infection and inflammation by various organisms, including C. acnes ,124,125.C. acnes  are commff model  as well in vitro sebocyte model [Another important cell type in the context of acne is the sebocyte, and isolation of human sebaceous glands and the culture of primary sebocytes were the first steps in the establishment of an te model . Howeverte model . In ordete model . Other ite model  and Seb-te model .in vivo-like morphology and more realistic environment [in vitro co-culture model combining HaCaT keratinocytes and SZ95 sebocytes in a ‘well and insert’ system was developed [C. acnes  cell types are combined. Two main types of co-culture models exist; i.e. mixed co-cultures and segregated co-cultures . These mironment . One of ironment ,135. Receveloped ,137. TheC. acnes . In thisC. acnes and its human host. First of all, the adhesion of C. acnes cells and biofilm aggregates to HaCaT keratinocytes  also produce high levels of porphyrins [C. acnes (sub)groups for the pathogenesis of acne are still unclear, they reinforce the notion that there are profound and biologically-relevant differences between these (sub)groups and illustrate the power of using these cell-culture based models.Several of the cell-culture based models described above were recently used to elucidate interactions between inocytes  and SZ95 strains . A signi strains . These aene deoR . Howeverrphyrins , indicat7.3C. acnes biofilms under physiologically relevant conditions, several more advanced models have been developed. These include various animal models in which implant-associated infections can be studied, e.g. a foreign-body infection model in which polytetrafluorethylene (Teflon) cages are subcutaneously implanted in the flanks of the guinea pigs [C. acnes and demonstrated low cure rates for daptomycin, vancomycin, levofloxacin and rifampicin, despite good activity against planktonic cells. For eradication of these in vivo C. acnes biofilms, combinations of rifampicin with daptomycin (or vancomycin) were required [C. acnes proteins, of which nine were exclusively produced by biofilm-grown C. acnes [In order to study nea pigs ,140, an nea pigs , a modelnea pigs , a rabbinea pigs  and a shnea pigs . The subrequired . Use of C. acnes .Drosophila melanogaster (fruit fly) model was developed to study C. acnes, C. avidum and Cutibacterium granulosum biofilms [Recently a germ-free biofilms . By main8C. acnes is a skin commensal that is also important in various infections, going from acne to device-related infections. This bacterium contains a wide range of (putative) virulence factors, and biofilm formation seems to be a common theme in many C. acnes infections. There is growing evidence that some C. acnes strains cause more damage to human cells and/or are more pro-inflammatory than others, but why that is the case is not entirely clear. While the increased production of certain virulence factors in biofilm grown C. acnes, as well as differences in the production of virulence factors (e.g. lipase) and pro-inflammatory mediators (e.g. porphyrins) between different C. acnes strains are likely to play a role, more research is needed. In addition, as a skin-associated organism, C. acnes frequently interacts with other organisms and how this influences biofilm formation, virulence, proinflammatory activity and cytotoxicity remains to be investigated in depth.The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TOM COENYE is senior editor of Biofilm. Given his role as Senior Editor, TOM COENYE had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to AKOS KOVACS.