Localization and symbiotic status of probiotics in the coral holobiont.

Corals establish symbiotic relationships with microorganisms, especially endosymbiotic photosynthetic algae. Although other microbes have been commonly detected in coral tissues, their identity and beneficial functions for their host are unclear. Here, we confirm the beneficial outcomes of the inoculation of bacteria selected as probiotics and use fluorescence in situ hybridization (FISH) to define their localization in the coral Pocillopora damicornis. Our results show the first evidence of the inherent presence of Halomonas sp. and Cobetia sp. in native coral tissues, even before their inoculation. Furthermore, the relative enrichment of these coral tissue-associated bacteria through their inoculation in corals correlates with health improvements, such as increases in photosynthetic potential, and productivity. Our study suggests the symbiotic status of Halomonas sp. and Cobetia sp. in corals by indicating their localization within coral gastrodermis and epidermis and correlating their increased relative abundance through active inoculation with beneficial outcomes for the holobiont. This knowledge is crucial to facilitate the screening and application of probiotics that may not be transient members of the coral microbiome. IMPORTANCE: Despite the promising results indicating the beneficial outcomes associated with the application of probiotics in corals and some scarce knowledge regarding the identity of bacterial cells found within the coral tissue, the correlation between these two aspects is still missing. This gap limits our understanding of the actual diversity of coral-associated bacteria and whether these symbionts are beneficial. Some researchers, for example, have been suggesting that probiotic screening should only focus on the very few known tissue-associated bacteria, such as Endozoicomonas sp., assuming that the currently tested probiotics are not tissue-associated. Here, we provide specific FISH probes for Halomonas sp. and Cobetia sp., expand our knowledge of the identity of coral-associated bacteria and confirm the probiotic status of the tested probiotics. The presence of these beneficial microorganisms for corals (BMCs) inside host tissues and gastric cavities also supports the notion that direct interactions with the host may underpin their probiotic role. This is a new breakthrough; these results argue against the possibility that the positive effects of BMCs are due to factors that are not related to a direct symbiotic interaction, for example, that the host simply feeds on inoculated bacteria or that the bacteria change the water quality.

Anxiety and burnout in young athletes: The mediating role of cognitive appraisal.

This study tested the relationship between trait anxiety, cognitive appraisal, and athletes' burnout proposing two hypotheses: (a) there is a direct relationship between athletes' trait anxiety and cognitive appraisal and burnout, and (b) cognitive appraisal mediates the relationship between trait anxiety and burnout, and this mediation occurs despite the competitive level and sport records of athletes. The study included 673 young athletes and provided measures of trait anxiety, cognitive appraisal, and burnout. Structural equation modeling indicated that cognitive appraisal mediates the relationship between trait anxiety and burnout, confirming hypothesis 2, and this model provided better fit than the direct model of hypothesis 1. However, the mediation also indicated that the direct relationship between trait anxiety and burnout should be considered. The mediating model was invariant according to competitive levels and sport records. In conclusion, cognitive appraisal is an important variable in explaining athletes' burnout.

Tracking the dynamics of heterotrophs and nitrifiers in moving-bed biofilm reactors operated at different COD/N ratios.

In this study, the impact of COD/N ratio and feeding regime on the dynamics of heterotrophs and nitrifiers in moving-bed biofilm reactors was addressed. Based on DGGE analysis of 16S rRNA genes, the influent COD was found to be the main factor determining the overall bacterial diversity. The amoA-gene-based analysis suggested that the dynamic behavior of the substrate in continuous and pulse-feeding reactors influenced the selection of specific ammonium-oxidizing bacteria (AOB) strains. Furthermore, AOB diversity was directly related to the applied COD/N ratio and ammonium-nitrogen load. Maximum specific ammonium oxidation rates observed under non-substrate-limiting conditions were observed to be proportional to the fraction of nitrifiers within the bacterial community. FISH analysis revealed that Nitrosomonas genus dominated the AOB community in all reactors. Moreover, Nitrospira was found to be the only nitrite-oxidizing bacteria (NOB) in the fully autotrophic system, whereas Nitrobacter represented the dominant NOB genus in the organic carbon-fed reactors.

Cartilage Repair Using Hydrogels: A Critical Review of in Vivo Experimental Designs.

This review analyzes the outcomes and technical aspects of in vivo studies published in the past decade using gels and hydrogels for cartilage repair. Using PubMed search engine, original research publications during the period of 2002/01/01 to 2015/04/30 identified 115 published papers. Of these, 3 studies failed to find a statistically significant improvement of treatment group as compared to control and 18 studies did not clearly identify hyaline-like cartilage formation in the treated groups. The most frequent repaired lesion was the rabbit acute full thickness trochlear defect, using a scaffold combining a gel or hydrogel and other material. One third of the scaffolds were cell-free (35%) and the majority of the studies did not use growth factors (71%). The present review may constitute a useful tool in design of future studies, as limitations of study designs are pointed and results in terms of translation to human application is discussed.

Neutrophil extracellular traps formation by bacteria causing endometritis in the mare.

Besides the classical functions, neutrophils (PMNs) are able to release DNA in response to infectious stimuli, forming neutrophil extracellular traps (NETs) and killing pathogens. The pathogenesis of endometritis in the mare is not completely understood. The aim was to evaluate the in vitro capacity of equine PMNs to secrete NETs by chemical activation, or stimulated with Streptococcus equi subspecies zooepidemicus (Szoo), Escherichia coli (Ecoli) or Staphylococcus capitis (Scap) strains obtained from mares with endometritis. Ex vivo endometrial mucus from mares with bacterial endometritis were evaluated for the presence of NETs. Equine blood PMNs were used either without or with stimulation by phorbol-myristate-acetate (PMA), a strong inducer of NETs, for 1-3h. To evaluate PMN ability to produce NETs when phagocytosis was impaired, the phagocytosis inhibitor cytochalasin (Cyt) was added after PMA. After the addition of bacteria, a subsequent 1-h incubation was carried out in seven groups. NETs were visualized by 4',6-diamidino-2-phenylindole (DAPI) and anti-histone. Ex vivo samples were immunostained for myeloperoxidase and neutrophil elastase. A 3-h incubation period of PMN + PMA increased NETs (p < 0.05). Bacteria + 25 nM PMA and bacteria + PMA + Cyt increased NETs (p<0.05). Szoo induced fewer NETs than Ecoli or Scap (p < 0.05). Ex vivo NETs were present in mares with endometritis. Scanning electron microscopy showed the spread of NETs formed by smooth fibers and globules that can be aggregated in thick bundles. Formation of NETs and the subsequent entanglement of bacteria suggest that equine NETs might be a complementary mechanism in fighting some of the bacteria causing endometritis in the mare.

Short communication: Antimicrobial resistance and virulence characterization of methicillin-resistant staphylococci isolates from bovine mastitis cases in Portugal.

Methicillin-resistant staphylococci (MRS) have already been reported as mastitis agents. Such bacterial species are a public health concern, and the characterization of their antimicrobial resistance and virulence profile is important to better control their dissemination. The present work evaluated the distribution of methicillin-resistance among 204 staphylococci from clinical (n=50) and subclinical (n=154) bovine mastitis. The presence ofthe mecA gene was determined by PCR. Phenotypic expression of coagulase, DNase, lipase, gelatinase, hemolytic enzymes, and biofilm production was evaluated. The presence of biofilm-related genes, icaA, icaD, and bap, was also determined. Antimicrobial resistance patterns for aminoglycosides, lincosamides, macrolides, fluoroquinolones, sulphonamides, tetracyclines, and fusidic acid were determined. Nineteen (9.3%) isolates were identified as MRS, and the presence of mecA in these isolates was confirmed by PCR. Virulence factors evaluation revealed that gelatinase was the most frequently detected (94.7%), followed by hemolysins (73.7%) and lipase (68.4%); 84.2% of the MRS isolates produced biofilm and icaA and icaD were detected in almost half of the MRS isolates (52.6%), but all were bap-negative. Resistance against other antimicrobial agents ranged from 0 (fusidic acid, ciprofloxacin, norfloxacin, enrofloxacin) to 100% (nalidixic acid). Resistance to nalidixic acid and nalidixic acid-tetracycline were the most common antimicrobial resistance profiles (31.6%). This study confirms that despite the low prevalence of MRS, isolates frequently express other virulence traits, especially biofilm, that may represent a serious challenge to clinicians.

Quantidade de metionina+cistina digestível para poedeiras semipesadas de 75 a 91 semanas de idade / Nutritional requirement of digestible methionine + cystine for brown-egg laying hens from 75 to 91weeks of age

Determinaram-se as exigências nutricionais de metionina+cistina digestível para poedeiras de linhagem comercial no segundo ciclo de produção de 75 a 91 semanas de idade, utilizando-se 150 aves semipesadas em delineamento inteiramente ao acaso. Estas foram distribuídas em cinco tratamentos, seis repetições e cinco aves por unidade experimental, alimentadas com uma dieta basal contendo 2.859kcal/kg de energia metabolizável e 16,30% de proteína bruta, suplementada com 0,132; 0,173; 0,215; 0,256 e 0,298% de DL-metionina (98%), de forma a proporcionar 0,588; 0,628; 0,669; 0,709 e 0,750% de metionina+cistina digestível na dieta. A inclusão de metionina+cistina obedeceu, respectivamente, às proporções de 67, 72, 77, 81 e 86% com a lisina fixada em 0,872%. Avaliaram-se os consumos de ração e de metionina+cistina, a conversão alimentar por dúzia e por massa de ovos, a taxa de postura, o peso e a massa de ovos, a porcentagem dos componentes dos ovos e a qualidade interna dos ovos e o ganho de peso. A inclusão de metionina+cistina digestível nas quantidades indicadas não exerceu efeito (P>0,05) sobre as características de produção e qualidade interna e externa dos ovos. A exigência estimada foi de 0,588% de metionina+cistina digestível, correspondendo ao consumo de 654,73mg/ave/dia.

This work determined the nutritional requirements for methionine + cystine for commercial hens in the second production cycle from 75 to 91 weeks of age, using 150 brown-egg layer hens in a completely randomized design, distributed in five treatments, six replicates of five hens each and fed a basal diet containing 2859kcal/kg of metobolizable energy, 16.30% crude protein supplemented with 0.132, 0.173, 0.215, 0.256 and 0.298% DL-methionine (98%), in order to provide 0.588, 0.628, 0.669, 0.709 and 0.750% methionine + cystine in the diet. The inclusion of methionine + cystine followed, respectively, the proportions of 67, 72, 77, 81 and 86% with lysine fixed at 0.872%. The feed intake, methionine + cystine intake, feed conversion per dozen eggs and egg mass, percentage of the eggs, egg weight, egg mass, percentage of egg components and internal quality of eggs and weight gain were evaluated. The inclusion methionine + cystine in the amounts indicated had no effect (P> 0.05) on the production characteristics and internal and external quality of eggs. The requirement was estimated at 0.588% methionine + cystine intake corresponding to 654.73mg/hen/day.

Effects of pine processionary caterpillar Thaumetopoea pityocampa contact in dogs: 41 cases (2002-2006).

The pine processionary caterpillar, Thaumetopoea pityocampa, is considered an emerging pine pest in Mediterranean countries, with high medical relevance. In recent years, adverse reactions reports in humans following contact with T. pityocampa have been increasingly reported. Dogs living in pinewood areas are also frequently exposed to the caterpillar. This work consisted on a retrospective study of 41 cases of lepidopterism. All dogs presented drooling, dysphagia, submandibular lymphadenomegaly and clinical signs of pain. The animals were distributed in three groups, according to the time span from exposure to the caterpillar until presentation: up to 2 h (group 1), 2-5 h (group 2) and more than 5 h (group 3). All animals from groups 2 (n = 5) and 3 (n = 9), and eight dogs from group 1 (n = 27) developed lingual necrosis. Lepidopterism coursed through a predictable clinical pattern. The evolution was mainly dependent on the time span between exposure to the caterpillar and medical intervention, which should take place earlier than 2 h from exposure.

Clinical and bacteriological survey of diabetic foot infections in Lisbon.

AIMS: An epidemiological survey of diabetic foot infections (DFIs) in Lisbon, stratifying the bacterial profile based on patient demographical data, diabetic foot characteristics (PEDIS classification), ulcer duration and antibiotic therapy. METHODS: A transversal observational multicenter study, with clinical data collection using a structured questionnaire and microbiological products (aspirates, biopsies or swabs collected using the Levine method) of clinically infected foot ulcers of patients with diabetes mellitus (DM). RESULTS: Forty-nine hospitalized and ambulatory patients were enrolled in this study, and 147 microbial isolates were cultured. Staphylococcus was the main genus identified, and methicillin-resistant Staphylococcus aureus (MRSA) was present in 24.5% of total cases. In the clinical samples collected from patients undergoing antibiotic therapy, 93% of the antibiotic regimens were considered inadequate based on the antibiotic susceptibility test results. The average duration of an ulcer with any isolated multi-drug resistant (MDR) organism was 29 days, and previous treatment with fluoroquinolones was statistically associated with multi-drug resistance. CONCLUSIONS: Staphylococcus aureus was the most common cause of DFIs in our area. Prevalence and precocity of MDR organisms, namely MRSA, were high and were probably related to previous indiscriminate antibiotic use. Clinicians should avoid fluoroquinolones and more frequently consider the use of empirical anti-MRSA therapy.

The hard-tick fauna of mainland Portugal (Acari: Ixodidae): an update on geographical distribution and known associations with hosts and pathogens.

This work is an updated revision of the available information on Portuguese ixodid tick species. It includes data on tick biology, ecology, taxonomy and host/pathogen-associations. The current list of Portuguese ixodid ticks comprises twenty species: Dermacentor marginatus (Sulzer, 1776), Dermacentor reticulatus (Fabricius, 1794), Haemaphysalis hispanica Gil Collado, 1938, Haemaphysalis inermis Birula, 1895, Haemaphysalis punctata Canestrini & Fanzago, 1878, Hyalomma lusitanicum Koch, 1844, Hyalomma marginatum Koch, 1844, Ixodes acuminatus Neumann, 1901, Ixodes bivari Dias, 1990, Ixodes canisuga Johnston, 1849, Ixodes frontalis (Panzer, 1798), Ixodes hexagonus Leach, 1815, Ixodes ricinus (Linnaeus, 1758), Ixodes simplex Neumann, 1906, Ixodes ventalloi Gil Collado, 1936, Ixodes vespertilionis Koch, 1844, Rhipicephalus (Boophilus) annulatus (Say, 1821), Rhipicephalus bursa Canestrini & Fanzago, 1878, Rhipicephalus pusillus Gil Collado, 1938, and Rhipicephalus sanguineus (Latreille, 1806).

Antimicrobial resistance and in vitro biofilm-forming ability of enterococci from intensive and extensive farming broilers.

Enterococci, major broiler intestinal colonizers, play a recognized role in antimicrobial resistance transmission. Several virulence mechanisms, such as biofilm expression, have been identified. Minimum inhibitory concentrations of vancomycin, enrofloxacin, oxytetracycline, streptomycin, and gentamicin and biofilm production of 34 isolates from intensive and extensive farming system broilers were evaluated. All isolates were susceptible to vancomycin. In extensive-reared broilers (n = 18), resistance to enrofloxacin, oxytetracycline, streptomycin, and gentamicin was high (83.33, 55.56, 100, and 83.33%, respectively). Intensive farming broilers (n = 16) showed a lower resistance level for enrofloxacin and streptomycin and a higher resistance level for oxytetracycline and gentamicin. The relation between antimicrobial susceptibility and farming system was not significant for all drugs tested (P > or = 0.05). Enterococci produced biofilm at 24 h (47.0%), 48 h (55.9%), and 72 h (58.8%). Resistance to gentamicin and streptomycin was related to biofilm production at all time points (P < or = 0.05), whereas resistance to enrofloxacin was only related to biofilm at 24 h (P < or = 0.05; Friedman's test). No relation was found between susceptibility to oxytetracyclin and biofilm formation at any of the 3 time points studied (P > or = 0.05). Poultry are colonized by biofilm-producing and antimicrobial-resistant enterococci, independently of the farming system. Results show a relation between resistance to the majority of the drugs tested and biofilm production, which reenforces the importance of these virulence factors in animal and public health.

Clinical and bacteriological assessment of dog-to-dog bite wounds.

Although dog-to-dog bite wounds are frequent, few studies correlate bacterial involvement to clinical aspects. This work aimed at relating clinical evolution and bacteriological data, with the evolution time (ET), i.e., the period of time elapsed from aggression until presentation. A total of 228 wounds from 83 cases of bitten dogs was evaluated; 48 of the wounds were sampled for bacteriology. Dogs with clinically infected wounds (N=29) were subjected to antimicrobial therapy and local disinfection. Dogs without clinical signs of infection were either subjected to the same treatment (N=43) or only subjected to daily wound saline irrigation (N=11), to evaluate the need for antimicrobial prophylaxis. The majority of wounds were laceration and puncture wounds with dermis penetration (41.2% and 23.2%, respectively). Only 17% of the wounds were clinically infected. The mean ET was 39h 30m and prolonged ET was significantly correlated to infection. None of the wounds from animals not given antibiotics became infected. Bacteriology was positive in 95.8% of the wounds sampled (N=46). A total of 125 isolates was obtained, mostly aerobes. Clinical infection was associated with the presence of strict anaerobes. Excluding antibiotics that should be preserved for life-threatening cases, the higher rates of in vitro antimicrobial susceptibility were observed for sulphamethoxazole-trimethoprim (94.4%) and amoxicillin-clavulanate (91.9%). This study identified the time lag from aggression to presentation as a risk factor for infection development. Further studies are required to evaluate the actual requirement for antimicrobial therapy when only dermis is affected in dog-to-dog bite wounds.

Molecular epidemiology and population structure of bovine Streptococcus uberis.

The molecular epidemiology and population structure of 30 bovine subclinical mastitis field isolates of Streptococcus uberis, collected from 6 Portuguese herds (among 12 farms screened) during 2002 and 2003, were examined by using pulsed-field gel electrophoresis (PFGE) for clustering of the isolates and multilocus sequence typing (MLST) to assess the relationship between PFGE patterns and to identify genetic lineages. The 30 isolates were clustered into 18 PFGE types, using a similarity cutoff of 80%, and 3 PFGE types accounted for almost half of the isolates (46.6%). These major types were herd specific, suggesting either cow-to-cow transmission or infection with isolates from the same environmental reservoirs. The remaining unrelated PFGE types of isolates were from different herds strongly suggesting environmental sources of Strep. uberis infection. All 30 isolates were analyzed by MLST and clustered into 14 sequence types (ST). These ST were found to be novel, either with 10 new alleles of 6 housekeeping genes or with different combinations of previously assigned alleles. Five of these ST were clustered into 3 clonal complexes (lineages), ST-143, ST-86, and ST-5, known to include bovine isolates from several geographic locations (Australia, New Zealand, United Kingdom, Sweden, and Denmark) and 9 singletons. To our knowledge, this is the first report that documents molecular typing studies of bovine isolates of Strep. uberis from Portugal, which were shown to represent novel genomic backgrounds of this pathogen.

Genomic characterization of Arcanobacterium pyogenes isolates recovered from the uterus of dairy cows with normal puerperium or clinical metritis.

Arcanobacterium pyogenes is considered to be the most relevant bacterium involved in the establishment of puerperal uterine infection in cattle due to its persistence in utero, resistance to treatment and synergic action with Gram negative anaerobes. Once the infection is established, A. pyogenes is responsible for the persistence of the infection. The objective of this study was to characterize A. pyogenes field isolates recovered from the uterus of cows with either normal puerperium or clinical metritis, in an attempt to identify factors that might be associated with the establishment and persistence of the disease. This characterization was based on BOX-PCR typing and on screening of eight virulence factor genes (plo, nanP, nanH, cbpA, fimA, fimC, fimE, fimG) by conventional PCR. Finally, a relationship between clonal types, virulence factors and presence of disease was investigated. A. pyogenes clonal types identified from isolates recovered from the uterus of postpartum dairy cows differed among herds. Although some clonal types were strictly associated with the development of clinical metritis, others were identified from isolates recovered from normal puerperium and clinical metritis cows. Moreover, the presence of the eight virulence factor genes was not related with the ability to induce clinical metritis, suggesting that the type of A. pyogenes may not be a determinant factor in the development of the disease. We suggest that host intrinsic factors, the synergism between A. pyogenes and other bacteria and the differential gene expression of virulence factor genes may play a more relevant role in the establishment of puerperal uterine infections.

First assessment of microbial diversity in faecal microflora of Eurasian otter (Lutra lutra Linnaeus, 1758) in Portugal.

The Eurasian otter (Lutra lutra Linnaeus, 1758), a predator from the Order Carnivora, Family Mustelidae, evolved the ability to swim and forage in water, being an important element of biodiversity. Otters are widely spread through Portugal, and scats have been extensively used in ecology studies; however, valid information on their microbiota is scarce. This work represents a first approach to characterise the otter faecal microflora in samples collected in river stretches of the Sado river basin (Portugal) during winter 2006. Eight sampling stretches of 8 km were selected, and from each, six to eight sampling sites were visited. A total of 31 scats were analysed. The microflora studied included aerobic bacteria, spore-forming anaerobic bacteria and viruses (coronavirus, parvovirus, adenovirus, parainfluenza virus). Bacterial isolates were identified based on morphology and metabolic pathways, and virus detection was performed by polymerase chain reaction. The results revealed the high degree of bacterial diversity in the faecal microflora of L. lutra. A total of 88 Gram-negative (23 genera) and 44 Gram-positive isolates (ten genera) were identified. The identification of four isolates was inconclusive, and their identification was performed by 16S ribosomal ribonucleic acid sequencing, which confirms the need for biochemical testing optimisation regarding animal isolates. None of the scats was positive for virus detection. Identification of otter faecal microflora and of potential pathogens is an important first step towards understanding and monitoring their importance in otter population health.

Technical note: Antimicrobial susceptibility of Portuguese isolates of Staphylococcus aureus and Staphylococcus epidermidis in subclinical bovine mastitis.

To evaluate the antimicrobial resistance traits of staphylococci responsible for subclinical bovine mastitis in Portugal, the minimum inhibitory concentrations (MIC) of 7 antimicrobial agents, frequently administered for mastitis treatment, were determined for 30 Staphylococcus aureus and 31 Staphylococcus epidermidis field isolates. Beta-lactamase production was detected through the use of nitrocefin-impregnated discs. The MIC that inhibited 90% of the isolates tested (MIC90) of penicillin, oxacillin, cefazolin, gentamicin, sulfamethoxazole/trimethoprim, oxytetracycline, and enrofloxacin were, respectively, 4, 0.5, 1, 1, 0.25, 0.25, and 0.06 microg/mL for Staph. aureus and > or = 64, 8, 1, 32, > or = 64, > or = 64, and 0.06 microg/mL for Staph. epidermidis. All Staph. aureus isolates showed susceptibility to oxacillin, cefazolin, gentamicin, sulphamethoxazole/trimethoprim, and enrofloxacin. Beta-lactamase production was detected in 20 of these isolates (66.7%), all of which were resistant to penicillin. Of the 31 Staph. epidermidis tested, 24 (77.4%) were beta-lactamase positive. All isolates were susceptible to both cefazolin and enrofloxacin. Nine Staph. epidermidis isolates were resistant to oxacillin, with MIC values ranging from 4 to 8 microg/mL. The MIC values of 5 antimicrobial agents tested were higher than those reported in other countries. Enrofloxacin was the only exception, showing lower MIC values compared with other reports. Overall, the antimicrobial agents tested in our study, with the exception of penicillin, were active against the 61 isolates studied.

Time course of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates.

Biofilm formation is considered a selective advantage for staphylococci mastitis isolates, facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers and enclosing in a polymeric matrix, being regulated by several loci. As biofilm formation can proceed through different pathways and time ranges, its detection may differ according to the time of observation. This study aimed at evaluating the time course evolution of biofilm production in Staphylococcus aureus (n = 26) and Staphylococcus epidermidis (n = 29) mastitis isolates by Fluorescent In Situ Hybridisation. Biofilm-forming ability increased with incubation time for both species: for S. aureus, 34.6%, 69.2% and 80.8% of the isolates were able to produce biofilm at 24, 48 and 72 h, respectively. For S. epidermidis, 44.8%, 62.1% and 75.9% of the isolates were biofilm-positive at 24, 48 and 72 h, respectively. No significant difference was found between species at each time point (Friedman's test, p > 0.05). For S. aureus, although a significant difference was found between 24 and 48 h (Wilcoxon matched paired test, p < 0.05), no significant difference was found between 24 and 48 h (p > 0.05). For S. epidermidis, significant differences were found between each time point (p < 0.05). Bacterial biofilms may impair eradication of chronic mastitis, rendering antibiotherapy less effective. Detection of biofilm-forming ability in mastitis isolates may provide useful information for the establishment of a more adequate therapeutic regimen, in view of the antimicrobial concentrations required for bacterial control. However, it is essential that biofilm formation time course is taken into consideration.

Identification of 14 novel mutations in the long isoform of USH2A in Spanish patients with Usher syndrome type II.

Mutations in USH2A gene have been shown to be responsible for Usher syndrome type II, an autosomal recessive disorder characterised by hearing loss and retinitis pigmentosa. USH2A was firstly described as consisting of 21 exons, but 52 novel exons at the 3' end of the gene were recently identified. In this report, a mutation analysis of the new 52 exons of USH2A gene was carried out in 32 unrelated patients in which both disease-causing mutations could not be found after the screening of the first 21 exons of the USH2A gene. On analysing the new 52 exons, fourteen novel mutations were identified in 14 out of the 32 cases studied, including 7 missense, 5 frameshift, 1 duplication and a putative splice-site mutation.

Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates.

Biofilm-forming ability has been increasingly recognized as an important virulence factor in Staphylococci, facilitating their persistence in the host, evading its defences and allowing bacterial survival at high antimicrobial concentrations. Staphylococcus aureus remains a major pathogen of chronic mastitis, but in the last years Staphylococcus epidermidis has emerged as a relevant mastitis pathogen. The present work aimed at the evaluation of the biofilm-forming ability of Staphylococci field isolates from bovine subclinical mastitis and at the development of a fluorescent in situ hybridisation (FISH) protocol that would allow the direct observation of biofilm formation in milk samples. The analysis of phenotypic expression in Congo Red Agar (CRA) and by FISH, showed that 37.5% of the S. aureus isolates produced biofilm, while by optical density measurement only 18.75% isolates revealed this phenotype. The results showed a fair agreement according to the kappa coefficient test (kappa = 0.259). Regarding S. epidermidis mastitis isolates, 37.5% revealed the ability to produce biofilm, but only four isolates were positive by all methods. This agreement was moderate (kappa = 0.467). The application of FISH to artificially contaminated milk samples allowed the direct observation of biofilm production by 37.5% isolates, showing total agreement with the CRA results. This method better mimics the in vivo conditions, especially in terms of the presence of calcium and iron, which in high concentrations, respectively, are known to inhibit or induce biofilm production.

Mutation profile of the MYO7A gene in Spanish patients with Usher syndrome type I.

Usher syndrome type I is the most severe form of Usher syndrome. It is an autosomal recessive disorder characterized by profound congenital sensorineural deafness, retinitis pigmentosa, and vestibular abnormalities. Mutations in the myosin VIIA gene (MYO7A) are responsible for Usher syndrome type 1B (USH1B). This gene is thought to bear greatest responsibility for USH1 and, depending on the study, has been reported to account for between 24% and 59% of USH1 cases. In this report a mutation screening of the MYO7A gene was carried out in a series of 48 unrelated USH1 families using single strand conformation polymorphism analysis (SSCP) and direct sequencing of those fragments showed an abnormal electrophoretic pattern. Twenty-five mutations were identified in 23 out of the 48 families studied (47.9%). Twelve of these mutations were novel, including five missense mutations, three premature stop codons, three frameshift, and one putative splice-site mutation. Based on our results we can conclude there is an absence of hot spot mutations in the MYO7A gene and that this gene plays a major role in Usher syndrome.