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.

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.

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.

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.

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.

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.

Bacteriological study of the liver in dogs.

This study aimed to confirm the possible presence of bacteria in the liver of healthy dogs. Laparotomy was performed in 20 animals admitted for routine abdominal surgery. To be selected for the study, dogs had to be healthy adults without clinical liver disease, signs of infection or macroscopic liver abnormalities. Biopsy samples were histologically and bacteriologically examined. Bacteriological analysis was negative for the livers of eight of the dogs. The remaining animals harboured a diverse bacterial flora in their liver. Twelve bacterial species were identified. Histology showed that the livers of 19 dogs had minor or no abnormalities, and only one animal had interstitial fibrosis and trabecular disarrangement. Histological changes were not related to the presence of bacteria. Thus the results showed that the liver of healthy dogs may harbour different bacterial species. These microorganisms did not cause any detectable manifestation of disease, despite being potential pathogens.

Feline pansteatitis revisited: hazards of unbalanced home-made diets.

Pansteatitis is caused by the consumption of high levels of unsaturated fatty acids and/or the insufficient intake of vitamin E, leading to inflammation of adipose tissue. This disease has been related to fish-based diets. However, non-conventional diets must also be considered. The authors present case records of two cats with pansteatitis, for which diet consisted mostly of pig's brain, comparing them with eight cases of disease in cats eating mainly oily fish. Cats fed pig's brain did not show clinical signs, while cats eating oily fish presented inappetence, depression, reluctance to move and subcutaneous nodules painful on palpation. Cats eating pig's brain did not show any change in blood parameters, while cats fed oily fish presented leukocytosis and anaemia. Histological examination confirmed pansteatitis in all cats, independently of the diet. All animals except one of the cats eating oily fish recovered after medical treatment and change of the feeding regime.