Diagnostic evaluation of a point-of-care test for culture and microbial susceptibility testing in canine dermatological infections in clinical practice

Background and Aim: Empirical antimicrobial therapy is frequently given in superficial bacterial folliculitis (SBF) and otitis externa (OE) in dogs, especially for the initial clinical presentation. Culture and subsequent antimicrobial susceptibility testing (AST) are generally limited to chronic cases with poor response to initial therapy. Several factors contribute to the failure to implement the use of AST in veterinary practice, i.e., long laboratory turnaround time or special requirements for sample shipping. Point-of-care (PoC) testing might reduce laboratory turnaround time and costs and the risk of emergence of multidrug-resistant pathogens. This study evaluated the Speed Biogram\u2122 PoC test in canine SBF and OE compared with conventional methods for culture and AST. Materials and Methods: Thirty-four canine samples were analyzed: eleven from SBF, seven from bacterial OE, four from mixed OE, six from Malassezia spp. OE, and six negative controls. Sensitivity (Se) and specificity (Sp) of the PoC test and the agreement between the PoC test and conventional methods were evaluated. Results: Se and Sp of PoC test in discriminating between healthy and unhealthy subjects were 100% (95% confidence interval [CI] 87.66-100.00) and 100% (95% CI 54.1-100.0), respectively. For bacterial identification, the k value was 0.532. Se and Sp of PoC tests for AST were 81.73% (95% CI 72.95-88.63) and 93.10% (95% CI 88.86-96.98), respectively with a total good agreement between tests (mean k=0.714), but major (8/27) and very major (19/27) errors were observed in 55% of bacterial conventional culture-positive samples. Conclusion: PoC test can identify dogs with SBF and OE, but AST is not sufficiently accurate. The lack of susceptibility testing for methicillin makes this test inappropriate for use in small animal practice

Biofilm-forming ability and virulence factors of methicillin-resistant Staphylococcus pseudintermedius from canine pyoderma

Staphylococcus pseudintermedius (SP) has been associated with high antibiotic-resistance rates (e.g. methicillin) in European countries [1,3]. This condition could be also related to the ability to produce biofilm [2]. The aim of this study was to investigate the presence of methicillin-resistant SP strains and determine their ability to produce biofilm and some crucial virulence factors. Forty-two SP strains, previously determined as multi drug resistant (MDR) by the disk diffusion method using a panel of 17 antimicrobial agents, were selected from our collection and tested phenotypically for the minimum inhibitory concentration (MIC) of methicillin and genotypically for the presence of mecA and blaZ genes. The ability to produce biofilm was assessed phenotypically by two different assays: the Congo Red Agar plates (CRA) and the Microtitre Plate test (MtP) and genetically by the amplification of icaA and icaD genes. Three virulence factors genes coding for bicomponent leukocidin and enterotoxins (luk-I, seC, se-int) were searched. Twenty-three strains revealed a value of MIC for the methicillin greater than 128 µg/mL. The 83% were mecA-positive and 86% resulted blaZ-positive; all the strains positive for mecA were also positive for blaZ. All SP strains resulted biofilm-producers by MtP assay and classified as weakly producers (4.7%), moderate producers (47.6 %) and strongly producers (47.6 %). In contrast, only 35.7% of all strains were considered biofilm-producers by CRA method. The amplification of icaA and icaD gene occurred respectively in 66.6% and 97.6%; only one strain was negative for both genes. Almost all strains were positive for luk-I (95%), seC (74%) and se-int (84%). Our data reveal the pathogenicity potential of SP strains from dogs, suggesting that they could be considered zoonotic potential agents and confirming other previous studies [3-5]. Moreover could be observed a clear linkage between antibiotic-resistance and ability to produce biofilm

Hematological, biochemical and microbiological evaluation of feline whole blood units collected using an open system and stored for 35 days

Despite the increasing availability of feline blood, which is collected and stored for transfusion purposes, few studies have assessed the effect of storage on feline whole blood (WB) units. The purpose of this study was to investigate selected hematologic and biochemical changes during storage of feline WB units and to determine when they occurred. Data from a quality control program for WB units was used in this study. Twelve feline WB units, collected using an open system, were sampled every 7 days from the point of collection to the end of storage at 35 days (D0, D7, D14, D21, D28, and D35). Measurements at each time point were: (1) hematologic parameters; (2) percentage hemolysis; (3) morphologic index scored at 0\u20133, based on echinocyte transformation of the erythrocytes; and (4) selected biochemical parameters. Aerobic and anaerobic culture was performed at D0 and D35. Results were compared statistically to D0 (statistical significance set at <0.01). Storage did not result in statistically significant changes in measured hematological parameters. There were statistically significant increases in percentage hemolysis and morphologic index, starting from D21 (P = 0.000 and P = 0.004, respectively). Glucose decreased significantly from D21 (P = 0.003); potassium increased significantly from D7 (P=0.001); and sodium increased significantly, starting from D28 (P = 0.009). Bacteria were not isolated. Blood in feline WB units collected using an open system underwent some significant storage changes that were time-dependent. As these changes could affect the quality and the utility of stored WB used in feline transfusion medicine, further study is required to determine their clinical importance

Evaluation of Feline Packed Red Blood Cell Units Obtained by Blood Sedimentation and Stored for 42 Days for Transfusion Purposes

Component therapy involves separation of whole blood (WB) into its components (packed red blood cells\u2014PRBCs\u2014and plasma), for specific replacement therapy and to reduce transfusion reactions. In cats, blood for transfusion is commonly collected using an open system and administered as WB, in part because of the challenge of preparing components from a small blood volume. Feline blood has a high erythrocyte sedimentation rate; therefore, if the syringe containing collected blood is placed upright, plasma can be removed from the red cells shortly after collection for separate storage of plasma and PRBCs. The aim of this study was to assess the characteristics of feline PRBC units obtained by blood sedimentation both at collection and after storage for 42 days. Blood was collected from fourteen feline blood donors into three 20-ml syringes pre-charged with CPDA-1:blood ratio of 1:7 using an open system. A pre-donation CBC was performed in each donor. The three syringes were allowed to sediment for approx. 1 hour at room temperature. Then plasma was aseptically expressed into plain transfer bags and RBC expressed into another transfer bag pre-charged with 10 ml of SAG-M. PRBCs units were stored in a blood-dedicated refrigerator and sampled using blood bag segments at preparation time (D0) and after 42 days storage (D42). On pre-donation blood and on PRBC units at D0 and D42 the following parameters were evaluated: I) hematological parameters (RBC, Hb, Hct, WBC, PLT); II) percentage hemolysis; III) morphological index (only for PRBC units), scored of 0 to 3 based on echinocyte transformation of the normal discocyte; IV) aerobic and anaerobic blood culture (only for PRBC units). From donor to PRBC units there was a significant increase in RBC count (mean increase +1886\ub1SD1399 \u3bcL/103), Hb concentration (+2.8\ub12.2 g/dl), Hct percentage (+8.3\ub15.5%). Significant reduction was found in PLT count (-249\ub1189 \u3bcL/103). Comparing PRBC at D0 and D42 a significant increase was found in percentage hemolysis (+1.2%), morphological index (+0.9) and a significant reduction in RBC count (-460\ub1679 \u3bcL/103), Hct percentage (-3.2\ub13.5%), WBC count (median -2589 \u3bcL/103), and PLT count (median -43 \u3bcL/10). All blood cultures were negative for bacterial growth. PRBC units obtained by sedimentation of donated blood appear to be a suitable blood component for treatment of normovolemic anemia. However, storage for 42 days, as suggested for canine and feline PRBC units, resulted in significant hematological changes that could reduce oxygen delivery after transfusion

Evaluation of feline red blood cells collected with an open system and stored for 35 days as whole blood units

Introduction: The increasing access to veterinary hospital blood banks and commercial sources of feline blood products means that transfusion therapy is more widely available to veterinarians and feline stored blood products are used more often. Despite the increasing availability of feline blood collected and stored for transfusion purposes, few studies have investigated storage lesions in feline whole blood (FWB) units and no study has evaluated hematological changes in FWB units. The objective of this study was to assess changes in feline RBCs collected and stored for transfusion purposes as FWB units. Methods:A prospective, laboratory invitrostudy wasconducted. Twelve nonleukoreduced FWB units were collected with an open system using three 20-mL syringes prefilled with citrate, phos- phate, dextrose, and adenine (CPDA-1) preservative-anticoagulant solution with ratio with blood of 1:7 from anesthetized feline blood donors. Units were stored in a blood bank dedicated refrigerator and sampled every 7 days (D7, D14, D21, D28) from collection (D0) to the end of storage (35 days, D35). At each time point, the following were evaluated: (1) hematological parameters (RBC, HGB, HCT, MCV, MCH, MCHC, RDW); (2) percentage of hemolysis; (3) morphological index, scored of 0 to 4 based on echinocyte transformation of the normal discocyte; and (4) aerobic and anaerobic blood culture. Results were statistically compared to D0, with t-test or Wilcoxon test, as appropriate with statistical significance set at P < 0.01. Results: There was no significant difference in hematological parameters at any time point with respect to D0. Significant increases were found in percentage of hemolysis and morphological index starting from 21 days of storage (P = 0.0002 and P = 0.0039, respectively). Mean hemolysis percentage value was less than 1% up to 21 days of storage. All blood cultures were negative for bacterial growth. Conclusion: RBCs in FWB units collected with an open system can undergo some significant hematological changes, but these results suggest that storage for up to 21 days is safe. In vivo studies are required to establish if these changes affect the ability of stored RBCs to circulate and provide adequate oxygen delivery after transfusion

In Vitro Efficacy of Essential Oils from Melaleuca Alternifolia and Rosmarinus Officinalis, Manuka Honey-based Gel, and Propolis as Antibacterial Agents Against Canine Staphylococcus Pseudintermedius Strains

Abstract: Essential oils (EOs) and honeybee products (e.g., honey and propolis) are natural mixtures of dierent volatile compounds that are frequently used in traditional medicine and for pathogen eradication. The aim of this study was to evaluate the antibacterial properties of tea tree (Melaleuca alternifolia) EO (TTEO), Rosmarinus ocinalis EO (ROEO), manuka-based gel, and propolis against 23 strains of Staphylococcus pseudintermedius (SP) isolated from canine pyoderma. Antimicrobial resistance screening was assessed using a panel of nine antimicrobial agents coupled with a PCR approach. An aromatogram was done for both EOs, using the disk diusion method. The minimum inhibitory concentration (MIC) was determined for all the compounds. Among the 23 SP strains, 14 (60.9%) were multidrug-resistant (MDR), 11 strains (47.8%) were methicillin-resistant (MRSP), and 9 (39.1%) were non-MDR. The mean diameter of the inhibition zone for Melaleuca and Rosmarinus were 24.5 8.8 mm and 15.2 8.9 mm, respectively, resulting as statistically dierent (p = 0.0006). MIC values of TTEO and ROEO were similar (7.6 3.2% and 8.9 2.1%, respectively) and no statistical significances were found. Honeybee products showed lower MIC compared to those of EOs, 0.22 0.1% for Manuka and 0.8 0.5% for propolis. These findings reveal a significant antibacterial eect for all the tested products

Effects of probiotic Lactobacillus acidophilus D2/CSL (CECT 4529) on the nutritional and health status of boxer dogs

Background The aim of the present study was to investigate the effects of Lactobacillus acidophilus D2/CSL (CECT 4529) probiotic strain on nutritional status and faecal and microbiological parameters in a group of purebred boxers. Methods Forty healthy adult boxer dogs were randomly assigned to a treated (LACTO) group receiving a commercial diet supplemented with L acidophilus D2/CSL (CECT 4529) to a final concentration of 5.0 x 109 colony-forming unit/kg of food, and a control (CTR) group receiving the same diet but without the probiotic (placebo). Nutritional status (body weight, skinfold thickness, body condition score) and faecal quality parameters were analysed. Results No differences in body weight and skin thickness were found during the whole experimental period. Dogs in the LACTO group showed a significantly higher body condition score than those in the CTR group (4.86\ub10.55 v 4.65\ub10.65), and no significant differences were recorded in body weight and skinfold thickness. The LACTO group showed a significantly lower faecal moisture (in per cent) compared with the CTR group (0.67\ub10.007 v 0.69\ub10.007). Faecal hardness (in kg) was higher in the LACTO group than in the CTR group (0.86\ub10.047 v 0.70\ub10.051), and faecal score also improved in the LACTO group (3.78\ub10.95 v 4.25\ub10.91). A significant difference in total Escherichia coli counts as well as in lactobacilli counts between the CTR and LACTO groups was only detected at 28 days. Conclusion Supplementation of L acidophilus D2/CSL (CECT 4529) significantly improved the nutritional status and faecal parameters of dogs

The bovine acute phase protein α1-acid glycoprotein (AGP) can disrupt Staphylococcus aureus biofilm

Staphylococcus aureus biofilm-related infections are of clinical concern due to the capability of bacterial colonies to adapt to a hostile environment. The present study investigated the capability of the acute phase protein alpha 1-acid glycoprotein (AGP) to a) disrupt already established S. aureus biofilm and b) interfere with the biofilm de novo production by using Microtiter Plate assay (MtP) on field strains isolated from infected quarters by assessing. The present study also investigated whether AGP could interfere with the expression of bacterial genes related to biofilm formation (icaA, icaD, icaB, and icaC) and adhesive virulence determinants (fnbA, fnbB, clfA, clfB, fib, ebps, eno) by quantitative real-time PCR (qPCR). The results provided the evidence that AGP could disrupt the biofilm structure only when it was already developed, but could not prevent the de novo biofilm formation. Moreover, AGP could interfere with the expression levels of genes involved in biofilm formation in a dose- and strain-dependent way, by upregulating, or downregulating, icaABC genes and fnbB, respectively. The results presented in this study provide new insights about the direct antibacterial activity of AGP in bovine milk. It remains to be demonstrated the molecular bases of AGP mechanism of action, in particular for what concerns the scarce capability to interact with the de novo formation of biofilm

Gut peculiarities of feed deprived white sturgeons (Acipenser transmontanus, Richardson 1836)

In the White sturgeon fish farms, some individuals have difficulty in getting access to food: such sturgeons are called \u201crunts\u201d, and they result in a slower growth rate than normally feeding fish. In this paper, we have studied the gut pecu- liarities of runt sturgeons. Utilizing in paralleling an analysis of diatom populations in both the fish gut tissues and the rearing tank waters, we hypothesized a causative relation between the occurrence of runt sturgeons and periodic diatom blooms. In fact, we have observed that the diatom species identified in the aquatic environment were also detected in organs (Fragilaria spp and Rhoicosfenia spp for both glandular body, mid-intestine) of the runt sturgeon\u2019s gut, but not in tissues of normally feeding individuals. Owing to their siliceous wall, diatoms can be responsible for areas of epithet- lial detachment in the mucosal surfaces of the alimentary canal and a catharral inflammation in both the gastric pits and intestinal folds which may be the cause of secondary bacterial diseases. We suggest that diatom blooms may contribute to the occurrence of runt sturgeons in the studied Italian fish farm

Proteomic study of antibiotic resistance in Escherichia coli strains

Enteropathogen Escherichia coli infection is the most common type of colibacillosis of young animals (primarily pigs and calves), and it is cause of diarrhoea among travellers and children in the developing world. The main virulence attributes of pathogens Escherichia coli are adhesins and enterotoxins, which are mostly regulated on large plasmids. In the current study, comparative proteomics was applied to identify changes in proteins responsible for antibiotic resistance in different in vivo isolates Escherichia coli. In particular it has been studied strains with same virulence factors, but a completely different antibiotic profile, obtained from different organs of the same anima