Proposal to reclassify Paenibacillus larvae subsp. pulvifaciens DSM 3615 (ATCC 49843) as Paenibacillus larvae subsp. larvae. Results of a comparative biochemical and genetic study

The bacterial pathogen Paenibacillus larvae subsp. larvae (P. l. larvae), is the etiological agent of American foulbrood, an extremely contagious and disastrous disease of honeybee brood. In case of American foulbrood the destruction of infected colonies is often considered the only workable control measure. Therefore, the ability to diagnose this disease properly is important to prevent unnecessary economic loss to beekeepers. The development of suitable methods for the early and reliable detection of P. l. larvae is hampered by the fact that the two subspecies of Paenibacillus larvae, P. l. larvae and Paenibacillus larvae subsp. pulvifaciens (P. l. pulvifaciens), seem to be indistinguishable by cultural characteristics as well as by PCR protocols. Here we present an extensive analysis of several P. larvae reference strains. We employed conventional culture techniques, morphological and biochemical identification, PCR-based methods and sequencing of the 16S rDNA. We found indeed that P. l. pulvifaciens strain DSM 3615 is indistinguishable from P. l. larvae (DSM 7030). We did not face any problems to discriminate between P. l. larvae and P. l. pulvifaciens strains DSM 8442 and DSM 8443. Therefore, classification of DSM 3615 as type strain of P. l. pulvifaciens seems not to be justified. We propose to reclassify this strain as P. l. larvae. Former problems in differentiating the two subspecies might have arisen from this misclassification. PCR-based methods as well as appropriate biochemical identification systems provide a reliable means for the discrimination between the two subspecies P. l. larvae and P. l. pulvifaciens.     

In vitro susceptibility of avian mycoplasmas to enrofloxacin, sarafloxacin, tylosin, and oxytetracycline.

In vitro susceptibility of avian Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) to enrofloxacin, sarafloxacin, tylosin, and oxytetracycline was determined by a serial broth dilution method. The minimum inhibitory concentration (MIC) was recognized by a conversion of the pH indicator phenol red in culture media to a yellow color. Each isolate or type strain of mycoplasma was tested in two replicates. The MICs of tylosin, enrofloxacin, sarafloxacin, and oxytetracycline against five isolates and two reference strains of MG (approximately 10(5) colony-forming units [CFU]/ml) were 0.05, 0.14, 0.37, and 1.30 microg/ml, respectively. The MICs of the four antimicrobial agents against six isolates and one reference strain of MS (approximate 10(5) CFU/ml) were 0.13, 1.82, 1.76, and 0.91 microg/ml, respectively. There were no differences (P > 0.05) between tylosin, enrofloxacin, and sarafloxacin against MG, but these three antibiotics were different (P < 0.05) from oxytetracycline. The MIC value of tylosin against MS was different (P < 0.05) from those of sarafloxacin and enrofloxacin, but it was not different (P > 0.05) from that of oxytetracycline.     

In vitro susceptibility of selected veterinary bacterial pathogens to ciprofloxacin, enrofloxacin and norfloxacin.

The minimum inhibitory concentrations (MIC) of ciprofloxacin, enrofloxacin, and norfloxacin were tested for approximately ten clinical isolates of each of Actinobacillus pleuropneumoniae, Actinobacillus suis, Actinomyces pyogenes, Corynebacterium pseudotuberculosis, Erysipelothrix rhusiopathiae, Haemophilus parasuis, Haemophilus somnus, Pasteurella haemolytica, Pasteurella multocida, Rhodococcus equi, Streptococcus equi, Streptococcus suis and Streptococcus zooepidemicus. Ciprofloxacin and enrofloxacin had similar activity and were more active than norfloxacin. All isolates had an MIC of 1.0 microgram/mL or less for ciprofloxacin and enrofloxacin, and these drugs had particularly marked activity against the gram-negative bacteria tested.     

In vitro susceptibility of field isolates of Francisella tularensis subsp. holarctica recovered in Spain to several antimicrobial agents

Forty-two recent (1997-1999) Spanish isolates of Francisella tularensis subsp.holarctica were tested in a broth microdilution method for their susceptibilities to 29 antimicrobial agents, including penicillins, cephalosporins, cephamicins, monobactams, penems, aminoglycosides, tetracyclines, macrolides, quinolones, chloramphenicol and fosfomycin. The isolates were resistant to beta-lactam antibiotics and susceptible to chloramphenicol, ciprofloxacin, levofloxacin and norfloxacin.     

In vitro antibiotic susceptibility of field isolates of Mycoplasma synoviae in Argentina

Minimum inhibitory concentrations (MICs) were determined in vitro for 7 antibiotics (aivlosin, enrofloxacine, tylosin, tiamulin, kitasamycin, chlortetracycline, and oxytetracycline) against eight recent local Argentinean isolates and two standard strains of Mycoplasma synoviae. Aivlosin (3-acetyl-4"-isovaleryl tylosin tartrate), tylosin, and tiamulin showed the lowest MICs with MIC90s of 0.006, 0.012, and 0.05 microg/ml, respectively. Except one strain that showed resistant values to chlortetracycline (> or = 12.5 microg/ml), all the analyzed strains were susceptible in different degrees to all the antibiotics tested. In this study, the improved activity of the tylosin-derived drug, aivlosin, was confirmed because it showed, in most strains, MIC values half those for tylosin.     

In vitro susceptibility of porcine respiratory pathogens to tilmicosin.

Bacterial isolates obtained from swine with various clinical diseases were tested for susceptibility to tilmicosin by minimum inhibitory concentration (MIC) and Kirby-Bauer disk diffusion tests using National Committee on Clinical Laboratory Standards methodology. The tilmicosin MIC90 was < or =0.125 microg/ml for Erysiopelothrix rhusiopathiae, < or = 1 microg/ml for Haemophilus parasuis isolates, 8 microg/ml for Actinobacillus suis and Pasteurella multocida type A, 16 microg/ml for toxigenic and nontoxigenic P. multocida type D, 64 microg/ml for Bordetella bronchiseptica, and >128 microg/ml for Staphylococcus hyicus and Streptococcus suis. The results of disk diffusion testing matched well with the MIC results for each pathogen. This in vitro survey of tilmicosin activity against various swine isolates suggests that further clinical evaluation of tilmicosin in swine may be warranted for disease associated with E. rhusiopathiae, H. parasuis, and A. suis but not B. bronchiseptica, S. suis, or S. hyicus.     

In vivo morphological and antigenic characteristics of Photobacterium damselae subsp. piscicida

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacterium''s structure and antigenicity when cultured in vivo is discussed.     

In vitro and in vivo characteristics of bacterial phytases and their efficacy in broiler chickens

1. Three bacterial phytases derived from Bacillus, Escherichia coli or Klebsiella were compared with a phytase derived from Aspergillus niger in vitro and in vivo. 2. The in vitro results indicated that Aspergillus, E. coli and Klebsiella phytase displayed their activity optima in an acid pH range while Bacillus phytase did so in neutral pH. 3. The trials also revealed that only Bacillus phytase is more resistant to heat treatments, while E. coli and Klebsiella phytases are more stable against proteolytic inactivation. 4. In vivo phytases derived from Aspergillus, Bacillus, E. coli, Klebsiella or a combination of Bacillus and E. coli improved the utilisation of phosphorus (P balance) significantly to 0.54, 0.54, 0.55, 0.55 or 0.58, respectively, compared to 0.42 in the negative control. 5. The phytases used in this study seemed to be equally effective in improving P utilisation regardless of proposed intestinal site of activity. Combination of phytases acting in the gizzard with phytases acting in the intestine seems to be a promising way to further improving in vivo efficacy of phytases in poultry.     

In vitro and in vivo characterization of avian Escherichia coli. I. Serotypes, metabolic activity, and antibiotic sensitivity

Over a 2 1/2-year period (January 1981 to June 1983), 177 Escherichia coli isolates were obtained from 145 field-reared broiler flocks in the Delmarva peninsula, and 20 were obtained from clinically normal day-old hatchery chickens representing an additional 17 flocks in Delmarva. Ninety-one isolates obtained from the field-reared birds between 2 and 8 weeks of age were associated with complicated air-sac disease. Serotyping efforts demonstrated a predominance of O2, O35, and O78 serogroups and a large number of untypable isolates. More than 50% of the isolates in each of the three dominant serogroups were collected from broilers with colibacillosis, but they were never detected in the yolk-sac samples of clinically normal day-old hatchery chickens. In vitro biochemical characterization of the E. coli isolates revealed variable rates of carbohydrate fermentation and amino acid decarboxylation. No common characteristics appeared to be shared by the predominant serogroups isolated from clinically affected birds, although several serogroup-specific reactions were noted. The majority of the isolates were sensitive to chloramphenicol, gentamicin, nalidixic acid, ormethoprim-sulfadimethoxine, spectinomycin, neomycin, and ampicillin. About half of the isolates were sensitive to nitrofurantoin and the sulfa compounds. Less than 25% of the isolates were sensitive to streptomycin, erythromycin, tetracyclines, novobiocin, penicillin, bacitracin, and lincomycin.     

In vitro and in vivo studies of the use of some medicinal herbals against the pathogen Aeromonas hydrophila in goldfish

Aeromonas hydrophila is a ubiquitous and opportunistic bacterial pathogen that produces ulcerative dermatitis under stress conditions and inflicts severe losses on global fisheries and fish culture. This study evaluates the antimicrobial potency of aqueous and ethanolic decoction (individual extract) and concoction (mixed extract) of three common medicinal herbs, turmeric Curcuma longa, Tulsi plant Ocimum sanctum, and neem Azadirachta indica, against the in vitro growth of A. hydrophila. Among the decoctions, A. indica exhibited the most potent antibacterial property (P < 0.05) against A. hydrophila. Among the concoctions, both the aqueous and ethanolic triherbal extracts mixed in the ratio of 1:1:1 had higher antibacterial activity (P < 0.05) than the other concoctions and decoctions. Goldfish Carassius auratus (10 +/- 2 g) were challenged with A. hydrophila intramuscularly in the caudal region with two separate doses (days 1 and 3) of 50 microL/fish (1.8 x 10(3) colony-forming units per milliliter). On days 9 (early) and 15 (late) of infection, fish were held in a net and dip treated for 5 min daily in a 1-L solution of 1% aqueous triherbal concoction. Red blood cell (RBC) count, hemoglobin, and hematocrit levels of the infected group were significantly higher (P < 0.05) than those of the control group. In the early treated group, all of the affected profile values returned to near normal, while the late-treated group registered a partial recovery, such as improved RBC count. The derived hematological values, such as mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, of the early and late-treated groups also significantly declined (P < 0.05) but were restored to near normal (P > 0.05) only in the early treated group. The results suggest that dip treatment of A. hydrophila-infected goldfish in an aqueous triherbal concoction had a synergistic restorative effect on the hematological variables.     

In vitro susceptibility to antimycotics of Microsporum canis isolates from cats.

One hundred thirty-four isolates of Microsporum canis, obtained from cats, were tested for in vitro susceptibility to various antifungal agents. The fungi were classified as susceptible, resistant, and intermediate by measuring the size of the zone of inhibited growth on yeast nitrogen base agar medium. Clotrimazole had the highest activity (99.2%), followed by tioconazole (89.6%), griseofulvin (88.8%), econazole (73.1%), ketoconazole (50.7%), miconazole (15.7%), and isoconazole (12.7%). We found 14.9% of the isolates to be susceptible to all the assayed drugs, whereas the highest resistance frequency (41.8%) was against 2 antimycotics. A simultaneous resistance to all the tested antimycotics was not found. The differences among the antifungal drugs activity were examined, and administration of drugs for which a simultaneous resistance was minimal is suggested.     

In vitro susceptibility of Pseudomonas mallei to antimicrobial agents

Pseudomonas mallei was isolated from pus samples obtained from 34 mallein-positive horses. The isolates were subjected to in vitro sensitivity test using 16 different antimicrobial discs. All isolates (34) were sensitive to sulfamethizole, gentamycin, tetracycline, sulfathiazole, kanamycin, tobramycin, streptomycin and a combination of trimethoprim and sulfamethoxazole while none of them were sensitive to cephalothin, colistin, ampicillin, penicillin and nitrofurantoin. Rifapicin, chloramphenicol and carbenicillin were effective against 32, 26 and 18 isolates respectively. The minimum inhibitory concentrations (MICs) of gentamycin, tetracycline, tobramycin, sulfamethizole, streptomycin, rifampicin and a combination of trimethoprim and sulfamethoxazole were 0.28, 0.38, 0.67, 1.40, 3.40, 5.86 and 5.30 micrograms/ml, respectively.     

In vitro susceptibility of canine and feline Escherichia coli to fosfomycin

Therapeutic options for multi-drug resistant (MDR) Escherichia coli in dogs or cats are limited. The objective of this study was to establish in vitro susceptibility of canine and feline E. coli to fosfomycin. Two sources of isolates were categorized based on susceptibility as to no resistance (NDR), single drug resistance (SDR), multidrug resistance (MDR) or extreme drug resistance (XDR). Clinical isolates were collected from throughout the US from dogs (n=157) or cats (n=43) with naturally occurring infection between March 2008 and January 2010. Experimental isolates were collected from fecal samples of dogs treated with no drug (NDR), amoxicillin (expressing SDR) or enrofloxacin (expressing MDR or XDR). Fosfomycin minimum inhibitory concentrations (MIC) were determined using E-Test(?). For clinical isolates, most (165/200) originated from the urinary tract, with the number of isolates per resistant category being: NDR (N=44, 22%), SDR (N=65, 32.5%), MDR (N=74, 37%), and XDR (N=17, 8.5%). Of these isolates, 99% (197/200) were susceptible to fosfomycin with the MIC(90) and MIC(50) being 2 and 1 μg/ml, respectively (range: 0.25-196 μg/ml). The number of experimental isolates in each category was NDR (3), SDR (23), MDR (38), and XDR (11) (29.3, 44, and 14.7%, respectively). Of these, 100% were susceptible to fosfomycin with MIC(90) and MIC(50) being 1.5 and 1 μg/ml (range: 0.38-4 μg/ml), respectively. The susceptibility of canine and feline MDR and XDR E. coli to fosfomycin at concentrations well below the susceptible breakpoint supports further investigation for its use when treating E. coli resistant to alternative antimicrobials.