Microbiological evaluation of dressing procedures for crocodile carcases in Queensland

SUMMARY Microbiological testing of crocodiles during the dressing procedure has shown that sanitising the skin before skinning reduces the bacterial count on the skin and that dipping crocodile meat in 1.3% acetic acid solution effectively reduces bacterial levels. The total bacterial count on the processed meat sample was comparable with that obtained in the beef, pork and lamb industries. Salmonellae were isolated from 14 of the 72 carcases. Most (65%) of these isolates were in Salmonella subspecies III, formerly classified as Arizona.     

Pharmacokinetics of ceftriaxone in freshwater crocodiles (Crocodylus siamensis) after intramuscular administration at two dosages

One of the major obstacles to the successful treatment of infectious disease in freshwater crocodile species is incorrect dosing of antibiotics. There are few reports on pharmacokinetics and dosage regimens of antimicrobial drugs in crocodiles. The purpose of the present study was to clarify the pharmacokinetic characteristics of ceftriaxone (CEF) in Siamese freshwater crocodiles (Crocodylus siamensis). Freshwater crocodiles, Crocodylus siamensis, in breeding farms were treated with a single intramuscular administration of CEF at two dosages, 12.5 and 25 mg/kg body weight (b.w.). Blood samples were collected at preassigned times up to 168 hr. The plasma concentrations of CEF were measured by a validated method through liquid chromatography tandem-mass spectrometry. CEF plasma concentrations were quantified up to 72 and 96 hr after low- and high-dose administration, respectively. The C_max values of CEF were 24.61 ± 5.15 µg/ml and 26.39 ± 2.81 µg/ml at dosages of 12.5 and 25 mg/kg b.w., respectively. The AUC_last values increased in a dose-dependent fashion. The half-life values were not statistically different between the groups (around 20 hr). The average binding percentage of CEF to plasma protein was 53.78 ± 2.11%. Based on the pharmacokinetic data, susceptibility break-point and the surrogate PK-PD index (T > MIC, 0.2 μg/ml), i.m. administration of CEF at a dose of 12.5 mg/kg b.w. might be appropriate for initiating treatment of susceptible bacterial infections in freshwater crocodiles.     

Selection and characterization of broad‐spectrum antibacterial substance‐producing Lactobacillus curvatus PA40 as a potential probiotic for feed additives

Lactic acid bacteria were screened for potential probiotics for use as feed additives. We obtained 3,000 isolates from feces of: cattle, dogs, goats, and infants; milk; yogurt; cheese; fermented sausages; Kimchi; and Cheonggukjang and tested their antibacterial activity toward indicator pathogens, including Bacillus cereus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica Enteritidis. We further tested their tolerance to artificial gastric juice (1% [w/v] pepsin, pH 2.5) and bile acid (0.1% [w/v] oxgall, pH 6.8). Six isolates exhibited strong antibacterial activity against indicator pathogens. The PA40 isolate from Kimchi exhibited marked resistance to artificial gastric juice and bile acid. The antibacterial substances produced by PA40 were stable to heat, pH, and enzymes. Strain PA40 was identified as a Lactobacillus curvatus strain using chemical tests and 16S rDNA sequencing and produced 248.4 mmol/L lactic acid after 48 hr of fermentative growth. The L. curvatus PA40 strain was also highly tolerant of the artificial gastrointestinal model system. Our results indicate that L. curvatus PA40 could be used as a potential probiotic feed additive.     

Characterization and partial purification of a bacteriocin‐like substance produced by thermophilic Bacillus licheniformis H1 isolated from cow manure compost

The production and partial characterization of bacteriocin‐like substances (BLSs) produced by bacteria isolated from cow manure compost were investigated. Eight BLS producers, which exhibited inhibitory activity against pathogenic bacteria, were isolated from cow manure compost at different stages of the composting process. The pile temperature ranged from 9.1°C to 73.2°C. The BLSs showed thermostability, but the BLS producers were not thermostable except for the H1 producer. Thermophilic Bacillus licheniformis H1 was further characterized. The culture supernatant of B. licheniformis H1 exhibited antagonistic activity against various species of Gram‐positive bacteria such as Listeria monocytogenes ATCC19111 but not against Gram‐negative bacteria except Pseudomonas fluorescens ATCC11251. Inactivation of bacteriocin‐like activity by α‐chymotrypsin, trypsin, and papain was highly significant (P < 0.001). The BLS was found to be stable under a pH range from 3 to 9 and at temperatures up to 75°C for 60 min, but it lost activity after being autoclaved at 121°C for 15 min. The optimum production of BLS by B. licheniformis H1 was obtained at a temperature of 55°C. Sodium dodecyl sulfate – polyacrylamide electrophoresis analysis of concentrated partially purified supernatants collected after resting the bacterial cells at 55°C revealed a bacteriocin‐like protein with a molecular mass of approximately 3.5 kDa. This study is the first report of a BLS from thermophilic B. licheniformis with an animal compost origin.     

Status of the Nile crocodile population in Pongolapoort Dam after river impoundment

The major Nile crocodile Crocodylus niloticus populations in South Africa are threatened by pollution, habitat alteration/destruction, and poaching. This has highlighted the importance of other minor populations. The Phongola River Nile crocodile population was previously considered as unsubstantial. Consequently, we investigated the Nile crocodile population numbers and status and the effects of the impoundment of the Phongola River on this. In 2009–2010 we determined a minimum population number of 281 Nile crocodiles in Pongolapoort Dam using a combination of survey methods. The population structure was identified as having a minimum of 116 (41.3%) juveniles (<1.2 m total length), 31 (11.0%) subadults (1.2–2.5 m total length) and 134 (47.7%) adults (>2.5 m total length). At the commencement of the breeding season in August, crocodiles congregated at a major basking site where the main tributary entered the dam. Three major nesting areas were identified, two of which were located on the river inlet to the dam. We identified approximately 30 nesting females during the 2009/10 nesting season. Several nests were predated by the Nile monitor Varanus niloticus. There was a total recruitment failure of nests along the river inlet to the dam due to a flash flood of the Phongola River in January 2010. This preliminary study suggests that the Pongolapoort Dam Nile crocodile population has a relatively high potential reproductive output, although their annual successes may vary greatly because of loss of nesting sites as a result of water-level fluctuations and predation. It appears that the river impoundment has generally had a positive impact on this Nile crocodile population, although suitable nesting sites may become limited. Continued long-term monitoring of the Nile crocodile population in Pongolapoort Dam is required to determine if the impoundment continues to support a viable population.     

东亚飞蝗抗菌活性物质的萃取及抗菌效应研究

试验研究东亚飞蝗成虫抗菌活性物质的萃取参数与抑菌效应。采用针刺蘸大肠杆菌菌悬液方法,以抗菌活性为指标,用杯碟法测定临床多重耐药菌的抑菌活性,确定最佳诱导时间,初步研究抗菌谱。结果表明,东亚飞蝗抗菌活性物质粗提液对革兰氏阳性菌(如金黄色葡萄球菌、枯草芽孢杆菌)或革兰氏阴性菌(如大肠杆菌、水稻白叶枯病原菌)均有一定的抑制作用。其抗菌谱为:金黄色葡萄球>水稻白叶枯病原菌>枯草芽孢杆菌>大肠杆菌,其中以对金黄色葡萄球菌的抑菌效果最明显。抗菌活性物质浓度为11.782mg/mL,蝗虫抗菌活性物质收率为8.623%。经纯化后的抗菌活性物质增强对革兰氏阳性菌或革兰氏阴性菌的抑制作用。采用针刺蘸大肠杆菌方式可成功诱导东亚飞蝗抗菌物质的表达,尤其以针刺蘸大肠杆菌诱导后饲养24h得到的抗菌活性物质抑菌活性最强。结果表明东亚飞蝗虫体内含有抗菌活性物质可被诱导表达,其抗菌作用机理有进一步研究。     

Effects of Salinity on Yersinia ruckeri Infection of Rainbow Trout and Brown Trout

Abstract

Juvenile rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta acclimated to freshwater or salinities of 9.0‰ or less were exposed to Yersinia ruckeri, the bacterial pathogen that causes enteric redmouth disease (ERM). Both species of fish were kept in the same recirculating systems after bacterial exposure. Rainbow trout mortality was significantly (P < 0.05) different in each salinity: 96.5% in freshwater, 89.5% in water of 1.1‰ salinity, 81.3% in 3.0‰ salinity, and 75.0% in 9.0‰ salinity (model SE = 1.0). All deaths occurred between 3 and 12 d after exposure to Y. ruckeri. Only 2.3% of brown trout in all salinities died, and differences among treatments were not significant. For both fish species, Y. ruckeri was isolated from liver, spleen, and trunk kidney of fish dying during this experiment, and lesions of rainbow trout were consistent with ERM. Yersinia ruckeri was not isolated from brown trout surviving for 21 d after bacterial exposure but was isolated from 3 of 24 surviving rainbow trout; a polymerase chain reaction assay detected the DNA of Y. ruckeri in 3 additional rainbow trout survivors. Neither the lesions of fish with ERM nor the percentage of surviving fish subclinically infected with Y. ruckeri was affected by salinity. Bacterial growth in vitro was not affected by low (≤9.0‰) salinity; however, bacterial adhesion to polystyrene was significantly reduced as salinity increased. Although mortality caused by Y. ruckeri was significantly lower for rainbow trout in water with slightly increased salinity, none of the salinities tested was effective in preventing serious losses caused by this pathogen in recirculating systems.     

An Antiviral Agent (46NW-04A) Produced by Pseudomonas sp. and Its Activity against Fish Viruses

Abstract

The antiviral agent 46NW-04A was isolated and characterized from cell-free culture fluid of Pseudomonas sp. 46NW-04 isolated from the aquatic environment. Production of the antiviral substance was maximal at 25°C during days 2–3 of bacterial incubation. Extraction from 30 L of culture fluid by ethyl acetate and purification by thin-layer chromatography on silica gel resulted in 709 mg of the purified antiviral material. Molecular weight of this substance was 1,126 by secondary ionization mass spectrometry, and chemical properties suggested that 46NW-04A was a peptide. Its antiviral activity, measured as the concentration causing 100% plaque reduction, was 25 μg/mL against Oncorhynchus masou virus and infectious hematopoietic necrosis virus. However, no antiviral activity was observed against infectious pancreatic necrosis virus at the concentrations tested. Pseudomonas sp. 46NW-04 was identified as Pseudomonas fluorescens biovar I.     

Identification and characterization of serum complement activity in the American crocodile (Crocodylus acutus)

Incubation of unsensitized sheep red blood cells with serum from the American crocodile (Crocodylus acutus) resulted in a concentration-dependent hemolysis. The hemolytic activity was heat-sensitive, and inhibited by EDTA in a concentration-dependent manner. The EDTA-inhibited SRBC hemolysis could be restored by the addition of excess Ca²⁺ or Mg²⁺, but not Ba²⁺ or Cu²⁺, revealing the specificity of this activity for these two divalent cations. The hemolytic activity of crocodile serum was titer-dependent, with 329 μL producing 50% of maximal SRBC hemolysis. The complement activity was also temperature-dependent, with decreased activity at lower temperatures (5–15 °C) and maximal activity occurred at 30–40 °C. The hemolysis occurred relatively slowly, with near zero activity after 10 min, 40% of activity observed within 15 min of exposure to SRBCs, and maximal activity at 30 min.     

The effect of lemongrass oil and its major components on clinical isolate mastitis pathogens and their mechanisms of action on Staphylococcus aureus DMST 4745

The aims of this study were to investigate the antibacterial activity of lemongrass oil (LG) and its major components which were citral, geraniol and myrcene, against four strains of clinically isolated bovine mastitis pathogens, including Staphylococcus aureus, Streptococcus agalactiae, Bacillus cereus and Escherichia coli by the broth microdilution method, as well as their activity on S. aureus biofilm formation. Attempts to clarify their mechanisms of action by investigation of the effects on intracellular material leakage and morphological changes of S. aureus DMST 4745 were also made. The results demonstrate that S. agalactiae and B. cereus are more susceptible to LG, citral and geraniol than S. aureus and E. coli. Moreover, they also inhibit S. aureus biofilm formation and exhibit effective killing activities on preformed biofilms. The LG appears to have multiple targets in the bacterial cell, depending on concentration used as well as the amount of its components.     

Equine pastern dermatitis

Bacteriological examination of pastern dermatitis in 12 cases resulted in a variable growth of bacterial species. Dermatophilus congolensis was found on direct smear in 2 cases, although only cultured in one of these. Eleven of the cases were treated with a new topical treatment, consisting of a combination of antibacterial agents and humectants ‐ agents that lower water activity (Mud Stop). Ten of these cases showed a good response to treatment, the other showing partial improvement before the treatment was changed.     

Pseudomonads from rabbits and their sensitivity to antibiotics and natural antimicrobials

The sensitivity/resistance of Pseudomonas spp. isolated from rabbits gastrointestinal tract and faeces to antibiotics, enterocins and herbal extracts was tested in this study. The counts of Pseudomonas-like bacteria were higher in faeces (3.23-6.16 log₁₀ CFU/mL/g) than in caecum (1.36-4.08 log₁₀ CFU/mL/g). Nineteen isolates (16 faecal, 3 caecal) were oxidase positive. The strains were allotted by phenotypization to Pseudomonas spp., Brevundimonas diminuta and Brevundimonasvesicularis. High percentage of resistant strains was observed to all antibiotics. The tested strains were more susceptible to natural substances, mainly to plant extracts oregano (95%) and sage extracts (58%). Comparing the antibacterial effect of antibiotics and enterocins against rabbits pseudomonads, enterocins were more effective; the strongest inhibitory activity was determined in the case of partially purified enterocins PPBs EF2019, EK13 and EF55.     

多黏类芽孢杆菌抗菌肽的分离纯化及特性研究

试验旨在分离纯化多黏类芽孢杆菌（Paenibacillus polymyxa）BLCC1-0402代谢产物中的抗菌肽,为抗菌肽制备及其制品检测提供参考。采用离心、不同分子质量卷式膜超滤浓缩、Superdex peptide 10/300GL凝胶过滤层析对多黏类芽孢杆菌发酵上清液进行逐级分离纯化,对不同时段的收集液做抑菌试验,以大肠杆菌O78标准菌株为指示菌,采用打孔法进行抑菌活性检测,比较评价分步层析效果,以Tricine-SDS-PAGE进行分子质量检测。结果显示,通过5和3 ku卷式膜超滤获得的3~5 ku组分蛋白质样品抗菌活性较强;对于3~5 ku组分经凝胶过滤层析分离纯化,纯化后的抗菌肽A3抑菌活性最强,经Tricine-SDS-PAGE小分子多肽电泳检测,已达到电泳纯,分子质量为4 ku;抑菌活性检测结果显示,该抗菌肽A3对大肠杆菌O78标准菌株具有较强的抑菌作用。同时,抗菌肽A3表现出较好的耐热性,90~100 ℃处理15 min,抑菌活性可保持在96%左右;具有较好的酸碱稳定性,在pH 2.0~9.0下,抑菌活性保持在90%以上;经胃蛋白酶作用后抗菌肽A3抑菌活性降低20%,胰蛋白酶作用后抗菌肽A3抑菌活性降低18%,蛋白酶K对抗菌肽A3的抑菌活性几乎无影响。本研究结果表明,分离得到的抗菌肽A3是一种对大肠杆菌O78具有抑菌活性的新型抗菌肽,具有一定的开发潜力,可为下一步抗菌肽的结构分析、理化特性分析等深入研究提供一定参考依据。     

Antibiotic Resistance of Aeromonas spp. Isolated from Tropical Fish Imported from Singapore

Abstract

To determine the scope of antibacterial resistance among Aeromonas spp., bacterial cultures were taken from a variety of tropical fish species imported from Singapore. The samples were plated on Rimler-Shotts medium for bacterial isolation and identified with both the API20E and Nonfermenter Test Strip systems. Aeromonas hydrophila, A. sobria, and A. caviae were identified in monomicrobic and concomitant infections. Following identification of bacterial isolates, 11 antimicrobials routinely used in the tropical fish industry and 1 new fluoroquinolone were tested for their effectiveness against Aeromonas spp. with the Kirby-Bauer disk-diffusion technique. Aeromonas sobria proved to be the most resistant, often showing susceptibility to only 3 of 12 test drugs. Aeromonas hydrophila was consistently the least resistant. Based on these results, antimicrobial resistance appears to be a rapidly emerging problem in the pet fish industry.     

救必应中药血清与抗菌药联合对产ESBLs细菌抑菌效果的研究

本试验主要探讨救必应中药血清与抗菌药联合诱导细菌传代的体外抑菌活性。制备救必应中药血清,采用二倍微量稀释法体外检测抗菌药的最小抑菌浓度(MIC),然后救必应中药血清与抗菌药联合诱导产超广谱β-内酰胺酶(extended spectrumβ-lactamases,ESBLs)细菌传代。救必应中药血清与抗菌药联合诱导细菌传代对耐药大肠杆菌有不同程度的抑菌活性。救必应中药血清可明显地增强β-内酰胺类抗菌药、氨基糖苷类抗菌药、喹诺酮类抗菌药和卡巴氧类抗菌药对耐药菌的抑制作用。     

Communications: Bacteria in the Hemolymph of the Freshwater Prawn Macrobrachium rosenbergii

Abstract

Bacteria from the hemolymph of the freshwater prawn Macrobrachium rosenbergii were identified and quantified. Total bacterial counts ranged from 0.0 to 4.6 × 10⁵ cells/1.0 mL of hemolymph. Predominant bacteria isolated included Aeromonas spp., Bacillus sp., and Pseudomonas spp. No bacteria were found in the hemolymph of prawns without lesions. The predominant species of bacteria isolated from water samples of prawn culture ponds was a chitinoclastic Bacillus sp.     

The fish community of the berg river estuary and an assessment of the likely effects of reduced freshwater inflows

Data concerning the species composition, abundance and distribution of fishes inhabiting the Berg River estuary are presented and used to assess the value of the estuary to fish, and the likely eflects of reduced freshwater inflows. A total of 31 species was recorded, eight of which were classified as estuarine residents, 11 as marine estuarine-opportunists, six as marine stragglers and six were freshwater species. Liza richard- sonii, an opportunist, was the most abundant (54% N) and widely distributed species encountered during the winter and summer seine-net surveys. Three other species (all residents) contributed more than 10% of the total catch; Atherina breviceps and Caffrogobius nudiceps occurring most abundantly in the lower estuary, and Gilchristella aestuaria in the middle estuary. The low number of species compared with estuaries in other regions of the South African coast reflect a well-established marine biogeographical trend. The higher proportions ol resident species, entirely dependent species and partially dependent species suggest, however, that west coast estuaries may be more important to the fish that inhabit them than are estuaries in other regions. It is concluded that the fish community of the estuary is already suffering the effects of habitat degradation and that further reductions in freshwater inllows are not desirable.     

Peptide derivatives of dermaseptin S4 in fresh bovine semen for bacterial contamination control: Physicochemical and structural characterization,antibacterial potency,and effects on red blood and sperm cells

The objectives of this study were to examine the physicochemical and structural properties of peptide derivatives of dermaseptin S4, investigate their detrimental effects on red blood and sperm cells and ascertain their antibacterial potency to control bacterial contaminants in fresh bovine semen. The dermaseptin S4 peptide derivatives used in this study were K4S4, S4(5–28), S4(5–28)a, K20S4(5–28), K4S4(1–16)a, K4S4(1–15)a and K4S4(1–15). Peptides K4S4, S4(5–28)a, K20S4(5–28), K4S4(1–15)a and K4S4(1–16)a, with a higher positive charge, were the most potent against the bacterial strains tested, with the lowest minimum inhibitory concentration (MIC), whereas S4(5–28) and K4S4(1–15), with a lower positive charge, showed the highest MIC (p < .01). Haemolysis percentage depended on peptide concentration (p < .01). The K4S4 was the most powerful haemolytic peptide, showing the highest haemolysis percentage at all peptide concentrations (p < .01). In contrast, S4(5–28), S4(5–28)a, K20S4(5–28) and K4S4(1–15) were not able to produce 50% cell lysis up to 100 µM (p < .01). All peptides reduced sperm motility in a dose-dependent manner when used in concentrations from 16 to 64 μM (p < .01). The highest reduction was seen due to K4S4 activity, and the lowest reductions of sperm motility were observed due to K4S4(1–16)a and K4S4(1–15)a activity (p < .01). Hence, we can conclude that K4S4(1–16)a and K4S4(1–15)a at a concentration of approximately 15 µM are the most promising peptides as antibacterial agents in fresh bovine semen, because at this concentration, they showed the most potent antibacterial activity against evaluated strains without significant effects on haemolysis or a reduction in sperm motility.     

Comparative Uptake of Orally Administered Difloxacin in Atlantic Salmon in Freshwater and Seawater

Abstract

Fluoroquinolones are synthetic antibacterials with potential use for treatment of fish diseases. This study examined the comparative uptake of difloxacin, administered as a single oral gavage, by juvenile Atlantic salmon Salmo salar in freshwater and in seawater. In separate freshwater and seawater experiments with fish of the same spawning cohort, the concentration of difloxacin in plasma and fillets was determined at 1, 2, 4, 8, 12, 18, 24, and 48 h after the fish received the dose. Although plasma difloxacin concentration began to plateau in the freshwater fish at about 24 h, the highest average, 1,195 ng/mL, was in fish sampled at 48 h; thus, the plasma levels continued to rise through the end of the test. A similar pattern was observed in fillets from fish held in freshwater, but the peak average fillet concentration at 48 h was 4,737 ng/g. Contrastingly, in the seawater study, the average peak plasma concentration of 678 ng/mL was observed at 8 h after gavage and the peak average fillet concentration of 924 ng/g was observed at 18 h. Although difloxacin concentrations were higher in the freshwater-held fish than in the seawateradapted fish, the results suggest that difloxacin could be efficacious in treating bacterial diseases of Atlantic salmon in both freshwater and seawater.     

Bacteriocin Activity of Enterococci from Rabbits

The antimicrobial spectrum of bacteriocin-producing enterococcal isolates from rabbits faeces, the biochemical characterization of bacteriocins, and their molecular mass and the presence of structural genes for bacteriocin production were investigated. Among enterococci selected from rabbit faeces, six strains of Enterococcus faecium (EF2019, EF1819, EF2119, EF1839, EF529, EF24/10) showed inhibitory activity against the indicators E. avium EA5, Listeria innocua LMG13568 and Listeria monocytogenes CCM4699 and against other enterococci and staphylococci tested. The molecular mass of bacteriocin-like substances ranged from 3 to 10 kDa. The presence of the structural genes for enterocins (ent) A, P and L50B was detected in all enterococci tested. However, no strain possessed the gene for ent B. E. faecium EF2019 showed the broadest inhibitory activity. A proteinaceous substance produced by the EF2019 strain was partially purified. This is a thermostable substance that is stable at pH 4.0, 7.0 and 9.0. Its production starts in the early logarithmic growth phase and culminates in the late logarithmic growth phase of the EF2019 strain. Partially purified bacteriocin (PPB) EF2019 added to the growing strain L. innocua LMG13568 (after 4 h) inhibited its growth as early as 1 h after addition with a decrease of 1.5 log cycles (5 h of cultivation). This effect was extended up to 24 h. The bacteriocinogenic E. faecium EF2019 strain and/or bacteriocin EF2019 could be utilized against contaminant bacteria, e.g. in rabbitries. More detailed studies under in vitro and in vivo conditions are in progress.