Detection and prevalence of carbapenem-resistant Gram-negative bacteria among European laboratories in the COMBACTE network: a COMBACTE LAB-Net survey.

Antimicrobial resistance (AMR) represents a global public health threat that jeopardises the progress medicine has made over the last century. To confront AMR, the Innovative Medicines Initiative (IMI) has supported the development of a large network of hospitals and laboratories in Europe as part of the New Drugs for Bad Bugs (ND4BB) programme and the COMBACTE projects. COMBACTE LAB-Net conducted a pilot survey on distribution and usage of carbapenem resistance detection methods among laboratories in the COMBACTE network in two clinical trials as part of the COMBACTE-CARE project. The survey was sent out to 211 laboratories in 20 European countries between May 2015 and June 2017. Answers were collected from 165 laboratories (78%). Sixty laboratories (36%) reported an outbreak of carbapenem-resistant (CR) Enterobacteriaceae during one of the two years preceding the completion of the survey. High rates of CR Acinetobacter spp. above 50% were reported by 74 laboratories (47%), particularly in the Western Balkan countries where the rates were sometimes higher than 90%. Apart from determining the antimicrobial susceptibility of isolates, laboratories also used various methods, such as Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF), Carbapenemase Nordmann-Poirel (Carba NP) test or molecular methods, to detect CR Gram-negative bacteria. The survey resulted in the selection of sites with high resistance rates that successfully recruited many patients in the EURECA observational clinical trial.

Non-toxic perfringolysin O and α-toxin derivatives as potential vaccine candidates against bovine necrohaemorrhagic enteritis.

Bovine necrohaemorrhagic enteritis is a fatal Clostridium perfringens type A-induced disease that is characterised by sudden death. Recently the involvement of perfringolysin O and α-toxin in the development of necrohaemorrhagic lesions in the gut of calves was suggested, and thus derivatives of these toxins are potentially suitable as vaccine antigens. In the current study, the perfringolysin O derivative PFOL491D, alone or in combination with α-toxin derivative GST-cpa247-370, was evaluated as possible vaccine candidate, using in vitro assays. PFOL491D showed no haemolytic effect on horse red blood cells and no cytotoxic effect on bovine endothelial cells. Furthermore, calves immunised with PFOL491D raised antibodies against perfringolysin O that could inhibit the perfringolysin O-associated haemolytic activity on horse red blood cells. Antisera from calves immunised with PFOL491D had a significantly higher neutralising capacity against the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells than serum from control calves (P <0.05). Immunisation of calves with PFOL491D in combination with GST-cpa247-370 elicited antibodies against perfringolysin O and α-toxin and consequently inhibited both the perfringolysin O-associated haemolytic activity and the α-toxin-associated lecithinase activity in vitro. Additionally, the neutralising ability of these antisera on the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells was significantly higher than that from calves immunised with PFOL491D (P <0.001). In conclusion, perfringolysin O derivative PFOL491D is an immunogenic antigen that can potentially be used to produce vaccine against bovine necrohaemorrhagic enteritis. Including α-toxin derivative GST-cpa247-370 has an additional protective effect and therefore vaccination of calves with a combination of both antigens seems even more promising.

Intestinal clostridial counts have no diagnostic value in the diagnosis of enterotoxaemia in veal calves.

Enterotoxaemia is an important cause of sudden death in veal calves. This study aimed to evaluate intestinal Clostridium perfringens counts as a diagnostic tool for enterotoxaemia. Field necropsies were conducted on 48 sudden death cases in Belgian Blue veal farms. In 31/48 suddenly deceased calves, the diagnosis of enterotoxaemia was made based on haemorrhagic lesions in the small intestines, while in seven of these cases, no clear-cut diagnosis could be made based on macroscopic appearance of the gut. In the 10 remaining calves, a definitive cause of death other than enterotoxaemia could be identified. Samples of the intestinal content were taken for quantification of C perfringens. After matching cases and controls for diet, and the interval between death and sampling, no significant differences could be detected between the mean C perfringens counts of the small intestines in enterotoxaemia cases and counts in the matching segments in the control group. These results indicate that intestinal C perfringens counts cannot be advised as a discriminative postmortem diagnostic tool for enterotoxaemia in veal calves, not even when sampled within three hours after death.

lesion development in a new intestinal loop model indicates the involvement of a shared Clostridium perfringens virulence factor in haemorrhagic enteritis in calves.

Clostridium perfringens-associated enterotoxaemia is a fatal disease in fast growing suckler and veal calves. An intestinal loop model was developed to study the pathogenesis of the disease. Loops were injected with stationary and logarithmic C. perfringens cultures with or without, a milk protein-based commercial milk replacer for calves. Isolates tested were from cases of bovine enterotoxaemia and from calves without signs of enterotoxaemia, in addition to netB-positive and -negative isolates from poultry, a type C isolate from piglets and the human isolate JIR325. All isolates induced necrohaemorrhagic lesions in combination with milk replacer, while all control loops (i.e. medium plus milk replacer) remained histologically normal. In addition, time-course experiments were conducted using an isolate from an outbreak of bovine enterotoxaemia. Histological examination showed that the earliest lesion was congestion of the capillaries, starting within 30 min of inoculation. Haemorrhage and mucosal necrosis began at the tips of the villi 3-4 h after bacterial inoculation. These lesions are similar to those observed in natural cases of bovine enterotoxaemia. Therefore, in this model, necrohaemorrhagic lesions can be induced by C. perfringens isolates from diverse origins, suggesting that the lesions may be caused by one or more virulence factors that are shared by these isolates.

Necrotic enteritis in broilers: an updated review on the pathogenesis.

Clostridium perfringens-induced necrotic enteritis and related subclinical disease have become economically significant problems for the broiler industry. Fortunately, scientific interest in this topic has grown: new C. perfringens virulence factors have been discovered and new insight gained about the pathogenesis of necrotic enteritis. It has been shown that alpha toxin, for a long time thought to be the key virulence factor, is not essential for the development of the disease. Moreover, it is now clearly established that only certain C. perfringens strains are capable of inducing necrotic enteritis under specific conditions that predispose to the disease and they constitute only a minority in the intestinal tract of healthy chickens. A novel pore-forming toxin, NetB, has been identified in these virulent avian C. perfringens strains. Using a gene knockout mutant, it has been shown that NetB is a critical virulence factor in the pathogenesis of necrotic enteritis in broilers. In addition to toxin production, other factors have been described that contribute to the ability of certain C. perfringens strains to cause necrotic enteritis in broilers. It has been suggested that proteolytic enzymes play an important role in the initial stages of necrotic enteritis since the villi are first affected at the level of the basement membrane and the lateral domain of the enterocytes. In field outbreaks of necrotic enteritis, a single clone of C. perfringens is dominant in intestines of all affected birds, as opposed to the mixture of different C. perfringens strains that can be isolated from healthy bird intestines. It has been proposed that bacteriocin production is responsible for the dominance of a single strain in necrotic enteritis cases. Furthermore, it has been shown that virulent strains are more able to adhere to extracellular matrix molecules than non-virulent strains. The current knowledge on the pathogenesis of the disease has been summarized in this short review.

Variable protection after vaccination of broiler chickens against necrotic enteritis using supernatants of different Clostridium perfringens strains.

Necrotic enteritis is an economically important disease of chickens caused by Clostridium perfringens. Immunity to necrotic enteritis is not fully characterized yet, but previous reports indicate that immunoprotective potential is present in the secreted component of C. perfringens. This study aimed to compare the vaccine potential of the supernatants of eight chicken strains of C. perfringens differing in origin, level of alpha toxin production and presence of netB gene. The supernatant of only one strain provided full protection, while one other strain provided partial protection against a severe infection challenge. Our results indicate that the protective characteristics of the supernatants are not solely based on the presence of NetB or alpha toxin.

Isolation of a clonal population of Clostridium perfringens type A from a Belgian Blue calf with abomasal ulceration.

A case of abomasal ulceration in a 3-month-old Belgian Blue calf is described. Microscopical examination revealed the ulcers to be demarcated by a band of neutrophilic inflammation that separated underlying healthy tissue from the superficial fibrinous necrotic material in which bacteria were present. Clostridium perfringens type A was isolated from multiple ulcers and from the intestinal contents of the animal and pulsed field gel electrophoresis confirmed that the isolates comprised a genetically clonal population.

The effect of commonly used anticoccidials and antibiotics in a subclinical necrotic enteritis model.

Necrotic enteritis poses an important health risk to broilers. The ionophore anticoccidials lasalocid, salinomycin, maduramicin, narasin and a combination of narasin and nicarbazin were tested in feed for their prophylactic effect on the incidence of necrotic enteritis in a subclinical experimental infection model that uses coccidia as a predisposing factor. In addition, drinking water medication with the antibiotics amoxicillin, tylosin and lincomycin was evaluated as curative treatment in the same experimental model. The minimal inhibitory concentrations (MICs) of all antibiotics and anticoccidials were determined in vitro against 51 Clostridium perfringens strains isolated from broilers. The strains examined appeared uniformly susceptible to lasalocid, maduramicin, narasin, salinomycin, amoxicillin and tylosin, whereas an extended frequency distribution range of MICs for lincomycin was seen, indicating acquired resistance in 36 isolates in the higher range of MICs. Nicarbazin did not inhibit the in vitro growth of the C. perfringens strains even at a concentration of 128 microg/ml. Supplementation of the diet from day 1 onwards with lasalocid, salinomycin, narasin or maduramicin led to a reduction in birds with necrotic enteritis lesions as compared with the non-medicated infected control group. A combination product of narasin and nicarbazin had no significant protective effect. Treatment with amoxicillin, lincomycin and tylosin completely stopped the development of necrotic lesions.

Control of Clostridium perfringens-induced necrotic enteritis in broilers by target-released butyric acid, fatty acids and essential oils.

The efficacy of target-released butyric acid, medium-chain fatty acids (C(6) to C(12) but mainly lauric acid) and essential oils (thymol, cinnamaldehyde, essential oil of eucalyptus) micro-encapsulated in a poly-sugar matrix to control necrotic enteritis was investigated. The minimal inhibitory concentrations of the different additives were determined in vitro, showing that lauric acid, thymol, and cinnamaldehyde are very effective in inhibiting the growth of Clostridium perfringens. The in vivo effects were studied in two trials in an experimental necrotic enteritis model in broiler chickens. In the first trial, four groups of chickens were fed a diet supplemented with butyric acid, with essential oils, with butyric acid in combination with medium-chain fatty acids, or with butyric acid in combination with medium-chain fatty acids and essential oils. In all groups except for the group receiving only butyric acid, a significant decrease in the number of birds with necrotic lesions was found compared with the infected, untreated control group. In the second trial the same products were tested but at a higher concentration. An additional group was fed a diet supplemented with only medium-chain fatty acids. In all groups except for that receiving butyric acid in combination with medium-chain fatty acids and essential oils, a significant decrease in the number of birds with necrotic lesions was found compared with the infected, untreated control group. These results suggest that butyric acid, medium-chain fatty acids and/or essential oils may contribute to the prevention of necrotic enteritis in broilers.

Generation of single-copy transposon insertions in Clostridium perfringens by electroporation of phage mu DNA transposition complexes.

Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in Clostridium perfringens has been restricted to the use of Tn916-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in C. perfringens, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of C. perfringens.

The use of organic acids to combat Salmonella in poultry: a mechanistic explanation of the efficacy.

Salmonella is a human pathogen that is commonly found in poultry products. It is possible to decrease chicken carcass and egg contaminations by adding organic acids to the feed or drinking water at appropriate times. Medium-chain fatty acids are more antibacterial against Salmonella than short-chain fatty acids. The antibacterial effect of these acids is species specific. Bacteria that are unable to decrease intracellular pH accumulate organic acid anions in accordance with the pH gradient across their cell membranes. The short-chain fatty acid butyrate specifically down-regulates expression of invasion genes in Salmonella spp. at low doses. Also medium-chain fatty acids and propionate decrease the ability of Salmonella spp. to invade epithelial cells, in contrast to acetic acid. Because not all bacteria are affected in a similar fashion by organic acids, it may be possible to use probiotic and prebiotic bacteria to achieve beneficial effects. If diets can be designed to stimulate organic acid production in the caecum, it may be possible to control Salmonella spp. via even easier and more cost-effective measures, compared with addition of acids to feed or drinking water.

Supplementation of coated butyric acid in the feed reduces colonization and shedding of Salmonella in poultry.

Short-chain fatty acids have been widely used as feed additives to control Salmonella in poultry. Data on the use of butyric acid in poultry are lacking. In this study, powder form and coated butyric acid were compared in their ability to reduce Salmonella colonization of ceca and internal organs shortly after infection of young chickens with Salmonella enteritidis. In the first trial, 4 groups of 25 specific pathogen free layer chickens were given feed either supplemented with powder form butyric acid, coated butyric acid, a combination of powder form and coated butyric acid (all groups received a total of 0.63 g of butyric acid/kg) or nonsupplemented feed. The specific pathogen free layer chickens were orally infected with 10(6) cfu of S. enteritidis. Coated butyric acid significantly decreased cecal colonization 3 d post-infection compared with control chickens, and powder form butyric acid had no effect. To study long-term shedding and colonization of Salmonella in broilers given coated butyric acid as feed additive (0.63 g of active product butyric acid/kg), 10 Ross broiler chickens were infected at d 5 with 10(5) cfu of S. enteritidis and housed together with 40 noninfected broilers. A control group received nonsupplemented feed. The group of broilers receiving coated butyric acid had a significantly lower number of broilers shedding Salmonella bacteria, but cecal colonization at slaughter age was equal for both groups. In conclusion, butyric acid decreases cecal colonization shortly after infection, decreases fecal shedding, and as a consequence, decreases environmental contamination by S. enteritidis-infected broilers. However, complete elimination can probably only be achieved with a combined approach using both hygienic measures and different protection measures, as the broilers still carried S. enteritidis bacteria in the ceca at slaughter age, although at enrichment level.