How to handle Swine Colibacillosis in the field? What kind of resistance do we have to expect?

Introduction

Colibacillosis is generally defined the infection with Escherichia coli characterized by many clinical forms. E. coli is a gram negative peritrichously flagellated bacteria belonging to the family Enterobatteriaceae and is the causative agent of a wide range of diseases in pigs, including neonatal diarrhoea, post-weaning diarrhoea (PWD), oedema disease (ED), septicaemia, polyserositis, coliform mastitis (CM) and urinary tract infection (UTI) (Fairbrother and Gyles, 2012).

Enteric colibacillosis may result in significant economic losses due to mortality, decreased weight gain, cost for treatments, vaccinations and feed supplements. In severe outbreaks of PWD a mortality rate up to 25% was reported (Fairbrother and Gyles, 2012). Depending on the severity of the disease, the cost of PWD was estimated to range from 40 to 314 per sow (Sjölund et al., 2014). Two main pathotypes are involved in enteric colibacillosis: enterotoxigenic E.coli (ETEC) and enteropathogenic E.coli (EPEC). ETEC is the most important pathotype in pig and include different virotypes (this term is used to describe strains characterized by different combinations of toxins and fimbriae). ETEC elaborate one or several enterotoxins that induce secretory diarrhoea, causing some of the most significant diseases in the pig industry worldwide, such as neonatal colibacillosis and PWD, the main subjects of this paper.

ETEC responsible of neonatal diarrhoea posses adhesins, surface proteins called fimbriae, identified as F4 (k88), F5 (k99), F6 (987P) and F41. The fimbriae allow the microorganism to adhere to specific receptors on the brush borders of the enterocytes of the small intestine. Growing up, piglets show a modification of their intestinal receptor for ETEC fimbriae and, as a consequence, their susceptibility to ETEC virotypes. For this reason PWD is most commonly caused by ETEC that usually have either F4 or F18 fimbriae (even though some exceptions are observed). A non-fimbrial adhesin identified as AIDA (adhesin involved in diffuse adherence) has been associated with ETEC strains recovered from weaned pigs with PWD and there is evidence that it plays a role in colonization of the intestine of pigs.

In general, ETEC adhere to the intestinal mucosa and produce enterotoxins causing changes of the flux of water and electrolytes in the enterocytes of the small intestine leading to diarrhoea when the excess of fluid in the lumen of the small intestine is not adsorbed in the large intestine. Excessive secretion and diarrhoea leads to dehydration, metabolic acidosis and death. Most ETEC of neonatal enteric colibacillosis produce heat-stable enterotoxin called STa. Based on the concentrations of the STa receptors, the posterior jejunum is the major site of hypersecretion in response to STa. ETEC strains responsible of PWD produce one or more of the enterotoxins STa, STb, LT for which the role in the development of diarrhoea has been defined. Enteroaggregative E.coli heat-stable enterotoxin (EAST1) was reported in ETEC isolated from pigs with diarrhoea, however its role in the development of diarrhoea has not been elucidated (Fairbrother and Gyles, 2012).

Managing enteric colibacillosis in pigs requires an understanding of the pathotypes and the virotypes of E.coli involved, as briefly reported above, and the conditions under which they are capable of causing disease, in order to implement appropriate diagnostics and strategies for prevention and control.

This paper is aimed at addressing some of the major questions that are frequently asked when facing with colibacillosis in the field, namely the diagnostic approach and the interpretation of the specific investigations, the epidemiology of the infection, the predisposing environmental conditions and host factors and the measures of prevention and control. An accurate diagnosis is the key element for approaching a clinical problem. Treatment and control programs for enteric diseases are pathogen specific and only if the “enemy” is known, appropriate measures of control can be implemented. Control measures are traditionally based on the use of antimicrobials for prophylactic, metaphylactic and therapeutic purposes. In particular, preventive feed medication is still applied in many countries despite others have significantly reduced or eliminated these wide range treatments, as consumer resistance and the pivotal issues associated with the selection of resistant bacteria. Antimicrobial resistance is a major problem in veterinary medicine and represents a treat to public health. For these reasons strategies for control of enterotoxigenic E.coli will be considered in the paper, focusing mainly on antibiotic therapy and antimicrobial resistance.

The diagnosis

1. The diagnostic approach to neonatal Colibacillosis and post-weaning diarrhoea in pig

When an outbreak of diarrhoea occurs in a pig herd, the starting point for a rational implementation of the control measures is the achievement of a diagnosis. The presumptive diagnosis is based on clinical presentation and the gross lesions.

Neonatal diarrhoea due to E.coli is observed most commonly in piglets aged from 0 to 4 days of life, and in general, in an endemic condition, litters from first-parity sows could be more involved, due to a lack of protection by colostral immunity.

PWD due to E.coli is commonly observed 2-3 weeks after weaning, even if not exceptionally it can be recorded at 6-8 weeks after weaning.

When E.coli sustains the enteritis, diarrhoea is characterized by yellowish, gray or slightly pink fluid with a characteristic smell, lasting in general one week.

Necropsy can provide useful information to orient the diagnosis, although it is not reliable based solely on the correlated findings. The most effective approach is to select a number of untreated pigs (3-5) suffering from diarrhoea since less than 12-24 hours (acute phase), to humanely euthanize them and perform an accurate necropsy in order to evaluate gross lesions (evaluating small intestine, colon, ileo-caecal valve, mesenteric lymph-nodes). Autolysis of the gut after death occurs promptly so also recently spontaneously death animals cannot be informative.

The small intestine is usually dilated, slightly oedematous and hyperaemic and the dilated stomach shows hyperaemia in the fundus. These lesions, even not pathognomonic are suggestive of enteric colibacillosis.

Samples of small (in particular ileum and jejunum) and large intestine should be taken for bacteriological and virological investigations and fixed in 10% buffered formalin. In fact, the definitive diagnosis requires the combination of several investigations. The identification of E.coli infections is based on bacteriological examination of samples of luminal content (first choice) or rectal swabs. The samples should be inoculated onto blood agar and McConckey agar or other media which are selective for Enterobatteriaceae such as Hektoen agar. These selective media allow differentiation of lactose fermenting (such as E.coli) from lactose non-fermenting Gram negative enteric bacilli. Colonies on solid media reach their full size within 1 day of incubation and vary from smooth to rough or mucoid. The characteristics of the colonies grown on blood agar and lactose fermentation on selective media gives a first diagnostic indication. In particular the presence of haemolytic colonies, both in neonatal diarrhoea and PWD, is often used as a rapid tool for the diagnosis of ETEC diarrhoea. This finding allows also a preliminary assessment on the fimbrial type involved, since colonies of F4 and F18 positive ETEC strains are almost always haemolytic on blood agar, while F5, F6, F41 and EPEC are not haemolytic (Fairbrother and Gyles, 2012). The interpretation of the results obtained with the bacteriological examination in treated animals is in many cases unreliable.

2. What are the next steps to reach a definitive diagnosis?

The E.coli strain, after the isolation on culture, should be identified as pathogenic, commonly by serotyping or genotyping. Serotyping is performed by tests of agglutination using polyvalent or monovalent sera to determine O (cell wall LPS) or F (fimbrial) antigens. In particular the determination of O antigens can be useful for diagnostic purposes since a small number of specific O groups have been associated with disease (table 1) (Fairbrother and Gyles, 2012).

Table 1: Important adhesins and serogroups of ETEC (modified from Fairbrother and Gyles, 2012).

Slide agglutination, for example, is a quick and cheap method commonly used in the past for the identification of F4 positive ETEC. This serotyping approach can lead to false classifications, mainly because of cross-reactions or lack of expression of fimbriae in vitro. For these reasons a complete serotyping of H (flagellar protein antigen) and O antigens, with the additional identification of K antigens (capsular polysaccharide), is the standard method for the definition of all serotypes, but in general is carried out in few reference laboratories (Fairbrother and Gyles, 2012).

Currently, genotypic analysis such as the polymerase chain reaction (PCR), for the detection of genes encoding for virulence factors is performed in many laboratories to characterize the strains isolated. Primers recognising different genes encoding for toxins (STa, STb, LT and EAST1) and fimbriae (F4, F5, F6, F18, F41) of ETEC, for the outer membrane protein Eae or intimin in enteropathogenic E.coli (EPEC) and for Stx2e toxin in STEC (E.coli strains involved in oedema diseases) strains, are available and can be used to perform PCR assays for daily routine diagnostic. Interestingly, certain F18 strains produce both enterotoxins and Stx2e. These strains are classified ETEC rather than STEC, since they produce clinical PWD more than oedema disease.

Histopathology in formalin-fixed, paraffin embedded tissue (ileum, jejunum and large intestine should be included) can be used as additional investigation for a definitive identification of E.coli that are observed adhering to the enterocyte brush border membrane of intestinal mucosa. E.coli F4 positive usually adhere to the cells of most of the jejunum and ileum, while other ETEC mainly colonize the distal jejunum or the ileum. Other changes include vascular congestion, haemorrhages and increased number of inflammatory cells (neutrophils and macrophages in the lamina propria and mild villous atrophy) (Fairbrother and Gyles, 2012). Interestingly, in EPEC infection the regions involved are usually the sides of the villi of the small intestine. The lesions are usually sparse and up to seven sections of intestine must be screened to observe the characteristic attaching and effacing (A/E) lesions.

3. Which differentials diagnosis should be considered in case of neonatal colibacillosis and PWD?

Disease outbreaks in large populations are multifactorial and focusing the diagnosis, as well as the following control strategies on single causes can misguide practitioners. The approach to enteric colibacillosis must consider the differential diagnosis and the potential different causes that can be involved at the same time in an outbreak. The predominant type of enteritis and the localisation of the lesions (small or large intestine, disseminated) are usually enough to reduce the potential causes to be listed in the diagnostic pathway. Diarrhoea in the pre-weaned piglet is probably straightforward to identify, treat, and prevent than post-weaning diarrhoea. In particular ETEC neonatal diarrhoea must be differentiated from other causes of diarrhoea, including EPEC, Clostridium difficile, Clostridium perfrigens type A and C, enteric coronavirus (TGEV, PEDV) and rotavirus groups A, B and C. In piglets older than 7 days coccidiosis due to Isospora suis should be also considered (Table 2).

ETEC PWD should be differentiated from other causes of diarrhoea already described in piglets such as EPEC, enteric coronavirus (TGEV, PEDV), rotavirus groups A, B and C, salmonellosis, proliferative enteropathy due to Lawsonia intracellularis and Brachyspira spp. (Table 3).

Table 2: Differential diagnosis of the main agents of neonatal diarrhoea (modified from Martelli et al., 2013).

Table 3: Differential diagnosis of the main agents of post-weaning diarrhoea (modified from Martelli et al., 2013).

4. Is the presence of intestinal ETEC sufficient to cause enteric colibacillosis?

Clinical manifestations of enteric colibacillosis obviously require the presence of enterotoxigenic E.coli but also environmental changes and recognized risk factors (Laine et al., 2008). Moredo et al. (2015) demonstrated that the percentage of ETEC positive non-diarrhoeic pigs was 16.6% during the lactation period, 66% in the nursery phase and 17.3% in the finisher population. These data demonstrates that this pathogen can also be shed in faeces from healthy animals as already reported by Osek, in 1999. This information must be considered for correct interpretation of diagnostic results. In particular the evaluation of diagnostic findings should be made only in consideration of both clinical signs and pathological lesions, as well as taking in account the concentration of the E.coli strain isolated, belonging to the identified pathotype and virotype.

Studies about phenotypic characterization of intestinal bacteria of pigs during suckling and postweaning period demonstrated that E.coli, streptococci of Lancefiled group D and K and Clostridium perfrigens are among the earliest bacteria to colonize the gut in piglets. These bacteria are considered normal inhabitants of the intestinal tract, with specific strains being an important cause of diarrhoea. The mean number of E.coli biochemical phenotypes in piglets increased as animals aged. After weaning the change of intestinal environment of piglets, mainly due to dietary changes, results in an alteration of the composition of the indigenous flora. The diversity of E.coli strains of intestinal flora is usually high in healthy pigs, while in enteric colibacillosis we observe an alteration of the balance between the bacteria present in the normal intestinal flora (Katouli et al., 1995). This condition lead to the proliferation of a dominating pathogenic strain, which colonize the small intestine (Hampson et al., 1988), rapidly reaching massive numbers to the order of 109/g of contents. This is the reason why frequently, if not always, samples collected in diarrhoeic pigs affected by colibacillosis, in the acute phase of the disease, allow the isolation of a pure culture of pathogenic E.coli.

5. Is the isolation of the pathogenic strain essential? What about the direct demonstration of virulence factors in the pathological samples (faeces, intestine, rectal swabs)?

Culture of the small intestine, faeces or rectal swabs of diarrhoeic pigs affected by enteric colibacillosis, as already mentioned, usually yields pure or nearly pure cultures of haemolytic or non haemolytic E.coli. ETEC isolated from cases of neonatal colibacillosis can appear as haemolytic (ETEC F4 positive) or non haemolytic (ETEC F4, F5, F6, F41) colonies on blood agar plates. ETEC isolated form cases of PWD are mostly haemolytic (ETEC F4 or F18) even if non haemolytic strains can be observed. For these reasons, although the presence of haemolytic colonies is frequently used as a rapid means for confirming a presumptive diagnosis of enteric colibacillosis, in particular in the post weaning period, the identification of virulence factors by PCR of E.coli isolates is of fundamental importance in order to correctly identify the pathogen. The use of PCR for the identification of virulence factors directly in samples from diseased pigs, without typing of individual isolates, can make the interpretation difficult and unreliable. This diagnostic approach, in fact, can give a mix of all the detectable virulence factors belonging to different E.coli strains present in the sample and, as a result, false combinations of these factors. In addition the presence of virulence factors of other intestinal Enterobacteriaceae, possessing similar genes, can be detected.

Usually outbreaks of F4 positive E.coli tend to involve only one strain at any one time, even if mixed infections with the isolation of different virotypes in the same outbreak were observed. In these cases probably one virotype predominates in any given outbreak (Fairbrother and Gyles, 2012). For these reasons in order to determine if more than one virotype is involved in an outbreak of enteric colibacillosis, it would be useful, compatibly with the costs for the examinations, to type more than one isolate (it might be advisable to test samples from 5 pigs with diarrhoea and typing 3 isolates previously chosen for their cultural and biochemical characteristics). Although this approach does not give absolute results, certainly it increases the reliability of the results obtained.

Back to the importance of the isolation of the strain responsible of the outbreak, this is a condition necessary to test in vitro the pathogen to different antimicrobial, in order to address veterinarians in the definition of an appropriate antimicrobial therapy.

6. Which methods are commonly used to evaluate the susceptibility to antibiotics? Do some molecules require specific methods for being tested? What about the interpretative criteria?

Two methods are mainly used to evaluate the susceptibility of bacteria to antibiotics: the disc diffusion method and the dilution susceptibility testing method.

The disc diffusion (Kirby-Bauer) method is widely used for antimicrobial susceptibility testing during the routinely diagnostic activity, it give a qualitative result and it is a quick and cheap method to evaluate the susceptibility of bacteria to antibiotics. Scientific organizations such as the Clinical Laboratory Standard Institute (CLSI) an international, interdisciplinary organization, promote accurate antimicrobial susceptibility testing (AST) and appropriate reporting by developing standard reference methods and interpretative criteria for the results of standard AST methods. Interpretative criteria of CLSI are developed based on international studies and are revised frequently. The disc diffusion test is not always a reliable method for detection of antimicrobial resistance. This is the case of colistin for which other methods, such as dilution-based methods (agar dilution test, broth dilution test, etc.), should be used (CLSI, 2008).

Dilution susceptibility testing methods are used to determine the minimal concentration of antimicrobial to inhibit (MIC) the microorganism, giving a quantitative result. This can be achieved by dilution of antimicrobial in either agar or broth media. The interpretation of the results is performed using clinical breakpoints (a discriminating concentration used in the interpretation of results of susceptibility testing to define isolates as susceptible, intermediate or resistant) developed again by Scientific Organization such as CLSI.

Interpretative breakpoints for susceptibility are described by clinical breakpoints that guide to the therapy, and epidemiological cut off. The epidemiological cut-off value (ECOFF) is the minimal inhibitory concentration/minimal effective concentration value that separates the bacteria population considered into those with and without acquired and/or mutational resistance based on their phenotypes (minimal inhibitory concentration). Any isolate presenting a MIC above this value is considered as resistant (non wild-type strain) irrespective of whether or not the achieved level of resistance compromises therapy. Isolates of a given bacterial species differing from the wild-type population might be considered resistant to the drug even if their MICs do not reach the clinical breakpoint, which predicts clinical success.

Back to colistin, the ECOFF calculated for E.coli is > 2 µg/ml (EUCAST 2009). The clinical breakpoints of colistin for Enterobacteriaceae have been reported by EUCAST (2013) and by the Comité de l’antibiogramme de la Société Française de Microbiology (CASFM 2014): susceptible ≤ 2 µg/ml; resistant > 2 µg/ml. These breakpoints are under continuous evaluation. Interestingly, Burch (2007) calculated that with feed concentration of 66 ppm, colistin can reach bactericidal concentrations, in the jejunum of pigs, for strains with MIC of 8 µg/ml, but not for strains of 16 µg/ml (Boyen et al., 2010).

The management of colibacillosis

1. What are the main preventive measures for neonatal colibacillosis?

Environmental temperature

One of the most important factors for preventing neonatal colibacillosis is the maintenance of piglets at an adequate environmental temperature. Ventilation should correctly create a dry and warm environment, reducing the moisture for bacterial growth. In particular sows need temperature not higher than 22°C, while a warmer creep area for piglets with constant temperature (30-34°C) must be considered (Fairbrother and Gyles, 2012).

Hygiene

Good hygiene in the farrowing crates reduces the environmental contamination by E.coli. This goal can be achieved through an all-in/all-out farrowing system and appropriate protocols of cleaning and disinfection of the farrowing room between batches.

Immunity

In general, most neonatal infections can be prevented by passive colostral and lactogenic immunity obtained by vaccination of the sow. However, as ETEC infections are non-invasive gastrointestinal infections, mucosal (i.e. lactogenic immunity) rather than systemic (i.e. colostral immunity) immunity will be important to fight the disease. Several killed whole cell bacterins or purified fimbrial vaccines are licensed to be administered parenterally in pregnant sow (Melkebeek al., 2013). Bacterins usually contain strains representing the most important serogroups and producing the fimbrial antigens F4, F5, F6 and F41, belonging to the virotypes most frequently responsible of neonatal diarrhoea. Vaccination is usually given parenterally at about 6 weeks and 2 weeks prior to parturition (Fairbrother and Gyles, 2012).

2. Which are the main preventive measures for ETEC PWD?

Good hygiene

Good hygiene in the farrowing area and nursery leads a reduction in the number of E.coli presented to the susceptible pigs. In particular, in PWD, environmental contamination with ETEC can be crucial for the infection of newly weaned pigs. Cleaning and sanitation are very important and in nursery with several weeks of production on the same site it is advisable additional measures other than the normal washing routine. In this case it can be recommended the use of NaOH soap, after the traditional cleaning (Rowles, 2014). This step provides additional impact on the removal of biofilm that can provide an excellent matrix for E.coli to reside and to resist to the disinfectants.

Sanitation of drinking water

Sanitation of water plays an important role too. Fresh, neutral or slightly acid drinking water, free of coliform and from high levels of iron, sulphates, magnesium, nitrates and manganese, contributes to prevent enteric diseases. Chlorination of drinking water is a cheap method to treat water lines and water systems even if it is not enough to remove the biofilm in water pipe. As a general rule, more chlorine will be needed to disinfect water with high levels of contamination (such as nitrite, iron, organic matter, etc.) or with a high pH, because the latter reduces the activity of chlorine.

Feeding regimen and dietary supplements

Restrictive feeding after weaning has been used as a preventive measure against PWD and overeating after weaning has been connected with the occurrence of PWD. In addition feed with decreased protein content was shown helpful against PWD outcomes. Reducing the nutritive value of the feed by increasing the fibre content and reducing crude protein and digestible energy is the first step for controlling PWD. Moreover, it is reported that the risk of PWD is higher in weaners fed only twice a day with restricted amount of feed than on the farms providing more than two meals per day with or without feed restriction or gave feed at libitum. So, low feed intake after weaning was considered a risk factor for diarrhoea in weaners (Laine et al., 2008).

Soybean-based feed was reported to favour PWD occurrence, but fermented soybeans or barley or rice diet can be of help in reducing PWD incidence.

Other supplements such as methionine, bovine lactoferrin peptides, glutamine, protease and lysozyme showed positive effect in PWD prevention. However, contradictory results are also reported in different studies, concluding that a reduced protein content level and diet supplementary intakes did not alter PWD outcomes.

Water acidification and organic acids

Normal water usually has a pH range of 7-8. To lower the pH of drinking water using for example organic acid, can reduce the faecal shedding of E.coli. Organic acids such as lactic, formic, propionic and acetic acid, contribute to the maintenance of the acid pH of the gastrointestinal tract, which may control potentially pathogenic bacteria. A study by Bosi et al. (1999) has shown that protected organic acid led to lower E. coli counts in the ileum and higher Lactobacillus counts in the colon indicating that protected organic acid is more effective in retarding absorption of dietary acids and allowing more effective delivery of acids to the distal ileum, caecum and colon of piglets. Medium chain fatty acids (MCFA) having 6 to 10 carbon atoms also have antimicrobial property. Dierick et al. (2002) reported that about 80% of the MCFA might exert bacteriostatic and bactericidal properties in the upper small intestine. Thus, it was assumed that including MCFA along with blends of organic acid would enhance its antimicrobial effects.

Probiotics and prebiotics

Probiotic treatments with yeast or bacterial strains, for example belonging to the genus Lactobacillus, which competitively inhibit adherence of ETEC have been reported to have some beneficial effects against PWD. Also in this field contradictory results are shown by different studies. Saccharomyces has been reported to stimulate intestinal immunity and to inhibit binding of bacterial toxins to enterocyte receptors.

Prebiotics, such as selectively fermented ingredients, selectively stimulate the proliferation of potentially beneficial microorganism in the gastrointestinal tract.

Passive immunoprophylaxis

Feeding of spray-dried porcine blood plasma (SDPP) to pigs determine a reduction of the occurrence and frequency of PWD (Fairbrother and Gyles, 2012). This is probably due to the presence of specific anti-ETEC antibodies in the SDPP. It was demonstrated as these specific antibodies, which protect F4-receptor-positive pigs against ETEC infection, inhibit ETEC excretion and reduce the E. coli-induced inflammatory response of pigs (Bosi et al., 2004).

In the past it was reported that a certain degree of protection, against the F4 and F18 ETEC, was attained by the addition to the feed of eggs from hens immunized with specific antigens.

Active immunoprophylaxis

The passive protection decreases with aging and lactogenic immunity suddenly stops by weaning and as a consequence the newly weaned piglets become highly susceptible to enteric pathogens.

To prevent the colonization of PWD ETEC in newly weaned piglets, an F4 and/or F18 specific IgA response is needed in the small intestine at the moment of infection. The oral route is the most logical route to obtain this type of response. In order to have active acquired antibodies ready at weaning, piglets need to be immunized during suckling period, ideally 10 to 14 days before weaning (Melkeebek, 2013).

Several oral vaccines have successfully performed in weaned pigs using subunit vaccines as well as live oral vaccines. A live oral vaccine against F4 positive ETEC is currently commercialized in Canada, Brazil, Mexico and Europe. The vaccine can be delivered via drinking water or by individual drenching in piglets from 18 days of age. Seven days after vaccination, the onset of immunity is detectable and the duration of immunity is 21 days after the vaccine administration.

Unlike F4 fimbriae that induce protective anti-F4 immunity, oral administration of vaccines based on E. coli bacteria expressing F18 fimbriae or purified F18 fimbriae do not induce protective immunity against F18 ETEC. However, recent studies demonstrated that a minor subunit of F18 (FedF) induces protective anti-F18 antibodies and could represent a new approach for an anti-F18 vaccine (Zhang, 2014).

The vaccines currently available protect against F4 positive ETEC, but not against F18 ETEC, so the identification of E. coli responsible of colibacillosis outbreak is imperative for controlling the disease.

Although progress has been made in developing effective vaccines against PWD, the control of enteric colibacillosis is somewhat difficult due to the complexity of the disease and the immunological heterogeneity among ETEC strains. New approaches such as toxoid antigens, toxoid fusion antigens and multiepitope fusion antigen, should be considered in order to develop multivalent vaccines for effective protection against ETEC associated PWD (Zhang, 2014).

Zinc oxide Zinc

Oxide can be considered an alternative to antimicrobials or can be used in association. Feed containing between 2400 and 3000 ppm of zinc reduce diarrhoea, mortality and improve growth. For long, it has been thought that zinc oxide must have an antibacterial effect, especially against E. coli. Several antimicrobial mechanisms of zinc oxide were proposed: 1) hydrogen peroxide, which is generated from the surface of zinc oxide, can penetrate through the cell membrane, produce some type of injury, and inhibit the growth of the cells; 2) the affinity between zinc oxide and bacterial cells is an important factor for antibacterial activity. Other investigators showed that zinc oxide reduced bacterial adherence of ETEC F4 and blocked bacterial invasion by preventing increased tight junction permeability and modulating cytokine gene expression (Roselli et al., 2003).

Zinc is poorly absorbed, so it becomes highly concentrated in manure with implications in terms of environmental pollution. The therapeutic use of zinc is currently debated. In general terms bacteria in animals may develop resistance to Zn as well as to other heavy metals such as Cu. Resistance genes to Zn are often located on plasmids, which may be transferable to other bacteria, intra- and inter-species. Exposure to trace metals may also contribute to antibiotic resistance, even in the absence of antibiotics themselves. Zn supplementation to animal feed may increase the proportion of multi-resistant E. coli in gut microbiota (Yazdankhah et al., 2014). Several studies have focused the attention on heavy metals used in animal farming and possible mechanisms that could promote the spread of antibiotic resistance via co-selection. One report associated zinc with methicillin-resistant Staphylococcus aureus (MRSA) CC398 in Denmark (Aarestrup et al., 2010), concluding that zinc compounds may be partly implicated in the emergence of MRSA clones. The co-selection mechanisms include co-resistance and cross-resistance. Co-resistance is defined as the close proximity of two or more genetic elements encoding for resistances. Sulphonamide resistance, for example, would follow the co-resistance path. The cross-resistance evolves when an antibacterial agent attacks the same target, for instance efflux systems that simultaneously transport two or more types of antibacterial agents. An example of cross resistance could be done with tetracycline, as zinc resistant strains would also expel tetracycline using the same efflux system (Vahjen et al., 2015).

Antibiotic therapy and the prevention/treatment of enteric collibacillosis

1. What is the state of the art?

The use of antimicrobials is widely practiced for prophylaxis, metaphylaxis and therapeutic purposes in preventing and controlling pig enteric colibacillosis. Preventive feed medication is currently used in many Countries despite serious drawbacks and the oral administration of antibiotics is usually carried out in a large number of animals at the same time. Under-dosing is frequent with oral administration in pigs and this condition can favour the selection of resistant bacteria (Burrow et al., 2014). Antimicrobials commonly used to prevent or treat enteric colibacilosis must be chosen for their ability to achieve therapeutic concentrations at intestinal level. Among them, fluoroquinolones, cephalosporins, apramycin, ceftiofur, neomycin, amoxicillin/clavulanic acid, trimethoprim/sulphonamide and colistin are the most frequently used.

Antimicrobial resistance to several antibiotics such as apramycin, neomycin, trimethoprim-sulphametoxazole, and to colistin has been increasingly observed in ETEC strains causing PWD (Zhang, 2014). The development of resistance to a wide range of antimicrobial drugs, as well as the demonstrated trend of resistance in ETEC strains to the most of the antibiotics used for the treatment of colibacillosis in pig, is nowadays reason of concern (Luppi et al., 2013).

2. What about the resistance of E. coli to antibiotics reported in different countries?

It is difficult, if not impossible, to provide general data on resistance, because the situation is variable in different Countries and pig populations, and mainly depends on the antimicrobial preferentially used.

In a study performed in Italy (Luppi et al., 2013) on E.coli F4 positive, aiming to evaluate the trend of resistance of ETEC isolated in a period of 10 years (2002-2011), isolates obtained from cases of colibacillosis were tested using the disc diffusion method to several antibiotics. Isolates showed a statistically significant increasing trend of resistance over the whole period of study to: enrofloxacin (from 14.5% to 89.3%), flumequine (from 49.1% to 92.9%), florfenicol (from 9.8% to 64.3%), thiamphenicol (from 50% to 92%) and cefquinome (from 3.8% to 44%). An increasing resistance (not statistically significant) was also observed to gentamicin (from 63.6% to 85.7%), apramycin (from 61.8% to 82.1%), trimethoprim-sulphametoxazole (from 75% to 89.3%), tetracycline (from 97% to 100%) and erythromycin (from 92.4% to 100%).

Resistance to enrofloxacin was described in E. coli strains isolated in Austria (Mayrhofer et al., 2004) and in Brazil, where nearly 30% of the isolates from cases of neonatal colibacillosis were resistant to this antibiotic (Costa et al., 2010). Fluorochinolones resistance has been strongly correlated with the quantity of the drug used to treat pigs and plasmid borne transfer of fluorochinolones resistance, in pig E. coli strains, has been demonstrated (Barton, 2014). Resistances to cefquinome and ceftiofur have been already described among E. coli isolates as demonstrated in a Swiss study (Stannarius et al. 2009). Relatively low levels of resistance to ceftiofur were reported in E. coli isolates from diseased pigs in Canada (11%) and Spain (4%) (Aarestrup et al., 2008). High levels of resistance to gentamicin were reported in pathogenic E. coli isolates in Belgium (46%), Poland (45%) and Spain (20%) (Aarestrup et al., 2008). Resistance to gentamicin and other aminoglycosides is usually transmissible, encoded on conjugative R-plasmids and often linked to resistance to other antimicrobials. The gene aac(3)-IV is the identified gene causing enzymatic cross resistance between gentamicin and apramycin (Jensen et al., 2006).

Resistance to florfenicol is reported in pathogenic E.coli. The increased resistance to this relatively new molecule should be a reason of concern, even if differences are observed between countries and/or between pathogenic and commensal strains. As an example, in a study performed in Switzerland, low resistance prevalence was found for florfenicol as well as for amoxicillin, amoxicillin/clavulanic acid, ampicillin, cefquinome, ciprofloxacin, colistin and gentamicin in E. coli isolated from healthy weaner pigs. The most frequently found resistances were against streptomycin (60.6%), sulphonamide (51.5%), tetracycline (35.2%) and trimethoprim (27.5%). With exception of colistin, most resistances were found for those antibiotics commonly used on the farms (Stannarius et al., 2009). Resistance to trimethoprim-sulphamethoxazole and to tetracycline are frequently observed among E. coli isolates as reported in UK, Spain, Canada, Denmark, France and Japan (Burch, 2005; Stannarius et al., 2000; Kozak et al., 2009; Aarestrup et al., 2008). High levels of resistance to streptomycin (88.3%), trimethoprim/sulphonamide (78.8%), tetracycline (57.3%) were described in E. coli strains isolated from faecal samples derived from swine in Poland (Mazurek et al, 2013). The high levels of resistance to tetracycline is probably due to the wide use of this antibiotic in the past for treating pig respiratory and enteric bacterial diseases, as described by Burch in 2005 in the UK.

The majority of the E. coli isolates from cases of PWD in Australia resulted resistant to streptomycin and tetracycline and just less than an half were resistant to spectinomycin, ampicillin and trimethoprim-sulphametoxazole. In the same study, a smaller number of isolates were resistant to neomycin and apramycin, and a proportion of these showed resistance to gentamicin, while few were resistant to florfenicol. None of the isolates were resistant to enrofloxacin or ceftiofur (Smith et al., 2010). A study performed in Korea showed as E. coli strains isolated from diarrhoeic pigs were multi-resistant (resistant to more than 4 antibiotics) whit high levels of resistance to several antibiotics: streptomycin (100%), tetracycline (97.3%), gentamicin (77%), trimethoprim-sulphametoxazole (75.7%), amoxicillin (75.7%), ampicillin (73%), chloramphenicol (64.9%), enrofloxacin (64.9%) and ciprofloxacin (59.5%) (Lee et al., 2009).

Multidrug resistance among ETEC isolates has been described and recently there has been an increasing tendency for porcine ETEC to express a multidrug-resistant phenotype (Smith et al., 2010). Multidrug-resistant pathogenic E. coli strains are often isolated from diarrhoeic pigs and resistance genes are frequently on plasmids.

3. What are the prospects for the use of colistin in the treatment of colibacillosis?

Colistin is commonly used, mainly in oral presentation, due to its excellent activity against E. coli. Colistin is a bactericidal drug that binds to lipopolysaccharide (LPS) and phospholipids in the outer membrane of Gram negative bacteria. Studies on the use of antibiotics in France showed that one-third of the antimicrobials used in pigs was attributable to colistin. In Belgium colistin is the most commonly used antibiotic to treat PWD and oedema disease in pigs. High colistin use was reported in Spain, while in Denmark, its use increase from 2003 to 2011. The usually recommended dose for therapy is 100,000IU/kg BW, but in some non-European countries, it is authorised the use of colistin at lower dosage, as feed additive for growth promotion. Even if colistin was characterized by a general low degree of resistance, in the last few years E. coli strains resistant to colistin is becoming more common. Strains of E. coli with acquired resistance are encountered among pathogenic isolates commonly in pigs suffering of diarrhoea (Kempf et al., 2013).

Table 4: Percentage of colistin resistance in E.coli isolated from healthy and diseased pigs (modified from Kempf et al., 2013).

Resistance to colistin is based on mutations responsible for modification of the LPS charge. Until now, polymyxin resistance has involved chromosomal mutations making the resistance mechanism unstable and incapable of spreading to other bacteria but has never been reported via horizontal gene transfer. A study performed in China (Liu et al., 2016) on antimicrobial resistance in commensal E. coli from food animals has shown an increase of colistin resistance and has described the emergence of the first transmissible, plasmid-mediated polymyxin resistance in the form of MCR-1. In terms of antibiotic resistance, plasmids play a central role as vehicles for resistance gene capture and subsequent dissemination.

Following a request from the European Commission (EC), in July 2013 the Committee for Medicinal Products for Veterinary Use (CVMP) and the Committee for Medicinal Products for Human use (CHMP) of EMA (the European Medicines Agency), adopted scientific advice and detailed considerations on colistin. This advice critically reviews recent information on the use of colistin in food-producing animals in the EU, its effect on the development of resistance to this category of antimicrobial agents in bacterial species that are of importance for human and animal health, and the possible impact on human and animal health.

The advice confirmed the importance of colistin in veterinary medicine for the treatment of enteric diseases in certain food-producing species for which there are few effective alternatives available. It was also highlighted that currently there is no evidence of spread of colistin resistance from food-producing animals to human patients, or vice versa. However, colistin is nowadays a last resort drug in human medicine in the context of treatment of infections caused by multi-drug resistant Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae such as E. coli and Klebsiella pneumoniae, for which mortality can be extremely high. Based on current evidence, the advice concluded that it is considered appropriate to maintain the use of colistin in veterinary medicine but to restrict indications to therapy or metaphylaxis and to remove all indications for prophylactic use in order to minimise any potential risk associated with a broader use. The advice give indications also about combinations of colistin with other antimicrobials and unless valid justification, combination products should be withdrawn. The advice gives also other responsible use principles:

1. to avoid the use of colistin as a substitute for good management practices

2. colistin should be only used based on susceptibility testing

3. the duration of the treatment should be limited to the minimum time necessary for the treatment of the disease, and that treatment should not exceed 7 days (these indications should be added in the summary of product characteristics or SPC)

4. use of the product deviating from the instructions given in the SPC may lead to treatment failures and increase the prevalence of bacteria resistant to colistin.

The final opinion was converted into a Decision by the European Commission on 16 March 2015. Recently the EMA has been asked by the European Commission to update its advice on the use of colistin in animals in the light of the recent discovery reported by Liu et al. (2016) described above.

Conclusion

Managing colibacillosis in pigs requires the understanding of the epidemiology of different pathotypes and virotypes and the conditions under which they are capable of causing disease. Before that, it is of fundamental importance to achieve a correct diagnosis and the laboratory is of great help to make a definitive diagnosis that allows the establishment of preventive and control measures. For these reasons routine diagnostics are needed to know what pathogens are involved in a hypothetic outbreak, in which the diarrhoea is the main symptom. A complete and accurate diagnosis considers an appropriate sampling for isolation, typing and testing the antimicrobial susceptibility of the pathogen. The use of antimicrobials is widely practiced for prophylaxis, metaphylaxis and therapeutic purposes. The growing concern about the increase of antimicrobial resistance among pathogenic E. coli strains is driving more attention to the alternatives to antibiotics such as vaccines, probiotics, prebiotics, additives, and good management practices. It is always more frequent to isolate multi-resistant E. coli strains from diarrhoeic pigs and in many cases there are very few alternatives, in terms of molecules that can be effective against these pathogens. These conditions force a more rational and judicious use of the antibiotics.

Presented at the 24th International Pig Veterinary Society Congress. For information on the next edition, click here.