The spread of multidrug resistance among avian Escherichia coli is usually attributed to the selective pressure exerted by the antimicrobials included in broiler feed for the past 60 years.1 Johnson et al.2 reinforced this suspicion since antibiotic-resistant and antibiotic-susceptible E. coli isolates from retail poultry products had similar phylogenetic background, and otherwise emerged from the same source population. Regarding antimicrobial exposure in poultry production, multidrug resistance among extraintestinal pathogenic E. coli (ExPEC) allied to community acquired infections is increasing in prevalence in many parts of the globe.3 The indiscriminate use of antimicrobials is high in developing countries, where human antibiotics are often accessible for non-therapeutic uses in healthy animals.4 Therefore, colonization of asymptomatic poultry by multidrug resistant ExPEC augments the probability of resistance gene acquisition by human strains through the food-chain.

Avian pathogenic E. coli (APEC) is commonly reported as an ExPEC pathotype, but its genotype is not clearly defined. According to Kwon et al.5 at least five virulence genes are present in APEC, many of them found in plasmid pTJ100.6 However, the increased survival gene (iss) has been identified as a virulence marker to distinguish between avian and human ExPEC.7 This gene exerts anti-complement resistance and has been found in conserved regions of ColV and ColBM plasmids,8,9 which are often identified in APEc strains.6,10 Recent findings also suggest that APEC and human neonatal meningitis E. coli (NMEC) are subpathotypes from ExPEC involved in pathogenesis of both avian and human infections.11 Therefore, hybrid plasmids (ColV- and ColBM-associated plasmids) harboring a number of distinct virulence genes and MDR-encoding islands can also be present in ExPEC isolates.11 Consequently, it is now clear that selected high-virulent and multidrug resistant ExPEC strains of avian origin represent subpathotypes of great danger to human public health.

Brazil has ranked first in the world in exports of poultry meat since 2004.12 Typical selections of poultry in Brazilian slaughterhouses take into account macroscopic alterations that can result in discarding carcasses.13 However, sub-clinical symptoms are not always perceived and often do not show a clue of the presence of pathogenic microorganisms. Thus, the risk of virulence and drug resistance gene transmission between avian E. coli obtained trough the food-chain and intestinal commensal E. coli is increasing. The goal of the present study was to investigate the pathogenesis to mammalian hosts of selected multidrug resistant ExPEC strains obtained from healthy poultry carcasses in Salvador, Brazil.

The antimicrobial resistance-susceptibility profiles of 27 extraintestinal pathogenic E. coli strains from carcasses of healthy and diseased poultry are shown in Table 3. The majority of ExPEC were resistant to at least four antibiotics from different classes. The most prevalent phenotypes were resistant to levofloxacin (51.8%), amoxicillin/clavulanic acid (70.4%), ampicillin (81.5%), cefalotin (88.8%), tetracycline (100%) and streptomycin (100%). The overall multidrug resistance varied from 4 to 11 antimicrobials and reached 92.6% of E. coli strains. In addition, 40.7% were simultaneously resistant to streptomycin, levofloxacin, ciprofloxacin and tetracycline. The proportion of highly multidrug resistant strains (8–11 antimicrobials) reached 22.2%. Conversely, the aminoglycoside amikacin of avian and human use were very effective against 89.9% of ExPEC. The MIC and MBC of typical antimicrobials used in Brazilian farms were determined for those ExPEC strains resistant to at least 4 antimicrobials. The level of resistance against doxycycline, neomycin, enrofloxacin and oxytetracycline reached, respectively, 88.0%, 100%, 75% and 91.7% of the strains (Table 4).

The complement resistance to goat serum was high for strains 2B and 46 and low for strains 4A and 41A, whereas resistance to human serum was high for strains 32, 46 and 2B and low for strains 44 and 4A (Fig. 1). Experiments carried out with laboratory animals have shown that Swiss mice challenged with strains 4A, 41A and 2B can survive seven days after infection. After this time, the bacterial load in the spleen and the liver reached respectively: 3,74×103 and 8,54×103CFU/g for strain 4A; 4,8×102 and 1,56×103CFU/g for strain 41A, and 4,0×101 and 1,2×104CFU/g for strain 2B. However, strain 2B provoked severe degeneration of hepatocytes besides lymphocytic and lipid infiltration in the liver, whereas spleen showed areas of degeneration of the white and red pulp (Fig. 2B and D). Likewise, the liver of mice challenged with strain 41A showed marked vacuolization of hepatocytes and lymphocytic infiltration, whereas the spleen showed leukocyte infiltration and white pulp without boundaries (data not shown). Conversely, strain 4A showed hepatocytes, portal space and lobular veins preserved with mild lymphocytic infiltration in the liver. Also, the spleen was morphologically preserved with capsule, and white pulp with central arteriole besides red pulp (Fig. 2A and C). These histological findings were generally perceptible in all mice from each animal group independently of the bacterial load in the organs.

Fig. 1.

Resistance to serum complement of multidrug resistant avian ExPEC strains harboring the iss gene.

(0.15MB).

Fig. 2.

Histological damage to laboratory animals after challenge with avian ExPEC. The slides of the liver (A and B) and spleen (C and D) of Swiss mice challenged with strain 4A (A and C) and 2B (B and D) are shown. The full arrow (A and B) indicates leukocyte infiltration whereas empty arrow shows degeneration of hepatocytes (B). The full star shows portal space (A) and empty star shows degeneration of red besides white pulp (D). The asterisk shows the white pulp of the spleen in normal morphology (C).

(1.4MB).

Poultry-associated diseases caused by ExPEC cause massive economic losses in the food industry.18–20 However, the broad use of antimicrobials by poultry farmers in recent decades has led to the emergence of multidrug resistant strains in many parts of the globe.21–24 Additionally, among multidrug resistant E. coli from chickens and piglets in China, 97% harbored the iss gene, suggesting this gene could also be used as a multidrug resistance marker.22 In Brazil, a previous study showed that the rates of multidrug resistance among avian E. coli reached 77.5%.25 Regarding ExPEC strains harboring the iss gene, we confirmed that the majority were resistant to several antimicrobials beyond those authorized for inclusion in broiler feed. Although the number of strains investigated was small, the levels of antimicrobial resistance to ampicillin, gentamicin, streptomycin, ciprofloxacin, chlorramphenicol and tetracycline were very high in comparison to the levels found in a recent study carried out with 101 E. coli strains from broilers and layer hens with colibacillosis infections in Bangladesh.26

Additionally, we have shown that a great number of avian ExPEC are probably β-lactamase producing strains, since resistance to amoxycillin/clavulanic acid was present in 70.4% of the isolates. Oteo et al.27 reported that resistance to amoxicillin-clavulanic acid is increasing among E. coli from human origin in Spanish hospitals, affecting 5,1% of 9090 blood isolates. Considering the food-chain, it is worrying that the only inhibitory drug against all avian ExPEC is carbapenem imipinem, restricted to human use. Also of particular concern are the resistance of avian ExPEC to fluoroquinoles such as enrofloxacin, which are similar to antibiotics being used in human medicine.28 For instance, the increasing resistance of E. coli strains to ciprofloxacin has been detected in several international studies.29,30 Enrofloxacin has been withdrawal from non-therapeutic use in the poultry industry in the United States.31 In Europe, the drug cannot be used in the animals from which eggs are produced for human consumption.32 However, in this study the level of antimicrobial resistance to doxycycline, neomycin, enrofloxacin and oxytetracycline reached alarming levels. Although we cannot confirm that Brazilian farmers restrict their use of such antimicrobials to treatment regimens, it was clear that these drugs should no longer be used on Brazilian poultry farms.

Avian ExPEC strains have been genetically associated with human E. coli causing urinary infections.6 Additionally, E. coli strains of the EcoR group B2 related to human and animal extraintestinal infections have been detected among APEC strains obtained from diseased and healthy chickens.33 In spite of the implications of avian ExPEC in carcasses of healthy poultry is unclear, a previous study in the UK showed that samples of imported chicken breasts were often positive for E. coli with CTX-M-2 genotype related to human infections in South America.34 Our data shows that resistance of avian E. coli to serum complement lyses in mammalian hosts was not directly related to the presence of the iss gene. This finding suggests that other genetic determinants among E. coli of avian origin must be related to human serum resistance. Additionally, multidrug-resistant ExPEC strains 41A and 2B were often more resistant to goat and human serum complement, and also more virulent to the experimentally infected mice than strain 4A, which is sensitive to the majority of antimicrobials.

Previous studies have shown the correlation between serum resistance and virulence of E. coli causing diseases in turkeys and chickens.35 Moreover, the characterization a transferable hybrid plasmid pAPEC-O103-ColBM encoding multidrug resistance and pathogenicity was recently described.11 Even though virulence and multidrug resistance genes are not uniformly distributed among conjugative plasmids, the diversity of avian ExPEC with zoonotic potential to cause human diseases can be more frequent than perceived. For example, in the present study the risk for human health was particularly observed for strain 2B, obtained from poultry carcasses approved for human consumption. Thus, an early diagnosis procedure should be followed on poultry farms and at slaughterhouses to identify hazardous microorganisms in asymptomatic poultry, and therefore eliminate carcasses that are not proper for human consumption. The data reinforce the general concern about the spread of multidrug-resistance and virulence genes between avian and human E. coli through the food-chain.

The authors have no conflict of interest to declare.