Extra-intestinal
The outburst of the antibiotic resistance phenomenon at global level has occurred due to the excessive and inappropriate use of antimicrobials in various fields, both in human medicine and in veterinary and zootechnical settings, strongly accelerating the development and diffusion of resistant strains. For instance, intensive livestock farming practices that compel farmers to rely more heavily on antibiotics have determined a dramatic increase in the prevalence of antibiotic-resistant bacteria in farm animals and food [
Quinolones in particular have long been the main choice of antimicrobial agent for the treatment of various Gram-negative infections, both in human and in veterinary medicine, ostensibly increasing the rate of resistant isolates all over the world [
Nontarget, commensal enteric bacteria are also exposed to this wide variety of antimicrobial substances, leading to an increase in resistance genes and, potentially, their horizontal transfer. Hence these bacteria may function as a reservoir of resistance though largely ignored [
According to the EFSA and ECDC report [
Furthermore, there is increasing evidence that
Between January and March 2017, 46 samples were analyzed at the Experimental Zooprophylactic Institute of Sicily “A. Mirri.” All the samples, in individually sealed packages, were purchased from different supermarkets in Palermo. They consisted of 23 poultry, 13 beef and 10 pork samples. According to the labels, all the samples came from intensive farms based in Sicily.
The samples were immediately sent to the laboratory on ice and subsequently processed in asepsis.
10 g of each sample was added to 90 ml of saline peptone solution (SSP). After homogenization by Stomacher and incubation for one hour at room temperature, samples were plated into Tryptone Bile X-Glucuronide (TBX) Agar. Colonies developed 18 to 24 hours after incubation at 44°C. They were tested by disk diffusion for resistance to fluoroquinolones, in particular to ciprofloxacin (CIP, 5
A single colony was suspended in 200
DNA extracts were analysed with multiplex PCR to ascertain their phylogenetic groups, as described by Clermont et al. [
Two multiplex PCRs were assayed to investigate the presence of eight virulence factors (VFs) in the
Strain genotypes were investigated with relation to the most common plasmid-mediated quinolone resistance genes:
All the strains were typed in order to screen for ST131-associated single nucleotide polymorphisms (SNPs) in
They were then subjected to Enterobacterial Repetitive Intergenic Consensus sequence PCR (ERIC-PCR), according to Versalovic et al. [
The fingerprints were photographed by the GelDoc (BIO-RAD) system and finally analyzed using the BIO-NUMERICS software (Applied Maths, Kortrijk, Belgium). Comparisons between band patterns were performed with the Dice similarity coefficient. The obtained matrices were combined using the UPGMA algorithm to produce a dendrogram, with a cut-off of 80% similarity.
Besides fluoroquinolones, all the strains were tested by disk diffusion for susceptibility to other antimicrobials, including amoxicillin–clavulanic acid (AUG, 20–10
This study analysed 46 samples. Almost all the strains isolated from poultry samples were resistant to fluoroquinolones (91.3%). A considerably lower percentage of the strains isolated from pigs and cattle showed resistance, namely, 20% and 15.3%, respectively. In total, we obtained 25 fluoroquinolone-resistant strains, to be further characterized in the following analyses.
As regards phylogenetic groups, D1 was the most prevalent (44%), followed by group A1 (28%), A0 (20%), and lastly B1 (8%); notably, no B2 group strains were observed (Figure
Percentage of strains by phylogenetic group.
In accordance with the absence of group B2 strains, ST131 was absent among our isolates, which all tested negative for the SNPs in the two genes of interest.
A limited number of virulence factors were observed, and these were concentrated in phylogenetic group D1. In fact, this group included all the strains with the
Therefore, 7 of the 25 isolates (28%) met the inclusion criteria for ExPEC; that is, they had at least two virulence factors, according to Johnson et al. [
Percentages of strains possessing VFs.
The genes
Phenotypic resistance patterns are summarized in Table
Resistance patterns observed in all the strains.
Resistance Pattern | N. isolates (%) |
---|---|
CIP, NOR, LVX (Fluoroquinolones only) | 3 (12%) |
CIP, NOR, LVX, AUG | 3 (12%) |
CIP, NOR, LVX, AUG, TE | 1 (4%) |
CIP, NOR, LVX, AUG, SXT | 1 (4%) |
CIP, NOR, LVX, AUG, SXT, TE | 16 (64%) |
CIP, NOR, LVX, AUG, SXT, TE, CN | 1 (4%) |
Finally, ERIC PCR showed quite a high level of heterogeneity, except for two pairs of strains (2 pork strains and 2 poultry strains), which shared the same patterns of bands and hence are displayed only once (Figure
Dendrogram obtained by ERIC-PCR of strains.
The steady increase in the prevalence of quinolone-resistant ExPEC isolates is particularly alarming due to their spread as opportunistic pathogens and suggests the need to deepen our knowledge of their source, reservoirs, and transmission pathways.
Poultry meat was highly contaminated with
The most common antibiotic classes used in bred chickens are penicillins, tetracyclines, sulfonamides and quinolones [
Although veterinary use of cephalosporins is permitted by law, notably our strains did not exhibit resistance to them. While the poultry industry in Italy renounced the use of III and IV generation cephalosporins in 2009, the other farming industries continue to use these antimicrobials for a rather wide range of diseases. Their consumption of this antibiotic class is one of the highest in Europe and has shown a slightly increasing trend since 2010 [
We found a higher prevalence of phylogenetic group D1 strains (44%), followed by A1 (28%), A0 (20%), and lastly B1 (8%); these latter groups (A and B1) are usually associated with environmental and commensal strains in humans [
Our isolates did not show a wide variety of VFs, as mainly the
However, Johnson et al.’s above-mentioned classification for determining the pathogenicity of microorganisms may not be exhaustive, as there may be other unexamined factors conferring pathogenic potential. For instance, in a study by Fasciana et al. [
As regards antibiotic resistance, since all the isolates in question exhibited phenotypic resistance to quinolones, other resistance mechanisms may explain the rather low prevalence of the PMQR genes investigated (20%). Indeed, as the most common mechanisms in animal isolates are chromosomal mutations in type II topoisomerase (
Genotyping by ERIC-PCR revealed 23 banding profiles; these results support a high genetic heterogeneity, which is an alarming fact, showing a multiple onset of MDR strains despite the restricted area of sampling.
This study, although numerically limited, emphasizes the already clear need to improve strategies to prevent the spread of antibiotic resistance and to reduce the amount of antibiotics used.
The high prevalence of resistant strains in this study, despite not all of them being classified as ExPEC, poses a direct risk, as these strains can subclinically colonize the consumer’s intestinal tract until advantageous circumstances favour an extraintestinal infection, or an indirect risk, potentially contributing resistance genes to human indigenous microbiota [
For instance, a subset of human ExPEC strains, isolated by Fasciana et al. [
Given the considerable public health threat that ExPEC represents, further long-term investigations are needed to give us more insight into the epidemiologic relationship between human and food-origin
With regard to animal production systems, a review of farm management is essential, especially as far as intensive farming is concerned, combining good practices and applying good hygiene measures and animal welfare in order to reduce the use of antimicrobials (i.e., an efficient antimicrobial stewardship), thus acting on reservoirs of antibiotic resistance. At present, intensive farming systems rely on a routine use of antibiotics, creating reservoirs of antimicrobial resistance genes that could spread in the environment or to different hosts. In fact, antibiotics are often used as prophylactic prevention measures, for mass treatment that is not associated with a specific diagnosis or for preventable diseases, in a way that is no longer sustainable. In order to deal with this antimicrobial resistance emergency, different levels of safety measures must be considered, including “tertiary prevention” (i.e., increasing the ability of the animals’ immune system to respond to infections) [
In addition, according to the farm-to-fork concept, it is important that also slaughterhouses and food handling practices are taken into account in the attempt to reduce foodborne transmission. Hence, an integrated implementation of GMP (Good Manufacturing Practices) and GHP (Good Hygienic Practices) should be applied throughout the production, processing, and consumption stages, and consumer awareness should be raised.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.