Amoxicillin and thiamphenicol treatments may influence the co-selection of resistance genes in the chicken gut microbiota

General description of sequences

After the quality filtering step, removal of chimeric fragments, and read merging, a total of 3,378,323 reads with 3007 different features was obtained, with an average of 27,244 sequences per individual sample. After quality filtering, none of the samples was excluded from the analysis of microbial communities.

Amoxicillin and thiamphenicol treatments influence microbial diversity and the abundance of specific taxa

Using 16S rRNA NGS, the gut microbial community composition of the chicks in each group was characterized at different time points. At phylum level, microbiota composition varied with age rather than with treatment (Supplementary Fig. S1). Proteobacteria were the most abundant phyla at 1 day of age (d.o.a.), Firmicutes became dominant at later stages, while Bacteroidota were highly abundant in caecum samples collected at 46 d.o.a. Similar dynamics were observed also at family level, since Enterobacteriaceae and Clostridiaceae were significantly more abundant at 1 d.o.a. in all groups, Lactobacillaceae, Lachnospiraceae, and Ruminococcaceae seemed to bloom at 8 d.o.a., and Rikenellaceae were the dominant family in the caecum samples collected at 46 d.o.a. (Fig. 1; Supplementary Fig. S2).

Early-age administration

In both α-diversity indices (Fig. 2A,B), there was a trend towards increasing diversity from early to late time points in all groups; however, the only significant differences were between the group treated with amoxicillin (AMX1) and the other groups on day 21 post treatment (p.t.), and within AMX1 group between day 21 p.t. and the other time points. PERMANOVA showed that the microbial community was significantly different between the group treated with thiamphenicol (THP1) and the other two groups (i.e. AMX1 and control) on day 1 p.t. (p < 0.001) and on day 12p.t. (p = 0.048), while there were no differences at the last time point. These findings are supported by the NMDS plots (Fig. 2C–E), in which a clear spatial separation between THP1 and the remaining groups at the first two time points was observed.

LEfSe analysis performed at the genus level at each time point showed an increased abundance of genus cc_115 after thiamphenicol administration (LDA = 4.40), and reduction of Helicobacter (LDA = 4.40) and Candidatus Arthomitus (LDA = 4.49) in the control group on day 1 p.t. (Supplementary Fig. 3A). At the following time point (12 d.p.t.), Bacteroides (LDA = 4.79) were reduced in the AMX1 group, while the genus Streptococcus was enriched in the control group (Supplementary Fig. 3B). On day 21 p.t. (Supplementary Fig. 3C), six taxa were enriched in the AMX1 group, including Sphingomonas (LDA = 4.11), Megamonas (LDA = 4.69) and Bacteroides (LDA = 4.31), one in the THP1 group, i.e. Gallibacterium (LDA = 4.35), and two in the control group, i.e. Streptococcus (LDA = 4.65) and Bifidobacterium (LDA = 4.13).

Middle-age administration

Middle-age administration of amoxicillin and thiamphenicol did not affect the α-diversity of the gut microbiota, as shown by both Shannon’s and Simpson’s indices (Fig. 3A,B). On the contrary, PERMANOVA and NMDS plot (Fig. 3C,D) showed differences in the microbial community composition (β-diversity) between the group treated with thiamphenicol (THP2) and the other two groups (i.e. amoxicillin treated (AMX2) and control), but only on day 1 p.t. (p < 0.001).

On day 1 p.t., Megamonas (LDA = 4.63) was enriched in the AMX2 group, Ruminococcus (LDA = 4.61) and cc_115 (LDA = 4.38) in THP2 group, whereas Helicobacter (LDA = 4.69) and Peptoniphilus (LDA = 4.52) were more abundant in the control (Supplementary Fig. 4A). At day 9 p.t. Gallibacterium was more abundant in the thiamphenicol treated group, while Peptoniphilus was less abundant in the control group (Supplementary Fig. 4B).

Amoxicillin and thiamphenicol influence the abundance of β-lactam and phenicol resistance genes

With the exception of bla_NDM, all the other ARGs investigated were detected in at least one sample. All of the detected ARGs were also identified in the control group, with the exception of bla_VIM-2 and bla_OXA-1. Genes conferring resistance to either β-lactams (i.e. bla_TEM-1, bla_SHV, bla_OXA-1 and bla_OXA-48) or phenicols (i.e. catA1, catB3, floR, and cmlA) were detected in chicks of 1 d.o.a. (Supplementary Fig. S5). In the caecum samples collected at the slaughter house, seven out of fourteen ARGs were identified (i.e. bla_TEM-1, bla_CMY-2, catA1, catA2, catB3, floR, and cmlA). All samples were characterized by at least one ARG (min = 1, max = 7, and mean = 3.83), and 89.84% of the samples showed resistance to both antimicrobial classes. Overall, the relative abundance of ARGs conferring resistance to β-lactams and phenicols was significantly increased after treatments; however, genes conferring resistance to β-lactams and phenicols showed different temporal fluctuations in the different groups.

Early-age treatment

The relative abundance of ARGs conferring resistance to phenicols was significantly increased in both AMX1 and THP1 groups compared to the control (Fig. 4A,B); however, while abundance was significantly higher at all time points in the THP1 group, it decreased on day 21 p.t. in the AMX1 group. Compared to the control group, ARGs against β-lactams were more abundant in the THP1 group only on day 1 p.t., while the relative abundance of these ARGs was higher in the AMX1 from day 12 p.t. onwards.

When considering individual ARGs, floR was more abundant in both AMX1 and THP1 groups than in the control group only on day 1 p.t. (Fig. 4C), while cmlA showed higher abundance in both AMX1 and THP1 groups until day 12 p.t. (Fig. 4D). None of the four remaining ARGs conferring resistance to phenicols was enriched after amoxicillin or thiamphenicol administration at any time point. On day 1 p.t., bla_TEM-1 was more abundant in both the AMX1 and THP1 groups compared to the control; however, while the abundance in the AMX1 group remained significantly higher at the following time points, it decreased in the THP1 group (Fig. 4E). bla_SHV was enriched in the AMX1 group on day 1 p.t. (Fig. 4F), while bla_CTX-M1-like was significantly more abundant in the THP1 group compared to the other two groups on day 12 p.t. (Fig. 4G). Of the remaining ARGs conferring resistance to β-lactams, none was enriched after both antimicrobial administrations.

Middle-age administration

The relative abundance of both β-lactam and phenicol ARGs was significantly increased in the treated groups on day 1 p.t.; however, on day 9 p.t. only genes conferring resistance to the class of the antimicrobial administered were enriched (Fig. 5A,B). floR and cmlA were more abundant after the administration of both AMDs but, while cmlA was enriched on day 1 post both treatments, floR was enriched on day 1 p.t. in the AMX2 group and on day 9 p.t. in the THP2 group (Fig. 5C,D). Interestingly, catA1, conferring resistance to phenicols, was enriched only after the administration of amoxicillin (1 d.p.t.), while bla_CMY-2, conferring resistance to β -lactams, was enriched in both the AMX2 (1 and 9 d.p.t.) and THP2 (9 d.p.t.) groups (Fig. 5E,F). None of the remaining ARGs was significantly enriched after either treatments at any time-point.

Microbiome and ARGs composition of caecum samples

Neither the type of AMDs administered nor the age of treatment seemed to have affected the microbiota diversity of the caecum samples collected at the slaughterhouse, since no significant differences in α- or β-diversity were detected (Fig. 6A–C). Similarly, there were no significant differences in the abundance of ARGs between treated and control groups (Fig. 6D,E).

Associations between microbial communities and ARGs and co-occurrence of ARGs

The associations between ARGs occurrence and specific taxa (genus level) are summarized in Table 1. Seventeen taxa were significantly associated with at least one ARG, Lactobacillus showing the highest number of associations (n = 3), while cmlA and bla_CMY-2 showed the highest number of associations with specific taxa. The occurrence of bla_CMY-2 showed positive associations with five genera (e.g. Subdoligranulum and Butyricicoccus) and negative associations with Streptococcus, Lactobacillus, and Enterococcus, while bla_SHV was positively correlated with five taxa, including Lactobacillus and Faecalibacterium. cmlA was positively associated with genera Lactobacillus, Bacteroides, and Subdolingranum, while negative associations were identified with eight taxa, including Escherichia/Shigella and Alistipes. Positive associations were also identified between floR and genera Escherichia/Shigella and Enterococcus. Both catA1 and catB3 were correlated with an increased abundance of Ruminococcus torques, while the latter gene was also associated with Clostridia.

Positive correlations were observed between genes conferring resistance to the same antimicrobial class and also between genes conferring resistance to β-lactams and phenicols. In detail, positive correlations were identified between bla_CMY-2 and catA2 (Spearman r = 0.2561, p = 0.003), between bla_TEM-1 and floR (Spearman r = 0.3662, p < 0.001) and cmlA (Spearman r = 0.4133, p < 0.001), and between bla_CTX-M1-LIKE and cmlA (Spearman r = 0.2286, p = 0.009). Data regarding the co-occurrence of ARGs are reported in Table 2.

Source: Ecology - nature.com