Salmonella grows massively and aerobically in chicken faecal matter

Abstract

The use of wastewater for irrigation and animal manure as fertilizer can cause transmission of intestinal pathogens, conditions frequently observed in low- and middle-income countries (LMICs). Here, we tested the ability of Salmonella to grow in the faecal matter. We inoculated freshly isolated Salmonella strains (from chickens) in chicken faecal matter and incubated for 1 to 12 days, under aerobic and anaerobic conditions. We found that both Salmonella and Escherichia coli multiplied massively in faecal matter outside a host and significantly higher in aerobic conditions. Our results have critical implications in waste management, as we demonstrate that aerobic treatments may not be the best to reduce the number of Salmonella in the environment.

Fig. 1

Scheme of experimental procedures. The experiments performed in trial 1 and in trial 2 are indicated. In the first trial, we determined the growth of Salmonella by plate counting in XLD and XLD with nitrofurantoin (NIT), and by molecular detection after 0, 24, 48 and 72 h of incubation; in the second trial, we performed Salmonella plate counting daily, from day 0 to day 12 of incubation.

Fig. 2

Growth of Salmonella Infantis and endogenous E. coli in chicken faecal matter, under aerobic and anaerobic conditions. Typical Salmonella colonies were counted in XLD and XLD with NIT (12 mg L⁻¹) (we took advantage of the Salmonella strain’s resistance to nitrofurantoin to facilitate Salmonella colony count), and E. coli was counted in 3M™ Petrifilm E. coli/Coliform Count Plates. Data shown are means ± SD. Asterisks indicate a statistically significant difference (t‐test, P < 0.05) between aerobic and anaerobic growth. The number of Petri dishes counted (replicate counts) is represented by n. For Salmonella 0 h, 72 h of aerobiosis and 48 h of anaerobiosis n = 14; for 24 h of aerobiosis n = 15; for 48 h of aerobiosis n = 6; for 24 h of anaerobiosis n = 16; and for 72 h of anaerobiosis n = 10. For E. coli 0 h, 24 h of anaerobiosis and 72 h of anaerobiosis n = 6; for 24 h of aerobiosis n = 7, for 48 and 72 h of aerobiosis n = 4; and for 48 h of anaerobiosis n = 8. These experiments were performed twice and correspond to the first trial.

Fig. 3

Specific growth rate for Salmonella Infantis, endogenous E. coli and total coliforms, under aerobic and anaerobic conditions. Specific growth rate, μ, was calculated with the formula: $\mu =2.3\log (N/No)/\mathrm{\Delta }t$, where N is the final population after a time interval of incubation, Δt, and No is the initial population. The incubation times were t1 = 0 h, t2 = 24 h, t3 = 48 h and t4 = 72 h. And the intervals were Δt1 = t2–t1, Δt2 = t3–t2 and Δt3 = t4–t3. Data shown are means ± SD. Asterisks indicate a statistically significant difference (t‐test, P < 0.05) between aerobic and anaerobic conditions. The number of Petri dishes counted is represented by n. For Salmonella Δt1 aerobiosis and anaerobiosis, and Δt2 anaerobiosis n = 14; for Δt2 and Δt3 aerobiosis n = 6; and for Δt3 anaerobiosis n = 8. For E. coli Δt1 aerobiosis and anaerobiosis, Δt2 anaerobiosis and Δt3 anaerobiosis n = 6; and for Δt2 and Δt3 aerobiosis n = 4. For total coliforms Δt1, Δt2 and Δt3 aerobiosis, and for Δt1 anaerobiosis n = 4; and for Δt2 and Δt3 anaerobiosis n = 3. These data correspond to the first trial.

Fig. 4

Growth curves of Salmonella serovars: Infantis, Heidelberg, Brandenburg and Stanley. Curves were obtained by 3M™ Molecular Detection Assay 2 – Salmonella (MDA2SAL). The blue line corresponds to the growth under aerobic conditions and the red one, to the growth under anaerobic conditions. Data shown are means ± SD. Asterisk indicates a statistically significant difference (t‐test, P < 0.05) between aerobic and anaerobic growth. The number of independent readings is represented by n; for all data points n = 4. The experiment was performed once and corresponds to the first trial.