Salmonella grows massively and aerobically in fecal matter

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 fecal matter; we inoculated freshly isolated Salmonella strains (from chickens) in chicken fecal matter and incubated for 24, 48 and 72 hrs under aerobic and anaerobic conditions. We found that both Salmonella and E. coli multiplied massively in fecal matter outside a host for 72 hrs, being their growth 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.


Introduction
Environmental transmission of intestinal pathogens is extremely important especially in Lowand Middle-Income Countries (LMICs) due to deficient sanitary infrastructure, unplanned urban growth, lack of wastewater treatment, etc. One of the main concerns in LMICs is the large proportion of untreated wastewater used for irrigation (Khalid et al., 2018) and the increasing use of animal manure as fertilizer without suitable treatment (Mandrell, 2009); these are problems that remain neglected in LMICs (Khalid et al., 2018). Reports of grave enteric infections caused by environmental contamination of produce are also commonplace nowadays in industrialized countries (Callejón et al., 2015). Some of these outbreaks have been associated with high mortality, morbidity and large economic losses (Mandrell, 2009). The incidence of these infections is exacerbated by the increasing appeal to consume natural, non-processed fresh products (Mandrell, 2009).
Salmonella contaminated water is responsible for a large number of outbreaks by ingestion of water or produce (Mandrell, 2009); the sources for this contamination are human and nonhuman fecal matter (Medrano-Félix et al., 2017). The use of animal waste as fertilizer constitutes a serious risk which can be controlled by appropriate composting technology (Tiquia et al., 1998;Szogi et al., 2015). Human waste contamination, however, is much more difficult to monitor or control in LMICs where wastewater treatment or toilets are not available (Khalid et al., 2018); the fate of Salmonella in these conditions is not understood completely, although some researchers indicate that Salmonella enters into a viable-non culturable state outside the host (Winfield and Groisman, 2003). The reduction of the risk of this type of transmission requires the understanding of every aspect of Salmonella physiology in the environment outside the host (Mandrell, 2009). It is worth mentioning that Salmonella's ability to grow in fecal matter has been ignored.
It is known that Salmonella and other Enterobacteriaceae survive in a fecal matter for some time and it has been shown that E. coli (another member of the Enterobacteriaceae) also grows massively in fecal matter (Russell and Jarvis, 2001;Vasco et al., 2015;Sharma et al., 2019). Here we tested Salmonella's ability to grow in fecal matter in aerobic conditions and discuss the potential implications for fecal waste management.

Overall approach
We inoculated fresh Salmonella isolates in chicken fecal matter and incubated for 24, 48 and 72 hrs under aerobic and anaerobic conditions; we counted Salmonella colonies number by culture and by the loop mediated isothermal amplification 3M™ Molecular Detection Assay 2 -Salmonella (MDA2SAL).
Five fresh Salmonella isolates, obtained from poultry, were identified as S. enterica serovars Infantis, Dublin, Heidelberg, Brandenburg and Stanley by means of a multiplex PCR (Kim et al., 2006). A S. Infantis resistant to nitrofurantoin was used for plate count tests.

Salmonella inoculation in chicken fecal matter
Each Salmonella strain was cultured in 4 mL of BHI, then the culture was centrifuged for 5 min at 4,000 xg; the supernatant was discarded, and the pellet was re-suspended in 500 μL of sterile saline solution. The process was repeated once to eliminate remnants of culture medium, and the resulting suspension was used to inoculate chicken feces. Fecal material was obtained from 3 2-week old broiler chickens free of Salmonella.
Fecal matter from the 3 chickens was pooled and split into seven 10 gram aliquots, placed in seven Petri dishes, mixed with 100 μL of each Salmonella strains, and three dishes were left in an aerobic environment and three in anaerobic conditions; all dishes were placed at room temperature and incubated for 24, 48 and 72 hrs (Vasco et al., 2015). Anaerobic atmosphere was created by means of BD GasPak™ EZ Anaerobe Gas Generating Pouch System with Indicator.

Colony counts
Two dishes containing fecal matter with S. Infantis (strain POL 398 B) suspension (1.5 x 10 9 cells per mL) incubated as before under aerobic and anaerobic conditions was subjected to colony count in culture media for Salmonella, E. coli and coliforms (0 hrs). After incubation, contents of the different dishes were diluted. Dilutions were made up to 10 -8 in buffered peptone water (BPW), and the dilutions 10 -6 , 10 -7 and 10 -8 were inoculated using plate pour method onto XLD and XLD with nitrofurantoin (NIT) (12 mg/L) (Sandegren et al., 2008) (we took advantage of the Salmonella strain´s resistance to nitrofurantoin to facilitate Salmonella colony count). Typical Salmonella colonies were counted in XLD and XLD with NIT. The three mentioned dilutions from each one of the fecal matters (aerobic or anaerobic) dishes were inoculated onto 3M™ Petrifilm E. coli/Coliform Count Plates (in duplicate) and incubated for 24 and 48 hrs at 37 °C. We counted red colonies (E. coli) from dishes incubated 24 and 48 hrs (from both aerobic and anaerobic). The number of coliforms corresponded to the sum of the red and blue colonies. For each treatment, we included and analyzed a fecal sample without Salmonella inoculation as a control.

Calculation of specific growth rate, µ
The specific growth rate (μ) was calculated using the formula: where N is the final population after a time interval of incubation, Δt, and No is the initial population (Maier, 2009;Montville et al., 2012).

Quantitative PCR
We determine the increase in Salmonella cells in chicken fecal matter at different time points by the loop mediated isothermal amplification method 3M™ Molecular Detection Assay 2 -Salmonella (MDA2SAL). For this purpose, we performed a calibration curve (Gandelman et al., 2010) as follows. We prepared a stool suspension with 1 g of chicken fecal matter and 9 mL of BPW (stool dilution). For the 10 -1 standard suspension we added to 900 µL of the stool dilution 100 µL of a Salmonella Infantis (strain POL 398 B) suspension whose cell concentration was determined by counting in a Petroff-Hausser chamber. For each of the following standard suspensions (10 -2 to 10 -10 ), we used 900 µL of stool dilution and 100 µL of successive dilutions of the S. Infantis suspension. Then we mix 20 µL of each standard suspension with the reagents of the kit 3M™ MDA2SAL and analyze them following manufacturer instructions.
Quantitative PCR was carried out after different incubation times (0, 24, 48 and 72 hrs); we made a 10 -1 dilution of the chicken feces (inoculated with each serovar: Infantis, Dublin, Heidelberg, Brandenburg and Stanley) and analyzed using 3M™ MDA2SAL. We used regression equation to calculate the concentration of Salmonella cells at each time point (Gandelman et al., 2010). Additionally, for each trial we run suspensions of the strains analyzed, whose concentration (number of cells per mL) was determined in the Petroff-Hausser chamber.

Statistic analysis
Using SPSS, we performed the U Mann-Whitney test, which compares independent samples that do not have a normal distribution (it is the non-parametric version of the Student's t-test).

Results and discussion
We found that Salmonella Infantis inoculated in chicken fecal matter multiplied, both in aerobic and anaerobic conditions, however, the aerobic growth was higher, notably from 48 to 72 hrs. Conversely, E. coli growth reached its peak after 48 hrs, from then on, the growth decreased (Fig. 1). Coliforms grew similarly but their numbers were higher (Supporting Information Fig. S1).

Fig. 1. Growth of Salmonella
Infantis and E. coli in chicken fecal matter, under aerobic and anaerobic conditions. Typical Salmonella colonies were counted in XLD and XLD with NIT (12 mg/L) (we took advantage of the Salmonella strain´s resistance to nitrofurantoin to facilitate Salmonella colony count). E. coli was counted in 3M™ Petrifilm E. coli/Coliform Count Plates (in duplicate). Bars indicate standard error. To determine whether this aerobic growth pattern could be extrapolated to other Salmonella serovars we inoculated chicken fecal matter with Salmonella strains (belonging to 5 different serovars), run the loop mediated isothermal amplification 3M™ Molecular Detection Assay 2 -Salmonella (MDA2SAL) at different times (under aerobiosis and anaerobiosis), and found that all strains showed similar behavior (Fig. 2). Salmonella spp. growth under aerobic conditions continued for 72 hrs (Fig. 3).  The specific growth rate (μ) under aerobic conditions showed that during the first 48 hrs E. coli had the highest value decreasing during the following 24 hrs, unlike Salmonella which started fast growth at this time (Fig. 4). We found that both Salmonella and E. coli multiplied in fecal matter outside a host. Our results indicate that Salmonella (as other Enterobacteriaceae) multiplies aerobically in fresh fecal matter (at higher levels than in the intestine) which may be a key step in the infective cycle. Our results show clear evidence that the fecal matter is a transient but very important component of the Enterobacteriaceae life cycle, where enterobacterial population expands (Russell and Jarvis, 2001;Vasco et al., 2015;Barrera et al., 2018), increasing the chances of reaching other hosts. Our study has an important limitation since we were unable to measure oxygen in fecal matter to confirm our results. , 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 hrs, t2 = 24 hrs, t3 = 48 hrs and t4 = 72 hrs. And the intervals were: Δt1 = t2 -t1, Δt2 = t3 -t2 and Δt3 = t4 -t3. Bars indicate standard error.
Microbiologists have struggled to explain why bacteria adapted to the anaerobic intestinal milieu possess energetically costly machinery to use oxygen (Govantes et al., 2000). Further, it has been shown that aerobic respiration is not important for Salmonella intestinal colonization (Barrow et al., 2015). We hypothesize that the reason for this apparent evolutionary mystery may be related to the enterobacterial ability to grow in fecal matter under aerobic conditions.
Enterobacteriaceae are facultative anaerobic which can synthesize ATP by different enzymatic pathways, depending on the external concentration of O2 and the redox changes in the environment. When O2 is available, the bacteria obtain energy by aerobic respiration, with O2 being the final acceptor of electrons. In shortage of O2, these bacteria generate ATP by one of the following mechanisms: i) synthesis of terminal oxidases that allow the bacteria to take advantage of traces of O2; ii) use of other inorganic molecules (such as NO3and S4O6 2-) as final electron acceptors (Yamamoto and Droffner, 1985;Bueno et al., 2012