Discussion

A retrospective study by Schulz et al. (2016) used the same sample set of sedimentation dust from poultry and pig farms as this research. The volume of 41 samples was too small to process, but the water activity (aw) of 78 samples was measured, revealing an average aw (standard deviation) of 0.567 (0.035) in pig farm dust, and 0.599 (0.038) in poultry barn dust (Schulz et al. 2016). It was lower than 0.65, the minimum growth requirement of xerophilic spore, for instance (Ponizovskaya et al., 2011). The aw-requirement of Enterococci was not reached. The minimum aw for Enterococcus faecalis growth was at least 0.928 at 26°C and it had to increase when the temperature dropped (Li & Torres, 1993). Besides, the genus Enterococcus grows at temperature between 5 and 50°C (Fisher & Phillips, 2009).

The dust samples in our study were sealed and stored in the dark at 4°C; hence, the growth of aerobic mesophilic bacteria was supposed to be inhibited during storage.

Figure 1 shows that the relative frequency of bacterial growing on BAA media was inversely proportional to the sampling age. The frequency of detection correlated well with the sampling periods. It could be expected that storage time reduced the cultivability of bacteria on BAA medium. The growth after 24 hours incubation increased more than that after 48 hours. The dust samples prior to 1990 showed no colony on BAA on the firstday of incubation (Table 1). Nevertheless, Enterococcus spp. could be identified after 48 hours (Table 2). This suggests that presumed Enterococci from old dust samples grow slower. Therefore, for quantification,

4. Long-term survival of Enterococci in dust samples from livestock buildings

presumed Enterococci colonies were counted after two days of incubation. Figure 2 shows the geometric mean CFU/g on TSA and BAA. The samples were divided into seven sampling periods like in Figure 1. Figure 2 shows that the more recent dust-sampling period tended to have higher CFU/g with both TSA and BAA. This is consistent with the study by Schulz et al. (2016) and can be explained by the loss of cultivability in older samples. Furthermore, the clear differences between the counts on the media in Figure 2 suggest that only a small part of bacteria (approx. 0.4% to 3.4%) can form potential colonies on BAA agar. This was expected because the azide in the selective media can inhibit the growth of gram-negative organisms (Facklam, 1973) and the Enterococci are able to hydrolyse esculin in the presence of bile (Murray, 1990; Manero & Blanch, 1999). The Staphylococcus spp., which predominates among cultivable bacteria in animal house dust (Hartung & Saleh, 2007), was suppressed by bile (Thermofisher.com, 2017).

Various species and enormous amounts of bacteria in dust made it difficult to accurately classify the bacteria. In our study, less than 30% (36/124) of suspected Enterococci on BAA were proved right on the level of genus. However, identified bacteria all belonged to the order Lactobacillales (lactic acid bacteria). A classification of the Enterococcus spp. proved difficult because others of this order also appeared. Aerococcus viridian, for instance, which is widely found in dust and animals, has similar phenotypic characteristics to Enterococci (Zhou et al., 2013).

Simply relying on catalase and oxidase tests, they are difficult to distinguish before

4. Long-term survival of Enterococci in dust samples from livestock buildings

performing API tests. Although the selective medium has obvious shortcomings, it is a cost-effective compromise. Combining catalase and oxidase tests offers an opportunity to differentiate the Enterococcifrom all growing bacteria.

According to the results of the API 20 STREP biochemical test, most of the isolated Enterococci were Enterococcus faecium (32/36). MALDI-TOF MS acknowledged this prevalence as well (30/36). The reason for this is because Enterococcus faecium is one of the most common isolates in the gastrointestinal tract of production animals (Fisher & Phillips, 2009). Five isolations of Enterococcus hirae identified by MALDI-TOF were all from the same one pig farm. Enterococcus hirae was first identified in young chickens (Bourafa et al., 2015). It is rare in humans but is a usual pathogen in mammals and birds (Savini et al., 2014). Enterococcus casseliflavus is commonly found in plants (Fisher & Phillips, 2009). The isolate may originate from organic materials such as feed in dust.

In the API tests, five isolates were identified as Leuconostoc spp.. However, these isolates could not have been identified using MALDI-TOF MS. Kulwichit et al. (2007) found that API systems have limitations in identifying Leuconostoc spp. as well. This is not only because some bacteria with overlapping phenotypes are not included in the list of API 20 STREP, but also the API system may give a false-positive result in the leucine arylamidase (LAP) and arginine dihydrolase (ADH) test (Kulwichit et al., 2007).

MALDI-TOF MS has a very high accuracy rate of identifying Enterococcus spp.,

4. Long-term survival of Enterococci in dust samples from livestock buildings

even at species level (Stępień-Pyśniak et al., 2017). The API system identified isolates accurately as belonging to the genus Enterococcus spp. Thirty-six isolates were considered as genus Enterococcus and this was by MALDI-TOF MS. At species level, 83% (30/36) Enterococcus spp. were consistently identified in API analyses and MALDI-TOF MS. Both methods are considered useful for identifying Enterococcus spp., MALDI-TOF MS showing a higher accuracy at species level.

Several previous studies on long-term survival of Enterococci mainly focused on a nosocomial environment over a period of only a few months (Noskin, et al. 1995;

Bonilla, et al. 1996; Wendt, et al. 1998; Neely & Maley, 2000). Our research proves for the first time that Enterococci can survive over a long period of time in sedimentation dust that was collected from animal farms. One isolate of Enterococcus faecium was kept viable more than 35 years in a pig farm dust sample. In the dust gathered from a broiler barn in 1994, cultivatable Enterococci were isolated. The composition of dust may have an influence on the survival of bacteria. However, storage in complex organic environments and under dry and cold conditions enabled an unexpected long-term survival of bacteria that commonly colonise in the gut of animals and humans. It is also proved that other species of the genus (Enterococcus hirae) can keep viable for over 26 years. Rapidly reducing the endogenous metabolic rate is a major survival strategy when bacteria face sparse nutrient conditions (Roszak

& Colwell, 1987). Enterococci have a good tolerance to high salt concentrations (Fisher & Phillips, 2009) and the sedimentation process may allow Enterococci to

4. Long-term survival of Enterococci in dust samples from livestock buildings

adapt to dryness. Enterococci possess a specific mechanism, namely that the membrane is more stable near the minimal growth temperature (Fisher & Philips, 2009). The aforementioned reasons explain why Enterococci can survive a long time in dust.

The tenacity of potential pathogens may prevail for years in livestock buildings when dust is not removed completely by hygienic measures. The dust could be a carrier of various microorganisms including potential pathogens, maintaining their significant genetic information such as pathogenicity and antibiotic resistance for a long time.

Therefore, dust in animal houses should be removed thoroughly. Furthermore, hygienic measures to avoid the transmission of animal house dust by people, vehicles or air during the housing of farm animals are recommended.

4. Long-term survival of Enterococci in dust samples from

livestock buildings