The choice of the fruit pomegranate for spoilage studies was based on two reasons. There are no studies on pomegranate fruits. The second reason is, it is a sturdy fruit and is available throughout the year in Maharashtra, India. The pomegranate fruits can be kept for up to 12 days at room temperature. Other fruits like mango, banana or papaya will spoil soon at room temperature and is difficult to detect spoilage because of microbes or the fruit physiology. Therefore, by using pomegranate fruit, it’s easier to evaluate the spoilage potential of individual bacteria or consortia and spoilage can also be proved by Koch’s postulates.
This study shows that the pomegranate surface is a habitat for a wide range of bacteria. The microbial load on the pomegranate surface was 103 cfu/g and the total yeast and mould count was 102 cfu/g. The microbial spectrum could vary with the source of fruit orchard, handling ways and season. Therefore, 20 fruits bought from different sources were used to collect the surface microflora. From each fruit, a swab was spread on the plate. Based on the colony characteristics (shape, colour and serration) total 17 unique colonies were isolated (Table 1). Most of the colonies (> 70%) were white, opaque, mucoid and round. Each of these bacterial colonies was identified by 16S rRNA sequence. The detailed identification is given in Table 1. Diverse bacterial genera were identified from the pomegranate surface as can be seen from phylogenetic tree (Fig. 1a).The phylogenentic tree was generated using Molecular Evolutionary Genetics Analysis version 11 (Tamura, Stecher, and Kumar 2021). These belong to Firmicutes, Enterobacteriaceae, Micrococcaceae, Microbacteriaceae, Xanthomonadaceae and Gammaproteobacteria family (Fig. 1b). Major genera associated with the surface were Acinetobacter, Micrococcus, Pantoea, Microbacterium, Strenotrophomonas, Bacillus, Staphylococcus and Exiguobacterium. The sequences were submitted to Genbank with accession number MN173078-MN173096. The maximum representation is from the Bacillus genus (9 species). There was no significant difference in bacterial diversity between individual fruits studied. However, we cannot rule out the possibility of variation in the bacterial diversity of pomegranate fruits sourced from different geological locations and time periods. But, the bacterial species isolated and reported here are most likely predominant species. Since numerous reports have reported a similar species composition on the other fruit surface (García et al. 2013 ; Leff et al. 2013; Mamphogoro et al. 2020).The metagenomic study across different time points and geographical locations may give much better insight into the microbial diversity of pomegranate.
Table 1
Microbes isolated from pomegranate surface and their potential to produce different enzymes and cause spoilage. “Positive” mean production of particular enzyme by the organism.
| Putative microbes | Cellulase | Amylase | Protease | Lactase | Pectinase | Spoilage Organism |
| Acinetobacter schindleri | | | | Positive | | Yes |
| Bacillus megaterium | Positive | Positive | | | Positive | Yes |
| Bacillus endophyticus | | Positive | Positive | | | Yes |
| Micrococcus luteus | | | | | Positive | No |
| Bacillus aryabhattai | Positive | Positive | Positive | | | Yes |
| Bacillus spp. | | Positive | Positive | | | Yes |
| Pantoea dispersa | | | | | | Yes |
| Bacilluszanthoxyli | Positive | | | | | Yes |
| Staphylococcus saprophyticus | | | | | | No |
| Bacillus acanthi | Positive | | Positive | | | Yes |
| Exiguobacterium enclense | | Positive | | | | Yes |
| Enterobacter cloacae | | | | | | No |
| Acinetobactercalcoaceticus | Positive | | | | | Yes |
| Bacillus cereus | Positive | Positive | | | Positive | Yes |
| Bacillus subtilis | Positive | Positive | | | | Yes |
| Strenotrophomonas maltophila | | | Positive | | | Yes |
| Microbacterium barkeri | Positive | | | | | Yes |
However, the aim of the current study is to isolate culturable bacteria and study spoilage physiology. Out of 17 species identified, 10 are known for food spoilage (Table 1), which gives us a clue that this normally present flora on the fruit surface can lead to spoilage of the fruit under certain conditions. Food-borne pathogens associated with genera were low; only Staphylococcus and Enterobacter were detected. It was shown that differences in relative abundance in microbial composition can also be attributed to changes in metabolites, physical interaction and symbiotic interactions between the host surface and other surface inhabitants (García et al. 2013). The bacteria on fruit or any other surface compete for space and nutrients. The complex dynamics of these bacterial communities decide the fruit spoilage. Since many of the bacteria cannot be cultured, it is difficult to understand the physiology of these bacterial interactions. However, in the current study, we could select four cultures with distinct colony morphology for interaction studies. In each set, two bacteria were co-inoculated in the Luria broth (LB) media and survival was observed by the plating method. Since we can distinguish the colonies, their survival could be monitored. A total of 12 sets of this co-culture were set up (Table 2) and observed for at least 10 days at room temperature. Exiguobacterium enclense out-competed all four tested species. Micrococcus luteus inhibited Pantoea dispersa and Bacillus megaterium. Pantoea dispersa survived better than Bacillus megaterium. Exiguobacterium enclense and Bacillus megaterium both are reported as the plant promoting bacteria in rhizosphere. However, there are no reports on the implications of these bacteria in food spoilage. Exiguobacterium enclense produces amylase and Bacillus megaterium produces cellulase, amylase and protease. Hence, both bacteria may contribute to fruit spoilage. Further, the four bacteria (Exiguobacterium enclense, Pantoea dispersa, Bacillus megaterium and Micrococcus luteus) have been reported to produce different bioactive compounds (Matar et al. 2009; Shanthakumar et al. 2015; Umadevi et al. 2013; Walterson et al. 2014). These bacteria may inhibit the quorum sensing and hence survive better than the other bacteria. These cultures may interact in different modes on the fruit surface. Since fruit surfaces may promote or inhibit a certain set of bacterial populations (Leff et al. 2013). However, these co-culture studies were not done on the fruit. Since the uniform distribution of competing cultures and retrieval of these cultures at a regular time point is difficult to reproduce. But the current co-culture studies give insights into inter-species interaction. However, multiple cultures with green florescent markers and continuous monitoring with time-lapse microscopy may shed more insights into the true nature of these bacterial interactions on the fruit surface.
Table 2
Competition among different microbes isolated from pomegranate surface and their survival in Luria broth in competition with other microbes for 10 days. The competition experiment suggest Exiguobactrium is most dominant species followed by Micrococcus, Pantoea and Bacillus. The order of dominance is Exiguobacterium > Mircococcus > Pantoea > Bacillus
| Competition between Organisms | Organism survived in competition after 10 days |
| Exiguobacterium + Micrococcus luteus enclense | Exiguobacterium enclense |
| Exiguobacterium + Pantoea dispersa enclense | Exiguobacterium enclense |
| Exiguobacterium + Bacillus megaterium enclense | Exiguobacterium enclense |
| Micrococcus luteus + Pantoea dispersa | Micrococcus luteus |
| Micrococcus luteus + Bacillus megaterium | Micrococcus luteus |
| Pantoea dispersa + Bacillus megaterium | Pantoea dispersa |
In the literature, many bacterial species are reported associated with food spoilage. Though seldom studies tried to prove Koch’s postulates and prove the causative organism. We attempted inoculating all 17 species, independently on the fruit surface. However, there was no significant spoilage observed after 10 days. Hence, we made 3 different combinations of 5 bacteria each (Table 3). These bacterial combinations were inoculated on the alcohol-sterilized pomegranate surface. All the 3 combinations were found to spoil the fruit within 10 days. The black spots appeared on the locations where bacterial cocktail was applied. We could also isolate inoculated microbes from the black spot region which proves that spoilage is caused by inoculated microbes and is not the result of any other microbial contamination (Fig. 2). The major reason of the spoilage could be the presence of Bacillus genus as most of the isolates belong to this genus, and the genus is already known for spoilage of fruits and vegetables (Tewari et al. 2015). All the Bacillus species isolated in the current study are producers of one or the other food spoilage enzymes (Table 1). But this is also possible that the combination of a set of bacteria may cause more damage than the other set. Often, food spoilage organisms produce various extracellular enzymes for the degradation of the food. In the current study, we found that 35% of the isolated bacteria produced cellulase, 41% produced amylase, 5% produced lactase, 17% produced pectinase and 26% were positive for the production of protease enzymes (Fig. 3). We found that Bacillus spp. were producing a maximum amount of amylase, cellulase and pectinase and this could be one reason for the predominant presence of the Bacillus genus on the fruit surface.
Table 3
Black spot produced by different combination of microbes isolated from surface of pomegranate. +ve indicate presence of black spot which means infection occurred.
| Isolate Combination | Black Spot Detection |
| Acinetobacter pitti + Micrococcus luteus + Bacillus megaterium + Pantoea + Staphylococcus saprophyticus | +ve |
| Exiguobacterium enclense + Micrococcus luteus + Bacillus megaterium + Microbacterium barkeri + Strenotophomonas maltophila | +ve |
| Pantoea dispersa + Microbacterium barkeri + Bacillus megaterium + Exiguobacterium enclense + Strenotrphomonas maltophila | +ve |
Hence, any bacterial species which has an inhibitory effect on the Bacillus species could be a good bio-control agent.
The food spoilage organism and antibiotic resistance are not related. However, there are many reports on an increase in the antibiotic resistance gene pool in the environmental microbiome. In 2021, Larsson et al., has indicated the importance of the environment in the evolution of resistance (Larsson et al. 2022). The fruits are often treated with pesticides and this also may lead to the selection of an antibiotic-resistant population (Jeadran et al. 2020). Another major concern is the horizontal gene transfer from these resistant isolates to the sensitive bacteria (Von et al. 2016; Larsson et al. 2022). Hence, bacterial isolates from the pomegranate were evaluated for different antibiotic resistance genes and antibiotic resistance profile is given in Table 4. A few of them were also tested positive for antibiotic resistance genes. These bacteria were found to contain blaTEM and aadA genes (Ramirez et al. 2010). blaTEM gene encodes resistance against β lactam antibiotics, e.g. Penicillin, cephalosporin etc. (Bajaj et al. 2015; Lachmayr et al. 2009). Among the tested microorganisms, 78.9% were blaTEM positive whereas aminoglycoside resistance genes were present in 10% of the tested microbes.
Table 4
Antibiotic resistance profile of microbes isolated from pomegranate surface. The zone of inhibition was measured in “cm”. The zone of inhibition indicate that organism is sensitive to the antibiotics where “R” represents resistance of microbe to that particular antibiotic.
| Organism | Ampicillin (cm) | Chloramphenicol (cm) | Vancomycin (cm) | Methicillin (cm) | Rifampicin (cm) | Ofloxacin (cm) | Streptomycin (cm) | Ticarcillin (cm) | Cephalothin (cm) | Ciprofloxacin (cm) |
| Acinetobacter schindleri | R | 2 | 0.8 | R | 1.5 | 2.1 | R | 3 | R | R |
| Bacillus megaterium | 1.7 | 2.6 | 2 | R | 1.9 | 2.6 | 4 | R | 3 | 3 |
| Bacillus endophyticus | 1.8 | 3 | 1.6 | R | 1.8 | 2.5 | 1 | R | 2 | 1 |
| Micrococcus luteus | 1 | 1 | 2 | R | 1 | 1 | 2 | R | 3 | 1.8 |
| Bacillus aryabhattai | 1 | 3 | 1.7 | R | 2.1 | 2.5 | 2.1 | R | 4 | 3 |
| Bacillus spp. | 1 | 1.8 | 1.9 | R | 1 | 1.7 | 2 | R | 3 | 2.8 |
| Pantoea dispersa | R | 2 | 3.5 | 1 | 2 | 1 | 2 | R | 2 | 2 |
| Bacillus zanthoxyli | 1.8 | 1.8 | 2.5 | R | 2 | 1.6 | 1 | R | 2 | 2.8 |
| Staphylococcus saprophyticus | 2.1 | 3 | 2.2 | R | 1.8 | 1.6 | 1 | R | 2 | 1 |
| Bacillus acanthi | 1 | 0.9 | 3 | R | 2 | 2.9 | 2 | R | 2 | 1.4 |
| Exiguobacterium enclense | 3 | 2.5 | 1.4 | R | 2 | 2 | 2 | R | 2.8 | 2.1 |
| Enterobacter cloacae | R | R | R | R | R | R | R | R | R | R |
| Acinetobacter calcoaceticus | 0.8 | 3 | 2 | 2 | 1.1 | R | R | R | R | 3 |
| Bacillus pseudomycoides | 2 | 1 | 1 | 1 | 1 | .6 | 2 | 1.1 | 1 | 0.9 |
| Bacillus subtilis | 2 | 2.1 | 1.9 | 0.4 | 1.1 | 1 | 2.3 | 2 | 1 | 0.9 |
| Strenotrophomas maltophila | 1 | 1.0 | 1.6 | 2 | 1.6 | 2 | 1.6 | .6 | .9 | 1.2 |
| Microbacterium barkeri | 1 | 0.6 | 0.2 | 1.1 | 1.2 | 1.9 | 2 | 2.1 | 2.2 | 2.1 |
The current study couldn’t address a few questions. Is the inter-species competition on the fruit similar to that observed in the growth medium? The number of permutations and combinations of the 17 culturable bacteria is huge. Therefore, all the combinations were not tested for fruit spoilage.
This study shed light on the ecology of the culturable microbiome of the pomegranate fruit, which may be responsible for the spoilage of pomegranate fruit. We attempted to assess competitive fitness among different spoilage-associated isolates and it was found that Exiguobacterium is the dominant species followed by Micrococcus, Pantoea and Bacillus. The reason for Exiguobacterium being a dominant species in a competitive environment might lie in its ability to survive in varying environments ranging from different pH, and temperature to high salt and metal stress. These isolates can also produce hydrolytic enzymes. The study of blaTEM gene containing environmental reservoirs of bacteria on the fruit surface will be helpful in devising strategies to protect the public from the menace of clinical risks linked with antimicrobial-resistant bacteria.