1. Introduction

“Rare actinobacteria” are non-Streptomyces actinobacteria whose isolation frequency is much lower than Streptomyces strains, commonly isolated by conventional methods [1,2]. Tiwari and Gupta [3,4] reported 120 new genera of “rare actinobacteria” in the first decade of the 21th century. A total of 40 out of 120 genera were isolated from soils, with comparatively lower percentages from other environments: marine and freshwater sediments, marine animals, plants, buildings, etc. A few reports included the rare genera Actinomadura, Nonomuraea, Micromonospora, Streptosporangium, Nocardiopsis, and Pseudonocardia as the most frequent in diverse environments [5-9]. It is noteworthy an abundance of “rare actinobacteria” in extreme environments, as exemplified in Atacama [7] and other deserts [10-13]. However, Crossiella has not been included among “rare actinobacteria” thus far. Researchers have found that Crossiella is an abundant genus in most studied Spanish caves, whether they are gypsum, karstic or volcanic [14-21], and in other terrestrial and aquatic environments.

2. The Genus Crossiella in Caves

The genus Crossiella contains two species, C. equi, causing nocardioform placentitis in horses, and C. cryophila, an environmental bacterium. Table 1 shows the occurrence of Crossiella in different Spanish caves. The high relative abundance of this genus is in moonmilks (Figure 1), either from karstic (Pindal) or volcanic (Fuente de la Canaria and Bucara II) caves, as well as in colored biofilms (Pindal, Altamira, Castañar, Covadura, Yeso) [17-21, and unpublished data]. The relative humidity is near 100% in these caves. In addition, other mineral/biological formations, such as a pink formation in Bucara II, exhibit high relative abundance (38.9%). Similarly, formations such as mucous formations or brown deposits also reach relatively high abundances (6.7-12.8%) [19]. Interestingly, low percentages of Crossiella found in the sediments under the moonmilk indicates an aerobic behaviour for this genus [22]. Crossiella was also found in phototrophic biofilms from Nerja Cave [20].

Figure 1. Scanning electron micrographs of moonmilk deposits in Pindal Cave, Spain. a) Longitudinal view of sediment covered by moonmilk. b-c) Crystalline calcite fibers (red arrow) and Actinomycetota filaments (blue arrow). d) Scanning electron micrographs and EDX spectra of crystalline calcite fibers. Note the swelling of filaments in c), similar to those reported for Crossiella cryophila [22].

Crossiella, at a relative abundance of 15.0% for was found in grey biofilms from Castañar Cave [21]. Similar grey biofilms were observed in Altamira Cave [23] and the Thyssen Museum, reaching a relative abundance of 64.2% [24]. Data from a geomicrobiological study of a Roman Nymphaeum located in the archaeological basement of Thyssen Museum, Malaga, Spain, were also included in Table 1 due to its interest.

The environmental conditions of this archaeological basement are special because they mix the characteristics of an environment heavily influenced by the natural underlying karst system with those of an enclosure located in a urban building. Apart from caves, it was remarkable that a subterranean environment, the Roman mortar pavement of the archaeological basement, was colonized by grey biofilms with a high relative abundance of Crossiella. This environment is characterized by permanent darkness, absence of visits and high relative humidity.

Table 1 shows the occurrence of Crossiella in moonmilks, grey, yellow, pink and white biofilms, and sediments from different caves and subterranean environments. Crossiella is abundant in different types of rocks, either in volcanic (Fuente de la Canaria, Bucara II), karstic (Pindal, Nerja, Castañar, Altamira) or gypsum (Covadura, Yeso) caves.

In addition to the studies in Table 1, authors have reported the occurrence of Crossiella using methodological approaches other than NGS. Stomeo et al. [25] found metabolically active Crossiella in white biofilms from Ardales Cave, Malaga, Spain; Portillo and Gonzalez [26] identified Crossiella as the major metabolically active bacterium in the black crust of a shelter located in Aragon, Spain, and Sanchez-Moral [27] reported Crossiella in Altamira Cave.

Table 2 shows the wide occurrence of Crossiella in caves in the USA, France and China. Less frequent records were found in caves in Italy, Pakistan, Portugal, Serbia and Thailand, among other countries [28-50].