Inter and intrapopulation clonality of S. aureus
In this study, the isoenzymatic electrophoresis profiles of S. aureus isolates from five distinct populations (A, B, C, D and E) were reproducible in three replicates of electrophoretic tests (≥95%). The high discriminatory power of MLEE (0.965–0.999) proves it to be a powerful and reliable tool for typing S. aureus in epidemiological studies (Supplemental Table 1). The reported results for the reproducibility and discriminatory power of MLEE accord with those previously reported in studies of medical relevance21,26,27,30; however, the discriminatory power described in this research was greater than those reported by other groups of researchers36,57.
S. aureus is a heterogeneous bacterium (polymorphic)58 with a clonal population structure40. Consequently, it is believed that S. aureus does not undergo extensive recombination, diversifies in large part by nucleotide mutations and displays a high degree of linkage disequilibrium (non-random associations among genic loci). In this study, qualitative and quantitative variations of polymorphic loci, average number of alleles per locus and average number of polymorphic alleles of S. aureus were observed across all 5 populations sampled. These variations have been observed in several studies of genetic diversity of S. aureus populations from human and bovine sources35,36,57-59. In addition, the identification of S. aureus strains in each population revealed polyclonal and monoclonal patterns within the populations. However, no monoclonal pattern was observed between the populations (A, B, C, D and E). These data suggest a high genetic diversity of S. aureus in every bacterial population studied (i.e., intrapopulation genetic heterogeneity) and among them, even without any correlation of interpopulation monoclonality. Specific and limited patterns of spread and transmission of S. aureus were also suggested to occur in every bacterial population. The evidence of interpopulation monoclonality and interpopulation polyclonality of S. aureus has been reported previously. Strains of S. aureus toxin 1 producers (Toxic Shock Syndrome – TSS) from ovine sources were genotypically characterized (tst gene) and considered distinct from those clones associated with cases of TSS in humans (approximately 90%), suggesting that allele differences exist (structural tst gene) among strains recovered from different host species60.
The host specificity among S. aureus clones was also demonstrated61,62. An extensive study from 2.077 S. aureus isolates from humans, cows and sheep revealed considerable genetic diversity among strains and found that most of the 252 identified clones were preferentially associated with a single species of host, whereas only 6 of 33 clonal strains were shared among cattle and humans. These results suggested that the ability of the bacterium to colonize humans or cows evolved several times during the differentiation of S. aureus populations and provided strong evidence for host specificity among clones62. The concept of host specialization among S. aureus strains is also reinforced by the fact that certain bacterial clones are predominantly associated with certain hosts, in other words, humans or animals (dairy cow), although some clones can be shared among both hosts. This fact indicates that the successful transfer of bacteria between humans and animals (dairy cow) is limited35.
Other evidence of genetic heterogeneity and host specificity comes from an analysis of S. aureus strains isolated from bovine teat skin, human skin, milking equipment and bovine milk. The analysis employed the PFGE method of genomic DNA digested by SmaI. Twenty-four main types and 17 subtypes among the isolates of 43 herds were identified, and there was still discrimination between isolates of bovine teats and milk. Although small in number, identical pulsotypes were found on human skin and on bovine teat skin, whereas the milking equipment harbored distinct pulsotypes; that is, there were both skin and milk strains. These results suggest that S. aureus strains from skin and milk can be transmitted via milking machines; however, there is no relationship with potential sources of intramammary infections caused by S. aureus in dairy cows18.
Genetic relationship of S. aureus clusters/taxa
The analysis of the genetic relationship among the S. aureus strains was determined satisfactorily using Nei’s genetic distance53 and an UPGMA dendrogram55, as demonstrated by correlation coefficient based on Pearson product-moment (rjk = 0.84949) [i.e., good agreement between dij (matrix of genetic distance) and elements Cjk (matrix of correlation derived from the UPGMA dendrogram)] (Supplemental Figure 1 and Supplemental Table 2). A high degree of genetic polymorphism (0.000 ≤ dij < 0.381) was found in the total population of bacterial isolates (i.e., on average, from zero to 38.1 allele substitutions were detected for each 100 existing loci from a common ancestral strain). Such isolates were distributed within 80 taxa (from I to LXXX), which were determined from a genetic distance of 0.1175 < dij ≤ 0.3810 (i.e., taxa genetically related to distance – strains/isolates not related). The taxon I presented a larger number of isolates, strains and bacterial clusters, followed by taxa IV, L, XXI, X, XXII, LXIV and LVII, LVI, V and II, XXIV, VI, XIII, III, XXIII and XXXVII, XXXVIII and LXVI, LXXIX, XVI, XLII and XVII, XII, XXXV and LXII, LIII, XLI and XXVI, LX, LII and LXVII, LIX, LXV, LXIX, XXIX, XLVI, LXIII and LXXIII, VIII, LI, IX, XI, XXX, XXXII and XXXIII, and others which had only one isolate/ET and no cluster. These results also suggest a high degree of genetic polymorphism because most isolates/strains of S. aureus (n = 365; 59.8%) can adapt to several population ecological niches (i.e., in two or even five distinct population origins: populations A, B, C, D and E), although allocated within few taxa (n = 16: taxa I, IV, V, VI, VIII, X, XI, XIII, XXII, XXIII, XXX, XXXIII, L, LVI, LIX and LXIV). However, a lower frequency of highly polymorphic isolates/bacterial strains (n = 245; 40.2%) can be specific to certain population groups best adapted to their ecological niches, genetically divergent and distributed in many taxa (n = 64).
The genetic relationship among clusters and between clusters and isolated/strain non-clustered, although allocated within a single taxon, was considered moderately related (0.0551 < dij ≤ 0.1175). In turn, each cluster has two or more isolates/strains that are identical or highly related (0.000 ≤ dij ≤ 0.0551). Highly related strains come from a common ancestor, that is, descendants have undergone microevolution and adaptations as a consequence of recombination (not extensive), nucleotide mutations and non-random associations among genetic loci (linkage disequilibrium)40,58. Therefore, these data suggest the possibility of intrapopulation microevolutionary processes for S. aureus (i.e., on average, from zero to 5.5 allele substitutions in 100 existing loci were detected from a common ancestral strain), as demonstrated in most of the clusters (n = 75 82.4% clusters harboring isolates/strains exclusively of populations A, B, C, D or E) and, consequently, the propagation of these microorganisms. However, epidemiological genotypic identity was suggested, that is, epidemiologically related populations maintain S. aureus strains (identical and/or highly related, moderately related, and completely unrelated) that genetically diverge from those epidemiologically unrelated populations. Interestingly, although occurring at a low frequency (n = 16 17.6% clusters), such microevolutionary changes or the co-existence of highly related strains (non-identical) were observed among bacterial populations of different origins [i.e., co-existence of highly related strains in populations A and B (clusters II and XI), in populations B and D (clusters XXX, XLVI and XLVIII), in populations B and E (clusters XVII, XVIII, XX, XXVIII, LX, LXII and LXIX), in populations D and E (cluster XLIV), in populations A, B and D (cluster XXIX), in populations B, D and E (cluster XLV) and in populations A, B, D and E (cluster I)]. These results suggest the hypothesis that some bacterial strains of S. aureus can adapt and colonize new habitats by spreading from indirect sources and are not epidemiologically related. Consequently, the occurrence of a genotypic identity can take an epidemiological dynamic character (i.e., spread to new habitat), although this is a low-frequency occurrence. The geographical location of bacterial isolates of S. aureus can partially explain this epidemiological genotypic identity. In fact, the habitat can have an important role on the adaptation of genotypically related bacterial groups and the occurrence of that identity, given that the populations A and B or C, D and E were geographically related.
The total genetic variability of populations of bacterial isolates, as proposed by Nei56, revealed a low index (<5%) attributable to differences “between” populations and a high index attributable to differences “within” populations (>95%). However, these data suggest a tendency of genetic divergence and microevolution between the S. aureus isolates of human origin (populations A and B) and bovine milkmaid origin (populations D and E), especially the mammary quarter animal anatomical site (population) (Figure 1). Another hypothesis suggests that populations from S. aureus strains of environmental and animal origin (populations D and E), as well as those of environmental and human origin (populations A and B), contribute little to the colonization (adaptation) and infection of certain animal anatomical sites (i.e., the mammary quarter, population C), regardless of the events of bacterial propagation, as previously described18,63,64. Under this hypothesis, intrinsic factors of the host could be involved in the selection and adaptation process or genetic convergence of groups of bacterial strains. This hypothesis should also be explored.
The molecular differentiation and clonal relationship (PFGE method and binary typing) of S. aureus (MRSA and MSSA) isolated from bovine mammary secretions, geographically unrelated bovines and humans (USA and the Netherlands), and the association of bacterial strains with the clinical observations in herds (clinical symptomatology and somatic cell counts – SCC) were analyzed65. Some PFGE (sub)types (A, F, G, B1, B2, E1 and E2) were identified only once, while PFGE types C, D and E were found in two, three, and four herds, respectively, as were 16 binary types. A limited number of prevalent types of S. aureus recovered from bovine mammary secretions, as well as the heterogeneity genetic, were found within and among herds65, suggesting that certain variants present in the environment may have a predilection for causing intramammary infections36,65-67. This genetic heterogeneity (subclonal) within the herds may be due to temporal evolution (longitudinal research over a year), allowing additional genetic diversification68,69. The cluster analysis (UPGMA method) associated with binary typing indicated clusters of bovine strains (n = 16) and human strains (n = 5) with 90 to 95% similarity. However, at the highest level of similarity (100%), all the clones were host specific. These results were also consistent with the concept of host specificity among S. aureus clones and suggest that the successful transfer of bacteria between humans and animals is not a common event35,65, although possible13-14,18,42,61-63,70-73.
S. aureus isolates (n = 227) from several herds of dairy cattle with mastitis, located in the southeastern region of Brazil (i.e., 18 dairy herds distributed among 9 municipal districts of the Rio de Janeiro State) were investigated using PFGE and MLST genotyping and analysis of genetic similarity (Dice coefficient and UPGMA algorithm)23. The PFGE method identified 60 pulsotypes (strains), which were distributed among 6 clonal complexes (CCs) (i.e., each clonal complex realized pulsotypes with SDice > 65%) characterized by MLST. The predominance of a limited number of closely related pulsotypes (suggesting common ancestry) responsible for bovine mastitis in distinct herds (different geographical locations) and within the herds suggested that these strains have a greater capacity to propagate and cause intramammary infections. The majority of pulsotypes belonged to CC126 (recovered from 13 herds and 8 municipal districts) and CC97 (recovered from 14 herds and 9 municipal districts), which was represented by 91% of the isolates23. Observations indicated that CC97 and CC126 were rarely or never detected among bacterial isolates from human population, respectively, suggesting the specificity of intramammary infections to ruminants23,40,42. In addition, important CCs associated with infections by S. aureus in humans (CC1, CC5 and CC30) were found in bacterial isolates from dairy cattle in 6 herds located in 5 municipal districts23.
A prospective analysis using PFGE typing (bacterial genomic DNA digested with SmaI), PCR of virulence genes (hemolysins – hla to hlg; leukocidins – lukED and lukM; superantigens – sea, sec, sed, of seg to seo, seu and tst; adhesins – fnbA and fnbB; and resistance to penicillin and methicillin – blaZ and mecA) and genetic relationship (coefficient of Dice and grouping analyses) of S. aureus, collected from intramammary infection sites and other extramammary sources (teat skin of dairy cows, teat channels and skin lesions; milking liners; hands and nostrils of milking staff) from two dairy herds independent establishments (herds I and II) of southern Finland, was performed to study the possible sources and reservoirs of bovine intramammary infections20. In this research, unique predominant bacterial genotypes were found in each herd, with the number of pulsotypes in the herd II (an open herd, including imported heifers from different regions of Finland) higher (n = 7) than that from herd I (a closed herd; n = 3), corroborating other reports on the high polymorphism (i.e., higher genetic heterogeneity) of isolates obtained from open herds17. Despite the existence of specific pulsotypes within each herd, the genotypes that were most likely better adapted to extramammary multiplication also caused intramammary infections, suggesting the existence of potential extramammary sources as the means of transfer. No correlation was found between specific genes for virulence and the source of the isolate. Still, identical pulsotypes in different herds were reported as being capable of harboring different virulence genes and resistance. In a herd, some pre-labor heifers harbored S. aureus strains in colostrum similar to those of lactating animals, and in both herds, the milking workers also displayed S. aureus strains identical to those of the animals, but the origin of colonization was considered uncertain20. Other factors may contribute to the selection and diversity of S. aureus strains in herds. The number and diversity of S. aureus strains will most likely rise if new strains are introduced via importing cattle, as previously shown17,20. Still, the successful control of infection of mastitis can increase the diversity of strains but decrease the spread of strains within the herd, whereas the failure to control can lead to the spread of only one or a few dominant strains throughout the herd. Pre-parturient heifers can also be an important reservoir of S. aureus, and strains originating from their skins were capable of causing intramammary infections in dairy cattle from the same herd17. However, herds may also harbor different strains of those lactating cattle18. Recent reports on epidemiological studies of S. aureus strains in humans and animals, including isolates from bovine mastitis, suggest host specificity although a high degree of variation within and among clonal strains of S. aureus originating from different host animals74-77.