Isoenzyme genotyping and phylogenetic analysis of oxacillin-resistance Staphylococcus aureus isolates

2 Universidade José do Rosário Vellano, Faculdade de Medicina, Laboratório de Farmacogenética e Biologia Molecular & Centro de Pesquisa em Ciência Animal, Departamento de Patologia e Farmacologia Animal, Alfenas, Minas Gerais, Brasil. Aim: The propagation of S. aureus in hospital and dental environments is considered an important public health problem since resistant strains can cause serious infections in humans. The genetic variability of 99 oxacillin-resistant S. aureus isolates (ORSA) from the dental patients (oral cavity) and environments (air) was studied by isoenzyme genotyping. Methods: S. aureus isolates were studied using isoenzyme markers (alcohol dehydrogenase, sorbitol dehydrogenase, mannitol-1-phosphate dehydrogenase, malate dehydrogenase, glucose dehydrogenase, D-galactose dehydrogenase, glucose-6-phosphate dehydrogenase, catalase and α/β-esterase) and genetic (Nei’s statistics) and cluster analysis (UPGMA algorithm). Results: A highly frequent polyclonal pattern was observed in this population of ORSA isolates, suggesting various sources of contamination or microbial dispersion. Genetic relationship analysis showed a high degree of polymorphism between the strains, and it revealed three taxa (A, B and C) distantly genetically related (0.653≤dij≤1.432) and fifteen clusters (I to XV) moderately related (0.282≤dij<0.653). These clusters harbored two or more highly related strains (0≤dij<0.282), and the existence of microevolutionary processes in the population of ORSA. Conclusion: This research reinforces the hypothesis of the existence of several sources of contamination and/or dispersal of ORSA of clinical and epidemiologically importance, which could be associated with carriers (patients) and dental environmental (air).


Introduction
The dissemination of S. aureus is considered an important public health problem because resistant strains can cause serious infections, especially in children and hospital patients [1][2][3] . Dentists treat a wide variety of patients, a fact that exposes these health professionals to people colonized or infected with resistant microorganisms 2,4 . High rates of resistance to antibiotics used during odontological prophylaxis have been detected in pathogens associated with bacterial endocarditis, for example, S. aureus [5][6][7][8] . Strains of S. aureus can be disseminated during dental treatment and occasionally lead to the contamination and infection of patients and dentists. Certain aspects of odontological practice can contribute to the dissemination of microorganisms 9,10 . The skin, environment and instruments can be contaminated with saliva, blood or debris during routine odontological treatment 10,11 . Several researchers have noted an increase in the amount of microorganisms present during clinical procedures in odontological environments, suggesting contamination from aerosols, especially when high-speed devices or ultrasonic scalers are used 12,13 . Among the species identified in microbiological studies, streptococci of the group viridans and Staphylococcus spp. are the most prevalent microorganisms found on surfaces of odontological equipment [12][13][14][15] , including methicillin-resistant S. aureus, which has been detected on odontological operatory surfaces, air-water syringes and recliner chairs 16 . Additionally, bacteria and fungi were significantly more frequent in dentist's hand with rings than those without rings, being Staphylococcus aureus, Escherichia coli and Candida albicans highly prevalent among the isolated potentially pathogenic microorganisms 10 .
Phenotypic methods (biotyping, serotyping, bacteriophage or bacteriocin typing and antimicrobial susceptibility profiles) and genotypic [pulsed-field gel electrophoresis (PFGE) and other methods based on the restriction of genomes, analysis of plasmids, typing methods based on polymerase chain reaction (PCR)] of microbiological characterization have elucidated the relationship and the distribution of human pathogens, which is considered essentially important for the epidemiology and control of hospital infections 17 . Isoenzymatic typing [multilocus enzyme electrophoresis (MLEE)] has been used for several decades as a "gold standard" in population genetics studies of eukaryotes [18][19][20] and systematic studies 21 , as well as in large-scale studies for determining the genetic diversity and structure of natural populations of a variety of bacteria species [22][23][24] and fungi [25][26][27] . This method represents an invaluable complement to the more recently developed molecular typing methods, particularly for largescale epidemiological studies 28 . In addition, MLEE possesses excellent typability (i.e., the percentage of different strains obtained) and reproducibility (i.e., the percentage of strains that display the same results in repetitive tests) and is associated with great discriminatory power (i.e., the ability to differentiate unrelated strains) [23][24][25][26][27][28][29][30][31][32][33] .
Epidemiological studies are necessary for the implementation of effective prevention measures. Genotyping of strains from patients in odontological clinical treatment and their environments can provide information that can potentially help control and prevent the spread of S. aureus involved in the processes of colonization and human infection. This scientific research evaluated the genetic diversity of natural populations of oxacillin-resistance S. aureus dental isolates (dental patients and environments). The frequency of strains and operational taxonomic groups (taxon and cluster) and possible epidemiological correlations were investigated by using isoenzymatic markers (MLEE) and genetic and grouping analysis.

Microbiological Sampling
A total of ninety-nine bacterial samples of oxacillin-resistant S. aureus (ORSA), from the bacteria collection of the  34 and antimicrobial susceptibility testing (i.e., diffusion disk and confirmatory triage for resistance to oxacillin) 35 .

Multilocus Enzyme Electrophoresis (MLEE)
Preparation of cell extracts, electrophoresis procedures, enzyme staining and genetic interpretation of MLEE patterns were performed according to methods previously reported 23,25,26,31 . To ensure reproducibility of the results, the cellular enzymes of the S. aureus ATCC ® 25.923 TM reference strain were systematically used. A total of nine metabolic enzymes (Table 1) was investigated using systems and solutions previously established for the MLEE analyses 23,25,26,31 . The discriminatory power of the MLEE method was determined using the numeric index of discrimination (D), in accordance with the probability that two unrelated isolates sampled from a test population are classified into different types (i.e., strains or ETs) 25,26 .

Grouping Analysis
The statistic of Nei (1972) 36 was used to estimate the genetic distance (d ij ) among the isolates/strains (ETs) of oxacillin-resistant S. aureus. The interpretation in terms of enzyme loci infers that, on average, from zero to an infinite number of allele substitutions are detected (for electrophoresis) for every 100 existing loci from a common ancestral strain. A tree with two-dimensional classification (dendrogram), based on the matrix d ij , was generated by the grouping SAHN method (Sequential, Agglomerative, Hierarchic, Nonoverlapping Clustering Methods) and the UPGMA algorithm (Unweighted Pair-Group Method Using an Arithmetic Average). Once MLEE provided all levels of relationship that must be solved by DNA fingerprinting methods (i.e., identification of the same strain between independent isolates, identification of microevolutionary changes in the same strain, identification of clusters of moderately related isolates and identification of completely unrelated isolates), a threshold (average value: d ij ) in the dendrogram was established to identify identical isolates and highly related isolates, clusters  (0 ≤ d ij < d ij ) and taxa (singular taxon, i.e., taxonomic group of any nature or rank) (d ij ≥ d ij ). Correlation coefficients based on the Pearson product-moment was used as a measure of agreement between the genetic distance values implicit in the UPGMA dendrogram and the original explicit values in the matrix of genetic distance d ij . All these analyses were performed using the NTSYS-pc program version 2.1 25,26,32 .
ET and [-] correspond to electrophoretic type (bacterial strain) and allele null, respectively. Continue  ET42

Discussion
In this study, the enzyme electrophoretic profiles of oxacillin-resistant S. aureus isolates on different gels were reproducible after three repetitions of each electrophoretic run. The discriminatory capacity (i.e., 99% probability that two unrelated isolates sampled from a population test are classified in different strains ETs ) of the MLEE method, based on genetic interpretation of electrophoretic enzyme patterns, was also observed (i.e., the combination of existing alleles on 30 enzyme loci revealed 79 ETs). Once again, MLEE proved to be a powerful tool for the typing of S. aureus in epidemiological studies. These results are in agreement with previously reported data on the discriminatory power and reproducibility of the MLEE method as applied to bacteria and yeasts of medical importance [23][24][25][26][27]31 , but the discriminatory power was higher than the values reported for S. aureus by other groups of researchers 29,30 .
Genetic polymorphism has been found in almost all natural populations and at all levels of genetic organization, from genotype characteristics to phenotypic traces. The possible reasons of its existence have been the subject of a long debate in the population genetics and molecular evolution fields 37,38 . S. aureus is a heterogeneous species (polymorphic) 39 that has been observed to have a clonal population structure 40 . Therefore, it is believed that S. aureus does not suffer extensive recombination, diversifies extensively by nucleotide mutations and displays a high degree of linkage disequilibrium (non-random asso-ciations between gene loci). A particular structural gene locus is defined as polymorphic when the frequency of its more common allele presents a value below 0.99 (99%). Some of the measures used to quantify this variability in populations of organisms are the allele and gene frequencies, the percentage of polymorphic loci, the average number of alleles per locus and heterozygosity 41 . In this study, quantitative and qualitative variations of polymorphic loci (30 100% polymorphic enzyme loci to one, two, three, four, five and six alleles) and the average number of alleles per polymorphic locus (3.16 ±1.62) were observed in the population of oxacillin-resistant S. aureus. These variations have been observed in several studies of genetic diversity of populations of S. aureus obtained from human and bovine sources 29,30,42,43 . In addition, the genetic polymorphism observed in the population of oxacillin-resistant S. aureus isolates revealed a highly frequent polyclonal pattern and infrequent monoclonal pattern, suggesting various sources of contamination or microbial dispersion from an epidemiological point of view.
The genetic relationship between the oxacillin-resistant S. aureus strains was determined by using the statistic d ij of Nei (1972) and the UPGMA dendrogram 25,26,32,36 , which displayed a value r jk acceptable (r jk ~ 0.8) based on the correlation coefficient of Pearson's product-moment [i.e., good agreement between the elements d ij (matrix of genetic distance) and C jk (correlation matrix derived from UPGMA dendrogram)]. A high degree of genetic polymorphism (0.000 ≤ d ij ≤ 1.705) was observed between the ORSA isolates (i.e., on average, from zero to 170.5 allele substitutions were detected in each 100 loci from a common ancestor strain). These isolates were allotted to three taxa (A, B and C), which were distantly genetically related (0.653 ≤ d ij ≤ 1.705). Taxon A presented a larger number of isolates, strains or clusters of bacteria (60 isolates 60% , 43 ETs 54.4% and eight clusters I-VIII ), followed by taxon B (33 isolates 33% , 30 ETs 37.9% and six clusters IX-XIV ) and taxon C (six isolates 6% , five ETs 6.3% and one cluster XV ). Each taxon presented one or more clusters and/or moderately related isolates (0.282 ≤ d ij < 0.653). In turn, these clusters harbored two or more identical and/or highly related isolates (0 ≤ d ij < 0.282). Considering that highly related isolates/strains highly come from a common ancestor [i.e., descendants have suffered microevolutions and adaptations as a result of recombination (not extensive), nucleotide mutations and non-random association between gene loci (linkage disequilibrium) 39,40 , these results suggest the existence of microevolutionary processes in the population of oxacillin-resistant S. aureus, as demonstrated in each cluster (i.e., on average, from zero to < 28.2 allele substitutions were detected in each 100 loci from a common ancestor strain). However, these data reinforce the hypothesis of the existence of several sources of contamination and/or dispersal of oxacillin-resistant S. aureus of clinical and epidemiologically importance, which could be associated with carriers (patients) and dental environmental (air). These epidemiological investigations have also been a goal of our research group and contribute to (i) knowledge about the dynamics of the spread and retention of S. aureus strains resistant to antibiotics in hospital and odontological environments (i.e., surgical devices, dental instrumentation, various surfaces, air and other) and (ii) the implementation or restructuring of containment barriers, use of personal protective equipment, means of identification and periodic treatment from professionals carriers of microorganisms (nasal cavities, oral and oropharyngeal, perineum and armpits), techniques and devices for air purification, hygiene and more efficient prophylaxis.
Certain aspects of practicing dentistry may contribute to the transmission of microorganisms 9 . The skin, environment, and instruments can be contaminated with saliva, blood or organic debris during routine dental treatment 11 . In the dental environment, investigators have observed an increase in the amount of microorganisms during clinical procedures, suggesting contamination by aerosols, especially when high-speed handpieces or ultrasonic scalers are used 12,13,15 . Among the species identified in microbiological studies, Streptococcus viridans and Staphylococcus spp. are the most prevalent microorganisms found on the surfaces of dental equipment [12][13][14][15] . In addition, the high-speed drills and cavitrons used in dental offices generate aerosols and droplets that are contaminated with blood and bacteria and may be a route for the transmission of diseases such as SARS (severe acute respiratory syndrome), tuberculosis, and Legionnaires' disease [44][45][46] . Methicillin-resistant S. aureus (MRSA) has frequently been detected on surfaces in dental operatories, including the air-water syringe and reclining chair 16 . Nosocomial infections or the colonization of MRSA occurred in eight out of 140 patients who displayed no evidence of MRSA upon admission to a clinic. Antibiogram tests revealed that the isolates from the eight patients were of the same strain as those from the surfaces of the dental operatory, suggesting S. aureus transmission between the patients and the dentist via the clinical environment 16 . The frequency of S. aureus isolated from the noses, hands, and tongues of students and patients and from the clinical environment of a pediatric dentistry clinic before and after dental treatment was determined 47 . The highest concentration of S. aureus was found in the noses and on the tongues of children and among the dental students, and the highest level of contamination was observed on gloved hands, which was followed by the tongue and hands without gloves before clinical care. At the end of dental treatment, S. aureus colonies isolated from the gloved hands of students decreased significantly. Considering the clinical environment, S. aureus dissemination increased at the end of dental procedures, and the most contaminated areas were the auxiliary table and the storeroom, which was located at the center of the clinic. Such results can be explained by the intense circulation of people in the clinic and the use of high-speed dental handpieces. However, it is still speculated that much of the S. aureus contamination detected in the clinical environment came from other sources, such as direct contact, skin exfoliation or the improper handling of plates, and it is concluded that the dental clinic is an appropriate environment for S. aureus cross-transmission.
Because molecular-based epidemiological studies are useful in identifying possible sources of the spread of microorganisms in hospitals and dental clinical settings, this study contributes to our knowledge on the dynamics of the spread of S. aureus strains resistant to antibiotics and points to the need for containment barriers, use of personal protective equipment, periodic identification and treatment of carriers among clinical staff, and installation of air purifiers. Thus, infection control guidelines and published research pertinent to dental infection control principles and practices must be applied by the dentist as a matter of routine in academic dental offices. This research showed a genetic polymorphism in the population of oxacillin-resistant S. aureus isolates (dental patients and air of the clinical environment) and a highly frequent polyclonal pattern of these bacterial strains, supporting the hypothesis of various sources of contamination or microbial dispersion in the dental clinic environment. The isoenzyme typing and genetic relationship analysis revealed also some taxa and clusters exhibiting different frequencies of strains and possibly microevolutionary changes. In addition to the genetic information of S. aureus, the present methodology potentially collaborates with measures of prevention, management, and tracking of S. aureus, especially in dental clinics with great workflow.