Infectious Diseases Diagnosis & Treatment (ISSN: 2577-1515)

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Hands as a Potential Vector for Transmission of Multidrug-Resistant Staphylococcus aureus Polyclonal Strains in Dental Clinical Environments

Mateus Cardoso Oliveira1 , João Pedro Cotrim Maia2 , Wagner Luís de  Carvalho Bernardo1 , Jeferson Júnior da Silva1 , Rodrigo Carlos Bassi1 ,  Manoel Francisco Rodrigues Netto1,4 , Isabel Celeste Caires Pereira  Gusmão2 , Carlos Tadeu dos Santos Dias3 , Marcelo Fabiano Gomes  Boriollo1*  

1 Department of Oral Diagnosis, Dental School of Piracicaba, State University of Campinas (FOP/UNICAMP), Brazil

2 Independent School of Nordeste (FAINOR), Brasil 

3 Department of Exact Sciences, College of Agriculture, University of São Paulo (ESALQ/USP), Brazil

4 UNA Faculty, Brazil 

*Corresponding author: Marcelo Fabiano Gomes Boriollo, Department of Oral Diagnosis, Dental School of Piracicaba, State University  of Campinas (FOP/UNICAMP), 901 Limeira Ave, Piracicaba, SP 13414-903, Brazil. 

Received Date: 14 July 2022

Accepted Date: 20 July 2022

Published Date: 25 July 2022

Citation: Oliveira MC, Maia JPC, Bernardo WLC, da Silva JJ, Bassi RC, et al. (2022) Hands as a Potential Vector for Transmission  of Multidrug-Resistant Staphylococcus aureus Polyclonal Strains in Dental Clinical Environments. Infect Dis Diag Treat 6: 194. DOI:  https://doi.org/10.29011/2577-1515.100194 

Abstract 

Background: Staphylococcus aureus has been considered one of the main opportunistic pathogens for humans, constituting a  relevant problem, considering its possible resistance to the main antibiotics used in medical practice. The aim of this study was to  detect the presence of oxacillin-resistant S. aureus (ORSA) in the hands of undergraduate dentistry students, throughout clinical  procedures (before and after dental procedures and hand hygiene). Methods: Hand samples were collected at different times (pre-asepsis; immediately after asepsis; and glove and hand surfaces immediately after dental procedures) and seeded in culture media  (PCA and MSA). S. aureus species were characterized by microbiological identification methods, antimicrobial susceptibility test  and genotyping by isoenzymatic markers. Results: A total of 14 students (73.7%) had S. aureus in both hands. Microbial resistance  was observed in 87.1% of S. aureus isolates: AMP (54.5%), AMX (26.5%), OXA (12.9%), TET (7.6%), CFE (7.6%), LEX (7.6%),  FOX (7.6%), CLI (15.2%) and ERY (44.7%). A total of 132 isolates of S. aureus with variable profiles of antimicrobial resistance  were identified, 26 of which were characterized as ORSA. Monoclonal and polyclonal patterns were observed in bacteria population.  Analyzes of genetic relationship revealed that two or more dentistry students share S. aureus isolates through their hands and gloves  during their academic clinical activities. Conclusion: The spread of multidrug-resistant S. aureus mono and polyclonal strains  requires a reevaluation of personal hygiene and environment (effective biosafety practices) as a means of preventing the spread and  cross-contamination between patients, professionals and academics, and dental clinical environments.

Keywords: Antimicrobial resistance; Dental clinic; MLEE  typing; Polyclonality; Staphylococcus aureus 

Introduction

Staphylococcus aureus are microorganisms especially important with regard to opportunistic infections, it is the most common agent in pyogenic infections and abscesses [1]. In immunocompromised individuals or who have suffered trauma and burns, this microorganism can cause more serious infections such as osteomyelitis and bacteremia, usually associated with metastatic abscesses, which in turn can cause endocarditis. Since the 1940s, there has been a great increase in the incidence of infections associated with hospital environment, caused by resistant strains of S. aureus, some showing resistance to more than 20 antimicrobial compounds, including antiseptics and disinfectants [2].

When contracting the potentially infective agent, the organism can behave in two ways: presenting a clinically diagnosable disease, or as an asymptomatic carrier, devoid of symptoms, despite being colonized. The carrier of S. aureus becomes a major problem, when in specific environments, such as hospitals, maternity units, outpatient clinics and dental offices [3], as staphylococcal diseases acquire characteristics of professional disease, being quite common clinical-dental infections [4]. The transmission routes are from person to person (cross-infection), by indirect contact (by air) or by direct contact, with this transfer depending on the presence of a source (patients or carriers) [5].

In general, the large number of studies aimed at this purpose seeks to detect carriers of that microorganism in nasal cavities and on the skin [6]. Hands have also been considered an important source of samples of S. aureus and one of main means of transmission of that bacteria in hospital environment, contributing significantly to the increase of sources and reservoirs of resistant samples [7].

The study of epidemiological role of hands in transmission of infections among professionals, engaged in dental activities, has shown potential importance of those as source of possible infections within the dental office as well as the possible relationship between samples isolated from different anatomical areas of a same individual, mainly between nasal cavity and hands, suggesting that most staphylococci in hands are of nasal origin [8].

Hand hygiene is a primary practice used to reduce the risk and spread of infection. Inadequate hygiene leaves microorganisms in hands that can be transferred to another patient. Hand hygiene must be performed before and after direct contact with patients, before and after the use of gloves, or after contact with inanimate objects in the patient’s immediate surroundings [9].

The detection of S. aureus in dental care environments is necessary, since the results of present studies provide the academic environment, dental class and other related areas, a significant contribution to future studies and in development of safe techniques in management of patients. The aim of this study was to detect the presence of oxacillin-resistant Staphylococcus aureus (ORSA) in the hands of undergraduate dentistry students, throughout clinical procedures (before and after dental procedures and hand hygiene). Species of S. aureus were genotyped by isoenzymatic markers and characterized in terms of antibacterial sensitivity profile.

Methods

Sampling

Samples of surfaces of hands and gloves were collected from 19 dentistry students, using swabs (Cral plast, Cotia, São Paulo) previously moistened with PBS solution (100 mM NaCl, 100 mM NaH2PO4, pH 7.2), and kept in 2 type Eppendorf tubes containing 1 ml of sterile PBS solution. Samples were obtained at three different times: 1) Pre-asepsis hand surfaces; 2) Hand surfaces immediately after asepsis; and 3) Glove and hand surfaces immediately after dental procedures. At the end of each collection, the samples were identified with date, time and place, then stored in Styrofoam boxes and transported to the microbiology laboratory. Polyvinylpyrrolidone-iodine soap (PVPI) is commonly used in clinics for hand asepsis. However, no training was done especially for research.

Bacterium

Aliquots of 100 µl (1:10 dilution) of the specimens collected were inoculated in PCA culture medium (Plate Count Agar, DifcoTM, São Paulo, Brazil) and in MSA selective culture medium (Mannitol Salt Agar, DifcoTM, São Paulo, Brazil), and incubated aerobically at 35 ºC for 24-48h, in order to determine the total microbial content and identify preliminarily pathogenic Staphylococcus or S. aureus, respectively. Mannitol positive colonies indicative of S. aureus (presence of yellow halo around the colonies) were grown in BHI culture medium (Brain Heart Infusion Broth, DifcoTM, São Paulo, Brazil.) at 37 °C for 24h. S. aureus species were characterized by microbiological identification methods (Gram staining, growth in CHROMagar Staphylococcus aureus chromogenic medium, biochemical tests such as catalase, coagulase [Coagu-Plasma, Laborclin Produtos para Laboratórios Ltda.], Factor A test agglutination [Staphy test, Probac do Brasil Produtos Bacteriológicos Ltda., Marnes La Coquette, France], mannitol fermentation test, DNAse test and Voges-Proskauer test) and antimicrobial susceptibility test (by the disk-diffusion method) [10, 11] and confirmatory screening for oxacillin resistance [12]. Ampicillin 10 µg (AMP), amoxicillin 10 µg (AMX), oxacillin 1 µg (OXA), tetracycline 30 µg (TET), vancomycin 30 µg (VAN), cephalexin 30 µg (LEX), cephalothin 30 µg (CEF), cefoxitin 30 µg (FOX), 2 µg clindamycin (CLI) and 15 µg erythromycin (ERY) (Control and Diagnostic Products Center - CECON, São Paulo, Brazil) were used for antimicrobial susceptibility testing. The reference strain Staphylococcus aureus subsp. aureus Rosenbach (ATCC 25923) was used to ensure the reproducibility and accuracy of disk diffusion susceptibility test.

MLEE Typing

MLEE method (multilocus enzyme electrophoresis) or isoenzymatic typing was used to examine the genetic relationship between two groups of organisms. Bacterial cultures, enzymatic extraction and electrophoresis and specific enzymatic staining were performed as previously described [13]. The enzymatic activities that will be analyzed included: alcohol dehydrogenase (EC 1.1.1.1), sorbitol dehydrogenase (EC 1.1.1.14), mannitol-1-phosphate dehydrogenase (EC 1.1.1.17), malate dehydrogenase (EC 1.1.1.37), glucose dehydrogenase (EC 1.1.1.47), D-galactose dehydrogenase (EC 1.1.1.48), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), catalase (EC 1.11.1.6) and α- and β-esterase (EC 3.1.1.1). S. aureus ATCC 25923 enzymes were included in each gel to ensure reproducible results.

The interpretation of patterns was carried out following the general rules commonly accepted in deduction of allelic composition for haploid organisms. The bands on gels were numbered in decreasing order of mobility, and the corresponding alleles were numbered using the same nomenclature. The lack of demonstrable activity for an enzyme was classified as a null allele at corresponding gene locus. Each unique combination of alleles on the examined enzyme locus has resulted in a subtype or electrophoretic (ET) type. The percentage of polymorphic locus, the average number of alleles per locus and the average number of alleles per polymorphic locus were also determined [13].

Data analysis methodology

Nei's statistical methods were used to estimate the genetic distance between S. aureus isolates and / or strains (ETs):

 

where I is the normalized gene identity between two populations (ranging from 0 to infinity), a measure of genetic distance based on gene identity (frequency of alleles for all loci, including monomorphic loci) between populations. That genetic distance measures the allelic differences accumulated by locus, which can also be estimated from the amino acid sequences of proteins and even for distantly related species. As a consequence, if sufficient data is available, the genetic distances between any pair of organisms can be measured in terms of dij. In addition, this measure can be applied to any part of the organism, regardless of the ploidy level or mating scheme. Its interpretation, in terms of enzymatic loci, implies that, on average, zero to an infinite number of allelic substitutions are detected (by electrophoresis) in every 100 loci of a common ancestral strain [14].

A tree with two-dimensional classifications, called dendrogram, was generated using the SAHN clustering method (Sequential, Agglomerative, Hierarchic, Nonoverlapping Clustering Methods) and the UPGMA algorithm (unweighted pair group method using arithmetic mean), based on their respective matrices dij. Since MLEE provides all levels of relationship, which must be resolved by DNA fingerprints (i.e., identifying the same strain in independent isolates, identifying microevolutionary changes in a strain, identifying groups of moderately related isolates and identi-fying completely isolates unrelated), a threshold (value: dij ) was established in the dendrograms to identify groups of identical and highly related isolates (0≤dij< dij ) and taxon (plural taxa; dijdij). Pearson’s product-moment correlation coefficient (ranging from–1 to +1),

 

[where Xij represents the value of the character state of the character i in OTU (Operational Taxonomy Unit) j, Xj is the average of all state values for OTU j and n is the number of characters sampled], was used as a measure of the agreement between the genetic distance values implied by the UPGMA dendrograms and those of the original dij genetic distance matrices. Those agreements have been interpreted as follows: 0.9£rjk, very good fit; 0.8£ rjk<0.9, good fit; 0.7£ rjk<0.8, poor fit; rjk<0.7, very poor fit. All of those analyzes were obtained using the NTSYS-pc 2.1 program.

The discriminatory power of MLEE method was established by the numerical index of discrimination (D) - Simpson’s diversity index -, according to the probability that two unrelated isolates sampled from the test population will be classified in different types [electrophoretic types (ETs), subtypes or strains] [13].

Results

Microbiological samples, from the hands of 19 dentistry students in academic clinical activities, were investigated for the presence of total bacteria (aerobic growth over PCA), fermenting bacteria — Staphylococcus sp. — and non-fermenting mannitol (aerobic growth over MSA), and S. aureus, in three different moments: 1) Pre-asepsis hand surfaces (A); 2) Hand surfaces immediately after asepsis; and 3) Glove surfaces (C) and hands (D) immediately after dental procedures (duration of procedures equivalent to ±2h30min) (Supplementary table 1).

Supplementary table 1: Colony forming unit (CFU/ml) of total bacteria (grown on PCA), mannitol- and mannitol+ bacteria (grown on SMA), and S. aureus (microbiological characterization) from hands of the 19 dentistry students before (A) and after (B) asepsis, and of gloves (C) and hands (D) after dental procedures.

Code of dentistry student (gender)

Dental procedures (Time)

Code of hands and gloves

Total bacteria

(CFU/ml)

Mannitol

(CFU/ml)

Mannitol +

(CFU/ml)

S. aureus

(CFU/ml)*

MLEE and antimicrobial susceptibility**

7 (♀)

Absence of

A-RH-7

620

70

 

procedure

A-LH-7

70

30

30

3

 

 

å

690

70

30

30

3

 

 

B-RH-7

10

10

10

1

 

 

B-LH-7

 

 

å

70

10

10

1

3 (♀)

Dental cleaning

A-RH-3

80

40

50

50

5

 

and restoration

A-LH-3

550

230

80

8

 

(1h20min)

å

630

40

280

130

13

 

 

B-RH-3

70

30

20

20

2

 

 

B-LH-3

110

110

80

8

 

 

å

180

30

130

100

10

 

 

D-RH-3

10

 

 

D-LH-3

 

 

å

10

5 (♀)

Endodontic

A-RH-5

140

10

30

30

3

 

procedures and

A-LH-5

390

30

90

80

8

 

dental restoration

å

530

40

120

110

11

 

(1h10min)

B-RH-5

 

 

B-LH-5

20

 

 

å

20

 

 

D-RH-5

30

20

 

 

D-LH-5

10

 

 

å

30

30

8 (♀)

Dental

A-RH-8

880

300

 

restoration:

A-LH-8

4,200

460

20

2

2

 

definitive

å

5,080

760

20

2

2

 

cementation of

B-RH-8

60

10

30

3

3

 

the dental crown

B-LH-8

110

30

 

(1h)

å

170

40

30

3

3

 

 

D-RH-8

 

 

D-LH-8

10

 

 

å

10

9 (♀)

Dental cleaning,

A-RH-9

10,840

20

40

10

1

 

prophylaxis and

A-LH-9

99,280

680

20

20

2

 

procedures of

å

110,120

700

60

30

3

 

dental prosthesis

B-RH-9

100

20

10

10

1

 

(1h10min)

B-LH-9

120

10

10

10

1

 

 

å

220

30

20

20

2

 

 

D-RH-9

 

 

D-LH-9

10

 

 

å

10

10 (♂)

Dental

A-RH-10

30

30

20

20

2

 

restoration:

A-LH-10

 

temporary

å

30

30

20

20

2

 

cementation of

B-RH-10

40

 

dental crown

B-LH-10

 

(1h25min)

å

40

 

 

D-RH-10

10

 

 

D-LH-10

 

 

å

10

4 (♀)

Dental

A-RH-4

1,550

10

30

30

3

 

restoration:

A-LH-4

860

50

170

80

8

 

temporary

å

2,410

60

200

110

11

 

cementation of

B-RH-4

710

 

dental crown

B-LH-4

10

 

(1h20min)

å

720

 

 

C-RH-4

80

20

 

 

C-LH-4

170

10

 

 

å

250

30

 

 

D-RH-4

560

10

20

20

2

 

 

D-LH-4

980

 

 

å

1,540

10

20

20

2

6 (♀)

Dental

A-RH-6

3,430

1,010

 

restoration and

A-LH-6

560

480

 

polishing

å

3,990

1,490

 

(1h)

B-RH-6

190

210

 

 

B-LH-6

880

700

 

 

å

1,070

910

 

 

C-RH-6

80

40

 

 

C-LH-6

310

70

 

 

å

390

110

 

 

D-RH-6

1,210

 

 

D-LH-6

20

250

 

 

å

1,230

250

11 (♀)

Restoration of

A-RH-11

360

80

10

10

1

 

dental resin

A-LH-11

2,900

20

120

80

8

 

temporary;

å

3,260

100

130

90

9

 

cementation of

B-RH-11

30

 

dental crown

B-LH-11

690

 

(1h25min)

å

720

 

 

C-RH-11

10

 

 

C-LH-11

 

 

å

10

 

 

D-RH-11

 

 

D-LH-11

 

 

å

12 (♀)

Restoration of

A-RH-12

10

10

10

10

1

 

dental resin

A-LH-12

40

20

10

10

1

 

temporary

å

50

30

20

20

2

 

(1h15min)

B-RH-12

 

 

B-LH-12

10

10

10

10

1

 

 

å

10

10

10

10

1

 

 

C-RH-12

 

 

C-LH-12

 

 

å

 

 

D-RH-12

 

 

D-LH-12

10

 

 

å

10

17 (♀)

Dental cleaning

A-RH-17

20

 

and polishing of

A-LH-17

70

70

 

restorations

å

70

90

 

(55min)

B-RH-17

10

 

 

B-LH-17

20

 

 

å

30

 

 

C-RH-17

 

 

C-LH-17

30

 

 

å

30

 

 

D-RH-17

 

 

D-LH-17

 

 

å

18 (♂)

Restoration of

A-RH-18

10

 

dental resin

A-LH-18

10

20

10

10

1

 

temporary;

å

10

30

10

10

1

 

cementation of

B-RH-18

 

dental crown

B-LH-18

10

 

(1h20min)

å

10

 

 

C-RH-18

40

30

 

 

C-LH-18

30

 

 

å

40

60

 

 

D-RH-18

10

 

 

D-LH-18

10

 

 

å

10

10

19 (♂)

Channel sealing,

A-RH-19

1,450

680

350

80

8

 

temporary

A-LH-19

1,280

20

560

80

8

 

restoration

å

2,730

700

910

160

16

 

(1h10min)

B-RH-19

70

10

 

 

B-LH-19

1,670

40

120

80

8

 

 

å

1,740

50

120

80

8

 

 

C-RH-19

490

10

30

30

3

 

 

C-LH-19

200

20

30

30

3

 

 

å

690

30

60

60

6

 

 

D-RH-19

60

10

10

10

1

 

 

D-LH-19

10

20

40

40

4

 

 

å

70

30

50

50

5

20 (♀)

Instrumentation,

A-RH-20

1,200

1,160

80

8

 

canal filling and

A-LH-20

10

20

 

temporary dental

å

1,210

20

1,160

80

8

 

restoration

B-RH-20

 

(3h05min)

B-LH-20

10

20

 

 

å

10

20

 

 

C-RH-20

30

10

 

 

C-LH-20

10

10

1

 

 

å

30

10

10

10

1

 

 

D-RH-20

10

10

1

 

 

D-LH-20

10

 

 

å

10

10

10

1

21 (♀)

Ultrasonic

A-RH-21

1,620

 

scraping of

A-LH-21

30

 

dental biofilms,

å

1,650

 

dental cleaning

B-RH-21

20

 

and prophylaxis

B-LH-21

20

 

(1h05min)

å

40

 

 

C-RH-21

290

10

 

 

C-LH-21

70

 

 

å

360

10

 

 

D-RH-21

700

40

 

 

D-LH-21

 

 

å

700

40

♀ and ♂ means gender female and mate, respectively. MD and ME means right hand and left hand, respectively. * Identification of S. aureus by microbiological methods: Gram staining, growth on chromogenic medium CHROMagar Staphylococcus aureus®, biochemical tests such as catalase test, coagulase test, clumping factor A test, mannitol fermentation test, DNAse test and Voges-Proskauer test. ** Number of isolates used for characterization by MLEE and antimicrobial susceptibility.

Analyzing the 19 students included in the present study, all (100%) showed contamination on the surfaces of hands pre-asepsis by total bacteria, 18 (94.7%) showed contamination by non-fermenting mannitol bacteria, 14 (73.7%) showed contamination by Staphylococcus sp. and 14 (73.7%) showed contamination by S. aureus. The results of contamination of the surfaces of hands, immediately after asepsis, revealed that 17 (89.5%) students still had total bacteria, 9 (47.4%) had non-mannitol fermenting bacteria, and 6 (31.6%) had both Staphylococcus sp. and S. aureus. Soon after dental clinical procedures, samples of surfaces of gloves and hands were evaluated in 13 and 18 of the 19 dental students, respectively. A total of 11 (84.6%) students showed contamination by total bacteria on the surface of their gloves, 9 (69.2%) students showed contamination by non-fermenting mannitol bacteria, and 3 (23.1%) students showed contamination by Staphylococcus sp. and S. aureus. Once those gloves were discarded, 13 (72.2%) students showed contamination by total bacteria on the surface of their hands, 9 (50.0%) students showed contamination by non-fermenting mannitol bacteria, and 3 (16.7%) students showed contamination by Staphylococcus sp. and S. aureus.

Data on the frequency of microbial contamination of the hands of dental students were submitted to multivariate statistical using the SAS® version 9.2 [principal component analysis (PCA) and interactive biplot construction; cluster analysis (CA) and dendrogram interpretation (threshold ≤ 0.5)], which revealed similarity profiles as follows (Table 1 and Fig. 1):

Table 1: The incidence of total bacteria, Staphylococcus sp. and S. aureus coming from hands and gloves of the dentistry students. The data obtained in the frequency were submitted to multivariate statistical (SAS® version 9.2), using principal component analysis (PCA) and interactive biplot construction, and using cluster analysis (CA) and dendrogram interpretation (threshold ≤ 0.5): sampling (A, B, C and D) versus total bacteria (PCA), mannitol bacteria (MSA), mannitol+ bacteria (MSA) and S. aureus.

 

Sampling

 

Surface

Number and frequency of dentistry students

Total bacteria

Mannitol

Mannitol+

S. aureus

n

%

n

%

n

%

n

%

A. Pre-aseptic hand Surfaces

Right hand

17

89.5

17

89.5

10

52.6

10

52.6

Left hand

18

94.7

15

78.9

12

63.2

12

63.2

Right and/or left

19

100.0

18

94.7

14

73.7

14

73.7

B. Hand surfaces immediately after asepsis

Right hand

13

68.4

6

31.6

4

21.1

4

21.1

Left hand

15

78.9

8

42.1

4

21.1

4

21.1

Right and/or left

17

89.5

9

47.4

6

31.6

6

31.6

C. Glove surfaces immediately after dental procedures

Right hand

10

76.9

9

69.2

2

15.4

2

15.4

Left hand

8

61.5

7

53.8

2

15.4

2

15.4

Right and/or left

11

84.6

9

69.2

3

23.1

3

23.1

D. Hand surfaces immediately after dental procedures

Right hand

8

44.4

5

27.8

3

16.7

3

16.7

Left hand

9

50.0

6

33.3

1

5.6

1

5.6

Right and/or left

13

72.2

9

50.0

3

16.7

3

16.7

PCA culture medium (Plate Count Agar): analysis of total bacteria. MSA selective culture medium (Mannitol Salt Agar): analysis of mannitol and mannitol+ microorganisms (preliminarily pathogenic Staphylococci and S. aureus, respectively).

S. aureus: species were characterized by Gram staining, growth in CHROMagar Staphylococcus aureus chromogenic medium, catalase, coagulase, Factor A test agglutination, mannitol fermentation test, DNAse test and Voges-Proskauer test.

Figure 1: Incidence profile of dentistry students displaying bacterial contamination on surfaces of the hands and gloves from different sampling moments: A. Pre-aseptic hand surfaces; B. Hand surfaces immediately after asepsis; C. Glove surfaces immediately after dental procedures; and D. Hand surfaces immediately after dental procedures. The data obtained in the frequency were submitted to multivariate statistical (SAS® version 9.2), using principal component analysis (PCA) and interactive biplot construction, and using cluster analysis (CA) and dendrogram interpretation (threshold 0.5): sampling (A, B, C and D) versus total bacteria (PCA), mannitol bacteria (MSA), mannitol+ bacteria (MSA) and S. aureus.

  1. Sampling A (pre-aseptic hand surfaces): right hand RH (A), left hand LH (A), and right and/or left hand RH and/or LH (A) were characterized by high values of total bacteria, mannitol and mannitol+ bacteria, and S. aureus;

  2. Sampling C (glove surfaces immediately after dental procedures): right hand RH (C), and right and/or left hand RH and/or LH (C) were characterized by high values of total bacteria and mannitol, but < sampling A;

  3. Sampling D (hand surfaces immediately after dental procedures): right hand RH (D) and left hand LH (D) were characterized by low values of total bacteria, mannitol and mannitol+ bacteria, and S. aureus;

  4. Sampling B (hand surfaces immediately after asepsis), and partial samplings C and D: right hand RH (B), left hand LH (B), right and/or left hand RH and/or LH (B), left hand LH (C) and right and/or left hand RH and/or LH (D) were characterized by intermediate values of those observed between sampling A (RH, LH, and RH and/or LH), C (RH, and RH and/or LH) and D (RH and LH).

Microbial density (CFU/ml) has proved to be variable and dependent on each dental student, taking into account their entry into the academic dental clinic, that is, prior to hand hygiene and asepsis. However, the average density of bacterial contamination prior to hand asepsis was shown to be significantly higher for total bacteria (7,216.3 ±24,962.5), followed by non-fermenting mannitol bacteria (330.0

±417.8 CFU/ml), Staphylococcus species (160.0 ±320.3 CFU/ml) and S. aureus (45.9 ±51.6 CFU/ml). That average density was shown to be lower on the surfaces of hands immediately after asepsis (total bacteria: 271.6 ±467.5 CFU/ml; non-mannitol fermenting bacteria: 62.1 ±206.5 CFU/ml; Staphylococcus sp.: 16.8 ±39.0 CFU/ml; and S. aureus 11.7 ±28.3 CFU/ml), and on the surfaces of gloves (total bacteria: 163.1 ±209.6 CFU/ml; non-mannitol fermenting bacteria: 56.9 ±77.3 CFU/ml; Staphylococcus sp.: 7.7 ±17.9; e S. aureus: 7.7

±17.9 CFU/ml) and hands (total bacteria: 258.3 ±495.9 CFU/ml; non-mannitol fermenting bacteria: 22.2 ±58.2 CFU/ml; Staphylococcus

sp.: 4.4 ±12.5 CFU/ml; e S. aureus: 4.4 ±12.5 CFU/ml) immediately after dental procedures.

Data on the average density (CFU/ml) of microbial contamination of the hands of dental students were submitted to multivariate statistical using the SAS® version 9.2 [principal component analysis (PCA) and interactive biplot construction; cluster analysis (CA) and dendrogram interpretation (threshold ≤ 0.5)], which revealed similarity profiles as follows (Table 2 and Fig. 2):

Table 2: Average density (CFU/ml) of bacterial contamination (total bacteria, mannitol- and mannitol+ bacteria, and S. aureus) on surfaces of the hands and gloves from dentistry students obtained in different sampling moments. The data obtained (average values of CFU/ml) were submitted to multivariate statistical (SAS® version 9.2), using principal component analysis (PCA) and interactive biplot construction, and using cluster analysis (CA) and dendrogram interpretation (threshold≤ 0.5): sampling (A, B, C and D) versus total bacteria (PCA), mannitol bacteria (MSA), mannitol+ bacteria (MSA) and S. aureus.

 

Sampling

 

Surface

CFU/ml

Total bacteria

Mannitol

Mannitol +

S. aureus

A

±SD

A

±SD

A

±SD

A

±SD

A. Pre-aseptic hand

Surfaces

Right hand

1,284.2

±2,477.3

150.5

±266.9

90.5

±270.7

17.9

±25.9

Left hand

5,932.1

±22,633.2

179.5

±246.1

69.5

±135.4

28.0

±33.8

7,216.3

±24,962.5

330.0

±417.8

160.0

±320.3

45.9

±51.6

B. Hand surfaces immediately after

asepsis

Right hand

72.6

±161.4

17.4

±48.5

3.7

±8.3

2.3

±5.3

Left hand

198.9

±430.3

44.7

±159.2

13.2

±36.1

9.5

±25.0

271.6

±467.5

62.1

±206.5

16.8

±39.0

11.7

±28.3

C. Glove surfaces immediately after dental procedures

Right hand

86.9

±143.5

18.5

±24.8

4.6

±11.3

4.6

±11.3

Left hand

76.2

±102.1

38.5

±62.7

3.1

±8.5

3.1

±8.5

163.1

±209.6

56.9

±77.3

7.7

±17.9

7.7

±17.9

D. Hand surfaces immediately after dental procedures

Right hand

143.9

±334.3

5.0

±10.4

2.2

±5.5

2.2

±5.5

Left hand

114.4

±318.6

17.2

±58.4

2.2

±9.4

2.2

±9.4

258.3

±495.9

22.2

±58.2

4.4

±12.5

4.4

±12.5

Sampling A and B consisted of 19 dental students and sampling C and D were composed of 13 and 18 students, respectively. CFU/ml correspond to colony forming unit per milliliter. PCA culture medium (Plate Count Agar): analysis of total bacteria.

MSA selective culture medium (Mannitol Salt Agar): analysis of mannitol and mannitol + microorganisms (preliminarily pathogenic Staphylococci and S. aureus, respectively). S. aureus: species were characterized by Gram staining, growth in CHROMagar Staphylococcus aureus chromogenic medium, catalase, coagulase, Factor A test agglutination, mannitol fermentation test, DNAse test and Voges-Proskauer test.

Figure 2: Average density (CFU/ml) of bacterial contamination (total bacteria, mannitol- and mannitol+ bacteria, and S. aureus) on surfaces of the hands and gloves from dentistry students obtained in different sampling moments: A. Pre-aseptic hand surfaces; B. Hand surfaces immediately after asepsis; C. Glove surfaces immediately after dental procedures; and D. Hand surfaces immediately after dental procedures. The data obtained (average values of CFU/ml) were submitted to multivariate statistical (SAS® version 9.2), using principal component analysis (PCA) and interactive biplot construction, and using cluster analysis (CA) and dendrogram interpretation (threshold ≤ 0.5): sampling (A, B, C and D) versus total bacteria (PCA), mannitol bacteria (MSA), mannitol+ bacteria (MSA) and S. aureus.

  1. Sampling A (pre-aseptic hand surfaces): left hand LH (A), and right and/or left hand RH and/or LH (A) were characterized by high values of average density (CFU/ml) of total bacteria, mannitol and mannitol+ bacteria, and S. aureus. Note: right hand RH (A) was only characterized by high value of average density (CFU/ml) of mannitol+ bacteria.

  2. Sampling B (hand surfaces immediately after asepsis), C (glove surfaces immediately after dental procedures), D (hand surfaces immediately after dental procedures), and A partial: right hand RH (ABCD), left hand LH (BCD), and right and/or left hand RH and/or LH (BCD) were characterized by low values of average density (CFU/ml) of total bacteria, mannitol and mannitol+ bacteria, and S. aureus.

Among the 132 isolates of S. aureus identified in the samples (A, B, C and D), 72 (54.5%), 35 (26.5%), 17 (12.9%), 10 (7.6%), 10 (7.6%), 10 (7.6%), 10 (7.6%), 20 (15.2%) and 59 (44.7%) isolates were resistant to AMP, AMX, OXA, TET, LEX, CEF, FOX, CLI and ERY, respectively. All isolates were sensitive to VAN.

Data on the frequency of antibacterial susceptibility profiles of 132 S. aureus isolates obtained from surfaces of the hands and gloves from fourteen dentistry students were submitted to multivariate statistical using the SAS® version 9.2 [principal component analysis (PCA) and interactive biplot construction; cluster analysis (CA) and dendrogram interpretation (threshold ≤ 0.5)], which revealed similarity profiles as follows (Table 3 and Fig. 3):

Table 3: Antibacterial susceptibility patterns of 132 S. aureus isolates obtained from surfaces of the hands and gloves from fourteen dentistry students in different sampling moments. The data obtained in the frequency were submitted to multivariate statistical (SAS® version 9.2), using principal component analysis (PCA) and interactive biplot construction, and using cluster analysis (CA) and dendrogram interpretation (threshold ≤ 0.5): antimicrobial versus antimicrobial susceptibility profile (resistant, intermediate and susceptible).

 

Antimicrobial

Antimicrobial susceptibility profile

Resistant

Intermediate

Susceptible

n

%

n

%

n

%

AMP

72

54.5

0

0.0

60

45.5

AMX

35

26.5

6

4.5

91

68.9

OXA

17

12.9

6

4.5

109

82.6

TET

10

7.6

11

8.3

111

84.1

VAN

0

0.0

0

0.0

132

100.0

LEX

10

7.6

0

0.0

122

92.4

CEF

10

7.6

0

0.0

122

92.4

FOX

10

7.6

1

0.8

121

91.7

CLI

20

15.2

11

8.3

101

76.5

ERY

59

44.7

16

12.1

57

43.2

å

243

18.4

51

3.9

1026

77.7

Ampicillin 10mg (AMP), amoxicillin 10mg (AMX), oxacillin 1mg (OXA), tetracycline 30mg (TET), vancomycin 30mg (VAN), cephalexin 30mg (LEX), cephalothin 30mg (CEF), cefoxitin 30mg (FOX), clindamycin 2mg (CLI), and erythromycin 15mg (ERY).

Figure 3: Antibacterial susceptibility patterns of 132 S. aureus isolates obtained from surfaces of the hands and gloves from fourteen dentistry students in different sampling moments: A. Pre-aseptic hand surfaces; B. Hand surfaces immediately after asepsis; C. Glove surfaces immediately after dental procedures; and D. Hand surfaces immediately after dental procedures. The data obtained in the frequency were submitted to multivariate statistical (SAS® version 9.2), using principal component analysis (PCA) and interactive biplot construction, and using cluster analysis (CA) and dendrogram interpretation (threshold≤ 0.5): antimicrobial versus antimicrobial susceptibility profile (resistant, intermediate and susceptible). Ampicillin 10μg (AMP), amoxicillin 10μg (AMX), oxacillin 1μg (OXA), tetracycline 30μg (TET), vancomycin 30μg (VAN), cephalexin 30μg (LEX), cephalothin 30μg (CEF), cefoxitin 30μg (FOX), clindamycin 2μg (CLI), and erythromycin 15μg (ERY).

  1. AMP and ERY: high frequency of S. aureus isolates displaying resistance to ampicillin and erythromycin;

  2. AMX: intermediate frequency of S. aureus isolates displaying resistance to amoxicillin;

  3. CEF, CLI, FOX, LEX, OXA, TET and VAN: high frequency of S. aureus isolates displaying susceptibility to cephalothin, clindamycin, cefoxitin, cephalexin, oxacillin, tetracycline and vancomycin.

A total of 26 isolates of oxacillin-resistant S. aureus (ORSA) were identified, 18 isolates resistant to multiple drugs (Supplementary table 2), as interpreted in CLSI documents:

Supplementary table 2: Antibacterial susceptibility patterns of 132 S. aureus isolates obtained from surfaces of the hands and gloves from fourteen dentistry students in different sampling moments: A. Pre-aseptic hand surfaces; B. Hand surfaces immediately after asepsis; C. Glove surfaces immediately after dental procedures; and D. Hand surfaces immediately after dental procedures. RH and LH means right hand and left hand, respectively.

Code of dentistry student

Code of hands and gloves

Code of S. aureus isolates

Antibacterial susceptibility *

AMP

AMX

OXA

TET

VAN

LEX

CEF

FOX

CLI

ERY

3 (♀)

A-RH-3

1

S

S

S

S

S

S

S

S

S

R

A-RH-3

2

S

S

S

S

S

S

S

S

S

R

A-RH-3

3

S

S

S

S

S

S

S

S

S

R

A-RH-3

4

S

S

S

S

S

S

S

S

S

S

A-RH-3

5

S

S

S

I

S

S

S

S

S

R

B-RH-3

6

R

R

S

S

S

S

S

S

S

R

B-RH-3

7

R

R

S

S

S

S

S

S

S

R

A-LH-3

8

S

S

S

I

S

S

S

S

S

R

A-LH-3

9

S

S

S

S

S

S

S

S

S

R

A-LH-3

10

S

S

S

I

S

S

S

S

S

R

A-LH-3

11**

S

S

S

S

S

S

S

R

S

R

A-LH-3

12

S

S

S

S

S

S

S

S

S

R

A-LH-3

13

S

S

S

I

S

S

S

S

S

R

A-LH-3

14

S

S

S

S

S

S

S

S

S

R

A-LH-3

15

S

S

S

S

S

S

S

S

S

R

B-LH-3

16

S

S

S

S

S

S

S

S

S

R

B-LH-3

17

R

S

S

S

S

S

S

S

S

R

B-LH-3

18

S

S

S

S

S

S

S

S

S

R

B-LH-3

19

S

S

S

S

S

S

S

S

S

R

B-LH-3

20

S

S

S

S

S

S

S

S

S

R

B-LH-3

21

S

S

S

S

S

S

S

S

S

R

B-LH-3

22

S

S

S

S

S

S

S

S

S

R

B-LH-3

23

S

S

S

S

S

S

S

S

S

R

4 (♀)

A-RH-4

24

R

R

S

S

S

S

S

S

S

I

A-RH-4

25

S

S

S

S

S

S

S

S

S

I

A-RH-4

26

R

R

S

S

S

S

S

S

S

R

D-RH-4

27

R

R

S

I

S

S

S

S

S

R

D-RH-4

28

S

S

S

S

S

S

S

S

S

R

A-LH-4

29

R

R

S

S

S

S

S

S

S

S

A-LH-4

30

R

R

S

S

S

S

S

S

S

S

A-LH-4

31

R

R

S

S

S

S

S

S

S

S

A-LH-4

32

R

R

S

S

S

S

S

S

S

S

A-LH-4

33

R

R

S

S

S

S

S

S

S

S

A-LH-4

34

R

S

S

S

S

S

S

S

S

S

A-LH-4

35

R

R

S

S

S

S

S

S

S

S

A-LH-4

36

R

R

S

S

S

S

S

S

S

S

5 (♀)

A-RH-5

37

S

S

S

S

S

S

S

S

S

R

A-RH-5

38

S

S

S

I

S

S

S

S

S

R

A-RH-5

39

S

S

S

I

S

S

S

S

S

R

A-LH-5

40

S

R

S

R

S

S

S

S

S

R

A-LH-5

41**

R

R

S

S

S

R

R

R

R

R

A-LH-5

42

R

R

S

R

S

S

S

S

S

I

A-LH-5

43

R

R

S

R

S

S

S

S

S

I

A-LH-5

44

R

R

S

R

S

S

R

S

S

I

A-LH-5

45

R

R

S

R

S

S

S

S

S

S

A-LH-5

46

S

R

S

R

S

S

S

S

I

I

A-LH-5

47

S

R

S

R

S

S

S

S

S

R

8 (♀)

B-RH-8

48

R

R

S

R

S

R

S

S

S

R

B-RH-8

49

R

R

S

I

S

S

S

S

S

R

B-RH-8

50

S

S

S

S

S

S

S

S

S

S

A-LH-8

51

S

S

S

S

S

S

S

S

S

S

A-LH-8

52

R

R

S

S

S

S

S

S

S

S

9 (♀)

A-RH-9

53

S

S

S

S

S

S

S

S

S

R

B-RH-9

54**

R

R

R

I

S

S

S

S

S

R

A-LH-9

55

R

R

I

S

S

S

S

S

S

R

A-LH-9

56**

S

S

R

S

S

S

S

S

S

R

B-LH-9

57

S

S

S

S

S

S

S

S

S

R

10 (♂)

A-RH-10

58

R

R

S

I

S

S

S

S

S

R

A-RH-10

59

R

R

S

I

S

S

S

S

S

S

11 (♀)

A-RH-11

60**

R

R

R

S

S

S

S

S

S

R

A-LH-11

61**

S

S

R

S

S

S

S

S

S

S

A-LH-11

62**

S

S

R

S

S

S

S

S

S

S

A-LH-11

63**

S

S

R

S

S

S

S

S

S

S

A-LH-11

64

S

S

S

S

S

S

S

S

S

S

A-LH-11

65**

S

S

R

S

S

S

S

S

S

S

A-LH-11

66**

S

S

R

S

S

S

S

S

S

S

A-LH-11

67**

S

S

R

S

S

S

S

S

S

S

A-LH-11

68**

S

S

R

S

S

S

S

S

S

S

12 (♀)

A-RH-12

69

S

S

S

S

S

S

S

S

R

R

A-LH-12

70**

R

R

S

S

S

R

R

R

R

R

B-RH-12

71

S

S

S

S

S

S

S

S

R

R

15 (♀)

A-RH-15

72

S

S

S

S

S

S

S

S

S

S

A-RH-15

73

S

S

S

S

S

S

S

S

S

S

C-RH-15

74

R

S

S

S

S

S

S

S

S

S

C-RH-15

75

R

S

S

S

S

S

S

S

S

S

C-RH-15

76

R

S

S

S

S

S

S

S

S

S

A-LH-15

77

R

S

I

S

S

S

S

S

R

R

A-LH-15

78

R

S

S

S

S

S

S

S

R

R

A-LH-15

79

S

S

S

S

S

S

S

S

S

s

A-LH-15

80

S

S

S

S

S

S

S

S

R

R

16 (♀)

A-LH-16

81**

R

R

I

S

S

R

R

R

R

R

A-LH-16

82**

R

S

S

S

S

R

R

R

R

R

18 (♂)

A-LH-18

83

S

S

S

S

S

S

S

S

I

I

19 (♂)

A-RH-19

84**

R

S

R

S

S

S

S

S

R

R

A-RH-19

85

R

S

S

S

S

S

S

S

S

S

A-RH-19

86

R

R

S

S

S

S

S

S

S

S

A-RH-19

87

R

S

S

S

S

S

S

S

S

S

A-RH-19

88**

R

S

R

S

S

S

S

S

R

R

A-RH-19

89

R

S

S

S

S

S

S

S

S

S

A-RH-19

90

R

R

I

S

S

S

S

S

R

R

A-RH-19

91

R

I

S

S

S

S

S

S

S

S

C-RH-19

92**

R

S

I

R

S

R

R

R

R

R

C-RH-19

93**

R

R

S

S

S

R

R

R

R

R

C-RH-19

94**

R

R

R

S

S

S

S

S

S

S

D-RH-19

95

R

S

S

S

S

S

S

S

R

R

A-LH-19

96

R

I

S

S

S

S

S

S

S

S

A-LH-19

97

R

S

S

S

S

S

S

S

S

S

A-LH-19

98

R

S

S

S

S

S

S

S

S

S

A-LH-19

99

R

S

S

S

S

S

S

S

S

S

A-LH-19

100

R

S

S

S

S

S

S

S

R

R

A-LH-19

101

R

S

S

S

S

S

S

S

S

S

A-LH-19

102

R

S

S

S

S

S

S

S

S

S

A-LH-19

103

R

I

S

S

S

S

S

S

S

S

B-LH-19

104

R

S

S

S

S

S

S

S

S

S

B-LH-19

105

R

S

S

S

S

S

S

S

S

S

B-LH-19

106

R

S

S

S

S

S

S

S

S

S

B-LH-19

107

R

S

S

S

S

S

S

S

S

S

B-LH-19

108

R

S

S

S

S

S

S

S

S

S

B-LH-19

109

R

S

S

S

S

S

S

S

S

S

B-LH-19

110

R

S

S

S

S

S

S

S

S

S

B-LH-19

111

R

S

S

S

S

S

S

S

S

S

C-LH-19

112**

R

S

S

S

S

R

R

R

R

R

C-LH-19

113**

R

S

R

S

S

R

R

R

R

R

C-LH-19

114**

S

S

S

S

S

R

R

R

R

R

D-LH-19

115**

S

I

R

S

S

S

S

S

S

S

D-LH-19

116

S

S

S

S

S

S

S

S

S

S

D-LH-19

117

R

R

S

S

S

S

S

S

S

S

D-LH-19

118**

R

S

R

S

S

S

S

I

I

I

20 (♀)

A-RH-20

119

S

S

I

S

S

S

S

S

I

I

A-RH-20

120

S

S

S

R

S

S

S

S

I

I

A-RH-20

121

S

S

S

S

S

S

S

S

R

R

A-RH-20

122

S

S

S

S

S

S

S

S

I

I

A-RH-20

123

S

S

S

S

S

S

S

S

I

I

A-RH-20

124

S

S

S

S

S

S

S

S

I

I

A-RH-20

125

S

S

S

S

S

S

S

S

I

I

A-RH-20

126

R

I

S

S

S

S

S

S

I

I

D-RH-20

127

S

S

S

S

S

S

S