Keywords
surgery infection - capsular contracture - nipple-sparing mastectomy - breast conservative
surgery - biofilm
Palavras-chave
infecção cirúrgica - contratura capsular - mastectomia com preservação do mamilo -
cirurgia conservadora da mama - biofilme
Introduction
Elective breast surgery, mastoplasty, and partial and total mastectomies are traditionally
considered clean surgeries, according to their potential for contamination.[1] However, the rate of infection in patients who undergo elective breast surgery ranges
between 3 and 15%,[2] which is above that expected for a surgery that is considered as clean. In patients
with risk factors for infection, such as smoking, neoadjuvant chemotherapy, and previous
radiotherapy, the infection rate may be as high as 25%.[3] The use of prophylactic antibiotics has reduced the infection rate from 15 to 9%,
but this percentage is still above that expected for a surgery that is considered
a clean one.[4]
In addition to postoperative clinical infections, another common but late complication
related to infection or to the presence of bacteria in the surgical area is capsular
contracture.[5] This may occur in up to 30% of patients undergoing plastic or reconstructive surgery
of the breast with implant placement.[6] The causes of capsular contracture are yet to be clarified, but the presence of
subclinical infection is one of the involved factors.[7] One hypothesis is that the presence of bacteria in the surgical area from the breast
ducts and parenchyma causes contamination of the implant, with the formation of a
biofilm that is resistant to antibiotics.[8] The formation of a biofilm leads to chronic inflammation, which causes capsular
contracture. In addition, this chronic inflammation around the implant covered by
biofilm may lead to the development of breast implant-associated large-cell lymphoma.[9]
The objective of the present study was to determine the presence of endogenous microbiota
in the breast tissue intraoperatively, at different locations in the breast. The presence
of endogenous bacteria in the breast tissue can explain why infection rates are higher
than expected in breast surgery.
Methods
Patients
The present prospective study was approved by the internal review board (IRP) of Universidade
Positivo. The study subjects were female patients undergoing elective breast surgery
(mastoplasty and partial and total mastectomies with immediate breast reconstruction)
at the Breast Unit of Hospital Nossa Senhora das Graças (Curitiba, PR); all patients
were operated on by the same surgical team. The patients received information about
the study and signed an informed consent form agreeing to participate in it. Forty-nine
patients were recruited, with a total of 78 breasts (29 bilateral and 20 unilateral
surgeries). Tissue samples were collected from each operated breast of the study patients.
The pieces of tissue were representative of the retroareolar or central areas, medial
, and lateral (axillary) quadrants, with the aim of comparing bacterial growth among
the various samples in different positions of the breast.
In addition, the influence of the following clinical variables on the results of the
cultures was investigated: diabetes mellitus, neoadjuvant chemotherapy, radiotherapy,
menopause, breastfeeding, body mass index (BMI), purpose of the surgery (oncologic,
cosmetic, or reconstructive), and presence of malignancy.
Sample Collection
All the patients received prophylactic antibiotics preoperatively (cefazolin 1 g,
intravenously during anesthesia induction). The skin was thoroughly prepared with
2% antimicrobial chlorhexidine (antisepsis) and with an alcohol-based solution of
0.5% chlorhexidine (asepsis) and subsequently covered with sterile surgical drapes,
exposing only the area of the skin involved in the surgery. Intraoperative samples
were collected from each breast in the first 30 min of surgery, from 3 different locations:
retroareolar tissue (region 1), medial gland tissue (region 2), and axillary gland
tissue (region 3). The samples consisted of pieces of tissue of at least 1 cm each,
excised by a cold scalpel. The samples were placed in sterile flasks with saline solution
(also sterile), stored in a cold room (4°C), and sent for culture in the microbiology
laboratory of Universidade Positivo.
The samples were incubated in brain heart infusion (BHI) broth at 37°C in a shaker
incubator, with constant shaking (150 RPM), for 7 days, and then they were subsequently
assessed for turbidity. The samples that became turbid due to microorganism growth
were inoculated in Petri dishes and placed in an incubator at 37°C for 7 days. Each
turbid broth was used to inoculate two dishes using different techniques: the pour-plate
technique for facultative/microaerophilic anaerobes, and the spread-plate technique
for aerobes. Different strains were isolated from each dish and were inoculated in
new dishes, with each strain placed in one quadrant of the dish. The cultures were
sent to a private clinical analysis laboratory to identify the species using a Vitek
2 automated counter.
Statistical Analysis
Data analysis started with the evaluation of the normality condition of the quantitative
variables using the D'Agostino-Pearson test. Subsequently, the Student t-test was
used for the quantitative variables that passed the normality test and Fisher exact
test was used for the qualitative variables to detect differences between patients
with positive and negative culture results (Zar, 2009).[10] The statistical analyses were performed using the GRAPHPAD PRISM statistical package,
and the level of significance was set at 5% (α = 0.05).
Results
The patients' ages ranged from 33 to 74 years (mean, 49 years; standard deviation,
8.48). Twenty-eight patients (57%) underwent oncologic surgery, 10 (20%) underwent
diagnostic surgery, 8 (16%) underwent delayed reconstructive surgery, 2 (4%) underwent
risk-reduction surgery, and 1 (2%) underwent one-stage oncologic surgery and risk-reduction
surgery (oncologic surgery in one breast and risk-reduction surgery in the contralateral
breast) ([Table 1]).
Table 1
Type of surgery performed
|
Type of surgery
|
Frequency
|
|
Oncologic surgery
|
28 (57.1)
|
|
Diagnostic surgery
|
10 (20.4)
|
|
Reconstructive surgery
|
8 (16.3)
|
|
Risk-reduction surgery
|
2 (4)
|
|
Oncologic + risk-reduction contralateral surgery
|
1 (2)
|
|
Total
|
49 (100)
|
A total of 218 pieces of breast tissue were removed and processed from 78 breasts
of 49 patients. Two patients had tumors in both breasts, 21 patients had a tumor in
the right breast, and 23 patients had a tumor in the left breast. Regarding neoadjuvant
therapies, 12 patients underwent chemotherapy before surgery and 6 underwent radiotherapy
before surgery. Three patients were diabetic, 17 were menopausal, and 31 had breastfed.
The patients' BMI varied between 18 kg/m2 and 36 kg/m2 (mean, 24.8 kg/m2; standard deviation, 5.66).
The culture media of 13 samples from 12 different patients exhibited turbidity (positive
result). Of these, two samples from two different patients did not exhibit bacterial
growth in the laboratory. Ten of the 49 patients (20.4%) exhibited bacterial growth
in at least one of the cultures of the sampled pieces. Eleven of the 218 samples (5%)
presented cultures with bacterial growth. The identified bacteria were Sphingomonas paucimobilis (three patients), Staphylococcus hominis (one patient), Staphylococcus lugdunensis (three patients), Staphylococcus epidermidis (two patients, one of them had the microorganism in two different samples), and Aeromonas salmonicida (one patient) ([Table 2]). No patient with positive samples for bacterial growth had clinical infection in
the postoperative period. There were no significant differences between patients with
and without a positive culture result with regard to the nine assessed variables ([Table 3]).
Table 2
Identified species
|
Species
|
Frequency (n = 10)
|
|
Staphylococcus lugdunensis
|
30.0%
|
|
Sphingomonas paucimobilis
|
30.0%
|
|
Staphylococcus epidermidis
|
20.0%
|
|
Staphylococcus hominis
|
10.0%
|
|
Aeromonas salmonicida
|
10.0%
|
Table 3
Relationship between the clinical findings and purpose of surgery and the presence
of endogenous microbiota in the breast
|
Variable analyzed
|
Positive (n = 10)
|
Negative (n = 38)
|
P-value
|
|
Age (years)
|
50.9 ± 9.2
|
48.6 ± 11.1
|
0.694
|
|
Purpose of surgery
|
Reconstruction
|
20.0%
|
15.8%
|
> 0.999
|
|
Diag + RR
|
0.0%
|
2.6%
|
|
Oncologic
|
80.0%
|
81.6%
|
|
Tumor location
|
Right
|
45.9%
|
44.4%
|
0.716
|
|
Left
|
51.4%
|
44.4%
|
|
Bilateral
|
2.7%
|
11.1%
|
|
DM (yes)
|
10.0%
|
5.4%
|
0.521
|
|
NC (yes)
|
10.0%
|
28.9%
|
0.414
|
|
RTX (yes)
|
20.0%
|
10.5%
|
0.591
|
|
BMI (kg/m2)
|
24.5 ± 1.6
|
24.9 ± 4.7
|
0.684
|
|
Menopause (yes)
|
44.4%
|
36.1%
|
0.711
|
|
Breastfeeding (yes)
|
70.0%
|
64.9%
|
> 0.999
|
Abbreviations: BMI, body mass index; Diag + RR, diagnostic and risk-reduction surgeries;
DM, diabetes mellitus; NC, neoadjuvant chemotherapy; RTX, radiotherapy.
Among the samples with bacterial growth, four were from retroareolar tissue (three
in the left breast and one in the right breast), four were from axillary tissue (one
left axillary, one right axillary plus right medial, and two right axillary), and
three were from medial tissue (one right medial, one left medial, and one right medial
plus right axillary) ([Table 4]).
Table 4
Locations of the positive cultures
|
Location of the positive culture
|
Frequency (n)
|
|
Retroareolar
|
4
|
|
Lateral
|
4
|
|
Medial
|
3
|
Discussion
Conceptually, clean surgeries are those performed in tissues that are sterile or susceptible
to decontamination, in the absence of a local infectious and inflammatory process
or gross technical errors, elective and traumatic surgeries with first-intention wound
healing and without drainage, and surgeries wherein there is no entry into the digestive,
respiratory, or urinary tracts. Breast surgeries are traditionally categorized as
clean surgeries. However, studies have reported higher rates of breast surgery infection
than expected for this category.[2]
[3] The routine use of prophylactic antibiotics has significantly reduced the rates
of infectious complications, but values that are higher than expected in clean operations
are still found. In a meta-analysis involving 2,395 patients, Zhang (2014)[11] concluded that the use of prophylactic antibiotics reduces the rates of infection
and prevents the development of other surgical complications, such as dehiscence,
ischemia, and necrosis.
Capsular contracture is a frequent cause of reoperations, and its etiology is still
unclear or lacking in consensus; however, it has been strongly associated with the
presence of bacteria in the surgical area,[5] which form a film over an implant, known as biofilm.[12] A biofilm is defined as an adhesion layer between bacterial cells and an extracellular
matrix, which is resistant to most antibiotics[5]
[13]
[14] and to physical and chemical methods of sterilization, because it blocks the penetration
of gases and liquids.[15] The presence of biofilms is confirmed by sonication and implant culture, even in
patients without signs of clinical infection.[16] Bacterial contamination is the factor with the greatest impact on capsular contracture
formation, regardless of the type of implant lining.[17] Bacterial contamination leads to the formation of a thicker capsule with greater
tissue reaction and a higher amount of inflammatory cells.[17]
[18]
Irrigation of the surgical area with antimicrobial substances reduces the risk of
developing capsular contracture,[19] another fact that supports the hypothesis that the presence of microbiota is a factor
for the development of capsular contracture, as reported by Yalanis et al. (2015)[19] in a meta-analysis with 5,153 patients. Another late complication of mastoplasty
with implants, anaplastic large-cell lymphoma, has been associated with the presence
of biofilms. Breast implant-associated anaplastic large-cell lymphoma is a rare T-cell
lymphoma that may develop around breast implants in plastic or reconstructive surgeries.[18]
[20] Chronic inflammation around the implant is thought to be the cause of lymphoma development,[18]
[20]
[21] and the presence of a biofilm around the implant is believed to promote inflammatory
reactions, which increase the probability of developing lymphoma.[22] Removal of the capsule is the primary treatment for this type of lymphoma.[18] However, the presence of the bacterial component alone does not appear to be sufficient
for the inflammatory stimulus required for the development of lymphoma; the surface
component of the silicone implant is also needed.[23] Therefore, Santanelli di Pompeo (2015)[24] suggests that the only safe treatment to avoid breast implant-associated lymphoma
is autologous flap breast reconstruction instead of implants.
Moreover, the presence of microbiota in nipple aspirate fluid has been demonstrated
through the amplification and sequencing of nucleic acids.[25] Chan (2016)[25] compared the microbiota of nipple aspirate fluid of patients with a history of breast
cancer with that of control patients and found abundant bacterial populations in both
groups. Similarly, different surgical techniques are associated with different complication
rates, with techniques involving incisions near the nipples and implants having higher
rates.[26]
[27]
[28] The use of a funnel-shaped device that assists in implant placing to avoid contact
between the implant and tissues during its positioning also reduces the rates of reoperation
due to capsular contracture.[29] These facts may be explained by the presence of bacteria inside the breast ducts
and by the intraoperative contamination of the implant by these bacteria.[7] A similar study with cultures of tissue samples collected from 50 breasts reported
bacterial growth in 19 of them, resulting in 38% of breasts with cultures positive
for microorganisms.[7] The authors of the study concluded that the breast harbors endogenous microbiota
that may be the source of spontaneous or postoperative infections.
In the present study, Sphingomonas paucimobilis, a gram-negative bacillus, was found in 30% of the positive cultures. This microorganism
is also present in the soil, plants, and potable water. It has been isolated from
distilled water tanks, respirators, and hemodialysis equipment in hospital settings.
Patients with chronic diseases or immunosuppression may be susceptible to infections
by this microorganism. Hospital and community infections have been described, including
sepsis, septic pulmonary embolism, peritonitis, septic arthritis, and endophthalmitis.[30]
Staphylococcus lugdunensis was also detected in 30% of the positive cultures in the present study. It was first
described in 1988 and was attributed an important role because of its capacity to
cause serious infections, such as endocarditis; intra-abdominal infections; as well
as infections of the skin and soft tissues, the central nervous system, and bones
and joints. Its penicillin-resistance rate can be as high as 76%, varying between
community and nosocomial strains.[31]
Staphylococcus epidermidis, a typical gram-positive commensal microorganism of the human skin microbiota, was
isolated in 20% of the positive cultures. It is a facultative anaerobe that is resistant
to various environmental conditions.[7] Its pathogenic capacity is closely related to the capacity of biofilm formation
of its strains, which makes it resistant to various hostile environments.[32]
Staphylococcus hominis was present in 10% of the positive cultures. It is another gram-positive microorganism
commonly present in the human skin, in animals of the human biome, and in the environment.
It is a causative agent of infections in immunocompromised individuals. It is capable
of facultative fermentation, as demonstrated by the isolation of lactate fermentation
genes.[33] Finally, Aeromonas salmonicida was also isolated in 10% of the positive cultures. The genus comprises gram-negative,
oxidase-positive, facultative anaerobes that are rod-shaped and widely distributed
in the aquatic environment. It was considered a pathogen in cold-blooded animals only
but has increasingly been reported as an opportunistic pathogen in humans, causing
mainly gastrointestinal infections, furunculosis, and septicemia.[34]
Conclusion
Despite the preoperative use of prophylactic antibiotics, rigorous skin antisepsis,
and adequate sterile surgical techniques, 20.4% of the patients in the present study
had positive cultures. This number, although in a limited sample, is similar to that
found in the literature, and the majority of the isolated species, which were staphylococci,
were the same as those detected in other studies. Some of the detected species are
associated with infections in immunocompromised patients. The locations with a higher
number of positive cultures were the retroareolar area and the lateral quadrant, which
is in line with the findings of other studies (not statistically significant). Thus,
the breast harbors endogenous microbiota that may be responsible for the formation
of biofilms and the contamination of implants and may even be associated with the
pathophysiology of implant-associated large-cell lymphoma. Further studies are necessary
to prove this hypothesis.
