From January 1st, 2008, to December 31st, 2010, we retrieved demographic data of patients diagnosed with mastitis based on International Classification of Diseases, 9th Revision - ICD-9 codes 610.1, 675.1, and 675.2. This data included age, type of visit (outpatient or hospitalization) when seeking medical aid, number and type of childbirth (normal spontaneous delivery [NSD] or cesarean section [C/S]), episodes with or without breastfeeding, and site of mastitis (unilateral or bilateral involvement). This information was sourced from the medical records of a 505-bed community hospital in central Taiwan.

The 36-month study was segmented into three distinct periods based on the incidence of mastitis and the implementation timing of an infection control bundle: the baseline period from January 1st, 2008, to August 31st, 2009 (20 months); the outbreak period from September 1st, 2009, to June 30th, 2010 (10 months); and the control bundle implementation period from July 1st to December 31st, 2010 (6 months).

For mastitis patients from whom S. aureus was isolated, data were also collected for molecular and phenotypic studies. These patients were further assessed for underlying diseases, birth rank, the interval between symptom onset and childbirth, lesion size, fever presence, suitability of empiric and target antibiotic therapies, and overall clinical outcomes.

Staphylococcus aureus isolates were cultivated and identified from mastitis foci through aspiration or surgical debridement using standard techniques. Each S. aureus isolate was confirmed with colonies exhibiting β-hemolysis on 5% sheep blood agar (BD Difco & BBL, NJ, USA), Gram-staining positive cocci, and giving positive results for catalase, coagulase, DNase, and mannitol fermentation tests. Staff from both the Department of Gynecology and Obstetrics and the Department of Pediatrics were subjected to three swabbing cultures: two from the surfaces of both hands and a third taken 1.5 cm deep into the nasal vestibule using sterile swabs (CultureSwab Transport System, Difco, Detroit, USA). Cultivated and identified colonized S. aureus isolates were obtained from these swabs. Environmental screening was conducted on surfaces like tables, desktops, computer mice, and work carts within the mentioned departments using sterile swabs rinsed with normal saline. The study protocol, encompassing isolate collection, staff and environmental screening, and the implementation of the infection control bundle, received approval from the hospital’s Institutional Review Board (CSMUH no. CS10218).

After screening with a 30 μg cefoxitin disc on the Műeller-Hinton agar (MHA) (BD Difco & BBL, NJ, USA) according to the Clinical and Laboratory Standards Institutes (CLSI) guidelines (10), the existence of the mecA gene, responsible for MRSA genotype, was performed using the polymerase chain reaction (PCR) (11). The antimicrobial susceptibility of S. aureus was assessed using disc diffusion against a range of antibiotics (erythromycin 15 μg, clindamycin 2 μg, tetracycline 30 μg, oxacillin 1 μg, vancomycin 30 μg, teicoplanin 30 μg, and tigecycline 15 μg) (BD Difco & BBL, NJ, USA) and fusidic acid (Thermo Fisher Scientific, MA, USA) following CLSI protocol (12) and manufacturer’s instructions. Resistance to three or more classes of antimicrobials was categorized as multidrug resistance. The minimum inhibitory concentrations (MICs) of vancomycin using reference standard compound (Sigma Aldrich Fine Chemicals Biosciences, MA, USA) against S. aureus were determined using agar dilution from vancomycin concentration of 0.0625 mg/L with 2-fold increase up to 64 mg/L in line with CLSI guidelines (12).

From July 1st, 2010 (the start of the third study period), all staff, patients, and their caregivers were instructed to rigorously follow the “My five moments for hand hygiene” approach recommended by the WHO (20). Measures to enhance breastfeeding management were given to patients and their caregivers, which included ensuring complete breast emptying, proper cleaning of the breast, milkers, and feeding bottles, mastering the breastfeeding technique, and being vigilant for signs of milk stasis or infection, such as continuous breast swelling and discomfort post-breastfeeding, with or without fever (6). Staff found to have S. aureus colonization during screening were treated with mupirocin applied to their anterior nares daily for 5 days (21). Surveillance and monitoring of patients with potential and confirmed mastitis continued until December 31st, 2010.

Differences between consecutive samples within the same population were assessed using the Mann–Whitney-U test for non-normally distributed populations (nonparametric). A p value of <0.05 was deemed indicative of a statistically significant difference between the samples.

During the three-year study period at the community hospital, 3,311 births were recorded, comprising 2,300 NSD and 1,011C/S. Concurrently, 249 patients received a mastitis diagnosis, with the majority (226, 90.8%) presenting during the puerperal period, equating to a prevalence of 6.8%. The patients’ average age was 30.4 years, ranging from 15 to 51 years, and all were female. The affected sites were the right side in 105 (42.2%) cases, the left side in 85 (34.1%) cases, and bilateral in 19 (7.6%) cases. The location was not recorded in 40 instances (16.1%). A majority (198, 79.5%) underwent outpatient treatment. Over the 3 years, the mastitis prevalence rates were 0.95 episodes per 1,000 patient-days of hospitalization and 7.52 episodes per 100 deliveries. The peak rates were observed in the second period, with 1.42 episodes per 1,000 patient-days and 13.01 episodes per 100 deliveries (p < 0.001 and 0.03 when compared to the first and third periods, respectively) (Table 1). The prevalence rates of mastitis during the second period were significantly different from the other two periods for both NSD and C/S patients (Table 1).

Of the 249 mastitis patients, only 61 (24.5%) underwent microbiologic surveys. From these, S. aureus was the sole microorganism isolated in 57 cases, which included 44 MRSA and 13 MSSA patients. During the second period, the prevalence rates of S. aureus-induced mastitis were 0.50 episodes per 1,000 patient-days and 4.67 episodes per 100 deliveries. The rates for MRSA were 0.40 episodes per 1,000 patient-days and 3.70 episodes per 100 deliveries. These rates were notably higher than those observed in the first and third periods (p = 0.02 and p < 0.001, respectively) (Table 1). Importantly, a marked decline in mastitis prevalence was observed in the third period following the adoption of the infection control bundle (Table 1 and Figure 1).

During the second period, 26 S. aureus isolates were collected from 26 mastitis patients, including 25 MRSA (M001 to M027, excluding M003 and M024) and one MSSA (M024). Additionally, one MRSA (M003) isolated from a patient with a buttock abscess served as a control. The demographic and clinical details of these mastitis patients are presented in Table 2. Among the 24 patients diagnosed with puerperal mastitis, the majority (16, 66.7%) experienced it during their first childbirth. Most patients (23, 88.5%) had no underlying medical conditions, with the average time from delivery to the onset of mastitis symptoms being 24.3 days (range, 5–78 days). The right breast was most frequently affected (14, 53.8%), followed by the left (8, 30.8%) and both breasts (4, 15.4%). Fever was observed in 14 patients (53.8%). The majority did not receive suitable initial antibiotic treatment (21, 80.8%), yet once susceptibility data were accessible, most (22, 84.6%) were given appropriate targeted antibiotics. All affected individuals underwent fine needle aspiration or surgical debridement. All but two patients, who were lost to follow-up, achieved clinical recovery (Table 2).

Thirty-one staff members (93 samples) from the Departments of Gynecology and Obstetrics and Pediatrics, along with 15 associated environmental sites, underwent swabbing culture screenings. From the mastitis patients, 26 S. aureus isolates were obtained, comprising 25 MRSA and 1 MSSA (M024). Additionally, 15 S. aureus isolates (10 MRSA and 5 MSSA – strains 1–3, 7–3, 20–1, 39–2, and 43–3) were procured from the hands and nostrils of nine staff members. Environmental screenings did not yield any S. aureus. All 35 MRSA isolates, 25 from mastitis patients and 10 from colonized staff, demonstrated resistance to a 30-μg cefoxitin disc in MHA and the presence of the mecA gene. The antibiogram revealed that a vast majority (23/25, 92%) of the clinical MRSA mastitis isolates exhibited multidrug resistance (Table 3). Two isolates (strains 24–3 and 42–3), which initially displayed satisfactory inhibitory zones against oxacillin discs, were subsequently verified to be MRSA due to cefoxitin resistance and the mecA gene presence. Conversely, the 6 MSSA isolates demonstrated lower resistance levels compared to MRSA. Vancomycin MICs, ascertained by agar dilution, indicated that the MICs for the 42 S. aureus isolates ranged from 1 to 2 mg/L, with a median of 1 mg/L (Table 3).

Figure 2 depicts the molecular profiles of 42 S. aureus isolates, as characterized by typing methods such as PFGE, SCCmec, agr, MLST, and spa typing. SCCmec was not able to type one clinical MSSA (M024) and 5 colonized MSSA isolates. All 25 clinical MRSA and 6 of the 10 colonized MRSA isolates were identified as SCCmecV_T-ST59. Four colonized MRSA isolates belonged to SCCmecIV-ST59. The spa types of all 25 clinical MRSA and 7 of the 10 colonized MRSA isolates were t437, while the remaining 3 colonized MRSA isolates were categorized as spa t529. All S. aureus isolates, with the exception of one (MSSA strain 39–2), fell under the agr group I; this exception was categorized as agr group III. The 6 MSSA isolates, which included 1 clinical isolate M024 and 5 colonized isolates, exhibited varied MLST and spa types (Figure 2). Thirteen distinct pulsotypes (ranging from A to M) were discerned among the 42 S. aureus isolates through PFGE, with their correlations illustrated in Figure 2. Pulsotype A encompassed 15 clinical MRSA isolates. Notably, two clinical (M020 and M027) and three colonized (11-3, 24-2, and 24-3) isolates exhibited identical pulsotypes, all classified as pulsotype B. The control strain, M003, sourced from a buttock abscess, was designated as SCCmecIV-ST59-spa t437-agr group I-pulsotype F.

Figure 2 and Table 3 reveal that 31 pvL + MRSA isolates (comprising 25 clinical MRSA and 6 colonized MRSA) were categorized as SCCmecV_T. The remaining 5 pvL- MRSA, inclusive of the control strain M003, were identified as SCCmecIV. None of the six MSSA isolates, which include the clinical strain M024 and 5 colonized strains, carried the pvl gene (as seen in Figure 2 and Table 3). The superantigenic toxin gene profiles indicated that the seb-selk-selr profile was predominant in clinical MRSA isolates (21, 84%), succeeded by seb-selk (3, 12%). A diverse range of toxin gene profiles was observed in the 6 MSSA and 10 colonized MRSA isolates (Table 3).