Staphylococcus aureus strains are part of the normal human flora. It is responsible for an impressive variety of diseases ranging from minor skin and soft tissue infections to major life-threatening and fatal infections such as bacteremia, endocarditis, pericarditis, pneumonia, empyema, osteomyelitis, myositis and septic arthritis.1 , 2 S. aureus is a leading cause of both community-associated and healthcare-associated invasive infections in children.3 , 4 S. aureus infections such as bacteremia are generally associated with high morbidity and mortality, and the acquisition of methicillin resistance further limits therapeutic options.5 , 6 S. aureus (MSSA) has an oxacillin minimum inhibitory concentration of ≤2 mcg/mL. Methicillin-resistant S. aureus (MRSA) has an oxacillin minimum inhibitory concentration ≥4 µg/mL.
Because MRSA was first identified at a hospital in the United Kingdom in 1961, it quickly became an important pathogen globally (referred to as hospital-associated MRSA [HA-MRSA]). Until the late 1980s and early 1990s, some cases of MRSA in young and otherwise healthy patients without hospital-related risk factors were reported. These isolates were called community-associated MRSA (CA-MRSA) and their emergence and spread increased the risk to public health. Although CA-MRSA strains can cause nosocomial infections, and HA-MRSA strains do circulate in the community, CA-MRSA is epidemiologically, clinically and genetically different from HA-MRSA compared with traditional HA-MRSA strains, CA-MRSA isolates harbor different types of SCCmec elements encoding methicillin (mec) resistance genes. CA-MRSA infections tend to occur in otherwise healthy younger patients with skin and soft tissue infections (SSTIs); such strains may be susceptible to a wider range of antibiotics, and usually harbor the staphylococcal cassette chromosome mec (SCCmec) types IV or V. To date, 13 SCCmec types (indicated by roman numerals I to XIII) and three mec (mecA/B/C) genes have been identified among S. aureus in the world. SCCmec types I, II and III are commonly reported in HA-MRSA, while types IV and V are frequently found in CA-MRSA. The majority of CA-MRSA isolates have SCCmec type IV or V, do not exhibit resistance to multiple antibiotics (except to b-lactams) and possess different exotoxin gene profiles.7–9
S. aureus elaborates a wide variety of extracellular toxins, many of which have potent biologic effects in the isolated state on intact animals, tissues, cells and membranes. These toxins generally are thought to be responsible for the virulence of S. aureus , and the role of several of these products in staphylococcal infections has been proved in humans. Panton-Valentine leukocidin (PVL) is the only known extracellular toxin that attacks the leukocyte exclusively. It consists of two protein components, LukS-PV and LukF-PV, encoded by the genes lukS-PV and lukF-PV carried on a bacteriophage. Leukocidin injected into rabbits causes a striking fall in levels of circulating and bone marrow leukocytes, followed by marked granulocytosis; these changes occur without death of the rabbits. Leukocidin interacts with the membrane phospholipid and causes depolarization, increased permeability and cell death. In early studies, only a fraction of S. aureus isolates recovered from humans carrying the PVL genes. Interest in PVL greatly increased after reports that S. aureus isolates carrying PVL genes were associated with severe furunculosis and particularly necrotizing pneumonia in children and adolescents that frequently was fatal.10 , 11
The aim of this study was to investigate the molecular profile, antimicrobial resistance and virulence genes associated with S. aureus isolates recovered from pediatric patients between 2010 and 2017 in 3 pediatric hospitals in Turkey.
MATERIAL AND METHODS
From January 2010 to December 2017, 132 S. aure us isolates were collected from pediatric patients (<18 years old) from 3 hospitals in Turkey. Hacettepe University Ihsan Dogramaci Children’s Hospital is a tertiary care, 269-bed pediatric referral hospital and other 2 hospitals are secondary and tertiary research and training hospitals with 120 and 330 bed capacity, respectively. Sociodemographic features, underlying diseases and clinical findings of the patients were recorded in medical records. The electronic records of the microbiology laboratory were screened and accessible isolates were taken to SCCmec and PVL gene analysis. S. aureus isolates were confirmed by classic microbiologic methods including Gram stain and catalase and coagulase activity. MRSA isolates were initially identified using cefoxitin screening and the presence of the mec A gene was confirmed by PCR. MRSA strains are characterized by the presence of a large heterologous mobile genetic element called the SCCmec , which includes the mecA gene, the gene complex (which comprises the mecA gene and its regulators, mecI and mecR1 ), SCCmec contains the ccr gene complex, which encodes recombinases responsible for the mobility of SCCmec . CA-MRSA was defined as an MRSA isolate that was obtained either from a patient within 48 hours of hospitalization, and without the patient having a medical history of MRSA infection or colonization, admission to a healthcare facility, dialysis, surgery or the insertion of indwelling devices in the past year. All strains were grown overnight on sheep blood agar plates at 37°C. This study was approved by the Ethics Committee of Hacettepe University Faculty of Medicine.
ANTIMICROBIAL SUSCEPTIBILITY TESTING
The antibiotic susceptibility profiles of S. aureus isolates were performed using the bioMérieux VITEK2 system following manufacturer’s instructions. Results were interpreted according to the definitions of the Clinical and Laboratory Standards Institute (2017). The following 16 drugs were tested: cefoxitin, linezolid, ciprofloxacin, clindamycin, erythromycin, trimethoprim-sulfamethoxazole, fucidic acid, vancomycin, tetracycline, penicillin, rifampin, mupirocin, oxacillin, gentamicin, teicoplanin, tigecycline and daptomycin. S. aureus ATCC 29213 was used for quality control.
SCCmec TYPING
MRSA isolates were subjected to SCCmec typing as described by Oliveira et al,9 mec types I–IV were assigned according to the combination of the cassette chromosome recombinase (ccr) type and mec class. MRSA isolates that could not be assigned to any expected type were defined as nontypable. PVL gen analysis was done as described before.12
STATISTICAL ANALYSIS
All statistical analyses were performed using the SPSS package program for Windows, version 17.0 (SPSS Inc., Chicago, IL). Values for numerical variables were provided as mean ± standard deviation or median (minimum–maximum) depending on normality of distribution. Categorical variables were provided as absolute values or percentages, the comparisons of which were made using the chi-square test. Antibiotic susceptibility pattern, region of isolate culture, PVL and SCCmec gen presence were compared between MSSA and MRSA strains. Two-way comparisons for numerical variables were made using the Mann–Whitney U test, whereas the Kruskal–Wallis test was used for comparison involving more than 2 groups. Factors associated with an increased mortality risk were identified using logistic regression analysis. A P value of <0.05 was considered an indicative of statistical significance.
RESULTS
A total of 132 S. aureus isolates—102 MSSA (81.8%) and 30 MRSA (18.2%)—were included in the study. Median age of the patients was 51 months (minimum–maximum: 0–263 months); 89 of the patients were male (67.4%) and 43 were female (32.6 %). The median ages of MRSA and MSSA isolate contributors were 51 (0–213 months), and 50 months (0–263 months), respectively (P = 0.636). There were 65 children with underlying diseases and recent use of hospital care, including oncologic malignancies (n = 20), neurologic diseases (n = 11), hematologic diseases (n = 8), genetic diseases (n = 8), cardiogenic diseases (n = 5), prematurity (n = 5), renal diseases (n = 4), diabetes mellitus (n = 2) and immune deficiency (n = 1).
Sixty (45.5%) S. aureus isolates were cultured from SSTIs, 50 (37.9%) from bloodstream infections, 11 (8.3%) from bone infections, and 11 (8.3%) from other sterile sites. Fifty-three (52%) MSSA isolates were cultured from SSTIs, 35 (34.3%) from bloodstream infections, 7 (6.9 %) from bone infections and 7 (6.9%) from other sterile sites (P = 0.083) (Table 1 , Figure 1 ). Fifteen (50%) MRSA isolates were cultured from blood cultures, 7 (23.3%) from SSTIs, 4 (13.3%) from bone infections and 4 (13.3%) from other sterile sites. Thirty (22.7%) isolates from cellulitis, 12 (9.1%) isolates from abscess and 11 isolates (8.3%) were isolated from osteomyelitis patients.
TABLE 1. -
Clinical and Molecular Characteristics of Patients According to Methicillin Resistance
MSSA
MRSA
P
Age, moTABLE 1.
50 (0–263)
51 (0–213)
0.636
Sex (male)TABLE 1.
69 (67.6%)
20 (66.7)
0.540
Hospital-acquiredTABLE 1.
50 (49%)
22 (73.39)
0.150
Community-acquiredTABLE 1.
52 (51%)
8 (26.7)
Hospitalization time, dTABLE 1.
16 (0–157)
37 (16–72)
<0.01
PVLTABLE 1.
8 (7.8)
4 (13.3)
0.270
MortalityTABLE 1.
8 (14)
0 (0)
0.120
Statistical significance is given as bold.
*Values are given as median (minimum–maximum).
†Values are given as n (%).
FIGURE 1.: Molecular, microbiologic and epidemiologic features of Staphylococcus aureus isolates.
Eight (7.8%) MSSA isolates and 4 (13.3%) MRSA isolates were PVL gene-positive (P = 0.276). PVL-positive S. aureus isolates were found in 7 males and 5 females (P = 0.500). Nine (75%) PVL gene-harboring S. aureus were isolated from SSTIs, 2 (16.7%) from blood culture and 1 (8.3%) from a sterile site (P = 0.360) (Table 2 ). One PVL-positive case was identified in a catheter-related blood stream infection and total parenteral nutrition intake correlated with PVL gene presence. Two PVL-positive cases (P = 0.280) had history of surgery in the last 1 year, and 4 patients with PVL-positive S. aureus (P = 0.176) had been hospitalized in the past year.
TABLE 2. -
Sociodemographic and Clinical Characteristics According to PVL Gene Presence
PVL Negative
PVL Positive
P
(n = 120)
(n = 12)
Epidemiologic associations with PVL status
Age in months, median (minimum–maximum)TABLE 2.
51 (0–263)
40 (0–136)
0.56
Male (%)TABLE 2.
82 (68.3)
7 (58.3)
0.342
MSSA (%)TABLE 2.
94 (78.3)
8 (66.7)
0.310
MRSA (%)TABLE 2.
26 (21.7)
4 (33.3)
Specimen type by PVL statusTABLE 2.
Skin and soft tissue infection (%)
51 (42.5)
9 (75)
0.240
Blood stream infection (%)
48 (40)
2 (16.7)
Bone infection (%)
11 (9.2%)
0 (0)
Other infection sites (%)
10 (8.3)
1 (8.3)
Hospitalization length (day)
20 (0–157)
17 (9–50)
0.860
Multidrug resistance (%)
15 (14)
0 (0)
0.239
High blood CRP (%)
63 (72.4)
7 (70)
0.565
Mortality, n (%)
8 (5.9)
0 (0)
1
SCCmec type III, n (%)
3 (1.799)
0 (0)
0.32
SCCmec type IV (n,%)
13 (10.8)
3 (25)
*Values are given as median (minimum–maximum).
†Values are given as n (%).
Of 25 MRSA isolates, 16 (53.3%), 3 (10%) and 11 (44%) cases were type IV, type III and nontypeable, respectively. Three SCCmec type IV positive MRSA isolates were also PVL-positive. Three MRSA isolates harboring SCCmec type III were isolated from blood culture. Eleven of 16 MRSA isolates harboring SCCmec type IV was isolated from blood culture, 3 isolates from bone infections and 2 isolates were isolated from SSTIs (P < 0.001). Twenty-two HA-MRSA and 50 HA-MSSA were isolated (P = 0.015). Twenty-two (30.6%) of the HA-S. aureus infections and 8 of 60 CA-S. aureus isolates (13.3%) were MRSA (P = 0.015). Five of 72 (6.9%) HA-S. aureus isolates and 7 of 60 (11.7%) CA-S. aureus isolates were PVL gene positive (P = 0.260). All of the 3 MRSA isolates harboring SCCmec III and 11 of 16 MRSA isolates with SCCmec IV were HA-MRSA infections (P = 0.066) (Table 3 ). Hospitalization in the past 1 year was found to increase by 3.95 times in MRSA infections (P = 0.038, 95% confidence interval [CI[: 1.078–14.48). Tetracycline, rifampin, clindamycin, ciprofloxacin, erythromycin or trimethoprim-sulfamethoxazole resistance were present in 2, 4, 7, 10, 16 and 4 MRSA isolates, respectively. None of MRSA isolates were resistant to gentamicin, daptomycin, linezolid, vancomycin, teicoplanin, mupirocin or tigecycline. Penicillin, tetracycline, rifampin, clindamycin, ciprofloxacin or erythromycin resistance were present in 83 (81.3%) MSSA isolates, 3 (2.9%) MSSA isolates, 1 (0.9%) MSSA isolates, 10 (9.8%) MSSA isolates, 3 (2.9%) MSSA isolates and 42 (41.1%) MSSA isolates, respectively. None of MSSA isolates were resistant to gentamicin, daptomycin, linezolid, vancomycin, teicoplanin, tigecycline nor trimethoprim-sulfamethoxazole. Eighty-three MSSA isolates were penicillin resistant. None of the PVL gene harboring S. aureus isolates was resistant to tetracycline, rifampin, ciprofloxacin, clindamycin or trimethoprim-sulfamethoxazole. HA-MRSA isolates were resistant to rifampin (33.3%), clindamycin (28.6%), ciprofloxacin (42.9%) and erythromycin (59.1%). CA-MRSA isolates were resistant to clindamycin (14.3%), ciprofloxacin (12.5%) and erythromycin (37.5%). Trimethoprim-sulfamethoxazole resistance was more common in CA-MRSA infections compared with HA-MRSA infections—60% vs. 5.3%, respectively (P = 0.018) (Table 4 ). Mortality was present in 8 (6%) patients and all were related to S.aureus bacteremia.
TABLE 3. -
Methicillin-resistant
Staphylococcus aureus Rate According to Clinical Diagnosis
Total S. aureus Isolates (n = 132)
MRSA Isolates (n = 30)
Clinical diagnosis
MSSA (n = 102)
MRSA (n = 30)
Community-associated
Hospital-associated
SSTITABLE 3.
53 (52)
7 (23.3)
6 (75)
5 (22.7)
Blood stream infectionTABLE 3.
35 (34.3)
15 (50%)
1 (12.5)
14 (63.7)
Bone infectionTABLE 3.
7 (6.9)
4 (13.3)
OthersTABLE 3. (cerebrospinal fluid, urine)
7 (6.9)
4 (13.3)
1 (12.5)
3 (13.6)
*Values are given as n (%).
TABLE 4. -
Antibiotic Susceptibility Patterns of CA-MRSA and HA-MRSA Isolates
CA-MRSA
HA-MRSA
P
Rifampicin resistanceTABLE 4.
1 (25)
3 (33.3)
0.40
Clindamycin resistanceTABLE 4.
1 (14.3)
6 (28.6)
0.41
Ciprofloxacin resistanceTABLE 4.
1 (12.5)
9 (42.9)
0.13
Erythromycin resistanceTABLE 4.
3 (37.5)
13 (59.1)
0.54
Trimethoprim/sulfamethoxazole resistanceTABLE 4.
3 (60)
1 (5.3)
0.018
Statistical significance is given as bold.
*Values are given as n (%).
DISCUSSION
This study described the epidemiologic and molecular characteristics of CA-MRSA and HA-MRSA isolates. We investigated the distribution of S. aureus isolates from pediatric patients according to the hospital wards, antibiotic susceptibility patterns, SCCmec types and the presence of PVL genes. SSTIs and blood stream infections were the most common clinical presentations in our study, as in other pediatric studies. To our knowledge, this is the first study that investigated the molecular characteristics of S. aureus isolates in pediatric patients in Turkey. Sixty of 132 (45.5%) S. aureus isolates were cultured from SSTIs, 50 (37.9%) from bloodstream infections, 11 (8.3%) from bone infections and 11 (8.3%) from other sterile sites. There was an underlying disease and recent use of hospital care in 49% of patients. MSSA isolates were more common in SSTIs and bone infections, and MRSA infections were more common in blood stream infections. The MRSA rate was 22.7%.
In 2019, Park et al13 S. aureus isolates from Korean children and found that the MRSA rate according to disease was 34.1% (14/41) for SSTIs and 23.1% (6/26) for bone and joint infections. These rates are higher in SSTI infections compared with those in our study. We observed an MRSA rate of 16% (4/11) in isolates from osteomyelitis and 10% (3/30) in cellulitis. Twelve S. aureus isolates were PVL gene positive (9.1%). PVL gene presence was more common in SSTIs. Nine PVL gene harboring S. aureus were isolated from SSTIs (75%), 2 from blood culture (16.7%) and 1 from a sterile site (8.3%). None of the isolates from bone infections was PVL gene positive. PVL gene presence was slightly more common in CA-MRSA infections than HA-MRSA infections, 7 and 5 isolates, respectively. Although PVL is known as a common virulence factor of CA-MRSA, HA-MRSA isolates in our study had a considerable rate of PVL positivity, pointing out the importance of surveillance of the changing epidemiology of MRSA. Although PVL-positive isolates predominated CA-MRSA infections, they were found in both community and hospital infections in our study. In a study conducted by Barrios López et al,14 S. aureus SSTIs was 32%. Eighty-nine (63%) were PVL-negative MSSA, 33 (23%) were PVL-positive MSSA, 13 (9%) were PVL-positive MRSA and 7 (5%) were PVL-negative MRSA.14
In an adult study performed in 2010 in Turkey, of the 110 S. aureus isolated, 61.8% harbored SCCmec type III, 34.5% SCCmec variant IIIB and 2.7% SCCmec type IV.15 mec type III S. aureus isolates than reported in the Turkey study. However, SCCmec type IV was more prevalent in our study (53.3%), similar to a 2019 pediatric study.16
The HA-MRSA distribution and drug resistance findings in the present study were similar to those reported in Europe—MSSA isolates ranging from 13% to 35% were susceptible to penicillin.17 Staphylococcus spp. is mediated by blaZ, the gene that encodes β-lactamase. As early as 1942, penicillin-resistant staphylococci were isolated from hospitals.18 S. aureus .19 20 S. aureus infections.
Until the mid-1990s, MRSA infections were limited to hospitals, infecting primarily the elderly, very young and patients with immune-deficiency or undergoing surgery. However, within the last 15 years, MRSA outbreaks were reported in healthy individuals without connection to health care institutions. It has become clear that these infections are due to the rise of new, distinct strains of MRSA, now called CA-MRSA strains.21 mec types IV or V genes.
Typically, the antibiotic resistance gene, mecA, harbored by SCCmec is responsible for the antibiotic resistance of MRSA. Recent studies revealed that CA-MRSA strains have increased virulence and fitness properties compared with traditional HA-MRSA strains. The contribution of toxins to CA-MRSA virulence has received attention, in part because these may represent promising targets for therapeutic intervention. PVL gene presence is known to be more prevalent in CA-MRSA infections, as in our findings. Our results show a high prevalence of PVL in community-onset S. aureus infections in children. Overall, SCCmec type IV was more common in hospital-acquired MRSA isolates and the PVL gene was frequently isolated in community-acquired S. aureus infections. Overall, we found no strict distinction between CA-MRSA and HA-MRSA in our molecular analysis. This highlights the need for differential studies to investigate HA-MRSA and CA-MRSA infections at molecular levels.
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