Abstract
Introduction
The objective of this study was to determine the prevalence of antibiotic resistance genes mecA, vanA, B, C and virulence genes Panton-Valentine Leucocidin (PVL) and fibronectin-binding protein (fnBPA) among S. aureus isolates from hospital-acquired sepsis from pediatric intensive care units.
Methods
The study was a retrospective cross-sectional study, including 250 unique isolates of S. aureus obtained from pediatric patients with hospital-acquired sepsis. The isolates were subjected to study of antibiotic susceptibility by disc diffusion method and molecular analysis of antibiotic resistance genes and certain virulence genes (PVL and fnBPA genes).
Results
Methicillin resistant S. aureus represented 178 (71%) of the isolated S. aureus and reduced susceptibility to vancomycin was detected by minimum inhibitory concentration in 39 (22%) isolates. It was found that there was a strong association between the MRSA strains and resistance to some antibiotics, devices association (p<0.001) and patient outcomes (p=0.003). There was a significant association between reduced vancomycin susceptibility (p=0.010), the presence of a central line catheter (p=0.000) and fnBPA gene (p<0.001) and mortality rate.
Conclusions
The present study highlights that major S. aureus strains isolated from sepsis in pediatric patients were methicillin resistant with a substantial proportion of reduced susceptibility to vancomycin. Although none of the isolates had van genes responsible for vancomycin resistance, this finding warrants a considerable attention for study as it was a risk factor for mortality in those patients. The virulence genes fibronectin-binding protein and Panton-Valentine Leucocidin were not uncommon in S. aureus.
Keywords: S. aureus, mecA, van, fnBPA, PVL
Introduction
Staphylococcus aureus (S. aureus) is a widespread pathogen associated with multiple infections in both healthcare facilities as well as community-acquired infections. The pathogen is responsible for multiple infections ranging from infections of the soft tissues to serious invasive infections such as sepsis and pneumonia. Its pathogenicity is due to many factors such as the resistance to antibiotics, production of enzymes and toxins. Methicillin resistant S. aureus (MRSA) has been a significant concern in healthcare infections worldwide.1
Methicillin resistant S. aureus reflects the antibiotic resistance pattern of this species which simply reflects the resistance to betalactam antibiotics and limits the options available for antibiotic therapy. Methicillin resistance is due to the presence of mecA gene. MecA gene codes for the penicillin-binding protein 2a (PBP2a) which reduces the binding affinity for the betalactam antibiotics, even the penicillinase-resistant penicillin. The mecA gene, known as the staphylococcal cassette chromosome mec (SCCmec) is present on a mobile genetic element. The mecA gene complex has plasmids and transposons insertion sites which are associated with the development of resistance genes to other antibiotics.2
Many studies since 2002 until now reported the emergence of S. aureus strains with reduced susceptibility to vancomycin, a glycopeptide antibiotic that is reserved as an alternative treatment of MRSA.3 Two different resistance mechanisms were present, intermediate resistance to S. aureus that occurs as a result of thickening in the cell wall, where many vancomycin molecules were trapped within the cell wall. The trapped molecules block the meshwork of peptidoglycan and eventually create a physical barrier to more incoming molecules of vancomycin;4 these isolates are called vancomycin-intermediate S. aureus (VISA) with MIC of 4–8 μg/mL.5 The second mechanism of resistance to vancomycin is due to the presence of vanA gene or other van resistance determinants, these isolates are called vancomycin-resistant S. aureus (VRSA) with MIC ≥ 16 μg/mL.5
Among the virulence factors expressed by S. aureus are fibronectin-binding protein A (fnBPA) and fibronectin-binding protein B (fnBPs), which are important virulence factors that mediate its action via S. aureus adhesion to the fibronectin, fibrinogen and elastin.6 Additionally, fnBPA is known to be an important coating component for S. aureus in its internalization by nonprofessional phagocytic cells to be protected against immune response and antibiotic treatment, the process that is associated with serious infections and septic death.7
Another factor of virulence expressed by S. aureus is the Panton-Valentine Leucocidin (PVL), which is a leucotoxin that mediates the destruction of leukocytes and tissue necrosis, it is encoded by two genes, lukS-PV and lukF-PV.8
The aim of this study was to investigate the prevalence of mecA, vanA, B, C antibiotic resistance genes and virulence genes Panton-Valentine Leucocidin (PVL) and fibronectin-binding protein (fnBPA) among S. aureus isolates from hospital-acquired sepsis of intensive-care units in pediatric patients.
Methods
Study design
The study was a retrospective cross-sectional study that included 250 unique isolates of S. aureus collected from pediatric patients from intensive care units, with hospital-acquired sepsis, from Mansoura University Children Hospital, Egypt, from January 2015 to March 2018. Hospital-acquired sepsis was diagnosed according to the center of disease control criteria.9 Their ages ranged between 20-65 months. The study was approved by the Mansoura ethical committee and approval to use isolates in future genetic studies of the microbes was received from each patient's parent.
Data collection
The resulting demographic and clinical data were obtained from the recorded electronic data system for each patient.
Bacterial isolates
Bacterial isolates were collected from each sample by standard microbiological techniques. S. aureus was identified by Gram stain, coagulase, catalase tests and mannitol fermentation.
Antimicrobial susceptibility test
Antibiotic disc diffusion method was used to detect antibiotic susceptibility according to Clinical and Laboratory Standards Institute guidelines (CLSI).10 The antibiotic discs used were cefoxitin, ciprofloxacin, clindamycin, erythromycin, gentamicin, amikacin, oxacillin, rifampin, tetracycline, sulfamethoxazole plus trimethoprim (Oxoid, Basingstoke, UK). Cefoxitin disc (30 μg) and oxacillin disc (1 μg) were used for the detection of methicillin-resistant isolates. Decreased susceptibility of the isolates to vancomycin was determined by observing the minimum inhibitory concentration (MIC) by agar dilution according to the CLSI guidelines.10
Following the definition of the Clinical and Laboratory Standards Institute, S. aureus isolates with vancomycin MIC 4–8 µg/mL were classified as vancomycin intermediate S. aureus, and those with MIC ≥16 µg/mL were classified as vancomycin resistant S. aureus, and S. aureus with reduced susceptibility to vancomycin with MIC from 2 μg/mL to 4 μg/mL.10
Detection of mecA, vanA, B, C, fnBPA, and PVL genes by PCR
DNA extraction
S. aureus was grown at 37°C for 18 hours on blood-agar plates. DNA was extracted by the use of DNeasy by Blood & Tissue Kit according to the manual procedures. Extracted DNA was kept frozen at -20°C before amplification procedures. The sequences of the primers used for all genes have been summarized in Table 1.
Table 1. The sequences used for the studied genes and the base pair (bp) of the amplified products.
| Gene | Sequences of the primers | bp | Reference |
|---|---|---|---|
| mecA | F:5/-AAAATCGATGGTAAAGGTTGGC-3\ R 5/-AGTTCTGGAGTACCGGATTTGC-3\ |
533 | 11 |
| vanA | F5/-ATGAATAGAATAAAAGTTGCAATAC R 5/-CCCCTTTAACGCTAATACGAT |
1029 | 12 |
| vanB | F: 5/-CCCGAATTTCAAATGATTGAAAA R 5/-CGCCATCCTCCTGCAAAA |
457 | 12 |
| vanC | F5/-GCTGAAATATGAAGTAATGACCA R 5/-CGGCATGGTGTTGATTTCGTT |
811 | 12 |
| PVL | Luks-PV: 5/AGTGAACTTATCTTTCTATTGAAAAACACTC-3\ Luks-PV: 5/-GCATCAASTGTATTGGATAGCAAAAGC-3\ |
433 | 13 |
| fnBPA | F: 5/-CACAACCAGCAAATATAG-3\ R: 5/-CTGTGTGGTAATCAATGTC-3\ |
1362 | 13 |
PCR detection of mecA
Amplification was achieved by using Qiagen ready to use mixture for amplification. A 1 µL volume of prepared DNA (0.5 µg) was applied to 25 µL PCR mixture with 0.7 µL of 0.8 µmol/L of each primer. The PCR thermal cycling protocol included first denaturation of 95°C for 3 min, followed by amplification for 33 cycles of 94°C for 1 min, 53°C for 30 s and 72°C for 1 min, with a final extension at 72°C for 6 min. Electrophoresis visualized the amplified products by staining with ethidium bromide and seen using UV transilluminator.11
PCR detection of vanA-C genes
The amplification was carried out using Qiagen amplification mixture. A 1 µL volume of prepared DNA (0.5 µg) was applied to 25 µL PCR mixture with 0.7 µL of 0.8 µmol/L of each primer. The PCR program consisted of initial denaturation step at 94°C for 3 min; followed by denaturation at 94°C for 30 seconds, annealing at 82°C, 59°C, and 58°C for vanA, vanB and vanC respectively for 2 min, and DNA extension at 72°C for 2 min. The reaction was terminated for 6 min after the last cycle by incubation at 72°C and the products were deposited at 4°C. PCR products (5.0 μL) were analyzed with electrophoresis after staining with ethidium bromide and seen using UV transilluminator.12
PCR detection of PVL gene
The PCR program consisted of initial denaturation step at 94°C for 1 min; this was followed by denaturation at 94°C for 30 s, primers annealing at 50 for 1 min, and DNA extension at 72°C for 2 min. After the last cycle, the reaction was terminated by incubation at 72°C for 6 min. PCR products (5.0 μL) were analyzed by 1% agarose gel electrophoresis and made visible by ethidium bromide staining and UV transillumination.13
PCR detection of fnBPA gene
After amplification for 30 cycles (30 s of denaturation at 94°C, 30 s of annealing at 57°C, and 1 min of extension at 72°C. PCR products were analysed by electrophoresis through 0.8% agarose gel stained by ethidium bromide staining and UV transillumination.13
Control strains
The control strains were used for the laboratory tests. For biochemical identification and antibiotics test susceptibility by disc diffusion method, S. aureus ATCC 29213 was used. For PCR for PVL gene, S. aureus ATCC 49775 was used. For detection of fnBPA gene, S. aureus ATCC was used. For mecA gene detection by PCR, S. aureus ATCC 33591 was used.14
Statistical analysis
The data were analyzed by the use of Statistical Package for Social Science (SPPS v24), (IBM, Armonk, NY, USA). The qualitative data were expressed as numbers and percentages and compared with the use of Chi-square test and p values were considered significant if <0.05. The quantitative data were analyzed as mean and standard deviation (SD). The risk factors assessment was measured by binary logistic regression and p values were considered significant if <0.05.
Results
The study included 250 children with mean age 39.9±13.1 months complaining of healthcare-acquired sepsis due to S. aureus.
The sepsis was diagnosed as primary sepsis with no obvious primary infection in 57% of the patients. The most frequent underlying disease was mainly renal diseases and diabetes mellitus (23% and 22% respectively), the mortality rate was 16% – Table 2.
Table 2. Demographic and clinical data of pediatric patients.
| Total patients (n=250) | ||
|---|---|---|
| No. | % | |
| Gender | ||
| Male | 123 | 49% |
| Female | 127 | 51% |
| Age, months (mean±SD) | 39.9 ± 13.1 | |
| Underlying etiology | ||
| Diabetes mellitus | 54 | 22% |
| Renal diseases | 57 | 23% |
| Respiratory infections | 46 | 18% |
| Hepatic disorders | 33 | 13% |
| Meningitis | 17 | 7% |
| Malignancy | 43 | 17% |
| Type of sepsis | ||
| Primary | 141 | 56% |
| Secondary | 109 | 44% |
| Type of device | ||
| Central venous line | 57 | 23% |
| Ventilator | 51 | 20% |
| Urinary catheter | 142 | 57% |
| Patient outcome | ||
| Discharge | 211 | 84% |
| Death | 39 | 16% |
Methicillin resistant S. aureus represented 178 (71%) of the isolated S. aureus and reduced sensitivity to vancomycin was detected by MIC in 39 (22%) isolates, data not shown.
The study of antibiotic resistance genes mecA and van genes revealed that mecA was detected in 71% of the isolated S. aureus while none of the isolated S. aureus had van genes. The prevalence of virulence genes was 24% for fnBPA and 4% for PVL.
The antibiotic resistance was more frequent among MRSA than non-MRSA strains and statistically significant increases were found in resistance to gentamicin (p=0.008) and clindamycin (p=0.013). There was a statistically significant increase in PVL (p=0.036) and statistically non-significant increase in fnBPA in MRSA than non-MRSA strains, (p=0.450). The most frequent devices associated with MRSA were urinary catheters and ventilators (p <0.001). Moreover, a significant statistical association with patient outcomes among MRSA strains (p=0.003) was seen – Table 3.
Table 3. Comparison between MRSA and non-MRSA regarding antibiotics resistance, device association and patient's outcome.
| MRSA (n=178) |
Non-MRSA (n=72) |
Chi-square value | df value | 95% confidence interval | P value | ||||
|---|---|---|---|---|---|---|---|---|---|
| No | % | No | % | Lower bound | Upper bound | ||||
| Oxacillin | 178 | 100 | 3 | 4 | 235.612 | 1 | 0.005 | 0.051 | p<0.001 |
| Gentamicin | 42 | 24 | 7 | 10 | 6.261 | 1 | 1.222 | 6.729 | 0.008 |
| Amikacin | 40 | 22 | 19 | 26 | 0.436 | 1 | 0.430 | 1.520 | 0.307 |
| Clindamycin | 34 | 19 | 8 | 11 | 5.373 | 1 | 1.132 | 5.735 | 0.013 |
| Vancomycin | 39 | 22 | 10 | 14 | 2.093 | 1 | 0.816 | 3.707 | 0.100 |
| Tetracycline | 25 | 14 | 7 | 10 | 0.858 | 1 | 0.625 | 3.683 | 0.240 |
| Erythromycin | 35 | 20 | 11 | 15 | 0.657 | 1 | 0.647 | 2.847 | 0.268 |
| Rifampicin | 63 | 35 | 21 | 29 | 0.891 | 1 | 0.735 | 2.409 | 0.214 |
| Trimethoprim/sulfamethoxazole | 81 | 45 | 25 | 35 | 2.441 | 1 | 0.890 | 2.770 | 0.077 |
| Ciprofloxacin | 34 | 19 | 7 | 10 | 3.289 | 1 | 0.924 | 5.205 | 0.048 |
| Cefoxitin | 178 | 100 | 0 | 0 | 250.000 | 1 | <0.001 | ||
| fnBPA | 44 | 46 | 19 | 26 | 0.076 | 1 | 0.490 | 1.711 | 0.450 |
| PVL | 4 | 2 | 6 | 8 | 4.945 | 1 | 0.069 | 0.925 | 0.036 |
| Gender (male) | 82 | 46 | 41 | 57 | 2.427 | 1 | 0.372 | 1.121 | 0.078 |
| Device | 19.269 | 2 | <0.001 | ||||||
| CLABSI | 28 | 16 | 29 | 40 | |||||
| Ventilator | 36 | 20 | 15 | 20 | |||||
| Urinary catheter | 114 | 64 | 28 | 38 | |||||
| Outcome | 0.003 | ||||||||
| Death | 20 | 11 | 19 | 26 | |||||
| Discharge | 158 | 89 | 53 | 73 | |||||
CLABSI – central line-associated bloodstream infection.
There was a significant association between male gender (p=0.032), reduced vancomycin susceptibility (p=0.010), the presence of a central line catheter (p<0.001) and fnBPA gene (p<0.001) and mortality – Table 4.
Table 4. Study of demographic, clinical and microbiological risk factors associated with mortality.
| Parameters | Died (n=39) | |||||
|---|---|---|---|---|---|---|
| No. | % | Odds ratio | 95% confidence interval | P value | ||
| Lower bound | Upper bound | |||||
| Gender | ||||||
| Male | 24 | 62% | 2.1 | 1.1 | 3.9 | 0.032 |
| Reduced vancomycin sensitivity | 16 | 41% | 2.6 | 1.4 | 4.4 | 0.010 |
| Device associated with sepsis | ||||||
| Central venous catheter versus other | 25 | 64% | 4.3 | 2.5 | 7.6 | <0.001 |
| mecA | 30 | 77% | 2.02 | 0.9 | 4.6 | 0.064 |
| fnBPA | 23 | 59% | 4.3 | 2.4 | 7.5 | <0.001 |
| PVL | 4 | 10% | 2.7 | 1.2 | 6.2 | 0.058 |
Discussion
S. aureus has emerged as a major pathogen in invasive infections affecting hospitalized patients. There is unceasing rise of MRSA strains among these patients, thus, influencing proper empiric antibiotic choice and requiring longitudinal control.15
In the present study, strains resistant to methicillin represented 71% of the isolated S. aureus. Previous analysis about MRSA frequency among S. aureus isolates denoted that the rates ranged from 13 up to 74%.16 These wide variations could be justified by the difference in geographical area from one country to another, different risk factors and the degree compliance with guidelines of infection control within the health system.17
The current findings indicate that there is a higher frequency of resistance to commonly used antibiotics for S. aureus treatment. A statistically significant association was reported between MRSA strains and resistance to gentamicin (p=0.008) and clindamycin (p=0.013). These findings are in line with previous data claiming that MRSA strains have emerged with concomitant resistance to many commonly used antibiotics from groups like aminoglycosides, macrolides, fluoroquinolones, chloramphenicol, and tetracycline.18 Once a S. aureus isolate is characterized as an MRSA, it is instantly classified as multiple drug resistant infection as it will be non-susceptible to all categories of β-lactam such as all categories of penicillins, cephalosporins, β-lactamase inhibitors, and carbapenems.19
The best antibiotic of choice for treatment of MRSA is vancomycin. However, there are reports about the emergence of intermediate resistance to vancomycin.4 The standard method for determination of vancomycin susceptibility is by MIC as accepted by Clinical and Laboratory Standard Institute.10
Using MIC to assess S. aureus susceptibility to vancomycin showed reduced susceptibility in 39 (22%) isolates. This was consistent with a previous study; it reported that 21.2% of isolated S. aureus had intermediate resistance to vancomycin.20 Inappropriate long-term vancomycin usage leads to gene mutation which results in changes in the thickness of the cell wall of S. aureus, that may be associated with reduced sensitivity to vancomycin.21 Nevertheless; the present data showed that none of the isolates had van genes. This result was in agreement with a previous study that evaluated the rare existence of van genes among the clinical isolates of S. aureus.22
Moreover, the frequency of virulence genes in the present study was found to be 24% for fnBPA and 4% for PVL. These findings are different from a previous study done in Saudi Arabia on 50 clinical isolates, it reported lack of PVL gene and lower prevalence of fnBPA gene (8%).14 This may be owed to the fact that PVL is a common gene in S. aureus isolated from community-acquired infections, with a lower prevalence in S. aureus isolated from hospital-acquired infections.22 It was found that its presence is statistically significant especially with MRSA strains (p=0.036). This finding came in agreement with other studies that focused on the spread of PVL positive MRSA strains in hospital-acquired infections.23
Furthermore, the invasive process of infection associated with S. aureus needs the presence of fibronectin-binding proteins that act as S. aureus invasins and the deletion of the gene encoding fnBPA in invasive laboratory strains, leading to a decrease in the invasive ability of these strains.24
The current results showed that the mortality rate reached 16% of the enrolled cases. This was comparable to an earlier study among children suffering from sepsis, which found the rate to reach 13%.25 Generally, the mortality rate from sepsis may vary according to different factors such as the gender, the development of septic shock and multiple organ dysfunctions and the etiological pathogen of the disease (especially antibiotic-resistant bacteria) and the presence of comorbidities affect the prognosis and explain the differences from one place to another.26
Male gender was one of the risk factors for mortality in the present study showing statistical significance (p=0.032). There was previous assumption regarding the sexual dimorphism in the immune responses to the infection that may have an impact on the mortality as the androgens may have immunosuppressive effects.27 Whether this hypothesis is applicable in children or not, needs further studies.
The other risk factors for grave outcomes were the association with central venous catheter (p<0.001). This is a well-known risk factor for invasive S. aureus infection and bacteremia.28 Moreover, there was an evident association between mortality and reduced sensitivity to vancomycin (p=0.010). This agreed with a previous study that demonstrated this association of reduced sensitivity to vancomycin in S. aureus and severe complications of sepsis.29 Consistently, in the present study, the presence of fnBPA gene was found to be associated with mortality (p<0.001). Further studies are required to validate these findings.
Conclusions
The present study highlights that major S. aureus strains isolated from sepsis in pediatric patients were methicillin resistant with a considerable proportion of these isolates with reduced sensitivity to vancomycin. Although none of the isolates had van genes responsible for vancomycin resistance, this finding warrants a considerable attention for study as it was a risk factor for mortality in those patients. The virulence genes fibronectin-binding protein and Panton-Valentine Leucocidin were not uncommon in S. aureus. This finding can be placed under more investigation to be used as a useful strategy in developing new treatment or vaccine for S. aureus infections. But also, prospective studies and continuous surveillance are needed to support these finding.
Acknowledgements
The authors would like to thank all participants and other healthcare workers who helped all through the process of the research.
Footnotes
Authors’ contributions statement: MZ designed the research plan, organized the study and participated in the main role of editing and revising the manuscript. SG, ARE, RAL carried out all laboratory tests and coordinated the data analysis. AM collected and supervised all clinical issues of patients. The corresponding author is DA and had a major contribution in writing of the manuscript and had a role in follow up of all steps of the study. All authors read and approved the final version of the manuscript.
Conflicts of interest: All authors – none to declare.
Funding: None to declare.
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