Figures
Abstract
Background
Pathways coordinated by innate pattern recognition receptors like mannose-binding lectin (MBL) and nucleotide-binding oligomerization domain 2 (NOD2) are among the first immune responses to Staphylococcus aureus (S. aureus) bloodstream infections (BSI) in animal models, but human data are limited. Here, we investigated the role of MBL deficiency and NOD2 mutations in the predisposition to and severity of S. aureus BSI.
Patients and Methods
A matched case-control study was undertaken involving 70 patients with S. aureus BSI and 70 age- and sex-matched hospitalized controls. MBL levels, MBL2 and NOD2 polymorphisms were analyzed.
Results
After adjusting for potential confounders, MBL deficiency (<0.5 µg/ml) was found less frequently in cases than controls (26 vs. 41%, OR 0.4, 95% confidence interval (CI) 0.20-0.95, p=0.04) as were low producing MBL genotypes (11 vs. 23%, OR 0.2, 95% CI 0.08-0.75, p=0.01), whereas NOD2 polymorphisms were similarly distributed. Cases with NOD2 polymorphisms had less organ dysfunction as shown by a lower SOFA score (median 2.5 vs. 4.5, p=0.02), whereas only severe MBL deficiency (<0.1 µg/ml) was associated with life-threatening S. aureus BSI (OR 5.6, 95% CI 1.25-24.85, p=0.02).
Conclusions
Contrary to animal model data, our study suggests MBL deficiency may confer protection against acquiring S. aureus BSI. NOD2 mutations were less frequently associated with multi-organ dysfunction. Further human studies of the innate immune response in S. aureus BSI are needed to identify suitable host targets in sepsis treatment.
Citation: Osthoff M, Au Yong HM, Dean MM, Eisen DP (2013) Significance of Mannose-Binding Lectin Deficiency and Nucleotide-Binding Oligomerization Domain 2 Polymorphisms in Staphylococcus aureus Bloodstream Infections: A Case-Control Study. PLoS ONE 8(9): e76218. https://doi.org/10.1371/journal.pone.0076218
Editor: Sunil K Ahuja, South Texas Veterans Health Care System and University Health Science Center San Antonio, United States of America
Received: May 28, 2013; Accepted: August 21, 2013; Published: September 27, 2013
Copyright: © 2013 Osthoff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the University of Melbourne, Department of Medicine funds. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Staphylococcus aureus (S. aureus) is a major cause of nosocomial and community-acquired bloodstream infections (BSI) accounting for up to 20% of hospital isolates [1]. S. aureus BSI is associated with a high morbidity and mortality compared to other BSI pathogens [2] and when it is caused by methicillin resistant isolates the mortality is even greater [3]. These infections place a huge burden on health care systems due to a longer duration of hospital stay and higher total treatment cost compared to bacteremia caused by any other pathogen [4]. In addition, the incidence of S. aureus BSI has steadily increased over the past 30 years as a consequence of frequent use of intravascular devices and invasive procedures [5]. General host risk factors for the acquisition of S. aureus BSI include staphylococcal colonization, surgical site infection, injection drug use, presence of immunosuppressive conditions and liver disease [2]. Central to the pathogenicity and immune evasion of S. aureus is the coordinated activity of several virulence factors including surface-expressed adhesins, complement inhibitors, exotoxins and exoenzymes that facilitate direct tissue destruction while avoiding activation of the innate immune system, particularly the complement system [6]. However, human studies examining the impact of the innate immune system on the susceptibility to and the severity of S. aureus BSI are limited [7,8].
Pattern recognition receptors (PRR) are crucially involved in the initial and immediate immune response against S. aureus (reviewed in [9]). In particular, nucleotide-binding oligomerization domain 2 (NOD2) and mannose-binding lectin (MBL) have been implicated in the pathogenesis of S. aureus infections in several experimental models. NOD2 is an intracellular sensor for both gram-positive and -negative bacterial cell wall components leading to a pro-inflammatory NF-κB and IL-1β mediated cytokine response (reviewed in [10]), although the exact mechanism and regulation of response in bacterial infections still remain to be fully elucidated. Animal model data on S. aureus and NOD2 are conflicting [11–13]. Results from two studies involving critically-ill sepsis patients suggest an increased risk of bacteremia and mortality in individuals with at least one NOD2 variant [14,15].
MBL, a liver-derived circulating lectin contributes to the efficient removal of pathogens and apoptotic cells by activating the lectin pathway of complement and promoting opsonophagocytosis [16], and has been implicated as an important defense mechanism in various infectious diseases [17]. Functional MBL deficiency is common in humans and is caused by polymorphisms within the coding and promoter regions of the MBL2 gene on chromosome 10 [18]. In vitro, MBL is able to bind to S. aureus [19] and evidence from animal models suggests that MBL deficiency significantly increases the susceptibility to and severity of S. aureus bacteremia [20,21]. However, its contribution to S. aureus induced complement activation and phagocytosis of S. aureus in adults is probably less than the antibody-mediated classical pathway activation [22–24]. Several clinical studies have reported a correlation between MBL deficiency and increased susceptibility to bacterial sepsis in children and adults [25–27].
Given these data on the potential role of NOD2 and MBL in human innate immune defences against severe S. aureus infection we hypothesized that MBL deficiency and NOD2 mutations might be associated with increased susceptibility to and severity of S. aureus BSI.
Patients and Methods
Ethics statement
The study had been approved by the Melbourne Health Human Research and Ethics Committee and all participants gave written informed consent for the study.
Participants
We conducted a matched prospective case-control study at two major tertiary hospitals involving 70 patients with S. aureus BSI and 70 age- and sex-matched hospitalized controls. Investigators were notified of all blood cultures positive for S. aureus by the central microbiology laboratory during the study period (September 2009 to September 2011). Case patients were enrolled with their first S. aureus BSI if they were >18 years old and had at least 1 positive blood culture for S. aureus. Hospitalised control patients were selected on the absence of infection as the cause for admission and were matched for age (within 2 years) and sex. Controls had to be admitted within 2 months of the case patient. To increase the power for the analysis of severity after S. aureus BSI, 30 patients with S. aureus BSI with similar epidemiology from a previous study were included only in this component of the study [25]. MBL levels and MBL2 genotype have been previously reported for these patients, and demographic and clinical data similar to the patients recruited in this study was available. We were able to use stored genomic DNA samples from these patients to determine NOD2 polymorphisms.
Risk factors for staphylococcal BSI
Demographic, clinical and microbiological data were collected by investigators blinded to MBL and/or NOD2 results including comorbidities and presence of intravenous (IV) lines or urinary catheters before the episode of S. aureus BSI. Liver disease was defined as cirrhosis, chronic hepatitis B and C, hepatocellular carcinoma or any other significant acute or chronic liver disease. Renal disease included acute and chronic renal impairment of various reasons excluding hemodialysis. Patients were regarded as immunosuppressed if they were receiving chemotherapy, corticosteroids (>7.5mg prednisolone equivalent per day), methotrexate, cyclosporine, tacrolimus, azathioprine or biologics such as TNF-α inhibitors.
The Sequential Organ Failure Assessment (SOFA) score was calculated for case patients on the day when the first positive blood culture was taken. A SOFA score of >7 was regarded as very severe disease being the mean score of non-survivors in the validation study of this score [28].
Determination of MBL plasma levels
EDTA blood samples which had been taken one to three days prior to the diagnosis of S. aureus BSI were accessed for further testing. Quantification of MBL plasma levels was performed by an investigator blinded to any patient data using a mannan-binding enzyme-linked immunosorbent assay as previously described [25,29]. Briefly, mannan-coated microtitre plates were incubated with samples at 1:25 and 1:100 dilutions for 90 min at room temperature followed by detection of bound MBL with a biotinylated monoclonal anti-MBL antibody (HYB 131-01, BioPorto Diagnostics, Denmark). MBL deficiency was defined as serum level < 0.5 µg/ml and, severe as < 0.1 µg/ml, respectively.
MBL2 and NOD2 genotyping
MBL2 promoter and first exon and NOD2 polymorphisms were determined by allele specific polymerase chain reaction (PCR) using TaqMan fluorescent probes (TaqMan genotyping assays, Life Technologies, Australia). For assay details, see Table S1. DNA lysates were prepared from 2µl of stored buffy coat according to the manufacturer’s instruction (TaqMan Sample-to-SNP, Life Technologies, Australia), and stored genomic DNA was used for 30 patients included in a previous study [25]. For all TaqMan assays, DNA amplification was carried out in 5µL volume reactions containing 1µl of DNA lysate or 20ng of genomic DNA, 0.25µl TaqMan genotyping assay mix, 2.5µl TaqMan GTXpress Master Mix (Life Technologies, Australia) and 1.25µl DNase-free water. All reactions were performed in 384-well plates and in the ViiA 7 thermocycler (Life Technologies, Australia) according to the manufacturer’s instructions. For allelic discrimination end-point fluorescence was read at 25°C, and the ViiA 7 software was used to analyze the results (Life Technologies, Australia).
MBL2 genotypes were classified as low (XA/YO, YO/YO), intermediate (XA/XA, YA/YO) or high (YA/YA, XA/YA) producing genotypes according to published literature [26] with exon variant alleles collectively designated as O and the wild-type gene as A, and the promoter variant allele and the wild-type gene designated as X and Y, respectively.
Definition of aims
The main aim of this study used to determine the sample size, was to compare the frequency of MBL deficiency in patients with S. aureus BSI with age/sex-matched, hospitalized control patients. We recruited 70 cases and controls in order to have an 80% chance of detecting an odds ratio of 3, with an expected frequency of MBL deficiency (defined as plasma concentration <0.5 µg/ml) in the control population of 24% [29] at the 5% level of significance. Additional aims included measuring the effect of NOD2 mutations on the risk of acquiring S. aureus BSI in cases compared to controls, and in cases alone, the influence of MBL levels and MBL2 and NOD2 mutations on the severity of S. aureus BSI as evaluated by the SOFA score and crude in-hospital mortality.
Statistical analysis
To investigate potential risk factors for acquiring S. aureus BSI, matched univariate analysis was performed by running conditional logistic regression on one variable at a time with S. aureus BSI as the dependent variable. In addition, Wilcoxon signed-rank test was applied to compare MBL levels in cases and matched controls. Multivariate conditional logistic regression models were used to estimate the effect of MBL deficiency on the risk of acquiring S. aureus BSI while adjusting for covariables with univariate p values less than 0.1 and which have been described in previous studies.
Regarding the severity of S. aureus BSI differences in outcome measures of cases according to patient characteristics, MBL levels and MBL2 or NOD2 mutations were first analyzed using the Fisher’s exact, the χ2 or the Mann-Whitney-U-Test where appropriate. Subsequently, stepwise binary logistic regression models were calculated to estimate the association of MBL levels and MBL2 or NOD2 mutations with predefined endpoints in multivariate analyses after adjustment for covariables with univariate p values less than 0.1. The Hardy-Weinberg equilibrium for MBL2 and NOD2 genotype frequencies was assessed by χ2 statistics. All testing was two-tailed. All analyses were performed using SPSS statistics, version 17.0 (SPSS Inc., USA).
Results
Demographic and clinical characteristics of cases and controls
The analyzed study population consisted of 70 S. aureus BSI cases and 70 age- and sex-matched, hospitalized controls. S. aureus BSI were nosocomially acquired (60%) and related to endovascular sources (49%) in the majority of cases. Controls were mainly admitted for trauma or elective surgery (Table 1). A median of 2 blood culture bottles were positive for S. aureus, and cultured isolates were methicillin resistant in 12/70 (17%) of cases (2/12 community-acquired). Antibiotic therapy appropriate for the susceptibility of the infective organism was received within 24 hours after blood cultures had been drawn in 59/70 (84%) cases.
| Cases (n=70) | |
|---|---|
| n (%) | |
| Infective source | |
| Intravascular line | 16 (23) |
| Skin or soft tissue | 11 (16) |
| Bone or joint | 13 (19) |
| Endocarditis | 7 (10) |
| Pneumonia | 8 (11) |
| Hemodialysis associated | 11 (16) |
| No source identified | 4 (6) |
| Microbiology | |
| PSSA | 15 (21) |
| MRSA | 12 (17) |
| Controls (n=70) | |
| n (%) | |
| Admission diagnosis, n (%) | |
| Trauma | 18 (26) |
| Medical condition | 29 (41) |
| Surgery (not trauma related) | 23 (33) |
Table 1. Clinical characteristics of S. aureus BSI cases and controls.
In terms of risk factors, S. aureus BSI cases were more likely to suffer from liver disease, to require hemodialysis and to have long-term IV lines when compared to controls (Table 2). In contrast, controls were more likely to have undergone recent surgery, with peripheral IV lines and urinary catheters used more frequently at the time of recruitment. There was no difference in terms of prevalence of diabetes mellitus, heart disease, cancer or immunosuppression.
| Variables | Controls | Cases | Univariate matched analysis | |
|---|---|---|---|---|
| (n=70) | (n=70) | OD (95% CI) P valuea | ||
| Age, mean (SD) | 61 (17) | 61 (18) | 1.09 (0.9-1.32) | 0.4 |
| Male sex, n (%) | 49 (70) | 51 (73) | 65 (0.01-5x106) | 0.45 |
| Diabetes, n (%) | 15 (21) | 20 (29) | 1.63 (0.67-3.92) | 0.28 |
| Heart disease, n (%) | 36 (51) | 30 (43) | 0.46 (0.16-1.31) | 0.14 |
| Cancer, n (%) | 10 (14) | 13 (19) | 1.36 (0.55-3.42) | 0.5 |
| Immunosuppressive treatment n (%) | 8 (11) | 15 (21) | 2.2 (0.82-5.70) | 0.12 |
| Liver disease, n (%) | 0 (0) | 15 (21) | 65 (1.03-4148.5) | <0.05 |
| Kidney disease, n (%) | 6 (9) | 10 (14) | 2.0 (0.60-6.64) | 0.26 |
| Hemodialysis, n (%) | 3 (4) | 16 (23) | 5.33 (1.55-18.3) | <0.01 |
| Illicit IV drug use, n (%) | 2 (3) | 6 (9) | 65 (0.02-2x105) | 0.31 |
| Recent surgery, n (%) | 41 (59) | 7 (10) | 0.08 (0.03-0.26) | <0.001 |
| Urinary catheter, n (%) | 30 (43) | 9 (13) | 0.22 (0.09-0.54) | 0.001 |
| Vascular lines, n (%) | ||||
| Long-term IV line | 14 (20) | 28 (40) | 2.1 (1.1-4.0) | 0.03 |
| Peripheral IV line | 57 (81) | 26 (37) | 0.11 (0.04-0.32) | <0.001 |
Table 2. Analysis of clinical characteristics as predisposing risk factors for S. aureus BSI.
Association of MBL and NOD2 variants with the risk of acquiring S. aureus BSI
MBL2 and NOD2 allele frequencies at all 7 positions were in agreement with the predicted Hardy-Weinberg equilibrium (data not shown). There was significant correlation between MBL2 genotypes and MBL levels (Kruskal-Wallis test, p<0.001, data not shown). As expected, patients with low producing genotypes all had MBL levels <0.5 µg/ml. The frequency distribution of MBL2 genotypes differed significantly among cases and controls with higher number of intermediate and low genotypes in controls (Table 3). In line with MBL2 genotypes, median MBL levels were significantly lower in controls compared to cases (0.9 (IQR 0.2-2.4) vs. 2.7 (IQR 0.5-4.6) µg/ml, p<0.001, Figure 1), and 18/70 of cases vs. 29/70 of controls had MBL levels <0.5 µg/ml. NOD2 mutations were exclusively heterozygous and were found in 10/70 and seven-seventieths of cases and controls, respectively (p=0.4). Only three individuals had both low producing MBL2 genotypes and a NOD2 mutation (2 cases, 1 control).
| Variables | Controls | Cases | Univariate matched analysis | |
|---|---|---|---|---|
| (n=70) | (n=70) | OD (95% CI) P valuea | ||
| MBL2 exon variants, n (%) | ||||
| A/A | 30 (43) | 44 (63) | Reference | |
| A/B | 22 | 16 | ||
| A/C | 5 | 1 | ||
| A/D | 8 | 5 | ||
| Total A/O | 35 (50) | 22 (31) | 0.44 (0.22-0.90) | 0.024 |
| B/B | 2 | 1 | ||
| B/C | 1 | 1 | ||
| B/D | 1 | 1 | ||
| C/D | 1 | 0 | ||
| Total O/O | 5 (7) | 4 (6) | 0.42 (0.10-1.79) | 0.24 |
| MBL2 promoter variants, n (%) | ||||
| Y/Y | 42 (60) | 52 (74) | Reference | |
| Y/X | 25 (36) | 17 (24) | 0.47 (0.20-1.12) | 0.09 |
| X/X | 3 (4) | 1 (1) | 0.27 (0.03-2.70) | 0.27 |
| MBL2 genotypes, n (%) | ||||
| YA/YA | 13 | 30 | ||
| XA/YA | 14 | 13 | ||
| Total high producing | 27 (39) | 43 (61) | Reference | |
| XA/XA | 3 | 1 | ||
| YA/YO | 24 | 18 | ||
| Total intermediate producing | 27 (39) | 19 (27) | 0.43 (0.20-0.94) | 0.034 |
| XA/YO | 11 | 4 | ||
| YO/YO | 5 | 4 | ||
| Total low producing | 16 (23) | 8 (11) | 0.31 (0.11-0.84) | 0.021 |
| MBL levels (µg/ml), median (IQR) | 0.9 (0.2-2.4) | 2.7 (0.5-4.6) | 1.32 (1.10-1.58)b | 0.002 |
| MBL <0.5µg/ml, n (%) | 29 (41) | 18 (26) | 0.50 (0.24-1.03) | 0.06 |
| NOD2 mutations, n (%) | 7 (10) | 10 (14) | 1.5 (0.53-4.21) | 0.44 |
| R702W C>T | 4 | 4 | ||
| G908R G>C | 2 | 2 | ||
| L1007fsinsC -/C | 1 | 4 | ||
Table 3. Analysis of MBL2 and NOD2 genotypes in S. aureus BSI cases and controls.
Differences in plasma mannose-binding lectin levels in S. aureus BSI cases and controls. Short horizontal lines (whiskers) represent minimum and maximum levels whereas horizontal lines inside the box-plot represent medians. Abbreviations: BSI, bloodstream infection; MBL, mannose-binding lectin. S. aureus, Staphylococcus aureus.
Contrary to our a priori hypothesis, MBL deficiency defined by low producing genotypes or lower MBL plasma levels (continuous variable) along with recent surgery, indwelling urinary catheters, and peripheral IV lines were associated with protection from S. aureus BSI. Previously described factors were associated with increased risk of S. aureus BSI (Tables 2 and 3).
Three risk factors that were highly correlated with protection from S. aureus BSI were not included in the multivariate model (recent surgery, indwelling urinary catheter and peripheral IV line) as their associations were in contrast with current literature and likely related to selection bias in the control group (trauma or elective surgical patients were more likely to be recruited as controls with urinary and peripheral IV catheters more frequently present in those patients at recruitment). After adjusting for potential confounders MBL deficiency as defined by MBL levels <0.5 µg/ml (OR 0.44, 95% confidence interval (CI) 0.20-0.95, p=0.04) or low producing genotypes (OR 0.24, 95% CI 0.08-0.75, p=0.01) remained independently associated with a decreased risk of acquiring a S. aureus BSI. Otherwise, only hemodialysis was an independent predictor of S. aureus BSI in a multivariate analysis (OR 6.01, 95% CI 1.70-21.54, p<0.01).
Association of MBL and NOD2 variants with severity of S. aureus BSI
One hundred cases were analyzed for associations between MBL2 or NOD2 mutations and severity of S. aureus. Clinical characteristics are displayed in Table 4. Regarding severity of S. aureus BSI the median SOFA score was 4 (IQR 2-6) and in-hospital mortality was 10% overall.
| Variables | S. aureus BSI | SOFA score | |||
|---|---|---|---|---|---|
| (n=100) | <2 (n=32) | 3-7 (n=52) | >7 (n=16) | P value | |
| Age, mean (SD) | 60.7 (18.7) | 63.7 (18.5) | 59.5 (19.0) | 58.5 (18.8) | 0.54 |
| Male sex, n (%) | 72 | 26 (81) | 38 (73) | 8 (50) | 0.07 |
| MBL2 genotypes, n (%) | |||||
| high (YA/YA, XA/YA) | 60 | 18 (56) | 33 (64) | 9 (56) | 0.84 |
| intermediate (YA/YO, XA/XA) | 29 | 9 (28) | 15 (29) | 5 (31) | |
| low (YO/YO, XA/YO) | 11 | 5 (16) | 4 (8) | 2 (13) | |
| MBL levels (µg/ml), median(IQR) | 2.4 (0.5-4.1) | 3.0 (0.7-6.0) | 2.4 (0.5-3.6) | 1.0 (0.1-4.0) | 0.38 |
| MBL <0.5µg/ml, n (%) | 25 | 7 (22) | 13 (25) | 5 (31) | 0.78 |
| MBL <0.1µg/ml, n (%) | 10 | 4 (13) | 2 (4) | 4 (25) | 0.04 |
| NOD2 mutation, n (%) | 16 | 8 (25) | 7 (14) | 1 (6) | 0.19 |
| Outcomes | |||||
| SOFA score, median (IQR) | 4 (2-6) | ||||
| ICU admission, n (%) | 22 | 1 (3) | 11 (21) | 10 (63) | <0.001 |
| In-hospital mortality, n (%) | 10 | 2 (6) | 5 (10) | 3 (19) | 0.4 |
Table 4. Clinical characteristics and outcomes in S. aureus BSI cases (n=100).
Organ dysfunction was less pronounced in patients with NOD2 mutations indicated by significantly lower SOFA scores (median 2.5 (IQR 0-3.75) vs. 4.5 (IQR 2-6), p=0.02) and the fact that the majority (8/16 (50%)) demonstrated a SOFA score of less than 3 and only 1/16 (6%) patient with a SOFA score >7 compared to 24/84 (29%) and 15/84 (18%) patients with NOD2 wild-type genotype, respectively (p=0.19). MBL deficiency (<0.5 µg/ml) had no influence on the severity overall as evaluated by the SOFA score (data not shown). However, severe MBL deficiency (<0.1 µg/ml) significantly increased the odds of a patient having severe organ dysfunction as defined by a SOFA score >7 (OR 5.57, 95% CI 1.25-24.85, p=0.02) after adjusting for age and gender. A similar non-significant trend was observed regarding severe MBL deficiency or NOD2 mutations and frequency of admission to ICU (4/10 (40%) vs. 18/90 (20%) and 2/16 (13%) vs. 20-84 (24%), respectively), whereas in-hospital mortality was not different.
Discussion
MBL and NOD2, two PRR of the innate immune system, have been implicated in the pathogenesis of S. aureus BSI in several experimental models [11–13,20,21]. This is the first human study designed to examine the effect of these two important first-line defense mechanisms on predisposition and severity of infection in S. aureus BSI patients, exclusively.
Despite previous experimental studies that were the basis for our a priori hypotheses, we did not demonstrate that MBL deficiency or NOD2 mutations predispose to S. aureus BSI. Interestingly, we found that rather the opposite is true in terms of MBL deficiency. MBL deficiency was associated with less than half the risk of acquiring S. aureus BSI. Previous studies which failed to demonstrate an effect of MBL deficiency [25,30] were likely underpowered as they had only examined a limited number of S. aureus BSI patients as part of larger sepsis trials, and controls were not matched. Recent data may help to resolve this apparent contradiction. It has been demonstrated that wildtype MBL2 genotypes are associated with persistent S. aureus nasal carriage in adults, a well known predictor for subsequent invasive disease [31]. Additionally, studies suggest that binding of MBL to S. aureus might be restricted to infancy due to inhibitory anti-wall teichoic acid antibodies in adults [23] and subsequently that anti-staphylococcal complement activation and opsonophagocytosis is dominated by the C1q-dependent classical pathway independent of MBL [22]. Finally, data that suggest a non-redundant role of MBL in staphylococcal infections in infancy come from two recent clinical studies that show that infants with MBL2 mutations are more susceptible to S. aureus colonization [32] and fatal invasive methicillin-resistant S. aureus co-infections after influenza [33].
It is unlikely that an acute phase elevation of MBL accounts for the higher levels in cases as the degree of elevation is usually mild and restricted to wild-type patients [34], and more importantly, genotypic data of our case patients were consistent with MBL levels demonstrating high producing haplotypes in a significantly greater proportion compared to controls (61 vs. 39%).
We also found no difference in the prevalence of NOD2 mutations in S. aureus BSI cases and controls with the frequency and presence of only heterozygous mutations being in line with previous reports from an Australian control population [35]. This finding is consistent with the clinical observation (and preliminary evidence [36]) that (untreated) Crohn’s disease patients, who have a higher prevalence of NOD2 mutations than healthy controls, are not at an increased risk of S. aureus BSI.
Overall then it seems that physical factors (hemodialysis or intravenous catheters) or comorbidities (liver failure) account more for susceptibility to S. aureus BSI than genetic defects in the innate immune proteins we studies, at least in an adult population.
Although MBL deficiency or NOD2 mutations had no significant impact on mortality, we could demonstrate important associations with our other a priori measure, the severity of S. aureus BSI as evaluated by the SOFA score. Interestingly, patients with NOD2 mutations had significantly lower SOFA scores and admissions to ICU with 50% showing a SOFA score <3 as compared to only 29% of patients lacking tested NOD2 polymorphisms. Less pulmonary inflammation and faster recovery has been shown in NOD2 knockout mice during S. aureus pneumonia [13]. Similarly, a diminished initial inflammatory response was demonstrated in NOD2 knockout mice after subcutaneous challenge with S. aureus [12] although the mice developed significantly larger ulcerations later on possibly related to an impaired bacterial clearance. In contrast, NOD2 knockout mice were more susceptible to S. aureus infection in a peritoneal challenge model [11]. Currently available data on human sepsis associations with NOD2 mutations indicate more prevalent bacteremia and higher sepsis-related mortality in ICU studies [14,15]. However, both human studies included only a minority of patients with S. aureus BSI, hence the ability to compare with our study is limited. In theory, heterozygous NOD2 mutations might impair the recognition of S. aureus to a limited degree but also attenuate the initial excessive and dysfunctional inflammatory response [37,38]. In summary, this might effectively result in less host damage overall in S. aureus BSI assuming removal of the pathogen by timely administration of effective antibiotic treatment.
Only severe MBL deficiency (<0.1 µg/ml) was associated with critical disease as evaluated by the SOFA score and admission to ICU, which is in line with knockout animal models [20,21] and previous sepsis studies including a variety of infections [25,26,39]. However, the significance of this observation is limited by the small sample size of patients with severe MBL deficiency.
Our study has some limitations including the fact that microbial virulence factors shown to influence the severity of community-acquired invasive S. aureus infections recently [40] were not examined. In addition, data on S. aureus colonization rate, a recognized risk factor for invasive infections were not available in cases and controls. Severity according to the SOFA score was only evaluated once on the day the first positive blood culture was drawn before antibiotic therapy was initiated. However, this approach eliminates possible confounders introduced later by differences in treatment (e.g. antibiotic management, timing of surgical intervention or infectious diseases consultation). We limited our analysis of the innate immune system to two key PRR, which have been shown to be significantly involved in S. aureus infection, previously. Ideally, future studies should include other important PRR like TLR-2 [41] which are also involved in the pathogenesis of S. aureus infections. Although our analysis of the importance of MBL and NOD2 in S. aureus BSI is the largest to date, its significance is limited in terms of mortality due to low event numbers.
In conclusion, this study does not support an important role for either MBL or NOD2 in protecting adults from acquiring S. aureus BSI. In fact, contrary to previous animal model data our results show that MBL deficiency seems to confer significant protection from S. aureus BSI. In addition, heterozygous NOD2 polymorphisms were less frequently associated with organ dysfunction in S. aureus BSI consistent with the notion that outcomes of infections are more driven by the host response to microorganisms than by their direct toxic effects [37,38]. Our present state of knowledge indicates that possible effects of innate immune system abnormalities are likely overwhelmed by conventional risks factors for staphylococcal BSI.
Supporting Information
Table S1.
Taqman genotyping assay details (Life Technologies, Australia).
https://doi.org/10.1371/journal.pone.0076218.s001
(DOC)
Author Contributions
Conceived and designed the experiments: MO HMAY MMD DPE. Performed the experiments: MO HMAY MMD. Analyzed the data: MO, MMD DPE. Contributed reagents/materials/analysis tools: HMAY MMD DPE. Wrote the manuscript: MO HMAY MMD DPE. Critically revised the manuscript: MO HMAY MMD DPE. Concur with the submission: MO HMAY MMD DPE.
References
- 1. Styers D, Sheehan DJ, Hogan P, Sahm DF (2006) Laboratory-based surveillance of current antimicrobial resistance patterns and trends among Staphylococcus aureus: 2005 status in the United States. Ann Clin Microbiol Antimicrob 5: 2. doi:https://doi.org/10.1186/1476-0711-5-2. PubMed: 16469106.
- 2. Naber CK (2009) Staphylococcus aureus bacteremia: epidemiology, pathophysiology, and management strategies. Clin Infect Dis 48 Suppl 4: S231-S237. doi:https://doi.org/10.1086/598189. PubMed: 19374578.
- 3. Blot SI, Vandewoude KH, Hoste EA, Colardyn FA (2002) Outcome and attributable mortality in critically Ill patients with bacteremia involving methicillin-susceptible and methicillin-resistant Staphylococcus aureus. Arch Intern Med 162: 2229-2235. doi:https://doi.org/10.1001/archinte.162.19.2229. PubMed: 12390067.
- 4. Noskin GA, Rubin RJ, Schentag JJ, Kluytmans J, Hedblom EC et al. (2005) The burden of Staphylococcus aureus infections on hospitals in the United States: an analysis of the 2000 and 2001 Nationwide Inpatient Sample Database. Arch Intern Med 165: 1756-1761. doi:https://doi.org/10.1001/archinte.165.15.1756. PubMed: 16087824.
- 5. Fowler VG Jr., Miro JM, Hoen B, Cabell CH, Abrutyn E et al. (2005) Staphylococcus aureus endocarditis: a consequence of medical progress. JAMA 293: 3012-3021. doi:https://doi.org/10.1001/jama.293.24.3012. PubMed: 15972563.
- 6. Jongerius I, Köhl J, Pandey MK, Ruyken M, van Kessel KP et al. (2007) Staphylococcal complement evasion by various convertase-blocking molecules. J Exp Med 204: 2461-2471. doi:https://doi.org/10.1084/jem.20070818. PubMed: 17893203.
- 7. Rose WE, Eickhoff JC, Shukla SK, Pantrangi M, Rooijakkers S et al. (2012) Elevated serum interleukin-10 at time of hospital admission is predictive of mortality in patients with Staphylococcus aureus bacteremia. J Infect Dis 206: 1604-1611. doi:https://doi.org/10.1093/infdis/jis552. PubMed: 22966128.
- 8. Fode P, Larsen AR, Feenstra B, Jespersgaard C, Skov RL et al. (2012) Genetic variability in beta-defensins is not associated with susceptibility to Staphylococcus aureus bacteremia. PLOS ONE 7: e32315. doi:https://doi.org/10.1371/journal.pone.0032315. PubMed: 22384213.
- 9. Fournier B, Philpott DJ (2005) Recognition of Staphylococcus aureus by the innate immune system. Clin Microbiol Rev 18: 521-540. doi:https://doi.org/10.1128/CMR.18.3.521-540.2005. PubMed: 16020688.
- 10. Moreira LO, Zamboni DS (2012) NOD1 and NOD2 Signaling in Infection and Inflammation. Front Immunol 3: 328. PubMed: 23162548.
- 11. Deshmukh HS, Hamburger JB, Ahn SH, McCafferty DG, Yang SR et al. (2009) Critical role of NOD2 in regulating the immune response to Staphylococcus aureus. Infect Immun 77: 1376-1382. doi:https://doi.org/10.1128/IAI.00940-08. PubMed: 19139201.
- 12. Hruz P, Zinkernagel AS, Jenikova G, Botwin GJ, Hugot JP et al. (2009) NOD2 contributes to cutaneous defense against Staphylococcus aureus through alpha-toxin-dependent innate immune activation. Proc Natl Acad Sci U S A 106: 12873-12878. doi:https://doi.org/10.1073/pnas.0904958106. PubMed: 19541630.
- 13. Kapetanovic R, Jouvion G, Fitting C, Parlato M, Blanchet C et al. (2010) Contribution of NOD2 to lung inflammation during Staphylococcus aureus-induced pneumonia. Microbes Infect 12: 759-767. doi:https://doi.org/10.1016/j.micinf.2010.05.003. PubMed: 20493961.
- 14. Brenmoehl J, Herfarth H, Glück T, Audebert F, Barlage S et al. (2007) Genetic variants in the NOD2/CARD15 gene are associated with early mortality in sepsis patients. Intensive Care Med 33: 1541-1548. doi:https://doi.org/10.1007/s00134-007-0722-z. PubMed: 17558494.
- 15. Henckaerts L, Nielsen KR, Steffensen R, Van Steen K, Mathieu C et al. (2009) Polymorphisms in innate immunity genes predispose to bacteremia and death in the medical intensive care unit. Crit Care Med 37: 192-201, e191-193 doi:https://doi.org/10.1097/CCM.0b013e31819263d8. PubMed: 19050632.
- 16. Ogden CA, deCathelineau A, Hoffmann PR, Bratton D, Ghebrehiwet B et al. (2001) C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med 194: 781-795. doi:https://doi.org/10.1084/jem.194.6.781. PubMed: 11560994.
- 17. Eisen DP, Minchinton RM (2003) Impact of mannose-binding lectin on susceptibility to infectious diseases. Clin Infect Dis 37: 1496-1505. doi:https://doi.org/10.1086/379324. PubMed: 14614673.
- 18. Garred P, Larsen F, Seyfarth J, Fujita R, Madsen HO (2006) Mannose-binding lectin and its genetic variants. Genes Immun 7: 85-94. doi:https://doi.org/10.1038/sj.gene.6364283. PubMed: 16395391.
- 19. Neth O, Jack DL, Dodds AW, Holzel H, Klein NJ et al. (2000) Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition. Infect Immun 68: 688-693. doi:https://doi.org/10.1128/IAI.68.2.688-693.2000. PubMed: 10639434.
- 20. Shi L, Takahashi K, Dundee J, Shahroor-Karni S, Thiel S et al. (2004) Mannose-binding lectin-deficient mice are susceptible to infection with Staphylococcus aureus. J Exp Med 199: 1379-1390. doi:https://doi.org/10.1084/jem.20032207. PubMed: 15148336.
- 21. Ip WK, Takahashi K, Moore KJ, Stuart LM, Ezekowitz RA (2008) Mannose-binding lectin enhances Toll-like receptors 2 and 6 signaling from the phagosome. J Exp Med 205: 169-181. doi:https://doi.org/10.1084/jem.20071164. PubMed: 18180310.
- 22. Brouwer N, Dolman KM, van Houdt M, Sta M, Roos D et al. (2008) Mannose-binding lectin (MBL) facilitates opsonophagocytosis of yeasts but not of bacteria despite MBL binding. J Immunol 180: 4124-4132. PubMed: 18322223.
- 23. Park KH, Kurokawa K, Zheng L, Jung DJ, Tateishi K et al. (2010) Human serum mannose-binding lectin senses wall teichoic acid Glycopolymer of Staphylococcus aureus, which is restricted in infancy. J Biol Chem 285: 27167-27175. doi:https://doi.org/10.1074/jbc.M110.141309. PubMed: 20592033.
- 24. Neth O, Jack DL, Johnson M, Klein NJ, Turner MW (2002) Enhancement of complement activation and opsonophagocytosis by complexes of mannose-binding lectin with mannose-binding lectin-associated serine protease after binding to Staphylococcus aureus. J Immunol 169: 4430-4436. PubMed: 12370377.
- 25. Eisen DP, Dean MM, Thomas P, Marshall P, Gerns N et al. (2006) Low mannose-binding lectin function is associated with sepsis in adult patients. FEMS Immunol Med Microbiol 48: 274-282. doi:https://doi.org/10.1111/j.1574-695X.2006.00144.x. PubMed: 17064281.
- 26. Eisen DP, Dean MM, Boermeester MA, Fidler KJ, Gordon AC et al. (2008) Low serum mannose-binding lectin level increases the risk of death due to pneumococcal infection. Clin Infect Dis 47: 510-516. doi:https://doi.org/10.1086/590006. PubMed: 18611155.
- 27. Fidler KJ, Wilson P, Davies JC, Turner MW, Peters MJ et al. (2004) Increased incidence and severity of the systemic inflammatory response syndrome in patients deficient in mannose-binding lectin. Intensive Care Med 30: 1438-1445. PubMed: 15127191.
- 28. Vincent JL, de Mendonça A, Cantraine F, Moreno R, Takala J et al. (1998) Use of the SOFA score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. Working group on "sepsis-related problems" of the European Society of Intensive Care Medicine. Crit Care Med 26: 1793-1800. doi:https://doi.org/10.1097/00003246-199811000-00016. PubMed: 9824069.
- 29. Minchinton RM, Dean MM, Clark TR, Heatley S, Mullighan CG (2002) Analysis of the relationship between mannose-binding lectin (MBL) genotype, MBL levels and function in an Australian blood donor population. Scand J Immunol 56: 630-641. doi:https://doi.org/10.1046/j.1365-3083.2002.01167.x. PubMed: 12472676.
- 30. Smithson A, Perelló R, Horcajada JP, Nicolas JM, Lozano F (2008) Is mannose-binding lectin deficiency associated with infection due to Gram-positive bacteria? Clin Infect Dis 47: 1492-1493; author reply: 10.1086/593106. PubMed: 18986266.
- 31. van Belkum A, Emonts M, Wertheim H, de Jongh C, Nouwen J et al. (2007) The role of human innate immune factors in nasal colonization by Staphylococcus aureus. Microbes Infect 9: 1471-1477. doi:https://doi.org/10.1016/j.micinf.2007.08.003. PubMed: 17913546.
- 32. Vuononvirta J, Toivonen L, Gröndahl-Yli-Hannuksela K, Barkoff AM, Lindholm L et al. (2011) Nasopharyngeal bacterial colonization and gene polymorphisms of mannose-binding lectin and toll-like receptors 2 and 4 in infants. PLOS ONE 6: e26198. doi:https://doi.org/10.1371/journal.pone.0026198. PubMed: 22022564.
- 33. Ferdinands JM, Denison AM, Dowling NF, Jost HA, Gwinn ML et al. (2011) A pilot study of host genetic variants associated with influenza-associated deaths among children and young adults. Emerg Infect Dis 17: 2294-2302. doi:https://doi.org/10.3201/eid1712.111002. PubMed: 22172537.
- 34. Herpers BL, Endeman H, de Jong BA, de Jongh BM, Grutters JC et al. (2009) Acute-phase responsiveness of mannose-binding lectin in community-acquired pneumonia is highly dependent upon MBL2 genotypes. Clin Exp Immunol 156: 488-494. doi:https://doi.org/10.1111/j.1365-2249.2009.03929.x. PubMed: 19438602.
- 35. Cavanaugh JA, Adams KE, Quak EJ, Bryce ME, O’Callaghan NJ et al. (2003) CARD15/NOD2 risk alleles in the development of Crohn’s disease in the Australian population. Ann Hum Genet 67: 35-41. doi:https://doi.org/10.1046/j.1469-1809.2003.00006.x. PubMed: 12556233.
- 36.
Elliott T, Hudspith B, Karaiskos C, Rayment N, Sanderson JD (2011) Prolonged survival of E coli but not Staphylococcus aureus in monocytes from patients with Crohn’s disease [abstract OC-123]. In: Abstracts of the British Society of Gastroenterology Annual General Meeting. March 14-17, 2011. Gut 60 Suppl 1: A61-62.
- 37. Rittirsch D, Flierl MA, Ward PA (2008) Harmful molecular mechanisms in sepsis. Nat Rev Immunol 8: 776-787. doi:https://doi.org/10.1038/nri2402. PubMed: 18802444.
- 38. Casadevall A, Pirofski LA (2003) The damage-response framework of microbial pathogenesis. Nat Rev Microbiol 1: 17-24. doi:https://doi.org/10.1038/nrmicro732. PubMed: 15040176.
- 39. Sutherland AM, Walley KR, Russell JA (2005) Polymorphisms in CD14, mannose-binding lectin, and Toll-like receptor-2 are associated with increased prevalence of infection in critically ill adults. Crit Care Med 33: 638-644. doi:https://doi.org/10.1097/01.CCM.0000156242.44356.C5. PubMed: 15753758.
- 40. Wehrhahn MC, Robinson JO, Pascoe EM, Coombs GW, Pearson JC et al. (2012) Illness severity in community-onset invasive Staphylococcus aureus infection and the presence of virulence genes. J Infect Dis 205: 1840-1848. doi:https://doi.org/10.1093/infdis/jis279. PubMed: 22492857.
- 41. Gillrie MR, Zbytnuik L, McAvoy E, Kapadia R, Lee K et al. (2010) Divergent roles of Toll-like receptor 2 in response to lipoteichoic acid and Staphylococcus aureus in vivo. Eur J Immunol 40: 1639-1650. doi:https://doi.org/10.1002/eji.200939929. PubMed: 20306471.