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Molecular characterization of virulence and resistance features in Staphylococcus aureus clinical strains isolated from cutaneous lesions in patients with drug adverse reactions
Irina Gheorghe#1, Iuliana Lupu2, 3, Othman Thamer1,4, Andreea Ion1, Ani Ioana Cotar5, Gratiela G Pircalabioru1, Marcela Popa1, Mariana Carmen Chifiriuc1#*, Veronica Lazar1
1Research Institute of the University of Bucharest- ICUB, Faculty of Biology, University of Bucharest, Bucharest, Romania, University of Bucharest.
2Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania.
3Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania; Department of Dermatology, “Elias” University Hospital, Bucharest, Romania.
4College of Health and Medical Technology, Baghdad, Iraq
5Cantacuzino National Institute of Research, Bucharest, Romania.
# these authors equally contributed to this work
*Correspondence: carmen_balotescu@yahoo.com.; Tel.: +40766728315
“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
**Department of Dermatology, “Elias” University Hospital, Bucharest, Romania
“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
**Department of Dermatology, “Elias” University Hospital, Bucharest, Romania
Academic Editor: name
Received: date; Accepted: date; Published: date
Abstract: Patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions that could be even more complicated by infectious processes, particularly those driven by specific pathogens, such as the community emergent methicillin resistant Staphylococcus aureus strains. This study was conducted on a total of 42 S. aureus clinical strains isolated in 2016 from acneiform reactions pustule and periungual lesions in patients with cutaneous drug adverse reactions. Multiplex PCR was performed on genomic DNA from isolates in order to identify the SCCmec central elements and the virulence genes: bbp (bone bound sialoprotein), ebpS (elastin-binding protein), fnbB, fnbA (fibronectin-binding proteins), fib, clfA,clfB (clumping factors A and B), cna (collagen-binding protein), luk-PV (Panton-Valentine leucocidin), hlg (gamma-haemolysin), tst (toxic shock syndrometoxin). The obtained results confirm the increased virulence potential and the high prevalence of SSCmec type VIII, followed by type IV and II in cutaneous isolates from patients with dermatologic toxic effects. More data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactam antibiotics.
Keywords: methicillin resistant Staphylococcus aureus; skin and soft tissue infection; antibiotic resistance; SCCmec
1. Introduction
Normal skin is colonized by large numbers of bacteria that live as commensals in its surface or in hair follicles. Sometimes, the overgrowth of these bacteria causes skin diseases, and in other occasions, bacteria that are normally found as part of the skin microbiota can cause diseases [1]. Methicillin-resistant S. aureus is a common problem in health care facilities, sports facilities, clinics, and the community. The MRSA strains associated with hospitals are referred to as hospital-acquired MRSA (HA-MRSA) and are the most common cause of hospital-acquired infections [2,3,4]. Methicillin-resistant S. aureus is the leading cause of skin and soft tissue infection (SSTI) in patients reporting to emergency departments for treatment [5] with a rising rate in primary care clinics and intensive care units [3].Recent epidemiological trends have shown an increase in the rate of skin and soft tissue infections caused both by healthcare-associated and CA-MRSA (MRSA acquired in the community)[6]. Many of the latter strains produce exotoxins and are epidemiologically distinct from healthcare-acquired strains. Factors that may affect the microbial cause include underlying disease such as diabetes or immune dysfunction; hospital attendance, injecting drug use, travel, animal contact and environmental contamination. These infections can be minor and self-limiting, such as furunculosis, to moderately severe, such as abscesses [7] to the life-threatening Staphylococcal scalded skin syndrome [8].
Recurrent infections, the use of injectable drugs, young age and being a member of the armed forces or an athlete are recognized risk factors for infection with CA-MRSA in the USA [9], but similar risk factors are published in Europe as well [10-12]. CA-MRSA infection occurs in younger patients and has a significant association with SSTI [10], while hospital attendance, surgery, dialysis, diabetes, indwelling devices and residence in a long-term care facility were risk factors associated with hospital-acquired (HA)-MRSA [6]. However, no clinical profile could reliably exclude MRSA [13]. In the Netherlands, people in contact with pigs have a higher risk of MRSA carriage than the general population. With the exception of a few small outbreaks [14], CA-MRSA remains focal and contained within Europe in 2010.The European physicians of infectious diseases consider that empirical therapy for community SSTI would have to be changed [15].
Although a number of criteria have been proposed to predict the likelihood of infection with CA-MRSA [6], the epidemiological and clinical criteria are rarely sufficient to distinguish accurately between MRSA and methicillin-susceptible S. aureus (MSSA) infection at initial presentation [16].The boundaries between HA-MRSA and CA-MRSA are becoming blurred due to the movement of patients and infections between hospitals and the community [17]. Nosocomial outbreaks of CA-MRSA following the admission of colonized or infected patients have occurred. In the USA, it is becoming increasingly difficult to distinguish between CA-MRSA and HA-MRSA on clinical and epidemiological grounds [15]. Since HA-MRSA and CA-MRSA strains exhibit often different genetic and phenotypic traits, the microbiological characteristics of the isolates may help to distinguish between the two types of infection [6].The evolution of strains causing SSTI and serious infection is rarely static and strains of apparently susceptible (at least phenotypically susceptible) but mecA gene-positive S. aureus strains have emerged to challenge diagnostic laboratories and clinicians [18].
The present study aimed to identify the types of SCCmec and virulence genes profiles in clinical S. aureus strains isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects in one hospital from Bucharest, Romania. There are several data regarding the presence of MRSA isolates in Romania, but only few hospitals have undertaken large scale studies correlating molecular, clinical and epidemiological data.
2. Results and discussion
The analysis of the distribution of virulence factors has revealed that from the analyzed strains, 90% of them were positive for the production of lecithinase (Table 1); 88% of the isolates were positives for lipase; 83% of the isolates expressed caseinase; a high percentage (76%) of the analyzed strains produced also gelatinase, which is a marker for the production of proteases with large-spectrum proteolytic activity.73% of the MRSA expressed esculinase, iron being an essential component for growing and virulence of the microorganisms. In the extracellular medium iron is not accessible for bacteria, therefore siderophores or other mechanisms to uptake iron from the transporter proteins, like transferrin are necessary to acquire it. It was proved that iron may be fixed by esculetol, so esculinase has an important role in ensuring Fe uptake necessary for the activation of bacterial genes and expression of some virulence factors [19]. Half of the isolates revealed DN- ase(Table 1), 40% presented γ -type haemolysiswhichcauselysisofa variety of cell types [20], 28% of MRSA (Table 1) revealed α-haemolysis which cause the lysis of erythrocytes and platelets damage, having a powerful action on vascular smooth muscle [20]; only 19% of the investigated strains presented β – haemolysis, an indicator for the presence of pore forming toxins that cause pores in the cell membrane, allowing the dissemination of infection. Numerous studies have shown that proteases produced by pathogenic organisms may contribute to the severity of the clinical symptoms of an infection.
Table 1.Phenotypic characterization for the presence of enzymatic virulence factors among the analyzed strains
Strain
Code/no Aesculin
hydrolisis DN-ase Lipase Caseinase Lecithinase Gelatinase Amilase Hemolysin
1 + – +++ + ++ – – γ
2 – – +++ + ++ – – γ
3 – – +++ – +++ – – β ++
4 – – + + + – – α
5 – – +++ + + – – β+
6 + – – + + – – β+
7 + + ++ + + – – α
8 – – +++ + + – – γ
9 + – +++ + ++ – – γ
10 – – +++ + ++ – – β+
11 + – ++ + ++ – – γ
12 + – ++ + + – – α
13 – – +++ + ++ – – α
14 + – ++ + ++ – – α
15 ++ + +++ + ++ ++ – γ
16 – + +++ ++ +++ + – γ
17 + – +++ ++ +++ + – γ
18 + + +++ ++ +++ + – γ
19 – + – ++ – – – γ
20 + + – ++ – – – γ
21 + – +++ ++ +++ + – β++
22 + – ++ + ++ ++ – β++
23 – + ++ ++ + – – –
24 + + ++ ++ + + – –
25 + + ++ ++ – – – α
26 – – +++ – + – – β+
27 + – +++ + ++ – – –
28 + ++ – – + + – α
29 + – + +++ + – – –
30 + – – – – – – –
31 + + ++ +++ ++ – – γ
32 + + + ++ + – – α
33 ++ + ++ + + – – γ
34 + + ++ + ++ ++ – γ
35 + + +++ + ++ + – γ
36 ++ – +++ +++ + – – α
37 – – +++ – ++ – – γ
38 + – +++ – +++ – – β+
39 ++ – + ++ ++ – – γ
40 + + +++ – +++ – – α
41 ++ + +++ ++ +++ – – α
42 + + +++ + +++ – – α
2.1. PCR assays for detection of S. aureus virulence genes
S. aureus produces two types of adhesins; one set has a characteristic LPXTG motif that anchors the adhesin to the components of the extracellular matrix (ECM); the representatives of this kind of adhesins are grouped in the family named microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) [21]. This family of adhesins includes: protein A, the fibronectin-binding proteins (Fnbps), collagen-binding protein (Cna), elastin-binding protein, clumping factors A and B (ClfA and B). The constituents of the second set of adhesins are non-covalently anchored to the cell surface, including the fibrinogen-binding protein (Fgbp) and coagulase. Fibrinogen is the most abundant host protein in endothelial lesions. Among fibrinogen-binding proteins expressed by S. aureus on bacterial cells, the clumping factors A and B (ClfA, ClfB) are responsible for typical S. aureus clumping in plasma, promoting the adherence to fibrinogen-coated surfaces [22].
The molecular analysis using PCR amplification of selected virulence genes showed that 36% of the strains presented the clfA gene, 36% of strains expressed clfB; 14% of the isolates presented fib expression whereas 7% of the strains harbored the hlg gene and 6% presented the bbp gene. No strain expressed ebpS, fnbB, fnbA, luk-PV and tst.A higher percent of virulence genes than those obtained in this study was showed by Machuca et al., in a study performed on 53 MRSA strains isolated from the hospital environments in Colombia, which showed that 89% of the strains carried the clfA gene, and 87% the clfB gene [23].
Figure 1. Distribution of expressed virulence genes in MRSA strains isolated from acneiform reactions pustule and periungual lesions.
The results of multiplex PCR for detection of bbp and ebpS genes showed that investigated MRSA strains have no ebpS gene, while six of them harbored the bbp gene. The presence of the bone bound sialoprotein encoding gene in some S. aureus analyzed strains can be correlated with resistance to methicillin and the diseases associated can be complicated and sometimes leading to sepsis staphylococci. The frequency of bbp gene in S. aureus strains has not been well described [24]. Previous studies have shown that a high percentage of nosocomial isolates from Institute for Cardiovascular Diseases “Prof. Dr. C.C. Iliescu” Bucharest, Romania are expressing both the clfA and clfB genes and SCCmecVJ1gene [24]. ClfA binds to platelet άIIbβ3 integrin [21]. On the other hand, ClfB binds to human cytokeratin 10 and to fibrinogen, as a bi-functional protein and acts as a key virulence factor, which leads to metastatic infection and/or development of sepsis [21]. Both ClfA and ClfB interact with and inhibit complement C3. Our results are in contrast with results obtained in other studies performed on 48 S. aureus strains isolated from atopic dermatitis cases and healthy individuals, which have indicated that the fib gene was expressed in 66.6% of the cases and ebpS gene in 70% of the cases [25].
The results of multiplex PCR for detection of genes encoding these toxins showed that hlg gene was present in 36% of the analyzed strains, whereas the luk-PV gene was absent (Figure 1). hlg gene was first described in 1938, including three ORFs designated hlgA, hlgC and hlgB genes. S. aureus is an excellent source of γ – haemolysin but does not produce α-, β- or δ- hemolysin [26].Gamma-hemolysin is produced by 97% of S. aureus strains and it induces the lysis of erythrocytes and other mammalian cells, as well as of some types of sub-cellular structures. This hemolysin was also shown to exhibit dermo-necrotic activity [27]. The luk-PV gene was frequently detected in S. aureus strains associated with necrotic lesions involving the skin, and in severe necrotic hemorrhagic pneumonia, whereas it is seldom observed in strains responsible for other infections, such as infective endocarditis and hospital-acquired staphylococcal infections.
2.2. PCR assays for detection of S. aureus methicilin resistance genes
The mecA gene, which encodes for the modified penicillin-binding protein 2a (PBP2a) [28], is primarily responsible for methicillin resistance in S. aureus. The mecA gene is carried on a mobile genetic element, named the staphylococcal cassette chromosome mec(SCCmec), which has been integrated into the S. aureus genome [29]. Currently at least 8 types of SCCmec elements (I-VIII) have been identified [30]. These SCCmec elements share similar characteristics, and contain a ccr (cassette chromosome recombinase), which is responsible forsite-specific insertion and excision of SSCmec into the S. aureus genome at the 3’ end of the open reading frame (orf X) [31].
Both Ha-MRSA and CA-MRSA are now becoming a major concern in dermatology outpatient clinics. Moreover, the increasing incidence of MRSA strains associated with community infections makes it even more important to analyze the horizontal transmission mechanisms of intra- and inter-species mobile genetic elements responsible for their genetic determinism as well as the resistance genes responsible for this resistance pattern. HA-MRSA possess the Type II-III SCC mec cassette genes which confer β-lactam and nonβ-lactam resistance (e.g. fluoroquinolones, clindamycin and gentamicin). In contrast CA-MRSA possess Type IV or V (the majority of CA-MRSA strains carrying a type 2 ccr-class B mec complex in a SCCmec IV cassette [6], are susceptible to almost all groups of antibiotics, express luk-PV gene which is uncommon in HA-MRSA.
Our molecular analysis through PCR arrays showed that 41% of the isolates belonged to SCC mecType VIII whereas 23% of the strains expressed the SCCmec type IV with subtypes IVa and IVd followed by SCCmec type II (10%). This distribution is similar to other studies [30].
Figure 2. Distribution of meticilin resistance genes among S. aureus isolates.
A similar percentage for the presence of SCC cassette type IV in clinical isolates of S. aureus was reported for strains isolated between 2014 and 2015 from patients hospitalized with various cardiovascular diseases in the Microbiology Laboratory at the Emergency Institute for Cardiovascular Diseases “Prof. Dr. C.C. Iliescu” Bucharest, Romania[33].
In contrast to our results, Ionescu et al. (2010) revealed the more frequent presence of leukocidin genes in the SSTI group of S. aureus isolates from patients in Iasi hospitals than in other groups [34] and concluded that the isolates belonged to CC80-MRSA-IV and USA300 possibly indicating an invasion of these normally community-associated strains into hospital settings. Another study performed in Tîrgu-Mureş Clinical Emergency Hospital has shown the presence of multidrug resistant HA-MRSA with SCCmec type III, luk-PV negative and enterotoxin A producing strains, a staphylococcal exotoxin with emetic potential and superantigenic activity, present for several years in their hospital suggesting the strain’s adaptation to the hospital environment and successful transmission strategies [35].
3. Materials and Methods
3.1. Bacterial strains
A total number of methicillin resistant S. aureus strains (n=42) was selected from a collection of S. aureus strains recently isolated from patients with drug cutaneous adverse reactions admitted during 2016 in the Department of Dermatology from Elias hospital in Bucharest (Romania). The selected strains were isolated from acneiform reactions pustule and periungual lesions. The strains identification was performed in the Microbiology Laboratory of the above mentioned hospital with automated VITEK 2 system. The antibiotic susceptibility testing of the respective strains was performed by diffusion method (Kirby -Bauer), following the recommendations of CLSI editions 2015 and 2016.The selected strains were tested for the virulence factors expression using specific substrata according to the method described by Lazar et al. [36]: haemolysins, CAMP factor, lecithinase, lipase, caseinase, gelatinase, esculin hydrolysis, amylase, DN-ase. In order to determine the presence of hemolysins, the strains were spotted on blood agar plates with 5% sheep blood. After 24h of incubation at 37°C the halo around the strains represented a positive test. Agar medium supplemented with 2.5% yolk and gelatin respectively, was used to determine the expression of lecithinase and gelatinase. After 3 days of incubation at 37°C the presence of the three afore mentioned virulence factors was determined by the presence of a clear area around the strains spot. For the lipase and caseinase activity, media containing 2.5% Tween 80 and 15% soluble casein respectively were used. The presence of the investigated virulence factors was indicated by the occurrence of a clear precipitation area around the strains spot. The DNA agar medium was used for the determination of DN-ase presence. After maximum 72h of incubation at 37°C, a HCl solution (1N) was added on the surface of the medium. A clear halo around the strain was considered a positive reaction. The presence of amylase was determined on a medium with starch substrate. After incubation up to 3 days at 37°C the presence of a precipitation around the strains was regarded as a positive result. For aesculin hydrolysis the medium with Fe 3+ citrate was used and inoculated by spotting and incubated for 24h at 37°C. A black precipitate around culture due to esculetol released under the action of beta-galactosidase was considered as positive reaction.
3.2. Molecular analysis
Genomic bacterial DNA was extracted using the alkaline extration method. One to five colonies of bacterial cultures were suspended in 1.5 ml tubes containing 20 µL solution of 0,05M NaOH (sodium hydroxide) and 0.25% SDS (sodium dodecyl sulphate). For the permabilization of the cell membrane the tubes were heated on a thermoblock at 95oC for 5 minutes. 180 µL TE buffer (Tris + EDTA) 1X was added and the tubes were centrifuged at 13000 rpm for 3 minutes. The DNA in the supernatant was kept and stored at -4oC before analysis. All PCR reactions were performed using a Thermal Cycler machine Corbet. The amplification products of each PCR reaction (multiplex /simplex) were visualized by electrophoresis on a 1% agarose gel, stained with ethidium bromide (10 g / ml) and identified based on their size using specific molecular weight markers (100pb, Mid Range DNA Ladder).
3.2.1. Detection of S. aureus virulence genes by PCR.
The detection of the specific virulence genes was performed by three simplex PCR and three multiplex PCR assays according with previous published protocols [37]. The obtained information was used to compare the prevalence of specific resistance and virulence genes amongst strains isolated from the hospital settings.
3.2.2. Detection of S. aureus resistance genes by PCR.
The genotypic characterization of the SCCmec cassette types present in the analysed strains was performed using PCR methods (simplex and multiplex) in order to elucidate the structure of these genetic elements and obtain the relevant epidemiological data. Two reactions were performed using the multiplex PCR with five and four pairs of specific primers respectively for the various sequences of the SCCmec cassette. Their classification and parameters used to conduct of reactions followed the protocol developed by Miheirico et al. [38] and Zhang et al. [39]. However, a simplex PCR was performed for detection of the ccrC gene, SCCmec cassette recombinase complex.
4. Conclusions
The obtained results showed that the cutaneous lesions of patients treated with epidermal growth factor inhibitors are infected with S. aureus strains exhibiting a large spectrum of virulence factors and increased methicillin resistance. The obtained results confirm the high prevalence of SSCmec type VIII, followed by type IV and II in these strains. More data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of infections and avoid the inappropriate administration of beta-lactam antibiotics.
Acknowledgments: The financial support of the research project PN-III-P2-2.1-BG-2016-0369 are gratefully acknowledged.
Author Contributions: IG and MCC conceived and designed the experiments; IG, OT, IL, IA and AIC performed the experiments; IL provided the S. aureus strains; VL, IG and MP analyzed the data; GGP, IG and MCC wrote the paper.
Abbreviations
MRSA Methicillin-resistant S. aureus
HA-MRSA Hospital acquired MRSA
CA-MRSA Community acquired MRSA
SSTI skin and soft tissue infection