LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY
Faculty of Medicine
Moa Josefin Berglund
PREVALENCE OF VIRULENCE AND METHICILLIN RESISTANCE MARKER mecA IN STAPHYLOCOCCUS AUREUS STRAINS ISOLATED FROM PATIENTS IN LITHUANIA
AND EPIDEMIOLOGICAL DATA COMPARISON WITH SWEDEN
MASTER THESIS of intergrated studies of Medicine
KAUNAS 2018
Supervisor: PhD Rita Plančiūnienė, Department of Microbiology, Lithuanian University of Health Science
TABLE OF CONTENTS
SUMMARY ... 3
SANTRAUKA ... 4
ACKNOWLEDGEMENT ... 5
CONFLICT OF INTEREST ... 5
CONFIRMATION OF INDEPENDENCE OF WORK ... 6
ABBREVIATIONS ... 7
TERMS ... 7
INTRODUCTION ... 8
AIM AND OBJECTIVES ... 9
1. LITERATURE REVIEW ... 10
1.1 Staphylococcus aureus and the rise of antimicrobial resistance ... 10
1.1.1 S. aureus structure and ecology ... 10
1.1.2 The rise of S. aureus antibiotic resistance ... 10
1.2 Virulence and resistance ... 11
1.2.1 S. aureus caused infections and the effect in the clinical sector ... 11
1.2.2 Importance of prevention and screening of MRSA ... 12
1.2.3 Mechanism of virulence and resistance in Staphylococcus aureus ... 13
1.2.4 Virulence and resistance of CA-MRSA and HA-MRSA ... 14
1.2.5 Diagnostic methods to confirm virulence and resistance ... 15
1.3 Epidemiology of MRSA in Lithuania compared to Sweden ... 18
1.3.1 MRSA in Lithuania ... 18
1.3.2 MRSA in Sweden ... 20
2. METHODOLOGY ... 23
2.1 Research workload, location and method ... 23
2.2 Bacterial culture preparation ... 23
2.3 Materials ... 23
2.4 Test Method ... 26
3. RESULTS ... 31
4. DISCUSSION ... 40
CONCLUSIONS ... 43
REFERENCES ... 44
SUMMARY
PREVALENCE OF VIRULENCE AND METHICILLIN RESISTANCE MARKER mecA IN STAPHYLOCOCCUS AUREUS STRAINS ISOLATED FROM PATIENTS IN LITHUANIA
AND EPIDEMIOLOGICAL DATA COMPARISON WITH SWEDEN
Moa Josefin Berglund Master Thesis
The Methicillin resistant Staphylococcus aureus (MRSA) is a major cause of illness and death. The magnitude of the problem of MRSA is an epidemic global treat and every third person is thought to carry MRSA [27]. The reason of resistance is through the expression of the mecA gene. MecA gene codes the penicillin-binding protein (PBP2a). The level of resistance is correlated to the amount of PBP2a production. The aim of this study was to obtain epidemiological data of MRSA in Lithuanian population and compare it to Sweden, a country with well-controlled prevalence of MRSA. As well too clinically investigate MRSA diagnostic methods, to analyse the results and to conclude if Lithuanian hospitals use sufficient diagnostic methods when confirming MRSA. This scientific work is based on 81 different strains of bacteria collected from central hospital laboratories in Lithuania. All these strains have previously been tested and confirmed as MRSA positive.
1. Table Diagnostic methods, manufactures and result obtained from LUHS clinical study.
Diagnostic Method Manufacturer Result
1. Mannitol salt agar Liofilchem 85% (+), 15 % (-)
2. Latex Agglutination test “spot A test” OXOID DR0100M 97% (+), 3 % (-)
3. Oxacillin resistant screening agar (ORSA) OXOID CM1008 91% (+), 1.5% (-), 7.5% (+/-) 4. Penicillin Binding Protein (PBP2) test OXOID DR0900 82%(+), 12 % weak (+), 6% (-) 5. Minimal inhibitory concentration (MIC) EUST B6034A 100 % (+) to Cefoxitin (FOX)
resistance 6. PCR of mecA gene and PVL coding gene Thermo Fisher
Scientific
mecA: 96% (+), 4 % weak (+).
PVL prevalence 13%.
7. Congo red agar OXOID, UK 100% (+) produced biofilms
Conclusion obtained from the tests of diagnostic methods confirmed a diagnostic error from central hospital laboratories of 3,7 %. The diagnostic methods are not sufficient in Lithuania. Better results are established with several diagnostic methods than solely one. The Lithuanian population resistance against methicillin is through the mecA mechanism as proved previously in other populations. PVL coding genes gave us an apparent prevalence of 13% and 100% of the strains produced biofilm.
SANTRAUKA
VIRULENTIŠKUMO ŽYMENŲ IR ATSPARUMO METICILINUI GENO mecA
PAPLITIMAS TARP STAPHYLOCOCCUS AUREUS PADERMIŲ, IŠSKIRTŲ IŠ LIETUVOS PACIENTŲ, IR EPIDEMIOLOGINIS DUOMENŲ PALYGINIMAS SU ŠVEDIJOS
DUOMENIMIS Moa Josefin Berglund Baigiamasis magistro darbas
Meticilinui atsparus Staphylococcus aureus (MASA) infekcijos yra viena iš pagrindinių ligų ir mirties priežasčių. Manoma, kad MASA problema yra epidemiologinė kontrolė ir tai, kad tarp S. aureus nešiotojų kas trečias turi MASA. Atsparumo priežastis yra mecA geno ekspresija. mecA genas koduoja pakitusį peniciliną jungiantį baltymą (PBP2a), o atsparumo lygis koreliuoja su sintezuojamu PBP2a kiekiu. Šio darbo tikslas buvo palyginti MASA paplitimą Lietuvos populiacijoje ir Švedijoje – šalyje, kuri pasižymi gera MASA kontrole. Taip pat išanalizuoti ir įvertinti MASA diagnostinius metodus ir jų patikimumą tvirtinant MASA infekcijas Lietuvos ligoninėse. Šio mokslinio darbo pagrindas yra 81 stafilokokų padermė, išskirtos pagrindinėse Lietuvos ligoninėse ir laboratorijų identifikuotos kaip MASA teigiamos.
1 lentelė
Diagnostiniai metodai Gamintojas Rezultatai
1. Manito druskos agaras Liofilchem 85% (+), 15 % (-)
2. Lateks agliutinacijos testas OXOID DR0100M 97% (+), 3 % (-) 3. Atsparumo oksacilinui atrankos agaras
(ORSA)
OXOID CM1008 91% (+), 1.5% (-), 7.5% (+/-) 4. Peniciliną jungiančio baltymo (PBP2) testas OXOID DR0900 82%(+), 12 % silpnai (+), 6%
(-) 5. Minimalios slopinančios koncentracijos
(MSK) nustatymas
EUST B6034A 100 % (+) atsparumas cefoksitinui (FOX) 6. mecA geno ir PVL koduojančio geno
nustatymas polimerazės grandinine reakcija (PGR)
Thermo Fisher Scientific
mecA: 96% (+), 4 % silpnai (+).
PVL paplitimas 13%.
7. Kongo raudonasis agaras OXOID, UK 100% (+) biofilmų produkcija
Palyginus įvairius MASA diagnostinius metodus, rezultatai parodė, kad ligoninių laboratorijų
diagnostinė paklaida sudaro 3,7 %. Lietuvos kai kurių ligoninių naudojami metodai yra nepakankami.
Geresni rezultatai gaunami naudojant kelis diagnostinius metodus. Lietuvoje cirkuliuojančių MASA atsparumą lemia mecA genas, kas įrodyta ir kitose šalyse. Panton-Valentine leukocidino (PVL) geno nustatymas parodė, kas jis paplitęs 13% visų tirtų MASA padermių tarpe. Visos tirtos padermės produkavo biofilmus.
ACKNOWLEDGEMENTS
I hereby would like to express my deepest appreciation and gratitude to my supervisor
Dr. Rita Plančiūnienė that helped me to collect all the strains and been working with me from the start, to the end in the laboratory with the scientific work and contributing with immense knowledge.
I also would like to thank Povilas Kavaliauskas (Institute of Infectious Diseases and Pathogenic Microbiology) for contributing with guidance and valuable aid during the master thesis research.
Finally, I would like to show my appreciation to Lithuanian University of Health science for contributing with all necessary equipment and tools that made this clinical research work possible.
CONFLICTS OF INTEREST
The author reports no conflicts of interest.
THE WORK WAS DONE IN THE DEPARTMENT OF MICROBIOLOGY CONFIRMATION OF THE INDEPENDENCE OF DONE WORK
I confirm that the presented Master Theses " PREVALENCE OF VIRULENCE AND METICILLIN RESISTANCE MARKER mecA IN STAPHYLOCOCCUS AUREUS STRAINS
ISOLATED FROM PATIENTS IN LITHUANIA AND EPIDEMIOLOGICAL DATA COMPARISON WITH SWEDEN ”
1. has been done by me;
2. has not been used in any other Lithuanian or foreign university;
3. I have not used any other sources not indicated in the work and I present the complete list of the used literature.
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CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE LITHUANIAN LANGUAGE IN THE DONE WORK
I confirm the correctness of the Lithuanian language in the done work.
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CONCLUSION OF THE SUPERVISOR REGARDING DEFENCE OF THE MASTER THESES
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THE MASTER THESES HAVE BEEN APPROVED IN THE INSTITUTE OF MICROBIOLOGY AND VIROLOGY
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Reviewers of the Master Theses 1)
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ABBREVIATIONS
MRSA- Methicillin resistant Staphylococcus aureus S. aureus- Staphylococcus aureus
HA-MRSA- Hospital acquired methicillin resistant Staphylococcus aureus CA-MRSA- Community acquired methicillin resistant staphylococcus aureus PMNs- polymorphonuclear leukocytes
MGE- mobile genetic element
SCCmec- staphylococcal cassette chromosome mec PBP2a- Penicillin binding protein
PSM- phenol-soluble modulins TSST- toxic shock syndrome toxin PVL- Panton-Valentine leukocidine Locus agr- locus accessory gene regulator CDC- Center of disease control
MIC- Minimal inhibitory concentration PCR- Polymerase chain reaction
LUHS- Lithuanian university of health science CLIS- Clinical and Laboratory standard institute MSSA- Methicillin sensitive Staphylococcus aureus PSP- Penicillinase stable penicillin
ORSA- Oxacillin resistant screening agar CRA- Congo red agar
TERMS
β- lactams: Antibiotics that contain β-lactam ring. They act by inhibiting the synthesis of the
peptidoglycan layer of the cell wall by targeting PBPs and thereby have bactericidal action. Groups of antibiotics that are included: Penicillin, Cephalosporin’s, Monobactams and Carbapenems [25, 26].
Methicillin: first semisynthetic Penicillin introduced after resistance was discovered against Penicillin [25].
INTRODUCTION
Staphylococcus aureus (S. aureus) is the most common human bacterial pathogen worldwide [3,2].
S. aureus colonize our skin and nasal passage, if any breaks occur in our protective barriers this can result in infections that range from uncomplicated skin and soft tissue infection till invasive infections such as pneumonia and sepsis [3, 1]. The mortality of people infected by S. aureus in the preantibiotic era override 80%. The first penicillin was introduced 1940 and improved the survival rate drastically.
Year 1942 resistant strains against β- lactams were acknowledged. Due to this, year 1959 semisynthetic penicillin know as methicillin were created to contain the spread of resistance, but already less than one year after, resistant strains against Methicillin were discovered [4]. Antimicrobial resistance (AMR) is a bacteria’s ability to resist the action of one or more antibiotic agents [8]. Today the term methicillin resistant S. aureus (MRSA) are used to define resistance against almost all β- lactam [3]. MRSA can be divided into, hospital acquired-MRSA (HA-MRSA) and community acquired-MRSA (CA-MRSA) [28].
S.aureus is an extraordinary pathogen that is capable to adapt and evolve its resistance and virulence and this is the reason that MRSA is a global treat of epidemic proportion [15]. The virulence of S.
aureus is multi-factorial and depends on, adhesions, toxins and its avoidance of host immune defence [15]. The European centre for disease prevention and control (ECDC) estimated that year 2009, resistant bacteria were accountable for 25000 deaths in Europe/year and the cost is minimum 1.5 billion euro. The reason resistances occur, is through the expression of the mecA gene. MecA gene codes the penicillin-binding protein (PBP2a). The level of resistance is correlated to the amount of PBP2a production.
The extent of MRSA in countries varies distinctly. Sweden has a prevalence of S. aureus septicemia at only 1%, compared to Southern Europe and Great Britain that have 25-50% [25].
When reading about the differences of epidemiology of MRSA in countries, the idea of the thesis were raised. To compare a well-controlled country, Sweden, with a less controlled country, Lithuania and to focus on clinically investigate the diagnostic methods used in the health sector for confirmation of MRSA. Therefore be able to analyze the results to conclude if the diagnostics methods in Lithuania are sufficient.
AIM AND OBJECTIVES
The aim of this study was to obtain epidemiological data of MRSA in Lithuanian population and compare it to a country with well-controlled prevalence of MRSA, Sweden. As well too clinically investigate MRSA diagnostic methods and to analyse the results, to conclude if Lithuanian hospitals use sufficient diagnostic methods when confirming MRSA. Further to confirm the correlation of mecA gene to MRSA from strains collected in the Lithuanian population and map prevalence of PVL coding genes as well as biofilm formation in MRSA confirmed strains.
Objectives:
1. To obtain and compare epidemiological data of MRSA in Lithuania and Sweden.
2. To analyze scientific reports about diagnostic methodology how Lithuania confirm methicillin resistant Staphylococcus aureus in hospitals.
3. To perform seven different diagnostic methods on strains collected from the Lithuanian population and compare and analyze the result from following methods:
- Mannitol salt agar media, to culture S. aureus - Latex agglutination test, to confirm S. aureus
- Oxacillin resistant screening agar, to evaluate MRSA
- Penicillin binding protein (PBP2) agglutination test, to evaluate MRSA
- Minimal inhibitor concentration antibiotic sensitivity test, to evaluate antimicrobial resistance
- PCR for detection of mecA, to confirm MRSA
- PCR for detection of PVL coding genes, to evaluate prevalence of PVL virulence
- PCR for detection of icaA gene, to evaluate if this is the mechanism of biofilm formation.
- Congo red agar, to evaluate production of biofilm, slime production.
1. LITERATURE REVIEW
1.1 Staphylococcus aureus and the rise of antimicrobial resistance
1.1.1. S. aureus structure and ecology
Staphylococcus aureus (S. aureus) is the most common human bacterial pathogen worldwide [3,2].
S.aureus is a Gram-positive cocci and grow in clusters and can be classified as coagulase positive (ability to clot blood) or coagulase-negative, salt tolerant and haemolytic [1]. The cell wall of S. aureus consists of thick layer of peptidoglycan that alternates N-acetylmuramic acid and N-glucosamine acid cross-linked by peptides. The process of cell wall cross-linking is catalysed by the Penicillin-binding proteins (PBPs). The function of PBPs is known to be the most important for S. aureus survival against β- lactams. Disturbances of the control mechanism usually lead to cell lysis [4, 5].
The adherence of the bacteria to its host is by surface proteins that attach to host proteins such as laminin and fibronectin that often is present on epithelial, endothelial and blood clots surface [1].
Another surface protein that is bounded to S.aureus is Protein A that binds immunoglobulin G, this will disrupt phagocytosis and enhances virulence [1].
S. aureus colonize our skin and nasal passage, if any breaks occur in our protective barriers this can result in infections that range from uncomplicated skin and soft tissue infection till invasive infections such as pneumonia and sepsis [3, 1].
S. aureus colonizes nasal membranes of around 50% of healthy population, while remaining 50 %, rarely or never is colonized. Nasal microbiota play an important role in host susceptibility [6].
Genetics do not significant contribute to the nasal microbiota composition, however it do impact the nasal bacterial density (total amount of nasal bacteria present). Nasal microbiota is environmentally acquired host phenotype, this mean that it is susceptible to environmental modification [7].
1.1.2. The rise of S. aureus antibiotic resistance
The mortality of people infected by S. aureus in the preantibiotic era override 80%. The first penicillin was introduced 1940 and improved the survival rate drastically. Year 1942 resistant strains against β- lactams were acknowledged. Due to this, year 1959 semisynthetic penicillin know as methicillin were created to contain the spread of resistance, but already less than one year after, resistant strains against methicillin were discovered [4]. Antimicrobial resistance (AMR) is a bacteria’s ability to resist the action of one or more antibiotic agents. The reason of AMR is due to mutation in bacterial genes and the spread of these genes between bacteria. [8] Today the term MRSA are used to define resistance against almost all β-lactam [3]. This result in reduction or complete loss in bacteriostatic or
bactericidal effect of the antibiotic treatment provided [21]. The reason this occur is through the expression of the mecA gene. MecA gene codes the penicillin-binding protein (PBP2a). The level of resistance is correlated to the amount of PBP2a production. Resistance of mecA positive genes can range from susceptible to complete resistance [21].
To diagnose if S. aureus is a Methicillin sensitive Staphylococcus aureus (MSSA) or MRSA, resistance to Oxacillin is determined. To clear out the terminology Cefoxitin, Oxacilin and Methicillin all belong to the same group and are used interchangeably. These are used for test of susceptibility of all β-lactam antimicrobials. Thereafter it can be classified as susceptible, intermediate or resistant according CLSI (clinical laboratory standard institute) breakpoint criteria [9].
MRSA can be suspected when an S.aureus strain, isolate with oxacillin minimum inhibitory concentration (MIC) of ≥4 mg/L or more but only confirmed by detecting a mecA gene [26].
The Methicillin resistant Staphylococcus aureus (MRSA) was at year 2011 predicted to colonize up to 53 million people worldwide [27] and the incidence of S. aureus is increasing in both healthy population and in those with underlying illness. This is divided into, hospital acquired-MRSA (HA- MRSA) and community acquired-MRSA (CA-MRSA) [28].
1.2 Virulence and resistance
1.2.1. S. aureus caused infections and the effect in the clinical sector
S. aureus is an extraordinary pathogen that is capable to adapt and evolve its resistance and virulence.
The capacity of the bacteria to evade the immunity of the host is high and it can cause a wide range of infections in both nosocomial and community acquired settings [15, 2]. Most common infection site is superficial soft tissue infections, skin lesions and localized abscesses, example: impetigo, mastitis, folliculitis and staphylococcal scalded skin syndrome [1,2]. More serious and deep infections such as osteomyelitis, pneumonia, toxic shock syndrome and endocarditis may also be the result of S. aureus [1,2].
The spread and extent of hospital acquired infections and surgical wound infections cost billion euros every year because of this capable pathogen [10]. The cost of Methicillin sensitive S.aureus (MSSA) compared to MRSA are estimated to be minimum 50% less in MSSA patients. The average length of hospitalisation of blood stream infected (BSI) CA-MRSA patients is 24 days while BSI HA-MRSA is 51 days [11]. Annually in EU, MRSA infections result in approximately 1 million extra days of hospitalization and additional cost for the hospitals around 380 million euro [12].
1.2.2. Importance of prevention and screening of MRSA
The most effective approach concerning both clinical effectiveness and cost-effectiveness is clinical risk assessment with target laboratory testing to detect MRSA. Screening of MRSA is estimated to save 935 lives and $231 million in medical cost only in USA annually. In Spain the cost of MRSA in one hospital was estimated to cost €101 000compared to the cost of a screening programme of €10 261. This can be concluded that a screening would be justified if only 4 MRSA infections were prevented annually in one hospital. The European centre for disease prevention and control (ECDC) estimated that year 2009, resistant bacteria were accountable for 25000 deaths in Europe/year and the cost is minimum 1.5 billion euros. Except from the economical reason that screening and prevention of MRSA infection would be beneficial also patient’s quality of life should be taken into consideration. The quality of a patient life that is infected by MRSA and has pneumonia is estimated to reduce life quality of 42%, a wound infection 36 % and an amputation that gets infected by 70% [12].
The number one prevention and precaution to stop the spread of CA-MRSA is accurate hand washing.
Information, education and practice to hand wash correctly should be actively encourage in families, schools, nursing-homes and so on, to control transmission of CA-MRSA in community. People at a higher risk to get CA-MRSA are: children younger than 2 years, athletes involved in contact sports, intravenous drug users, military persons, men who have sex with men, inmates, people with chronic skin disorders, places with overcrowding and veterinarians [17,20]
Table 2: Centres for Disease Control and Prevention (CDC), Public health institute of the United States [19].
Guidelines to prevent spread of MRSA in hospitals by CDC:
Careful hand hygiene and use of disposable gloves when in contact of mucous membranes, body fluids and non-intact skin
When risk of sprays of body fluid, use moth, nose and eye protection Wear a gown when there is risk of contamination of clothes
Reusable equipment should be cleaned and disinfected between every patient Frequently touched surfaces should be disinfected regularly
The laundry should be appropriate handled
Additionally guidelines how to handle a patient that carry MRSA by CDC:
Patient should if possible be placed in a single bedroom and isolated from contact with other patients
Always wear gloves when in contact with patient or in the surrounding area Always dress in a gown when entry the room and remove it before exit Avoid all unnecessary transport of patient to reduce the spread
Do not share equipment between patient until it is thoroughly disinfected Environment should be cleaned and disinfected daily
1.2.3. Mechanism of Virulence and resistance in Staphylococcus aureus
The organism causes infections that in majority of cases have multifactorial pathogenesis. This includes resistance to human immune system as polymorphonuclear leukocytes (PMNs) and other white blood cells [1]. Virulence of S. aureus is multi-factorial and depends on adhesions, toxins and its avoidance of host immune defence [15].
S. aureus is also notorious known for its capacity to produce several toxins [16]. Majority of S. aureus strains produces α-toxin, γ-toxin, leukotoxins and phenol-soluble modulins (PSMs). The alpha toxin that is secreted by majority of S. aureus strains is a pore-forming protein with cytotoxic activity. This protein is linked to pathogenesis of pneumonia caused by S. aureus [29]. The alpha toxin lyses cells as erythrocytes and macrophages, it contribute to the penetration of the epithelial barrier and have a significant role in morbidity and mortality.
The γ-toxin and the leukocidins are protein toxins that damage membranes by acting together. The γ- toxin has hemolytic and weakly leukotoxic activity [1].
PSM is peptides that increase the cytolytic capacities, especially to human neutrophils. This triggers the inflammatory response. PSM furthermore contributes to the biofilm formation [16]. S. aureus is very good at forming biofilms. Biofilm is a surface-attached bacterial collection that is embedded in extracellular matrix. This provides an important role in protection from antibiotics and host defense.
Also it allows the bacteria to remain attached to the surface. By doing so, biofilms contribute to colonization, prolonged infection and spread of MRSA [15]. There are five known intracellular adhesion cluster (ica) genes known to be responsible for the biofilm production. The biofilm formation is divided into three stages. First stage, attachment occurs when staphylococcal surface proteins establish interactions with host tissue. Second stage, proliferation and maturation of the biofilm, increasing the production of extracellular matrix that consist of biofilm polysaccharide intercellular adhesins (PIA), proteins, teichoic acids and eDNA. The result is channels form to provide nutrient delivery to all layer of the biofilm. Third stage, the biofilm detach and disperse to distal areas [14].
Other toxins, such as superantigens; toxic shock syndrome toxin (TSST), exfoliative toxins and Panton-Valentine leukocidine (PVL) varies a lot between different strains [1].
The Panton Valentine leukocidin (PVL) has a potent leukotoxicity and are non-hemolytic. PVL isolates are found in total 2% of all S.aureus infections but in 90% of isolates from dermonecrotic lesions do express this toxin and therefore it is mainly correlated as an important factor in necrotizing skin infections [1]. The PVL toxin act by releasing inflammatory mediators from human neutrophils that lead to degranulation, it lyses the neutrophils, monocytes and macrophages by its cytolytic pore forming activity, and it result in damaged the cell membrane. PVL also have substantial pro-
inflammatory effects, it is associated with higher mortality and osteomyelitic complications. The PVL is an extracellular product that is encoded by lukS-PV and lukF-PV genes [16,17].
S. aureus express two types of superantigens; enterotoxins and toxic shock syndrome toxin (TSST-1).
Ingested enterotoxins are the underlying cause of S.aureus food poisoning and are responsible for vomiting and diarrhea, if enterotoxins is systemically released it can cause toxic shock syndrome (TSS) [1].
Exfoliative toxins cause in neonates the scalded skin syndrome. The clinical manifestation is widespread of blistering and loss of epidermis. The toxin express protease and esterase activity and this is thought to split proteins involved to keep epidermis intact [1]
1.2.4. Virulence and resistance of CA-MRSA and HA-MRSA
There are mainly three characteristics that differ in between CA-MRSA and HA-MRSA strains when it comes to antibiotic resistance. First is the different in SCCmec elements [16]. MRSA genetic resistance is due to a mobile genetic element (MGE) called staphylococcal cassette chromosome mec (SCCmec). SCCmec is a DNA fragment. There are several types of SCCmec and there is a constant discovery of new types and elements. Today we are aware of eleven different types of SCCmec [15].
SCCmec type I, II and III is associated with HA-MRSA and type IV and V with CA-MRSA [1]. All SCCmec DNA fragments have in common that it include the mecA gene. MecA code the penicillin binding protein (PBP2a). This factor is absolutely necessary for methicillin resistance. PBP2a inhibits β-lactam antibiotics to bind to the bacteria [15]. This impairs the ability of the antibiotics to destroy the cell wall of the bacteria [1]. Second is that CA-MRSA usually are sensitive to most antibiotics except β-lactams, compared to HA-MRSA that more often is multi-resistant. Third, Minimal inhibitory concentration (MIC) of CA-MRSA is often lower than those of HA-MRSA [16].
Virulence characteristics are also different; by starter CA-MRSA have the capacity to infect young and healthy people with no previous connection to hospitals. This indicates a highly increased virulence compared to HA-MRSA. The reason is specific strains that enable CA-MRSA to spread free in population between healthy individuals.
In almost all CA-MRSA clones there are PVL genes. PVL is associated with aggressive skin infections, such as furunculosis and abscess formation [15]. PVL is found in CA-MRSA and not in HA-MRSA and thought to be the cause of the high virulence of CA-MRSA.
The success of spread of CA-MRSA depends on several factors, not only the virulence itself that is mainly controlled by locus accessory gene regulator (locus agr) [18], but also due to the capacity of S.
aureus to grow and persist in the human host. This attribute is known as “fitness” of S. aureus. Genes that promote growth and persistence during an infection ultimately increase CA-MRSA virulence [16].
SCCmec type IV is known to increase the fitness of CA-MRSA. The alpha toxin have a significant role in morbidity and mortality in CA-MRSA, USA300 strain. The alpha toxin is produced in higher amounts in CA-MRSA than HA-MRSA and as well PSM is particularly produced in high amounts in CA-MRSA compared to average lower amounts in HA-MRSA [16].
Table 3: Characteristics of CA-MRSA compared to HA-MRSA [16,17]
CA-MRSA HA-MRSA Age characteristics Younger patients Older patients
SCCmec type IV, V I, II, III
PVL virulence factor Common Unusual Predominant strains USA300 (CMRSA-10)
USA400 (CMRSA-7) USA100 (CMRSA-1)
USA200 (CMRSA-3,6) Multidrug resistance
(Other than β-lactam)
Rare Common
α-Toxin High amount Lower amount
PSM High amount Lower amount
Table 4: The five main Community acquired-MRSA strains that is responsible for majority of CA- MRSA worldwide [17].
Strains responsible for most of CA-MRSA worldwide:
Number: Strain: Additionally name of strain:
1 ST1 Midwest clone
2 ST30 Southwest Pacific/ Oceania clone
3 ST80 European clone
4 ST59 Pacific clone
5 ST8 USA 300
Strain USA300 is exceptionally infectious and can cause infections in healthy humans with no predisposing risk factors [1]. Almost all CA-MRSA infections is caused by USA300 clone [8]
The strain USA300, SCCmec IV was first reported year 2000 in USA as the underlying cause of outbreaks of skin and soft tissue infections in football players and prisoners. Year 2005 USA300 was the predominant strain of all CA-MRSA infections. The USA300 strain commonly carries the genes lukF-PV and lukS-PV that encode PVL. The isolate develop more resistance against antibiotics and have broadened the profile of resistance to a notably large extent including Erythromycin, Levofloxacin, Mupirocin and Tetracycline. The strain is widespread and exists in Europe, South America and Australia. Today the USA300 strain is cause of global epidemic SSTI and moreover it can cause very severe diseases as necrotizing pneumonia, endocarditis and osteomyelitis. The virulence of USA300 is high and contains several factors such as PVL, PSMs, α-toxin and high amount of secreted proteases [30].
1.2.5. Diagnostic methods to confirm virulence and resistance
Guidelines used in Europe to confirm and discover breakpoints of microbial resistance follow CLSI or EUCAST standardized diagnostic of antimicrobial resistance. The method is based on Kirby-Bauer approach with Müller Hinton agar and inoculum at 0.5 McFarland [8].
The reason resistance occur is through the expression of the mecA gene. As previously mentioned, mecA gene codes the penicillin-binding protein (PBP2a). The level of resistance is correlated to the amount of PBP2a production. The gold standard method to detect MRSA is polymerase chain reaction (PCR). PCR is done to detect the presence of mecA gene. Not all laboratories have access to provide PCR detection. Traditionally the detection of MRSA is done with Kirby-Bauer Oxacillin disc diffusion test 6 µg/ml,in Mannitol-salt agar, Oxacillin MIC or Oxacillin screening methods [22].
2008, Journal of medical microbiology concluded after a study, which compared the effectiveness of MRSA diagnostic methods, that at least two different methods should be used to avoid false negative and false positive result. Preferably one method with high sensitivity and one with high specificity should be used [22]. For identification of S. aureus there is no single test that guarantee reliable result, therefore several methods always are recommended [31].
Table 5: Description of few different diagnostic methods used to identify S. aureus and MRSA
Method: Description: Aim: Sensitivity/specificity:
Mannitol salt media test:
This is a medium with a high salt concentration that is mainly used to detect gram positive Staphylococcus. The S. aureus grow by the typical golden clusters and have a positive Mannitol-fermentation. This gives yellow halos around the colonies due to the reaction of the acidic that S. aureus is producing and the Phenol red that is used as a pH indicator on the plates.
Identify S. aureus
24 h incubation 94% sensitivity [31]
Latex
agglutination test ”spot A test”:
Protein A and polysacchariders detection method.
Protein A is found on surface of 95% of all S. aureus. Some MRSA may express protein A in undetectable low levels, but all these strains contain
polysaccharides. Both protein A and these specific
polysaccharides clump together and cause agglutination with
Identify S. aureus
Rapid and reliable, with a sensitivity of 98,4% and specificity of 96.9%.
Results can be
concluded in 20 seconds [32].
latex particles with fibrinogen, IgG and specific antibodies against S.aureus.
ORSA (oxacillin resistant screening agar):
Screening agar with the antibiotic Oxacillin 6 µg/mlin Manitol-Salt agar used to evaluate MRSA. MRSA can be suspected when an S. aureus strain, isolate with Oxacillin minimum inhibitory
concentration (MIC) of ≥ 4 mg/L. This medium
incorporates Aniline blue for detection of mannitol
fermentation that in presence of S. aureus result in intense blue colour.
Identify antimicrobial Resistance to Oxacillin and MRSA detection No growth After 24-48 hour indicate
Meticillin resistance.
24 h incubation:
specificity 92 % sensitivity 100 % 48h incubation:
specificity 66 % sensitivity 100%
[33, 34]
PCR: DNA-based assay.
Fast, accurate diagnostic tool for detection of mecA gene.
MSSA strains do not possess mecA gene.
Confirm MRSA by detection of mecA gene.
Can also detect virulent genes as lukF-PV and lukS-PV
Gold standard method, Distinguish low level resistant bacteria from true resistant strains that harbour mecA gene [35]
PBP2: PBP2 is a simple and rapid slide Latex agglutination test; it’s a rapid test that detects the PBP2 that is produced by the mecA gene and present in membranes of MRSA. An agglutination reaction between PBP2 and monoclonal antibodies on the slide leads to fast result.
Identify MRSA by detection of PBP2
Sensitivity: 100%
Specificity: 96%
[34]
MIC (Minimal inhibitory concentration) determination:
Defined as lowest concentration of antibiotic (µg/ml)that will inhibit growth of
microorganism after 12-24 hour incubation in liquid medium.
Confirm Resistance and guide Physician to Choose Correct Antimicrobial treatment
12-24 hours Used in diagnostic Laboratories and as a research tool.
Demand experience User [36].
1.3 Epidemiology of MRSA in Lithuania compared to Sweden
1.3.1. MRSA in Lithuania
2008 Institute of hygiene in Lithuania performed a study of MRSA control in Lithuanian general hospitals. The aim was to analyse and evaluate the detection methods in Lithuanian acute care hospitals. 40 different hospitals were invited to take part in the survey, 30 choose to participate and provided response to the study. These hospitals reported out of 7807 S. aureus strains that were isolated, 721 MRSA positive. Result is MRSA prevalence of 9.2 %.
The detection of MRSA showed a higher rate in large-scale hospitals with more than 600 hospital beds. The majority of hospitals (85%) restricted diagnostic method to the disc diffusion method to detect MRSA. The use of media with oxacillin was low, 25 % and 20% used minimal inhibitory concentration (MIC) determination method [23].
Fig 1: Summarizes the incidence of Preventive measurements taken by Lithuanian hospitals 2008, to prevent spread of MRSA according study provided by Institute of hygiene in Lithuania [23].
46.2 % of the hospitals always isolated the patients that were MRSA positive, 53.8% did not take these precaution measurements to minimize the spread. The most used prevention was hand antiseptic that was offered in the entrance in 73.1% of the hospitals. Gloves were located at entrance in 69.2% of the cases and gowns in 65.4%. The use of individual equipment was reported a statistic in 42.3% and separate staff was provided in 11.5% by the hospitals. The screening for prevention of MRSA was only performed in 23.3% of the hospitals and this was only when patients was in contact with MRSA.
Screening of the staff was done if personnel were in contact with MRSA in 46.4% of the hospitals.
Fig 2: Incidence of Preventive measurements taken by Lithuanian hospitals 2008, to prevent spread of MRSA according study provided by Institute of hygiene in Lithuania [23].
The level of antimicrobial resistance (AMR) in Lithuania is one of the highest in EU. Year 2014 Lithuania induced a two-year plan to improve rational use of antibiotics. The government of Lithuania aim to develop a national strategic action plan for AMR based on European commission recommendation. To handle the high levels of antimicrobial resistance in Lithuania, there is a necessity to cooperate with multiple sectors to achieve declined levels of resistance and to be more selective in use of antibiotics for both humans and livestock. From 2005-2015 have the health budget in Lithuania increased from 5.6% to 6.5 %. The expenditure Per capita is 1406 euro compared to EU average on 2797 euro. Total health expenditure in Lithuania is the sixth lowest in EU and the total health expenditure per capita is half of EU average. [38]
Fig 3: Health expenditure per capita in EU, by European Commission 2017 [38].
0 0.2 0.4 0.6 0.8 1 1.2
Negative Positive
1.3.2. MRSA in Sweden
All MRSA cases in south Sweden have been followed since year 2000. In south of Sweden there is 1,2 million habitants. This area has very low prevalence for MRSA, 25 out of 100.000 in 2010. During this time the epidemiology of MRSA been studied in detail. Between 2000-2010 all cases have been
included. The numbers of MRSA infections have increased from 31 in 2000 to 315 in 2010. Majority of cases was CA-MRSA. 32% was diagnosed upon a clinical infection, 35% that was positive been close household contacts and solely 24% acquired the infection in Sweden and was of Swedish origin.
58% of the MRSA positive people were asymptomatic carrier [25].
In Sweden MRSA is regarded as a public threat to health and controlled therefore by the Swedish communicable disease act. Since 2000 it is mandatory to report and register all detected MRSA cases.
Also tracing of the infection is done in all new cases. Everyone that been hospitalized or employed at a hospital or nursing home abroad within 6 months and seeking job or hospital care have to be screened for MRSA [25].
These guidelines and a very strict hygiene policy have resulted that Sweden have a prevalence of S.
aureus septicemia at only 1%, compared to Southern Europe and Great Britain that have 25-50%. The microbiological methods used to identify S. aureus were performed on blood agar and on selective plates. Colonies confirmed coagulase positive were tested for sensitivity or resistance by disk diffusion with Oxacillin. Test of PBP2a and/or polymerase chain reaction (PCR) to detect mecA genes and PVL genes are standard to verify MRSA [25].
Patients tested positive, are followed with monthly cultures until declared free of MRSA. All household contacts are screened minimum two times. Eradication treatment is not routinely provided to MRSA carriers in Sweden, but to all healthcare workers or to patients with a lot of healthcare contact [25].
The annual rapport of European commission 2017, confirm that 84% of all health expenditure is financed by tax payments. Sweden is top five in EU on health expenditures spent per capita, 2015 this number was 3932 Euro [39].
When it comes to the fight against antimicrobial resistance Sweden is in lead. Sweden is one of the countries that use least antibiotics in livestock industries and continuously work towards a rational use of antibiotics in human healthcare sector. Sweden emphasizes national clinical guidelines, education and information to control spread of AMR [39].
The picture below show the situation of MRSA in Lithuania compared to Sweden year 2015. There are only few countries: Netherlands, Norway, Denmark, Finland, Island and Sweden that have achieved to keep MRSA rate low due to careful screening, prevention guidelines, and strict control over antibiotic overuse [16].
Fig 4: Staphylococcus Aureus antimicrobial resistance 2015 in Lithuania compared to Sweden provided by the annual rapport of ECDC [24].
Fig 5: Staphylococcus aureus isolates tested (N) and the percentage of methicillin resistance in EU countries from year 2012-2015, (95% CI) [37].
2. METHODOLOGY
2.1 Research workload, location and method
This research work was carried out in the Microbiology and virology institute of Lithuanian university of health science (LUHS) in Lithuania, Kaunas. The clinical research started October 2017 and was continued to April 2018. 81 samples of different S. aureus bacterial strains were used from Institute of Infectious diseases and Pathogenic microbiology. The samples have been collected between year 2013-2015 from central hospital laboratories in Klaipeda, Šiauliai, Kaunas and Vilnius. The samples have all been tested previously at the laboratories and been confirmed as MRSA positive. The Methicillin resistant samples have thereafter been evaluated by diagnostic methods; Mannitol salt agar media test, Latex agglutination test “spot A test”, Oxacillin resistant screening agar (ORSA) medium, Penicillin binding protein (PBP2), Minimal inhibitory concentration (MIC) antibiotic sensitivity test, Polymerase chain reaction (PCR) of mecA gene and PVL coding gene, Biofilm formation test.
2.2 Bacterial culture preparation
Isolates of S. aureus was activated to grow in Thioglycollate fluid medium and then incubated in a heater with 37 degrees for 48 hours. After the bacteria with confirmed positive growth were transferred from the Thioglycollate medium to Mannitol salt agar plates. All Mannitol salt agar plates were then placed in the incubator with 37 degrees.
2.3 Materials
Table 6: All diagnostic methods performed in the clinical research and its manufactures.
Diagnostic Method Manufacturer
1. Mannitol salt agar Liofilchem
2. Latex Agglutination test “spot A test” OXOID Dryspot TM staphytect plus, DR0100M 3. Oxacillin resistant screening agar
(ORSA)
OXOID CM1008
4. Penicillin Binding Protein (PBP2) Latex agglutination test
OXOID DR0900
5. Minimal inhibitory concentration (MIC) antibiotic sensitivity test
EUST B6034A, Sensititre Staphylococci plate
6. Singuplex PCR detection of mecA gene and PVL coding gene
Thermo Fisher Scientific
7. Congo red agar OXOID, UK
Table 7: Composition of Mannitol salt agar media
2.3.1. Mannitol salt agar culture materials
Formula Amount (g/l) Manufacturer
Pancreatic Digest of casein 5.0 Liofilchem
Peptic Digest of animal tissue 5.0 Liofilchem
Beef Extract 1.0 Liofilchem
D-Mannitol 10.0 Liofilchem
Sodium Chloride 75.0 Liofilchem
Phenol Red 0.025 Liofilchem
Agar 15 Liofilchem
Table 8: Composition of Latex agglutination “ spot A” test
2.3.2. Latex agglutination test “spot A” materials
Components/Material Manufacturer
Blue latex particles coated with both porcine fibrinogen and rabbit IgG together with specific polyclonal antibodies raised against capsular polysaccharide of S. aureus (Test reaction area)
OXOID Dryspot TM staphytect plus, DR0100M
Blue latex particles sensitised with non- reactive globulin (control reaction area)
OXOID Dryspot TM staphytect plus, DR0100M
Cards and moister absorbent sachet OXOID Dryspot TM staphytect plus, DR0100M
Table 9: Composition of Oxacillin resistant screening agar
2.3.3. Oxacillin resistant screening agar materials
Formula Amount gm/litre Manufacturer
Peptone 11.8 Oxoid
Yeast Extract 9.0 Oxoid
Mannitol 10.0 Oxoid
Sodium Chloride 55.0 Oxoid
Lithium chloride 5.0 Oxoid
Aniline Blue 0.2 Oxoid
Agar 12.5 Oxoid
Table 10: Composition of Penicillin protein binding (PBP2) latex agglutination test
2.3.4 Penicillin Binding Protein (PBP2) Latex Agglutination Test, OXOID DR0900 Materials
Components/Material Manufacturer
Test Latex sentisitised with monoclonal antibody against PBP2
OXOID DR0901
Control Latex Sentisitised with monoclonal antibody of the same IgG subclass showing no reactivity with proteins of S. aureus
OXOID DR0902
Extraction reagent 1 OXOID DR0903
Extraction reagent 2 OXOID DR0904
Test Cards OXOID DR0900
Mixing Sticks OXOID DR0900
Table 11: Composition and antimicrobial agents of Minimal inhibitory concentration (MIC) test 2.3.5. Minimal inhibitory concentration (MIC) antibiotic sensitivity test Materials
Sensititre staphylococci plate-EUST
Antimicrobics Concentration Manufacturer
Clindamycin (CLI) 0.12µg, 0.25 µg , 0.5 µg, 1 µg, 2 µg, 4 µg
EUST
Tetracycline (TET) 0.5 µg, 1 µg, 2 µg, 4 µg, 8 µg, 16 µg
EUST
Rifampin (RIF) 0.016 µg, 0.03 µg, 0.06 µg, 0.12 µg, 0.25 µg, 0.5 µg
EUST
Streptomyocin (STR) 4 µg, 8 µg, 16 µg, 32 µg EUST
Fusidate (FUS) 0.5 µg, 1 µg, 2 µg, 4 µg EUST
Penicillin (PEN) 0.12 µg, 0.25 µg, 0.5 µg, 1 µg, 2 µg
EUST
Chloramphenicol (CHL) 4 µg, 8 µg, 16 µg, 32 µg, 64 µg EUST Kanamycin (KAN) 4 µg, 8 µg, 16 µg, 32 µg, 64 µg EUST
Tiamulin (TIA) 0.5 µg, 1 µg, 2 µg, 4 µg EUST
Quinupristin/ dalfopristin (SYN)
0.5 µg, 1 µg, 2 µg, 4 µg EUST
Vancomycin (VAN) 1 µg, 2 µg, 4 µg, 8 µg, 16 µg EUST Gentamicin (GEN) 1 µg, 2 µg, 4 µg, 8 µg, 16 µg EUST Trimethoprim (TMP) 2 µg, 4 µg, 8 µg, 16 µg, 32 µg EUST Erythromycin (ERY) 0.25 µg, 0.5 µg, 1 µg, 2 µg, 4
µg, 8 µg
EUST
Ciprofloxacin (CIP) 0.25 µg, 0.5 µg, 1 µg, 2 µg, 4 µg, 8 µg
EUST
Cefoxitin (FOX) 0.5 µg, 1 µg, 2 µg, 4 µg, 8 µg, 16 µg
EUST
Linezolid (LZD) 1 µg, 2 µg, 4 µg, 8 µg EUST
Mupirocin (MUP) 0.5 µg, 1 µg, 2 µg, 256 µg EUST
Sulfamethoxazole (SMX) 64 µg, 128 µg, 256 µg, 512 µg EUST
Control Negative EUST
Control Positive EUST
Table 12: Composition PCR for mecA detection
2.3.6 Singuplex PCR detection of mecA gene and PVL coding gene Materials
Component mecA PVL Manufacturer
Green Master Mix 10 µl 10 µl Thermo Fisher Scientific
Primer F 1 µl 1 µl Thermo Fisher Scientific
Primer R 1 µl 1 µl Thermo Fisher Scientific
Prepared DNA 4 µl 3 µl Thermo Fisher Scientific
Nuclease free water 4 µl 5 µl. Thermo Fisher Scientific
Final reaction volume 20 µl 20 µl Thermo Fisher Scientific
Table 13: Composition of Congo red agar media
2.3.7 Biofilm formation assay on Congo red agar (CRA) Materials
CRA Composition Amount Manufacturer
Congo red dye 0.8 g Oxoid, UK
Sucrose 36 g Oxoid, UK
Brain heart infusion agar (BHIA)
52 g Oxoid, UK
Water 1000 ml Oxoid, UK
2.4 Test methods
2.4.1 Mannitol salt agar method
Mannitol salt agar is a selective medium used for isolating pathogenic Staphylococci.
Mannitol is fermentable carbohydrate. The high salt concentration of 7.5% inhibits most bacteria other than Staphylococci. pH indicator is phenol red. Agar is used as solidifying agent. Rest of formula ingredients are for growth of organism. Final pH 7.4 ± 0.2 at 25 °C. Plates was inoculated after 81 isolated suspected S. aureus strains been swept over the agar surface. Incubated at 35 °C ± 2°C for 24- 48 hours.
Table 14: Reference control of Mannitol salt agar
Quality Control Staphylococcus aureus ATCC 25923 Staphylococcus aureus ATCC 6538
2.4.2 Latex agglutination test “spot A” method
Latex slide agglutination for detection of S. aureus by detecting protein A and specific polysaccharides found on surfaces of all S. aureus.
81 isolated strains of suspected S. aureus were investigated by the “spot A” method. Reagent on the card was mixed with 2-3 mm of suspected Staphylococcus aureus colonies emulsified in 50-µl of saline. Result was evaluated in 1 minute. The reference strains for test of reaction provided agglutination and the control test with non-reactive globulin were phenotypically homogenous and gave no sign of agglutination.
2.4.3 Oxacillin screening agar method
ORSA is a medium for screening for methicillin resistant Staphylococcus aureus. Based on Mannitol salt agar with reduction in salt concentration (5,5%). ORSA contain oxacillin at 2 mg/litre to inhibit MSSA. The suspected bacteria’s of the 81 isolated strains is swept on surface of the agar plate and incubated at 37 °C for 24 hours. Mannitol fermentation will colour the agar blue.
Table 15: Reference control of Oxacillin screening agar Quality Control
Control Strain Expected results
Positive MRSA ATCC 43300 Good growth, blue coloured colonies
Negative MSSA ATCC 25923 No growth
2.4.4 Penicillin Binding Protein (PBP2) latex agglutination method
78 isolated and suspected MRSA strains were evaluated by PBP2 method. Latex particles sensitised with monoclonal antibody against PBP2 will react with methicillin resistant Staphylococci and cause agglutination.
Table 17: Method of PBP2 supernatant in stepwise manner
Extraction of PBP2 supernatant
Step Component/ amount Action
1. 4 drops Reagent 1 Microcentrifuge tube
2. 3-5 µl cells of suspected
isolated colonies
Microcentrifuge tube
3. Tube with turbid suspension Heat over 95 °C for 3 min
And then cool down in room temperature
4. Add 1 drop of Reagent 2 Into tube, mix and centrifuge
1500 x g for 5 minutes
All the 78 isolated strains of suspected MRSA supernatant were placed at test circle, the latex and supernatant were mixed and within 3 minutes the result could be recorded. Quality control strains confirmed expected result.
Table 18: Reference control for PBP2
Quality control strain
Positive MRSA ATCC 43300
Negative MSSA ATCC 25923, ATCC 29213
2.4.5. Minimal inhibitory concentration (MIC) test method
50 randomly selected MRSA samples from the total of 78 strains were collected for MIC test determination. MIC test is performed to find out the susceptibility of the organism to the antimicrobial agent. The nephelometer was calibrated with the McFarland standard. Mix of 3-4 colonies and a suspension of 4 ml saline, vortex mix. Suspension was set at McFarland 0.5. 50 µl of the suspension was added to 10 ml of Muller Hinton broth. 50 µl per well in the panels were inoculated, inoculums = 5 x 105 CFU/ml. Panel was sealed and Incubated at 37 degrees for 20 hours.
2.4.6. PCR assay for detection of the mecA gene and PVL coding gene method
78 isolated strains of MRSA, previously tested positive for methicillin resistance by other conventional diagnostic methods were analyzed by PCR for both mecA gene, PVL coding genes and for icaA gene, that is one of many genes that may be responsible for production of adhezins.
DNA template preparation: 2-3 colonies of bacteria (one sterile 10 µl loop) were collected from cultures on Tryptic Soy Agar (Liofilshem) that been inoculated for 24 h. The colonies were suspended in 500 µl of sterile nuclease free water (Thermo Fischer Scientific). Tubes of the suspension was placed in a heating block and heated for 10 min (over 95ºC). Then centrifuged for 10 min. at 10000 rpm/min.
Table 19: Primers design and PCR amplification of mecA, PVL coding gene and icaA gene.
PRIMER SEQUENCE (5´-3´) PRODUCT SIZE (bp)
F-MecA
R-MecA
GTA GAA ATG ACT GAA CGT CCG ATA A
CCA ATT CCA CAT TGT TTC GGT CTA A
310 bp
F-PVL
R-PVL
ATC ATT AGG TAA AAT GTC TGG ACA TGA TCC A GCA TCA AGT
433 bp
F-icaA
R-icaA
ACA GTCGCT ACG AAA AGA AA
GGA AAT GCCATA ATG ACA AC
188 bp
Table 20: Control reference PCR Negative Control for mecA, icaA and lucPV
ATCC25923, ATCC29213
Table 21: mecA Thermocycling conditions sets
Degree Time Cycles
94˚C 2 min 2
94˚C 30 sec 4
59˚C 30 sec 30
72˚C 1 min 1
75˚C 5 min 1
Table 22: PVL Thermocycling conditions sets
Degree Time Cycles
94˚C 2 min 2
94˚C 30 sec 4
61˚C 30 sec 30
72˚C 1 min 1
72˚C 5 min 1
Table 23: icaA Thermocycling conditions sets
Degree Time Cycles
94˚C 2 min 2
94˚C 30 sec 4
50˚C 30 sec 30
72˚C 1 min 1
75˚C 5 min 1
PCR Electrophoresis 5 V/cm were performed in 1,5 % agarose gel with 0.5 µg/ml of ethidium bromide in TAE buffer ("Roth"). Amplification bands were analyzed in UV at 302 nm. For the marker lines 100-pb DNA ladder was used
2.4.7 CRA method
The slime production was studied by cultivation of 78 MRSA isolates strains for biofilm production on Congo red agar. The negative control strain ATCC 25923, was confirmed negative. Inoculated agar was incubated for 48 h at 37 °C.
Table 24: Reference control Congo red agar
Negative control ATCC 25923
3. RESULTS
3.1 Results from Mannitol screening agar, Latex agglutination “spot A” test and Oxacillin resistant screening agar
The collection of total 81 isolated suspected methicillin resistant strains were first cultured by
Mannitol salt agar. Positive S. aureus growth were present in 69 strains, negative growth by Mannitol were confirmed in 12 strains with the strain number: 16, 17, 24, 42, 44, 46, 64, 73, 78, 91, 102 and 108. One strain, 62 showed a non-phenotypically growth with orange-yellow colonies and strain number 21 had a very weak positive growth.
Table 25: Interpretation of Results of Mannitol salt agar medium
S. aureus Cultivates with yellow or white colonies
surrounded by a yellow zone.
Coagulase-negative Staphylococci Small colourless to red colonies with no colour change to the medium
Negative No growth
The same 81 collected strains were thereafter tested by Latex agglutination “spot A” method which implicated that 78 strains were positive as Staphylococcus aureus and the two other strains were another bacteria than S. aureus and negative by “spot A” test. The negative strain number was: 62 and 78.
Table 26: Interpretation of phenotypically result of “spot A” test Positive (S. aureus) Negative (Not S. aureus)
Blue agglutination Homogenous structure, no clumping
Fig.6. Mannitol salt agar results compared to Spot “A” test results
85% 97%
15%
3%
11%
0 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Mannitol salt agar "spot A" test Positive Negative False negative
The third step was to test the same 81 strains with Oxacillin resistant screening agar method.
Phenotypically 76 strains were confirmed positive and 4 strains were Negative by Mannitol
fermentation but had positive growth, strain number: 16, 17, 24 and 62. Strain number 78 was negative with no growth.
Table 27: Interpretation ORSA result
Culture medium Colony colour
ORSA CM1008
Positive (MRSA) Negative (MSSA) Intense blue on colourless
media
No growth
Table 28: Results from 14 of 81 strains that did show various results by diagnostic methods; MSA,
“spot A” test and ORSA.
Results Strain
number
Mannitol salt agar Latex
agglutination
“spot A” test
ORSA Comment
16 (-) False negative (+) (+/-) Growth but
mannitol negative fermentation
S.aureus and resistant but no typical blue fermentation on ORSA.
17 (-) False negative (+) (+/-) Growth but
mannitol negative fermentation
S.aureus and resistant but no typical blue fermentation on ORSA.
21 (+) Weak positive (+) (+)
24 (-) False negative (+) (+/-) Growth but
mannitol negative fermentation
S.aureus and resistant but no typical blue fermentation on ORSA.
42 (-) False negative (+) (+)
44 (-) False negative (+) (+)
46 (-) False negative (+) (+)
62 Very yellow, not
typical growth
(-) (+/-) Growth but
mannitol negative fermentation
Not S. aureus but resistant against Methicillin
64 (-) False negative (+) (+)
73 (-) Sporogenic
bacterium
Sporogenic bacterium.
Not S. aureus
78 (-) (-) (-) Not S. aureus and no resistance against methicillin
91 (-) False negative (+) (+)
102 (-) False negative (+) (+)
108 (-) False negative (+) (+)
After the MSA, Latex agglutination “spot A” test and ORSA result, it was concluded that strain number 62 is not Staphylococcus aureus but a methicillin resistant strain. Strain number 73 is not S.
aureus but in fact a sporogenic bacterium and strain number 78 is neither Stapylococcus aureus bacterium or methicillin resistant. Therefore these three strains were excluded from further diagnostic testing and the remaining 78 strains were proceeded with for further diagnostic methods.
3.2 PBP2 Latex agglutination test result
Table 29: Interpretation of result of PBP2
Negative (MSSA) No visible clumping
Weak Positive Small clumps against clouded background
Strong Positive (MRSA) Large and small clumps against clouded
background
Total number of 78 suspected isolated MRSA strains was tested. Negative reference strains ATCC 25923, ATCC 29213 were both found to be with no agglutination reaction and positive reference strain ATCC 43300 were with strong agglutination. Among the 78 MRSA strains investigated, 64 phenotypically confirmed strong positive result, nine was weak positive and five strains were negative, strain number: 19, 61, 99, 100 and 101.
82%
6%
12%
PBP2 Result
Strong positive Negative Weak positive
3.3 MIC test result
From the total of 78 suspected methicillin resistant strains, 50 strains were randomly collected for minimal inhibitory concentration antibiotic sensitivity test. The most important parameter is resistance against Cefoxitin (FOX) above 4 µg, which represent methicillin resistance.
Table 30: Antimicrobial resistance pattern by 50 strains, observe that Rifampin (RIF), Streptomycin (STR), Fusidate (FUS), Tiamulin (TIA), Quinipristin/ Daifopristin (SYN), Mupirocin (MUP) and Sulfamethoxazole (SMX) are not represented in the table. The table represent results of Clindamycin (CLI), Tetracycline (TET), Penicillin (PEN), Chloramphenicol (CHL), Kanamycin (KAN), Vancomycin (VAN), Gentamicin (GEN), Trimetoprim (TMP), Erythromycin (ERY), Ciprofloxacin (CIP), Cefoxitin (FOX) and Linezolid (LZD).
Sensitive (S), Resistance (R), all numbers is in µg. Empty space represents complete sensitivity.
Resistance is indicated with a red colour.
Antibiotic
Strain Name
CLI S≤0.25 R> 0.5
TET S≤ 1 R>2
PEN S≤0.125 R>0.125
CHL S≤8 R> 8
KAN S≤8 R>16
VAN S≤2 R> 2
GEN S≤1 R> 1
TMP S≤2 R>4
ERY S≤1 R>2
CIP S≤1 R>1
FOX S≤4 R>4
LZD S≤4 R>4
2 0.25 ≤0.5 >2 >64 >64 ≤1 >16 ≤ 2 > 8 8 >16 ≤ 1
10 ≤ 0.125 64 0.5 8 16 2
11 >16 1 8 >64 16 0.5 4 16 2
12 4 >2 32 2 0.5 8 >16
14 0.5 >32 0.5 4 16
15 >16 0.5 0.5 8 16 2
16 0.25 >64 >16 0.5 8 >16 2
17 >2 >64 >16 0.5 8 16 2
18 >2 8 >64 >16 0.5 8 >16 2
19 >2 64 >64 >16 0.5 8 16 2
20 >2 64 >64 >16 0.5 8 >16 2
21 >4 >16 >2 64 >64 >16 >8 4 16 2
22 >2 64 >64 >16 0.5 8 16 2
23 >2 64 >64 >16 0.5 4 >16 2
24 >2 8 >64 >16 1 8 >16 2
25 >2 64 >64 >16 0.5 8 >16
29 >16 1 64 0.5 8 >16 2
30 >16 1 64 0.5 4 16 2
31 >16 0.5 64 1 8 16 2
32 >16 >2 8 >64 0.5 16 2
33 >2 64 >64 >16 0.5 8 16 2
34 >4 >16 >2 64 16 >8 4 >16 2
36 >2 64 >64 >16 0.5 8 16 2
3 >2 64 >64 >16 4 0.5 8 >16 2
51 >16 >2 8 >64 2 2 8 16 2
53 >16 >2 8 >64 0.5 16 2
55 >2 64 >64 >16 8 >16
57 >2 64 >64 >16 1 8 >16
60 >4 >16 >2 8 >64 16 >8 >16 2
61 4 >2 >64 8 >32 >8 >8 >16
64 >2 8 >64 >8 >8 >16 2
65 >2 >64 >16 0.5 4 >16 2
58 >4 >16 >2 8 >64 16 >8 >16
59 >2 64 >64 >16 0.5 8 >16
66 >2 8 1 4 >16 2
67 >2 8 >64 >16 0.5 8 >16 2
68 >2 64 >64 >16 0.5 8 >16 2
70 >2 64 >64 >16 0.5 8 >16 2
74 >2 8 0.5 0.5 16 2
79 >16 >2 8 >64 16 0.5 8 16 2
85 >16 >2 8 >64 >16 8 0.5 >8 >16 2
87 8 8 16
91 1 8 >64 0.5 16 2
92 >2 8 >64 >16 0.5 8 >16 2
95 >2 8 >64 >16 4 8 >16
97 >2 8 0.5 16 2
47 0.12 0.5 >2 4 4 1 1 2 0.5 0.25 16 2
48 >2 8 >64 >16 0.5 8 >16 2
49 >2 >64 >16 0.5 8 >16 2
50 >16 >2 8 >64 0.5 16
Fig .7. The pattern of resistance presented by MIC test from 50 MRSA suspected isolated strains from the Lithuanian population. The table represent results of Clindamycin (CLI), Tetracycline (TET), Penicillin (PEN), Chloramphenicol (CHL), Kanamycin (KAN), Vancomycin (VAN), Gentamicin (GEN), Trimetoprim (TMP), Erythromycin (ERY), Ciprofloxacin (CIP), Cefoxitin (FOX) and Linezolid (LZD).
The result from MIC test presented a 100% Methicillin resistance among the 50 controlled strains.
3.4 Single target PCR assay result 3.4.1 mecA
According European guidelines Polymerase chain reaction is the gold standard of diagnostic methods to confirm antimicrobial resistance by identification of the mecA gene. All 78 strains were investigated by the PCR method and the result was unanimously positive, all strains contained the mecA gene. Four strains gave a weak positive result, strain number: 99, 100, 101 and 97.
0%
20%
40%
60%
80%
100%
120%
CLI TET PEN CHL KAN VAN GEN TMP ERY CIP FOX LZD
Resistant Sensitive
