RESISTANCE PHENOTYPIC OF GRAM-NEGATIVE BACTERIAL STRAINS ISOLATED FROM HOSPITALWisam abdulameer najm, Mariana-Carmen Chifiriuc*, Irina Gheorghe, , Otilia Banu, Mihailescu Dan

2.Materials and methodsa. Blood cultureIs a method of qualitative identification of microorganisms reached the blood of bacteremia and sepsis . Blood dissemination ( bacteremia) is when bacteria come from a focus on septic or injured mucosa . Status may not have bacteremia or clinical expression may be accompanied by chills and fever that can add symptoms lesion that caused bacteremia . Sepsis is an aggravated bacteremia with clinical outcome and is manifested by chills, fever, irregular , toxemia , hypotension , rash . Pathogens are Staphylococcus aureus involved in sepsis ( blood reach the cutaneous barrier when it affected mucosa or catheters or drainage ) , coagulase-negative staphylococci ( CNS ) , Escherichia coli and other Enterobacteriaceae , Pseudomonas , Streptococcus , anaerobic bacteria , yeasts .Blood is drawn aseptically and inoculated into blood culture bottle whose composition favors the development of germs aerobic , anaerobic and microaerophilic . There Inmediu imbogttire factors that favor the development of germs : peptone , casein, gelatin , hemin , vitamin B6 , CO2 . In the culture there also an anticoagulant , because otherwise they risk being captured microorganisms in fibrin clot and not to develop colonies. At the same time, some anticoagulants may be toxic to certain pathogens. This uses sodium polyanetholsulfonat (SPS ), a non-toxic anticoagulant that favors bacterial proliferation by neutralizing human serum bactericidal activity by inhibiting the action of antibiotics ( streptomycin , kanamycin , gentamicin , Polymixina B). SPS 's role may inhibit some strains of Peptostreptococcus and Neisseria effect neutralized by the presence of gelatin in the culture medium . For analyzes hemoculturii use semiautomatic or automatic computerized systems . Blood culture bottle can examine and macroscopically for signs of bacterial growth. If the liquid in the bottle when environmental turbidity present are Gram negative bacteria, Staphylococcus ssp, Bacteroides ssp if there haemolysis when involving Streptococcus ssp, Staphylococcus ssp, Lyster ssp, ssp Clostridium, Bacillus ssp if there gas occurs aerobic and anaerobic Gram-negative bacilli and blood clots occur if there is S. aureus.If blood culture is positive ( in blood were discovered pathogens that have multiplied in culture medium ) , the expansion chamber will pass and will rise above its green sleeve . Then it will be taken from the bottom of the expansion chamber a small amount of medium to be used for smears , which will be examined microscopically after Gram staining . In relation to the outcome readings are subculturing on solid and liquid media : blood agar , Columbia blood agar chocolat , Levine, Sabouraud Chloramphenicol , thioglycolate broth with resazurin . Plates were incubated aerobically at 37 C in 5-7 % CO2 atmosphere for 48-72 hours . Examine colonies isolated by identification tests : catalase , identification kit Staphylococcus , Streptococcus identification kit discs Optochin factors X , V and XV polytrope media for enterobacteria , oxidase reaction , identification of yeasts by Candifast test .Were tested for sensitivity to antibiotics or antifungal agents. If that were isolated several bacterial species in a single bottle or bottles of the same patient different , they are kept in observation in the following situations: immunosuppressed patient , comatose or cater maintained for long . Depending on the period of growth of the bacteria they are divided into: those that grow in the first 1-2 days as Streptococcus pneumoniae, Enterobacteriaceae; the growing bacilli after 3 day non-fermentative Gram-negative and anaerobic bacteria; 4-5 days grow Haemophilus and Staphylococcus coagulase-negative; 6-7 days ssp Candida species; 9 days corynebacteria

b. Urine culture Is analysis that uncovers urinary tract infections . Urinary tract infections are caused by various microorganisms inflammatory diseases reaching the urinary tract , where it multiplies and causes as changes in normal kidneys and urinary tract. After location, infections may be low when the urethra and bladder are affected and high when covered and ureters and kidneys. Urinary infections can be asymptomatic or symptomatic (presence of pain and / or burning on urination , pain in the lower abdomen , cloudy and smelly urine ) may be common in boys during the first year of life in women with sexual activity in people diabetic with cancer , renal failure , with urethral climb and the elderly ( especially in men, due to prostate problems ) . To have a threshold bacterial urinary infection must be 105 CFU ( colony forming units ) for Gram negative bacilli / ml urine for 5x104UFC/ml like Staphylococcus and Candida albicans 104 CFU / ml . Pathogenic bacteria most commonly found in the etiology of urinary tract infections are Gram negative bacilli glucose fermentation (E. coli, Klebsiella , Proteus , Enterobacter , Citrobacter , Morganella , Providencia ) , Gram-negative bacilli nefermentativi glucose ( Pseudomonas , Acinetobacter ) , cocci Gram positive ( Enterococcus spp , Staphylococcus aureus, Staphylococcus saprophyticus , Streptococcus hemolytic group B). Corynebacterium urealyticum causes urinary tract infections in patients with prolonged antibiotic treatment or urological surgery . Candida albicans determines metabolic uretrocistite uncontrolled diabetics .Secretions from the wound produced by damaged tissue and the composition of epidermal cell death, leukocytes, bacteria. Visual analysis , chemical and bacteriological these secretions are important to prevent wound infection or discover the healing time and track patient progress . The main pathogen of wound infection is S. aureus , Pseudomonas aeruginosa in burns .

c. Nasal exudate Is the microbiological examination of nasal secretions for determining upper respiratory pathogens . Normal bacterial flora of the nose presents a small number of bacterial species are dominated by Staphylococcus albus , Staphylococcus aureus and difteromorfi . Rarely can be identified hemolytic streptococci , enterococci , neiserii nonpathogenic , Acinetobacter and Moraxella . The relationship between etiologic agents and various diseases of the nose is not specific only in few cases . The most common inflammatory lesions encountered in the nose are folliculitis and furunculosis of staphylococcal origin , eczema and impetigo streptococci vestibular origin , commonly caused by streptococcal infection staphylococcal . In rinitele bacteriene sau in rinitele virale suprainfectate sunt izolai frecvent stafilococi Neisseria menigitidis coagulazopozitivi occasionally. In purulent rhinitis often is found association with staphylococcus pneumonia and those with false membrane, association staphylococcus, pneumococcus and Corynebacterium diphtheriae. Bolilecronice Ozen and rinoscleromul nose and are usually caused by Klebsiella rhinoscleromatis. Sampling is done by one sterile swab moistened with saline, different for each nostril, which rotates nazali intoduce cavity easy to detach secretion. Bacteriological examination of nasal secretion involves performing cultures, microscopic examination is relevant only in case of porting the diphtheria bacillus. In this case recolteazdou pads, one serving to conduct a smear, and the other to the sowing of crops. Usually, nasal swab seeded on blood agar. Pharyngeal exudate is bacteriological examination of secretions from the pharynx. Discharge sample is taken from the pharyngeal cavity with a sterile swab will then be placed in a recipientce presents a solution that will stimulate the growth of microorganisms, and all the buffer that will inoculate the culture medium.d. Pathogenic bacterial strains. In this study we have used pathogenic bacterial strains E. coli and K. pneumoniae. E. coli, 66 Klebsiella pneumonia and Proteus mirabilis and Serratia marcescens and Aeromonas hydrophila and coagulase-negative staphylocci and Enterococcus faecium, Enterococcus faecalis, Streptococcus agalactiae and Streptococcus spp and Candida albicans and non-C. albicans strains, provided Microbiology Laboratory of the Institute of Cardiovascular Diseases "( Institutul Clinic Fundeni", Bucharest, in 2013)The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that normally inhabits the soil and surfaces in aqueous environments. Its adaptability and highintrinsic antibiotic resistance enable it to survive in a wide range of other natural and artificial settings, including surfaces in medical facilities. Serious P. aeruginosa infections are often nosocomial, and nearly all are associated with compromised host defenses such as in neutropenia, severe burns, or cystic fibrosis (CF; Table 1; Lyczak et al., 2000). Therapeutic options are increasingly limited due to the continued emergence and spread of antimicrobialresistant strains; as a result, P. aeruginosa infections demonstrate high morbidity and mortality. In the United States, P. aeruginosa is among the most common hospital pathogens and is the second most common pathogen isolated from patients with ventilator-associated pneumonia (VAP; Hidron et al., 2008). Given the severity of P. Aeruginosa infections and the limited antimicrobial arsenal with which to treat them, finding alternative prevention and treatment strategies is an urgent priority.

InfectionsMajor risk factors

Soft tissueBurns, open wounds, postsurgery

Urinary tractUse of urinary catheter

BacteremiaImmunocompromised

Diabetic footOld age, COPD, cystic fibrosis,mechanical ventilation

Otitis externa (swimmers ear)Tissue injury, water blockagein ear canal

Keratitis (corneal infectionExtended contact lens wear,contaminated contact lens solution

Otitis media folliculitis(hot tub rash)Otitis media folliculitis(hot tub rash)

Table 1

The most prevalent bacterial infections were Acinetobacter spp. (26%), Pseudomonas spp. (18%), and Klebsiella spp. (9%), which represented a 1% decrease in the Pseudomonas spp. prevalence rate and a 4% and 11% increase in prevalence of Acinetobacter spp. And Klebsiella spp.

e. The Pseudalert test detects the presence of Pseudomonas aeruginosa in bottled, pool, and spa water samples. The test is based on a bacterial enzyme detection technology that signals the presence of Pseudomonas aeruginosa through the hydrolysis of a substrate present in the Pseudalert reagent. Pseudomonas aeruginosa cells rapidly grow and reproduce using the rich supply of amino acids, vitamins, and other nutrients present in the Pseudalert reagent. Actively growing strains of Pseudomonas aeruginosa have an enzyme that cleaves the substrate to produce a blue fluorescence under UV light. Pseudalert detects Pseudomonas aeruginosa at 1 cfu in either 100 mL or 250 mL samples within 24 hours for non-carbonated water samples and within 26 hours for carbonated samples.

The susceptibility of 104 isolates of P. aeruginosa to 13 different antibiotics was determined by agar disk diffusion method. The alkaline lysis method was used for plasmid extraction. PFGE technique was optimized for DNA fingerprinting of isolatesPseudomonas aeruginosa is a Gram-negative, aerobic, monoflagelat, asporogenous, nonfermentativ opportunist that is found in soil, water, plant surface, have minimal nutritional requirements and can withstand low temperatures. Is able to use food as a wide range of organic products that adapt and thrive in many ecological niches, even in fossil fuel. The water increases as biofilm or free cells. Although aerobic environments may increase without O2, NO3 or arginine only if there is medium. The cell has a size of 1-5 long and 0.5 to 1 in diameter (B. Iglewski,1996). Pseudomonas aeruginosa is commonly found in contaminated water, sewage system, the fountains, the swimming pools. Pseudomonas groups to form biofilms tend to adhere to a wide variety of substrates, making them the same resistance to environmental factors and antibiotics.This bacterium is an opportunistic pathogen because it causes serious infections in immunocompromised patients , causing nosocomial infections as found on most medical equipment , toilets , showers , sinks, and even the potent disinfectant soaps . Clinical isolates secrete certain pigments and piocianin ( colonies that secrete the pigment acquires a greenish blue tint ) , pioverdin ( green pigment is secreted especially in environments lacking Fe ) piorubin ( purple pigment ) and piomelanin ( brown pigment ) .P. aeruginosa can cause suppurative infections in patients with burns or surgery can cause pneumonia, bacteremia , endocarditis , meningitis and brain abscess , otitis externa and media keratitis , endoftalmii , osteomyelitis , urinary tract infection , enterocolitis , chronic infections patient with cystic fibrosis .In vitro Pseudomonas colonies are usually smooth, fried egg may look flattened edges and higher middle or secretaries may look mucoid alginate due mainly to strains isolated from tracheal secretions and urine. Colonies also producing a characteristic odor of linden flowers and green apple . P. aeruginosa grow in environments with an optimum temperature of 37 C , but may grow to reach normal temperature and 42 C

. 3. Results and DiscussionPseudomonas aeruginosaFrom a total of 197 strains of P. aeruginosa, isolated during the year 2012, 79 strains were isolated tracheal secretions (representing a 40%), 47 (24%) of the wound secretions, 25 (13%) of urocultures , 22 (11%) from blood cultures, 17 (9%) of the various secretions (bronchial secretions of eschar prosthesis iliac vein catheter, dialysis catheter, necrosis), 5 sputum and nasal exudate 3 of of 2% and 1 % of isolates (fig.1).

Fig. 1. Graphical representation of the sources of isolation of strains Pseudomonas aeruginosa.

Section with the highest prevalence of infection with P. aeruginosa was polling Surgery Cardiovascular (100 cases), followed by vascular surgery department (57 cases), Cardiology 1 (24 cases), Anesthesia and Intensive Care (7 cases), Cardiology 2 (4 cases), Cardiology 4 (3 cases) and Cardiology 3 in two cases. The highest number of deaths was recorded in cardiovascular surgery department (63 cases). (fig. 2)

Fig. 2. Graphical representation of the prevalence of infections caused by Pseudomonas aeruginosa in different wards of the hospital.

Most patients who suffered infections with P. aeruginosa were over the age of 65 years (109 cases), 71 of them were aged between 50 and 64 years, 15 were aged between 35 and 49 years, 2 were classified in the category 20-34 years, there was no patient younger than 20 years (fig. 3). The average age of all patients was 66 years.

Fig. 3. Graphical representation of patients with infections caused by Pseudomonas aeruginosa by age.

Of all patients who have infections caused by P. aeruginosa 151 (77%) belonged to the male gender and 46 (23%) female gender. (fig. 4)

Fig. 4. Graphical representation of patients of patients caused by Pseudomonas aeruginosa by sexes.

The mean duration of hospitalization days in patients whose tracheal secretions was isolated P. aeruginosa was 62 days, and their average age was 65 years. Of these 62 patients (78%) were male, and 17 (22%) of the female. 15 (20%) of the patients had more than 100 days of hospitalization, 28 (35%) had between 50 and 100 days of hospitalization and 36 (45%) stayed hospitalized less than 50 days. From Figure 5 it can be seen that the days of hospitalization were the more as the age of the patients was mad The majority of patients who were hospitalized for less than 21 days were under the age of 60 years.

Fig. 5. Plotting the number of days of hospitalization correlated with age in patients whose tracheal secretions was isolated Pseudomonas aeruginosa.

Whose average age apacienilor of wound secretions were sampled P. aeruginosa is 65 years and the average duration of their hospital stay is 16 days. 37 (79%) of the patients with infections caused by P. aeruginosa wounds were male and 10 (21%) were female. Most patients were under 20 days of hospitalization, eight people were hospitalized between 20 and 35 days, but there were two people that were hospitalized 87, and 110 days. Most patients with wound infections were aged between 40 and 65 years (48%), 7 (14%) patients were over 80 years (fig. 6)

Fig. 6. Plotting the number of days of hospitalization correlated with age in patients whose wound secretions of Pseudomonas aeruginosa was isolated.

Patients whose secretions various (eschar bronchial prosthesis iliac venous catheter, dialysis catheter, necrosis) was isolated bacillus Pseudomonas had an average of 63 days of hospitalization days, most being hospitalized for a period longer than 40 days, and 4 of them in the hospital for more than 100 days. The mean age of patients was 61 years, most were over 55. (fig. 7)

Fig. 7. Plotting the number of days of hospitalization correlated with the age of patients whose discharge various (eschar bronchial prosthesis iliac venous catheter, dialysis catheter, necrosis) were isolated . aeruginosa.

The mean duration of hospitalization days in patients whose blood cultures were isolated P. aeruginosa was 57 days, and their average age was 67 years. Of these 13 patients (59%) were male and nine (41%) of the female. 10 (45%) of the patients had more than 80 days of hospitalization, 7 (32%) had between 20 and 60 days of hospitalization and four (18%) stayed hospitalized for less than 10 days. Patients who were hospitalized more than 80 days had over 65 years. (fig.8).

Fig. 8. Plotting the number of days of hospitalization correlated with age in patients whose blood cultures was isolated Pseudomonas aeruginosa.

In patients with urinary tract infections caused by P. aeruginosa, the average days of hospitalization was 54 days, 6 (24%) of them internal state between 90 and 130 days, 5 (20%) between 60 and 70 days, 9 (36%) of 10 to 30 days, and 3 (12%) in 10 days. The median age was 65 years. In patients with urinary tract infections 20 (80%) were male and 5 were female.(fig. 9)

Fig. 9. Plotting the number of days of hospitalization correlated with age in patients whose urine cultures was isolated Pseudomonas aeruginosa.

Among patients whose sputum P. aeruginosa was isolated 3 had an age of 75 years and 2 had an age of 40 years. Their length of stay not exceeding 21 days, average days of hospitalization was 12 days.

Fig. 10. Plotting the number of days of hospitalization correlated with age in patients whose sputum Pseudomonas aeruginosa was isolated.

The results showed widespread resistance (51.1 91.2 %) of the isolates to all the antibiotics, except nitrofurantoin with resistance rate of 7.3 % (Figure 10). Among the fluoroquinolones, ofloxacin showed the least resistance (51.1 %), followed by Ciprofloxacin (65.7 %) and Norfloxacin (86.9 %). The result also showed that the E. coli isolates were multiply-resistant with the highest percentage of multiresistant isolates skewed toward the highest number of antibiotics. Up to 50 (36.5%) strains were resistant to 10 out of the 11 antibiotics employed. A total number of 20 (14.6 %), 18 (14.14 %) and 13 (9.5 %) isolates were resistant to 7, 8 and 9 antibiotics respectively, while 8 (5.8 %) of the isolates were resistant to all the antibiotics employed.

ConclusionsDuring 2013 were isolated 337 strains of S. aureus isolation sources are the following : 51 % of wound secretions , nasal exudate 30% , 8% from blood cultures , 5% of tracheal secretions , 5% of secretions ( aneurysm , discharge valves, ganglion , abscess , mediastinal fluid , vascular prosthesis , venous catheter , dialysis catheter , necrosis) and 1% of sputum .This study revealed the presence of ESBL producing Eschericiha coli in Cotonou. It demonstrated also high resistance rate to antibiotics commonly used for infections treatment. Continuous monitoring and judicious antibiotic usage are required. Of the 197 strains of P. aeruginosa isolated during 2012, 40 % tracheal secretions , 24% of wound secretions , 13% of urine cultures , 11% of blood cultures , 9% of secretions ( bronchial secretions of eschar prosthesis iliac venous catheter , dialysis catheter , necrosis) , 2% and 1% sputum nasal exudate .The study showed a high prevalence of infection with P. aeruginosa in the department of cardiovascular surgery . Nososcomiale infections with P. aeruginosa species etiologic agent caused the highest mortality rate ( 51%).P.aeruginosa has been reported to have an innate resistance to several antibiotics due to the presence of lipopolysaccharides in the outer membrane, but persistent administration of antimicrobial agents, has resulted in the emergence of multi-resistant strains of P.aeruginosa (Van Eldere, 2003).

Resistance patterns of Staphylococcus aureus from Bucharest

Wisam abdulameer najm, Mariana-Carmen Chifiriuc*, Irina Gheorghe, , Otilia Banu, Mihailescu Dan

1. Materials and methods

A total number of S. aureus strains (n=20) were isolated from hospitalized patients with various cardiovascular diseases from different clinical sources, mostly from wound secretions during 2014 and 2015 from Cardiovascular Disease Institute Prof. C.C. Iliescu in Bucharest (Romania). Other sources of isolation were: nasal exudates, respiratory secretions, blood cultures venous, peritoneal fluid, pleural fluid, tracheal aspirates, pharyngeal exudates, only one strain was isolated from a venous catheter. The strains identification was performed in the Microbiology Laboratory of the Cardiovascular Diseases Institute Prof. C.C. Iliescu with the use of the coagulase test, the Mannitol enriched medium and the automated VITEK 2 system.The susceptibility analysis of the strains was performed by diffusion method (Kirby -Bauer), following the recommendations of CLSI editions 2013 and 2014.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 L solution of NaOH (sodium hydroxide) and SDS (sodium dodecyl sulphate). For the permabilization of cell membrane the tubes were heated on thermoblock at 95oC for 5 minutes. 180 L TE buffer (Tris + EDTA)1X was added in the tubes 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 Thermal Cycler machine Corbet. The amplification products of each PCR reaction (multiplex / simplex) were visualized by electrophoresis on a 2% agarose gel, stained with ethidium bromide (10 g / ml) and identified based on their typical dimensions by means of specific molecular weight markers (100pb, I Lader Bench Top 100bp DNA, Promega, USA). Genotypic-characterized SCCmec cassette types present in analysed strains was performed using PCR methods (simplex and multiplex) in order to elucidate the structure of these genetic elements and obtaining the relevant data from the epidemiological point of view. Two reactions were performed using the multiplex PCR five and four pairs of specific primers for the various sequences at the SCCmec cassette to detect simultaneously the necessary constituents of the primer acestora. Their classification and parameters used to conduct of reactions followed the protocol developed by Miheirico et al. (2007). Sequences of primers used, their specificity and amplification programs used are listed in

Tables 1 and 2 and the components used in these reactions are shown in Table 3. PrimeriSecvena de nucleotideMarime amplicon (pb)Primeri Specificitate (tipul de caset mec, regiune)

CIF2 F2CIF2 R25-TTC GAG TTG CTG ATG AAG AAG G-3 5-ATT TAC CAC AAG GAC TAC CAG C-3

495I, regiunea J1

RIF5 F10RIF5 R135- TTC TTA AGT ACA CGC TGA ATC G-3 5- GTC ACA GTA ATT CCA TCA ATG C-3 414III, regiunea J3

ccrB2 F2ccrB2 R25-AGT TTC TCA GAA TTC GAA CG-3 5-CCG ATA TAG AAW GGG TTA GC-3 311II i IV, complexul ccr

mecI P2mecI P3 5-ATC AAG ACT TGC ATT CAG GC-3 5-GCG GTT TCA ATT CAC TTG TC-3209II i III, complexul mec

mecA P4mecA P75-TCC AGA TTA CAA CTT CAC CAG G-3 5-CCA CTT CAT ATC TTG TAA CG-3 162gena mecA

SCCmecV J1 FSCCmecV J1 R5-TTC TCC ATT CTT GTT CAT CC-3 5-AGA GAC TAC TGA CTT AAG TGG-3377V, regiunea J1

dcs F2dcs R15-CAT CCT ATG ATA GCT TGG TC-3 5-CTA AAT CAT AGC CAT GAC CG-3 342I, II, IV i VI, regiunea J3

kdp F1kdp R15-AAT CAT CTG CCA TTG GTG ATG C-3 5-CGA ATG AAG TGA AAG AAA GTG G-3 284II, regiunea J1

SCC mec III J1 FSCCmec III J1 R5-CAT TTG TGA AAC ACA GTA CG-3 5-GTT ATT GAG ACT CCT AAA GC-3 243III, regiunea J1

Table 1 Nucleotide sequences of primers used, their specificity and the size of the amplicons obtained (after Milheirico et al. 2007).

The amplification program

temperature94C94C53C72C72C

Duration4 min30 sec30 sec1 min4 min

Number of cycles1301

Table 2 PCR conditions used to amplify the SCCmec element (after Milheirico et al. 2007).

Volum primers(10M)Volum PCR Master Mix*Volume ultra pure waterVolum ADNThe reaction volume

0,3 l10 l6,5 l0,5 l20 l

Table 3 Reaction components used in the PCR reactions.The two reactions were performed using the multiplex PCR of four pairs of specific primers in order to distinguish types and subtypes I V SCCmec cassettes. Sequences of primers used and deployment parameters of reactions followed the protocol developed by Zhang et al. (2005). However, a simplex PCR gene detection followed CCRC, SCCmec cassette recombinase complex. Sequences of primers used, their specificity and amplification programs used are listed in Tables 4, 5 and 6 and the components used in these reactions are shown in Table 3.

PrimersThe nucleotide sequence ofamplicon size (pb)Specific primers (cassette mec type)

Type I-FType I-R5-GCT TTA AAG AGT GTC GTT ACA GG-3 5-GTT CTC TCA TAG TAT GAC GTC C-3 613SCCmec I

Type II-FType II-R5-CGT TGA AGA TGA TGA AGC G-3 5-CGA AAT CAA TGG TTA ATG GAC C-3 398SCCmec II

Type III-FType III-R5-CCA TAT TGT GTA CGA TGC G-3 5-CCT TAG TTG TCG TAA CAG ATC G-3 280SCCmec III

Type IVa-FType IVa-R5-GCC TTA TTC GAA GAA ACC G-3 5-CTA CTC TTC TGA AAA GCG TCG-3 776SCCmec IVa

Type IVb-FType IVb-R5-TCT GGA ATT ACT TCA GCT GC-3 5-AAA CAA TAT TGC TCT CCC TC-3 493SCCmec IVb

Type IVc1-FType IVc1-R5-TCT ATT CAA TCG TTC TCG TAT T-3 5-TCG TTG TCA TTT AAT TCT GAA CT-3 200SCCmec IVc

Type IVd1-FType IVd1-R5-AAT TCA CCC GTA CCT GAG AA-3 5-AGA ATG TGG TTA TAA GAT AGC TA-3 881SCCmec IVd

Type V-FType V-R5-GAA CAT TGT TAC TTA AAT GAG CG-3 5-TGA AAG TTG TAC CCT TGA CAC C-3 325SCCmec V

ccrC-FccrC-R5-CGT CTA TTA CAA GAT GTT AAG GAT AAT-3 5-CCT TTA TAG ACT GGA TTA TTC AAA ATA T-3 495ccr Tip 5

Table 4The nucleotide sequences of primers used, their specific nature and size of amplicons produced (by Zhang et al. 2005

The amplification program

Temperature94C94C65C72C94C55C72C72C

Duration5 min45 sec45 sec1,5 min45 sec45 sec1,5 min10 min

Number of cycle110251

Table 5 The conditions used for PCR amplification of the genetic elements according to the types of SCCmec cassettes (by Zhang et al. 2005)

The amplification of program

Temperature94C94C50C72C72C

Durattion5 min1 min1 min2 min10 min

Number of cycles1301

Table 6: PCR conditions used to amplify gene CCRC (by Zhang et al. 2005).*DreamTaqGreen(ThermoScientific,SUA),2x.All PCR reactions were performed using Thermal Cycler machine Corbet. The amplification products of each PCR reaction (multiplex / simplex) were visualized by electrophoresis on a 2% agarose gel, stained with ethidium bormur (10 g / ml) and identified based on their typical dimensions by means of specific molecular weight markers (100pb, I Lader Bench Top 100bp DNA, Promega, USA).

2. Results and discussion

Given the increased number of genetic polymorphisms detected in the SCCmec cassette is necessary to know the description and classification of them in order to understand the molecular epidemiology of S. aureus resistant to methicillin (MRSA), a powerful pathogen prevalent in both nosocomial and community. Given the increased number of genetic polymorphisms detected in the SCCmec cassette is necessary to know the description and classification of them in order to understand the molecular epidemiology of S. aureus resistant to methicillin (MRSA), a powerful pathogen prevalent in both nosocomial and community. Table 4 The results obtained in these experiments.

Bacterial strainsSCCmec cassetetes genes

ccrB2mecImecACIF2ccrCSCCmecVJ1TypeIVaTypeIVbTypeIVcTypeIITypeIVd

S.a.2+++

S.a 3+

S.a.4++

S.a.5+++

S.a.6++

S.a.7++

S.a.8+++

S.a.9+++

S.a.10+

S.a.11+

S.a.12

S.a.13+

S.a.14

S.a.15

S.a.16

S.a.17

S.a.18

S.a.19

S.a.20

The molecular analysis through PCR arrays showed that 8 strains revealed the ccrB2 gene, only 1 strain expressed the mecI gene (Tab4, Fig 1). In the case of the ccr genes the genotypic analysis pointed out that 9 strains presented the ccrC gene (Tab4, Fig 2). Regardingthe types of SCCmec cassettes 9 strains expressed the Type Iva (Tab4, Fig 3).

ccrB2-311 bp

mecl209 bp

Figure 1 . Gel electrophoresis with primers for corresponding elements of SCCmec cassettes: ccrB2, mecI, mecA, CIF2, RIF5. The figure shows that MRSA isolates 2, 3, 4, 5, 6, 7, 8, 9 express the ccrB gene and only strain no.2 revealed mecI gene. Well 1(top and bottom) marker gm: 100pb

ccrC-495 bp

Figure 2 Gel electrophoresis with primers for corresponding ccrC gene. Well 1(top and bottom) marker gm: 100pb. (Promega , USA). Wells 2-20: S.aureus strain analyzed . Wella: 2, 4, 5, 6, 8, 9, 14, 18, 19: positive.

Type IVa-776 bp

Figure 3. Gel electrophoresis with primers for corresponding Type Iva, Type IVb, zzType IVc, and Type II genes. Well 1 (top and bottom) marker gm : 100pb.(Promega, USA).Wells 3-22: S.aureus strain analysed. The wells: 5, 7, 8, 9, 10, 11, 13, 17, 19: positive for Type Iva element(776 bp)3. ConclusionIn the context of the emergence of a significant number of multi-resistant staphylococcal strains to antibiotics, the MRSA strains dominating [(In Romania, in 2010, between 25 and 50% of S. aureus strains isolated from blood cultures and CSF showed MRSA phenotype (ECDC Surveillance report, 2012)], understanding mecansimelor responsible for resistance is essential. emergence of MRSA strains associated with community infection makes it even more important to understand the genetic basis of phenotypic MRSA and horizontal transmission mechanisms, intra-and inter-species genetic elements responsible for these characteristics. Staphylococcal chromosomal SCCmec cassettes are carriers of genetic elements determining the Resistance so - lactam antibiotics and to other classes of antimicrobial substances. Genetic polymorphisms associated with these structures (Ito, T., Y. Katayama, and K. Hiramatsu. 1999), their variability and their acquisition mecansimele are particularly important to understand the structure and functionality, their correct classification in order to establish accurate data on the molecular epidemiology of MRSA strains. Bacterial strains analyzed is characterized by structural heterogeneity of SCCmec cassettes, most can, however, be included in boxes SCCmec type III and IV, the latter category, although characteristic of staphylococcal strains of Community origin are becoming more frequently reported in the context clinical. The strains considered non-tipabile by the methods used in the present study, further investigation is required to establish proper ownership SCCmec cassettes contained. Analysed strains is characterized by structural heterogeneity of SCCmec cassettes, most can,however, be included in boxes SCCmec type III and IV, the latter category, although characteristic of staphylococcal strains of Community origin are becoming more frequentlyreported in the context clinical