[Investigation of Streptococcus pyogenes virulence factors and typing by multiple locus variable number tandem repeat fingerprinting (MLVF) method]. / Streptococcus pyogenes izolatlarinin virülans faktörlerinin arastirilmasi ve "multiple locus variable number tandem repeat fingerprinting (MLVF)" yöntemi ile tiplendirilmesi.

Streptococcus pyogenes is an important bacterial pathogen that colonizes the throat and skin of human beings and causes a wide variety of diseases ranging from mild infections like pharyngitis, tonsillitis and impetigo to severe invasive infections such streptococcal toxic shock syndrome, septicemia, and necrotizing fasciitis, and produces a wide variety of virulence factors. The aim of this study was to investigate the antibiotic resistance, virulence genes; [pyrogenic exotoxin genes (speA, C, G, H, I, J, K, L, M, smeZ and ssa), deoxyribonuclease genes (sdaB, spd3, sdc ve sdaD), protease genes (speB, spyCEP ve scpA) and inhibitor genes (mac and sic)] of S.pyogenes strains isolated from throat cultures of patients with symptomatic tonsillo-pharyngitis and typing by multiple locus variable number tandem repeat fingerprinting (MLVF) method. One hundred and fifty S.pyogenes isolates were identified by conventional methods and streptococcus group A latex kit (Biomerieux, France). Antibiotic susceptibility tests were performed by Kirby-Bauer disk diffusion method as recommended by Clinical and Laboratory Standards Institute. DNA isolation was performed by using a commercial DNA isolation kit (Qiagen, Germany) in accordance with manufacturer's recommendations. The virulence genes were determined by multiplex PCR. MLVF method was performed with multiplex PCR using specific primers for repeated sequences within bacterial genome. All of the S.pyogenes isolates were susceptible to penicillin G, cefotaxime, ceftriaxone, chloramphenicol, clindamycin, erythromycin, levofloxacin, vancomycin and linezolid. Among streptococcal pyrogenic exotoxin genes the most frequent gene was smeZ (90.0%) followed by speG (88.0%), speC (58.7%), ssa (42.7%), speA (33.3%), speJ (24.0%), speK (18.7%), speH (14.0%), speI (13.3%), speL and speM (9.3%). Of the DNase genes, sdaB was detected in all strains (100%), spd3, sdc, sdaD genes were determined as 64.7%, 36.0%, 24.7% respectively. Protease genes (speB, spyCEP, scpA) and mac gene from the inhibitor genes were positive in all strains, and sic gene was positive in only 3 (2.0%) of the isolates. Thirty-two different patterns that contained two or more isolates were determined by MLVF analysis. Ninety one isolates were included in any of the 32 different patterns, while 59 isolates were defined as sporadic isolates. In conclusion, S.pyogenes isolates collected from throat cultures of patients with symptomatic tonsillo-pharyngitis in Konya/Turkey were susceptible to all antibiotics studied and have carried a very high rate of virulence factors. However the isolates were mostly clonally unrelated and sporadic. This study is the first report in Turkey, in which S.pyogenes isolates were typed by the MLVF method and a large number of virulence factors were investigated.

Two outbreaks of ESBL-producing Klebsiella pneumoniae in a neonatal intensive care unit.

BACKGROUND: In the present study, two epidemic episodes of extended spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in the neonatal intensive care unit (NICU) were evaluated. METHODS: Routine and surveillance culture samples were taken from seven neonates with signs of infection in the NICU of Selcuk University Faculty of Medicine between 10 March and 25 April 2011, and between 11 June and 30 September 2011. RESULTS: ESBL-producing K. pneumoniae strains were isolated in six different samples (one wound, one blood, and four cerebrospinal fluid cultures) of the three neonates in the first episode and in 11 different samples (seven blood and four cerebrospinal fluid cultures) of the four neonates in the second episode. ESBL-producing K. pneumoniae was isolated from inguinal, axillar region, and stool samples of the nine colonized neonates in the second episode. It was determined on pulse field gel electrophoresis that all strains originated from two clones. CONCLUSIONS: The deficiencies in the infection control measures in an NICU may transform into an epidemic rapidly. Therefore, periodic training, observation, and monitoring of compliance are important.

[In vitro synergistic activity of sulbactam in combination with imipenem, meropenem and cefoperazone against carbapenem-resistant Acinetobacter baumannii isolates]. / Karbapeneme dirençli Acinetobacter baumannii izolatlarina karsi sulbaktam ile imipenem, meropenem ve sefoperazon kombinasyonlarinin in vitro sinerjistik aktivitesi.

Acinetobacter baumannii which is an opportunistic pathogen leading to nosocomial epidemics, exhibit high rates of antimicrobial resistance. Treatment of Acinetobacter infections is a challenge since most of the isolates are multiple antibiotic resistant. The aim of this study was to investigate minimum inhibitory concentrations (MICs) of sulbactam, imipenem, meropenem, and cefoperazone and in vitro synergistic activity of sulbactam in combination with imipenem, meropenem and cefoperazone against A.baumannii isolates of hospitalized patients. Forty A.baumannii strains isolated from various clinical specimens and found to be resistant to carbapenems by disc diffusion method, were included in the study. The isolates were identified by conventional methods and VITEK 2 (bioMerieux, France) automated identification system. MICs of sulbactam, imipenem, meropenem, and cefoperazone were determined by the broth microdilution method according to the standards of CLSI and in vitro synergy test was performed using the checkerboard microdilution method. Synergistic, partial synergistic, additive, indifferent and antagonistic effects of drug combinations were evaluated with the fractional inhibitory concentration index (FICI). Interpretation of the FICI was as follows: ≤ 0.5 synergy; > 0.5 to < 1 partial synergy; 1 additive; > 1 to < 4 indifference; and ≥ 4 antagonism. Forty A.baumannii isolates were resistant to imipenem and cefoperazone, but two were susceptible, seven were moderately susceptible and 31 were resistant to meropenem with the microdilution method. MIC values of the isolates for sulbactam were found to be 4 µg/ml in two, 8 µg/ml in five, 16 µg/ml in three, 32 µg/ml in 13, 64 µg/ml in three, 128 µg/ml in six and > 128 µg/ml in eight isolates. According to the FICI; imipenem/sulbactam combination exhibited synergy in 18 (45%), partial synergy in 4 (10%) and indifferent effect in 2 (5%) isolates, the combination of meropenem and sulbactam showed synergy in 19 (48%), partial synergy in 3 (7.5%), and indifferent effect in 3 (7.5%) isolates, the combination of cefoperazone/sulbactam demonstrated synergy in 18 (45%), partial synergy in 2 (5%), and indifferent effect in 2 (5%) isolates. There was no antagonistic effect with the tested combinations. In conclusion, MIC values of sulbactam were generally high in carbapenem-resistant A.baumannii strains. However, synergistic effect was detected in approximately half of the strains with the sulbactam/carbapenem combinations. The data obtained in this study should be supported by further advanced in vitro and clinical studies to predict the accurate clinical efficacy of sulbactam containing combinations on A.baumannii infections.

[Distribution of emm genotypes and antibiotic susceptibility of Streptococcus pyogenes strains: analogy with the vaccine in development]. / Streptococcus pyogenes suslarinin emm genotiplerinin dagilimi ve antibiyotik duyarliligi: Gelistirilmekte olan asi ile karsilastirma.

Streptococcus pyogenes is the most common bacterial pathogen causing pharyngotonsillitis, and also can lead to diseases such as otitis media, impetigo, necrotizing fasciitis, bacteremia, sepsis and toxic shock-like syndrome. M protein encoded by emm gene is an important virulence factor of S.pyogenes and it is used for genotyping in epidemiological studies. The aims of this study were to determine the M protein types of group A streptococci (GAS) by using emm gene sequence analysis method, to compare the M types in terms of analogy with the vaccine in development and to determine the antibiotic susceptibilities of the isolates. A total of 35 GAS strains isolated from various clinical specimens in our laboratory were included in the study. Strains growing in blood culture were considered as invasive, strains growing in throat and abscess cultures were considered as non-invasive. The isolates have been identified by conventional methods and 16S rRNA sequence analysis at species level. emm genotyping of strains identified as S.pyogenes, was performed by PCR method as proposed by the CDC. Amplicons were obtained and sequenced in 23 out of 35 isolates. The results were compared with CDC emm sequence database. Antibiotic susceptibility of the isolates was performed by agar dilution method and evaluated as recommended by CLSI. Twenty-three out of 35 isolates could be typed and 15 different emm genotypes were detected. The most common emm types were emm1 (22%), emm89 (13%), emm18 (9%) and emm19 (9%). The detection rate of other emm types (emm5, 12, 14, 17, 26, 29, 37, 74, 78, 92, 99) was 47%. Types emm1, 12, 19, 74, 89 and 99 were observed in strains isolated from blood cultures. It was detected that nine of the 15 (60%) emm types are within the contents of 26 valent vaccine (emm 1, 5, 12, 14, 18, 19, 29, 89, 92). It was also observed that 17 (74%) of the 23 cases were infected by vaccine types and the four emm types (emm1, 12, 19, 89) identified in blood samples were among the vaccine types. All of the strains were found susceptible to penicillin, ampicillin, erythromycin, lincomycin, gentamicin, chloramphenicol, vancomycin and linezolid, however six isolates were resistant to levofloxacin (MIC= 4 and 16 µg/ml) and one isolate was resistant to tetracycline (MIC= 16 µg/ml). In conclusion, this preliminary local study with limited number of invasive and non-invasive S.pyogenes isolates, emphasized the need for larger scale multi-center studies to determine the analogy and efficacy of the vaccine in development.

[MLST types of vancomycin-resistant Enterococcus faecium strains isolated from blood cultures]. / Kan kültürlerinden izole edilen vankomisine dirençli Enterococcus faecium suslarinin MLST tipleri.

Enterococci, particularly vancomycin-resistant enterococci (VRE), are important nosocomial pathogens with limited treatment options. Enterococci have low-level resistance to penicillins and aminoglycosides and are intrinsically resistant to cephalosporins. In addition, they can acquire high-level resistance to beta-lactam antibiotics, aminoglycosides and glycopeptides. The aim of this study was to determine glycopeptide resistance mechanisms and genetic relationships of vancomycin-resistant E.faecium strains isolated from blood cultures between 2003-2009 years by molecular epidemiologic methods. A total of 38 VRE strains isolated from blood cultures were included in this study. The isolates were identified by conventional methods and Phoenix 100 BD automated system (Becton Dickinson Diagnostic Systems, USA) and confirmed by sequence analysis of 16S rRNA amplicons. Antibiotic susceptibility tests were performed by the Kirby-Bauer disk diffusion method accor-ding to the CLSI standards. MIC values of vancomycin were determined in vancomycin resistant strains by E-test (AB Biodisk, Sweden) method. Vancomycin resistance genes included vanA, vanB, vanC, and vanD were investigated by polymerase chain reaction (PCR) method. Clonal relationship between strains was determined by pulsed-field gel electrophoresis (PFGE). Sequence analysis was performed for examples selected for multilocus sequence typing (MLST) of each pulsotype and subtype. Thirty eight strains of enterococci isolated from blood cultures were defined as E.faecium by phenotypic methods and confirmed by 16S rRNA sequence analysis. Vancomycin MIC values of strains were determined as > 256 µg/ml by E test. The vanA gene was detected in all isolates. Clonal relationship of 38 isolates E.faecium carrying the vanA gene was determined by PFGE and MLST methods. PFGE detected four pulsotypes (A-D) and one sporadic isolate. Twenty nine strains belonged to A pulsotype, three strains belonged to B pulsotype, two strains belonged to C pulsotype and three strains belonged to D pulsotype. Out of 29 isolates, eight strains were type A1, nine strains were type A2, six strains were type A3, two strains were type A4 and four strains were type A5. MLST identified four different sequence types (STs). Twenty nine A pulsotype and its subtypes belonged to ST117 (76.3%), three B pulsotype belonged to ST280 (7.9%), two C pulsotype belonged to ST18 (5.2%) and three D pulsotype belonged to ST17 (7.9%). In conclusion, bloodstream infections caused by VRE in our hospital arose from a dominant strain belonged to ST117. However, presence of different pulsotypes of this strain indicated that the strain had been present in the hospital for a long time and had accumulated genetic variations. In addition, infections caused by minor pulsotypes were also detected. Therefore for prevention and control of the spread of nosocomial infections caused by VRE, it is crucial to identify resistance patterns and clonal relationship of these organisms.

Serratia marcescens sepsis outbreak in a neonatal intensive care unit.

BACKGROUND: Contaminated parenteral nutrition (PN) is an important source of infection in neonates. Many organisms have been reported to cause contamination resulting in outbreaks in intensive care units. The aim of the present study was to investigate an outbreak caused by Serratia marcescens in a neonatal intensive care unit (NICU). METHODS: This was a descriptive study of an outbreak of sepsis in an NICU of a university teaching hospital. The outbreak was detected in seven patients from 10 to 12 December 2005 following the administration of PN. Extensive environmental samplings for culture were performed. The clonal relationship among isolates was tested using pulsed-field gel electrophoresis, random amplification of polymorphic DNA-polymerase chain reaction and plasmid DNA typing. RESULTS: Serratia marcescens was found in blood cultures from infected newborns and from in-use PN solutions. Gestational age of the seven babies ranged from 28 to 34 weeks (median, 32 weeks), birthweight ranged from 1000 g to 2190 g (median, 1469 g), and postnatal age ranged from 8 to 22 days. The mortality rate was 14.3%. All these strains of S. marcescens had the same antibiotic susceptibility pattern and the same genomic DNA profile. Plasmid typing, as well as RAPD-PCR showed that all isolates had the same profile. CONCLUSION: The source of the nosocomial sepsis in seven neonates was the PN solution. Contamination may occur during storage or repeated handling during PN preparation.

[Investigation of the clonality and Panton-Valentine leukocidin toxin among nosocomial methicillin-resistant Staphylococcus aureus strains]. / Hastane kaynakli metisiline dirençli Staphylococcus aureus suslari arasinda klonalitenin ve Panton-Valentin lökosidin toksininin arastirilmasi.

Methicillin-resistant Stapyhlococcus aureus (MRSA) is one of the major causes of morbidity and mortality in hospitalized patients. This study was aimed to investigate the clonality of the MRSA strains isolated from patients with nosocomial infection and also to determine the presence of Panton-Valentine leukocidin (PVL) toxin in these isolates. A total of 37 samples (31 isolated from surgical wound samples, 2 them from abscess and 4 from drainage samples) obtained from patients hospitalized at surgery, internal medicine and intensive care units, were included to the study. The clonality among MRSA strains was demonstrated by pulsed-field gel electrophoresis (PFGE) and the presence of PVL by polymerase chain reaction using luk-PV-1 and luk-PV-2 primers. PFGE revealed that 31 of 37 strains were A pulsotype and subtypes, 3 strains were B pulsotype and the last 3 were C pulsotype. Pulsotype A has been isolated especially from cardiovascular surgery and other surgery departments and intensive care units, pulsotype B from orthopedic and pulsotype C from neurology and neurosurgery wards. PVL gene was not identified in any of the isolates. These results indicated the presence of a dominant clone among MRSA strains in our hospital, however, different pulsotypes may also be present in different surgery units. Continuous molecular epidemiological surveillance of nosocomial MRSA strains and their PVL positivity supply valuable clinical and epidemiological data for infection control and patient follow-up.

First case report of neurobrucellosis associated with hydrocephalus.

Brucellosis is a common zoonosis in many parts of the world, including Mediterranean and Middle Eastern countries. The disease is primarily related to occupations at risk, such as veterinarians, farmers, laboratory technicians, abattoir workers, and others working with animals and their products. Neurologic complications of brucellosis are quite rare, ranging from 1.7 to 10% of those infected. To date, no cases of neurobrucellosis with hydrocephalus have been reported. A 38-year-old right-handed farmer complained of headaches, nausea, vomiting, gait disturbance, and sweating for 2 days. He also complained of bilateral hearing loss of 4 months duration. On neurologic examination, dysmmetry, dysdiadochokinesis, ataxia on the left, and bilateral sensorineural hearing loss existed. On cranial MRI, a communicating hydrocephalus was noted. Because the patient consumed fresh sheep cheese and was a farmer, brucellosis was considered in the differential diagnosis. Brucella agglutination was positive with a 1/320 titer in the blood and a 1/80 titer in the cerebrospinal fluid. Ceftriaxone, doxycycline, and rifampicin were administered and by the fourth week of treatment, the ataxia was markedly improved, and the patient was able to walk without support. His cranial MRI demonstrated a total regression of the hydrocephalus. As a result, we suggest that neurobrucellosis should be considered in patients with hydrocephalus, especially if they live in an endemic area for brucellosis, even in the absence of other systemic signs.

[Short communication: Extended-spectrum beta-lactamase production in Klebsiella pneumoniae strains isolated from blood cultures and their antibiotic susceptibilities]. / Kisa bildiri: Kan kültürlerinden izole edilen Klebsiella pneumoniae suslarinda genislemis spektrumlu beta-laktamaz varligi ve antibiyotik duyarliliklari.

This study was carried out to detect the extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae strains isolated from blood cultures of hospitalized patients, and to determine their antimicrobial susceptibilities. A total of 102 K. pneumoniae strains isolated from blood samples were taken in the study, and ESBL production and susceptibilities to amikacin, gentamicin, imipenem, ciprofloxacin, amoxicillin/clavulonate (AMX/CA), ceftazidime, ceftriaxone, trimethoprim/ sulphametoxazole (TMP-SMX), piperacilin-tazobactam (PIP/TAZ) and chloramphenicol were investigated by using E-test (AB Biodisk, Sweden). ESBL positivity was observed in 65 (63.7%) of the isolates, and all of the strains were found susceptible to imipenem. The resistance rates of ESBL-producing isolates were detected as 27.7% for amikacin, 41.5% for chloramphenicol, 49.2% for TMP-SMX, 55.4% for ciprofloxacin and 60% for PIP/TAZ; whereas these rates for ESBL non-producers were 2.7%, 5.4%, 5.4%, 2.7%, and 13.5%, respectively. Both the resistance rates and MIC values (MIC50 and MIC90) of the tested antimicrobial agents except imipenem, were found higher in ESBL positive strains than the ESBL negative strains (p < 0.05). The results of this study, in accordance with the previous national and international reports, indicated high rate of ESBL positive K. pneumoniae and also increased rate of antimicrobial resistance in such strains. Clinical microbiology laboratories should put ESBL detection tests into practice and each hospital should determine their antibiotic treatment policies according to their data.

Pleuropulmonary and soft tissue Nocardia cyriacigeorgici infection in a patient with Behcet's disease.

Infections with Nocardia species are generally seen in immunocompromised subjects. In this report, we present a case of pleuropulmonary and skin Nocardia cyriacigeorgici infection in a male patient with Behcet's disease who used corticosteroids and immunosupressives for a long period of time. He died before the diagnosis of Nocardia infection was made.

[Salmonella enterica serotypes and Salmonella infections: a multicenter study covering ten provinces in Turkey]. / Salmonella enterica serotipleri ve Salmonella enfeksiyonlari: Türkiye'de on ili kapsayan çok merkezli bir çalisma.

In order to find the distinctive features of Salmonellae and Salmonella infections in Turkey, 620 Salmonellae strains, isolated from various clinical samples (481 stool, 108 blood, 12 urine, 3 bone marrow, 3 cerebrospinal fluid, 9 pus, and one from each of the bile, pleural fluid, wound, catheter samples) in 13 clinical microbiology laboratories of 10 provinces in Turkey (Ankara, Antalya, Bursa, Edirne, Eskisehir, Istanbul, Izmir, Kayseri, Konya and Trabzon) between July 1, 2000 and June 30, 2002, were serotyped. Among the patients 43% were female, 57% were male, 63.2% were from outpatient clinics and 36.8% were hospitalized patients. Seventy eight percent of the patients had gastroenteritis, 10.7% had septicemia/local infection, 9.8% had typhoid/paratyphoid fever and 1.5% were carriers. Incidence of gastroenteritis was higher in 0-5 years age group (p<0.001). Of the 620 Salmonella enterica isolates, 47.7% were S. Enteritidis, 34.7% S. Typhimurium, 6% S. Paratyphi B, 2.9% S. Typhi, 0.2% S. paratyphi A, 6.1% serogroup C1, and 2.4% serogroup C2. S. Enteritidis was the most common serotype in all provinces except for Kayseri, where S. Typhimurium was found to be the most common serotype (68.2%). Overall, the most frequently isolated serotype was S. Enteritidis, also being the most common serotype in stool and blood cultures. During the surveillance period two outbreaks have occurred, the first one by S. Enteritidis strains in Edirne, and the second one by S. Typhimurium strains in Kayseri. As a result, Salmonella infections are still a common health problem in Turkey, and active surveillance of Salmonella infections has vital importance.

Late-onset Streptococcus pasteurianus sepsis in a preterm baby in a neonatal intensive care unit.

Apnea, cyanosis, lethargy and prolongation in capillary filling time developed on the postnatal 37(th) day in a preterm baby who was born at the 30(th) gestational week with a birth weight of 1 300 g. Acute phase reactants and immature/total neutrophil count ratio were found to be high. The patient who was diagnosed with sepsis was successfully treated with meropenem which was started empirically. In his blood culture Streptococcus pasteurianus grew. S. pasteurianus is in the subgroup of streptococcus bovis which is one of the D group streptococci and its previous name is S. bovis type II/2. In the literature, there are very few cases of neonatal infection related with this bacterium. As far as we know, this is first case of neonatal sepsis caused by S. pasteurianus in Turkey. In addition, we tried to determine the clinical properties of neonatal infections arising from S. pasteurianus by reviewing the literature.

Streptococcus zooepidemicus meningitis and bacteraemia.

Group C streptococci are common causative agents of epidemic infections in animals and a rare cause of meningitis in humans. The case is reported of a 75-y-old man with meningitis caused by a group C streptococcus (Streptococcus zooepidemicus). He had frequent contact with horses, which were a possible source of infection. In spite of treatment with a third generation cephalosporin, the outcome was fatal.