Epidemiologic and Molecular Characteristics of Staphylococcus aureus Strains Isolated From Hospitalized Pediatric Patients.

BACKGROUND: We aimed to determine molecular characteristics of Staphylococcus aureus isolates cultured from hospitalized pediatric patients. METHODS: All accessible S. aureus isolates cultured from hospitalized pediatric patients were analyzed for staphylococcal cassette chromosome mec (SCCmec) types, Panton-Valentine Leukocidin (PVL) encoding genes and antibiotic resistance patterns. RESULTS: A total of 132 S. aureus isolates, 102 methicillin-susceptible S. aureus (MSSA) (81.8%), 30 methicillin-resistant S. aureus (MRSA) (18.2%) were included in the study. Sixty of 132 (45.5%) S. aureus isolates were cultured from skin and soft tissue infections (SSTIs), 50 (37.9%) from bloodstream infections, 11 (8.3%) from bone infections and 11 (8.3%) from other sterile sites. Fifty-three of 102 (52%) MSSA isolates were cultured from SSTI, 35 (34.3%) from bloodstream infections, 7 (6.9%) from bone infections and 7 (6.9%) from other sterile sites (P = 0.083). Fifteen MRSA isolates (50%) were cultured from blood culture, 7 from (23.3%) SSTI, 4 (13.3%) from bone infections and 4 from (13.3%) other sterile sites. Nine PVL gene harboring S. aureus isolates were isolated from SSTI (75%), 2 from blood culture (16.7%) and 1 from other sterile site (8.3%). Three MRSA (6.7%) isolates were found to be positive for SCCmec type III and 16 MRSA isolates (53.3%) were found to be positive for SCCmec type IV. Three MRSA isolates harboring SCCmec type III was isolated from blood culture, 11 of 16 MRSA isolates harboring SCCmec type IV was isolated from blood culture, 3 isolates were isolated from bone infections and 2 isolates were isolated from SSTI (P < 0.001). Five of 72 (6.9%) hospital-acquired S. aureus isolates and 7 of 60 (11.7%) community-acquired S. aureus isolates were PVL gene positive. Twenty-two of 72 (30.6%) hospital-acquired S. aureus infections and 8 of 60 (13.3%) community-acquired S. aureus isolates were MRSA (P = 0.015). All of the 3 SCCmec III harboring MRSA isolates and 11 of 16 SCCmec IV carrying MRSA isolates were hospital acquired. Hospitalization in the past 1 year was found to increase MRSA infections 3.95 times (P = 0.038, 95% confidence interval: 1.078-14.48). CONCLUSIONS: As distribution of virulence genes differs among S. aureus isolates from different regions, it is necessary to monitor the emergence of genes encoding PVL, SCCmec in both MRSA and MSSA throughout the world. Our results show a high prevalence of PVL in community-onset S. aureus infections in children. SCCmec type IV was more commonly isolated in hospital-acquired MRSA isolates, and PVL gene was more commonly isolated in community-acquired S. aureus infections.

Absence of the mecC gene in methicillin-resistant Staphylococcus aureus isolated from various clinical samples: The first multi-centered study in Turkey.

BACKGROUND: mecA is a predefined gene causing methicillin resistance in Staphylococcus aureus (S. aureus) isolates; however, it has been shown that some methicillin-resistant S. aureus (MRSA) strains do not carry this gene. Recently, in isolates found to be MRSA-positive but mecA-negative, a new resistance gene called mecC, which is a homolog of mecA, has been reported. This study aimed to investigate the mecC and mecA genes in MRSA strains isolated from different geographic regions in Turkey. METHODS: The sample of the study consisted of 494 MRSA strains isolated from seven geographical regions in Turkey between 2013 and 2016. The strains were obtained from 17 centers, comprising 13 university hospitals, three education and research hospitals, and one state hospital. Methicillin resistance in S. aureus strains was determined using the agar disk diffusion method with a cefoxitin disk and the agar dilution method with oxacillin. The mecC and mecA genes in MRSA strains was investigated by Polymerase Chain Reaction (PCR). RESULTS: Of the MRSA strains investigated, 47.9% were isolated from intensive care units. Concerning sample type, 36.7% were detected in the respiratory tract (tracheal aspirate, sputum, etc.), 24.8% in blood, 18.7% in skin and soft tissues, 9.3% in nasal swabs, 5.4% in urine, 4.1% in ears, and 1% in sterile body fluid. Using PCR, mecC was not identified in any of the S. aureus strains isolated from different clinical microbiology laboratories. mecA gene positivity was found in 315 of the MRSA strains (63.8%). Staphylococcal Cassette Chromosome mec (SCCmec) type was identified in 232 strains (46.9%), of which 136 (58.7%) were type II, 75 (32.4%) were type IV, 12 (5.1%) were type IIIb, six (2.5%) were type I, and three (1.3%) were type III. CONCLUSION: This is the first multi-centered study to investigate MRSA strains isolated from different regions in Turkey. The mecC gene was not detected in any of the MRSA strains. We believe that this study will constitute an important basis for monitoring possible future changes.

[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.

Real-time PCR Detection of the Most Common Bacteria and Viruses Causing Meningitis.

BACKGROUND: Central nervous system (CNS) infections require prompt diagnosis, as the clinical condition progresses rapidly and may lead to severe permanent sequelae or death. The causative agents include viruses, bacteria, fungi, and parasites. In this study, samples with the diagnosis of CNS infection based on cerebrospinal fluid (CSF) sent to us from other hospitals/labs, were studied by multiplex real-time Polymerase Chain Reaction (PCR) method. The purpose of this study is to demonstrate, retrospectively, the most common bacteria and viruses causing meningitis and seasonal distribution of these agents using the multiplex real-time PCR method in CSF samples. METHODS: This study retrospectively evaluated the results of 470 CSF specimens that had been sent to the Molecular Unit of our hospital with a pre-diagnosis of CNS infection and had been tested with the PCR method between January 2014 and December 2015. Specimens were tested using multiplex real-time PCR assay for Adenovirus (AdV), Cytomegalovirus (CMV), Enteroviruses (EV) (Polioviruses, Coxackieviruses, Echoviruses, and other enteroviruses), Epstein- Barr virus (EBV), Herpes simplex virus 1 and 2, Human Herpes virus 6 and 7, Varicella-zoster virus (VZV), Human Parechoviruses and Parvovirus B19, Hemophilus influenzae, Streptococcus pneumoniae or Neisseria meningitidis. (FTD NEURO9 and FTD Bacterial meningitis, multiplex real-time PCR Kit). RESULTS: A bacterial or viral agent was identified in 98 (21%) of the 470 CSF samples. Of the patients, 85% were children and 15% were adults. Of the 98 positive samples, 22 (22.5%) patients were 15 years or older, and the remaining 76 (77.5%) were younger than 15 years. While Enterovirus (25%) was the most frequently identified agent, Adenovirus ranked second (22%) and Streptococcus pneumoniae ranked third (15%) in total. Positivity was highest in the 0 - 5-year age range. Bacteria were detected with the PCR method in 22 patients: S. pneumonia in 14, and N. meningitidis in 8. In cultures, S. pneumonia grew only in 7 and N. meningitidis in one. EV and AdV were seen in the summer months. The two coexisted in 3 (3%) patients. CONCLUSIONS: Early diagnosis and treatment of meningitis are very important for reducing its mortality and morbidity. In patients with suspected meningitis, early detection of the responsible agents may be possible with molecular methods, such as PCR. Significant economic benefits may be obtained by preventing unnecessary antibiotic use and hospitalizations through the early detection of the microbial agents.

Serotype distribution of Streptococcus pneumoniae in children with invasive diseases in Turkey: 2008-2014.

Successful vaccination policies for protection from invasive pneumococcal diseases (IPD) dependent on determination of the exact serotype distribution in each country. We aimed to identify serotypes of pneumococcal strains causing IPD in children in Turkey and emphasize the change in the serotypes before and after vaccination with 7-valent pneumococcal conjugate vaccine (PCV-7) was included and PCV-13 was newly changed in Turkish National Immunization Program. Streptococcus pneumoniae strains were isolated at 22 different hospitals of Turkey, which provide healthcare services to approximately 65% of the Turkish population. Of the 335 diagnosed cases with S. pneumoniae over the whole period of 2008-2014, the most common vaccine serotypes were 19F (15.8%), 6B (5.9%), 14 (5.9%), and 3 (5.9%). During the first 5 y of age, which is the target population for vaccination, the potential serotype coverage ranged from 57.5 % to 36.8%, from 65.0% to 44.7%, and from 77.4% to 60.5% for PCV-7, PCV-10, and PCV-13 in 2008-2014, respectively. The ratio of non-vaccine serotypes was 27.2% in 2008-2010 whereas was 37.6% in 2011-2014 (p=0.045). S. penumoniae serotypes was less non-susceptible to penicillin as compared to our previous results (33.7 vs 16.5 %, p=0.001). The reduction of those serotype coverage in years may be attributed to increasing vaccinated children in Turkey and the increasing non-vaccine serotype may be explained by serotype replacement. Our ongoing IPD surveillance is a significant source of information for the decision-making processes on pneumococcal vaccination.

The seroprevalence of both hepatitis B and hepatitis C at the first-step health organizations and the difference between the urban and rural areas.

BACKGROUND: Hepatitis B virus (HBV) and hepatitis C virus (HCV) are very important infectious agents for public health. The aim of this retrospective study was to assess the seroprevalence of hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (anti-HBs) and anti-HCV test results of patients who admitted to first-step health organizations in central and peripheral districts of Konya, the central region of Turkey during the period 2005-2010. METHODS: In this study, HBsAg, anti-HBs and anti-HCV screening test results of patients who admitted to first-step health organizations in Konya during the period 2005-2010 were retrospectively investigated from the laboratory records. This study was approved by the Konya Health Directorate. All screening tests were performed on the automatic third-generation enzyme-linked immunosorbent assay (MEIA). This immunoassay method was carried out according to the instructions of the manufacturer. Borderline and positive results were retested. RESULTS: Konya is the largest city of Turkey in terms of surface area and one of the economically developed cities. For HBsAg, anti-HBs and anti-HCV screening, whole test results of 5 years are given in Table 1 and Figure 1. The differences between the urban and rural for HBsAg (p = 0.062 > 0.05) and anti-HCV(p = 0.874 > 0.05) were not statistically significant. Among the markers only for anti-HBs, the difference between the urban and rural was statistically significant (P = 0.042 < 0.05). Of them, 4.15 % were positive for HBsAg, 36.46 % were positive for anti-HBs and 1.16 % were positive for anti-HCV. CONCLUSION: In this study, Konya has been evaluated as two regions: central and peripheral. Our study showed us that distribution of the diseases vary from one region to another. We consider that difference in social diversity is one of the factors. These infections are major health problems. So the results of immunodiagnostic tests for HBsAg, anti-HBs and anti-HCV will be useful for guiding control actions and for new preventive strategies.

[Investigation of Brucella canis Seroprevalence in Brucellosis Suspected Cases]. / Bruselloz Süpheli Olgularda Brucella canis Seroprevalansinin Arastirilmasi

Brucella canis which is the main etiologic agent of brucellosis in dogs, can be transmitted to man. It causes mild or asymptomatic infection in human compared with other Brucella species. B.canis can be transmitted to man either by laboratory accidents or contact with infected dogs. Since B.canis infections in humans are not routinely investigated in hospitals in Turkey, the data are limited to reveal the current status of B.canis infections in people in our country. The purpose of this study was to determine the seroprevalence of B.canis infection in brucellosis-suspected cases. The study was conducted at Konya Education and Research Hospital, (located at Central Anatolia of Turkey) during March-August 2010 period. Serum samples were obtained from 1000 patients (age range: 15-65 years; 652 of them were women) presented with brucellosis-like symptoms, including fever, headache, night sweats, appetite loss, weakness, arthralgia and myalgia. Rose Bengal Plate Tests (Seromed, Turkey) for smooth Brucella species were negative in all serum samples. Rough type B.canis antigen was prepared with B.canis NCTC 10854 strain for serodiagnosis. Antibody responses to B.canis in the serum samples were investigated by rapid slide agglutination test (SAT) and modified plate agglutination test (MPAT). Of the 1000 sera tested, 34 (0.34%) were found to be positive with SAT while the remaining were found negative. MPAT was used for the detection of antibody titer and 22 (0.22%) out of 1000 sera were found positive with MPAT (one had 1/48, five had 1/96, six had 1/192, six had 1/384, four had 1/768 titers). Among 22 positive patients, 17 were female and five were male, and the difference between the genders was found statistically significant (p< 0.05). It was concluded the use of both S and R antigens in the serological tests applied for the diagnosis of brucellosis in our country will supplement both diagnosis and seroepidemiological data related to brucellosis.

A comparison of immuncapture agglutination and ELISA methods in serological diagnosis of brucellosis.

BACKGROUND: Different serological tests are used in serologic diagnosis of brucellosis. The most widely used of these are Standard Tube Agglutination and Coombs anti-brucella tests. Whereas ELISA Ig M and Ig G tests have been in use for a long time, immuncapture agglutination test has been recently introduced and used in serological diagnosis. The aim of this study was to compare diagnostic values of ELISA Ig M and Ig G and immuncapture agglutination tests with Coombs anti-brucella test. METHODS: Sera from 200 patients with presumptive diagnosis of brucellosis were included into the study. Coombs anti-brucella test, ELISA Ig M and Ig G tests and Immuncapture test were investigated in these sera. Then, sensitivity, specificity, negative predictive and positive predictive values were calculated. RESULTS: Sensitivity, specificity, negative predictive and positive predictive values were found to be 90.6%, 76.3%, 94.2%, and 65.9% respectively for the Immuncapture test, whereas they were found to be 73.7%, 58.9%, 84.2%, and 42.8% for Ig G and 72.2%, 67.8%, 85.2%, and 48.7% for Ig M. The Immuncapture test was found to be compatible with ELISA Ig M and Ig G tests but it was statistically incompatible with Coombs anti-brucella test. CONCLUSIONS: Immuncapture agglutination test yields similar results to those of Coombs anti-brucella test. This test is a useful test by virtue of the fact that it determines blocking antibodies in the diagnosis and follow-up of brucellosis.

[Molecular epidemiology and antifungal susceptibility of Candida species isolated from urine samples of patients in intensive care unit]. / Yogun Bakim Ünitesinde Yatan Hastalarin Idrarlarindan Izole Edilen Candida Türlerinin Moleküler Epidemiyolojisi ve Antifungal Duyarliliklari

The aims of this study were to analyse the amphotericin B and fluconazole susceptibility and molecular epidemiology of Candida strains (Candida albicans, Candida tropicalis and Candida glabrata) isolated from the urine samples of patients hospitalized in the intensive care unit. Identification of the isolates was done according to microscopic morphology (chlamydospor, blastospor, pseudohyphae and true hyphae) on cornmeal agar, germ tube formation and carbohydrate assimilation patterns (API ID 32C bioMérieux, France). Antifungal susceptibilities of the isolates were determined by in vitro broth microdilution method recommended by Clinical and Laboratory Standards Institute (CLSI). To investigate the clonal relationship of the isolates, randomly amplified polymorphic DNA (RAPD) analysis was performed by using Cnd3 primer. Of the 56 Candida isolates minimum inhibitory concentration (MIC) ranges, MIC50 and MIC90 values for amphotericin B were 0.125-1 µg/ml, 0.125 and 0.5 µg/ml for C.albicans, 0.125-1 µg/ml, 0.25 and 1 µg/ml for C.tropicalis and 0.125-1 µg/ml, 0.25 and 1 µg/ml for C.glabrata, respectively. Fluconazole MIC ranges, MIC50 and MIC90 values were 0.25-4 µg/ml, 0.25 and 0.5 µg/ml for C.albicans, 0.25-16 µg/ml, 0.5 and 1 µg/ml for C.tropicalis and 0.5-64 µg/ml, 8 and 16 µg/ml for C.glabrata, respectively. For amphotericin B, none of the isolates had high MIC values (MIC > 1 µg/ml). While one of the C.glabrata isolates was resistant to fluconazole (MIC ≥ 64 µg/ml), one C.tropicalis and two C.glabrata isolates were dose-dependent susceptible (MIC: 16-32 µg/ml). The results of RAPD analysis indicated an exogenous spread from two clones for C.albicans, one clone for C.glabrata and one clone for C.tropicalis. This study underlines the importance of molecular epidemiological analysis of clinical samples together with hospital environmental samples in terms of Candida spp. To determine the exogenous origin for the related strains and to prevent nosocomial Candida infections.

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.