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The Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex consists of phenotypically very similar nosocomial species; A.baumannii, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii and Acinetobacter djikshoorniae and one environmental species A.calcoaceticus. The rapid and accurate identification of the members of Acb complex is critical as these nosocomial pathogens can show differences in antimicrobial susceptibility and clinical outcomes. The conventional phenotypic methods are slow, unreliable and less efficient for the differentiation of Acb complex species, including the A.baumannii species within the Acb complex. Although various molecular methods are available, such as amplified ribosomal DNA restriction analysis (ARDRA) and blaOXA-51-like gene specific PCR, they are usually inconvenient for the routine diagnostic laboratories. Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers an opportunity for rapid, cost-effective and convenient bacterial identification in routine diagnostic procedures conducted in clinical laboratory. In this study, we aimed to evaluate the diagnosis performance of MALDI-TOF MS system to identify blood isolates of A.baumannii. A total of 180 nonduplicate carbapenem resistant Acb complex (strain numbers; TR1-TR60) and A.baumannii (TR61-TR180) blood isolates were collected from the intensive care units of the three university hospitals in Turkey from January 2016 to December 2016. All isolates were evaluated by using blaOXA-51-like gene specific real time (Rt-PCR) analysis, ARDRA (restriction enzymes-AluI, CfoI, MboI, MspI, RsaI) method and MALDI-TOF MS (VITEK® MS, bioMerieux, France) system. All the strains except TR10, TR31, TR35 and TR52 were identified as A.baumannii by ARDRA method. Out of 177 of all the isolates, presence of blaOXA-51-like gene was found except for TR10, TR31 and TR52 isolates. However, TR31 without the presence of blaOXA-51-like gene was identified as A.pittii using the ARDRA. Totally 176 isolates which were identified as A.baumannii by both of the methods, ARDRA and Rt-PCR- blaOXA-51-like, were accepted as a reference for the evaluation of the diagnosis performance capacity of the MALDI-TOF MS. Overall, for all 176 isolates tested, the sensitivity obtained with the MALDI-TOF MS were 99.4% with 75% specificity. The accuracy value of the method was determined as 98.9% for the identification of A.baumannii to the species level. MALDI-TOF MS is increasingly used in diagnostic microbiology for the routine identification of bacteria to the genus, species or subspecies level with high rates of sensitivity and specificity. In future, by expanding the database, MALDI-TOF MS system would possibly become the ideal method for routine diagnostic laboratories that could potentially identify more species and even determine some characteristics of antimicrobial resistance and virulence determinants.

Salmonella infections can be seen in four clinical types, namely gastroenteritis, bacteremia/sepsis, enteric fever and carriage. These infections can result in uncomplicated diarrhea in most cases, but can lead to invasive disease requiring antimicrobial therapy and can be life-threatening in elderly or immunocomprimised patients. Broad-spectrum cephalosporins and fluoroquinolones are crucial options in the treatment of the invasive infections. Ciprofloxacin resistance is rarely seen in non-typhoid Salmonella enterica isolates, and only in S. Typhimurium, S. Choleraesuis and S. Schwarzengrund. In this report, we aimed to discuss a patient infected with ciprofloxacin-resistant Salmonella Kentucky under the light of data from our country and the world. A 52-year-old male patient wih acute myocardial infarction was hospitalized in intensive care unit of cardiovasculer surgery for left ventricular assist device (LVAD) implantation for the treatment of left ventricular disfunction. On the seventh day of LVAD and coronary artery bypass grafting (CABG), the patient presented high fever and productive cough. His physical examination revealed hyperemia around the insertion point of right jugular central venous catheter (CVC) and a serous discharge from the insertion point of LVAD located just below the inferior edge of sternum. Empiric IV cefoperazone/sulbactam (SCF) therapy was started with the prediagnosis of pneumonia and bloodstream infection. The blood samples taken from peripheral veins and CVC, and swabs taken from LVAD insertion point for culture when the patient was febrile, revealed the growth of bacteria with S type and lactose-negative colonies on EMB and SS media. Biochemical characteristics of the isolate were as follows: lactose fermentation negative, H2S positive, IMVIC (-,+,-,+), urease negative, lysine/ornithine decarboxylase positive and motile. The bacteria was then identified as Salmonella enterica serotype Kentucky (8,20;i;z6) by agglutination tests. Antibiotic susceptibility tests were conducted according to CLSI guidelines and it was found as ampicillin- and ciprofloxacin-resistant. Ciprofloxacin resistance of the isolate was confirmed with E-test. Stool culture was performed to investigate the source of infection, and S. Kentucky was isolated. On the 15th day of SCF treatment, LVAD was taken out, and tissue cultures taken from the fibrillar tissues between pericardial layers during surgery, also yielded S. Kentucky growth. On the second day of SCF therapy the patient's fever returned normal and on the seventh day, CBC and CRP values were normalized. Nevertheless, the clinical situation of the patient worsened gradually and on the 40th day he was intubated due to low oxygen saturation and pleural effusion. His antibiotherapy was stopped on 42nd day as the blood cultures were negative and his clinical situation was attributed to cardiac failure. The patient died four days after the antibiotherapy has stopped due to cardiac reasons. To our knowledge, this is the first reported case infected with ciprofloxacin-resistant Salmonella Kentucky in our country.

The diagnosis of human brucellosis requires culture or serological tests for the conformation of the clinical findings. Isolation of the bacteria is used as a gold standard, however it is time consuming and processing of positive cultures has a potential risk for laboratory acquired human brucellosis. Polymerase chain reaction (PCR) based methods have offered new approaches for early diagnosis of brucellosis and reduce the risk of laboratory acquired human brucellosis. A major limitation of the PCR method is the difficulty to remove the inhibitors in specimens. The aim of this study was to determine the performance of two DNA extraction kits by using two separate PCR master mixes and to determine appropriate "extraction kit - PCR master mix" combination for the diagnosis of Brucella from whole blood samples and blood culture bottles. Two commercial DNA extraction kits, NORGEN Blood DNA isolation kit (Norgen) and Thermo Scientific GeneJet Whole blood genomic DNA purification kit (Thermo Fisher Scientific, USA) and two PCR master mixes, QuantiTect multiplex PCR (QuentiTect, Qiager, Almanya) and Ampliqon Multiplex TEMPase (Amliqon, Denmark) were assessed on 30 simulated blood samples with known concentrations (102-104 cfu/ml) of Brucella melitensis ATCC 23456 strain and 10 blood culture bottles that gave positive signal. By using different combinations of extraction kits and PCR master mixes, a total of 160 different multiplex real-time PCR (Rt-PCR) trials were performed with probes and primers specific to Brucella spp., B.melitensis, and the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the 120 Rt-PCR trials performed on the DNA samples extracted from blood samples gave positive results with GAPDH probe/primers. The rate of positive PCR results for Brucella spp. was 96.7% for the combination of Norgen-QuantiTect, 93.3% for Thermo-Ampliqon, 93.3% for Thermo-QuantiTect, and 86.7% for Norgen-Ampliqon. The frequency of positive B.melitensis results for these combinations were 96.7%, 93.3%, 56.7% and 90%, respectively. In the samples with the bacterial density of 102 cfu/ml, Brucella spp. detection rates were 80% for Thermo-Ampliqon and Norgen-Ampliqon, and 90% for Thermo-QuantiTect and Norgen-QuantiTect; and for B.melitensis positivite rates were 90%, 70%, 20%, and 90%, respectively. Rt-PCR assays with the DNA samples extracted from blood culture bottles using Norgen isolation kit yielded 80% positivite result. However, the frequency of PCR positivite results was only 20% in the DNA samples extracted by Thermo DNA extraction kit. PCR result for GAPDH gene was also negative in 80% of the samples extracted by Thermo kit. Our results revealed that for the removal of inhibitors and detection of even low number of Brucella spp./B.melitensis in blood samples and blood culture bottles, NORGEN Blood DNA isolation kit can be used with a combination of QuantiTect multiplex PCR or Ampliqon Multiplex TEMPase.