ACTA Microbiologica Hellenica (Volume 64, Issue 3)

Βasic principles of sonication method for detaching viable microorganisms embedded in implant biofilm

Nikolaos D. Frangoulis, Antonios Stylianakis
Microbiology Department, General Hospital of Athens KAT, Athens, Greece

Ultrasound performance has been used over the last decade for diagnosis of implants related infections, a serious complication of device surgery. It’s a relatively novel method, fast developing and much promising on solving diagnostic and therapeutic problems of Clinical Microbiology,and is well known as sonication or ultrasonication. The basic idea of this method is the detachment of alive bacteria from their covering implant biofilm.
Formation of biofilm creates serious difficulties in the detection of the causative bacteria of implant related infections by the conventional methods of microbiology and in consequent antimicrobial chemotherapy as well. Detachment is achieved by the use of low frequency ultrasound waves that causes asymmetric cavitation to sonication fluid’s microbubbles on the surface of the examined implant. Sonication fluid is then cultured quantitatively or examined by molecular techniques. Description and analysis of the basic principles of the ultrasound method will increase our knowledge about it and help a lot to its improvement and evolution. In this study we revise the basic biological, physicsand technical principles of the assay, as well as its research aspects, and we extract conclusions about its value and perspectives, focusing on diagnosis of implant associated infections.

Keywords: implant associated infections, biofilm, sonication, microbubbles,asymmetric cavitation.

Prevalence and molecular characterization of Mycobacterium tuberculosis resistance to aminoglycosides in the Northeast of Iran

Fatemeh Askarizadeh 1,2, Ehsan Aryan1,2, Seyed Isaac Hashemy3, Masoud Youssefi1,2, Hadi Farsiani1,2, Kiarash Ghazvini1,2

1.Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
2.Department of Microbiology and Virology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
3.Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

Background: Tuberculosis is an important cause of death with an increased antibiotic resistance.Kanamycin and amikacin are among the most effective aminoglycosides for multidrug-resistant strains. We investigated the resistance to these two antibiotics in 111 susceptible and 9 resistant specimens.
Materials and Methods: Phenotypic method was used to assess the resistance, PCR method was performed for rrs and eis genes and the results confirmed by sequencing.
Results: No resistance against kanamycin and amikacin was observed in susceptible specimens and only three resistant samples had such resistance.
Two had mutations at A1401G nucleotide, and the nucleotide change of G>A was observed at 1484 position in one sample. Mutations at A514C, A907C, G1332A positions were observed in one sample. eis gene sequencing revealed that one sample had mutation in promoter region at A-13G position.
Conclusion: The most important mutation was at nucleotide A1401G in rrs gene and a low prevalence of aminoglycoside resistance was observed in northeastern Iran.

Keywords: Tuberculosis, Resistance, Aminoglycosides, MDR-TB

Temperature, osmotic and pH stress are associated with antibiotic susceptibility changes in Staphylococcus aureus

Iraj Nikokar1,2, Haleh Ganjian2
1.Department of Microbiology, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran
2.Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences,
Rasht, Iran

The role of bacteria, especially Staphylococcus aureus in health and processing of food are inevitable.During food processing, bacteria are exposed to a variety of stresses and effect on bacterial physiology, which could be varied based on pathogen and nature of stress. The present study aimed to investigate the probable effect of some environmental stresses on the antibiotic susceptibility of S. aureus toward different antibiotic.Resistant subpopulation of S. aureus ATCC 25923 was selected by exposure to different environmental stresses and subjected for further analysis. The antibiotic susceptibility of unstressed (control) and stressed S. aureus strain was performed by the disk diffusion method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the pooled proteins from each condition. Antibiotic susceptibility results revealed all unstressed strains were susceptible to tested agents, while stressed isolated showed a different susceptibility pattern. In general, acid, alkaline and cold stresses were associated with increasing the resistance to tested antibiotics. However, high temperature and osmotic pressure by glucose concentration were mostly associated with increased susceptibility to antibacterial agents. Analysis of SDS-PAGE results showed protein profile changes in stressed cells compared to unstressed strain. In summary, our results indicated that S. aureus strains could undeniably tolerant wide range of environmental stresses and successfully growth after stress elimination. Moreover, we foundthat survived stressed S. aureus are mostly associated with changes in antibiotic susceptibility.

Key words: Staphylococcus aureus, Antibiotic susceptibility, Environmental stress, SDS-PAGE

Composition of children pharyngeal microbiome by 16S rRNA deep sequencing

Georgia Gioula, Angeliki Melidou, Panayotis Siasios, Fani Minti, Nikolaos Malisiovas
Microbiology Department, School of Medicine, Faculty of Health Science, Aristotle University of Thessaloniki,
Thessaloniki, Greece

The upper respiratory tract (URT) is colonised by a large variety of bacteria that constitute the respiratory microbiota. Most do not have a role and may even be protective. The aim of the present study was to analyse the microbiome and characterize the relative abundance of microbialcommunities of the pharynx by next generation sequencing of the 16S rRNA gene of bacteria in healthy children and children with a respiratory infection. Ten phyla were identified in the 8 study subjects. The most abundant phyla detected, were Firmicutes, Proteobacteria and Bacteroidetes, while the relative abundance of each was highly variable across the subjects. At the family, genus and species level, 24 families, 19 genera and 71 species respectively were common both in patients and healthy subjects, while some (28 families and 24 genera) were identified only in healthy subjects and few (7 families, 8 genera and 9 species) were identified only in patients.No statistically significant differences were observed in relation to the age or gender ofthe subjects. Interestingly, the most abundant bacteria detected in healthy children were Streptococcus, Prevotella, Moraxella, Veillonella and Leptotrichia, while in young patients Moraxella was not detected among the most prevalent bacteria, supporting the notion that it may play a protectiverole in infection. Protective and pathogenic bacteria have been identified in healthy childrenand in patients. Such studies can form the basis for new approaches to fight diseases responding poorly to traditional interventions.

Key words: microbiome, 16SrRNA, respiratory, children, deep sequencing

Epidemiological data of Coxsackie and Echo viruses in N. Greece

K. Sidiropoulou 1, S. Mamalis 1, M. Christoforidi 2, M. Exindari 3
1.Medical Doctor, Department of Microbiology, School of Medicine, Aristotle University of Thessaloniki
2.Biologist, Department of Microbiology, School of Medicine, Aristotle University of Thessaloniki
3. Associate Professor of Microbiology, MD, Department of Microbiology, School of Medicine, Aristotle University of

The circulation of Coxsackie and Echo viruses is investigated in a period of fourteen years. Clinical data were reported and IgM and IgG specific antibodies were detected by ELISA and IFA techniques for 6654 specimens (5368 for Coxsackie, 1286 for Echo) collected in hospitals of N. Greece. The results revealed 653 IgM positive and 2234 IgG positive specimens for Coxsackie as well as 98 IgM positive and 189 IgG positive specimens for Echo viruses. Coxsackie and Echo infections affected both genders and a statistically significant increase of IgM Coxsackie antibodies in females was found. Regarding the Coxsackie infections, 34.6% happened in spring, while Echo infections happened mainly in autumn and winter (62.2%). The infection rate generally decreased with age except for Echo viruses that infected the 19-60y individuals more than the 0-6y ones. Coxsackie viruses most often caused respiratory illness (IgM 19.9%) presenting statistically significant difference from the rest of clinical manifestations, while Echo mostly caused gastroenteritis (20.4%) though without total statistical significance. No statistically significant difference was observed for clinical manifestations between males and females.

Key words: Coxsackie, ECHO, enteroviruses, N. Greece, epidemiology

Evaluation of Polyomavirus BK infection in oncogenic patients

Ashraf Kariminik
Department of Microbiology, Kerman Branch, Islamic Azad University, Kerman, Iran

Polyomavirus BK, a double-stranded DNA virus, is a member of the Polyomaviridae family that is known to infect humans. Transmission of Polyomavirus BK occurs during childhood via oral or respiratory tracts. Over 80% of adult population is seropositive, but the virus remains latent in the renourinary tract, brain, liver, lymphoid tissues. Certain conditions of the host, especially immunosuppression, can allow Polyomavirus BK to reactivate from the persistent subclinical state to an active lytic infection, resulting in serious disease such as tumors of cells that do not support productive infections. The goal of this cross-sectional study was to evaluate BK Polyomavirus in the cerebrospinal fluid of oncogenic hospitalized patients. 120 cerebrospinal fluid specimens were screened for the BK Polyomavirus DNA. Polymerase chain reaction with specific primers was carried out. PCR products were electrophoresed in 2% gel agarose and the appearance of a 173 base band determined BKV positivity.
Positive PCR for BK virus was seen in four patients (3.33%). This finding is very important. Based on the findings, causes of cancer in the patients can be due to the presence of the virus, or the reactivation of the virus after immunosuppression.Therefore, further studies are needed to investigate the population studied and to evaluate the virus in urine samples and blood samples.

Key words: Polyomavirus BK, Oncogenic patients, Cerebrospinal fluid