Phenotypic and genetic relationship of Acinetobacter Baumannii isolates.

UNLABELLED: The interest in Acinetobacter continues to rise. One of the main reasons is the emergence of multi-resistant strains, which cause outbreaks of infection involving several patients in a ward, in the intensive care unit and in different areas of the hospital. Many outbreaks of its infection or colonization in surgical, neonatal and burn intensive care units have been reported, but the epidemiology of these infections remains unclear. AIM: To investigate the relationship among the isolates of Acinetobacter baumannii, comparing some of their phenotypic and genetic features. MATERIAL AND METHODS: A total of 20 Acinetobacter baumanni isolates were included in the study. 12 strains of Acinetobacter baumannii were obtained within a week in July 2010, from neonates hospitalized at the paediatric intensive care unit and on the neonatal ward. Three strains were isolated from neonates at the paediatric intensive care unit three months ago. All the Acinetobacter baumannii strains were isolated from tracheal aspirates obtained from neonates with infection of the lower respiratory tract. Five additional Acinetobacter baumannii strains were included in the study as controls. They were isolated from wound swabs taken from adult patients with wound infection, hospitalized at the University Traumatology Clinic. Susceptibility of the bacterial strains to 13 different antimicrobial agents was determined by the disk diffusion method (Kirby-Bauer). Additional testing of the susceptibility was performed by the VITEK 2 system. RAPD-PCR fingerprinting was carried out using the following primer (5' GAAACAGCTATGACCATG -3'). RESULTS: All A. baumannii isolates were multi-drug resistant. Antibiotic susceptibility-testing by the disk-diffusion method and automated VITEK 2 system showed 3 and 2 antimicrobial susceptibility patterns, respectively. RAPD-PCR assay of A. baumannii strains revealed two different RAPD-fingerprints. All the strains of A. baumannii isolated within a week in July 2010 from tracheal aspirates taken from neonates in the paediatric intensive care unit and neonates in the paediatric ward revealed the same RAPD-fingerprint, as well as 3 strains of A. baumannii isolated from tracheal aspirates taken from neonates in the paediatric intensive care unit three months ago. 5 strains of A. baumannii isolated from wound swabs of patients hospitalized at the Traumatology Clinic revealed a different RAPD-fingerprint. CONCLUSION: All the strains of A. baumannii isolated from neonates in the paediatric intensive care unit and paediatric ward were multi-drug resistant. Investigating the resistance patterns in multi-resistant isolates of Acinetobacter is a useful method which can predict the strain relationship. This method could be completed by at least one molecular method, such as the RAPD-PCR technique, which has shown itself to be a convenient and more reliable in interpreting the strain relationship of the A. baumannii isolates. Good infection control procedures, including phenotypic and molecular typing of A. baumannii isolates, are essential for preventing outbreaks of multi-drug resistant A. baumannii infections in our hospitals.

Isolation, identification and antimicrobial susceptibility of brucella blood culture isolates.

UNLABELLED: Isolation of slowly growing and fastidious Brucella spp strains from clinical specimens is difficult, because of varying factors, including species specificities, stadium of disease, and previous antibiotic treatment of the patients. The use of automated blood culture systems has overcome some cultivation problems. The automated identification system such as VITEK 2 compact allows more precise identification, as well. AIM: To present our own experience in the isolation of Brucella species from blood cultures, by the Bact/Alert automated system, identification by the VITEK 2 compact system and antimicrobial susceptibility of isolated strains. MATERIAL AND METHODS: Patients from various regions of Macedonia hospitalized in the University Infectious Diseases and Febrile Condition Clinic in Skopje. FAN blood culture bottles (aerobic and anaerobic) of the Bact/Alert system were used, inoculated with 5-10 ml of blood, incubated under continuous agitation and monitored for up to 5 days or until they became positive (in our cases for 2-3 days). Confirmations of all isolates were made by the VITEK 2 automated system on GN cards. RESULTS: During a period of three years, 113 blood cultures from patients with diagnosis of brucellosis hospitalized at the above-mentioned clinic were examined. A total of 16 blood cultures from different patients were positive (14.2%), showing Gram negative bacilli, oxidase positive small colonies on Columbia agar media. The isolates were identified as four biochemically different types of B. mellitensis, mainly within 8 hours. Susceptibility testing by the disk diffusion method on Muller Hinton agar showed sensitivity of all strains to cephalosporin, tetracycline, aminoglycoside and quinolone antibiotic groups. CONCLUSION: With the BacT/Alert system Brucella spp. were isolated in 14.2% of suspected cases of brucellosis. Isolation was done within 2-3 days. Only B. melitensis from the Brucella genus could be identified by the VITEK 2 system and some biochemical differences could be detected. The VITEK 2 system is not able to determine the susceptibility of B. melitensis. The Disk-diffusion method used in this study showed sensitivity to all tested antibiotics, although not recommended by CLSI for the Brucella genus.

Phenotypes and genes of resistance of pneumococci to penicillin isolated from children.

UNLABELLED: (Full text is available at http://www.manu.edu.mk/prilozi). In recent decades, the increase of Streptococcus pneumoniae strains resistant to beta-lactams, to other classes of antimicrobial drugs and especially to penicillin (penicillin-resistant pneumococcus - PRP) has further complicated the treatment of pneumococcal infection. Penicillin resistance in pneumococci is due to the development of altered penicillin-binding proteins (PBPs) in the bacterial cell wall. PBPs are known as six different variants (PBP1a, 1b, 2x, 2a, 2b and 3). AIM: to compare the presence and types of genes responsible for penicillin resistance in Streptococcus pneumoniae isolates with the minimal inhibitory concentrations (MIC) of penicillin as well as their correlation within the period of childhood. MATERIAL AND METHODS: A total of 45 pneumococci obtained from nasal swabs and tracheal aspirates of children treated at the University Paediatric Clinic in Skopje were examined. According to age, the children were grouped as 1-3, 4-6 and 7-10 years. the oxacillin test (1microg) was used as a rapid screening test for the detection of PRP. MIC of penicillin were determined using the agar dilution method and interpreted according to NCCLS as resistant (if MIC are > 2 microg/ml), intermediate resistant (between 0,12-1.0 microg/ml) and susceptible (< 0,06 microg/ml). The genes pbp2b and pbp 2x, which are the genes mainly responsible for the onset of PRP, were detected using polymerase chain reaction (PCR). RESULTS: the oxacillin test showed that 38 pneumococci were resistant and 7 susceptible to penicillin. MIC of penicillin showed that 7 strains were resistant, 33 strains were intermediate resistant (12, 18, and 3 with MIC of 0.5 microg/ml, 0.25 microg/ml and 0.12 microg/ml, respectively) and 5 susceptible. According to MIC, of the total 40 resistant/intermediate resistant pneumococci, in 22 genes pbp2b and/or pbp2x, were confirmed (3 resistant strains with both genes; 7 intermediate resistant and 3 resistant strains with pbp2x genes; whereas 8 intermediate resistance and 1 susceptible strain with pbp2b). In a total of 11 strains (10 intermediate resistant and one resistant according to MIC), pbp2b and/or pbp2x genes were not detected, and their resistance is probably due to some other mechanisms or other genes that code PBP. The largest number of the examined pneumococci (32) were isolated from children aged 1-3 years and in 18 of them either pbp2b or pbp2x genes were detected. CONCLUSION: the oxacillin test is not suitable for discriminating the intermediate resistant and resistant pneumococci, while it is relevant for the detection of susceptible strains. Penicillin resistance of pneumococci that were causes of infection in children was on a lower level (15.5% resistant strains with MIC 1double dagger2 mg/ml and 73.3% intermediate resistant strains with MIC 0.12double dagger1 microg/ml). Pbp2b and/or pbp2x genes were detected in 22 of the examined strains and all of them except one were intermediate resistant or resistant. The Pbp2b gene is mostly present in the intermediate resistant strains and because it was detected in one susceptible strain, this gene is responsible for a low level of resistance. The pbp2x gene was detected in all the resistant strains and that is why we could conclude that it was coding the high level of resistance. Streptococcus pneumoniae was predominantly isolated from the age group 1-3 years where the PRP were not significant (Chi square; p > 0.05). Key words: Streptococcus pneumoniae, Penicillin resistance, Minimal Inhibitory Concentration (MIC), Genes of Resistance.

Virulence factors and antibiotic resistance in Enterococcus faecalis isolated from urine samples.

Haemolysin, enterococcal surface protein (Esp), aggregation substance and gelatinase are some markers that have been proposed as possible enterococci virulence factors. The aim of this study was to detect the presence of haemolysin, gelatinase and enterococcal surface protein in enterococci isolated from urine and to determine their susceptibility to antimicrobial agents. A total of 50 strains of Enterococcus faecalis isolated from urine samples was examined. UTI agar (Oxoid) was used for the isolation and identification of the strains as Enterococcus spp. The differentiation of the species was done by the Vitek automated system (GPI-card). Haemolysin production was detected phenotypically on Columbia CNA agar as a zone of beta haemolysis around the streak. Production of gelatinase was determined as a clear halo around the colonies on tripticase soy agar supplemented with 1.5% skim milk. Esp was proved by detection of the esp gene using PCR after DNA extraction. Antibiotic sensitivity to ampicillin, ceftriaxone, vancomycin, nitrofurantoin and ciprofloxacin was examined by the agar diffusion method. In 16 Enterococcus faecalis strains (32%) all the virulence factors were present. Two factors were found in 19 (38%) strains and only one in 11 strains. There were only 4 strains without any virulence factor. Esp was the most frequently determined factor (in 38 isolates). All the strains were susceptible to vancomycin and nitrofurantoin; 12 isolates were resistant to ampicillin, 17 to ceftriaxone and 14 to ciprofloxacin. No relationship was found between virulence factors and resistance to an antibiotic.

Methicillin-resistant Staphylococcus aureus: comparison of susceptibility test methods with mecA gene analysis for determining oxacillin (methicillin) resistance in our clinical isolates.

The aim of the study was to determine which of the following susceptibility test methods, using recommended or modified NCCLS, best detects oxacillin resistance: disk diffusion, agar screen, and broth dilution. PCR for mecA was used as "gold standard". We studied 120 Staphylococcus aureus isolates received from different patients hospitalized at the Clinical center in Skopje from May 2001 to November 2003. There were no two isolates from the same patient. Methicillin resistance in Staphylococcus aureus strains was performed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS). PCR appears to be promising. Since variations among the methods exist and no acceptable guidelines are formulated, a combination of conventional methods, including either 3 microg of oxacillin/ml. in Mueller Hinton broth or one of the screen agar plates (6 microg/ml), alone or with PCR should be the method of choice for the detection of MRSA. (Tab. 4, Ref. 12.)

Quantitative changes in macrophage distribution in normal mouse ovary over the course of the estrous cycle examined with an image analysis system.

PROBLEM: The current study considered the distribution of macrophages within the major ovarian structures throughout the estrous cycle. METHODS: Immunohistochemical analyses were carried out using an avidin-biotin-peroxidase staining method and the rat anti-mouse macrophage monoclonal antibody anti-Mac-l was applied to stain macrophages. A computer-assisted image analysis system was used to quantify and compare the distribution of macrophages within individual ovarian structures during the estrous cycle. The following morphological structures were analyzed: primordial, preantral, antral, pre-Graafian, and atretic follicles; first-, second-, and third-generation corpora lutea; and the interstitium. The analysis included follicular and corpus luteum substructures: theca, granulosa cells, and interstitium. The system allows the estimation of macrophage distribution as a macrophage density per microns2 of the defined area. RESULTS: Primordial and preantral follicles did not contain macrophages during all stages of the estrous cycle. In antral, pre-graafian, and graafian follicles, macrophages were located and quantified only in the theca and were not detected in the granulosa cell layer. In contrast, atretic follicles showed macrophage localization in both thecal and granulosa cell layers. Macrophages were present in small numbers in the granulosa luteal cell layer and in high numbers in the thecal layer of newly developing corpora lutea. In the second generation of corpus luteum, macrophages followed the same pattern of distribution, while old corpora lutea contained significantly higher numbers of macrophages in both thecal and luteal cell layers. Surprisingly, significant quantitative changes in the macrophages distribution were detected over the course of the estrous cycle. Macrophage density was significantly higher in proestrus and metestrus when compared with the density in diestrus and estrus in most of the studied substructures with the exception of atretic follicles. Atretic follicles showed high macrophage density throughout the cycle with a two-fold higher density at metestrus. CONCLUSION: Macrophages were present in the mouse ovary over the course of the estrous cycle. The greatest numbers of macrophages appearing in corpora lutea and in atretic follicles suggest a role for macrophages in corpus luteum differentiation and follicular atresia. Their patterns of distribution at proestrus and metestrus within microenvironmental compartments suggests a functional correlation with the events of ovarian development.

Effects of products of activated immune cells and recombinant cytokines on spontaneous and ionophore-induced acrosome reaction.

PROBLEM: The purpose of this study was to examine whether the products of activated immune cells influence spontaneous and ionophore-induced sperm acrosome reaction. METHOD: The spontaneous and ionophore-induced acrosome reaction were evaluated by staining with fluorescein isothiocyanate (FITC) Pisum sativam agglutinin after incubation in capacitating media supplemented with either supernatants from Con-A activated leukocyte cultures or human recombinant (r) IL-1 beta, TNF-alpha, and INF-gamma. RESULTS: The supernatants from Con A-activated peripheral blood leukocyte cultures at 1:1 and 1:10 dilution significantly increased the rate of spontaneous acrosome reaction (P < 0.001 and P < 0.01). Along with displayed abnormally elevated levels of spontaneous acrosome loss, sperm cells showed an insufficient ability to undergo acrosome reaction in response to the ionophore treatment. Recombinant IL-1 beta at increasing concentrations from 30 to 3 x 10(4) U/ml did not have an effect on spontaneous and ionophore-induced acrosome reaction. In contrast, spermatozoa that underwent capacitation in media with 7 x 10(3), 7 x 10(4), and 7 x 10(5) U/ml of rINF-gamma showed a significant increase in spontaneous and induced acrosome reaction compared to the control (P < 0.001). Recombinant TNF-alpha at concentrations of 3.5 x 10(3) U/ml and 3.5 x 10(4) U/ml significantly inhibited ionophore-induced acrosome reaction (P < 0.001). Both rINF-gamma and rTNF-alpha together revealed an effect on the acrosome reaction similar to Con-A generated supernatants (1:1 and 1:10 dilution) only at the highest concentrations. CONCLUSIONS: Some cases of infertility may result from a defective acrosome reaction (premature acrosome loss or insufficient acrosome response to the stimulants) caused by products of activated lymphocytes and macrophages that are released into the male and female reproductive tracts.

[Dynamics of the distribution of lymphocyte subpopulations in the human menstrual corpus luteum]. / Dynamika zastoupení subpopulací leukocytu v lidském menstruacním zlutém telísku (lmCL).

Recent evidence suggests that immunoendocrine interactions play a definitive role during development and regression of the human menstrual corpus luteum (hmCL). We studied the distribution of immune cells within individual structures of hmCL during various stages of its development. Immunoperoxidase-stained ultra-thin frozen sections were evaluated, using light microscopy fitted with an image analysis system. The results suggest that monocytes/macrophages and MHC class II positive cells are the most prominent immune cells within the hmCL throughout its lifespan. Both cell types are concentrated within the trabeculae. In addition, MHC class II positive cells are abundant also within the granulosa-luteal layer. T helper/inductor (Th/i) and T cytotoxic/suppressor (Tc/s) cells were detected only in minor amounts within the thecal trabeculae of mature tissue. Possible links between the occurrence and functional roles of the immune cells studied are discussed.

Development and distribution of the white blood cells within various structures of the human menstrual corpus luteum examined using an image analysis system.

PROBLEM: Emerging evidences suggest that immunoendocrine interactions play definitive roles during development and regression of the human menstrual corpus luteum (hmCL). We have studied the distribution of immune cells within individual structures of hmCL during various stages of its development. METHOD: Immunoperoxidase-stained ultra-thin frozen sections were evaluated using light microscopy fitted with an image analysis system. RESULTS: The results suggest that monocytes/macrophages and MHC class II positive cells are the most prominent immune cells within the hmCL throughout its whole lifespan. Both cell types are concentrated within the trabeculae. In addition, MHC class II positive cells are abundant also within the granulosa-luteal layer. T helper/inductor (Th/i) and T cytotoxic/suppressor (Tc/s) cells were detected only in minor amounts within the thecal trabeculae of mature tissue. CONCLUSIONS: Possible links between the occurrence and functional roles of the immune cells studied are discussed.