Genetic relatedness and recombination analysis of Allorhizobium vitis strains associated with grapevine crown gall outbreaks in Europe.

AIMS: To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8-year period and to assess the relative contribution of mutation and recombination in shaping their diversity. METHODS AND RESULTS: By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed. CONCLUSIONS: The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis.

First Report of Agrobacterium vitis as the Causal Agent of Grapevine Crown Gall in Serbia.

In November 2010, a serious outbreak of crown gall disease was observed on 3-year-old grapevine (Vitis vinifera L.) cv. Cabernet Sauvignon grafted onto Kober 5BB rootstock in two commercial vineyards located in the South Banat District in Serbia. Large, aerial tumors were visible above the grafting point on grapevine trunks, and in most cases, the tumors completely girdled the trunk. From the gall tissues, white, circular, and glistening bacterial colonies were isolated on yeast mannitol agar medium. Eight, nonfluorescent, gram-negative, and oxidase-positive strains were isolated from seven tumor samples and selected for further identification. PCR assays with A/C' (1) and VCF3/VCR3 (4) primers corresponding to the virD2 and virC genes yielded 224- and 414-bp fragments, respectively, confirming that the strains harbored the plasmid responsible for pathogenicity. The strains were differentiated to the species/biovar level with a multiplex PCR assay targeting 23S rRNA gene sequences (3) and were identified as Agrobacterium vitis. The 16S rDNA gene sequence from one isolate (GenBank Accession No. JN185718) showed 99% identity to the sequences of A. vitis previously deposited in NCBI GenBank database. The physiological and biochemical test results corresponded to the results of genetic analysis (2). The strains grew at 35°C and in nutrient broth supplemented with 2% NaCl. They were negative in 3-ketolactose, acid clearing on PDA supplemented with CaCO3, and ferric ammonium citrate tests; nonmotile at pH 7.0; pectolytic at pH 4.5; utilized citrate; produced acid from sucrose and alkali from tartarate. Pathogenicity was confirmed by inoculation of three plants per bacterial strain on grapevine cv. Cabernet Franc and on a local cultivar of tomato (Lycopersicon esculentum L.). The plants were inoculated on the stem by pricking one to three times through a drop of inoculum (108 CFU/ml) at three inoculation sites. Sterile distilled water was used as a negative control. Inoculated plants were maintained in a greenhouse at 24 ± 3°C. Typical tumors developed at the inoculation sites on tomatoes 3 weeks after inoculation and on grapevine 6 weeks after inoculation. No symptoms were observed on the control plants. Bacteria were reisolated from tumorigenic tissues and identified as pathogenic A. vitis by PCR. Crown gall disease was sporadically observed in vineyards in Serbia in previous years, but did not cause significant damage. Therefore, the causal agent was not studied in detail. To our knowledge, this is the first report of A. vitis determined as the causal agent of grapevine crown gall in Serbia. References: (1) J. H. Haas et al. Appl. Environ. Microbiol. 61:2879, 1995. (2) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (3) J. Pulawska et al. Syst. Appl. Microbiol. 29:470, 2006. (4) K. Suzaki et al. J. Gen. Plant Pathol. 70:342, 2004.

Comparison of two simple microaerobic atmospheres for cultivation of thermophilic campylobacters.

A comparison of a modification of the spirit burn method and one based on the Fortner principle was carried out on 3474 faecal samples from patients with diarrhoea. Of the 96 isolates of Campylobacter, 47 showed equal abundance of growth, regardless of the method used. By using the spirit burn method, however, growth of 33 isolates was significantly increased, and 18 out of those 33 isolates grew only in the spirit burn method atmosphere. The spirit burn method is more effective than the one based on the Fortner principle, it is easy to perform, quick, and cheap.

Penicillin-resistant and multidrug-resistant Streptococcus pneumoniae in a pediatric hospital in Zagreb, Croatia.

Sixty-four penicillin-resistant Streptococcus pneumoniae isolates [benzylpenicillin minimal inhibitory concentrations (MICs) between 0.05 and 1.6 micrograms/ml] recovered at the Pediatric Hospital "Dr. Fran Mihaljevic" in Zagreb, Croatia between October 1990 and March 1993 were analyzed for serotype, antibiotic susceptibility patterns, and chromosomal relatedness using pulsed-field gel electrophoretic (PFGE) analysis of chromosomal DNA fragmented by digestion with the SmaI endonuclease. Hospital "Dr. Fran Mihaljevic" services the capital of Croatia and its vicinity. Most of the isolates were from nasopharyngeal carriage, but several isolates were from otitis media, sinusitis, and meningitis. Most isolates belonged to either serotype 23F (36/64) or 19F (12/64); the rest, including three 15C isolates, were in 11 additional distinct serotypes. The overwhelming majority (25/36) of the serotype 23F isolates had penicillin MIC values of 1-2 micrograms/ml and shared variants of a common PFGE pattern, closely related to the PFGE identified in multiresistant pneumococci of the same serotype with wide geographic spread to Spain, Portugal, France, and the United States. This group of bacteria was also resistant to tetracycline, chloramphenicol, and sulfamethoxazole/trimethoprim. In contrast to the relative genetic and phenotypic homogeneity of the more highly penicillin resistant isolates, pneumococci with penicillin MICs between 0.5 and 0.4 microgram/ml (29/64) were distributed in 13 different serotypes and as many as 20 distinct PFGE patterns.

Antibiotic resistance among gram-negative nosocomial pathogens in the intensive care unit: results of 6-year body-site monitoring.

Results of 6-year body-site monitoring in an intensive care unit (ICU) are presented and antimicrobial resistance of gram-negative isolates analyzed. The study included 622 patients. Six hundred thirty-five bacterial isolates-causes of nosocomial sepsis, pneumonia, and urinary tract infections (UTIs)-were tested during the study. Gram-negative bacteria were the predominant isolates, causing 65% of cases of sepsis, 78.7% of pneumonias, and 70.2% of UTIs. Gram-negative isolates (454) were highly resistant to antimicrobials commonly used in the ICU, with the exception of imipenem. Resistance was 1.1% among pathogens responsible for UTIs, 6.7% among those causing sepsis, and 13.6% among those responsible for pneumonia. Klebsiella pneumoniae associated with pneumonia and sepsis was significantly less resistant to ciprofloxacin than were isolates from urine (22.8% and 13.9%, respectively, vs 44.4%). Pseudomonas aeruginosa strains responsible for pneumonia were less resistant to ceftazidime than were isolates causing sepsis and UTI (35.7% vs 51.3% and 51.5%, respectively). Acinetobacter calcoaceticus strains associated with UTI were significantly more resistant to netilmicin than were strains responsible for sepsis and pneumonia (83.3% vs 40.3% and 42.6%, respectively). The study confirmed that in addition to focused microbiologic surveillance, multiple-body-site monitoring can provide unique information about the sensitivity of the pathogens involved. The results suggest that antimicrobial resistance among nosocomial pathogens depends on the site of infection or the type of microbiologic specimen.

[Ceftazidime in the therapy of gram-negative meningitis in childhood]. / Ceftazidim u terapiji gram-negativnih meningitisa u djecjoj dobi.

Six cases of neonatal meningitis due to E. coli (3 cases), K. pneumoniae (1 case), P. aeruginosa (1 case) and S. marcescens (1 case), and eleven cases of suckling and little child meningitis caused by M. influenzae (10 cases) and N. meningitidis (1 case) were treated with ceftazidime. The susceptibility of agents was qualitatively tested according to the disk-diffusion method, and quantitatively according to biological dilution method on liquid broth. Ceftazidime concentrations in cerebrospinal fluid and sera were determined by the modified microbiological method using diffusion on agar. Efficacy of ceftazidime therapy was assessed by quickness of cerebrospinal fluid "sterilization", duration of antimicrobial therapy and outcome of the disease. In spite of very good agents susceptibility to ceftazidime determined by disk-diffusion method, notable differences were found in quantitatively determined susceptibility (minimal inhibitory and minimal bactericidal concentration). Antibiotic penetrability was various in proportion with individual intensity of blood brain barrier break down. Bactericidal effect and prompt "sterilization" of cerebrospinal fluid within 48 hours after the beginning of ceftazidime therapy was achieved in those patients in whom ceftazidime cerebrospinal fluid concentration was 10 and several times higher than the minimal bactericidal concentration (all cases due to H. influenzae, N. meningitidis and E. coli). In these cases the issue of the disease was also favourable and none of the patients died.(ABSTRACT TRUNCATED AT 250 WORDS)

[Duration of the Neisseria meningitidis serogroup B serotype 2 carrier state]. / Trajanje kliconostva Neisseria meningitidis serogrupe B serotipa 2.

Of 168 examined secondary school children from Zagreb, Neisseria meningitidis, serogroup B, serotype 2 was isolated from the pharynx smear taken from 14 healthy carriers, aged 15-18. Carriership was observed during the period of 19 months. During that period the pharynx smear was taken four times so that in total there were five samples from each examinee, with the purpose of finding Neisseria. Neisseria meningitidis serogroup B, serotype 2 was isolated and identified by using the standard microbiological methods. Serogroup and serotype were identified by the slide agglutination method, and were used group and type specific antisera. Of 14 identified carriers two persons had continuous duration of carriership of N. meningitidis, serogroup B, serotype 2 during the period of nineteen months. In 6 persons over the period of 9 months, in all five samples of pharynx smear the same meningococcus was identified. This epidemiological study has determined the longest duration of carriership of Neisseria meningitidis serogroup B, serotype 2 described so far in the literature. The role of carriership in the occurrence of the meningococcal disease has not been fully explained.

[Antimicrobial treatment of Haemophilus influenzae meningitis in childhood]. / Antimikrobno lijecenje Haemophilus influenzae meningitisa u djecjoj dobi.

Antimicrobial treatment of bacterial meningitis should be done by antibiotic to which the causative agent is susceptible, which attained serum levels ensure adequate penetration across the blood-brain barrier and which bactericidal levels in cerebrospinal fluid are achieved. A total number of 61 child in age from 2 months to 7 years with bacteriologically proved Haemophilus influenzae meningitis was included in the study. The possibility and usefulness of application of various antibiotics used in the treatment of this disease as well as the results attained have been discussed. A total number of 40 patients was treated with only one antibiotic (ampicillin 6, chloramphenicol 6, cefuroxime 3, cefotaxime 18, ceftazidime 7) and 21 patients were treated with combinations of antibiotics (ampicillin + chloramphenicol 14, cefuroxime + chloramphenicol 7). The causative agent was susceptible to the applied antibiotic in all cases. The agent susceptibility was estimated qualitatively by disk diffusion method and quantitatively by methods of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Even 31.1% of H. influenzae strains were resistant to ampicillin. Penetration across the blood-brain barrier was monitored by estimation of antibiotic concentration in sera and liquor. Antibiotic concentrations in liquor attained the bactericidal levels. The same good results were attained with any of single applied antibiotics. In cases of delayed initiation of the antimicrobial therapy the appearance of neurological complications was more frequent, the outcome of the disease was worse and the duration of treatment was longer.(ABSTRACT TRUNCATED AT 250 WORDS)

[Bacteremia caused by beta-hemolytic group A streptococci]. / Bakteriemije uzrokovane betahemolitickim streptokokom grupe A.

The epidemiological, clinical and laboratory characteristic of group A beta-hemolytic streptococcal (GABHS) bacteremia in patients treated at the University Hospital of Infectious Diseases in Zagreb were analyzed. Of 51 cases of bacteremia due to GABHS seen over the past 15 years, 15 (29%) have been registered since 1987. The mean age of our patients was 25 years (range, 5 months to 87 years); and 29 (57%) were under 18. Ten (19%) patients died, 6 being less than 18 years of age. Forty-seven (92%) cases were community-acquired and 16 (32%) had underlying disease. Primary foci of infection, defined as a sites of inflammation that precedes bacteremia, included pharyngitis in 15 and erysipelas/cellulitis in 16 patients. Six patients had no focus of infection. Shock was recorded in 4 patients, all of whom died. Seven patients had a rash, in 5 the rash was typical of scarlet fever. Of the 6 children who died 4 were previously healthy and they all died within 24 hours following admission, and were sick at least 48 hours before admission to our hospital. Our experience suggests that serious GABHS infections may appear in children, that it requires prompt recognition and treatment and that a worldwide change in the virulence of GABHS may have occurred.

[Hospital infections and resistance to new antibiotics in intensive care units: relation to diagnostic and therapeutic procedures]. / Bolnicke infekcije i rezistencija na neke novije antibiotike u jedinici intenzivne skrbi: ovisnost o dijagnosticko-terapijskim zahvatima.

The results of hospital infection surveillance over an eight-month period in the Intensive Care Unit (ICU) of the University Hospital of Infectious Diseases "Dr Fran Mihaljevic", Zagreb, are presented together with the results of the antibiotic resistance of isolated gram-negative bacteria in relation to the clinical material taken for culture. Of 110 strains of gram-negative bacteria isolated, 103 (93.6%) were resistant to ampicillin, 80 (72%) to gentamicin, 74 (67%) to cefotaxime, 50 (45.5%) to pefloxacin. Imipenem, ciprofloxacin, ceftazidime and amikacin were the most active representatives of their respective groups with 5, 25, 49 and 50% of resistant strains. Strains isolated in urinary tract infections were significantly less resistant to amikacin and ceftazidime (p < 0.05) than strains isolated from other sources. Our findings stress the need for close surveillance of antibiotic resistance in these selected groups of patients, and are particularly relevant for planning empirical anmtibiotic therapy of hospital infection in the ICU.

A case of fatal sepsis in a child due to highly resistant Streptococcus pneumoniae.

Infection with Streptococcus pneumoniae continues to be a significant cause of morbidity and mortality. Most of the pneumococci remain exquisitely sensitive to penicillin. However, S. pneumoniae with a reduced susceptibility to penicillin has been reported. To our knowledge, we present the first case in Croatia of fatal sepsis in a child due to Streptococcus pneumoniae that was highly resistant to penicillin.

[Monitoring bacterial resistance to antibiotics in the CroatianRepublic]. / Pracenje rezistencije bakterija na antibiotike u Republici Hrvatskoj.

In 1996 a Committee for antibiotic resistance surveillance in Croatia was founded by the Croatian Academy of Medical Sciences. In this study antibiotic surveillance results for the period June 1-December 31, 1997 from 12 microbiology laboratories throughout Croatia are presented. Sensitivity to antibiotics was determined by disk diffusion method for the following bacteria: Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus spp., Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa. In general, high proportion of resistant isolates was recorded throughout Croatia, although some regional variations were noticed. Mean resistance of pneumococci to penicillin was 38%, in S. aureus resistance to methicillin was 47%, and 3rd generation cephalosporin-resistance in E. coli was 6% and in Klebsiella spp. 21%. In P. aeruginosa resistance to gentamicin averaged 50%, to imipenem 13% and to ceftazidim 8%. Future aims of the Committee are to continue routine antibiotic resistance surveillance during certain periods every year, and to estimate clinical significance of resistant bacteria, detect mechanisms of resistance and improve the quality of laboratory work through education and quality control projects.

Comparison of clinical characteristics of group A streptococcal bacteremia in children and adults.

This report reviews 55 cases of bacteremia due to group A streptococci (GAS) in patients treated at the University Hospital of Infectious Diseases, in Zagreb, Croatia, during the period 1974-1994. Clinical findings for 27 children (age, < 18 years) were compared with those for 28 adults. A clear predominance of the respiratory tract as a primary focus of infection in younger patients was observed (P = .00006). Children were more frequently colonized by GAS in their throats than were adults (P = .04). Suppurative osteoarticular metastatic foci were more often found in children (P = .02). Acute underlying conditions were more common in children (P = .04), whereas chronic underlying conditions were more common in adults (P = .00005). The case fatality rate was 23% among children (6 of 27) and 50% among patients aged > 60 years (5 of 10). The course of bacteremia due to GAS was benign in eight children (29.6%) and 12 adults (42.8%). Our experience suggests that there are significant differences in the presentation of bacteremia due to GAS in children and adults.

Asymptomatic pharyngeal carriage of beta-haemolytic streptococci and streptococcal pharyngitis among patients at an urban hospital in Croatia.

In a one-year study at the University Hospital of Infectious Diseases in Zagreb, Croatia the rate of asymptomatic throat carriage of beta-haemolytic streptococci was investigated. Throat carriage was investigated in 1796 patients, none of whom had a sore throat nor signs and/or symptoms of a respiratory tract infection. The carrier rate of beta-haemolytic streptococci was 8.3%, for group A streptococci 6%, group B 1.3%, group C 0.3% and group F 0.1%. The highest rate was observed in the 6 to 14 year age group: 13.8% for all streptococcal groups, 11.7% for group A alone. The proportion of non-A streptococci was higher in older age groups. Tonsillectomised individuals were less frequently carriers. No sex or season-dependent variations were observed. In a four-month study of 629 patients with pharyngitis the throat cultures yielded: group A streptococci in 44.7%, group B in 1.7%, group C in 0.8%, and group G in 0.6% of the patients. Group A streptococci in 44.7%, group B in 1.7%, group C in 0.8%, and group G in 0.6% of the patients. Group A streptococcal rates in carriers compared to rates in patients with pharyngitis suggest that approximately one fourth of the schoolchildren with culture-positive pharyngitis actually are not truly infected, but only carriers currently having a sore throat of non-streptococcal etiology. Antibiotics were administrated to 605 (96.2%) patients with pharyngitis; in 571 (94.4%) of the cases before culture results became available. After culture-negative results were obtained the therapy was discontinued in only 28.5% of the cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Short-term treatment of pertussis with azithromycin in infants and young children.

A prospective, open, noncomparative study was conducted to assess the efficacy and safety of azithromycin given once daily for 3 or 5 days to eradicate Bordetella pertussis from the upper respiratory tract of infants and young children. Seventeen children received azithromycin in a dose of 10 mg/kg on day 1 followed by 5 mg/kg once daily for four consecutive days, and 20 were given 10 mg/kg once daily for 3 days. Seven days after the initiation of therapy, 33 of 35 (94.3%) patients had negative cultures for Bordetella pertussis. On day 14, cultures from all 34 evaluable patients were negative. These findings suggest that a controlled, comparative study of erythromycin versus short-term administration of azithromycin is justified.

Virulence characteristics of Escherichia coli strains causing asymptomatic bacteriuria.

BACKGROUND: The objective of this study was to examine the expression of Escherichia coli virulence-associated factors among the strains isolated from a group of women with a history of recurrent urinary tract infections (UTIs), in whom asymptomatic bacteriuria (ABU) was detected at follow-up, and from a group of children without a history of previous UTI, in whom ABU was detected during the screening. Possible differences between the virulence potential of these strains were investigated. MATERIALS AND METHODS: Hemolysin production, the ability to adhere to Buffalo green monkey cell line and hemagglutination (HA) ability of the ABU-associated E. coli strains were tested. E. coli strains isolated from patients with acute recurrent UTIs served as a comparison. RESULTS: The well-known low virulence of strains isolated from patients with ABU was demonstrated. In contrast to strains isolated from recurrent uncomplicated UTIs, the ABU-associated strains were mostly nonhemolytic (75%), nonadherent (70%) and lacked HA ability (61%). HA ability was significantly more common among the strains isolated from children without a history of UTI than among the strains isolated from women with recurrent UTIs (chi2 = 9.97, p < 0.01), whereas the adherence and hemolytic abilities did not differ between the two ABU groups. CONCLUSION: A further prospective study is needed to determine whether the HA ability is the predictor of subsequent symptomatic UTI.