Tuberculosis outbreak in a nursing home involving undocumented migrants and Israeli citizens.

ABSTRACK: OBJECTIVES: Israel has absorbed > 60,000 migrant from the horn of Africa (MHOA) since 2006. No cross-transmission of Mycobacterium tuberculosis from MOHA to Israeli citizens has yet been reported. This study describes the results of contact investigation and laboratory work-out of a unique mixed cluster which included both MOHA and Israeli citizens. METHODS: Description of the results of epidemiological investigation including laboratory confirmation. RESULTS: This unique Mycobacterium tuberculosis strain included 29 patients: 26 were MOHA and three citizens who immigrated to Israel from the former Soviet Union. This is the first mixed cluster described in Israel, which has not been represented in the SITVIT international database of genotyping markers. The transmission from non-citizens to citizens occurred in a nursing institution, when MOHA infected three other contacts- two of whom were retarded residents, one of them died. The index case was screened before employment, and was permitted to return to wok although his chest X-ray demonstrated radiological findings compatible with tuberculosis. Epidemiological links were found in other 12 MOHA members of the cluster. CONCLUSION: This report describes cross-transmission of Mycobacterium tuberculosis from non-citizens MOHA to Israeli citizens who were residents of a nursing home, which may be the first sign for an epidemiological shift. Although cross-ethnical transmission is still rare in Israel, medical settings should employ efficient infection control measures to protect both patients and staff from Mycobacterium tuberculosis.

Drug-resistant tuberculosis in Israel: risk factors and treatment outcomes.

SETTING: All culture-positive tuberculosis (TB) isolates in Israel. OBJECTIVES: To outline the magnitude of drug-resistant TB in Israel, describe treatment outcomes and identify risk factors. DESIGN: Retrospective study of laboratory data of all strains of adult TB patients tested for resistance to first- and second-line anti-tuberculosis drugs between 1999 and 2010. RESULTS: Of 4652 reported TB cases, 3552 (76.3%) underwent culture (annual range 73-81%); 445 (12.5%) were resistant to one or more first-line drugs, while 207 (5.8%) had multidrug-resistant TB (MDR-TB). Risk factors for MDR-TB included being male, age 30-59 years, migrants (mainly from the former Soviet Union [FSU]) who had stayed in Israel >2 years, and having pulmonary TB, human immunodeficiency virus (HIV) infection and sputum smear positivity. Of all MDR-TB patients, 71.0% achieved treatment success, while 19.8% died. Twelve Israeli citizens had extensively drug-resistant TB (5.8% of MDR-TB cases). All had emigrated from the FSU and had pulmonary TB; 1 was HIV-infected. Seven (58.4%) achieved treatment success and 5 (41.6%) died. CONCLUSIONS: Drug-resistant TB in Israel is influenced by migration, especially from the FSU, where the patients were probably infected. Rapid sputum sampling performed in the early stages of the disease, patient isolation and drug susceptibility testing should be the standard of care to avoid further transmission and improve TB control.

Molecular epidemiology and mapping of tuberculosis in Israel: do migrants transmit the disease to locals?

SETTING: Israel receives migrants from various countries, some of which have high tuberculosis (TB) prevalence. OBJECTIVE: To assess the predominant Mycobacterium tuberculosis strains in Israel isolated during 2008-2010 among Israeli-born and migrant patients, and to investigate possible transmission of TB from migrants to the local population. METHODS: Molecular characterisation employed 43-spacer spoligotyping and 16-loci mycobacterial interspersed repetitive units-variable number of tandem repeats typing. All patients were classified according to those who were members of a cluster and those who were not. RESULTS: Among 684 M. tuberculosis strains isolated from new patients genotyped and assigned to their specific cohort populations during the study period, major spoligotype families were Central Asian (CAS) (n = 140, 20%), Beijing (n = 101, 15%) and T (n = 160, 23%). Most Beijing strains (66%) were isolated from patients from the former Soviet Union (FSU), while CAS strains were mainly (74%) from Ethiopia, Eritrea and Sudan (EES). For the heterogeneous T-clade, patient countries of origin were 38% EES and 33% FSU. CONCLUSIONS: Predominant M. tuberculosis genotypes in Israel in 2008-2010 were similar to genotypes endemic to the migrants' countries of origin. Epidemiological investigations did not demonstrate transmission between migrants and Israeli-born patients sharing the same cluster.

Testing a Rapid Diagnostic Medium for Erwinia amylovora and Development of a Procedure for Sampling Blossoms in Pear Orchards.

ABSTRACT The coliform agar produced by Merck was tested for rapid diagnosis of Erwinia amylovora (the causal agent of fire blight) in pear blossoms. The medium enabled the diagnosis to be completed within 36 h. Diagnoses performed with the medium were confirmed by the BIOLOG and the fatty-acid profile methods. The diagnostic medium was used to determine the spatial distribution of colonized blossoms in the orchards and it was found that E. amylovora may be distributed both in clusters and at random. These findings were used in the development of a statistical model for sampling blossoms in the orchard. The model determines the number of trees to be sampled in the orchard and the number of blossoms be taken from each tree, which would enable the true colonization incidence of blossoms in the orchard to be estimated at desired levels of accuracy and confidence. Parameters included in the model are: the total number of trees in the orchard (T), the number of trees to be sampled in the orchard (t), the number of blossoms to be sampled from each tree (n), the true colonization incidence of blossoms (pi), a coefficient of aggregation (rho), the required level of confidence (1 - alpha), and the required level of accuracy (L). Sensitivity analyses revealed that the parameter governing sample size is the required level of accuracy. Sampling of 20 blossoms from each of several hundred trees is required to achieve an accuracy of +/-1%, but only a few single trees are needed for an accuracy level of +/-10%. A sampling procedure then was developed, validated with an independent data set, and found to be accurate. It was concluded that sampling of pear blossoms and estimation of the incidence of blossom colonization by E. amylovora could improve fire blight management, but not in all cases.

Evaluation of local and imported fire blight warning systems in Israel.

ABSTRACT The possibility of using local and imported warning systems for the management of fire blight (caused by the bacterium Erwinia amylovora) in pears was tested in Israel from 1997 to 2000. Three imported systems (MARYBLYT 4.3, BIS95, and Cougarblight 98C) and one local system (Fire Blight Control Advisory [FBCA]) were used. All systems were tested in simulation experiments; MARYBLYT 4.3 and FBCA were also tested in orchard experiments under natural infections. Simulation experiments included 193 orchard-plots in which the time of disease onset enabled us to determine the date of infection. Thirty-five experiments were conducted in commercial orchards; in 10 of these, fire blight developed naturally. The performance of the imported warning systems was too variable to be accurately used under Israeli conditions. In the simulation experiments, the success rate (i.e., the capacity of the systems to predict the exact date of the occurrence of infection episodes) of the imported systems was low (3 to 55%) with considerably large variability among years (CV = 30 to 67%). Similar results were obtained in the orchard experiments for MARYBLYT 4.3: in only two of five experiments where plots were managed according to that system was disease severity significantly lower than that recorded in untreated control plots. In comparison, the local system, FBCA, predicted most infection episodes in the simulation experiments with low variability (99%, CV = 1.0%). In the orchard experiments, adequate disease suppression was achieved in all eight experiments in which FBCA recommendations were followed. We concluded that it was not possible to import and successfully implement fire blight warning systems in Israel that have been developed in regions with dissimilar environmental conditions.

New Considerations for Pruning in Management of Fire Blight in Pears.

The efficacy of pruning infected pear tissues to combat fire blight (caused by Erwinia amylovora) was evaluated in two sets of experiments conducted during 1999 to 2001 in Israel. In the first set of two experiments, diseased tissues were removed soon after the observation of blossom infections. Pruning was effective in 0 to 50% of the treated trees, and resulted in complete eradication of E. amylovora. In the remaining trees, pruning not only did not result in eradication of the bacteria from the tree tissues, it made the situation worse, as the disease had invaded the main branches and limbs of a significantly larger proportion of pruned trees than of non-pruned ones, because of alteration of the physiological status of the host plant by pruning. In the five experiments of the second set, the efficacy of pruning fire blight infections on main branches and limbs was studied; the time of pruning varied among the experiments. Effectiveness of cutting and removing infected branches and limbs was linearly related to time of treatment: the efficacy of pruning improved significantly with lateness of the treatment. The best results were obtained when pruning was carried out while the trees were dormant, in December: none of these trees had a severely infected canopy the following spring. Based on the results obtained in this study, it was concluded that factors related to all three components of the disease triangle (i.e., pathogen, host, and environment), rather than only the actual presence of diseased tissues, should be taken into account in considering the need for cutting and removing fire blight-diseased tissues. Accordingly, recommendations for Israeli growers were revised and updated.

Changes in the Sensitivity of Erwinia amylovora Populations to Streptomycin and Oxolinic Acid in Israel.

A survey of streptomycin resistance in the fire blight pathogen, Erwinia amylovora, conducted in pear, apple, and quince orchards in Israel during 1998 to 2001 revealed a decrease in the frequency of locations with streptomycin-resistant strains, from 57% in 1998 to 15% in 2001. In 2001, streptomycin-resistant strains were detected in only five locations in two restricted areas in western Galilee and the Golan Heights, compared with 16 locations found in 1998 throughout the northern part of the country. Since the use of streptomycin for fire blight control was terminated in 1997, this antibiotic has been replaced with oxolinic acid (Starner) in commercial orchards. Strains resistant to oxolinic acid were isolated from two pear orchards in the northern part of Israel in 1999. In a nationwide survey conducted during the spring and winter of 2000 and 2001, 51 and 47 pome fruit orchards, respectively, were sampled. Oxolinic acid-resistant strains were detected in several orchards located in two restricted areas in northern Galilee. Strains with resistance to both streptomycin and oxolinic acid were not found during 2000 to 2001. Results of this survey are used in managing fire blight with bactericides.

Beta subunit of DNA polymerase III holoenzyme is induced upon ultraviolet irradiation or nalidixic acid treatment of Escherichia coli.

Exposure of Escherichia coli to UV irradiation or nalidixic acid, which induce both the SOS and heat shock responses, led to a 3-4-fold increase in the amount of the beta subunit of DNA polymerase III holoenzyme, as assayed by Western blot analysis using anti-beta antibodies. Such an induction was observed also in a delta rpoH mutant lacking the heat shock-specific sigma 32 subunit of RNA polymerase, but it was not observed in recA13 or lexA3 mutants, in which the SOS response cannot be induced. Mapping of transcription initiation sites of the dnaN gene, encoding the beta subunit, using the S1 nuclease protection assay showed essentially no induction of transcription upon UV irradiation, indicating that induction is regulated primarily at the post-transcriptional level. Analysis of translational gene fusions of the dnaN gene, encoding the beta subunit, to the lacZ reporter gene showed induction of beta-galactosidase activity upon UV irradiation of cells harboring the fusion plasmids. Elimination of a 5' flanking DNA sequence in which the dnaN promoters P1 and P2 were located, did not affect the UV inducibility of the gene fusions. Thus, element(s) present from P3 downstream were sufficient for the UV induction. The induction of the dnaN-lacZ gene fusions was dependent on the recA and lexA gene products, but not on the rpoH gene product, in agreement with the immunoblot analysis. The dependence of dnaN induction on the SOS regulators was not mediated via classical repression by the LexA repressor, since the dnaN promoter does not contain a sequence homologous to the LexA binding site, and dnaN mRNA was not inducible by UV light. This suggests that SOS control may be imposed indirectly, by a post-transcriptional mechanism. The increased amount of the beta subunit is needed, most likely, for increased replication and repair activities in cells which have been exposed to UV radiation.

Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid.

A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed. The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage. Mutations arose spontaneously during growth of E. coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6). Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements. Most of the insertions were caused by IS1, but IS5 insertions were observed too. In strains harboring Tn10, IS10 was responsible for most insertions. Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions. The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements. During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation. Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event. In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements.

RecA protein inhibits in vitro replication of single-stranded DNA with DNA polymerase III holoenzyme of Escherichia coli.

Purified RecA protein from Escherichia coli inhibited 5-10-fold the rate of in vitro replication of both unirradiated and UV-irradiated single-stranded DNA (ssDNA) with DNA polymerase III holoenzyme. Maximal inhibition occurred at a ratio of 1 molecule of RecA per 2-4 nucleotides of DNA, and it affected primarily the initiation of elongation on primed ssDNA. Adding single-strand DNA-binding protein (SSB) caused a relief of inhibition. Under conditions when there was enough SSB to cover the ssDNA completely, RecA protein had no effect on initiation, elongation or dissociation steps of replication. These observations together with data from in vivo studies suggest a role for RecA protein in the arrest of DNA replication observed in cells exposed to UV-radiation and a variety of chemical carcinogens.

The role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro. Significance for SOS-targeted mutagenesis.

The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated. RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs. In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication. The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed. Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level (Livneh, Z. (1986) J. Biol. Chem. 261, 9526-9533), inhibition at the DNA level also did not increase bypass of photodimers. Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs. Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association. Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected. However, reactivation of lesion-terminated primers was achieved by pretreatment with a 3'----5' exonuclease which excised 200 nucleotides or more, generating new 3'-OH termini located away from the UV lesions. When these exonuclease-treated products were subjected to a second round of replication, an increased level of DNA synthesis was observed including additional bypass of photodimers. These results suggest the possibility that 3'----5' exonuclease processing might be required at least transiently during one of the stages of trans-lesion DNA replication, which is believed to be the mechanism of SOS-targeted mutagenesis.

Dynamics of termination during in vitro replication of ultraviolet-irradiated DNA with DNA polymerase III holoenzyme of Escherichia coli.

During in vitro replication of UV-irradiated single-stranded DNA with Escherichia coli DNA polymerase III holoenzyme termination frequently occurs at pyrimidine photodimers. The termination stage is dynamic and characterized by at least three different events: repeated dissociation-reinitiation cycles of the polymerase at the blocked termini; extensive hydrolysis of ATP to ADP and inorganic phosphate; turnover of dNTPs into dNMP. The reinitiation events are nonproductive and are not followed by further elongation. The turnover of dNTPs into dNMPs is likely to result from repeated cycles of insertion of dNMP residues opposite the blocking lesions followed by their excision by the 3'----5' exonucleolytic activity of the polymerase. Although all dNTPs are turned over, there is a preference for dATP, indicating that DNA polymerase III holoenzyme has a preference for inserting a dAMP residue opposite blocking pyrimidine photodimers. We suggest that the inability of the polymerase to bypass photodimers during termination is due to the formation of defective initiation-like complexes with reduced stability at the blocked termini.

Studies toward the biosynthesis of vasoactive intestinal peptide (VIP).

In view of the potential biological importance of VIP, we have begun to examine the regulation of its biosynthesis. For this purpose we have, as a first step, searched for an enriched source of VIP biosynthesis. By a combination of chromatographic procedures and radioimmunoassays we discovered an as yet unknown source for VIP production, namely a human buccal tumor, containing 0.67 +/- 0.05 ng VIP/micrograms protein which is greater than the richest source in brain (the cerebral cortex). Thus, we decided to use the tumor tissue for VIP-mRNA purification and characterization. To identify VIP-mRNA we are using as hybridization probes, synthetic oligodeoxynucleotides with relatively unambiguous nucleotide sequence complementary to the predicted VIP-mRNA sequence. These probes are synthesized, using the deoxynucleoside phosphoramidite approach, to a length of 17 bases each, and contain all the possible DNA sequences according to the genetic code. These specific probes are then radioactively labelled using the reaction catalyzed by the enzyme polynucleotide kinase and afterwards hybridized to mRNA, which had been resolved on denaturing agarose gels. Employing this approach, we identified a single putative VIP-mRNA band which was then partially purified by sucrose gradient centrifugation. Upon in vitro translation in a rabbit reticulocyte lysate cell free system, this mRNA was found to code for VIP immunoreactive proteins. In conclusion, our studies suggest the existence of high molecular weight precursors to VIP cross-reactive with anti-VIP antibodies, that are coded for by a partially purified mRNA containing VIP sequences.