Structure and variation of CRISPR and CRISPR-flanking regions in deleted-direct repeat region Mycobacterium tuberculosis complex strains.

BACKGROUND: CRISPR and CRISPR-flanking genomic regions are important for molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) strains, and potentially for adaptive immunity to phage and plasmid DNA, and endogenous roles in the bacterium. Genotyping in the Israel National Mycobacterium Reference Center Tel-Aviv of over 1500 MTBC strains from 2008-2013 showed three strains with validated negative 43-spacer spoligotypes, that is, with putatively deleted direct repeat regions (deleted-DR/CRISPR regions). Two isolates of each of three negative spoligotype MTBC (a total of 6 isolates) were subjected to Next Generation Sequencing (NGS). As positive controls, NGS was performed for three intact-DR isolates belonging to T3_Eth, the largest multiple-drug-resistant (MDR)-containing African-origin cluster in Israel. Other controls consisted of NGS reads and complete whole genome sequences from GenBank for 20 intact-DR MTBC and for 1 deleted-DR MTBC strain recognized as CAS by its defining RD deletion. RESULTS: NGS reads from negative spoligotype MTBC mapped to reference H37Rv NC_000962.3 suggested that the DR/CRISPR regions were completely deleted except for retention of the middle IS6110 mobile element. Clonally specific deletion of CRISPR-flanking genes also was observed, including deletion of at least cas2 and cas1 genes. Genomic RD deletions defined lineages corresponding to the major spoligotype families Beijing, EAI, and Haarlem, consistent with 24 loci MIRU-VNTR profiles. Analysis of NGS reads, and analysis of contigs obtained by manual PCR confirmed that all 43 gold standard DR/CRISPR spacers were missing in the deleted-DR genomes. CONCLUSIONS: Although many negative spoligotype strains are recorded as spoligotype-international-type (SIT) 2669 in the SITVIT international database, this is the first time to our knowledge that it has been shown that negative spoligotype strains are found in at least 4 different 24 loci MIRU-VNTR and RD deletion families. We report for the first time negative spoligotype-associated total loss of CRISPR region spacers and repeats, with accompanying clonally specific loss of flanking genes, including at least CRISPR-associated genes cas2 and cas1. Since cas1 deleted E.coli shows increased sensitivity to DNA damage and impaired chromosomal segregation, we discussed the possibility of a similar phenotype in the deleted-DR strains and Beijing family strains as both lack the cas1 gene.

Tuberculosis outbreak in an immigrant reception center in Israel: a clinical and epidemiologic perspective.

Tuberculosis (TB) outbreaks in congregate settings pose a public health concern and a clinical challenge. We report a TB outbreak involving 6 cases of active TB among 28 recent Ethiopian immigrants (EIs) in an immigrant reception center in Israel. The outbreak erupted several weeks after a meticulous pre-immigration TB screening of this group. All five culture-positive TB patients were infected with the CAS1_DELHI family, SIT 25 strain. Pulmonary involvement manifested as only a persistent cough without systemic symptoms. This outbreak occurred because of miscommunication among healthcare staff and between healthcare staff and the EI index case. It was fuelled by the staff ignorance of the social bonds within the group, and the sluggish once-monthly schedule of the on-site TB clinic operated at the reception center, which further lacked radiography facilities. This outbreak highlights the challenges of screening for active TB among immigrants and hard to reach groups.

Polymorphic exact tandem repeat A (PETRA): a newly defined lineage of mycobacterium tuberculosis in israel originating predominantly in Sub-Saharan Africa.

As part of the Israel National Program for Prevention and Control of Tuberculosis, the molecular epidemiology of new tuberculosis cases is monitored. Prospective screening showed that about 20% of all new cases of culture-positive tuberculosis (43 of 222) in Israel in the year 2008 were caused by certain Mycobacterium tuberculosis strains of the central Asian (CAS) spoligotype lineage. The identity and similarity of these strains by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing form a lineage we call PETRA for polymorphic at locus ETR A. The name PETRA was given to 79 strains we have found since the year 2000, because the largest number of strains with MIRU-VNTR profiles identical other than at locus A formed three groups, including 5 of 10 strains that had deleted the ETR A region from their genomes. No PETRA strain was found to be multiple drug resistant (resistant to both isoniazid and rifampin [rifampicin]). Most patients (75% [58 of 77 patients of known origin]) infected with PETRA were of sub-Saharan African origins. The genotypes associated with the 79 PETRA lineage strains presented in this paper suggest that the PETRA lineage is a large, major contributor to new tuberculosis cases in Israel.