Insertion Sequence IS6110 Research Articles

Improved Detection of Mycobacterium tuberculosis in Sudanese Children by use of Insertion Sequence IS6110

This study was carried out in Khartoum state during the period from January 2010 to December 2013 to detect Mycobacterium tuberculosis in children with symptoms of tuberculosis infection using different conventional and advanced diagnostic techniques. One hundred ninety seven specimens of gastric lavage and sputum were collected from different hospital in Khartoum state.specimens were decontaminated and inoculated on Lowenstein Jensen media according to modified Pettrof’s method, two smears were prepared and stained by Ziehl Neelsen stainbacterial DNA was extracted from each specimen by using phenol chloroform method, and then the Polymerase Chain Reaction technique was adopted to detect Insertion Sequence IS6110 gene of M. tuberculosisin in these specimens. This study showed that the positive results for ZN, Culture and PCR were 16 (8.1%), 32 (16.2%) and 35(17.8%) respectively. The study concluded that the PCR technique is a most sensitive and specific technique for a fast identification of M. tuberculosis in gastric lavage and sputum from children who are unable to expectorate good quality sputum sample or diagnosed as negative using conventional diagnostic methods.

Establishment and multi drug resistance evolution of ST235 Pseudomonas aeruginosa strains in the intensive care unit of a Colombian hospital

Drug resistant Pseudomonas aeruginosa represents a therapeutic challenge. To assess the diversity of P. aeruginosa antibiotic resistant variants, isolates were recovered from hospital patients in Colombia. Thirty of 60 isolates contained class 1 integrons and five were of Sequence Type ST235 having appeared in a single intensive care unit. All five possessed an unusual integron but showed differences in gene cassette content and the presence/absence of insertion sequence IS26. This showed that differences can arise rapidly, even within a single ICU. Also, the emergence of IS26 in P. aeruginosa is contributing to the evolution of resistance in this bacterium.

IS6110-5′3′FP: an automated typing approach for Mycobacterium tuberculosis complex strains simultaneously targeting and resolving IS6110 5′ and 3′ polymorphisms

Fingerprinting of Mycobacterium tuberculosis complex strains based on the IS6110 insertion sequence would considerably gain in terms of discriminatory power and versatility if both 5' and 3' polymorphisms were simultaneously targeted, and if it benefited from automated capillary electrophoresis. In response to these requirements, we developed IS6110-5'3'FP (IS6110 5' and 3' fluorescent polymorphisms). IS6110-5'3'FP involves the construction of an M. tuberculosis genomic library in a plasmid vector using HincII endonuclease, which cuts within the IS6110 sequence. After amplification in Escherichia coli, the library is subjected to selective and simultaneous PCR amplification of IS6110 5' and 3' polymorphic fragments, using differentially labeled fluorescent primers. The resulting amplicons are then fractionated on a capillary sequencer and the signal peaks analyzed as digital data. IS6110-5'3'FP consistently detected and resolved both 5' and 3' IS6110 polymorphic fragments (35% and 65%, respectively) with a high level of reproducibility. The method differentiated all M. tuberculosis strains, as did IS6110 restriction fragment length polymorphism (RFLP), the gold standard of IS6110-based typing. Strikingly, the potential of IS6110-5'3'FP to resolve more polymorphic fragments than IS6110 RFLP was demonstrated. IS6110-5'3'FP demonstrated sufficient potential to be a promising automated alternative to IS6110 RFLP, amenable to high throughput analysis and inter-laboratory comparison.

Polymerase Chain Reaction targeting insertion sequence IS6110 for the diagnosis of pulmonary tuberculosis among Sudanese children and young adults

This study was carried out in Khartoum State during the period from January 2011 to December 2013 to improve the rate of detection of Mycobacterium tuberculosis (MTB) in children with symptoms of tuberculosis (TB) infection using different conventional and advanced diagnostic techniques. One hundred and ninety-seven specimens of gastric lavage and sputum were collected from different hospitals in Khartoum State, including Elbolok Hospital, Jafar Ibn Owf Hospital, Elasha’ab Teaching Hospital, Soba University Hospital and Academy Charity Hospital.All children participating in the study were subjected to the Mantoux test after obtaining appropriate consent injected by 5 tuberculin units of tuberculin purified protein derivative, and the results were recorded after three days. Specimens were decontaminated and inoculated on Lowenstein–Jensen media according to the modified Petroff’s method. Two smears were prepared and stained by Ziehl–Neelsen stain and Auramine fluorescent dye; bacterial DNA was extracted from each specimen by using phenol chloroform method, and then the Polymerase Chain Reaction technique was adopted to detect Insertion Sequence IS6110 gene of MTB in these specimens. This study showed that the positive results for TST, ZN, Auramine, Culture and PCR were 86 (43.7%), 16 (8.1%), 22 (11.2%), 32 (16.2%) and 35(17.8%), respectively. The study concluded that the PCR technique is the most sensitive and specific technique for a quick identification of MTB in gastric lavage and sputum from children who are unable to expectorate a good quality sputum sample or who are diagnosed as negative using conventional diagnostic methods.

Investigating the role of free-living amoebae as a reservoir for Mycobacterium ulcerans.

BackgroundThe reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment.Methodology/Principal FindingsWe investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127).Conclusion/SignificanceThis study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.

Performance of newer and conventional diagnostic methods in detection of drug sensitive and resistant tuberculous meningitis

ObjectiveTo compare the performance of traditional and newer diagnostic methods for diagnosis of tuberculous meningitis (TBM) along with detection of drug resistant TBM. MethodsSingle cerebrospinal fluids sample from 281 suspected TBM patients was processed during August 2011 to July 2012 for acid fast bacilli (AFB) examination by Ziehl-Neelsen and auramine staining methods, AFB culture on Lowenstein-Jensen culture media, by microscope observation of drug susceptibility testing assay, and nucleic acid amplification tests by in-house conventional PCR and in-house real time PCR targeting IS6110 insertion sequence. All the isolates were subjected to drug susceptibility testing for isoniazid, rifampicin, ethambutol and streptomycin by 1% proportion method. ResultsThe percentage positivity by AFB smear examination and culture on Lowenstein-Jensen media was 21% (59/281), 11.4% (32/281) respectively. The detection rate by conventional PCR was 33.1% (93/281). The real time PCR showed positivity rate of 44.8% (126/281). The extra detection by real time PCR was 11.7%. Only one isolate was multiple drug resistant and 22 (68.8%) were pan-susceptible. Remaining eight isolates showed either mono/poly drug resistant to first line antitubercular drugs. ConclusionsReal time PCR is a more sensitive and rapid method if it is appropriately adopted in clinical practice for diagnosis of TBM. Prevalence of multiple drug resistance Mycobacterium tuberculosis causing TBM is low.

An electrochemical DNA biosensor for the detection of Mycobacterium tuberculosis, based on signal amplification of graphene and a gold nanoparticle-polyaniline nanocomposite.

Due to its low growth rate and its fastidious nature, Mycobacterium tuberculosis is difficult to identify. Its rapid and sensitive detection is, however, critical for the control of tuberculosis. Molecular biology, and more recently electrochemical technology, have been exploited for the detection of this pathogen. In the present study, a novel DNA biosensor was developed for the highly sensitive detection of the specific DNA insertion sequence IS6110 of M. tuberculosis, using reduced graphene oxide-gold nanoparticles (rGO-AuNPs) as a sensing platform and gold nanoparticles-polyaniline (Au-PANI) as a tracer label for amplification. Reduced graphene oxide, which has a large surface area, provided a biocompatible matrix. Gold nanoparticles were electrodeposited on the surface of the rGO modified electrode, which not only increased immobilisation of the capture probe but also promoted electronic transfer. The Au-PANI nanocomposite exhibited good biocompatibility and excellent electrochemical activity. It was therefore used as a tracer label for electrochemical detection, which provided a simple preparation process for a signal-on DNA biosensor. With the excellent electroactivity of the Au-PANI nanocomposite, the resulting DNA biosensor exhibited high sensitivity for the detection of M. tuberculosis over a broad linear range, between 1.0 × 10(-15) and 1.0 × 10(-9) M. The DNA biosensor showed good stability and high specificity and provides a new strategy for clinical M. tuberculosis diagnostics and probably also for pathogenic bacteria in general.

Pheno- and genotyping of Brucella abortus biovar 5 isolated from a water buffalo (Bubalus bubalis) fetus: First case reported in the Americas

An isolate of Brucella spp. from an aborted water buffalo (Bubalus bubalis) fetus was characterized based on its pheno- and genotype. The phenotype was defined by carbon dioxide requirement, hydrogen sulfide production, sensitivity to thionin and basic fuchsin and agglutination with Brucella A and M monospecific antisera. The genotype was based on the amplification of the following genes: bcsp31, omp2ab, and eri and the species-specific localization of the insertion sequence IS711 in the Brucella chromosome via B. abortus–B. melitensis–B. ovis–B. suis (AMOS)-PCR. Unexpectedly, the isolate showed a phenotype different from B. abortus bv 1, the most prevalent strain in cattle in Argentina, and from vaccine strain 19, currently used in bovines and water buffaloes. Genotyping supported the phenotypic results, as the analysis of the omp2ab gene sequence showed an identical pattern to either B. abortus bv 5 or B. melitensis. Finally, the AMOS PCR generated a 1700-bp fragment from the isolate, different than those amplified from B. abortus bv 1 (498bp) and B. melitensis (731bp), confirming the presence of B. abortus bv 5. The OIE/FAO Reference Laboratory for Brucellosis confirmed this typing. This is the first report of B. abortus bv 5 from a water buffalo in the Americas.

Occurrence of 3 Bordetella Species During an Outbreak of Cough Illness in Ohio

An increase in laboratory diagnosis of pertussis was noted in central Ohio during 2010. Diagnosis was made using a polymerase chain reaction assay targeting the multicopy insertion sequence IS481, which is found in both Bordetella pertussis (Bp) and Bordetella holmesii (Bh). An increase in specimens testing positive for Bordetella parapertussis (Bpp) using insertion sequence IS1001 was also noted. Nasopharyngeal swab specimens submitted April 1, 2010, to March 31, 2011, were tested using a multiplex polymerase chain reaction assay for Bp/Bh (IS481) and Bpp followed by singleplex assays for Bp and Bh. A subgroup of specimens was also cultured for Bordetella species, and antimicrobial susceptibility testing was performed on recovered organisms. Demographic and clinical features were compared for patients with Bp, Bh and Bpp. Of 520 IS481-positive specimens, 214 (41.1%) were positive for Bp, 79 (15.2%) were positive for Bh and 5 (1.0%) were positive for both Bp and Bh; 222 (42.7%) were negative for both targets. An additional 220 specimens were positive for Bpp. Among a sample of 155 IS481-positive specimens, 40, 15 and 0 were culture positive for Bp, Bh and Bpp, respectively. Among a sample of 55 BparaIS1001-positive (Bpp) specimens, 22, 0 and 0 were culture positive for Bpp, Bp and Bh, respectively. All Bordetella species were susceptible to macrolide antibiotics. Patients with Bh were older than patients with Bp, who were older than those positive for Bpp (mean ages: 12.0, 8.0 and 4.2 years, respectively; P < 0.001). One or more classic signs of pertussis (ie, paroxysmal cough, whoop, post-tussive emesis) were seen in 55.9% of 263 patients (59 Bp, 24 Bh, 80 Bpp and 100 negative for Bordetella species), but did not differ statistically among the groups (χ = 5.1, P = 0.17). All 3 Bordetella species, Bp, Bh and Bpp, were detected during on outbreak of pertussis-like cough illness. There were noted differences in age and seasonality, but clinical features at the time of presentation did not allow clear differentiation of these infections. All Bordetella species recovered from culture and tested were susceptible in vitro to macrolide antibiotics. Additional study is necessary to further characterize epidemiologic and clinical characteristics of Bh-associated cough illness and to determine potential co-occurrence of Bordetella species with other bacterial and viral respiratory tract pathogens.

SOME FEATURES OF GENOME STRUCTURE AND EVOLUTION OF MYCOBACTERIUM TUBERCULOSIS

Abstract. Mycobacterium tuberculosis complex includes both human pathogens (M. tuberculosis, M. africanum and M. canettii), rodent paghogens (M. microti), as well as Mycobacterium bovis with wide range of hosts and related M. caprae and M. pinnipedii. In spite of phenotypic and host differences these species present a highly homogeneous genospecies with 99.7–99.9% of genome homology and extremely low level of horizontal gene transfer. Recent genetic research in the last decade permitted to revisit and revise old and new dogmas about genome and evolution of M. tuberculosis. In particular, a classical theory about bovine origin of human tuberculosis during domestication process was rejected. It was demonstrated that genomes of the related species of M. tuberculosis complex evolved through large unidirectional deletions leading to origin of M. tuberculosis sensu stricto, M. bovis and other species (M. canettii, M. microti, M. pinnipedii, M. caprae) from the same progenitor species. Large deletions influence the pathogenic potential of different clonal lineages within M. tuberculosis. At the same time, genetic variation within the short time frames is achieved via changes in the repetitive DNA and transposition of the insertion sequences IS6110 across the genome. Furthermore, M. tuberculosis may adapt to the selective pressure of the host immune system and antituberculosis drugs via specific point mutations. In the last few years a higher level of SNP variation between closely related strains was demonstrated with opens new perspectives for full-genome and multilocus sequence typing of M. tuberculosis.

Short communication: Viable Mycobacterium avium subspecies paratuberculosis in retail artisanal Coalho cheese from Northeastern Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis and it potentially plays a role in Crohn’s disease. In humans, the main route of transmission of MAP might be the intake of contaminated milk and dairy products. Considering that MAP has already been detected in many types of cheese in different counties, and that Coalho cheese is an important dairy product in northeastern Brazil, the aim of this study was to report the first detection of MAP in retail Coalho cheese in Brazil by PCR and culture. Of 30 retail Coalho cheese samples, 3 (10%) amplified fragments of a similar size to that expected (626 bp) were obtained and viable MAP was recovered by culture from 1 (3.3%) sample. The DNA from the positive culture sample was sequenced and showed 99% identity with the insertion sequence IS900 deposited in GenBank. It was possible to identify the presence of MAP-specific DNA in the analyzed samples for the first time in Brazil, and to recover viable cells from retail Coalho cheese.

Prevalence of asymptomatic Bordetella pertussis and Bordetella parapertussis infections among school children in China as determined by pooled real-time PCR: A cross-sectional study

Studies have documented that older children and adolescents act as a reservoir of Bordetella pertussis infection for young infants who have not yet completed their primary immunization schedule. Asymptomatic pertussis infection has been reported during outbreaks. This cross-sectional study aimed to investigate whether B. pertussis and Bordetella parapertussis can colonize the nasopharynx of healthy school children, using culture and pooled real-time PCR with targets for insertion sequences IS481 and IS1001. Nasopharyngeal (NP) swabs were taken from 629 asymptomatic school children aged 7 to 15 y in 4 counties of China during the period July-September 2011. The number of subjects included in each county ranged from 153 to 165. The 4 counties selected are located in the north, south, east, and southwest regions of China. NP swabs were inoculated onto Regan-Lowe agar for isolation of suspected Bordetella organisms. Pooled real-time PCRs were used to detect B. pertussis and B. parapertussis based on the IS481 and IS1001 targets separately. Of the 629 subjects, 2 (0.3%) and 30 (4.8%) were confirmed to be culture-positive and PCR-positive, respectively, for B. pertussis, and 1 (0.2%) and 13 (2.1%) were confirmed to be culture-positive and PCR-positive, respectively, for B. parapertussis. All culture-positive samples were also PCR-positive. Furthermore, positive B. pertussis and B. parapertussis samples were found in all counties. Our results indicate that asymptomatic B. pertussis infections are common in school children in China, and asymptomatic B. parapertussis infections are more prevalent than previously documented.

Direct and Rapid Diagnosis of Extra-Pulmonary Tuberculosis in Archival Tissue by Nested and Touchdown PCR

Aims: To identify and differentiate mycobacterium spp. in archival formalin-fixed, paraffinembedded tissue by PCR to supply additional differential diagnostic method for Mycobacterium infections, where tuberculosis had been tested for by histopathological methods but without culturing. Place and Duration of Study: Department of Medical Laboratory Sciences, Department of Pathology and Microbiology (Faculty of Medicine) and King Abdulla University Hospital, between January 2004 and July 2010. Methodology: Fifty-six extra-pulmonary specimens of formalin-fixed, paraffin-embedded tissue obtained from patients showing granulomatus inflammation and/or other histopathologic features. Specimens were analyzed by a hemi-nested PCR assay targeting the gene encoding for 16S ribosomal RNA, which is common to all mycobacteria spp. The PCR positive specimens were amplified by a touch-down PCR targeting a fragment in the insertion sequence IS6110 specific to Mycobacterium tuberculosis complex. Results were compared to acid fast bacilli stain and with histopathology of each specimen. Results: M. tuberculosis complex DNA was detected in 27 (48.2%) specimens, and nonOriginal Research Article Article British Microbiology Research Journal, 4(5): 530-539, 2014 531 tuberculous mycobacteria in four specimens, compared to 10 (18%) specimens that were positive by acid fast staining. The positive cases were observed more in the lymph nodes, and pleural specimens. Conclusions: This study is among few studies to use the touch-down PCR assay as a promising auxiliary tool for the diagnosis of extra-pulmonary tuberculosis in archival tissue specimens. It could be used in conjunction with routine laboratory tests (e.g., cultures, acid fast staining) and clinical criteria of the patient to increase the accurate diagnosis of such cases. It is also recommended that culture be routinely done for all tuberculosis suspected cases.

Polymerase chain reaction based detection ofMycobacterium tuberculosisIS6110 in cattle faeces

PCR amplification of insertion element IS6110 of Mycobacterium tuberculosis in fecal samples was evaluated in the diagnosis of intestinal tuberculosis (ITB). Single intradermal tuberculin test was carried in 125 cattle from organized dairy farms, off which, 36 animals gave a positive reaction. Blood and faecal samples were collected from all the tuberculin positive (36) animals. Demonstration of acid fast bacilli was done in the faecal samples by using the Ziehl - Neelsen staining. DNA was extracted and PCR was carried out in the blood and faecal samples by using IS6110 insertion sequence. Acid fast bacilli were observed in 10 (27.77%) faecal smears whereas, 18 (50%) faecal samples and 4 (11.11%) blood samples were found positive for the insertion sequence IS6110 of Mycobacterium tuberculosis complex. This result shows that IS6110 insertion sequence-PCR assay may be applied to detect tuberculous bacilli directly from faecal sample of dairy cows and therefore could be a valuable diagnostic or screening test for herds.

Evaluation of amplification targets for the specific detection of Bordetella pertussis using real-time polymerase chain reaction.

Bordetella pertussis infections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detect B pertussis are typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity. A novel B pertussis real-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481, ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including different Bordetella species and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products. Analytical sensitivity was highest for the assay targeting the IS481 element; however, the assay lacked specificity for B pertussis in the reference panel and in the clinical samples. False-positive results were also observed with assays targeting the ptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains of B pertussis. However, a novel assay targeting the porin gene demonstrated high specificity for B pertussis both in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted all B pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction. A novel porin assay for B pertussis demonstrated superior performance and may be useful for improved molecular detection of B pertussis in clinical specimens.

Development of a multiplex real-time PCR assay for the detection of Bordetella pertussis and Bordetella parapertussis in a single tube reaction

Pertussis is an infectious respiratory disease caused by the fastidious bacterium Bordetella pertussis, which may infect unvaccinated, previously vaccinated children, and adults in whom immunity has waned. Infants are at a particular risk for severe disease and complications. Bordetella parapertussis may cause a similar illness, however the symptoms are less severe and of shorter duration. Pertussis is a highly contagious disease and early diagnosis is essential. Studies have shown that PCR is 2–4 times more likely than culture to detect Bordetella pertussis. We developed a multiplex, real-time PCR assay using analyte-specific reagent (ASR) primers and probes dispensed in a convenient lyophilized bead format that targeted the multi-copy insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively. These specific ASRs were used in conjunction with Cepheid Smartmix. Included in the ASRs is a competitive internal control to evaluate the performance of the PCR reaction. After DNA extraction, amplification and detection were done on the Smart Cycler System, which performs integrated amplification and detection automatically in a single step. Specificity of the assay was confirmed using multiple distinct bacterial strains. Sensitivity of the assay and extraction efficiency were evaluated on DNA isolated from pure bacterial cultures and on spiked respiratory specimens. We also spiked different swab types and transport media to evaluate for interfering substances. To assess accuracy, we studied different patient specimen types received from two outside laboratories that used similar or different methods to detect B. pertussis and B. parapertussis. The sensitivity and the specificity of the assay for B. pertussis were 90% and 96%, respectively, and for B. parapertussis 71% (only 7 positive specimens were available for testing) and 100%, respectively. Our assay was found to be a valid method for the simultaneous detection of B. pertussis and B. parapertussis.

Novel Class 1 Integrons in Multi-drug Resistant Isolates from Eastern China.

Integrons are mobile genetic elements able to capture, express and excise resistance genes, playing an important role in the spread of bacterial resistance. The present study was to investigate the occurrence and diversity of integrons in 120 clinical multi-drug resistant Gram-negative isolates from eastern China. Screening of integrons was performed by PCR and gene cassettes were further characterized by PCR-RFLP and sequencing. Class 1 integrons were detected in 70.8% of isolates and no class 2 and class 3 integrons were detected in any isolates. A total of 19 resistant gene cassettes were identified, four representative of novel gene cassettes: an aacA3 variant (aacA3c), an aacA4 variant (aacA4'-17), a bla OXA variant (bla OXA-251 ), and a catB8 gene cassette interrupted by an insertion sequence IS10 (catB8::IS10). In addition, 14 cassette arrays were detected, including three novel integrons: gcuD1-aacA4'-17-gcu38B-catB8::IS10 (In712), aacA3c-aadA13-bla OXA-251 (In713) and dfrA1-gcu37-aadA5 (In714). The presence of novel integron structures in clinical isolates suggests hospital environments may favor the formation of novel combination of gene cassettes. Moreover, the high prevalence of integrons in multi-drug resistant isolates highlights the urgent need to employ effective means to avoid dissemination of drug-resistant bacteria.

Formation of the High-Affinity Calcium Binding Site in Pro-subtilisin E with the Insertion Sequence IS1 of Pro-Tk-subtilisin

Subtilisin E is activated from its inactive precursor Pro-subtilisin E by autoprocessing and degradation of the propeptide. Subtilisin E has two calcium binding sites, the high-affinity Ca1 site and the low-affinity Ca2 site. The Ca1 site is conserved in various subtilisin-like proteases and is important for stability. This site is not formed in Pro-subtilisin E, because the structural rearrangement of the N-terminal region of the subtilisin domain upon autoprocessing is necessary for the formation of this site. As a result, Pro-subtilisin E is not fully folded. In contrast, Pro-Tk-subtilisin from Thermococcus kodakarensis is fully folded, because it does not require the structural rearrangement upon autoprocessing for the formation of the Ca1 site due to the presence of the insertion sequence IS1 between the propeptide and subtilisin domains. To examine whether the Ca1 site is formed in Pro-subtilisin E by inserting IS1 between the propeptide and subtilisin domains, the Pro-subtilisin E mutant with this insertion, IS1-Pro-subtilisin E, and its active site mutants, IS1-Pro-S221A and IS1-Pro-S221C, were constructed and characterized. The crystal structure of IS1-Pro-S221A revealed that this protein is fully folded and the Ca1 site is formed. In this structure, IS1 serves as a linker that brings the N-terminus of the subtilisin domain near the Ca1 site. IS1-Pro-S221A in a calcium-bound form was more stable than that in a calcium-free form by 13.1 °C. IS1-Pro-S221C was more rapidly autoprocessed than Pro-S221C. These results suggest that IS1 facilitates the formation of the Ca1 site and the complete folding of Pro-subtilisin E and thereby accelerates its autoprocessing.

PREVALENCE OF BORDETELLA HOLMESII AND BORDETELLA BRONCHISEPTICA IN RESPIRATORY TRACT SAMPLES FROM BELGIAN PATIENTS WITH PERTUSSIS-LIKE SYMPTOMS BY SENSITIVE CULTURE METHOD AND MASS SPECTROMETRY

Insertion sequences IS481 and IS1001 are targets for molecular detection of respectively Bordetella pertussis and Bordetella parapertussis. There is a raising concern about specificity of these targets due to sequence similarity with Bordetella holmesii and Bordetella bronchiseptica. The likelihood of false (para)pertussis diagnoses should be correlated with the prevalence of these organisms in the respiratory tract (RT). From October 2010 until September 2011, 2,207 RT samples were submitted to the Belgian reference laboratory for pertussis diagnosis. End-point IS481/IS1001 PCR and culture were performed for B. pertussis and B. parapertussis. We developed a sensitive culture method followed by screening with matrix-assisted laser desorption/ionisation- time of flight mass spectrometry (MALDI-TOF MS) to look for B. holmesii and B. bronchiseptica in our samples,. Only one B. bronchiseptica and no B. holmesii were detected in RT samples from Belgian patients with pertussis-like symptoms.

First-Time Detection of Mycobacterium bovis in Livestock Tissues and Milk in the West Bank, Palestinian Territories

BackgroundBovine tuberculosis, bTB, is classified by the WHO as one of the seven neglected zoonontic diseases that cause animal health problems and has high potential to infect humans. In the West Bank, bTB was not studied among animals and the prevalence of human tuberculosis caused by M. bovis is unknown. Therefore, the aim of this study was to estimate the prevalence of bTB among cattle and goats and identify the molecular characteristics of bTB in our area.Methodology/principal findingsA total of 208 tissue samples, representing 104 animals, and 150 raw milk samples, obtained from cows and goats were examined for the presence of mycobacteria. The tissue samples were collected during routine meat inspection from the Jericho abattoir. DNA was extracted from all samples, milk and tissue biopsies (n = 358), and screened for presence of TB DNA by amplifying a 123-bp segment of the insertion sequence IS6110. Eight out of 254 animals (3.1%) were found to be TB positive based on the IS6110-PCR. Identification of M. bovis among the positive TB samples was carried out via real time PCR followed by high resolution melt curve analysis, targeting the A/G transition along the oxyR gene. Spoligotyping analysis revealed a new genotype of M. bovis that was revealed from one tissue sample.SignificanceDetection of M. bovis in tissue and milk of livestock suggests that apparently healthy cattle and goats are a potential source of infection of bTB and may pose a risk to public health. Hence, appropriate measures including meat inspection at abattoirs in the region are required together with promotion of a health campaign emphasizing the importance of drinking pasteurized milk. In addition, further studies are essential at the farm level to determine the exact prevalence of bTB in goats and cattle herds in the West Bank and Israel.