IS6110 restriction fragment length polymorphism of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in Campinas,Brazil: evidence of intercontinental distribution of strains

Tuberculosis (TB) is a major concern in developing countries. In Brazil, few genotyping studies have been conducted to verify the number of IS6110 copies present in local prevalent strains of Mycobacterium tuberculosis, the distribution and clustering of strains. IS6110 DNA fingerprinting was performed on a sample of M. tuberculosis isolates from patients with AFB smear-positive pulmonary TB, at a hospital in Brazil. The IS6110 profiles were analyzed and compared to a M. tuberculosis database of the Houston Tuberculosis Initiative, Houston, US. Seventy-six fingerprints were obtained from 98 patients. All M. tuberculosis strains had an IS6110 copy number between 5-21 allowing for differentiation of the isolates. Human immunodeficiency virus infection was confirmed in nearly half the patients of whom data was available. Fifty-eight strains had unique patterns, while 17 strains were grouped in 7 clusters (2 to 6 strains). When compared to the HTI database, 6 strains matched isolates from El Paso, Ciudad de Juarez, Houston, and New York. Recently acquired infections were documented in 19% of cases. The community transmission of infection is intense, since some clustered strains were recovered during the four-year study period. The intercontinental dissemination of M. tuberculosis strains is suspected by demonstration of identical fingerprints in a distant country.     

Genotyping of Mycobacterium tuberculosis isolates from a low-endemic setting in northwestern state of Paraná in Southern Brazil

The purpose of this study was to provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis in a low-endemic setting in northwestern state of Paraná in Southern Brazil. We employed spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) techniques to genotype M. tuberculos isisolates from patients with pulmonary tuberculosis (TB). The 93 isolates analyzed by spoligotyping were divided into 36 different patterns, 30 of which were described in the SITVIT database. Latin American and Mediterranean, Haarlem and T families were responsible for 26.9%, 17.2% and 11.8% of TB cases, respectively. From the 84 isolates analyzed by MIRU-VNTR, 58 shared a unique pattern and the remaining 26 belonged to nine clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. A combination of MIRU-VNTR and spoligotyping resulted in 85.7% discriminatory power (Hunter-Gaston index = 0.995). Thus, combining spoligotyping and MIRU-VNTR typing proved to be most useful for epidemiological study in this low-endemic setting in Southern Brazil. The current study demonstrated that there is significant diversity in circulating strains in the city of Maringá and the surrounding regions, with no single genotype of M. tuberculosis predominating.     

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.     

Fitness study of the RDRio lineage and Latin American–Mediterranean family of Mycobacterium tuberculosis in the city of Rio Grande, Brazil

RD^Rio is a novel Mycobacterium tuberculosis lineage of the Latin American–Mediterranean (LAM) family. LAM has been found worldwide but is more predominant in South America. The aim of this study was to assess the presence of the RD^Rio lineage and LAM family in the city of Rio Grande, Brazil, and to investigate the fitness of these strains based on determination of their growth rate. Fifty clinical isolates of M. tuberculosis were genotyped and 43 different patterns were found by spoligotyping and mycobacterial interspersed repetitive units–variable number of tandem repeats. The predominant genotypes belonged to the LAM family (54% of the strains) followed by clade T (22%) and Haarlem (16%). The RD^Rio lineage represented 38% of the total strains and 70.4% of the LAM strains found in this study. Strains belonging to the LAM family showed a fitness advantage when comparing their rate of growth with that of non-LAM strains, but a significant difference between RD^Rio and non-RD^Rio strains was not confirmed.     

The Beijing genotype and drug resistant tuberculosis in the Aral Sea region of Central Asia

Background

After the collapse of the Soviet Union, dramatically increasing rates of tuberculosis and multidrug-resistant tuberculosis (MDR-TB) have been reported from several countries. This development has been mainly attributed to the widespread breakdown of TB control systems and declining socio-economic status. However, recent studies have raised concern that the Beijing genotype of Mycobacterium tuberculosis might be contributing to the epidemic through its widespread presence and potentially enhanced ability to acquire resistance.

Methods

A total of 397 M. tuberculosis strains from a cross sectional survey performed in the Aral Sea region in Uzbekistan and Turkmenistan have been analysed by drug susceptibility testing, IS6110 fingerprinting, and spoligotyping.

Results

Fifteen isolates showed mixed banding patterns indicating simultaneous infection with 2 strains. Among the remaining 382 strains, 152 (40%) were grouped in 42 clusters with identical fingerprint and spoligotype patterns. Overall, 50% of all isolates were Beijing genotype, with 55% of these strains appearing in clusters compared to 25% of non-Beijing strains. The percentage of Beijing strains increased with increasing drug resistance among both new and previously treated patients; 38% of fully-susceptible isolates were Beijing genotype, while 75% of MDR-TB strains were of the Beijing type.

Conclusion

The Beijing genotype is a major cause of tuberculosis in this region, it is strongly associated with drug resistance, independent of previous tuberculosis treatment and may be strongly contributing to the transmission of MDR-TB. Further investigation around the consequences of Beijing genotype infection for both tuberculosis transmission and outcomes of standard short course chemotherapy are urgently needed.     

Molecular epidemiology of drug-resistant tuberculosis in Sweden

Drug-resistant tuberculosis (TB), including the more severe forms of multidrug- and extensively drug-resistant forms, is an increasing public health concern globally. In Sweden the majority of patients with TB are immigrants from countries with a high incidence of TB including the drug-resistant forms. In this study, the spread of resistant TB in Sweden was investigated by molecular fingerprinting. Isolates resistant to at least one of the drugs, isoniazid, rifampicin, ethambutol or streptomycin, from 400 patients collected between 1994 and 2005, were studied by restriction fragment length polymorphism (RFLP) and by spoligotyping. Thirty-five clusters of patients infected with strains with identical RFLP and spoligotyping patterns (2-96 patients per cluster), comprising a total of 203 patients, were found. One large outbreak of isoniazid resistant tuberculosis was identified, involving 96 patients, mainly from the Horn of Africa. To identify chains of transmission, molecular epidemiological characterization of TB isolates should, if possible, be performed on isolates from all new TB patients.     

Multidrug Resistant Mycobacterium tuberculosis: A Retrospective katG and rpoB Mutation Profile Analysis in Isolates from a Reference Center in Brazil

Background

Multidrug resistance is a critical factor in tuberculosis control. To gain better understanding of multidrug resistant tuberculosis in Brazil, a retrospective study was performed to compare genotypic diversity and drug resistance associated mutations in Mycobacterium tuberculosis isolates from a national reference center.

Methods and Findings

Ninety-nine multidrug resistant isolates from 12 Brazilian states were studied. Drug-resistance patterns were determined and the rpoB and katG genes were screened for mutations. Genotypic diversity was investigated by IS6110-RFLP and Luminex 47 spoligotyping. Mutations in rpoB and katG were seen in 91% and 93% of the isolates, respectively. Codon 315 katG mutations occurred in 82.8% of the isolates with a predominance of the Ser315Thr substitution. Twenty-five isolates were clustered in 11 groups with identical IS6110-RFLP patterns while 74 showed unique patterns with no association between mutation frequencies or susceptibility profiles. The most prevalent spoligotyping lineages were LAM (47%), T (17%) and Haarlen (12%). The Haarlen lineage showed a higher frequency of codon 516 rpoB mutations while codon 531 mutations prevailed in the other isolates.

Conclusions

Our data suggest that there were no major multidrug resistant M. tuberculosis strains transmitted among patients referred to the reference center, indicating an independent acquisition of resistance. In addition, drug resistance associated mutation profiles were well established among the main spoligotyping lineages found in these Brazilian multidrug resistant isolates, providing useful data for patient management and treatment.     

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.     

Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Transmission of tuberculosis in Havana, Cuba: a molecular epidemiological study by IS6110 restriction fragment length polymorphism typing

The combination of molecular and conventional epidemiological methods has improved the knowledge about the transmission of tuberculosis in urban populations. To examine transmission of tuberculosis in Havana, Cuba, with DNA fingerprinting, we studied 51 out of 92 Mycobacterium tuberculosis strains isolated from tuberculosis patients who resided in Havana and whose infection was culture-confirmed in the period from September 1997 to March 1998. Isolates from 28 patients (55%) had unique IS6110 restriction fragment length polymorphism (RFLP) patterns, while isolates from 23 others (45%) had identical patterns and belonged to 7 clusters. Three clusters consisting of six, five and two cases were each related to small outbreaks that occurred in a closed setting. Three other clustered cases were linked to a large outbreak that occurred in another institution. Younger patients were more correlated to clustering than older ones. The finding that 45% of the isolates had clustered RFLP patterns suggests that recent transmission is a key factor in the tuberculosis cases in Havana. The IS6110 RFLP typing made it possible to define the occurrence of outbreaks in two closed institutions.     

IS6110-mediated deletion polymorphism in the direct repeat region of clinical isolates of Mycobacterium tuberculosis

This study investigates the phenomenon of IS6110-mediated deletion polymorphism in the direct repeat (DR) region of the genome of Mycobacterium tuberculosis. Clinical isolates and their putative predecessors were compared using a combination of DR region restriction fragment length polymorphism, IS6110 DNA fingerprinting, spoligotyping, and DNA sequencing, which allowed the mapping of chromosome structure and deletion junctions. The data suggest that adjacently situated IS6110 elements mediate genome deletion. However, in contrast to previous reports, deletions appear to be mediated by inversely oriented IS6110 elements. This suggests that these events may occur via mechanisms other than RecA-mediated homologous recombination. The results underscore the important role of IS6110-associated deletion hypervariability in driving M. tuberculosis genome evolution.     

Molecular characterisation of <Emphasis Type="Italic">Mycobacterium bovis</Emphasis>isolated from cattle slaughtered at the Bamako abattoir in Mali

Background

Mali is one of the most important livestock producers of the Sahel region of Africa. A high frequency of bovine tuberculosis (BTB) has been reported but surveillance and control schemes are restricted to abattoir inspections only. The objective of this study was to conduct, for the first time, molecular characterisation of Mycobacterium bovis strains isolated from cattle slaughtered at the Bamako abattoir. Of 3330 animals screened only 60 exhibited gross visible lesions. From these animals, twenty strains of M. bovis were isolated and characterised by spoligotyping.

Results

Organ lesions typical of BTB were most often detected in the liver, followed by the lung and the peritoneum. M. bovis was isolated from 20 animals and 7 different spoligotypes were observed among these 20 strains; three of the patterns had not been previously reported. Spoligotype patterns from thirteen of the strains lacked spacer 30, a characteristic common in strains of M. bovis found in Chad, Cameroon and Nigeria. However, unlike the other three Central African countries, the majority of spoligotype patterns observed in Mali also lacked spacer 6. Of the remaining seven strains, six had spoligotype patterns identical to strains commonly isolated in France and Spain.

Conclusion

Two groups of M. bovis were detected in cattle slaughtered at the Bamako abattoir. The spoligotype pattern of the first group has similarities to strains previously observed in Chad, Cameroon and Nigeria. The additional absence of spacer 6 in the majority of these strains suggests a Mali specific clone. The spoligotype patterns of the remaining strains suggest that they may have been of European origin.

     

Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela

Background

Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD).

Results

Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised. Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis.

Conclusion

This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.     

A DNA chip-based spoligotyping method for the strain identification of Mycobacterium tuberculosis isolates

The clinical efficacy of a spoligotyping method for discriminating Mycobacterium tuberculosis strains was evaluated. Among the strains other than Beijing or Beijing like family, 30 different spoligotypes out of 39 strains were produced, which included 4 strains not having IS6110 sequence. The oligonucleotide chip-based spoligotyping technique is useful for early screening and detection of clonal proximity of M. tuberculosis isolates.     

Molecular characterization of Mycobacterium tuberculosis isolates from Izmir, Turkey

In recent years, molecular typing methods have been used in epidemiologic studies of Mycobacterium tuberculosis isolates in various areas of the world. However, there have been few data on this issue in Turkey. We describe the molecular characterization of 56 Mycobacterium tuberculosis isolates recovered from individual patients in Izmir and the surrounding area by three different molecular methods. Isolated M. tuberculosis strains were characterized by IS6110 RFLP, spoligotyping and major genetic group designation. In total, 51 RFLP and 35 spoligopatterns were identified. Fourteen (25%) isolates were indicated as low copy number. Based on three genotypic characterization methods together, five clusters with two isolates each were identified. Most of the isolates (98.2%) were assigned as genetic groups 2 or 3. Only one isolate was identified as Beijing family strain (principal genetic group 1). The shared international clades were found to be Beijing-family, var T1 (ST 37), LAM (Latin-American-Mediterranean) 7 (ST 41), LAM 9 (ST 42), Haarlem 1 (ST 47), Haarlem 3 (ST 50) and T1 (ST 53). In this study, IS6110 RFLP, spoligotyping and major genetic group designation were found to be useful methods for molecular epidemiologic studies.     

R-plasmid transfer frequencies from environmental isolates of Escherichia coli to laboratory and fecal strains.

Multiple-drug-resistant strains of Escherichia coli were isolated from the water at an estuarine site. They represented about 8.3% of the total E. coli population. Fifty-five strains, representing each of the 32 resistance patterns identified, were mated with an E. coli K-12 F- strain. Matings were performed on membrane filters, and the cells were washed to remove any colicins produced by the donors. Thirty-one strains, about 5% of the mean E. coli density in the samples, transferred drug resistance and, hence, posessed conjugative R plasmids. Of these, 80% transferred drug resistance at a frequency of about 10(-4) or less. Nine environmental R+ strains were mated with three fecal recipients. The R-plasmid transfer frequencies to the fecal strains from the environmental donors correlated well with those from a derepressed K-12 R+ laboratory donor. The R+ X K-12 F- lac- transconjugants from 16 environmental strains were "backcrossed" to a lac+ K-12 F- strain. All transfer frequencies were higher in the backcrosses than in the original matings from the environmental donor. Furthermore, 7 of 13 different transconjugants, which accepted plasmids at repressed frequencies of less than 10(-3), donated them at frequencies greater than 10(-2). This suggests that these were derepressed plasmids in a repressed host.     

Identification of unique genetic markers in Rv0927c among Mycobacterium tuberculosis W-Beijing strains

Fifty-six clinical isolates of Mycobacterium tuberculosis were analyzed by spoligotyping to determine the prevalence of W-Beijing strains. Forty-nine of the 56 isolates belonged to W-Beijing strains and 7 isolates were non-Beijing strains. Comparative two-dimensional gel electrophoresis analysis of protein patterns between the W-Beijing and non-Beijing strains identified a unique protein Rv0927c that is absent in the former but present in the latter and the reference strain M. tuberculosis H37Rv. Compared with 7 non-Beijing clinical isolates and H37Rv, all 49 W-Beijing strains had two characteristic mutations, a deletion of AGC at nucleotide position 421 of Rv0927c gene encoding a putative short dehydrogenase/reductase, causing deletion of serine codon at amino acid position 141 and a -127 G-->A mutation in Rv0927c-pstS3 intergenic region, resulting in failure to express Rv0927c. Western blot analysis indicated that polyclonal antibody raised against H37Rv Rv0927c overexpressed in Escherichia coli reacted with non-Beijing strains and H37Rv but not W-Beijing strains. Characteristic mutations of Rv0927c that are present in W-Beijing strains can be used as a novel genetic marker for rapid molecular typing of M. tuberculosis W-Beijing strains.     

Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria

The direct repeat region in Mycobacterium tuberculosis complex strains is composed of multiple direct variant repeats (DVRs), each of which is composed of a 36-bp direct repeat (DR) plus a nonrepetitive spacer sequence of similar size. It has been shown previously that clinical isolates show extensive polymorphism in the DR region by the variable presence of DVRs, and this polymorphism has been used in the epidemiology of tuberculosis. In an attempt to better understand the evolutionary scenario leading to polymorphic DR loci and to improve strain differentiation by spoligotyping, we characterized and compared the DNA sequences of the complete DR region and its flanking DNA of M. tuberculosis complex strains. We identified 94 different spacer sequences among 26 M. tuberculosis complex strains. No sequence homology was found between any of these spacers and M. tuberculosis DNA outside of the DR region or with any other known bacterial sequence. Although strains differed extensively in the presence or absence of DVRs, the order of the spacers in the DR locus was found to be well conserved. The data strongly suggest that the polymorphism in clinical isolates is the result of successive deletions of single discrete DVRs or of multiple contiguous DVRs from a primordial DR region containing many more DVRs than seen in present day isolates and that virtually no scrambling of DVRs took place during evolution. Because the majority of the novel spacer sequences identified in this study were confined to isolates of the rare Mycobacterium canettii taxon, the use of the novel spacers in spoligotyping led only to a slight improvement of strain differentiation by spoligotyping.     

Spoligotyping of clinical Mycobacterium tuberculosis isolates from the state of Minas Gerais, Brazil

We performed spoligotyping on 114 strains of the Mycobacterium tuberculosis (Mtb) complex that had been isolated from patients in Minas Gerais Health Units during 2004. A total of 82/114 (72%) clinical isolates were clustered and 32/114 (28%) were unique. Seven shared types containing nine strains were newly created. A total of nine patterns corresponded to unreported orphan strains, as evaluated against all of the strains recorded in the SITVIT2 proprietary database in the Institut Pasteur de la Guadeloupe. The major clades were composed of isolates that belong to the following genotypes: Latin-America and Mediterranean (63/114, 55.3%) (the ill-defined T superfamily) (12/114, 10.5%), Haarlem (8/114, 7%), X clade (6/114, 5.3%), S clade (3/114, 2.6%) and the East-African Indian and Manu types, each with 1/114 (0.9%) isolates. A considerable number of strains (n = 20, 17.5%) showed patterns that did not fall within any of the previously described major clades. We conclude the bulk of tuberculosis (TB) (92/114, 80.7%) in our location is recent evolutionary strains that belong to the principal genetic groups 2/3. Further studies on epidemiology of TB are required to understand Mtb biodiversity and TB transmission in this region.     

Escherichia coli bacteriocins: antimicrobial efficacy and prevalence among isolates from patients with bacteraemia

Bacteriocins are antimicrobial peptides generally active against bacteria closely related to the producer. Escherichia coli produces two types of bacteriocins, colicins and microcins. The in vitro efficacy of isolated colicins E1, E6, E7, K and M, was assessed against Escherichia coli strains from patients with bacteraemia of urinary tract origin. Colicin E7 was most effective, as only 13% of the tested strains were resistant. On the other hand, 32%, 33%, 43% and 53% of the tested strains exhibited resistance to colicins E6, K, M and E1. Moreover, the inhibitory activity of individual colicins E1, E6, E7, K and M and combinations of colicins K, M, E7 and E1, E6, E7, K, M were followed in liquid broth for 24 hours. Resistance against individual colicins developed after 9 hours of treatment. On the contrary, resistance development against the combined action of 5 colicins was not observed. One hundred and five E. coli strains from patients with bacteraemia were screened by PCR for the presence of 5 colicins and 7 microcins. Sixty-six percent of the strains encoded at least one bacteriocin, 43% one or more colicins, and 54% one or more microcins. Microcins were found to co-occur with toxins, siderophores, adhesins and with the Toll/Interleukin-1 receptor domain-containing protein involved in suppression of innate immunity, and were significantly more prevalent among strains from non-immunocompromised patients. In addition, microcins were highly prevalent among non-multidrug-resistant strains compared to multidrug-resistant strains. Our results indicate that microcins contribute to virulence of E. coli instigating bacteraemia of urinary tract origin.