Targeted Chromosomal Barcoding Establishes Direct Genotype-Phenotype Associations for Antibiotic Resistance in Mycobacterium abscessus.

A bedaquiline-resistant Mycobacterium abscessus isolate was sequenced, and a candidate mutation in the atpE gene was identified as responsible for the antibiotic resistance phenotype. To establish a direct genotype-phenotype relationship of this mutation which results in a Asp-to-Ala change at position 29 (D29A), we developed a recombineering-based method consisting of the specific replacement of the desired mutation in the bacterial chromosome. As surrogate bacteria, we used two M. abscessus bedaquiline-susceptible strains: ATCC 19977 and the SL541 clinical isolate. The allelic exchange substrates used in recombineering carried either the sole D29A mutation or a genetic barcode of silent mutations in codons flanking the D29A mutation. After selection of bedaquiline-resistant M. abscessus colonies transformed with both substrates, we obtained equivalent numbers of recombinants. These resistant colonies were analyzed by allele-specific PCR and Sanger sequencing, and we demonstrated that the presence of the genetic barcode was linked to the targeted incorporation of the desired mutation in its chromosomal location. All recombinants displayed the same MIC to bedaquiline as the original isolate, from which the D29A mutation was identified. Finally, to demonstrate the broad applicability of this method, we confirmed the association of bedaquiline resistance with the atpE A64P mutation in analysis performed in independent M. abscessus strains and by independent researchers. IMPORTANCE Antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of infections caused by microorganisms. On the other hand, infections caused by Mycobacterium abscessus affect people with chronic lung diseases, and their incidence has grown alarmingly in recent years. Further, these bacteria are known to easily develop AMR to the few therapeutic options available, making their treatment long-lasting and challenging. The recent introduction of new antibiotics against M. abscessus, such as bedaquiline, makes us anticipate a future when a plethora of antibiotic-resistant strains will be isolated and sequenced. However, in the era of whole-genome sequencing, one of the challenges is to unequivocally assign a biological function to each identified polymorphism. Thus, in this study, we developed a fast, robust, and reliable method to assign genotype-phenotype associations for putative antibiotic-resistant polymorphisms in M. abscessus.

Genetics of Mexico Jamiltepec Oaxaca Mixtec Amerindians according to HLA genes.

Mexican Mixtec population from coastal Jamiltepec (Oaxaca) Amerindians was studied for its HLA profile. They show genetic characteristics close to Pacific Islanders and other Mexican Isthmus Amerindians (Mazatecans, Zapotecans and Mayas). Interestingly, this coastal Oaxaca Mixtec population is genetically closer to Mazatecans than to Oaxaca Mixtec from mountains according to HLA genes. Mixtec HLA frequent extended haplotype A*24:02-B*35:14-DRB1*16:02 has been also found in Jaidukama North Colombia forest Amerindians and in Guatemala Mayas; A*24:02, DRB1*04:03, DRB1*04:04 and DRB2*16:02 are frequent alleles also common to Pacific Inhabitants. Notwithstanding, Mixtecs show deep cultural and genetic roots with Mesoamerican Amerindians and all of them probably contributed to construct Monte Alban culture around an important Pyramid Complex close to Oaxaca City.

HLA study in Bolivian Quechua Amerindians from Titikaka Lake Area.

Quechua Amerindians established Inca Empire and chose Cuzco as their capital. Their language is closely related to that of Aymara ethnic group and both of them were originated from Titikaka Lake Altiplano area. In the present study we have analyzed Bolivian Quechua HLA profile and found that it has common characters with other Andean and Pacific Amerindians (Uros, Aymaras, Lamas, Mapuches, Athabascan), and Pacific Islanders, including Easter Islanders: relatively high frequency of HLA-A*24 (:02), class II haplotypes DRB1*08:02-DQB1*04:02, and DRB1*04:03-DQB1* 03:02. Titikaka Lake area prehistoric populations: Quechua, Aymaras and Uros are closely related according to HLA Nei DA genetic distances and other HLA traits: they built up Tiwanaku culture, which resembles that of Easter Island (i.e.: similar giant heads); later, Quechuas also moved to Cuzco. This genetic reletedness together with Easter Island and Titikaka Lake Tiwanaku (Bolivia, Peru) cultural common similarities support a prehistoric Pacific people/Amerindians gene flow.

HLA study in Amerindian Bolivia La Paz Aymaras.

Aymara people has been a relatively homogeneous group since Spanish Conquest by 1,532 CE, even if previously represented a group of various cultural defined populations who gave rise to them. They were and are established in Andean Altiplano around Titikaka Lake (Bolivia, Peru), Argentina and Chile neighborhood, speak Aymara language and have been maintained after Europeans arrival at a lower social status than Quechua (Inca) speaking people. However, both Aymara and Quechua populations acknowledge Titikaka Lake as center of their origins; both languages are also related. Specific high frequencies of HLA-A*02, -A*24 and -A*68, HLA-B*35, -B*39 and -B*48, HLA-DRB1*08:02, -DRB1*09:01, and -DRB1*14:02, and HLA-DQB1*04:02, -DQB1*03:02 and -DQB1*03:01 alleles are found in Aymaras and HLA class II haplotypes common to Andean Amerindians (DRB1*08:02-DQB1*04:02 and DRB1*04:03-DQB1*03:02), like Quechua, Aymara, Uros, Lamas and Mapuche are also found in Easter and other Pacific Islands. Giant human head stone statues at Tiwanaku (Titikaka Lake, Bolivia) are also found at Easter Island. Thus, it is possible a gene and cultural flow between Andean Amerindians and Easter and other Pacific Islands, as it was demonstrated by Thor Heyerdahl in his Kon-Tiki expedition which reached Pacific Islands sailing from El Callao Harbour (Lima, Peru).

Study of HLA genes in Mexico Mayo/Yoremes Amerindians: Further support of gene exchange with Pacific Islanders.

Mexican Mayo Amerindians live in southern Sonora and North Sinaloa states. They probably come from North or are related to First American Inhabitants established further North. A non-related sample of them have volunteered to HLA study in order to achieve a profile useful for their epidemiology and future transplant interstate programs, in addition to ascertain ancestry and anthropological studies. HLA typing was carried out by a standard methodology. HLA-B*48 allele(s) was found, which is characteristic of Pacific Amerindians and Pacific Islanders/southern Asians. Also, HLA-A*24 (most likely HLA-A*24:02) shows specific high frequencies in this population and also in indigenous people, like Aleuts, Alaska Yupik, Japan, Taiwan, Australia, New Zealand, Papua New Guinea, southern China and other Pacific Islands. Other Andean Amerindians also show a high HLA-A*24:02 frequencies. This confirms our previous results of a possible direct gene flow between Pacific Islanders/southern Asians and Amerindians. In addition, typical Amerindian haplotypes have been found in high frequency like HLA-A*24-B*39-DRB1*04:07-DQB1*03:02, HLA-A*02-B*35-DRB1*04:07-DQB1*03:02 and HLA-A*24-B*35-DRB1*04:07-DQB1*03:02, and new haplotypes are also described like HLA-A*02-B*35-DRB1*14:06-DQB1*03:01, HLA-A*02-B*48-DRB1*04:04-DQB1*03:02, and HLA-A*02-B*08-DRB1*04:07-DQB1*03:02. This study also supports that Americas peopling was not only carried out through Bering Strait but also through Pacific and Atlantic Oceans in an earlier time than proposed.

HLA genes in Amerindians from Mexico San Vicente Tancuayalab Teenek/Huastecos.

Huastecos or Teenek Amerindians are presently living at North East Mexico (San Luis Potosi State). They have probably one of the most ancient culture of Mexico and Central America together with Mayas and Olmec groups with which also show close relationships. Proximity to Atlantic Ocean/Mexican Gulf originated that Spaniards had very early contact with them at about 1519 CE or before. In the present paper we have aimed to study HLA gene profile which may be useful for HLA and disease epidemiology and transplant programs in Teeneks. HLA-DRB1*04:07, -DRB1*14:06 and -DRB1*04:11 have been found in high frequency like in other Amerindian groups. High frequency typical Amerindians HLA extended haplotypes have been found, such as A*02-B*35-DRB1*04:07-DQB1*03:02; A*68-B*39-DRB1*04:07-DQB1*03:02 and A*02-B*39-DRB1*04:07-DQB1*03:02; also new haplotypes have been described, like A*02-B*52-DRB1*04:11-DQB1*03:02, A*68-B*35-DRB1*14:02-DQB1*03:01 and A*68-B*40-DRB1*16:02-DQB1*03:01. Genetic proximity is observed not only to linguistically close Mayans, but also to Mazatecans, Mixtecans and Zapotecans, who speak an altogether different languages; it shows once more that genes and languages do not correlate. This population was greatly diminished after European contact between 1500 and 1600 years CE; in fact, North and South America First Inhabitants population was brought from 80 down to 8 million people because of diseases (i.e.: measles, smallpox or influenza), slavery and war.