New Variants of Pseudomonas aeruginosa High-Risk Clone ST233 Associated with an Outbreak in a Mexican Paediatric Hospital.

Recent multidrug resistance in Pseudomonas aeruginosa has favoured the adaptation and dissemination of worldwide high-risk strains. In June 2018, 15 P. aeruginosa strains isolated from patients and a contaminated multi-dose meropenem vial were characterized to assess their association to an outbreak in a Mexican paediatric hospital. The strains were characterized by antibiotic susceptibility profiling, virulence factors' production, and biofilm formation. The clonal relationship among isolates was determined with pulse-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) sequencing. Repressor genes for the MexAB-OprM efflux pump were sequenced for haplotype identification. Of the strains, 60% were profiled as extensively drug-resistant (XDR), 33% as multidrug-resistant (MDR), and 6.6% were classified as sensitive (S). All strains presented intermediate resistance to colistin, and 80% were sensitive to aztreonam. Pyoverdine was the most produced virulence factor. The PFGE technique was performed for the identification of the outbreak, revealing eight strains with the same electrophoretic pattern. ST235 and ten new sequence types (STs) were identified, all closely related to ST233. ST3241 predominated in 26.66% of the strains. Twenty-five synonymous and seventeen nonsynonymous substitutions were identified in the regulatory genes of the MexAB-OprM efflux pump, and nalC was the most variable gene. Six different haplotypes were identified. Strains from the outbreak were metallo-ß-lactamases and phylogenetically related to the high-risk clone ST233.

Nucleotide substitutions in the mexR, nalC and nalD regulator genes of the MexAB-OprM efflux pump are maintained in Pseudomonas aeruginosa genetic lineages.

Pseudomonas aeruginosa has different resistant mechanisms including the constitutive MexAB-OprM efflux pump. Single nucleotide polymorphisms (SNPs) in the mexR, nalC, and nalD repressors of this efflux pump can contribute to antimicrobial resistance; however, it is unknown whether these changes are mainly related to genetic lineages or environmental pressure. This study identifies SNPs in the mexR, nalC, and nalD genes in clinical and environmental isolates of P. aeruginosa (including high-risk clones). Ninety-one P. aeruginosa strains were classified according to their resistance to antibiotics, typified by multilocus sequencing, and mexR, nalC, and nalD genes sequenced for SNPs identification. The mexAB-oprM transcript expression was determined. The 96.7% of the strains were classified as multidrug resistant. Eight strains produced serine carbapenemases, and 11 strains metallo-ß-lactamases. Twenty-three new STs and high-risk clones ST111 and ST233 were identified. SNPs in the mexR, nalC, and nalD genes revealed 27 different haplotypes (patterns). Sixty-two mutational changes were identified, 13 non-synonymous. Haplotype 1 was the most frequent (n = 40), and mainly identified in strains ST1725 (33/40), with 57.5% pan drug resistant strains, 36.5% extensive drug resistant and two strains exhibiting serin-carbapenemases. Haplotype 12 (n = 9) was identified in ST233 and phylogenetically related STs, with 100% of the strains exhibiting XDR and 90% producing metallo-ß-lactamases. Haplotype 5 was highly associated with XDR and related to dead when compared to ST1725 and ST233 (RRR 23.34; p = 0.009 and RRR 32.01; p = 0.025). A significant relationship between the mexR-nalC-nalD haplotypes and phylogenetically related STs was observed, suggesting mutational changes in these repressors are highly maintained within genetic lineages. In addition, phylogenetically related STs showed similar resistant profiles; however, the resistance was (likely or partly) attributed to the MexAB-OprM efflux pump in 56% of the strains (only 45.05% showed mexA overtranscription), in the remaining strains the resistance could be attributed to carbapenemases or mechanisms including other pumps, since same SNPs in the repressor genes gave rise to different resistance profiles.

Identification of extensive drug resistant Pseudomonas aeruginosa strains: New clone ST1725 and high-risk clone ST233.

Several microorganisms produce nosocomial infections (NIs), among which Pseudomonas aeruginosa stands out as an opportunist pathogen with the capacity to develop multiresistance to first-choice antibiotics. From 2007 to 2013, forty-six NIs produced by P. aeruginosa were detected at a pediatric tertiary care hospital in Mexico with a significant mortality rate (17.39%). All isolates (n = 58/46 patients) were characterized by evaluating their response to several antibiotics as panresistant (PDR), extensively resistant (XDR), multiresistant (MDR) or sensitive (S). In addition, all isolates were typified through multilocus sequencing of seven genes: acsA, aroE, guaA, mutL, nuoD, ppsA and trpE. Furthermore, to establish the genetic relationships among these isolates, we carried out a phylogenetic inference analysis using maximum likelihood to construct a phylogenetic network. To assess evolutionary parameters, recombination was evaluated using the PHI test, and the ratio of nonsynonymous to synonymous substitutions was determined. Two of the strains were PDR (ST1725); 42 were XDR; four were MDR; and ten were S. Twenty-one new sequence types were detected. Thirty-three strains exhibited novel sequence type ST1725. The ratio of nonsynonym to synonym substitutions was 1:1 considering all genes. Phylogenetic analysis showed that the genetic relationship of the PDR, XDR and MDR strains was mainly clonal; however, the PHI test and the phylogenetic network suggest that recombination events occurred to produce a non-clonal population. This study aimed not only to determine the genetic diversity of clinical P. aeruginosa but also to provide a warning regarding the identification and spreading of clone ST1725, its ability to cause outbreaks with high mortality rates, and to remain in the hospital environment for over seven years. These characteristics highlight the need to identify clonal outbreaks, especially where high resistance to most antibiotics is observed, and control measures are needed. This study also represents the first report of the PDR ST1725.

Abnormality of adipokines and endothelial dysfunction in Mexican obese adolescents with insulin resistance.

PURPOSE: The aim of this study was to investigate the possible relationship among insulin resistance (IR), endothelial dysfunction, and alteration of adipokines in Mexican obese adolescents and their association with metabolic syndrome (MetS). MATERIALS AND METHODS: Two hundred and twenty-seven adolescents were classified according to the body mass index (BMI) (control: N=104; obese: N=123) and homeostasis model of the assessment-insulin resistance index (HOMA-IR) (obese with IR: N=65). The circulating concentrations of leptin, adiponectin, soluble intercellular adhesion molecule-1 (sICAM-1), and IR were determined by standard methods. RESULTS: The obese adolescents with IR presented increased presence of MetS and higher circulating concentrations in sICAM-1 in comparison with the obese subjects without IR. The lowest concentrations of adiponectin were observed in the obese with IR. In multivariate linear regression models, sICAM-1 along with triglycerides, total cholesterol, and waist circumference was strongly associated with HOMA-IR (R2=0.457, P=0.008). Similarly, after adjustment for age, BMI-SDS, lipids, and adipokines, HOMA-IR remained associated with sICAM-1 (R2=0.372, P=0.008). BMI-SDS was mildly associated with leptin (R2=0.176, P=0.002) and the waist circumference was mild and independent determinant of adiponectin (R2=0.136, P=0.007). CONCLUSIONS: Our findings demonstrated that the obese adolescents, particularly the obese subjects with IR exhibited increased presence of MetS, abnormality of adipokines, and endothelial dysfunction. The significant interaction between IR and endothelial dysfunction may suggest a novel therapeutic approach to prevent or delay systemic IR and the genesis of cardiovascular diseases in obese patients.

Polymorphism analysis and new JAG1 gene mutations of Alagille syndrome in Mexican population.

Alagille syndrome is a multisystem disorder with an autosomic dominant pattern of inheritance that affects the liver, heart, eyes, kidneys, skeletal system and presents characteristic facial features. Mutations of the JAG1 gene have been identified in 20-89% of the patients with Alagille syndrome, this gene encodes for a ligand that activates the Notch signaling pathway. In the present study we analyzed 9 Mexican patients with Alagille syndrome who presented the clinical criteria for the classical presentation of the disease. By using the denaturing high performance liquid chromatography mutation analysis we were able to identify different mutations in 7 of the patients (77.77%), importantly, we found 5 novel mutations in JAG1 gene. The allelic frequency distribution of 13 polymorphisms in Mexican population is also reported. The overall results demonstrated an expanding mutational spectrum of JAG1 gene in the Mexican population.

Importancia del diagnóstico de mutaciones en el gen de la conexina 26 en el manejo integral de la sordera congénita no sindrómica / Importance of the diagnosis of connexin 26 gene mutations in the integral management of non-syndromic congenital deafness

Introducción. La sordera congénita es un problema de salud pública. Su incidencia en México es de 2-3 por cada 1000 recién nacidos. El diagnóstico oportuno con el tamiz auditivo neonatal es fundamental para un mejor pronóstico funcional. Aproximadamente 70% de las sorderas congénitas son de origen genético, con herencia autosómica recesiva. La mayoría de estos casos se asocia con mutaciones en el gen GJB2 , que codifica para la proteína conexina 26. Hay tres mutaciones reportadas como las más frecuentes en este gen: c.35delG, c.167delT y c.235delC. Métodos. Previo consentimiento informado de los pacientes, se obtuvo 1 ml de sangre periférica para la extracción de ADN. Mediante las técnicas de PCR-RFLP o PCR seguida de secuenciación, se buscaron las tres mutaciones más frecuentes del gen GJB2 . Resultados. Se realizó el estudio molecular en 11 pacientes: Se encontró un cambio en la secuencia codificante en cinco de ellos. Un paciente fue homocigoto para c.35delG; otro resultó heterocigoto para c.35insG, mutación no reportada previamente; un tercero fue heterocigoto para c.34G>T y dos más fueron heterocigotos para el polimorfismo c.79G>A (p.V27I). En ningún caso se hallaron las mutaciones c.167delT y c.235delC. Conclusiones. Se encontraron cambios de secuencias que correspondieron a dos polimorfismos y a tres mutaciones. La frecuencia de las tres mutaciones investigadas fue menor a lo reportado en la literatura y se encontró una mutación no reportada previamente. Este estudio evidencia la importancia del diagnóstico oportuno con manejo integral, incluyendo el asesoramiento genético con base en estudios moleculares, y resalta la importancia de conocer el perfil genotípico de este grupo de pacientes.

Background. Congenital deafness is a public health problem affecting 2-3:1000 newborns in Mexico. Neonatal audiologic screening allows early detection with important implications for the functional prognosis. About 70% of cases of congenital deafness are associated with a genetic etiology with an autosomal recessive pattern of inheritance. Most cases are caused by mutations in the GJB2 gene, which codifies conexin 26. The three most commonly reported mutations in this gene are c.35delG, c.167delT and c.235delC. Methods. After obtaining informed consent, DNA was extracted from a blood sample, and the three previously mentioned mutations were searched for using PCR-RFLP or PCR followed by sequencing. Results. Molecular analysis was carried out in 11 patients. In five of these patients, a change in sequence was observed. In none of the patients were c.167delT and c.235delC mutations found. One patient was homozygous for c.35delG and another patient was heterozygous for c.35insG, which is a mutation not previously reported. A third patient was heterozygous for c.34G>T. Two additional patients had the c.79G>A (p.V27I) polymorphism. Conclusions. Frequency of the three mutations analyzed was lower compared to other populations. Five sequence changes were observed, two polymorphisms and three mutations, one of them novel. This study also demonstrates the relevance of early diagnosis and multidisciplinary management and the importance of determining the genetic basis of this disease in pediatric patients with congenital deafness.

CYP3A5 polymorphism in Mexican renal transplant recipients and its association with tacrolimus dosing.

BACKGROUND AND AIMS: Variability in CYP3A5 expression associated with differences in tacrolimus bioavailability has been documented. The wild-type allele CYP3A5*1 expresses the functional protein, whereas the CYP3A5*3 allele is a splice variant with a premature stop codon and encodes a truncated nonfunctional protein. The aim of the study was to determine the frequency of CYP3A5*1 and CYP3A5*3 in 291 (124 adults, 167 pediatric) Mexican renal transplant recipients, evaluate the tacrolimus dose requirements by genotype and compare genotype frequency data with that of other populations. METHODS: We carried out a multicenter study. Patients were recruited from three institutions located in Mexico City. Genotyping of the CYP3A5*1 and CYP3A5*3 alleles was performed by direct DNA sequencing. RESULTS: Eighteen patients (6.2%) were CYP3A5*1*1 homozygous carriers or functional protein expresser homozygous, 121 patients (41.6 %) were CYP3A5*1*3 were heterozygous carriers or heterozygous expressers, and 152 patients (52.2%) were CYP3A5*3*3 homozygous carriers or homozygous nonexpressers. There was a statistically significant difference in frequency of the functional and nonfunctional expresser phenotypes from those reported for Black and Caucasian, but not for South Asian populations. The CYP3A5 phenotype had a significant impact in tacrolimus bioavailability, as wild-type carriers required higher dosing compared to mutated carriers to achieve similar drug trough levels. Patients with CYP3A5*1*1 genotype had a median dose requirement of 0.16 mg/kg/day, CYP3A5*1*3 patients had a median tacrolimus dose of 0.13 mg/kg/day and CYP3A5*3*3 had a median dose of 0.07 mg/kg/day (Kruskal-Wallis, p <0.0001). CONCLUSIONS: Of the Mexican transplant recipients, 52.2% were CYP3A5*3*3 and required significantly lower tacrolimus dose than those with CYP3A5*1 allele.

Prevalencia de polimorfismos del receptor de vitamina D en pacientes pediátricos mexicanos con enfermedad renal crónica / Prevalence of vitamin D receptor gene polymorphisms in Mexican children with chronic kidney disease

Introducción. La posible influencia de los diferentes polimorfismos del receptor de vitamina D sobre la regulación del eje calcio-hormona paratiroidea -vitamina D es particularmente importante en pacientes con enfermedad renal crónica, ya que cursan con trastornos en el metabolismo mineral óseo. El objetivo del trabajo fue describir la frecuencia de los polimorfismos rs7975232 (ApaI), rs731236 (TaqI) y rs1544410 (BsmI) en el receptor de vitamina D y su relación con el calcio, el fósforo, la fosfatasa alcalina y la hormona paratiroidea en pacientes pediátricos con enfermedad renal crónica. Métodos. Se incluyeron 31 pacientes con enfermedad renal crónica. Se tomaron muestras de sangre a cada uno para la determinación de marcadores bioquímicos y muestras de ADN para la genotipificación del gen del receptor a vitamina D utilizando el método de la reacción en cadena de la polimerasa y restricción enzimática. Resultados. La edad promedio de los pacientes fue de 15.6 ± 3.6 años. Para ApaI, fueron homocigotos AA siete pacientes (22.58%); Aa, 18 pacientes (58.06%) y AA, seis pacientes (19.35%). Para BsmI, 14 pacientes fueron BB (45.16%) y 17 pacientes fueron Bb (54.83%). Los pacientes BB tuvieron calcio sérico mayor que los Bb (p = 0.04). Para TaqI, fueron TT 19 pacientes (61.3%); Tt, once pacientes (35.48%) y uno, tt (3.22%). Aquellos con alelo t (Tt o tt) presentaron concentraciones de calcio sérico significativamente más bajas que el homocigoto TT. Conclusiones. Se reportaron las frecuencias de genotipos y alelos del receptor de vitamina D en niños mexicanos. Se obtuvieron valores de calcio sérico más elevados en aquellos con alelo BB o TT.

Background. The possible influence of vitamin D receptor (VDR) gene polymorphisms on the regulation of the calcium-parathyroid hormone-vitamin D axis is particularly important in end-stage renal disease (ESRD) patients due to alterations in bone mineral metabolism. The aim of the study was to describe the frequency of VDR gene polymorphisms rs7975232 (ApaI), rs731236 (TaqI), rs1544410 (BsmI) and their relationship with serum calcium, phosphate, alkaline phosphatase and parathyroid hormone in children with ESRD. Methods. Thirty one patients were included in the study. Blood samples were drawn for biochemical determinations and DNA extraction. VDR genotypes were analyzed as restriction fragment length polymorphisms using ApaI, BsmI and TaqI. Results. Mean age of the patients was 15.6 ± 3.6 years. For ApaI, seven patients were AA (22.58%), 18 patients were Aa (58.06%) and six patients were aa (19.35%). For BsmI, 14 patients were BB (45.16%) and 17 were Bb (54.83%). Patients who were homozygous for BB had higher serum calcium concentrations than Bb patients (p = 0.04). For TaqI, 19 patients were TT (61.3%), 11 were Tt (35.48%) and one was tt (3.22%). Those with t allele (Tt or tt) had serum calcium concentrations significantly lower than TT patients. Conclusions. Genotype and allele frequency of the VDR gene is reported in Mexican patients with ESRD. Those with BB or TT genotype had higher serum calcium concentrations.