MOLECULAR EPIDEMIOLOGY OF MULTIDRUG RESISTANT ENTEROBACTER CLOACAE BLOOD ISOLATES FROM A UNIVERSITY HOSPITAL

urpose: to evaluate the epidemiological relationship between 3rd generation cephalosporin resistant Enterobacter cloacae blood isolates collected from patients in the University Hospital in Varna city during the period March 2014 and January 2017 and to characterize the ESBLs production in these isolates. Materials and methods: a total of 47 consecutive (nonduplicate) 3rd generation cephalosporin resistant isolates of Enterobacter cloacae, obtained from blood samples of patients admitted in different wards in Varna University Hospital, were investigated. Antimicrobial susceptibility to set of antimicrobial agents was tested by disc diffusion method and Phoenix (BD), and the results were interpreted according to EUCAST guidelines 2017. Identification of ESBL encoding genes was performed by PCR and sequencing. Isolates were genotyped by ERIC PCR. Results: The antimicrobial susceptibility in the whole collection of isolates, shown in decreasing order, is as follows: amikacin, 97.8% < levofloxacin, 76.6% < trimethoprime/ sulphometoxazole, 40.4% < ciprofloxacin, 19% < gentamicin, 8.4% < cefepime, 4.2% < piperacillin/ tazobactam, tobramycin, 2.1%. Multidrug resistance was detected in 70.2% of the isolates. The most widespread enzyme was CTX-M-15, found in 95.5% (n=43). Nine different ERIC types were detected. The dendrogram of similarity revealed three main clones of E. cloacae: Clone I, comprising two closely related subclones (ERIC type A and Aa) (similarity coefficient 0.92), was predominant, detected in Haematology (n=9), Haemodialysis (n=8), ICU (n=6), Cardio surgery (n=3), Pulmonology (n=4) and Gastroenterology (n=1); Clones II (ERIC type C) and III were presented by 5, and 3 isolates with identical profiles, obtained from patients, hospitalized in different wards. The ERIC profiles K, L, M and P, were found in single isolates only and were interpreted as sporadic. Conclusions: multi-drug resistance in E. cloacae was associated with successful intrahospital dissemination of three CTX-M-15 producing E. cloacae clones. Clone I was predominant, demonstrating high cross-transmission, epidemic and invasive potential. BlaCTX-M-15 was identified as a major mechanism of resistance to 3rd generation cephalosporins in E. cloacae

First Report of DHA-1 Producing Enterobacter cloacae Complex Isolate in Bulgaria

The aim of the present study was to reveal the characteristics of an Enterobacter cloacae complex isolate producing DHA-1 AmpC enzyme recovered from a patient hospitalized in St Marina Hospital, Varna.Materials and methods: Susceptibility testing, conjugation experiments, isoelectric focusing, PCR and sequencing were carrying out.Results: Of 176 Enterobacter spp. isolates only one isolate was positive for blaDHA. The sequencing revealed the presence of blaDHA-1 and blaCTX-M-3. The antimicrobial susceptibility testing showed higher resistance rates to almost all beta-lactams (ceftazidime, cefotaxime, cefepime, amoxicillinclavulanic acid, piperacillin/tazobactam), tobramycin, gentamycin, trimethoprim/sulphomethoxazole and quinolones (ciprofloxacin and levofloxacin). The isolate was susceptible to imipenem, meropenem and amikacin. The isoelectric focusing showed a band at pI 5.4 without ceftazidime and cefotaxime activity; a band at pI 7.8 with cefoxitin activity and another - with pI 8.4 with cefotaxime activity. Conjugation experiments were successful only for blaCTX-M-3 carrying determinants. Conclusions: To the best of our knowledge this is the first report of DHA-1 producing isolate in Bulgaria. The emergence of DHA-1 producing E. cloacae complex demonstrates the possibility for further dissemination of the gene encoding this enzyme. Infectious control measures are needed for the prevention of this phenomenon

Pseudoautosomal Region 1 length polymorphism in the human population

The human sex chromosomes differ in sequence, except for the pseudoautosomal regions (PAR) at the terminus of the short and the long arms, denoted as PAR1 and PAR2. The boundary between PAR1 and the unique X and Y sequences was established during the divergence of the great apes. During a copy number variation screen, we noted a paternally inherited chromosome X duplication in 15 independent families. Subsequent genomic analysis demonstrated that an insertional translocation of X chromosomal sequence into the Y chromosome generates an extended PAR [corrected].The insertion is generated by non-allelic homologous recombination between a 548 bp LTR6B repeat within the Y chromosome PAR1 and a second LTR6B repeat located 105 kb from the PAR boundary on the X chromosome. The identification of the reciprocal deletion on the X chromosome in one family and the occurrence of the variant in different chromosome Y haplogroups demonstrate this is a recurrent genomic rearrangement in the human population. This finding represents a novel mechanism shaping sex chromosomal evolution.status: publishe

Pseudoautosomal Region 1 Length Polymorphism in the Human Population

<div><p>The human sex chromosomes differ in sequence, except for the pseudoautosomal regions (PAR) at the terminus of the short and the long arms, denoted as PAR1 and PAR2. The boundary between PAR1 and the unique X and Y sequences was established during the divergence of the great apes. During a copy number variation screen, we noted a paternally inherited chromosome X duplication in 15 independent families. Subsequent genomic analysis demonstrated that an insertional translocation of X chromosomal sequence into theMa Y chromosome generates an extended PAR. The insertion is generated by non-allelic homologous recombination between a 548 bp LTR6B repeat within the Y chromosome PAR1 and a second LTR6B repeat located 105 kb from the PAR boundary on the X chromosome. The identification of the reciprocal deletion on the X chromosome in one family and the occurrence of the variant in different chromosome Y haplogroups demonstrate this is a recurrent genomic rearrangement in the human population. This finding represents a novel mechanism shaping sex chromosomal evolution.</p></div

Reciprocal deletion.

<p>(A) 180K Custom Microarray aCGH results with the upper track displaying an overview of the log2-ratio of the fluorescent signal across the entire X chromosome and the central track displaying a zoomed in portion of the X chromosome, with the log2-ratio of the fluorescent signals on the Y Axis. (B) PCR bands across the deletion region for the deletion carrying patient (P), father (F), mother (M), sister (S), female control (fc), male control (mc), and negative control (neg). (C) Sequencing of the amplicon in part B for the patient. Red letters indicate X specific reference sequence, yellow letters indicate LTR6B reference sequence, yellow letters highlighted in red indicate sequence specific for X specific LTR6B, yellow letters highlighted in purple indicate sequence specific for pseudoautosomal LTR6B, purple letters highlighted in purple indicate sequence originated from PAR1 that was not sequenced but contains the forward primer site, and yellow letters highlighted in yellow indicate LTR6B sequence that was not sequenced but contains the forward primer site. Highlighted in black are annotated SNPs.</p

Sequencing to validate the insertion and demonstrate recurrence.

<p>(A) Illustrations of a reference Y chromosome, a reference X chromosome, and a Y chromosome with an X insertion. X specific sequence is indicated in red, Y specific sequence in blue, PAR1 reference sequence in purple, and LTR6B's in yellow. Arrows indicate primer pairs, with a bar representing an amplifiable product. The position of the SNPs of this study is shown in the order found in the amplicon. (B) PCRs using the Sanger.Junc primers shows bands for patients (P) and fathers (F), but not mothers (M), male controls (mc), female control (fc) or negative controls (neg), confirming the presence of an X specific insertional translocation in Y. (C) Sequenced amplicons of PCRs from part B, excluding reference upstream/downstream sequence. Red letters are from the X specific reference sequence. Yellow letters are from LTR6B reference sequence with red highlights indicating X specific LTR6B sequence and purple highlights indicating sequence specific for pseudoautosomal LTR6B. Purple letters indicate pseudoautosomal reference sequence. The gap underlined in red indicate bases missing from the X specific LTR6B. In black are annotated SNPs/Indels. In order from the beginning to the end of sequences, green boxes indicate SNP positions for rs2534625/rs12843082, rs2316283, rs2534627, and rs2857320. This Sanger sequencing identified two junction types, indicated as Junc1 and Junc2. (D) Phased haplotypes found through PacBio amplicon sequencing of the PacBio Duplication amplicons, with haplotypes assigned numbers indicated by gray boxes. Families in which both the patient and father were sequenced are color coded. No color indicates a sample in which the father was not sequenced. * Each individual has two haplotypes in the figure, except patients 10 and 15, which had a second unillustrated haplotype with many more variants that more closely resembled Y chromosome sequence.</p

Sample overview.

<p>If appropriate, each sample has indicated family and relationship: P -patient, B -brother, F -father, Sanger.Junc is the sequencing results of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004578#pgen-1004578-g002" target="_blank">Figure 2</a> C. PacBio.Junc indicates the breakpoint haplotype deduced from the PacBio amplicons. 1c is the same as junction 1, with the addition of SNP rs211656. Duplication.Haplotype indicates the PacBio phased alleles from the duplicated region found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004578#pgen-1004578-g002" target="_blank">Figure 2</a> D (o  =  other haplotype). * indicates the deduced allele of paternal origin in father-son-(brother) pairings. Y-chr haplogroup is the main haplotype groups from chromosome Y SNP-based analyses (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004578#pgen.1004578.s002" target="_blank">Figure S2</a>).</p><p>Sample overview.</p

Identification of an X specific insertional translocation in Y.

<p>(A) Determining a duplication by array-CGH using a 180K Custom Microarray. The upper track shows an overview of the log2-ratio of the fluorescent signal across the entire X chromosome. The central track shows a zoomed in portion of the X chromosome, with the log2-ratio of the fluorescent signals on the Y Axis. (B) Further zooming in on the duplication, including the location of genes <i>XG</i> and <i>GYG2</i>. (C) FISH results for a father carrier (1, 3) and male control (2, 4) using probes targeting PAR1 (1, 2) and the duplicated region (3, 4). Note the presence of PAR1 on X and Y chromosomes in the carrier father and control, the PAR flanking probe on the X chromosome in both individuals, but the X insertion signal is found only on the Y chromosome of the carrier father. (D) The heterozygous SNP profile from Illumina sequencing of patient P1 for hg19 chrX:2,680,000-2,830,000. The dashed vertical gray line indicates the pseudoautosomal boundary, the yellow vertical lines illustrate LTR6B positions, gray diamonds illustrate heterozygote SNPs, and the black horizontal lines indicate mean frequencies of all depicted SNPs in the span of the line. Across the top is illustrated chromosome X, with unique X sequence in red, PAR1 reference sequence in purple, and LTR6B's in yellow. Note the presence of heterozygote SNPs in the X specific region, and the SNPs featuring frequencies of 0.33 in the proximal PAR1.</p

Expanded phenotypic spectrum of neurodevelopmental and neurodegenerative disorder Bryant-Li-Bhoj syndrome with 38 additional individuals.

Bryant-Li-Bhoj syndrome (BLBS), which became OMIM-classified in 2022 (OMIM: 619720, 619721), is caused by germline variants in the two genes that encode histone H3.3 (H3-3A/H3F3A and H3-3B/H3F3B) [1-4]. This syndrome is characterized by developmental delay/intellectual disability, craniofacial anomalies, hyper/hypotonia, and abnormal neuroimaging [1, 5]. BLBS was initially categorized as a progressive neurodegenerative syndrome caused by de novo heterozygous variants in either H3-3A or H3-3B [1-4]. Here, we analyze the data of the 58 previously published individuals along 38 unpublished, unrelated individuals. In this larger cohort of 96 people, we identify causative missense, synonymous, and stop-loss variants. We also expand upon the phenotypic characterization by elaborating on the neurodevelopmental component of BLBS. Notably, phenotypic heterogeneity was present even amongst individuals harboring the same variant. To explore the complex phenotypic variation in this expanded cohort, the relationships between syndromic phenotypes with three variables of interest were interrogated: sex, gene containing the causative variant, and variant location in the H3.3 protein. While specific genotype-phenotype correlations have not been conclusively delineated, the results presented here suggest that the location of the variants within the H3.3 protein and the affected gene (H3-3A or H3-3B) contribute more to the severity of distinct phenotypes than sex. Since these variables do not account for all BLBS phenotypic variability, these findings suggest that additional factors may play a role in modifying the phenotypes of affected individuals. Histones are poised at the interface of genetics and epigenetics, highlighting the potential role for gene-environment interactions and the importance of future research