Multiple STs Research Articles

Extensively Drug-Resistant Klebsiella pneumoniae Associated with Complicated Urinary Tract Infection in Northern India.

Klebsiella pneumoniae (Kp), which is associated with hospital-acquired infections, is extensively drug-resistant (XDR), making treatment difficult. Understanding the genetic epidemiology of XDR-Kp can help determine its potential to be hypervirulent (hv) through the presence of siderophores. We characterized the genomes of 18 colistin-resistant XDR-Kp isolated from 14 patients with complicated tract infection at an Indian healthcare facility. The 18 organisms comprised the following sequence types (STs): ST14 (n = 9), ST147 (n = 5), ST231 (n = 2), ST2096 (n = 1), and ST25 (n = 1). Many patients in each ward were infected with the same ST, suggesting a common source of infection. Some patients had recurrent infections with multiple STs circulating in the ward, providing evidence of hospital transmission. β-lactamase genes (blaCTX-M-1, blaSHV, and blaampH) were present in all isolates. blaNDM-1 was present in 15 isolates, blaOXA-1 in 16 isolates, blaTEM-1D in 13 isolates, and blaOXA-48 in 3 isolates. Disruption of mgrB by various insertion sequences was responsible for colistin resistance in 6 isolates. The most common K-type among isolates was K2 (n = 10). One XDR convergent hvKp ST2096 mutation (iuc+ybt+blaOXA-1+blaOXA-48) was associated with prolonged hospitalization. Convergent XDR-hvKp has outbreak potential, warranting effective antimicrobial stewardship and infection control.

Long-read sequencing-based in silico phage typing of vancomycin-resistant Enterococcus faecium

BackgroundVancomycin-resistant enterococci (VRE) are successful nosocomial pathogens able to cause hospital outbreaks. In the Netherlands, core-genome MLST (cgMLST) based on short-read sequencing is often used for molecular typing. Long-read sequencing is more rapid and provides useful information about the genome’s structural composition but lacks the precision required for SNP-based typing and cgMLST. Here we compared prophages among 50 complete E. faecium genomes belonging to different lineages to explore whether a phage signature would be usable for typing and identifying an outbreak caused by VRE. As a proof of principle, we investigated if long-read sequencing data would allow for identifying phage signatures and thereby outbreak-related isolates.ResultsAnalysis of complete genome sequences of publicly available isolates showed variation in phage content among different lineages defined by MLST. We identified phage present in multiple STs as well as phages uniquely detected within a single lineage. Next, in silico phage typing was applied to twelve MinION sequenced isolates belonging to two different genetic backgrounds, namely ST117/CT24 and ST80/CT16. Genomic comparisons of the long-read-based assemblies allowed us to correctly identify isolates of the same complex type based on global genome architecture and specific phage signature similarity.ConclusionsFor rapid identification of related VRE isolates, phage content analysis in long-read sequencing data is possible. This allows software development for real-time typing analysis of long-read sequencing data, which will generate results within several hours. Future studies are required to assess the discriminatory power of this method in the investigation of ongoing outbreaks over a longer time period.

Epidemiological Characterization of Colistin and Carbapenem Resistant Enterobacteriaceae in a Tertiary: A Hospital from Anhui Province.

PurposeAntimicrobial resistance, especially carbapenem resistance Enterobacteriaceae and plasmid mediated mobile colistin resistance, is a serious issue worldwide. This study was designed to determine the epidemiological characteristics of plasmid mediated colistin resistance and carbapenem resistant Enterobacteriaceae from tertiary A hospital located in Hefei, China.MethodsTotally, 158 carbapenems resistant Enterobacteriaceae (CRE) were screened for antibiotic susceptibility, mcr-1, extended spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), and fosfomycin resistance genes using PCR and sequencing. The sequence types were identified by multilocus sequence typing (MLST). Plasmid profiles were determined by PCR based replicon typing (PBRT), and the plasmid sizes were confirmed by southern blotting.ResultsThe isolates showed high MIC50 and MIC90 for all antimicrobials, except tigecycline, meropenem, and colistin. The main Carbapenemase genes were blaKPC-2 (90.5%), blaNDM-1(3.7%), blaOXA-48(5.6%) and fosA3 (14.5%). The blaCTXM-15 found 36.7%, mcr-1 (3.7%) recorded in six isolates. PBRT revealed blaKPC-2 in K. pneumoniae on IncR, IncFII, and IncA/C. blaNDM-1 in E. coli on IncFII, whereas in E. cloacae noticed on IncHI2 plasmid. mcr-1 was recorded among IncFIIK, IncFII, and IncF in E. coli, K. pneumoniae, and E. cloacae. Resistance genes (mcr-1, blaNDM-1, blaKPC-2) harboring plasmids are successfully trans-conjugant to EC-600. A high incidence of ST11 was observed in K. pneumoniae carbapenem resistant isolates. While in E. coli, multiple STs were identified. However, mcr-1 in ST23 was identified for the first time in Anhui Province. Among Enterobacter cloacae, ST270 detected carrying blaNDM-1. Southern-hybridization confirmed the plasmid sizes 35–150kb.ConclusionThis study indicates the co-carrying of mcr-1, blaKPC-2, and blaNDM-1 among clinical isolates, the prevalence of different Enterobacteriaceae STs is alarming, especially in E. coli. Holding such a resistance profile is a threat for humans and animals, which may be transferred between the strains through plasmid transfusion. Persistent control actions are immediately necessary to combat this hazard.

Systematic characterization of Bacillus Genetic Stock Center Bacillus thuringiensis strains using Multi-Locus Sequence Typing.

The goal of this work was to perform a systematic characterization of Bacillus thuringiensis (Bt) strains from the Bacillus Genetic Stock Center (BGSC) collection using Multi-Locus Sequence Typing (MLST). Different genetic markers of 158 Bacillus thuringiensis (Bt) strains from 73 different serovars stored in the BGSC, that represented 92% of the different Bt serovars of the BGSC were analyzed, the 8% that were not analyzed were not available. In addition, we analyzed 72 Bt strains from 18 serovars available at the pubMLST bcereus database, and Bt strains G03, HBF18 and Bt185, with no H serovars provided by our laboratory. We performed a systematic MLST analysis using seven housekeeping genes (glpF, gmK, ilvD, pta, pur, pycA and tpi) and analyzed correlation of the results of this analysis with strain serovars. The 233 Bt strains analyzed were assigned to 119 STs from which 19 STs were new. Genetic relationships were established by phylogenetic analysis and showed that STs could be grouped in two major Clusters containing 21 sub-groups. We found that a significant number of STs (101 in total) correlated with specific serovars, such as ST13 that corresponded to nine Bt isolates from B. thuringiensis serovar kenyae. However, other serovars showed high genetic variability and correlated with multiple STs; for example, B. thuringiensis serovar morrisoni correlated with 11 different STs. In addition, we found that 16 different STs correlated with multiple serovars (2-4 different serovars); for example, ST12 correlated with B. thuringiensis serovar alesti, dakota, palmanyolensis and sotto/dendrolimus. These data indicated that only partial correspondence between MLST and serotyping can be established.

KPC enzymes in the UK: an analysis of the first 160 cases outside the North-West region.

Klebsiella pneumoniae carbapenemases (KPCs) have been increasingly reported in the UK since 2003. We analysed patient and isolate data for KPC-positive bacteria confirmed by the national reference laboratory from UK laboratories from August 2003 to August 2014, excluding North-West England, where the epidemiology has previously been studied. MICs were determined by BSAC agar dilution. Carbapenem-resistant isolates lacking imipenem/EDTA synergy were tested by PCR for blaKPC. MLST and blaKPC sequencing were performed on a subset of isolates. Plasmid analysis was performed by transformation, PCR-based replicon typing and, in some cases, whole-plasmid sequencing. Patient data provided by the sending laboratories were reviewed. Two hundred and ten isolates with KPC enzymes were submitted from 71 UK laboratories outside North-West England, representing 160 patients. All were Enterobacteriaceae, predominantly K. pneumoniae (82%; 173/210), and most (91%; 191/210) were from hospitalized patients. Analysis of 100 isolates identified blaKPC-2 (62%), blaKPC-3 (30%) and blaKPC-4 (8%). Clonal group (CG) 258 was dominant among K. pneumoniae (64%; 54/84), but 21 unrelated STs were also identified. Plasmid analysis identified a diverse range of plasmids representing >11 different replicon types and found in multiple STs and species. Most (34/35) plasmids with IncFIB/FIIK replicons exhibited >99% sequence identity to pKpQIL. KPC enzymes are increasingly detected in Enterobacteriaceae in the UK, albeit without the major outbreaks seen in North-West England. K. pneumoniae CG258 are the dominant hosts, but plasmid spread plays a major role in KPC dissemination between other K. pneumoniae STs and enterobacterial species.

Screening for Y chromosome azoospermia factor region microdeletions using modified multiplex PCR

Objective To establish an universal primer-multiplex PCR system for diagnosis of Y chromosome AZF region microdeletions in 262 patients with non-obstructive azoospermic and severe oligozoospermic male infertility. Methods In each panel of multiplex PCR, YUP and YCP containing a fragment of non-human DNA sequence at their 5' ends were designed. The universal primers and chimiric primers were employed for the amplification at the same multiplex PCR system to screen for the Y chromosome AZF region ( a, b and c) microdeletions in 262 non-obstructive azoospermic and severe oligozoospermic male infertility patients. Results Thirty-three out of 262 patients (12. 60% ) were detected with Y chromosome AZF microdeletions. Among them, 27 cases were AZF c microdeletions and 6 ones were AZF b + c microdeletions. These results were in agreement with the results from EMQN method. There was no false-positivity. The gel electrophoresis for detection of multiple STS from both methods showed that the sY84,sY86, sY127, sY134, sY254, sY255, SRY bands were homogeneous and clear with similar brightness. Conclusion The modified multiplex PCR is suitable for screening of Y chromosome AZF microdeletions in non-obstructive azoospermic and severe digozoospermic male infertility patients. Key words: Infertility,male; Y chromosome; Chromosome deletion; Polymerase chain reaction

A Common Deletion at Chromosomal Region 17q21 in Sporadic Prostate Tumors Distal to BRCA1

Prostate cancer is the most common cancer in males in the United States, yet the etiology of this disease is still poorly understood. In previous work from our laboratory, one or more deleted regions were found in prostate tumors distal to the breast and ovarian cancer susceptibility gene (BRCA1) on chromosome 17. This suggested that genes at 17q21 may play a pivotal role in prostate cancer progression, and there may be new tumor suppressor genes at this locus. We now present a physical map built with P1, P1 artificial chromosome, and bacterial artificial chromosome clones encompassing a DNA sequence anchored by multiple STS markers. The analysis of prostate tumors indicated an 85-kb novel commonly deleted interval flanked by D17S1184–D17S183–D17S1203–D17S1860, which is at least 470 kb distal to the BRCA1 gene. Fifty-four of 126 prostrate cancer cases (43%) showed a deletion by a direct FISH technique using P1 probes in this region. Searching with clone end sequences in the sequence database BLAST, the deleted clone covered genomic DNA sequence that contained upstream binding factor (UBF), EPB3 genes, SHCL1, ASB-4-like sequence, and acidic protein-like sequence. PCR for the ESTs confirmed that these genes or ESTs are within the deletion region. Our results will be helpful for finding candidate tumor suppressor genes in prostate cancer.