Detection of Multi-drug Resistant \u3cem\u3eEscherichia coli\u3c/em\u3e in the Urban Waterways of Milwaukee, WI

Urban waterways represent a natural reservoir of antibiotic resistance which may provide a source of transferable genetic elements to human commensal bacteria and pathogens. The objective of this study was to evaluate antibiotic resistance of Escherichia coli isolated from the urban waterways of Milwaukee, WI compared to those from Milwaukee sewage and a clinical setting in Milwaukee. Antibiotics covering 10 different families were utilized to determine the phenotypic antibiotic resistance for all 259 E. coli isolates. All obtained isolates were determined to be multi-drug resistant. The E. coli isolates were also screened for the presence of the genetic determinants of resistance including ermB (macrolide resistance), tet(M) (tetracycline resistance), and β-lactamases (blaOXA, blaSHV, and blaPSE). E. coli from urban waterways showed a greater incidence of antibiotic resistance to 8 of 17 antibiotics tested compared to human derived sources. These E. coli isolates also demonstrated a greater incidence of resistance to higher numbers of antibiotics compared to the human derived isolates. The urban waterways demonstrated a greater abundance of isolates with co-occurrence of antibiotic resistance than human derived sources. When screened for five different antibiotic resistance genes conferring macrolide, tetracycline, and β-lactam resistance, clinical E. coli isolates were more likely to harbor ermB and blaOXA than isolates from urban waterway. These results indicate that Milwaukee’s urban waterways may select or allow for a greater incidence of multiple antibiotic resistance organisms and likely harbor a different antibiotic resistance gene pool than clinical sources. The implications of this study are significant to understanding the presence of resistance in urban freshwater environments by supporting the idea that sediment from urban waterways serves as a reservoir of antibiotic resistance

On the isolation of TI-plasmid from Agrobacterium tumefaciens

An efficient lysis method for Agrobacterium cells was developed, which allows a reproducible isolation of the tumor inducing (TI)-plasmid. The lysis method is based on the sensitivity of this bacterium to incubation with lysozyme, n-dodecylamine,EDTA, followed by Sarkosyl, after growth in the presence of carbenicillin. We also present a procedure for the isolation of the TI-plasmid on a large scale, that might be used for the mass isolation of other large plasmids which like the TI-plasmid, can not be cleared with earlier described procedures. The purity of the plasmid preparations was determined with DNA renaturation kinetics, which method has the advantage that the plasmid need not to be in the supercoiled or open circular form

Use of whole-genus genome sequence data to develop a multilocus sequence typing tool that accurately identifies Yersinia isolates to the species and subspecies levels

The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica

Development of a multilocus sequence typing scheme for the molecular typing of Mycoplasma pneumoniae

This work was funded by Public Health England. These studies were supported by funding initiatives by the National Institute for Social Care and Health Research (NISCHR; research support from the Welsh Government) via the registered research group Microbial and Infection Translational Research Group (MITReG) and Children and Young Persons Research Network (CYPRN).Mycoplasma pneumoniae is a major human respiratory pathogen causing both upper and lower respiratory disease in humans of all ages, and it can also result in other serious extrapulmonary sequelae. A multilocus sequence typing (MLST) scheme for M. pneumoniae was developed based on the sequences of eight housekeeping genes (ppa, pgm, gyrB, gmk, glyA, atpA, arcC, and adk) and applied to 55 M. pneumoniae clinical isolates and the two type strains M129 and FH. A total of 12 sequence types (STs) resulted for 57 M. pneumoniae isolates tested, with a discriminatory index of 0.21 STs per isolate. The MLST loci used in this scheme were shown to be stable in 10 strains following 10 sequential subculture passages. Phylogenetic analysis of concatenated sequences of the eight loci indicated two distinct genetic clusters that were directly linked to multilocus variable-number tandem repeat analysis (MLVA) type. Genetic MLST clustering was confirmed by genomic sequence analysis, indicating that the MLST scheme developed in this study is representative of the genome. Furthermore, this MLST scheme was shown to be more discriminatory than both MLVA and P1 typing for the M. pneumoniae isolates examined, providing a method for further and more detailed analysis of observed epidemic peaks of M. pneumoniae infection. This scheme is supported by a public Web-based database (http://pubmlst.org/mpneumoniae).PostprintPeer reviewe

Інформаційний бюлетень як засіб зв'язків із громадкістю протестантської церкви

Shigella sonnei, an emerging global cause of shigellosis, consists of four distinct lineages and the current pandemic involves several geographically associated, multidrug- resistant clones that belong to lineage III (1-3). A typing scheme based on high resolution melting (HRM) of six chromosomal single nucleotide polymorphisms (SNPs) has been described to identify all lineages/sub-lineages (4)

Multicenter Clinical Evaluation of the Xpert GBS LB Assay for Detection of Group B Streptococcus in Prenatal Screening Specimens

Neonatal infection with Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis and meningitis in newborns. Recent guidelines have recommended universal screening of all pregnant women to identify those colonized with GBS and administration of peripartum prophylaxis to those identified as carriers to reduce the risk of early-onset GBS disease in neonates. Enriched culture methods are the current standard for prenatal GBS screening; however, the implementation of more sensitive molecular diagnostic tests may be able to further reduce the risk of early-onset GBS infection. We report a clinical evaluation of the Xpert GBS LB assay, a molecular diagnostic test for the identification of GBS from broth-enriched vaginal/rectal specimens obtained during routine prenatal screening. A total of 826 specimens were collected from women undergoing prenatal screening (35 to 37 weeks' gestation) and tested at one of three clinical centers. Each swab specimen was tested directly prior to enrichment using the Xpert GBS assay. Following 18 to 24 h of broth enrichment, each specimen was tested using the Xpert GBS LB assay and the FDA-cleared Smart GBS assay as a molecular diagnostic comparator. Results obtained using all three molecular tests were compared to those for broth-enriched culture as the gold standard. The sensitivity and specificity of the Xpert GBS LB assay were 99.0% and 92.4%, respectively, compared to those for the gold standard culture. The Smart GBS molecular test demonstrated sensitivity and specificity of 96.8% and 95.5%, respectively. The sensitivities of the two broth-enriched molecular methods were superior to those for direct testing of specimens using the Xpert GBS assay, which demonstrated sensitivity and specificity of 85.7% and 96.2%, respectively

Multicenter Evaluation of the Portrait Staph ID/R Blood Culture Panel for Rapid Identification of Staphylococci and Detection of the mecA Gene

Bloodstream infections are a leading cause of morbidity and mortality in the United States and are associated with increased health care costs. We evaluated the Portrait Staph ID/R blood culture panel (BCP) multiplex PCR assay (Great Basin Scientific, Salt Lake City, UT) for the rapid and simultaneous identification (ID) of Staphylococcus aureus, Staphylococcus lugdunensis, and Staphylococcus species to the genus level and the detection of the mecA gene directly from a positive blood culture bottle. A total of 765 Bactec bottles demonstrating Gram-positive cocci in singles or clusters were tested during the prospective trial at 3 clinical sites. The Portrait Staph ID/R BCP results were compared with results from conventional biochemical and cefoxitin disk methods performed at an independent laboratory. Discordant ID and mecA results were resolved by rpoB gene sequencing and mecA gene sequencing, respectively. A total of 658 Staphylococcus species isolates (S. aureus, 211 isolates; S. lugdunensis, 3 isolates; and Staphylococcus spp., 444 isolates) were recovered from monomicrobial and 33 polymicrobial blood cultures. After discrepant analysis, the overall ratios of Portrait Staph ID/R BCP positive percent agreement and negative percent agreement were 99.4%/99.9% for Staphylococcus ID and 99.7%/99.2% for mecA detection

Evaluation of spectra VRE, a new chromogenic agar medium designed to screen for vancomycin-resistant Enterococcus faecalis and Enterococcus faecium

Spectra VRE (Remel, Lenexa, KS) is a chromogenic medium designed to recover and differentiate vancomycin-resistant Enterococcus faecium and Enterococcus faecalis (VRE). This medium was compared to bile esculin azide agar (BEAV) and was 98.2% sensitive and 99.3% specific compared to BEAV, which was 87.6% sensitive and 87.1% specific at 24 h

A multi-mRNA host-response molecular blood test for the diagnosis and prognosis of acute infections and sepsis: Proceedings from a clinical advisory panel

Current diagnostics are insufficient for diagnosis and prognosis of acute infections and sepsis. Clinical decisions including prescription and timing of antibiotics, ordering of additional diagnostics and level-of-care decisions rely on understanding etiology and implications of a clinical presentation. Host mRNA signatures can differentiate infectious from noninfectious etiologies, bacterial from viral infections, and predict 30-day mortality. The 29-host-mRNA blood-based InSe