•There are few data in the literature regarding sepsis or septic shock due to extended-spectrum β-lactamases (ESBL) strains.•The INCREMENT project is the largest international cohort study on ESBL ...infections.•Septic shock due to ESBL strains is associated with high 30-day mortality.•Escalation of antibiotic therapy is an important determinant of mortality.•Carbapenems should be used only in strains with resistance to ß-lactam/ß-lactamase inhibitors.
There are few data in the literature regarding sepsis or septic shock due to extended-spectrum β-lactamases (ESBL)-producing Enterobacteriaceae (E). The aim of this study was to assess predictors of outcome in septic patients with bloodstream infection (BSI) caused by ESBL-E.
Patients with severe sepsis or septic shock and BSI due to ESBL-E were selected from the INCREMENT database. The primary endpoint of the study was the evaluation of predictors of outcome after 30 days from development of severe sepsis or septic shock due to ESBL-E infection. Three cohorts were created for analysis: global, empirical-therapy and targeted-therapy cohorts.
367 septic patients were analysed. Overall mortality was 43.9% at 30 days. Escherichia coli (62.4%) and Klebsiella pneumoniae (27.2%) were the most frequent isolates. β-lactam/β-lactamase inhibitor (BLBLI) combinations were the most empirically used drug (43.6%), followed by carbapenems (29.4%). Empirical therapy was active in vitro in 249 (67.8%) patients, and escalation of antibiotic therapy was reported in 287 (78.2%) patients. Cox regression analysis showed that age, Charlson Comorbidity Index, McCabe classification, Pitt bacteremia score, abdominal source of infection and escalation of antibiotic therapy were independently associated with 30-day mortality. No differences in survival were reported in patients treated with BLBLI combinations or carbapenems in empirical or definitive therapy.
BSI due to ESBL-E in patients who developed severe sepsis or septic shock was associated with high 30-day mortality. Comorbidities, severity scores, source of infection and antibiotic therapy escalation were important determinants of unfavorable outcome.
In order to estimate herd-level prevalence of extended-spectrum β-lactamase/AmpC β-lactamase (ESBL/AmpC)- and carbapenemase-producing commensal
in ruminants in the Basque Country (northern Spain), a ...cross-sectional survey was conducted in 2014 to 2016 in 300 herds using selective isolation. ESBL-/AmpC-producing
was isolated in 32.9% of dairy cattle herds, 9.6% of beef cattle herds, and 7.0% of sheep flocks. No carbapenemase-producing
was isolated. Phenotypic antimicrobial susceptibility determined by broth microdilution using EUCAST epidemiological cutoff values identified widespread coresistance to extended-spectrum cephalosporins and other antimicrobials (110/135 isolates), particularly tetracycline, sulfamethoxazole, trimethoprim, and ciprofloxacin. All isolates were susceptible to tigecycline, imipenem, meropenem, and colistin. The genomes of 66 isolates were sequenced using an Illumina NovaSeq 6000 and screened for antimicrobial resistance determinants against ResFinder and PointFinder. The plasmid/chromosomal locations of resistance genes were predicted with PlasFlow, and plasmid replicons were identified using PlasmidFinder. Fifty-two acquired resistance genes and point mutations in another four genes that coded for resistance to 11 antimicrobial classes were identified. Fifty-five genomes carried ESBL-encoding genes,
being the most common, and 11 carried determinants of the AmpC phenotype, mostly the
gene. Additionally, genes coding for β-lactamases of the CTX-M group 9 were detected as well as the sporadic presence of
,
, and a point mutation in the
promoter. Only a bovine isolate coharbored more than one ESBL/AmpC genetic determinant (
and a mutation in the
promoter), confirming its ESBL- and AmpC β-lactamase-producing phenotype. Most ESBL/AmpC genes were located in IncI1 plasmids, which also carried a great variety of other antimicrobial resistance genes.
Extended-spectrum β-lactamase (ESBL)- and AmpC β-lactamase (AmpC)-producing
isolates have emerged in recent years as some of the fastest spreading antimicrobial resistance determinants in humans and food-producing animals, becoming a concern for animal and public health. This study provided insight into the prevalence of cefotaxime-resistant
in cattle and sheep in the Basque Country and the associated genetic determinants of antimicrobial resistance. These constituted an important contribution to the limited repository of such data for cattle in the region and for sheep worldwide. Antimicrobial susceptibility testing by phenotypic and molecular methods is key in surveillance programs to enhance early detection of resistance development, monitor resistance trends, and provide guidance to clinicians in selecting the adequate therapy.
To rapidly identify carbapenemase producers in Enterobacteriaceae, we developed the Carba NP test. The test uses isolated bacterial colonies and is based on in vitro hydrolysis of a carbapenem, ...imipenem. It was 100% sensitive and specific compared with molecular-based techniques. This rapid (<2 hours), inexpensive technique may be implemented in any laboratory.
pET expression plasmids are widely used for producing recombinant proteins in
Escherichia coli.
Selection and maintenance of cells harboring a pET plasmid are possible using either a Tn3.1-type ...genetic fragment (which encodes a ß-lactamase and confers resistance to ß-lactam antibiotics) or a Tn903.1-type genetic fragment (which encodes an aminoglycoside-3’-phosphotransferase and confers resistance aminoglycoside antibiotics). Herein we have investigated how efficiently pET plasmids are maintained using these two fragments. The study reveals that pET plasmids are efficiently maintained with both Tn3.1 and Tn903.1 genetic fragments prior to the induction of recombinant protein production, and over short induction times (i.e., 2 h). However, over longer induction times (i.e., 20 h), the efficiency of plasmid maintenance depends on the host strain used, and the type of antibiotic selection cassette used. Based on our collective observations, we have 2 general tips for efficiently maintaining pET plasmids during recombinant production experiments.
Tip #1: Use a strain with lowered levels of the T7 RNA polymerase, such as C41(
DE3
). pET plasmids will be efficiently maintained over long induction times with both the Tn3.1 and Tn903.1 genetic fragments, regardless of whether antibiotics are present during cultivation.
Tip #2: If a strain with higher levels of T7 RNA polymerase strain is necessary, such as BL21(
DE3
)), keep induction times short or use a plasmid containing a Tn903.1-type fragment and select with kanamycin.
A few reports indicate that livestock might represent a new reservoir for carbapenemase-producing Enterobacteriaceae (CPE). In 2015, VIM-1-producing
Escherichia coli
were detected at slaughter in ...colon contents of animals from a German fattening pig farm within the national monitoring on ESBL-producing
E. coli.
In this study, pooled faces samples from pigs, as well as samples from the barn surrounding environment of this fattening farm were taken, to evaluate the dissemination of CPEs. Several modifications of the culture-dependent detection procedure were investigated for their potential to improve the sensitivity of the CPE isolation method. The current reference procedure was adapted by adding a real-time PCR pre-screening and additional enrichment steps. It was possible to isolate 32 VIM-1-producing
E. coli
from four fecal samples of three different barns using two serial enrichment steps in combination with real-time PCR and selective agar plates. By genetic typing, we confirmed the presence of two
E. coli
clonal lineages circulating on this particular farm: one was harboring the
bla
VIM–
1
on an IncHI2 plasmid while the second lineage carried the gene on the chromosome. Despite its different locations, the
bla
VIM–
1
gene was harbored on a class 1 integron in both clonal lineages. Whole-genome sequencing revealed that the VIM-1-carrying plasmids exhibited only slight variability in its compositions and sizes. We assume that the prevalence of CPEs in animal production in Germany and other European countries might be underestimated and there is a concern of further spread of VIM-1-producing bacteria in German livestock and food.
Abstract
Background
The Democratic Republic of the Congo (DRC) has one of the highest neonatal death rates (between 14% and 28%) in the world. In the DRC, neonatal sepsis causes 15.6% of this ...mortality, but data on the bacterial etiology and associated drug susceptibility are lacking.
Methods
Hemocultures of 150 neonates with possible early-onset neonatal sepsis (pEOS) were obtained at the Hôpital Provincial Général de Référence de Bukavu (Bukavu, DRC). The newborns with pEOS received an empirical first-line antimicrobial treatment (ampicillin, cefotaxime, and gentamicin) based on the synopsis of international guidelines for the management of EOS that are in line with World Health Organization (WHO) recommendations. Isolates were identified using matrix-assisted laser desorption/ ionization time-of-flight mass spectrophotometry. Antibiotic resistance was assessed using the disk diffusion method.
Results
Fifty strains were obtained from 48 patients and identified. The 3 most prevalent species were Enterobacter cloacae complex (42%), Klebsiella pneumoniae (18%), and Serratia marcescens (12%). Enterobacter cloacae isolates were resistant to all first-line antibiotics. All K. pneumoniae and S. marcescens isolates were resistant to ampicillin, and the majority of the K. pneumoniae and half of the S. marcescens isolates were resistant to both cefotaxime and gentamicin. All E. cloacae complex strains, 89% of K. pneumoniae, and half of S. marcescens had an extended-spectrum ß-lactamase phenotype.
Conclusions
The most prevalent pathogens causing EOS in Bukavu were E. cloacae complex, K. pneumoniae, and S. marcescens. Most of these isolates were resistant to the WHO-recommended antibiotics.
The early-onset neonatal sepsis case fatality rate was 25.3% among hospitalized neonates in Bukavu. Enterobacter cloacae, Klebsiella pneumoniae, and Serratia marcescens were the pathogens most commonly retrieved. A total of 100%, 88.9%, and 86.1% of these isolates were resistant to ampicillin, cefotaxime, and gentamicin, respectively.
Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that blaTEM gene plays a key role in the P/T-R ...acquisition, no clinical in vivo study has yet confirmed the role of blaTEM or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4 g/0.5 g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of blaTEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and blaTEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of blaTEM gene in P/T-R isolates, generated by three different genetic events: (1) IS26-mediated duplication of the blaTEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying blaTEM, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms.
•IS26-mediated duplication of the blaTEM gene is involved in the acquisition of P/T-R by E. coli.•Generation of a small multicopy plasmid carrying blaTEM is involved in the acquisition of P/T-R by E. coli.•Adaptive evolution via plasmid size reduction, leading to a higher plasmid copy number is involved in the P/T-R by E. coli.•Reduction of OmpC expression is associated with P/T-R in E. coli.
The emerging environmental spread of antibiotic-resistance genes (ARGs) and their subsequent acquisition by clinically relevant microorganisms is a major threat to public health. Animal manure has ...been recognized as an important reservoir of ARGs; however, the dissemination of manure-derived ARGs and the impacts of manure application on the soil resistome remain obscure. Here, we conducted a microcosm study to assess the temporal succession of total bacteria and a broad spectrum of ARGs in two contrasting soils following manure application from cattle that had not been treated with antibiotics. High-capacity quantitative PCR detected 52 unique ARGs across all the samples, with β-lactamase as the most dominant ARG type. Several genes of soil indigenous bacteria conferring resistance to β-lactam, which could not be detected in manure, were found to be highly enriched in manure-treated soils, and the level of enrichment was maintained over the entire course of 140 days. The enriched β-lactam resistance genes had significantly positive relationships with the relative abundance of the integrase intI1 gene, suggesting an increasing mobility potential in manure-treated soils. The changes in ARG patterns were accompanied by a significant effect of cattle manure on the total bacterial community compositions. Our study indicates that even in the absence of selective pressure imposed by agricultural use of antibiotics, manure application could still strongly impact the abundance, diversity and mobility potential of a broad spectrum of soil ARGs. Our findings are important for reliable prediction of ARG behaviors in soil environment and development of appropriate strategies to minimize their dissemination.
The study indicates that even in the absence of selective pressure imposed by agricultural use of antibiotics, manure application could still strongly impact the abundance, diversity and mobility potential of a broad spectrum of soil ARGs.
Graphical Abstract Figure.
The study indicates that even in the absence of selective pressure imposed by agricultural use of antibiotics, manure application could still strongly impact the abundance, diversity and mobility potential of a broad spectrum of soil ARGs.
Extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) are a serious threat among emerging antibiotic resistant bacteria. Particularly, the number of cases of ESBL-E infections reported in ...children has been increasing in recent years, and approved antibiotic treatments for this age group are limited. However, information regarding the prevalence of colonization in European children, risk factors associated with colonization, and the characteristics of the colonizing strains is scarce. The aims of this study were to determine the prevalence of ESBL-E colonization in fecal samples of apparently healthy schoolchildren, to identify lifestyle routines associated with colonization, and to characterize clonal relationships and mechanisms of resistance in ESBL-E isolates.
A cohort of 887 healthy children (3-13 years old) from seven primary and secondary schools in the Madrid metropolitan area was recruited between April-June 2018, and sociodemographic information and daily habits were collected. Fecal samples were screened for ESBL-E carriage in selective medium. ESBL-E isolates were further characterized by assessing molecular epidemiology (PFGE and MLST), ESBL gene carriage, and antibiotic resistance profile. This information was analyzed in conjunction with the metadata of the participants in order to identify external factors associated with ESBL-E carriage.
Twenty four ESBL-E, all but one
were detected in 23 children (prevalence: 2.6%; 95% CI: 1.6-3.6%). Of these, seven contained the
allele, five the
, five the
, three the
, three the
, and one the
. Significant clonal diversity was observed among the isolates that grouped into 22 distinct clusters (at <85% similarity of PFGE profile). ESBL-producing
isolates belonged to 12 different STs, with ST10 (25%) and ST131 (17%) being the most frequent. Apart from ß-lactams, resistance to trimethoprim/sulfamethoxazole (46%), ciprofloxacin (33%), levofloxacin (33%), tobramycin (21%), and gentamicin (8%) were the most frequently detected.
The prevalence of ESBL-E in the studied cohort of children was lower than the average colonization rate previously detected in Europe for both children and adults.
was the main ESBL-producing species detected and CTX-M were the most frequently identified ESBLs. High ST diversity suggests polyclonal dissemination. Compared to other STs, ST131 isolates were associated with resistance to various antimicrobials.
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