Multi-drug-resistant Gram-negative bacteria are of major clinical concern. The increasing prevalence of carbapenemase-producing Enterobacteriaceae (CPE), resistant to all beta-lactams including ...carbapenems and able to colonize the large intestine, represents a key threat. Rapid, accurate detection of intestinal CPE colonization is critical to minimize transmission, and hence reduce costly, difficult-to-treat CPE infections. There is currently no ‘gold standard’ CPE detection method. A survey of diagnostic laboratories in England found considerable heterogeneity in diagnostic CPE testing methods and procedures.
Long-term-care facilities (LTCFs) are reservoirs of resistant bacteria. We undertook a point-prevalence survey and risk factor analysis for specific resistance types among residents and staff of a ...Bolzano LTCF and among geriatric unit patients in the associated acute-care hospital. Urine samples and rectal, inguinal, oropharyngeal and nasal swabs were plated on chromogenic agar; isolates were typed by pulsed-field gel electrophoresis; resistance genes and links to insertion sequences were sought by PCR; plasmids were analysed by PCR, restriction fragment length polymorphism and incompatibility grouping. Demographic data were collected. Of the LTCF residents, 74.8% were colonized with ≥1 resistant organism, 64% with extended-spectrum β-lactamase (ESBL) producers, 38.7% with methicillin-resistant Staphylococcus aureus (MRSA), 6.3% with metallo-β-lactamase (MBL) producers, and 2.7% with vancomycin-resistant enterococci. Corresponding rates for LTCF staff were 27.5%, 14.5%, 14.5%, 1.5% and 0%, respectively. Colonization frequencies for geriatric unit patients were lower than for those in the LTCF. Both clonal spread and plasmid transfer were implicated in the dissemination of MBL producers that harboured IncN plasmids bearing blaVIM-1, qnrS, and blaSHV-12. Most (44/45) ESBL-producing Escherichia coli isolates had blaCTX-M genes of group 1; a few had blaCTX-M genes of group 9 or blaSHV-5; those with blaCTX-M-15 or blaSHV-5 were clonal. Risk factors for colonization of LTCF residents with resistant bacteria included age ≥86 years, antibiotic treatment in the previous 3 months, indwelling devices, chronic obstructive pulmonary disease, physical disability, and the particular LTCF unit; those for geriatric unit patients were age and dementia. In conclusion, ESBL-producing and MBL-producing Enterobacteriaceae and MRSA were prevalent among the LTCF residents and staff, but less so in the hospital geriatric unit. Education of LTCF employees and better infection control are proposed to minimize the spread of resistant bacteria in the facility.
CTX-M: changing the face of ESBLs in Europe Livermore, David M.; Canton, Rafael; Gniadkowski, Marek ...
Journal of antimicrobial chemotherapy,
02/2007, Letnik:
59, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Since around 2000—earlier in Poland and Spain and later in France and the UK—dramatic shifts have occurred in the prevalence and types of extended-spectrum β-lactamases (ESBLs) in Europe. Before this ...watershed, most producers were nosocomial isolates, often Klebsiella spp. or Enterobacter spp. from specialist care units, and had mutant TEM or SHV ESBLs. Subsequently, CTX-M ESBLs have become dominant, with much greater penetration into Escherichia coli, and with many infections in ‘complicated community’ patients, usually with underlying disease, recent antibiotic usage, or healthcare contact. The degree of clonality among producers varies with the country, as does the enzyme type produced, with group 9 (CTX-M-9 and -14) enzymes dominant in Spain and group 1 enzymes (particularly CTX-M-3 and -15) dominant elsewhere. Irrespective of the particular enzyme, most producers are multiresistant. These changing patterns present major therapeutic and infection control challenges, with the public health intervention points unclear.
Objectives Recently, a CTX-M-15 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli O25b-ST131 clone, belonging to the B2 phylogenetic group and with a high virulence potential, has been ...reported all over the world, representing a major public health problem. The present study was carried out to develop a rapid and simple detection assay that identifies members of this clone. Methods A total of 627 E. coli isolates of which 373 produced an ESBL, collected across four continents, were screened using a O25b-ST131 clone allele-specific PCR for the pabB gene. Results One hundred and forty-three ESBL isolates were found positive with the assay. These isolates were all of O25b type and, when studied by multilocus sequence typing (25 cases), were all of ST131. The O25b-ST131 clone was found to produce ESBLs other than CTX-M-15, specifically CTX-M-2, -3, -14, -27, -32 and -61 as well as TEM-24. This clone represents 3% of non-ESBL B2 isolates originating from urinary tract infections in Paris. Conclusions We have developed a PCR-based assay that easily identifies a clone with high likelihood of producing ESBLs, including CTX-M-15.
The diazabicyclooctane β-lactamase inhibitor OP0595 (RG6080) also acts as an antibiotic, targeting PBP2 in Enterobacteriaceae, but this activity is vulnerable to mutational resistance. We used WGS to ...investigate the basis of this resistance.
Twenty OP0595-selected mutants, comprising four derived from each of five different Escherichia coli strains, were sequenced on Illumina HiSeq. Reads from each mutant were mapped to the assembled genome of the corresponding parent. A variant-calling file generated with Samtools was parsed to determine genetic alterations.
Besides OP0595, the mutants consistently showed decreased susceptibility to mecillinam, which likewise targets PBP2, and grew as stable round forms in the presence of subinhibitory concentrations of OP0595. Among the 20 mutants, 18 had alterations in genes encoding tRNA synthase and modification functions liable to induce expression of the RpoS sigma factor through activation of the stringent response or had mutations suppressing inactivators of RpoS or the stringent response signal-degrading enzyme, SpoT. TolB was inactivated in one mutant: this activates RcsBC regulation and was previously associated with mecillinam resistance. The mechanism of resistance remained unidentified in one mutant. Both the RpoS and RcsBC systems regulate genes of cell division, including ftsAQZ that can compensate for loss or inhibition of PBP2, allowing survival of the challenged bacteria as stable round forms, as seen.
WGS identified the global stringent response signal, entailing induction of RpoS, as the main mediator of mutational resistance to OP0595 in E. coli.
The expanding global distribution of multi-resistant Klebsiella pneumoniae demands faster antimicrobial susceptibility testing (AST) to guide antibiotic treatment. Current ASTs rely on time-consuming ...differentiation of resistance and susceptibility after initial isolation of bacteria from a clinical specimen. Here we describe a flow cytometry workflow to determine carbapenem susceptibility from bacterial cell characteristics in an international K. pneumoniae isolate collection (n = 48), with a range of carbapenemases. Our flow cytometry-assisted susceptibility test (FAST) method combines rapid qualitative susceptible/non-susceptible classification and quantitative MIC measurement in a single process completed shortly after receipt of a primary isolate (54 and 158 minutes respectively). The qualitative FAST results and FAST-derived MIC (MIC
) correspond closely with broth microdilution MIC (MIC
, Matthew's correlation coefficient 0.887), align with the international AST standard (ISO 200776-1; 2006) and could be used for rapid determination of antimicrobial susceptibility in a wider range of Gram negative and Gram positive bacteria.
Nowadays, six types of acquired vancomycin resistance in enterococci are known; however, only VanA and to a lesser extent VanB are widely prevalent. Various genes encode acquired vancomycin ...resistance and these are typically associated with mobile genetic elements which allow resistance to spread clonally and laterally. The major reservoir of acquired vancomycin resistance is Enterococcus faecium; vancomycin-resistant Enterococcus faecalis are still rare. Population analysis of E. faecium has revealed a distinct subpopulation of hospital-acquired strain types, which can be differentiated by molecular typing methods (MLVA, MLST) from human commensal and animal strains. Hospital-acquired E. faecium have additional genomic content (accessory genome) including several factors known or supposed to be virulence-associated. Acquired ampicillin resistance is a major phenotypic marker of hospital-acquired E. faecium in Europe and experience has shown that it often precedes increasing rates of VRE with a delay of several years. Several factors are known to promote VRE colonisation and transmission; however, despite having populations with similar predispositions and preconditions, rates of VRE vary all over Europe.
Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were first seen in the UK in 2003 and have been increasingly reported since 2010, largely owing to an ongoing outbreak in ...North-West England. We examined the role of clonal spread and plasmid transmission in their emergence.
Isolates comprised KPC-positive K. pneumoniae ( n = 33), Escherichia coli ( n = 7) and Enterobacter spp. ( n = 4) referred to the national reference laboratory between 2008 and 2010 from 17 UK centres, including three in North-West England. Isolates were typed by MLST. Plasmids were transferred by electroporation and characterized by PCR or sequencing. PCR screening assays were developed to distinguish plasmid pKpQIL variants.
The K. pneumoniae isolates included 10 STs, of which three belonged to clonal group (CG) 258. CG258 ( n = 19) isolates were detected in 13 centres but accounted for only 7/19 (36.8%) of those from North-West England. Most KPC-producers (37/44, 84.1%), including 16/19 CG258 K. pneumoniae , carried bla KPC on IncFII K2 plasmids. Sequencing of a subset of these plasmids ( n = 11) revealed similarities with published pKpQIL. One variant, pKpQIL-UK identified in K. pneumoniae CG258 ( n = 5) and ST468 ( n = 1) isolates from distinct centres had only a few nucleotide changes from classical pKpQIL, whereas pKpQIL-D1 ( n = 1) and pKpQIL-D2 ( n = 4), from isolates of various species in the North-West, harboured large variations, reflecting replacement of the partitioning and replication functions and potentially thereby facilitating spread. PCR revealed that 36/37 (97.3%) IncFII K2 -type plasmids in KPC-positive isolates had pKpQIL markers.
pKpQIL-like plasmids played a major role in the early dissemination of KPC enzymes in the UK.
Recent EUCAST advice asserts that, with low breakpoints, susceptibility results for cephalosporins and carbapenems can be reported 'as found', even for strains with extended-spectrum β-lactamases ...(ESBLs) and carbapenemases. The CLSI has similar advice, but with higher ceftazidime and cefepime breakpoints than those of EUCAST. Pharmacodynamic and animal data are used to support these views, along with some analysis of clinical case series. We contend that such advice is misguided on three counts. First, whilst there are cases on record where cephalosporins and carbapenems have proved effective against infections due to low-MIC ESBL producers and low-MIC carbapenemase producers, respectively, there are similar numbers of cases where such therapy has failed. Second, routine susceptibility testing is less precise than in research analyses, meaning that ESBL and carbapenemase producers with 'real' MICs of 1-8 mg/L will oscillate between susceptibility categories according to who tests them and how. Third, although EUCAST continues to advocate ESBL and carbapenemase detection for epidemiological purposes, the likely consequence of not seeking these enzymes for treatment purposes is that some laboratories will not seek them at all, leading to a loss of critical infection control information. In short, it is prudent to continue to seek ESBLs and carbapenemases directly and, where they are found, generally to avoid substrate drugs as therapy.
Escherichia coli is the most common agent of bacteraemia, bacterial gastroenteritis and urinary tract infections (UTIs). Lineages causing UTIs and gastrointestinal disease are well defined, but less ...is known about those causing bacteraemia. We therefore investigated the population structure of E. coli from bacteraemia in the UK and Ireland between 2001 and 2010.
E. coli isolates (n = 2166) were submitted to the BSAC Bacteraemia Surveillance Programme from 18 UK and Irish centres from 2001 to 2010. Genotypes were analysed by MLST using the Achtman scheme; MICs, blaCTX-M group and patient demographics were previously determined in the BSAC surveillance.
Four hundred and forty-eight STs were identified, but five of these, and their associated clonal complexes (CCs), accounted for 58.4% (1264 of 2166) of isolates: CC73 was the most common (20.7%), followed by CC131 (13.9%), CC95 (11.3%), CC69 (6.9%) and CC12 (5.5%). All these, except CC69 (group D), belong to phylogenetic group B2. CC131 isolates were much more often MDR than other STs were: they rose from 2.9% of isolates in 2001 to 20.5%-20.7% in 2007-08 and then declined to 14.3% in 2010. Resistance rates to cephalosporins, aminoglycosides and fluoroquinolones remained below 10% in other major CCs throughout.
The five most prevalent bacteraemia STs have all been associated previously with UTIs. They dominated in all years, but their proportions fluctuated, most notably for ST131, a globally disseminated high-risk clone that is often MDR.
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