Species of the Enterobacter cloacae complex are widely encountered in nature, but they can act as pathogens. The biochemical and molecular studies on E. cloacae have shown genomic heterogeneity, ...comprising six species: Enterobacter cloacae, Enterobacter asburiae, Enterobacter hormaechei, Enterobacter kobei, Enterobacter ludwigii and Enterobacter nimipressuralis, E. cloacae and E. hormaechei are the most frequently isolated in human clinical specimens. Phenotypic identification of all species belonging to this taxon is usually difficult and not always reliable; therefore, molecular methods are often used. Although the E. cloacae complex strains are among the most common Enterobacter spp. causing nosocomial bloodstream infections in the last decade, little is known about their virulence-associated properties. By contrast, much has been published on the antibiotic-resistance features of these microorganisms. In fact, they are capable of overproducing AmpC β-lactamases by derepression of a chromosomal gene or by the acquisition of a transferable ampC gene on plasmids conferring the antibiotic resistance. Many other resistance determinants that are able to render ineffective almost all antibiotic families have been recently acquired. Most studies on antimicrobial susceptibility are focused on E. cloacae, E. hormaechei and E. asburiae; these studies reported small variations between the species, and the only significant differences had no discriminating features.
Highlights • Glycopeptide-resistant Staphylococcus aureus is a growing clinical problem. • We compiled cases of daptomycin non-susceptibility to better understand resistance. • Large variability in ...time to development of resistance and previous antibiotic treatment was seen. • It is important to determine the vancomycin MIC when treating severe S. aureus infections. • MRSA strains may become hVISA in bacteraemia predisposing to daptomycin resistance.
Even though colistin-based treatment represents the antimicrobial-regimen backbone for the management of multidrug-resistant Gram-negative infections, colistin resistance is still rare, at least as a ...full resistance, in
(
). We investigated the genomics and transcriptomics of two clinical Extensively Drug Resistance (XDR) colistin-susceptible/resistant (COL-S/R)
strain-pairs in which COL-resistance was developed after exposure to colistin therapy. The molecular characterization of the strains showed that all strains belonged to PFGE-A, ST-281, OXA-23 producers, Global Clone-II, and were resistant to imipenem, meropenem, ampicillin/sulbactam, ciprofloxacin, gentamicin, amikacin, trimethoprim/sulfamethoxazole, and susceptible to tigecycline, in agreement with NGS-acquired resistome. COL-R vs. COL-S
comparative genomics, mapping on
ATCC 17978 and
ACICU Reference Genomes, revealed a closely related genomic phylogeny, especially between strain-pair isolates, and distinctive common genomic non-synonymous SNPs (nsSNPs) in COL-R
strains. Furthermore,
and
nsSNPs were found. Notably we recovered, for the first time,
and
nsSNPs previously described only in "
" mutants and associated with colistin resistance in a clinical COL-R
. COL-R vs. COL-S
comparative transcriptomics evidenced a strain-dependent response to the colistin resistance onset highly variable among the single COL-R strains vs. their COL-S parents and merely seven common over-expressed transcripts, i.e. the PgaB lipoprotein for biofilm-matrix production, the diacylglycerol kinase for the lipid recycling in the membrane-derived oligosaccharide cycle, a membrane non-ribosomal peptide synthetase, the Lipid A phosphoethanol aminotransferase PmrC, and three hypothetical proteins. The transcript analysis of the "COL-R related genes" and the RNA-seq data confirmed
over-expression responsible for a greater positive net cell-charge, and
under-expression in COL-R causing a decreased LPS production, as main mechanisms of colistin resistance. Our study reports the COL-R
genomic and transcriptomic signatures reflecting the interplay between several direct and indirect potential adaptations to antimicrobial pressure, including the occurrence of SNP accumulation hotspot loci in genes related to intrinsic or adaptive colistin resistance, surface adhesion proteins and porins, and over-expressed genes involved in different pathways, i.e. biofilm production, oxidative stress response, extensive drug and COL resistance.
•88.6% ESBL E. coli and 76.0% carbapenemase K. pneumoniae susceptible to fosfomycin.•For ESBL-producing E. coli, the AD method showed CA of 100% with BMD and GT.•For KPC-producing K. pneumoniae, ...there were CA of 92% and 94 with BMD and GT.•The BD Phoenix system exhibits a CA > 90% for all isolates.•The AD method is the only reference method for carbapenem-resistant K. pneumoniae.
The increasing emergence and diffusion of multidrug-resistant (MDR) pathogenic bacteria, both in hospital and community settings, is inducing clinicians to reconsider old antibiotics, such as fosfomycin, to overcome the difficulties posed by these microorganisms. Recent studies have reported good in vitro activity of fosfomycin against extended spectrum ß-lactamases (ESBL) and carbapenem-resistant Enterobacteriaceae. The aim of this study was to assess thein vitro activity of fosfomycin by different methods against 120 clinical MDR isolates.
Fosfomycin minimum inhibitory concentrations were determined using the agar dilution reference method (AD), gradient test (GT), broth microdilution method (BMD), according to CLSI recommendations, and automated systems (VITEK 2 and BD Phoenix) against 85 carbapenem-resistant Klebsiella pneumoniae and 35 ESBL-producing Escherichia coli. Agreement and discrepancies between the evaluated methods and the reference method were calculated.
Fosfomycin showed very good activity against ESBL-producing E. coli (88.6%). Excellent agreement (100%) between the three (AD, BMD and GT) susceptibility methods was found for E. coli. No major errors were observed. The fosfomycin resistance rate ranged from 24% (KPC-producing) to 100% (NDM-OXA-48 co-producing) K. pneumoniae. For all carbapenem-resistant K. pneumoniae strains, categorical agreement was >90% for all methods except for VITEK 2, which was 84%.
When ESBL E. coli isolates are found to be susceptible to fosfomycin with automated systems, it is not necessary to verify these results with the AD reference method; while for resistant strains, the GT can be used. In cases of KPC K. pneumoniae resistant to fosfomycin, the AD method is the only reference method.
•Metallo-beta-lactamase-producing Pseudomonas aeruginosa osteomyelitis is hard to treat.•Aztreonam/ceftazidime–avibactam combined with amikacin was strongly synergic.•Time-kill curves showed that ...triple combination achieved 99.9% killing until 48 h.•Antipseudomonal synergistic agents plus debridement resolved the infection.
We describe a challenging case of patient with metallo-beta-lactamase-producing Pseudomonas aeruginosa sternal osteomyelitis following aortic valve replacement with biological prosthesis. The strain exhibited a multidrug-resistance phenotype carrying the blaVIM-1 gene and belonged to the high-risk clone sequence type ST235. The patient was successfully treated with surgical debridement plus antibiotic therapy with ceftazidime/avibactam, aztreonam, and amikacin. Time-kill curves showed that this triple antibiotic combination at 1 × MIC was strongly synergic after 8 h, achieving 99.9% killing and maintaining this until 48 h.
Ceftazidime-avibactam (CZA) is one of the best therapeutic options available for infections caused by
carbapenemase (KPC)-producing bacteria. However, sporadic reports of CZA-resistant strains have ...been rapidly increasing in patients. Herein, we provide detailed case reports of the emergence of ceftazidime-avibactam resistance to identify their resistome and virulome using genomic molecular approaches. Sixteen isolates were collected from 13 patients at three hospitals in Catania and Catanzaro (Italy) between 2020-2021. Antimicrobial susceptibility was determined by broth microdiluition. The samples included in study were analyzed for resistome, virulome and Sequence Type (ST) using Whole Genome Sequencing (WGS). All strains were resistant to ceftazidime/avibactam, ciprofloxacin, extended-spectrum cephalosporins and aztreonam, 13/16 to meropenem, 8/16 to colistin and 7/16 to fosfomycin; 15/16 were susceptible to meropenem/vaborbactam; all strains were susceptible to cefiderocol. Molecular analysis showed circulation of three major clones: ST101, ST307 and ST512. In 10/16 strains, we found a
gene; in 6/16 strains, four different
variants (
) were detected. A plethora of other beta-lactam genes (
,
,
and
) was observed;
was found in ST307 and ST512, instead
in one out four ST101 strains. With regard to membrane permeability,
K35 and
K36 harbored frameshift mutations in 15/16 strains; analysis of
K37 gene revealed that all strains harbored a non-functional protein and carry wild-type PBP3. There is an urgent need to characterize the mechanisms underlying carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance. Furthermore, it is becoming increasingly important to explore feasible methods for accurate detection of different KPC enzymes.
(
) is one of the most threatening nosocomial pathogens. The implementation of novel and more effective surveillance and diagnostic strategies is mandatory to prevent the occurrence of legionellosis ...outbreaks in hospital environments. On these bases, the present review is aimed to describe the main clinical and molecular features of
focusing attention on the latest findings on drug resistance mechanisms. In addition, a detailed description of the current guidelines for the disinfection and surveillance of the water systems is also provided. Finally, the diagnostic strategies available for the detection of
spp. were critically reviewed, paying the attention to the description of the culture, serological and molecular methods as well as on the novel high-sensitive nucleic acid amplification systems, such as droplet digital PCR.
The aim of this study was to ascertain the incidence and clinical significance of metallo-β-lactamases among Enterobacter strains isolated from patients with nosocomial infections. We prospectively ...collected data on patients with Enterobacter infection during a 13-month period. All of the strains were investigated for antibiotic susceptibility, the presence and expression of metallo-β-lactamases, and clonality. Of 29 infections (11 involving the urinary tract, 7 pneumonias, 3 skin/soft tissue infections, 3 intra-abdominal infections, 3 bacteremias, and 2 other infections), 7 (24%) were caused by Enterobacter cloacae strains harboring a blaVIM₋₁ gene associated or not with a blaSHV₁₂ gene. Infections caused by VIM-1-producing strains were more frequently associated with a recent prior hospitalization (P = 0.006), cirrhosis (P = 0.03), relapse of infection (P < 0.001), and more prolonged duration of antibiotic therapy (P = 0.01) than were other infections. All of the isolates were susceptible to imipenem and meropenem and had blaVIM₋₁ preceded by a weak P1 promoter and inactivated P2 promoters. Most VIM-1-producing Enterobacter isolates belonged to a main clone, but four different clones were found. Multiclonal VIM-1-producing E. cloacae infections are difficult to diagnose due to an apparent susceptibility to various beta-lactams, including carbapenems, and are associated with a high relapse rate and a more prolonged duration of antibiotic therapy.
In the present study, the in vitro activity of the sulbactam–durlobactam (SUL–DUR) combination was evaluated against 141 carbapenem-resistant A. baumannii (CRAb) clinical strains collected from six ...Italian laboratories. Over half (54.6%) of these isolates were resistant to colistin. The SUL–DUR combination was active against these CRAb isolates with MIC50 and MIC90 values of 0.5 mg/L and 4 mg/L, respectively. Only eleven isolates were resistant to SUL–DUR with MIC values ranging from 8 to 128 mg/L. The SUL–DUR resistant A. baumannii exhibited several antimicrobial resistance genes (ARGs) such as blaOXA-20, blaOXA-58, blaOXA-66, blaADC-25, aac(6′)-Ib3 and aac(6′)-Ib-cr and mutations in gyrA (S81L) and parC (V104I, D105E). However, in these isolates, mutations Q488K and Y528H were found in PBP3. Different determinants were also identified in these CRAb isolates, including adeABC, adeFGH, adeIJK, abeS, abaQ and abaR, which encode multidrug efflux pumps associated with resistance to multiple antibacterial agents. This is the first report on the antimicrobial activity of SUL–DUR against carbapenem-resistant A. baumannii isolates selected from multiple regions in Italy.
•Robust consensus was confirmed between AD fosfomycin panel and reference AD.•S. aureus showed 100% CA and 91.25% EA.•Enterobacterales showed 94% CA and 98% EA.•No evaluation errors were observed ...among S. aureus isolates.•AD fosfomycin panel can be a rapid gold standard method for fosfomycin MICs.
Many clinical laboratories have difficulty in routinely performing in vitro fosfomycin susceptibility testing using the agar dilution (AD) method, considered to be the gold standard method. The objective of our work was to evaluate a rapid commercial fosfomycin agar dilution panel against clinical Staphylococcus aureus and Enterobacterales strains, in two different centres located in Italy and in the UK.
A total of 99 Enterobacterales (mostly Escherichia coli and Klebsiella pneumoniae) and 80 S. aureus clinical isolates was used to evaluate the commercial device, a 12-well panel containing fosfomycin incorporated into CA-MH agar supplemented with 25mg/L of glucose-6-phosphate (Liofilchem S.r.l., Roseto degli Abruzzi, Italy). Testing was performed in two centres (Italy and UK) and kit results were compared against the gold standard in-house AD MIC method.
According to the EUCAST breakpoints, fosfomycin inhibited 61% of the S. aureus strains, and 76% of the Enterobacterales isolates tested by the AD reference method. There was a Categorical Agreement (CA) of 100% and an Essential Agreement (EA) of 91.25% for S. aureus; while the Enterobacterales strains showed a CA of 94% and an EA of 97%. No evaluation errors were observed among S. aureus, while 5% Major Error and 1% Very Major Error were observed for the Enterobacterales.
Our results confirmed the feasibility of determining fosfomycin susceptibility using a commercial AD panel as a routine substitution for the AD test. The few differences observed were only in strains with MICs around the breakpoint used.
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