C-terminal deletion of RelA protein is suggested as a possible cause of infective endocarditis recurrence with Enterococcus faecium.

Infective endocarditis (IE) caused by Enterococcus spp. represents the third most common cause of IE, with high rates of relapse compared with other bacteria. Interestingly, late relapses (>6 months) have only been described in Enterococcus faecalis, but here we describe the first reported IE relapse with Enterococcus faecium more than a year (17 months) after the initial endocarditis episode. Firstly, by multi locus sequence typing (MLST), we demonstrated that both isolates (EF646 and EF641) belong to the same sequence type (ST117). Considering that EF641 was able to overcome starvation and antibiotic treatment conditions surviving for a long period of time, we performed bioinformatic analysis in identifying potential genes involved in virulence and stringent response. Our results showed a 13-nucleotide duplication (positions 1638-1650) in the gene relA, resulting in a premature stop codon, with a loss of 167 amino acids from the C-terminal domains of the RelA enzyme. RelA mediates the stringent response in bacteria, modulating levels of the alarmone guanosine tetraphosphate (ppGpp). The relA mutant (EF641) was associated with lower growth capacity, the presence of small colony variants, and higher capacity to produce biofilms (compared with the strain EF646), but without differences in antimicrobial susceptibility patterns according to standard procedures during planktonic growth. Instead, EF641 demonstrated tolerance to high doses of teicoplanin when growing in a biofilm. We conclude that all these events would be closely related to the long-term survival of the E. faecium and the late relapse of the IE. These data represent the first clinical evidence of mutations in the stringent response (relA gene) related with E. faecium IE relapse.

Usefulness of sonication in the microbiological diagnosis of cardiovascular implantable electronic device infections: systematic review, meta-analysis and meta-regression.

BACKGROUND: Multiple studies have demonstrated the utility of sonication to improve culture yield in patients with cardiovascular implantable electronic device (CIED) infections. OBJECTIVE: To analyze the usefulness of sonication in the microbiological diagnosis of CIED infections in comparison with traditional cultures. METHODS: Systematic database searches were performed to identify studies that provided enough data concerning both sensitivity and specificity of traditional (non-sonicated) and sonicated cultures from CIED samples. The diagnostic accuracy measures were obtained by three different statistical approaches: (i) The univariate model; (ii) The bivariate random; and (iii) The Bayesian bivariate hierarchical model. Heterogeneity was assessed using meta-regression. FINDINGS: Nine studies met the criteria for inclusion in the meta-analysis (1684 cultures). The summary estimates of sensitivity were higher for sonicated cultures (0.756) in comparison with non-sonicated cultures (0.446). On meta-regression, sonication of CIEDs significantly increased the sensitivity (p = 0.001) as well as the rates of false positive results (p = 0.003). The final model also showed that the studies that used a threshold for positivity were associated with lower rates of false positive results (p < 0.001). INTERPRETATION: Our results suggest that sonication improves the microbiological diagnosis of CIED infections in comparison with traditional cultures, but a standardization of processes is necessary.

Implementation of a PCR-based strategy to control an outbreak by Serratia marcescens in a Neonatal Intensive Care Unit.

OBJECTIVES: To evaluate the clinical and epidemiological impact of a new molecular surveillance strategy based on qPCR to control an outbreak by Serratia marcescens in a Neonatal Intensive Care Unit (NICU). METHODS: We design a specific qPCR for the detection of S. marcescens in rectal swabs of patients admitted to a NICU. We divided the surveillance study into two periods: (a) the pre-PCR, from the outbreak declaration to the qPCR introduction, and (b) the PCR period, from the introduction of the qPCR until the outbreak was solved. In all cases, S. marcescens isolates were recovered and their clonal relationship was analysed by PFGE. Control measures were implemented during the outbreak. Finally, the number of bloodstream infections (BSI) was investigated in order to evaluate the clinical impact of this molecular strategy. RESULTS: Nineteen patients colonized/infected by S. marcescens were detected in the pre-PCR period (October 2020-April 2021). On the contrary, after the PCR implementation, 16 new patients were detected. The PFGE revealed 24 different pulsotypes belonging to 7 different clonal groups, that were not overlapping at the same time. Regarding the clinical impact, 18 months after the qPCR implementation, no more outbreaks by S. marcescens have been declared in the NICU of our hospital, and only 1 episode of BSI has occurred, compared with 11 BSI episodes declared previously to the outbreak control. CONCLUSIONS: The implementation of this qPCR strategy has proved to be a useful tool to control the nosocomial spread of S. marcescens in the NICU.

Aliidiomarina shirensis as Possible Source of the Integron- and Plasmid-Mediated Fosfomycin Resistance Gene fosC2.

In-silico analysis and cloning experiments identified a fosC2-like fosfomycin resistance gene in the chromosome of Aliidiomarina shirensis, with our data suggesting that this bacterium might be added to the list of species identified as reservoirs of fos-like genes that were subsequently acquired by other Gram-negative species. Indeed, the fosC2 gene was identified as acquired in Providencia huaxinensis and Aeromonas hydrophila isolates, with this gene being located in class 1 integron structures in the latter cases. Biochemical characterization and site-directed mutagenesis showed a higher catalytic efficiency for the intrinsic FosC2AS (from A. shirensis) than for the acquired FosC2 (from P. huaxinensis) enzyme due to a single substitution in the amino acid sequence (Gly43Glu). Notably, this study constitutes the first identification of the likely natural reservoir of a complete gene cassette (including its attC site).

Impact of Acquired Broad-Spectrum ß-Lactamases on Susceptibility to Cefiderocol and Newly Developed ß-Lactam/ß-Lactamase Inhibitor Combinations in Escherichia coli and Pseudomonas aeruginosa.

The ability of broad-spectrum ß-lactamases to reduce the susceptibility to ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam (AZA), and cefiderocol (FDC) was evaluated both in Pseudomonas aeruginosa and in Escherichia coli using isogenic backgrounds. Although metallo-ß-lactamases conferred resistance in most cases, except for AZA, several clavulanic-acid-inhibited extended-spectrum ß-lactamases (PER, BEL, SHV) had a significant impact on the susceptibility to CZA, C/T, and FDC.

NDM-35-Producing ST167 Escherichia coli Highly Resistant to ß-Lactams Including Cefiderocol.

A multidrug-resistant (carbapenems, aztreonam + avibactam, and cefiderocol) ST167 Escherichia coli clinical isolate recovered from a patient hospitalized in Switzerland produced NDM-35 showing ca. 10-fold increased hydrolytic activity toward cefiderocol compared to NDM-1. The isolate co-produced a CMY-type ß-lactamase, exhibited a four amino-acid insertion in PBP3, and possessed a truncated iron transporter CirA protein. Our study identified an association of unrelated resistance mechanisms leading to resistance to virtually all ß-lactams in a high-risk E. coli clone.

Reduced Chlorhexidine Susceptibility Is Associated with Tetracycline Resistance tet Genes in Clinical Isolates of Escherichia coli.

Chlorhexidine is a widely used antiseptic in hospital and community health care. Decreased susceptibility to this compound has been recently described in Klebsiella pneumoniae and Pseudomonas aeruginosa, together with cross-resistance to colistin. Surprisingly, few data are available for Escherichia coli, the main species responsible for community and health care-associated infections. In order to decipher chlorhexidine resistance mechanisms in E. coli, we studied both in vitro derived and clinical isolates through whole-genome sequence analysis. Comparison of strains grown in vitro under chlorhexidine pressure identified mutations in the gene mlaA coding for a phospholipid transport system. Phenotypic analyses of single-gene mutants from the Keio collection confirmed the role of this mutation in the decreased susceptibility to chlorhexidine. However, mutations in mlaA were not found in isolates from large clinical collections. In contrast, genome wide association studies (GWAS) showed that, in clinical strains, chlorhexidine reduced susceptibility was associated with the presence of tetA genes of class B coding for efflux pumps and located in a Tn10 transposon. Construction of recombinant strains in E. coli K-12 confirmed the role of tetA determinant in acquired resistance to both chlorhexidine and tetracycline. Our results reveal that two different evolutionary paths lead to chlorhexidine decreased susceptibility: one restricted to in vitro evolution conditions and involving a retrograde phospholipid transport system; the other observed in clinical isolates associated with efflux pump TetA. None of these mechanisms provide cross-resistance to colistin. This work demonstrates the GWAS power to identify new resistance mechanisms in bacterial species.

Selective Culture Medium for Screening of Fosfomycin Resistance in Enterobacterales.

A selective medium for screening fosfomycin (FOS)-resistant Enterobacterales was developed. Performances of this medium were first evaluated by using cultures of a collection of 84 enterobacterial clinical strains (42 FOS susceptible and 42 FOS resistant). The SuperFOS medium showed excellent sensitivity and specificity of detection (100%) in those conditions. Then, by testing spiked stool and spiked urine specimens, it revealed excellent performances, with lower limits of identification ranging from 101 to 102 CFU/ml. This screening medium allows easy and accurate detection of FOS-resistant isolates regardless of their resistance mechanisms.

Antioxidant Molecules as a Source of Mitigation of Antibiotic Resistance Gene Dissemination.

Escherichia coli is the most commonly identified human pathogen and a prominent microorganism of the gut microbiota. Acquired resistance to antibiotics in this species is driven mainly by horizontal gene transfer and plasmid acquisition. Currently, the main concern is the acquisition of extended-spectrum ß-lactamases of the CTX-M type in E. coli, a worldwide-observed phenomenon. Plasmids encoding CTX-M enzymes have different scaffolds and conjugate at different frequencies. Here, we show that the conjugation rates of several plasmid types encoding broad-spectrum ß-lactamases are increased when the E. coli donor strain is exposed to subinhibitory concentrations of diverse orally given antibiotics, including fluoroquinolones, such as ciprofloxacin and levofloxacin, but also trimethoprim and nitrofurantoin. This study provides insights into underlying mechanisms leading to increased plasmid conjugation frequency in relation to DNA synthesis inhibitor-type antibiotics, involving reactive oxygen species (ROS) production and probably increased expression of genes involved in the SOS response. Furthermore, we show that some antioxidant molecules currently approved for unrelated clinical uses, such as edaravone, p-coumaric acid, and N-acetylcysteine, may antagonize the ability of antibiotics to increase plasmid conjugation rates. These results suggest that several antioxidative molecules might be used in combination with these "inducer" antibiotics to mitigate the unwanted increased resistance plasmid dissemination.

Pathogenicity Genomic Island-Associated CrpP-Like Fluoroquinolone-Modifying Enzymes among Pseudomonas aeruginosa Clinical Isolates in Europe.

Many transferable quinolone resistance mechanisms have been identified in Gram-negative bacteria. The plasmid-encoded 65-amino-acid-long ciprofloxacin-modifying enzyme CrpP was recently identified in Pseudomonas aeruginosa isolates. We analyzed a collection of 100 clonally unrelated and multidrug-resistant P. aeruginosa clinical isolates, among which 46 were positive for crpP-like genes, encoding five CrpP variants conferring variable levels of reduced susceptibility to fluoroquinolones. These crpP-like genes were chromosomally located as part of pathogenicity genomic islands.

Occurrence of NDM-1-producing Morganella morganii and Proteus mirabilis in a single patient in Portugal: probable in vivo transfer by conjugation.

OBJECTIVES: To decipher the genetics of acquisition of carbapenemase-encoding genes identified in two carbapenem-resistant Enterobacteriaceae recovered from a single patient in Portugal. METHODS: Carbapenemase genes were searched by PCR assays and mating-out assays were performed to further characterize the plasmid support of the carbapenemase genes. Genetic characterization of the plasmid supports was performed by whole-plasmid sequencing using the Illumina technology. RESULTS: We identified here two NDM-1-producing isolates, namely a Morganella morganii and a Proteus mirabilis, sharing the same blaNDM-1-positive plasmid. This 154 kb plasmid belonged to the IncA/C2 type, recently renamed IncC, and co-harboured two AmpC ß-lactamase genes, namely blaCMY-4 and blaDHA-1, in addition to the 16S rRNA methylase gene armA encoding high-level resistance to aminoglycosides. In addition, the M. morganii isolate produced the CTX-M-33 extended-spectrum ß-lactamase possessing weak carbapenemase activity, encoded by another plasmid. CONCLUSIONS: We showed here that, in addition to KPC-type and OXA-181 carbapenemases, which have been identified as widespread in this country, another concern is the emergence of NDM-1-producing enterobacterial isolates in Portugal. We demonstrated here the in vivo plasmid transfer of a blaNDM-1-positive plasmid leading to dissemination of this carbapenemase gene within different enterobacterial species in a single patient.

Epidemiology of Carbapenemase-Producing Klebsiella pneumoniae in a Hospital, Portugal.

We aimed to provide updated epidemiologic data on carbapenem-resistant Klebsiella pneumoniae in Portugal by characterizing all isolates (N = 46) recovered during 2013-2018 in a 123-bed hospital in Lisbon. We identified blaKPC-3 (n = 36), blaOXA-181 (n = 9), and blaGES-5 (n = 8) carbapenemase genes and observed co-occurrence of blaKPC-3 and blaGES-5 in 7 isolates. A single GES-5-producing isolate co-produced the extended-spectrum ß-lactamase BEL-1; both corresponding genes were co-located on the same ColE1-like plasmid. The blaOXA-181 gene was always located on an IncX3 plasmid, whereas blaKPC-3 was carried on IncN, IncFII, IncFIB, and IncFIIA plasmid types. The 46 isolates were distributed into 13 pulsotypes and 9 sequence types. All isolates remained susceptible to ceftazidime/avibactam, but some exhibited reduced antimicrobial susceptibility (MIC = 3 mg/L).

Acquisition of Extended-Spectrum ß-Lactamase GES-6 Leading to Resistance to Ceftolozane-Tazobactam Combination in Pseudomonas aeruginosa.

A clinical Pseudomonas aeruginosa isolate resistant to all ß-lactams, including ceftolozane-tazobactam and carbapenems, was recovered. It belonged to sequence type 235 and produced the extended-spectrum ß-lactamase (ESBL) GES-6 differing from GES-1 by two amino acid substitutions (E104K and G170S). GES-6 possessed an increased hydrolytic activity toward carbapenems and to ceftolozane and a decreased susceptibility to ß-lactamase inhibitors compared to GES-1, except for avibactam. We show here that resistance to ceftolozane-tazobactam may occur through acquisition of a specific ESBL in P. aeruginosa but that ceftazidime-avibactam combination remains an effective alternative.

CTX-M-33, a CTX-M-15 derivative conferring reduced susceptibility to carbapenems.

CTX-M-type extended-spectrum ß-lactamases (ESBL) are widespread among Enterobacterales worldwide. The most common variant is CTX-M-15 hydrolyzing ceftazidime at high rate, but sparing carbapenems. We identified here CTX-M-33, a point mutant derivative of CTX-M-15 (Asp to Ser substitution at Ambler position 109), exhibiting a low carbapenemase activity. ß-Lactamase CTX-M-33 was identified in a Klebsiella pneumoniae isolate belonging to ST405, lacking the outer membrane protein OmpK36, that was resistant to broad-spectrum cephalosporins and ß-lactam/ß-lactamase inhibitor combinations, and displayed a decreased susceptibility to carbapenems. Comparative hydrolytic activity assays showed that CTX-M-33 hydrolyzed ceftazidime at a lower level than CTX-M-15, but significantly hydrolyzed meropenem. In addition, CTX-M-33 showed higher Mutant Prevention Concentration values and wider mutant selection window in presence of meropenem, in accordance with its observed hydrolytic properties. We identified here the very first CTX-M enzyme possessing a weak carbapenemase activity, that may correspond to an emerging phenomenon when considering its possibility to evolve from the widespread ESBL CTX-M-15.

mcr-9, an Inducible Gene Encoding an Acquired Phosphoethanolamine Transferase in Escherichia coli, and Its Origin.

The plasmid-located mcr-9 gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal Escherichia coli strain belonging to a very rare phylogroup, the D-ST69-O15:H6 clone. This MCR-9 protein shares 33% to 65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1 to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a function similar to that of MCR-1 by adding a phosphoethanolamine group to lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once the mcr-9 gene was cloned and produced in an E. coli K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9 Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate silently and remain undetected unless induced by colistin.

ESBLs and resistance to ceftazidime/avibactam and ceftolozane/tazobactam combinations in Escherichia coli and Pseudomonas aeruginosa.

OBJECTIVES: To evaluate the efficacy of the recently launched ß-lactam/ß-lactamase inhibitor combinations ceftazidime/avibactam and ceftolozane/tazobactam against ESBL-producing Escherichia coli and Pseudomonas aeruginosa strains. METHODS: A series of ESBL-encoding genes (blaTEM, blaSHV, blaCTX-M, blaVEB, blaPER, blaGES and blaBEL) was cloned and expressed in E. coli or P. aeruginosa recipient strains. Cultures of E. coli TOP10 harbouring recombinant plasmids and therefore producing the different ESBLs tested were grown in order to perform measurements of catalytic activities, using benzylpenicillin, ceftazidime and ceftolozane as substrates. IC50s were additionally determined for clavulanic acid, tazobactam and avibactam. RESULTS: We showed here an overall better activity of ceftazidime/avibactam compared with ceftolozane/tazobactam toward ESBL-producing E. coli and P. aeruginosa. Several ESBLs of the GES, PER and BEL types conferred resistance to ceftolozane/tazobactam in E. coli and P. aeruginosa. For GES-6 and PER-1 producers, resistance to ceftolozane/tazobactam could be explained by a high hydrolysis of ceftolozane and a low activity of tazobactam as an inhibitor. On the other hand, PER-producing P. aeruginosa also exhibited resistance to ceftazidime/avibactam. CONCLUSIONS: Altogether, the results show that the ESBL PER-1, which is widespread worldwide, may be a source of resistance to both ceftolozane/tazobactam and ceftazidime/avibactam. Excellent activity of ceftazidime/avibactam was highlighted for both ESBL-producing E. coli and ESBL-producing P. aeruginosa.

Survival of infection with TEM ß-lactamase-producing Escherichia coli with Pan-ß-lactam resistance.

BACKGROUND: Antimicrobial resistance is a critical global health issue, significantly contributing to patient mortality. Recent antibiotic developments have aimed to counteract carbapenemase-producing Enterobacterales; however, the impact of their use on the emergence of antibiotic resistance is unknown. This study investigates the first case of a non-carbapenemase-producing, pan-ß-lactam-resistant Escherichia coli strain from a patient previously treated with ceftolozane-tazobactam and cefiderocol. METHODS: This study describes the clinical progression of a 39-year-old ICU patient who developed multiple infections, culminating in the isolation of a pan-ß-lactam-resistant E. coli strain (EC554). The resistance profile was characterised through MIC determination, whole-genome sequencing, the use of the ß-lactam inactivation method, RT-qPCR, efflux pump inhibition assays, outer membrane protein analysis, and blaTEM transformation. FINDINGS: The EC554 isolate displayed resistance to all tested ß-lactams and ß-lactam-ß-lactamase inhibitor combinations. Whole-genome sequencing revealed four plasmids in EC554, with the only ß-lactamase gene being blaTEM-252 on the pEC554-PBR-X1-X1 plasmid. We found that the extremely resistant phenotype was attributable to a combination of different mechanisms: a high expression of TEM-252, efflux pump activity, porin loss, and PBP3 mutations. INTERPRETATION: The findings illustrate the complex interplay of multiple resistance mechanisms in E. coli, highlighting the potential for high-level resistance even without carbapenemase production. This study underscores the importance of comprehensively characterising resistance mechanisms in order to inform effective treatment strategies and mitigate the spread of resistant strains.

Activity of cefepime, carbapenems and new ß-lactam/ß-lactamase inhibitor combinations on Enterobacter cloacae complex and Klebsiella aerogenes in Spain (SMART 2016-2022).

Objectives: To analyse the susceptibility profile to cefepime, carbapenems and new ß-lactam/ß-lactamase inhibitor combinations in Enterobacter cloacae complex and Klebsiella aerogenes isolated from intra-abdominal, urinary, respiratory and bloodstream infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain. Methods: The susceptibilities of 759 isolates (473 E. cloacae complex and 286 K. aerogenes) collected in 11 Spanish hospitals from 2016 to 2022 were analysed following the EUCAST 2023 criteria. Molecular characterization looking for ß-lactamase genes was performed through PCR and DNA sequencing analysis. Results: E. cloacae complex showed resistance to third-generation cephalosporins in 25% of the cases, whereas K. aerogenes was resistant in 35%. Regarding cefepime, resistance in E. cloacae was higher (10%) than in K. aerogenes (2%). Carbapenems showed >85% activity in both microorganisms. Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam had good activity against these microorganisms (>95%). In contrast, the activity of ceftolozane/tazobactam was lower (80%). A high proportion of the isolates resistant to new ß-lactam/ß-lactamase inhibitor combinations carried a carbapenemase, mainly OXA-48-like and VIM-1. Conclusions: Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam show high activity against both E. cloacae complex and K. aerogenes isolates recovered in the SMART-Spain study. In contrast, differences have been found in the case of cefepime, showing more activity against K. aerogenes than E. cloacae complex. These results are useful for antimicrobial stewardship programmes and for the implementation of local and national guidelines.