KSA-1, a naturally occurring Ambler class A extended spectrum ß-lactamase from the enterobacterial species Kosakonia sacchari.

BACKGROUND: Several bacterial species belonging to the Gammaproteobacteria possess intrinsic class A ß-lactamase genes that may represent a source of further dissemination and acquisition to other Gram-negative species. Here we characterised KSA-1 class A ß-lactamase, the gene of which was identified within the chromosome of an environmental Enterobacterales species, namely Kosakonia sacchari, which was also recently identified as the progenitor of an MCR-like colistin-resistance determinant. METHODS: In silico analysis using the GenBank database identified a class A ß-lactamase gene within the chromosome of K. sacchari SP1 (GenBank accession no. WP_017456759). The corresponding protein KSA-1 shared 63% amino acid identity with the intrinsic CKO-1 from Citrobacter koseri and 53% with TEM-1. Using the K. sacchari DSM 100203 reference strain as a template, blaKSA-1 was amplified, cloned into the plasmid pUCp24 and expressed in Escherchia coli TOP10. Minimal inhibitory concentrations and kinetic parameters were obtained from the purified enzyme. RESULTS: K. sacchari strain SP1 conferred resistance to amino-, carboxy- and ureido-penicillins only. Once produced within E. coli, KSA-1 showed a typical clavulanic acid-inhibited extended spectrum ß-lactamase associated with a peculiar temocillin resistance profile. Kinetic assays were performed using a purified extract of KSA-1 and demonstrated a high hydrolysis rate for benzylpenicillin and piperacillin, as well as weakly extended spectrum cephalosporins. Determination of inhibitory constants showed 50% inhibitory concentration values of 2.2, 3 and 1.8 nM for clavulanic acid, tazobactam and avibactam, respectively. Analysis of sequences surrounding the blaKSA-1 gene did not reveal any mobile element that could have been involved in the acquisition of this ß-lactamase gene in that species. CONCLUSION: KSA-1 is a class A extended spectrum ß-lactamase distantly related to known extended spectrum or broad-spectrum Ambler class A ß-lactamases, which is highly resistant to temocillin. The blaKSA-1 gene could be considered as intrinsic within the species.

Role of blaTEM and OmpC in the piperacillin-tazobactam resistance evolution by E. coli in patients with complicated intra-abdominal infection.

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 ß-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.

High-level delafloxacin resistance through the combination of two different mechanisms in Staphylococcus aureus.

Delafloxacin is a new fluoroquinolone indicated for the treatment of complicated bacterial skin infections caused by Staphylococcus aureus. Despite its recent approval by the US Food and Drug Administration, the emergence of S. aureus-resistant strains has been reported. As such, this study aimed to investigate the activity of delafloxacin against a collection of S. aureus, and to determine the mechanisms of resistance. The activity of delafloxacin was measured in 59 S. aureus clinical isolates [40 methicillin-resistant S. aureus (MRSA) and 19 methicillin-susceptible S. aureus (MSSA)]. Whole-genome sequencing (WGS) was performed in the isolates resistant to delafloxacin. The minimum inhibitory concentrations required to inactivate 50% and 90% of the isolates (MIC50 and MIC90, respectively) were higher in MRSA (0.19 mg/L and 0.75 mg/L, respectively) than in MSSA (0.008 mg/L and 0.25 mg/L, respectively). Furthermore, 10 S. aureus clinical isolates (16.9%) were categorized as resistant to delafloxacin. Regarding the WGS data, several mutations were found in the quinolone resistance-determining region. Nevertheless, a mutation in the same position (E84K and E84V) of topoisomerase IV (ParC) was found exclusively in the four high-level delafloxacin-resistant isolates. Interestingly, a plasmid-encoded qacC gene (efflux pump) was found to be harboured by the isolate with the highest delafloxacin MIC value (32 mg/L). The use of a wide-spectrum efflux pump inhibitor revealed an important contribution of this system to the acquisition of delafloxacin resistance. In conclusion, delafloxacin has activity against S. aureus, including MRSA. However, this study showed that mutations in position 84 of ParC and the acquisition of a QacC efflux pump are key factors for the development of delafloxacin resistance in S. aureus.