<i>In Vitro</i> Activity of Two Cefepime-Based Novel Combinations, Cefepime/Taniborbactam and Cefepime/Zidebactam, against Carbapenemase-Expressing <i>Enterobacterales </i>Collected in India

In recent times, discovery efforts for novel antibiotics have mostly targeted carbapenemase-producing Gram-negative organisms. Two different combination approaches are pertinent: b-lactam-b-lactamase inhibitor (BL/BLI) or b-lactam-b-lactam enhancer (BL/ BLE). Cefepime combined with a BLI, taniborbactam, or with a BLE, zidebactam, has been shown to be promising. In this study, we determined the in vitro activity of both these agents along with comparators against multicentric carbapenemase-producing Enterobacterales (CPE). Nonduplicate CPE isolates of Escherichia coli (n = 270) and Klebsiella pneumoniae (n = 300), collected from nine different tertiary-care hospitals across India during 2019 to 2021, were included in the study. Carbapenemases in these isolates were detected by PCR. E. coli isolates were also screened for the presence of the 4-Amino-Acid insert in penicillin binding protein 3 (PBP3). MICs were determined by reference broth microdilution. Higher MICs of cefepime/taniborbactam (.8 mg/L) were linked to NDM, both in K. pneumoniae and in E. coli. In particular, such higher MICs were observed in 88 to 90% of E. coli isolates producing NDM and OXA-48-like or NDM alone. On the other hand, OXA-48-like-producing E. coli or K. pneumoniae isolates were nearly 100% susceptible to cefepime/taniborbactam. Regardless of the carbapenemase types and the pathogens, cefepime/ zidebactam showed potent activity (.99% inhibited at#8mg/L). It seems that the 4-amino-Acid insert in PBP3 (present universally in the study E. coli isolates) along with NDM adversely impact the activity of cefepime/taniborbactam. Thus, the limitations of the BL/BLI approach in tackling the complex interplay of enzymatic and nonenzymatic resistance mechanisms were better revealed in whole-cell studies where the activity observed was a net effect of b-lactamase inhibition, cellular uptake, and target affinity of the combination. IMPORTANCE The study revealed the differential ability of cefepime/taniborbactam and cefepime/zidebactam in tackling carbapenemase-producing Indian clinical isolates that also harbored additional mechanisms of resistance. NDM-expressing E. coli with 4-Amino-Acid insert in PBP3 are predominately resistant to cefepime/taniborbactam, while the b-lactam enhancer mechanism-based cefepime/zidebactam showed consistent activity against single-or dual-carbapenemase-producing isolates including E. coli with PBP3 inserts.</p

Laboratory detection and clinical implication of oxacillinase-48 like carbapenemase: The hidden threat

Carbapenemase producing Gram-negative pathogen is of great concern for physician. The challenging aspects are treatment option and infection control. Monitoring of respective carbapenemase resistance mechanism is necessary to prevent the outbreaks. Currently, the rapid emergence of oxacillinase (OXA-48) like is alarming. Increasing frequency of OXA-48 is seen than the classical carbapenemase (KPC, NDM, IMP, and VIM) across the world. The blaOXA-48 gene is commonly identified in Escherichia coli and Klebsiella pneumoniae. The transferrable plasmid of OXA-48 is associated with rapid spread and inter-species dissemination. In general, OXA-48-like enzymes weakly hydrolyzes both carbapenem and broad spectrum cephalosporins. Except OXA-163, which effectively hydrolyze cephalosporin. This poor hydrolytic profile obscures the detection of OXA-48-like. It may go undetected in routine diagnosis and complicates the treatment option. Co-production of OXA-48-like with CTX-M-15 and other carbapenemase (NDM, VIM) leads to the emergence of multidrug resistant strains

Evolving rapid methicillin-resistant Staphylococcus aureus detection: Cover all the bases

The dissemination of methicillin-resistant (MR) Staphylococcus aureus (SA) in community and health-care settings is of great concern and associated with high mortality and morbidity. Rapid detection of MRSA with short turnaround time can minimize the time to initiate appropriate therapy and further promote infection control. Early detection of MRSA directly from clinical samples is complicated by the frequent association of MRSA with methicillin-susceptible SA (MSSA) and coagulase-negative Staphylococcus (CoNS) species. Infection associated with true MRSA or MSSA is differentiated from CoNS, requires target specific primers for the presence of SA and mec A or nuc or fem A gene for confirmation of MR. Recently, livestock-associated MRSA carrying mec C variant complicates the epidemiology of MRSA further. Several commercial rapid molecular kits are available with a different combination of these targets for the detection of MRSA or MSSA. The claimed sensitivity and specificity of the currently available commercial kits is varying, because of the different target combination used for detection of SA and MR

In-Vitro Selection of Ceftazidime/Avibactam Resistance in OXA-48-Like-Expressing Klebsiella pneumoniae: In-Vitro and In-Vivo Fitness, Genetic Basis and Activities of β-Lactam Plus Novel β-Lactamase Inhibitor or β-Lactam Enhancer Combinations

Ceftazidime/avibactam uniquely demonstrates activity against both KPC and OXA-48-like carbapenemase-expressing Enterobacterales. Clinical resistance to ceftazidime/avibactam in KPC-producers was foreseen in in-vitro resistance studies. Herein, we assessed the resistance selection propensity of ceftazidime/avibactam in K. pneumoniae expressing OXA-48-like β-lactamases (n = 10), employing serial transfer approach. Ceftazidime/avibactam MICs (0.25–4 mg/L) increased to 16–256 mg/L after 15 daily-sequential transfers. The whole genome sequence analysis of terminal mutants showed modifications in proteins linked to efflux (AcrB/AcrD/EmrA/Mdt), outer membrane permeability (OmpK36) and/or stress response pathways (CpxA/EnvZ/RpoE). In-vitro growth properties of all the ceftazidime/avibactam-selected mutants were comparable to their respective parents and they retained the ability to cause pulmonary infection in neutropenic mice. Against these mutants, we explored the activities of various combinations of β-lactams (ceftazidime or cefepime) with structurally diverse β-lactamase inhibitors or a β-lactam enhancer, zidebactam. Zidebactam, in combination with either cefepime or ceftazidime, overcame ceftazidime/avibactam resistance (MIC range 0.5–8 mg/L), while cefepime/avibactam was the second best (MIC: 0.5–16 mg/L) in yielding lower MICs. The present work revealed the possibility of ceftazidime/avibactam resistance in OXA-48-like K. pneumoniae through mutations in proteins involved in efflux and/or porins without concomitant fitness cost mandating astute monitoring of ceftazidime/avibactam resistance among OXA-48 genotypes

Genomic insights of optrA-carrying linezolid-resistant Enterococcus faecium using hybrid assembly: first report from India

ABSTRACT: Objectives: Linezolid resistance in Enterococcus faecium is emerging worldwide. In this study, we aimed to characterise two linezolid-resistant E. faecium isolates using whole-genome sequencing. Methods: Antimicrobial susceptibility testing was performed by the broth microdilution method. A hybrid assembly approach of IonTorrent and MinION sequencing reads was used to generate the complete genome of linezolid-resistant E. faecium isolates VB3025 and VB3240. Results: VB3025 and VB3240 had minimum inhibitory concentration (MICs) for linezolid of 1024 μg/mL and 512 μg/mL, respectively. In addition, VB3025 was found to be resistant to both vancomycin and teicoplanin, while VB3240 was susceptible to these antibiotics. A hybrid assembly approach was used to generate the complete genome of VB3025 and VB3240 isolates harbouring the optrA gene. Notably, VB3025 carried two copies of optrA (chromosomal and plasmid), while in VB3240 optrA was identified on the chromosome. Interestingly, the plasmid pVB3025_2 co-carried the resistance gene clusters aph(3)-IIIa–sat4–ant(6)-Ia–ermB, the vanHAX operon and a copy of the optrA gene. Moreover, the optrA gene inserted into a Tn554 transposon carrying the ermA gene was identified in both VB3025 and VB3240 isolates. Furthermore, mutation analysis revealed the presence of a G2592T mutation in the 23S rRNA of both isolates. Conclusion: This is the first study reporting optrA-positive linezolid-resistant E. faecium from India. A novel plasmid co-carrying vancomycin and linezolid resistance determinants highlights the threat for potential dissemination

Can isepamicin be a potential option for extended spectrum beta-lactamases and carbapenemases expressing Escherichia coli?

Background: Beta-lactamase expressing Enterobacterales are challenging to treat. The potency of aminoglycosides as a bactericidal agent has increased interest in using them in combination regimens. In the present study, we evaluated the in-vitro activity of isepamicin and its comparator against phenotypically confirmed ESBL and carbapenemase expressing E. coli isolates. Methods: ESBL-E. coli (n = 103) and carbapenemase expressing E. coli (n = 105) collected from various clinical samples were included in this study. The minimum inhibitory concentrations (MICs) of ceftazidime, meropenem, gentamicin, amikacin, and isepamicin were determined using the broth microdilution method according to CLSI guidelines. Results: Isepamicin potently inhibited 98% of ESBL expressing E. coli isolates and the MIC90 for isepamicin was two-fold lower than that of amikacin. Isepamicin retained its inhibitory activity against 95% of amikacin resistant and 97% of gentamicin resistant ESBL expressing E. coli isolates. There were 31% of amikacin-resistant and 22% of gentamicin-resistant, carbapenemase-expressing E. coli isolates were susceptible to isepamicin. Conclusion: The findings suggest that isepamicin may be useful for treating community acquired urinary tract infection as monotherapy and systemic ESBL-E. coli infection as combination therapy, due to its minimal nephrotoxicity and ototoxicity, compared to other aminoglycosides

Genomic insights on heterogeneous resistance to vancomycin and teicoplanin in Methicillin-resistant Staphylococcus aureus: A first report from South India

Methicillin-resistant Staphylococcus aureus (MRSA) infection is an important clinical concern in patients, and is often associated with significant disease burden and metastatic infections. There is an increasing evidence of heterogeneous vancomycin-intermediate S. aureus (hVISA) associated treatment failure. In this study, we aim to understand the molecular mechanism of teicoplanin resistant MRSA (TR-MRSA) and hVISA. A total of 482 MRSA isolates were investigated for these phenotypes. Of the tested isolates, 1% were identified as TR-MRSA, and 12% identified as hVISA. A highly diverse amino acid substitution was observed in tcaRAB, vraSR, and graSR genes in TR-MRSA and hVISA strains. Interestingly, 65% of hVISA strains had a D148Q mutation in the graR gene. However, none of the markers were reliable in differentiating hVISA from TR-MRSA. Significant pbp2 upregulation was noted in three TR-MRSA strains, which had teicoplanin MICs of 16 or 32 μg/ml, whilst significant pbp4 downregulation was not noted in these strains. In our study, multiple mutations were identified in the candidate genes, suggesting a complex evolutionary pathway involved in the development of TR-MRSA and hVISA strains

Genomic insights on heterogeneous resistance to vancomycin and teicoplanin in Methicillin-resistant Staphylococcus aureus: A first report from South India.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is an important clinical concern in patients, and is often associated with significant disease burden and metastatic infections. There is an increasing evidence of heterogeneous vancomycin-intermediate S. aureus (hVISA) associated treatment failure. In this study, we aim to understand the molecular mechanism of teicoplanin resistant MRSA (TR-MRSA) and hVISA. A total of 482 MRSA isolates were investigated for these phenotypes. Of the tested isolates, 1% were identified as TR-MRSA, and 12% identified as hVISA. A highly diverse amino acid substitution was observed in tcaRAB, vraSR, and graSR genes in TR-MRSA and hVISA strains. Interestingly, 65% of hVISA strains had a D148Q mutation in the graR gene. However, none of the markers were reliable in differentiating hVISA from TR-MRSA. Significant pbp2 upregulation was noted in three TR-MRSA strains, which had teicoplanin MICs of 16 or 32 μg/ml, whilst significant pbp4 downregulation was not noted in these strains. In our study, multiple mutations were identified in the candidate genes, suggesting a complex evolutionary pathway involved in the development of TR-MRSA and hVISA strains