Clinical Resistance Research Articles

Clinical, molecular, and drug resistance epidemiology of HIV in Jordan, 2019-2021: A national study

BackgroundLimited epidemiologic studies have been conducted in Jordan describing the HIV epidemic. This study aimed to address this gap to inform HIV prevention and control. MethodsA nationally-representative cross-sectional study was conducted among adults living with HIV in Jordan. Laboratory testing included HIV viral load and next-generation-sequencing-based clinical genotype. Log-binomial regression estimated risk ratios (RRs) and 95% confidence intervals (CIs). ResultsAmong 231 (70%) participants, most were male (184/80%), and from Jordan (217/94%). Among 188 treatment-experienced-participants (>6 months), 165 (88%) were virally suppressed. High-level resistance was most frequent against nucleoside reverse transcriptase inhibitor (13/81%), and integrase-strand transfer inhibitor (INSTI) (10/62%) drugs among viremic (≥1000 HIV copies/mL) treatment-experienced participants with drug-resistant mutations (DRMs, n = 16). Common HIV subtypes (n = 43) were B (6/14%), A1 (5/12%), and CRF01_AE (5/12%); additionally, novel recombinant forms were detected. In multivariate analysis, independently higher risk for late diagnosis (n = 49) was observed with diagnosis through blood donation (vs check-up: RR 2.20, 95%CI 1.16-4.17) and earlier time-period of diagnosis (1986-2014 vs 2015-2021: RR 2.87, 95%CI 1.46-5.62). ConclusionsLate diagnosis and INSTI resistance endanger national HIV prevention and treatment in Jordan—high-level resistance to INSTI suggests therapeutic drug monitoring is needed for treatment efficacy and conservation of treatment options.

Efficacy and Safety of Amenamevir, a Helicase-Primase Inhibitor for the Treatment of Acyclovir-Resistant Herpes Simplex Virus 1 Keratitis.

The purpose of this study was to describe the efficacy and tolerance of amenamevir (AMNV), an inhibitor of the viral helicase-primase, for the treatment of recalcitrant herpes simplex keratitis (HSK) caused by acyclovir-resistant (ACVR) herpes simplex virus 1 strains. In this retrospective case series, 6 consecutive patients with HSK caused by an ACVR herpes simplex virus 1 strain with a failure of conventional antiviral therapy were included after having been treated with AMNV (there was no control group of comparable patients for whom previous treatment would have been continued despite its inefficacy). Medical files were assessed for clinical data including reason(s) for AMNV introduction (frequent recurrences despite appropriate preventive antiviral treatment and/or clinical resistance to suppressive antiviral treatment of an ongoing clinical relapse), genotypical resistance to herpes simplex virus 1 documentation, immune status, clinical types and number of HSK episodes before and during AMNV treatment, adverse effects observed during AMNV treatment, and best corrected visual acuity. Of 6 patients, 4 (66%) did not experience a single recurrence during AMNV therapy while 2 others had recurrences (1 over 24 months of treatment and 2 over 23 months, ie two-fold less frequently than with conventional preventive treatment). On the overall history of these 6 patients, AMNV appeared to be associated with a reduction in HSK recurrences, with a mean of only 0.02 ± 0.04 episodes/month during follow-up under AMNV as compared to 0.14 ± 0.04 episodes/month in the year preceding AMNV introduction (P = 0.03). Improvement in vision acuity was also observed (mean best corrected visual acuity 0.17 ± 0.12 logarithm of the minimum angle of resolution at the end of follow-up vs. 0.30 ± 0.35 before AMNV onset), albeit nonsignificant probably due to the limited number of patients (P = 0.38). Neither clinical nor biological adverse effects were observed while under AMNV during the follow-up (16.5 ± 5.8 months). Although there was no control group, AMNV may be a valuable option to reduce ACVR HSK recurrences.

Epithelial Expressed B7-H4 Drives Differential Immunotherapy Response in Murine and Human Breast Cancer.

This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target.

Heterogeneity of SOS response expression in clinical isolates of Escherichia coli influences adaptation to antimicrobial stress

In recent years, new evidence has shown that the SOS response plays an important role in the response to antimicrobials, with involvement in the generation of clinical resistance. Here we evaluate the impact of heterogeneous expression of the SOS response in clinical isolates of Escherichia coli on response to the fluoroquinolone, ciprofloxacin. In silico analysis of whole genome sequencing data showed remarkable sequence conservation of the SOS response regulators, RecA and LexA. Despite the genetic homogeneity, our results revealed a marked differential heterogeneity in SOS response activation, both at population and single-cell level, among clinical isolates of E. coli in the presence of subinhibitory concentrations of ciprofloxacin. Four main stages of SOS response activation were identified and correlated with cell filamentation. Interestingly, there was a correlation between clinical isolates with higher expression of the SOS response and further progression to resistance. This heterogeneity in response to DNA damage repair (mediated by the SOS response) and induced by antimicrobial agents could be a new factor with implications for bacterial evolution and survival contributing to the generation of antimicrobial resistance.

BCR::ABL1 kinase N-lobe mutants confer moderate to high degrees of resistance to asciminib

AbstractSecondary kinase domain mutations in BCR::ABL1 represent the most common cause of resistance to tyrosine kinase inhibitor (TKI) therapy in patients with chronic myeloid leukemia. The first 5 approved BCR::ABL1 TKIs target the adenosine triphosphate (ATP)–binding pocket. Mutations confer resistance to these ATP-competitive TKIs and those approved for other malignancies by decreasing TKI affinity and/or increasing ATP affinity. Asciminib, the first highly active allosteric TKI approved for any malignancy, targets an allosteric regulatory pocket in the BCR::ABL1 kinase C-lobe. As a non–ATP-competitive inhibitor, the activity of asciminib is predicted to be impervious to increases in ATP affinity. Here, we report several known mutations that confer resistance to ATP-competitive TKIs in the BCR::ABL1 kinase N-lobe that are distant from the asciminib binding pocket yet unexpectedly confer in vitro resistance to asciminib. Among these is BCR::ABL1 M244V, which confers clinical resistance even to escalated asciminib doses. We demonstrate that BCR::ABL1 M244V does not impair asciminib binding, thereby invoking a novel mechanism of resistance. Molecular dynamic simulations of the M244V substitution implicate stabilization of an active kinase conformation through impact on the α-C helix as a mechanism of resistance. These N-lobe mutations may compromise the clinical activity of ongoing combination studies of asciminib with ATP-competitive TKIs.

Drug-Resistant Bone, Joint and Spine Tuberculosis: Evolution of Diagnosis and Treatment.

Drug resistant (DR) osteoarticular TB (OATB) is a challenge in view of it being deep seated lesion and paucibacillary disease. Case definition, investigation protocol, treatment of proven DR and those cases where DR could not be demonstrated lacks clarity and evidence. Hence, a series of studies were conducted to develop an algorithm to investigate and treat therapeutically refractory disease (TRD) or presumptive drug resistance (PDR) cases of OATB. 6 studies were conducted. Study one and two evaluated criteria to label TRD/PDR. Three subsequent studies were conducted where TDR/PDR or fresh cases of OATB cases were investigated by AFB smear, Bactec/liquid culture, histology and genotypic DST by CBNAAT & LPA. Sixth study was a retrospective evaluation of all DR cases treated for proven or clinical drug resistance (CDR). Patient of bone/spine TB on ATT for 5 months or more show poor clinico-radiological treatment response as worsening of lesion, increased spinal deformity, persistent discharging sinus/ulcer, appearance of fresh lesion, recurrence of previous lesion, wound dehiscence of post-operative surgical scar cab labelled as PDR cases. These cases on histology ascertained TB and were proven DR on genotypic and phenotypic DST and are treated successfully. The patients of histologically ascertained TB and no/indeterminate phenotypic and genotypic DST were successfully treated as clinical drug resistance on MDR protocol. We described an algorithm. We must suspect PDR(TRD) based on criteria described. The tissue must be procured and submitted for AFB smear, histology and phenotypic and genotypic DST for diagnosis of TB. Genotypic and phenotypic DST will be useful to prove (90% instances) type of drug resistance. Remaining on strong clinical suspicion of DR and yet inconclusive on phenotypic/genotypic DST (<10%), may be treated as CDR as MDR. The adverse drug reactions and hepatic side-effects should be monitored diligently and these cases to be treated till healed status is demonstrated.

Regulatory interactions between daptomycin- and bacitracin-responsive pathways coordinate the cell envelope antibiotic resistance response of Enterococcus faecalis.

Enterococcal infections frequently show high levels of antibiotic resistance, including to cell envelope-acting antibiotics like daptomycin (DAP). While we have a good understanding of the resistance mechanisms, less is known about the control of such resistance genes in enterococci. Previous work unveiled a bacitracin resistance network, comprised of the sensory ABC transporter SapAB, the two-component system (TCS) SapRS and the resistance ABC transporter RapAB. Interestingly, components of this system have recently been implicated in DAP resistance, a role usually regulated by the TCS LiaFSR. To better understand the regulation of DAP resistance and how this relates to mutations observed in DAP-resistant clinical isolates of enterococci, we here explored the interplay between these two regulatory pathways. Our results show that SapR regulates an additional resistance operon, dltXABCD, a known DAP resistance determinant, and show that LiaFSR regulates the expression of sapRS. This regulatory structure places SapRS-target genes under dual control, where expression is directly controlled by SapRS, which itself is up-regulated through LiaFSR. The network structure described here shows how Enterococcus faecalis coordinates its response to cell envelope attack and can explain why clinical DAP resistance often emerges via mutations in regulatory components.

Whole-Genome Sequencing and Phenotypic Analysis of Streptococcus equi subsp. zooepidemicus Sequence Type 147 Isolated from China.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the important zoonotic and opportunistic pathogens. In recent years, there has been growing evidence that supports the potential role of S. zooepidemicus in severe diseases in horses and other animals, including humans. Furthermore, the clinical isolation and drug resistance rates of S. zooepidemicus have been increasing yearly, leading to interest in its in-depth genomic analysis. In order to deepen the understanding of the S. zooepidemicus characteristics and genomic features, we investigated the genomic islands, mobile genetic elements, virulence and resistance genes, and phenotype of S. zooepidemicus strain ZHZ 211 (ST147), isolated from an equine farm in China. We obtained a 2.18 Mb, high-quality chromosome and found eight genomic islands. According to a comparative genomic investigation with other reference strains, ZHZ 211 has more virulence factors, like an iron uptake system, adherence, exoenzymes, and antiphagocytosis. More interestingly, ZHZ 211 has acquired a mobile genetic element (MGE), prophage Ph01, which was found to be in the chromosome of this strain and included two hyaluronidase (hyl) genes, important virulence factors of the strain. Moreover, two transposons and two virulence (virD4) genes were found to be located in the same genome island of ZHZ 211. In vitro phenotypic results showed that ZHZ 211 grows faster and is resistant to clarithromycin, enrofloxacin, and sulfonamides. The higher biofilm-forming capabilities of ZHZ 211 may provide a competitive advantage for survival in its niche. The results expand our understanding of the genomic, pathogenicity, and resistance characterization of Streptococcus zooepidemicus and facilitate further exploration of its molecular pathogenic mechanism.

Unveiling a Listeria monocytogenes Outbreak in a Rabbit Farm: Clinical Manifestation, Antimicrobial Resistance, Genomic Insights and Environmental Investigation.

Listeria monocytogenes poses a threat to both human and animal health. This work describes an L. monocytogenes outbreak in a Portuguese rabbit farm, detailing the isolates' clinical manifestations, necropsy findings, and phenotypic and genomic profiles. Clinical signs, exclusively observed in does, included lethargy and reproductive signs. Post-mortem examination of does revealed splenomegaly, hepatomegaly with a reticular pattern, pulmonary congestion, and haemorrhagic lesions in the uterus, with thickening of the uterine wall and purulent greyish exudates. Positive L. monocytogenes samples were identified in fattening and maternity units across different samples, encompassing does and environmental samples. Core-genome Multi Locus Sequence Typing (cgMLST) analysis confirmed the outbreak, with the 16 sequenced isolates (lineage II, CC31, and ST325) clustering within a ≤2 allelic difference (AD) threshold. Antimicrobial susceptibility testing for five antibiotics revealed that 15 out of 19 outbreak isolates were resistant to sulfamethoxazole-trimethoprim (SXT). Concordantly, all SXT-resistant sequenced isolates were found to exclusively harbour a plasmid containing a trimethoprim-resistance gene (dfrD), along with loci linked to resistance to lincosamides (lnuG), macrolides (mphB), and polyether ionophores (NarAB operon). All sequenced outbreak isolates carried the antibiotic resistance-related genes tetM, fosX, lin, norB, lmrB, sul, and mprF. The outbreak cluster comprises isolates from does and the environment, which underscores the ubiquitous presence of L. monocytogenes and emphasizes the importance of biosecurity measures. Despite limited data on listeriosis in rabbit farming, this outbreak reveals its significant impact on animal welfare and production.

Structure-Based Optimization of Pyrazinamide-Containing Macrocyclic Derivatives as Fms-like Tyrosine Kinase 3 (FLT3) Inhibitors to Overcome Clinical Mutations.

Secondary mutations in Fms-like tyrosine kinase 3-tyrosine kinase domain (FLT3-TKD) (e.g., D835Y and F691L) have become a major on-target resistance mechanism of FLT3 inhibitors, which present a significant clinical challenge. To date, no effective drugs have been approved to simultaneously overcome clinical resistance caused by these two mutants. Thus, a series of pyrazinamide macrocyclic compounds were first designed and evaluated to overcome the secondary mutations of FLT3. The representative 8v exhibited potent inhibitory activities against FLT3D835Y and FLT3D835Y/F691L with IC50 values of 1.5 and 9.7 nM, respectively. 8v also strongly suppressed the proliferation against Ba/F3 cells transfected with FLT3-ITD, FLT3-ITD-D835Y, FLT3-ITD-F691L, FLT3-ITD-D835Y-F691L, and MV4-11 acute myeloid leukemia (AML) cell lines with IC50 values of 12.2, 10.5, 24.6, 16.9, and 6.8 nM, respectively. Furthermore, 8v demonstrated ideal anticancer efficacy in a Ba/F3-FLT3-ITD-D835Y xenograft model. The results suggested that 8v can serve as a promising macrocycle-based FLT3 inhibitor for the treatment of AML.

Clinical manifestations, antimicrobial resistance and genomic feature analysis of multidrug-resistant Elizabethkingia strains

BackgroundElizabethkingia is emerging as an opportunistic pathogen in humans. The aim of this study was to investigate the clinical epidemiology, antimicrobial susceptibility, virulence factors, and genome features of Elizabethkingia spp.MethodsClinical data from 71 patients who were diagnosed with Elizabethkingia-induced pneumonia and bacteremia between August 2019 and September 2021 were analyzed. Whole-genome sequencing was performed on seven isolates, and the results were compared with a dataset of 83 available Elizabethkingia genomes. Genomic features, Kyoto Encyclopedia of Genes and Genomes (KEGG) results and clusters of orthologous groups (COGs) were analyzed.ResultsThe mean age of the patients was 56.9 ± 20.7 years, and the in-hospital mortality rate was 29.6% (21/71). Elizabethkingia strains were obtained mainly from intensive care units (36.6%, 26/71) and emergency departments (32.4%, 23/71). The majority of the strains were isolated from respiratory tract specimens (85.9%, 61/71). All patients had a history of broad-spectrum antimicrobial exposure. Hospitalization for invasive mechanical ventilation or catheter insertion was found to be a risk factor for infection. The isolates displayed a high rate of resistance to cephalosporins and carbapenems, but all were susceptible to minocycline and colistin. Genomic analysis identified five β-lactamase genes (blaGOB, blaBlaB, blaCME, blaOXA, and blaTEM) responsible for β-lactam resistance and virulence genes involved in stress adaptation (ureB/G, katA/B, and clpP), adherence (groEL, tufA, and htpB) and immune modulation (gmd, tviB, cps4J, wbtIL, cap8E/D/G, and rfbC). Functional analysis of the COGs revealed that “metabolism” constituted the largest category within the core genome, while “information storage and processing” was predominant in both the accessory and unique genomes. The unique genes in our 7 strains were mostly enriched in KEGG pathways related to microRNAs in cancer, drug resistance (β-lactam and vancomycin), ABC transporters, biological metabolism and biosynthesis, and nucleotide excision repair mechanisms.ConclusionThe Elizabethkingia genus exhibits multidrug resistance and carries carbapenemase genes. This study presents a comparative genomic analysis of Elizabethkingia, providing knowledge that facilitates a better understanding of this microorganism.

Pan-azole- and multi-fungicide-resistant Aspergillus fumigatus is widespread in the United States.

Aspergillus fumigatus is an important global fungal pathogen of humans. Azole drugs are among the most effective treatments for A. fumigatus infection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistant A. fumigatus has been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance in A. fumigatus are tandem repeats (TR) in the cyp51A promoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistant A. fumigatus has been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistant A. fumigatus in the U.S., we collected isolates from agricultural sites in eight states and tested 202 isolates for sensitivity to azoles. We found azole-resistant A. fumigatus in agricultural environments in seven states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades, and TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow indicating recombination between clades highlighting the potential for azole-resistance to continue spreading in the U.S.IMPORTANCEAspergillus fumigatus is a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris, and compost. Azoles are the first line of defense antifungal drugs against A. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistant A. fumigatus has been a problem in Europe and Asia for 20 years and has recently been reported in patients in the United States (U.S.). Until this study, we did not know much about azole-resistant A. fumigatus in agricultural settings in the U.S. In this study, we isolated azole-resistant A. fumigatus from multiple states and compared it to isolates from around the world. We show that A. fumigatus which is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.

A Retrospective Observational Study of the Microbial Etiology and Antimicrobial Susceptibility Patterns of Blood Cultures From ICU Patients at a Healthcare Facility in North India.

Introduction Bloodstream infections (BSI) are a leading source of fatalities and morbidity in hospitals. However, the clinical spectrum and antimicrobial resistance differ globally. Identifying the pathogenic spectrum and variations in antibiotic resistance is crucial for controlling BSI and preventing inappropriate antibiotic use. Material and methods This retrospective observational study was conducted at the Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, UP, India, for one year between June 2022 and June 2023. A total of 669 adult patients' blood cultures were obtained from ICUs. Blood culture was done using a BacT/Alert 3D (BioMérieux SA,Marcy-l'Étoile,France) automated system. Identification of the bacterial as well as fungal isolates was done using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), and the antimicrobial susceptibility profile was analyzed using the VITEK 2 Compact system (BioMérieux SA). Results Of the 669 blood culture samples, 213 (31.8%) showed bacterial or fungal growth. Of these 213 isolates, the most common isolate was coagulase-negativeStaphylococci(21.6%), followed byKlebsiella pneumoniae(19.3%)and Acinetobacterspp.(17.8%). The majority of gram-negative bacteria were resistant to most drugs, and vancomycin and linezolid were both effective against the majority of gram-positive bacteria. Conclusion The current study found that septicemia was more frequently caused by gram-negative bacteria than by gram-positive bacteria. Blood cultures are always necessary in cases of suspected septicemia, and once the antimicrobial susceptibility profile of the pathogen causing septicemia has been determined, suitable antimicrobials should be prescribed and used to lower the antimicrobial resistance burden.

Clinical Significance of Upregulation of EZH1 Expression in Hepatocellular Carcinoma Tissues.

The incidence and mortality of hepatocellular carcinoma (HCC) are increasing. It is urgent to develop more effective HCC biomarkers for diagnosis and treatment. This project intends to verify the expression of enhancer of zeste 1 polycomb repressive complex 2 subunit (EZH1) and its mechanism in HCC. This study integrates global microarray and high-throughput sequencing datasets, combined with internal immunohistochemistry, to analyze the expression and prognostic value of EZH1 in HCC. Functional enrichment analysis was conducted to investigate transcriptional targets, which were achieved by intersecting HCC over-expressed genes, EZH1 co-expressed genes and putative transcriptional targets. The relationship between EZH1 and anticancer drugs was detected by drug sensitivity analysis. In this study, 84 datasets from 40 platforms (3,926 HCC samples and 3,428 non-cancerous liver tissues) were included to show the high expression of EZH1 in HCC. Immunohistochemistry with 159 HCC samples and 62 non-HCC samples confirmed the high expression level. HCC patients with high EZH1 expression had worse survival prognoses. Gene ontology and Reactome analysis revealed that metabolism-related pathways, including autophagy, are critical for HCC. Interestingly, as one of the EZH1 potential transcriptional targets, autophagy-related 7 (ATG7) appeared in the above pathways. ATG7 was positively correlated with EZH1, upregulated in HCC, and mediated poor prognosis. Upregulation of EZH1 was found to be in contact with HCC anti-tumor drug resistance. The upregulation of EZH1 expression can promote the occurrence of HCC and lead to poor clinical progression and drug resistance; these effects may be mediated by regulating ATG7.

A retrospective study on the effect of statins on mortality and antimicrobial resistance among patients with Staphylococcus aureus bloodstream infection.

There is insufficient evidence in statin on the treatment of Staphylococcus aureus (SA) infection, we observe and analyze the clinical outcomes and antibiotic resistance of SA bloodstream infections in patients who received statins. A retrospective study was carried out in SA bloodstream infection of hospitalized patients from January 2018 to August 2023. The 30-day attributable mortality, 30-day all-cause mortality and clinical data of patients who received statins and non-statins were compared. A total of 74 patients with SA bloodstream infection were included, 32 (43.2%) patients received treatment with statins and 42 (56.8%) with non-statins. The incidence of methicillin-resistant SA (MRSA) was significantly lower in the statins group (15.6% vs. 38.1%, p = 0.034), however, no significant differences were observed in the mortality rate (p = 0.410). This study revealed the superiority of statins in reducing incidence of MRSA among SA bloodstream infection patients, but statins do not improve the 30-day mortality rate.

Incidence of carbapenem-resistant Escherichia coli ST2437 of clinical origin harbouring blaOXA-144 gene: a report from India.

Carbapenem-resistant Escherichia coli has been categorized as a pathogen of critical priority by the World Health Organization as it is highly infectious with high mortality and morbidity rates and widespread transmission potential. Carbapenem resistance is primarily mediated by carbapenemase-encoding genes and, additionally, through intrinsic factors. In India, over the years, carbapenemase-encoding genes have been reported from diverse clinically significant pathogens. The present study identifies E. coli of clinical origin that harbours blaOXA-144. The study isolate was obtained from a tertiary referral hospital in northeast India. Carbapenemase production was investigated through culture on chromogenic agar and Rapidec Carba NP test as per manufacturer's instructions. Susceptibility of the isolate was performed by the Kirby-Bauer disc diffusion method and agar dilution method following CLSI guidelines. PCR targeting carbapenemase-encoding genes was performed, followed by transformation and conjugation experiments. Whole-genome sequencing of the isolate was done through the Illumina sequencing platform and the data were analysed using the Centre for Genomic Epidemiology database. BJD_EC180 is 6 919 180 bp in length and consists of six rRNA operons, 111 tRNA, and 6849 predicted protein-coding sequences. BJD_EC180 belonged to ST2437 and harboured the carbapenemase-encoding gene blaOXA-144 with ISAba1 upstream, along with multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, amphenicols, sulphonamides, tetracyclines, trimethoprim, and rifampin. Carbapenem-resistant E. coli harbouring blaOXA-144 associated with insertion sequence pose a serious health threat as their mobilization into carbapenem non-susceptible strains that will contribute to the resistance burden and therefore, needs urgent monitoring.

Evaluation of culture conditions for sewage-based surveillance of antibiotic resistance in Klebsiella pneumoniae

BackgroundRecent studies have shown promise in predicting clinical antibiotic resistance rates from sewage data. Few have focused on Klebsiella pneumoniae, despite its virulence and importance as carrier of antibiotic resistance. Several media have been suggested for the isolation of K. pneumoniae from complex samples. However, comprehensive evaluations of culture protocols for isolation of K. pneumoniae from sewage are lacking. MethodsHere, influent samples from a major Swedish sewage treatment plant were used to evaluate ten culture conditions in parallel: cultivation on Brilliant green containing Inositol-Nitrate-Deoxycholate agar (BIND), Bruce agar, Klebsiella ChromoSelect Selective agar®, MacConkey-Inositol-Carbenicillin, or Simmons Citrate Agar with Inositol (SCAI) incubated at either 37°C or 42°C for 44 h. The culture conditions were compared based on colony counts of presumed K. pneumoniae and identification precision assessed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. ResultsThe sensitivity was lowest for BIND, whereas it was similar for the other media irrespective of incubation temperature. For four media, a better precision was observed after incubation at 42°C compared to 37°C, to a large extent explained by a lower frequency of captured Klebsiella oxytoca. SCAI incubated at 42°C showed the highest precision (84.4%). By combining this protocol with subsequent antibiotic resistance screening of collected isolates, low resistance rates in sewage K. pneumoniae were revealed, potentially reflecting the local resistance landscape. ConclusionWhen combined with downstream analyses, SCAI incubated at 42°C could be a valuable culture protocol for sewage-based studies on various aspects of K. pneumoniae epidemiology including antibiotic resistance prevalence.

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis.

Daptomycin is a last-line antibiotic commonly used to treat vancomycin-resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol-modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here, we performed invitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH, resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the ΔmprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced extracellular acidification and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies.

Abstract 2009: Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance

Abstract Taxanes are microtubule (MT)-stabilizing drugs widely used for the treatment of solid tumors, including the highly prevalent cancers of the breast, the prostate and gastric cancer. However, despite their initial activity, taxane resistance invariably occurs, leaving patients with limited therapeutic options. Currently, the molecular determinants of clinical taxane resistance remain poorly understood. We recently identified CLIP-170S, a novel truncated variant of the MT plus-end binding protein CLIP-170, that confers taxane resistance in vitro and is associated with clinical taxane resistance in patients with gastric cancer. We further showed that CLIP-170S is aberrantly localized along the MT lattice, thus impairing taxane access to its binding site in the MT lumen. However, the mechanism underlying the resistance-inducing extensive lattice localization of CLIP-170S is unknown. The activity and MT binding properties of canonical CLIP-170 are regulated by conformational changes mediated by the interaction of its N- and C-terminal domains. In its inactive, “closed” conformation, CLIP-170 is soluble as the N- and C-terminal domains interact with each other. In the active “open” conformation, CLIP-170’s N- and C-terminal domains are apart, thus exposing its MT-binding domain and allowing MT plus-end binding. As CLIP-170S is missing part of its N-terminal domain, we hypothesized that CLIP-170S is constitutively displayed in an open conformation that promotes its extensive and aberrant localization to the MT lattice. To prove this hypothesis, we used the FKBP-rapalog-FRB system to “force” CLIP-170S to assume a “closed” conformation. We generated a fusion CLIP-170S protein tagged with FKBP and FRB domains in its N- and C-terminal, respectively. The addition of rapalog induced FKBP (N-ter) and FRB (C-ter) domains to interact, promoting CLIP-170S to adopt a “closed” conformation. CLIP-170S in this “closed” conformation resulted in its release from the MT-lattice and restored taxane binding to MTs. We next extended our search to other CLIP-170 clinically prevalent variants with sequence alteration predicted to bind to the MT-lattice and potentially interfere with taxane sensitivity. This search identified CLIP-170IR (an intron retention variant) and several other truncation mutants prevalent in gastric, breast and lung cancer cell lines. Experimentally, we showed that these variants not only phenocopy CLIP-170S binding to the MT- lattice but also confer taxane-resistance. Together, these data demonstrate a novel mechanism of taxane resistance mediated by CLIP-170 mutations that induce CLIP-170 to adopt a constitutively “open” MT-lattice binding conformation, which renders them susceptible to causing taxane resistance. Citation Format: Urko del Castillo, Kiran Kumari Sahu, William G. Stone, Manish A. Shah, Paraskevi Giannakakou. Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2009.

Abstract 6424: A patient centric full spectrum flow cytometry assay for comprehensive phenotyping of CAR T cells in clinical trials

Abstract Purpose While there is a strong desire to fully understand next generation CAR-T product characteristics in clinical trials, limited amounts of blood specimens are available as the enrolled patients are very sick and lymphodepleted. To address this high unmet need, we developed a single tube 20-color full spectrum flow cytometry assay for an in-depth characterization of CAR-T cells that support fulfilment of five key clinical trial endpoints, namely, manufacturing success (fitness and memory state), pharmacokinetics, response or resistance (ICIs), efficacy (gross tumor burden) and safety (B cell regeneration) for use across a broad range of CAR-T therapies and clinical indications. Study Design: The single tube 20-parameter full spectrum flow cytometry (FC) assay was designed to include CD45, HLA-DR, CD3, CD4, CD8, CD56 as backbone markers, CD45RA, CCR7, CD95, CD25 and CD127 antibodies allowed for the identification of all major subsets of CD4 and CD8 T cells. CXCR3, CD38, PD1, LAG3, and TIM3 were used for identification of activation/exhaustion phenotypes, while CD20 and HLA-DR were useful for identification of B cells and monocytes respectively. Additionally, we left two parameters (PE and AF647) open for customized CAR-T detection reagents or other customized markers. This assay was developed using Cytek® Northern Lights™ and frozen PBMCs. Results This robust 20-color full spectrum FC assay allowed a thorough analysis of the major T cell populations cells. FMO (Fluorescence-Minus-One) control studies confirmed the specificity of the antibodies and of the panel in identifying Naïve, EMRA, CM, EM, Th17, T stem cell memory and regulatory T cells with minimum background on all parameters. This method showed to be highly reproducible and can be adapted for use with many CAR-T detection reagents. Conclusion In summary, we developed a robust 20-parameter multiparameter full spectrum flow cytometry assay focused on different populations of T cells in a single tube. The inclusion of drop ins for prospective CAR-T cell detection reagents allows for easy integration of future advances in CAR-T cell research. This universal panel shows great potential for usage across CAR-T clinical trials, as it serves as a valuable tool understand product fitness, PK, biomarker monitoring and disease burden simultaneously. Citation Format: Keegan Vaughan, Selvam Muthusamy, Coral Zhao, Austin Carbajal, Charlie Fu, Naveen Dakappagari, Zeni Alfonso. A patient centric full spectrum flow cytometry assay for comprehensive phenotyping of CAR T cells in clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6424.