Adaptation to a Commercial Quaternary Ammonium Compound Sanitizer Leads to Cross-Resistance to Select Antibiotics in Listeria monocytogenes Isolated From Fresh Produce Environments

The effective elimination of Listeria monocytogenes through cleaning and sanitation is of great importance to the food processing industry. Specifically in fresh produce operations, the lack of a kill step requires effective cleaning and sanitation to mitigate the risk of cross-contamination from the environment. As facilities rely on sanitizers to control L. monocytogenes, reports of the development of tolerance to sanitizers and other antimicrobials through cross-resistance is of particular concern. We investigated the potential for six L. monocytogenes isolates from fresh produce handling and processing facilities and packinghouses to develop cross-resistance between a commercial sanitizer and antibiotics. Experimental adaptation of isolates belonging to hypervirulent clonal complexes (CC2, CC4, and CC6) to a commercial quaternary ammonium compound sanitizer (cQAC) resulted in elevated minimum inhibitory concentrations (2–3 ppm) and minimum bactericidal concentrations (3–4 ppm). Susceptibility to cQAC was restored for all adapted (qAD) isolates in the presence of reserpine, a known efflux pump inhibitor. Reduced sensitivity to 7/17 tested antibiotics (chloramphenicol, ciprofloxacin, clindamycin, kanamycin, novobiocin, penicillin, and streptomycin) was observed in all tested isolates. qAD isolates remained susceptible to antibiotics commonly used in the treatment of listeriosis (i.e., ampicillin and gentamicin). The whole genome sequencing of qAD strains, followed by comparative genomic analysis, revealed several mutations in fepR, the regulator for FepA fluoroquinolone efflux pump. The results suggest that mutations in fepR play a role in the reduction in antibiotic susceptibility following low level adaptation to cQAC. Further investigation into the cross-resistance mechanisms and pressures leading to the development of this phenomenon among L. monocytogenes isolates recovered from different sources is needed to better understand the likelihood of cross-resistance development in food chain isolates and the implications for the food industry.

Activity of imipenem/relebactam on Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility

Background and Objectives: Imipenem/relebactam (IMP/R) is a newly FDA approved β-lactam/β-lactamase inhibitor combination. Relebactam ability to restore IMP activity could differ according to the cause of imipenem non-susceptibility. Therefore, we investigated the in-vitro activity of IMP/R against Klebsiella pneumoniae with different mechanisms of imi- penem non-susceptibility. Materials and Methods: Imipenem-nonsusceptible (IMP-NS) K. pneumoniae isolates were collected and characterized for β-lactamase encoding genes by multiplex PCR. For IMP-NS carbapenemase-negative isolates, study of Ompk35 & Ompk36 gene expression was performed by reverse transcription-PCR while efflux pump activity was studied by minimum inhibitory concentration (MIC) reduction assay using efflux pump inhibitor. Susceptibility testing of K. pneumoniae to IMP and IMP/R were achieved by broth microdilution (BMD) method. Results: During the study period, 140 isolates of IMP-NS K. pneumoniae were collected. BMD method showed that relebac- tam restored IMP susceptibility in 100%, 60% and 49% of isolates that only harbor AmpC, extended spectrum beta lactamase (ESBL) and carbapenemases, respectively. IMP/R was most potent against all bla KPC and 50% of bla _producing isolates. No demonstrable activity of IMP/R against K. pneumoniae harboring metallo-β-lactamases (MBLs). Out of 18 isolates with IMP non-suceptibility due to porins loss with overproduction of ESBL and/or AmpC, 14 (77.7%) isolates were IMP/R sus- ceptible. IMP/R showed no activity against isolates with only efflux pump hyperactivity. Conclusion: Relebactam could restore IPM activity in KPC or AmpC-producing IMP/NS K. pneumoniae but with no ac- tivity against MBL- producing isolates. Relebactam activity against isolates harbouring-bla OXA-48 or with altered Ompk35 & Ompk36 gene expression and efflux pump hyperactivity need further studies. Therefore, using IMP/R antibiotic in the treat- ment of infections caused by IMP/NS K. pneumoniae should be based on its molecular profile of IMP resistance to optimize the utility of IMP/R.

The effect of the efflux pump inhibitor Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP) on the susceptibility to imipenem and cefepime in clinical strains of Acinetobacter baumannii

Introduction In the last years the rapid expansion of multidrug-resistant A. baumannii strains have become a major health problem. Efflux pumps are a group of transport proteins that contribute to the development of antibiotic resistance. The aim of this study was to evaluate the effect of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) on the antimicrobial action of imipenem and cefepime on clinical strains of A. baumannii. Materials and methods A total of 49 non-duplicate clinical samples were collected during January through December of 2018 from patients hospitalized in the Hospital Regional Docente de Cajamarca. Of the 49 samples obtained, the confirmatory identification of A. baumannii was performed on 47 samples by molecular methods. The amplification of the blaOXA-51-like gene was carried out by polymerase chain reaction (PCR). The determination of the minimum inhibitory concentration (MIC) was calculated using the microdilution method in culture broth. The susceptibility to both antibiotics (cefepime and imipenem) was evaluated in the presence and absence of the inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Results A total of 47 strains of A. baumannii were isolated: 97.87% (46/47) were resistant to Imipenem, 2.13% (1/47) of them were classified as intermediate and none of these strains were susceptible. On the other hand, 51.06% (24/47) of isolates were resistant to cefepime; 19.15% (9/47) intermediate and 29.79% (14/47) susceptible. We considered a significant difference in antibiotic susceptibility if the MIC changed at least 4 dilutions, after the addition of the inhibitor. In the case of CCCP in addition to imipenem, 2.1% (1/47) had a significant change of 4 or more reductions in MIC, 59.6% (28/47) achieved a change equal or less than 3 dilutions and 17.0% (8/47) did not have any change. In the case of CCCP with cefepime the percentage of strains with the significant change of MIC was 8.5% (4/47). On the other hand, 53.2% (24/47) presented a reduction equal or less than 3 dilutions and 12.8% (6/47) did not show changes. Conclusion In conclusion, our results demonstrate that the use of CCCP may improve the antibiotic effect of imipenem and cefepime on clinical strains of A. baumannii. The relevance of this study is that it provides evidence that this efflux pump inhibitor may be an alternative treatment against multidrug-resistant A. baumannii.

Extensive Drug-Resistant Salmonella enterica Isolated From Poultry and Humans: Prevalence and Molecular Determinants Behind the Co-resistance to Ciprofloxacin and Tigecycline

The emergence of extensive drug-resistant (XDR) Salmonella in livestock animals especially in poultry represents a serious public health and therapeutic challenge. Despite the wealth of information available on Salmonella resistance to various antimicrobials, there have been limited data on the genetic determinants of XDR Salmonella exhibiting co-resistance to ciprofloxacin (CIP) and tigecycline (TIG). This study aimed to determine the prevalence and serotype diversity of XDR Salmonella in poultry flocks and contact workers and to elucidate the genetic determinants involved in the co-resistance to CIP and TIG. Herein, 115 Salmonella enterica isolates of 35 serotypes were identified from sampled poultry (100/1210, 8.26%) and humans (15/375, 4.00%), with the most frequent serotype being Salmonella Typhimurium (26.96%). Twenty-nine (25.22%) Salmonella enterica isolates exhibited XDR patterns; 25 out of them (86.21%) showed CIP/TIG co-resistance. Exposure of CIP- and TIG-resistant isolates to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) efflux pump inhibitor resulted in an obvious reduction in their minimum inhibitory concentrations (MICs) values and restored the susceptibility to CIP and TIG in 17.24% (5/29) and 92% (23/25) of the isolates, respectively. Molecular analysis revealed that 89.66% of the isolates contained two to six plasmid-mediated quinolone resistance genes with the predominance of qepA gene (89.66%). Mutations in the gyrA gene were detected at codon S83 (34.62%) or D87 (30.77%) or both (34.62%) in 89.66% of XDR Salmonella. The tet(A) and tet(X4) genes were detected in 100% and 3.45% of the XDR isolates, respectively. Twelve TIG-resistant XDR Salmonella had point mutations at codons 120, 121, and 181 in the tet(A) interdomain loop region. All CIP and TIG co-resistant XDR Salmonella overexpressed ramA gene; 17 (68%) out of them harbored 4-bp deletion in the ramR binding region (T-288/A-285). However, four CIP/TIG co-resistant isolates overexpressed the oqxB gene. In conclusion, the emergence of XDR S. enterica exhibiting CIP/TIG co-resistance in poultry and humans with no previous exposure to TIG warrants an urgent need to reduce the unnecessary antimicrobial use in poultry farms in Egypt.

Identified Seaweed Compound Diphenylmethane Serves as an Efflux Pump Inhibitor in Drug-Resistant Escherichia coli

Drug efflux pumps are one of the major elements used by antibiotic-resistant bacteria. Efflux pump inhibitors (EPIs) are potential therapeutic agents for adjunctive therapy, which can restore the activity of antibiotics that are no longer effective against pathogens. This study evaluated the seaweed compound diphenylmethane (DPM) for its EPI activity. The IC50 and modulation results showed that DPM has no antibacterial activity but can potentiate the activity of antibiotics against drug-resistant E. coli. Time-kill studies reported that a combination of DPM and erythromycin exhibited greater inhibitory activity against drug-resistant Escherichia coli. Dye accumulation and dye efflux studies using Hoechst 33342 and ethidium bromide showed that the addition of DPM significantly increased dye accumulation and reduced dye efflux in drug-resistant E. coli, suggesting its interference with dye translocation by an efflux pump. Using MALDI-TOF, it was observed that the addition of DPM could continuously reduce antibiotic efflux in drug-resistant E. coli. Additionally, DPM did not seem to damage the E. coli membranes, and the cell toxicity test showed that it features mild human-cell toxicity. In conclusion, these findings showed that DPM could serve as a potential EPI for drug-resistant E. coli.

EXTH-31. BRAIN DISTRIBUTION, CLEARANCE AND TOXICITY EVALUATION OF SMALL-MOLECULE INHIBITORS FOLLOWING CONVECTION-ENHANCED DELIVERY TO THE BRAINSTEM

BACKGROUND Diffuse midline gliomas (DMGs) harboring the H3K27M mutation are highly aggressive, fatal brainstem tumors that primarily occur in children. The blood-brain barrier (BBB) prevents numerous drugs from reaching CNS tumors, like DMG, at cytotoxic concentrations. Convection-enhanced delivery (CED) has emerged as a drug delivery technique that bypasses the BBB through a direct interstitial infusion under a pressure gradient. However, drug distribution and clearance from the brain following CED is poorly understood and has been cited as a potential reason for the lack of efficacy observed in prior clinical trials. OBJECTIVE The objective of this study was to understand how two small molecule inhibitors (alisertib, ponatinib) that inhibit cell growth and proliferation in DMG cells in vitro distribute and clear from the brain following CED to the brainstem. METHODS Sprague-dawley rats underwent a single 60mL CED infusion of drug to the brainstem (200mM alisertib, 10mM ponatinib) and were sacrificed 0.083, 1, 2, 4, 8 and 24 hours following the completion of the infusion. Brains were dissected and drug concentration was determined via HPLC analysis. RESULTS No rats showed any clinical or neurological signs of toxicity post-infusion. Both drugs showed significant differences in drug concentration based on anatomical brain region where higher concentrations were observed in the pons and cerebellum compared to the cortex. Drug half-life in the brain was ~0.5 hours for alisertib and ~1 hour for ponatinib, but this was not significantly increased following co-administration of elacridar, a BBB efflux pump inhibitor. CONCLUSIONS These results suggest that elimination of drugs from the brain in a complex, multifactorial mechanism that warrants further preclinical investigation prior to the initiation of a clinical trial.

PHENOTYPIC DETECTION OF EFFLUX MECHANISM IN AMIKACIN-RESISTANT ACINETOBACTER ISOLATES FROM TWO DIFFERENT STATES IN SOUTH INDIA

Acinetobacter has already gained resistance to the majority of antibiotics available. Aminoglycosides are commonly used to treat invasive infections. Aminoglycoside resistance is associated with decreased drug absorption, aminoglycoside modification, and aminoglycoside efflux. The aim of this study was to detect the presence of an efflux mechanism in amikacin-resistant Acinetobacter isolated from hospital wards using Carbonyl Cyanide 3- Chlorophenylhydrazone (CCCP). One hundred isolates of Acinetobacter were isolated from tertiary care hospitals in two distinct South Indian states. Antibacterial susceptibility patterns were discovered between 2017 and 2019. Amikacin minimum inhibitory concentration (MIC) for resistant Acinetobacter isolates was determined using Clinical and Laboratory Standards Institute (CLSI) standards. The efflux system activity was determined using CCCP. Among 100 Acinetobacter baumannii isolates, 49 isolates with amikacin resistance were found. The MIC’s of Acinetobacter ranged between 2 – 1024 μg/mL for the amikacin studied. After treatment with the efflux pump inhibitor, 38.77% of isolates became less resistant to amikacin, as determined by phenotypic detection of efflux pumps, showing a decrease in antibiotic MICs of at least four fold. The data demonstrated the importance of efflux pump activity conferring amikacin resistance on Acinetobacter clinical isolates.

Efflux Pump Activity and Mutations Driving Multidrug Resistance in Acinetobacter baumannii at a Tertiary Hospital in Pretoria, South Africa

Acinetobacter baumannii (A. baumannii) has developed several resistance mechanisms. The bacteria have been reported as origin of multiple outbreaks. This study aims to investigate the use of efflux pumps and quinolone resistance-associated genotypic mutations as mechanisms of resistance in A. baumannii isolates at a tertiary hospital. A total number of 103 A. baumannii isolates were investigated after identification and antimicrobial susceptibility testing by VITEK2 followed by PCR amplification of blaOXA-51. Conventional PCR amplification of the AdeABC efflux pump (adeB, adeS, and adeR) and quinolone (parC and gyrA) resistance genes were performed, followed by quantitative real-time PCR of AdeABC efflux pump genes. Phenotypic evaluation of efflux pump expression was performed by determining the difference between the MIC of tigecycline before and after exposure to an efflux pump inhibitor. The Sanger sequencing method was used to sequence the parC and gyrA amplicons. A phylogenetic tree was drawn using MEGA 4.0 to evaluate evolutionary relatedness of the strains. All the collected isolates were blaOXA-51-positive. High resistance to almost all the tested antibiotics was observed. Efflux pump was found in 75% of isolates as a mechanism of resistance. The study detected parC gene mutation in 60% and gyrA gene mutation in 85%, while 37% of isolates had mutations on both genes. A minimal evolutionary distance between the isolates was reported. The use of the AdeABC efflux pump system as an active mechanism of resistance combined with point mutation mainly in gyrA was shown to contribute to broaden the resistance spectrum of A. baumannii isolates.

Pseudomonas aeruginosa mexR and mexEF Antibiotic Efflux Pump Variants Exhibit Increased Virulence

Antibiotic-resistant Pseudomonas aeruginosa infections are the primary cause of mortality in people with cystic fibrosis (CF). Yet, it has only recently become appreciated that resistance mutations can also increase P. aeruginosa virulence, even in the absence of antibiotics. Moreover, the mechanisms by which resistance mutations increase virulence are poorly understood. In this study we tested the hypothesis that mutations affecting efflux pumps can directly increase P. aeruginosa virulence. Using genetics, physiological assays, and model infections, we show that efflux pump mutations can increase virulence. Mutations of the mexEF efflux pump system increased swarming, rhamnolipid production, and lethality in a mouse infection model, while mutations in mexR that increased expression of the mexAB-oprM efflux system increased virulence during an acute murine lung infection without affecting swarming or rhamnolipid gene expression. Finally, we show that an efflux pump inhibitor, which represents a proposed novel treatment approach for P. aeruginosa, increased rhamnolipid gene expression in a dose-dependent manner. This finding is important because rhamnolipids are key virulence factors involved in dissemination through epithelial barriers and cause neutrophil necrosis. Together, these data show how current and proposed future anti-Pseudomonal treatments may unintentionally make infections worse by increasing virulence. Therefore, treatments that target efflux should be pursued with caution.