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.

CVB3-Mediated Mitophagy Plays an Important Role in Viral Replication via Abrogation of Interferon Pathways

Coxsackievirus B3 (CVB3) is a common enterovirus that causes systemic inflammatory diseases, such as myocarditis, meningitis, and encephalitis. CVB3 has been demonstrated to subvert host cellular responses via autophagy to support viral replication in neural stem cells. Mitophagy, a specialized form of autophagy, contributes to mitochondrial quality control via degrading damaged mitochondria. Here, we show that CVB3 infection induces mitophagy in human neural progenitor cells, HeLa and H9C2 cardiomyocytes. In particular, CVB3 infection triggers mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin/LC3 translocation to the mitochondria. Rapamycin or carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment led to increased CVB3 RNA copy number in a dose-dependent manner, suggesting enhanced viral replication via autophagy/mitophagy activation, whereas knockdown of PTEN-induced putative kinase protein 1(PINK1) led to impaired mitophagy and subsequent reduction in viral replication. Furthermore, CCCP treatment inhibits the interaction between mitochondrial antiviral signaling protein (MAVS) and TANK-binding kinase 1(TBK1), thus contributing to the abrogation of type I and III interferon (IFN) production, suggesting that mitophagy is essential for the inhibition of interferon signaling. Our findings suggest that CVB3-mediated mitophagy suppresses IFN pathways by promoting fragmentation and subsequent sequestration of mitochondria by autophagosomes.

Parkinson’s disease–associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy

Background Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson’s disease (PD), and several genes linked to familial PD, including PINK1 (encoding PTEN-induced putative kinase 1 [PINK1]) and PARK2 (encoding the E3 ubiquitin ligase Parkin), are directly involved in processes such as mitophagy that maintain mitochondrial health. The dominant p.D620N variant of vacuolar protein sorting 35 ortholog (VPS35) gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2. Methods To better mimic and study the patient situation, we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35. These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce the PINK1/Parkin-mediated mitophagy, which was assessed using biochemical and microscopy approaches. Results Mitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state. As a result, the mitochondria of these cells were desensitized to the CCCP-induced collapse in mitochondrial potential, as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult. Consequently, Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired. Conclusion Our findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35, PINK1 and Parkin in PD.

Unraveling Secretory Mechanisms that Control Pentraxin 3 Secretion in Adipocytes During Inflammation

As a soluble pattern recognition receptor, Pentraxin 3 (PTX3) plays an important role in innate immunity and obesity-associated metabolic inflammation. PTX3 is abundantly expressed and secreted in adipocytes in response to lipopolysaccharide (LPS) stimulation. Appropriate regulation of PTX3 secretion is critical for maintaining inflammatory homeostasis. This study aims to unravel the mechanisms that control PTX3 secretion in adipocytes during LPS-induced inflammation. Upon 6h treatment of LPS, PTX3 expression and secretion were significantly induced in 3T3-L1 and stromal-vascular (SV) differentiated adipocytes, but to a lesser extent in SV cells or 3T3-L1 fibroblasts. However, LPSdoes not significantly stimulate PTX3 expression and secretion in macrophages. Using chemical inhibitors of conventional and unconventional protein secretion, we explored the mechanisms for controlling LPS-stimulated PTX3 secretion. 3T3-L1 adipocytes were treated with LPS for 6h in the presence or absence of various inhibitors blocking protein secretion from the Golgi complex (Monensin and Brefeldin A), mitochondrial oxidation (carbonyl cyanide 3-chlorophenylhydrazone [CCCP]), autophagy-lysosome (chloroquine and 3-methyladenine) and inflammasome (Bay 11–7082 and wedelolactone) activation, or exosome synthesis and trafficking (GW4869, manumycin A, calpeptin, and Y-27632). There were no significant effects of all inhibitors except for Monensin, Brefeldin A, and CCCP on intracellular and secreted levels of PTX3 in adipocytes. We found that Monensin and Brefeldin A significantly blocked LPS-stimulated PTX3 secretion, resulting in cellular PTX3 accumulation in adipocytes. Disrupting mitochondrial membrane potential by CCCP caused the reduction in PTX3 secretion from adipocytes. Additionally, we detected PTX3 in exosomes isolated from LPS-treated adipocytes. Inhibiting exosome synthesis by Manumycin A attenuated LPS-stimulated PTX3 secretion in both adipocyte culture media and isolated exosomes but not in the non-exosomal fraction of media, suggesting the involvement of the exosomal pathway in PTX3 secretion. However, the levels of exosomal PTX3 were significantly lower than that of the non-exosomal PTX3, and only 4.3% of secreted PTX3 was detected in the exosomal fraction of cultural media. Inhibiting the Golgi complex pathway blocked both the exosomal and non-exosomal secretion of PTX3 in adipocytes. After further fractionation of isolated crude exosomes by the iodixanol density gradient centrifugation, we showed that the majority of PTX3 was found in the non-extracellular vesicular (EV) fractions; only a small portion of secreted PTX3 overlapped with the exosomal marker CD63 in the small EV fractions. We conclude that PTX3 is secreted mainly through the conventional protein secretion pathway and minimally through the exosomal or EV pathway in response to LPS stimulation.

The cyanobacterial taxis protein HmpF regulates type IV pilus activity in response to light

Motility is ubiquitous in prokaryotic organisms including the photosynthetic cyanobacteria where surface motility powered by type 4 pili (T4P) is common and facilitates phototaxis to seek out favorable light environments. In cyanobacteria, chemotaxis-like systems are known to regulate motility and phototaxis. The characterized phototaxis systems rely on methyl-accepting chemotaxis proteins containing bilin-binding GAF domains capable of directly sensing light, and the mechanism by which they regulate the T4P is largely undefined. In this study we demonstrate that cyanobacteria possess a second, GAF-independent, means of sensing light to regulate motility and provide insight into how a chemotaxis-like system regulates the T4P motors. A combination of genetic, cytological, and protein–protein interaction analyses, along with experiments using the proton ionophore carbonyl cyanide m-chlorophenyl hydrazine, indicate that the Hmp chemotaxis-like system of the model filamentous cyanobacterium Nostoc punctiforme is capable of sensing light indirectly, possibly via alterations in proton motive force, and modulates direct interaction between the cyanobacterial taxis protein HmpF, and Hfq, PilT1, and PilT2 to regulate the T4P motors. Given that the Hmp system is widely conserved in cyanobacteria, and the finding from this study that orthologs of HmpF and T4P proteins from the distantly related model unicellular cyanobacterium Synechocystis sp. strain PCC6803 interact in a similar manner to their N. punctiforme counterparts, it is likely that this represents a ubiquitous means of regulating motility in response to light in cyanobacteria.

Phenoxyalkylimidazoles with an oxadiazole moiety are subject to efflux in Mycobacterium tuberculosis

The phenoxyalkylimidazoles (PAI) are an attractive chemical series with potent anti-tubercular activity targeting Mycobacterium tuberculosis respiration. Our aim was to determine if the PAI compounds are subject to efflux. Two analogs containing an oxadiazole had improved potency in the presence of the efflux inhibitors reserpine and carbonyl cyanide m-chlorophenylhydrazine, whereas the potency of analogs with a diazole was not affected.

Detecting nitrile-containing small molecules by infrared photothermal microscopy

We demonstrated IR photothermal imaging of trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) in cells. The result indicates that a nitrile group can be used as a vibrational tag to image target molecules.