Multidrug-resistant Gram-negative Pathogens Research Articles

Transcriptomic Mapping of Neurotoxicity Pathways in the Rat Brain in Response to Intraventricular Polymyxin B.

Intraventricular or intrathecal administration of polymyxins are increasingly used to treat multidrug-resistant (MDR) Gram-negative bacteria caused infections in the central nervous system (CNS). However, our limited knowledge of the mechanisms underpinning polymyxin-induced neurotoxicity significantly hinders the development of safe and efficacious polymyxin dosing regimens. To this end, we conducted transcriptomic analyses of the rat brain and spinal cord 1h following intracerebroventricular administration of polymyxin B into rat lateral ventricle at a clinically relevant dose (0.5mg/kg). Following the treatment, 66 differentially expressed genes (DEGs) were identified in the brain transcriptome while none for the spinal cord (FDR ≤ 0.05, fold-change ≥ 1.5). DEGs were enriched in signaling pathways associated with hormones and neurotransmitters, including dopamine and (nor)epinephrine. Notably, the expression levels of Slc6a3 and Gabra6 were decreased by 20-fold and 4.3-fold, respectively, likely resulting in major perturbations of dopamine and γ-aminobutyric acid signaling in the brain. Mass spectrometry imaging of brain sections revealed a distinct pattern of polymyxin B distribution with the majority accumulating in the injection-side lateral ventricle and subsequently into third and fourth ventricles. Polymyxin B was not detectable in the left lateral ventricle or brain tissue. Electrophysiological measurements on primary cultured rat neurons revealed a large inward current and significant membrane leakage following polymyxin B treatment. Our work demonstrates, for the first time, the key CNS signaling pathways associated with polymyxin neurotoxicity. This mechanistic insight combined with pharmacokinetic/pharmacodynamic dosing strategies will help guide the design of safe and effective intraventricular/intrathecal polymyxin treatment regimens for CNS infections caused by MDR Gram-negative pathogens.

Drug Repurposing Approaches towards Defeating Multidrug-Resistant Gram-Negative Pathogens: Novel Polymyxin/Non-Antibiotic Combinations.

Multidrug-resistant (MDR) Gram-negative pathogens remain an unmet public health threat. In recent times, increased rates of resistance have been reported not only to commonly used antibiotics, but also to the last-resort antibiotics, such as polymyxins. More worryingly, despite the current trends in resistance, there is a lack of new antibiotics in the drug-discovery pipeline. Hence, it is imperative that new strategies are developed to preserve the clinical efficacy of the current antibiotics, particularly the last-line agents. Combining conventional antibiotics such as polymyxins with non-antibiotics (or adjuvants), has emerged as a novel and effective strategy against otherwise untreatable MDR pathogens. This review explores the available literature detailing the latest polymyxin/non-antibiotic combinations, their mechanisms of action, and potential avenues to advance their clinical application.

Chromosomally and Plasmid-Located mcr in Salmonella from Animals and Food Products in China.

This study aimed to investigate the prevalence and genomic characteristics of the colistin resistance gene mcr in Salmonella enterica in China. In total, 445 S. enterica isolates from animals and food products were screened through PCR and sequencing for the presence of mcr. The mcr genes were detected in nine Salmonella strains (2.02%), with complete mcr-1 in S. enterica serovar Indiana (n = 1) and an S. Typhimurium monophasic variant (S. 4,[5],12:i:-; n = 1), mcr-4.3 in S. enterica serovar London (n = 1), and an incomplete mcr-1 in S. Indiana (n = 6). They exhibited MIC values of 0.25 to 8 mg/L to colistin and showed resistance to multiple antimicrobial agents. Whole-genome sequencing was performed on mcr-positive Salmonella strains using Illumina HiSeq or PacBio single-molecule real-time sequencing. The complete mcr-1 gene was located on conjugative IncN1-IncHI2 plasmid and IncX4 plasmid, respectively, with high similarity to other mcr-1-bearing plasmids belonging to the same incompatibility type. Together with an additional 13 antimicrobial resistance genes, the incomplete mcr-1 was embedded in an 81,442-bp multiresistance region on the chromosome in S. Indiana YZ20MCS6. The Δmcr-1-pap2 segment and a set of tellurite resistance determinants (terYXWZABCDEF) in six S. Indiana strains were similar to other IncHI2 plasmid backbones. The mcr-4.3 gene was located on an untyped plasmid pYULZMPS10. Although low prevalence of mcr was observed in Salmonella, continuous surveillance of this gene in Salmonella is required. Plasmids play an important role in mcr transmission, and mcr-1, although incomplete, can be captured by chromosomes with the help of mobile elements. IMPORTANCE Colistin is a last-resort antibiotic for severe infections caused by multidrug-resistant (MDR) Gram-negative pathogens. Colistin resistance genes mcr, particularly mcr-1, have been found in Enterobacteriaceae around the world, mainly in Escherichia coli and Salmonella. Salmonella enterica is a major foodborne pathogen, with MDR Salmonella being considered a "Serious Threat Level pathogen" by the Centers for Disease Control and Prevention. Therefore, the prevalence of mcr in Salmonella strains must be monitored. In this study, a low mcr prevalence (2.02%) was observed in Salmonella strains from animals and food products, with plasmid-borne mcr-1 in S. enterica serovar Indiana and an S. Typhimurium monophasic variant (S. 4,[5],12:i:-) and chromosomally located mcr-1 in S. Indiana. The mcr-4.3 gene was first identified in S. enterica serovar London associated with an untyped plasmid. Although this study reports a low mcr prevalence in Salmonella, the transmission ability of mcr-positive Salmonella strains to humans via the food chain is a public health concern.

Synthesis and Evaluation of Polymyxins Bearing Reductively Labile Disulfide-Linked Lipids.

Polymyxins are a class of lipopeptide anti-infective agents with potent and specific activity against Gram-negative bacteria. While toxicity concerns associated with polymyxin B and E (colistin) have historically limited their clinical application, today they are increasingly used as last-resort antibiotics given the rise of multidrug-resistant Gram-negative pathogens. The adverse side effects of polymyxins are well known, particularly as related to their nephrotoxicity. Here, we describe the synthesis and evaluation of a novel series of polymyxin analogues, aimed at reducing their nephrotoxic effects. Using a semisynthetic approach, we explored modifications of the exocyclic part of the polymyxin scaffold, namely, the terminal amino acid and lipophilic tail. By incorporating a reductively labile disulfide linkage in the lipid tail, we obtained novel polymyxins that exhibit potent antibacterial activity on par with polymyxin B but with reduced toxicity toward human renal proximal tubular epithelial cells.

Fluorescence polarization immunoassay based on fragmentary hapten for rapid and sensitive screening of polymyxins in human serum

Polymyxin B (PMB) and polymyxin E (PME, or named colistin) are old polypeptide antibiotics and recently reintroduced as the last-line therapy for serious infections caused by multidrug-resistant Gram-negative pathogens. A rapid and reliable screening method for PMB and PME in human serum is urgently required due to their narrow therapeutic windows and individual differences. To develop fluorescence polarization immunoassay (FPIA) with high sensitivity and uniform specificity for PMB and PME, one novel fragmentary polymyxins hapten, named H1, was designed only containing the fatty acid acylated tripeptides and intendedly introducing sulfhydryl group for selective conjugation purpose. The H1 induced significantly higher titer and affinity antibody response than the PMB as hapten and produced five monoclonal antibodies (mAbs) with IC50 of 1.8–6.6 ng/mL for PMB and cross-reactions of 75.0–105.0 % for PME. To investigate the influence of tracer structure on the FPIA sensitivity, 12 tracers differing in spacer arm lengths, fluorophore types and hapten structures were fully paired with five mAbs, showing a safety length of spacer arm ranged from 4 to 17 Å. Under optimal conditions, the IC50 of FPIA was 3.7 ng/mL in buffer with a limit of detection of 1.4 μg/L in human serum for PMB and PME. The recoveries in spiked human serum were 73.8–91.8 % with CVs less than 12.8 % and whole assay time less than 25 min including sample preparation. These results showed that the FPIA was an efficient, accurate, and sensitive method for rapid screening of polymyxins and suitable for therapeutic drug monitoring.

Trends in Stenotrophomonas maltophilia antibiotic resistance rates in the United States Veterans Affairs Health System.

Introduction. Stenotrophomonas maltophilia is an important multidrug-resistant Gram-negative pathogen. While largely a hospital-acquired pathogen, there have been increasing reports of the pathogen in the community.Gap Statement. Trends in S. maltophilia prevalence and resistance rates that include outpatient isolates are unknown.Aim. We described recent trends in prevalence and resistance of S. maltophilia in the national Veterans Affairs (VA) Healthcare system.Methodology. The study identified positive S. maltophilia clinical cultures among VA adult patients from 2010 to 2018 across all VA hospitals, long-term care facilities/units, and outpatient settings. Annual S. maltophilia resistance rates were evaluated. Multidrug resistant (MDR) was defined as resistance to sulfamethoxazole/trimethoprim (SMX/TMP) and minocycline or levofloxacin. Time trends were assessed with regression analyses to estimate annual average percent changes (AAPC) with 95 % confidence intervals using Joinpoint software.Results. Over the 9 year study period, 18 285 S. maltophilia cultures were identified (57 % hospital, 3 % long-term care, 40 % outpatient). The most common source of S. maltophilia cultures were respiratory cultures (34.6 %) followed by urine cultures (30.4 %). In VA hospitals and long-term care facilities, the number of S. maltophilia cultures decreased significantly (by 5.4% and 8.4 % per year respectively). Overall, 3.1 % of isolates were MDR which remained stable over the study period. Resistance to other antibiotics assessed mostly remained stable, except SMX/TMP resistance decreased significantly by 8.5 % (2010, 15 %; 2018, 6 %) per year in VA hospitals.Conclusion. While previous work has recognized S. maltophilia as primarily a nosocomial pathogen, the present study found that 40 % of cultures collected were among outpatients. Between 2010 and 2018, the number of positive S. maltophilia cultures decreased significantly in the national VA Healthcare System. Resistance to SMX/TMP decreased over the study period in VA hospitals and now more closely reflects previously reported resistance rates worldwide (0-10 %). MDR S. maltophilia remained stable and low in the national VA Healthcare System.

In vitro Activity of Cefiderocol and Comparators against Carbapenem-Resistant Gram-Negative Pathogens from France and Belgium.

Infections with carbapenem-resistant (CR) Gram-negative (GN) pathogens have increased in many countries worldwide, leaving only few therapeutic options. Cefiderocol (CFDC) is approved in Europe for the treatment of aerobic GN infections in adults with limited treatment options. This study evaluated the in vitro activity of cefiderocol and comparators against multidrug-resistant (MDR) bacteria including meropenem-resistant (MR) or pandrug-resistant (PR) GN clinical isolates from France and Belgium. The minimum inhibitory concentrations (MICs) of CFDC were determined by broth microdilution, using iron-depleted cation-adjusted Mueller–Hinton broth, and were compared to those of 10 last-line antibiotics. The MICs were interpreted according to EUCAST and CLSI breakpoints, and in the absence of species-specific breakpoints, non-species-related pharmacokinetic/pharmacodynamic breakpoints were used. Among the 476 isolates tested, 322 were carbapenemase producers (CP), 58 non-CP-CRs, 52 intrinsically CR, 41 expanded-spectrum cephalosporin resistant and 5 were multi-susceptible. Susceptibility to CFDC was high using EUCAST breakpoints 81%, 99% and 84%, and was even higher using CLSI breakpoints to 93%, 100% and 88% for Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii, respectively. Susceptibility to cefiderocol using non-species-related breakpoints for Stenotrophomonas maltophilia, Achromobacter xylosoxydans and Burkholderia cepacia, was 100%, 100% and 92.3%, respectively. The susceptibility rates were lower with the NDM producers, with values of 48% and 30% using EUCAST breakpoints and 81% and 50% using CLSI breakpoints for Enterobacterales and Acinetobacter spp, respectively. CFDC demonstrated high in vitro susceptibility rates against a wide range of MDR GN pathogens, including MR and PR isolates.

Preferential Selection of Low-Frequency, Lipopolysaccharide-Modified, Colistin-Resistant Mutants with a Combination of Antimicrobials in Acinetobacter baumannii.

ABSTRACTColistin, which targets lipopolysaccharide (LPS), is used as a last-resort drug against severe infections caused by drug-resistant Acinetobacter baumannii. However, A. baumannii possesses two colistin-resistance mechanisms. LPS modification caused by mutations in pmrAB genes is often observed in clinical isolates of multidrug-resistant Gram-negative pathogens. In addition to LPS modification, A. baumannii has a unique colistin resistance mechanism, a complete loss of LPS due to mutations in the lpxACD genes, which are involved in LPS biosynthesis. This study aimed to elucidate the detailed mechanism of the emergence of colistin-resistant A. baumannii using strains with the same genetic background. Various colistin-resistant strains were generated experimentally using colistin alone and in combination with other antimicrobials, such as meropenem and ciprofloxacin, and the mutation spectrum was analyzed. In vitro selection of A. baumannii in the presence of colistin led to the emergence of strains harboring mutations in lpxACD genes, resulting in LPS-deficient colistin-resistant strains. However, combination of colistin with other antimicrobials led to the selection of pmrAB mutant strains, resulting in strains with modified LPS (LPS-modified strains). Further, the LPS-deficient strains showed decreased fitness and increased susceptibility to many antibiotics and disinfectants. As LPS-deficient strains have a higher biological cost than LPS-modified strains, our findings suggested that pmrAB mutants are more likely to be isolated in clinical settings. We provide novel insights into the mechanisms of resistance to colistin and provide substantial solutions along with precautions for facilitating current research and treatment of colistin-resistant A. baumannii infections.IMPORTANCE Acinetobacter baumannii has developed resistance to various antimicrobial drugs, and its drug-resistant strains cause nosocomial infections. Controlling these infections has become a global clinical challenge. Carbapenem antibiotics are the frontline treatment drugs for infectious diseases caused by A. baumannii. For patients with infections caused by carbapenem-resistant A. baumannii, colistin-based therapy is often the only treatment option. However, A. baumannii readily acquires resistance to colistin. Many patients infected with colistin-resistant A. baumannii undergo colistin treatment before isolation of the colistin-resistant strain, and it is hypothesized that colistin resistance predominantly emerges under selective pressure during colistin therapy. Although the concomitant use of colistin and carbapenems has been reported to have a synergistic effect in vitro against carbapenem-resistant A. baumannii strains, our observations strongly suggest the need for attention to the emergence of strains with a modified lipopolysaccharide during treatment.

In Vitro Activity of Robenidine Analogues NCL259 and NCL265 against Gram-Negative Pathogens.

Multidrug-resistant (MDR) Gram-negative pathogens, especially Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Enterobacter spp., are recognized by the World Health Organization as the most critical priority pathogens in urgent need of drug development. In this study, the in vitro antimicrobial activity of robenidine analogues NCL259 and NCL265 was tested against key human and animal Gram-negative clinical isolates and reference strains. NCL259 and NCL265 demonstrated moderate antimicrobial activity against these Gram-negative priority pathogens with NCL265 consistently more active, achieving lower minimum inhibitory concentrations (MICs) in the range of 2–16 µg/mL. When used in combination with sub-inhibitory concentrations of polymyxin B to permeabilize the outer membrane, NCL259 and NCL265 elicited a synergistic or additive activity against the reference strains tested, reducing the MIC of NCL259 by 8- to 256- fold and the MIC of NCL265 by 4- to 256- fold. A small minority of Klebsiella spp. isolates (three) were resistant to both NCL259 and NCL265 with MICs > 256 µg/mL. This resistance was completely reversed in the presence of the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PAβN) to yield MIC values of 8–16 µg/mL and 2–4 µg/mL for NCL259 and NCL256, respectively. When NCL259 and NCL265 were tested against wild-type E. coli isolate BW 25113 and its isogenic multidrug efflux pump subunit AcrB deletion mutant (∆AcrB), the MIC of both compounds against the mutant ∆AcrB isolate was reduced 16-fold compared to the wild-type parent, indicating a significant role for the AcrAB-TolC efflux pump from Enterobacterales in imparting resistance to these robenidine analogues. In vitro cytotoxicity testing revealed that NCL259 and NCL265 had much higher levels of toxicity to a range of human cell lines compared to the parent robenidine, thus precluding their further development as novel antibiotics against Gram-negative pathogens.

Emergence and Transmission of Plasmid-Mediated Mobile Colistin Resistance Gene mcr-10 in Humans and Companion Animals.

ABSTRACTMobile colistin resistance (mcr) genes mediated by plasmids have widely disseminated throughout the world. Recently, 10 mcr genes (mcr-1 to mcr-10) and a large number of variants have been identified in more than 60 countries. However, only a few instances of Enterobacter cloacae complex (ECC) bearing mcr-10 from animal origin have been reported globally. The aim of this study was to fill a knowledge gap in mcr-10-positive ECC of animal origin and analyze the potential transmission trend and different characteristics between human and companion animal isolates. The mcr-10 gene was identified on a self-transmissible plasmid in the human isolate and non-transmissible plasmids in other three animal strains. mcr-10 was adjacent to a XerC-type tyrosine recombinase-gene, and various insertion sequences were located on the downstream of core conservative structure xerC-mcr-10, thus indicating this region might be a candidate for insertions of mobile genetic elements and mcr-10 might be mobilized by IS-mediated mechanisms. Moreover, phylogenetic analysis found that mcr-10-positive isolates were mainly distributed in the clade of Enterobacter roggenkampii, exhibiting significant species specificity. These findings indicated that mcr-10 has emerged among Enterobacter spp. within humans and companion animals, highlighting that the importance of taking effective control measures to monitor the dissemination and evolution of mcr genes.IMPORTANCE Colistin was considered as the last-resort drug against severe clinical infections caused by multidrug-resistant Gram-negative pathogens. Mobile colistin resistance (mcr) genes and its variants carried by plasmids have been reported in diverse niches in recent years, and yet few studies reported carriage of mcr-10 in ECC strains of companion animal origin. How plasmid-borne mcr-10 transmitted in opportunistic pathogens and different characteristics of mcr-10-bearing strains isolated from humans and companion animals are not well understood. In this study, we discovered mcr-10-harboring strains in multidrug-resistant ECC isolates of companion animal origin for the first time and conducted a comprehensive analysis of the genetic environment of mcr-10 from multiple countries around the world, providing the potential basis for formulating control measures to slow down the spread of colistin resistance.

Early and Appropriate Use of Ceftazidime-Avibactam in the Management of Multidrug-Resistant Gram-Negative Bacterial Infections in the Indian Scenario.

The increasing prevalence of antibiotic-resistant pathogens exerts a substantial burden on the healthcare infrastructure worldwide. The World Health Organization (WHO) has declared that multidrug-resistant (MDR) Gram-negative pathogens, especially, carbapenem-resistant Enterobacterales (CRE), Acinetobacter baumannii, and Pseudomonas aeruginosa as the topmost priority while developing newer antimicrobials. The increasing prevalence of infectious diseases caused by MDR Gram-negative bacteria also poses a challenge when choosing the empiric antimicrobial therapy for seriously ill hospitalized patients. The infections caused by MDR Gram-negative organisms ultimately result in increased mortality, morbidity, prolonged hospital stay, and increased cost of management. To tackle these challenges, newer antimicrobials like ceftazidime-avibactam were explored. The article also discusses the in vitro activity and therapeutic efficacy of ceftazidime-avibactam along with its pharmacokinetic properties and the role it will play in the management of MDR Gram-negative organisms in the Indian setting. Several studies have highlighted the role of early and appropriate antibiotic use in the reduction of mortality in patients with Gram-negative infections. Timely initiation of appropriate antibiotic therapy for serious infections leads to favorable clinical outcomes. Early and appropriate use of ceftazidime-avibactam while treating MDR Gram-negative infections has been associated with improved clinical outcomes. The aim of this review is to highlight the efficacy of ceftazidime-avibactam in the treatment of MDR Gram-negative infections. We have also summarized the information on outcomes achieved by early and appropriate use of ceftazidime-avibactam.

A Lysozyme Murein Hydrolase with Broad-Spectrum Antibacterial Activity from Enterobacter Phage myPSH1140.

ABSTRACTBacteriophages and bacteriophage-derived peptidoglycan hydrolases (endolysins) present promising alternatives for the treatment of infections caused by multidrug resistant Gram-negative and Gram-positive pathogens. In this study, Gp105, a putative lysozyme murein hydrolase from Enterobacter phage myPSH1140 was characterized in silico, in vitro as well as in vivo using the purified protein. Gp105 contains a T4-type lysozyme-like domain (IPR001165) and belongs to Glycoside hydrolase family 24 (IPR002196). The putative endolysin indeed had strong antibacterial activity against Gram-negative pathogens, including E. cloacae, K. pneumoniae, P. aeruginosa, S. marcescens, Citrobacter sp., and A. baumannii. Also, an in vitro peptidoglycan hydrolysis assay showed strong activity against purified peptidoglycans. This study demonstrates the potential of Gp105 to be used as an antibacterial protein to combat Gram-negative pathogens.

Polymyxin B in Combination with Glycerol Monolaurate Exerts Synergistic Killing against Gram-Negative Pathogens.

The rapid emergence and spread of multidrug-resistant (MDR) bacterial pathogens pose a serious danger to worldwide human health, and resistance to last-resort drugs, such as polymyxins, is being increasingly detected in MDR Gram-negative pathogens. There is an urgent need to find and optimize combination therapies as an alternative therapeutic strategy, with a dry pipeline in novel antibiotic research and development. We found a monoester formed from the combination of lauric acid and glycerol, glycerol monolaurate (GML), possessing prominent antibacterial and anti-inflammatory activity. However, it is still unclear whether GML in combination could increase antimicrobial activity. Here, we reported that polymyxin B (PMNB) combined with GML exhibited a synergistic antimicrobial impact on Gram-negative strains in vitro, including clinical MDR isolates. This synergistic antimicrobial activity correlated with the destruction of bacterial cell structures, eradication of preformed biofilms, and increased reactive oxygen species (ROS) accumulation. We also showed that PMNB synergized with GML effectively eliminated pathogens from bacterial pneumonia caused by Klebsiella pneumoniae to rescue mice. Our research demonstrated that the PMNB and GML combination induced synergistic antimicrobial activity for Gram-negative pathogens in vitro and in vivo. These findings are of great importance for treating bacterial infections and managing the spread of infectious diseases.

Multidrug-Resistant Gram-Negative ESKAPE Pathogens from a Tertiary-Care Hospital: Prevalence and Risk Factors

Multidrug-Resistant Gram-Negative ESKAPE Pathogens from a Tertiary-Care Hospital: Prevalence and Risk Factors

Comparative Evaluation of Vitek 2 and Etest versus Broth Microdilution for Ceftazidime/Avibactam and Ceftolozane/Tazobactam Susceptibility Testing of Enterobacterales and Pseudomonas aeruginosa.

Ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T) are novel antibiotics with activity against multidrug-resistant Gram-negative pathogens. Nevertheless, resistance to both agents has been reported emphasizing the need for accurate and widely accessible susceptibility testing. In the present study, Vitek 2 and Etest CAZ and C/T MIC results for 100 non-repetitive clinical isolates (83 Enterobacterales and 17 P. aeruginosa, whereof 69 challenge isolates) were compared to the standard broth microdilution (BMD) method. EUCAST breakpoints were used for assessing the categorical (CA) and essential (EA) agreement between the methods along with the corresponding error rates. The Vitek 2 performance was comparable to that of BMD for testing both antimicrobial agents exceeding the ISO requirements (CA 98–99%, EA 96–100%, major errors (MEs) 0–1%, very major error (VMEs) 1%). Likewise, the Etest provided accurate results for CZA and C/T testing against Enterobacterales and P. aeruginosa, respectively (CA 100%, EA 97–100%, MEs 0%, VMEs 0%). On the contrary, EA of 85% and 6% VME rate were found for CZA Etest and P. aeruginosa. Overall, Vitek 2 measurements of CZA and C/T susceptibility correlated closely with the reference BMD, indicating that it can represent a suitable alternative to BMD for susceptibility testing of Enterobacterales and P. aeruginosa. The Etest did not fulfill the ISO performance criteria of EA and VME for CZA and P. aeruginosa. Further studies are needed to assess whether the Etest allows a reliable assessment of CZA and C/T EUCAST MICs.

Otilonium bromide boosts antimicrobial activities of colistin against Gram-negative pathogens and their persisters

Colistin is the last-line antibiotic against Gram-negative pathogens. Here we identify an FDA-approved drug, Otilonium bromide (Ob), which restores the activity of colistin against colistin-resistant Gram-negative bacteria in vitro and in a mouse infection model. Ob also reduces the colistin dosage required for effective treatment of infections caused by colistin-susceptible bacteria, thereby reducing the toxicity of the drug regimen. Furthermore, Ob acts synergistically with colistin in eradicating multidrug-tolerant persisters of Gram-negative bacteria in vitro. Functional studies and microscopy assays confirm that the synergistic antimicrobial effect exhibited by the Ob and colistin involves permeabilizing the bacterial cell membrane, dissipating proton motive force and suppressing efflux pumps, resulting in membrane damages, cytosol leakage and eventually bacterial cell death. Our findings suggest that Ob is a colistin adjuvant which can restore the clinical value of colistin in combating life-threatening, multidrug resistant Gram-negative pathogens.

Bactericidal Effect of Cecropin A Fused Endolysin on Drug-Resistant Gram-Negative Pathogens.

The rapid spread of superbugs leads to the escalation of infectious diseases, which threatens public health. Endolysins derived from bacteriophages are spotlighted as promising alternative antibiotics against multi-drug resistant bacteria. In this study, we isolated and characterized the novel Salmonella typhimurium phage PBST08. Bioinformatics analysis of the PBST08 genome revealed putative endolysin ST01 with a lysozyme-like domain. Since the lytic activity of the purified ST01 was minor, probably owing to the outer membrane, which blocks accessibility to peptidoglycan, antimicrobial peptide cecropin A (CecA) was fused to the N-terminus of ST01 to disrupt the outer membrane. The resulting CecA::ST01 has been shown to have increased bactericidal activity against gram-negative pathogens including Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Enterobacter cloacae and the most affected target was A. baumannii. In the presence of 0.25 μM CecA::ST01, A. baumannii ATCC 17978 strain was completely killed and CCARM 12026 strain was wiped out by 0.5 μM CecA::ST01, which is a clinical isolate of A. baumannii and resistant to multiple drugs including carbapenem. Moreover, the larvae of Galleria mellonella could be rescued up to 58% or 49% by the administration of CecA::ST01 upon infection by A. baumannii 17978 or CCARM 12026 strain. Finally, the antibacterial activity of CecA::ST01 was verified using 31 strains of five gram-negative pathogens by evaluation of minimal inhibitory concentration. Thus, the results indicate that a fusion of antimicrobial peptide to endolysin can enhance antibacterial activity and the spectrum of endolysin where multi-drug resistant gram-negative pathogens can be efficiently controlled.

NDM-5-Producing Escherichia coli Co-Harboring mcr-1 Gene in Companion Animals in China.

Simple SummaryThis is a study related to NDM-5-producing E. coli in companion animals in China. Notably, an E. coli isolate possessing both blaNDM-5-bearing plasmid and mcr-1-bearing plasmid was identified in a dog from the same veterinary clinic, where we previously found the mobile IncX3–X4 hybrid plasmid encoding both blaNDM-5 and mcr-1 in a cat. This observation highlights the diversity of blaNDM-5- and mcr-1-harboring plasmids and the transferability of such resistant pathogens from companion animals to humans. Given that colistin is the last-resort antibiotic for treating human infections caused by carbapenem-resistant Enterobacteriaceae, the co-transfer of resistance to both antibiotics may seriously compromise the effectiveness of clinical therapy.Carbapenem and colistin are important antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. Here, we isolated the blaNDM-5-harboring Escherichia coli in companion animals in healthy or diseased companion animals from veterinary clinics in six cities in China from July to November 2016. A total of 129 rectal swabs of healthy or diseased dogs and cats were collected from veterinary clinics in six different cities in China, and the isolates were subjected to carbapenem and colistin susceptibility testing. Resistance genes were confirmed using PCR. Conjugation experiments were conducted to determine the transferability of antibiotic resistance genes (ARGs) in the strains. The isolated rate of blaNDM-5-harboring E. coli strains was 3.88% (five strains). These five strains were multidrug resistant to at least three antibiotics and corresponded to four sequence types including ST101. The blaNDM-5 gene was located on 46 kb IncX3 plasmids in these five strains, and the genetic contexts were shared and were nearly identical to the K. pneumoniae plasmid pNDM5-IncX3 from China. In addition, one strain (CQ6-1) co-harbored blaNDM-5-encoding-IncX3 plasmid along with a mcr-1-encoding-IncX4 plasmid, and their corresponding genetic environments were identical to the blaNDM-5-IncX3 and mcr-1-IncX4 hybrid plasmid reported previously from the same area and from the same clinic. The results indicated that the similar genetic contexts were shared between these isolates from companion animals, and the IncX3-type plasmids played a key role in the spread of blaNDM-5 among these bacteria.

Challenges of Antimicrobial Resistance and Stewardship in Solid Organ Transplant Patients.

Purpose of ReviewWithout effective antimicrobials, patients cannot undergo transplant surgery safely or sustain immunosuppressive therapy. This review examines the burden of antimicrobial resistance in solid organ transplant recipients and identifies opportunities for antimicrobial stewardship.Recent FindingsAntimicrobial resistance has been identified to be the leading cause of death globally. Multidrug-resistant pathogens are associated with significant morbidity and mortality in transplant recipients. Methicillin-resistant S. aureus affects liver and lung recipients, causing bacteremia, pneumonia, and surgical site infections. Vancomycin-resistant enterococci is a nosocomial pathogen primarily causing bacteremia in liver recipients. Multidrug-resistant Gram-negative pathogens present urgent and serious threats to transplant recipients. Extended-spectrum beta-lactamase-producing E. coli and K. pneumoniae commonly cause bacteremia and intra-abdominal infections in liver and kidney recipients. Carbapenemase-producing Enterobacterales, mainly K. pneumoniae, are responsible for infections early-post transplant in liver, lung, kidney, and heart recipients. P. aeruginosa and A. baumannii continue to be critical threats. While there are new antimicrobial agents targeting resistant pathogens, judicious prescribing is crucial to minimize emerging resistance. The full implications of the COVID-19 global pandemic on antimicrobial resistance in transplant recipients remain to be understood. Currently, there are no established standards on the implementation of antimicrobial stewardship interventions, but strategies that leverage existing antimicrobial stewardship program structure while tailoring to the needs of transplant recipients may help to optimize antimicrobial use.SummaryClinicians caring for transplant recipients face unique challenges tackling emerging antimcirobial resistance. Coordinated antimicrobial stewardship interventions in collaboration with appropriate expertise in transplant and infectious diseases may mitigate against such threats.

Leaks in the Pipeline: a Failure Analysis of Gram-Negative Antibiotic Development from 2010 to 2020.

ABSTRACTThe World Health Organization (WHO) has warned that our current arsenal of antibiotics is not innovative enough to face impending infectious diseases, especially those caused by multidrug-resistant Gram-negative pathogens. Although the current preclinical pipeline is well stocked with novel candidates, the last U.S. Food and Drug Administration (FDA)-approved antibiotic with a novel mechanism of action against Gram-negative bacteria was discovered nearly 60 years ago. Of all the antibiotic candidates that initiated investigational new drug (IND) applications in the 2000s, 17% earned FDA approval within 12 years, while an overwhelming 62% were discontinued in that time frame. These “leaks” in the clinical pipeline, where compounds with clinical potential are abandoned during clinical development, indicate that scientific innovations are not reaching the clinic and providing benefits to patients. This is true for not only novel candidates but also candidates from existing antibiotic classes with clinically validated targets. By identifying the sources of the leaks in the clinical pipeline, future developmental efforts can be directed toward strategies that are more likely to flow into clinical use. In this review, we conduct a detailed failure analysis of clinical candidates with Gram-negative activity that have fallen out of the clinical pipeline over the past decade. Although limited by incomplete data disclosure from companies engaging in antibiotic development, we attempt to distill the developmental challenges faced by each discontinued candidate. It is our hope that this insight can help de-risk antibiotic development and bring new, effective antibiotics to the clinic.