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Detection of haemolytic activity of campylobacters by agarose haemolysis and microplate assay

Tay ST, Devi S, Puthucheary SD, Kautner IM

J Med Microbiol, 1995 Mar;42(3):175-80.
PMID: 7884798

There are several methods for the detection of haemolytic activity in campylobacters. However, we found the haemolytic effect of campylobacters on conventional blood agar plates to be variable, inconsistent and difficult to interpret. Blood agarose plates showed campylobacter haemolytic activity more clearly. The incubation conditions (temperature and gaseous) appear to be important for the expression of this activity. Ninety four percent of the Campylobacter isolates examined were found to be haemolytic by the microplate assay with minimal haemolytic units that ranged from 1 to 64. Haemolytic activity was detected only from live bacterial cultures and not from any of the 50 bacterial culture supernates, which suggests that campylobacters may possess a cell-associated haemolysin. The identification of such haemolytic activity in a large number of campylobacters (94%) suggests its potential role as a virulence factor in campylobacter gastroenteritis.

Matched MeSH terms: Polymyxin B/pharmacology
Role of the cell envelope in the antibacterial activities of polymyxin B and polymyxin B nonapeptide against Escherichia coli

Sahalan AZ, Dixon RA

Int J Antimicrob Agents, 2008 Mar;31(3):224-7.
PMID: 18083010

The role of membrane permeabilisation and disruption in the mechanism of action of some polymyxin analogues against Gram-negative organisms is contentious. The effects of polymyxin B (PMB) and its analogue polymyxin B nonapeptide (PMBN) on Escherichia coli envelopes should correlate, but previous work by other workers suggests that PMBN has a different mode of action. This study has reassessed the biochemical techniques used previously and has shown that, in contrast to previous studies, PMBN (a well-characterised antibacterial synergist) readily releases periplasmic proteins and lipopolysaccharide from treated E. coli at subinhibitory concentrations in normal physiological buffer conditions. We conclude that, when tested with appropriate methodology, PMBN closely correlates with the early effects of PMB on the cell envelope of E. coli and this study shows that it is now consistent with the accepted interactions of membrane-active agents against Gram-negative cells.

Matched MeSH terms: Polymyxin B/pharmacology*
Fulltext Novel Polymyxin Combination with the Antiretroviral Zidovudine Exerts Synergistic Killing against NDM-Producing Multidrug-Resistant Klebsiella pneumoniae

Lin YW, Abdul Rahim N, Zhao J, Han ML, Yu HH, Wickremasinghe H, et al.

Antimicrob Agents Chemother, 2019 04;63(4).
PMID: 30670431 DOI: 10.1128/AAC.02176-18

Polymyxins are used as a last-line therapy against multidrug-resistant (MDR) New Delhi metallo-β-lactamase (NDM)-producing Klebsiella pneumoniae However, polymyxin resistance can emerge with monotherapy; therefore, novel strategies are urgently needed to minimize the resistance and maintain their clinical utility. This study aimed to investigate the pharmacodynamics of polymyxin B in combination with the antiretroviral drug zidovudine against K. pneumoniae Three isolates were evaluated in static time-kill studies (0 to 64 mg/liter) over 48 h. An in vitro one-compartment pharmacokinetic/pharmacodynamic (PK/PD) model (IVM) was used to simulate humanized dosage regimens of polymyxin B (4 mg/liter as continuous infusion) and zidovudine (as bolus dose thrice daily to achieve maximum concentration of drug in broth [Cmax] of 6 mg/liter) against K. pneumoniae BM1 over 72 h. The antimicrobial synergy of the combination was further evaluated in a murine thigh infection model against K. pneumoniae 02. In the static time-kill studies, polymyxin B monotherapy produced rapid and extensive killing against all three isolates followed by extensive regrowth, whereas zidovudine produced modest killing followed by significant regrowth at 24 h. Polymyxin B in combination with zidovudine significantly enhanced the antimicrobial activity (≥4 log10 CFU/ml) and minimized bacterial regrowth. In the IVM, the combination was synergistic and the total bacterial loads were below the limit of detection for up to 72 h. In the murine thigh infection model, the bacterial burden at 24 h in the combination group was ≥3 log10 CFU/thigh lower than each monotherapy against K. pneumoniae 02. Overall, the polymyxin B-zidovudine combination demonstrates superior antimicrobial efficacy and minimized emergence of resistance to polymyxins.

Matched MeSH terms: Polymyxin B/pharmacology*
Cytotoxicity of Vibrio cholerae on Madin Darby Bovine Kidney cells

Haque QM, Mohamad NF, Helaluddin AB, Saeed M

Pak J Pharm Sci, 2010 Oct;23(4):393-7.
PMID: 20884452

The cytotoxicity of cell-free culture filtrates of 31 isolates of Vibrio cholerae O1 and O139, 5 reference strains and 26 clinical isolates, was tested on Madin Darby Bovine Kidney (MDBK) cells and Vero cells. The 3-[4,5-dimethylthiazol-2-y]-2, 5-diphenyltetrazolium bromide (MTT) test was used to detect the effect of the filtrates on the proliferation and viability of cultured cell populations. The filtrates were prepared from serial ten-fold dilutions of inoculated AKI and APW broth media with and without the addition of polymyxin B. The APW culture filtrates of both V. cholerae O1 and O139 with and without added polymyxin B showed greater toxicity to MDBK cells as compared to AKI filtrates. The cytotoxicity of AKI-grown V. cholerae O139 to MDBK cells was greater than that of V. cholerae O1 grown in the same medium. The cytotoxicity of APW filtrates on Vero cells was low and only noted when polymyxin was added to the medium.

Matched MeSH terms: Polymyxin B/pharmacology
Antimicrobial susceptibility profiling and genomic diversity of multidrug-resistant Acinetobacter baumannii isolates from a teaching hospital in Malaysia

Kong BH, Hanifah YA, Yusof MY, Thong KL

Jpn J Infect Dis, 2011;64(4):337-40.
PMID: 21788713

The resistance phenotypes and genomic diversity of 185 Acinetobacter baumannii isolates obtained from the intensive care unit (ICU) of a local teaching hospital in Kuala Lumpur from 2006 to 2009 were determined using antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Antibiogram analyses showed that the isolates were fully resistant to β-lactam antimicrobials and had high resistance rates to the other antimicrobial agents tested. However, the isolates were susceptible to polymyxin B. Resistance to cefoperazone/sulbactam was only detected in strains isolated from 2007 to 2009. Some environmental isolates and an isolate from the hands of a healthcare worker (HCW) had identical resistance profiles and PFGE profiles that were closely related to patient isolates. Cluster analyses based on the PFGE profiles showed there was a persistent clone of endemic isolates in the ICU environment. The transmission route from HCWs to fomites to patients, which caused a long-term infection in the ICU of the University Malaya Medical Centre, was observed in this study. These data provide a better understanding of A. baumannii epidemiology within the hospital and the possible transmission routes. Knowledge of changes in the resistance rates of A. baumannii in our local hospital will improve antimicrobial therapy.

Matched MeSH terms: Polymyxin B/pharmacology
Fulltext The Development of Drugs against Acanthamoeba Infections

Siddiqui R, Aqeel Y, Khan NA

Antimicrob Agents Chemother, 2016 11;60(11):6441-6450.
PMID: 27600042 DOI: 10.1128/AAC.00686-16

For the past several decades, there has been little improvement in the morbidity and mortality associated with Acanthamoeba keratitis and Acanthamoeba encephalitis, respectively. The discovery of a plethora of antiacanthamoebic compounds has not yielded effective marketed chemotherapeutics. The rate of development of novel antiacanthamoebic chemotherapies of translational value and the lack of interest of the pharmaceutical industry in developing such chemotherapies have been disappointing. On the other hand, the market for contact lenses/contact lens disinfectants is a multi-billion-dollar industry and has been successful and profitable. A better understanding of drugs, their targets, and mechanisms of action will facilitate the development of more-effective chemotherapies. Here, we review the progress toward phenotypic drug discovery, emphasizing the shortcomings of useable therapies.

Matched MeSH terms: Polymyxin B/pharmacology
Modifications in the pmrB gene are the primary mechanism for the development of chromosomally encoded resistance to polymyxins in uropathogenic Escherichia coli

Phan MD, Nhu NTK, Achard MES, Forde BM, Hong KW, Chong TM, et al.

J Antimicrob Chemother, 2017 10 01;72(10):2729-2736.
PMID: 29091192 DOI: 10.1093/jac/dkx204

Objectives: Polymyxins remain one of the last-resort drugs to treat infections caused by MDR Gram-negative pathogens. Here, we determined the mechanisms by which chromosomally encoded resistance to colistin and polymyxin B can arise in the MDR uropathogenic Escherichia coli ST131 reference strain EC958.
Methods: Two complementary approaches, saturated transposon mutagenesis and spontaneous mutation induction with high concentrations of colistin and polymyxin B, were employed to select for mutations associated with resistance to polymyxins. Mutants were identified using transposon-directed insertion-site sequencing or Illumina WGS. A resistance phenotype was confirmed by MIC and further investigated using RT-PCR. Competitive growth assays were used to measure fitness cost.
Results: A transposon insertion at nucleotide 41 of the pmrB gene (EC958pmrB41-Tn5) enhanced its transcript level, resulting in a 64- and 32-fold increased MIC of colistin and polymyxin B, respectively. Three spontaneous mutations, also located within the pmrB gene, conferred resistance to both colistin and polymyxin B with a corresponding increase in transcription of the pmrCAB genes. All three mutations incurred a fitness cost in the absence of colistin and polymyxin B.
Conclusions: This study identified the pmrB gene as the main chromosomal target for induction of colistin and polymyxin B resistance in E. coli.

Matched MeSH terms: Polymyxin B/pharmacology*
Fulltext A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity

Deris ZZ, Akter J, Sivanesan S, Roberts KD, Thompson PE, Nation RL, et al.

J Antibiot (Tokyo), 2014 Feb;67(2):147-51.
PMID: 24169795 DOI: 10.1038/ja.2013.111

Polymyxin B and colistin were examined for their ability to inhibit the type II NADH-quinone oxidoreductases (NDH-2) of three species of Gram-negative bacteria. Polymyxin B and colistin inhibited the NDH-2 activity in preparations from all of the isolates in a concentration-dependent manner. The mechanism of NDH-2 inhibition by polymyxin B was investigated in detail with Escherichia coli inner membrane preparations and conformed to a mixed inhibition model with respect to ubiquinone-1 and a non-competitive inhibition model with respect to NADH. These suggest that the inhibition of vital respiratory enzymes in the bacterial inner membrane represents one of the secondary modes of action for polymyxins.

Matched MeSH terms: Polymyxin B/pharmacology*
Arginase activity in a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

Sosroseno W, Musa M, Ravichandran M, Fikri Ibrahim M, Bird PS, Seymour GJ

Oral Microbiol. Immunol., 2006 Jun;21(3):145-50.
PMID: 16626370

The aim of the present study was to determine whether or not lipopolysaccharide from Actinobacillus actinomycetemcomitans could stimulate arginase activity in a murine macrophage cell line (RAW264.7 cells).

Matched MeSH terms: Polymyxin B/pharmacology
Nitric oxide production by a murine macrophage cell line (RAW264.7) stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

Sosroseno W, Barid I, Herminajeng E, Susilowati H

Oral Microbiol. Immunol., 2002 Apr;17(2):72-8.
PMID: 11929552

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could stimulate a murine macrophage cell line (RAW264.7 cells) to produce nitric oxide (NO). The cells were treated with LPS-A. actinomycetemcomitans or Escherichia coli LPS (LPS-Ec) for 24 h. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B and cytokines (IFN-gamma, TNF-alpha, IL-4 and IL-12) on the production of NO were also determined. The role of protein tyrosine kinase, protein kinase C and microtubulin organization on NO production were assessed by incubating RAW264.7 cells with genistein, bisindolylmaleide and colchicine prior to LPS-A. actinomycetemcomitans stimulation, respectively. NO levels from the culture supernatants were determined by the Griess reaction. The results showed that LPS-A. actinomycetemcomitans stimulated NO production by RAW264.7 cells in a dose-dependent manner, but was slightly less potent than LPS-Ec. NMMA and polymyxin B blocked the production of NO. IFN-gamma and IL-12 potentiated but IL-4 depressed NO production by LPS-A. actinomycetemcomitans-stimulated RAW264.7 cells. TNF-alpha had no effects on NO production. Genistein and bisindolylmalemaide, but not colchicine, reduced the production of NO in a dose-dependent mechanism. The results of the present study suggest that A. actinomycetemcomitans LPS, via the activation of protein tyrosine kinase and protein kinase C and the regulatory control of cytokines, stimulates NO production by murine macrophages.

Matched MeSH terms: Polymyxin B/pharmacology