A demonstration of athermal effects of continuous microwave irradiation on the growth and antibiotic sensitivity of Pseudomonas aeruginosa PAO1.

Stress, caused by exposure to microwaves (2.45GHz) at constant temperature (37± 0.5(o) C), alters the growth profile of Pseudomonas aeruginosa PAO1. In the absence of microwave treatment a simple, highly reproducible growth curve was observed over 24 hours or more. Microwave treatment caused no reduction in growth during the first 6 hours, but at a later stage (>12hours) the growth was markedly different to the controls. Secondary growth, typical of the presence of persisters clearly became apparent, as judged by both the dissolved oxygen and the cell density profiles. These treated cells showed distinct morphological changes, but on re-growth these cells reverted to normal. The Microwave Induced Persisters were subject to antibiotic challenge (tobramycin) and showed increased sensitivity when compared to the un-stressed planktonic cells. This is in marked contrast to antibiotic induced persisters which show increased resistance. This provides evidence for both a non-thermal effect of microwaves and a previously undescribed route to a novel form of antibiotic susceptible persister cells. This article is protected by copyright. All rights reserved

Nitro-Carba test, a novel and simple chromogenic phenotypic method for rapid screening of carbapeneamase-producing Enterobacteriaceae.

OBJECTIVES: The present study developed Nitro-Carba Test (NCT), a rapid and simple chromogenic method for screening of Carbapenemase-producing Enterobacteriaceae (CPE). METHODS: The NCT was validated with a total of 31 carbapenemase-producing isolates (9 KPCs, 11 MBLs and 11 OXA-48s) and with 56 non-carbapenemase-producing strains. The assay relies on the hydrolysis of nitrocefin in the presence of carbapenems. The carbapenemases were extracted with lysis buffer prior to addition to wells with and without imipenem (IPM), meropenem (MEM) and ertapenem (ETP). Following the addition of nitrocefin, a change in colour from yellow to red, indicating carbapenemase production, was observed within 20min. The susceptibility profiles of each bacterial strain were also investigated. RESULTS: A NCT detected all 31 CPEs within a timeframe of only 10seconds to 12min. All carbapenemase producers hydrolyzed nitrocefin in all wells. No colour change in the wells with carbapenems was observed in non-carbapenemase producers. The sensitivity for all three carbapenems was 100%, while specificity of IPM, MEM and ETP were 64.29%, 91.07% and 100%, respectively. IPM, MEM and ETP against all carbapenemase-producing strains had MIC values ranging from 0.5 to ≥256μg/mL, 0.25 to ≥256μg/mL and 1 to ≥256μg/mL, respectively. OXA-48-producing isolates showed lower MIC values compared with producers of MBL and KPC. CONCLUSION: This assay is a promising method detecting CPE rapidly. The NCT is a simple and reliable method, capable of detecting CPE in even carbapenem-susceptible strains

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended-spectrum-β-lactamases, AmpC β-lactamases and co-β-lactamases in Enterobacteriaceae.

A resazurin chromogenic agar (RCA) along with combined disc method has been developed as a promising method for rapid screening of extended-spectrum-β-lactamase (ESBL), AmpC β-lactamase, and co-production of ESBL and AmpC. Cefpodoxime (CPD) discs supplemented with and without clavulanic acid (CA), cloxacillin (CX), or CA+CX were evaluated against 86-molecularly confirmed β-lactamase-producing Enterobacteriaceae, including 15 ESBLs, 32 AmpCs, 9 co-producers of ESBL and AmpC, and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also exhibited a good performance in the screening for the co-existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is a simple and inexpensive method that allows observation of results within 7 h. It can be applicable in any microbiological laboratory, especially in the endemic areas of ESBL, AmpC, or co-β-lactamase-producing Enterobacteriaceae

A nitrocefin disc supplemented with ertapenem for rapid screening of carbapenemase-producing Enterobacteriaceae

Reliable, simple and rapid methods for laboratory detection of carbapenemases are important for an appropriate antibiotic administration. A nitrocefin disc containing ertapenem for rapid screening of carbapenemase production among Enterobacteriaceae is developed in the present study. A total of 87 molecularly-confirmed Enterobacteriaceae including 31 carbapenemase producers and 56 non-carbapenemase producers, were tested with nitrocefin discs supplemented with and without ertapenem (20 μg/disc). Nitrocefin discs with ertapenem successfully discriminated all 31 carbapenemase and all non-carbapenemase producers within 30 minutes. The sensitivity and specificity of the method were 100%. The minimum inhibitory concentrations (MICs) of ertapenem against all carbapenemase-producing isolates ranged from 1 to ≥ 256 μg/mL. This simple test could help to minimize the treatment failure and control the dissemination of infections caused by carbapenemase-associated resistant bacteria. It is a promising approach that could be performed routinely in any laboratory

Boesenbergia rotunda (L.) Mansf. extract potentiate the antibacterial activity of some β-lactams against β-lactam-resistant Staphylococci.

OBJECTIVE: The purpose of this study was to investigate the effect of Boesenbergia rotunda (L.) Mansf. Extract (BRE) and peptidoglycan inhibitor antibiotics, either used alone or in combination against β-lactam resistant Staphylococci. METHODS: Antibacterial and synergistic activities of BRE alone and in combination with ampicillin (AMP), cloxacillin (CLX), cefazolin (CFZ) or vancomycin (VAN) were evaluated against two β-lactam-resistant Staphylococcus aureus (BRSA) and one β-lactam-resistant Staphylococcus epidermidis (BRSE). The activities were confirmed by killing curve assays. The preliminary antimicrobial action was elucidated by transmission electron microscopy (TEM) and cytoplasmic membrane (CM) permeability. RESULTS: All tested Staphylococci were inhibited by BRE at a MIC of 16μg/mL. Two BRSA strains showed high resistance to CLX, AMP, and CFZ, while BRSE was resistant to CLX and AMP. All tested isolates remained susceptible to VAN. The chequerboard assay demonstrated the fractional inhibitory concentration index (FICI) at 0.502 of the BRE and CLX combination against the BRSA strains. Killing curve determinations confirmed the antibacterial and synergistic activities. The TEM study revealed collapse of the CM in BRE-treated cells and damage of both CM and peptidoglycan in BRE plus CLX-treated cells. The CM permeability assay showed that either BRE or nisin alone, and BRE plus CLX significantly induced leakage of OD260-absorbing materials. CONCLUSIONS: The BRE potentiated β-lactams, particularly CLX against β-lactam-resistant Staphylococci by damaging the CM and peptidoglycan layer, leading to leakage of intracellular materials. The combination of BRE and β-lactams provides a potential way forward in developing a novel antistaphylococcal agent

Speciation of common Gram-negative pathogens using a highly multiplexed high resolution melt curve assay

The identification of the bacterial species responsible for an infection remains an important step for the selection of antimicrobial therapy. Gram-negative bacteria are an important source of hospital and community acquired infections and frequently antimicrobial resistant. Speciation of bacteria is typically carried out by biochemical profiling of organisms isolated from clinical specimens, which is time consuming and delays the initiation of tailored treatment. Whilst molecular methods such as PCR have been used, they often struggle with the challenge of detecting and discriminating a wide range of targets. High resolution melt analysis is an end-point qPCR detection method that provides greater multiplexing capability than probe based methods. Here we report the design of a high resolution melt analysis assay for the identification of six common Gram-negative pathogens; Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, Salmonella Sp, and Acinetobacter baumannii, and a generic Gram-negative specific 16S rRNA control. The assay was evaluated using a well characterised collection of 113 clinically isolated Gram-negative bacteria. The agreement between the HRM assay and the reference test of PCR and sequencing was 98.2% (Kappa 0.96); the overall sensitivity and specificity of the assay was 97.1% (95% CI: 90.1–99.7%) and 100% (95% CI: 91.78–100%) respectively

Genome analysis of secondary metabolitebiosynthetic gene clusters of Photorhabdus akhurstii subsp. akhurstii and its antibacterial activity against antibiotic-resistant bacteria

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent