Mark I. Garvey

The Efflux Pump Inhibitor Reserpine Selects Multidrug-Resistant Streptococcus pneumoniae Strains That Overexpress the ABC Transporters PatA and PatB

ABSTRACT One way to combat multidrug-resistant microorganisms is the use of efflux pump inhibitors (EPIs). Spontaneous mutants resistant to the EPI reserpine selected from Streptococcus pneumoniae NCTC 7465 and R6 at a frequency suggestive of a single mutational event were also multidrug resistant. No mutations in pmrA (which encodes the efflux protein PmrA) were detected, and the expression of pmrA was unaltered in all mutants. In the reserpine-resistant multidrug-resistant mutants, the overexpression of both patA and patB, which encode ABC transporters, was associated with accumulation of low concentrations of antibiotics and dyes. The addition of sodium orthovanadate, an inhibitor of ABC efflux pumps, or the insertional inactivation of either gene restored wild-type antibiotic susceptibility and wild-type levels of accumulation. Only when patA was insertionally inactivated were both multidrug resistance and reserpine resistance lost. Strains in which patA was insertionally inactivated grew significantly more slowly than the wild type. These data indicate that the overexpression of both patA and patB confers multidrug resistance in S. pneumoniae but that only patA is involved in reserpine resistance. The selection of reserpine-resistant multidrug-resistant pneumococci has implications for analogous systems in other bacteria or in cancer.

Natural and synthetic compounds such as trimethoprim behave as inhibitors of efflux in Gram-negative bacteria

OBJECTIVES We hypothesized that small heterocyclic or nitrogen-containing compounds could act as RND efflux pump inhibitors (EPIs). To ascertain possible EPIs, we sought to identify compounds that synergized with substrates of RND efflux pumps for wild-type bacteria and those that overexpress an efflux pump, but had no synergistic activity against strains in which a gene encoding a component of the AcrAB-TolC efflux pump had been inactivated. METHODS Twenty-six compounds plus L-phenylalanyl-L-arginyl-beta-naphthylamide (PAbetaN) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were screened by bioassay to identify compounds that synergized with ciprofloxacin for a range of Enterobacteriaceae and Pseudomonas aeruginosa. The MICs of ciprofloxacin, tetracycline, chloramphenicol, erythromycin and ethidium bromide+/-synergizing compounds were determined, and the ability to inhibit the efflux of Hoechst 33342 was measured. RESULTS Two compounds, trimethoprim and epinephrine, consistently showed synergy with antibiotics for most strains. The combinations did not show synergy for Salmonella enterica serovar Typhimurium in which the AcrAB-TolC efflux pump was inactive. Both compounds inhibited the efflux of Hoechst 33342. CONCLUSIONS Two compounds, trimethoprim and epinephrine, which are already licensed for use in man, may warrant further analysis as EPIs. The combination of trimethoprim with another antibiotic is a well-used combination in anti-infective chemotherapy, and so combination with another agent, such as a quinolone, may be a viable option and further studies are now required.

Medicinal plant extracts with efflux inhibitory activity against Gram-negative bacteria

It was hypothesised that extracts from plants that are used as herbal medicinal products contain inhibitors of efflux in Gram-negative bacteria. Extracts from 21 plants were screened by bioassay for synergy with ciprofloxacin against Salmonella enterica serotype Typhimurium, including mutants in which acrB and tolC had been inactivated. The most active extracts, fractions and purified compounds were further examined by minimum inhibitory concentration testing with five antibiotics for activity against Enterobacteriaceae and Pseudomonas aeruginosa. Efflux activity was determined using the fluorescent dye Hoechst 33342. Eighty-four extracts from 21 plants, 12 fractions thereof and 2 purified molecules were analysed. Of these, 12 plant extracts showed synergy with ciprofloxacin, 2 of which had activity suggesting efflux inhibition. The most active extract, from Levisticum officinale, was fractionated and the two fractions displaying the greatest synergy with the five antibiotics were further analysed. From these two fractions, falcarindiol and the fatty acids oleic acid and linoleic acid were isolated. The fractions and compounds possessed antibacterial activity especially for mutants lacking a component of AcrAB-TolC. However, no synergism was seen with the fractions or purified molecules, suggesting that a combination of compounds is required for efflux inhibition. These data indicate that medicinal plant extracts may provide suitable lead compounds for future development and possible clinical utility as inhibitors of efflux for various Gram-negative bacteria.

Antibacterial terpenes from the oleo-resin of Commiphora molmol (Engl.).

Two octanordammaranes, mansumbinone (1) and 3,4‐seco‐mansumbinoic acid (2), and two sesquiterpenes, β‐elemene (3) and T‐cadinol (4) have been isolated from the oleo‐resin of Commiphora molmol (Engl.). The structures of these compounds were established unambiguously by a series of 1D and 2D‐NMR analyses. We have also unambiguously assigned all 1H and 13C NMR resonances for 2 and revised its 13C data. The crude extract of the oleo‐resin of C. molmol displayed potentiation of ciprofloxacin and tetracycline against S. aureus, several Salmonella enterica serovar Typhimurium strains and two K. pneumoniae strains. The antibacterial activity of terpenes 1–4 was determined against a number of Staphylococcus aureus strains: SA1199B, ATCC25923, XU212, RN4220 and EMRSA15 and minimum inhibitory concentration (MIC) values were found to be in the range of 4–256 µg/ml. The highest activity was observed by the seco‐A‐ring octanordammarane 2 with an MIC of 4 µg/ml against SA1199B, a multidrug‐resistant strain which over‐expresses the NorA efflux transporter, the major characterized antibiotic pump in this species. This activity compared favorably to the antibiotic norfloxacin with an MIC of 32 µg/ml. Compound 2 also displayed weak potentiation of ciprofloxacin and tetracycline activity against strains of Salmonella enterica serovar Typhimurium SL1344 and L10. Copyright

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification

OBJECTIVES The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-TolC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide. METHODS The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT-PCR, ability to form a biofilm and motility assays. RESULTS Despite important differences in the triclosan-dependent transcriptomes of the two species, increased expression of efflux pump component genes was seen in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent. CONCLUSIONS These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents.

Overexpression of patA and patB, Which Encode ABC Transporters, Is Associated with Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae

ABSTRACT Fifty-seven clinical isolates of Streptococcus pneumoniae were divided into four groups based on their susceptibilities to the fluoroquinolones ciprofloxacin and norfloxacin and the dyes ethidium bromide and acriflavine. Comparative reverse transcription-PCR was used to determine the level of expression of the genes patA and patB, which encode putative ABC transporters. Overexpression was observed in 14 of the 15 isolates that were resistant to both fluoroquinolones and dyes and in only 3 of 24 of those resistant to fluoroquinolones only. Isolates overexpressing patA and patB accumulated significantly less of the fluorescent dye Hoechst 33342 than wild-type isolates, suggesting that PatA and PatB are involved in efflux. Inactivation of patA and patB by in vitro mariner mutagenesis conferred hypersusceptibility to ethidium bromide and acriflavine in all isolates tested and lowered the MICs of ciprofloxacin in the patAB-overproducing and/or fluoroquinolone-resistant isolates. These data represent the first observation of overexpression of patA and patB in clinical isolates and show that PatA and PatB play a clinically relevant role in fluoroquinolone resistance.

A Year in the Life of a Contaminated Heater-Cooler Unit With Mycobacterium chimaera?

OBJECTIVE Heater-cooler units (HCUs) have been shown to be a source of Mycobacterium chimaera infections. For the past year, weekly water samples have been taken from HCUs used at University Hospitals Birmingham (UHB) NHS Foundation Trust. We report the microbial contamination of the HCUs over a year detailing the decontamination regimes applied at UHB to reduce the microbial load. DESIGN Observational study SETTING UHB is a tertiary referral teaching hospital in Birmingham, United Kingdom, that provides clinical services to nearly 1 million patients every year. The UHB Cardiac department is one of the largest in the United Kingdom and provides treatment for adult patients with a wide range of cardiac diseases. METHODS Water samples taken from HCUs used at UHB for cardiopulmonary bypass surgery were sampled over a year to determine the number of microorganisms by membrane filtration. Various decontamination processes were employed throughout the year. RESULTS Varying total viable counts containing a wide variety of microorganisms were obtained from water inside the HCUs. No M. chimaera were isolated after replacement of the HCU internal tubing. Stringent decontamination regimes resulted in degradation of the HCUs and increased TVCs after several months. CONCLUSION More work is required to ensure effective decontamination processes to reduce the microbial load within the HCUs. Our studies indicate that weekly water sampling for TVC will be required indefinitely to monitor the water quality in these units as well as regular replacement of the tubing to control the build-up of biofilm. Infect Control Hosp Epidemiol 2017;38:705-711.

The Effect of Universal Decolonization With Screening in Critical Care to Reduce MRSA Across an Entire Hospital

OBJECTIVE To describe the effect of universal methicillin-resistant Staphylococcus aureus (MRSA) decolonization therapy in a large intensive care unit (ICU) on the rates of MRSA cases and acquisitions in a UK hospital. DESIGN Descriptive study. SETTING University Hospitals Birmingham (UHB) NHS Foundation Trust is a tertiary referral teaching hospital in Birmingham, United Kingdom, that provides clinical services to nearly 1 million patients every year. METHODS A break-point time series analysis and kernel regression models were used to detect significant changes in the cumulative monthly numbers of MRSA bacteremia cases and acquisitions from April 2013 to August 2016 across the UHB system. RESULTS Prior to 2014, all ICU patients at UHB received universal MRSA decolonization therapy. In August 2014, UHB discontinued the use of universal decolonization due to published reports in the United Kingdom detailing the limited usefulness and cost-effectiveness of such an intervention. Break-point time series analysis of MRSA acquisition and bacteremia data indicated that break points were associated with the discontinuation and subsequent reintroduction of universal decolonization. Kernel regression models indicated a significant increase (P<.001) in MRSA acquisitions and bacteremia cases across UHB during the period without universal decolonization. CONCLUSION We suggest that routine decolonization for MRSA in a large ICU setting is an effective strategy to reduce the spread and incidence of MRSA across the whole hospital. Infect Control Hosp Epidemiol 2017;38:430-435.

Unmasking leading to a healthcare worker Mycobacterium tuberculosis transmission

BACKGROUND Mycobacterium tuberculosis is a major health burden worldwide. The disease may present as an individual case, community outbreak, or more rarely as a nosocomial outbreak. Even in countries with a low prevalence such as the UK, tuberculosis (TB) presents a risk to healthcare workers (HCWs). AIM To report an outbreak which manifested 12 months after a patient with pulmonary tuberculosis was admitted to Queen Elizabeth Hospital Birmingham. METHODS We present the epidemiological and outbreak investigations; the role of whole genome sequencing (WGS) in identifying the outbreak and control measures to prevent further outbreaks. FINDINGS Subsequent to a diagnosis of open TB in a patient, transmission was confirmed in one HCW who had active TB; HCWs with latent TB infection (LTBI) were also identified among seven HCW contacts of the index patient. Of note, all the LBTI patients had other risk factors for TB. Routine use of WGS identified the outbreak link between the index patient and the HCW with active TB disease, and informed our investigations. CONCLUSION Exposure most likely occurred during an aerosol-generating procedure (AGP) which was done in accordance with national guidance at that time without using respiratory protection. Enhanced control measures were implemented following the outbreak.

Can a toxin gene NAAT be used to predict toxin EIA and the severity of Clostridium difficile infection

BackgroundDiagnosis of C. difficile infection (CDI) is controversial because of the many laboratory methods available and their lack of ability to distinguish between carriage, mild or severe disease. Here we describe whether a low C. difficile toxin B nucleic acid amplification test (NAAT) cycle threshold (CT) can predict toxin EIA, CDI severity and mortality.MethodsA three-stage algorithm was employed for CDI testing, comprising a screening test for glutamate dehydrogenase (GDH), followed by a NAAT, then a toxin enzyme immunoassay (EIA). All diarrhoeal samples positive for GDH and NAAT between 2012 and 2016 were analysed. The performance of the NAAT CT value as a classifier of toxin EIA outcome was analysed using a ROC curve; patient mortality was compared to CTs and toxin EIA via linear regression models.ResultsA CT value ≤26 was associated with ≥72% toxin EIA positivity; applying a logistic regression model we demonstrated an association between low CT values and toxin EIA positivity. A CT value of ≤26 was significantly associated (p = 0.0262) with increased one month mortality, severe cases of CDI or failure of first line treatment. The ROC curve probabilities demonstrated a CT cut off value of 26.6.DiscussionsHere we demonstrate that a CT ≤26 indicates more severe CDI and is associated with higher mortality. Samples with a low CT value are often toxin EIA positive, questioning the need for this additional EIA test.ConclusionsA CT ≤26 could be used to assess the potential for severity of CDI and guide patient treatment.