Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones

Our previous paper (Wilkinson et al, 2016) used high-resolution cryo-electron microscopy to solve the structure of the Escherichia coli RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in E. coli or Klebsiella pneumoniae increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in K. pneumoniae clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy

Comparison of standardised versus non-standardised methods for testing the in vitro potency of oxytetracycline against mannheimia haemolytica and pasteurella multocida

The in vitro pharmacodynamics of oxytetracycline was established for six isolates of each of the calf pneumonia pathogens Mannheimia haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and bacterial time-kill curves were determined in two matrices, Mueller Hinton broth (MHB) and calf serum. Geometric mean MIC ratios, serum:MHB, were 25.2:1 (M. haemolytica) and 27.4:1 (P. multocida). The degree of binding of oxytetracycline to serum protein was 52.4%. Differences between serum and broth MICs could not be accounted for by oxytetracycline binding to serum protein. In vitro time-kill data suggested a co-dependent killing action of oxytetracycline. The in vitro data indicate inhibition of the killing action of oxytetracycline by serum factor(s). The nature of the inhibition requires further study. The outcome of treatment with oxytetracycline of respiratory tract infections in calves caused by M. haemolytica and P. multocida may not be related solely to a direct killing action

Prevalence and Predictors of Urinary Tract Infection and Severe Malaria Among Febrile Children Attending Makongoro Health Centre in Mwanza City, North-Western Tanzania.

In malaria endemic areas, fever has been used as an entry point for presumptive treatment of malaria. At present, the decrease in malaria transmission in Africa implies an increase in febrile illnesses related to other causes among underfives. Moreover, it is estimated that more than half of the children presenting with fever to public clinics in Africa do not have a malaria infection. Thus, for a better management of all febrile illnesses among under-fives, it becomes relevant to understand the underlying aetiology of the illness. The present study was conducted to determine the relative prevalence and predictors of P. falciparum malaria, urinary tract infections and bacteremia among under-fives presenting with a febrile illness at the Makongoro Primary Health Centre, North-Western Tanzania. From February to June 2011, a cross-sectional analytical survey was conducted among febrile children less than five years of age. Demographic and clinical data were collected using a standardized pre-tested questionnaire. Blood and urine culture was done, followed by the identification of isolates using in-house biochemical methods. Susceptibility patterns to commonly used antibiotics were investigated using the disc diffusion method. Giemsa stained thin and thick blood smears were examined for any malaria parasites stages. A total of 231 febrile under-fives were enrolled in the study. Of all the children, 20.3% (47/231, 95%CI, 15.10-25.48), 9.5% (22/231, 95%CI, 5.72-13.28) and 7.4% (17/231, 95%CI, 4.00-10.8) had urinary tract infections, P. falciparum malaria and bacteremia respectively. In general, 11.5% (10/87, 95%CI, 8.10-14.90) of the children had two infections and only one child had all three infections. Predictors of urinary tract infections (UTI) were dysuria (OR = 12.51, 95% CI, 4.28-36.57, P < 0.001) and body temperature (40-41 C) (OR = 12.54, 95% CI, 4.28-36.73, P < 0.001). Predictors of P. falciparum severe malaria were pallor (OR = 4.66 95%CI, 1.21-17.8, P = 0.025) and convulsion (OR = 102, 95% CI, 10-996, P = 0.001). Escherichia coli were the common gram negative isolates from urine (72.3%, 95% CI, 66.50-78.10) and blood (40%, 95%CI, and 33.70-46.30). Escherichia coli from urine were 100% resistant to ampicillin, 97% resistant to co-trimoxazole, 85% resistant to augmentin and 32.4% resistant to gentamicin; and they were 100%, 91.2% and 73.5% sensitive to meropenem, ciprofloxacin and ceftriaxone respectively. Urinary tract infection caused by multi drug resistant Escherichia coli was the common cause of febrile illness in our setting. Improvement of malaria diagnosis and its differential diagnosis from other causes of febrile illnesses may provide effective management of febrile illnesses among children in Tanzania

Extended-spectrum ß-lactamase and carbapenemaseproducing Aeromonas species in wild animals from Portugal

Aeromonas are Gram-negative, facultative-anaerobic, non-spore-forming, glucose-fermenting, oxidase- and catalase-positive rods (Martin-Carnahan and Joseph 2005). Apart from fish, which are widely reported hosts for Aeromonads, insects, crustaceans, reptiles, birds and mammals were also found to harbour Aeromonas species, both in healthy and disease state (Pearson and others 2000, Turutoglu and others 2005, Evangelista-Barreto and others 2006, Ceylan and others 2009). An increase in resistance levels of the genus, particularly to -lactam antimicrobial agents, has been observed not only in clinical isolates, but also in environmental strains (Saavedra and others 2004, 2007).C Dias was the recipient of grant SFRH/BD/90120/2012 from the Fundação para a Ciência e Tecnologia (FCT) under QREN – POPH- Potencial Humano, Tipologia 4.1 – Formação avançada da União Europeia, and strategic research project PEst-OE/AGR/ UI0772/2014

Repurposing drugs to fast-track therapeutic agents for the treatment of cryptococcosis

Many infectious diseases disproportionately affect people in the developing world. Cryptococcal meningitis is one of the most common mycoses in HIV-AIDS patients, with the highest burden of disease in sub-Saharan Africa. Current best treatment regimens still result in unacceptably high mortality rates, and more effective antifungal agents are needed urgently. Drug development is hampered by the difficulty of developing effective antifungal agents that are not also toxic to human cells, and by a reluctance among pharmaceutical companies to invest in drugs that cannot guarantee a high financial return. Drug repurposing, where existing drugs are screened for alternative activities, is becoming an attractive approach in antimicrobial discovery programs, and various compound libraries are now commercially available. As these drugs have already undergone extensive optimisation and passed regulatory hurdles this can fast-track their progress to market for new uses. This study screened the Screen-Well Enzo library of 640 compounds for candidates that phenotypically inhibited the growth of Cryptococcus deuterogattii. The anthelminthic agent flubendazole, and L-type calcium channel blockers nifedipine, nisoldipine and felodipine, appeared particularly promising and were tested in additional strains and species. Flubendazole was very active against all pathogenic Cryptococcus species, with minimum inhibitory concentrations of 0.039-0.156 μg/mL, and was equally effective against isolates that were resistant to fluconazole. While nifedipine, nisoldipine and felodipine all inhibited Cryptococcus, nisoldipine was also effective against Candida, Saccharomyces and Aspergillus. This study validates repurposing as a rapid approach for finding new agents to treat neglected infectious diseases

Colistin-sparing regimens against Klebsiella pneumoniae carbapenemase-producing K. pneumoniae isolates: Combination of tigecycline or doxycycline and gentamicin or amikacin

AbstractBackground/PurposeIn vitro studies of the combination of an aminoglycoside with tigecycline or doxycycline against Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae isolates are rarely published. The goal of this study was to evaluate the antibacterial activity of the combination regimens.MethodsThirteen genetically different KPC-producing K. pneumoniae isolates were randomly selected. Drug concentrations of amikacin, gentamicin, tigecycline, and doxycycline were adjusted to 1-, 1/2-, and 1/4-fold of respective minimum inhibitory concentrations (MICs). Each drug alone or the combinations of amikacin or gentamicin with tigecycline or doxycycline were tested by combination studies.ResultsTreatment with the 1× MIC concentration in combinations of amikacin or gentamicin and tigecycline or doxycycline for 24 hours resulted in bactericidal activity of 84–100% in the isolates. Treatment with 1/2× MIC combinations resulted in synergism of 69–100% in the isolates. Notably, doxycycline plus gentamicin or amikacin was synergistic for all tested isolates. However, bactericidal or synergistic effect was barely evident following 1/4× MIC combinations. There was no antagonism in any of the combination regimens.ConclusionEnhanced activity was noted following treatment with doxycycline combined with gentamicin or amikacin against KPC-producing K. pneumoniae isolates, warranting further in vitro and animal investigations before clinical application

ICEApl1, an integrative conjugative element related to ICEHin1056, identified in the pig pathogen Actinobacillus pleuropneumoniae

ICEApl1 was identified in the whole genome sequence of MIDG2331, a tetracycline-resistant (MIC = 8 mg/L) serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. PCR amplification of virB4, one of the core genes involved in conjugation, was used to identify other A. pleuropneumoniae isolates potentially carrying ICEApl1. MICs for tetracycline were determined for virB4 positive isolates, and shotgun whole genome sequence analysis was used to confirm presence of the complete ICEApl1. The sequence of ICEApl1 is 56083 bp long and contains 67 genes including a Tn10 element encoding tetracycline resistance. Comparative sequence analysis was performed with similar integrative conjugative elements (ICEs) found in other members of the Pasteurellaceae. ICEApl1 is most similar to the 59393 bp ICEHin1056, from Haemophilus influenzae strain 1056. Although initially identified only in serovar 8 isolates of A. pleuropneumoniae (31 from the UK and 1 from Cyprus), conjugal transfer of ICEApl1 to representative isolates of other serovars was confirmed. All isolates carrying ICEApl1 had a MIC for tetracycline of 8 mg/L. This is, to our knowledge, the first description of an ICE in A. pleuropneumoniae, and the first report of a member of the ICEHin1056 subfamily in a non-human pathogen. ICEApl1 confers resistance to tetracycline, currently one of the more commonly used antibiotics for treatment and control of porcine pleuropneumonia

Wild Morchella conica Pers. from different origins: a comparative study of nutritional and bioactive properties

Morchella conica Pers. is a species of fungus that belongs to the Morchellaceae family and was studied in order to obtain more information about this species, by comparing Portuguese and Serbian wild samples. Free sugars, fatty acids, tocopherols, organic and phenolic acids were analysed by chromatographic techniques. M. conica methanolic extracts were tested regarding antioxidant and antimicrobial properties. The absence of hepatotoxicity was confirmed in porcine liver primary cells.The authors are grateful to Fundação para a Ciência e Tecnologia (FCT, Portugal) and COMPETE/QREN/EU for financial support to this work (research project PTDC/AGR-ALI/110062/2009 and BPD/4609/2008 to L. Barros) and to CIMO (strategic project PEst-OE/AGR/UI0690/2011). The authors also thank Serbian Ministry of Education, Science and Technological Development for financial support (grant number 173032)