Hinton Broth Research Articles

Synthesis, characterization and biological evaluation of 4-oxo-thiazolidine compounds

A series of compounds 4-oxo-thiazolidine derivatives AJ5a–j were synthesized by condensation reactions. The structure of synthesized compounds was characterized by IR, 1H NMR, 13C NMR and mass spectroscopy. The present paper deals with newly synthesized thiazolidine compounds AJ5a–AJ5j that were screened for their antimicrobial activity against different strains of bacteria and fungi using serial broth dilution method (Mueller–Hinton broth dilution method). In vitro antitubercular activity of compounds AJ5a–j was carried out against Mycobacterium tuberculosis H37Rv. Compounds AJ5a, AJ5d, AJ5e, AJ5f and AJ5g were found most active against selected bacterial strains (MIC=62.5μg/mL) and compounds AJ5a–j were found moderately active against M. tuberculosis.

Evaluation of deodorized yellow mustard concentrations for control of Escherichia coli O157:H7 viability in dry fermented sausage

Although 6% (w/w) deodorized mustard powder can achieve the mandatory 5 Log CFU/g reduction of Escherichia coli O157:H7 in 18 d during dry-fermented sausage manufacture, sensory analysis showed that mustard concentrations lower than 6% were more acceptable to consumers. In the present work the antimicrobial effectiveness of lower levels of deodorized (autoclave-treated) mustard was evaluated against E. coli O157:H7. Concentrations of 2% and 4% (w/w) mustard treated at 115 °C for 15 min were first tested in Mueller–Hinton broth (14 d) and then during fermented sausage manufacture (42 d) against E. coli O157:H7. During tests in broth, 4% deodorized mustard yielded a significantly greater reduction in E. coli O157:H7 numbers than 2% at 14 d. Mixtures of autoclave-treated and hot mustard at 1% or 2% (w/w) each were equally or slightly less antimicrobial against E. coli O157:H7 in sausage than their corresponding concentrations (2% or 4%) of deodorized mustard. While 4% deodorized mustard in sausage reduced E. coli O157:H7 viability by 5 Log at 28 d, 2% deodorized mustard did not achieve the reduction target. Mustard addition influenced starter culture recovery by causing a reduction in Staphylococcus carnosus numbers, but did not change Pediococcus pentosaceus viability during tests. It is probable that a minimum threshold between 3 and 4% (w/w) deodorized mustard may be required to assure control of E. coli O157:H7 viability in dry-fermented sausages.

Effect of Seawater on the Activity of Antibiotics Against Vibrios Isolated from the Hemolymph of Cultured Pacific White Shrimp

The purpose of this study was to evaluate the effect of seawater (SW) on the activity of antibiotics belonging to 10 families (aminoglycosides, aminopenicillins, carbapenems, β‐lactams, chloramphenicols, monobactams, nitrofurans, quinolones, sulfonamides, and tetracyclines) against Vibrio strains isolated from hemolymph of Pacific white shrimp, Litopenaeus vannamei, farmed in Northeastern Brazil and standard strain Vibrio cholera ATCC 19582. Susceptibility of the strains to antibiotics was determined by disk diffusion method and the minimum inhibitory concentration was determined by macrodilution method. The media Mueller–Hinton agar and broth used in the above methods were diluted in distilled water (control, 1% NaCl, pH 7.5) and SW (2.5% NaCl, pH 7.5). The antibiotics most affected by dilution in SW were tetracycline, penicillin, cephalothin, aztreonam, ampicillin, and imipenem, as indicated by a considerable increase in the number of strains classified as intermediate or resistance. Thus, in this study, the efficiency of these antibiotics on Vibrio strains was found to be reduced by contact with SW.

Clinical significance of infections caused by plasmid-mediated AmpC β-lactamases and extended-spectrum β-lactamase-producing Escherichia coli

Escherichia coli frequently causes both nosocomial and community-acquired infections, and antimicrobial resistance in E. coli is a growing problem worldwide. Resistance to broad-spectrum antibiotics in E. coli is often mediated by the production of extended-spectrum b-lactamases (ESBLs) [1]. In recent years, some E. coli strains have been isolated that harbor plasmid-mediated AmpC genes imported from other Enterobacteriaceae carrying chromosomal AmpC genes, such as Citrobacter freundii, Enterobacter cloacae, and Aeromonas species [2]. Recent studies on plasmid-mediated AmpC b-lactamase (PAB)producing E. coli have shown only the prevalence of PAB [3–6]. Furthermore, most previous studies regarding PABproducing E. coli have involved all clinical isolates, including colonized pathogens [1, 4–6]. Thus, we investigated the clinical features and clinical outcomes of patients with PAB-producing E. coli bacteremia. Also, we conducted this study in order to delineate the differences in the clinical manifestations and outcome of E. coli bacteremia between ESBL and non-ESBL groups, and to evaluate the impact of ESBL-producing organisms on outcome in E. coli bacteremia. We reviewed the medical records of individuals diagnosed with E. coli bacteremia from July 2006 to August 2007 at Samsung Medical Center, Seoul, Republic of Korea, a 1,950-bed tertiary-care university hospital. Patients were included in the study if their blood culture results were positive for E. coli. Only the first bacteremic episode for each patient was included in this study. E. coli bacteremia was defined as a growth of E. coli in a blood culture specimen. Nosocomial infection was defined as an infection that occurred [72 h after admission to the hospital. Community-onset infections occurring B72 h after admission were further classified as healthcare-associated if any of the established criteria were present [7]. We considered antimicrobial therapy to be inappropriate if the drugs used did not have in vitro activity against the isolates or if the patient did not receive antimicrobial therapy. The bacterial isolates were obtained from the Asian Bacterial Bank (ABB) of the Asia Pacific Foundation for Infectious Disease (APFID). In vitro antimicrobial susceptibility testing of the isolates was performed in Mueller–Hinton broth (Becton–Dickinson, Sparks, MD) by the broth microdilution method in accordance with the guidelines established by the Clinical and Laboratory Standards Institute (CLSI). Isolates with resistance to cefoxitin were considered to be positive for AmpC b-lactamase. Cefoxitin-positive isolates were further tested using the previously described polymerase chain reaction (PCR) primers and conditions [6]. ESBL production was confirmed in screening-positive isolates via a double-disk synergy test using BD BBL Sensi-Disc (Becton–Dickinson). Doubledisk synergy test-positive isolates were further tested by PCR and sequence analyses to determine the gene This study was presented, in part, at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), San Francisco, CA, USA, September 2009 (abstract 2413).

Survival of human pathogenic bacteria in different types of natural mineral water

The aim of this study was to determine the survival of human pathogens (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) in five natural mineral waters (NMWs) with different properties and mineralization levels. Five NMWs from four Spanish spas with different dry residue at 110 °C were used: A = 76,935 mg/L; B = 1,827 mg/L; C = 808.4 mg/L; D = 283.8 mg/L; and E = 170.4 mg/L. An initial inoculum of 1 × 10(6) colony forming units (cfu)/mL was used for survival studies. Distilled water, chlorinated tap water and Mueller-Hinton broth were used as controls. Colony counts in all different waters were lower than those achieved with Mueller-Hinton broth over all incubation periods. A direct effect between the bacterial survival and the level of mineralization water was observed. The NMW E with low mineralization level along with the radioactive properties showed the highest antibacterial activity among all NMWs.

Modified resazurin microtiter assay for in vitro and in vivo assessment of sulfamonomethoxine activity against the fish pathogen Nocardia seriolae

Resazurin microtiter assay (REMA) was carried out using four sulfonamides, three culture media, and four inoculum sizes as a first screening step to establish an easy-to-interpret sulfonamides susceptibility testing method for Nocardia seriolae. The in vitro activity of sulfamonomethoxine (SMM) against 190 clinical N. seriolae isolates was then examined, and in vivo experimental treatment was performed. When the culture medium and the inoculum size were considered in tandem, a 0.5× the original concentration of cation-adjusted Mueller–Hinton broth and an inoculum size of 102 CFU/well showed the clearest endpoint reading for all tested drugs, and the REMA-generated data were in excellent agreement with those generated by the reference Etest method. SMM activity showed minimum inhibitory concentration (MIC) values of 4–32 μg/ml against all tested N. seriolae isolates. Treatment of amberjack groups experimentally infected with N. seriolae isolates having SMM MICs of 4 and 32 μg/ml, resulted in survival rates of 100% and 87.5% in the two groups, respectively. In this study, we developed a simple visual method to test SMM activity against N. seriolae.

Antimicrobial activity of Amazonian oils against Paenibacillus species

The Gram-positive, spore-forming bacterium Paenibacillus larvae is the primary bacterial pathogen of honeybee brood and the causative agent of American foulbrood disease (AFB). One of the feasible alternative treatments being used for their control of this disease is essential oils. In this study in vitro antimicrobial activity of Andiroba and Copaíba essential oils against Paenibacillus species, including P. larvae was evaluated. Minimal inhibitory concentration (MIC) in Mueller–Hinton broth by the microdilution method was assessed. Andiroba registered MIC values of 1.56–25%, while the MICs values obtained for Copaíba oil were of 1.56–12.5%. In order to determine the time-response effect of essential oils on P. larvae, this microorganism was exposed to the oils for up to 48h. After 24h treatment with Andiroba oil and after 48h treatment with Copaíba oil no viable cells of P. larvae ATCC 9545 were observed. The possible toxic effect of essential oils were assessed by the spraying application method of the same concentrations of MICs. Bee mortality was evident only in treatment with Andiroba oil and the Copaíba oil shows no toxic effects after 10days of observation. Taking together ours results showed for the first time that these oils presented a high activity against Paenibacillus species showing that Copaíba oil may be a candidate for the treatment or prevention of AFB.

Determination of minimum inhibitory and minimum bactericidal concentrations of tiamulin against field isolates of Actinobacillus pleuropneumoniae

Tiamulin activity was measured against 19 UK field isolates of Actinobacillus pleuropneumoniae collected between 2003 and 2009 and the type strain ATCC 27090 as a control, with the intention of comparing broth with serum as growth media. Broth microdilution MIC/MBC tests were performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline M31-A3, in ‘Veterinary Fastidious Medium’ (VFM) (supplemented Mueller–Hinton broth at pH 7.3) and in 100% swine serum. For improved precision, a modified, overlapping doubling-dilution series was used (tiamulin concentration range 0.3–72 μg/ml). The MBC was reported as the lowest concentration producing a 99.9% reduction in bacterial density in the sub-cultured well contents, relative to the starting inoculum. The mean MBC/MIC ratio for tiamulin against A. pleuropneumoniae in VFM was low (1.74:1), even though tiamulin is classed as a bacteriostatic drug. Only three of the 19 isolates and the reference strain grew in 100% serum and their MICs were higher than those determined in VFM. It is postulated that this difference was due to differences in pH of the matrices or binding of tiamulin to serum proteins or a combination of both factors.

Effect of multidrug resistance-conferring mutations on the fitness and virulence of Pseudomonas aeruginosa

Multidrug resistance has become a quandary in the treatment of bacterial infections. The effect of resistance mutations and the fitness cost on the pathogenicity of Pseudomonas aeruginosa is not well established. The objective of this study was to examine the impact of multidrug resistance on the fitness and virulence of P. aeruginosa. Fourteen P. aeruginosa strains with various resistance mechanisms were used. In vitro growth of these isolates was investigated in full-strength and 0.25-strength Mueller-Hinton broth (MHB). Exponential growth rates were estimated from serial bacterial burden over 24 h. In vitro growth of two multidrug-resistant strains (PAO1ΔmexRΔoprD and PA9019) was studied when each was grown in co-culture with wild-type strain PAO1. In vivo growth was compared between PAO1 and PAO1ΔmexRΔopD using a murine pneumonia model; virulence over 10 days was studied in six isolates. Significant reduction in growth rate was observed in selected mutants (P < 0.01). PAO1 out-competed PAO1ΔmexRΔoprD and PA9019 in vitro, and in vivo growth of PAO1 was faster than PAO1ΔmexRΔoprD. Compared with PAO1, PAO1ΔmexR and PAO1ΔoprD showed a slight reduction in mortality rate; significantly lower mortality was seen in PAO1ΔmexRΔoprD (P < 0.01). However, virulence of PA9019 was not significantly different from that of PAO1. Specific resistance mutations were associated with fitness cost in P. aeruginosa, and accumulation of such mutations was associated with a reduction in virulence. However, it was difficult to predict the impact in clinical isolates. Knowledge of multidrug resistance mechanisms and compensatory mutations would likely be helpful.

Activity of simulated serum concentrations of daptomycin versus vancomycin during the first 24 h of treatment in the presence of physiological albumin concentrations against vancomycin-susceptible, -tolerant or -intermediate-resistant Staphylococcus aureus

In order to determine whether reduced susceptibility or tolerance to vancomycin in Staphylococcus aureus influences the activity of daptomycin by simulating serum concentrations in the first 24h of treatment in the presence of physiological concentrations of human albumin, a computerised pharmacodynamic simulation was performed using Mueller–Hinton broth with 4g/dL human albumin concentrations. For daptomycin, the media was adjusted to physiological ionised calcium concentrations by adding 100μg/mL Ca2+. Protein binding was measured. Six S. aureus isolates were used, comprising one vancomycin-susceptible S. aureus (VSSA), three vancomycin-tolerant strains, one heteroresistant vancomycin-intermediate S. aureus (hVISA) and one homogeneous vancomycin-intermediate S. aureus (VISA). Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of daptomycin increased eight times when determined in the presence of albumin (MICALB and MBCALB, respectively). Measured protein binding was 86.6% (Cmax) and 86.5% (Cmin) for daptomycin and 51.6% (Cmax) and 42.2% (Cmin) for vancomycin. Similar values were obtained for fAUC/MIC (where fAUC is the area under the concentration–time curve obtained with extrapolated concentrations using the highest protein binding rate experimentally obtained) and AUC/MICALB for each antibiotic. Daptomycin showed early (≤6h) bactericidal activity [maximal effect (Emax) >4log10 reductions in initial inocula] against all strains. Vancomycin produced an Emax of 2.3log10 reductions at 8h against the VSSA and reductions ≤1.8log10 for the other strains in the 8–24h period. Pharmacodynamic parameters were fAUC/MBC from 8.0 to 15.6 (vancomycin) and from 56.0 to 111.6 (daptomycin) for tolerant strains, and fAUC/MIC of 126.8 and 63.3 for vancomycin and 222.6 and 113.2 for daptomycin against hVISA and VISA strains, respectively. Against the study strains (vancomycin-susceptible, -tolerant, heteroresistant or intermediate), daptomycin, in contrast to vancomycin, exhibited early bactericidal activity despite its high protein binding.

Impact of pH on bacterial growth and activity of recent fluoroquinolones in pooled urine

Acidification of urine is widely recommended for prevention and treatment of urinary tract infections. We set out to describe the effect of modification of pH on bacterial growth of relevant bacteria as well as on activity of modern fluoroquinolones in urine in vitro. Bacterial growth of Escherichia coli ATCC 25922 and Klebsiella oxytoca ATCC 700324 was determined in pooled human urine adjusted to pH levels between 5.0 and 8.0. Minimal inhibitory concentrations (MICs) and time–kill curves were performed for ciprofloxacin, levofloxacin and moxifloxacin in pH-adjusted urine and Mueller–Hinton Broth (MHB). Uptake of radioactive labeled [C 14]-ciprofloxacin into bacterial cells was investigated at different pHs. While no difference in bacterial growth of E. coli and K. oxytoca was observed at pH values between 5.0 and 8.0, acidification of urine led to major impairment of antimicrobial activity of all tested fluoroquinolones, indicated by an up to 40-fold increase in MIC compared to MHB and nearly total neutralization of activity in time–kill experiments. The most probable mechanism behind this observation may have been reduced uptake of fluoroquinolones into bacterial cells, as indicated by bacterial uptake of [C 14]-ciprofloxacin and a reversibility of the effect. The observed reduction in activity of modern fluoroquinolones confirms previous observations from older compounds.

Plasma protein binding may reduce antimicrobial activity by preventing intra-bacterial uptake of antibiotics, for example clindamycin

although plasma protein binding (PPB) is accepted to be an essential factor in reducing antimicrobial activity, little is known about the underlying mechanisms. One possibility includes impaired penetration of an antimicrobial into bacterial cells in the presence of PPB. As a prerequisite for testing this hypothesis an optimized medium displaying high protein binding without impairing bacterial growth had to be identified for our model compound clindamycin. determination of PPB, bacterial growth and antimicrobial killing was performed in Mueller-Hinton broth (MHB) containing various amounts of human albumin or serum. [(3)H]clindamycin was used to investigate clindamycin penetration into Staphylococcus aureus. of all investigated media only MHB(50%serum) and MHB(70%serum) achieved protein binding comparable to pure serum. In contrast, MHB(20%serum) and most media containing only albumin demonstrated considerably lower protein binding. Pure serum resulted in bacterial growth inhibition compared with MHB while MHB(16%albumin) and MHB(50%serum) did not result in significant differences in bacterial count after 24 h. However, in both MHB(16%albumin) and MHB(50%serum) the antimicrobial activity of clindamycin was reduced by >2 log(10) cfu/mL compared with pure MHB. The radioactive signal after administration of [(3)H]clindamycin to S. aureus was significantly decreased in pure serum as well as in MHB(16%albumin) and MHB(50%serum), while no significant difference was observed for MHB(4%albumin) and MHB(20%serum). reduction of the intracellular radioactive signal in the presence of serum proteins correlated both with the degree of protein binding and reduction of antimicrobial activity supporting the hypothesis of impairment of activity by PPB by reducing intra-bacterial antimicrobial concentrations.

Measuring antimicrobial susceptibility of Pseudomonas aeruginosa using Poloxamer 407 gel

Pseudomonas aeruginosa is a Gram-negative bacterium that causes various opportunistic infections. Chronic and intractable infections with P. aeruginosa are closely related to the high levels of resistance displayed by this organism to antimicrobial agents and its ability to form biofilms. Although the standard method for examining antimicrobial resistance involves susceptibility testing using Mueller-Hinton agar or broth, this method does not take into account the influence of biofilm formation on antimicrobial susceptibility. Poloxamer 407 is a hydrophilic, nonionic surfactant of the more general class of copolymers that can be used to culture bacteria with similar properties as cells in a biofilm environment. Therefore, the aim of this study was to compare the antimicrobial susceptibility of bacteria cultured in Poloxamer 407 gel to those grown on Mueller-Hinton agar using the Kirby-Bauer disk diffusion method with 24 strains of P. aeruginosa. Antimicrobial sensibility differed between the two mediums, with >60% of the strains displaying increased resistance to β-lactams when cultured on Poloxamer 407 gel. In addition, scanning electron microscopy revealed that typical biofilm formation and extracellular polymeric substance production was only observed with bacteria grown on Poloxamer 407 gel. Therefore, antimicrobial susceptibility test using Poloxamer 407 gel may provide more accurate information and allow the selection of suitable antimicrobial agents for treating patients infected with biofilm-forming pathogens.

Human infection associated with methicillin-resistant Staphylococcus pseudintermedius ST71

Sir, Staphylococcus pseudintermedius is one of the most common pathogens isolated from skin and post-operative infections in dogs and cats and can also occasionally cause infections in humans. People working or living with animals are more likely to be colonized with S. pseudintermedius in their nasal cavities and human infection acquired from dogs has been recently reported. In recent years, there have been increasing numbers of infections in dogs and cats caused by methicillin-resistant S. pseudintermedius (MRSP) and a predominant MRSP clone is disseminating in dogs and cats throughout Europe. This MRSP clone belongs to the multilocus sequence type (MLST) ST71, spa type t02 and SmaI PFGE group J (ST71-t02-J) and contains the staphylococcal cassette chromosome element SCCmec II–III. It displays resistance to many classes of antibiotics and represents a challenge for therapy. Here, we report, to our knowledge, the first case of a human infection caused by MRSP ST71 emphasizing its zoonotic potential and therapeutic challenge. An adult patient presented to the doctor in 2009 with headache, watery eyes and a small oedema over the right eye. The patient had a history of recurrent rhinosinusitis and three surgical interventions had been performed over the previous 6 years, the last one in 2005. CT illustrated an obliteration of the right sinus frontalis with an expansive mucocele. Yet another surgical intervention was absolutely essential and post-operative treatment for 3 weeks with 500 mg of ciprofloxacin twice per day and 300 mg of clindamycin three times per day was given. Five weeks post-operatively, a purulent infection appeared. After 10 days of treatment with 875 mg of amoxicillin/125 mg of clavulanic acid twice daily and 300 mg of clindamycin three times daily and no improvement, a sample was sent for bacteriological analysis. The laboratory identified a multidrug-resistant Staphylococcus belonging to the Staphylococcus intermedius group (SIG) using the ID 32 STAPH system (bioMerieux, Marcy l’Etoile, France) and disc diffusion susceptibility testing (Oxoid Ltd, Basingstoke, UK). Based on these results, a local treatment with fusidic acid gauze and application of 2% mupirocin ointment four times a day for 8 days followed by a topical glucocorticoid/antibiotic combination therapy (0.5 mg/g fluocinonide, 2.5 mg/g neomycin, 0.25 mg/g gramicidin and 100000 IU/g nystatin) packing for 4 days improved the local condition. Six weeks after the purulent infection, the wound had healed with scarring and no bacterium was detectable in a repeat smear. Local follow-up treatment with steroids and routine check-ups confirmed the absence of residual infection. The multidrug-resistant Staphylococcus isolate was further identified as MRSP ST71-t02-J containing SCCmec II–III using MLST typing, spa typing, PFGE and SCCmec typing methods described previously. MICs of antibiotics were determined in Mueller–Hinton broth using custom Sensititre susceptibility NLV73 plates (Trek Diagnostics System, East Grinstead, UK) except for rifampicin, trimethoprim, kanamycin and fusidic acid, which were tested using home-made microbroth dilution plates. Antibiotic resistance genes were detected using a microarray. Mutations in topoisomerase genes gyrA and grlA were identified by sequencing of PCR products (Table 1). The isolate was susceptible to the antibiotics amikacin, chloramphenicol, fusidic acid, linezolid, nitrofurantoin, the combination quinupristin/dalfopristin, rifampicin and vancomycin (Table 1). The patient owned a male dog that needed home care because of various clinical problems such as diabetes, recurrent warts and abdominal tumour. The dog had consultations in various clinics and underwent several antibiotic treatments (details of substances could not be ascertained). The dog was euthanized before samples could be taken to determine whether it was a carrier of the strain. The patient also owned horses and cats; all of them were in good health without clinical signs. Even if the ultimate source of the MRSP infection could not be identified, the patient was infected with the same MRSP clone (ST71-t02-J with SCCmec II–III) that has been disseminating in dogs over Europe in recent years. A case similar to the one reported here has been described in the USA, where a woman developed sinusitis caused by a methicillinresistant SIG whose origin could be attributed to her pet dog. In that case, vancomycin and linezolid were used for treatment. The increasing number of dogs carrying MRSP constitutes a risk for pet owners to become colonized with MRSP, and MRSP infections in humans may also increase in the near future. It is therefore important to recognize MRSP as a zoonotic pathogen and have antibiotics available for the treatment of MRSP infections in humans. Therefore, antibiotics such as mupirocin, linezolid, quinupristin/dalfopristin, rifampicin and vancomycin that are used for decolonization or as ‘last resort antibiotics’ against methicillin-resistant staphylococci in humans should not be used for treatment in animals.

In vitro activity of oritavancin against community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin-non-susceptible S. aureus (DNSSA)

Isolates of community-associated meticillin-resistant Staphylococcus aureus (CA-MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA) and daptomycin non-susceptible S. aureus (DNSSA) are increasing in frequency and new antistaphylococcal therapies are needed. Microdilution testing using Mueller–Hinton broth was used to determine the minimal inhibitory concentrations (MICs) of oritavancin and nine additional antimicrobial agents against 92 CA-MRSA, 23 VISA, 7 DNSSA and 10 VRSA isolates. Minimal bactericidal concentrations were also determined. Pulsed-field gel electrophoresis (PFGE) was performed. Staphylococcal cassette chromosome mec (SCC mec) typing as well as assays for Panton–Valentine leukocidin (PVL) and arginine catabolic mobile element (ACME) genes were performed. Oritavancin was more bactericidal than any of the other comparators against CA-MRSA and demonstrated excellent activity against VRSA and VISA.

Squalamine, an original chemosensitizer to combat antibiotic-resistant Gram-negative bacteria

Sir, The worldwide dissemination of multidrug-resistant (MDR) pathogens has severely reduced the efficacy of our antibiotic arsenal and increased the frequency of therapeutic failure. To reduce the intracellular accumulation of antibiotics, a key bacterial response is to stringently control membrane transporters involved in the diffusion of drugs through the envelope into the cell and those involved in the expulsion of antibiotics, the so-called ‘in and out’ flux. In Gram-negative bacteria, the outer membrane is an additional barrier to antibiotic penetration. Numerous antibiotics that are active against Grampositive are much less active against Gram-negative bacteria. An attractive approach to circumvent bacterial resistance is the development of a chemosensitizer agent to promote an increase of the internal antibiotic concentration in resistant strains. This agent will be used in combination with classic antibiotics to bypass the mechanical and enzymatic barriers that reduce the intracellular concentration of the active antibacterial drug. Squalamine exhibits interesting antimicrobial activity, suggesting its potential application to combat resistant pathogens. It modifies membrane integrity by increasing permeability, as demonstrated by ATP release and dye entry in Gram-negative bacteria, and it has a moderate cytotoxicity at doses that kill MDR bacterial pathogens. The aim of this study was to evaluate the effect of subinhibitory concentrations of squalamine on the activity of various antibiotic classes against different resistant bacteria. The chemosensitizer activity of squalamine was studied by determining the MICs of various usual antibiotics in the presence of subinhibitory concentrations of squalamine. These concentrations corresponded to 1/5 and 1/10 of the respective squalamine MICs determined for each bacterial strain. A control was included by using the well-described efflux pump inhibitor phenylalanine arginine b-naphthylamide (PAbN) to evaluate the involvement of efflux. The strains were grown on Mueller–Hinton broth (Becton Dickinson) at 378C. The Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae and Pseudomonas aeruginosa strains used have been previously described. Results were read after 18 h at 378C and the MIC values are the means of three independent experiments. The results for documented E. coli strains exhibiting various porins and efflux pump profiles indicated that, even at the low subinhibitory concentration tested (MIC/10), squalamine had an effect on the activity of various different antibiotic classes (Table 1). A significant reduction in chloramphenicol, tetracycline and ciprofloxacin MICs was observed for AG100 and AG100Atet in the presence of squalamine. AG100Atet is an acrAB2 strain that overproduces several drug efflux pumps and down-regulates porin expression. An effect was also obtained on cefepime susceptibility, indicating that squalamine may favour the uptake of this b-lactam molecule via the permeabilizing effect previously reported. It is worthwhile mentioning that squalamine also increases the activity of tested antibiotics against susceptible cells, AG100 and AG100A, demonstrating that the effect is not focused on a resistance mechanism but is related to increased drug penetration. The MICs of erythromycin were not significantly modified by the addition of squalamine, indicating the prominent role of efflux mechanisms for this antibiotic group. In the K. pneumoniae KP55 clinical isolate, the MICs determined in the presence of squalamine were significantly decreased, e.g. 16–32-fold for chloramphenicol, ciprofloxacin, tetracycline and cefepime (Table 1). Squalamine was more efficient at increasing tetracycline and cefepime activity against E. aerogenes than PAbN, highlighting the role of uptake in susceptibility to the two antibiotics. The effect of squalamine on strains devoid of porin (KP63 and KP55) indicated an increase in penetration for cefepime and ciprofloxacin, which normally use porin channels to pass the membrane barrier. Regarding P. aeruginosa and E. aerogenes strains, the results similarly suggest that when the mechanical barrier, restricted uptake or efficient efflux pump participates in resistance, squalamine is able to significantly increase bacterial susceptibility. Squalamine has an effect on the susceptible strains (E. coli, E. aerogenes and P. aeruginosa) tested in this study, suggesting that squalamine may chemosensitize the membrane of nonresistant bacteria and promote the use of a decreased amount of antibiotics. We have previously demonstrated that the alteration of lipopolysaccharide involved in polymyxin-resistant clinical isolates only moderately changes squalamine susceptibility. Consequently, the use of squalamine against polymyxin-resistant isolates selected during colistin treatment, a therapy that is now proposed for the MDR phenotype, may be an attractive hypothesis for the development of future drug combinations. In the bacterial resistance context, and because of its action and its relative insensitivity to efflux resistance mechanisms, squalamine may be a fruitful partner for the development of combinations, such as b-lactams/b-lactamase inhibitors, to combat MDR pathogens. This use allows us to complete our new strategies by promoting the penetration (influx) of antibiotics, in parallel with inhibitors of efflux pumps andb-lactamase inhibitors. This aspect is especially important, since some recent molecules having an original antibacterial spectrum exhibit restricted diffusion through the Research letters

In vitro antibacterial properties of magnesium metal against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus

Bacterial infections are a costly sequela in any wound. The corrosion properties of 0.15, 0.30, 0.45 and 0.60 g of Mg metal were determined in Mueller–Hinton broth by serially measuring the Mg 2+ concentrations and pH over 72 h. In addition, the effect of Mg metal, increased Mg 2+ concentration and alkaline pH on the in vitro growth of Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were evaluated in three separate experiments. The primary outcome measure for culture studies was colony-forming units/ml compared to appropriate positive and/or negative controls. Regardless of the mass of Mg added, there was a predictable increase in pH and Mg 2+ concentration. The addition of Mg and an increase of pH resulted in antibacterial effects similar to the fluoroquinolone antibiotic; however, a simple increase in Mg 2+ concentration alone had no effect. The results demonstrate an antibacterial effect of Mg on three common aerobic bacterial organisms, the mechanism of which appears to be an alkaline pH.

Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller-Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline's activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.

Effects of thymol and diphenyliodonium chloride against Campylobacter spp. during pure and mixed culture in vitro

To determine if the purported deaminase inhibitors diphenyliodonium chloride (DIC) and thymol reduce the growth and survivability of Campylobacter. Growth rates of Campylobacter jejuni and Camp. coli were reduced compared to unsupplemented controls during culture in Muellar-Hinton broth supplemented with 0.25 micromol DIC or thymol ml(-1) but not with 0.01 micromol monensin ml(-1) or 1% ethanol. Recovery of Camp. jejuni and Camp. coli was reduced >5 log(10) CFU from controls after 24 h pure culture in Bolton broth supplemented with 0.25 or 1.0 micromol DIC ml(-1) or with 1.0 micromol thymol ml(-1). Similarly, each test Campylobacter strain was reduced >3 log(10) CFU from controls after 24 h mixed culture with porcine faecal microbes in Bolton broth supplemented with 0.25 or 1.0 micromol DIC ml(-1) or with 1.0 micromol thymol ml(-1). Treatments with 0.25 micromol thymol ml(-1), 0.01 micromol monensin ml(-1) or 1% ethanol were less effective. Ammonia production during culture or incubation of cell lysates was reduced by 0.25 or 1.0 micromol DIC ml(-1) but only intermittently reduced, if at all, by the other treatments. Diphenyliodonium chloride and thymol reduced growth, survivability and ammonia production of Camp. jejuni and Camp. coli. Results suggest a potential physiological characteristic that may be exploited to develop interventions.

Synergistic interactions between cefalexin and kanamycin in Mueller-Hinton broth medium and in milk

This study investigated the in vitro bactericidal activity of an intramammary drug product by comparing the kill kinetics of cefalexin and kanamycin, alone and in fixed ratio combination, against Streptococcus uberis, Staphylococcus aureus and Escherichia coli strains isolated from field cases of bovine mastitis. The effect of milk as a diluent on the rate of bacterial killing was also assessed. Antibacterial kill kinetics was determined against each bacterial strain in Mueller-Hinton broth (MHB) and in milk. In MHB, the fixed cefalexin : kanamycin combination (1.5 : 1 w/w) exhibited a clear synergistic bactericidal activity against the strains tested. The combination also showed an enhanced killing activity in milk, as compared to either agent alone. The data show the occurrence of synergistic interactions between cefalexin and kanamycin, resulting in a faster and enhanced bactericidal activity against major mastitis pathogens. The study demonstrated that the combination exhibited a larger and faster rate of kill of S. aureus, S. uberis and E. coli compared to either cefalexin or kanamycin alone, while using a lower total amount of antibiotic. Synergistic and additive effects were also observed when milk was used as a medium. The results support the use of this combination of narrow spectrum antibiotics to treat clinical mastitis via the intramammary route and provide data on its killing kinetics.