Design of Antibacterial Prochelators to Target Drug-Resistant Bacteria

Transition metals such as iron and copper are valued in biology for their redox activities because they are able to access various oxidation states. However, these transition metals are also implicated in a number of human disease states and play a role in bacterial infections. The ability to manipulate and monitor metal ions has vast implications on the fields of biology and human health. As such, the research described here covers two related goals: to manipulate metals in specific biological circumstances and to visualize this disturbance in cellular metal homeostasis.

Antibiotic resistance necessitates the development of drugs that exploit new mechanisms of action such as the disruption of metal homeostasis. In order to manipulate metals at the site of bacterial infection, two prochelators were developed around a β-lactam core such that the active chelator is released in the presence of bacteria that produce the resistance-causing β-lactamase enzyme. Both prochelators display enhanced activity toward resistant bacteria compared to clinical antibiotics.

Fluorescent sensors are a powerful tool for detecting small concentrations of biological analytes. Two analogs of a ratiometric fluorescent sensor were designed and synthesized to monitor cellular concentrations of copper and iron. These sensors were found to operate as designed in vitro; however the fluorescence intensity necessary for quantification of cellular metal pools has not yet been achieved.

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. RIF-resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes ≤2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-susceptible and RIF-resistant bacteria, is reader dependent and limits its clinical use. The aim of this study was to develop an objective, reader-independent method to define rpoB mutants using beacon qPCR. This would facilitate the transition from a research protocol to the clinical setting, where high-throughput methods with objective interpretation are required. For this, DNAs from 107 M. tuberculosis clinical isolates with known susceptibility to RIF by culture-based methods were obtained from 2 regions where isolates have not previously been subjected to evaluation using molecular beacon qPCR: the Texas–Mexico border and Colombia. Using coded DNA specimens, mutations within an 81-bp hot spot region of rpoB were established by qPCR with 5 beacons spanning this region. Visual and mathematical approaches were used to establish whether the qPCR cycle threshold of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and RIF-resistant bacteria. Only then had the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF-resistance by culture phenotype and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.^

Bacterial stimulation of copper phytoaccumulation by bioaugmentation with rhizosphere bacteria

Copper contaminated areas pose environmental health risk to living organisms. Remediation processes are thus required for both crop production and industrial activities. This study employed bioaugmentation with copper resistant bacteria to improve phytoremediation of vineyard soils and copper mining waste contaminated with high copper concentrations. Oatmeal plant (Avena sativa L) was used for copper phytoextraction. Three copper resistant bacterial isolates from oatmeal rhizosphere (Pseudomonas putida A1 Stenotrophomonas maltophilia A2 and Acinetobacter calcoaceticus A6) were used for the stimulation of copper phytoextraction. Two long-term copper contaminated vineyard soils (Mollisol and Inceptisol) and copper mining waste from Southern Brazil were evaluated. Oatmeal plants substantially extracted copper from vineyard soils and copper mining waste. As much as 1549 mg of Cu kg(-1) dry mass was extracted from plants grown in Inceptisol soil. The vineyard Mollisol copper uptake (55 mg Cu kg(-1) of dry mass) in the shoots was significantly improved upon inoculation of oatmeal plants with isolate A2 (128 mg of Cu kg(-1) of shoot dry mass). Overall oatmeal plant biomass displayed higher potential of copper phytoextraction with inoculation of rhizosphere bacteria in vineyard soil to the extent that 404 and 327 g ha(-1) of copper removal were respectively observed in vineyard Mollisol bioaugmented with isolate A2 (S. maltophilia) and isolate A6 (A. calcoaceticus). Results suggest potential application of bacterial stimulation of phytoaccumulation of copper for biological removal of copper from contaminated areas. (C) 2010 Elsevier Ltd. All rights reserved.

Resistance to β-lactams in bacteria isolated from different types of Portuguese cheese

The purpose of this study was to investigate the presence of beta-lactam-resistant bacteria in six different types of Portuguese cheese. The numbers of ampicillin resistant (AMP(r)) bacteria varied from 4.7 x 10(2) to 1.5 x 10(7) CFU/g. Within 172 randomly selected beta-lactam-resistant bacteria, 44 resistant phenotypes were found and 31.4% were multidrug resistant. The majority (85%) of the isolates identified belonged to the Enterobacteriaceae family. The presence of the bla(TEM) gene was detected in 80.9% of the tested isolates. The results suggest that without thermal processing of the milk and good hygienic practices, cheese may act as a vehicle of transfer of beta-lactam-resistant bacteria to the gastrointestinal tract of consumers.

Antimicrobial use, incidence, etiology and resistance patterns in bacteria causing ventilator-associated pneumonia in a clinical-surgical intensive care unit

INTRODUCTION : Antimicrobial resistance is an increasing threat in hospitalized patients, and inappropriate empirical antimicrobial therapy is known to adversely affect outcomes in ventilator-associated pneumonia (VAP). The aim of this study was to evaluate antimicrobial usage, incidence, etiology, and antimicrobial resistance trends for prominent nosocomial pathogens causing ventilator-associated pneumonia in a clinical-surgical intensive care unit (ICU). METHODS : Gram-negative bacilli and Staphylococcus aureus causing VAP, as well as their antimicrobial resistance patterns and data on consumption (defined daily dose [DDD] per 1,000 patient days) of glycopeptides, extended-spectrum cephalosporins, and carbapenems in the unit were evaluated in two different periods (A and B). RESULTS: Antimicrobial use was high, mainly of broad-spectrum cephalosporins, with a significant increase in the consumption of glycopeptides (p < 0.0001) and carbapenems (p < 0.007) in period B. For Acinetobacter baumannii and members of the Enterobacteriaceae family, 5.27- and 3.06-fold increases in VAPs, respectively, were noted, and a significant increase in resistance rates was found for imipenem-resistant A. baumannii (p = 0.003) and third-generation cephalosporins-resistant Enterobacteriaceae (p = 0.01) isolates in this same period. CONCLUSIONS: Our results suggest that there is a link between antibiotics usage at institutional levels and resistant bacteria. The use of carbapenems was related to the high rate of resistance in A. baumannii and therefore a high consumption of imipenem/meropenem could play a major role in selective pressure exerted by antibiotics in A. baumannii strains.

Antibacterial potentiality of Argemone mexicana solvent extracts against some pathogenic bacteria

The sensitivity of two Gram positive (Staphylococcus aureus and Bacillus subtilis) and two Gram negative (Escherichia coli and Pseudomonas aeruginosa) pathogenic multi-drug resistant bacteria was tested against the crude extracts (cold aqueous, hot aqueous, and methanol extracts) of leaves and seeds of Argemone mexicana L. (Papaveraceae) by agar well diffusion method. Though all the extracts were found effective, yet the methanol extract showed maximum inhibition against the test microorganisms followed by hot aqueous extract and cold aqueous extract.

Vancomycin-resistant enterococcus outbreak in a pediatric intensive care unit: report of successful interventions for control and prevention

The objective of this study is to retrospectively report the results of interventions for controlling a vancomycin-resistant enterococcus (VRE) outbreak in a tertiary-care pediatric intensive care unit (PICU) of a University Hospital. After identification of the outbreak, interventions were made at the following levels: patient care, microbiological surveillance, and medical and nursing staff training. Data were collected from computer-based databases and from the electronic prescription system. Vancomycin use progressively increased after March 2008, peaking in August 2009. Five cases of VRE infection were identified, with 3 deaths. After the interventions, we noted a significant reduction in vancomycin prescription and use (75% reduction), and the last case of VRE infection was identified 4 months later. The survivors remained colonized until hospital discharge. After interventions there was a transient increase in PICU length-of-stay and mortality. Since then, the use of vancomycin has remained relatively constant and strict, no other cases of VRE infection or colonization have been identified and length-of-stay and mortality returned to baseline. In conclusion, we showed that a bundle intervention aiming at a strict control of vancomycin use and full compliance with the Hospital Infection Control Practices Advisory Committee guidelines, along with contact precautions and hand-hygiene promotion, can be effective in reducing vancomycin use and the emergence and spread of vancomycin-resistant bacteria in a tertiary-care PICU.

Heavy metal tolerance patterns of total heterotrophic bacteria isolated from the soils of Mahatma Gandhi University campus, Kottayam, Kerala

Industrialization of our society has led to an increased production and discharge of both xenobiotic and natural chemical substances. Many of these chemicals will end up in the soil. Pollution of soils with heavy metals is becoming one of the most severe ecological and human health hazards. Elevated levels of heavy metals decrease soil microbial activity and bacteria need to develop different mechanisms to confer resistances to these heavy metals. Bacteria develop heavy-metal resistance mostly for their survivals, especially a significant portion of the resistant phenomena was found in the environmental strains. Therefore, in the present work, we check the multiple metal tolerance patterns of bacterial strains isolated from the soils of MG University campus, Kottayam. A total of 46 bacterial strains were isolated from different locations of the campus and tested for their resistant to 5 common metals in use (lead, zinc, copper, cadmium and nickel) by agar dilution method. The results of the present work revealed that there was a spatial variation of bacterial metal resistance in the soils of MG University campus, this may be due to the difference in metal contamination in different sampling location. All of the isolates showed resistance to one or more heavy metals selected. Tolerance to lead was relatively high followed by zinc, nickel, copper and cadmium. About 33% of the isolates showed very high tolerance (>4000μg/ml) to lead. Tolerance to cadmium (65%) was rather low (<100 μg/ml). Resistance to zinc was in between 100μg/ml - 1000μg/ml and the majority of them shows resistance in between 200μg/ml - 500μg/ml. Nickel resistance was in between 100μg/ml - 1000μg/ml and a good number of them shows resistance in between 300μg/ml - 400μg/ml. Resistance to copper was in between <100μg/ml - 500μg/ml and most of them showed resistance in between 300μg/ml - 400μg/ml. From the results of this study, it was concluded that heavy metal-resistant bacteria are widely distributed in the soils of MG university campus and the tolerance of heavy metals varied among bacteria and between locations

High prevalence of multidrug-tolerant bacteria and associated antimicrobial resistance genes isolated from ornamental fish and their carriage water

Background: Antimicrobials are used to directly control bacterial infections in pet (ornamental) fish and are routinely added to the water these fish are shipped in to suppress the growth of potential pathogens during transport. Methodology/Principal Findings: To assess the potential effects of this sustained selection pressure, 127 Aeromonas spp. isolated from warm and cold water ornamental fish species were screened for tolerance to 34 antimicrobials. Representative isolates were also examined for the presence of 54 resistance genes by a combination of miniaturized microarray and conventional PCR. Forty-seven of 94 Aeromonas spp. isolates recovered from tropical ornamental fish and their carriage water were tolerant to >= 15 antibiotics, representing seven or more different classes of antimicrobial. The quinolone and fluoroquinolone resistance gene, qnrS2, was detected at high frequency (37% tested recent isolates were positive by PCR). Class 1 integrons, IncA/C broad host range plasmids and a range of other antibiotic resistance genes, including floR, blaTEM21, tet(A), tet(D), tet(E), qacE2, sul1, and a number of different dihydrofolate reductase and aminoglycoside transferase coding genes were also detected in carriage water samples and bacterial isolates. Conclusions: These data suggest that ornamental fish and their carriage water act as a reservoir for both multi-resistant bacteria and resistance genes.

The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig

Aims: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracyclineresistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig. Methods and Results: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E. coli. Higher numbers of S. Typhimurium DT104 were shed from treated pigs than untreated pigs. This lasted up to 6 weeks post-treatment in the high-dose group. In this group, there was a 30% increase in E. coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l(-1) and a 10% increase in E. coli with an MIC > 50 mg l(-1) during and 2 weeks post-treatment. This effect was less-pronounced in the low-dose group. PCR identified the predominant tetracycline resistance genes in the E. coli as tetA, tetB and tetC. The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 mug g(-1) faeces during treatment. Conclusion: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time. Significance and Impact of the Study: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man.

Sources of variation in the ampicillin-resistant Escherichia coli concentration in the feces of organic broiler chickens

Currently, there are limited published data for the population dynamics of antimicrobial-resistant commensal bacteria. This study was designed to evaluate both the proportions of the Escherichia coli populations that are resistant to ampicillin at the level of the individual chicken on commercial broiler farms and the feasibility of obtaining repeated measures of fecal E. coli concentrations. Short-term temporal variation in the concentration of fecal E. coli was investigated, and a preliminary assessment was made of potential factors involved in the shedding of high numbers of ampicillin-resistant E. coli by growing birds in the absence of the use of antimicrobial drugs. Multilevel linear regression modeling revealed that the largest component of random variation in log-transformed fecal E. coli concentrations was seen between sampling occasions for individual birds. The incorporation of fixed effects into the model demonstrated that the older, heavier birds in the study were significantly more likely (P = 0.0003) to shed higher numbers of ampicillin-resistant E. coli. This association between increasing weight and high shedding was not seen for the total fecal E. coli population (P = 0.71). This implies that, in the absence of the administration of antimicrobial drugs, the proportion of fecal E. coli that was resistant to ampicillin increased as the birds grew. This study has shown that it is possible to collect quantitative microbiological data on broiler farms and that such data could make valuable contributions to risk assessments concerning the transfer of resistant bacteria between animal and human populations.

Antimicrobial resistance of heterotrophic marine bacteria isolated from seawater and sands of recreational beaches with different organic pollution levels in southeastern Brazil: evidences of resistance dissemination

Antimicrobial resistance of marine heterotrophic bacteria to different antimicrobials agents were evaluated in seawater, dry and wet sands from three marine recreational beaches with different pollution levels. In all studied beaches, the greatest frequencies of resistance were found in relation to penicillin. on Gonzaguinha, the most polluted beach, 72.3% of all isolated strains showed simple resistance, whilst 8.33% had multiple resistance. The values found on Ilha Porchat beach, were 70.8% and 6.9% for simple and multiple resistances, respectively. on GuaraA(0), the less polluted beach, only 35.3% of isolated strains had simple resistance. Multiple resistance was not observed. While samples from Gonzaguinha and Ilha Porchat beach showed isolated strains resistant to seven and six different antimicrobial agents, respectively, samples from GuaraA(0) beach were resistant only to penicillin and erytromicin. The positive correlations obtained between the degree of seawater contamination and frequency and variability of bacterial resistance indicate that polluted marine recreational waters and sands are sources of resistant bacteria contributing thus, to the dissemination of bacterial resistance.

Effect of low-dose gaseous ozone on pathogenic bacteria

Background: Treatment of chronically infected wounds is a challenge, and bacterial environmental contamination is a growing issue in infection control. Ozone may have a role in these situations. The objective of this study was to determine whether a low dose of gaseous ozone/oxygen mixture eliminates pathogenic bacteria cultivated in Petri dishes. Methods: A pilot study with 6 bacterial strains was made using different concentrations of ozone in an ozone-oxygen mixture to determine a minimally effective dose that completely eliminated bacterial growth. The small and apparently bactericidal gaseous dose of 20 mu g/mL ozone/oxygen (1: 99) mixture, applied for 5min under atmospheric pressure was selected. In the 2nd phase, eight bacterial strains with well characterized resistance patterns were evaluated in vitro using agar-blood in adapted Petri dishes (10(5) bacteria/dish). The cultures were divided into 3 groups: 1-ozone-oxygen gaseous mixture containing 20 mu g of O-3/mL for 5 min; 2- 100% oxygen for 5 min; 3- baseline: no gas was used. Results: The selected ozone dose was applied to the following eight strains: Escherichia coli, oxacillin-resistant Staphylococcus aureus, oxacillin-susceptible Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii, Acinetobacter baumannii susceptible only to carbapenems, and Pseudomonas aeruginosa susceptible to imipenem and meropenem. All isolates were completely inhibited by the ozone-oxygen mixture while growth occurred in the other 2 groups. Conclusion: A single topical application by nebulization of a low ozone dose completely inhibited the growth of all potentially pathogenic bacterial strains with known resistance to antimicrobial agents.

Vancomycin-resistant enterococcus outbreak in a pediatric intensive care unit: report of successful interventions for control and prevention

The objective of this study is to retrospectively report the results of interventions for controlling a vancomycin-resistant enterococcus (VRE) outbreak in a tertiary-care pediatric intensive care unit (PICU) of a University Hospital. After identification of the outbreak, interventions were made at the following levels: patient care, microbiological surveillance, and medical and nursing staff training. Data were collected from computer-based databases and from the electronic prescription system. Vancomycin use progressively increased after March 2008, peaking in August 2009. Five cases of VRE infection were identified, with 3 deaths. After the interventions, we noted a significant reduction in vancomycin prescription and use (75% reduction), and the last case of VRE infection was identified 4 months later. The survivors remained colonized until hospital discharge. After interventions there was a transient increase in PICU length-of-stay and mortality. Since then, the use of vancomycin has remained relatively constant and strict, no other cases of VRE infection or colonization have been identified and length-of-stay and mortality returned to baseline. In conclusion, we showed that a bundle intervention aiming at a strict control of vancomycin use and full compliance with the Hospital Infection Control Practices Advisory Committee guidelines, along with contact precautions and hand-hygiene promotion, can be effective in reducing vancomycin use and the emergence and spread of vancomycin-resistant bacteria in a tertiary-care PICU.