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1.  Antibiotic Prescriptions in Critically-Ill Patients: A Latin American Experience 
Background:
It is widely acknowledged that the presence of infection is an important outcome determinant for intensive care unit (ICU) patients. In fact, antibiotics are one of the most common therapies administered in the ICU settings.
Aim:
To evaluate the current usage of antibiotics in Latin American ICUs.
Subjects and Methods:
A one-day p-oint prevalence study to investigate the patterns of antibiotic was undertaken in 72 Latin American (LA) ICUs. Data was analyzed using the Statistix 8 statistical software, version 2.0 (USA). Results were expressed as proportions. When applicable, two tailed hypothesis testing for difference in proportions was used (Proportion Test); a P value of <0.05 was considered significant.
Results:
Of 704 patients admitted, 359 received antibiotic treatment on the day of the study (51%), of which 167/359 cases (46.5%) were due to hospital-acquired infections. The most frequent infection reorted was nosocomial pneumonia (74/359, 21%). Only in 264/359 patients (73.5%), cultures before starting antibiotic treatment were performed. Thirty-eight percent of the isolated microorganisms were Enterobacteriaceae extended-spectrum β-lactamase-producing, 11% methicillin-resistant Staphylococcus aureus and 10% carbapenems-resistant non-fermentative Gram-negatives. The antibiotics most frequently prescribed were carbapenems (125/359, 35%), alone or in combination with vancomycin or other antibiotic. There were no significant differences in the “restricted” antibiotic prescription (carbapenems, vancomycin, piperacillin–tazobactam, broad-spectrum cephalosporins, fluoroquinolones, tigecycline and linezolid) between patients with APACHE II score at the beginning of the antibiotic treatment <15 [83/114 (72.5%)] and ≥15 [179/245 (73%)] (P = 0.96). Only 29% of the antibiotic treatments were cultured directed (104/359).
Conclusion:
Carbapenems (alone or in combination) were the most frequently prescribed antibiotics in LA ICUs. However, the problem of carbapenem resistance in LA requires that physicians improve the use of this class of antibiotics. Our findings show that our web-based method for collection of one-day point prevalence was implemented successfully. However, based on the limitations of the model used, the results of this study must be taken with caution.
doi:10.4103/2141-9248.113666
PMCID: PMC3728867  PMID: 23919194
Antibiotics; Carbapenems; Intensive care unit; Usage
2.  Mortality and Hospital Stay Associated with Resistant Staphylococcus aureus and Escherichia coli Bacteremia: Estimating the Burden of Antibiotic Resistance in Europe 
PLoS Medicine  2011;8(10):e1001104.
The authors calculate excess mortality, excess hospital stay, and related hospital expenditure associated with antibiotic-resistant bacterial bloodstream infections (Staphylococcus aureus and Escherichia coli) in Europe.
Background
The relative importance of human diseases is conventionally assessed by cause-specific mortality, morbidity, and economic impact. Current estimates for infections caused by antibiotic-resistant bacteria are not sufficiently supported by quantitative empirical data. This study determined the excess number of deaths, bed-days, and hospital costs associated with blood stream infections (BSIs) caused by methicillin-resistant Staphylococcus aureus (MRSA) and third-generation cephalosporin-resistant Escherichia coli (G3CREC) in 31 countries that participated in the European Antimicrobial Resistance Surveillance System (EARSS).
Methods and Findings
The number of BSIs caused by MRSA and G3CREC was extrapolated from EARSS prevalence data and national health care statistics. Prospective cohort studies, carried out in hospitals participating in EARSS in 2007, provided the parameters for estimating the excess 30-d mortality and hospital stay associated with BSIs caused by either MRSA or G3CREC. Hospital expenditure was derived from a publicly available cost model. Trends established by EARSS were used to determine the trajectories for MRSA and G3CREC prevalence until 2015. In 2007, 27,711 episodes of MRSA BSIs were associated with 5,503 excess deaths and 255,683 excess hospital days in the participating countries, whereas 15,183 episodes of G3CREC BSIs were associated with 2,712 excess deaths and 120,065 extra hospital days. The total costs attributable to excess hospital stays for MRSA and G3CREC BSIs were 44.0 and 18.1 million Euros (63.1 and 29.7 million international dollars), respectively. Based on prevailing trends, the number of BSIs caused by G3CREC is likely to rapidly increase, outnumbering the number of MRSA BSIs in the near future.
Conclusions
Excess mortality associated with BSIs caused by MRSA and G3CREC is significant, and the prolongation of hospital stay imposes a considerable burden on health care systems. A foreseeable shift in the burden of antibiotic resistance from Gram-positive to Gram-negative infections will exacerbate this situation and is reason for concern.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Antimicrobial resistance—a consequence of the use and misuse of antimicrobial medicines—occurs when a microorganism becomes resistant (usually by mutation or acquiring a resistance gene) to an antimicrobial drug to which it was previously sensitive. Then standard treatments become ineffective, leading to persistent infections, which may spread to other people. With some notable exceptions such as TB, HIV, malaria, and gonorrhea, most of the disease burden attributable to antimicrobial resistance is caused by hospital-associated infections due to opportunistic bacterial pathogens. These bacteria often cause life-threatening or difficult-to-manage conditions such as deep tissue, wound, or bone infections, or infections of the lower respiratory tract, central nervous system, or blood stream. The two most frequent causes of blood stream infections encountered worldwide are Staphylococcus aureus and Escherichia coli.
Why Was This Study Done?
Although hospital-associated infections have gained much attention over the past decade, the overall effect of this growing phenomenon on human health and medical services has still to be adequately quantified. The researchers proposed to fill this information gap by estimating the impact—morbidity, mortality, and demands on health care services—of antibiotic resistance in Europe for two types of resistant organisms that are typically associated with resistance to multiple classes of antibiotics and can be regarded as surrogate markers for multi-drug resistance—methicillin-resistant S. aureus and third-generation cephalosporin-resistant E. coli.
What Did the Researchers Do and Find?
Recently, the Burden of Resistance and Disease in European Nations project collected representative data on the clinical impact of antimicrobial resistance throughout Europe. Using and combining this information with 2007 prevalence data from the European Antibiotic Resistance Surveillance System, the researchers calculated the burden of disease associated with methicillin-resistant S. aureus and third-generation cephalosporin-resistant E. coli blood stream infections. This burden of disease was expressed as excess number of deaths, excess number of days in hospital, and excess costs. Using statistical models, the researchers predicted trend-based resistance trajectories up to 2015 for the 31 participating countries in the European region.
The researchers included 1,293 hospitals from the 31 countries, typically covering 47% of all available acute care hospital beds in most countries, in their analysis. For S. aureus, the estimated number of blood stream infections totaled 108,434, of which 27,711 (25.6%) were methicillin-resistant. E. coli caused 163,476 blood stream infections, of which 15,183 (9.3%) were resistant to third-generation cephalosporins. An estimated 5,503 excess deaths were associated with blood stream infections caused by methicillin-resistant S. aureus (with the UK and France predicted to experience the highest excess mortality), and 2,712 excess deaths with blood stream infections caused by third-generation cephalosporin-resistant E. coli (predicted to be the highest in Turkey and the UK). The researchers also found that blood stream infections caused by both methicillin-resistant S. aureus and third-generation cephalosporin-resistant E. coli contributed respective excesses of 255,683 and 120,065 extra bed-days, accounting for an estimated extra cost of 62.0 million Euros (92.8 million international dollars). In their trend analysis, the researchers found that 97,000 resistant blood stream infections and 17,000 associated deaths could be expected in 2015, along with increases in the lengths of hospital stays and costs. Importantly, the researchers estimated that in the near future, the burden of disease associated with third-generation cephalosporin-resistant E. coli is likely to surpass that associated with methicillin-resistant S. aureus.
What Do These Findings Mean?
These findings show that even though the blood stream infections studied represent only a fraction of the total burden of disease associated with antibiotic resistance, excess mortality associated with these infections caused by methicillin-resistant S. aureus and third-generation cephalosporin-resistant E. coli is high, and the associated prolonged length of stays in hospital imposes a considerable burden on health care systems in Europe. Importantly, a possible shift in the burden of antibiotic resistance from Gram-positive to Gram-negative infections is concerning. Such forecasts suggest that despite anticipated gains in the control of methicillin-resistant S. aureus, the increasing number of infections caused by third-generation cephalosporin-resistant Gram-negative pathogens, such as E. coli, is likely to outweigh this achievement soon. This increasing burden will have a big impact on already stretched health systems.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001104.
The World Health Organization has a fact sheet on general antimicrobial resistance
The US Centers for Disease Control and Prevention webpage on antibiotic/antimicrobial resistance includes information on educational campaigns and resources
The European Centre for Disease Control provides data about the prevalence of resistance in Europe through an interactive database
doi:10.1371/journal.pmed.1001104
PMCID: PMC3191157  PMID: 22022233
3.  Update on the appropriate use of linezolid in clinical practice 
Multi-antibiotic resistant Gram-positive cocci, which include Staphylococcus aureus, the coagulase-negative staphylococcal group, Enterococcus faecalis and Enterococcus faecium, and other streptococci, represent emerging pathogens especially in the setting of the immunocompromised, hospitalized patients, in particular when surgery, invasive procedures, or prosthetic implants are of concern, patients are admitted in intensive care units, or underlying chronic disorders and immunodeficiency are of concern, and broad-spectrum antibiotics or immunosuppressive drugs are widely administered. During the recent years, the phenomenon of multiresistant Gram-positive cocci is spreading to the community, where the retrieval of such microorganism is progressively increasing. The spectrum of available antimicrobial compounds for an effective management of these relevant infections is significantly impaired in selection and clinical efficacy by the emerging and spread of methicillin-resistant and more recently glycopeptide-resistant Gram-positive microbial strains. The first oxazolidinone derivative linezolid, together with the recently licensed quinupristin–dalfopristin, daptomycin, and tigecycline, followed by a number of glycopeptides, fluoroquinolones, and other experimental compounds on the pipeline, represent an effective response to the great majority of these concerns, due to their innovative mechanisms of action, their maintained or enhanced activity against multiresistant pathogens, their effective pharmacokinetic/pharmacodynamic properties, their frequent possibility of synergistic activity with other compounds effective against Gram-positive pathogens, and a diffuse potential for a safe and easy administration, also when compromised patients are of concern. The main problems related to the epidemiological and clinical features of multiresistant Gram-positive infection, the potential clinical indications of all recently available compounds compared with the standard of care of treatment of resistant Gram-positive infections, and updated data on efficacy and tolerability of linezolid as the golden standard compound for vancomycin-resistant Gram-positive cocci in multiple clinical situations, are outlined and updated on the ground of an extensive review of all the available, recent evidences coming from the international literature.
PMCID: PMC1936365  PMID: 18360656
resistant Gram-positive organisms; staphylococci; enterococci; oxazolidinones; linezolid
4.  New antibiotics for bad bugs: where are we? 
Bacterial resistance to antibiotics is growing up day by day in both community and hospital setting, with a significant impact on the mortality and morbidity rates and the financial burden that is associated. In the last two decades multi drug resistant microorganisms (both hospital- and community-acquired) challenged the scientific groups into developing new antimicrobial compounds that can provide safety in use according to the new regulation, good efficacy patterns, and low resistance profile. In this review we made an evaluation of present data regarding the new classes and the new molecules from already existing classes of antibiotics and the ongoing trends in antimicrobial development. Infectious Diseases Society of America (IDSA) supported a proGram, called “the ′10 × ´20′ initiative”, to develop ten new systemic antibacterial drugs within 2020. The microorganisms mainly involved in the resistance process, so called the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and enterobacteriaceae) were the main targets. In the era of antimicrobial resistance the new antimicrobial agents like fifth generation cephalosporins, carbapenems, monobactams, β-lactamases inhibitors, aminoglycosides, quinolones, oxazolidones, glycopeptides, and tetracyclines active against Gram-positive pathogens, like vancomycin-resistant S. aureus (VRSA) and MRSA, penicillin-resistant streptococci, and vancomycin resistant Enterococcus (VRE) but also against highly resistant Gram-negative organisms are more than welcome. Of these compounds some are already approved by official agencies, some are still in study, but the need of new antibiotics still does not cover the increasing prevalence of antibiotic-resistant bacterial infections. Therefore the management of antimicrobial resistance should also include fostering coordinated actions by all stakeholders, creating policy guidance, support for surveillance and technical assistance.
doi:10.1186/1476-0711-12-22
PMCID: PMC3846448  PMID: 23984642
New antibiotics; Resistance; Bacteria; FDA; EMA
5.  Current Epidemiology and Growing Resistance of Gram-Negative Pathogens 
In the 1980s, Gram-negative pathogens appeared to have been beaten by oxyimino-cephalosporins, carbapenems, and fluoroquinolones. Yet these pathogens have fought back, aided by their membrane organization, which promotes the exclusion and efflux of antibiotics, and by a remarkable propensity to recruit, transfer, and modify the expression of resistance genes, including those for extended-spectrum β-lactamases (ESBLs), carbapenemases, aminoglycoside-blocking 16S rRNA methylases, and even a quinolone-modifying variant of an aminoglycoside-modifying enzyme. Gram-negative isolates -both fermenters and non-fermenters-susceptible only to colistin and, more variably, fosfomycin and tigecycline, are encountered with increasing frequency, including in Korea. Some ESBLs and carbapenemases have become associated with strains that have great epidemic potential, spreading across countries and continents; examples include Escherichia coli sequence type (ST)131 with CTX-M-15 ESBL and Klebsiella pneumoniae ST258 with KPC carbapenemases. Both of these high-risk lineages have reached Korea. In other cases, notably New Delhi Metallo carbapenemase, the relevant gene is carried by promiscuous plasmids that readily transfer among strains and species. Unless antibiotic stewardship is reinforced, microbiological diagnosis accelerated, and antibiotic development reinvigorated, there is a real prospect that the antibiotic revolution of the 20th century will crumble.
doi:10.3904/kjim.2012.27.2.128
PMCID: PMC3372794  PMID: 22707882
Enterobacteriaceae; Pseudomonas; Acinetobacter; β-lactamase; Carbapenemase
6.  Antibiotic use and resistance in emerging economies: a situation analysis for Viet Nam 
BMC Public Health  2013;13:1158.
Background
Antimicrobial resistance is a major contemporary public health threat. Strategies to contain antimicrobial resistance have been comprehensively set forth, however in developing countries where the need for effective antimicrobials is greatest implementation has proved problematic. A better understanding of patterns and determinants of antibiotic use and resistance in emerging economies may permit more appropriately targeted interventions.
Viet Nam, with a large population, high burden of infectious disease and relatively unrestricted access to medication, is an excellent case study of the difficulties faced by emerging economies in controlling antimicrobial resistance.
Methods
Our working group conducted a situation analysis of the current patterns and determinants of antibiotic use and resistance in Viet Nam. International publications and local reports published between 1-1-1990 and 31-8-2012 were reviewed. All stakeholders analyzed the findings at a policy workshop and feasible recommendations were suggested to improve antibiotic use in Viet Nam.
Here we report the results of our situation analysis focusing on: the healthcare system, drug regulation and supply; antibiotic resistance and infection control; and agricultural antibiotic use.
Results
Market reforms have improved healthcare access in Viet Nam and contributed to better health outcomes. However, increased accessibility has been accompanied by injudicious antibiotic use in hospitals and the community, with predictable escalation in bacterial resistance. Prescribing practices are poor and self-medication is common – often being the most affordable way to access healthcare. Many policies exist to regulate antibiotic use but enforcement is insufficient or lacking.
Pneumococcal penicillin-resistance rates are the highest in Asia and carbapenem-resistant bacteria (notably NDM-1) have recently emerged. Hospital acquired infections, predominantly with multi-drug resistant Gram-negative organisms, place additional strain on limited resources. Widespread agricultural antibiotic use further propagates antimicrobial resistance.
Conclusions
Future legislation regarding antibiotic access must alter incentives for purchasers and providers and ensure effective enforcement. The Ministry of Health recently initiated a national action plan and approved a multicenter health improvement project to strengthen national capacity for antimicrobial stewardship in Viet Nam. This analysis provided important input to these initiatives. Our methodologies and findings may be of use to others across the world tackling the growing threat of antibiotic resistance.
doi:10.1186/1471-2458-13-1158
PMCID: PMC4116647  PMID: 24325208
Antibiotic resistance; Bacterial diseases; Health policy; Health systems; Legislation (health); Resource constrained; Antibiotic consumption
7.  Outcome of infections due to pandrug-resistant (PDR) Gram-negative bacteria 
Background
The increasing problem of infections due to multidrug-resistant Gram-negative bacteria has led to re-use of polymyxins in several countries. However, there are already clinical isolates of Gram-negative bacteria that are resistant to all available antibiotics, including polymyxins.
Methods
We present a case series of patients with infections due to pathogens resistant to all antimicrobial agents tested, including polymyxins. An isolate was defined as pandrug-resistant (PDR) if it exhibited resistance to all 7 anti-pseudomonal antimicrobial agents, i.e. antipseudomonal penicillins, cephalosporins, carbapenems, monobactams, quinolones, aminoglycosides, and polymyxins.
Results
Clinical cure of the infection due to pandrug-resistant (PDR) Gram-negative bacteria, namely Pseudomonas aeruginosa or Klebsiella pneumoniae was observed in 4 out of 6 patients with combination of colistin and beta lactam antibiotics.
Conclusion
Colistin, in combination with beta lactam antibiotics, may be a useful agent for the management of pandrug-resistant Gram-negative bacterial infections. The re-use of polymyxins, an old class of antibiotics, should be done with caution in an attempt to delay the rate of development of pandrug-resistant Gram-negative bacterial infections.
doi:10.1186/1471-2334-5-24
PMCID: PMC1087841  PMID: 15819983
8.  Whole-Animal Chemical Screen Identifies Colistin as a New Immunomodulator That Targets Conserved Pathways 
mBio  2014;5(4):e01235-14.
ABSTRACT
The purpose of this study was to take advantage of the nematode Caenorhabditis elegans to perform a whole-animal chemical screen to identify potential immune activators that may confer protection against bacterial infections. We identified 45 marketed drugs, out of 1,120 studied compounds, that are capable of activating a conserved p38/PMK-1 mitogen-activated protein kinase pathway required for innate immunity. One of these drugs, the last-resort antibiotic colistin, protected against infections by the Gram-negative pathogens Yersinia pestis and Pseudomonas aeruginosa but not by the Gram-positive pathogens Enterococcus faecalis and Staphylococcus aureus. Protection was independent of the antibacterial activity of colistin, since the drug was administered prophylactically prior to the infections and it was also effective against antibiotic-resistant bacteria. Immune activation by colistin is mediated not only by the p38/PMK-1 pathway but also by the conserved FOXO transcription factor DAF-16 and the transcription factor SKN-1. Furthermore, p38/PMK-1 was found to be required in the intestine for immune activation by colistin. Enhanced p38/PMK-1-mediated immune responses by colistin did not reduce the bacterial burden, indicating that the pathway plays a role in the development of host tolerance to infections by Gram-negative bacteria.
IMPORTANCE
The innate immune system represents the front line of our defenses against invading microorganisms. Given the ever-increasing resistance to antibiotics developed by bacterial pathogens, the possibility of boosting immune defenses represents an interesting, complementary approach to conventional antibiotic treatments. Here we report that the antibiotic colistin can protect against infections by a mechanism that is independent of its microbicidal activity. Prophylactic treatment with colistin activates a conserved p38/PMK-1 pathway in the intestine that helps the host better tolerate a bacterial infection. Since p38/PMK-1-mediated immune responses appear to be conserved from plants to mammals, colistin may also activate immunity in higher organisms, including humans. Antibiotics with immunomodulatory properties have the potential of improving the long-term outcome of patients with chronic infectious diseases.
doi:10.1128/mBio.01235-14
PMCID: PMC4145682  PMID: 25118236
9.  Infections Caused by Stenotrophomonas maltophilia in Recipients of Hematopoietic Stem Cell Transplantation 
Frontiers in Oncology  2014;4:232.
Stenotrophomonas maltophilia (S. maltophilia) is a globally emerging Gram-negative bacillus that is widely spread in environment and hospital equipment. Recently, the incidence of infections caused by this organism has increased, particularly in patients with hematological malignancy and in recipients of hematopoietic stem cell transplantation (HSCT) having neutropenia, mucositis, diarrhea, central venous catheters or graft versus host disease and receiving intensive cytotoxic chemotherapy, immunosuppressive therapy, or broad-spectrum antibiotics. The spectrum of infections in HSCT recipients includes pneumonia, urinary tract and surgical site infection, peritonitis, bacteremia, septic shock, and infection of indwelling medical devices. The organism exhibits intrinsic resistance to many classes of antibiotics including carbapenems, aminoglycosides, most of the third-generation cephalosporins, and other β-lactams. Despite the increasingly reported drug resistance, trimethoprim-sulfamethoxazole is still the drug of choice. However, the organism is still susceptible to ticarcillin-clavulanic acid, tigecycline, fluoroquinolones, polymyxin-B, and rifampicin. Genetic factors play a significant role not only in evolution of drug resistance but also in virulence of the organism. The outcome of patients having S. maltophilia infections can be improved by: using various combinations of novel therapeutic agents and aerosolized aminoglycosides or colistin, prompt administration of in vitro active antibiotics, removal of possible sources of infection such as infected indwelling intravascular catheters, and application of strict infection control measures.
doi:10.3389/fonc.2014.00232
PMCID: PMC4142553  PMID: 25202682
Stenotrophomonas maltophilia; bacteremia; neutropenia; hematopoietic stem cell transplantation; drug resistance
10.  New Delhi Metallo – beta lactamase – 1 containing Enterobacteriaceae: Origin, Diagnosis, Treatment and Public health concern 
One of the biggest problems associated with the antibiotic therapy is resistance. Recently published studies have revealed that enterobacteriaceae, like E. coli and Klebsiella, isolated from several Indian centers are resistant to many antibiotics including some highly potent antibiotics like carbapenems. It has been proposed that this resistance is because of a carbapenemase enzyme called NDM- 1 (New Delhi Metallo-betalactamase-1). This carbapenemase is class B carbapenemase also called metallolactamases as they require zinc at their active site. This enzyme is coded by a gene called bla - NDM -1 or gene NDM -1. NDM -1 containing enterobacteriaceae can be screened in laboratory by few techniques. Metallolactamase production can be detected by disk approximation test or Modified Hodge test and NDM -1 gene can be detected by polymerase chain reaction by the use of specific primer targeting the gene. Infections caused by such bacteria are associated with high morbidity and mortality. Two classes of antibiotics i.e., polymyxins (colistin) and glycylcyclines (tigecyclines), have shown in vitro activity against NDM -1 harboring enterobacteriaceae. The safety profile of both of these antibiotics is questionable. There is a need for active screening of microorganisms for NDM -1 and research should be directed towards the development of safe antibiotics for the treatment of these kinds of infections.
PMCID: PMC3325060  PMID: 22514756
Antibiotics resistance; Enterobacteriaceae; New Delhi Beta metallo lactamase; carbapenemases
11.  Determination of MIC Distribution of Arbekacin, Cefminox, Fosfomycin, Biapenem and Other Antibiotics against Gram-Negative Clinical Isolates in South India: A Prospective Study 
PLoS ONE  2014;9(7):e103253.
Objectives
To determine the in vitro activity of antibiotics, including arbekacin, cefminox, fosfomycin and biapenem which are all still unavailable in India, against Gram-negative clinical isolates.
Methods
We prospectively collected and tested all consecutive isolates of Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. from blood, urine and sputum samples between March and November 2012. The minimum inhibition concentration (MIC) of 16 antibiotics was determined by the broth micro-dilution method.
Results
Overall 925 isolates were included; 211 E. coli, 207 Klebsiella spp., 153 P. aeruginosa, and 354 Acinetobacter spp. The MIC50 and MIC90 were high for cefminox, biapenem and arbekacin for all pathogens but interpretative criteria were not available. The MIC50 was categorized as susceptible for a couple of antibiotics, including piperacillin/tazobactam, carbapenems and amikacin, for E. coli, Klebsiella spp. and P. aeruginosa. However, for Acinetobacter spp., the MIC50 was categorized as susceptible only for colistin. On the other hand, fosfomycin was the only antibiotic that inhibited 90% of E. coli and Klebsiella spp. isolates, while 90% of P. aeruginosa isolates were inhibited only by colistin. Finally, 90% of Acinetobacter spp. isolates were not inhibited by any antibiotic tested.
Conclusion
Fosfomycin and colistin might be promising antibiotics for the treatment of infections due to E. coli or Klebsiella spp. and P. aeruginosa, respectively, in India; however, clinical trials should first corroborate the in vitro findings. The activity of tigecycline should be evaluated, as this is commonly used as last-resort option for the treatment of multidrug-resistant Acinetobacter infections.
doi:10.1371/journal.pone.0103253
PMCID: PMC4113358  PMID: 25068396
12.  Identification of a Novel Antimicrobial Peptide from Human Hepatitis B Virus Core Protein Arginine-Rich Domain (ARD) 
PLoS Pathogens  2013;9(6):e1003425.
The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I–IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II–IV (HBc153-176) and ARD I–III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I–IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that HBc ARD could be a new promising antimicrobial peptide.
Author Summary
Antibiotics-resistant pathogens have been a major problem to our public health. Recently, in our studies of human hepatitis B virus (HBV), we accidentally discovered potent and broad spectrum antimicrobial peptides from HBV core protein (HBc) arginine-rich domain (ARD). The peptides are mainly composed of SPRRR repeats and are effective against both Gram-positive and Gram-negative bacteria, as well as fungi. We found different bactericidal mechanisms of the ARD peptides, which involved LPS binding, DNA binding and membrane permeabilization in various tested bacteria, such as P. aeruginosa, K. pneumoniae, E. coli and S. aureus. We also found that this ARD peptide was effective for colistin-resistant A. baumannii. The peptides exhibited no hemolysis activity to human red blood cells and no cytotoxicity to human hepatoma cells and kidney cells. Furthermore, the ARD peptide was shown to be safe and protective in the animal model. Recently, intestinal flora was found to influence the development of immunity. We discussed here the potential involvement of the antimicrobial activity of HBc ARD in the establishment of HBV chronic infection in the newborns. We proposed here that the HBc ARD peptides could serve as an alternative to the conventional antibiotics in clinical medicine.
doi:10.1371/journal.ppat.1003425
PMCID: PMC3681751  PMID: 23785287
13.  In Vitro Antimicrobial Susceptibility to Isepamicin of 6,296 Enterobacteriaceae Clinical Isolates Collected at a Tertiary Care University Hospital in Greece 
The reevaluation of “forgotten” antibiotics can identify new therapeutic options against extensively drug-resistant Gram-negative pathogens. We sought to investigate isepamicin in this regard. We retrospectively evaluated the antimicrobial susceptibility to isepamicin of Enterobacteriaceae sp. isolates from unique patients, collected at the microbiological laboratory of the University Hospital of Heraklion, Crete, Greece, from 2004 to 2009. Susceptibility testing was done with the automated Vitek 2 system. The breakpoints for susceptibility to isepamicin, tigecycline, and other antibiotics were those proposed by the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM), the FDA, and the CLSI, respectively. A total of 6,296 isolates were studied, including primarily 3,401 (54.0%) Escherichia coli, 1,040 (16.5%) Klebsiella pneumoniae, 590 (9.4%) Proteus mirabilis, and 460 (7.3%) Enterobacter sp. isolates. Excluding the species with intrinsic resistance to each antibiotic, antimicrobial susceptibility was highest for colistin (5,275/5,441 isolates [96.9%]) and isepamicin (6,103/6,296 [96.9%]), followed by meropenem (5,890/6,296 [93.6%]), imipenem (5,874/6,296 [93.3%]), and amikacin (5,492/6,296 [87.2%]). The antimicrobial susceptibility of the 1,040 K. pneumoniae isolates was highest for isepamicin (95.3%), followed by colistin (89.3%) and meropenem (63.0%). Regarding resistant K. pneumoniae isolates, susceptibility to isepamicin was observed for 91.1% of the 392, 87.7% of the 375, and 85.6% of the 111 isolates that were nonsusceptible to the carbapenems, all other aminoglycosides, and colistin, respectively. Isepamicin exhibited high in vitro activity against almost all of the Enterobacteriaceae species. It could particularly serve as a last-resort therapeutic option for carbapenem-resistant K. pneumoniae in our region, where it is endemic, as it does not show considerable cross-resistance with other aminoglycosides.
doi:10.1128/AAC.06358-11
PMCID: PMC3370768  PMID: 22391548
14.  Dissemination of Cephalosporin Resistance Genes between Escherichia coli Strains from Farm Animals and Humans by Specific Plasmid Lineages 
PLoS Genetics  2014;10(12):e1004776.
Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.
Author Summary
The rapid global rise of infections caused by Escherichia coli that are resistant to clinically relevant antimicrobials, including third-generation cephalosporins, is cause for concern. The intestinal tract of livestock, in particular poultry, is an important reservoir for drug resistant E. coli, but it is unknown to what extent these bacteria can spread to humans. Food is thought to be an important source because drug-resistant E. coli have been detected in animals raised for meat consumption and in meat products. Previous studies that used traditional, low-resolution, genetic typing methods found that drug resistant E. coli present in humans and poultry were indistinguishable from each other, suggesting dissemination of these bacteria through the food-chain to humans. However, by applying high-resolution, whole-genome sequencing methods, we did not find evidence for such transmission of bacteria through the food-chain. Instead, by employing a novel approach for the reconstruction of mobile genetic elements from whole-genome sequence data, we discovered that genetically unrelated E. coli isolates from both humans and animal sources carried nearly identical plasmids that encode third-generation cephalosporin resistance determinants. Our data suggest that cephalosporin resistance is mainly disseminated via the transfer of mobile genetic elements between animals and humans.
doi:10.1371/journal.pgen.1004776
PMCID: PMC4270446  PMID: 25522320
15.  History and evolution of antibiotic resistance in coagulase-negative staphylococci: Susceptibility profiles of new anti-staphylococcal agents 
Coagulase-negative staphylococci (CNS) are a heterogenous group of Gram-positive cocci that are widespread commensals among mammalia. Unlike their coagulase-positive counterpart, Staphylococcus aureus, CNS produce few virulence patterns and normally refrain from invading tissue. Yet, not only can CNS cause infections in normal host tissue, but modern medicine has also seen their rise as opportunists that display adherence to medical device materials to produce a protective biofilm. CNS have historically been more resistant to antimicrobials, including the β-lactam antibiotics, than S. aureus and some hospitals reveal rates of oxacillin resistance in CNS approaching 90%. Cross resistance to non-β-lactam agents has been a recurrent theme over the past 40 years in the CNS. Thus, there has been a pressing need for newer antimicrobial agents with good antistaphylococcal activity. Those new agents tend to have excellent antistaphylococcal activity include daptomycin, linezolid, oritavancin, telavancin, tigecycline, dalbavancin, new quinolones, and ceftibiprole, several of which have unique mechanisms of action. The MIC90 for these new compounds typically ranges from 0.5–4 μg/mL. Staphylococcal biofilm formation is quite common in CNS infections and markedly increases the MIC for most older antimicrobials. Several of the newer agents offer some promise of penetration of biofilm to inhibit or kill adherent staphylococci. CNS will likely remain a major cause of infections in the modern age, evolve further antimicrobial resistance mechanisms, and require development of newer antimicrobials for curative therapy.
PMCID: PMC2387300  PMID: 18516271
coagulase-negative staphylococcus; Staphylococcus epidermidis; Staphylococcus haemolyticus; Staphylococcus lugdenesis; biofilm; new antibiotics; antibiotic resistance
16.  Drugs of Last Resort? The Use of Polymyxins and Tigecycline at US Veterans Affairs Medical Centers, 2005–2010 
PLoS ONE  2012;7(5):e36649.
Multidrug-resistant (MDR) and carbapenem-resistant (CR) Gram-negative pathogens are becoming increasingly prevalent around the globe. Polymyxins and tigecycline are among the few antibiotics available to treat infections with these bacteria but little is known about the frequency of their use. We therefore aimed to estimate the parenteral use of these two drugs in Veterans Affairs medical centers (VAMCs) and to describe the pathogens associated with their administration. For this purpose we retrospectively analyzed barcode medication administration data of parenteral administrations of polymyxins and tigecycline in 127 acute-care VAMCs between October 2005 and September 2010. Overall, polymyxin and tigecycline use were relatively low at 0.8 days of therapy (DOT)/1000 patient days (PD) and 1.6 DOT/1000PD, respectively. Use varied widely across facilities, but increased overall during the study period. Eight facilities accounted for three-quarters of all polymyxin use. The same statistic for tigecycline use was twenty-six VAMCs. There were 1,081 MDR or CR isolates during 747 hospitalizations associated with polymyxin use (1.4/hospitalization). For tigecycline these number were slightly lower: 671 MDR or CR isolates during 500 hospitalizations (1.3/hospitalization) (p = 0.06). An ecological correlation between the two antibiotics and combined CR and MDR Gram-negative isolates per 1000PD during the study period was also observed (Pearson’s correlation coefficient r = 0.55 polymyxin, r = 0.19 tigecycline). In summary, while polymyxin and tigecycline use is low in most VAMCs, there has been an increase over the study period. Polymyxin use in particular is associated with the presence of MDR Gram-negative pathogens and may be useful as a surveillance measure in the future.
doi:10.1371/journal.pone.0036649
PMCID: PMC3353942  PMID: 22615789
17.  Antibiotic susceptibility and molecular epidemiology of Acinetobacter calcoaceticus–baumannii complex strains isolated from a referral hospital in northern Vietnam 
Acinetobacter calcoaceticus–baumannii complex is a common cause of hospital-acquired infections (HAIs) globally, remarkable for its high rate of antibiotic resistance, including to carbapenems. There are few data on the resistance of A. baumannii in Vietnam, which are essential for developing evidence-based treatment guidelines for HAIs. Antibiotic susceptibility testing was conducted by VITEK®2, and pulsed-field gel electrophoresis (PFGE) was performed on 66 clinical A. baumannii complex isolates recovered during 2009 at the National Hospital of Tropical Diseases (NHTD), a referral hospital in Hanoi, Vietnam. Basic demographic and clinical data were collected and analysed using descriptive statistics. Most isolates came from lower respiratory tract specimens (59; 89.4%) from intensive care unit (ICU) patients [64/65 (98.5%) with available data] who had been admitted to NHTD for ≥2 days [42/46 (91.3%) with available data]. More than 90% of the isolates were resistant to the tested β-lactamase/β-lactamase inhibitors, cephalosporins, carbapenems, fluoroquinolones and trimethoprim/sulfamethoxazole. Moreover, 25.4% (16/63) were resistant to all tested β-lactams, quinolones and aminoglycosides. All isolates remained sensitive to colistin and 58.7% were susceptible to tigecycline. Of the 66 isolates, 49 could be classified into eight PFGE types (A–H). Every PFGE type, except D, had cluster(s) of three or more isolates with a temporal relationship. In conclusion, these data suggest a significant rise in A. baumannii antibiotic resistance in Vietnam. Clustering within PFGE types supports cross-transmission of A. baumannii within the ICU at NHTD. Increased research and resources in optimising treatment, infection control and antibiotic stewardship are needed.
doi:10.1016/j.jgar.2014.05.003
PMCID: PMC4270437  PMID: 25540720
Acinetobacter baumannii; Ventilator-associated pneumonia; Hospital-acquired infection; Antibiotic resistance; Genotype
18.  Profile of infective microorganisms causing ventilator-associated pneumonia: A clinical study from resource limited intensive care unit 
Background:
Ventilator-associated pneumonia (VAP) is the most common cause of hospital acquired infection and death among patients admitted in ICU. Microorganisms responsible for VAP vary from place to place. Gram-negative bacteria (GNB) have emerged as a major group of pathogen causing VAP and over the years carbapenem group of antibiotics has emerged as one of the important antibiotics used in the critically ill patients. There have been reports of increased occurrence of infection by carbapenem-resistant bacteria in health care settings in recent times.
Aim:
The aim of the study was to assess the incidence of VAP, their microbiological profile with reference to carbapenemase producing GNB in the intensive care unit of a tertiary care hospital, their relation to initial emperical antibiotic therapy, sensitivity patterns, and outcome.
Materials and Methods:
This prospective study was carried out over the period of 1 year (July 2010-June 2011) on 100 randomly selected patients above the age of 18 years admitted in the emergency/ICU and requiring intubation and mechanical ventilation for more than 72 hours. The diagnosis of VAP was established on the basis of clinical and radiological parameters as per Centre of Disease Centres (CDC) guidelines. A baseline sample was obtained after initial endotracheal intubation. Thereafter, the culture sent on the first day of occurrence of clinical sign of VAP. Culture was done on blood agar and MacConkey agar. All imipenem-resistant strains were further confirmed by Modified Hodge test and combined disc for confirmation of respective carbapenemase.
Results:
Incidence of VAP was found to be 51%. GNB mainly Citrobacter 28 (52.83%) and Klebsiella pneumoniae 7 (13.21%), were the most commonly isolated pathogens. The prevalence of carbapenemase-producing GNB was alarmingly high 24/50 (48%). The entire carbapenemase producers showed high degree of cross resistance to antibiotics with some sensitivity to Polymyxin B (94 %) and Tigecycline (96%)
Conclusion:
High incidence of VAP and the potential carbapenemase-producing GNB are real threat in our ICU. The emergence of microorganisms known for its inherent resistance among most of the common first-line antibiotics calls for a alarm in all upcoming tertiary care hospitals.
doi:10.4103/0970-9185.117111
PMCID: PMC3788236  PMID: 24106362
Carbapenemase; multidrug-resistant organisms; nosocomial pneumonia; ventilator-associated pneumonia
19.  Antibiotic rotation strategies to reduce antimicrobial resistance in Gram-negative bacteria in European intensive care units: study protocol for a cluster-randomized crossover controlled trial 
Trials  2014;15:277.
Background
Intensive care units (ICU) are epicenters for the emergence of antibiotic-resistant Gram-negative bacteria (ARGNB) because of high rates of antibiotic usage, rapid patient turnover, immunological susceptibility of acutely ill patients, and frequent contact between healthcare workers and patients, facilitating cross-transmission.
Antibiotic stewardship programs are considered important to reduce antibiotic resistance, but the effectiveness of strategies such as, for instance, antibiotic rotation, have not been determined rigorously. Interpretation of available studies on antibiotic rotation is hampered by heterogeneity in implemented strategies and suboptimal study designs. In this cluster-randomized, crossover trial the effects of two antibiotic rotation strategies, antibiotic mixing and cycling, on the prevalence of ARGNB in ICUs are determined. Antibiotic mixing aims to create maximum antibiotic heterogeneity, and cycling aims to create maximum antibiotic homogeneity during consecutive periods.
Methods/Design
This is an open cluster-randomized crossover study of mixing and cycling of antibiotics in eight ICUs in five European countries. During cycling (9 months) third- or fourth-generation cephalosporins, piperacillin-tazobactam and carbapenems will be rotated during consecutive 6-week periods as the primary empiric treatment in patients suspected of infection caused by Gram-negative bacteria. During mixing (9 months), the same antibiotics will be rotated for each consecutive antibiotic course. Both intervention periods will be preceded by a baseline period of 4 months. ICUs will be randomized to consecutively implement either the mixing and then cycling strategy, or vice versa. The primary outcome is the ICU prevalence of ARGNB, determined through monthly point-prevalence screening of oropharynx and perineum. Secondary outcomes are rates of acquisition of ARGNB, bacteremia and appropriateness of therapy, length of stay in the ICU and ICU mortality. Results will be adjusted for intracluster correlation, and patient- and ICU-level variables of case-mix and infection-prevention measures using advanced regression modeling.
Discussion
This trial will determine the effects of antibiotic mixing and cycling on the unit-wide prevalence of ARGNB in ICUs.
Trial registration
ClinicalTrials.gov NCT01293071 December 2010.
doi:10.1186/1745-6215-15-277
PMCID: PMC4227018  PMID: 25011604
20.  Clonal Expansion during Staphylococcus aureus Infection Dynamics Reveals the Effect of Antibiotic Intervention 
PLoS Pathogens  2014;10(2):e1003959.
To slow the inexorable rise of antibiotic resistance we must understand how drugs impact on pathogenesis and influence the selection of resistant clones. Staphylococcus aureus is an important human pathogen with populations of antibiotic-resistant bacteria in hospitals and the community. Host phagocytes play a crucial role in controlling S. aureus infection, which can lead to a population “bottleneck” whereby clonal expansion of a small fraction of the initial inoculum founds a systemic infection. Such population dynamics may have important consequences on the effect of antibiotic intervention. Low doses of antibiotics have been shown to affect in vitro growth and the generation of resistant mutants over the long term, however whether this has any in vivo relevance is unknown. In this work, the population dynamics of S. aureus pathogenesis were studied in vivo using antibiotic-resistant strains constructed in an isogenic background, coupled with systemic models of infection in both the mouse and zebrafish embryo. Murine experiments revealed unexpected and complex bacterial population kinetics arising from clonal expansion during infection in particular organs. We subsequently elucidated the effect of antibiotic intervention within the host using mixed inocula of resistant and sensitive bacteria. Sub-curative tetracycline doses support the preferential expansion of resistant microorganisms, importantly unrelated to effects on growth rate or de novo resistance acquisition. This novel phenomenon is generic, occurring with methicillin-resistant S. aureus (MRSA) in the presence of β-lactams and with the unrelated human pathogen Pseudomonas aeruginosa. The selection of resistant clones at low antibiotic levels can result in a rapid increase in their prevalence under conditions that would previously not be thought to favor them. Our results have key implications for the design of effective treatment regimes to limit the spread of antimicrobial resistance, where inappropriate usage leading to resistance may reduce the efficacy of life-saving drugs.
Author Summary
Staphylococcus aureus is a major cause of human disease, made even more notable due to the spread of antibiotic resistance. We used a combination of animal models to study the spread of bacteria between organs during an infection and the resulting effect of antibiotic intervention. We found that S. aureus infection is highly clonal, following a “bottleneck” in which very few bacterial cells found each abscess. Despite previous in vitro research, the effect of antibiotics on S. aureus infection was poorly understood. We utilized our systemic infection models to study intervention with sub-curative antibiotic doses, such as one might encounter upon failing to complete an antibiotic course. We have shown that such doses are able to support the preferential expansion of antibiotic-resistant organisms during a mixed infection. This selection is due to the clonal pattern of infection, occurring despite a lack of effect on growth rate or on the spontaneous generation of resistance. Furthermore, it is generic to multiple pathogen species, including Pseudomonas aeruginosa, and antibiotic classes, such as with methicillin-resistant S. aureus (MRSA) in the presence of oxacillin. Given the current debate in the field, our results have important implications for the design of properly-controlled treatment regimes.
doi:10.1371/journal.ppat.1003959
PMCID: PMC3937288  PMID: 24586163
21.  Structural and Molecular Basis for Resistance to Aminoglycoside Antibiotics by the Adenylyltransferase ANT(2″)-Ia 
mBio  2015;6(1):e02180-14.
ABSTRACT  
The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme-mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2″)-Ia, which confers resistance to gentamicin, tobramycin, and kanamycin. Despite the importance of this enzyme in drug resistance, its structure and molecular mechanism have been elusive. This study describes the structural and mechanistic basis for adenylylation of aminoglycosides by the ANT(2″)-Ia enzyme. ANT(2″)-Ia confers resistance by magnesium-dependent transfer of a nucleoside monophosphate (AMP) to the 2″-hydroxyl of aminoglycoside substrates containing a 2-deoxystreptamine core. The catalyzed reaction follows a direct AMP transfer mechanism from ATP to the substrate antibiotic. Central to catalysis is the coordination of two Mg2+ ions, positioning of the modifiable substrate ring, and the presence of a catalytic base (Asp86). Comparative structural analysis revealed that ANT(2″)-Ia has a two-domain structure with an N-terminal active-site architecture that is conserved among other antibiotic nucleotidyltransferases, including Lnu(A), LinB, ANT(4′)-Ia, ANT(4″)-Ib, and ANT(6)-Ia. There is also similarity between the nucleotidyltransferase fold of ANT(2″)-Ia and DNA polymerase β. This similarity is consistent with evolution from a common ancestor, with the nucleotidyltransferase fold having adapted for activity against chemically distinct molecules.
Importance   To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular problem among Gram-negative human pathogens, since there are very few therapeutic options for infections caused by these organisms. In order to successfully circumvent or inhibit the activity of aminoglycoside-modifying enzymes, and to thus rejuvenate the activity of the aminoglycoside antibiotics against Gram-negative pathogens, structural and mechanistic information is crucial. This study reveals the structure of a clinically prevalent aminoglycoside resistance enzyme [ANT(2″)-Ia] and depicts the molecular basis underlying modification of antibiotic substrates. Combined, these findings provide the groundwork for the development of broad-spectrum inhibitors against antibiotic nucleotidyltransferases.
Importance  
To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular problem among Gram-negative human pathogens, since there are very few therapeutic options for infections caused by these organisms. In order to successfully circumvent or inhibit the activity of aminoglycoside-modifying enzymes, and to thus rejuvenate the activity of the aminoglycoside antibiotics against Gram-negative pathogens, structural and mechanistic information is crucial. This study reveals the structure of a clinically prevalent aminoglycoside resistance enzyme [ANT(2″)-Ia] and depicts the molecular basis underlying modification of antibiotic substrates. Combined, these findings provide the groundwork for the development of broad-spectrum inhibitors against antibiotic nucleotidyltransferases.
doi:10.1128/mBio.02180-14
PMCID: PMC4313920  PMID: 25564464
22.  Pharmacology of polymyxins: new insights into an ‘old’ class of antibiotics 
Future microbiology  2013;8(6):10.2217/fmb.13.39.
Increasing antibiotic resistance in Gram-negative bacteria, particularly in Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, presents a global medical challenge. No new antibiotics will be available for these ‘superbugs’ in the near future due to the dry antibiotic discovery pipeline. Colistin and polymyxin B are increasingly used as the last-line therapeutic options for treatment of infections caused by multidrug-resistant Gram-negative bacteria. This article surveys the significant progress over the last decade in understanding polymyxin chemistry, mechanisms of antibacterial activity and resistance, structure–activity relationships and pharmacokinetics/pharmacodynamics. In the ‘Bad Bugs, No Drugs’ era, we must pursue structure–activity relationship-based approaches to develop novel polymyxin-like lipopeptides targeting polymyxin-resistant Gram-negative ‘superbugs’. Before new antibiotics become available, we must optimize the clinical use of polymyxins through the application of pharmacokinetic/pharmacodynamic principles, thereby minimizing the development of resistance.
doi:10.2217/fmb.13.39
PMCID: PMC3852176  PMID: 23701329
colistin; lipid A; lipopolysaccharide; pharmacokinetic/pharmacodynamic; polymyxin; resistance; structure–activity relationship
23.  Identification of novel antimicrobial resistance genes from microbiota on retail spinach 
BMC Microbiology  2013;13:272.
Background
Drug resistance genes and their mobile genetic elements are frequently identified from environmental saprophytic organisms. It is widely accepted that the use of antibiotics in animal husbandry selects for drug resistant microorganisms, which are then spread from the farm environment to humans through the consumption of contaminated food products. We wished to identify novel drug resistance genes from microbial communities on retail food products. Here, we chose to study the microbial communities on retail spinach because it is commonly eaten raw and has previously been associated with outbreaks of bacterial infections.
Results
We created metagenomic plasmid libraries from microbiota isolated from retail spinach samples. We identified five unique plasmids that increased resistance to antimicrobial drugs in the E. coli host. These plasmids were identified in E. coli that grew on plates that contained ampicillin (pAMP), aztreonam (pAZT), ciprofloxacin (pCIP), trimethoprim (pTRM), and trimethoprim-sulfamethoxazole (pSXT). We identified open reading frames with similarity to known classes of drug resistance genes in the DNA inserts of all 5 plasmids. These drug resistance genes conferred resistance to fluoroquinolones, cephalosporins, and trimethoprim, which are classes of antimicrobial drugs frequently used to treat human Gram negative bacterial infections. These results show that novel drug resistance genes are found in microbiota on retail produce items.
Conclusions
Here we show that microbiota of retail spinach contains DNA sequences previously unidentified as conferring antibiotic resistance. Many of these novel sequences show similarity to genes found in species of bacteria, which have previously been identified as commensal or saprophytic bacteria found on plants. We showed that these resistance genes are capable of conferring clinically relevant levels of resistance to antimicrobial agents. Food saprophytes may serve as an important reservoir for new drug-resistance determinants in human pathogens.
doi:10.1186/1471-2180-13-272
PMCID: PMC3890574  PMID: 24289541
Antibiotic resistance; Gram negative bacteria; Metagenomic library
24.  Infections Caused by Acinetobacter baumannii in Recipients of Hematopoietic Stem Cell Transplantation 
Frontiers in Oncology  2014;4:186.
Acinetobacter baumannii (A. baumannii) is a Gram-negative, strictly aerobic, non-fermentative coccobacillus, which is widely distributed in nature. Recently, it has emerged as a major cause of health care-associated infections (HCAIs) in addition to its capacity to cause community-acquired infections. Risk factors for A. baumannii infections and bacteremia in recipients of hematopoietic stem cell transplantation include: severe underlying illness such as hematological malignancy, prolonged use of broad-spectrum antibiotics, invasive instrumentation such as central venous catheters or endotracheal intubation, colonization of respiratory, gastrointestinal, or urinary tracts in addition to severe immunosuppression caused by using corticosteroids for treating graft versus host disease. The organism causes a wide spectrum of clinical manifestations, but serious complications such as bacteremia, septic shock, ventilator-associated pneumonia, extensive soft tissue necrosis, and rapidly progressive systemic infections that ultimately lead to multi-organ failure and death are prone to occur in severely immunocompromised hosts. The organism is usually resistant to many antimicrobials including penicillins, cephalosporins, trimethoprim–sulfamethoxazole, almost all fluoroquinolones, and most of the aminoglycosides. The recently increasing resistance to carbapenems, colistin, and polymyxins is alarming. Additionally, there are geographic variations in the resistance patterns and several globally and regionally resistant strains have already been described. Successful management of A. baumannii infections depends upon appropriate utilization of antibiotics and strict application of preventive and infection control measures. In uncomplicated infections, the use of a single active beta-lactam may be justified, while definitive treatment of complicated infections in critically ill individuals may require drug combinations such as colistin and rifampicin or colistin and carbapenem. Mortality rates in patients having bacteremia or septic shock may reach 70%. Good prognosis is associated with presence of local infection, absence of multidrug resistant strain, and presence of uncomplicated infection while poor outcome is associated with severe underlying medical illness, bacteremia, septic shock, multi-organ failure, HCAIs, admission to intensive care facilities for higher levels of care, and culture of certain aggressive genotypes of A. baumannii.
doi:10.3389/fonc.2014.00186
PMCID: PMC4095644  PMID: 25072028
Acinetobacter baumannii; hematological malignancy; hematopoietic stem cell transplantation; virulence; drug resistance
25.  Discovery and preclinical development of new antibiotics 
Upsala Journal of Medical Sciences  2014;119(2):162-169.
Antibiotics are the medical wonder of our age, but an increasing frequency of resistance among key pathogens is rendering them less effective. If this trend continues the consequences for cancer patients, organ transplant patients, and indeed the general community could be disastrous. The problem is complex, involving abuse and overuse of antibiotics (selecting for an increasing frequency of resistant bacteria), together with a lack of investment in discovery and development (resulting in an almost dry drug development pipeline). Remedial approaches to the problem should include taking measures to reduce the selective pressures for resistance development, and taking measures to incentivize renewed investment in antibiotic discovery and development. Bringing new antibiotics to the clinic is critical because this is currently the only realistic therapy that can ensure the level of infection control required for many medical procedures. Here we outline the complex process involved in taking a potential novel antibiotic from the initial discovery of a hit molecule, through lead and candidate drug development, up to its entry into phase I clinical trials. The stringent criteria that a successful drug must meet, balancing high efficacy in vivo against a broad spectrum of pathogens, with minimal liabilities against human targets, explain why even with sufficient investment this process is prone to a high failure rate. This emphasizes the need to create a well-funded antibiotic discovery and development pipeline that can sustain the continuous delivery of novel candidate drugs into clinical trials, to ensure the maintenance of the advanced medical procedures we currently take for granted.
doi:10.3109/03009734.2014.896437
PMCID: PMC4034554  PMID: 24646082
Candidate drug; efficacy; hit molecule; lead molecule; liability testing; medicinal chemistry

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