Switching from membrane disrupting to membrane crossing, an effective strategy in designing antibacterial polypeptide.

Drug-resistant bacterial infections have caused serious threats to human health and call for effective antibacterial agents that have low propensity to induce antimicrobial resistance. Host defense peptide-mimicking peptides are actively explored, among which poly-ß-l-lysine displays potent antibacterial activity but high cytotoxicity due to the helical structure and strong membrane disruption effect. Here, we report an effective strategy to optimize antimicrobial peptides by switching membrane disrupting to membrane penetrating and intracellular targeting by breaking the helical structure using racemic residues. Introducing ß-homo-glycine into poly-ß-lysine effectively reduces the toxicity of resulting poly-ß-peptides and affords the optimal poly-ß-peptide, ßLys50HG50, which shows potent antibacterial activity against clinically isolated methicillin-resistant Staphylococcus aureus (MRSA) and MRSA persister cells, excellent biosafety, no antimicrobial resistance, and strong therapeutic potential in both local and systemic MRSA infections. The optimal poly-ß-peptide demonstrates strong therapeutic potential and implies the success of our approach as a generalizable strategy in designing promising antibacterial polypeptides.

Detection of beta-lactamase-producing Enterobacteriaceae in a veterinary hospital environment / Detecção de Enterobactérias produtoras de beta-lactamases em ambiente hospitalar veterinário

Due to the strong selective pressure resulting from the misuse of antibiotics, the natural process of bacterial resistance has been accelerated, leading to the increasingly constant appearance of multiresistant isolates. The high number of multi-resistant bacteria is a one health problem. Enterobacteriaceae are usually commensal bacteria of the gastrointestinal tract. However, they can cause infections, and the most important resistance characteristic among them is the production of ß-lactamases. This study aimed to identify ESBL-producing Enterobacteriaceae of types of TEM, SHV, and the CTX-Mgroups. To isolate the enterobacteria, swabs were collected by swiping objects that had contact with the patients and professionals, and the water of the hospital environment. Ten collections were carried out, yielding 306 samples, from which 118 enterobacteria were identified: Escherichia coli, Enterobacter spp., Klebsiella spp., Proteus mirabilis, Serratiaspp., and Citrobacter spp. Isolates. The genes TEM and CTX-M, for the production of ß-lactamases, were detected in 12.7% of the 118 enterobacterial isolates. It is very important to know the bacterial population circulating in the veterinary hospital environment and its resistance to antimicrobials so that professionals can take appropriate measures to minimize the risks of transmission, especially from cages and consultation tables. In addition, the correct control of the microbiological quality of the supply water, as well as environmental cleaning procedures, are essential to prevent the transmission of these microorganisms.(AU)

Devido à grande pressão seletiva decorrente do uso indevido de antibióticos, tem se acelerado o processo natural de resistência das bactérias, levando ao aparecimento cada vez mais constante de isolados multirresistentes. O elevado número de bactérias multirresistentes identificadas é um problema da saúde única. As enterobactérias são bactérias geralmente comensais do trato gastrointestinal, entretanto podem causar infecções, e a característica de resistência mais importante entre elas é a produção de ß-lactamases. Buscando caracterizar melhor os microrganismos circulantes e potencialmente causadores de infecções em ambiente hospitalar veterinário, este estudo objetivou identificar as enterobactérias produtoras de ESBL do tipo TEM, SHV e os cinco grupos de CTX-M presentes em isolados circulantes em hospital veterinário. Foi realizada coleta de suabes de arrasto de objetos que entram em contato com os pacientes e com os profissionais que ali trabalham, bem como de água, para a identificação das enterobactérias. Foram realizadas 10 coletas, obtendo-se 306 amostras, dessas, 118 enterobactérias foram identificadas: Escherichia coli, Enterobacter, Klebsiella, Proteus mirabilis, Serratia e Citrobacter. Dentre as enterobactérias identificadas, alguns isolados possuíam genes para a produção de ß-lactamases, do tipo TEM e CTX-M. É de grande importância conhecer a população bacteriana circulante no ambiente hospitalar veterinário, e a sua resistência aos antimicrobianos, para que os profissionais possam tomar medidas apropriadas para minimizar os riscos de transmissão, principalmente a partir de gaiolas e mesas de atendimento. Além disso, o correto controle da qualidade microbiológica da água de abastecimento, bem como dos procedimentos de higienização do ambiente, são fundamentais para evitar a transmissão destes microrganismos.(AU)

Resistance mechanisms to inhibitors of p53-MDM2 interactions in cancer therapy: can we overcome them?

Since the discovery of the first MDM2 inhibitors, we have gained deeper insights into the cellular roles of MDM2 and p53. In this review, we focus on MDM2 inhibitors that bind to the p53-binding domain of MDM2 and aim to disrupt the binding of MDM2 to p53. We describe the basic mechanism of action of these MDM2 inhibitors, such as nutlin-3a, summarise the determinants of sensitivity to MDM2 inhibition from p53-dependent and p53-independent points of view and discuss the problems with innate and acquired resistance to MDM2 inhibition. Despite progress in MDM2 inhibitor design and ongoing clinical trials, their broad use in cancer treatment is not fulfilling expectations in heterogenous human cancers. We assess the MDM2 inhibitor types in clinical trials and provide an overview of possible sources of resistance to MDM2 inhibition, underlining the need for patient stratification based on these aspects to gain better clinical responses, including the use of combination therapies for personalised medicine.

[Pathogens' distribution and changes of antimicrobial resistance in the bile of acute biliary tract infection patients].

Objective: To investigate the pathogens' distribution and antimicrobial resistance in the bile of acute biliary tract infection patients. Methods: The data of bile bacterial culture and drug sensitivity test of 223 acute biliary tract infection patients who underwent gallbladder puncture or endoscopic retrograde cholangiopancreatography drainage from July 2009 to July 2019 were analyzed retrospectively at Department of General Surgery,Xinhua Hospital,Affiliated to Shanghai Jiao Tong University School of Medicine.There were 141 males and 82 females with age of 67.3 years(range:28 to 93 years).Three to five milliliter of bile was extracted from each patient and sent to the laboratory for bacterial culture,identification and drug sensitivity test.The patients were divided into two groups according to the visiting time: the former group (n=124) was admitted from July 2009 to July 2014,and the latter group(n=99) was admitted from August 2014 to July 2019.The distribution of pathogenic bacteria and the changing trend of drug resistance rate of common bacteria in the two groups were compared.The results of drug sensitivity test were analyzed by WHONET software provided by WHO bacterial surveillance network.The drug resistance rates in different time periods were compared by χ2 test. Results: In this study,there were 147 cases of acute cholangitis and 76 cases of acute cholecystitis.A total of 376 strains of pathogenic bacteria were cultured.Among them,98 strains(26.1%) were gram-positive bacteria,269 strains(71.5%) were gram-negative bacteria and 9 strains(2.4%) were fungi.The top three gram-positive bacteria were Enterococcus faecium (49.0%,48/98),Enterococcus faecalis(20.4%,20/98),and Enterococcus luteus(7.1%,7/98).The top 5 gram-negative bacteria were Escherichia coli(33.5%,90/269),Klebsiella pneumoniae(13.8%,37/269),Pseudomonas aeruginosa(13.0%,35/269),Acinetobacter baumannii (12.6%,34/269),and Enterobacter cloacae(4.8%,13/269).From 2009 to 2019,there was no significant change in the proportion of gram-positive bacteria (former group vs. latter group: 25.3% vs. 28.2%) and gram-negative bacteria(former group vs.latter group: 74.7% vs. 71.8%) in the bile of patients with acute biliary tract infection.Gram-positive bacteria were mainly Enterococci(85.7%,84/98) and gram-negative bacteria were Escherichia coli(33.5%,90/269).Acinetobacter baumannii accounted for 7.8%(11/142) of gram-negative bacteria in the former group and 18.1%(23/127) in the latter group,an increase of 10.3% over previous five years.Pseudomonas aeruginosa had a downward trend,16.9% in the former group(24/142) and 8.7% in the latter group (11/127),the proportion decreased by 8.2%,and the other changes were not significant.The drug resistance rates of common gram-positive bacteria were relatively stable,and the drug resistance rates of Enterococcus faecium to many antibiotics were higher than those of Enterococcus faecalis.The resistance rates of gram-negative bacteria to most antibiotics showed an upward trend,among which Klebsiella pneumoniae showed an upward trend to most of antibiotics(former group: 0/15－4/13, latter group: 55.0%－70.0%; χ2=3.996－16.942, P=0.000－0.046).The drug resistance rates of Acinetobacter baumannii was generally higher,but there were no significant changes in the drug resistance rates of Acinetobacter baumannii between the two groups.The drug resistance rates of Pseudomonas aeruginosa to most antibiotics increased,and the overall drug resistance rates of Escherichia coli were stable and showed a slight upward trend. Conclusions: The main pathogens in bile of patients with acute biliary tract infection are gram-negative bacteria.The constituent ratio of various gram-negative bacteria had no significant change from 2009 to 2019,but the drug resistance rates shows an upward trend.Among the gram-negative bacteria, Escherichia coli is the most important pathogen,and the proportion has no significant change.The proportion of Acinetobacter baumannii in the former group was significantly higher than that in the former group.And the proportion of Pseudomonas aeruginosa has a decreased trend.

The Many Roles of the Bacterial Second Messenger Cyclic di-AMP in Adapting to Stress Cues.

Bacteria respond to changes in environmental conditions through adaptation to external cues. Frequently, bacteria employ nucleotide signaling molecules to mediate a specific, rapid response. Cyclic di-AMP (c-di-AMP) was recently discovered to be a bacterial second messenger that is essential for viability in many species. In this review, we highlight recent work that has described the roles of c-di-AMP in bacterial responses to various stress conditions. These studies show that depending on the lifestyle and environmental niche of the bacterial species, the c-di-AMP signaling network results in diverse outcomes, such as regulating osmolyte transport, controlling plant attachment, or providing a checkpoint for spore formation. c-di-AMP achieves this signaling specificity through expression of different classes of synthesis and catabolic enzymes as well as receptor proteins and RNAs, which will be summarized.

Overview of Host Defense Peptides and Their Applications for Plastic and Reconstructive Surgeons.

BACKGROUND: Host defense peptides are a family of endogenous short peptides that are found in all living beings and play a critical role in innate immunity against infection. METHODS: A nonsystematic review of host defense peptides was conducted with specific interest in properties and applications relevant to plastic and reconstructive surgery. RESULTS: In addition to their direct antimicrobial actions against pathogens, including multidrug-resistant bacteria, they also demonstrate important functions in immunomodulation, tumor cell lysis, and tissue regeneration. These properties have made them a topic of clinical interest for plastic surgeons because of their potential applications as novel antibiotics, wound healing medications, and cancer therapies. The rising clinical interest has led to a robust body of literature describing host defense peptides in great depth and breadth. Numerous mechanisms have been observed to explain their diverse functions, which rely on specific structural characteristics. However, these peptides remain mostly experimental, with limited translation to clinical practice because of numerous failures to achieve acceptable results in human trials. CONCLUSIONS: Despite the broad ranging potential of these peptides for use in the field of plastic and reconstructive surgery, they are rarely discussed in the literature or at scientific meetings. In this review, the authors provide a summary of the background, structure, function, bacterial resistance, and clinical applications of host defense peptides with the goal of stimulating host defense peptide-based innovation within the field of plastic and reconstructive surgery.

The in vitro antibacterial effect of permethrin and formaldehyde on Staphylococcus aureus.

Antibiotic-resistant strains of bacteria such as methicillin-resistant Staphylococcus aureus are a threat to human health, and effective treatment options against them are needed. This study aimed to determine whether the insecticide permethrin was capable of inhibiting the growth of S. aureus or if some other component of a permethrin cream was responsible for a decrease in scabies associated bacterial infection previously observed. Ten S. aureus strains were grown in the presence of permethrin and formaldehyde both alone and in combination with percent inhibition determined by viable counts. Also, a time-kill assay was conducted on S. aureus exposed to the same conditions. Finally, the morphology of S. aureus grown in the presence of permethrin was examined by scanning electron microscopy. Bacterial inhibition by permethrin ranged from 0% to 41% whereas inhibition by formaldehyde was 100%. The time-kill curves of permethrin exposed cells were very similar to the positive growth control while the formaldehyde and combination exposure showed complete inhibition even at the 0-hr time point. The scanning electron micrographs of permethrin grown S. aureus showed healthy cocci cells with no sign of cell damage. Our results show that permethrin is not capable of inhibiting the growth of bacteria enough for it to be termed bactericidal. Formaldehyde is a known antiseptic and therefore was responsible for the antibacterial effect observed after the use of permethrin cream.

Prostate biopsy techniques and pre-biopsy prophylactic measures: variation in current practice patterns in the Netherlands.

BACKGROUND: The clinical landscape of prostate biopsy (PB) is evolving with changes in procedures and techniques. Moreover, antibiotic resistance is increasing and influences the efficacy of pre-biopsy prophylactic regimens. Therefore, increasing antibiotic resistance may impact on clinical care, which probably results in differences between hospitals. The objective of our study is to determine the (variability in) current practices of PB in the Netherlands and to gain insight into Dutch urologists' perceptions of fluoroquinolone resistance and biopsy related infections. METHODS: An online questionnaire was prepared using SurveyMonkey® platform and distributed to all 420 members of the Dutch Association of Urology, who work in 81 Dutch hospitals. Information about PB techniques and periprocedural antimicrobial prophylaxis was collected. Urologists' perceptions regarding pre-biopsy antibiotic prophylaxis in an era of antibiotic resistance was assessed. Descriptive statistical analysis was performed. RESULTS: One hundred sixty-one responses (38.3%) were analyzed representing 65 (80.3%) of all Dutch hospitals performing PB. Transrectal ultrasound guided prostate biopsy (TRUSPB) was performed in 64 (98.5%) hospitals. 43.1% of the hospitals (also) used other image-guided biopsy techniques. Twenty-three different empirical prophylactic regimens were reported among the hospitals. Ciprofloxacin was most commonly prescribed (84.4%). The duration ranged from one pre-biopsy dose (59.4%) to 5 days extended prophylaxis. 25.2% of the urologists experienced ciprofloxacin resistance as a current problem in the prevention of biopsy related infections and 73.6% as a future problem. CONCLUSIONS: There is a wide variation in practice patterns among Dutch urologists. TRUSPB is the most commonly used biopsy technique, but other image-guided biopsy techniques are increasingly used. Antimicrobial prophylaxis is not standardized and prolonged prophylaxis is common. The wide variation in practice patterns and lack of standardization underlines the need for evidence-based recommendations to guide urologists in choosing appropriate antimicrobial prophylaxis for PB in the context of increasing antibiotic resistance.

Structures of lipoprotein signal peptidase II from Staphylococcus aureus complexed with antibiotics globomycin and myxovirescin.

Antimicrobial resistance is a major global threat that calls for new antibiotics. Globomycin and myxovirescin are two natural antibiotics that target the lipoprotein-processing enzyme, LspA, thereby compromising the integrity of the bacterial cell envelope. As part of a project aimed at understanding their mechanism of action and for drug development, we provide high-resolution crystal structures of the enzyme from the human pathogen methicillin-resistant Staphylococcus aureus (MRSA) complexed with globomycin and with myxovirescin. Our results reveal an instance of convergent evolution. The two antibiotics possess different molecular structures. Yet, they appear to inhibit identically as non-cleavable tetrahedral intermediate analogs. Remarkably, the two antibiotics superpose along nineteen contiguous atoms that interact similarly with LspA. This 19-atom motif recapitulates a part of the substrate lipoprotein in its proposed binding mode. Incorporating this motif into a scaffold with suitable pharmacokinetic properties should enable the development of effective antibiotics with built-in resistance hardiness.

The Role of Ciprofloxacin Resistance and Extended-spectrum beta-lactamase (ESBL) Positivity in Infective Complications Following Prostate Biopsy.

PURPOSE: To evaluate ciprofloxacin resistance (CR) and extended-spectrum beta-lactamase (ESBL) positivity in the rectal flora, antibiotic prophylaxis received, and post-biopsy infectious complications in patients undergoing prostate biopsy. MATERIAL & METHODS: Rectal swab samples collected from 99 patients suspected of prostate cancer two days before prostate biopsy were tested for microbial susceptibility and ESBL production. All patients were given standard ciprofloxacin and ornidazole prophylaxis. Ten days post-biopsy, the patients were contacted by phone and asked about the presence of fever and/or symptoms of urinary tract infection. RESULTS: Escherichia coli (E.coli) was the most common isolate detected in 82 (75%) of the rectal swab samples. Ciprofloxacin resistance was detected in 33% and ESBL positivity in 22% of the isolated E.coli strains. No microorganisms other than E.coli were detected in blood, urine, and rectal swab cultures of patients who developed post-biopsy complications. CR E.coli strains also showed resistance to other antimicrobial agents. The lowest resistance rates were to amikacin (n = 2, 7.4%) and nitrofurantoin (n = 1, 3.7%). Seven patients (7.6%) developed infectious complications. There was no significant difference in probability of hospitalization between patients with CR strains (14.3%) and those with ciprofloxacin-susceptible strains (14.3% vs. 4.7%; p = 0.194). However, strains that were both CR and ESBL-positive were associated with significantly higher probability of hospitalization compared to ciprofloxacin-susceptible strains (28.6% vs. 3.8%; p = 0.009). CONCLUSION: The higher rate of infectious complications with CR and ESBL-positive strains suggests that the agents used for antibiotic prophylaxis should be reevaluated. It is important to consider local resistance data when using extended-spectrum agents to treat patients presenting with post-biopsy infectious complications.

Detection of efflux pump CmeABC in enrofloxacin resistant Campylobacter spp. strains isolated from broiler chickens (Gallus gallus domesticus) in the state of Rio de Janeiro, Brazil / Detecção da bomba de efluxo CmeABC em cepas de Campylobacter spp. resistentes à enrofloxacina isoladas de frangos de corte (Gallus gallus domesticus) no Estado do Rio de Janeiro, Brasil

Fowls are the main reservoirs of the highly important food-originating pathogen called Campylobacter spp. and broilers' meat and byproducts are the main vehicles of this microorganism. Increasing of Campylobacter spp. resistant strains to fluorquinolones, an antimicrobial class often employed in poultry farming and in human medicine has become a great concern to poultry breeders. In fact, several studies evaluated increasing bacterial resistance against these antimicrobial agents. The role of CmeABC efflux system has been underscored among the resistance mechanisms in Campylobacter spp. to fluorquinolones. This study investigated the occurrence of CmeABC efflux pump in 81 and 78 enrofloxacin resistant strains of Campylobacter jejuni and C. coli respectively, isolated from broilers collected from six abattoirs situated at São José do Vale do Rio Preto/RJ poultry center and from two commercial abattoirs situated at Metropolitan Region of Rio de Janeiro, from 2013 to 2016. The resistance to enrofloxacin was assessed by agar dilution to determine minimum inhibitory concentration (MIC). The CmeABC efflux system was investigated through the detection of genes genes cmeA, cmeB and cmeC by PCR. The activity of CmeABC efflux pump was investigated in 20 strains by using the efflux pump inhibitor Phenylalanine-Arginine ß-Naphthylamide (PAßN). The three genes cmeA, cmeB and cmeC were detected in 94.3% of the strains (C. jejuni = 80 and C. coli = 70), whereas the system was absent or incomplete in 5.7% of strains (C. jejuni = 1 and C. coli = 8). MIC varied between 0.5µg/ml and 64µg/ml, and 88.7% of strains were enrofloxacin resistant and 11.3% featuring intermediate resistance. The inhibition of the efflux pump by PAßN reduced the MIC to enrofloxacin up to eight times in fifteen strains (75%). These results indicate that this system is frequent and active in Campylobacter spp. Resistant strains in the presence of enrofloxacin.(AU)

As aves são os principais reservatórios de Campylobacter spp., importante patógeno de origem alimentar e a carne de frango e produtos derivados são os principais veículos desse microrganismo. O aumento de cepas de Campylobacter spp. resistentes às fluorquinolonas, uma classe antimicrobiana frequentemente empregada na avicultura e na medicina humana, tornou-se uma grande preocupação para os produtores de aves e vários estudos avaliaram o aumento da resistência bacteriana a esses antimicrobianos. O papel do sistema de efluxo CmeABC tem sido enfatizado entre os mecanismos de resistência em Campylobacter spp. à fluorquinolonas. O presente estudo investigou a ocorrência da bomba de efluxo CmeABC em 81 cepas de Campylobacter jejuni e 78 cepas de Campylobacter coli resistentes à enrofloxacina, isoladas de frangos de corte coletados em seis abatedouros situados no polo avícola de São José do Rio Preto/RJ e de dois abatedouros comerciais situados na Região Metropolitana do Rio de Janeiro, de 2013 a 2016. A resistência à enrofloxacina foi avaliada pelo método de diluição em ágar para determinar a concentração inibitória mínima (CIM). O sistema de efluxo CmeABC foi investigado através da detecção dos genes cmeA, cmeB e cmeC por PCR. A atividade da bomba de efluxo CmeABC foi investigada em 20 cepas utilizando o inibidor da bomba de efluxo Phenylalanine-Arginine ß-Naftilamida (PAßN). Os três genes cmeA, cmeB e cmeC foram detectados em 94,3% das cepas (C. jejuni = 80 e C. coli = 70), enquanto o sistema estava ausente ou incompleto em 5,7% das cepas (C. jejuni = 1 e C coli = 8). A CIM variou entre 0,5µg/ml e 64µg/ml e 88,7% das cepas foram resistentes à enrofloxacina, enquanto 11,3% apresentaram resistência intermediária. A inibição da bomba de efluxo pelo PAßN reduziu a CIM da enrofloxacina até oito vezes em quinze cepas (75%). Estes resultados indicam que este sistema é frequente e ativo em cepas resistentes de Campylobacter spp. na presença de enrofloxacina.(AU)

Rise and rise of the ABC transporter families.

This review will inevitably be influenced by my personal experience and personal view of the progression of this amazing family of proteins. This has generated a huge literature in over nearly five decades, some ideas have bloomed and faded while others have persisted, other contributions simply become redundant, overtaken by better techniques. At the outset, the pioneers had no idea of the magnitude of the topic they were working on, then a very rough idea of the significance emerged and, progressively, the picture becomes sharper and finally extraordinary. I have tried to produce at least an outline of that progression. My apologies for the also inevitable omissions, especially relating to the mass of biochemical and spectroscopy and genetical studies. I decided to prioritise structural biology because structures when successful are definitive and of course provide a 'visual' image. However, I tried to limit the structural aspects to the proteins that reflected the main advances.

The role of procalcitonin outside of the Intensive Care Unit (ICU): a multidisciplinary approach.

OBJECTIVE: Biochemical markers are commonly used in medicine to guide diagnostic investigation or therapy duration and/or monitor treatment efficacy. Due to the emergence and spread of antimicrobial resistance, markers able to prompt a more rational use of antimicrobial therapy are regarded with the greatest attention. Procalcitonin (PCT) certainly stands out among others, yet its role must be better established especially outside of the critical care area. Data about PCT utilization in non-critical patients, optimal negativity cut-offs as well as a protocol for measurement timing are all lacking. MATERIALS AND METHODS: To address these issues, a focus group was set up to propose and endorse shared statements regarding the most beneficial use of PCT in real life as infection marker for non-critical patients, based on the authors' experience and a review of recent literature. RESULTS: A group of nine experts in the fields of Infectious Diseases, Internal Medicine, Microbiology, Clinical Chemistry, Surgery and Medical Economics participated in the discussion of nine pre-specified statements. CONCLUSIONS: The potential role for PCT in differentiating infectious and non-infectious clinical syndromes and guiding antimicrobial therapy discontinuation was acknowledged. Moreover, a shared measurement protocol and desirable cut-offs for the non-critical area were proposed. Finally, observations were made about a reasonable selection of the patient population to be tested.

Acid-happy: Survival and recovery of enteropathogenic Escherichia coli (EPEC) in simulated gastric fluid.

BACKGROUND: Gastric fluid pH serves an important function as an ecological filter to kill unwanted microbial taxa that would otherwise colonise the intestines, thereby shaping the diversity and composition of microbial communities found in the gut. The typical American-based diet causes the gastric pH to increase to pH 4 to 5, and it takes ∼2 h to return to pH 1.5 (normal). This window of increased gastric pH may allow potential pathogens to negotiate the hostile environment of the stomach. Another factor to consider is that in developing countries many people experience hypochlorhydria related to malnutrition and various gastric diseases. Enteropathogenic E. coli (EPEC) is a leading cause of infantile diarrhoea and has a high incidence in the developing world. The aim of this study was to assess the survival and recovery of non-acid adapted EPEC exposed to simulated gastric fluid (SGF) over a period of 180 min. RESULTS: EPEC were grown in nutrient-rich medium and acid challenged in SGF at pH 1.5, 2.5, 3.5 and 4.5. Culturability was evaluated using a standard plate count method, and metabolic viability was assessed via cellular energy (adenosine triphosphate [ATP] assay) and respiratory activity (3-bis(2-methyloxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide [XTT] assay), and recovery and proliferation by means of optical density in liquid cultures. Sampling was performed at 0, 30, 60, 120, and 180 min post-SGF exposure. The results of this study showed that EPEC is remarkably acid resistant and was able to survive a simulated gastric environment for up to 3 h (180 min) at various pH (1.5, 2.5, 3.5, and 4.5). EPEC was culturable at all pH (1.5, 2.5, 3.5 and 4.5) at the higher inoculum size of 5.4-7.1 × 106 CFU/ml, and at all pH except pH 1.5 at the lower inoculums of 5.4-7.1 × 103 CFU/ml or 5.4-7.1 × 101 CFU/ml. The organism remained metabolically viable at pH 1.5, 2.5, 3.5, and 4.5 and was able to recover and proliferate once placed in a neutral, nutrient-rich environment. CONCLUSION: In this study, EPEC demonstrated remarkable acid resistance and recovery at low pH without prior acid adaptation, which could prove to be problematic even in healthy people. In individuals with decreased gastric acidity, there is a higher probability of pathogen colonization and a resulting change in the gut microbiome. The results highlight the potential increase of food- and waterborne diseases in persons with compromised gastric function, or who are malnourished or immunocompromised. The data herein may possibly help in calculating more precisely the risk associated with consuming bacterial contaminated food and water in these individuals.

A switch in the poly(dC)/RmlB complex regulates bacterial persister formation.

Bacterial persisters are phenotypic variants that tolerate exposure to lethal antibiotics. These dormant cells are responsible for chronic and recurrent infections. Multiple mechanisms have been linked to persister formation. Here, we report that a complex, consisting of an extracellular poly(dC) and its membrane-associated binding protein RmlB, appears to be associated with persistence of the opportunistic pathogen Pseudomonas aeruginosa. Environmental stimuli triggers a switch in the complex physiological state (from poly(dC)/RmlB to P-poly(dC)/RmlB or RmlB). In response to the switch, bacteria decrease proton motive force and intracellular ATP levels, forming dormant cells. This alteration in complex status is linked to a (p)ppGpp-controlled signaling pathway that includes inorganic polyphosphate, Lon protease, exonuclease VII (XseA/XseB), and the type III secretion system. The persistence might be also an adaptive response to the lethal action of the dTDP-L-rhamnose pathway shutdown, which occurs due to switching of poly(dC)/RmlB.

Can light-based approaches overcome antimicrobial resistance?

The relentless rise of antibiotic resistance is considered one of the most serious problems facing mankind. This mini-review will cover three cutting-edge approaches that use light-based techniques to kill antibiotic-resistant microbial species, and treat localized infections. First, we will discuss antimicrobial photodynamic inactivation using rationally designed photosensitizes combined with visible light, with the added possibility of strong potentiation by inorganic salts such as potassium iodide. Second, the use of blue and violet light alone that activates endogenous photoactive porphyrins within the microbial cells. Third, it is used for "safe UVC" at wavelengths between 200 nm and 230 nm that can kill microbial cells without damaging host mammalian cells. We have gained evidence that all these approaches can kill multidrug resistant bacteria in vitro, and they do not induce themselves any resistance, and moreover can treat animal models of localized infections caused by resistant species that can be monitored by noninvasive bioluminescence imaging. Light-based antimicrobial approaches are becoming a growing translational part of anti-infective treatments in the current age of resistance.

Eukaryotic Initiation Factor 4E (eIF4E) sequestration mediates 4E-BP1 response to rapamycin.

The cap dependent translation initiation is a tightly controlled process of cooperative ternary complex formation by 4E-BP1, eIF4E and the 5' cap of eukaryotic mRNA in response to environmental cues like glucose, nutrients and growth factor levels. Based on the well-described effects of mTORC1/rapamycin complex on 4E-BP1 phosphorylation/s, it is generally accepted that rapamycin is a global inhibitor of cap-dependent translation. We have previously shown that 4E-BP1 resistance to rapamycin was overcome by the stoichiometric abundance of S6K1. Now we present evidence that the TOS-bearing amino terminal domain of S6K1 is sufficient to relieve the rapamycin resistance of 4E-BP1 as TOS deleted variants of S6K1, active or inactive with regard to S6K1 activity failed to bring about relief of 4E-BP1 resistance to rapamycin. We also show that the reciprocal inactivation of S6K1 by abundance of 4E-BP1 gets accomplished only with intact TOS motif in the protein. The data presented in this study identifies eIF4E and not Raptor as a cellular factor responsible to regulate rapamycin sensitivity of 4E-BP1 suggesting that the phosphorylation dynamics and rapamycin sensitivity of 4E-BP1 and S6K1 are regulated independently.

An EmrB multidrug efflux pump in Burkholderia thailandensis with unexpected roles in antibiotic resistance.

The antibiotic trimethoprim is frequently used to manage Burkholderia infections, and members of the resistance-nodulation-division (RND) family of efflux pumps have been implicated in multidrug resistance of this species complex. We show here that a member of the distinct Escherichia coli multidrug resistance B (EmrB) family is a primary exporter of trimethoprim in Burkholderia thailandensis, as evidenced by increased trimethoprim sensitivity after inactivation of emrB, the gene that encodes EmrB. We also found that the emrB gene is up-regulated following the addition of gentamicin and that this up-regulation is due to repression of the gene encoding OstR, a member of the multiple antibiotic resistance regulator (MarR) family. The addition of the oxidants H2O2 and CuCl2 to B. thailandensis cultures resulted in OstR-dependent differential emrB expression, as determined by qRT-PCR analysis. Specifically, OstR functions as a rheostat that optimizes emrB expression under oxidizing conditions, and it senses oxidants by a unique mechanism involving two vicinal cysteines and one distant cysteine (Cys3, Cys4, and Cys169) per monomer. Paradoxically, emrB inactivation increased resistance of B. thailandensis to tetracycline, a phenomenon that correlated with up-regulation of an RND efflux pump. These observations highlight the intricate mechanisms by which expression of genes that encode efflux pumps is optimized depending on cellular concentrations of antibiotics and oxidants.

Marine bacteria and fungi as promising source for new antibiotics.

Natural products and derivatives thereof are of considerable importance in the discovery of new pharmaceuticals, for example, for the treatment of cancer, diabetes, inflammation diseases, and infection diseases caused by bacteria, fungi, viruses, or parasites. The great biodiversity of marine microorganisms is reflected in their huge chemical diversity, which provides a rich source of biologically active compounds. An increasing interest in marine microorganisms as promising producers of new compounds with potential medical applications has raised increasing interest in the sustainable exploration of marine microbial resources for the discovery of new antibiotics, which is highlighted. The bottlenecks in the development of drugs using the large marine natural product pipeline are also discussed.

Novel Antibiotics in the Treatment of Urinary Tract Infections.

Several new antibiotic agents for urinary tract infections include ß-lactam/ß-lactamase inhibitor combinations with cephalosporins and carbapenems. Siderophore cephalosporins, novel aminoglycosides, fluoroquinolones, and tetracyclines are also in clinical development. Nevertheless, antimicrobial stewardship remains important in preserving the utility of the antibiotic agents currently available.