Detection of persistent resistance to antibacterial drugs in various strains of Francisella tularensis]

Under natural conditions, the Francisella tularensis strains AE-261 and P-13864 capable of forming the persist type of resistance to antibacterial drugs and being the cause of the infection in laboratory animals not responding to monotherapy with antibiotics were detectable. The antibioticograms of strains AE-261 and P-13864 under the in vitro conditions did not differ from those of the other studied strains responding to the antibiotic therapy. The observed phenomenon could be associated with individual peculiarities of the strains and their phenotypic variation in the host. Combinations of aminoglycoside antibiotics (streptomycin, gentamicin and amikacin) with rifampicin were shown to be highly active in the treatment of general forms of the infection due to such strains. The combined therapy of tularemia was also considered promising because of its high efficacy when the treatment was started at late periods as well as because unlike the monotherapy with the aminoglycoside antibiotics it provided complete elimination of the pathogen from the host.     

New immunofluorescent method for the rapid determination of microbial antibiotic sensitivity

A new procedure for rapid determination of the levels of antibiotic sensitivity in pathogenic microorganisms with the use of fluorescent antibodies is described. The procedure was developed with the use of a model of the vaccinal strains of Bacillus anthracis. It is based on determination of the microbial antibiotic resistance with the method of serial dilutions on solid media. Still, the medium with an antibiotic is inoculated instead of the pathogen with the native material subject to the analysis. The antibiotic effect on the microorganism is estimated with the method of fluorescent antibodies. The replica preparations obtained as a result of the pathogen growth in a mixed culture on nutrient media containing definite concentrations of the antibiotic are examined with the method of luminescence microscopy. The modification of the immunofluorescent procedure for rapid determination of the microbial sensitivity to antibiotics does not require obligatory isolation of the pathogen as a pure culture. This makes the procedure more economic with respect to the time necessary for the analysis. The following conditions for performing rapid analysis with respect to Bacillus anthracis are required: the minimal concentration of the pathogen in the specimen (2.10(5) spores/ml), preliminary thermal treatment of the specimen for destroying the spore microflora, additional cultivation for 6-8 hours at 37 degrees C. The presence of the accompanying sporulating microflora, i.e. common microorganisms present in the atmosphere, soil and open water bodies does not prevent the performance of the analysis.     

Antibiotic production by bacterial biocontrol agents

Interest in biological control of plant pathogens has been stimulated in recent years by trends in agriculture towards greater sustainability and public concern about the use of hazardous pesticides. There is now unequivocal evidence that antibiotics play a key role in the suppression of various soilborne plant pathogens by antagonistic microorganisms. The significance of antibiotics in biocontrol, and more generally in microbial interactions, often has been questioned because of the indirect nature of the supporting evidence and the perceived constraints to antibiotic production in rhizosphere environments. Reporter gene systems and bio-analytical techniques have clearly demonstrated that antibiotics are produced in the spermosphere and rhizosphere of a variety of host plants. Several abiotic factors such as oxygen, temperature, specific carbon and nitrogen sources, and microelements have been identified to influence antibiotic production by bacteria biocontrol agents. Among the biotic factors that may play a determinative role in antibiotic production are the plant host, the pathogen, the indigenous microflora, and the cell density of the producing strain. This review presents recent advances in our understanding of antibiotic production by bacterial biocontrol agents and their role in microbial interactions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Resistance to antibiotics of Lactobacillus isolated from the intestines of healthy persons

Lactobacilli constitute a significant part of the microbial population in the gastrointestinal tract of humans and animals. Sensitivity of intestinal lactobacilli to 11 chemotherapeutic drugs was tested with the method of serial dilutions in the solid medium MRS under aerobic and anaerobic conditions. Criteria for dividing the organisms into sensitive and resistant ones with respect to the drugs are proposed. Prevalence of lactobacilli polyresistant strains in the intestine contents of healthy persons at the age of 25 to 50 years being in prolonged constant contact with antibiotics was shown. On the whole 77 various combinations of the antibiotic resistance markers were detected in 141 tested strains. The most frequent were isolated simultaneously resistant to 6 or 7 antibiotics. Different levels of the antibiotic resistance in the strains tested under the aerobic and anaerobic conditions were observed.     

Sensitivity of the wound microflora of oncological patients to some new antibiotics]

Sensitivity of the microflora of the oncological patients' wounds to the new antibiotics, such as gentamicin, kanamycin, oxacillin, ampicillin and lincomycin was studied with the help of the disc method. The discs with the above antibiotics were prepared under laboratory conditions in accordance with the respective instructions in the WHO. Sensitivity of 429 bacterial cultures, including 98 cultures of pathogenic staphylocci, 45 cultures of Enterococci, 43 hemolytic streptococci, 143 cultures of Escherichia, 50 cultures of Ps. aeruginosa and 50 cultures of Proteus was determined. The studies showed that gentamicin was the most active antibiotic aganist all the microbial species isolated from the surgical and other wounds of oncological patients. It may be used in treatment of the infections caused by association of the microbes belonging to different species, as well as in treatment of purulent processes before elucidating their etiology, 16.7 per cent of the Enterococcal isolates were resistant to gentamicin. Monomycin, kanamycin, oxacillin, lincomycin and macrolide antibiotics at present are sufficiently active against pathogenic staphylococci and hemolytic streptococci.     

Variations in antibiotic resistance profile in Enterobacteriaceae isolated from wild Australian mammals

We carried out a retrospective analysis of 946 strains of Enterobacteriaceae isolated from wild Australian mammals between 1993 and 1997. The prevalence of resistance to fixed concentrations of 32 antimicrobial agents was determined, and the respective roles that taxonomic family of the host, state of origin and bacterial species play in defining prevalence and range of resistance were investigated. Our results demonstrated a low but widespread prevalence of antimicrobial resistance in wild isolates. Only amikacin, ciprofloxacin, meropenem and gentamicin inhibited growth in all 946 samples. There was extensive variation in the combination of antibiotics to which isolates were resistant, and multiple antibiotic resistance was common. Geographical location and host group significantly influenced the antibiotic resistance profile of an isolate, whereas bacterial species influenced both the resistance profile of an isolate and the number of antibiotics it was resistant to. The role of these factors in determining observed antibiotic resistance profiles suggests that any study measuring resistance in wild isolates should include the broadest possible range of bacterial species, host species and sampling locations. As such, this study provides an important new baseline for future measurements of antibiotic resistance in the Australian environment.     

Coselection for microbial resistance to metals and antibiotics in freshwater microcosms

Bacterial resistances to diverse metals and antibiotics are often genetically linked, suggesting that exposure to toxic metals may select for strains resistant to antibiotics and vice versa. To test the hypothesis that resistances to metals and antibiotics are coselected for in environmental microbial assemblages, we investigated the frequency of diverse resistances in freshwater microcosms amended with Cd, Ni, ampicillin or tetracycline. We found that all four toxicants significantly increased the frequency of bacterioplankton resistance to multiple, chemically unrelated metals and antibiotics. An ampicillin-resistant strain of the opportunistic human pathogen Ralstonia mannitolilytica was enriched in microcosms amended with Cd. Frequencies of antibiotic resistance were elevated in microcosms with metal concentrations representative of industry and mining-impacted environments (0.01-1 mM). Metal but not antibiotic amendments decreased microbial diversity, and a weeklong exposure to high concentrations of ampicillin (0.01-10 mg l-1) and tetracycline (0.03-30 mg l-1) decreased microbial abundance only slightly, implying a large reservoir of antibiotic resistance in the studied environment. Our results provide first experimental evidence that the exposure of freshwater environments to individual metals and antibiotics selects for multiresistant microorganisms, including opportunistic human pathogens.     

The antibacterial resistance of the strains making up the components of probiotic preparations

The spectrum of the antibiotic resistance of 21 strains of normal microflora, mainly forming constituents of widely used probiotics to 25 antibacterial preparations. Lactobacillus spp. were prevalent in the strains under test. The spectrum of the antibiotic resistance of lactobacilli varied, the gradation of resistance being more pronounced with respect to strains and not species. Both highly sensitive and highly resistant Lactobacillus strains were found: L. acidophilus (a component of biopreparation "Linex"), L. plantarum 8RA3, L. fermentum 90T4C (probiotic "Lactobacterin"), L. fermentum BL96. Bacteria used as the components of combined preparation "Linex" exhibited the highest resistance to a number of modern antibiotics. Strains of bifidobacteria were found to be highly sensitive to antibiotics.     

Intestinal dysbacteriosis in antibacterial therapy and perspectives in treatment by antibiotic-resistant probiotics]

Effect of different antibiotics and standard antibacterial therapy regimes on intestine microflora was investigated. Lincozamides demonstrated the most negative effect. Early addition of probiotics to the treatment with antibacterials had positive effect. Susceptibility of 21 strains of normal microflora to 25 antibiotics was tested. Resistance to antibiotics of lactobacilli varied significantly (more among strains and less among species). It was shown that L. acidophilus (probiotic "Acilact") was resistant to metronidazole only. High resistance to antibiotics was shown for L. plantarum 8RA3, L. fermentum 90T4C (components of probiotic "Lactobacterin"), L. fermentum BL96, L. acidophilus BL and L. acidophilus (component of "Linex"). Susceptibility of microorganisms in complex formulation "Linex" to the modern antibiotics was low. It is concluded that the use of stable antibiotic-resistant strains of normal microflora is favorable as addition to antibiotic therapy.     

Utilization of microbial iron assimilation processes for the development of new antibiotics and inspiration for the design of new anticancer agents

Pathogenic microbes rapidly develop resistance to antibiotics. To keep ahead in the “microbial war”, extensive interdisciplinary research is needed. A primary cause of drug resistance is the overuse of antibiotics that can result in alteration of microbial permeability, alteration of drug target binding sites, induction of enzymes that destroy antibiotics (ie., beta-lactamase) and even induction of efflux mechanisms. A combination of chemical syntheses, microbiological and biochemical studies demonstrate that the known critical dependence of iron assimilation by microbes for growth and virulence can be exploited for the development of new approaches to antibiotic therapy. Iron recognition and active transport relies on the biosyntheses and use of microbe-selective iron-chelating compounds called siderophores. Our studies, and those of others, demonstrate that siderophores and analogs can be used for iron transport-mediated drug delivery (“Trojan Horse” antibiotics) and induction of iron limitation/starvation (Development of new agents to block iron assimilation). Recent extensions of the use of siderophores for the development of novel potent and selective anticancer agents are also described.     

Evolution of antibiotic resistance by human and bacterial niche construction

Antibiotic treatment by humans generates strong viability selection for antibiotic-resistant bacterial strains. The frequency of host antibiotic use often determines the strength of this selection, and changing patterns of antibiotic use can generate many types of behaviors in the population dynamics of resistant and sensitive bacterial populations. In this paper, we present a simple model of hosts dimorphic for their tendency to use/avoid antibiotics and bacterial pathogens dimorphic in their resistance/sensitivity to antibiotic treatment. When a constant fraction of hosts uses antibiotics, the two bacterial strain populations can coexist unless host use-frequency is above a critical value; this critical value is derived as the ratio of the fitness cost of resistance to the fitness cost of undergoing treatment. When strain frequencies can affect host behavior, the dynamics may be analyzed in the light of niche construction. We consider three models underlying changing host behavior: conformism, the avoidance of long infections, and adherence to the advice of public health officials. In the latter two, we find that the pathogen can have quite a strong effect on host behavior. In particular, if antibiotic use is discouraged when resistance levels are high, we observe a classic niche-construction phenomenon of maintaining strain polymorphism even in parameter regions where it would not be expected.     

Altering host resistance to infections through microbial transplantation

Host resistance to bacterial infections is thought to be dictated by host genetic factors. Infections by the natural murine enteric pathogen Citrobacter rodentium (used as a model of human enteropathogenic and enterohaemorrhagic E. coli infections) vary between mice strains, from mild self-resolving colonization in NIH Swiss mice to lethality in C3H/HeJ mice. However, no clear genetic component had been shown to be responsible for the differences observed with C. rodentium infections. Because the intestinal microbiota is important in regulating resistance to infection, and microbial composition is dependent on host genotype, it was tested whether variations in microbial composition between mouse strains contributed to differences in "host" susceptibility by transferring the microbiota of resistant mice to lethally susceptible mice prior to infection. Successful transfer of the microbiota from resistant to susceptible mice resulted in delayed pathogen colonization and mortality. Delayed mortality was associated with increased IL-22 mediated innate defense including antimicrobial peptides Reg3γ and Reg3β, and immunono-neutralization of IL-22 abrogated the beneficial effect of microbiota transfer. Conversely, depletion of the native microbiota in resistant mice by antibiotics and transfer of the susceptible mouse microbiota resulted in reduced innate defenses and greater pathology upon infection. This work demonstrates the importance of the microbiota and how it regulates mucosal immunity, providing an important factor in susceptibility to enteric infection. Transfer of resistance through microbial transplantation (bacteriotherapy) provides additional mechanisms to alter "host" resistance, and a novel means to alter enteric infection and to study host-pathogen interactions.     

The effect of different oral antibiotics on the gastrointestinal microflora of a wild rodent (Aethomys namaquensis)

Gut sterilization via the oral administration of antibiotics facilitates physiological studies of the nutritionally important relationship between intestinal microflora and the host. However, the composition of gut flora is extremely variable, and as a result, the efficacy of antibiotics in achieving gut sterilization varies considerably between species. We tested the effectiveness of three antibiotic cocktails in sterilizing the gut of a rodent pollinator, the Namaqua rock mouse (Aethomys namaquensis). The cocktails were (1) streptomycin sulfate and bacitracin (previously used with domestic mice and rats), (2) chloramphenicol and bacitracin (based on antibiotic screening tests performed on faecal flora) and (3) Baytril 10% oral solution (a veterinary antimicrobial agent containing enrofloxacin). We tested for antibiotic inactivation by determining bacterial viability through fluorescence staining of faecal samples. We also tested techniques to maintain sterility during antibiotic treatment without the benefit of a laminar flow cabinet. Antibiotics were administered orally in food and water consumed ad libitum over 4 consecutive days. Antibiotic effectiveness was assessed by culturing anaerobic bacteria from faecal samples collected before and after each antibiotic treatment. Treatment with Baytril 10% oral solution eliminated or significantly reduced faecal flora, whereas other antibiotics did not. This study clearly demonstrates the importance of testing the effectiveness of antibiotics before their use in studies that involve antibiotic-treated subjects, particularly if these are species previously untested.     

Synthetic peptide vaccine and antibody therapeutic development: prevention and treatment of Pseudomonas aeruginosa

Pseudomonas aeruginosa and Pseudomonas maltophilia account for 80% of opportunistic infections by pseudomonads. Pseudomonas aeruginosa is an opportunistic pathogen that causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, and a variety of systemic infections, particularly in patients with severe burns, and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa is notable for its resistance to antibiotics, and is therefore a particularly dangerous pathogen. Only a few antibiotics are effective against Pseudomonas, including fluoroquinolones, gentamicin, and imipenem, and even these antibiotics are not effective against all strains. The difficulty treating Pseudomonas infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with a strain that is so resistant that it cannot be treated. Since antibiotic therapy has proved so ineffective as a treatment, we embarked on a research program to investigate the development of a synthetic peptide consensus sequence vaccine for this pathogen. In this review article we will describe our work over the last 15 years to develop a synthetic peptide consensus sequence anti-adhesin vaccine and a related therapeutic monoclonal antibody (cross-reactive to multiple strains) to be used in the prevention and treatment of P. aeruginosa infections. Further, we describe the identification and isolation of a small peptide structural element found in P. aeruginosa strain K (PAK) bacterial pili, which has been proven to function as a host epithelial cell-surface receptor binding domain. Heterologous peptides are found in the pili of all strains of P. aeruginosa that have been sequenced to date. Several of these peptide sequences have been used in the development of an consensus sequence anti-adhesin vaccine targeted at the prevention of host cell attachment and further for the generation of a monoclonal antibody capable of prevention and treatment of existing infections.     

Comparative proteomics to evaluate multi drug resistance in Escherichia coli

Drug resistance in food-borne bacterial pathogens is an almost inevitable consequence of the use of antimicrobial drugs, used either therapeutically or to avoid infections in food-producing animals. In the past decades, the spread and inappropriate use of antibiotics have caused a considerable increase of antibiotics to which bacteria have developed resistance and, moreover, bacteria are becoming resistant to more than one antibiotic simultaneously. Understanding mechanisms at the molecular level is extremely important to control multi-resistant strains and to develop new therapeutic strategies. In the present study, comparative proteomics was applied to characterize membrane and cytosolic proteome in order to investigate the regulation of protein expression in multi-resistance E. coli isolated from young never vaccinated water buffalo. Results highlighted differentially expressed proteins under multi drug resistance conditions giving new insights about mechanisms involved in resistance, as quorum sensing mechanisms, and suggesting possible novel bacterial targets to develop alternative antibiotic drugs.     

Antimicrobial resistance of Listeria monocytogenes

Listeria monocytogenes is an opportunistic pathogen that causes rare but frequently fatal infections, termed listerioses. In general, strains of L. monocytogenes are susceptible to a wide range of antibiotics, except for the cephalosporins, fluorochinolones and fosfomycin (Hof, 1991). The current therapy of choice is a combination of ampicillin and aminoglycoside, usually gentamicin (Lorber, 1997). In cases when it is not possible to use a beta-lactam antibiotic, second-choice therapy involves the use of an association of trimethoprim with a sulfonamide, such as in co-trimoxazole, in which the more active in the combination seems trimethoprim, synergized by the sulfa compound. Other second line agents for listeriosis include erythromycin and vancomycin (Temple and Nahata, 2000). The first strains of L. monocytogenes resistant to antibiotics were reported in 1988 (Poyart-Salmeron et al. 1990) The present paper reviews the current state of affairs with regard to the resistance of L. monocytogenes isolated from food products and clinical material to different antibiotics, with particular emphasis on those used in the therapy of listeriosis.     

Frequency of virulence genes and antibiotic resistances in Enterococcus spp. isolates from wastewater and feces of domesticated mammals and birds, and wildlife

Enterococci are gastrointestinal tract residents and also an important cause of nosocomial infections. To understand which species, virulence determinants, and antibiotic resistances are prevalent in enterococci shed by various hosts groups, a total of 1460 strains isolated from 144 fecal samples obtained from wastewater, domesticated mammals and birds, and wildlife were characterized. Identification of isolates to the species level showed that Enterococcus faecalis was dominant in domesticated mammals and birds and wildlife feces, whereas Enterococcus faecium was dominant among wastewater isolates, and that no single Enterococcus species could be associated with a specific host group. The frequency of 12 virulence determinants was evaluated among isolates, but no single virulence determinant could be associated with a specific host group. Resistance to 12 antibiotics was evaluated among isolates, and it was shown that the highest frequency of resistance at breakpoint concentration was found in domesticated mammals and birds (P?≤ 0.05 for 4 antibiotics). Our results suggests that (1) species identification and virulence typing of Enterococcus spp. isolates are not useful for the identification of the host groups responsible for fecal contamination of water by microbial source tracking and that (2) antibiotic use for clinical, veterinary, or animal husbandry practices is promoting resistance.     

Ecology and evolution of antimicrobial resistance in bacterial communities

Accumulating evidence suggests that the response of bacteria to antibiotics is significantly affected by the presence of other interacting microbes. These interactions are not typically accounted for when determining pathogen sensitivity to antibiotics. In this perspective, we argue that resistance and evolutionary responses to antibiotic treatments should not be considered only a trait of an individual bacteria species but also an emergent property of the microbial community in which pathogens are embedded. We outline how interspecies interactions can affect the responses of individual species and communities to antibiotic treatment, and how these responses could affect the strength of selection, potentially changing the trajectory of resistance evolution. Finally, we identify key areas of future research which will allow for a more complete understanding of antibiotic resistance in bacterial communities. We emphasise that acknowledging the ecological context, i.e. the interactions that occur between pathogens and within communities, could help the development of more efficient and effective antibiotic treatments.Subject terms: Microbial ecology, Antibiotics, Bacterial evolution     

The global regulator Crc modulates metabolism,susceptibility to antibiotics and virulence in Pseudomonas aeruginosa

The capacity of a bacterial pathogen to produce a disease in a treated host depends on the former's virulence and resistance to antibiotics. Several scattered pieces of evidence suggest that these two characteristics can be influenced by bacterial metabolism. This potential relationship is particularly important upon infection of a host, a situation that demands bacteria adapt their physiology to their new environment, making use of newly available nutrients. To explore the potential cross‐talk between bacterial metabolism, antibiotic resistance and virulence, a Pseudomonas aeruginosa model was used. This species is an important opportunistic pathogen intrinsically resistant to many antibiotics. The role of Crc, a global regulator that controls the metabolism of carbon sources and catabolite repression in Pseudomonas, was analysed to determine its contribution to the intrinsic antibiotic resistance and virulence of P. aeruginosa. Using proteomic analyses, high‐throughput metabolic tests and functional assays, the present work shows the virulence and antibiotic resistance of this pathogen to be linked to its physiology, and to be under the control (directly or indirectly) of Crc. A P. aeruginosa strain lacking the Crc regulator showed defects in type III secretion, motility, expression of quorum sensing‐regulated virulence factors, and was less virulent in a Dictyostelium discoideum model. In addition, this mutant strain was more susceptible to beta‐lactams, aminoglycosides, fosfomycin and rifampin. Crc might therefore be a good target in the search for new antibiotics.     

Multi-site Analysis Reveals Widespread Antibiotic Resistance in the Marine Pathogen <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

Vibrio vulnificus is a serious opportunistic human pathogen commonly found in subtropical coastal waters, and is the leading cause of seafood-borne mortality in the USA. This taxon does not sustain prolonged presence in clinical or agricultural settings, where it would undergo human-induced selection for antibiotic resistance. Therefore, few studies have verified the effectiveness of commonly prescribed antibiotics in V. vulnificus treatment. Here we screened 151 coastal isolates and 10 primary septicaemia isolates against 26 antimicrobial agents representing diverse modes of action. The frequency of multiple resistances to antibiotics from all sources was unexpectedly high, particularly during summer months, and a substantial proportion of isolates (17.3%) were resistant to eight or more antimicrobial agents. Numerous isolates demonstrated resistance to antibiotics routinely prescribed for V. vulnificus infections, such as doxycycline, tetracycline, aminoglycosides and cephalosporins. These resistances were detected at similar frequencies in virulent and non-virulent strains (PCR-based virulence typing) and were present in septicaemia isolates, underlying the public health implications of our findings. Among environmental isolates, there were no consistent differences in the frequency of resistance between pristine and anthropogenically impacted estuaries, suggesting natural rather than human-derived sources of resistance traits. This report is the first to demonstrate prevalent antibiotic resistance in a human pathogen with no clinical reservoirs, implying the importance of environmental studies in understanding the spread, evolution and public health relevance of antibiotic resistance factors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.