Presence of a Polymicrobial Endometrial Biofilm in Patients with Bacterial Vaginosis

Objective

To assess whether the bacterial vaginosis biofilm extends into the upper female genital tract.

Study Design

Endometrial samples obtained during curettage and fallopian tube samples obtained during salpingectomy were collected. Endometrial and fallopian tube samples were analyzed for the presence of bacteria with fluorescence-in-situ-hybridisation (FISH) analysis with probes targeting bacterial vaginosis-associated and other bacteria.

Results

A structured polymicrobial Gardnerella vaginalis biofilm could be detected in part of the endometrial and fallopian tube specimens. Women with bacterial vaginosis had a 50.0% (95% CI 24.0–76.0) risk of presenting with an endometrial Gardnerella vaginalis biofilm. Pregnancy (AOR  = 41.5, 95% CI 5.0–341.9, p<0.001) and the presence of bacterial vaginosis (AOR  = 23.2, 95% CI 2.6–205.9, p<0.001) were highly predictive of the presence of uterine or fallopian bacterial colonisation when compared to non-pregnant women without bacterial vaginosis.

Conclusion

Bacterial vaginosis is frequently associated with the presence of a structured polymicrobial Gardnerella vaginalis biofilm attached to the endometrium. This may have major implications for our understanding of the pathogenesis of adverse pregnancy outcome in association with bacterial vaginosis.     

Mathematical Model and Mechanisms for Biofilm Wastewater Treatment Systems

Summary Fundamental theoretical depiction is still lacking on biofilm wastewater treatment systems up to today. A mathematical model of biofilm wastewater treatment systems, taking account of suspended microorganisms and some factors influencing biofilm formation and stabilization, is developed in this paper. By theoretical and numerical analyses, the factors influencing biofilm formation and stabilization, such as the dilution rate, influent organic concentration, detachment and initial inoculum concentration etc, are discussed. Qualitative investigations were carried out and suggestions on industrial applications are then proposed. This paper not only plays an important role in understanding the physical mechanisms of biofilm dynamics, but also has far-reaching implications for industrial practices.     

细菌抗生素和重金属协同选择抗性机制研究进展

随着各类抗生素和新型复合金属材料的不断开发和使用,环境中抗生素和重金属离子协同污染的机率不断提高,对环境选择最为敏感的细菌通过自身的进化和发展形成了二者协同选择的抗性机制,如协同抗性、交叉抗性、协同调控和生物膜诱导机制。     

Long-Term Succession of Structure and Diversity of a Biofilm Formed in a Model Drinking Water Distribution System

In this study, we examined the long-term development of the overall structural morphology and community composition of a biofilm formed in a model drinking water distribution system with biofilms from 1 day to 3 years old. Visualization and subsequent quantification showed how the biofilm developed from an initial attachment of single cells through the formation of independent microcolonies reaching 30 μm in thickness to a final looser structure with an average thickness of 14.1 μm and covering 76% of the surface. An analysis of the community composition by use of terminal restriction fragment length polymorphisms showed a correlation between the population profile and the age of the sample, separating the samples into young (1 to 94 days) and old (571 to 1,093 days) biofilms, whereas a limited spatial variation in the biofilm was observed. A more detailed analysis with cloning and sequencing of 16S rRNA fragments illustrated how a wide variety of cells recruited from the bulk water initially attached and resulted in a species richness comparable to that in the water phase. This step was followed by the growth of a bacterium which was related to Nitrospira, which constituted 78% of the community by day 256, and which resulted in a reduction in the overall richness. After 500 days, the biofilm entered a stable population state, which was characterized by a greater richness of bacteria, including Nitrospira, Planctomyces, Acidobacterium, and Pseudomonas. The combination of different techniques illustrated the successional formation of a biofilm during a 3-year period in this model drinking water distribution system.     

Escherichia coli Population Structure and Antibiotic Resistance at a Buffalo/Cattle Interface in Southern Africa

At a human/livestock/wildlife interface, Escherichia coli populations were used to assess the risk of bacterial and antibiotic resistance dissemination between hosts. We used phenotypic and genotypic characterization techniques to describe the structure and the level of antibiotic resistance of E. coli commensal populations and the resistant Enterobacteriaceae carriage of sympatric African buffalo (Syncerus caffer caffer) and cattle populations characterized by their contact patterns in the southern part of Hwange ecosystem in Zimbabwe. Our results (i) confirmed our assumption that buffalo and cattle share similar phylogroup profiles, dominated by B1 (44.5%) and E (29.0%) phylogroups, with some variability in A phylogroup presence (from 1.9 to 12%); (ii) identified a significant gradient of antibiotic resistance from isolated buffalo to buffalo in contact with cattle and cattle populations expressed as the Murray score among Enterobacteriaceae (0.146, 0.258, and 0.340, respectively) and as the presence of tetracycline-, trimethoprim-, and amoxicillin-resistant subdominant E. coli strains (0, 5.7, and 38%, respectively); (iii) evidenced the dissemination of tetracycline, trimethoprim, and amoxicillin resistance genes (tet, dfrA, and bla_TEM-1) in 26 isolated subdominant E. coli strains between nearby buffalo and cattle populations, that led us (iv) to hypothesize the role of the human/animal interface in the dissemination of genetic material from human to cattle and toward wildlife. The study of antibiotic resistance dissemination in multihost systems and at anthropized/natural interface is necessary to better understand and mitigate its multiple threats. These results also contribute to attempts aiming at using E. coli as a tool for the identification of pathogen transmission pathway in multihost systems.     

Embedded Biofilm,a New Biofilm Model Based on the Embedded Growth of Bacteria

A variety of systems have been developed to study biofilm formation. However, most systems are based on the surface-attached growth of microbes under shear stress. In this study, we designed a microfluidic channel device, called a microfluidic agarose channel (MAC), and found that microbial cells in the MAC system formed an embedded cell aggregative structure (ECAS). ECASs were generated from the embedded growth of bacterial cells in an agarose matrix and better mimicked the clinical environment of biofilms formed within mucus or host tissue under shear-free conditions. ECASs were developed with the production of extracellular polymeric substances (EPS), the most important feature of biofilms, and eventually burst to release planktonic cells, which resembles the full developmental cycle of biofilms. Chemical and genetic effects have also confirmed that ECASs are a type of biofilm. Unlike the conventional biofilms formed in the flow cell model system, this embedded-type biofilm completes the developmental cycle in only 9 to 12 h and can easily be observed with ordinary microscopes. We suggest that ECASs are a type of biofilm and that the MAC is a system for observing biofilm formation.     

Structure of the Antibiotic Resistance Factor Spectinomycin Phosphotransferase from Legionella pneumophila

Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3′) isozymes and an APH(2″) enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binary complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3′) and APH(2″) enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.     

Performance and Microbial Structure of a Combined Biofilm Reactor

A novel combined biofilm reactor was established and applied as a single treatment unit for carbon and nitrogen removal of wastewater. The nitrogen removal performance of the reactor at different levels of organic carbon (COD) loading was investigated when the influent total nitrogen (TN) loading was 0.74 g TN/m² day. Continuous experimental results demonstrated that 80% nitrogen was eliminated when the influent COD loading ranged between 2.06 g and 3.92 g COD/m² day. Microbial composition in the reactor was analyzed using fluorescent in situ hybridization (FISH) and conventional batch tests. The relative abundance of ammonia-oxidizing bacteria in the aerobic zone of the reactor measured by FISH was consistent with the result from conventional batch tests.     

Effect of Periodic Disinfection on Persisters in a One-Dimensional Biofilm Model

It is well known that disinfection methods that successfully kill suspended bacterial populations often fail to eliminate bacterial biofilms. Recent efforts to understand biofilm survival have focused on the existence of small, but very tolerant, subsets of the bacterial population termed persisters. In this investigation, we analyze a mathematical model of disinfection that consists of a susceptible-persister population system embedded within a growing domain. This system is coupled to a reaction-diffusion system governing the antibiotic and nutrient. We analyze the effect of periodic and continuous dosing protocols on persisters in a one-dimensional biofilm model, using both analytic and numerical method. We provide sufficient conditions for the existence of steady-state solutions and show that these solutions may not be unique. Our results also indicate that the dosing ratio (the ratio of dosing time to period) plays an important role. For long periods, large dosing ratios are more effective than similar ratios for short periods. We also compare periodic to continuous dosing and find that the results also depend on the method of distributing the antibiotic within the dosing cycle.     

Antibiotic Resistance in Bacteria and Its Future for Novel Antibiotic Development

Since the first introduction of the sulfa drugs and penicillin into clinical use, large numbers of antibiotics have been developed and hence contributed to human health. But extensive use of antibiotics has raised a serious public health problem due to multiantibiotic resistant bacterial pathogens that inevitably develop resistance to every new drug launched in the clinic. Consequently, there is a pressing need to develop new antibiotics to keep pace with bacterial resistance. Recent advances in microbial genomics and X-ray crystallography provide opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics respectively. To prevent and control infectious diseases caused by multiantibiotic resistant bacteria, we need to understand more about the molecular aspects of the pathogens’ physiology and to pursue ways to prolong the life of precious antibiotics.     

Origins and Evolution of Antibiotic Resistance

Summary: Antibiotics have always been considered one of the wonder discoveries of the 20th century. This is true, but the real wonder is the rise of antibiotic resistance in hospitals, communities, and the environment concomitant with their use. The extraordinary genetic capacities of microbes have benefitted from man''s overuse of antibiotics to exploit every source of resistance genes and every means of horizontal gene transmission to develop multiple mechanisms of resistance for each and every antibiotic introduced into practice clinically, agriculturally, or otherwise. This review presents the salient aspects of antibiotic resistance development over the past half-century, with the oft-restated conclusion that it is time to act. To achieve complete restitution of therapeutic applications of antibiotics, there is a need for more information on the role of environmental microbiomes in the rise of antibiotic resistance. In particular, creative approaches to the discovery of novel antibiotics and their expedited and controlled introduction to therapy are obligatory.     

Clonality and Occurrence of Genes Encoding Antibiotic Resistance and Biofilm in Methicillin-Resistant Staphylococcus epidermidis Strains Isolated from Catheters and Bacteremia in Neutropenic Patients

Thirty methicillin-resistant Staphylococcus epidermidis strains isolated from catheters and blood cultures from neutropenic patients were studied. They were classified into 17 multidrug-resistance patterns. Polymerase cahin reaction analysis revealed that methicillin resistance was encoded by the mecA gene in all strains, and aminoglycosides resistance was due to aac(6')-Ie-aph(2')-Ia (23 strains), ant(4')-Ia (13), and aph(3')-IIIa (1) genes. The aac(6')-Ie-aph(2')-Ia gene was detected concomitantly with aph(3')-IIIa, and ant(4')-Ia genes in one and nine strains, respectively. Erythromycin resistance was encoded by the ermC (11 strains), ermA (6), and msrA (2) genes. The ermC gene was inducibly expressed in five strains, whereas the ermA was exclusively constitutively expressed. The icaA and icaC genes were detected in 19 strains; however, biofilm production was observed in only 16 strains. Most strains harbored multiple plasmids of variable sizes ranging from 2.2 to 70 kb, and two strains were plasmid-free. PFGE identified 15 distinct PFGE types, and five predominant genotypes were found. Our study showed the occurrence of complex genetic phenomenons. In unrelated strains, evidence of horizontal transfer of antibiotic-encoding genes and/or ica operon, and in indistinguishable strains, there is a quite good likelihood of independent steps of loss and/or gain of these genes. This genome dynamicity might have enhanced the invasiveness power of these methicillin-resistant S epidermidis strains.     

The Contribution of Antibiotic Resistance Mechanisms in Clinical Burkholderia cepacia Complex Isolates: An Emphasis on Efflux Pump Activity

Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.     

Transfer and Expression of a Multiple Antibiotic Resistance Plasmid in Marine Bacteria

Conjugal transfer of a multiresistance plasmid from Pseudomonas fluorescens to halophilic and halotolerant bacteria was studied under in vitro and in situ conditions. Mating conducted in broth as well as on plates yielded a plasmid transfer frequency of as high as 10⁻³. Among these two, plate mating facilitated conjugal transfer of plasmid, because the cell-to-cell contact is more in plate mating. When P. fluorescens was incubated in seawater, the organism progressively lost its colony forming activity within 15 days. Microscopic examination revealed the presence of very short rods, indicating that the cells have become viable but nonculturable (VNC). Mating conducted in natural seawater without any added nutrients revealed that the conjugal transfer is influenced by the physical state of the donor and the recipients as well as the availability of nutrients. But a plasmid transfer frequency of 10⁻⁷ was obtained even after the donor cells have become VNC suggesting that the nonculturable state and nutrient deprived condition may not limit plasmid transfer. The results suggest that the terrestrial bacteria entering into the seawaters with antibiotic resistance plasmids may be responsible for the prevalence of resistance genes in the marine environment. Received: 4 May 1998 / Accepted: 18 June 1998     

Transfer of Antibiotic Resistance in Staphylococcus aureus

Download video (34MB)Help with mp4 files     

Transfer of Antibiotic Resistance Marker Genes between Lactic Acid Bacteria in Model Rumen and Plant Environments

Three wild-type dairy isolates of lactic acid bacteria (LAB) and one Lactococcus lactis control strain were analyzed for their ability to transfer antibiotic resistance determinants (plasmid or transposon located) to two LAB recipients using both in vitro methods and in vivo models. In vitro transfer experiments were carried out with the donors and recipients using the filter mating method. In vivo mating examined transfer in two natural environments, a rumen model and an alfalfa sprout model. All transconjugants were confirmed by Etest, PCR, pulsed-field gel electrophoresis, and Southern blotting. The in vitro filter mating method demonstrated high transfer frequencies between all LAB pairs, ranging from 1.8 × 10⁻⁵ to 2.2 × 10⁻² transconjugants per recipient. Transconjugants were detected in the rumen model for all mating pairs tested; however, the frequencies of transfer were low and inconsistent over 48 h (ranging from 1.0 × 10⁻⁹ to 8.0 × 10⁻⁶ transconjugants per recipient). The plant model provided an environment that appeared to promote comparatively higher transfer frequencies between all LAB pairs tested over the 9-day period (transfer frequencies ranged from 4.7 × 10⁻⁴ to 3.9 × 10⁻¹ transconjugants per recipient). In our test models, dairy cultures of LAB can act as a source of mobile genetic elements encoding antibiotic resistance that can spread to other LAB. This observation could have food safety and public health implications.Lactic acid bacteria (LAB) form a taxonomically diverse group of gram-positive, catalase-negative microorganisms, which share the capacity to ferment sugars into lactic acid. Due to their aerotolerant, anaerobic nature, they are found widespread in a variety of different environments. Traditionally LAB are economically important given their use in the manufacture and preservation of fermented foods, such as milk, meat, vegetables, and cereals, in addition to their use as starter cultures. Over the last 2 decades, there has been an increased focus on the health-promoting properties associated with increased ingestion of probiotic LAB. As a result of these health claims, there is an increased availability of commercially prepared probiotic products, including yogurts, milk, cheeses, and even probiotic supplements in tablet form.The global spread of antibiotic resistance, including the emergence of multiresistant bacterial “super bug” strains, has created a public health problem of potentially crisis proportions. The very success of antibiotics accounts for part of the resistance problem; overuse of antibiotic treatments in both humans and animals has selected for a rapid increase of resistant bacterial strains. Acquired resistance genes may transfer by conjugation, transformation, or transduction. However, with regard to horizontal gene transfer (HGT), conjugation (which involves the use of plasmids or conjugative transposons as vehicles for resistance determinants) is thought to have the most significant impact on the spread of resistance genes in the environment (5).Genes conferring acquired resistance to antibiotics such as tetracycline, erythromycin, and vancomycin have been detected in LAB isolated from fermented meat and milk products (3, 6, 8, 9, 11, 22, 37). Conjugative plasmids and transposons are common in LAB (1, 4), and due to their wide environmental distribution, it is possible that these commensal bacteria act as vectors for the dissemination of antibiotic resistance determinants to the consumer via the food chain (8, 24, 32). Such evidence has raised questions regarding LAB''s traditionally accepted safety status and initiated investigations in the biosafety of probiotic products (35). However, no consensus for testing the safety of LAB probiotic products exists at the European level.To date, most of the research assessing the risk posed by the dissemination of resistance genes by LAB has been laboratory-based studies using in vitro mating models. Knowledge concerning HGT in the natural environment is limited (23, 39), and evidence is often circumstantial and extrapolated from laboratory-based studies (4). In order to fully understand the extent to which LAB strains transfer resistance genes in the natural environment, it is essential to study genetic exchange in this context. The rumen may be considered a site for potential conjugal gene transfer due to the following features: (i) its high bacterial density (10¹⁰ cells ml⁻¹); (ii) available surfaces suitable for the attachment of bacteria, including substrate particles and the rumen wall; and (iii) frequent seeding of the rumen with soil and plant microorganisms. Similarly, alfalfa sprouts provide a suitable plant model to investigate in vivo conjugal transfer between LAB strains due to their basic growth requirements (for instance, no soil is involved in growing, so therefore, background flora is eliminated), and natural LAB strains are known to colonize sprouts, so there is a good chance of survival once inoculated (16).The aim of this study was to examine the horizontal transfer of tetracycline and erythromycin resistance determinants from three wild-type LAB strains, using both an in vitro mating method and in vivo models. Impacts of this transfer are discussed in the light of food safety and potential effects on public health.