Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation

Background

Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.

Methods

Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.

Results

Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.

Conclusion

We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.     

Genomic and proteomic profiles of <Emphasis Type="Italic">Acholeplasma laidlawii</Emphasis> strains differing in sensitivity to ciprofloxacin

As a result of comparative analysis of complete genomes as well as cell and vesicular proteomes of A. laidlawii strains differing in sensitivity to ciprofloxacin, it was first shown that the mycoplasma resistance to the antibiotic is associated with the reorganization of genomic and proteomic profiles, which concerns many genes and proteins involved in fundamental cellular processes and realization of bacterial virulence.     

Escherichia coli-induced productions of pro-inflammatory cytokines are regulated by MAP kinases and G-protein but not by Akt: Relationship with phylogenetic groups and resistance patterns

Introduction

We investigated the role of PI3-K, MAP kinases, and heterotrimeric G proteins in inducing cytokines production in human whole blood cultures stimulated by viable Escherichia coli (E. coli) clinical strains.

Materials and methods

We used eight E. coli strains that belong to different phylogenetic groups and presented by different antibiotic resistance patterns. Whole blood from healthy volunteers was incubated at 37 °C for 150 min, with lipopolysaccharide (LPS) from E. coli O111:B4 or selected viable E. coli clinical strains, with or without SB202190 (p38 inhibitor), PD98059 (ERK inhibitor), PTX (pertussis toxin; heterotrimeric G proteins inhibitor), wortmaninn (PI3-K inhibitor). The TNF-α, IL-1β, IL-10 and IFN-γ concentrations were measured in culture supernatants (ELISA).

Results

IL-10 and IFN-γ were not detectable. Susceptible strains induced higher TNF-α and IL-1β productions than β-lactam resistant strains (p < 0.05), with no difference between phylogenetic groups. A transformed strain carrying a plasmid-mediated AmpC-β-lactamase gene (CMY-2) induced lower TNF-α and IL-1β production than the parent wild type strain (p < 0.05). SB202190 (p38 inhibitor) and PD98059 (ERK inhibitor) reduced TNF-α concentrations by, respectively, 80% (p < 0.05) and 50% (p < 0.05). Wortmaninn (PI3-K inhibitor) had no significant effect. PTX (heterotrimeric G proteins inhibitor) altered TNF-α production after viable bacteria stimulation (1.7-fold increase; p < 0.05) but not after LPS (TLR-4) stimulation. Regarding IL-1β, wortmaninn, SB202190 and PTX had no significant effect whereas PD98059 significantly decreased production in whole cell cultures (p < 0.05).

Conclusion

Susceptible strains induce greater TNF-α and IL-1β productions than resistant strains. ERK kinase plays a major role in viable E. coli strains inducing TNF-α and IL-1β production. E. coli exerts an effect on the pertussis toxin-sensitive G-protein through a TLR-4-independent mechanism.     

<Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> glucose uptake in biofilm versus planktonic cells

The aim of this work was to compare the glucose uptake of biofilms formed by four different Staphylococcus epidermidis strains as well as to compare between sessile and planktonic cells of the same strain. Biofilm cells showed a lower level of glucose uptake compared to planktonic cells. Moreover, glucose uptake by cells in the sessile form was strongly influenced by biofilm composition. Therefore, this work helps to confirm the phenotypic variability of S. epidermidis strains and the different behaviour patterns between sessile and free cells.     

Deciphering the multifactorial nature of Acinetobacter baumannii pathogenicity

Background

Acinetobacter baumannii is an emerging bacterial pathogen that causes a broad array of infections, particularly in hospitalized patients. Many studies have focused on the epidemiology and antibiotic resistance of A. baumannii, but little is currently known with respect to its virulence potential.

Methodology/Principal Findings

The aim of this work was to analyze a number of virulence-related traits of four A. baumannii strains of different origin and clinical impact for which complete genome sequences were available, in order to tentatively identify novel determinants of A. baumannii pathogenicity. Clinical strains showed comparable virulence in the Galleria mellonella model of infection, irrespective of their status as outbreak or sporadic strains, whereas a non-human isolate was avirulent. A combined approach of genomic and phenotypic analyses led to the identification of several virulence factors, including exoproducts with hemolytic, phospholipase, protease and iron-chelating activities, as well as a number of multifactorial phenotypes, such as biofilm formation, surface motility and stress resistance, which were differentially expressed and could play a role in A. baumannii pathogenicity.

Conclusion/Significance

This work provides evidence of the multifactorial nature of A. baumannii virulence. While A. baumannii clinical isolates could represent a selected population of strains adapted to infect the human host, subpopulations of highly genotypically and phenotypically diverse A. baumannii strains may exist outside the hospital environment, whose relevance and distribution deserve further investigation.     

Genetic Architecture of Intrinsic Antibiotic Susceptibility

Background

Antibiotic exposure rapidly selects for more resistant bacterial strains, and both a drug''s chemical structure and a bacterium''s cellular network affect the types of mutations acquired.

Methodology/Principal Findings

To better characterize the genetic determinants of antibiotic susceptibility, we exposed a transposon-mutagenized library of Escherichia coli to each of 17 antibiotics that encompass a wide range of drug classes and mechanisms of action. Propagating the library for multiple generations with drug concentrations that moderately inhibited the growth of the isogenic parental strain caused the abundance of strains with even minor fitness advantages or disadvantages to change measurably and reproducibly. Using a microarray-based genetic footprinting strategy, we then determined the quantitative contribution of each gene to E. coli''s intrinsic antibiotic susceptibility. We found both loci whose removal increased general antibiotic tolerance as well as pathways whose down-regulation increased tolerance to specific drugs and drug classes. The beneficial mutations identified span multiple pathways, and we identified pairs of mutations that individually provide only minor decreases in antibiotic susceptibility but that combine to provide higher tolerance.

Conclusions/Significance

Our results illustrate that a wide-range of mutations can modulate the activity of many cellular resistance processes and demonstrate that E. coli has a large mutational target size for increasing antibiotic tolerance. Furthermore, the work suggests that clinical levels of antibiotic resistance might develop through the sequential accumulation of chromosomal mutations of small individual effect.     

Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions

The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains.The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains (B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains.     

Quinine sulfate and bacterial invasion

Background

As many patients who receive antimalarial drugs for treatment of noninfectious, inflammatory diseases are also immunosuppressed and might have a concomitant bacterial infection, we studied the effectiveness of these drugs against bacterial infections, to find out whether they could protect against (and even treat) such conditions and obviate the need for an additional antibiotic drug.

Methods

Effect of QS on bacterial growth: Escherichia coli (E. coli) HB101 pRI203 were cultured overnight at 37°C in TSB and inoculated (approx 1 × 10⁷ cells /ml) in MEM in the presence of QS at various concentrations (0, 50 and 100 μM).The effect of QS at concentration of 50 and 100 μM on the entry process of E. coli HB101 pRI203 into HeLa cells was studied under different experimental conditions: 1. QS was incubated with 3 × 10⁵ HeLa cells for 60 min at 37°C prior to infection. 2. QS was added to HeLa cell monolayers during the infection period.

Results

QS showed no antibacterial activity after 24 h of incubation.The invasive efficiency of the bacteria was significantly inhibited at a dose-dependent manner, when QS was added to HeLa cells for 60 min at 37°C prior to infection (condition 1), and to a lesser extent when added during the period of infection (condition 2).

Conclusions

Although the antimalarials are generally regarded as being inactive against most extracellular bacterial species, our results indicate that QS significantly inhibited the internalization/invasion efficacy of E. coli in the host cells.

     

In vitro activity of the antimicrobial peptide AP7121 against the human methicillin-resistant biofilm producers Staphylococcus aureus and Staphylococcus epidermidis

Abstract

In vitro activity against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis biofilm producers from blood cultures of patients with prosthetic hip infections was evaluated. The Minimum Inhibitory Concentration (MIC) for AP7121 was determined and the bactericidal activity of AP7121 (MICx1, MICx4) against planktonic cells was studied at 4, 8 and 24?h. The biofilms formed were incubated with AP7121 (MICx1, MICx4) for 1 and 24?h. The anti-adhesion effect of an AP7121-treated inert surface over the highest MIC isolate was studied with scanning electron microscopy (SEM). The bactericidal activity of AP7121 against all the planktonic staphylococcal cells was observed at 4?h at both peptide concentrations. Dose-dependent anti-biofilm activity was detected. AP7121 (MICx4) showed bactericidal activity at 24?h in all isolates. SEM confirmed prevention of biofilm formation. This research showed the in vitro anti-biofilm activity of AP7121 against MRSA and S. epidermidis and the prevention of biofilm formation by them on an abiotic surface.     

Inducible disruption of autophagy in the lung causes airway hyper-responsiveness

Objective

Central adiposity and inflammation play key roles in the development of insulin resistance through the effects of pro-inflammatory adipokines such as IL-6, but the effect of infiltrating adipocytes in skeletal muscle tissues is not known. Communications between muscle cells and fat cells may contribute to the inflammatory response associated with insulin resistance.

Methods

In this study we used a co-culture system of skeletal muscle (L6) and adipocyte (3T3-L1) cell lines to study expression of the inflammatory cytokine IL-6 and changes in insulin signaling. This model could mimic the adipocytes infiltrating myocytes that is commonly seen in obese patients.

Results

When plated alone the L6 cells express IL-6 mRNA and secrete IL-6 protein, both of which are increased when the cells are challenged with the bacterial lipopolysaccharide (LPS). In contrast, the 3T3-L1 cells had very little expression of IL-6 mRNA or protein. Co-culture of 3T3-L1 pre-adipocytes with L6 cells, at a density ratio of 1:10, respectively, increased IL-6 expression significantly and decreased insulin-stimulated Akt phosphorylation. To examine the role of IL-6 in insulin sensitivity we incubated the L6 cells with IL-6. A brief challenge of L6 cells with IL-6 enhanced insulin-stimulated Akt phosphorylation. In contrast, incubation of the L6 cells with IL-6 for 96 h markedly decreased insulin-stimulated Akt phosphorylation.

Conclusion

The enhanced IL-6 mRNA expression and IL-6 release in L6 myocytes co-cultured with 3T3-L1 cells indicate an important interaction between adipocytes and myocytes. This observation may shed some light on the long-standing enigma of obesity-induced insulin resistance where infiltration of the skeletal muscle by preadipocytes/adipocytes is evident.     

Salmonella enterica serovar Typhimurium-induced internalization and IL-8 expression in HeLa cells does not have a direct relationship with intracellular Ca levels

The invasion-associated type III secretion system (T3SS-1) of S. Typhimurium is required to initiate and sustain an acute inflammatory response in the intestine. We investigated the relationship of S. Typhimurium T3SS-1-induced IL-8 expression and invasion with intracellular Ca²⁺ mobilization in HeLa cells. Compared to the sipAsopABDE2 mutant, strains carrying a mutation in sipA, or mutations in sopABDE2 induced higher levels of IL-8 and greater bacterial internalization despite the fact that these mutants elicited similarly low intracellular concentrations of Ca²⁺. Likewise, complemented sipAsopABDE2 mutant with sopE2 did not affect intracellular Ca²⁺ concentrations or IL-8 expression, but significantly increased bacterial internalization. Treating HeLa cells with the calcium chelator BAPTA-AM or with D-BAPTA-AM, a derivative with greatly reduced Ca²⁺ chelating activity, yielded strong evidence that BAPTA-AM does not affect invasion and inhibits IL-8 secretion by a calcium-dependent mechanism. These findings suggest that, although wild-type S. Typhimurium-induced IL-8 expression and bacterial internalization in HeLa cells coincides with increased cytosolic Ca²⁺, the differing levels of IL-8 and invasion induced by strains carrying different effector proteins are unrelated to levels of intracellular Ca²⁺.     

Tetracycline and Chloramphenicol Efficiency Against Selected Biofilm Forming Bacteria

Despite the constantly increasing need for new antimicrobial agents, antibiotic drug discovery and development seem to have greatly decelerated in recent years. Presented with the significant problem of advancing antimicrobial resistance, the global scientific community has attempted to find alternative solutions; one of the most promising ones is the evaluation and use of old antibiotic compounds. A number of old antibiotic compounds, such as aminoglycosides, chloramphenicol, and tetracycline, are re-emerging as valuable alternatives for the treatment of difficult-to-treat infections. This study examined the in vitro potency for biofilm formation of five isolates (Klebsiella sp., Pseudomonas aeruginosa, Achromobacter sp., Klebsiella pneumoniae, and Bacillus pumilis) and the effects of antibiotics on these biofilms. Furthermore the quantitative analysis of planktonic, loosely attached cells, and their susceptibility to antibiotics was also determined. Twitching motility was observed to determine any effect in the biofilm forming capability of the isolates. All the isolates tested were efficient biofilm-forming strains in the polypropylene and borosilicate test tubes. Standard bacterial enumeration technique and CV staining produced equivalent results both in biofilm and planktonic assays. The biofilm formation of all the strains was affected in the presence of tetracycline or chloramphenicol. Highly significant decrease (P < 0.01) in biofilm formation was observed by treatment with chloramphenicol compared to tetracycline. In addition, the two antibiotics also affected adversely the planktonic and loosely attached cells of all isolates. Thus, testing the effects of older antibiotics on biofilms may supply useful information in addition to standard in vitro testing, particularly in diseases where biofilm formation is involved in the pathogenesis.     

Tracking Acquired Antibiotic Resistance in Commensal Bacteria of Galápagos Land Iguanas: No Man,No Resistance

Background

Antibiotic resistance, evolving and spreading among bacterial pathogens, poses a serious threat to human health. Antibiotic use for clinical, veterinary and agricultural practices provides the major selective pressure for emergence and persistence of acquired resistance determinants. However, resistance has also been found in the absence of antibiotic exposure, such as in bacteria from wildlife, raising a question about the mechanisms of emergence and persistence of resistant strains under similar conditions, and the implications for resistance control strategies. Since previous studies yielded some contrasting results, possibly due to differences in the ecological landscapes of the studied wildlife, we further investigated this issue in wildlife from a remote setting of the Galapagos archipelago.

Methodology/Principal Findings

Screening for acquired antibiotic resistance was carried out in commensal enterobacteria from Conolophus pallidus, the terrestrial iguana of Isla Santa Fe, where: i) the abiotic conditions ensure to microbes good survival possibilities in the environment; ii) the animal density and their habits favour microbial circulation between individuals; and iii) there is no history of antibiotic exposure and the impact of humans and introduced animal species is minimal except for restricted areas. Results revealed that acquired antibiotic resistance traits were exceedingly rare among bacteria, occurring only as non-dominant strains from an area of minor human impact.

Conclusions/Significance

Where both the exposure to antibiotics and the anthropic pressure are minimal, acquired antibiotic resistance traits are not normally found in bacteria from wildlife, even if the ecological landscape is highly favourable to bacterial circulation among animals. Monitoring antibiotic resistance in wildlife from remote areas could also be a useful tool to evaluate the impact of anthropic pressure.     

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.

Results

Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.

Conclusions

The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1599-9) contains supplementary material, which is available to authorized users.     

Biofilm formation as an extra gear for Apilactobacillus kunkeei to counter the threat of agrochemicals in honeybee crop

The alteration of a eubiosis status in honeybees’ gut microbiota is directly linked to the occurrence of diseases, and likely to the honeybees decline. Since fructophilic lactobacilli were suggested as symbionts for honeybees, we mechanistically investigated their behaviour under the exposure to agrochemicals (Roundup, Mediator and Reldan containing glyphosate, imidacloprid and chlorpyrifos-methyl as active ingredients respectively) and plant secondary metabolites (nicotine and p-coumaric acid) ingested by honeybees as part of their diet. The effects of exposure to agrochemicals and plant secondary metabolites were assessed both on planktonic cells and sessile communities of three biofilm-forming strains of Apilactobacillus kunkeei. We identified the high sensitivity of A. kunkeei planktonic cells to Roundup and Reldan, while cells embedded in mature biofilms had increased resistance to the same agrochemicals. However, agrochemicals still exerted a substantial inhibitory/control effect if the exposure was during the preliminary steps of biofilm formation. The level of susceptibility resulted to be strain-specific. Exopolysaccharides resulted in the main component of extracellular polymeric matrix (ECM) in biofilm, but the exposure to Roundup caused a change in ECM production and composition. Nicotine and p-coumaric acid had a growth-promoting effect in sessile communities, although no effect was found on planktonic growth.     

Interaction of fosfomycin with the Glycerol 3-phosphate Transporter of Escherichia coli

Background

Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections of bacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterial Glycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycin resistance.

Methods

The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia coli reconstituted in liposomes has been studied. IC₅₀ and the half-saturation constant of the transporter for external fosfomycin (K_i) were determined by transport assay of [¹⁴C]glycerol-3-phosphate catalyzed by recombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed with recombinant GlpT was measured.

Results

Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC₅₀ of 6.4 mM. A kinetic analysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with the substrate-binding site and the K_i measured was 1.65 mM. Transport assays performed with proteoliposomes containing internal fosfomycin indicate that it was not very well transported by GlpT. Complementation study, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, beside deficiency in antibiotic transporter, could be due to other gene defects.

Conclusions

The poor transport observed in a reconstituted system together with the high value of K_i and the results of complementation study well explain the usual high dosage of this drug for the treatment of the urinary tract infections.

General significance

This is the first report regarding functional analysis of interaction between fosfomycin and GlpT.     

A study of the competitive properties of the aluminum-tolerant strain <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii</Emphasis> 9-4A by the antibiotic resistance method

The virulence, competitive ability, and symbiotic efficiency of 2 Rhizobium leguminosarum bv. trifolii strains—the wild aluminum tolerant strain 9-4A and the commercial strain 348a—were compared when introducting their variants marked with antibiotic resistance into the rhizosphere of red clover (Trifolium pratense L.) plants. High virulence and competitive ability of the strain tolerant to aluminum was demonstrated by a concurrent inoculation of the seeds with these two strains. The resistance acquisition by the commercial strain was accompanied by a decrease in its symbiotic efficiency. Presumably, the resistant variant of aluminum-tolerant isolate retains its symbiotic properties due to its adaptation to acidity factors at the level of membrane function.