Antibacterial Effects of Green Tea Polyphenols on Clinical Isolates of Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The antibacterial effects of tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. Characterization of the minimal inhibitory concentration (MIC) of oxacillin for 30 S. aureus strains isolated from patients treated with oxacillin identified 13 strains with an oxacillin MIC ≥ 4 μg/mL as methicillin-resistant Staphylococcus aureus (MRSA) (range: 8 to 512 μg/mL), while 17 strains were methicillin-susceptible Staphylococcus aureus (MSSA) (range: 0.25–0.5 μg/mL). The MICs of TPP ranged from 50 to 180 μg/mL for both the MSSA and the MRSA strains. The MICs of oxacillin for each of the 13 MRSA strains were reduced between 8- and 128-fold when these strains were coincubated with sub-MIC (≤0.5× MIC) levels of TPP, demonstrating that the combination of TPP plus oxacillin was synergistic for all of the clinical MRSA isolates. Two-dimensional polyacrylamide gel electrophoresis identified 14 extracellular proteins of MRSA-13 down-regulated and 3 proteins up-regulated by exposure to TPP. These studies demonstrate that TPP can differentially stimulate the expression of various proteins in these bacteria and synergize the bactericidal activity of oxacillin for MRSA.     

Susceptibility of some clinical isolates of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> to fractions from the aerial parts of <Emphasis Type="Italic">Leuzea carthamoides</Emphasis>

The antimicrobial activity of the dichloromethane extract from aerial parts of Leuzea carthamoides DC. was tested in vitro against 19 Staphylococcus aureus strains (ATCC 25923, CNCTC Mau 43/60, clinical isolates). The extract was fractionated by column chromatography on silica gel into six fractions (petroleum ether, toluene, dichloromethane, ethyl acetate, methanol and water). The minimum inhibitory concentrations (MICs) of the fractions ranged from 64 to 1024 μg/mL. An ethyl acetate fraction (EA 1) with the widest range of activity inhibited all of the strains with MIC in the range 128–512 μg/mL. This fraction exhibited potent activity against strains which showed associated resistance to oxacillin, ciprofloxacin and erythromycin.     

Aminoglycoside-Resistance Mechanisms in Multidrug-Resistant <Emphasis Type="Italic">Staphylococcus </Emphasis><Emphasis Type="Italic">aureus</Emphasis> Clinical Isolates

Aminoglycoside resistance in six clinically isolated Staphylococcus aureus was evaluated. Genotypical examination revealed that three isolates (HLGR-10, HLGR-12, and MSSA-21) have aminoglycoside-modifying enzyme (AME) coding genes and another three (GRSA-2, GRSA-4, and GRSA-6) lacked these genes in their genome. Whereas isolates HLGR-10 and HLGR-14 possessed bifunctional AME coding gene aac(6′)-aph(2′′), and aph(3′)-III and showed high-level resistance to gentamycin and streptomycin, MSSA-21 possessed aph(3′)-III and exhibited low resistance to gentamycin, streptomycin, and kanamycin. The remaining three isolates (GRSA-2, GRSA-4, and GRSA-6) exhibited low resistance to all the aminoglycosides because they lack aminoglycoside-modifying enzyme coding genes in their genome. The transmission electron microscopy of the three isolates revealed changes in cell size, shape, and septa formation, supporting the view that the phenomenon of adaptive resistance is operative in these isolates.     

Emergence of vancomycin-intermediate <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> from predominant methicillin-resistant <Emphasis Type="Italic">S. aureus</Emphasis> clones in a Korean hospital

The genetic and epidemiological features of four vancomycin-intermediate Staphylococcus aureus (VISA) isolates obtained from a Korean hospital were evaluated in this study. The VISA isolates were genotyped as sequence type (ST) 5-staphylococcal cassette chromosome mec (SCCmec) II variant (n=2) and ST239-SCCmec III (n=2), which were derived from the predominant methicillin-resistant S. aureus (MRSA) clones in Korean hospitals. One VISA isolate was acquired during vancomycin treatment, whereas three VISA isolates were obtained from the patients who had not previously been exposed to glycopeptides. As VISA is likely to arise from the predominant MRSA clones and may then possibly spread between patients, the emergence of VISA should be monitored with great care in hospitals.     

Typing of Staphylococcal Cassette Chromosome <Emphasis Type="Italic">mec</Emphasis> Encoding Methicillin Resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Strains Isolated at the Bone Marrow Transplant Centre of Tunisia

Staphylococcal Cassette Chromosome mec (SCCmec) is a mobile genetic element that carries the gene mecA mediating the methicillin resistance in staphylococci. It is composed of mec and ccr gene complexes. Six SCCmec types have been defined so far. SCCmec typing of 13 methicillin-resistant Staphylococcus aureus (MRSA) out of 72 (18%) non redundant S. aureus strains recovered in 1998–2007 at the Bone Marrow Transplant Centre of Tunis was carried out. The isolates were identified by conventional methods. Antibiotic susceptibility was determined by oxacillin and cefoxitin disks and oxacillin MIC by E-test. Methicillin resistance was detected by mecA PCR. The SCCmec complex types were determined by PCR. The epidemiology of MRSA has been investigated by PFGE. Among 13 mecA positive strains, 12 were resistant to oxacillin (MIC = 3 to >256 μg/μl) and to cefoxitin and one strain was pre-resistant: susceptible to oxacillin (MIC = 0.19 μg/μl) and to cefoxitin. Hospital-acquired MRSA (HA-MRSA) strains had essentially SCCmec type IV (nine strains) or III (two strains) or I (one strain). One strain shown to carry ccrAB1 and ccrAB2 genes in combination with class B mec. Seven of 13 MRSA strains isolated from 2000 to 2006 were classified with major similarity group A harbored SCCmec type IV.     

Comparison of <Emphasis Type="Italic">mecA</Emphasis> Polymerase Chain Reaction With Phenotypic Methods for the Detection of Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In the present study, several conventional methods to detect methicillin-resistant Staphylococcus aureus (MRSA) were compared with polymerase chain reaction (PCR) detection of mecA gene–positive isolates. Cefoxitin E-test was also evaluated as a possible phenotypic method of MRSA detection. Oxacillin agar screen and PBP2′ latex agglutination methods were found to be more sensitive than oxacillin and cefoxitin disk-diffusion methods. Cefoxitin disk diffusion was found to be the most specific. A combination of oxacillin agar screening with cefoxitin disk diffusion, or oxacillin disk diffusion with PBP2′, improved sensitivity and specificity. Cefoxitin E-test with the current break points had low sensitivity and specificity (33.3% and 75%, respectively) for the detection of MRSA. However, changing the break points to ≤ 4 μg/ml and to ≥ 6 μg/ml for sensitive and resistant, respectively, greatly improved both. Changing the 30-μg cefoxitin disk-diffusion break points to ≤ 21 mm for resistant slightly improved sensitivity but had no effect on specificity. It was therefore concluded that the use of more than one screening method is necessary to detect all MRSA isolates in clinical settings.     

Vancomycin-resistance Transferability from VanA Enterococci to <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In last decade methicillin-resistant Staphylococcus aureus with high level of vancomycin-resistance (VRSA) have been reported and generally the patients with VRSA infection were also infected with a vancomycin-resistant Enterococcus (VRE). Considering that the high level of vancomycin-resistance in VRSA isolates seems to involve the horizontal transfer of Tn1546 transposon containing vanA gene from coinfecting VRE strains, the authors have studied the “in vitro” conjugative transfer of this resistance from VanA enterococci to S. aureus. Out of 25 matings performed combining five vancomycin-resistant enterococci as donors (three Enterococcus faecalis and two Enterococcus faecium), and five S. aureus as recipients, all clinical isolates, two have been successful using E. faecalis as donor. The transfer of vancomycin-resistance was confirmed by vanA gene amplification in both transconjugants and the resistance was expressed at lower levels (MIC 32 μg/ml) in comparison with the respective VRE donors (MIC > 128 μg/ml). The vancomycin-resistance of trasconjugants was maintained even after subsequent overnight passages on MSA plates containing subinhibitory levels of vancomycin. This study shows that the vanA gene transfer can be achieved through techniques “in vitro” without the use of laboratory animals employed, in the only similar experiment previously carried out by other authors, as substrate for the trasconjugant growth. Moreover, in that previous experiment, contrary to this study, the vancomycin resistant S. aureus trasconjugants were selected on erythromycin agar and not by direct vancomycin agar selection.     

Susceptibility of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains toward combinations of oxacillin-2,4-dihydroxychalcone

In order to determine the existence of synergism of the bacteriostatic action of flavonoids against G⁺ bacteria between a clinically interesting conventional antibiotic and a flavonoid, combinations of oxacillin (OXC) and 2,4-dihydroxychalcone (DCH) as enhancer were assayed against methicillin-sensitive Staphylococcus aureus ATCC 29 213 and methicillin-resistant S. aureus ATCC 43 300. Using a kinetic-turbidimetric method, growth kinetics was monitored in a broth containing variable amounts of OXC alone and combinations of variable OXC-constant DCH. The minimum inhibitory concentrations (MIC) of OXC alone and in combination with DCH were evaluated. For the 29 213 strain, OXC MIC was 25 μg/mL, while combinations of 2–8 μg/mL OXC with 10 μg/mL of DCH totally inhibited growth and showed synergism. The resistance of the 43 300 strain in the presence of OXC was verified; OXC-DCH combinations decreased bacterial growth by 35 %. DCH augments the action of OXC against methicillin-susceptible S. aureus and therefore constitutes a good bacteriostatic agent for methicillin-resistant S. aureus.     

Extracellular protease in <Emphasis Type="Italic">Actinomycetes</Emphasis> culture supernatants inhibits and detaches <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> biofilm formation

Bacterial biofilms are associated with chronic infections due to their resistance to antimicrobial agents. Staphylococcus aureus is a versatile human pathogen and can form biofilms on human tissues and diverse medical devices. To identify novel biofilm inhibitors of S. aureus, the supernatants from a library of 458 Actinomycetes strains were screened. The culture supernatants (1% v/v) of more than 10 Actinomycetes strains inhibited S. aureus biofilm formation by more than 80% without affecting the growth. The culture supernatants of these biofilm-reducing Actinomycetes strains contained a protease (equivalent to 0.1 μg proteinase K ml⁻¹), which both inhibited S. aureus biofilm formation and detached pre-existing S. aureus biofilms. This study suggests that protease treatment could be a feasible tool to reduce and eradicate S. aureus biofilms.     

Molecular characterization of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> nasal isolates from Turkey

Methicillin-resistant Staphylococcus aureus (MRSA) colonize most frequently in the anterior nares of the nose and cause serious infections all over the world. The aim of this study was to determine the nasal carriage rate of S. aureus and MRSA strains in Turkish elementary school children. We also analyzed molecular characterizations of MRSA strains by using pulse field gel electrophoresis (PFGE), multi locus sequence typing (MLST), staphylococcal chromosomal cassette mec (SCCmec) typing, and detection of the Panton-valentine leucocidin (PVL) gene. The nasal swabs were obtained from 4,050 children during a 4 month period in Ankara. In vitro antimicrobial susceptibility testing to 1 μg oxacillin and 30 μg cefoxitin was determined by a disk diffusion method. We found that the 1,001 of 4,050 (24.7%) children were colonized with S. aureus. Three S. aureus strains were resistant to oxacillin and cefoxitin. The rate of MRSA among all children was 0.07%. The MRSA strains revealed three different PFGE pattern. All MRSA isolates by harbored the SCCmec type IV element, but not the PVL gene. The two MRSA isolate belonged to sequence type (ST) 30, whereas the other one was a unique type. The results of this study demonstrated that S. aureus nasal carriage rate was consistent with previous studies. However, MRSA carriage rate was low. This study also indicated that the ST30-type IV without PVL gene MRSA clone may be expected to spread in Turkish community.     

Synergistic effect between dieckol from <Emphasis Type="Italic">Ecklonia stolonifera</Emphasis> and β-lactams against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We have been attempting for some time to discover a compound evidencing antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The dieckol isolated from Ecklonia stolonifera has been shown to exhibit antibacterial activity against methicillin-susceptible S. aureus (MSSA) and MRSA. The minimum inhibitory concentrations (MICs) of dieckol were determined in a range of 32 to 64 μg/mL against standard MSSA and MRSA strains. Furthermore, dieckol clearly reversed the high-level ampicillin and penicillin resistance of MRSA. The MICs of ampicillin against two standard strains of MRSA were dramatically reduced from 512 to 0.5 μg/mL in combination with 1/4 MIC of dieckol (16 μg/mL). The fractional inhibitory concentration (FIC) indices of ampicillin and penicillin were measured from 0.066 to 0.266 in combination with 8 or 16 μg/mL of dieckol against all tested MRSA strains, thereby suggesting that dieckol-ampicillin or dieckol-penicillin combinations exert a synergistic effect against MRSA. The results of this study indicate that dieckol, administered in combination with β-lactams, may prove effective in the treatment of MRSA infections. Our finding may also contribute to the development of an alternative phytotherapeutic anti-MRSA agent.     

Increase in cell size and acid tolerance response in a stepwise-adapted methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> mutant

A methicillin-susceptible strain of Staphylococcus aureus (MSSA) showed stepwise adaptation when grown in increasing concentrations of oxacillin, eventually reaching a maximum of 35 μg/ml. The resultant oxacillin resistant mutant strain was stable and did not revert to susceptibility on frequent subculturing. The response of the cells to different concentrations of oxacillin was examined by scanning electron microscopy, which showed that the size of the bacterium increased with increasing concentrations of oxacillin. These changes in cell size were dependent on the concentration of oxacillin and occurred only after addition of non-lethal concentrations. In the presence of lethal concentrations (≥35 μg/ml) that completely inhibited cell growth, the cell sizes were smaller than those of wild-type cells and irregular in shape. This stepwise-adapted methicillin (oxacillin) resistant S. aureus (MRSA) mutant showed a greater acid tolerance response (ATR) to lactic and citric acids than the parent susceptible strain. These data indicates that methicillin resistance alters the morphology and ATR in stepwise-adapted MRSA mutant cells.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.     

The catalase gene differentiates between some strains of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> ssp. <Emphasis Type="Italic">anaerobius</Emphasis>

Staphylococcus aureus ssp anaerobius strain S10 was isolated from an outbreak of sheep abscess disease. Sequence of the catalase gene of this strain showed 99 % identity to the catalase gene (katB) sequence of the reference strain (S. aureus ssp. anaerobius strain MVF213) with mismatching of three base pairs. An important substitution located 1036 nucleotides upstream of the initiation codon from “C” in katB to “T” in the catalase gene of strain S10 originated a stop codon. The deduced protein (345 amino acids) is 105 amino acids shorter than that of katB. Partial sequence of the catalase gene of other 8 local isolates in addition to another reference strain (DSM 20714/ATCC 35844) revealed the same mutations in all local (African) strains, whereas the sequence of the reference (European) strain was typical to that of katB. Sequence of the catalase gene of S. aureus ssp. anaerobius strain S10 was deposited in GenBank under accession no. EU281993.     

Antimicrobial resistance and presence of <Emphasis Type="Italic">ileS-2</Emphasis> gene encoding mupirocin resistance in clinical isolates of methicillin-resistant <Emphasis Type="Italic">Staphylococcus</Emphasis> sp.

Seventy-eight staphylococcal strains were isolated from surgical-site, blood-stream and other hospital-acquired infections. Eighteen isolates were determined as methicillin (MET)-resistant S. aureus (MRSA), while the remaining were MET-resistant coagulase-negative staphylococci (CoNS). Fifty percent of CoNS strains were multiresistant, while 10 % of isolates were resistant only to β-lactams. Clinical isolates of CoNS were generally more resistant to antimicrobial agents than S. aureus strains. Thirty-nine % of S. aureus strains were resistant only to β-lactams. None of the MRSA strains carried ileS-2 gene; this gene was found in two strains of S. epidermidis.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Vancomycin heteroresistance in coagulase negative <Emphasis Type="Italic">Staphylococcus</Emphasis> blood stream infections from patients of intensive care units in Mansoura University Hospitals,Egypt

Background

Vancomycin heteroresistance in coagulase negative Staphylococci (CoNS) is a recent health concern especially in serious infections like bloodstream infections as it may lead to failure of therapy. Little information is available about the prevalence vancomycin heteroresistance in CoNS causing bloodstream infections in intensive care units (ICUs) patients of Mansoura University Hospitals (MUHs).

Methods

This prospective study enrolled 743 blood samples collected from ICUs patients presented with clinical manifestations of bloodstream infections over the period extending from January 2014 to March 2016. Samples were processed, coagulase negative Staphylococci were identified by routine microbiological methods and the absence of coagulase activity. Species were identified by API Staph 32. Oxacillin resistant CoNS were identified by cefoxitin disc diffusion method. Susceptibility testing of isolated CoNS to vancomycin was carried out using vancomycin agar dilution method. Mec A gene detection by PCR was done for oxacillin resistant isolates. Screening for vancomycin heteroresistance was done on brain heart infusion (BHI) agar containing 4 μg/mL vancomycin. Confirmation of vancomycin heteroresistance was carried out by population analysis profile (PAP).

Results

A total of 58 isolates were identified as CoNS from patients of clinically suspected bloodstream infections. The identified species were 33 (56.9%) Staphylococcus epidermidis, 12 (20.7%) Staphylococcus capitis, 7 (12.1%) Staphylococcus haemolyticus, and 3 isolates (5.2%) Staphylococcus lugdunesis. Three isolates were unidentified by API Staph 32. Forty-four (75.9%) isolates were oxacillin resistant. Mec A gene was detected in all oxacillin resistant isolates. All isolates had susceptible vancomycin MICs by agar dilution. Nine isolates (15.5%) could grow on BHI agar containing 4 μg/mL vancomycin. These isolates showed heterogeneous profile of resistance to vancomycin by population analysis profile.

Conclusions

Vancomycin heteroresistant CoNS causing bloodstream infections is growing unrecognized health hazard in ICUs patients. These isolates have susceptible vancomycin MICs. Screening methods are recommended and should be considered to improve clinical outcome in these high risk patients.

     

Rapid detection of methicillin resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates; evaluation of colorimetric Quicolor ES agar and determination of breakpoint inhibition zone diameters of cefoxitin

In this study, it was aimed to evaluate colorimetric Quicolor ES agar for the rapid detection of methicillin resistance and to determine susceptibility and resistance breakpoint zone diameters for cefoxitin by using 51 methicillin susceptible Staphylococcus aureus (MSSA) and 63 methicillin resistant S. aureus (MRSA) isolates. In the study, while oxacillin and cefoxitin results were obtained within 4–7 h (5.5 h in average) for MSSA isolates, the results of MRSA isolates were obtained within 5.5–9 h (6.6 h in average) for both antibiotics on QC ES agar. QC ES agar is an inexpensive medium for rapid detection (4–9 h) of methicillin resistance by disc diffusion method using oxacillin or cefoxitin. Additional studies for further evaluation of the efficiency of QC-ES agar in rapid determination of methicillin resistance in S. aureus may be beneficial.     

Multidrug-Resistant Enterococci in Animal Meat and Faeces and Co-Transfer of Resistance from an <Emphasis Type="Italic">Enterococcus durans</Emphasis> to a Human <Emphasis Type="Italic">Enterococcus faecium</Emphasis>

Forty-eight isolates resistant to at least two antibiotics were selected from 53 antibiotic-resistant enterococci from chicken and pig meat and faeces and analysed for specific resistance determinants. Of the 48 multidrug-resistant (MDR) strains, 31 were resistant to two antibiotics (29 to erythromycin and tetracycline, 1 to erythromycin and vancomycin, 1 to vancomycin and tetracycline), 14 to three (erythromycin, tetracycline and vancomycin or ampicillin) and 3 to four (erythromycin, vancomycin, ampicillin and gentamicin). erm(B), tet(M), vanA and aac (6′)-Ie aph (2′′)-Ia were the antibiotic resistance genes most frequently detected. All 48 MDR enterococci were susceptible to linezolid and daptomycin. Enterococcus faecalis (16), Enterococcus faecium (8), Enterococcus mundtii (2) and Enterococcus gallinarum (1) were identified in meat, and E. faecium (13) and Enterococcus durans (13) in faeces. Clonal spread was not detected, suggesting a large role of gene transfer in the dissemination of antibiotic resistance. Conjugative transfer of resistance genes was more successful when donors were enterococcal strains isolated from faeces; co-transfer of vanA and erm(B) to a human E. faecium occurred from both E. faecium and E. durans pig faecal strains. These data show that multidrug resistance can be found in food and animal species other than E. faecium and E. faecalis, and that these species can efficiently transfer antibiotic resistance to human strains in inter-specific matings. In particular, the occurrence of MDR E. durans in the animal reservoir could have a role in the emergence of human enterococcal infections difficult to eradicate with antibiotics.