Digital antimicrobial susceptibility testing using the MilliDrop technology

We present the MilliDrop Analyzer (MDA), a droplet-based millifluidic system for digital antimicrobial susceptibility testing (D-AST), which enables us to determine minimum inhibitory concentrations (MICs) precisely and accurately. The MilliDrop technology was validated by using resazurin for fluorescence readout, for comparison with standard methodology, and for conducting reproducibility studies. In this first assessment, the susceptibility of a reference Gram-negative strain Escherichia coli ATCC 25922 to gentamicin, chloramphenicol, and nalidixic acid were tested by the MDA, VITEK®2, and broth microdilution as a reference standard. We measured the susceptibility of clinically relevant Gram-positive strains of Staphylococcus aureus to vancomycin, including vancomycin-intermediate S. aureus (VISA), heterogeneous vancomycin-intermediate S. aureus (hVISA), and vancomycin-susceptible S. aureus (VSSA) strains. The MDA provided results which were much more accurate than those of VITEK®2 and standard broth microdilution. The enhanced accuracy enabled us to reliably discriminate between VSSA and hVISA strains.     

In vitro antibacterial effects of statins against bacterial pathogens causing skin infections

With financial considerations impeding research and development of new antibiotics, drug repurposing (finding new indications for old drugs) emerges as a feasible alternative. Statins are extensively prescribed around the world to lower cholesterol, but they also possess inherent antimicrobial properties. This study identifies statins with the greatest potential to be repurposed as topical antibiotics and postulates a mechanism of action for statins’ antibacterial activity. Using broth microdilution, the direct antibacterial effects of all seven parent statins currently registered for human use and three selected statin metabolites were tested against bacterial skin pathogens Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens. Simvastatin and pitavastatin lactone exerted the greatest antibacterial effects (minimum inhibitory concentrations of 64 and 128 μg/mL, respectively) against S. aureus. None of the statins tested were effective against E. coli, P. aeruginosa, or S. marcescens, but simvastatin hydroxy acid acid might be active against S. aureus, E. coli, and S. marcescens at drug concentrations >?256 μg/mL. It was found that S. aureus may exhibit a paradoxical growth effect when exposed to simvastatin; thus, treatment failure at high drug concentrations is theoretically probable. Through structure-activity relationship analysis, we postulate that statins’ antibacterial action may involve disrupting the teichoic acid structures or decreasing the number of alanine residues present on Gram-positive bacterial cell surfaces, which could reduce biofilm formation, diminish bacterial adhesion to environmental surfaces, or impede S. aureus cell division.     

Microbiology of chronic rhinosinusitis

Most sinus infections are viral and only a small percentage develop bacterial infection. Rhino-, influenza, and para-influenza viruses are the most frequent viral causes of sinusitis. The most common bacterial isolates from children and adult patients with community-acquired acute bacterial sinusitis are Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pyogenes. Staphylococcus aureus and anaerobic organisms (Prevotella and Porphyromonas, Fusobacterium, and Peptostreptococcus spp.) are the commonest isolates in chronic rhinosinusitis (CRS). Aerobic and anaerobic beta lactamase-producing bacteria (BLPB) were recovered from over a third of these patients. Methicillin-resistant S. aureus (MRSA) accounted for over 60 % of S. aureus isolates. Pseudomonas aeruginosa and other aerobic and facultative Gram-negative rods are frequently recovered in nosocomial sinusitis, the immunocompromised host, individuals with human immunodeficiency virus infection, and in cystic fibrosis. The CRS infection evolves the formation of a biofilm that might play a significant role in the pathogenesis and persistence of CRS. The microbiology of sinusitis is influenced by previous antimicrobial therapy, vaccinations, and the presence of normal flora capable of interfering with the growth of pathogens. Recognition of the unique microbiology of CRS and their antimicrobial susceptibility is of great importance when selecting antimicrobial therapy.     

Antimicrobial effects of new designed 2-amino-tetrahydro-4<Emphasis Type="Italic">H</Emphasis>-chromene-3-carbonitrile derivatives against some pathogenic microbial strains

Chromenes, also called benzopyrane derivatives and chromene-4-one, are natural compounds which have several biological effects. The aims of the present study were to synthesize 2-amino-tetrahydro-4H-chromene-3-carbonitrile derivatives and to evaluate their antibacterial effects against selected bacterial strains. Nine 2-amino-tetrahydro-4H-chromene-3-carbonitrile derivatives were designed and synthesized. Each synthesized derivative was dissolved in dimethyl sulfoxide and diluted using distilled water. Then, serial dilutions of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/ml were prepared and added to the Mueller-Hinton agar medium. The minimum inhibitory concentration (MIC) of derivatives was determined against routine pathological strains of bacteria including Staphylococcus epidermidis, Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Serratia marcescens, Pseudomonas aeruginosa, and Klebsiella pneumoniae on the basis of growth on the each plate. Only two compounds of 2-amino-5,6,6,1-tetrahydro-5-oxo-4-(3-pyridinyl)-4H-chromene-3-carbonitrile and 2-amino-5,6,6,1-tetrahydro-6,6-dimethyl-5-oxo-4-(3-pyridinyl)-4H-chromene-3-carbonitrile showed antibacterial activity especially against M. luteus and B. subtilis. Some of 2-amino-tetrahydro-4H-chromene-3-carbonitrile derivatives had stronger antibacterial effects which may be due to substituted pyridine ring. Therefore, these compounds are a candidate as the new choices of antibacterial drugs.     

Reduced pro-inflammatory responses to <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> bloodstream infection and low prevalence of enterotoxin genes in isolates from patients on haemodialysis

Patients with end-stage renal failure undergo regular haemodialysis (HD) and often develop episodes of Staphylococcus aureus bloodstream infection (BSI), which can re-occur. However, clinically, patients on HD, with S. aureus BSI, respond well to treatment, rarely developing overt signs of sepsis. We investigated the contributions of bacterial virulence and cytokine responses to the clinical course of S. aureus BSI in HD and non-HD patients. Seventy patients were recruited, including 27 (38.6 %) patients on HD. Isolates were spa-typed and virulence and antimicrobial resistance gene carriage was investigated using DNA microarray analysis. Four inflammatory cytokines, IL-6, RANTES, GROγ and leptin, were measured in patient plasma on the day of diagnosis and after 7 days. There was no significant difference in the prevalence of genotypes or antimicrobial resistance genes in S. aureus isolates from HD compared to non-HD patients. The enterotoxin gene cluster (containing staphylococcal enterotoxins seg, sei, sem, sen, seo and seu) was significantly less prevalent among BSI isolates from HD patients compared to non-HD patients. Comparing inflammatory cytokine response to S. aureus BSI in HD patients to non-HD patients, IL-6 and GROγ were significantly lower (p?=?0.021 and p?=?0.001, respectively) in HD patients compared to other patients on the day of diagnosis and RANTES levels were significantly lower (p?=?0.025) in HD patients on day 7 following diagnosis. Lowered cytokine responses in HD patients and a reduced potential for super-antigen production by infecting isolates may partly explain the favourable clinical responses to episodes of S. aureus BSI in HD patients that we noted clinically.     

Mammary candidiasis: molecular-based detection of <Emphasis Type="Italic">Candida</Emphasis> species in human milk samples

In this prospective and monocentric study, we investigated the performance of a commercialized real-time polymerase chain reaction (RT-PCR) test system for the specific detection of DNA from Candida albicans, C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis in human milk samples of patients suspicious of mammary candidiasis. For this purpose, 43 breast-feeding women with characteristic symptoms of mammary candidiasis and 40 asymptomatic controls were enrolled. By culture, Candida spp. were detected in 8.8 % (4/46) and 9.3 % (4/43) of patient and control samples, respectively. Candida albicans (2/46), C. parapsilosis (1/46), and C. guilliermondii (1/46) were present in patient samples, and C. lusitaniae (3/43) and C. guilliermondii (1/43) were present in the controls. After RT-PCR was applied, Candida spp. were found to be present in 67.4 % (31/46) and 79.1 % (34/43) of patient and control samples investigated, respectively. PCR detection of C. albicans and C. parapsilosis revealed only a low sensitivity and specificity of 67.4 % and 41.9 %, respectively. Our data do not support the use of Candida RT-PCR for sensitive and specific diagnosis of mammary candidiasis.     

Incidence and antimicrobial resistance trends in bloodstream infections caused by ESKAPE and <Emphasis Type="Italic">Escherichia coli</Emphasis> at a large teaching hospital in Rome,a 9-year analysis (2007–2015)

The proportion of antimicrobial resistance (AMR) among the ESKAPE and Escherichia coli (ESKAPEEc) pathogens causing bloodstream infection (BSI) increased worldwide. We described longitudinal trends in ESKAPEEc BSI and AMR over 9 years (2007–2015) at a large teaching hospital in Italy. Of 9720 unique BSI episodes, 6002 (61.7%) were caused by ESKAPEEc pathogens. The majority of these episodes (4374; 72.9%) were hospital-onset infections. The most frequent pathogen was E. coli (32.8%), followed by Staphylococcus aureus (20.6%), Klebsiella pneumoniae (16.1%), and Pseudomonas aeruginosa (11.6%). There was a significant increase of hospital-onset K. pneumoniae (from 2.3 to 5.0 per 10,000 patient-days; P =?0.001) and community-onset E. coli (from 3.3 to 9. 1 per 10,000 emergency admissions; P =?0.04) BSIs. Among hospital-onset BSIs, increases of extended-spectrum β-lactamase (ESBL)-producing E. coli (from 25.4 to 35.2%, P?= 0.006), carbapenemase-producing K. pneumoniae (from 4.2 to 51.6%, P <?0.001), and methicillin-resistant S. aureus (from 33.9 to 44.4%, P <?0.001) BSIs were observed between the 2007–2009 and 2010–2012 study periods. In contrast, a decrease of BSIs caused by P. aeruginosa resistant to ceftazidime (from 45.5 to 28.2%, P <?0.001), ciprofloxacin (from 46 to 36.3%, P =?0.05), and meropenem (from 55 to 39.9%, P =?0.03) was observed through all 9 years of the study period. Among community-onset BSIs, increases of BSIs caused by ESBL-producing E. coli (from 28.6 to 42.2%, P =?0.002) and carbapenemase-producing K. pneumoniae (from 0 to 17.6%) were observed between the 2007–2009 and 2010–2012 study periods. Our findings show increased rates of BSI and relative AMR for specific pathogen-health care setting combinations, and call for continued active surveillance and infection control policies.     

The ability of various strains of <Emphasis Type="Italic">Staphylococcus</Emphasis> to create biofilms and their effect on cells of the human body

Research on staphylococci research is important because of their ability to cause such severe infections as soft tissue infections, endocarditis, sepsis, toxic shock syndrome, and food poisoning. Coagulase-positive Staphylococcus aureus is the main infection agent of intrahospital infections. This agent has many factors of pathogenicity, which are well known. Among the coagulase-negative staphylococcus (CNS) strains, S. haemolyticus and S. epidermidis are clinically important, because they cause infections in patients with weakened immune system. The mechanisms of the CNS pathogenicity are understood insufficiently. The goal of this work was to evaluate the potential pathogenicity of clinical CNS strains based on their capacity to form biofilms and their character of interaction with the human cells through an example of HT-29 cell culture. The research was carried out on the laboratory strain S. aureus ATCC 29213 and clinical strains S. haemolyticus SH39 and S. epidermidis SE 36-1, which were isolated from neonatal autopsy materials. The visual tests of biofilm formation by each strain and testing the impact of the strains on HT-29 cell culture were carried out in this work. Two CNS species form biofilms with a higher rate than S. aureus. Upon incubation for 2 h of HT-29 cells with the staphylococcus strains tested in this work, adhesion of bacteria on the cells’ surfaces was observed. The adhesion was most pronounced in the case of S. aureus ATCC 29213 and S. haemolyticus SH39. Upon 3 h of incubation with S. aureus ATCC 29213 and S. haemolyticus SH39, the destruction of a monolayer of HT-29 cells was observed. The incubation for 24 h with three strains tested in this work caused the complete destruction of the monolayer of HT-29 cells. The maximal toxic effect on HT-29 cells was inherent in S. haemolyticus SH39 strain. The cumulative results obtained in this work indicate the presence of the pathogenicity factors in S. haemolyticus SH39 strain, and their molecular nature needs to be researched further.     

Mutations in Hotspot Regions of <Emphasis Type="Italic">ERG11</Emphasis> Gene in Fluconazole Resistant Isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> in Guilan Province,Northern Iran

Background

Widespread use of azoles has resulted in rapid development of azole resistance in Candida albicans strains. Mutations in ERG11, a target enzyme of azoles, alter the binding ability of azoles to this enzyme and result in the development of resistant strains. In this study, we evaluated ERG11 mutations in fluconazole resistant isolates of C. albicans.

Materials and methods

In this study, 60 clinical samples were isolated from Guilan hospitals. Then differential tests were used to identify C. albicans strains. Disc diffusion and MIC tests were used to the analyze fluconazole susceptibility. Then, the resistant isolates were evaluated by PCR and sequencing methods for ERG11 mutations.

Results

Of 60 clinical samples, 40 C. albicans strains were identified through specific symptoms. Susceptibility tests showed that four C. albicans strains were resistant to high dose fluconazole (≥512 μg/mL). In all resistant isolates was found missense mutations such as K291N, C470G and Q474R and three isolates had premature nonsense mutation (Y477stop).

Discussion

Our study indicates that the level of fluconazole resistance in C. albicans strains is high in Guilan province and other drugs should be used in resistant infections. It seems that missense mutations in four isolates play role in azole resistance. However in three isolates premature stop codon may be involved in high dose resistance. And it is suggested that in fourth isolates another mechanisms introduce increase of resistance dose in combination with missense mutation in ERG11. Results of this study suggest that in patients by high dose of resistance do not use azole because of mutations that decrease azole effects.

     

Clinical and microbiological characteristics of peritoneal dialysis-related peritonitis caused by <Emphasis Type="Italic">Escherichia coli</Emphasis> in southern Taiwan

Peritonitis is a serious complication and major cause of treatment failure in patients undergoing peritoneal dialysis (PD). Escherichia coli is the major pathogen in extraintestinal Gram-negative infections, including PD-related peritonitis. The outcomes of E. coli peritonitis in PD varied from relatively favorable outcomes to a higher incidence of treatment failure. The aim of this study was to investigate the impact of bacterial virulence and host characteristics on the outcomes of PD-related peritonitis caused by E. coli. From January 2000 to June 2016, a total of 47 episodes of monomicrobial and 10 episodes of polymicrobial E. coli PD-related peritonitis, as well as 89 episodes of monomicrobial Gram-positive (56 Staphylococcus spp. and 33 Streptococcus spp.) PD-related peritonitis cases, were retrospectively enrolled. Clinical features, E. coli bacterial virulence, and outcomes were analyzed. Compared to Streptococcus spp. peritonitis, E. coli peritonitis had a higher peritoneal catheter removal rate (38 versus 12%; P =?0.0115). Compared to the monomicrobial group, patients in polymicrobial group were older and had higher peritoneal catheter removal rate (80 versus 38%; P =?0.0324). Treatment failure of E. coli peritonitis was associated with more polymicrobial peritonitis and immunocompromised comorbidity, longer duration of PD therapy, and more antimicrobial resistance. E. coli isolates with more iron-related genes had higher prevalence of phylogenetic group B2 and papG II, iha, ompT, and usp genes. This study demonstrates the important roles of clinical and bacterial characteristics in the outcomes of monomicrobial and polymicrobial E. coli PD-related peritonitis.     

Estimation of flavoniods,antimicrobial, antitumor and anticancer activity of <Emphasis Type="Italic">Carissa opaca</Emphasis> fruits

Background

Carissa opaca Stapf ex Hanes fruits is traditionally used in the treatment of asthma, hepatitis and microbial infections. The present study was arranged to investigate the antimicrobial, cytotoxic and antitumor activity of various fractions of C. opaca extract and its bioactive metabolites responsible for that activity.

Methods

To characterize various fractions of C.opaca antibacterial, antifungal, cytotoxic and antitumor assays are used. Eight strains of bacteria including Bacillus subtilis, Enterobactor aerogenes, Escherichia coli, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeroginosa, Salmonella typhy, and Staphylococcus aureus and four strains of fungal viz: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Fusarium solani are used. Brine shrimps and potato dics are used for anticancer and antitumor potency of extract. High performance liquid chromatography (HPLC) is utilized for determination of bioactive metabolites responsible for the activity.

Results

HPLC chromatogram revealed the presence of orientin, isoquercetin, myricetin and apigenin. Various fractions of C. oapca showed significant antibacterial, antitumor and anticancer activity. In case of C. opaca fruit inhibition growth of Aspergillus niger was ranged between 23.2?±?1.36% to 43.3?±?2.39%, Aspergillus flavus ranged between 27.6?±?1.39% to 65.6?±?3.44%, Aspergillus fumigatus ranged between 13.2?±?1.00% to 52.4?±?1.54% and Fusarium solani ranged between 10.5?±?1.02% to 14.6?±?1.74%.

Conclusion

It can be concluded that, various fractions of C.opaca are accessible source of ethno pharmacy as they are consumed in different areas of Pakistan with ultimate health compensations.

     

Development of EUCAST zone diameter breakpoints and quality control criteria for ceftazidime-avibactam 10-4 μg

Ceftazidime-avibactam disk studies were performed for disk mass selection and for establishing EUCAST quality control ranges and zone diameter breakpoints. The disk mass study included disk diffusion testing with ceftazidime-avibactam 10-4 and 10-6 μg disks and broth microdilution MIC testing for challenge set of 94 Enterobacteriaceae and 45 Pseudomonas aeruginosa. EUCAST SOP 9.0-based QC and MIC-disk correlations studies were followed for development of ceftazidime-avibactam 10-4 μg ranges for Escherichia coli ATCC 25922, P. aeruginosa ATCC 27583, and Klebsiella pneumoniae ATCC 700603 and for zone diameter breakpoint determination. The ceftazidime-avibactam 10-4 and 10-6 μg disks performed similar in comparison to broth microdilution, with zones ≤?14 mm for all resistant strains. The 10-4 μg disk was selected and used in QC and breakpoint studies. There was minimal variation of ceftazidime-avibactam 10-4 μg QC study results between disks, media, and sites. The QC ranges were within 7 mm for all strains. The zone diameter breakpoint study demonstrated good correlation of MIC and disk results. The established zone diameter breakpoints resulted in false susceptible rates of 1.6 and 4.0% for Enterobacteriaceae and P. aeruginosa. EUCAST selected the ceftazidime-avibactam 10-4 μg disk and established QC ranges for E. coli 25922 of 24–30 mm, P. aeruginosa ATCC 27853 of 21–27 mm, and K. pneumoniae ATCC 700603 of 18–24 mm. The zone diameter breakpoints that correlated best with the MIC breakpoints of susceptible ≤?8 mg/L and resistant >?8 mg/L were Enterobacteriaceae (S?≥?13, R?<?13 mm) and P. aeruginosa (S?≥?17, R?<?17 mm).     

Synergy of antibacterial and antioxidant activities from crude extracts and peptides of selected plant mixture

Background

A plant mixture containing indigenous Australian plants was examined for synergistic antimicrobial activity using selected test microorganisms. This study aims to investigate antibacterial activities, antioxidant potential and the content of phenolic compounds in aqueous, ethanolic and peptide extracts of plant mixture.

Methods

Well diffusion, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were used to test antibacterial activity against four pathogenic bacteria namely Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Pseudomonas aeruginosa. DPPH (2, 2-diphenyl-1- picrylhydrazyl) and superoxide dismutase (SOD) assays were used to evaluate antioxidant activity. HPLC and gel filtration were used for purification of the peptides. Scanning electron microscope was applied to investigate the mode of attachment of the peptides on target microbial membranes.

Results

Aqueous extraction of the mixture showed no inhibition zones against all the test bacteria. Mean diameter of inhibition zones for ethanol extraction of this mixture attained 8.33 mm, 7.33 mm, and 6.33 mm against S. aureus at corresponding concentrations of 500, 250 and 125 mg/ml while E .coli showed inhibition zones of 9.33 mm, 8.00 mm and 6.66 mm at the same concentrations. B. cereus exhibited inhibition zones of 11.33 mm, 10.33 mm and 10.00 mm at concentrations of 500, 250 and 125 mg/ml respectively. The peptide extract demonstrated antibacterial activity against S. aureus, E. coli and B. cereus. The MIC and MBC values for ethanol extracts were determined at 125 mg/ml concentration against S. aureus and E. coli and B. cereus value was 31.5 mg/ml. MIC and MBC values showed that the peptide extract was significantly effective at low concentration of the Australian plant mixture (APM). Phenolic compounds were detected in hot aqueous and ethanolic extracts of the plant mixture. Hot aqueous, ethanol and peptides extracts also exhibited antioxidant activities.

Conclusions

It was concluded that APM possessed good antibacterial and antioxidant activities following extraction with different solvents. The results suggest that APM provide a new source with antibacterial agents and antioxidant activity for nutraceutical or medical applications.

     

Antimicrobial combination treatment including ciprofloxacin decreased the mortality rate of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> bacteraemia: a retrospective cohort study

Ineffective antimicrobial therapy of Pseudomonas aeruginosa bacteraemia increases mortality. Recent studies have proposed the use of antimicrobial combination therapy composed of a beta-lactam with either ciprofloxacin or tobramycin. To determine if combination therapy correlates to lower mortality and is superior compared to monotherapy, we investigated the effect of antimicrobial treatment regimens on 30-day mortality in a cohort with Pseudomonas aeruginosa bacteraemia. All cases of P. aeruginosa bacteraemia (n?=?292) in southwest Skåne County, Sweden (years 2005–2010, adult population 361,112) and the whole county (2011–2012, 966,130) were identified. Available medical and microbiological records for persons aged 18 years or more were reviewed (n?=?235). Antimicrobial therapy was defined as empiric at admission or definitive after culture results and was correlated to 30-day mortality in a multivariate regression model. The incidence and mortality rates were 8.0 per 100,000 adults and 22.9% (67/292), respectively. As expected, multiple comorbidities and high age were associated with mortality. Adequate empiric or definitive antipseudomonal treatment was associated with lower mortality than other antimicrobial alternatives (empiric p?=?0.02, adj. p?=?0.03; definitive p?<?0.001, adj. p?=?0.007). No difference in mortality was seen between empiric antipseudomonal monotherapy or empiric combination therapy. However, definitive combination therapy including ciprofloxacin correlated to lower mortality than monotherapy (p?=?0.006, adj. p?=?0.003), whereas combinations including tobramycin did not. Our results underline the importance of adequate antipseudomonal treatment. These data also suggest that P. aeruginosa bacteraemia should be treated with an antimicrobial combination including ciprofloxacin when susceptible.     

High frequency of occupied <Emphasis Type="Italic">attB</Emphasis> regions in Norwegian <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates supports a two-step MRSA screening algorithm

Rapid nucleic acid amplification tests for methicillin-resistant Staphylococcus aureus (MRSA) diagnostics commonly target the mec resistance gene, genes specific for S. aureus, and the integration site for the SCCmec resistance cassette, orfX. Due to poor specificity when these target genes are used individually, additional culture is required to verify positive results. The combination of these targets is useful, but the optimal algorithm may depend on the presence of the genetic markers in S. aureus isolates, as well as the prevalence of MRSA in a population. The aim of the present study was to identify a rapid, low-cost, and functional screening algorithm in order to reduce the response time for MRSA diagnostics. An in-house orfX-SCCmec polymerase chain reaction (PCR) assay was established and evaluated. The results were compared with an existing mec/nuc PCR assay and traditional culture. Methicillin-sensitive S. aureus (MSSA) that tested false-positive in the orfX-SCCmec PCR assay were further investigated with full genome sequencing using the Ion PGM? System to verify results and causality. Based on these data, a two-step screening algorithm with initial mec/nuc PCR followed by orfX-SCCmec PCR on positive samples was suggested and tested on 1443 patient samples. 22.5 % of MSSA isolates tested false-positive with the orfX-SCCmec PCR. Full genome sequencing of these isolates identified genetic variation in the attB region of S. aureus, including empty cassette variants and non-mec SCC. The suggested two-step MRSA screening algorithm allowed us to report MRSA results for 95.6 % of all samples and 99 % of MRSA-negative samples after one day.     

Patterns and trends of pediatric bloodstream infections: a 7-year surveillance study

We characterize the epidemiology of pediatric bloodstream infections (BSIs) in Switzerland. We analyzed pathogen distribution and resistance patterns in monomicrobial and polymicrobial BSIs in children from 2008 to 2014 using data from the Swiss antibiotic resistance centre (ANRESIS). A confirmatory statistical analysis was performed comparing pathogens and resistance across 20 acute care hospitals. We identified 3,067 bacteremia episodes, of which 1,823 (59 %) were considered true BSI episodes. Overall, S. aureus (16.5 %, 300) was the most frequent pathogen, followed by E. coli (15.1 %, 276), coagulase-negative staphylococci (CoNS, 12.9 %, 235), S. pneumoniae (11.1 %, 202) and non-E. coli Enterobacteriaceae (8.7 %, 159). S. aureus and E. coli showed similar frequencies in all of the variables analyzed (e.g., hospital acquisition, hospital type, medical specialty). The proportion of these microorganisms did not change over time, resistance rates remained low (4.3 % methicillin resistance in S. aureus; 7.3 % third-/fourth-generation cephalosporin resistance in E. coli), and no significant resistance trends were observed. We observed a 50 % increase of CoNS BSIs from 2008 (9.8 %, 27) to 2014 (15.2 %, 46, p value for trend?=?0.03). S. pneumoniae decreased from 17.5 % (48) to 6.6 % (20) during that timeframe (p for trend?=?0.007). S. aureus and E. coli remained the most significant pathogens among pediatric BSIs in Switzerland, exhibiting low resistance rates. CoNS accounted for a greater proportion of BSIs over time. The decrease in bacteremic pneumococcal infections can likely be attributed to the introduction of the 13-valent conjugate vaccine in 2011.     

Novel Nystatin A<Subscript>1</Subscript> derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis

Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A₁, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A₁ had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A₁ was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C. albicans and tissue cell viability after RHOE infection, Nystatin A₁ derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A₁. Furthermore, compared with Nystatin A₁, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion.     

Antimicrobial activity of HL-60 cells compared to primary blood-derived neutrophils against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Background

The human leukemia cell line HL-60 is considered an alternative cell culture model to study neutrophil differentiation and migration. The aim of this study was to characterize the suitability of HL-60 cells differentiated to neutrophil-like cells (nHL-60) as substitute for blood-derived human neutrophils to investigate the interaction of neutrophils with Staphylococcus aureus.

Methods

For this purpose, antimicrobial activity, bacterial uptake, production of reactive oxygen species and the release of neutrophil extracellular traps (NETs) by nHL-60 cells were analyzed and compared to primary blood-derived neutrophils using Staphylococcus aureus as important human and animal pathogen.

Results

Overall, the antimicrobial activities of nHL-60 cells were distinctly lower compared to blood-derived neutrophils. Furthermore, production of reactive oxygen species as well as NET formation was clearly impaired in nHL-60 cells.

Conclusion

This study indicates that HL-60 cells are of limited usage as an alternative model to study antimicrobial functions of neutrophils against Staphylococcus aureus.

     

Toe web intertrigo in Kaposi’s sarcoma patients: a microbiological study in a large cohort of patients

Kaposi ‘s sarcoma (KS) is a rare multifocal angioproliferative disease associated with human herpes virus 8 (HHV-8) infection, characterized by cutaneous nodules or plaques especially on the lower limbs. Some skin modifications, such as chronic lymphedema, plantar hyperkeratosis and interdigital desquamation, may be associated with consequent impairment of the local immunosurveillance and increased risk of some bacterial or mycotic infections. With the objective of evaluating if bacterial or mycotic infections in KS patients are supported by different microorganisms compared to control patients, we performed an observational retrospective study, comparing positive cultural swabs of interdigital intertrigo of KS patients with positive cultural swabs of interdigital intertrigo of patients admitted to our dermatologic unit during the last 10 years. One hundred KS patients and 84 control patients were admitted to this study. Some of the skin swabs from interdigital spaces were positive for more than one microorganism, and therefore we found 187 microorganisms among the KS group and 182 microorganisms in the control group. The most common microrganisms among KS patients were T. mentagrophytes (16%), S. aureus (14.9%), P. aeruginosa (13.9%), S. marcescens (5,9%), while among non-KS patients were S. aureus (26,9%), C. albicans (22%), S. agalactiae (7.7%) and E. coli (9.9%). These differences are statistically significant (p < 0.01). KS patients may be more affected by toe web intertrigo due to other bacteria and dermatophytes than the general population. During clinical examination, a careful inspection is necessary for an early diagnosis of toe web intertrigo, in order to prevent serious complications, such as cellulitis and sepsis. Consequently, a cultural examination with antibiogram is required to identify the causative agent of intertrigo and guide antimicrobial therapy.     

Genomic insights into the TTSS island of enteropathogenic <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">Salmonella</Emphasis> and its conjugational transfer

Whole genome sequences of the non-pathogenic K12 and pathogenic O127:H6 strains of Escherichia coli were analyzed using Mauve software. The genomes showed 80% similarity with few desert regions including a 35.99 kb region in pathogenic strain. This region contained Locus of Enterocyte Effacement (LEE) and Type III Secretion System (TTSS) genes. Whole genome alignment of this E. coli pathogenic strain and Salmonella enterica subsp. enterica serovar Newport str. SK254 showed 40% homology. Intimin protein coding escU gene of E. coli and ssaU gene of S. enterica were analyzed by BLASTn and ClustalW and cross referred with Pathogenicity Island Database (PDI DB) to check similarity with other foodborne bacterial species. The ssaU gene of Salmonella was found to be designated as escU in E. coli database. Comparison of these genes at nucleotide and amino acid sequence level revealed possible codon redundancies. Six clinical isolates of E. coli (designated as SBANU 1 to 6) were screened for salt aggregation and hemolysin production abilities followed by PCR analysis of escU gene. The E. coli isolate SBANU 6 was further used in induced conjugation assay with S. enterica as donor. PCR analysis and sequencing of the amplified DNA in E. coli transconjugant cells revealed the possible acquiring of ssaU gene from S. enterica.