Analysis of the bacterial biofilm formation in different models of the in vitro culture

Introduction. Microtiter plate assay (MPA) remains one of workhorses of in vitro biofilm research but it requires optimization of experimental conditions to fulfill the biofilm formation requirements of different bacterial pathogens. Aim. The aim was to determine the effect of TSB and RPMI1640 culture media and selected culture variables (O2 vs. 5% CO2 , extended incubation time) on the biofilm production by bacteria commonly involved in biofilm-related infections: Enterococcus faecalis (EF), Escherichia coli (EC), Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP). Material and methods. The investigation was performed using the MPA with crystal violet. Results. Statistically significant (p<0.05) increase in biofilm production between 24h and 72h time points was observed for EF (TSB o2, RPMIo2 and RPMIco2 ), EC (TSBo2 ) , SA (TSBo2 , TSBco2 ), KP (TSBo2 , TSBco2 ), PA (RPMIco2, TSBco2 ). The TSB caused a significantly greater stimulation of biofilm production compared to RPM1640. It outcompeted RPMI1640 irrespective of the atmospheric conditions for SA and KP and under aerobic conditions for EF. Conclusion. Although the TSB provided the most optimal conditions for biofilm production, the process was influenced by the strain type, atmospheric conditions and period of cultivation which limits the ability to design a single universal model of the in vitro biofilm investigation

The role of opportunistic Corynebacterium spp. in human infections

Introduction. The non-diphtherial corynebacteria (diphtheroids, “coryneform” bacteria) have been increasingly recognized as causative agents of human infections. Aim. To provide an overview of the role of non-diphtherial Corynebacterium species in human infections. Material and methods. Analysis of the literature data found in the PubMed database. Results. The role of diphtheroids - inherently low-virulent microorganisms considered members of the human microbiota – as potential pathogens has been linked to specific risk factors including immunosuppression, implantation of biomaterials and invasive medical procedures. Their pathogenic potential is primarily associated with frequent multidrug resistance, the ability to adhere to biotic and abiotic surfaces and/or to form biofilm as well as with internalization, intracellular survival and persistence within human cells. The most common infections include bacteremia, sepsis, endocarditis, meningitis, urinary tract infections, respiratory tract infections, wound and skin infections, and endophthalmitis. The leading species are C. jeikeium, C. striatum, C. urealyticum, C. amycolatum, and C. pseudodiphtheriticum. Conclusion. Opportunistic corynebacteria can be responsible for a wide range of infections which can be expected to increase in frequency in the future due to an enlarging population of patients with predisposing risk factors but also due to the increasing problem of antibiotic resistance in this group of bacteria

The role of opportunistic Corynebacterium spp. in human infections

Introduction. The non-diphtherial corynebacteria (diphtheroids, “coryneform” bacteria) have been increasingly recognized as causative agents of human infections. Aim. To provide an overview of the role of non-diphtherial Corynebacterium species in human infections. Material and methods. Analysis of the literature data found in the PubMed database. Results. The role of diphtheroids - inherently low-virulent microorganisms considered members of the human microbiota – as potential pathogens has been linked to specific risk factors including immunosuppression, implantation of biomaterials and invasive medical procedures. Their pathogenic potential is primarily associated with frequent multidrug resistance, the ability to adhere to biotic and abiotic surfaces and/or to form biofilm as well as with internalization, intracellular survival and persistence within human cells. The most common infections include bacteremia, sepsis, endocarditis, meningitis, urinary tract infections, respiratory tract infections, wound and skin infections, and endophthalmitis. The leading species are C. jeikeium, C. striatum, C. urealyticum, C. amycolatum, and C. pseudodiphtheriticum. Conclusion. Opportunistic corynebacteria can be responsible for a wide range of infections which can be expected to increase in frequency in the future due to an enlarging population of patients with predisposing risk factors but also due to the increasing problem of antibiotic resistance in this group of bacteria

Intraphagolysosomal conditions predispose to Staphylococcus epidermidis small colony variants persistence in macrophages.

Staphylococcus epidermidis small colony variants can survive inside macrophages and their survival has been proposed as a pivotal process in the pathogenesis of biomaterial associated infections. In the present study the intracellular location of clinical isolates of SCV and parental wild type strains inside macrophages was determined. Furthermore, the effect of IFN-γ and rapamycin on the level of SCV/WT as well as lysosomes colocalisation and iNOS induction in THP-activated macrophages in response to WT and SCV strains of Staphylococcus epidermidis were examined. It was demonstrated that SCV strain of S. epidermidis can survive and persist inside macrophages and its intracellular survival is supported by the induction of phagosomal acidification. The ability to reduce the high proportion of LysoTracker positive SCV containing phagosomes was exclusively found when IFN-γ was used. The findings suggest that IFN-γ mediates SCV killing via two distinct mechanisms, phagosome alkalisation and an increased iNOS synthesis, so the cytokine may control S. epidermidis WT and SCV infection in macrophages. Staphylococcus epidermidis SCV is a less potent stimulus of iNOS than the WT strain and the feature may help SCV to persist in hostile environment of macrophages. Rapamycin treatment did not influence the iNOS synthesis but reduced the percentage of both bacterial strains within acidic organelles. However, the percentage of SCV within LysoTracker positive organelles, even though reduced comparing to non-primed cells, was higher than in the WT strain indicating that Staphylococcus epidermidis possesses unique metabolic features allowing SCV to survive within macrophages

Charakterystyka izolatów Staphylococcus epidermidis wyhodowanych od pacjentów z zakażeniami endoprotez stawu biodrowego

Background. Coagulase negative staphylococci are at the forefront of etiologic agents of periprosthetic joint infections (PJIs). The purpose of the study was to characterise causative isolates (n=19) of Staphylococcus epidermidis (SE) – with emphasis on their phenotypic and genotypic heterogeneity. Material and methods. The isolates were cultured from multiple samples obtained perioperatively during revision surgery from 14 patients with clinically and/or microbiologically proven PJI. Phenotypic heterogeneity included variations of colony morphologies, drug resistance patterns and/or the capability of the biofilm formation and was verified by the DNA fingerprinting assay. Results. Phenotypic discrepancies were observed between isolates cultured from 5 patients (35.7%). The genotyping assay identified 3 pairs of isolates as unrelated; single pairs were genetically related and indistinguishable. The biofilm production was detected in 17 isolates, among which 5 (29.4%) were proficient biofilm formers harbouring the icaADBC genes. Additionally, one ica-positive isolate produced a moderate, protease-sensitive biofilm. The remaining isolates were moderate biofilm producers among which four developed protease-sensitive biofilms. Conclusions. The majority of PJIs are monoclonal; nevertheless, phenotypic diversity of SE is a frequent phenomenon which can complicate the diagnostic proceeding. Adherence ability is an important pathogenic trait of SE although the chemical composition of the resultant biofilm, its intensity and regulation of development can vary.Wprowadzenie. Gronkowce koagulazo-ujemne są wiodącymi czynnikami etiologicznymi zakażeń okołoprotezowych. Celem pracy była charakterystyka izolatów (n=19) Staphylococcus epidermidis (SE) ze szczególnym uwzględnieniem ich fenotypowej i genotypowej heterogenności. Materiał i metody. Izolaty gronkowców zostały wyhodowane z materiałów klinicznych pobranych śródoperacyjnie od 14 pacjentów, u których zakażenie endoprotezy stawu biodrowego zostało potwierdzone klinicznie i/lub mikrobiologicznie. Fenotypową heterogenność definiowano w oparciu o odmienne cechy morfologii kolonii bakteryjnych, profile lekowrażliwości izolatów i/lub ich zdolność tworzenia biofilmu w warunkach in vitro i weryfikowano ją na postawie genotypowania. Wyniki. Fenotypowe rozbieżności zaobserwowano pomiędzy izolatami SE wyhodowanymi od 5 (35.7%) pacjentów. W oparciu o wyniki genotypowania za genetycznie niespokrewnione uznano 3 pary izolatów, pojedyncze pary zaś za spokrewnione i nieodróżnialne/klonalne. Wytwarzanie biofilmu zostało potwierdzone dla 17 badanych izolatów, wśród których 5 (29.4%) wytwarzało intensywny biofilm i posiadało geny icaADBC. Ponadto, jeden izolat SE ica+ wytwarzał umiarkowany, wrażliwy na działanie proteazy biofilm. Pozostałe izolaty wytwarzały biofilm umiarkowany. Cztery z nich charakteryzowały się tworzeniem biofilmu wrażliwego na działanie proteazy. Wnioski. Większość PJIs ma charakter monoklonalny, niemniej jednak zmienność fenotypowa SE pozostaje częstym zjawiskiem, co może utrudniać diagnostykę zakażeń wywoływanych przez te drobnoustroje. Zdolność adherencji jest ważną cechą warunkującą patogenność SE, aczkolwiek skład chemiczny powstającego w jej wyniku biofilmu oraz jego intensywność i regulacja mechanizmów wpływających na jego tworzenie mogą różnić się pomiędzy izolatami

Community-acquired methicillin-resistantStaphylococcus aureus (CA-MRSA) in Poland:further evidence for the changing epidemiologyof MRSA.

This study reports the isolation of CA-MRSA strain which was found to colonize the nasal mucosa of a patient undergoing haemodialysis treatment. The MRSA was subjected to molecular analysis by Pulsed Field Gel Electrophoresis (PFGE), multiplex PCR assay for staphylococcal cassette chromosome mec (SCCmec) typing, and PCR detection of the pvl gene encoding for Panton-Valentine leukocidin. The analyzed MRSA harbored the SCCmec type IV and the pvl gene-two unique genetic markers of CA-MRSA. The PFGE pattern of the strain corresponded to the common European CA-MRSA (MLST Type ST80). Moreover, the strain was only resistant to beta-lactam agents and tetracycline. This study adds further evidence for the changing epidemiology of MRSA and indicates the ability of CA-MRSA to affect persons with established risk factors in addition to previously healthy individuals

Community-acquired methicillin-resistantStaphylococcus aureus (CA-MRSA) in Poland:further evidence for the changing epidemiologyof MRSA.

This study reports the isolation of CA-MRSA strain which was found to colonize the nasal mucosa of a patient undergoing haemodialysis treatment. The MRSA was subjected to molecular analysis by Pulsed Field Gel Electrophoresis (PFGE), multiplex PCR assay for staphylococcal cassette chromosome mec (SCCmec) typing, and PCR detection of the pvl gene encoding for Panton-Valentine leukocidin. The analyzed MRSA harbored the SCCmec type IV and the pvl gene-two unique genetic markers of CA-MRSA. The PFGE pattern of the strain corresponded to the common European CA-MRSA (MLST Type ST80). Moreover, the strain was only resistant to beta-lactam agents and tetracycline. This study adds further evidence for the changing epidemiology of MRSA and indicates the ability of CA-MRSA to affect persons with established risk factors in addition to previously healthy individuals

Characterisation of Staphylococcus aureus nasaland skin carriage among patients undergoinghaemodialysis treatment.

The aim of the study was to investigate the rate of Staphylococcus aureus nasal and skin carriage in patients undergoing haemodialysis. The cultured staphylococcal isolates were subsequently characterized by molecular methods. The study group comprised 43 haemodialysed patients from whom nasal and skin swabs from the vascular access sites were collected. The identification of staphylococcal isolates and antibiotic susceptibility testing were performed on the basis of conventional diagnostic procedures. The staphylococci were further characterized using Pulsed-Field Gel Electrophoresis (PFGE). S. aureus was cultured from 12 (27.9%) patients. Only one (8.3%) patient was colonized with the microorganism both in the anterior nares and the vascular access site representing a single strain, as evidenced by PFGE analysis. Antibiotic susceptibility testing identified one (7.6%) methicillin-resistant S. aureus (MRSA) strain. PFGE typing identified several S. aureus genotypes with the lack of one specific strain responsible for colonization. However, it should be noted that among two (A and D) PFGE patterns genetically indistinguishable and closely related isolates (two isolates for each pattern) were identified. The obtained results revealed a relatively low rate of S. aureus carriage accompanied by low methicillin resistance rate and a significant genetic diversity of cultured isolates with the lack of one predominant strain responsible for colonization