Multifactorial mechanisms of the pathogenesis of methicillin-resistant Staphylococcus hominis isolated from bloodstream infections.

Staphylococcus hominis is a species of the coagulase-negative staphylococci. It has been designated as a potential pathogen but so far the pathogenic mechanisms of this bacterium have not been determined. We studied 30 clinical isolates of methicillin-resistant S. hominis, which were previously examined for biofilm forming properties. The results of this study revealed that all these S. hominis strains had the ability to adhere to HeLa cells. Over 40% of the S. hominis strains invaded epithelial cells. The invasion index ranged from 0 to 41.5%. All isolates exhibited the cytotoxic activity of extracellular factors, which caused the destruction of epithelial cells. More than 90% of these methicillin-resistant strains contained at least one aminoglycosides resistance gene. The ant(4')-I gene was found in 63% of the isolates, aac(6')/aph(2″) in 20% and aph(3')-IIIa in 47%. Two strains were assigned to SCCmec type VIII and three to SCCmec type III. The remaining isolates (83%) harboured a non-typeable SCCmec type. The mec complex A was predominant in this species. The results indicate that the pathogenicity of S. hominis may be multifactorial, involving adhesion, invasion and the activity of extracellular toxins, which cause damage to the host epithelium.

Effect of subinhibitory concentrations of tigecycline and ciprofloxacin on the expression of biofilm-associated genes and biofilm structure of Staphylococcus epidermidis.

Staphylococcus epidermidis is a leading cause of foreign body-associated infections. This is related to the bacterium's ability to form biofilms on synthetic materials. Bacteria within a biofilm may be exposed to subinhibitory concentrations (sub-MICs) of antibiotics because of an agent's limited penetration into the biofilm core. Here, we investigated the effect of sub-MICs of tigecycline and ciprofloxacin on the expression of biofilm-associated genes, i.e. icaA, altE and sigB, and the biofilm structure of five clinical isolates of S. epidermidis. For most tested isolates, the expression of these genes increased after exposure to 0.25 MIC and 0.5 MIC tigecycline. A slight decrease in icaAmRNA levels was observed only in two isolates in the presence of 0.25 MIC tigecycline. The effect of ciprofloxacin exposure was isolate-dependent. At 0.5 MIC, ciprofloxacin induced an increase of sigB and icaAmRNA levels in three of the five tested isolates. At the same time, expression of the altE gene increased in all isolates (from 1.3-fold to 42-fold, depending on the strain). Confocal laser scanning microscopy analysis indicated that sub-MIC ciprofloxacin decreased biofilm formation, whereas tigecycline stimulated this process. Our data suggest that sub-MIC tigecycline may have bearing on the outcome of infections.

Characterization of Staphylococcal Cassette Chromosome mec (SCCmec) in Methicillin-Resistant Staphylococcus epidermidis Strains Isolated from Biomaterial-Associated Infections and their Antibiotic Resistance Patterns.

This work aims to provide an insight into staphylococcal cassette chromosome mec elements and antibiotic resistance in clinical isolates of Staphylococcus epidermidis. The dominating type was SCCmec - IV. Fifteen isolates were assigned to SCCmec type III, two isolates to SCCmec type II. Most isolates were resistant to at least three of the non-ß-lactam antibiotics tested. None of the strains exhibited resistance to new generation antibiotics, such as daptomycin and linezolid. Also, none of these strains showed resistance to tigecycline and only four strains were resistant to rifampin i.e. antibiotics which are very efficient in treating biofilm-associated infections.

Virulence and the presence of aminoglycoside resistance genes of Staphylococcus haemolyticus strains isolated from clinical specimens.

We examined thirty methicillin-resistant Staphylococcus haemolyticus isolates cultured from clinical specimens for antibiotic resistance, various important interactions of the bacteria with epithelial cells and putative virulence determinants. All strains were resistant to oxacillin and carried the mecA gene. Aminocyclitol-3'-phosphotransferase (aph(3')-IIIa) gene encoding nucleotidyltransferases was detected in 43 %, aminocyclitol-6'-acetyltransferase-aminocyclitol-2″-phosphotransferase (aac(6')/aph(2″)) gene encoding bifunctional acetyltransferases/phosphotransferases in 33 %, aminocyclitol-4'-adenylyltransferase (ant(4')-Ia) gene encoding phosphotransferases in 20 %. The coexistence of resistance to methicillin and aminoglycosides was investigated in multi-resistant strains. Coexisting (aac(6')/aph(2″)) and (aph(3')-IIIa) genes were detected in 33 % of isolates, whereas 63 % of isolates had at least one of these genes. All strains revealed adherence ability and most of them (63 %) were invasive to epithelial cells. Electron microscopy revealed that the bacteria were found in vacuoles inside the cells. We observed that the contact of the bacteria with host epithelial cells is a prerequisite to their cytotoxicity at 5 h-incubation. Culture supernatant of the strains induced a low effect of cytotoxicity at the same time of incubation. Cell-free supernatant of all isolates expressed cytotoxic activity which caused destruction of HEp-2 cells at 24 h. None of the strains was cytotonic towards CHO cells. Among thirty strains, 27 % revealed lipolytic activity, 43 % produced lecithinase and 20 % were positive for proteinase activity. Analyses of cellular morphology and DNA fragmentation exhibited typical characteristic features of those undergoing apoptosis. The Pearson linear test revealed positive correlations between the apoptotic index at 24 h and percentage of cytotoxicity. Our results provided new insights into the mechanisms contributing to the development of S. haemolyticus-associated infections. The bacteria adhered and invaded to non-professional phagocytes. The invasion of epithelial cells by S. haemolyticus could be similar to phagocytosis that requires polymerization of the actin cytoskeleton. The process is inhibited by cytochalasin D. Moreover, they survived within the cells by residing in membrane bound compartments and induced apoptotic cell death.

In Vitro Activity of Rifampicin Combined with Daptomycin or Tigecycline on Staphylococcus haemolyticus Biofilms.

Staphylococcus haemolyticus is of increasing concern as a cause of several biofilm-associated infections, and today, it represents the second most common organism among clinical isolates of coagulase-negative staphylococci. However, little is known regarding the treatment of infections caused by these bacteria. In this study, we characterize the biofilm formed by S. haemolyticus strains isolated from bloodstream infections and assess in vitro the activity of rifampicin combined with daptomycin or tigecycline against bacteria growing in a biofilm. The results of our studies indicated that the majority (78 %) of methicillin-resistant Staphylococcus haemolyticus strains have the ability to form a biofilm in vitro. None of these strains carried icaADBC genes indicating that they form biofilm via ica-independent mechanisms. The molecular characterization of the biofilm showed that proteins are the predominant matrix component and play a major role in biofilm structure. Extracellular DNA and polysaccharides, other than polysaccharide intercellular adhesin, are also present in the biofilm matrix, but they play a minor role. The images obtained by confocal laser scanning microscopy showed that most S. haemolyticus strains formed a dense biofilm with a low number of dead cells. In vitro study demonstrated excellent activity of tigecycline in combination with rifampicin against cell growth in the proteinous biofilm. The BIC (biofilm inhibitory concentration) value for tigecycline/rifampicin ranged from 0.062 to 1 µg/ml, whereas for daptomycin/rifampicin from 0.125 to 2 µg/ml. These results indicated that the tigecycline/rifampicin combination was more effective against ica-independent biofilm, formed by S. haemolyticus strains, than the daptomycin/rifampicin combination.

Activity of vegetative insecticidal proteins Vip3Aa58 and Vip3Aa59 of Bacillus thuringiensis against lepidopteran pests.

Vegetative insecticidal proteins (Vips) secreted by some isolates of Bacillus thuringiensis show activity against insects and are regarded as insecticides against pests. A number of B. thuringiensis strains harbouring vip3A genes were isolated from different sources and identified by using a PCR based approach. The isolates with the highest insecticidal activity were indicated in screening tests, and their vip genes were cloned and sequenced. The analysis revealed two polymorphic Vip protein forms, which were classified as Vip3Aa58 and Vip3Aa59. After expression of the vip genes, the proteins were isolated and characterized. The activity of both toxins was estimated against economically important lepidopteran pests of woodlands (Dendrolimus pini), orchards (Cydia pomonella) and field crops (Spodoptera exigua). Vip3Aa58 and Vip3Aa59 were highly toxic and their potency surpassed those of many Cry proteins used in commercial bioinsecticides. Vip3Aa59 revealed similar larvicidal activity as Vip3Aa58 against S. exigua and C. pomonella. Despite 98% similarity of amino acid sequences of both proteins, Vip3Aa59 was significantly more active against D. pini. Additionally the effect of proteolytic activation of Vip58Aa and Vip3Aa59 on toxicity of D. pini and S. exigua was studied. Both Vip3Aa proteins did not show any activity against Tenebrio molitor (Coleoptera) larvae. The results suggest that the Vip3Aa58 and Vip3Aa59 toxins might be useful for controlling populations of insect pests of crops and forests.

Class 1 integrons and antibiotic resistance of clinical Acinetobacter calcoaceticus-baumannii complex in Poznan, Poland.

Sixty-three clinical isolates of Acinetobacter calcoaceticus-baumannii complex were analyzed for the presence of integrons and antimicrobial resistance. Class 1 integrons were detected in 40 (63.5 %) isolates. None of them had class 2 or class 3 integrons. The majority of the integrons contained aacC1-orfA-orfB-aadA1 gene cassette array. The presence of integrons was associated with the increased frequency of resistance to 12 of 15 antimicrobials tested, multi-drug resistance phenotype, and the overall resistance ranges of the strains.

Clonal analysis of Staphylococcus hominis strains isolated from hospitalized patients.

Staphylococcus hominis is a part of normal skin flora, but it is also a cause of nosocomial infections. The aim of this study was to investigate the genetic relatedness of 62 strains of S. hominis obtained from hospitalised patients during an 11-year period. For the discrimination of these clinical strains we used repetitive sequence-based PCR method (BOX-PCR) and multiple-locus variable-number tandem repeat analysis (MLVA). BOX-PCR analysis revealed a large genetic diversity among clinical strains and we did not find a predominant clone with the ability to persist in a hospital environment. MLVA is not as discriminatory as BOX fingerprinting and would not be a useful method for epidemiological studies.

Association between the presence of class 1 integrons, virulence genes, and phylogenetic groups of Escherichia coli isolates from river water.

Ninety-six class 1 integron-positive and 96 integron-negative Escherichia coli isolates cultured from the water of the Warta River, Poland, were characterized for their phylogenetic group affiliation and for the presence of genes associated with virulence. Most strains belonged to phylogenetic group A, but phylogenetic group affiliation was not related with the presence of integrons. The occurrence of heat-stable toxin gene of enterotoxigenic E. coli, S fimbriae subunit gene sfaS, and siderophore receptor genes, fyuA and iutA, was associated with the presence of class 1 integrons. Moreover, virulence factor score (the total number of virulence-associated genes) was associated with the presence of integrons in groups. The results bring new insight into relations between the presence of integrons in E. coli, virulence traits, as well as phylogenetic group affiliation.

Increased frequency of integrons and ß-lactamase-coding genes among extraintestinal Escherichia coli isolated with a 7-year interval.

We analyzed the level of antimicrobial resistance, and the presence of integrons and ß-lactamase-coding genes in 69 clinically relevant Escherichia coli strains originating from extraintestinal infections isolated in 1999-2001 and 2008-2010. Comparison of the two groups showed significant differences in drug resistance frequency, and the presence of integron and ß-lactamase-coding genes. The frequency of resistance to all antimicrobials beside imipenem, streptomycin, piperacillin/tazobactam, and sulfamethoxazole increased significantly, especially towards aminoglycosides, ß-lactams and fluoroquinolones. Similarly, we noticed an increase in the number of strains with integrons from 31.6 to 80.7 %. The presence of integrase genes was associated with elevated frequency of resistance to each antimicrobial tested besides imipenem, piperacillin/tazobactam and ceftazidime. The presence of integrons was also associated with multidrug resistance phenotype. The genetic content of integrons comprised genes determining resistance toward aminoglycosides, sulfonamides and trimethoprim. Moreover, we noticed a significant increase in the frequency of bla (CTX-M) ß-lactamases, with appearance of bla (CTX-M-15) variant and newer plasmid-encoded ß-lactamases like CMY-15 and DHA. The emergence of strains resistant to several classes of antimicrobials and carrying integrons, ESBL and AmpC ß-lactamase-coding genes may predict the spread of isolates with limited treatment options.

Staphylococcus haemolyticus strains target mitochondria and induce caspase-dependent apoptosis of macrophages.

The aim of this study was to investigate the interaction of Staphylococcus haemolyticus strains with a macrophage cell line. Infection with the strains resulted in macrophage injury. All strains exhibited cytotoxic effects towards J774 cells. Moreover, the bacteria triggered apoptosis of the cells. The lowest apoptotic index did not exceed 21 %, whereas the highest reached 70 % at 24 h and 85 % at 48 h after infection. Incubation with the bacteria caused loss of mitochondrial membrane potential (ΔΨm) in macrophages. The pro-apoptotic activity of the strains was blocked by a pan-caspase inhibitor z-VAD-fmk, indicating the involvement of caspases in the bacteria-mediated cell death. We observed that the induction of macrophage apoptosis could constitute an important mechanism of pathogenesis by which S. haemolyticus strains evade host immune defences and cause disease.

Aeromonas spp.-mediated cell-contact cytotoxicity is associated with the presence of type III secretion system.

In the study we examined the production of cytotonic and cytotoxic toxins and the presence of a type III secretion system (TTSS) in 64 Aeromonas spp. strains isolated from fecal specimens of patients with gastroenteritis. We observed that contact of the bacteria with host epithelial cells is a prerequisite for their cytotoxicity at 3 h incubation. Cell-contact cytotoxic activity of the strains was strongly associated with the presence of the TTSS. Culture supernatants of the strains induced low cytotoxicity effects at the same time of incubation. Cell-free supernatants of 61 (95%) isolates expressed cytotoxic activity which caused the destruction of HEp-2 cells at 24 h. Moreover, 44% strains were cytotonic towards CHO cells and 46% of strains invaded epithelial cells.

Acinetobacter calcoaceticus-baumannii complex strains induce caspase-dependent and caspase-independent death of human epithelial cells.

We investigated interactions of human isolates of Acinetobacter calcoaceticus-baumannii complex strains with epithelial cells. The results showed that bacterial contact with the cells as well as adhesion and invasion were required for induction of cytotoxicity. The infected cells revealed hallmarks of apoptosis characterized by cell shrinking, condensed chromatin, and internucleosomal fragmentation of nuclear DNA. The highest apoptotic index was observed for 4 of 10 A. calcoaceticus and 4 of 7 A. baumannii strains. Moreover, we observed oncotic changes: cellular swelling and blebbing, noncondensed chromatin, and the absence of DNA fragmentation. The highest oncotic index was observed in cells infected with 6 A. calcoaceticus isolates. Cell-contact cytotoxicity and cell death were not inhibited by the pan-caspase inhibitor z-VAD-fmk. Induction of oncosis was correlated with increased invasive ability of the strains. We demonstrated that the mitochondria of infected cells undergo structural and functional alterations which can lead to cell death. Infected apoptotic and oncotic cells exhibited loss of mitochondrial transmembrane potential (ΔΨ(m)). Bacterial infection caused generation of nitric oxide and reactive oxygen species. This study indicated that Acinetobacter spp. induced strain-dependent distinct types of epithelial cell death that may contribute to the pathogenesis of bacterial infection.

Insecticidal activity of Bacillus thuringiensis strains isolated from soil and water.

We attempted to search novel Bacillus thuringiensis strains that produce crystals with potential utility in plant protection and with higher activity than strains already used in biopesticide production. Seven B. thuringiensis soil and water isolates were used in the research. We predicted the toxicity of their crystals by cry gene identification employing PCR method. The isolate MPU B63 with interesting, according to us, genes content was used in evaluating its crystal toxicity against Cydia pomonella caterpillars. The strain MPU B63 was cultured from water sample and had cry1Ab, cry1B, and cry15 genes. The LC50 crystals of MPU B63 were compared to LC50 of commercial bioinsecticide Foray determined against C. pomonella (codling moth). The activity of MPU B63 inclusions against codling moth larvae was approximately 24-fold higher than that of Foray. The results are a promising introduction for further study evaluating the potential usefulness of isolate MPU B63 crystals in plant protection.

Apoptosis of epithelial cells and macrophages due to nonpigmented Serratia marcescens strains.

Serratia marcescens strains are opportunistic pathogens that are increasingly recognized as a cause of severe nosocomial infections. In this study we observed interactions between nonpigmented strains with human epithelial and macrophage-like cells. The strains revealed hemolytic activity only after the contact of the cells with erythrocytes. The contact of the bacteria with the host cells was also essential to their cytotoxicity. Moreover, all strains revealed adherence ability and were invasive to epithelial cells. Analyses of cellular morphology and DNA fragmentation of the HEp-2 and J774 cells exhibited typical features of cells undergoing apoptosis. We observed morphological changes, including condensation of nuclear chromatin and formation of membrane-bound apoptotic bodies. The lowest apoptotic index in HEp-2 cells did not exceed 25%, whereas the highest reached 59% at 24 h and 72% at 48 h after infection. Most of the strains (60%) induced fragmentation of nuclear DNA. The process depended on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. This study provided new insights into the mechanisms of nonpigmented S. marcescens pathogenesis. The results revealed that the strains produce cell-contact toxins that facilitate bacterial invasion, induce hemolysis, cytotoxicity, and apoptosis of host cells.

The Yersinia high-pathogenicity island in Escherichia coli and Klebsiella pneumoniae isolated from polymicrobial infections.

We examined 12 pairs of strains of Escherichia coli and Klebsiella pneumoniae isolated from mixed infections in human for the presence of the Yersinia high-pathogenicity island (HPI). In one case both isolates carried the HPI, whereas in 11 cases one strain of the pair was HPI-positive. Although there were differences in the organization of the Yersinia HPI, all HPI-positive isolates were able to produce yersiniabactin. The presence of the Yersinia HPI may enhance the capability of strains involved in mixed infections to replicate in iron-deprived conditions in the host.

Transferable integrons of Gram-negative bacteria isolated from the gut of a wild boar in the buffer zone of a national park.

The aim of this study was to determine the presence of integron-bearing Gram-negative bacteria in the gut of a wild boar (Sus scrofa L.) shot in the buffer zone of a national park. Five Gram-negative strains of Escherichia coli, Serratia odorifera, Hafnia alvei and Pseudomonas sp. were isolated. Four of these strains had class 2 integrase (intI2), and one harbored class 1 integrase (intI1). The integron-positive strains were multiresistant, i.e., resistant to at least three unrelated antibiotics. All of the integrons were transferred to E. coli J-53 (Rif(R)) in a conjugation assay. The results showed that a number of multiresistant, integron-containing bacterial strains of different genera may inhabit a single individual of a wild animal, allowing the possibility of transfer of antimicrobial resistance genes.

Phylogenetic groups, virulence genes and quinolone resistance of integron-bearing Escherichia coli strains isolated from a wastewater treatment plant.

We investigated phylogenetic affiliation, occurrence of virulence genes and quinolone resistance in 109 integron-containing strains of Escherichia coli isolated from a wastewater treatment plant. Selection for integron-bearing strains caused a shift toward phylogroup D, which was most numerous, followed by A, B1 and B2. Phylogroups D and B2, both of which are reported to include virulent extraintestinal pathotypes, made up 50.5% of all isolates and were present in every stage of wastewater treatment, including final effluent. Diarrheagenic pathotypes made up 21% of the strains. The average virulence factor genes score was low (1.40) and the range was from 0 to 5. Quinolone and fluoroquinolone resistance was observed in 56.0% and 50.4% of the strains, respectively; however, it was not associated with virulence factor score. Although the average virulence factor score was low, 17.4% of strains had three and more virulence genes. They were isolated mostly from raw sewage, but 30% of them were cultured from final effluent. Release of multiresistant integron-bearing E. coli strains with virulence traits into the environment may create potential threat and be of public health concern.

[Antimicrobial sensivity and ability to slime production of koagulaze-negative staphylococci]. / Wrazliwosc na antybiotyki i zdolnosc wytwarzania sluzu przez szczepy gronkowców koagulazo-ujemnych.

The aim of this study was to assess the ability of slime production ofcoagulase-negative staphylococci (CONS) and evaluate the susceptibility of bacteria to antibiotics. Strains were isolated from clinical specimens obtained from hospitalized patients. The most frequently isolated species were S. epidermidis (51%), S. hominis (18%), S. haemolyticus (13%). The result of this study shows that 61% of S.epidermidis produce slime on CRA (Congo red agar), whereas none of the tested S. haemolyticus strains has this ability. All examined strains were susceptible to vancomycin, linezolid and quinupristin/ dalfopristin. The majority of strains were susceptible to minocycline, fusid acid, nitrofurantoin and rifampicin. Sixty six percent of isolates were determined as methicillin-resistant coagulase-negative staphylococci.

A novel Bacillus thuringiensis Cry9Ea-like protein with high insecticidal activity towards Cydia pomonella larvae.

BACKGROUND: The host specificity of known Bacillus thuringiensis Cry and Vip pesticidal proteins still needs extensive investigation and the proteins currently used in crop protection are not effective against many pest species. Cydia pomonella (L.) is a widespread and economically important pest of apples, very difficult to control, since the larvae spend most of their life inside a fruit. Currently, large amounts of broad-spectrum, detrimental synthetic agents are used to combat this herbivore and therefore biopesticides with high activity against C. pomonella are very much needed. RESULTS: The toxicity of B. thuringiensis Cry9Ea along with five distinct pesticidal proteins (Cry1Aa, Cry1Ca, Cry1Ia, Cry2Ab and Vip3Aa) has been determined towards the first-instar larvae of C. pomonella. Cry9Ea has much higher activity than the remaining tested proteins (30-1200-fold lower LC50 ) and possibly is the most potent B. thuringiensis pesticidal protein bioassayed against C. pomonella so far. In contrast, Cry9Ea is not toxic towards Spodoptera exigua (Hübn.), indicating a potentially narrow spectrum of activity. Both insect species show high variability in susceptibility to the remaining Cry/Vip proteins. CONCLUSIONS: The obtained results extend the existing knowledge regarding B. thuringiensis pesticidal protein host range and indicate Cry9Ea as a promising candidate for successful biological control of C. pomonella. © 2020 Society of Chemical Industry.