Evaluation of The Pathogenic Potential of Insecticidal Serratia marcescens Strains to Humans.

We observed the death of insect caterpillars of Spodoptera exigua in the laboratory culture line and identified Serratia marcescens as the bacterial causative agent of the insect death. We confirmed that S. marcescens had insecticidal activity against S. exigua and caused high mortality of larvae. The LC50 values of S. marcescens CFU per 1 cm2 of insect diet surface were similar for all isolates. Our research reports novel strains with high pesticidal activity as candidates for future research on a new bioinsecticide. As bioinsecticides cannot be harmful to non-target organisms, we determined the pathogenic properties of S. marcescens to humans. We proved the ability of S. marcescens to damage mammalian epithelial cells. All strains had cytopathic effects to Vero cells with a cytotoxic index ranging from 51.2% ± 3.8% to 79.2% ± 4.1%. We found that all of the strains excreted catecholate siderophore - enterobactin. All isolates were resistant to sulfamethoxazole, tobramycin, gentamicin, cefepime, and aztreonam. We did not observe the ESBL phenotype and the integrons' integrase genes. Resistance to sulfamethoxazole was due to the presence of the sul1 or sul2 gene. The use of resistant S. marcescens strains that are pathogenic to humans in plant protection may cause infections difficult to cure and lead to the spread of resistance genes. The results of our study emphasize the necessity of determination of the safety to vertebrates of the bacteria that are proposed to serve as biocontrol agents. The novelty of our study lies in the demonstration of the indispensability of the bacteria verification towards the lack of hazardous properties to humans.We observed the death of insect caterpillars of Spodoptera exigua in the laboratory culture line and identified Serratia marcescens as the bacterial causative agent of the insect death. We confirmed that S. marcescens had insecticidal activity against S. exigua and caused high mortality of larvae. The LC50 values of S. marcescens CFU per 1 cm2 of insect diet surface were similar for all isolates. Our research reports novel strains with high pesticidal activity as candidates for future research on a new bioinsecticide. As bioinsecticides cannot be harmful to non-target organisms, we determined the pathogenic properties of S. marcescens to humans. We proved the ability of S. marcescens to damage mammalian epithelial cells. All strains had cytopathic effects to Vero cells with a cytotoxic index ranging from 51.2% ± 3.8% to 79.2% ± 4.1%. We found that all of the strains excreted catecholate siderophore ­ enterobactin. All isolates were resistant to sulfamethoxazole, tobramycin, gentamicin, cefepime, and aztreonam. We did not observe the ESBL phenotype and the integrons' integrase genes. Resistance to sulfamethoxazole was due to the presence of the sul1 or sul2 gene. The use of resistant S. marcescens strains that are pathogenic to humans in plant protection may cause infections difficult to cure and lead to the spread of resistance genes. The results of our study emphasize the necessity of determination of the safety to vertebrates of the bacteria that are proposed to serve as biocontrol agents. The novelty of our study lies in the demonstration of the indispensability of the bacteria verification towards the lack of hazardous properties to humans.

In vitro virulence characteristics of rare serovars of Salmonella enterica isolated from sand lizards (Lacerta agilis L.).

The aim of this study was to estimate virulence potential of Salmonella enterica strains colonizing the gut of free-living sand lizards (Lacerta agilis L.). The strains belonged to three Salmonella serovars: Abony, Schleissheim, and Telhashomer. Adhesion and invasion abilities of the strains were determined in quantitative assays using the gentamicin protection method. Induction of apoptosis was assessed using HeLa cell monolayers. PCR assays were used for detection of 26 virulence genes localised within mobile elements: pathogenicity islands, virulence plasmids, and prophage sequences. In vitro studies revealed that all strains had adhesion and invasion abilities to human epithelial cells. The isolates were cytotoxic and induced apoptosis of the cells. The serovars differed in the number of virulence-associated genes: up to 18 genes were present in Salmonella Schleissheim, 17 in Salmonella Abony, whereas as few as six genes were found in Salmonella Telhashomer. Generally, Salmonella Abony and Salmonella Schleissheim did not differ much in gene content connected with the presence SPI-1 to -5. All of the strains lacked genes localised within bacteriophages and plasmids. The presence of virulence-associated genes and in vitro pathogenicity assays suggest that Salmonella sp. strains originating from autochthonous, free-living lizards can potentially infect and cause disease in humans.

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.

Clonality, virulence and the occurrence of genes encoding antibiotic resistance among Staphylococcus warneri isolates from bloodstream infections.

Coagulase-negative Staphylococcuswarneri is an opportunistic pathogen capable of causing several infections, especially in patients with indwelling medical devices. We evaluated the virulence-associated properties of 23 clinical isolates recovered from blood specimens. In addition, the carriage of biofilm-associated genes, as well as antibiotic-resistant genes, was identified. S. warneri isolates appeared to be clonally unrelated and revealed a high degree of genetic diversity. All isolates revealed adhesion to epithelial cells, and 43.5 % of strains invaded the cells. Moreover, 52 % of isolates formed biofilm in vitro. PCR analysis demonstrated the presence of the ica operon, in two of the 12 biofilm-positive isolates. This indicated that biofilm formation, in this species, is not restricted to strains harbouring icaADBC genes, encoding polysaccharide intercellular adhesion. Analysis by confocal laser scanning microscopy revealed that biofilm-forming strains formed a three-dimensional structure, composed of mainly living cells. All strains revealed cell-contact cytotoxicity that was strongly associated with biofilm formation. Moreover, cell-free supernatants, of 95 % of the isolates, expressed a cytotoxic activity which caused the destruction of HeLa cells. S. warneri capable of forming biofilm carried significantly more genes encoding resistance to beta-lactams, aminoglicosides and macrolide-lincosamide streptogramin B antibiotics than biofilm-negative isolates. We have shown that tigecycline/rifampicin is effective against bacteria growing as a biofilm. The biofilm inhibitory concentration range of tigecycline/rifampicin was ≤1 µg ml-1. Results indicated that S. warneri have the ability to adhere, form biofilm, invade and destroy epithelial cells, which could be important mechanisms contributing to the development of diseases.

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.

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.

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.

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.

Aeromonas spp. induce apoptosis of epithelial cells through an oxidant-dependent activation of the mitochondrial pathway.

We investigated interactions of Aeromonas caviae, Aeromonas veronii biotype sobria and Aeromonas hydrophila strains, isolated from faecal specimens of humans with gastroenteritis, with HT29 intestinal epithelial cells. All strains were found to be cytotoxic to the cells. Bacterial infection caused generation of reactive oxygen species (ROS) and nitric oxide radical (NO(·)). The maximal levels of ROS and NO(·) were 14 and 35 times, respectively, greater in cells infected with Aeromonas spp. than in those incubated with non-pathogenic Escherichia coli. The cells incubated with cytolytic enterotoxin isolated from A. veronii biotype sobria induced the highest level of ROS and caused the highest cytotoxicity. We observed that increased accumulation of intracellular ROS leads to a loss of mitochondrial membrane potential (ΔΨ(m)). Analyses of cellular morphology and DNA fragmentation revealed characteristic features of cells undergoing apoptosis. The process was dependent on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. Treatment of infected HT29 cells with three distinct antioxidants prevented intracellular ROS production, mitochondrial damage and apoptosis. The Pearson linear test revealed positive correlations between apoptotic index at 24 h and percentage cytotoxicity, ROS production, NO(·) production and loss of ΔΨ(m). This study has provided new insights into the mechanisms contributing to the development of Aeromonas-associated gastroenteritis. The results indicate that bacteria-induced apoptosis of epithelial cells results from mitochondrial depolarization due to oxidative stress.

Virulence properties and integron-associated antibiotic resistance of Klebsiella mobilis strains isolated from clinical specimens.

This study examined Klebsiella mobilis isolates cultured from clinical specimens for virulence-associated properties and antibiotic resistance. The strains produced a number of siderophores, including enterobactin, aerobactin and yersiniabactin. All isolates were able to adhere to and invade epithelial cells. They had cytotoxic activity, which caused destruction of human laryngeal epithelial HEp-2 cells and evoked lysis of murine macrophage J774 cells. Analyses of HEp-2 and J774 cellular morphology and DNA fragmentation in the cells showed features typical of cells undergoing apoptosis. Some K. mobilis strains harboured class 1 integrons carrying the aadA1 gene encoding an aminoglycoside adenyltransferase.

Cytotoxic activity of Serratia marcescens clinical isolates.

Twenty Serratia marcescens isolates from clinical specimens were examined for their cytotoxic activity on four cell lines (HEp-2, Vero, CHO, J774). Most of the isolates were found to be cytotoxic to CHO (70%), Vero (75%) and HEp-2 cells (90%). CHO cells were the most sensitive to cell-free supernatants, followed by HEp-2 and Vero cells. Two strains produced cytotonic toxins which caused elongation of CHO cells. Moreover, twelve isolates (60%) revealed cytotoxic potential to macrophage cell line J774. The results indicate that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.

Isolates of the Enterobacter cloacae complex induce apoptosis of human intestinal epithelial cells.

Strains of the Enterobacter cloacae complex are becoming increasingly important human pathogen. The aim of the study was to identify, by sequencing the hsp60 gene, the species of clinical isolates phenotypically identified as E. cloacae and to examine them for virulence-associated properties: the ability of adhesion, invasion to HEp-2 cells and the induced apoptosis of infected epithelial cells. The majority of the strains were identified as Enterobacter hormaechei with E. hormaechei subsp. steigerwaltii being the most frequent subspecies. Other strains belonged to E. hormaechei subsp. oharae, E. cloacae cluster III, and E. cloacae cluster IV. The strains were examined for virulence-associated properties: the ability to adhesion and invasion to HEp-2 cells and the apoptosis induction of infected epithelial cells. All strains revealed adherence ability and most of them (71%) were invasive to epithelial cells. Analyses of cellular morphology and DNA fragmentation in the HEp-2 cells exhibited typical features of cells undergoing apoptosis. We observed morphological changes, including condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of cell membrane. The lowest apoptotic index did not exceed 6%, whereas the highest reached 49% at 24h and 98% at 48 h after infection. Forty strains (73%) induced fragmentation of nuclear DNA and characteristic intranucleosomal pattern with the size of about 180-200 bp in DNA extracted from infected cells at 48 h after infection. The results indicated that the bacteria of the E. cloacae complex may adhere to and penetrate into epithelial cells and induce apoptosis, which could be an important mechanism contributing to the development diseases.

Cytotoxic activity of Enterobacter cloacae human isolates.

We examined 55 Enterobacter cloacae isolates from clinical specimens for the production of cytotonic and cytotoxic toxins and the presence of the type III secretion system (TTSS). Twelve isolates (22%) revealed cytotoxic activity that caused destruction of Vero cells, whereas 28 (51%) strains induced lysis of the murine macrophage J774 cell line. TTSS genes were present in 27% of the isolates. The results indicated that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.

Aeromonas spp. human isolates induce apoptosis of murine macrophages.

Interactions of Aeromonas caviae, Aeromonas veronii biotype sobria, and Aeromonas hydrophila strains isolated from fecal specimens of humans with gastroenteritis on murine macrophages, J774 cells, were investigated. Analyses of cellular morphology and DNA fragmentation in phagocytes infected with these strains exhibited typical characteristic features of cells undergoing apoptosis. We observed the morphological changes, including condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of cell membrane, and fragmentation of nuclear DNA into oligonucleosomal fragments. The lowest apoptotic index did not exceed 25%, whereas the highest reached 78% at 24 h and 96% at 48 h after infection. After incubation of J774 cells with cytotoxic enterotoxin isolated from A. veronii biotype sobria strain, we noted that the toxin was able to trigger cytotoxicity and apoptosis of macrophages. The results indicate that apoptosis could be one of the mechanisms contributing to the development of Aeromonas-associated diarrheal disease.

Interaction of Aeromonas spp. human isolates with murine macrophages.

This study aimed to evaluate phagocytic and bactericidal activity of macrophages originated from murine J774 macrophage cell line against 26 strains of Aeromonas caviae, Aeromonas veronii biotype sobria and Aeromonas hydrophila isolated from human fecal specimens. The analysis of phagocytic index (PI) indicated these strains were phagocytosed in low numbers, below 1.1 x l0(-3) for 65% strains of A. veronii biotype sobria and A. hydrophila. The highest PI value (0.019) was noted in murine macrophages infected with 19% of A. caviae isolates. Analysis of the number of viable bacteria in phagocytes revealed that 31% of Aeromonas spp. strains could replicate for an initial 3 h after gentamicin treatment. Opsonisation of the bacteria with non-immune serum did not affect phagocytic activity and bacterial killing. Stimulation of macrophages with gamma-interferon (IFN-gamma) at a concentration of 10 IU/ml caused the highest increase in phagocytic activity but had no effect on the rate of bacteria elimination by murine macrophages. Aeromonas spp. isolates showed an invasive ability toward non-phagocytic epithelial cells. These findings indicate that Aeromonas spp. strains have evolved mechanisms to avoid phagocytosis, suggesting that murine macrophages, even activated by IFN-gamma, have a limited ability to eliminate these bacteria, which may invade epithelial cells and spread in the illeum.

Purification and characterization of cytolytic toxins produced by Aeromonas hydrophila and Aeromonas veronii biotype sobria strains.

Cytolytic toxins produced by Aeromonas hydrophila and Aeromonas veronii biotype sobria strains were partially purified from culture filtrates by two steps of purification: ammonium sulfate precipitation and hydrophobic chromatography using Phenyl-Sepharose CL-4B. Hemolytic activity was detected in one or two peaks in elution profile. Purified toxins were also cytotoxic to Vero and CHO cells. Moreover, these toxins revealed cytotonic activity to CHO cells.

Enteropathogenic activity and invasion of HEp-2 cells by Aeromonas caviae clinical isolates.

Twenty Aeromons caviae isolates from stool of children with diarrhea symptoms were examined for virulence-associated properties: production of cytotoxic and cytotonic toxins, and invasive ability. Most of A. caviae strains were cytotoxic to Vero and CHO cells and produced cytotonic toxins which caused elongation of CHO cells. Moreover, five of A. caviae strains revealed invasive ability towards HEp-2 cells.

Enhancement of the virulence of Aeromonas caviae diarrhoeal strains by serial passages in mice.

Thirteen clinical Aeromonas caviae isolates from the faeces of 13 children with mild to severe diarrhoea were tested for enhancement of mouse lethality, adhesion ability, siderophore and cholera toxin cross-reactive (CTC) factor production, by four consecutive passages through mice by intraperitoneal injection of A. caviae suspensions. The passaged A. caviae strains were re-isolated from monomicrobic cardiac blood samples and inocula were prepared for the next passage. All A. caviae isolates possessed the ability to adhere to the mucosal epithelial surface of the rabbit small intestine. Serial passage in mice showed that the virulence of some isolates for mice was increased in terms of percentage mortality and a lowering of the LD50. For some of the isolates, but not all, serial passage appeared to increase siderophore production and adhesion to rabbit small intestinal cells. For the A. caviae isolates tested, increased values of the CTC factor were observed after passage. A clear correlation was observed between the lowering of LD50 and the enhancement of CTC factor production after passage in mice. These results indicate that the A. caviae isolates possessed virulence factors.