- Email: [email protected]
Isolates of the Enterobacter cloacae complex induce apoptosis of human intestinal epithelial cells
Microbial Pathogenesis 49 (2010) 83e89
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Isolates of the Enterobacter cloacae complex induce apoptosis of human intestinal epithelial cells ska*, Ryszard Koczura, Joanna Mokracka, Tomasz Puton, Adam Kaznowski Sylwia Krzymin , ul. Umultowska 89, Poland Department of Microbiology, Faculty of Biology, A. Mickiewicz University, 61-614 Poznan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 March 2010 Received in revised form 22 April 2010 Accepted 23 April 2010 Available online 6 May 2010
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 24 h and 98% at 48 h after infection. Forty strains (73%) induced fragmentation of nuclear DNA and characteristic intranucleosomal pattern with the size of about 180e200 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. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Enterobacter cloacae complex Genetic diversity Adhesion Invasion Apoptosis
1. Introduction Bacteria of Enterobacter cloacae occur in water, sewage, soil, food, and as commensal microflora in the intestinal tracts of humans and animals [1]. Molecular studies on E. cloacae recognized on the biochemical characteristics have showed genomic heterogeneity of this taxonomic complex comprising six species: E. cloacae, Enterobacter hormaechei, Enterobacter asburiae, Enterobacter kobei, Enterobacter ludwigii, and Enterobacter nimipressuralis. Identification of these species upon phenotypic traits is usually difficult and not reliable; therefore, molecular methods are used [2]. Upon the sequence of the hsp60 gene, the E. cloacae complex has been divided into 12 genetic clusters and one sequence crowd. Nine of the clusters correspond to species: E. asburiae, E. kobei, E. ludwigii, E. hormaechei subsp. oharae, E. hormaechei subsp. hormaechei, E. hormaechei subsp. steigerwaltii, E. nimipressuralis, E. cloacae subsp. cloacae, and
* Corresponding author. Tel.: þ48 61 829 5935; fax: þ48 61 829 5636. ska). E-mail address: [email protected] (S. Krzymin 0882-4010/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2010.04.003
E. cloacae subsp. dissolvens. Three clusters do not have specific names and are referred to as E. cloacae cluster III, E. cloacae cluster IV and E. cloacae cluster IX [3]. Similar results were obtained on using rpoB genotyping, multi-locus sequence analysis and comparative genomic hybridization [2]. Some strains phenotypically identified as E. cloacae are opportunistic pathogens implicated as the causative agent of local and systemic infections in humans. They are important nosocomial pathogens responsible for bacteremia, lower respiratory tract, skin, soft tissue, urinary tract, intra-abdominal and ophthalmic infections, endocarditis, septic arthritis and osteomyelitis [1]. They are etiological agents of outbreaks of septicemia in neonatal intensive care [4]. Among the E. cloacae complex, E. hormaechei subsp. steigerwaltii, E. hormaechei subsp. oharae, and E. cloacae cluster III have been reported to be the bacteria most frequently recovered from clinical specimens [5,6]. E. nimipressuralis is a plant pathogen and has not been associated with human diseases [3]. Although the E. cloacae complex strains are among the most common Enterobacter species causing nosocomial bloodstream infections in the last decade, still little is known about their virulence-associated properties [1]. Among the most common risk
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
84
factors for developing E. cloacae bloodstream infections are prolonged hospitalization, severity of illnesses, and exposure to invasive procedures. Additional predisposing factors are the usage of central venous catheter, prolonged antibiotic therapy, parenteral nutrition and immunosuppressive therapy [7,8]. A requirement for successful colonization and development of disease by microbial pathogens is the ability to adhere to the surface of host epithelial cells [9]. Bacterial adhesion to the cells may result in internalization, either by phagocytosis or by bacteria-induced invasion. These processes are associated with the initiation of infection by many pathogenic bacteria and are therefore considered as essential virulence factors. There is increasing evidence that microbial pathogens induce oxidative stress in the infected cells, and this may represent an important mechanism leading to epithelial injury [10]. During bacterial infection, reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radicals (OH), superoxide anion (O 2 ) and reactive nitrogen intermediates in the form of nitric oxide may be involved in chromosomal DNA degradation leading to cell death through apoptosis. There is growing evidence that apoptosis of host cells plays an important role in modulating the pathogenesis of a variety of infectious diseases [11]. Paraje et al. [12] have suggested that E. cloacae strains produce toxin that increases the production of ROS in leucocytes and lead to oxidative stress with subsequent cell death by apoptosis. In our study, we focused on the interaction of the E. cloacae complex isolates with epithelial cells. We investigated their ability to adhere to and invade HEp-2 cells, a well established and frequently used model for studying interactions between bacteria and human cells [13,14]. Moreover, we studied the induction of apoptosis of HEp-2 cells by the E. cloacae complex.
2. Results 2.1. Molecular differentiation of strains by hsp60 sequence analysis PCR amplification of a part of the hsp60 heat shock protein gene resulted in a 341-bp product for all strains. The PCR products were sequenced and 272-nt sequences were compared to the sequences of type and reference strains of Enterobacter spp. All of the 53 strains phenotypically identified as E. cloacae were clustered within the E. cloacae complex. The majority of the strains were identified as E. hormaechei (81%) with E. hormaechei subsp. steigerwaltii being the most frequent subspecies (72%). Five (9%) belonged to E. hormaechei subsp. oharae and E. cloacae cluster III. Three isolates and the negative, nonpathogenic control MPU E326/7 belonged to E. cloacae cluster IV. One strain was identified as E. cloacae subsp. cloacae and one as E. kobei. All strains and the sources of their origin are listed in Table 1.
Table 1 Strains of the E. cloacae complex used in the study. Species
Source of origin (number of strains)
Strain No.
E. hormaechei subsp. steigerwaltii
Urine (20)
Catheter (1) Feces (1) Blood (1)
MPU E: 2, 4, 7, 8, 9, 11, 13, 32, 33, 35, 37, 38, 40a, 47a, 48, 49, 51, 53, 54a, 56a MPU E: 14, 17, 21, 43, 44, 59 MPU E: 1, 19, 20 MPU E: 16, 41, 46, 52, 55, 62 MPU E: 45 MPU E: 57 MPU E: 58
Urine (1) Blood (1) Secretion (2) Wound (1) Urine (1)
MPU MPU MPU MPU MPU
E: E: E: E: E:
3 18 12, 15 63a 42
Blood (1) Secretion (4) Urine (1) Blood (1) Secretion (1) Water (1) Catheter (1)
MPU MPU MPU MPU MPU MPU MPU
E: E: E: E: E: E: E:
23 6, 28, 30, 31 34 26 22 326/7 63b
Blood (6) Secretion (3) Wound (6)
E. hormaechei subsp. hormaechei E. hormaechei subsp. oharae
E. cloacae subsp. cloacae E. cloacae cluster III E. cloacae cluster IV
E. kobei
MPU e bacterial strains of the collection of Department of Microbiology, A. Mi , Poland. ckiewicz University, Poznan
2.2. Adhesion and invasion assay Adhesion and invasion to HEp-2 cells were quantified by gentamicin survival assay. Preliminary experiments showed that all the E. cloacae complex strains were gentamicin sensitive and unable to grow in media containing 0.2 mg/ml of the antibiotic. The results of adhesion activity are presented in Table 2. All strains revealed higher efficiency of adhesion in comparison with noninvasive controls. The adhesion index (AdI) ranged from 0.21 105 to 31 105 CFU per 100 HEp-2 cells. The negative controls of nonpathogenic E. cloacae MPU E326/7 and E. coli K12C600 showed indexes of 0.29 105 CFU and 0.15 104, respectively, whereas that of Yersinia enterocolitica O:8/1B, the positive control, was 19.6 105 CFU per ml. The highest index (17.8 105e36.9 105) was obtained for 30% strains isolated from blood, 20% from secretions and 38% from wounds. Isolates of the E. cloacae complex were invasive towards HEp-2 cells (Table 3). Four strains (7.5%) showed the highest invasion activity. The invasion index ranged between 16.2 and 52.9%, whereas for Y. enterocolitica O:8/1B reached 61.3%. For 34 isolates
Table 2 Adhesion of the E. cloacae complex strains to HEp-2 cells. Adhesion index range (105)a
Number of strains (%)
Enterobacte hormaechei
E.cloacae III
E. cloacae IV
17.8e36.9b 8.5e14.3
8 (15.1) 13 (24.5)
23 28
22, 26
5.4e7.9
11 (20.7)
3.1e5.3 1.4e2.9 0.21e0.79
11 (20.7) 5 (9.4) 5 (9.4)
62, 55, 43, 20, 21 8, 4, 11, 17, 18, 44, 45, 46, 37, 19, 63a 52, 1, 40a, 2, 47a, 14, 58, 59, 3, 33, 41 7, 32, 38, 12, 26, 51, 48, 35, 30 49, 53, 54a, 9 57, 13, 15, 56a
a
E. cloacae s. cloacae
E. kobei
42
63b
6, 31 30 34
The mean number of associated (CFU) bacteria/100 HEp-2 cells. Mean CFU/ml of two separate assays performed in triplicate. Means in the group did not differ significantly at P < 0.05 according Tukey’s HSD test. Adhesion index of E. coli K12C600 ¼ 0.15 104, E. cloacae 326/7 ¼ 0.29 105 and Y. enterocolitica O:8/1B ¼ 19.6 105. b
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
85
Table 3 Invasion ability of the E. cloacae complex strains to HEp-2 cells. Invasion index rangea
Number of strains (%)
Enterobacter hormaechei
32.1e52.9b 16.2e31.2 4.1e7.1 1.1e2.7 0.1e0.9
2 2 5 8 20
0.01e0.09
16 (30.2)
21, 15 57, 16 7, 2, 13, 54a 40a, 19, 49, 38, 12, 35 53, 48, 11, 20, 56a, 4, 41, 17, 33, 43, 14, 59, 47a, 52, 18, 55, 63a, 23, 44 62, 51, 58, 46, 3, 37, 32, 9, 34, 1, 8, 45
(3.8) (3.8) (9.4) (15.1) (37.7)
E. cloacae III
E. cloacae IV
30 31
E. kobei
42 26
6, 28
E. cloacae s. cloacae
63b
22, 34
a
The percentage of internalized bacteria/100 HEp-2 cells in comparison with the number of adhering bacteria of two separate experiments performed in triplicate. Means in the group did not differ significantly at P < 0.05 according Tukey’s HSD test. Invasion index of E. cloacae 326/7 ¼ 0.036%, E. coli K12C600 ¼ 0.013% and Y. enterocolitica O:8/1B ¼ 61.3%. b
(64%) the index ranged between 0.1 and 7.1% and was higher than those of the negative controls. Sixteen isolates (30%) showed the lowest invasive ability, comparable to that of nonpathogenic negative controls. The invasion index for E. cloacae MPU E326/7 and E. coli K12C600 strains was 0.036% and 0.013%, respectively. Treatment of epithelial cells with cytochalasin D had no effect on E. cloacae adhesion, but reduced bacteria uptake by 91e96% after 24 h. 2.3. Quantification of epithelial cell death 2.3.1. Trypan blue exclusion assay Trypan blue exclusion assay was used to assess the damage of epithelial cells. The analysis was performed 24 and 48 h after infection. Trypan blue is excluded from viable cells with functional cellular membranes, but penetrates membranes of apoptotic and necrotic cells, thereby suggesting that such assay is appropriate for staining dead cells. At 24 h after infection cell viability remained high (82e94%) for 60.4% strains and ranged from 68% to 80% for 40% strains. At 48 h, the viability declined by 53% to 78% for 29% isolates and from 12% to 49% for 71% of isolates. 2.3.2. Morphological assessment of apoptosis The apoptotic activity was expressed as apoptotic index (ApI) and was determined by ethidium bromide and acridine orange staining and observation under fluorescence microscope. In this
assay, ApI and cell membrane integrity can be assessed simultaneously. Acridine orange permeates cell membrane and makes the nuclei appear green, whereas ethidium bromide is only taken up by cells with lost cytoplasmic membrane integrity and stains nuclei red. Live cells had a normal green nucleus (Fig. 1a), early apoptotic cells had bright green nucleus with condensed chromatin, late apoptotic cells displayed condensed or fragmented orange chromatin, and cells that died from necrosis had a structurally normal orange nucleus (Fig. 1b). Strains of the E. cloacae complex provoked apoptosis of HEp-2 cells, which displayed such morphological changes as cell shrinkage, loss of normal cell-to-cell contacts and blebbing at the cell surface. The analysis of mean percentage of apoptotic cells (Table 4) at 24 h showed five groups with statistically significant differences between them (ANOVA, F23,120 ¼ 115.407, P < 0.001). The highest apoptotic indexes ranging from 26.4% to 38.7% were observed for 27% strains. The lowest (6.7e10.3%) were expressed by 9.4% strains of E. cloacae. The percentage of apoptotic cells increased at 48 h post infection. We found statistically significant differences between six groups of strains (ANOVA, F23,120 ¼ 147.513, P < 0.001). The highest apoptotic indexes (83.1e98.3%), were observed in cells infected with 14.5% of E. cloacae complex strains. The lowest (18.7e25.4%) were revealed by 7.3% strains. The apoptotic indexes were reduced to 3.7e8.1% after treatment of HEp-2 monolayer with 1 mg ml1 of cytochalasin D prior infection. Some E. cloacae strains also caused necrotic
Fig. 1. Apoptosis of HEp-2 cells. The cells were stained with propidium iodide and acridine orange (100 mg/ml) and observed in fluorescence microscope. The cells were infected with (a) Escherichia coli K12C600; (b) Enterobacter hormaechei susp. hormaechei MPU E17; the arrows point to: A e live, B e apoptotic, C e necrotic cells. Magnifications: (a) 200; (b) 250.
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
86
Table 4 Apoptotic index of the Enterobacter cloacae complex strains at 24 h and 48 h after infection of HEp-2 cells. Group
Apoptotic index rangea (percentage of strains)
Enterobacter hormaechei
E. cloacae III
E. cloacae IV
At 24 h Ab B
6.7e10.3 (9.4) 11.2e17.6 (35.8)
31 23, 28
22 34, 26
C D
19.3e25.8 (15.1) 26.4e38.7 (26.4)
E
39.7e49.1 (13.2)
18, 16, 48 17, 40a, 3, 38, 37, 47a, 1, 21, 56a, 19, 49, 57, 51, 41, 32 15, 53, 35, 44, 20, 59 35, 9, 11, 52, 14, 54a, 58, 13, 7, 2, 8, 55, 45 43, 4, 12, 46, 62, 33, 63a
At 48 h A B C D
18.7e25.4 27.8e36.4 38.7e47.6 51.8e65.1
E
66.4e81.7 (32.1)
F a b
(5.7) (15.1) (7.5) (29.1)
83.1e98.3 (11.3)
38, 40a, 15 18, 56a, 41, 7, 20 51, 19, 8, 49 16, 52, 13, 48, 57, 9, 32, 2, 35, 11, 1 33, 44, 59, 12, 47, 37, 53, 45, 14, 62, 58, 54, 43, 3, 63a 17, 46, 55, 4, 21
30 6
E. cloacae s. cloacae
E. kobei
42
63b
30, 28
22
6, 31, 23
26, 34 42 63b
Mean percentage of apoptotic cells from two experiments in triplicate. Within each column, means designated by the same letter did not differ significantly at P < 0.05 according to Tukey’s post-hoc test.
changes. The highest necrotic indexes (39e41%) were observed for 11.3% strains at 24 h. The value of indexes increased to 45% to 69%, for 15.1% strains at 48 h.
isolates were able to adhere to HEp-2 cells. For 19% of the isolates, the adhesion index was comparable to that of Y. enterocolitica O:8/ 1B, the enteroinvasive positive control. The highest adhesion ability was observed for strains isolated from blood: two E. hormaechei
2.3.3. DNA fragmentation The apoptotic effect of E. cloacae strains on HEp-2 cells was also confirmed by the analysis of DNA fragmentation. Characteristic patterns of DNA (180e200 bp) were obtained for 11.3% strains 24 h after infection (Fig. 2). The percent of strains that caused DNA fragmentation in HEp-2 cells increased to 75.5% after 48 h of infection. These patterns were similar to that obtained for the positive control. DNA fragmentation was observed when apoptotic index exceeded 47%.
3. Discussion The phenotypic similarity of strains belonging to different E. cloacae genomic groups creates many problems with their identification. As a consequence, our knowledge regarding epidemiology, disease spectrum and understanding of virulence mechanisms of these bacteria is limited. In our study we used sequencing of the hsp60 gene, which shows good discriminatory power to identify species and subspecies within the E. cloacae complex [2]. The majority of clinical strains were identified as E. hormaechei, with E. hormaechei subsp. steigerwaltii as a predominant group. E. hormaechei strains have been isolated from a variety of samples, including blood, stool, urine, and wounds. However, strains of E. cloacae III and IV clusters can also be pathogenic to humans. The ability of the strains to adhere and invade host cells and their capability to induce apoptosis suggest that these mechanisms may be considered as contributing factors in disease development by the E. cloacae complex. Therefore, this study was important for clarifying the contribution of adhesion and internalization to the pathogenicity of the E. cloacae complex strains. Although evidence has suggested that these bacteria also invade epithelial cells [14], the fate of these cells after bacterial internalization is not known. In our study, we provided evidence that the E. cloacae complex strains may induce apoptosis of HEp-2 cells. In order to colonize a host, bacteria must display the ability to adhere to epithelial cells, which are an integral component of the mucosal immune system [9]. Our results demonstrated that all
Fig. 2. (a) Analysis of intranucleosomal DNA fragmentation of HEp-2 cells infected with different strains. Lane 1: molecular size marker. DNA from cells infected with: Lane 2: strain of E. coli K-12 C200 (negative control). Lane 3: E. cloacae MPU E48 at 24 h after infection. Lane 4: E. cloacae MPU E48 (48 h). Lane 5: Y. enterocolitica O:8/1B (48 h). Lane 6: E. cloacae MPU E4 (48 h). Lane 7: E. cloacae MPU E63a (48 h). (b) Quantification of apoptotic and necrotic cells as apoptotic and necrotic indexes expressed in percentages.
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
subsp. hormaechei, one E. cloacae cluster III and IV, one isolate of E. hormaechei subsp. hormaechei and E. cloacae cluster III from aspirate, three E. hormaechei subsp. hormaechei from bedsore and wound infections. The interaction of enteropathogens with epithelial cells is the first stage of successive bacterial invasion of the host [9]. In our study, 70% of the E. cloacae complex strains were invasive with invasion index higher than that of nonpathogenic control. Most of them (62%) belonged to E. hormaechei cluster. The highest indexes, comparable to that of Y. enterocolitica O:8/1B, were observed for 4 E. hormaechei (7.5%) strains originated from pharynx, blood, feces, and lower leg ulcer. Previously, Keller et al. [14] have reported that all E. cloacae strains isolated from specimens isolated from humans were adherent and invasive to HEp-2 cells. The strains were much less invasive than the enteroinvasive E. coli control strain. Townsend et al. [15] suggested that the ability to invade epithelial cells might be a strong indication of potential virulence of Enterobacter spp. strains. Invasion of the host cells by pathogenic bacteria may occur via phagocytosis, which involves polymerization of actin microfilaments [16]. This polymerization may be prevented by cytochalasin D. In our study, the E. cloacae complex invasion to HEp-2 cells was inhibited when the monolayer was pretreated with cytochalasin D. The results suggest that the bacteria invade the epithelial cells by a microfilament dependent endocytosis mechanism followed by a rearrangement of the cytoskeletal proteins. We examined the interaction of the E. cloacae complex strains with HEp-2 cells and demonstrated that one of the consequences of cell adhesion and invasion by the bacteria is injury to the epithelial cells and cell death. Using three different criteria (trypan blue exclusion assay, acridine orange and bromide ethidium staining and analysis of DNA fragmentation), we observed that the E. cloacae complex infection induced apoptosis of the cells. The morphological changes included condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of the cell membrane. The highest apoptotic activity was expressed by E. hormaechei strains. We also observed that 73% strains caused fragmentation of nucleosomal DNA of infected cells to multimers which are a biochemical hallmark of apoptosis. Analysis of morphological changes and internucleosomal cleavage of host cell DNA indicated that 14 strains isolated from urine, 9 from blood, 5 from secretions and all strains isolated from wounds caused apoptosis of epithelial cells. High level of adhesion was consistent with the ability to induce HEp-2 cells death. Moreover, isolates for which high invasion indexes (16e52%) were observed induced apoptosis. In contrast, infection with nonpathogenic E. cloacae MPU E326/7, E. coli K12C600 and the E. cloacae copmlex isolates with low invasion indexes did not trigger apoptosis. The ability of the E. cloacae complex with high invasive index to induce cell death was inhibited by cytochalasin D, indicating that the bacteria must have been internalized. Previous studies have shown that epithelial cells undergo apoptosis after adhesion or invasion by different pathogens. Enteropathogenic strains of Enterobacter sakazakii and Escherichia coli (EPEC) that revealed adhesion to epithelial cells induced apoptosis [17,18]. Species of several genera within the family Enterobacteriaceae, including Salmonella, Shigella and Yersinia have been found to trigger cell death of host epithelial cells after bacterial invasion [11,19]. Apoptosis-inducing signals could include increase of intracellular calcium, oxidative damage, and membrane changes resulting from introduction of bacterial proteins into the host cell via the type III secretion system (TTSS) [11]. In our previous study, we have observed that TTSS genes were present in 27% of the Enterobacter spp. isolates [20]. In this study all strains with the secretion system caused apoptosis of HEp-2 cells. Four (7.5%) of them revealed high invasion indexes and 11 isolates (21%) demonstrated high adhesion activity. It is possible that the type
87
III-mediated secretion is responsible for the breakdown of membrane integrity, like that observed in response to Salmonella spp. and Yersinia spp. infections [21]. A number of microbial factors that can trigger the induction of apoptosis in host cells have been identified. We have previously demonstrated that 22% E. cloacae complex strains used in this study were cytotoxic to epithelial cells [20]. The activity was revealed after toxin activation with 2-mercaptoethanol. Barnes et al. [22] and Paraje et al. [12,23,24] have isolated thiol-activated cytotoxic toxin from E. cloacae strains that exhibited hemolytic and leucotoxic effect. High concentration of the toxin generated reactive oxygen species, which led to oxidative stress and subsequent apoptotic cell death. It was unlikely that in our study the toxin played a significant role in causing the apoptosis of HEp-2 cells, because it requires culture conditions different from those used in our studies. We did not activate cytotoxins with 2mercaptoethanol; therefore, the strains revealed low apoptotic indexes. We observed that the interaction of eight E. hormaechei strains (15%) with epithelial cells led to necrotic damage. The highest necrotic activity was observed for three strains isolated from urine and one from blood and stool. Candy and Devane [25] have reported that E. cloacae strains isolated from infants in neonatal ward caused outbreak of necrotizing enterocolitis (NE). So far, there has been no evidence about necrotizing factors produced by E. cloacae isolates. Hunter et al. [17] demonstrated that E. sakazakii induced clinical and histological NE in newborn rats. The strains were found to bind to enterocytes without direct invasion. They induced apoptosis and increased production of inflammatory cytokine IL-6. The results of this study lead to a better understanding of the E. cloacae complex infections and demonstrate that adhesion and invasion of the strains to epithelial cells induce apoptotic cell death. The processes may be a primary strategy of the strains, resulting in tissue destruction, bacterial spreading, and, in consequence, invasive disease or systemic infection. 4. Materials and methods 4.1. Bacterial strains and their molecular differentiation by hsp60 sequence analysis Fifty-three strains identified by a biochemical test kit (API 20E, bioMerieux) as E. cloacae were further differentiated by sequence analysis of the hsp60 gene. Bacterial DNA was isolated by using Novabeads Bacterial Genomic DNA kit (Novazym). Primers Hsp60-F and Hsp60-R were used to amplify a 341-bp fragment of hsp60 in a PCR reaction that involved an initial denaturation for 3 min at 94 C, followed by 30 cycles of denaturation (30 s, 94 C), annealing (30 s, 57 C) and elongation (60 s, 72 C) with a final elongation step (5 min, 72 C). The reactions were performed in a MyCycler thermal cycler (BioRad). PCR products were purified with ExoSAP-IT (Affymetrix) and sequenced in a 3130 l Genetic Analyzer (Applied Biosystems). A 272-nt fragment of the sequence was compared to the sequences of type and reference strains by using ClustalW algorithm and neighbor-joining method [6]. The hsp60 sequences of the following type and reference strains were used: E. cloacae subsp. cloacae, strain ATCC 13047T, E. cloacae subsp. dissolvens ATCC 23373T, E. cloacae cluster III EN114, E. cloacae cluster IV EN117, E. cloacae cluster IX EN25, E. asburiae ATCC 35953T, E. kobei ATCC BAA260T, E. ludwigii CIP 108491T, E. hormaechei subsp. oharae CIP 108490t, E. hormaechei subsp. hormaechei ATCC 49162T, E. hormaechei subsp. steigerwaltii CIP108489T, E. nimipressuralis ATCC 9912T, E. cancerogenus ATCC 33241T, E. amnigenus ATCC 3072T, E. cowanii ATCC 107300T, E. gergoviae ATCC 33028T, E. pyrinus ATCC 49851T, and E. sakazakii ATCC 29544T [3,6].
88
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
4.2. Cell cultures
4.6. Assessment of apoptosis of HEp-2 cells
Human epidermoid carcinoma cells from the larynx (HEp-2) were cultured in Eagle Minimum Essential Medium (EMEM, Sigma) with 5% fetal calf serum (FCS, Sigma) containing 2 mM glutamine, 50 IU of penicillin per milliliter, streptomycin (100 mg/ml) and nystatin (1 mg/ml). The cells were seeded with 100 ml of suspension containing 2 105 cells per well and incubated at 37 C in an atmosphere with 5% CO2 [14].
Three methods were used for the analysis of the cells: (a) trypan blue exclusion assay, (b) acridine orange and bromide ethidium staining and (c) analysis of DNA fragmentation. Infected cells viability was determined by trypan blue exclusion assay, in which a suspension of trypsynized cells was stained with 0.1% trypan blue (Sigma) for 3 min at room temperature. The cells stained blue were considered nonviable, whereas those that excluded the stain e viable. The percentage of dead cells was calculated by dividing the mean number of stained cells by the total number of cells in 50 microscopic fields, and multiplying it by 100 [27]. Infected HEp-2 cells were stained with acridine orange and ethidium bromide [19]. The monolayer was detached using 0.25% trypsin and 0.25% EDTA in PBS, and the suspension was stained with acridine orange (100 mg/ml) and ethidium bromide (100 mg/ml) solution, and examined under fluorescence microscope (Nikon Eclipse TE-2000). At least 100 cells were counted at random and were grouped into three distinct categories: viable, apoptotic and necrotic. This procedure was repeated three times and the number of cells in each category was expressed in percentages. Apoptotic index (ApI) is presented as mean and represents two independent experiments performed in triplicate. A one-way analysis of variance ANOVA with Tukey’s post-hoc test at significance level P< 0.05 was performed. The analysis of DNA fragmentation was carried out as described previously [28]. Infected monolayer of HEp-2 cells was incubated at 24 and 48 h after treatment with gentamicin. The cells were washed with PBS and resuspended in lysis buffer containing 100 mM NaCl, 10 mM TriseHCl, 1 mM EDTA, 1% SDS pH 7.5 and 200 mg ml1 proteinase K for 16 h at 37 C. The lysates were extracted with an equal volume of phenol-chloroform (1:1, vol/vol) and with an equal volume of chloroform-isoamyl alcohol (24:1, vol/vol) before precipitation with ethanol. The precipitates were dried, solubilized in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) and digested with 2 mg/ml of RNase. Electrophoresis was performed in a 1.5% agarose gel (Basica LE GQT, Prona) at 120 V for 3 h. Gene Ruler 100 bp DNA Ladder (MBI Fermentas) was used as a molecular weight marker. DNA was stained with ethidium bromide, visualized under UV light and digitalized with a Bio-Print V.99 system (Vilbert-Lourmat, France).
4.3. Infection conditions For each experiment, HEp-2 cells at a concentration of 2 106/ml in EMEM were seeded into 96-well plates (Nunc) and allowed to attach overnight. Incubation with bacteria was performed at a multiplicity of infection (MOI) 1:50 (HEp-2 cells in number 2 10 6 incubated with approximately of 1 108 of bacteria) for 2 h at 37 C to allow for adhesion and invasion of them to HEp-2 cells (infection period). Next, the medium was replaced with EMEM containing gentamicin (0.2 mg/ml) for 2 h at 37 C to kill extracellular bacteria. After three-time washing in PBS, the cells were incubated in the medium without gentamicin [14]. 4.4. Adhesion, invasion of HEp-2 cells assay Invasion assay was performed using gentamicin protection assay according to Keller et al. [14] with modifications. In samples in which the adhesion and invasion were analyzed, 200 ml of EMEM with 2% FCS and 2% D-mannose was added and HEp-2 cells were incubated for additional 4 h (multiplication period). After washing with PBS, the integrity of the monolayer was checked and 100 ml of 0.01% Triton X-100 in PBS was added to each well and the cells were incubated for 5 min at room temperature. The total number of bacteria attached to the cells and the intracellular bacteria was determined by plating the lysates onto tryptic soy agar (TSA, Difco). The number of attached bacteria was determined by subtracting the number of intracellular bacteria from total counts. The results were expressed as adhesion index (AdI), i.e. the mean number of associated bacteria per 100 HEp-2 cells. Invasion index (InI) was expressed as the percentage of internalized bacteria per 100 HEp-2 cells in comparison with the number of adhering bacteria [19]. As a control, an invasive strain of Yersinia enterocolitica O:8/1B (pYVþ), a nonpathogenic E. cloacae MPU E326/7 isolated from sewage, and E. coli K12C600 were included. Adhesion and invasion indexes are presented as means, they represent two independent experiments performed in triplicate. A one-way analysis of variance ANOVA with Tukey’s post-hoc test at significance level P < 0.05 was performed. 4.5. Inhibition of E. cloacae invasion The effect of cytochalasin D, a potent inhibitor of actin-dependent internalization, was used to determine the contribution of cytoskeleton rearrangement to E. cloacae invasion of epithelial cells. The HEp-2 monolayer was pretreated with 1 mg/ml of cytochalasin D (SigmaeAldrich) in EMEM 60 min prior to bacterial inoculation [26]. The cells were washed three times with PBS and the invasion assay and counting of the apopototic cells were performed, as described above. Addition of cytochalasin D had no effect on the bacterial viability and toxicity to HEp-2 cells as determined by plating onto TSA and performing trypan blue exclusion assay, respectively.
References [1] Sanders Jr WE, Sanders CC. Enterobacter spp.: pathogens poised to flourish at the turn of the century. Clin Microbiol Rev 1997;10:220e41. [2] Paauw A, Caspers MPM, Schuren FHJ, Leverstein-van Hall MA, Deletoile A, Montijn RC, et al. Genomic diversity within the Enterobacter cloacae complex. PloS ONE 2008;3:1e11. [3] Hoffmann H, Roggenkamp A. Population genetics of the nomenspecies Enterobacter cloacae. Appl Environ Microbiol 2003;69:5306e15. [4] Yu WL, Cheng HS, Lin HC, Peng CT, Tsai CH. Outbreak investigation of nosocomial Enterobacter cloacae bacteraemia in a neonatal intensive care unit. Scan J Infect Dis 2000;32:293e8. [5] Hoffmann H, Stindl S, Ludwig W, Stumpf A, Mehlen A, Monget D, et al. Enterobacter hormaechei subsp. oharae subsp. nov., Enterobacter hormaechei subsp. hormaechei comb. nov., Enterobacter hormaechei subsp. steigerwaltii subsp. nov., three new subspecies of clinical importance. J Clin Microbiol 2005;43:3297e303. [6] Morand PC, Billoet A, Rottman A, Sivadon-Tardy V, Eyrole L, Jeanne L, et al. Specific distribution within Enterobacter cloacae complex of strains isolated from infected orthopedic implants. J Clin Microbiol 2009;47:2489e95. [7] Grimont F, Grimont PAD. The genus Enterobacter. In: Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. Procaryotes, vol. 6. , Berlin: Springer; 2006. p. 197e214. [8] Yogaraj JS, Elward AM, Fraser VJ. Rate, risk factors, and outcomes of nosocomial primary bloodstream infection in pediatric intensive care units patiens. Pediatrics 2002;110:481e5. [9] Niemann HH, Schubert W-D, Heinz DW. Adhesins and invasins of pathogenic bacteria: a structural view. Microbes Infect 2004;6:101e12. [10] Higuchi Y. Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress. Biochem Pharmacol 2003;66:1527e35. [11] Gao LY, Kwaik YA. The modulation of host cell apoptosis by intracellular bacterial pathogens. Trends Microbiol 2000;8:306e13.
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin [12] Paraje MG, Barnes AI, Albesa I. An Enterobacter cloacae toxin able to generate oxidative stress and to provoked dose-dependent lysis of leucocytes. Int J Med Microbiol 2005;295:109e16. [13] Gladstone P, Jesudason MV, Sridharan G. Invasive properties of South Indian strains of Streptococcus pyogenes in a HEp-2 cell model. Clin Microbiol Infect 2003;9:1031e4. [14] Keller R, Pedroso MZ, Ritchmann R, Silva RM. Occurrence of virulence-associated properties in Enterobacter cloacae. Infect Immun 1998;66:645e9. [15] Townsend S, Hurrell E, Forsythe S. Virulence studies of Enterobacter sakazakii isolates associated with a neonatal intensive care unit outbreak. BMC Microbiol 2008;8:64e72. [16] Bonazzi M, Cossart P. Bacterial entry into cells: a role for the endocytic machinery. FEBS Lett 2006;580:2962e7. [17] Hunter CJ, Singamsetty VK, Chokshi NK, Boyle P, Camerini V, Grishin AV, et al. Enterobacter sakazakii enhances epithelial cell injury by inducing apoptosis in a rat model of necrotizing enterocolitis. J Infect Dis 2008;198:586e93. [18] Abul-Milh M, Wu Y, Lau B, Lingwood CA, Foster DB. Induction of epithelial cell death including apoptosis by enteropathogenic Escherichia coli expressing boundle-forming pili. Infect Immun 2001;69:7356e64. [19] Superti F, Pietrantoni A, Di Base AM, Longhi C, Valenti P, Tinari A. Invmediated apoptosis of epithelial cells infected with enteropathogenic Yersinia: A protective effect of lactoferrin. Res Microbiol 2005;156:728e37.
89
ska S, Mokracka J, Koczura R, Kaznowski A. Cytotoxic activity of [20] Krzymin Enterobacter cloacae human isolates. FEMS Immunol Med Microbiol 2009;56:248e52. [21] Moss JE, Idanpaan-Heikkila I, Zychlinsky A. Induction of apoptosis by microbial pathogen. In: Cossard P, Bouquet P, Normark S, Rappuoli R, editors. Cellular microbiology; 2000. Washington, DC. [22] Barnes AI, Paraje MG, Battan Paolo del C, Albesa I. Molecular properties and metabolic effect on blood cells produced by a new toxin of Enterobacter cloacae. Cell Biol Toxicol 2001;17:409e18. [23] Paraje MG, Iribarren P, Albesa I. Interaction of bacterial toxin with leukocytes measured by flow cytometry. Curr Microbiol 2002;45:171e4. [24] Paraje MG, Eraso AJ, Albesa I. Pore formation, polymerization, hemolytic and leucotoxic effects of a new Enterobacter cloacae toxin neutralized by antiserum. Microbiol Res 2005;160:203e11. [25] Candy DCA, Devane SP. Role of microorganisms in necrotizing enterocolitis. Seminars Neonatol 1997;2:255e62. [26] Dogan B, Klaessig S, Rishniw M, Almeida RA, Oliver SP, Simpson K, et al. Adherent and invasive Escherichia coli are associated with persistent bovine mastitis. Vet Microbiol 2006;116:270e82. [27] Marouni MJ, Sela S. Fate of Streptococcus pyogenes and epithelial cells following internalization. J Med Microbiol 2004;53:1e7. ska S, Kaznowski A, Chodysz M. Aeromonas spp. induced apoptosis of [28] Krzymin murine macrophages. Curr Microbiol 2009;58:252e7.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Isolates of the Enterobacter cloacae complex induce apoptosis of human intestinal epithelial cells ska*, Ryszard Koczura, Joanna Mokracka, Tomasz Puton, Adam Kaznowski Sylwia Krzymin , ul. Umultowska 89, Poland Department of Microbiology, Faculty of Biology, A. Mickiewicz University, 61-614 Poznan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 March 2010 Received in revised form 22 April 2010 Accepted 23 April 2010 Available online 6 May 2010
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 24 h and 98% at 48 h after infection. Forty strains (73%) induced fragmentation of nuclear DNA and characteristic intranucleosomal pattern with the size of about 180e200 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. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Enterobacter cloacae complex Genetic diversity Adhesion Invasion Apoptosis
1. Introduction Bacteria of Enterobacter cloacae occur in water, sewage, soil, food, and as commensal microflora in the intestinal tracts of humans and animals [1]. Molecular studies on E. cloacae recognized on the biochemical characteristics have showed genomic heterogeneity of this taxonomic complex comprising six species: E. cloacae, Enterobacter hormaechei, Enterobacter asburiae, Enterobacter kobei, Enterobacter ludwigii, and Enterobacter nimipressuralis. Identification of these species upon phenotypic traits is usually difficult and not reliable; therefore, molecular methods are used [2]. Upon the sequence of the hsp60 gene, the E. cloacae complex has been divided into 12 genetic clusters and one sequence crowd. Nine of the clusters correspond to species: E. asburiae, E. kobei, E. ludwigii, E. hormaechei subsp. oharae, E. hormaechei subsp. hormaechei, E. hormaechei subsp. steigerwaltii, E. nimipressuralis, E. cloacae subsp. cloacae, and
* Corresponding author. Tel.: þ48 61 829 5935; fax: þ48 61 829 5636. ska). E-mail address: [email protected] (S. Krzymin 0882-4010/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2010.04.003
E. cloacae subsp. dissolvens. Three clusters do not have specific names and are referred to as E. cloacae cluster III, E. cloacae cluster IV and E. cloacae cluster IX [3]. Similar results were obtained on using rpoB genotyping, multi-locus sequence analysis and comparative genomic hybridization [2]. Some strains phenotypically identified as E. cloacae are opportunistic pathogens implicated as the causative agent of local and systemic infections in humans. They are important nosocomial pathogens responsible for bacteremia, lower respiratory tract, skin, soft tissue, urinary tract, intra-abdominal and ophthalmic infections, endocarditis, septic arthritis and osteomyelitis [1]. They are etiological agents of outbreaks of septicemia in neonatal intensive care [4]. Among the E. cloacae complex, E. hormaechei subsp. steigerwaltii, E. hormaechei subsp. oharae, and E. cloacae cluster III have been reported to be the bacteria most frequently recovered from clinical specimens [5,6]. E. nimipressuralis is a plant pathogen and has not been associated with human diseases [3]. Although the E. cloacae complex strains are among the most common Enterobacter species causing nosocomial bloodstream infections in the last decade, still little is known about their virulence-associated properties [1]. Among the most common risk
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
84
factors for developing E. cloacae bloodstream infections are prolonged hospitalization, severity of illnesses, and exposure to invasive procedures. Additional predisposing factors are the usage of central venous catheter, prolonged antibiotic therapy, parenteral nutrition and immunosuppressive therapy [7,8]. A requirement for successful colonization and development of disease by microbial pathogens is the ability to adhere to the surface of host epithelial cells [9]. Bacterial adhesion to the cells may result in internalization, either by phagocytosis or by bacteria-induced invasion. These processes are associated with the initiation of infection by many pathogenic bacteria and are therefore considered as essential virulence factors. There is increasing evidence that microbial pathogens induce oxidative stress in the infected cells, and this may represent an important mechanism leading to epithelial injury [10]. During bacterial infection, reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radicals (OH), superoxide anion (O 2 ) and reactive nitrogen intermediates in the form of nitric oxide may be involved in chromosomal DNA degradation leading to cell death through apoptosis. There is growing evidence that apoptosis of host cells plays an important role in modulating the pathogenesis of a variety of infectious diseases [11]. Paraje et al. [12] have suggested that E. cloacae strains produce toxin that increases the production of ROS in leucocytes and lead to oxidative stress with subsequent cell death by apoptosis. In our study, we focused on the interaction of the E. cloacae complex isolates with epithelial cells. We investigated their ability to adhere to and invade HEp-2 cells, a well established and frequently used model for studying interactions between bacteria and human cells [13,14]. Moreover, we studied the induction of apoptosis of HEp-2 cells by the E. cloacae complex.
2. Results 2.1. Molecular differentiation of strains by hsp60 sequence analysis PCR amplification of a part of the hsp60 heat shock protein gene resulted in a 341-bp product for all strains. The PCR products were sequenced and 272-nt sequences were compared to the sequences of type and reference strains of Enterobacter spp. All of the 53 strains phenotypically identified as E. cloacae were clustered within the E. cloacae complex. The majority of the strains were identified as E. hormaechei (81%) with E. hormaechei subsp. steigerwaltii being the most frequent subspecies (72%). Five (9%) belonged to E. hormaechei subsp. oharae and E. cloacae cluster III. Three isolates and the negative, nonpathogenic control MPU E326/7 belonged to E. cloacae cluster IV. One strain was identified as E. cloacae subsp. cloacae and one as E. kobei. All strains and the sources of their origin are listed in Table 1.
Table 1 Strains of the E. cloacae complex used in the study. Species
Source of origin (number of strains)
Strain No.
E. hormaechei subsp. steigerwaltii
Urine (20)
Catheter (1) Feces (1) Blood (1)
MPU E: 2, 4, 7, 8, 9, 11, 13, 32, 33, 35, 37, 38, 40a, 47a, 48, 49, 51, 53, 54a, 56a MPU E: 14, 17, 21, 43, 44, 59 MPU E: 1, 19, 20 MPU E: 16, 41, 46, 52, 55, 62 MPU E: 45 MPU E: 57 MPU E: 58
Urine (1) Blood (1) Secretion (2) Wound (1) Urine (1)
MPU MPU MPU MPU MPU
E: E: E: E: E:
3 18 12, 15 63a 42
Blood (1) Secretion (4) Urine (1) Blood (1) Secretion (1) Water (1) Catheter (1)
MPU MPU MPU MPU MPU MPU MPU
E: E: E: E: E: E: E:
23 6, 28, 30, 31 34 26 22 326/7 63b
Blood (6) Secretion (3) Wound (6)
E. hormaechei subsp. hormaechei E. hormaechei subsp. oharae
E. cloacae subsp. cloacae E. cloacae cluster III E. cloacae cluster IV
E. kobei
MPU e bacterial strains of the collection of Department of Microbiology, A. Mi , Poland. ckiewicz University, Poznan
2.2. Adhesion and invasion assay Adhesion and invasion to HEp-2 cells were quantified by gentamicin survival assay. Preliminary experiments showed that all the E. cloacae complex strains were gentamicin sensitive and unable to grow in media containing 0.2 mg/ml of the antibiotic. The results of adhesion activity are presented in Table 2. All strains revealed higher efficiency of adhesion in comparison with noninvasive controls. The adhesion index (AdI) ranged from 0.21 105 to 31 105 CFU per 100 HEp-2 cells. The negative controls of nonpathogenic E. cloacae MPU E326/7 and E. coli K12C600 showed indexes of 0.29 105 CFU and 0.15 104, respectively, whereas that of Yersinia enterocolitica O:8/1B, the positive control, was 19.6 105 CFU per ml. The highest index (17.8 105e36.9 105) was obtained for 30% strains isolated from blood, 20% from secretions and 38% from wounds. Isolates of the E. cloacae complex were invasive towards HEp-2 cells (Table 3). Four strains (7.5%) showed the highest invasion activity. The invasion index ranged between 16.2 and 52.9%, whereas for Y. enterocolitica O:8/1B reached 61.3%. For 34 isolates
Table 2 Adhesion of the E. cloacae complex strains to HEp-2 cells. Adhesion index range (105)a
Number of strains (%)
Enterobacte hormaechei
E.cloacae III
E. cloacae IV
17.8e36.9b 8.5e14.3
8 (15.1) 13 (24.5)
23 28
22, 26
5.4e7.9
11 (20.7)
3.1e5.3 1.4e2.9 0.21e0.79
11 (20.7) 5 (9.4) 5 (9.4)
62, 55, 43, 20, 21 8, 4, 11, 17, 18, 44, 45, 46, 37, 19, 63a 52, 1, 40a, 2, 47a, 14, 58, 59, 3, 33, 41 7, 32, 38, 12, 26, 51, 48, 35, 30 49, 53, 54a, 9 57, 13, 15, 56a
a
E. cloacae s. cloacae
E. kobei
42
63b
6, 31 30 34
The mean number of associated (CFU) bacteria/100 HEp-2 cells. Mean CFU/ml of two separate assays performed in triplicate. Means in the group did not differ significantly at P < 0.05 according Tukey’s HSD test. Adhesion index of E. coli K12C600 ¼ 0.15 104, E. cloacae 326/7 ¼ 0.29 105 and Y. enterocolitica O:8/1B ¼ 19.6 105. b
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
85
Table 3 Invasion ability of the E. cloacae complex strains to HEp-2 cells. Invasion index rangea
Number of strains (%)
Enterobacter hormaechei
32.1e52.9b 16.2e31.2 4.1e7.1 1.1e2.7 0.1e0.9
2 2 5 8 20
0.01e0.09
16 (30.2)
21, 15 57, 16 7, 2, 13, 54a 40a, 19, 49, 38, 12, 35 53, 48, 11, 20, 56a, 4, 41, 17, 33, 43, 14, 59, 47a, 52, 18, 55, 63a, 23, 44 62, 51, 58, 46, 3, 37, 32, 9, 34, 1, 8, 45
(3.8) (3.8) (9.4) (15.1) (37.7)
E. cloacae III
E. cloacae IV
30 31
E. kobei
42 26
6, 28
E. cloacae s. cloacae
63b
22, 34
a
The percentage of internalized bacteria/100 HEp-2 cells in comparison with the number of adhering bacteria of two separate experiments performed in triplicate. Means in the group did not differ significantly at P < 0.05 according Tukey’s HSD test. Invasion index of E. cloacae 326/7 ¼ 0.036%, E. coli K12C600 ¼ 0.013% and Y. enterocolitica O:8/1B ¼ 61.3%. b
(64%) the index ranged between 0.1 and 7.1% and was higher than those of the negative controls. Sixteen isolates (30%) showed the lowest invasive ability, comparable to that of nonpathogenic negative controls. The invasion index for E. cloacae MPU E326/7 and E. coli K12C600 strains was 0.036% and 0.013%, respectively. Treatment of epithelial cells with cytochalasin D had no effect on E. cloacae adhesion, but reduced bacteria uptake by 91e96% after 24 h. 2.3. Quantification of epithelial cell death 2.3.1. Trypan blue exclusion assay Trypan blue exclusion assay was used to assess the damage of epithelial cells. The analysis was performed 24 and 48 h after infection. Trypan blue is excluded from viable cells with functional cellular membranes, but penetrates membranes of apoptotic and necrotic cells, thereby suggesting that such assay is appropriate for staining dead cells. At 24 h after infection cell viability remained high (82e94%) for 60.4% strains and ranged from 68% to 80% for 40% strains. At 48 h, the viability declined by 53% to 78% for 29% isolates and from 12% to 49% for 71% of isolates. 2.3.2. Morphological assessment of apoptosis The apoptotic activity was expressed as apoptotic index (ApI) and was determined by ethidium bromide and acridine orange staining and observation under fluorescence microscope. In this
assay, ApI and cell membrane integrity can be assessed simultaneously. Acridine orange permeates cell membrane and makes the nuclei appear green, whereas ethidium bromide is only taken up by cells with lost cytoplasmic membrane integrity and stains nuclei red. Live cells had a normal green nucleus (Fig. 1a), early apoptotic cells had bright green nucleus with condensed chromatin, late apoptotic cells displayed condensed or fragmented orange chromatin, and cells that died from necrosis had a structurally normal orange nucleus (Fig. 1b). Strains of the E. cloacae complex provoked apoptosis of HEp-2 cells, which displayed such morphological changes as cell shrinkage, loss of normal cell-to-cell contacts and blebbing at the cell surface. The analysis of mean percentage of apoptotic cells (Table 4) at 24 h showed five groups with statistically significant differences between them (ANOVA, F23,120 ¼ 115.407, P < 0.001). The highest apoptotic indexes ranging from 26.4% to 38.7% were observed for 27% strains. The lowest (6.7e10.3%) were expressed by 9.4% strains of E. cloacae. The percentage of apoptotic cells increased at 48 h post infection. We found statistically significant differences between six groups of strains (ANOVA, F23,120 ¼ 147.513, P < 0.001). The highest apoptotic indexes (83.1e98.3%), were observed in cells infected with 14.5% of E. cloacae complex strains. The lowest (18.7e25.4%) were revealed by 7.3% strains. The apoptotic indexes were reduced to 3.7e8.1% after treatment of HEp-2 monolayer with 1 mg ml1 of cytochalasin D prior infection. Some E. cloacae strains also caused necrotic
Fig. 1. Apoptosis of HEp-2 cells. The cells were stained with propidium iodide and acridine orange (100 mg/ml) and observed in fluorescence microscope. The cells were infected with (a) Escherichia coli K12C600; (b) Enterobacter hormaechei susp. hormaechei MPU E17; the arrows point to: A e live, B e apoptotic, C e necrotic cells. Magnifications: (a) 200; (b) 250.
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
86
Table 4 Apoptotic index of the Enterobacter cloacae complex strains at 24 h and 48 h after infection of HEp-2 cells. Group
Apoptotic index rangea (percentage of strains)
Enterobacter hormaechei
E. cloacae III
E. cloacae IV
At 24 h Ab B
6.7e10.3 (9.4) 11.2e17.6 (35.8)
31 23, 28
22 34, 26
C D
19.3e25.8 (15.1) 26.4e38.7 (26.4)
E
39.7e49.1 (13.2)
18, 16, 48 17, 40a, 3, 38, 37, 47a, 1, 21, 56a, 19, 49, 57, 51, 41, 32 15, 53, 35, 44, 20, 59 35, 9, 11, 52, 14, 54a, 58, 13, 7, 2, 8, 55, 45 43, 4, 12, 46, 62, 33, 63a
At 48 h A B C D
18.7e25.4 27.8e36.4 38.7e47.6 51.8e65.1
E
66.4e81.7 (32.1)
F a b
(5.7) (15.1) (7.5) (29.1)
83.1e98.3 (11.3)
38, 40a, 15 18, 56a, 41, 7, 20 51, 19, 8, 49 16, 52, 13, 48, 57, 9, 32, 2, 35, 11, 1 33, 44, 59, 12, 47, 37, 53, 45, 14, 62, 58, 54, 43, 3, 63a 17, 46, 55, 4, 21
30 6
E. cloacae s. cloacae
E. kobei
42
63b
30, 28
22
6, 31, 23
26, 34 42 63b
Mean percentage of apoptotic cells from two experiments in triplicate. Within each column, means designated by the same letter did not differ significantly at P < 0.05 according to Tukey’s post-hoc test.
changes. The highest necrotic indexes (39e41%) were observed for 11.3% strains at 24 h. The value of indexes increased to 45% to 69%, for 15.1% strains at 48 h.
isolates were able to adhere to HEp-2 cells. For 19% of the isolates, the adhesion index was comparable to that of Y. enterocolitica O:8/ 1B, the enteroinvasive positive control. The highest adhesion ability was observed for strains isolated from blood: two E. hormaechei
2.3.3. DNA fragmentation The apoptotic effect of E. cloacae strains on HEp-2 cells was also confirmed by the analysis of DNA fragmentation. Characteristic patterns of DNA (180e200 bp) were obtained for 11.3% strains 24 h after infection (Fig. 2). The percent of strains that caused DNA fragmentation in HEp-2 cells increased to 75.5% after 48 h of infection. These patterns were similar to that obtained for the positive control. DNA fragmentation was observed when apoptotic index exceeded 47%.
3. Discussion The phenotypic similarity of strains belonging to different E. cloacae genomic groups creates many problems with their identification. As a consequence, our knowledge regarding epidemiology, disease spectrum and understanding of virulence mechanisms of these bacteria is limited. In our study we used sequencing of the hsp60 gene, which shows good discriminatory power to identify species and subspecies within the E. cloacae complex [2]. The majority of clinical strains were identified as E. hormaechei, with E. hormaechei subsp. steigerwaltii as a predominant group. E. hormaechei strains have been isolated from a variety of samples, including blood, stool, urine, and wounds. However, strains of E. cloacae III and IV clusters can also be pathogenic to humans. The ability of the strains to adhere and invade host cells and their capability to induce apoptosis suggest that these mechanisms may be considered as contributing factors in disease development by the E. cloacae complex. Therefore, this study was important for clarifying the contribution of adhesion and internalization to the pathogenicity of the E. cloacae complex strains. Although evidence has suggested that these bacteria also invade epithelial cells [14], the fate of these cells after bacterial internalization is not known. In our study, we provided evidence that the E. cloacae complex strains may induce apoptosis of HEp-2 cells. In order to colonize a host, bacteria must display the ability to adhere to epithelial cells, which are an integral component of the mucosal immune system [9]. Our results demonstrated that all
Fig. 2. (a) Analysis of intranucleosomal DNA fragmentation of HEp-2 cells infected with different strains. Lane 1: molecular size marker. DNA from cells infected with: Lane 2: strain of E. coli K-12 C200 (negative control). Lane 3: E. cloacae MPU E48 at 24 h after infection. Lane 4: E. cloacae MPU E48 (48 h). Lane 5: Y. enterocolitica O:8/1B (48 h). Lane 6: E. cloacae MPU E4 (48 h). Lane 7: E. cloacae MPU E63a (48 h). (b) Quantification of apoptotic and necrotic cells as apoptotic and necrotic indexes expressed in percentages.
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
subsp. hormaechei, one E. cloacae cluster III and IV, one isolate of E. hormaechei subsp. hormaechei and E. cloacae cluster III from aspirate, three E. hormaechei subsp. hormaechei from bedsore and wound infections. The interaction of enteropathogens with epithelial cells is the first stage of successive bacterial invasion of the host [9]. In our study, 70% of the E. cloacae complex strains were invasive with invasion index higher than that of nonpathogenic control. Most of them (62%) belonged to E. hormaechei cluster. The highest indexes, comparable to that of Y. enterocolitica O:8/1B, were observed for 4 E. hormaechei (7.5%) strains originated from pharynx, blood, feces, and lower leg ulcer. Previously, Keller et al. [14] have reported that all E. cloacae strains isolated from specimens isolated from humans were adherent and invasive to HEp-2 cells. The strains were much less invasive than the enteroinvasive E. coli control strain. Townsend et al. [15] suggested that the ability to invade epithelial cells might be a strong indication of potential virulence of Enterobacter spp. strains. Invasion of the host cells by pathogenic bacteria may occur via phagocytosis, which involves polymerization of actin microfilaments [16]. This polymerization may be prevented by cytochalasin D. In our study, the E. cloacae complex invasion to HEp-2 cells was inhibited when the monolayer was pretreated with cytochalasin D. The results suggest that the bacteria invade the epithelial cells by a microfilament dependent endocytosis mechanism followed by a rearrangement of the cytoskeletal proteins. We examined the interaction of the E. cloacae complex strains with HEp-2 cells and demonstrated that one of the consequences of cell adhesion and invasion by the bacteria is injury to the epithelial cells and cell death. Using three different criteria (trypan blue exclusion assay, acridine orange and bromide ethidium staining and analysis of DNA fragmentation), we observed that the E. cloacae complex infection induced apoptosis of the cells. The morphological changes included condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of the cell membrane. The highest apoptotic activity was expressed by E. hormaechei strains. We also observed that 73% strains caused fragmentation of nucleosomal DNA of infected cells to multimers which are a biochemical hallmark of apoptosis. Analysis of morphological changes and internucleosomal cleavage of host cell DNA indicated that 14 strains isolated from urine, 9 from blood, 5 from secretions and all strains isolated from wounds caused apoptosis of epithelial cells. High level of adhesion was consistent with the ability to induce HEp-2 cells death. Moreover, isolates for which high invasion indexes (16e52%) were observed induced apoptosis. In contrast, infection with nonpathogenic E. cloacae MPU E326/7, E. coli K12C600 and the E. cloacae copmlex isolates with low invasion indexes did not trigger apoptosis. The ability of the E. cloacae complex with high invasive index to induce cell death was inhibited by cytochalasin D, indicating that the bacteria must have been internalized. Previous studies have shown that epithelial cells undergo apoptosis after adhesion or invasion by different pathogens. Enteropathogenic strains of Enterobacter sakazakii and Escherichia coli (EPEC) that revealed adhesion to epithelial cells induced apoptosis [17,18]. Species of several genera within the family Enterobacteriaceae, including Salmonella, Shigella and Yersinia have been found to trigger cell death of host epithelial cells after bacterial invasion [11,19]. Apoptosis-inducing signals could include increase of intracellular calcium, oxidative damage, and membrane changes resulting from introduction of bacterial proteins into the host cell via the type III secretion system (TTSS) [11]. In our previous study, we have observed that TTSS genes were present in 27% of the Enterobacter spp. isolates [20]. In this study all strains with the secretion system caused apoptosis of HEp-2 cells. Four (7.5%) of them revealed high invasion indexes and 11 isolates (21%) demonstrated high adhesion activity. It is possible that the type
87
III-mediated secretion is responsible for the breakdown of membrane integrity, like that observed in response to Salmonella spp. and Yersinia spp. infections [21]. A number of microbial factors that can trigger the induction of apoptosis in host cells have been identified. We have previously demonstrated that 22% E. cloacae complex strains used in this study were cytotoxic to epithelial cells [20]. The activity was revealed after toxin activation with 2-mercaptoethanol. Barnes et al. [22] and Paraje et al. [12,23,24] have isolated thiol-activated cytotoxic toxin from E. cloacae strains that exhibited hemolytic and leucotoxic effect. High concentration of the toxin generated reactive oxygen species, which led to oxidative stress and subsequent apoptotic cell death. It was unlikely that in our study the toxin played a significant role in causing the apoptosis of HEp-2 cells, because it requires culture conditions different from those used in our studies. We did not activate cytotoxins with 2mercaptoethanol; therefore, the strains revealed low apoptotic indexes. We observed that the interaction of eight E. hormaechei strains (15%) with epithelial cells led to necrotic damage. The highest necrotic activity was observed for three strains isolated from urine and one from blood and stool. Candy and Devane [25] have reported that E. cloacae strains isolated from infants in neonatal ward caused outbreak of necrotizing enterocolitis (NE). So far, there has been no evidence about necrotizing factors produced by E. cloacae isolates. Hunter et al. [17] demonstrated that E. sakazakii induced clinical and histological NE in newborn rats. The strains were found to bind to enterocytes without direct invasion. They induced apoptosis and increased production of inflammatory cytokine IL-6. The results of this study lead to a better understanding of the E. cloacae complex infections and demonstrate that adhesion and invasion of the strains to epithelial cells induce apoptotic cell death. The processes may be a primary strategy of the strains, resulting in tissue destruction, bacterial spreading, and, in consequence, invasive disease or systemic infection. 4. Materials and methods 4.1. Bacterial strains and their molecular differentiation by hsp60 sequence analysis Fifty-three strains identified by a biochemical test kit (API 20E, bioMerieux) as E. cloacae were further differentiated by sequence analysis of the hsp60 gene. Bacterial DNA was isolated by using Novabeads Bacterial Genomic DNA kit (Novazym). Primers Hsp60-F and Hsp60-R were used to amplify a 341-bp fragment of hsp60 in a PCR reaction that involved an initial denaturation for 3 min at 94 C, followed by 30 cycles of denaturation (30 s, 94 C), annealing (30 s, 57 C) and elongation (60 s, 72 C) with a final elongation step (5 min, 72 C). The reactions were performed in a MyCycler thermal cycler (BioRad). PCR products were purified with ExoSAP-IT (Affymetrix) and sequenced in a 3130 l Genetic Analyzer (Applied Biosystems). A 272-nt fragment of the sequence was compared to the sequences of type and reference strains by using ClustalW algorithm and neighbor-joining method [6]. The hsp60 sequences of the following type and reference strains were used: E. cloacae subsp. cloacae, strain ATCC 13047T, E. cloacae subsp. dissolvens ATCC 23373T, E. cloacae cluster III EN114, E. cloacae cluster IV EN117, E. cloacae cluster IX EN25, E. asburiae ATCC 35953T, E. kobei ATCC BAA260T, E. ludwigii CIP 108491T, E. hormaechei subsp. oharae CIP 108490t, E. hormaechei subsp. hormaechei ATCC 49162T, E. hormaechei subsp. steigerwaltii CIP108489T, E. nimipressuralis ATCC 9912T, E. cancerogenus ATCC 33241T, E. amnigenus ATCC 3072T, E. cowanii ATCC 107300T, E. gergoviae ATCC 33028T, E. pyrinus ATCC 49851T, and E. sakazakii ATCC 29544T [3,6].
88
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin
4.2. Cell cultures
4.6. Assessment of apoptosis of HEp-2 cells
Human epidermoid carcinoma cells from the larynx (HEp-2) were cultured in Eagle Minimum Essential Medium (EMEM, Sigma) with 5% fetal calf serum (FCS, Sigma) containing 2 mM glutamine, 50 IU of penicillin per milliliter, streptomycin (100 mg/ml) and nystatin (1 mg/ml). The cells were seeded with 100 ml of suspension containing 2 105 cells per well and incubated at 37 C in an atmosphere with 5% CO2 [14].
Three methods were used for the analysis of the cells: (a) trypan blue exclusion assay, (b) acridine orange and bromide ethidium staining and (c) analysis of DNA fragmentation. Infected cells viability was determined by trypan blue exclusion assay, in which a suspension of trypsynized cells was stained with 0.1% trypan blue (Sigma) for 3 min at room temperature. The cells stained blue were considered nonviable, whereas those that excluded the stain e viable. The percentage of dead cells was calculated by dividing the mean number of stained cells by the total number of cells in 50 microscopic fields, and multiplying it by 100 [27]. Infected HEp-2 cells were stained with acridine orange and ethidium bromide [19]. The monolayer was detached using 0.25% trypsin and 0.25% EDTA in PBS, and the suspension was stained with acridine orange (100 mg/ml) and ethidium bromide (100 mg/ml) solution, and examined under fluorescence microscope (Nikon Eclipse TE-2000). At least 100 cells were counted at random and were grouped into three distinct categories: viable, apoptotic and necrotic. This procedure was repeated three times and the number of cells in each category was expressed in percentages. Apoptotic index (ApI) is presented as mean and represents two independent experiments performed in triplicate. A one-way analysis of variance ANOVA with Tukey’s post-hoc test at significance level P< 0.05 was performed. The analysis of DNA fragmentation was carried out as described previously [28]. Infected monolayer of HEp-2 cells was incubated at 24 and 48 h after treatment with gentamicin. The cells were washed with PBS and resuspended in lysis buffer containing 100 mM NaCl, 10 mM TriseHCl, 1 mM EDTA, 1% SDS pH 7.5 and 200 mg ml1 proteinase K for 16 h at 37 C. The lysates were extracted with an equal volume of phenol-chloroform (1:1, vol/vol) and with an equal volume of chloroform-isoamyl alcohol (24:1, vol/vol) before precipitation with ethanol. The precipitates were dried, solubilized in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) and digested with 2 mg/ml of RNase. Electrophoresis was performed in a 1.5% agarose gel (Basica LE GQT, Prona) at 120 V for 3 h. Gene Ruler 100 bp DNA Ladder (MBI Fermentas) was used as a molecular weight marker. DNA was stained with ethidium bromide, visualized under UV light and digitalized with a Bio-Print V.99 system (Vilbert-Lourmat, France).
4.3. Infection conditions For each experiment, HEp-2 cells at a concentration of 2 106/ml in EMEM were seeded into 96-well plates (Nunc) and allowed to attach overnight. Incubation with bacteria was performed at a multiplicity of infection (MOI) 1:50 (HEp-2 cells in number 2 10 6 incubated with approximately of 1 108 of bacteria) for 2 h at 37 C to allow for adhesion and invasion of them to HEp-2 cells (infection period). Next, the medium was replaced with EMEM containing gentamicin (0.2 mg/ml) for 2 h at 37 C to kill extracellular bacteria. After three-time washing in PBS, the cells were incubated in the medium without gentamicin [14]. 4.4. Adhesion, invasion of HEp-2 cells assay Invasion assay was performed using gentamicin protection assay according to Keller et al. [14] with modifications. In samples in which the adhesion and invasion were analyzed, 200 ml of EMEM with 2% FCS and 2% D-mannose was added and HEp-2 cells were incubated for additional 4 h (multiplication period). After washing with PBS, the integrity of the monolayer was checked and 100 ml of 0.01% Triton X-100 in PBS was added to each well and the cells were incubated for 5 min at room temperature. The total number of bacteria attached to the cells and the intracellular bacteria was determined by plating the lysates onto tryptic soy agar (TSA, Difco). The number of attached bacteria was determined by subtracting the number of intracellular bacteria from total counts. The results were expressed as adhesion index (AdI), i.e. the mean number of associated bacteria per 100 HEp-2 cells. Invasion index (InI) was expressed as the percentage of internalized bacteria per 100 HEp-2 cells in comparison with the number of adhering bacteria [19]. As a control, an invasive strain of Yersinia enterocolitica O:8/1B (pYVþ), a nonpathogenic E. cloacae MPU E326/7 isolated from sewage, and E. coli K12C600 were included. Adhesion and invasion indexes are presented as means, they represent two independent experiments performed in triplicate. A one-way analysis of variance ANOVA with Tukey’s post-hoc test at significance level P < 0.05 was performed. 4.5. Inhibition of E. cloacae invasion The effect of cytochalasin D, a potent inhibitor of actin-dependent internalization, was used to determine the contribution of cytoskeleton rearrangement to E. cloacae invasion of epithelial cells. The HEp-2 monolayer was pretreated with 1 mg/ml of cytochalasin D (SigmaeAldrich) in EMEM 60 min prior to bacterial inoculation [26]. The cells were washed three times with PBS and the invasion assay and counting of the apopototic cells were performed, as described above. Addition of cytochalasin D had no effect on the bacterial viability and toxicity to HEp-2 cells as determined by plating onto TSA and performing trypan blue exclusion assay, respectively.
References [1] Sanders Jr WE, Sanders CC. Enterobacter spp.: pathogens poised to flourish at the turn of the century. Clin Microbiol Rev 1997;10:220e41. [2] Paauw A, Caspers MPM, Schuren FHJ, Leverstein-van Hall MA, Deletoile A, Montijn RC, et al. Genomic diversity within the Enterobacter cloacae complex. PloS ONE 2008;3:1e11. [3] Hoffmann H, Roggenkamp A. Population genetics of the nomenspecies Enterobacter cloacae. Appl Environ Microbiol 2003;69:5306e15. [4] Yu WL, Cheng HS, Lin HC, Peng CT, Tsai CH. Outbreak investigation of nosocomial Enterobacter cloacae bacteraemia in a neonatal intensive care unit. Scan J Infect Dis 2000;32:293e8. [5] Hoffmann H, Stindl S, Ludwig W, Stumpf A, Mehlen A, Monget D, et al. Enterobacter hormaechei subsp. oharae subsp. nov., Enterobacter hormaechei subsp. hormaechei comb. nov., Enterobacter hormaechei subsp. steigerwaltii subsp. nov., three new subspecies of clinical importance. J Clin Microbiol 2005;43:3297e303. [6] Morand PC, Billoet A, Rottman A, Sivadon-Tardy V, Eyrole L, Jeanne L, et al. Specific distribution within Enterobacter cloacae complex of strains isolated from infected orthopedic implants. J Clin Microbiol 2009;47:2489e95. [7] Grimont F, Grimont PAD. The genus Enterobacter. In: Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. Procaryotes, vol. 6. , Berlin: Springer; 2006. p. 197e214. [8] Yogaraj JS, Elward AM, Fraser VJ. Rate, risk factors, and outcomes of nosocomial primary bloodstream infection in pediatric intensive care units patiens. Pediatrics 2002;110:481e5. [9] Niemann HH, Schubert W-D, Heinz DW. Adhesins and invasins of pathogenic bacteria: a structural view. Microbes Infect 2004;6:101e12. [10] Higuchi Y. Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress. Biochem Pharmacol 2003;66:1527e35. [11] Gao LY, Kwaik YA. The modulation of host cell apoptosis by intracellular bacterial pathogens. Trends Microbiol 2000;8:306e13.
ska et al. / Microbial Pathogenesis 49 (2010) 83e89 S. Krzymin [12] Paraje MG, Barnes AI, Albesa I. An Enterobacter cloacae toxin able to generate oxidative stress and to provoked dose-dependent lysis of leucocytes. Int J Med Microbiol 2005;295:109e16. [13] Gladstone P, Jesudason MV, Sridharan G. Invasive properties of South Indian strains of Streptococcus pyogenes in a HEp-2 cell model. Clin Microbiol Infect 2003;9:1031e4. [14] Keller R, Pedroso MZ, Ritchmann R, Silva RM. Occurrence of virulence-associated properties in Enterobacter cloacae. Infect Immun 1998;66:645e9. [15] Townsend S, Hurrell E, Forsythe S. Virulence studies of Enterobacter sakazakii isolates associated with a neonatal intensive care unit outbreak. BMC Microbiol 2008;8:64e72. [16] Bonazzi M, Cossart P. Bacterial entry into cells: a role for the endocytic machinery. FEBS Lett 2006;580:2962e7. [17] Hunter CJ, Singamsetty VK, Chokshi NK, Boyle P, Camerini V, Grishin AV, et al. Enterobacter sakazakii enhances epithelial cell injury by inducing apoptosis in a rat model of necrotizing enterocolitis. J Infect Dis 2008;198:586e93. [18] Abul-Milh M, Wu Y, Lau B, Lingwood CA, Foster DB. Induction of epithelial cell death including apoptosis by enteropathogenic Escherichia coli expressing boundle-forming pili. Infect Immun 2001;69:7356e64. [19] Superti F, Pietrantoni A, Di Base AM, Longhi C, Valenti P, Tinari A. Invmediated apoptosis of epithelial cells infected with enteropathogenic Yersinia: A protective effect of lactoferrin. Res Microbiol 2005;156:728e37.
89
ska S, Mokracka J, Koczura R, Kaznowski A. Cytotoxic activity of [20] Krzymin Enterobacter cloacae human isolates. FEMS Immunol Med Microbiol 2009;56:248e52. [21] Moss JE, Idanpaan-Heikkila I, Zychlinsky A. Induction of apoptosis by microbial pathogen. In: Cossard P, Bouquet P, Normark S, Rappuoli R, editors. Cellular microbiology; 2000. Washington, DC. [22] Barnes AI, Paraje MG, Battan Paolo del C, Albesa I. Molecular properties and metabolic effect on blood cells produced by a new toxin of Enterobacter cloacae. Cell Biol Toxicol 2001;17:409e18. [23] Paraje MG, Iribarren P, Albesa I. Interaction of bacterial toxin with leukocytes measured by flow cytometry. Curr Microbiol 2002;45:171e4. [24] Paraje MG, Eraso AJ, Albesa I. Pore formation, polymerization, hemolytic and leucotoxic effects of a new Enterobacter cloacae toxin neutralized by antiserum. Microbiol Res 2005;160:203e11. [25] Candy DCA, Devane SP. Role of microorganisms in necrotizing enterocolitis. Seminars Neonatol 1997;2:255e62. [26] Dogan B, Klaessig S, Rishniw M, Almeida RA, Oliver SP, Simpson K, et al. Adherent and invasive Escherichia coli are associated with persistent bovine mastitis. Vet Microbiol 2006;116:270e82. [27] Marouni MJ, Sela S. Fate of Streptococcus pyogenes and epithelial cells following internalization. J Med Microbiol 2004;53:1e7. ska S, Kaznowski A, Chodysz M. Aeromonas spp. induced apoptosis of [28] Krzymin murine macrophages. Curr Microbiol 2009;58:252e7.