Research Article For reprint orders, please contact: [email protected]
Carbapenem resistance confers to Klebsiella pneumoniae strains an enhanced ability to induce infection and cell death in epithelial tissue-specific in vitro models Laura Leone1, Salvatore Raffa1,2, Daniela Martinelli2, Maria Rosaria Torrisi1,2 & Iolanda Santino*,1,2 Abstract Background: Carbapenem-resistant Klebsiella pneumoniae strains (KPC-Kp) are emerging worldwide causing different nosocomial infections including those of the urinary tract, lung or skin wounds. For these strains, the antibiotic treatment is limited to only few choices including colistin, whose continuous use led to the emergence of carbapenem-resistant KPC-Kp strains resistant also to this treatment (KPC-Kp Col-R). Aim: Very little is known about the capacity of the different strains of KPC-Kp to invade the epithelial cells in vitro. To verify if the acquisition of carbapenem-resistant and the colistin-resistant phenotypes are correlated with a different ability to infect a series of epithelial cell lines of various tissutal origin and with a different capacity to induce cellular death. Materials & methods: We used Klebsiella pneumoniae (KP), KPC-Kp and KPC-Kp Col-R strains, isolated from different patients carrying various tissue-specific infections, to infect a series of epithelial cell lines of different tissutal origin. The invasive capacity of the strains and the extent and characteristics of the cell damage and death induced by the bacteria were evaluated and compared. Conclusion: Our results show that both KPC-Kp and KPC-Kp Col-R display a greater ability to infect the epithelial cells, with respect to KP, and that the bacterial cell invasion results in a nonprogrammed cell death. Klebsiella pneumoniae (KP) is one of the most common Gram-negative bacteria causing communityacquired infections and in particular nosocomial infections [1,2] . Similarly to other Gram-negative bacteria which constitute the normal intestinal flora, KP is able to colonize different districts causing infections of the urinary tract, lung or skin wounds. The intensive use of carbapenem antibiotics, such as imipenem and meropenem, which are recommended as first-line therapy for severe infections caused by Enterobacteriaceae producing extended spectrum β-lactamases (ESBLs), including KP, caused the worldwide emergence in recent years of carbapenem-resistant K. pneumoniae strains (KPC-Kp). From a public health point of view, the spread of KPC-Kp is worrying especially because these bacteria are the source of many hospital-acquired infections and the antimicrobial treatment options are very restricted [3–5] . Antibiotic treatment is limited to a few choices, typically including colistin, tigecycline and one or more aminoglycoside. Recent studies suggest that combination treatment with colistin, tigecycline and meropenem might improve survival among bacteremic patients [6] . Unfortunately, the heavy and increasing use of colistin as important component of the therapeutic treatments has led to the emergence of colistin-resistant KPC-Kp strains (KPC-Kp Col-R) in many countries [7–10] . KP uses different mechanisms to invade and colonize host cells and its virulence factors are multiple, including surface-exposed lipopolysaccharides, iron acquisition systems, adhesins, fimbriae
Keywords
• cell death • epithelial cell lines • infection ability • Klebsiella pneumoniae • KPC-Kp
Department of Clinical & Molecular Medicine, ‘Sapienza’ University of Rome, V.le Regina Elena, 324, 00161 Rome, Italy Azienda Ospedaliera S. Andrea, Rome, Italy *Author for correspondence: Tel.: +39 06 33775488; [email protected] 1 2
10.2217/FMB.14.145 © 2015 Future Medicine Ltd
Future Microbiol. (2015) 10(5), 743–761
part of
ISSN 1746-0913
743
Research Article Leone, Raffa, Martinelli, Torrisi & Santino and the polysaccharide capsule (CPS), which is involved in protection from opsonization and phagocytosis [11,12] . Several strains are characterized by a different ability to adhere and invade the epithelium [13] . It is thought that adherence of the bacterium to the plasma membrane of epithelial cells is a necessary event to induce internalization and colonization in the various districts. In fact, although traditionally considered an extracellular pathogen, recent studies have demonstrated the ability of KP to enter into different human-cultured epithelial cell lines as airway, liver, intestinal and bladder cell lines [2,14,15] . It has been also described that KP invades bladder and ileocecum cells, through a receptor-mediated endocytosis [16] and that the polysaccharide capsule interferes with the internalization of KP [15] . KP internalization was analyzed also in airway epithelial cells in which a different virulence of the strain depending on the level of CPS was observed [2,11] . The bacterium’s ability to enter in host cells is closely related with the capacity to induce cellular death [17] . Recent in vitro studies have shown that KP infections can trigger cell death in epithelial cells. The cytotoxicity seems to depend on the duration time and on the different multiplicity of infection (MOI) [2,14] . The ability to induce a cellular damage seems to be related to death mediators as proapoptotic factors and executioners of programmed cell death [14,18] . Although there are some studies about the interaction between KP and epithelial cells, very little is known about the capacity of KPC-Kp strains to invade the cells in vitro. A few reports suggest an association between resistance to antibiotics, like aminoglycosides and/or β-lactams, and adhesion to human intestinal epithelial cells [19,20] . Regarding the structure of the bacterial membrane and the colistin-resistance, in a recent report [21] , the authors have compared genome sequences of different KPC-Kp strains isolated in patients and have identified mutations in the KPC-Kp genome that are associated with colistin-resistance. Some of these mutations result in aminoacid changes in membrane proteins and seem to alter membrane function possibly reducing drug influx [21] . In the present study, we compared KPC-Kp and KPC-Kp Col-R strains, clinically isolated from patients with different types of infections, with KP in order to verify if the acquisition of colistin-resistant phenotype is correlated in vitro with a different ability to infect epithelial cells
744
Future Microbiol. (2015) 10(5)
and with a different capacity to induce cellular death. To this aim, KPC-Kp and KPC-Kp Col-R strains isolated from urine, wound swab and sputum samples were used to infect colon epithelial cells. Moreover, kidney, lung and skin epithelial cell lines were infected with the KPC-Kp and KPC-Kp Col-R strains isolated from the corresponding tissue sites and the invasive capacity of the different strains and the extent as well as the type of the cell damage induced by the bacteria were evaluated and compared not only on the same cell line but also on epithelial cells of various origins. Materials & methods ●●Bacterial strains
The carbapenem-resistant clinical strains of K. pneumoniae (KPC-Kp) belonging to the sequence type 512 clone (ST512) used in this study were previously characterized [22] . Briefly, bacterial identification and drug susceptibility of Kp isolates were tested using the Vitek 2 automated system (bioMérieux, Craponne, France). E. coli ATCC 25922 was used as quality control strain. Susceptibly for colistin was also confirmed using the E-test (bioMérieux, France) according to the manufacturer’s instructions and interpreted using the EUCAST cut-off. Imipenem resistance was defined as an MIC of ≥8 μg/ml, and colistin resistance was defined as an MIC of ≥2 μg/ml. Phenotypic screening for carbapenemases was carried out on all isolates by using the modified Hodge test using disks of meropenem 10 μg and ertapenem 10 μg according to the manufacturer’s instructions. The molecular typing of KPC-type β-lactamase was identified by polymerase chain reaction and sequencing and the clonality of the isolates was investigated by multilocus sequence typing MLST, as previously described [22] . KPC-Kp isolates were obtained from urine (n = 1), wound swab (n = 1) and sputum (n = 1) samples of the patients. KPC-Kp clinical strains belonging to the clone ST512 and showing resistance also to colistin (KPC-Kp Col-R) were isolated from urine (n = 3), wound swab (n = 3) and sputum (n = 3) samples of the patients. K. pneumoniae clinical strains isolated (n = 3) from nonhospitalized patients were identified and characterized for drug susceptibility as above. The demographic and clinical features of the patients are shown in Table 1.
future science group
Klebsiella pneumoniae strains induce infection & cell death in epithelial cells ●●Cell cultures & bacterial infection
●●Immunofluorescence
The human epidermal keratinocyte HaCaT cell line and the human intestinal epithelial Caco2 cell line were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented 10% fetal bovine serum (FBS) and antibiotics. The human lung epithelial NCI-H460 cell line and the human kidney epithelial 786–0 cell line were cultured in RPMI-1640 medium supplemented with 10% FBS. Fresh bacteria previously grown overnight at 37°C in brain heart infusion broth (BD, Diagnostic Systems, Franklin Lakes, NJ, USA) were used for all the experiments. For the bacterial infection, all the different cells were grown on coverslips until approximately 80% of confluence. Before infection, cells were maintained overnight in basal medium without serum or antibiotics and subsequently were infected with the different strains of overnight-grown bacteria normalized by spectrometry at an MOI ranging from 100 to 1000:1 and centrifuged for 4 min at 200 × g at 25°C. Infected plates were then incubated for 2 h at 37°C/5% CO2 in humidified incubator, washed three-times with PBS to remove unbound bacteria and then incubated again for 2 h at 37°C to allow bacterial internalization [2] . Subsequently, the supernatants (SNs) obtained from the different infected epithelial cells were collected and frozen at -80°C until use. For all the experiment, the MOI 1000:1 was chosen because it gave the best yield of infection.
Infected cells were fixed with 4% paraformaldehyde followed by treatment with 0.1 M glycine for 20 min at 25°C and with 0.1% Triton X-100 for additional 5 min at 25°C to allow permeabilization. Cells were then incubated with the following primary antibodies: EpCAM FITC (1:10 in PBS; Miltenyi Biotec, Bergish Gladbach, Germany) and integrin β1 (1:500 in PBS; TS2/16, Santa Cruz Biotechnology Inc., TX, USA). The primary antibody was visualized, after appropriate washing with PBS, by using goat antimouse IgG–FITC (1:50 in PBS; Cappel MP, CA, USA) for 30 min at 25°C. Bacteria and nuclei were stained with propidium iodide (Miltenyi Biotec). Coverslips were finally mounted with Mowiol in PBS for observation. Internalization of KP strains on the different epithelial cells was analyzed by confocal microscopy. The infected cells were observed under an Axioskop 2 microscope equipped with Pascal LSM5 confocal laser scan (Zeiss, Oberkochen, Germany). Fluorescence images from optical sections (thickness
Carbapenem resistance confers to Klebsiella pneumoniae strains an enhanced ability to induce infection and cell death in epithelial tissue-specific in vitro models Laura Leone1, Salvatore Raffa1,2, Daniela Martinelli2, Maria Rosaria Torrisi1,2 & Iolanda Santino*,1,2 Abstract Background: Carbapenem-resistant Klebsiella pneumoniae strains (KPC-Kp) are emerging worldwide causing different nosocomial infections including those of the urinary tract, lung or skin wounds. For these strains, the antibiotic treatment is limited to only few choices including colistin, whose continuous use led to the emergence of carbapenem-resistant KPC-Kp strains resistant also to this treatment (KPC-Kp Col-R). Aim: Very little is known about the capacity of the different strains of KPC-Kp to invade the epithelial cells in vitro. To verify if the acquisition of carbapenem-resistant and the colistin-resistant phenotypes are correlated with a different ability to infect a series of epithelial cell lines of various tissutal origin and with a different capacity to induce cellular death. Materials & methods: We used Klebsiella pneumoniae (KP), KPC-Kp and KPC-Kp Col-R strains, isolated from different patients carrying various tissue-specific infections, to infect a series of epithelial cell lines of different tissutal origin. The invasive capacity of the strains and the extent and characteristics of the cell damage and death induced by the bacteria were evaluated and compared. Conclusion: Our results show that both KPC-Kp and KPC-Kp Col-R display a greater ability to infect the epithelial cells, with respect to KP, and that the bacterial cell invasion results in a nonprogrammed cell death. Klebsiella pneumoniae (KP) is one of the most common Gram-negative bacteria causing communityacquired infections and in particular nosocomial infections [1,2] . Similarly to other Gram-negative bacteria which constitute the normal intestinal flora, KP is able to colonize different districts causing infections of the urinary tract, lung or skin wounds. The intensive use of carbapenem antibiotics, such as imipenem and meropenem, which are recommended as first-line therapy for severe infections caused by Enterobacteriaceae producing extended spectrum β-lactamases (ESBLs), including KP, caused the worldwide emergence in recent years of carbapenem-resistant K. pneumoniae strains (KPC-Kp). From a public health point of view, the spread of KPC-Kp is worrying especially because these bacteria are the source of many hospital-acquired infections and the antimicrobial treatment options are very restricted [3–5] . Antibiotic treatment is limited to a few choices, typically including colistin, tigecycline and one or more aminoglycoside. Recent studies suggest that combination treatment with colistin, tigecycline and meropenem might improve survival among bacteremic patients [6] . Unfortunately, the heavy and increasing use of colistin as important component of the therapeutic treatments has led to the emergence of colistin-resistant KPC-Kp strains (KPC-Kp Col-R) in many countries [7–10] . KP uses different mechanisms to invade and colonize host cells and its virulence factors are multiple, including surface-exposed lipopolysaccharides, iron acquisition systems, adhesins, fimbriae
Keywords
• cell death • epithelial cell lines • infection ability • Klebsiella pneumoniae • KPC-Kp
Department of Clinical & Molecular Medicine, ‘Sapienza’ University of Rome, V.le Regina Elena, 324, 00161 Rome, Italy Azienda Ospedaliera S. Andrea, Rome, Italy *Author for correspondence: Tel.: +39 06 33775488; [email protected] 1 2
10.2217/FMB.14.145 © 2015 Future Medicine Ltd
Future Microbiol. (2015) 10(5), 743–761
part of
ISSN 1746-0913
743
Research Article Leone, Raffa, Martinelli, Torrisi & Santino and the polysaccharide capsule (CPS), which is involved in protection from opsonization and phagocytosis [11,12] . Several strains are characterized by a different ability to adhere and invade the epithelium [13] . It is thought that adherence of the bacterium to the plasma membrane of epithelial cells is a necessary event to induce internalization and colonization in the various districts. In fact, although traditionally considered an extracellular pathogen, recent studies have demonstrated the ability of KP to enter into different human-cultured epithelial cell lines as airway, liver, intestinal and bladder cell lines [2,14,15] . It has been also described that KP invades bladder and ileocecum cells, through a receptor-mediated endocytosis [16] and that the polysaccharide capsule interferes with the internalization of KP [15] . KP internalization was analyzed also in airway epithelial cells in which a different virulence of the strain depending on the level of CPS was observed [2,11] . The bacterium’s ability to enter in host cells is closely related with the capacity to induce cellular death [17] . Recent in vitro studies have shown that KP infections can trigger cell death in epithelial cells. The cytotoxicity seems to depend on the duration time and on the different multiplicity of infection (MOI) [2,14] . The ability to induce a cellular damage seems to be related to death mediators as proapoptotic factors and executioners of programmed cell death [14,18] . Although there are some studies about the interaction between KP and epithelial cells, very little is known about the capacity of KPC-Kp strains to invade the cells in vitro. A few reports suggest an association between resistance to antibiotics, like aminoglycosides and/or β-lactams, and adhesion to human intestinal epithelial cells [19,20] . Regarding the structure of the bacterial membrane and the colistin-resistance, in a recent report [21] , the authors have compared genome sequences of different KPC-Kp strains isolated in patients and have identified mutations in the KPC-Kp genome that are associated with colistin-resistance. Some of these mutations result in aminoacid changes in membrane proteins and seem to alter membrane function possibly reducing drug influx [21] . In the present study, we compared KPC-Kp and KPC-Kp Col-R strains, clinically isolated from patients with different types of infections, with KP in order to verify if the acquisition of colistin-resistant phenotype is correlated in vitro with a different ability to infect epithelial cells
744
Future Microbiol. (2015) 10(5)
and with a different capacity to induce cellular death. To this aim, KPC-Kp and KPC-Kp Col-R strains isolated from urine, wound swab and sputum samples were used to infect colon epithelial cells. Moreover, kidney, lung and skin epithelial cell lines were infected with the KPC-Kp and KPC-Kp Col-R strains isolated from the corresponding tissue sites and the invasive capacity of the different strains and the extent as well as the type of the cell damage induced by the bacteria were evaluated and compared not only on the same cell line but also on epithelial cells of various origins. Materials & methods ●●Bacterial strains
The carbapenem-resistant clinical strains of K. pneumoniae (KPC-Kp) belonging to the sequence type 512 clone (ST512) used in this study were previously characterized [22] . Briefly, bacterial identification and drug susceptibility of Kp isolates were tested using the Vitek 2 automated system (bioMérieux, Craponne, France). E. coli ATCC 25922 was used as quality control strain. Susceptibly for colistin was also confirmed using the E-test (bioMérieux, France) according to the manufacturer’s instructions and interpreted using the EUCAST cut-off. Imipenem resistance was defined as an MIC of ≥8 μg/ml, and colistin resistance was defined as an MIC of ≥2 μg/ml. Phenotypic screening for carbapenemases was carried out on all isolates by using the modified Hodge test using disks of meropenem 10 μg and ertapenem 10 μg according to the manufacturer’s instructions. The molecular typing of KPC-type β-lactamase was identified by polymerase chain reaction and sequencing and the clonality of the isolates was investigated by multilocus sequence typing MLST, as previously described [22] . KPC-Kp isolates were obtained from urine (n = 1), wound swab (n = 1) and sputum (n = 1) samples of the patients. KPC-Kp clinical strains belonging to the clone ST512 and showing resistance also to colistin (KPC-Kp Col-R) were isolated from urine (n = 3), wound swab (n = 3) and sputum (n = 3) samples of the patients. K. pneumoniae clinical strains isolated (n = 3) from nonhospitalized patients were identified and characterized for drug susceptibility as above. The demographic and clinical features of the patients are shown in Table 1.
future science group
Klebsiella pneumoniae strains induce infection & cell death in epithelial cells ●●Cell cultures & bacterial infection
●●Immunofluorescence
The human epidermal keratinocyte HaCaT cell line and the human intestinal epithelial Caco2 cell line were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented 10% fetal bovine serum (FBS) and antibiotics. The human lung epithelial NCI-H460 cell line and the human kidney epithelial 786–0 cell line were cultured in RPMI-1640 medium supplemented with 10% FBS. Fresh bacteria previously grown overnight at 37°C in brain heart infusion broth (BD, Diagnostic Systems, Franklin Lakes, NJ, USA) were used for all the experiments. For the bacterial infection, all the different cells were grown on coverslips until approximately 80% of confluence. Before infection, cells were maintained overnight in basal medium without serum or antibiotics and subsequently were infected with the different strains of overnight-grown bacteria normalized by spectrometry at an MOI ranging from 100 to 1000:1 and centrifuged for 4 min at 200 × g at 25°C. Infected plates were then incubated for 2 h at 37°C/5% CO2 in humidified incubator, washed three-times with PBS to remove unbound bacteria and then incubated again for 2 h at 37°C to allow bacterial internalization [2] . Subsequently, the supernatants (SNs) obtained from the different infected epithelial cells were collected and frozen at -80°C until use. For all the experiment, the MOI 1000:1 was chosen because it gave the best yield of infection.
Infected cells were fixed with 4% paraformaldehyde followed by treatment with 0.1 M glycine for 20 min at 25°C and with 0.1% Triton X-100 for additional 5 min at 25°C to allow permeabilization. Cells were then incubated with the following primary antibodies: EpCAM FITC (1:10 in PBS; Miltenyi Biotec, Bergish Gladbach, Germany) and integrin β1 (1:500 in PBS; TS2/16, Santa Cruz Biotechnology Inc., TX, USA). The primary antibody was visualized, after appropriate washing with PBS, by using goat antimouse IgG–FITC (1:50 in PBS; Cappel MP, CA, USA) for 30 min at 25°C. Bacteria and nuclei were stained with propidium iodide (Miltenyi Biotec). Coverslips were finally mounted with Mowiol in PBS for observation. Internalization of KP strains on the different epithelial cells was analyzed by confocal microscopy. The infected cells were observed under an Axioskop 2 microscope equipped with Pascal LSM5 confocal laser scan (Zeiss, Oberkochen, Germany). Fluorescence images from optical sections (thickness
