Elsevier

Microbial Pathogenesis

Volume 49, Issue 3, September 2010, Pages 83-89
Microbial Pathogenesis

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

https://doi.org/10.1016/j.micpath.2010.04.003Get rights and content

Abstract

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 180–200 bp in DNA extracted from infected cells at 48 h after infection. The results indicated that the bacteria of the E. cloacae complex may adhere to and penetrate into epithelial cells and induce apoptosis, which could be an important mechanism contributing to the development diseases.

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 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 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 (O2) 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.

Section snippets

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

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.

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

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