Detection of Curli Biogenesis Genes Among Enterobacter cloacae Isolated From Blood Cultures

1Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran 2Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IR Iran *Corresponding author: Bita Bakhshi, Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran. Tel: +98-2182884558, Fax: +98-2182884555, E-mail: b.bakhshi@modares.ac.ir


Background
Enterobacter cloacae is the most commonly isolated species of genus Enterobacter, which has been accepted as the etiologic agent of many infections in hospitalized and enfeebled patients and has been known as a significant bacterial pathogen in recent years (1).E. cloacae are often isolated from nosocomial infections, including pneumonia, urinary tract and bloodstream infections.Particularly, E. cloacae are responsible for 3% -6% of bloodstream infections, with approximate mortality rates ranging from 27% to 61% (2).Curli is a new class of bacterial surface structures which is expressed in E. cloacae, Escherichia coli and Salmonella spp.and is specified by its ability to bind to serum protein fibronectin (3).Curli fimbriae is involved in bacterial adherence to surfaces, cell accumulation and is a significant part of the extracellular matrix essential for the establishment of developed biofilms.Curli fimbriae is also regarded as significant virulence elements as it interact with a wide range of host proteins, which are suggested to help bacterial spreading in the host.These include extracellular matrix proteins and contact-phase proteins.Curli is known by toll-like receptors, leading to activation of the innate immune system.Curli is so regarded pathogen-associated molecular patterns (PAMPs) (4).
An extremely control pathway involving two divergently expressed operons is essential for curli biosynthe-sis.The csgBAC operon encodes the major curli subunit, csgA, and its homolog csgB.The csgDEFG operon encodes csgD, a transcriptional activator of the csgBAC operon, together with csgE and csgF, which act as chaperones and are needed for efficient Curli construction (5,6).Curli homologous have formerly been recognized in some, but far from all genera of the Enterobacteriales (Escherichia, Shigella, Salmonella, Citrobacter and Enterobacter) (4).

Objectives
In the present investigation, we intended to detect the E. cloacae isolates in blood cultures of patients with sepsis and to study the presence of major Curli biogenesis genes; csgD, csgA.

Bacterial Strains
Nine E. cloacae isolates were collected from blood cultures of inpatients admitted to three major academic and governmental hospitals in the Tehran, Iran during December 2012 to November 2013.Samples were systematically and prospectively collected and stored.Confirmation of identity of the infecting organism as E. cloacae was performed with phenotypic identification systems; API20E (BioMerieux, Marcy l'Etoile,France).The results were interpreted with the Analytical Profile Index (API) database of the ApiLab Plus software (api web stand alone V 1.2.1;BioMerieux, Marcy l'Etoile, France).The E. cloacae PTCC 1003 was used as positive control.
The presence of a csgA and csgD genes was determined by PCR using csgA and csgD-specific primers listed in Table 1.Chromosomal DNA was prepared for PCR analysis by boiling method in which fresh bacteria colonies were suspended in distilled water molecular grade and boiled for 10 minutes.The suspension was centrifuged (10 minutes at 10,000 rpm) and the supernatant transferred into a new tube and used directly for PCR assay.

Results
All of E. cloacae strains which were identified by API 20E had the code of 3.305.573.57, and at least 95% identity with E. cloacae species.Sequencing of hsp60 gene and alignment of 341 bp sequence with BLAST software of GenBank database confirmed the identity of isolates.The csgD gene showed a band of expected size (355bp) in all 9 E. cloacae under study (100%).Seven of nine E. cloacae isolates produced 245bp amplification band of csgA gene (77.75%).The result of PCR analyses for csgA and csgD genes is shown in Figure 1.Lane.M: 1 kb ladder, lane.1, positive control of csgA gene and lane 2 positive control of csgD gene, lane3 and 4 negative control of csgA and csgD genes, lanes 6, 8, 10, 12 and 14 csgA gene of strains that isolated from blood culture.Lanes 7, 9, 11 and 13 csgD gene of strains that isolated from blood culture.

Discussion
E. cloacae bacteremia infection is a serious cause of morbidity and mortality in both developing and developed countries, especially Iran (1,8).In a prospective study of Karambin et al. (2010) on 611 newborns admitted with the probable diagnosis of septicemia, 64 (10.6%) cases had positive blood culture and commonest pathogens identified were Enterobacter spp.(78.1%) and Klebsiella spp.(6.2%) (9).In a 2-year retrospective study of Rahbar et al. (2005) on 6,492 patients in various wards, 593 (9.1%) had positive blood cultures, that among which gram-negative bacilli were responsible for 42.3% of isolates and Enterobacter spp. was in the second (10).
All of E. cloacae bloodstream strains in this study harbored csgD gene which shows the wide distribution of this adhesin among blood isolates.Seven of isolates (77.75%) carried csgA gene and its absence in remaining 32.25% of isolates may be probably due to the absence of entire csgA gene or point mutations within its coding sequence which correlates with the primer annealing site.Studies of Barnhart et al. (2006) have shown that mutants of csgA show non-curliated phenotype (11).Bian et al. (2000) showed evidence that curli is expressed in vivo in human sepsis and suggested a possible role for curli and csgA in the induction of proinflammatory cytokines during E.coli sepsis (3).Biesecker (2012) showed a rise within the survival of curliproducing E. coli in vivo, and advised that curli defends against complement killing (12).Therefore, probably isolates of E. cloacae that produce the csgA are more virulant than those with no csgA synthesis and curli product.This may the reason for the extensive presence of csgA and csgD in E. cloacae strains isolated from bloodstream.
In conclusion, this study showed that most of the E. cloacae strains isolated from blood cultures have csg genes and probably are able to express curli and suggest that this virulant factor may play a significant role in invasiveness and pathogenesis of E. cloacae.