The relationship between biofilm formation and presence of fimH and mrkD genes among E. coli and K. pneumoniae isolated from patients in Mosul

Background and objectives: Biofilms have a major medical significance as they decrease susceptibility to antimicrobial agents. The decreased susceptibility to microbial agents within a biofilm arises from multiple factors, including physical impairment of diffusion of antimicrobial agents, reduced bacterial growth rates, and local alterations of the microorganisms (M.O) that may impair activity of the antimicrobial agent. The aim of the study was to investigate the biofilm formation among coliforms and study the relationship between biofilm formation and the presence of fimH and mrkD genes among E. coli and K. pneumonia isolates respectively . Materials and method: One hundred and seventy three clinical samples were collected from both gender who visited or admitted to ALSalam General Teaching Hospital and ALWafa‘a center for diabetic patients in Mosul city from April 2013 to February 2014, (110) urine samples from patients suffering from urinary tract infections (UTIs) and (63 ) from patients with diabetic foot infections (DFIs). All samples were cultured on selective media as MacConkey and blood agar aerobically. Coliform isolates are identified to species level depending on biochemical and physiological tests and using Rapid TM ONE panel kit to be conformed to the diagnosis. The ability of coliforms spp. for biofilm formation was assessed using qualitative and quantitative assay. The Congo red agar (CRA) method was used in the qualitative biofilm assay. Results: Out of (85) bacterial spp. , 44(51.8%) species were biofilm producers. The tissue culture plate (TCP) method was used in the quantitative biofilm formation assay. The results showed that 46(54.1%) spp. produced biofilm strongly. All biofilm producing E. coli and K. pneumoniae spp. which gives strong biofilm formation by CRA method and non-biofilm producer spp. were subjected to PCR for determining the relation between biofilm formation and the presence of fimH gene in E. coli isolates and mrkD gene in K. pneumoniae isolates. The results revealed that all biofilm producing E. coli 15(100%) isolated from UTI samples were positive for fimH gene. While all biofilm producing K. pneumoniae 9(100%) and 5(100%) isolated from UTIs and DFIs respectively were positive for mrkD gene.


INTRODUCTION
Infection caused by pathogenic E. coli and other coliforms spp. are often initiated by binding of the bacteria to the host cell surface via specific bacterial adhesins with fimbriae . Fimbrial adhesins are thread-like structures that reach out from the bacterial surface enabling bacteria to adhere to host cells ( Heydari et al., 2013 ). Most clinical isolates of coliforms express type 1 (mannose-sensitive) and type 3 (mannose-resistant) fimbrial adhesins. Among adhesions of uropathogenic E. coli ,the adhesive subunit of type 1 fimbriae ,FimH, is a major determinant ,which has high tropism for urinary tract receptors ;thus, FimH adhesion is important in colonizing different niches of E. coli ( Hojati et al., 2015 ). Type 3 fimbrial adhesins are able to mediate the binding of K. pneumonia to various human cells , such as endothelial cells , epithelial cells of the respiratory tract and urinary tract . MrkD protein is an important factor in binding of the microorganism to collagen molecules (Melo et al.,2014 ).
The presence of adhesins (fimH and mrkD) related to biofilm formation were also investigated (Bellifa et al., 2013 andJarjees , 2014). Different microorganisms combine together to form micro-communities within a matrix of extracellular polymeric substances (EPS) and are called biofilm . The ability of a microorganisms to form biofilm is an important virulence factor and such biofilms are the main cause of many chronic infections ( Swarna et al., 2012 ). Biofilm formation is common in chronic wounds such as diabetic foot infections (DFIs), and urinary tract infections (Zubair et al., 2011 andPoovendran andRamanathan , 2014).
Biofilms have a major medical significance as they decrease susceptibility to antimicrobial agents. The decreased ___________________________________________________________________________________________________________________________________________________________ * PhD Candidate / College of Nursing / University of Mosul. ** Professor / College of Science / University of Mosul. susceptibility to microbial agents within a biofilm arises from multiple factors , including physical impairment of diffusion of antimicrobial agents , reduced bacterial growth rates , and local alterations of the microorganisms(M.O) that may impair activity of the antimicrobial agent (Pramodhini et al., 2012). The aim of the study was to investigate the biofilm formation among coliforms and study the relationship between biofilm formation and the presence of fimH and mrkD genes among E. coli and K. pneumonia isolates respectively .

MATERIAL AND METHOD Isolation and identification of coliforms bacteria
One hundred and seventy three clinical samples were collected from both gender who visited or admitted to AL-Salam General Teaching Hospital and AL-Wafa'a center for diabetic patients in Mosul city from April 2013 to February 2014 , (110) urine samples from patients suffering from urinary tract infections (UTIs) and (63 ) from patients with diabetic foot infections (DFIs). All samples were cultured on selective media as MacConkey and blood agar aerobically. Coliform isolates are identified to species level depending on biochemical and physiological tests and using Rapid ™ ONE panel kit to be confirmed to the diagnosis.             Nursing Journal, Vol.3, No.1, 2015 Biofilm formation was conducted by growing bacteria isolates in a flat bottom tissue culture plates ( 96 well ) as previously described by Zubair et al ., ( 2011 ). Biofilm production was estimated based on the Absorbance 570 nm as in (Table 1).

Phenotypic detection of the type 1 and 3 fimbriae among coliforms
A suspension of human blood and PBS was mixed on a clean microscopic slide with a single colony of fresh bacterial cultures . The slide was gently rotated until agglutination was visible . Agglutination assays were performed in the presence or absence of (5%) mannose ( Stahlhut et al ., 2012 ) . The phenotypic expression of type 1 fimbriae was measured by the occurrence of mannose-sensitive hemagglutination (MSHA), while expression of type 3 fimbriae was detected as mannoseresistant hemagglutination ( MRHA ) , which not inhibited by addition of mannose (Li et al ., 2009)

DNA extraction
The total DNA ( chromosomal and plasmid ) was extracted from all isolates using Wizard Genomic DNA purification kit supplemented by (Promega \ USA) according to manufacture instructions. The purity and concentration of genomic DNA were measured using Biodrop spectrophotometer. The isolated DNA was checked by (0.7%) agarose gel electrophoresis and visualized by exposed to UV light using UV transilliuminator.

Primers used
Primers sequences were taken from previous articles( Table 2) . All primers were synthesized by Alpha DNA company , Canada .

Polymerase chain reaction ( PCR ) assay PCR reaction
All PCR reactions were performed in 25μl volumes in Eppendorf tube (Table 3) . Laminar flow cabinet with UV lamp was used for the preparation of reaction mixture , all the reaction components were prepared separately in ice and used with optimum concentration . Detection of the type 1 pili encoding gene PCR conditions program depending on Karimian et al ., ( 2012 ) , was used for detection of fimH gene (Table 4). After the reaction was complete , the PCR products were visualized by exposed to UV light using UV transilliuminator , then photographed by using digital camera .

Detection of the type 3 pili encoding gene
Amplification of mrkD gene was done by using PCR program depending on Melo et al ., ( 2014 ) , as shown in (Table 5 ).

Statistical analysis
All statistical analysis was conducted using the Statistical Package for Social Sciences (S.P.S.S.) version 19 from IBM Company, USA. The x2 test was used for statistical comparison of groups , values < 0.05 were regarded as significant .
The result showed that the isolated bacteria belonged to five genera ; the number and percentage of coliforms isolated from UTIs and DFIs are listed in Table (

Biofilm formation among coliform bacteria
Several methods of biofilm formation have been developed over the years using numerous types of M.O. for different applications (Agostinho et al., 2011). The ability of each coliform bacteria isolated from UTIs and DFIs into biofilm formation was assessed using qualitative and quantitative assay . The Congo red agar (CRA) method is used in the qualitative biofilm assay. The results showed that out of (85) bacterial isolates , 44 (51.8%) isolates were biofilm producers which showed black colonies with crystalline appearance. While 41 (48.2%) isolates gave pink colonies indicating nonbiofilm production as in (Table 7 and Figure 1).

Detection of the type 1 fimbriae gene (fimH) and the type 3 fimbriae gene (mrkD) in biofilm and non-biofilm producing species.
In the present study , the most frequent biofilm and non-biofilm producing coliforms (E. coli and K. pneumoniae) , which gives strong biofilm formation by CRA method , were subjected to PCR for determining the relation between biofilm formation and the presence of fim H gene in E. coli isolates , and mrkD gene in K. pneumoniae isolates . Moreover, type 1 fimbria (MSHA) , and type 3 fimbriae (MRHA) were investigated phenotypically as in (Tables 9  and 10).
The fimH gene was amplified using specific primers and appeared as a band of (559 bp) on agarose gel as shown in (Figures 3 and  4). It was found that all biofilm producing E. coli 15(100%) isolated from UTI samples were positive for fimH gene, while only 9(62.2%) non-biofilm producing isolates were carried fimH gene. Regarding E. coli isolated from DFI samples, only 7(70%) biofilm producing E. coli isolates had fimH gene .The prevalence of fimH gene was well studied by many researcher in different countries. Locally, Jarjees (2014) found that (93%) of UPEC isolates were positive for fimH gene in the study of patients with UTI , attended at the Hospitals of Erbil city / Iraq. The fimH gene was( 65.9%) positive in UPEC isolated from Egyptian childrens. Hojati et al, (2015) described the presence of fimH gene in E. coli strains isolated from hospitalized and nonhospitalized patients with UTIs as (92.8%) in the Iranian study. Also, Arabi et al (2012) investigated the frequency of fimH and other adhesion genes in UPEC isolated from some private clinical laboratories located in Tehran / Iran , and determined the fim H gene frequency as( 87.7%) . Another study conducted in Malaysia by Lai et al,(2015) revealed that (92.5%) of UPEC isolates were positive for fimH gene . The type 1 fimbriae is commonly found among UPEC as well as non-UPEC strains (Heydari et al., 2013). However, in this study the fimH gene was detected in E. coli isolated from patients with DFIs . But, we did not found any previous study about the relation between the presence of fimH gene and biofilm formation in patients with DFIs to compare our results.
In the present study, there was a relation between biofilm formation and the presence of fimH gene as shown in (Table 9). Recently, Cargole-Novella (2015) reported that biofilm formation by extraintestinal E. coli can be associated with the expression of different adhesins such as type 1 fimbriae, and there was significant correlation between biofilm formation on glass and the presence of fimH gene . Furthermore, Moreira et al,(2006) found that adhesive structures , such as type 1 fimbriae (fim H) was expressed during the initial stages of biofilm development .
As shown in (Table 9). The presence of type 1 fimbriae (MSHA) was identified in all biofilm and non-biofilm producing E. coli phenotypically by MSHA assays . In our results type 1 fimbriae were detected in (73.3%) and (46.2%) of biofilm and non-biofilm producing E. coli respectively . While, only (60%) and (42.9%) of biofilm and non-biofilm producing E. coli isolated from DFIs respectively were positive for test. Some isolates did not give any agglutination , they may loss their fimbriae during culture process, or some strains of bacteria lacking these fimbriae ( Madigan et al., 2000 ). Another study conducted by Najar et al, (2007) found that type 1 fimbriae (MSHA) was seen in (71%) of isolates of UPEC and this finding is similar to our results.
Various fimbrial adhesins have been shown to play a role in biofilm formation . Most K. pneumoniae isolates express type 3 fimbrial adhesin ( Bandeira et al., 2014 ). In this study, the mrkD gene was amplified by PCR and appeared as a band of 228bp on agarose gel. Electrophoresis of mrk D gene is shown in (Figures 5 and 6).
Many studies have revealed that type 3 fimbriae (mrkD gene) are important in K. pneumoniae biofilm formation (Jagnow and Clegg , 2003 ;Ong et al., 2010 andBellifa et al., 2013). Therefore, the mrkD gene may play significant roles in forming biofilm . In the present study, all biofilm and non-biofilm producing K. pneumoniae were examined for the presence of mrkD gene . It was found that all biofilm producing K. pneumoniae 9(100%) and 5(100%) isolated from UTIs and DFIs respectively were positive for mrkD gene, thus, demonstrating a relationship between biofilm formation and the presence of mrkD gene as in (Table10). Same the fimH gene , the mrkD gene was also detected in K. pneumoniae isolated from DFIs . But, we did not found any previous study about the relation between formation of biofilm and the presence of mrkD gene to compare our results . However, the presence of type 3 fimbriae (MRHA) was identified in all K. pneumoniae isolates phenotypically. As shown in (Table 10). not all isolates show positive mrkD gene by PCR gives positive test by MRHA assays, they may lose their fimbriae during culturing, or some isolates lacking these fimbriae as mention later in fimH gene detection.We can conclude from the results that type 1-encoding gene (fimH), and type 3-encoding gene (mrkD) are associated with biofilm formation in coliforms spp.