*1.1.7. Health care Associated Infections (HAI) and biofilm*

Catheter Associated Urinary Tract Infection( CA-UTI) is the commonest (>40%) HAI [41]. Nosocomial bacteriuria or candiduria develops in 25% of patients having urinary catheter for >7 days with a daily risk of 5% [42]. Most infected urinary catheters are covered by a thick biofilm containing infecting microorganisms. A biofilm forms intraluminally or extraluminally or both ways.

With the increasing use of vascular access devices, catheter related bloodstream infection (CR-BSI), septic thrombophlebitis, endocarditis and other metastatic infections e.g.lung abscess osteomyelitis and endophthalmitis etc. are also increasing. In the United States out of 5 million Central Venous cathetes used each year, 3-8% lead to BSI [43]. The initiation of catheter colonization occurs with the formation of a biofilm in the catheter lumen. Moreove the resistance levels of biofilm associated organisms may be much higher than those of planktonic organisms [44]. After stoppage of antimicrobial therapy, the biofilm associated organisms resurge and cause another clinical infectios. A recent approach to reduce CR-BSI is bundles of preventive measures, which means a group of preventive measures, when executed together, result in better outcomes than when implemented alone [45]. This included handwashing, using full barrier precautions during insertion of central venous line, cleaning the skin with chlorhexidine. The femoral site should be avoided if possible and catheters should be removed as early as possible.

Hospital acquired pnumoniais are the second most common cause of HAI and has the highest morbidity and mortality of all HAIs [46]. The initial step in pathogenesis of HAP is colonization of patient's oropharynx with resistant hospital pathogen. The endotracheal tube lumen is a nidus for the growth of bacteria within the biofilm. Hand washing and Personal protective equipment (PPE) must be used to reduce the incidence of HAP/ Ventilator assaociated pneumonia (VAP).

#### *1.1.8. Resistance of biofilm to antimicrobials and disinfectants*

It has been observed that biofilms are not easily eradicated even by cidal antimicrobials, quarternary ammonium compounds, halogens and halogen release agents. The crux of the problem is the presence of persisters within the biofilms that can rebound when antibiotic concentration falls. The causes are multifactorial – i) restricted penetration of antimicrobials within the biofilm architecture, ii) decreased growth rate of bacterial cells forming the biofilm, iii) expression of resistance gene by the bacterial cells within the biofilm etc [47]. Restricted penetration of antimicrobials may occur as negatively charged exopolysaccharide restrict permeation of positively charged antibiotics e.g. Aminoglycoside and exopolymer matrix also

restrict diffusion of antimicrobial within the biofilm. Synergy between retarded diffusion and degradation by enzymes (e.g. β-lactamase) also provide effective resistance to antimicrobials. Fluoroquinolones are very effective in stopping the growth of a biofilm but restricted diffusion can protect the microbial cells within the biofilm [48]. All antimicrobials are more effective in killing rapidly growing cells. Penicillin & ampicillin do not kill non-growing cells as rate of killing is directly proportional to rate of growth for these two antibiotics. Even cephalosporins, aminoglycosides & fluroquinolones can kill rapidly dividing cells more effectively. Multiple drug resistance (MDR) pumps may play a role in biofilm resistance at low antibiotic concen‐ tration. Sometimes unknown MDR pumps might be over expressed in biofilm e.g. for chlor‐ amphenicol in *E. coli* biofilm. Moreover, the biofilms increase the opportunity of gene transfer beteen the microorganisms and can convert a previously avirulent commensal organism to a highly virulent pathogen. The enhanced efficiency of gene transfer in biofilms also fascilitates the spread of antibiotic resistance and virulence factors [49]. Though most of the research works deals with single species biofilms, multispecies biofilm amongst different bacteria and interkingdom biofilms between fungus Candida albicans and various bacterial species are also gaining importance in causing different diseases [50]. Biofilm formation is a major virulence factor for Candida albicans and Candida biofilms are difficult to eradicate due to their high resistance to antifungals. A recent study has reported that within the biofilm Staphylococcus aureus was attached uniquely with the pseudohyphae of Candida albicans. This synergistic interaction resulted in differential protein expressions which are actually virulence factors for Staphylcoccus aureus. This indicate C. albicans may enhance S.aureus pathogenesis [51]. Recently it has been reported that co-existence of S.aureus and C.albicans in a biofilm resulted in increased Vancomycin resistance in S.aureus [52] However antagonistic interaction has been reported between *Pseudomonas aeruginosa and Candida albicans* [53].

It is not possible to detect the antimicrobial resistance of biofilms by conventional methods of disc diffusion and broth microdilution as per CLSI guideline because these methods are only meant for planktonic cells.

#### *1.1.9. Biofilms and altruism*

*In S. epidermidis* biofilm initial adherence is by polysaccharide adhesin (PSA) and accumulation of cells is due to production of polysaccharide intercellular adhesin (PIA).) PIA is encoded by *ica* (intercellular adhesin) operon *ica* ADBC [39]. The *icaR* gene regulates *ica* operon. Production of PIA is also subject to ON - OFF switching (phase variation). Majority of clinical isolates of

Catheter Associated Urinary Tract Infection( CA-UTI) is the commonest (>40%) HAI [41]. Nosocomial bacteriuria or candiduria develops in 25% of patients having urinary catheter for >7 days with a daily risk of 5% [42]. Most infected urinary catheters are covered by a thick biofilm containing infecting microorganisms. A biofilm forms intraluminally or extraluminally

With the increasing use of vascular access devices, catheter related bloodstream infection (CR-BSI), septic thrombophlebitis, endocarditis and other metastatic infections e.g.lung abscess osteomyelitis and endophthalmitis etc. are also increasing. In the United States out of 5 million Central Venous cathetes used each year, 3-8% lead to BSI [43]. The initiation of catheter colonization occurs with the formation of a biofilm in the catheter lumen. Moreove the resistance levels of biofilm associated organisms may be much higher than those of planktonic organisms [44]. After stoppage of antimicrobial therapy, the biofilm associated organisms resurge and cause another clinical infectios. A recent approach to reduce CR-BSI is bundles of preventive measures, which means a group of preventive measures, when executed together, result in better outcomes than when implemented alone [45]. This included handwashing, using full barrier precautions during insertion of central venous line, cleaning the skin with chlorhexidine. The femoral site should be avoided if possible and catheters should be removed

Hospital acquired pnumoniais are the second most common cause of HAI and has the highest morbidity and mortality of all HAIs [46]. The initial step in pathogenesis of HAP is colonization of patient's oropharynx with resistant hospital pathogen. The endotracheal tube lumen is a nidus for the growth of bacteria within the biofilm. Hand washing and Personal protective equipment (PPE) must be used to reduce the incidence of HAP/ Ventilator assaociated

It has been observed that biofilms are not easily eradicated even by cidal antimicrobials, quarternary ammonium compounds, halogens and halogen release agents. The crux of the problem is the presence of persisters within the biofilms that can rebound when antibiotic concentration falls. The causes are multifactorial – i) restricted penetration of antimicrobials within the biofilm architecture, ii) decreased growth rate of bacterial cells forming the biofilm, iii) expression of resistance gene by the bacterial cells within the biofilm etc [47]. Restricted penetration of antimicrobials may occur as negatively charged exopolysaccharide restrict permeation of positively charged antibiotics e.g. Aminoglycoside and exopolymer matrix also

*S. aureus* also possess *ica* structural genes [40].

or both ways.

62 Infection Control

as early as possible.

pneumonia (VAP).

*1.1.7. Health care Associated Infections (HAI) and biofilm*

*1.1.8. Resistance of biofilm to antimicrobials and disinfectants*

Biofilms are like small cities and encourage altruism. Microbial cells within biofilm often sacrifice their maximum growth rate to use the available community resources more efficient‐ ly. In a biofilm atleast some of the microbial cells experience nutrient limitation and exist in a slow growing state [54]. In this process while individual cells are disadvantaged, the microbial community as a whole is benefited. Hence, it is said that biofilms are the colonial way of life of microorganisms.

Detection of biofilms can be done by both phenotypic methods and genotypic methods. In phenotypic methods biofilms are detected by Congo red agar method(CRA),Plastic tube method(TM), Tissue culture plate method(TCP) and Confocal Laser Scanning Microscopy (CLSM). In genotypic method, usually Polymerase chain reaction (PCR) for amplification of microbial DNA, coding for biofilm formation is done. The phenotypic methods are easy and cheap compared to genotypic method.

Hence, the present study was undertaken to detect the biofilm producing organisms, isolated from different clinical specimens in our laboratory.

**12.** Then the optical density of each well containing 125μl solution was measured at a wavelength of 545nm in an ELISA reader. As each strain was put in triplicate the average

Biofilms: A Challenge to Medical Fraternity in Infection Control

http://dx.doi.org/10.5772/55649

65

The biofilm formation of different strains were classified in three groups according to the cut off OD. The cut off OD (ODc) for the microtiter plate test was defined as three standard deviations above the mean OD of the negative control. Isolates were classified into four groups as nonadheremt, weakly adherent, moderately adherent and strongly adherent according to

Out of 350 bacterial strains studied, 90 were *Pseudomonas aeuginosa*, 80 were *E. coli*, 35 were *Klebsiella pneumoniae*, 80 were *Coagulase positive Staphylococci*, 30 were Coagulase negative 35 included Proteus sp(5), *Vibrio cholerae*(3), *Acinetobacter baumanii*(4), Enterococcus sp.(23). Out of 50 Candida strains 23 were *Candida albicans*, 16 were *Candida tropicalis*, 2 were *Candida dubliensis*, 6 were *Candida krusei* and 3 were *Candida glabrata*. Amongst 350 bacterial strains, 153(43.7%) and out of 50 *Candida* species 28(56%) were biofilm producers respectively. Amongst 50 Candida species, 11 (22%) were strong biofilm producers, and 6/11 (54.5%) were

of the three readings were taken.

Stepanovi et al [57].

*Candida albicans*.

**3. Observation and results**

**Figure 1.** Microtitre plate biofilm assay for detection of microbial attachment

Maximum 65( 72.2%) of *Pseudomonas aeruginosa* strains produced biofilms. 51(33.3% ) biofilm producing bacterial strains were isolated from catheterized urine samples or patients having
