Antimicrobial susceptibility of Clostridium difficile isolated from different sources of Imam Reza Hospital, Tabriz

The aim of this study was to determine antibiotic susceptibility of Clostridium difficile isolated from different sources. Two hundred stool samples of staff, patients (at admission to the wards and the same patients after seven days of hospitalization), and 135 samples of hospital environment were collected. Three standard methods including direct plating onto cycloserine-cefoxitin fructose agar, alcohol shock and enrichment culture with 0.1% sodium taurocholate were used to isolate C. difficile. All C. difficile isolates identified by biochemical tests and were tested by disk diffusion agar using 15 antibiotic disks. MIC of isolates was determined for vancomycin and metronidazol by Etest. Seventy C. difficile were isolated from different sources. No resistant isolates to vancomicin and metronidazol were detected by disk diffusion or Etest. The rate of recovery by 0.1% sodium taurocholate enrichment method and alcohol shock was significantly higher than those by CCFA (pv 0.02, pv 0.04).


INTRODUCTION
Clostridium difficile is a Gram-positive spore forming strict anaerobic bacteria and is considered as the main etiological agent of hospital aquired diarrhoea. In the hospital environment, spores resistant to acid, chemical, and physical factors are common contaminants from hospital staff and patients who harbour C. difficile (Fawley and Wilcox, 2001). Approximately 20% of individuals who are hospitalized acquire C. difficile during hospitalization by different routes including healthy staff carrier, and more than 37% of these patients develop diarrhoea after antibiotic therapy has made the bowel susceptible to infection (McDonald et al., 2006;Macfarland et al., 1989).
The severity of infection ranges from self-limiting diarrhoea to life threatening pseudomembranous colitis *Corresponding author. E-mail: t_pirzad@yahoo.com. Tel/fax: 98-411-3364661. (Mobile): 09141014516.1 (PMC), accounting for 15 to 25% of antibiotic-associated diarrhoea without colitis and 90 to 100% of pseudomembranous colitis associated with antibiotic use (Poxton et al., 2001;Bartlett, 1994). While treatment of asymptomatic carriers is not recommended, patients with more serious infections may require antibiotic therapy immediately. Susceptibility testing is rarely performed for C. difficile because of complexity and cost. Vancomycin and metronidazole are still the two primary antibiotics used in the treatment of C. difficile infection (Noren, 2010). Although susceptible C. difficile to vancomycin and metronidazole have been reported from many countries (Huang et al., 2009) but resistant and reduced sensitivity strains of this bacterium to these clinically important antibiotics have also been isolated (Pelaez et al., 2008(Pelaez et al., , 2005Dworczynski et al., 1991).
The aim of the present study was to determine recovery rate of C. difficile from different sources by three methods and to find out susceptibility patterns of C. difficile isolated from different sources.

Hospital setting
The Imam Reza hospital is situated in Tabriz city with 520 beds in 26 clinical wards, providing services for two thousand patients monthly. The hospital environment is regularly cleaned with detergent, water, and disinfectants such as Descocide by special team. Tables are usually cleaned with big spray. Hand washing with soap and water is the most common way for staff hand hygiene but Manorapid is also used. All disinfectants used are produced in Germany, the Anticeptica chem. Pharm. GmbH.

Collection of specimens
From 22 December 2009 to 20 April 2010, ten out of 26 wards of Imam Reza hospital including, nephrology, gastroenterology, pulmonology, endocrine and rheumatology, neurology, infectious disease, ICU of neurology, ICU of neurology surgery, ICU of general surgery and ICU of pulmonary were selected based on the length of patients hospitalization.
One hundred stool samples were collected from staff who had been working for at least three years in these wards. Hundred stool samples were obtained from 50 consecutive patients at first day of admission to these wards and from the same patients after seven days of hospitalization (each patient two faeces). One hundred and thirty-five samples were collected from different regions (100-cm 2 areas) of the hospital wards comprising patients' room floors (n = 30), beds (n = 30), doorknobs (n = 30), corridors (n = 10), toilets (n = 16) and keyboard of computers (n = 19) with thioglycolate moistened sterile cotton wool swabs in to 1 mL thioglycollate.

RESULTS
Out of 70 C. difficile isolates which were cultured as a first time in north west of Iran, 18% (18/100), 10.37% (14/135), 32% (16/50) and 44% (22/50), were isolated from Staff, hospital environment, patients at first day of admission and the same patients after seven days of hospitalization respectively. Six patients (12%) were colonized by C. difficile during seven days of hospitalizations. Fourteen isolates (10.37%) of C. difficile was obtained from various region of pulmonary (n = 5), infectious disease (n = 3), ICU of neurology (n = 3), ICU of pulmonary (n = 2) and endocrine and rheumatology (n = 1) wards. C. difficile was recovered from 11.1, 38.9, and 50% of the specimens by using CCFA, alcohol shock and 0.1% sodium taurocholate enrichment methods, respectively. The rate of recovery by 0.1% sodium taurocholate enrichment method and alcohol shock was significantly higher than those by CCFA (pv 0.02, pv 0.04). There was no significant difference between alcohol shock and sodium taurocholate enrichment methods (PV 0.39). All isolates were found to be susceptible to piperacillin / tazobactam, metronidazole, imipenem, chloramphenicol, amoxicillin + clavulanic acid, vancomycin, ampicillin and resistant to ciprofloxacin, gentamicin and colistin. The isolates were susceptible to cefotaxime (85.7%), erythromycin (87.1%), rifampin (74.2%), tetracycline (41.4%), and clindamycin (32.8%). The MIC of vancomycin varied from 0.19 to 2 µg/mL, whereas that of metronidazole varied from 0.38 to 2 µg/mL. Seventeen (24.28%) and 53 (75.72%) isolates were resistant to three and four or more antibiotics, respectively. Phenotypic typing of 70 C. difficile isolates based on seven resistance patterns are shown in Table 1.

DISCUSSION
The most commonly drugs used to treat diseases associated with C. difficile are metronidazole and vancomycin. Most clinical laboratories assume that all C. difficile strains are susceptible to metronidazole and vancomycin. However, in Spain a 6.3% rate resistance to metronidazole (16 µg/mL) among isolates of C. difficile was reported (Pelaez et al., 2002). Wong  HIV-infected patients. The new antibiotics against C. difficile with reduced susceptibility to vancomycin were also tested (Pelaez et al., 2005). In 1991, the first isolate of C. difficile not susceptible to vancomycin appeared in Poland but the study was performed by a disk diffusion method (Dworczynski et al., 1991). The first isolates of C. difficile not susceptible to vancomycin appeared in Spain in 1996, although none showed full resistance to these agents (Pelaez et al., 2002). On the other hand, among 193 C. difficile strains isolated in Poland between 1998 and 2003, resistance to metronidazole and vancomycin was not observed (Pituch et al., 2005). All C. difficile isolates were reported to be sensitive to vancomycin and metronidazole (Poilane et al., 2007). Pituch et al also did not report resistance to vancomycin and metronidazole in 2003. In our study, all isolates were found to be susceptible to metronidazole and vancomycin by both Etest and disc diffusion methods but prevalence of resistant to other antimicrobial agents is highly variable like other studies in different populations and different countries, from 0 to 100% (Poilane et al., 2007(Poilane et al., , 2008Huang et al., 2009;Cattoir et al., 2008). In this research a total of seven different antibiotic resistant patterns were identified, among which type P3 was the commonest type overall and was isolated from each group of samples (41.43%), followed by resistance type P2 (24.28%), P4 (10%), P6 and P1 (7.15%), P5 (5.72%) and P7 (4.28%) ( Table 1), indicating that, transmission and colonization of C. difficile could happen among patients, staff and environment but the severity of the infection will depends on the own patient's vulnerability to infection.
Among patients who are being hospitalized, some new strains are usually introduced by colonized patient and transmitted to other patients or to the environment, thus, colonized and infected patients are an important reservoir of C. difficile (Fawley and Wilcox, 2001;Titov et al., 2000). Although it can occasionally spread through the air (Best et al., 2010), the main type of transmission is via the hands of hospital staff. Our results showed that, 32% of newly hospitalized patients and 18% of staff were healthy carrier in this hospital. Indiscriminate consumption of antibiotics by patients and people who can easily buy any antibiotics without physician's prescription can be the main factor in overgrowth of C. difficile in their bowels, while most of staff are aware of antibiotic side effects and avoid using antibiotics freely. In addition 10.37% of environmental samples were contaminated with this bacterium, thus, 12% of hospitalized patients were colonized during seven days staying in the various wards. Although there is minimal literature regarding the transmission of C. difficile from hospital staff to patients, and none looking solely at doctors but the hands of hospital personnel caring for patients with C. difficile often become colonized with the organism, facilitating transmission among hospital inpatients (Macfarland et al., 1987). In one study, the risk of colonization was found to increase in direct proportion to the length of hospital stay, ranging from 13% among patients admitted for less than 1 week to as high as 50% among patients admitted for more than 4 weeks; this suggests that ongoing exposure to C. difficile occurs throughout the hospital stay (Conly, 2000). Rotimi et al in 2002 in Kuwait reported acquisition rate of C. difficile from 5.9 to 36% during 4 to 53 days of hospitalisation in various wards. The reason for low rate of colonization (12%) in this research could be the kind of wards that had been studied.
The enrichment broth was chosen to provide the optimal recovery of C. difficile spores, based on the premise that spores would be the main form of C. difficile to survive suboptimal handling conditions. Bile salts such as sodium cholate and taurocholate have previously been shown to enhance the recovery of C. difficile by facilitating spore germination (Buggy et al., 1985) and may be particularly useful when this procedure is followed by alcohol shock method, especially if samples are handled under suboptimal anaerobic conditions (Marler et al., 1992). In this study the rate of recovery by 0.1% sodium taurocholate enrichment method and alcohol shock was significantly higher than those by CCFA (pv 0.02, pv 0.04) as several studies have shown nearly the same results (Arroyo et al., 2005;Marler et al., 1992).