Characterization of Bacterial Strains and their Resistance Status in Hospital Environment

Characterization of Bacterial Strains and their Resistance Status in Hospital Environment Meriem EL Bakkali1,2, Khadija Hmid3, Khalid El Kari2, Mimoune Zouhdi3, Mohammed EL Mzibri2 and Amin Laglaoui1* 1Equipe de Recherche Biotechnologies et Génie des Biomolécules (ERBGB) FSTTanger, Morocco 2Unité de Biologie et Recherche Médicale, CNESTEN-Rabat, Morroco 3Service de Microbiologie de l’Hôpital Ibn Sina de Rabat, Morroco


Introduction
Worldwide, the control of the hospital environment is a key of success of health care quality [1]. However, the increasing emergence and spread of pathogenic bacteria, without distinguishing between Gram-positive and Gram-negative organisms, in hospitals is of great concern and continues to challenge infection prevention and hospital epidemiology practice [2,3]. Hospital environments are responsible of the dissemination of microorganisms for different distances and progressive contamination of various supports, including surfaces, hands [4][5][6][7], air and water [8], and constitute therefore a major source of infections. Environmental microorganisms like Legionella pneumophila, E. coli, Mycobacterium xenopi or acynobacter, were the principal causes of serious recent epidemic infections, by their easier access to sterile body sites. They were the origin of increased lengths of hospital stay, severe illness, death and increased care cost [9,10]. Pathogens were found in all hospital units but the interest was usually focused on intensive care and surgery units, especially due to the vulnerability of patients in these units [11,12]. Health care professionals have established rigorous isolation guidelines; which give current recommendations comprising an infection control plan of the general measures to prevent and limit pathogen dissemination. That may provide an effective way to standardize and increase reliability the criteria of application of infection control methods in hospitals [13,14]. During the last decades, a bleaker picture has emerged with the appearance of multi-drug resistant pathogens, especially resistant strains to new generations of antibiotics, raising concerns of a future epidemic of virtually untreatable infections [9,15]. Micro-organisms in the inanimate hospital environment are said to contribute only negligibly to endemic nosocomial infections [16,17]. However, it is still difficult to document the relationship between the contamination of the hospital environment and nosocomial infections, given the difficulty of capturing such data and the limited studies made in this way, with the exception of some studies covering some co-morbidities, associated to Legionella sp, Aspergillus sp, atypical Mycobacteria or Staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterobaciteria (VRE) [3,18]. Even more, nosocomial infections is the second most common cause of death after cancer diseases, with around 1.4 million newly infection cases and over 500 000 deaths estimated to occur annually in the European Union [19]. In Morocco, as other developing countries, data is limited to the number of cases registered in some medical centers, like Ibn Sina hospital in Rabat, which highlighted the presence of pathogens in distinct areas of the hospital environment like Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella sp and various Gram-negative bacilli. Therefore, the present study is planned to evaluate the rate of pathogen bacteria in the environment of the university hospital Ibn Sina in Rabat and to characterise their resistance status for better management of the hospital environment quality.

Patients
A total of 139 patients admitted for hospitalisation at the Central university hospital Ibn Sina in Rabat in January 2010 were recruited. These patients were selected among patients developing nosocomial infections occurring 48 hrs post admission, according to the definitions described by the Centers for Disease Control [20]. The specimen sources included blood, urine, cerebrospinal fluid, pleural space, respiratory tract (collected during bronchoscopy or endotracheal suction), tip of central venous catheters, bedsores and surgical incision sites. All specimens were collected at the bed site, transferred to the laboratory immediately for microbiological analysis and were inoculated on proper culture media within two hours.

Environmental sampling
Sampling was performed on hands of hospital personnel and admitted patients before disinfection. Environmental sampling was performed during the morning after the regular daily cleaning. The date, time, conditions and sites of sampling were noted. Different surfaces and locations were included (Bed covers, bed rails, masks,  oxygen bubbler, carriage of care, laboratory bench, bedside table,  surgical operation table, breathing tubes, infusion pump, aspirators, baby incubators, wash-hand basins, water taps, shower fitting, door handles, floors and others). Basically, 20 swabs were used, at least, for each sampling site, giving a total of 470 samples. For sampling, sterile polyester fibre-tipped applicator swabs (Becton Dickinson, Basel, Switzerland) were moistened in 2 ml sterile saline solution and rolled several times over a surface area of around 25 cm 2 .

Bacteriological testing
Spits were inoculated onto the Chocolate enriched or bacitracin agar in 5% CO 2 , and for Neisseria sp, the 5% sheep blood agar was used. Blood was injected into two or more "blood bottles" with specific media for aerobic and anaerobic organisms and sub cultured onto 5% sheep blood agar for Streptococcus pneumoniae, Chapman agar plates for Gram-positive staphylococcus, DCL Desoxycholate citrate lactose agar plates for Gram-negative bacteria and chocolate agar for exigent bacteria. Cerebrospinal fluid was inoculated onto Chocolate enriched / bacitracin agar in 5% CO 2 . The urine specimens were inoculated onto phosphate buffered saline agar, Cystine Lactose Electrolyte Deficient agar and Mac Conkey agar. The other Clinical specimens were inoculated onto DCL agar, 5% sheep blood agar, Selenite broth for Salmonella species, Chocolate enriched / bacitracin agar in 5% CO 2 for Neisseria sp and Haemophilus sp. Swabs were vortexed and subcultured on Chapman agar plates for Gram-positive bacteria, DCL Desoxycholate citrate lactose agar plates for Gram-negative bacteria and chocolate agar for exigent bacteria. The plates were incubated for 18-24 h at 37 °C and visible colonies were further sub-cultured and incubated for 24 h at 37 °C. Isolation and identification of microorganisms were done according to standard procedures. Bacteria were identified by examination of colonial morphology, haemolytic characteristics on appropriate agar media, Gram staining, rapid tests (catalase, oxidase, coagulase/Dnase, optochin disc, bile solubility, spot indole, latex agglutination), and classic and API galleries (BioMérieux, France) [21].

Drug susceptibility testing
Drug susceptibility testing was performed by the method of disk diffusion according to the guidelines of the National Committee for Clinical Laboratory Standards [22]. The culture of each isolate was diluted to have turbidity around 0.5 McFarland standard, then plated onto Muller-Hinton agar plate (Difco, France). Antibiotic disks (Oxoid, France) were applied to each plate. After incubation at 35°C for 24 h, the zone of inhibition diameter was measured. Data analyses were performed using the susceptibility cut points according to the Clinical and Laboratory Standards Institute guidelines [23]. Multiresistance of Gram-negative bacteria was defined as resistance to at least three antibiotics of the following antibiotic classes: penicillins, third-generation cephalosporins, carbapenems, quinolones and aminoglycosides.

Statistical Analysis
Statistical analysis was performed using SPSS 17.1 software that uses directly the Yates' chi-square test for small sample size. Differences were considered statistically significant for P ≤ 0.05.

Isolation of pathogen strains from patients, hospital surfaces and hands
A total of 814 sampling were made, 139 from patients, 470 from surfaces and 205 from personnel and patients' hands. Personnel and patients' hands were the most infected samples. Indeed, 98.5% of samples from personnel and patients' hands were infected with pathogen bacteria (202/205). Whereas only 46% of patients (64/139) and 26.8% of surfaces' samples (126/470) were positives. Among the 392 positive samples, 2182 pathogen strains were isolated, comprising 1112 Gram-positive and 1070 Gram-negative bacteria.

The distribution of strains according to samples' origins
The distribution of strains isolated from patients, surfaces and hands, and their detection rates was reported in Table 1 and showed that 67 bacteria were isolated from patients, 865 from surfaces of various locations in different services and 1250 from personnel and patients' hands. Characterisation of strains isolated from patients showed clearly a high prevalence of Gram-negative bacteria. Bacterial identification showed a predominance of Klebsiella sp (16,42%), E. coli (16,42), Enterobacter sp (11.94%) and Pseudomonas sp (10.45%). Pathogen bacteria were also isolated from personnel hands Table 1. Globally, isolation of Gram-positive bacteria was higher than Gramnegative bacteria. Gram-positive bacteria were isolated from 130 of 202 (64.36%) of hands, however Gram-negative bacteria were isolated only from 35.64% of hands (72/202). Moreover, no significant difference was obtained on sampling from personnel' and patients' hands either for Gram-negative or Gram-positive bacteria (p=0.9656). The distribution of strains isolated and their detection rates on surfaces is also reported in Table 1. Results clearly demonstrate the presence of both Grampositive and Gram-negative bacteria with the predominance of Pseudomonas sp, Klebsiella sp, Staphylococcus aureus and coagulase negative Staphylococcus, Pseudomonas sp was isolated from 23.12 % and 11.60% of surfaces and hands respectively. Klebsiella was collected from 16.76% of surfaces and 16% of total hands. Staphylococcus aureus represented 12.72% and 20.8% of total isolates from surfaces and hands respectively. Interestingly, coagulase negative Staphylococcus were the most predominant bacteria isolated from the hospital environment and represented 30.64% and 30.80% of total strains isolated from surfaces and personnel and patients' hands, respectively. Moreover, the percentages of detection of Gram-positive and Gram-negative isolated at different locations is summarised in Figure 1. For both Gram-negative and Gram-positive bacteria, the main infected surfaces were the bed covers and bathroom representing the major reservoirs of pathogens. Moreover, Gram-negative bacteria were predominant in wash-hand basins and the patient's masks whereas Gram-positive bacteria prevail in samples taken from tables.

Isolation and distribution of drug resistant strains
The resistance status of isolated bacteria was also evaluated. Among the 2182 isolates, 322 were drug resistant strains. Significant difference of the distribution of theses strains was observed depending on the origin of sampling (p<0.0001). Indeed, 37 strains of 67 isolates from patients were drug resistant giving the highest rate of multi drug resistance (55.22%). However, only 15.51% (150/865) and 9.53% (135/1250) strains isolated from surfaces and hands were drug resistant respectively. Table 2 illustrates the rate of multi-drug resistance of isolated strains. Drug resistant rate ranged mainly from 7.69 to 66.67%. However, all strains belonged to providencia genus and only 0.31% of  Table 3. Drug resistant strains belonging to Citrobacter, Morganella and coagulase negative Staphylococcus were isolated only from patients whereas the other drug resistant strains were isolated from patients and from surfaces and/or hands.

Drug resistance status
The Tables 4 and 5 show the resistance profile of different drug resistant pathogens isolated from hospital during this study. The majority of gram-negative isolated strains were characterised by the high resistance to ampicillin and the association amoxicillin-clavulanic acid. Klebsiella sp and Pseudomonas sp showed high rates of resistance to 3 rd generation of cephalosporins. Indeed, resistance to ceftriaxone was observed in 90.70 % of drug resistant Klebsiella strains whereas resistance to ceftazidime and cefotaxime was observed in all drug resistant Klebsiella strains. Furthermore, 71.43%, 100% and 93.51% of drug resistant Pseudomonas strains were resistant to ceftazidime, cefotaxime and ceftriaxone respectively. The 4 strains demonstrated a particularly high resistance to aminosids antibiotics, especially        amikacin and nethilmycin. S. auresu strains showed a high resistance to quinolone drugs (>66%), including ciprofloxacin and ofloxacin, low resistance to Sulfamethoxazole + trimethoprim and fusidic acid, whereas no strain exhibited resistance to vancomycin. Moreover, more than 47% of Staphylococcus aureus strains were MRSA as determined by the susceptibility testing to oxacillin.

Discussion
In this study, we have clearly demonstrated that microorganisms isolated from the hands of personnel or patients are much higher than those isolated from surfaces and patients. That ensures the possibility of disseminating microorganisms by professions who neglect to wash their hands after touching patients [24,25]. Several studies have reported the importance of frequent and adequate hand washing to reduce rates of Hospital-acquired infections [26][27][28][29], showed that hands regularly acquire bacterial pathogens, after contact with patients and the environmental surfaces near hospitalised patients [16]. Moreover, many pathogens responsible of nosocomial infections can survive on dry surfaces for several weeks [30]. Characterisation of pathogen strains showed the predominance of gram-negative bacteria from patients and surfaces and Gram-positive bacteria from hands. Previous studies have reported different rates of viable microorganisms on healthcare personnel hands, Acinetobacter sp. 3-15%, Klebsiella sp. 17%, MRSA up to 16.9%, Pseudomonas sp. 1.3-25% [31]. Gram-positive pathogens such as Staphylococci strains show much higher transmission rates compared to Gram-negatives. That could be explained by diminished survival time of Gram-negatives in the environment [32]. In fact, Gram-negative bacteria other than Acinetobacter sp [33], survive on dry surfaces for few hours only, while the survival time can be several days for Staphylococci [34,35]. In this study strains isolated from patients are basically the same as those isolated from surfaces of various hospital locations and hands, indicating the persistence of these strains in the hospital environment. However interestingly, investigations into epidemics have not confirmed that patients were infected specifically due to surrounding environmental contamination [2,36], but depend also on the patient health status.  [39]. Different studies have reported low bacterial counts of multi-resistant organisms in the environment of colonised patients [2,40,41]. This was confirmed by our findings reporting that only 15.51% of strains isolated from surfaces and 9.53% of strains isolated from hands were multidrug resistant, in contrast, 55.22% of strains isolated from patients were drug resistant. Our results clearly demonstrated that the rate of drug resistant Gram-negative bacteria was much higher than Gram-positive bacteria, from both patients and the hospital environment. Previous studies showed converse results with a high degree of Gram-positive drug resistant isolates from the hospital environment [2].
Basically, resistance profiles of strains colonised hospital environment were similar to those isolated from patients; this suggests that patients could be contaminated from hospital surfaces or through healthcare workers. In this field, strain typing using molecular approaches will be of a great interest to compare bacteria from patients and the hospital environment. Thus, the implementation and/or reinforcement of effective cleaning measures is necessary to limit the dissemination of pathogens in the hospital environment and to contaminate newly admitted patients [3,[42][43][44]. Inappropriate use of antibiotics allows bacterial pathogens, or opportunistic strains (from non-clinical environments) to acquire new resistance mechanisms [45,46]. β-lactams are the most widely used antibiotics leading to the emergence of high number of resistant strains. Currently, the most used antibiotics in Morocco are 3 rd generation of cephalosporins, carbapenems, quinolones and aminoglycosides. Unfortunately, the excessive and not efficient use of theses antibiotics might be associated with increased risk of microbial resistance. In the current study, the frequency of resistance to the most used antimicrobial agents, including cephalosporins, was relatively higher. The frequency of resistance to the main antimicrobial agents was 60.53 % to 100% for ceftriaxone, 59.26% to 100% for ceftazidime, 54.54% to 100% for cefotaxime, 47.37% to 77.78% for ciprofloxacin and 6.86% to 33.77% for imipenem. Overall, our results corroborate with other studies conducted in different countries. In previous studies conducted in Iran, Oman and Turkey, the resistance to ceftriaxone ranged from 68 to 98.1% [47][48][49]. Conversely, the resistance to ceftriaxone was much lower in Belgian as reported by Glupczynski et al. That could be due to adequate empirical treatment using this antibiotic [50]. In the current study, the mean resistance to ceftazidime was 79.1%. It is almost similar to an Iranian study with resistance ranging from 60 to 88 % [47,49]. Once again, this resistance is higher than reported results for Belgian study [50]. In our study, resistance to cefotaxim ranged from 54.54% to 100%, this in agreement with reported data from Turkey with percentage of resistance ranging between 59.4% and 96.2% [49]. During the last decades, susceptibility to quinolones has decreased more than other antibiotics worldwide, and this might be a consequence of the wide usage of these antibiotics. Quinolone-resistant strains might therefore spread more easily than the strains resistant to other antimicrobial agents [51]. The published data on the resistance of hospital bacterial strains to ciprofloxacin are controversial. Indeed, in an Iranian study resistance to ciprofloxacin was 56% -77% [47], 20 to 59.2% in Turkey [49]. However, it was much higher in Argentina exceeding 80% and is less than 25.5%, in a Brazilian study [52,53]. In the present study, the mean resistance to imipenem is 16.73% (6.85% -33.77%).The frequency of resistance to imipenem was 14% in an Iranian study, 13% in a Belgian study and 8% in a Polish study, and this is in agreement with our findings [47,50,54]. In conclusion, the increasing antimicrobial resistance rate in hospitals and the possible dissemination of resistant bacteria in the inanimate surfaces or the hands of health professionals and patients, reinforce the need for knowledge and control of the sources of pathogens in the hospital environment. The evaluation of the environmental role in the acquisition of healthcare associated infections is needed to collaborate with infection control committees. The establishment of a control system is also required in hospitals for the reduction of the length of stay, costs and morbidmortality. Such a surveillance system should continuously report the prevalence of microorganisms and their resistance pattern to hospital wards; this information will be used in defining policies for control of hospital environments, and building awareness about the diminished efficacy of antibiotics with the wide-scale use, especially in Moroccan hospitals where antimicrobial prescription is sometimes inappropriate. Thus, more attention must be given to the implementation of effective approaches to optimize antimicrobial use.