Multidrug-resistant bacteria isolated from patients hospitalized in Intensive Care Unit in University Hospital of Constantine , Algeria ( 2011-2015 )

The incidence of infections caused by multidrug resistant bacteria is increasing worldwide. The frequent misuse of antibiotic drug has greatly contributed to worldwide dissemination of antibiotics resistance. Multi-drug resistance in Gram-negative and Gram-positive bacteria causes a wide range of infections, particularly in the Intensive Care Unit (ICU) settings leading to an increased impact on morbidity, mortality and costs. This study was undertaken to determine the prevalence of multidrug resistant of bacterial isolates in patients admitted in ICU of university hospital of Constantine. We analyzed a 5-year period, from 2011 to 2015. Over five years period, 7472 clinical samples were collected in the Clinical Microbiology Laboratory of Benbadis University Hospital in Constantine. Identification of the isolates was performed by API automated systems (bioMérieux, Marcy l’Etoile, France) and automate microscan walkaway 96 (Siemens). Antibiotic resistance was determined by the Clinical and Laboratory Standards Institute (CLSI) disk diffusion test on Mueller-Hinton Agar. Multidrugresistant isolates included in this study were methicillin-resistant Staphylococcus aureus, vancomycinresistant enterococci, Enterobactericeae that produce extended-spectrum beta lactamases and/or carbapenemases, multidrug-resistant Acinetobacter baumannii and multidrug-resistant Pseudomonas aeruginosa. A total of 3528 isolates were collected from various specimens such as blood (47.05%).The Staphylococcus sp, Klebsiella sp, Acinetobacter sp, P. aeruginosa and E. coli are the most common isolates recovered from clinical specimens in ICU (26.3, 18.7, 14.3, 11.9 and 9.2% respectively). MRSA strains constituted over 65% of all S. aureus isolates and 30.3% of E. faecium were found to be vancomycin resistant. Extended spectrum β-lactmase producers were expressed in 53.2% and 50.6 from K. pneumoniae and E. coli. Carbapenem resistance among K. pneumoniae improved slightly from 2.89 to 4.21%. A. baumannii isolates exhibited extremely high levels of resistance to all antibiotics except colistin (100% sensitive). In addition, 80.4% of A. baumannii isolates were found to be resistant to imipenem. Imipenem resistant P. aeruginosa isolates showed 36.4%.


INTRODUCTION
The increase and spread of multidrug resistant (MDR bacteria have become a major concern worldwide.The hospital acquired infections caused by MDR infections caused by MDR bacteria have led not only to an increase in mortality, morbidity, and cost of treatment, but also continue to endanger the life of patients (Martin and Yost, 2011;Delle Rose et al., 2015).
MDR bacteria can cause a wide range of infections, including bacteremia, pneumonia, urinary tract infection, peritonitis etc., which can lead to substantial morbidity and mortality, particularly in the ICU settings (Chen et al., 2016).
The risk of acquiring MDR bacteria in the ICU is increased by severity of illness, length of stay, use of intravascular devices, exposure of ICU patients to invasive therapeutic procedures like endotracheal intubation, the intensity of exposure to infected patients and the frequent misuse of antibiotic drug (Khan et al., 2014;Royer et al. 2015;Wroblewska et al., 2006).
Infection and colonization with MRSA may be more frequent in the ICU than in general wards (Fridkin and Gaynes, 1999) and its resistance is conferred by the acquisition of one of several staphylococcal cassette chromosome mec elements that carry a gene (mecA) that encodes a penicillin-binding protein (PBP2a) with low affinity for β-lactam antibiotics (Katayama et al., 2000).Vancomycin-resistant enterococci (VRE) have emerged as a nosocomial pathogen especially in ICUs worldwide (Austin et al., 1999).VRE are among the major health care-associated MDR organisms causing a serious problem in choosing an appropriate therapy (Schouten et al., 2008).
Infections caused by Gram-negative bacteria have features that are of particular concern.Specifically, the rate of infections related to MDR gram-negative bacteria (MDR-GNB) in ICU is increasing.These organisms are highly efficient at up-regulating or acquiring genes that code for mechanisms of antibiotic drug resistance, especially in the presence of antibiotic selection pressure (Russotto et al., 2015).
The emergence of resistance in Enterobacteriaceae is considered an alarming health threat.During this last decade, a growing number of K. pneumoniae and E. coli have developed resistance against third-generation cephalosporin, due to extended-spectrum β-lactamases (ESBLs) (Pitout and Laupland, 2008).Carbapenems are the first line treatment of ESBLs producing bacteria, and the emergence of carbapenem-resistant isolates conferred by New Delhi Metallo-β-lactamase-1 (NDM-1) leaves limited therapeutic options.
Non -fermentative Gram-negative bacilli (NF-GNB) mainly A. baumannii and P. aeruginosa, have emerged worldwide and the resistance of these organisms to antibiotics, particularly to carbapenems, has posed important therapeutic challenges.
Currently, carbapenems are considered the antimicrobials of choice for treatment of serious infections caused by A. baumannii and P. aeruginosa but their efficacy is increasingly compromised by resistance as reported worldwide (Al Jarousha et al., 2009).This resistance has been attributed to the production of carbapenemhydrolysing-lactamase enzymes of Ambler molecular class D (oxacillinases) and B (metallo-lactamases) (Woodford et al., 2011).
Despite its importance, a few studies on the MDR bacteria isolated from patients hospitalized in ICU were investigated in Algeria.
The aim of the present study was to determine the prevalence of MDR Gram positive and Gram negative organisms isolated from patients hospitalized in ICU of a university hospital in Constantine, Algeria.

RESULTS
Among the 7472 samples collected, 3528 were positive (47.21% of total samples), 426 were contaminated and represented 5.70% of total samples and 47.08% of all samples were negative (Table 1).
Klebsiella sp.ranked second overall among ICU patients (18.73%) and was the most frequently isolated Gram-negative organism (27.56%).K. pneumoniae was most common specie 608/661 (91.98).For K. pneumoniae, the highest resistance rate was for ticarcillin, cefazolin and amoxicillin/clavulinat (100, 85.29 and 79.82%, respectively).All strains of K.In the present study, imipenem resistant was detected in 80.42% of A. baumanii, and 36.84% of P. aeruginosa.We recorded an increase of imipenem-resistant strains of A. baumannii during the study period from 60.39% in 2011 to 94.2% in 2014.The proportion of imipenem resitance increased slightly from 33.33% in 2011 to 50% in 2015 for P.aeruginosa (Table 6).

DISCUSSION
Most isolates were recovered from the blood specimens (47.05%).This finding corroborated the results reported by other investigator in India (Jitendra et al., 2012).While in the ICU of Kingdom of Saudi Arabia (KSA), most isolates were recovered from the respiratory specimens (38.8%), followd by the blood specimens (33%) (Saeed et al., 2010).
Staphylococci constituted the group of bacteria most commonly isolated from ICU patients (Johnson et al., 2003).The commonest organism isolated from all samples was Staphylococci (26.38%) in our study.
The most common GP cocci in this study were SCN, followed by S.aureus, whereas in the study of Fridkin et al. (1999), the most common GP cocci were S. aureus, followed by SCN.
In the present study, MRSA strains constituted over 65% of all S. aureus isolates.MRCNS strains represented 90.15% of CNS strains.These are high percentages from MRSA and MRCNS compared with data from other reports (Khan et al., 2014;Wang et al., 2011).
MRSA, first described in the 1960, is now endemic in many hospitals, infection and colonization with MRSA may be more frequent in the ICUs than in general wards (Thompson and FRCP, 2004).
Antibiotic susceptibility profile of MRSA showed that these strains exhibited the highest sensitivity to vancomycin (100%) In our study, Enterococci are important pathogens of patients hospitalized in the ICU, particularly in view of the increasing frequency of resistance to vancomycin.In this study, strains of enterococci comprised 3.31% of all bacterial isolates and 10.35% of Gram-positive isolates, this observation agreed with the finding of Johnson et al. (2003).Among enterococci isolates, 71.79% were E. faecalis and 25.46% were E. faecium.In the study of Sood et al. (2008), E. faecalis has been the predominant enterococcal species accounting for 80-85% of clinical isolates, followed by E. faecium which accounts for about 10-15% of clinical isolates.
In the present study, 10 (8.54%) isolates were found to be vancomycin resistant, 10 out of 33 isolates (30.3%) of E. faecium but no strains of vancomycin-resistant E. faecalis were detected.It has also been found in various studies that E. faecium accounts for far fewer clinical enterococcal isolates than E. faecalis, but it is far more resistant to glycopeptides.
In a study conducted by Saeed et al. (2010), less than 7.1% of E. faecalis were found to be resistant to vancomycin, whereas 40.1% of the E. faecium isolates were resistant to vancomycin.Zhanel et al. (2003) found that most of the VRE were E. faecium (88%), while only 12% were of E. faecalis.The Gram-negative organisms most frequently isolated in our study were Klebsiella  Over the past decade there has been a dramatic increase in MDR-Gram-negative rods, particularly among isolates recovered from ICU patients.In our study, ESBL production was detected in 53.28 and 50.61% of strains of K. pneumoniae and E. coli respectively.These data are similar to the proportion of 56.2 and 43.6% of ESBLproducing isolates amongst E. coli and K. pneumoniae isolated in clinical samples in China (Wang et al., 2008) but it is far higher than the same proportions reported in France (8 and 13%, respectively) (Arnaud et al., 2013).Whereas it is a less percentage compared with others reports in the literature.An Algerian's report of the resistance of bacteria to antibiotics (2012) have reported 76.73% of ESBL-producing isolates amongst K. pneumoniae in 2012.Saeed et al. (2010) found that ESBL producing E. coli were approximately 100% among the tested isolates, which were more common than that seen in K. pneumoniae (92%).
Carbapenem-resistant K. pneumoniae has emerged during recent years in several intensive care unit (Debby et al., 2012).Carbapenem resistance among K. pneumoniae improved slightly in 2013 and 2015 from 2.89% to 4.21% in the present study.Increasing rates of carbapenem resistance among Enterobacteriaceae have been reported by other investigators in various European countries (Mouloudi et al., 2010;Nordmann et al., 2011;Walsh, 2010).Rates of 2.3% and 38% of carbapenem resistance among K. pneumoniae have been reported (Sękowska et al., 2014;Chaudhary and Payasi (2013).KPC-producing strains appear to have the widest distribution, but a rising number of OXA-48-producing strains has been reported (Glasner et al., 2013).
It constituted 17.59% of Gram-negative strains in our study.This data is almost similar to the 24% reported from ICUs in Europe (Hanberger et al., 1999).
In the present study, the percentage of imipenemresistant strains of P. aeruginosa was 36.84%.The proportion of imipenem resitance increased from 33.33% in 2011 to 50% in 2015.This is high compared with reports in the literature ranging from 2 to 20% (Glupczynski et al., 2001;Hsueh et al., 2001).
Strains of Acinetobacter sp.constituted 14.34% of all isolates and 21.10% of GN organisms.This is a high percentage compared with data from USA ICUs (Sader et al., 2014), but it is similar to others reports (Saeed et al., 2010;Tan et al., 2014).In present study, A. baumanii showed very high resistance rates to most antimicrobial agents and an important decrease in the susceptibility was observed for imipenem during the five years study period (60.39% in 2011 to 94.2% in 2015).This is very high compared with reports in the literature ranging from 9.6% to 23.8% (Wroblewska et al., 2006).While, among the four hundred and fifty four isolates of A.baumani isolated in India's ICU, 81.71% isolates were found to be carbapenemase producing (Chaudhary and Payasi, 2013).
The global spread of carbapenem-resistant P. aeruginosa and A. baumannii is of great concern (Hanberger et al., 1999).The significantly low susceptibility of ICU-acquired strains to carbapenems may be related to the increasing use of carbapenems in ICU.The most active compound against these organisms was colistin.

Conclusion
This study showed that Staphylococcus sp, Klebsiella sp, Acinetobacter sp, P. aeruginosa and E. coli were the most common isolates recovered from clinical specimens in the ICU of Constantine.Blood specimens represented 47.06% of all specimens collected in the ICU.This study demonstrated that most of these pathogens isolated from clinical samples were MDR.We conclude that the incidence of high rates of resistance is alarming high and is continuously increasing and spreading.Therefore surveillance of bacterial prevalence and susceptibility patterns of the isolates from ICU is crucial in determining optimum empirical therapy of infections in critically ill patients.

Figure 1 .
Figure 1.Prevalence of Gram positive and Gram negative isolates.

Table 1 .
Number and rate of positive culture from all samples.

Table 2 .
Nature of the analyzed samples.
pneumoniae were sensitive to colistin (Table5).ESBL production was detected in 53.28% of strains of K.pneumoniae.

Table 4 .
Prevalence of multidrug resistant staphylococci and enterococci stratified by year.

Table 6 .
Prevalence of multidrug resistant of major four isolates Gram negative, stratified by year.