Resistance of Gram-Negative Bacilli in Lebanon

Several studies have reported the isolation of resistant Gram-Negative Bacilli in Lebanon.However, those studies are new and scarce as compared to worldwide data and mostly restricted to single center studies. In this review, we attempt to provide a reliable and comprehensive report describing the current situation and providing prospects for bacterial resistance in Lebanon. Several studies have shown that Extended Spectrum ββ-Lactamase-producing E. coli and K. pneumoniae strains are being increasingly reported. Moreover, 2.15% of E. coli and 7.84% of K. pneumoniae isolates have shown carbapenem resistance and up to 30% of isolated E. coli strains were found to be Multi-Drug Resistant. Molecular studies showed that the most widespread ββ-Lactamases in Lebanon are of the CTX-M-15 and SHV types. In addition, K. pneumoniae strains producing metallo-ββ-Lactamase and Klebsiella pneumoniae Carbapenemase have been reported. Resistant Pseudomonas aeruginosa and Acinetobacter baumannii caused several nosocomial infections and some Acinetobacter baumannii strains were found to produce OXA-58 type ESBL. e few data addressing the rate of antibiotic consumption in Lebanon show a high rate of antibiotic misuse and abuse. In conclusion, there is a need for antibiotic stewardship programs and additional studies that go beyond the scope of single-center studies in Lebanon.


Introduction
Bacterial resistance is an issue that has cultivated major worldwide concern in clinicians and researchers alike [1].Resistant bacterial strains are increasing in prevalence [2,3] and their acquired ability to avoid the action of one or more antibacterial agents is causing a serious clinical threat [4].Of the many resistant bacteria, Gram-Negative Bacilli (GNB) are increasingly isolated [5][6][7].Two major families of GNB with considerable resistant strains are the Enterobacteriaceae and the nonfermenters [8].
One mechanism by which GNB acquire resistance is via the production of -lactamases.ese -lactamases include, among others, the Extended Spectrum -Lactamases (ESBLs), the Metallo -Lactamases (MBLs), and the OXA -lactamases [7].Moreover, some organisms have acquired resistance to more than one antibacterial agent.ese organisms are classi�ed as Multidrug Resistant (MDR) if they are resistant to more than 3 classes of antibacterial agents, Extensive Drug Resistant (XDR) if they are resistant to all but 1 or 2 classes of antibacterial agents, and Pan-Drug Resistant (PDR) if they are resistant to all antibacterial agents [9].
Several studies show that the rate of antibiotic consumption is one factor that is directly correlated to the emergence of resistance in bacteria [10,11].Borg et al. [12] have shown that there is a high rate of wide spectrum antibiotic consumption in the Mediterranean region.More speci�cally, in Lebanon, alarming rates of misuse and abuse of antimicrobial agents have been reported [11,13,14].is high rate of antibiotic consumption, coupled by a lack of judicious and rationale use, creates an environment for resistance to emerge.However, data regarding the spread of resistant GNB in Lebanon are relatively new and scarce when compared to worldwide data, and are mostly restricted to single-center studies [15][16][17].
In light of the signi�cant clinical threat that resistant GNB are causing and the absence of a national surveillance center to provide reliable data, we intend to review the most important studies related to bacterial resistance in Enterobacteriaceae and nonfermenters in Lebanon.Our aim is to present a reliable and comprehensive report describing the current situation and providing prospects for bacterial resistance in a country where antibiotics are easily accessible and widely abused.
1.1.Escherichia coli and Klebsiella pneumoniae.Strains of E. coli and K. pneumoniae with high rates of antibiotic resistance are increasingly reported worldwide [18,19].It has been shown that countries in the Eastern Mediterranean region have a higher prevalence of resistant strains when compared to several European countries [12].In Lebanon, by the year 1994, as much as 65% of clinical isolates of E. coli were found to be resistant to ampicilin at the American University of Beirut Medical Center [20].Around ten years later, this percentage had increased to 72% in the same institute [21].Another study at the American University of Beirut Medical Center over a period of 4 years showed that the rate of ESBL producing E. coli increased from 3% to 5% whereas that of K. pneumoniae increased from 6.4% to 13% [22].is high rate of increase in incidence is higher than that reported in other countries of the world, such as USA [23,24].e screening of 4399 clinical isolates of E. coli and 1248 isolates of K. pneumoniae from the Saint George Hospital, University Medical Center, in Beirut over a period of 5 years showed that 2% of the E. coli strains and 20% of the K. pneumoniae were ESBL producers [25].Moreover, ESBL production in organisms isolated from Urinary Tract Infections (UTI) in that same hospital increased from 2.3% to 16.8% over a period of 9 years [6].Another study investigating the intestinal carriage of ESBL producing E. coli and K. pneumoniae of hospitalized patients across 5 Lebanese tertiary care facilities reported that 80.5% of E. coli and 13.6% of K. pneumoniae were ESBL producers [14].ese studies show that the rate of ESBL production is increasing in both E. coli and K. pneumoniae isolates in this country and threatens to escalate if not dealt with promptly and appropriately.
E. coli isolated from prostate gland biopsies from the American University of Beirut Medical Center showed that 5 out of 8 organisms produced ESBL [26].At that same hospital, 40% of E. coli and 33% of K. pneumoniae isolated from patients with ventilator-associated pneumonia were shown to be positive for ESBL production [27].ese rates of resistance are higher than those reported in other regions of the world [24,28], but remain lower than those detected in other Mediterranean countries such as Egypt [29].Nevertheless, these high rates warrant immediate attention due to the great risk they impose on hospitalized patients.
On the other hand, the production of statistical pamphlets by different Lebanese hospitals reporting the different susceptibility pro�les of bacterial strains encountered throughout the year is becoming a general trend.ese pamphlets are a valuable tool used mainly by clinical microbiologists, infection control personnel, and infectious disease clinicians for the monitoring of the patterns of resistance of different bacterial strains.e lack of a national surveillance center in Lebanon gives an even greater value for these pamphlets and encourages more and more hospitals to produce them.Although these pamphlets may lack speci�city and might include nonstatistically signi�cant data, or even may erroneously report duplicate strains (what would give a false increase or decrease in resistance), they remain a needed �rst step for the creation of a national surveillance center of studies.
Susceptibility testing of E. coli strains isolated from patients with hospital acquired Urinary Tract Infections (UTI) at the American University of Beirut Medical Center between January and December of 2001 showed that 64% of these isolates were resistant to Trimethoprim/Sulfomethoxazole (TM/SFX) and 46% were resistant to quinolones.However, only 1 isolate was found to produce ESBL [13].At that same hospital, intestinal carriage of E. coli showed 100% susceptibility to ertapenem, 83% susceptibility to piperacillin/tazobactam, and 96% susceptibility to amikacin.In the same study, the susceptibility pro�le of K. pneumoniae strains in the intestines were 100% to ertapenem, 91% to piperacillin/tazobactam, 82% to amikacin, 72-75% to quinolones and 88% to cefepime [30].In 2008, E. coli isolates were 100% susceptible to tigecycline, whereas 16% of K. pneumoniae strains were intermediately resistant and 3% were fully resistant to tigecycline in the same hospital [21].A study by Borg et al. conducted at the Saint George Hospital, University Medical Center showed that 18.9% of clinical isolates of E. coli were resistant to third generation cephalosporins, 21% were resistant to quinolones, and 68% were resistant to aminopenicillin [12].ese reported pro�les show that resistance in both intestinally carried and clinical isolates of E. coli and K. pneumoniae is evident; however, they were still reported to be susceptible to carbapenems.Nevertheless, 2.15% of E. coli and 7.84% of K. pneumoniae isolates were found to be resistant to carbapenems in a recent study [31].Moreover, two other studies have reported that up to 30% of isolated E. coli strains were MDR [15,32].e observed resistance to carbapenems and tigecycline and the emergence of MDR strains impose a great threat that needs immediate attention and prompt action.
At the molecular level, the analysis on 72 ESBL producing GNB (of which 77% were E. coli) showed that all CTX-M type ESBL producers (83% of total strains) harbored the  CTX-M-15 gene, while the SHV-type ESBL producers (18% of total strains) harbored the  SHV-5a gene.All the isolates were found to carry the  TEM gene.25 E. coli strains were also found to harbor the  OXA-1 gene and only one E. coli isolate harbored both the  CTX-M-15 and the  SHV-5a genes at the same time [17].A separate study at the American University of Beirut Medical Center showed that 96% of the ESBL producing E. coli had the  CTX-M gene, 67% had the  SHV , and 57% had the  TEM genes.Analysis on K. pneumoniae strains showed that 40% of ESBL producing K. pneumoniae had the  CTX-M gene, 82% had the  TEM , and 84% had the  SHV genes [33].Taken together, these studies suggest that the most widespread -lactamases in Lebanon are of the CTX-M-15 and the SHV type.Trans-continental comparison of the CTX-M-15 E. coli isolates in Lebanon revealed that these strains were genetically related to the O25: H4 clones found in France, Canada, and Portugal, suggesting a common ancestry [34].
In 2007, the �rst metallo--lactamase producing K. pneumoniae in Lebanon was isolated and found to harbor the  IMP-1 and  CTX-M genes [35].Another carbapenemresistant K. pneumoniae strain was isolated later on in 2008, mediated via the  OXA-48 gene [36].is type of carbapenem resistance is consistent with �ndings from Turkey, Belgium, Egypt, and France [37].Moreover, Klebsiella pneumonia carbapenemase (KPC) was detected in a single isolate [38].ese carbapenem-resistant strains greatly limit the choice of antibiotic treatment and therefore impose a huge threat to hospitalized patients.
In conclusion, resistance in E. coli and K. pneumoniae isolates is increasing in the Lebanese population, with the major types of -lactamases produced in these isolates being CTX-M-15-and SHV-type ESBLs.e high rate reported is expected to increase even more if the current lack of control over antibiotic consumption persists.Moreover, the emergence of multidrug resistance and carbapenem resistance is a major threat because of these strains' ability to render a large number of antibiotics obsolete.erefore, antibiotic stewardship programs are urgently needed in order to control antibiotic consumption and limit the wide-spread antibiotic abuse and misuse in the Lebanese population [12] and attempt to decrease the rate of emergence of resistant strains.

Other Enterobacteriaceae
Salmonella species have been isolated from Lebanese fast food [39] and incriminated in food poisoning outbreaks [40], suggesting therefore some degree of exposure of the community to this organism.ESBL production among Salmonella isolates has also been reported in Lebanon.e major type of ESBL found in Salmonella species in Lebanon is of the TEM and SHV type [23].In 2003, Daoud et al. noted that all 49 Salmonella strains isolated at the Saint George Hospital were susceptible to ceazidime and cefotaxime (Z.Daoud, personal communication).In January 2004, a CTX-M-15type ESBL producing Salmonella eneterica was isolated from the stool of a 6-year-old child in Lebanon [41], reporting for the �rst time a detrimental change in the resistance pro�le of this organism.Molecular analysis revealed that this type of ESBL is similar to those reported in India and Turkey [42].e relatively high prevalence of Salmonella in Lebanese fast food and its ability to produce ESBL makes it a public health issue that concerns the whole community.
A study at the American University of Beirut reported that 60% of isolated Shigella species were resistant to ampicilin, chloramphenicol, and co-trimoxazole [20].In 2005, three individually unique ESBL producing Shigella sonnei species were isolated [43].ese isolates were resistant to amoxicillin, cefotaxime, ceazidime, aztreonam, trimethoprim/sulfamethoxazole, gentamycin, and kanamycin.Two of these isolates harbored the  TEM1 gene and all three isolates harbored the plasmid-encoded  CTX-M gene [43].Moreover, in 2009, 4 ESBL producing Shigella sonnei species harboring the  CTX-M-15 gene were isolated from the stool of patients in a single tertiary care center.is �nding suggests that the transfer of resistant genes occurred via horizontal plasmid transfer [44].In addition, it has been noted that Lebanese immigrants living in Sweden had high incidence rates of traveler-associated shigellosis aer visiting Lebanon, suggesting that Shigella species are highly prevalent in Lebanon [45] and making resistance in this organism a very serious problem.
2.1.Pseudomonas aeruginosa and Acinetobacter baumannii.Pseudomonas aeruginosa is a bacterium that poses a threat to immunocompromised patients [46] due to its high prevalence in tap water, medical equipment, hospital personnel, and hospital patients [47].A study in 2003 showed that out of 38 bacterial species isolated from patients with ventilator-associated pneumonia, 8 where Pseudomonas aeruginosa [27].In 2005, a single Pseudomonas aeruginosa clone of genotype 1 was found to be responsible for 18% of nosocomial infections in various medical units at the American University of Beirut Medical Center [48].is �nding shows the potential of Pseudomonas aeuroginosa of causing serious nosocomial infection outbreaks.However, deeper investigation into the spread of virulent Pseudomonas aeruginosa strains across multiple tertiary care centers in Lebanon is needed.
Acinetobacter baumannii has caused several outbreaks throughout the Mediterranean area [49].In Lebanon, a single Acinetobacter baumannii strain was responsible for an outbreak in a major tertiary care center in Beirut.Analysis on this strain showed that it produced the OXA-58 type ESBL [50].A multidrug resistant Acinetobacter baumannii harboring the  OXA-58 gene caused another outbreak at the Saint George Hospital of Beirut between the years 2004 and 2005 [51].Even though the Acinetobacter baumannii strains causing these outbreaks were found to be multidrug resistant, they were still fully susceptible to tigecycline [21].Nevertheless, the heavy dependence of several patients on multiple antibiotics in the ICU [52] may result in increased emergence of resistance among Acinetobacter baumannii strains.

Antimicrobial Consumption and Conclusion
Very few data is available regarding antibiotic consumption in Lebanon.A study by the ARMed group has shown that broad spectrum antibiotics (such as 3rd generation cephalosporins and quinolones) are extensively consumed in this country, where more than 15% of the total national consumption consists of quinolones [12].A high rate of consumption of 3rd generation cephalospirins has been shown to be a major risk factor for the development of resistance [11].erefore, the high rates detected in Lebanon poses a great threat, where resistant strains will keep on increasing and making treatment progressively harder [53].Moreover, the haphazard distribution, easy availability of antibiotics, lack of stewardship programs, and proper record keeping in Lebanese hospitals aggravates this problem even more [16].One step towards limiting the emergence of resistant bacterial strains would be to encourage the hospitals to calculate their consumption of antibiotics and study its possible correlation with bacterial resistance.Many sowares and applications such as "ABCcalc" and "WHOnet" are made available for this purpose.ese programs are easily installed and allow hospitals to track the antibiotic consumption and the emergence of resistant strains throughout the year.is information may prove to be extremely valuable in the constant struggle to decrease the emergence of resistance in Lebanon.
In conclusion, the widespread antibiotic abuse and misuse has been a major driving factor for the high rate of resistance found in Gram-Negative Bacilli isolated in Lebanon [16,24].Antibiotic stewardship programs are urgently needed since their continued absence would only result in sustaining the increasing rates of resistance and an ever increasing difficulty in their treatment.Several additional studies that stretch beyond the scope of single-center studies are needed as well in order to shed light on the prevalence of resistance across the country.Nevertheless, few novel studies targeting endogenous plants are being conducted in Lebanon in an attempt to discover new molecules that exert antibiotic activity [54,55].Whether these attempts would be successful and helpful in the ongoing effort to combat resistant strains remains to be unveiled.