Fecal carriage of extended-spectrum β-lactamases and AmpC-producing Escherichia coli in a Libyan community

The emergence of extended-spectrum-β-lactamase (ESBL)-producing E. coli in the community has been one of the most significant epidemiologic changes in infectious disease in recent years. The epidemiology of ESBL-associated infections is complex and combines the expansion of mobile genetic elements with specific clonal dissemination [1, 2]. In the last decades, worldwide, Enterobacteriaceae producing ESBLs of the CTX-M type have shown a rapid and alarming dissemination and have been recognized as the most prevalent causative agents of hospital- and community-acquired infections [3–5]. The CTX-M- β lactamases, of which there are now over 150 different types detected in very distant regions such as Europe, the Near and Far East, South America, North America, and more recently in Africa are responsible for the recent huge increment in ESBLs throughout the world [3, 6–10]. With the wide distribution of these enzymes worldwide, it seems that there are predominant enzymes in each region (3). However, there is limited data regarding the molecular epidemiology of ESBL-producing Enterobacteriaceae in the Middle East and North Africa (MENA) [11, 12].

Several studies have demonstrated the high prevalence and diverse clones of CTX-M-producing E.coli from healthy humans [13, 14] and have reported the rate of intestinal colonization by commensal E. coli isolates harboring ESBLs in healthy volunteers [15–18]. Despite the serious threat encountered by the transmission of these potentially harmful organisms in the community among healthy members of the same family unit, the rate of fecal carriage of ESBL-producing Enterobacteriaceae has mainly been investigated as part of nosocomial outbreak investigations [19] and the number of prospective longitudinal studies conducted in the hospital or in community settings remains rare [20]. In Libya, there is no a single report on the spread of bacterial resistance due to ESBL-producing isolates from either the hospital or community setting. We conducted the present study to investigate the occurrence of ESBL-positive E. coli isolates in human fecal flora and to characterize their encoding genes, and the clonal relationships.

In this study we investigated the occurrence of ESBLs in human fecal flora from Libyan children living in two cities during two time periods. Overall, the percentage of fecal ESBL-producing E. coli recovered is comparable to those reported from other parts of the world including Saudi Arabia [30] and Tunisia [14], but lower than those found in Egypt [18], Thailand [17], China [13], and Japan [31]. In our study, the majority of ESBL-producing E. coli was isolated from patients with community-acquired infections and not from hospitalized patients with diarrhea which suggests the ability of ESBL-producing isolates to spread in the community beyond the hospital environment and potentially exclude their limitation to nosocomial infections, thus helping to exacerbate public health concerns [5]. Leflont et al. [32] have recently reported the increased rate of ESBL fecal carriage in healthy subject populations living in Paris from a 0.6% to 6% over a five year period and indicated that acquisition from a common source related to food and person-to-person transmission may contribute to the spread of ESBL-producing E. coli within families.

The presence of these organisms in the bowel in high rates was suggested to be a risk factor for infections with these bacteria and their dissemination in fecal flora [33]. In developing countries most patients received antimicrobial treatment without prescription, such common practices in nearly all developing countries exert a selective pressure on E. coli organisms, known to be a common cause of community-acquired infections, [34] to develop such resistance that leads to a potential sustainable source for nosocomial infections. In our study, bla CTX-M gene was detected in all 18 ESBL-producing E. coli isolates and bla CTX-M-1 was the most commonly identified resistance mechanism in samples from the earliest surveillance point (April 2000 to March 2001). Of particular interest in this region, the existence of three types of CTX-M families identified during the first year of study with a preponderance of CTX-M-1 in previous year and a persistence of CTX-M-15 later.

This finding supports work by others indicating that a specific CTX-M enzyme can dominate in a geographical region [3]. A possible explanation could be due to the dynamic nature of organisms harboring these enzymes; over time the dominant mechanism will change, as the dominant clonal group changes. While CTX-M-15 is the most common CTX-M type in the Middle East region and North Africa, other investigators have reported that CTX-M-15 was not more prevalent than CTX-M-1 among the fecal isolates [16, 35]. The difference in distribution of CTX-M types was also attributed by some studies to the various sources of the CTX-M-producing E. coli isolates carried in the gut of patients [15]. Some previous reports suggested a possible transmission of ESBL-producing E. coli through the food chain [36]; interestingly, it was shown, particularly in France, that CTX-M-1 is the predominant CTX-M among food-producing animals [37].

In general, CTX-M-producing E. coli have predominantly been identified from the community as a cause of urinary tract infections. However, the high rate of their fecal shedding in the outpatient setting in Libya is an emerging epidemiological problem that requires application of updated control measures like routine rectal screening of patients admitted from the community, even though routine fecal examination sounds impractical and is not required even if the patients were admitted to an intensive care unit in most hospitals and institutions around the world [38].

It is worth-mentioning that CTX-M β-lactamase genes are widely known to be carried on a plasmid linked to mobile genetic elements that are utilized as vehicles for resistance genes horizontal movement, in addition to carry on resistance to other drug like aminoglycoside and fluoroquinolone [15, 39, 40]. CTX-M isolates from the current study demonstrated resistance to non-β –lactam classes including the aminoglycoside and fluoroquinolone families.

The coexistence of AmpC phenotype in an ESBL isolate is a serious phenomenon that can mask the accurate detection of the ESBL phenotype [41]; this might have a critical effect on the success of clinical treatment. In our study, nine CTX-M positive isolates exhibited AmpC phenotype and CMY-2 was the main gene detected. Other AmpC genes previously reported (23) were not tested for the remaining negative samples. A recent study from Egypt has reported the occurrence of both resistance mechanisms for ESBL Enterobacteriaceae from outpatients and hospitalized cases, recommending the proper use of modified double-disc synergy test (MDDST), in addition to confirmatory PCR testing in order to avoid the unsuccessful identification of such isolates when they coexist [23].

Multiple studies have associated the dissemination of ESBL-CTX-M-15-producing isolates with the spread of the epidemic ST131 E. coli strain belonging to phylogenic group B2 [12, 33]. Interestingly, our phylogenetic group analysis demonstrated that most of CTX-M-15 positive isolates did not fall under B2 group, and according to the MLST typing system, epidemic ST131 clone, most globally ST associated with CTX-M-15, was not identified, indicating that this clone is less represented in fecal ESBL-producing E. coli, which is in agreement with other recent studies from Tunisia [15] and Spain [36]. In contrast, the present study identified seven ST clones associated with the dissemination of CTX-M-15 that are not previously implicated in isolates producing-ESBL, in addition to two emerged ST reported earlier in Spain (ST155) and Tunisia (ST23) from fecal E. coli with carbapenamases and ESBL activites, respectively. This particular characteristic of Libyan CTX-M-15 isolates indicates that their dissemination occurred by heterogeneous clones with a different genetic background.

In summary, this is the first detailed description of fecal CTX-M-producing E. coli in Libya. The dynamic nature and high clonal diversity of fecal CTX-M-producing isolates amplify the importance of the intestinal tract to act as a reservoir for ESBL-producing organisms, and a trafficker of antibiotic resistance genes. The increased prevalence of these organisms in human fecal flora and their establishment in the community represent an opportunity for these clones to become persistent. Because of the significant public health implications, the spread of organisms producing CTX-M-producing isolates merits close monitoring with enhanced surveillance efforts [31], with the introduction of molecular diagnostic procedures in a clinical or reference laboratory to track their spread in the community and hospital settings.