β-Lactamase production is the predominant mechanism for resistance to β-lactams in Enterobacteriaceae. Extended-spectrum β-lactamases (ESBLs) have been reported globally, most often in Escherichia coli and Klebsiella pneumoniaee¹. ESBL-producing K. pneumoniaee have spread quickly and pose a serious risk of healthcare-associated infections. There is limited data regarding the molecular epidemiology of ESBL-producing Enterobacteriaceae in the Middle East and North Africa [1, 2]. AmpC production is one of the mechanisms of resistance to β-lactams in enterobacteria, conferring resistance to all β-lactams except fourth-generation cephalosporins and carbapenems, and is typically associated with multidrug resistance (MDR) [3]. Treatment options are severely limited because AmpC is often associated with other multiple resistance genes, such as those of resistance to quinolones as well as other β-lactamase genes [3, 4]. The genes encoding these enzymes are chromosome or plasmid borne [5]. In particular, K. pneumoniaee have acquired plasmid-mediated AmpC β-lactamases [6]. Based on the sequence similarities with species-specific AmpC enzymes, plasmid AmpC variants are classified into five evolutionary groups: the CIT variants (CMY-2 types) originating in Citrobacter freundii, the Enterobacter sp. EBC variants (ACT-1 type, MIR-1), the Morganella morganii DHA variants, the Hafnia alvei ACC variants, and the Aeromonas sp. FOX and MOX variants [3, 4]. The geographic scattering of the different types of AmpC shows that the CMY-2 type is the most frequent, particularly in Europe [7], and in North Africa [8-10]. In Libya, only a few reports on AmpC production in Enterobacteriaceae strains were published [11, 12]. The aim of this study was to investigate the prevalence and molecular epidemiology of cefoxitin resistance bla_AmpC genes among K. pneumoniaee and E. coli isolates recovered from hospitalized patients in Tripoli, Libya.

Of the K. pneumoniae and E. coli tested, 75% and 16% were resistant to gentamicin, 74% and 1.3% to imipenem, 71% and 12% to cefoxitin, 80% and 12% to Cefepime, 69% and 22.6% to ciprofloxacin, respectively. The isolates remained susceptible to colistin (Table 1). None of the E. coli isolates were defined as MDR compared with K. pneumoniae (50/76; 65.8%). The incidence of ESBL producers was significantly higher among K. pneumoniae 65/76 (85.5%) compared with 13/75 (17.3%) of E. coli isolates (P <0.0001, OR=4.93). Using PCR, plasmid-mediated AmpC genes were detected in 7.9% (6/76) of K. pneumoniae, and 4% (3/75) in E. coli isolates Fig. (1). Therefore, phenotypic detection of AmpC was only presented for these nine isolates, the origin and characteristics of clinical interest of these isolates are summarized in Table 2. Only 4/9 (44.4%) were positive using E-test and 3/9 (33.3%) for AmpC screen test disk test. These methods failed to detect one isolate (false-negative) even though the isolates was AmpC positive using PCR. These results demonstrate that phenotypic testing assays on these isolates underestimated detection of AmpC enzyme production and did not correlate well with molecular results. The performance of different AmpC confirmatory tests in combination with different antibiotic and inhibitor combinations is shown in Table 2.

Fig. (1). Amplification of plasmid-mediated AmpC gene in ESBL producing K. pneumoniae isolates (1-6) by single PCR.

Table 3 shows the distribution of antibiotic resistance among AmpC positive isolates. All AmpC positive strains were resistant to cefoxitin and the majority were resistant (7/9; 77.8%) to cefepime and recovered from patients hospitalized in different intensive care units (ICUs). The majority of AmpC positive isolates 6/9; 66.6% (4 in K. pneumoniae, and 2 in E. coli) were found to carry the CMY encoding gene. A single AmpC gene was detected in 5/9 isolates, i.e, bla_CMY (n=3),bla_MOX (n=1), bla_DHA (n=1) however, multiple AmpC genes were detected in 3/9 isolates with the following distribution: bla_CMY and bla_EBC (n=1), and bla_CMY and bla_MOX (n=2). Neither bla_FOX nor bla_ACC was detected in all tested isolates. None of the tested six encoding gene variants were detected in one isolate (Table 4).

Fig. (1). Amplification of plasmid-mediated AmpC gene in ESBL producing K. pneumonia isolates (1-6) by single PCR Lane M 50bp ladder; Lanes 1 to 6 AmpC-producing isolates, lane 7 is positive control. The amplified amplicon size is 481bp.

The occurrence, types and rate of dissemination of AmpC enzymes has increased worldwide, their early detection is crucial and critical since AmpC β-lactamases show marked variation in geographic distribution [17]. Detection of pathogens producing AmpC β-lactamases is often associated with potentially fatal laboratory reports of false susceptibility to β-lactams phenotypically [18]. Thus, their accurate, authentic and valid detection are important from epidemiological, clinical, laboratory, and infection control methods, especially in developing countries. In the present study, we investigated the incidence of plasmid-mediated AmpC among K. pneumoniae and E. coli clinical isolates from Tripoli hospitals in Libya.

K. pneumoniae isolates were found less susceptible to all antimicrobial agents tested and none of E. coli isolates were defined as MDR compared with K. pneumoniae (65.8%). In contrast to the previous study, we found that 33.2% of E. coli and 42% of K. pneumoniae were defined as MDR [12]. This study showed that the incidence of ESBL producers was significantly higher among K. pneumoniae (85.5%) compared with (17.3%) of E. coli isolates. These results indicate that K. pneumoniae strains represent a major therapeutic and epidemiological threat and require the implementation of strict hygiene procedures and regular surveillance studies to determine the genetic basis of resistance.

There was low agreement between genotypic and phenotypic methods used in this study, only 4/9; 44.4% and 3/9; 33.3% of genetically identified AmpC producers were found phenotypically positive using E-test for combined disc diffusion method respectively. Therefore, the genotypic and phenotypic methods used for detection of AmpC did not correlate well. Detecting plasmid mediated AmpC with co-existing ESBL, ampC gene was not functional and/or expressed at low levels is very challenging. Given these difficulties in detecting plasmid mediated AmpC β-lactamases, their prevalence is currently being underestimated. Bolmstrom and colleagues showed that the overall sensitivity and specificity were 88 to 93% using E-test strips for detection of AmpC [19, 20]. Similar to our technique using cefepime alone and in combined with β-lactam inhibitor has been evaluated previously and the authors found that this method was the most sensitive test (66.1%) for AmpC co-producers [21]. It has been suggested that the most convenient method for detection of AmpC was the double-disk test [22, 23]. Cefoxitin insusceptibility is a useful screen for Klebsiella spp., Salmonella spp., C. koseri, P. mirabilis, and E. coli in areas where the ACC-1 and ACC-4 enzymes are not encountered (so far not detected in Libya). Phenotypic detection of AmpC in E. coli does not indicate if the enzyme is chromosomal or plasmid mediated, but as a crude guide, lack of multiple drug resistance is suggestive of a chromosomal AmpC whereas multiple drug resistance is consistent with either plasmid-mediated or chromosomal AmpC production [24]. Therefore, phenotypic tests cannot distinguish between the various families of plasmid-mediated AmpC enzymes and may also overlook chromosomally determined AmpC β-lactamases with an extended spectrum and hence, it usually poses a problem due to misleading results [25, 26]. Recently, khari and colleagues evaluated different AmpC confirmatory testes showed that there was low agreement between the genotypic and phenotypic detection of AmpC β-lactamases, and suggested that the phenotypic detection of AmpC β-lactamase production has been hampered by the lack of validated methods [27]. Furthermore, phenotypic detection of plasmid-mediated AmpC β-lactamases has been described to have poor specificity and is not advisable for routine detection of these β-lactamases [28]. In contrast to these authors, Reuland and co-authors found not the E-test but double disk combination test cloxacillin as the best test, with the best sensitivity and specificity after the combination of screening criteria, the authors suggested that the difference might be due to differences in the selection of strains [29].

Overall, AmpC gene was detected in 7.9% of K. pneumoniae, and 4% in E. coli isolates, all isolates were recovered from different ICU patients and mainly from Pediatric hospital. The majority of AmpC positive isolates 66.6% were found to carry CMY encoding gene followed by MOX; DHA and EBC. Our findings are consistent with previous reports noting the predominance of CMY worldwide [30-32]. In accordance with the earlier study in Tunisia reported the coexistence of various bla_AmpC genes in a single strain and of such coorcurrence in several species in Enterobacteriaceae [33], multiple AmpC genes were detected in one-third of isolates: one isolate expressing bla_CMY and bla_EBC genes co-exist together, the other two isolates co-expressing bla_CMY and bla_MOX genes. However, in 5/9 isolates only one AmpC gene was detected bla_CMY, bla_MOX or bla_DHA. Similar to our findings neither bla_FOX nor bla_ACC were detected in all tested isolates in Algiers hospitals [34]. Two studies in Egypt reported that no genes belonging to ACC were detected in all tested isolates 35-37.None of the targeted six encoding gene variant primers used in the present study were detected in one isolate or indicates they were most likely AmpC hyperproducers that showed positive results phenotypically using one technique because of overexpression of the chromosomal AmpC gene. To our knowledge, this is the first description of these genes in Libyan hospitals.

The shortcoming of the study was multiplex PCR was not performed; detection of plasmid mediated AmpC β-lactamases and other AmpC variants were not investigated. This emphasizes the need for such enzymes detection for preventing this emerging resistance into hospitals and for controlling its spread within the com-munity. That will avoid therapeutic failures and nosocomial outbreaks.

PCR is the gold standard method for detection of AmpC β-lactamase. The dissemination of cefoxitin resistance genes within the hospitals may indicate nosocomial healthcare issue. The most prevalent AmpC gene belongs to CMY followed by MOX; DHA and EBC. Hence, identification of types of AmpC may help the physician to prescribe the most appropriate antibiotic, thus decreasing the selective pressure, which generates antibiotic resistance.