Molecular and Epidemiological Characterization of Enterobacterial Multidrug-Resistant Strains in Tlemcen Hospital (Algeria) (2008–2010)

Introduction

CTX-M β-lactamases are recognized as the most widespread extended-spectrum β-lactamases (ESBLs) among clinical isolates of Enterobacteriaceae. They are able to compromise efficacy of all β-lactams, except cephamycins and carbapenems. ¹¹ More than 65 bla_CTX-M genotypes have been described in the world (www.lahey.org/studies/inc_webt.asp). Among them, bla_CTX-M-15 gene has recently emerged as the dominant type. It was originally characterized in India in 2000 ²¹ and involved in several nosocomial outbreaks.^5,28,31 Its association with mobile elements such as ISEcp1 can explain the ease with which its spreading among bacteria in clinical setting.^37,40 bla_CTX-M-15 gene is commonly found on transferable plasmids that additionally carried other antibiotic resistance genes with recent reports from Canada, Europe, and North Africa, particularly Central Maghreb (Tunisia, Algeria, Morocco).^{4,5,12,13,23,26,28,29,33}

In Algeria, the first report of the presence of CTX-M-producing Enterobacteriaceae came in 2005, from Bejaia, east of Algeria. ⁴² Four isolates (3 Escherichia coli and 1 Klebsiella pneumoniae) were found to produce CTX-M-15 and one isolate (Enterobacter cloacae) was found to produce CTX-M-3. Since then, few Algerian surveys have reported the presence of ESBLs and associated resistance to non β-lactam markers in clinical isolates.^{2,22,23,29,30,39,43}

More recently, the class D carbapenemase bla_OXA-48 gene has been reported in Morocco and in Tunisia. ³⁸ These countries can be considered to be important reservoirs of OXA-48 producers. Currently, no data are available from Algeria. However, Poirel et al. suggested that Algeria could be also a country where OXA-48 may be endemic, following positive observations in patients transferred from that country. The OXA-48 carbapenemase could confer a slight decrease of susceptibility to carbapenems (when no decrease of permeability was associated), which makes its phenotypic detection difficult. Moreover, OXA-48 is very often associated to ESBLs, such as CTX-M-15, CTX-M-14, or SHV-12.

Tlemcen is located at 600 km from the center of Algeria and 76 km east of the Moroccan town of Oujda. Tlemcen is a town with ∼850,000 inhabitants. In the university-affiliated Hospital of Tlemcen, a public institution with 800 acute-care beds, we have observed an increase in the resistance of Enterobacteriaceae isolates. The purpose of this study was to characterize these isolates to (1) determine the specific type of β-lactamases (ESBLs and class D carbapenemase), (2) identify the resistant genes to non β-lactam antibiotic, and (3) analyze their clonal relationship.

Materials and Methods

Results

Seventy-one collected isolates (17 E. coli, 50 K. pneumoniae and 4 E. cloacae) were defined as ESBL producers according to their resistance to the markers antibiotics and DDST results. All of them were highly resistant to ticarcilline (MIC>512 μg/ml) and piperacilline (MIC=512 μg/ml). The cefotaxime MICs were ranged from (64 to >512 μg/ml), ceftazidime from (8 to 128 μg/ml), gentamicin from (32 to >512 μg/ml) and those of tobramycin from (8 to >512 μg/ml). All isolates remained susceptible to imipenem (MICs 0.125 to 0.5 μg/ml). Ertapenem and meropenem were not tested because they are not available in Algeria. Sixty-four strains (except 7 E. coli) were resistant to ciprofloxacin (MICs 8 to 64 μg/ml), and 57 to trimethoprim-sulfamethoxazole (diameter=7 mm).

Epidemiological investigation showed that the 71 enterobacterial isolates yielded 25 distinct rep-PCR patterns: 11 patterns among 17 E. coli, 12 patterns among 50 K. pneumonia, and 2 patterns among 4 E. cloacae. Each clones of E. coli included one to four isolates; four clones belonged to phylogenetic group A1, three to phylogenetic group B2 (1 B2-2 and 2 B2-3), and four to phylogenetic group D1 (Table 1). Only one strain of E. coli B2-3 belonged to the ST131. Two major clones were observed among K. pneumoniae isolates and were designed CKp1 (11 isolates, 22%) and CKp5 (25 isolates, 50%). The clone CKp1 was recovered only in 2008 in ICU, whereas CKp5 predominated in 2009 and was recovered in both surgery and ICU. CKp1 and CKp5 corresponded to two different sequence types (STs), respectively, ST931 (clone CKp1) and ST15 (clone CKp5). Isolates from both major clones possessed virulence genes known to be frequent in this bacterial species, such as fimbrial and nonfimbrial adhesins (ycfM, mrkD) and the iron-scavenging system entB. In contrast, only ST15 strains (clone CKp5) yielded yersiniabactin, a siderophore encoded by the Yersinia high-pathogenicity island that is less frequent in K. pneumoniae and is implicated in respiratory tract infections.

CTX-M-encoding genes were detected in all isolates and in their transconjugants. The results of PCR and sequence analysis are summarized in Table 1. The deduced amino acid sequences corresponded to CTX-M-15 in 69 (97%) isolates (16 E. coli, 50 K. pneumoniae, and 3 E. cloacae) and CTX-M-3 in 2 (3%) isolates (E. coli and E. cloacae). The bla_TEM-1 gene was identified in 7 E. coli, 33 K. pneumoniae and 4 E. cloacae. The bla_OXA-1 gene was detected in 44 isolates (8 E. coli, 33 K. pneumoniae, and 3 E. cloacae). Among the 69 bla_CTX-M-15 positive, 39 (56%) isolates possessed both bla_TEM-1 and bla_OXA-1. None of the studied strains produced the class D carbapenemase OXA-48.

The aminoglycosides modifying enzymes encoding genes aac(3′)-II and aac(6′)-lb-cr were found in 43 (60%) of the isolates. Their simultaneous production was found in 41 (58%) of the isolates. The qnrB2 gene was found in 7 (10%) isolates and was always associated to the aac(6′)-lb-cr gene. Concerning the resistance to sulphonamides, PCR analysis indicated that 50 isolates carried the sul2 gene, 22 the sul1 gene; 15 carried both sul1 and sul2. Isolates with sul1 gene positive were associated with class 1 integron structures. The combination of genes cassettes identified in those 22 isolates were dfrA1-aadA2 (n=2), dfrA12-aadA2 (n=17), and aadA2 alone (n=3).

Mating experiments carried out on the 25 representatives ESBL isolates allowed the transfer of bla_CTX-M genes from 18 isolates (8 E. coli, 8 K. pneumonia, and 2 E. cloacae). Nine transconjugants were resistant to aminoglycosides (GM/GM-TM), five to ciprofloxacin and six to trimethoprim-sulfamethoxazole. The spacer region between ISEcp1 and bla_CTX-M gene was characterized by W and V+W sequences in 15 and 10 isolates, respectively. All isolates with V+W sequence were positive for IncL/M incompatibility group of plasmids, whereas all isolates with W spacer sequence were positive for IncF group or not determined.

Discussion

At Tlemcen hospital, 71 enterobacterial strains producing ESBLs were collected between April 2008 and March 2010. All of the isolates carried the bla_CTX-M determinant. Sixty-nine of them produced CTX-M-15 and 2 CTX-M-3. Our results are in accordance with those of previous studies in Algeria.^39,42 Surprisingly, OXA-48 was not found in our collection of strains recovered between 2008 and 2010. These results suggest that this gene was not endemic of this Algerian area, although it is near a Moroccan town.

The genotypic analysis of E. coli strains by rep-PCR showed a high diversity: 11 different rep-PCR patterns were individualized among the 17 examined isolates. Four patterns corresponded to phylogenetic group A1, three to phylogenetic group B2 (1 B2-2 and 2 B2-3), and four to phylogenetic group D1. Only one strain belonged to ST131. This clone with a high virulence potential has been reported all over the world.^9,32

For K. pneumoniae strains, the rep-PCR analysis showed two major clones producing CTX-M-15: CKp1 (ST931, n=11) and CKp5 (ST15, n=25). In contrast with CKp5 (ST15), CKp1 (ST931) was only found in 2008. This finding tends to suggest that CKp5 has replaced CKp1 in ICU and disseminated in other wards of Tlemcen hospital. This suggestion is reinforced by the fact that the K. pneumoniae (ST15) producing CTX-M-15 ESBL is an epidemic clone which has been spread across Europe ¹⁵ and Morocco. ³⁶ It was also the only one to harbor the yersiniabactin gene, which has been described among KPC-producing K. pneumoniae isolates of ST258. ³ This can explain their greatest virulence (number of patients) and therefore, their persistence during the period of the study. Further investigations are needed to establish a link between ybtS and the spread of multiresistant K. pneumoniae isolates.

Multiresistance of Enterobacteriaceae has often been described for ESBL and particularly CTX-M-producing clinical isolates.^5,6,17,26 Most of our E. coli isolates (including the ST131) also contained the bla_TEM-1 and/or bla_OXA-1 genes, whose occurrence has been previously described in CTX-M-15-producing strains in the world. In addition, the aminoglycoside (aac(3′)-II) resistance genes as well as aminoglycoside-quinolone (aac(6′)-Ib-cr) resistance genes were present in 8 (50%) and 6 (35%) isolates, respectively. In the case of K. pneumoniae, the strains of CKp1 clone (n=11, 20%) were resistant to sulphonamides and trimethoprim, whereas those belonging to the CKp5 clone (n=25, 50%) expressed resistance aminoglycosides, ciprofloxacin, and sulphonamides, and possessed bla_TEM-1 and bla_OXA-1 genes. This multiresistance could also, in part, explain the persistence of the clone CKp5 (ST15) over the study period.

The high level of resistance to ciprofloxacin (MIC=64 mg/L) in our isolates (except in seven E. coli) made treatment very difficult. It has been postulated that plasmid-mediated low-level resistance to fluoroquinolones may encourage the selection of gyrA mutants with high-level resistance. ²¹ In contrast with the previous report in Algeria,^23,29,43 the qnrB2 allele was found in this study. It was less frequent than the aac(6′)-Ib-cr gene and was absent in E. coli strains. The larger dissemination of AAC (6′)-Ib-cr compared to Qnr determinants has been previously described. ²⁷ Four E. coli (23%), 16 K. pneumoniae (36%), and one E. cloacae were resistant to ciprofloxacine without production of plasmid-encoded quinolone resistance genes. This result and the high level of resistance to ciprofloxacin observed also in strains producing the aac(6′)-Ib-cr gene suggest that the main mechanism of resistance to fluoroquinolone in our strains is probably due to punctual mutations in gyrases or topoisomerases encoding genes.

The sul1 gene was less frequent than sul2 gene in our isolates. This finding is in accordance with previous studies that report sul2 gene to be more widespread among clinical isolates. ²⁵ However, it differs from the distribution of sulfamethoxazole-resistant allele observed in Tunisia. ¹⁴ Among the studied isolates, 7 E. coli, 12 K. pneumonia, and 3 E. cloacae harbored class 1 integrons, which included in their variable region the aadA2 and the dfr (A1 and A12) gene cassettes related to streptomycin and trimethoprim resistance, respectively. The aadA2 alone or in association (dfrA1-aadA2, dfrA12-aadA2) has been previously described in different bacterial isolates.^1,19,20 The dfrA12 gene cassette was the most prevalent in our study. This observation does not correlate with previous findings in African Enterobacteriaceae isolates.^14,18

The spacer region between ISEcp1 and bla_CTX-M-15 in 9 of the 25 representative isolates was characterized by the V+W sequence. In 7 of them, bla_CTX-M-15 was positively mobilized by conjugation with plasmid of incompatibility group IncL/M. This result is in accordance with findings of Messai et al. ³⁰ which showed that the CTX-M-15 enzyme in Algeria was developed from CTX-M-3 under clinical context. However, in 15 of the representative isolates, the spacer region was characterized by the W sequence between ISEcp1 and bla_CTX-M-15 and by V+W between ISEcp1 and bla_CTX-M-3. This organization was described by Eckert et al. ¹⁷ In the remaining CTX-M-3 E. coli E9 producer, the spacer region was characterized by the W sequence. This result suggests that the E. coli E9 could be a revertant.

In conclusion, we identified two bacterial populations of CTX-M-15 producers in our hospital, one that derived from the CTX-M-3 under Algerian clinical context and one that is universally found. The current dissemination of bla_CTX-M-15 was assigned to the epidemic clones K. pneumoniae ST15 and ST931 and to genetic transit of plasmids among unrelated strains. The absence of carbapenemase OXA-48 in the period of the study reinforces the idea that the epidemiology of beta-lactam resistance in Algeria could be different from that observed in the neighboring countries, such as Morocco and Tunisia.