Cephalosporin resistant Escherichia coli from cancer patients in Cairo, Egypt

Authors


Correspondence

Caterina Mammina, Department of Sciences for Health Promotion and Mother-Child Care “G. D'Alessandro”, Via del Vespro 133, Palermo I-90127, Italy.

Tel: +39 0916553623; fax: +39 0916553641; email: caterina.mammina@unipa.it

ABSTRACT

Cephalosporin-resistant Escherichia coli has been increasingly reported worldwide. In this study, 32 cephalosporin resistant E. coli isolates identified from cancer patients in Cairo, Egypt in 2009–2010 were analyzed. Twenty-three were of phylogenetic group D, seven A and one each B1 and B2. By rep-PCR 15 phylogroup D isolates were grouped in four clusters, one with sequence type (ST) 405 and three ST68. Seventeen isolates showed single patterns. blaCTX-M-15 and aac(6')-Ib-cr were the most common resistance determinants. blaOXA-48 and blaVIM were also detected. Multidrug resistant E. coli seriously affects healthcare, especially in immunocompromised hosts, such as cancer patients.

List of Abbreviations
ESBL

extended spectrum β-lactamase

MIC

minimum inhibitory concentration

MLST

multilocus sequence typing

PMQR

plasmid-mediated quinolone resistance

ST

sequence type

UPGMA

unweighted pair group method with arithmetic mean

Escherichia coli is a major cause of both community and healthcare-associated infections [1, 2]. Extra-intestinal infections due to E. coli increase morbidity, mortality, and healthcare costs in hospitalized patients [3]. Their impact can be especially severe in immunocompromised patients, such as cancer patients receiving chemotherapy [4].

Extended spectrum β-lactamases, AmpC and carbapenemase-producing E. coli have been reported worldwide [2]. PMQR genes have also been increasingly reported [5, 6]. To date, at least three types of PMQR determinants, namely qnr families, aac(6′)-Ib-cr and quinolone efflux pump (qepA and oqxAB) have been extensively described in E. coli [3, 5, 6]. In particular, qnr genes have been frequently detected among isolates producing ESBLs [6]. Additionally, a close association between aac(6′)-Ib-cr and CTX-M-15, an ESBL that has emerged worldwide, has been reported by many epidemiological studies [6]. Recent studies in Egypt have reported a high prevalence of CTX-M-15 encoding genes among different E. coli clones in community and hospital settings [7, 8]. The aim of this study was to investigate the molecular epidemiology and resistance determinants pattern of cephalosporin resistant E. coli isolates identified from cancer patients in Cairo, Egypt in 2009–2010.

A retrospective analysis of E. coli isolates from clinical samples was performed at the National Cancer Institute, Cairo, Egypt, from January 2009 to June 2010. Identification and antimicrobial susceptibility testing of gram-negative isolates had been performed in the microbiology laboratories of the hospitals of origin by routine methods. Thirty-two of 73 viable isolates (43.8%) were selected after ESBL production screening according to the following MIC breakpoints: cefotaxime, ≥8 mg/L; ceftazidime, ≥2 mg/L and aztreonam, ≥8 mg/L [9]. Duplicate isolates from the same patient with indistinguishable susceptibility patterns were excluded. Basic demographic and clinical data were obtained from the databases of the microbiology laboratories. Because the study consisted of a retrospective review of routine microbiological data that were analyzed anonymously, approval by the Ethics Committee and informed consent were not required.

Minimum inhibitory concentrations of amoxicillin–clavulanic acid, cefotaxime, ceftazidime, imipenem, meropenem, gentamicin and ciprofloxacin of the 32 selected isolates were assessed by E-test (Biomérieux, Marcy l'Etoile, France). Assignment of E. coli phylogenetic groups was performed by the triplex PCR assay described by Clermont et al. [10]. Clonal relationships were established by rep-PCR amplification using the DiversiLab Escherichia fingerprinting kit (BioMérieux) according to the manufacturer's instructions [11]. Rep-PCR products were detected and sized using microfluidic LabChips placed on an Agilent 2100 bioanalyzer (Agilent Technologies, Diegem, Belgium). DNA fragment patterns were then analyzed by using Pearson correlation coefficient pairwise pattern matching and the UPGMA clustering algorithm. Representative E. coli isolates of the four rep-PCR clusters, unclustered phylogroup D isolates and two additional isolates of special epidemiological interest were characterized by MLS) using the Achtman typing scheme (mlst.ucc.ie/mlst/dbs/Ecoli) according to the protocols published on the website.

A set of six multiplex PCRs was used to screen the isolates for OXA-1-like broad-spectrum β-lactamases, CTX-M, SHV and TEM, plasmid-mediated AmpC β-lactamases and class A, B and D carbapenemases [12]. New Delhi metallo-β-lactamase 1 was searched for using specific primers [13]. PMQR genes qnrA, qnrB, qnrC, qnrD, qnrS, qepA and aac(6')-Ib-cr were investigated by PCR as previously described [5]. Identity of the β-lactamase and quinolone resistance genes was confirmed by DNA sequence analysis.

Twenty-seven of the 31 isolates for which information was available were from adult and four from pediatric cases. All but one patient were hospitalized and 24 were receiving imipenem treatment. There was only one instance of two isolates with different susceptibility patterns from the same patient. A high proportion of isolates was from fecal samples (14/31), followed by exudates and blood (6 and 5, respectively) and other normally sterile sites.

All isolates were confirmed by E-test to be resistant to cefotaxime and/or ceftazidime. Only one isolate was resistant to carbapenems. Fourteen and 24 isolates were resistant to gentamicin and ciprofloxacin, respectively.

The E. coli isolates were unevenly distributed into the four phylogenetic groups, 23 belonging to group D, 7 to A and 1 each to B1 and B2 (Table 1). Consistent with previous reports from Egypt and other low-resource countries, phylogroups A and D were predominant, whereas the hyperepidemic strain B2-ST131 was under-represented [8].

Table 1. Phenotypic and genetic resistance patterns of 32 extended spectrum cephalosporin resistant Escherichia coli isolates from cancer patients, Cairo, Egypt, 2009–2010
No. of isolatesTime of isolationMIC (µg/mL)*,bla genes*Presence of*Phylogroup*ST
AMCCTXCAZIPMMEMGENCIPqnrSaac(6')-Ib-cr
  • *in parentheses number of isolates;
  • MIC values were obtained by E-test;
  • non-clustered isolates are grouped by pattern of resistance determinants.
  • AMC, amoxicillin–clavulanic acid; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; GEN, gentamicin; IPM, imipenem; MEM, meropenem; ND, none detected; NT, not tested; ST, sequence type.
Clustered isolates
6—Cluster 1May 2009–May 20108–12>25632–>2560.250.047–0.0941.5 (1), 64–96 (5)0.19 (1), >32 (5)ND (1)− (1)D405
         CTX-M-15 (15) + (5)  
2—Cluster 2June 2009, November 20096–8>25632–480.250.0321.5>32CTX-M-15 D68
2—Cluster 3June 2009, May 20108>25632–480.250.0320.75>32CTX-M-15+D68
5—Cluster 4May 2009–October 20096–8>256320.250.0321.5>32CTX-M-15+D68
Non-clustered isolates
1May 20091696320.190.02332>32CTX-M-15+A
1June 2009123220.190.0230.500.19CTX-M-15NTNTDST68
3July–October 20096–864–9616–240.023–0.190.016–0.0320.50–1.50.19–0.38CTX-M-15NTNTD (2)ST68
            A
            B2131
5June 2009–May 20106–1218–>2568–>2560.125–0.250.023–0.03212–96>32CTX-M-15+D (3)ST68
            A
            B2131
1May 20094180.1250.016320.38CTX-M-15, SHV-12+A
1October 20096>256960.250.09464>32CTX-M-15, SHV-12+DST68
1February 2009122.580.190.0160.75>32CMY-2A
1June 200962160.190.03232>32SHV-12+A
1August 20092>56>2561.50.501.5>32CMY-2, SHV-12, VIM-1+A
1December 2009128>256>25624>3232>32CMY-2, OXA-48, VIM-29B1101
120096424160.380.25240.19CTX-M-15, OXA-48NTNTDST648

Rep-PCR fingerprinting enabled the identification of four clusters, including 15 phylogroup D isolates, and 17 single patterns (Fig. 1). This suggests that the observed over-representation of phylogroup D might be at least partially explained by intra-hospital cross-transmission. In contrast, the heterogeneity of group A isolates which, along with group B1, are reportedly frequently associated with commensal organisms, suggests a prominent epidemiological role for this phylogroup in the region under study. According to MLST one cluster belonged to ST405 and the remaining three to ST68. All but one of the non-clustered phylogroup D isolates were also attributed with ST68. Isolates D/ST405 have been repeatedly reported to express a multiresistant phenotype [2, 8]. In contrast, isolates D/ST68 carrying blaCTX-M-15 and aac(6')-Ib-cr were an unexpected finding. Indeed, only two D/ST68 isolates containing blaCMY-2 have been reported recently, both from wild coastline birds in Miami Beach, Florida, USA [14]. The B2 strain belongs to the worldwide spread ST131 [2].

Figure 1.

Dendrogram showing banding patterns and clonal relationships of ESBL-Escherichia coli by rep-PCR, interpreted by the Pearson method (8). Molecular subtype clusters were defined as having a minimum of 95% similarity.

All but one isolate in cluster 1 and 13 non clustered isolates showed a blaCTX-M-15 gene, which was consistent with the global predominance of this ESBL [2]. SHV-12 and CMY-2 were detected in only four and three non-clustered isolates, respectively. Three isolates co-produced OXA-48 and/or VIM carbapenemases (Table 1). Although carbapenemases have been infrequently detected in E. coli, recently some reports have highlighted the emergence of blaOXA-48 and blaVIM carrying isolates [15, 16]. Recently, OXA-48-producing E. coli identified in France from patients transferred from Egypt were described [16]. Our findings thus confirm the hypotheses about a likely endemic circulation of OXA-48 in Egypt and other north African countries [16].

Of special interest, the carbapenem-resistant isolate of phylogroup B1 containing blaCMY-2, blaOXA-48 and blaVIM-29 was attributed with ST101. This supports the concerning evidence of a previous study by Mushtaq et al. who reported that 9/18 isolates of NDM-producing E. coli from England, Pakistan and India were B1-ST101 [17].

Finally, ciprofloxacin resistance was associated with the presence of qnrS in only two phylogroup A isolates, whereas in all the remaining strains aac(6')-Ib-cr was detected (Table 1). Twenty of 27 ciprofloxacin resistant E. coli isolates showed an association with blaCTX-M-15 and aac(6')-Ib-cr genes. Thus, the genetic makeup which has driven the success of the ST131 pandemic clone appears to be diffuse among E. coli strains of different lineages and habitats.

Acquisition of multidrug resistance gene traits by a widely disseminated human commensal organism on a global scale may seriously affect human health and healthcare resources by causing difficult-to-treat infections in both community and healthcare settings, thus increasingly fueling the antibiotic crisis [1, 2]. The impact may be devastating in limited resource countries and immunocompromised hosts, such as cancer patients. A previous report from Egypt described rates of resistance to third generation cephalosporins of approximately 60%in bloodstream isolates of E. coli from five hospitals in Cairo, Egypt in 1999–2000 [18]. Our findings confirm an alarming picture of multidrug resistance in E. coli and highlight acquisition of a variety of resistance genetic determinants in association with PMQR genes and the emergence of resistance to carbapenems.

ACKNOWLEDGMENTS

This work was financially supported by Institutional funds of the Department of Sciences for Health Promotion and Mother-Child Care “G. D'Alessandro.”

DISCLOSURE

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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