Eur J Clin Microbiol Infect Dis (2012) 31:1511–1516 DOI 10.1007/s10096-011-1471-z
ARTICLE
Prevalence and characterisation of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in healthy volunteers in Tunisia R. Ben Sallem & K. Ben Slama & V. Estepa & A. Jouini & H. Gharsa & N. Klibi & Y. Sáenz & F. Ruiz-Larrea & A. Boudabous & C. Torres
Received: 26 July 2011 / Accepted: 18 October 2011 / Published online: 8 November 2011 # Springer-Verlag 2011
Abstract The objective of this investigation was to analyse the carriage rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faecal samples of healthy humans in Tunisia and to characterise the recovered isolates. One hundred and fifty samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 μg/ml) for ESBL-positive E. coli recovery. The characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, multilocus sequence typing (MLST) and phylogroup typing were performed by polymerase chain reaction (PCR) and sequencing. The presence and characterisation of integrons and virulence factors were studied by PCR and sequencing. ESBL-positive E. coli isolates were detected in 11 of 150 faecal samples (7.3%) and one isolate/sample was further characterised. These isolates contained the blaCTX-M-1 (ten isolates) and blaTEM-52c genes (one isolate). The ISEcp1 (truncated by IS10 in four strains) and orf477 sequences R. Ben Sallem : K. Ben Slama : A. Jouini : H. Gharsa : N. Klibi : A. Boudabous Laboratoire des Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Université Tunis-El Manar, 2092 Tunis, Tunisia V. Estepa : F. Ruiz-Larrea : C. Torres (*) Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios, 51, 26006 Logroño, Spain e-mail:
[email protected] Y. Sáenz : C. Torres Área de Microbiología Molecular, Centro de Investigación Biomédica de La Rioja, Logroño, Spain
were found upstream and downstream, respectively, of all blaCTX-M-1 genes. Seven different sequence types (STs) and three phylogroups were identified among CTX-M-1-producing isolates [ST/phylogroup (number of isolates)]: ST58/B1 (3), ST57/D (2), ST165/A (1), ST155/B1 (1), ST10/A (1), ST398/A (1) and ST48/B1 (1). The TEM-52-producing isolate was typed as ST219 and phylogroup B2. Six ESBL isolates contained class 1 integrons with the gene cassettes dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one). Healthy humans in the studied country could be a reservoir of CTX-M-1-producing E. coli.
Introduction An important increase in the prevalence of clinical Escherichia coli isolates harbouring extended-spectrum beta-lactamases (ESBLs), especially of the CTX-M class, has been observed worldwide in the last several years [1]. Furthermore, commensal ESBL-positive E. coli isolates from humans, food-producing animals and food of animal origin have also been reported [2–6]. Similarities between the type of ESBLs and plasmids detected in E. coli isolates from humans, animals and food have suggested the potential transference of these ESBL genes from animals to humans, most likely through the food chain [6]. CTX-M15 is the most frequent type of ESBL detected among clinical E. coli isolates in Tunisia [7–9]. However, this type of beta-lactamase has not been detected in food isolates in this country, in which the CTX-M-1 variant was frequently identified [3, 4]. No data exist about the prevalence of ESBLs in healthy humans in the Maghreb and very scarce data in other African countries [10]. The purpose of this study was to evaluate the carriage level of ESBL-positive
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E. coli isolates in healthy humans in Tunisia and to characterise their encoding genes in order to correlate data with those previously obtained in clinical and food isolates in this country.
Materials and methods Isolates and susceptibility testing One hundred and fifty faecal samples were collected during the period from December 2009 to March 2010 from healthy human volunteers (age range from 6 months to 72 years), living in five different urban areas of Tunisia (city/location/number of samples): Tunis/North-Eastern/ 109, Sfax/South-Eastern/16, El Kef/North-Western/12, Gafsa/South-Eastern/10, Kairouan/North-Central/3. None of the individuals included in the study had been exposed to antibiotics during the 3 months prior to the sample collection and all of them gave their consent for the participation in this study. Nine of the individuals were farmers or veterinarians and seven were medical doctors or sanitary personnel related with hospitals. The remaining 139 healthy individuals did not have any relation with the animal or the hospital environment. Samples were seeded onto MacConkey agar plates supplemented with cefotaxime (CTX, 2 μg/mL). After incubation at 37°C for 24 h, colonies showing E. coli morphology were recovered, identified by classical biochemical methods and by species-specific polymerase chain reaction (PCR) (amplification of the uidA gene) [3], and screened for ESBL phenotype by the double disk test [11]. One ESBL-positive E. coli isolate per positive sample was further studied. Susceptibility testing to 17 antibiotics was carried out by the disk diffusion method [11]. The antibiotics tested were as follows: ampicillin, cefoxitin, ceftazidime, cefotaxime, imipenem, aztreonam, gentamicin, amikacin, tobramycin, streptomycin, nalidixic acid, ciprofloxacin, sulphonamides, trimethoprim–sulfamethoxazole, tetracycline, rifampicin and chloramphenicol. E. coli ATCC 25922 was used as a control strain. Genetic typing of isolates E. coli isolates were characterised by multilocus sequence typing (MLST) [12] in order to ascertain the corresponding sequence type (ST) and clonal complex (CC) (http://mlst.ucc.ie/). The clonal relationship among the 11 E. coli strains was also determined by pulsed field gel electrophoresis (PFGE) using XbaI enzyme, as previously described [13]. The isolates were assigned to the phylogenetic groups A, B1, B2 or D using a PCR strategy with specific primers for chuA, yjaA and TspE4.C2 determinants [14].
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Serotyping and virulence genotyping of E. coli isolates All isolates were screened for the O25b serotype and for the afa operon [15, 16]. In addition, sxt, fimA, papG allele III, hlyA, cnf1, papC, aer, eae and bfp genes, encoding virulence factors often found in pathogenic E. coli (ExPEC) isolates, were tested by PCR [17]. Detection and characterisation of beta-lactamase genes and other resistance genes. The genetic environment of blaCTX-M genes The genes encoding TEM, SHV, OXA-1 and CTX-M type beta-lactamases and the genetic environment of blaCTX-M genes were analysed by PCR and sequencing [3]. The presence of genes associated with resistance to tetracycline [tet(A) and tet(B)], sulphonamides [sul1, sul2 and sul3], gentamicin [aac(3)-II and aac(3)-IV], streptomycin [strA and strB] and quinolones [qnr, qepA and aac(6′)-Ib-cr] was determined by PCR [8]. Detection and characterisation of integrons The presence of intI1 and intI2 genes (encoding class 1 and class 2 integrases, respectively) was examined by PCR [8]. The variable regions of class 1 and class 2 integrons were characterised by PCR and sequencing in all intI1- or intI2positive isolates. The presence of qacEΔ1-sul1 genes in the 3′-conserved segment of class 1 integrons was also investigated in all intI1-positive isolates [8].
Results ESBL-producing E. coli isolates were detected in 11 of the 150 (7.3%) analysed faecal samples. Three of the 11 positive samples corresponded to a poultry farmer, a veterinarian (had scarce contact with marine fish but not with other animals) and a medical doctor (working in the urgency service of a hospital). The other seven ESBLpositive individuals did not have any relationship with the hospital or the animal environment. All positive individuals had no relationship among them. If we consider the healthy individuals with no contact with hospitals or the animal environment, the prevalence of ESBL carriers would be 5.7% (8 out of 139 individuals). Additionally, ESBL carriers were detected in two of nine farmers/veterinarians and in one of seven medical doctors/sanitary staff. Table 1 shows the characteristics of these 11 isolates recovered from the 11 ESBL carriers. The ESBL genes found were the following: blaCTX-M-1 (nine isolates), blaCTX-M-1 +blaTEM-1b (one isolate) and blaTEM-52c (one isolate). All PCR tests performed to detect the presence of
Tunis Tunis
Tunis
C3139 C3138c
C3142e
e
d
c
b
a
Tunis Tunis Tunis Tunis Tunis Tunis Kairouan
ST219
ST48 ST398
ST57 ST57 ST155 ST58 ST58 ST58 ST10 B1 A
D D B1 B1 B1 B1 A
Singleton B2
CC10 CC398
CC350 CC350 CC155 CC155 CC155 CC155 CC10
A
blaTEM-52c
ISEcp1-blaCTX-M-1-orf477 ISEcp1-blaCTX-M-1-orf477 fimA-aer
fimA fimA-aer
SXT−SUL−STR SXT−SUL−TET−NAL SUL−TET−NAL SUL−TET SXT−SUL−STRI SXT−SUL SXT−SUL SXT−SUL−TET− NAL−CIP−STR SXT−SUL−TET−STRI SXTI−SUL−TET− CHL−NAL −
−/−
None
−
+/− −/−
dfrA17-aadA5 − − − dfrA17-aadA5 dfrA17-aadA5 dfrA17-aadA5 dfrA1-aadA1
Other resistance genes detected outside integron
sul2 tet(A), sul2 tet(A), sul2 tet(A), sul2 sul2 sul2 sul2 blaTEM-1b, tet(A), strA-strB, sul2 dfrA17-aadA5 tet(A), tet(B), sul2 − tet(A), sul2
+/− −/− −/− −/− +/− +/− +/− +/+
Class 1 integron Virulence Resistance phenotype to factors non-beta-lactam antibioticsa intI1/qacEΔ1+ Integron sul1 structure
ISEcp1b-blaCTX-M-1-orf477 − ISEcp1-blaCTX-M-1-orf477 fimA-aer ISEcp1-blaCTX-M-1-orf477 fimA-aer ISEcp1-blaCTX-M-1-orf477 fimA-aer ISEcp1b-blaCTX-M-1-orf477 fimA-aer ISEcp1b-blaCTX-M-1-orf477 fimA ISEcp1b-blaCTX-M-1-orf477 fimA-aer ISEcp1-blaCTX-M-1-orf477 fimA-aer
Phylogroup ESBL and genetic environment
This strain was obtained from the faecal sample of a medical doctor working in a hospital
This strain was obtained from the faecal sample of a veterinarian with scarce contact with animals
This strain was obtained from the faecal sample of a poultry farmer
ISEcp1 disrupted by the IS10 element in the opposite orientation
SXT, trimethoprim–sulfamethoxazole; SUL, sulphonamides; STR, streptomycin; TET, tetracycline; CHL, chloramphenicol; NAL, nalidixic acid; CIP, ciprofloxacin; I intermediate resistance
P8
P7 P6
P2a P2b P3 P4 P4 P4 P5
CC165
C3133 C3136 C3134c C3135 C3140 C3141 C3137d
ST165
Tunis
C3132
P1
Region of PFGE MLST the sample pattern Sequence Clonal type complex
E. coli isolates
Table 1 Characteristics of the 11 extended-spectrum beta-lactamase (ESBL)-positive Escherichia coli isolates recovered from the faecal samples of healthy humans
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other bla genes were negative. The ISEcp1 and orf477 sequences were identified upstream and downstream of the blaCTX-M-1 gene, respectively, in all ten isolates that harboured this gene. The ISEcp1 sequence was truncated in four of these strains by the IS10 element, located in the opposite position (Table 1). Six ESBL-positive isolates contained class 1 integrons with the following gene cassette arrangements: dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one isolate). Five of those integrons lacked the qacEΔ1 and sul1 genes. No class 2 integrons were detected among the studied isolates. Seven different sequence types ascribed to five clonal complexes were identified by MLST among CTX-M-1producing strains: CC155 (four strains of sequence types ST155 and ST58), CC10 (two strains of ST10 and ST48), CC350 (two strains of ST57), CC165 (one strain of ST165) and CC398 (one strain of ST398). The phylogroups A, B1 and D (three, five and two strains, respectively) were identified among CTX-M-1-positive strains. The TEM-52producing strain was typed as ST219 (considered as a singleton) and to the phylogroup B2 (Table 1). The PFGE analysis demonstrated eight unrelated pulsotypes among the 11 ESBL-producing strains. The two ST57-D strains showed a closely related pulsotype. On the other hand, the three ST58-B1 strains showed an indistinguishable PFGE pattern (Table 1). The fimA and aer virulence genes were detected in ten and eight strains, respectively, but none of the ESBLproducing strains harboured the sxt, papG-III, papC, hly, cnf1, eae and bfp virulence genes or were ascribed to the O25b or O157 serotypes.
Discussion The detection of ESBL-positive E. coli in 7.3% of the analysed human samples remarks the wide dissemination of these resistant bacteria among healthy population in Tunisia. It seems that prevalence is higher when we consider the group of individuals with specific risk factors as is the case of farmers/veterinarians or medical doctors/sanitary staff, although the number of individuals in this groups was too low to obtain clear conclusions. Resistant commensal bacteria of the intestinal tract can be implicated, on some occasions, in human infections or they can transfer resistance genes to pathogenic bacteria. The existence of a reservoir of ESBL-containing bacteria is of relevance and its future evolution should be tracked. Similar studies have been performed in other countries with different prevalence rates (prevalence/year of sampling): Perú and Bolivia (1.7%/2005), Lebanon (2.4%/2003), Spain (6%/2007), Japan (7%/2009– 2010) and Thailand (58%/2008) [18–22]. In our case, the sampling was performed in the period 2009–2010.
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The present study corresponds to the first evaluation of the carriage level of ESBL-positive E. coli isolates in healthy humans in the Maghreb and one of the first ones in Africa. As a matter of fact, there is only one previous reference that reports the carriage of ESBL-containing E. coli isolates (specifically, CTX-M-15) in the subdominant faecal flora of two of 20 healthy children from a remote Senegalese village [10]. An important debate at this point is the one related to the origin of these resistant isolates. In Tunisia, it has been reported that CTX-M-15 is the main type of ESBL in clinical E. coli isolates recovered in hospitals [7–9, 23], and CTX-M-1 is the most frequent type among food E. coli isolates [3, 4]. These data may suggest the potential food origin of the isolates recovered in the healthy human population, and the potential implication of the food chain in the transmission processes. We cannot discard the potential future emergence of the CTX-M-1 variant in the hospital setting in Tunisia due to its wide dissemination in the community. Some previous reports suggested a possible transmission of ESBL-producing E. coli from poultry to humans, most likely through the food chain [6]. Moreover, the high clonal diversity detected among the blaCTX-M-1carrying strains of our study (eight unrelated pulsotypes and seven different STs belonging to five CCs) suggests that a horizontal ESBL-containing plasmid transmission among different genetic lineages of E. coli may be responsible for the recent and fast spread of this variant in this country, which should be evaluated in the future. The five individuals that carried similar clones detected in this study (ST58-B1 and ST57-D) had no relationship among them. The blaCTX-M-1 gene has been previously found to be associated to the ISEcp1 sequence. This IS element contains typical -35 and -10 putative promoter regions and could mobilise such a gene [4]. In our study, we found the presence of ISEcp1 upstream of blaCTX-M-1 in the ten strains containing this gene. Interestingly, this IS was found to be disrupted by the IS10 element in the opposite orientation in four of these strains. A similar organisation was reported for blaCTX-M-14 and blaCTX-M-15 [24], but never for blaCTX-M-1. It is interesting to note that the presence of IS10 flanking ISEcp1 may affect the impact of this insertion sequence on the mobilisation and expression of the blaCTX-M-1 gene. To our knowledge, this is the first time that TEM-52producing E. coli strains have been detected in Tunisia. The blaTEM-52 gene has also been detected among foodproducing, wild animals, pets and healthy humans from Belgium, France, Greece, Portugal and Spain [2, 16, 25, 26]. Most ESBL-producing E. coli isolates belonged to phylogroups B1 (5/11) and A (3/11), which are known to be associated with animal or human commensal E. coli strains. Phylogroups D (2/11) and B2 (1/11), which are
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mainly associated with extraintestinal pathogenic E. coli, were less frequently represented. This is in agreement with other studies on commensal E. coli of human origin [19, 27]. According to a search of the E. coli MLST database, ST57 has been detected only in animal isolates, ST58, ST48, ST155, ST165, ST10 and ST398 appear to be common types in isolates of human and animal origins, and ST219 only in human isolates. In a recent French study, CC10 isolates were found to be the most prevalent among faecal samples from healthy carriers of nalidixic acidresistant (but ESBL-negative) E. coli [28]. Sixty percent of CTX-M-1-producing E. coli strains carried integrons and, in most of the cases, these integrons lacked the qacEΔ1 and sul1 genes (5 of 6 cases). It is interesting to remark that this type of integron lacking the 3′-conserved region and with similar gene cassette arrangements has been previously reported among most of the integron-positive CTX-M-1-producing E. coli isolates of food products in Tunisia [4]. The intestinal tract of healthy humans in Tunisia seems to be a reservoir of ESBL-producing E. coli isolates. The detection of the blaCTX-M-1 gene in isolates of different genetic lineages, and the previous detection of blaCTX-M-1 in food isolates but not in clinical isolates in this country, may suggest the potential implication of the food chain as the vehicle of transmission. Future studies will allow determining whether this type of ESBL variant can be transferred to isolates of the hospital environment, as has occurred in other countries. Acknowledgements This study was financed by an Integrated Action of the Spanish Agency of International Collaboration (AECID) of Spain grant and a grant from the Tunisian Ministry of Higher Education and Scientific Research. V. Estepa has a predoctoral fellowship from the University of La Rioja (Spain). Part of the results of this manuscript have been presented at the 21st ECCMID/27th ICC Joint Congress of the European Society of Clinical Microbiology and Infectious Diseases and the International Society of Chemotherapy, Milan, Italy, 7–10 May 2011.
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