Abstract
This study aimed to characterize third-generation cephalosporin (3GC)-resistant Klebsiella pneumoniae isolated from fresh fruits and vegetables purchased at Bejaia city, Algeria. K. pneumoniae isolates were identified by MALDI-TOF. Susceptibility to antibiotics was tested by the disk diffusion method. Whole genome sequencing (WGS) was carried out to determine sequence type (ST), plasmid incompatibility group (Inc.), and acquired antimicrobial resistance gene presence. A total of 13 3GC-resistant K. pneumoniae strains were isolated. WGS identified blaCTX-M-15 in 11 extended-spectrum-beta-lactamases (ESBL)-K. pneumoniae and blaDHA-1 in 2 AmpC-K. pneumoniae. The aac(6’)lb-cr gene was identified in 8 out of 13 isolates. Multilocus sequence typing (MLST) evidenced five different STs, namely ST14, ST45, ST219, ST236, and ST882. MDR K. pneumoniae contaminated fresh fruits and vegetables, often eaten raw and inappropriately washed, may represent an underestimated public health threat. This study highlights that hygiene measures during harvesting and retail process are of utmost importance to limit further ESBL/AmpC spread to the consumer households.
Introduction
K
Extended-spectrum-beta-lactamases (ESBL)-producing K. pneumoniae isolates have been described in various countries6–9 and their associated resistance to antibiotics varies between countries. According to Navon-Venezia et al., 1 data retrieved from the European Antimicrobial Resistance Surveillance Network for the years 2005–2015 show that eastern and southwestern European countries, as well as Mediterranean countries, are endemic for ESBL-producing K. pneumoniae, exceeding 50%–60% of nonsusceptibility to third-generation cephalosporins (3GCs), fluoroquinolones, and aminoglycosides.
The role of food in human exposure to such antimicrobial-resistant bacteria is still unclear; nevertheless it is becoming a growing food safety issue. Enterobacteriaceae strains ingested through food may contain ESBL and plasmidic AmpC (pAmpC) genes carried by mobile genetic elements and colonize human commensal flora. K. pneumoniae, being a common contaminant of either plant- or animal-based foods, is likely to play a key role in introducing into the human gut environmental strains. 10
This study aimed to characterize by whole genome sequencing (WGS) a collection of K. pneumoniae strains resistant to 3GCs isolated from fruits and vegetables purchased at Bejaia city, Algeria.
Materials and Methods
Sampling
A total of 310 samples of fruits and vegetables were purchased in five markets located inside the city of Bejaia, northeastern Algeria, between April 2013 and March 2014. Sampling procedure has been previously described. 11 During a total of seven visits to markets, the sampler behaved as an average consumer and shopped seasonal fruits and vegetables from the regular Algerian regimen. Most of the products were domestically produced: 294 of 310 fruit and vegetable samples were originating from Algeria. The province of origin, also called wilaya—Algeria is divided into 48 wilayas—of each sample was recorded. Detailed information on the 310 samples is recorded in Supplementary Table 2 from Mesbah-Zekar et al. 11
Characterization of MDR Klebsiella pneumoniae Isolated from Fresh Fruits and Vegetables Purchased at Bejaia City
Genes indicated in black and bold were identified with 100% length coverage and 100% identity, genes indicated in black were identified with 100% coverage length and identity ≥90%, genes marked with # were identified with covered length ≥60% and <100%, identity range between 90% and 100% of the partially aligned sequence.
MLS, macrolides–lincosamides–streptogramins; NF, not found.
Microbiological analysis and antimicrobial susceptibility tests
Bacterial isolation procedure has been previously described. 11 In brief, 25 g of fruits and vegetables samples were placed aseptically in a sterile plastic bag containing 225 mL of buffered peptone water (BPW), vigorously shaken, and the suspensions were then incubated at 37°C for 18 hrs. After pre-enrichment, 10 μL of BPW was streaked onto MacConkey's agar plates (Fluka) supplemented with 8 mg/L of ceftazidime and incubated for 18–24 hrs at 37°C for isolation of 3GC-resistant K. pneumoniae. The isolates were further identified by MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) using a Microflex LT® and Biotype 3.0 software (Bruker Daltonik, GmbH, Germany).
Each K. pneumoniae isolate was tested for antimicrobial susceptibility against 29 antibiotics from seven classes by the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) recommendations, as detailed by Mesbah Zekar et al. 11 Isolates were classified as susceptible, intermediate, or resistant according to the clinical interpretative criteria recommended by the CLSI. 12 Multidrug resistance (MDR) was considered when the isolate was resistant to three or more antimicrobial classes. 13 ESBL production was detected by double-disk synergy test as previously described. 14 Presence of carbapenemase activity was explored by two methods.15,16
Genomic analysis of K. pneumoniae isolates
Total DNA was extracted using E.Z.N.A.® Bacterial DNA Kit (Omega Bio-tek, Norcross, GA) as per the manufacturer's instructions.
WGS of K. pneumoniae strains was performed at ICM platform, Hôpital Pitié-Salpétrière, Paris, France. Nucleic acid fragmentation was performed by ultrasound, the DNA library was prepared using KAPA kit (Roche Sequencing Solutions, Pleasanton, CA), and the sequencing was performed using MiSeq technology (Illumina, San Diego, CA). Raw reads were trimmed (minimum length, 35 bp; quality score, 0.03) and assembled de novo in CLC Genomics Workbench 7.5.1.
The CGE server* was used to reassess K. pneumoniae identification (KmerFinder) 17 to identify acquired antimicrobial resistance genes (Resfinder), 18 plasmid presence (PlasmidFinder), 19 plasmids typing (pMLST), 19 and strains typing (MLST). 20
Results
ESBL-producing K. pneumoniae collection
Thirteen K. pneumoniae strains resistant to 3GCs were recovered from fruits and vegetables usually consumed raw (mint, tomato, parsley, lettuce, beet, peach, carrot, and celery) purchased at different markets in Bejaia city (Table 1).
Characteristics of MDR Klebsiella pneumoniae Isolated from Fresh Fruits and Vegetables Purchased in Bejaia City
AMP, ampicillin; TIC, ticarcillin; AMC, amoxicillin clavulanic acid; CEF, cephalotin; FAM, cefamandole; CXM, cefuroxime; FOX, cefoxitin; CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; FEP, cefepime; ATM, aztreonam; GEN, gentamicin; KAN, kanamycin; STR, streptomycin; TET, tetracycline; TGC, tigecycline; CIP, ciprofloxacin; PEF, pefloxacin; NAL, nalidixic acid; SXT, trimethoprim sulfamethoxazole; TMP, trimethoprim; SSS, sulfonamides; CHL, chloramphenicol.
Antimicrobial susceptibility profiles tests are summarized in Table 1. All 13 Klebsiella pneumoniae isolates were resistant to some penicillins (ampicillin [AMP] and ticarcillin [TIC]), some first-generation cephalosporins (cephalothin [CEF]), some second-generation cephalosporins (cefuroxime [CXM] and cefamandole [FAM] and some 3GCs (cefotaxime [CTX], ceftazidime [CAZ], and ceftriaxone [CRO]); 85% of them were resistant to fourth-generation cephalosporins (cefepime [FEP]), 69% were resistant to amoxicillin clavulanate (AMC), and 92% to aztreonam (ATM) (Table 1). Only 15% were resistant to cefoxitin (FOX). All isolates were susceptible to imipenem and ertapenem. Nonetheless, absence of carbapenemase activity was double checked by both Carba NP test and CIM test.
Resistance to non-beta-lactam antibiotics was also observed, including resistance to sulfonamides (trimethroprim sulfamethoxazole [SXT] = 77%, trimethoprim [TMP] = 85%, and sulfonamides [SSS] = 54%), aminoglycosides (gentamicin [GEN] = 54%, kanamycin [KAN] = 77%, and streptomycin [STR] = 54%), tetracyclines (tetracycline [TET] = 31%, tigecycline [TGC] = 23%), (fluoro)quinolones (pefloxacin [PEF] = 92%; ciprofloxacin [CIP] = 54%, and nalidixic acid [NAL] = 15%), and phenicols (chloramphenicol [CHL] = 23%) (Table 1).
Among the 13 isolates, 11 of them were found to be ESBL producers and the 2 other strains were pAmpC producers.
Genomic analysis of K. pneumoniae isolates
The 11 ESBL-producing K. pneumoniae isolates harbored multiple beta-lactamases: blaSHV-1, blaCTX-M-15, blaOXA-1, blaSHV-101, blaSHV-28, and blaTEM-1B. They also acquired resistance genes able to impair effectiveness of fluoroquinolones (qnrS1, aac(6’)Ib-cr, qnrB66), aminoglycosides (aph(3’)-Ia, aadA2, strB, strA, aac(6’)Ib-cr, aac(3)-IIa), sulfonamides (sul1, sul2), trimethoprim (dfrA12, dfrA14), tetracyclines (tet(A)), fosfomycins (fosA), macrolides–lincosamides–streptogramins (MLS) (mph(A)), and phenicols (catA2) (Table 2).
The two pAmpC-producing K. pneumoniae isolates harbored aac(6’)Ib-cr, aph(3’)-Ia, aadA1, strB, and strA genes encoding resistance to aminoglycosides, blaDHA-1, blaOXA-1, blaSHV-101, blaTEM-1D, and blaSHV-60 encoding resistance to beta-lactams, aac(6’)Ib-cr and qnrB4 encoding resistance to fluoroquinolones, fosA to fosfomycin, mph(A) to MLS, catB3 to phenicols, aee-3 to rifampicin, sul1 to sulfonamides, dfrA1 to trimethoprim, and tet(A) to tetracyclines (Table 2).
Plasmid replicons were identified in all the 13 isolates. IncFII plasmid replicon type was the most frequently present (11 of 13). Only one isolate harbored an IncR replicon.
Multilocus sequence typing of the 13 K. pneumoniae isolates leads to the identification of five different sequence types (STs): ST14, ST219, ST882, ST45, and ST 236 (Table 2). The same CTX-M-15/SHV-28/OXA-1-producing ST14 K. pneumoniae clone was found in different vegetables purchased from five different sellers (4, 7, 8, 13, and 16) in the same market (Idimco). At Ihaddadn market, CTX-M-15-producing ST219 K. pneumoniae clone was found in vegetables purchased from three different sellers (25, 27, and 33), whereas a ST882 K. pneumoniae clone was recovered from seller 26 (DHA-1/OXA-1) and an ST45 clone from seller 40 (CTX-M-15/TEM-1B/SHV-1/OXA-1). A different ST45 clone (CTX-M-15/SHV-1/OXA-1) was also detected at Royal market, which is located ∼500 m from Ihaddadn. At Lekhmis market, only an ST236 clone was isolated.
In other words, according to vender's location, ST14 appeared to be specific to Idimco market, ST236 to Lekhmis market, ST882 and ST219 to Ihaddadn market, and finally ST45 to both Ihaddadn market and Royal market.
Interestingly, different varieties of fruits and vegetables were contaminated with several ESBL/AmpC-producing K. pneumoniae, in particular, those purchased at Idimco (ST14) and Ihaddadn markets (ST45, ST219, and ST882). No ST was associated with any specific fruit or vegetable. Half of the 3GC-resistant K. pneumoniae contaminated fruits and vegetables (7 of 13) were produced in Sétif Wilaya, where ST14 (n = 4) was the most frequent ST isolated. Remarkably, outside Sétif Wilaya, no ST was isolated twice from two different items produced in the same Wilaya.
Discussion
The contribution of fruits and vegetables, often eaten raw, to human exposure to antimicrobial-resistant bacteria is considered as relevant due to the high consumption frequency. 21 However, data are still scarce compared with meat contamination and it remains difficult to identify the source of contamination of fruits and vegetables.
One of the aims of this study was to characterize 3GC-resistant K. pneumoniae isolated from fresh fruits and vegetables purchased at Bejaia city, Algeria.
All K. pneumoniae strains in this study produced beta-lactamases conferring resistance to almost all beta-lactam antibiotics, including 3GCs and fourth-generation cephalosporins. All of them were considered multidrug resistant (≥3 drugs belonging to different classes 13 ). Veldman et al. also reported the identification of such MDR K. pneumoniae, in fresh culinary herbs imported from Southeast Asia, highlighting the potential human health risk associated with their consumption. 22 The ESBL/pAmpC genes identified in this study were associated with other genes conferring resistance to quinolones, aminoglycosides, fosfomycin, tetracyclines, phenicols, sulfonamides, and/or trimethoprim. The emergence of multiple resistances toward different antibiotic classes is frequently found in hospital-adapted K. pneumoniae isolates due to the accumulation of antibiotic resistance genes that may be encoded on multiple plasmids.1,10
The blaCTX-M-15 was predominant in our study: this observation is in line with the worldwide expansion of this ESBL enzyme. 23 Of note, CTX-M-15-producing K. pneumoniae had been reported in Algerian hospitals. 8 ESBL gene variants detected in our study also correspond to the previously most frequently found variants in Algeria among clinical K. pneumoniae isolates8,24–26 as well as in animals. 27 Moreover, such isolates had also been detected in food.22,28
Herein, all isolates harbored one or more plasmid mediated quinolone resistance (PMQR) genes (aac(6)-Ib-cr and/or qnrS1). The aac(6)-Ib-cr gene, conferring both resistance to aminoglycosides and quinolones, was detected in 8 of 13 isolates. The aac(6)-Ib-cr gene had already been associated with ESBL-encoding genes in clinical K. pneumoniae isolated in Algerian hospitals.26,29–31 Even if colocation of PMQR and ESBL genes on the same IncF plasmid type was not demonstrated here, colocation of blaCTX-M and quinolone resistance mechanisms on the same plasmid has already been described in K. pneumoniae isolated from culinary herbs. 22 Carattoli et al. already described in 2009 blaCTX-M-15 genes, often associated with blaTEM-1, blaOXA-1, and aac(6′)-Ib-cr resistance genes and located on plasmids belonging to the IncF group. 32 IncF plasmids are conjugative and range between 45 and 200 kb. They are associated in the literature with the global spread of blaCTX-M-15 in human E. coli. 33
On several instances, the same major clone was found on the same date at different sellers located in the same market, which may reflect immediate cross-contamination, through handling practices by sellers or consumers. Indeed, the consumer can touch different fruits and vegetables before buying, which makes the consumer a possible vehicle of transmission of bacteria to fruits and vegetables or from one fruit or vegetable to another. The contamination may also have been mediated through transport equipment, or even through environmental factors such as dust. More precisely, the detection of ST45 at different markets (Ihaddadn and Royal) may be due to the transport equipment or dust or the seller's handling practices. ST14 was detected on two different plants (mint and parsley) at the same booth (seller 8): contamination might be associated with improper handling practices or contaminated environment. ST219 and ST14 were found at Ihaddadn market and Idimco market, respectively, on different fruits and vegetables from different sellers, who are working not far from each other: this may reflect immediate cross-contamination, through poor hygiene on the booth or when the consumers are handling the produce while shopping.
Of note, ST14 K. pneumoniae carrying blaCTX-M-15 had been isolated in a Tanzanian hospital in the context of neonatal sepsis. 34 A ST45 had been also isolated in an Algerian hospital environment. 26 An ST219 ESBL and carbapenemase-producing K. pneumoniae had been isolated from wound infection in a burn unit in Annaba, Algeria in 2015. 35 Furthermore, ST219 and ST882 isolates had already been detected on sandwiches in Bejaia city 28 ; one may emphasize that these sandwiches are composed of meat and vegetables and both studies were conducted at the same period of time in the same city. The detection of the same ST in different environments (fresh fruits and vegetable, sandwich, and hospital) raises concerns and encourages further investigations. Thus, to be able to assess a clonal link between these MDR K. pneumoniae detection events, MLST is not discriminatory enough. Indeed, to accurately trace back sources of contamination, WGS-based approaches such as cgMLST or wgMLST or pairwise single nucleotide polymorphism distance typing would have been more powerful than MLST.36,37 Unfortunately WGS data of isolates from previous Algerian studies were not available, whereas MLST was.26,28,35
The diversity of ST detected among these isolates of MDR K. pneumoniae indicates that the dissemination of resistance is not associated with the spread of a unique clone but rather with the dissemination of plasmids carrying these genes. Ben Said et al. reported the same conclusion. 38 Moreover, due to the high diversity in acquired resistance genes, high plasmid load, variability of GC content, and broad ecological range, Wyres and Holt qualified K. pneumoniae as “a key trafficker in drug resistance genes.” 10
Although foods of animal origin are mostly eaten cooked or transformed, the fruits and vegetables tested herein are very often eaten raw and improperly washed. The possibility of transfer of ESBL/AmpC-producing bacteria to the human gut through raw food consumption needs to be explored. Despite the fact that no direct evidence of human colonization by a single K. pneumoniae clone originating from ingested food exists so far, 10 different authors have also highlighted the potential health hazard derived from consumption of raw food containing multidrug-resistant bacteria.22,38–41
Conclusion
Detection of such MDR K. pneumoniae suggests that fruits and vegetables may constitute an underestimated threat to public health, as this source of household contamination is poorly studied. It is of interest to note that fruits and vegetables contaminated by these MDR K. pneumoniae are often eaten raw and improperly washed, which may cause transfer of ESBL/AmpC producers to the human gut. As a matter of fact, antimicrobial resistance surveillance programs mostly focused to date on food of animal origin. Monitoring antimicrobial resistance reservoirs in food from nonanimal origin should also be considered.
Finally, formal identification of the source(s) of contamination of fruits and vegetables detected at retail is foreseen. Further studies at each step of the production chain are needed to be able to promote science-based hygiene measures to limit further spread of ESBL/AmpC carrying bacteria to our households.
Nucleotide Sequence Accession Numbers
Whole genome assemblies were deposited in Genbank under BioProject accession number PRJNA482637.
Footnotes
Acknowledgments
The authors thank Muriel Marault for technical assistance and Sabine Delannoy for assistance and advice on genomic data analysis
Disclosure Statement
The authors declare no competing financial interests linked to this study.
Funding Information
This study was financially supported by “Soutien de Programme—Direction Générale de l'Enseignement et de la Recherche” from the French Ministry for Agriculture and by “Programme National Exceptionnel (P.N.E)” from the Algerian Ministry of Higher Education and Scientific Research.