Antibiotic Resistanceof Food-Borne Enterobacterial Strains Isolated in Bucharest, Romania

Antibiotic resistanceof food-borne enterobacterial strains isolated in Bucharest, Romania

Andreea L. Mateescu1, Mihaela M. Mitache2, Mariana C. Chifiriuc1, Tatiana Vassu Dimov1

1University of Bucharest, Faculty of Biology, 1-3 Portocalelor Ale., District 6, 60101, Bucharest, Romania

2Division of Public Health, 104-124 Dudeşti Path, District 3, 020321, Bucharest, Romania

Contact information for Corresponding Author

[Mariana Carmen Chifiriuc, Ale. Portocalelor 1-3, 060101 Bucharest, 0040766728315, 00402111577,

Abstract

The aim of this study was the investigation of antibiotic resistance features in 66 enterobacterial strains isolated from different food products. Antibiotic susceptibility testing was carried out by diskdiffusion method. The detection of ampC genes (6 families: FOX, MOX, CIT, DHA, ACC, EBC) was carried out by multiplex PCR. Simplex PCR was used for the detection of blaTEM, blaCTX-M, tetA, tetB, tetC and tetD resistance genes. Phenotypic studies showed that most of the analyzed strains were resistant to ampicillin (66.6%), cefuroxime (43.9%), tetracycline (40.9%), amoxicillin/clavulanic acid (37.8%), piperacillin (33.3%) and cefazolin (31.8%). A small percentage of the enterobacterial strains were resistant to trimethoprim/sulfamethoxazole (9%), and aminoglycosides (7.5%). Five Escherichiacoli and one Salmonella sp. isolates produced extended-spectrum β-lactamases (ESBL), mediated by blaCTX-M and/or blaTEM genes. The ampC gene types in Salmonella sp. strains, harboring the ACC and CIT genes, were distinct from those encountered inEscherichiacoli strains, which exhibited the FOX and EBC genes. Most of the tetracycline resistant strains (55.5%) harbored the tetB gene.The presence of ESBL and AmpC producing strains in food products is a cause of concern, because these phenotypes are usually accompanied by a low susceptibility to other classes of antibiotics.

Keywords:antibiotic, β-lactamase, enterobacteria, food-borne, genes, multi-drug resistance.

Introduction

The globalization of the food industry and the development of extensive food distribution networks have increased the risk of foodborne disease outbreaks involving multiple states or countries. In particular, outbreaks associated with fresh products have emerged as an important public health concern. During July and August 1998, eight restaurant-associated outbreaks of shigellosis caused by a common strain of Shigella (S.) sonnei occurred in the United States and Canada(Naimi al. [1]).The risks of outbreaks caused by multiresistant pathogens are even greater in developingcountries, due to inadequate hygienic-sanitary measures.

Food-borne diseases caused by members of Enterobacteriaceae family, like non-typhoid Salmonella (S.), Shigella or pathogenic Escherichia (E.) coli represent an important public health problem and an economic burden in many parts of the world (Miko & al.[2]).The main sources are foods of animal origin, such as poultry, eggs, milk, beef and pork, but in addition fruits and vegetables have been implicated as vehicles for bacteria dissemination.

E. coli strains are among the most frequently isolated bacteria from food products, reason for which this species is used to assess the hygienic quality of food and food ingredients. In addition, the level of antibiotic resistance in E. coli is considered to be a good indicator of the selection pressure exerted by antibiotic use(Lei al.[3]).

Antimicrobial drug resistance is spreading among enterobacteria, limiting the utility of traditionally used agents (Kelesidis & al.[4]).In addition to human and veterinary medicine, antibiotics are extensively used in agricultural settings, such as for the treatment of infections, growth enhancement, and prophylaxis in food animals, leading to the selection of drug and multidrug-resistant bacteria (Burgos & al.[5]).

Molecular analysis of antibiotic resistance genes and antibiotic-resistant mobile elements, such as plasmids, transposons, and the more recently explored integrons, has shown that identical elements were found in bacteria that colonize both animals and humans, suggesting a role for raw foods in the dissemination of resistant bacteria and resistance genes to humans via the food chain (Carattoli & al. [6]; Hao Van & al. [7]).

The aim of this study was the phenotypic and genotypic investigation of antibiotic resistance makers and multidrug resistanceprofiles in enterobacterial strains isolated from different food products in Bucharest, Romania.

Materials and Methods

Strains isolation and identification

The isolation and enumeration of E. colistrains was carried out according to ISO 16649-2/2007and isolation of the other enterobacterial strains was done according to ISO 21528-2/2007. For the identification of the strains we used API 20E rapid identification system.

Antimicrobial susceptibility assay

The disk diffusion method was used in order to assess the resistance profiles of the analyzed strains. The susceptibility testing was performed using following antibiotics: ampicillin (AMP), piperacillin (PIP), amoxicillin/clavulanic acid (AMC), cefazolin (KZ), cefoxitin (FOX), cefuroxime (CXM), aztreonam (ATM), cefepime (FEP), imipenem (IMP), cefotaxime (CTX), gentamicin (GM), amikacin (AK), tetracycline (TE), fosfomycin (FOS), ciprofloxacin (CIP), norfloxacin (NOR), trimethoprim/sulfamethoxazole (SXT). Antibiotic disks were obtained from the Oxoid Company (UK). Enterobacterial strainsisolates were evaluated based on the diameter of the growth inhibition zones and classified assusceptible (S), intermediate resistant (I) or resistant (R) according to the CLSI criteria (2011).The multi-drug resistance (MDR) phentypes were defined as simultaneous resistance to at least one antibiotic from threedifferent classes (Falagas al.[8]).

PCR assay

Preparation of bacterialcelllysates.

Bacterial strains were grownin 1 ml BHI overnight then centrifuged and resuspended in 1 ml of sterile water for washing (2x).Cell lysis was performed by boiling at 100oC for 10 min, followed by cooling at -20oC.

The enterobacterial strains were tested for the presence of the following resistance genes: ampC genes (FOX, MOX, CIT, DHA, ACC, EBC), blaTEM, blaCTX-M, tetA, tetB, tetC, tetD. The primers used in this study and their corresponding genes are listed in Table 1.

Table 1. Synthetic oligonucleotides used as primers for PCR

Target(s) / Sequence (5’to 3’ , as synthesized) / Expected amplicon
size (bp) / Source
MOX-1, MOX-2, CMY-1,
CMY-8 to CMY-11 / MOXMF GCT GCT CAA GGA GCA CAG GAT
MOXMR CAC ATT GAC ATA GGT GTG GTG C / 520 / Pérez and Hanson, 2002[9]
LAT-1 to LAT-4, CMY-2
to CMY-7, BIL-1 / CITMF TGG CCA GAA CTG ACA GGC AAA
CITMR TTT CTC CTG AAC GTG GCT GGC / 462 / Pérez and Hanson, 2002[9]
DHA-1, DHA-2 / DHAMF AAC TTT CAC AGG TGT GCT GGG T
DHAMR CCG TAC GCA TAC TGG CTT TGC / 405 / Pérez and Hanson, 2002[9]
ACC / ACCMF AAC AGC CTC AGC AGC CGG TTA
ACCMR TTC GCC GCA ATC ATC CCT AGC / 346 / Pérez and Hanson, 2002[9]
MIR-1T ACT-1 (EBC cluster) / EBCMF TCG GTA AAG CCG ATG TTG CGG
EBCMR CTT CCA CTG CGG CTG CCA GTT / 302 / Pérez and Hanson, 2002[9]
FOX-1 to FOX-5b / FOXMF AAC ATG GGG TAT CAG GGA GAT G
FOXMR CAA AGC GCG TAA CCG GAT TGG / 190 / Pérez and Hanson, 2002[9]
BlaCTX-M / CTX-M-F 5’-CGC TGT TGT TAG GAA GTG TG-3’
CTX-M-R 5’-GGC TGG GTG AAG TAA GTG AC-3 / 730 / Bali and others, 2010[10]
BlaTEM / TEM-F ATA AAA TTC TTG AAG ACG AAA
TEM-R GTC AGT TAC CAA TGC TTA ATC / 1080 / Eftekhar and others, 2005[11]
TetA / TetA-F GCG CGA TCT GGT TCA CTC G
TetA-R AGT CGA CAG YRG CGC CGG C / 164 / Aminov and others, 2002[12]
TetB / TetB-F TAC GTG AAT TTA TTG CTT CGG
TetB-R ATA CAG CAT CCA AAG CGC AC / 206 / Aminov and others, 2002[12]
TetC / TetC-F GCG GGA TAT CGT CCA TTC CG
TetC-R GCG TAG AGG ATC CAC AGG ACG / 207 / Aminov and others, 2002[12]
TetD / TetD-F GGA ATA TCT CCC GGA AGC GG
TetD-R CAC ATT GGA CAG TGC CAG CAG / 187 / Aminov and others, 2002[12]

For the purpose of detecting family-specific AmpC β-lactamase genes (FOX, MOX, CIT, DHA, ACC, EBC) a multiplex PCR was used. The PCR used six sets of ampC-specific primers resulting in amplicons that ranged from 190 bp to 520 bp, easily distinguished by gel electrophoresis.Amplification was performed in a total reaction volume of 25 µl. The reaction mixture contained 12.5 µl PCR Master Mix 2x(Promega), 0.5µl/0.25µl/0.3µl each primer (10 M) (fox/mox, cit, dha/accm, ebcm), 1µl of template represented by bacterialcelllysates and double-distilled H2O was added to a total volume of 25 µl. The parameters for the amplification cycles were as follows: denaturation for 30s at 94oC, annealing of primers for 30 s at 64oC, and primer extension for 1 min at 72oC (25x). Prior to the first cycle, the PCR mixture was incubated for 3 min at 94oC. After the last cycle, the mixture was incubated for 7 min at 72oC for the final elongation.

bla-CTX-M and bla-TEM genes were detected by simplex PCR. The reaction mixture contained 4µl of 10x PCR amplification buffer (Promega), 1.2µl MgCl (25 mM), 2µl dNTP (2 mM), 0.2 µl Taq polymerase(1 U), 0.6 µl of each primer(10 M), 1µl of template, double-distilled H2O was added to a total volume of 20 µl. Amplification cycles were run as follows: denaturation for 2 min at 95oC, annealing of primers for 30 s at 60oC (blaCTX-M) and 58oC (blaTEM), and primer extension for 1 min at 72oC (35x). Prior to the first cycle, the PCR mixture was incubated for 5 min at 95oC. After the last cycle, the mixture was incubated for 10 min at 72oC for the final elongation.

The identification of tetracycline resistance genes (tetA, tetB,tetC, tetD) was carried out by simplex PCR. Reaction mixture: 12.5 µl PCR Master Mix 2x (Promega), 0.5 µl each primer(10 M), 1µl of template and double-distilled H2O was added to a total volume of 25 µl. Amplification parameters: denaturation for 30s min at 94oC, annealing of primers for 30 s at 60oC (tetA, tetC, tetD) and 58oC (tetB), and primer extension for 30s at 72oC (30x). Prior to the first cycle, the PCR mixture was incubated for 2 min at 94oC. After the last cycle, the mixture was incubated for 10 min at 72oC for the final elongation. The PCR products were analyzed by electrophoresis in 1.5% agarose gel.

Results and Discussions

Sixty-six food-borne bacterial strains were isolated form different sources and identified using API identification system (Table 2).

Table 2. Summary of the analyzed bacterial strains and of their isolation source

Isolation source / Number of strains
Mincemeat / E. coli-32 strains
Salmonella sp.-7 strains
Enterobacter (E.) cloacae-1 strain
Salad / E. coli-2 strains
Sausages / S. arizonae-1strain
E. coli-4 strains
Pork chop / E. coli-1 strain
Cheese / E. coli-1 strain
Klebsiella (K.) pneumoniae-1 strain
E (E.) aerogenes-1 strain
Cream / K. oxytoca-1 strain
Liver pate / Citrobacter (C.) farneri- 1 strain
Butter and cheesespreads / K. ornithynolitica- 1 strain
Butter / Serratia (S.) odorifera-1 strain
Rice with milk / C. farneri -1 strain
Well water / S. marcescens-1 strain
Milk / E. coli-1 strain
K. oxytoca-1 strain
Yogurt / E. coli-2 strains
Hafnia (H.) alvei-1 strain
Fresh potatoes / C. youngae-1 strain
Almonds / Pantoea ( )sp.-1 strain
Meat case / Shimwellia (S.) blattae-2 strains

A large percentage (66.6%) of the analyzed strains showed resistance to ampicillin. These results are not surprising, as it is a well known fact that Klebsiella, Enterobacter and Citrobacter species have intrinsic resistance to ampicillin.

Also, many of the analyzed strains were resistant to cefuroxime (43.9%), tetracycline (40.9%), amoxicillin/clavulanic acid (37.8%), piperacillin (33.3%), cefazolin (31.8%) and cefoxitin (21.2%).Only a small percentage of the enterobacterial strains were resistant to trimethoprim/sulfamethoxazole (9%), and aminoglycosides (7.5%) (Table 3).

Table 3 Distribution of antibiotic resistance levels for isolated strains belonging to the main genera of Enterobacteriaceae family

Antibiotics / Escherichia / Salmonella / Enterobacter / Klebsiella / Citrobacter / Serratia / Schimwellia / Hafnia
n=43 / n=8 / n=2 / n=4 / n=3 / n=2 / n=2 / n=1
n / % / n / % / n / % / n / % / n / % / n / % / n / % / n / %
AMC / 12 / 28 / 5 / 62 / 2 / 100 / 1 / 25 / 1 / 33 / 1 / 50 / 2 / 100 / 1 / 100
AMP / 25 / 58 / 6 / 75 / 2 / 100 / 4 / 100 / 2 / 66 / 2 / 100 / 2 / 100 / 1 / 100
PIP / 13 / 30 / 5 / 62 / 1 / 50 / 1 / 25 / 1 / 33 / 1 / 50 / 0 / 0 / 0 / 0
KZ / 9 / 21 / 5 / 62 / 1 / 50 / 1 / 25 / 1 / 33 / 1 / 50 / 2 / 100 / 1 / 100
CXM / 21 / 49 / 2 / 25 / 2 / 100 / 0 / 0 / 1 / 33 / 1 / 50 / 2 / 100 / 0 / 0
ATM / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
IMP / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
FEP / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
CTX / 1 / 2 / 1 / 12 / 0 / 0 / 0 / 0 / 1 / 33 / 0 / 0 / 0 / 0 / 0 / 0
GM / 4 / 9 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
AK / 3 / 7 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
TE / 19 / 44 / 4 / 50 / 1 / 50 / 1 / 25 / 1 / 33 / 1 / 50 / 0 / 0 / 0 / 0
CIP / 2 / 4 / 0 / 0 / 0 / 0 / 0 / 0 / 1 / 33 / 0 / 0 / 0 / 0 / 0 / 0
NOR / 2 / 4 / 0 / 0 / 0 / 0 / 0 / 0 / 1 / 33 / 0 / 0 / 0 / 0 / 0 / 0
SXT / 4 / 9 / 0 / 0 / 0 / 0 / 1 / 25 / 1 / 33 / 0 / 0 / 0 / 0 / 0 / 0
FOS / 1 / 2 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0 / 0
FOX / 5 / 11 / 2 / 25 / 2 / 100 / 0 / 0 / 2 / 66 / 0 / 0 / 2 / 100 / 1 / 100

n= total number of isolated strains

It is also noteworthy that resistance to ampicillin and tetracycline is often accompanied by resistance to cephalosporins of 1st and 2nd generation, such as cefazolin and cefuroxime. Out of the tested strains, 10 E. colistrains,5 Salmonella sp.strains, 1 S.marcescensstrain and 1 E.aerogenesstrain, were simultaneously resistant to AMP, KZ/CXM, TE, some of them featuring resistance to both cephalosporins. This phenotype is completedby resistance to cephamycins(cefoxitin) in six of the analyzed strains, 3 Salmonella sp. strains, 2 E. colistrains and 1 E.Aerogenesstrain.

Other studies have shown that many of the bacterial strains resistant to ampicillin and to least one other antibiotic harbored plasmids (Ash & al.[13]; Pasquali & al. [14]). It has been noted that, most often, enterobacterial strains resistant to ampicillin also present resistance to tetracycline. In our study out of the 44 ampicillin-resistant tested strains, 22 have beenalso resistant to tetracycline. This phenotype may suggest the presence of plasmids containing both genes for resistance to ampicillin and tetracycline. For instance, the plasmid pFPTB1 from S. typhimurium, carries blaTEM-1 gene conferring resistance to ampicillin and tetA gene for resistance to tetracycline (Pasquali & al. [14]).

We identified 9 multidrug resistant strains(defined as simultaneous resistance to antibiotics from at least three different classes),which represent 13.6% of the analyzed strains, 7 of them belonging toE. colispecie, 1 K. oxytocastrain and 1 C. farneristrain. The most common MDR phenotype was represented by BL+TE+SXT, with SXT being replaced by GM resistance in 3 of the isolates(Table. 4).

Table 4. Antibioresistance phenotypes of the analyzed strains

STRAINS / Beta-lactam antibiotics / Aminoglycosides / TE / SXT / Quinolones / FOS
AMP / PIP / AMC / KZ / FOX / CXM / CTX / ATM / FEP / IMP / GM / AN / CIP / NOR
E. coli1989 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli2446 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli32 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli33 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli 22 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli64 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli3481 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli2590 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli3261 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli3299 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli3115 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli3058 / I / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli79 / S / S / S / S / S / S / S / S / S / S / S / S / R / S / S / S / S
E.coli2421 / S / S / S / S / S / S / S / S / S / S / S / S / R / S / S / S / S
E. coli3441 / S / S / S / S / S / S / S / S / S / S / S / S / R / S / S / S / S
E. coli2568 / S / S / S / S / S / S / S / S / S / S / S / R / R / S / S / S / S
E. coli3445 / S / S / S / S / S / S / S / S / S / S / R / R / S / S / S / S / S
E. coli2382 / R / S / S / S / S / S / S / S / S / S / S / S / R / S / S / S / S
E. coli3068 / R / S / S / S / S / S / S / S / S / S / S / S / R / S / S / S / S
E. coli2531 / R / S / S / S / S / S / S / S / S / S / S / S / R / S / S / S / S
E. coli2611 / R / S / S / S / S / R / S / S / S / S / S / S / S / S / S / S / S
E. coli2660 / R / S / S / S / S / R / S / S / S / S / S / S / S / S / S / S / S
E. coli2449 / R / S / S / S / S / R / S / S / S / S / S / S / S / S / S / S / S
E. coli2460 / R / S / S / S / S / R / S / S / S / S / S / S / S / S / S / S / S
E. coli82 / R / R / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
E. coli14009 / S / R / S / S / S / R / S / S / S / S / S / S / R / S / S / S / S
E. coli2431 / R / S / S / S / R / R / S / S / S / S / S / S / R / S / S / S / S
E. coli3329 / R / I / S / R / S / R / S / S / S / S / S / S / R / S / S / S / S
E. coli1991 / R / R / R / S / S / R / S / S / S / S / S / S / S / S / S / S / S
E. coli2448 / R / R / R / S / S / R / S / S / S / S / S / S / S / S / S / S / S
E. coli78 / R / S / R / R / S / R / S / S / S / S / S / S / S / S / S / S / S
E. coli80 / R / R / R / S / S / S / S / S / S / S / S / S / R / R / S / S / S
E. coli77 / R / S / R / S / R / R / S / S / S / S / S / S / S / S / S / S / S
E. coli76 / R / S / R / S / S / R / S / S / S / S / S / S / R / R / S / S / S
E. coli2429 / R / R / R / S / S / R / S / S / S / S / S / S / R / S / S / S / S
E. coli2471 / R / R / R / S / S / R / S / S / S / S / S / S / R / R / S / S / S
E. coli3330 / R / R / S / R / S / R / S / S / S / S / R / S / R / S / S / S / S
E. coli44 / R / S / R / R / R / R / S / S / S / S / S / S / S / S / S / S / R
E. coli3129 / R / R / R / R / S / R / S / S / S / S / S / S / R / S / S / S / S
E. coli2403 / R / R / R / R / R / R / S / S / S / S / S / S / S / S / S / S / S
E. coli3019 / R / R / S / R / R / R / S / S / S / S / R / R / R / S / S / S / S
E. coli14057 / R / R / S / R / S / R / R / I / S / S / S / S / R / R / R / R / S
E. coli3138 / R / R / R / R / S / R / S / S / S / S / R / S / R / S / R / R / S
H. alvei81 / R / S / R / R / R / S / S / S / I / S / S / S / S / S / S / S / S
K. pneumoniae 3262 / R / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
K. ornithynolitica323 / R / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
K. oxytoca83 / R / S / R / R / S / S / S / S / S / S / S / S / S / S / S / S / S
K. oxytoca3249 / R / R / S / S / S / S / S / S / S / S / S / S / R / R / S / S / S
S.odorifera329 / R / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
C. youngae68 / R / S / R / S / R / S / S / S / S / S / S / S / S / S / S / S / S
C. farneri3845 / R / R / S / R / R / R / R / S / S / S / S / S / R / S / S / R / S
C. farneri322 / S / S / S / S / S / S / S / S / S / S / S / S / S / R / R / R / S
S. marcescens13959 / R / R / R / R / S / R / S / S / S / S / S / S / R / S / S / S / S
Salmonella sp. 1845 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
S. arizonae75 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
S. arizonae2326 / R / R / R / R / R / I / S / S / S / S / S / S / S / S / S / S / S
S. pullorum2128 / R / R / R / R / S / I / R / S / S / S / S / S / S / S / S / S / S
S. enterica 2524 / R / R / R / S / S / R / S / S / S / S / S / S / R / S / S / S / S
S. enterica 72 / R / R / R / R / S / S / S / S / I / S / S / S / R / S / S / S / S
Salmonella sp. 66 / R / S / R / R / R / S / S / S / S / S / S / S / R / S / S / S / S
Salmonella sp. 90 / R / R / I / R / S / R / S / S / S / S / S / S / R / S / S / S / S
E. cloacae67 / R / S / R / R / R / R / S / S / S / S / S / S / S / S / S / S / S
E. aerogenes71 / R / R / R / S / R / R / S / S / S / S / S / S / R / S / S / S / S
Pantoea spp. 69 / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S / S
S.blattae74 / R / S / R / R / R / R / S / S / S / S / S / S / S / S / S / S / S
S. blattae73 / R / S / R / R / R / R / S / S / S / S / S / S / S / S / S / S / S

The MDR phenotypes in the analyzed strains could suggest the presence of integrons, because previous studies have shown that they have a major role in the spread of this phenotype among enterobacteria(Leverstein–van Hall & al. [15]).Integrons are genetic elements able to acquire and rearrange open reading frames (ORFs) embedded in gene cassette units and convert them to functional genes by ensuring their correct expression (Cambray & al.[16]).

We have also found that five E. coli strains and one Salmonella sp. strain produced Extended-Spectrum Beta-Lactamase (ESBL). Molecular tests revealed the presence of both blaCTX-M and blaTEM genes in two of the ESBL producing strains, the rest harboring only blaTEM (Fig. 1).The presence of blaCTX-M and blaTEM genes in food borne enterobacteria is not verysurprising, other studies show that CTX-M and TEM enzymes are the predominant ESBLs in many European countries, with E. coli joining K. pneumoniae as major hosts, and with producers increasingly isolated from community patients (Livermore & al. [17]).

Fig. 1 PCR products of blaCTX-M (A) and blaTEM (B). M: 100bp Lanes (A): 1-E. coli 14057; 2-E. coli 14009; 3-K. oxytoca 3249; 4-C. farneri 322; 5-C. farneri 3845; 6-E. coli 14057; 7-E. coli 14009; 8-E. coli 3138; 9-E. coli 3058; Lanes (B) 1-Ladder 100 bp; 2-E. coli 14057; 3-E. coli 14009; 4- C. farneri 3845 5-E. coli 3330; 6-E. coli 3329; 7-E. coli 3138.

Plasmid-mediated class C β-lactamases have been discovered worldwide. Most of these enzymes are cephalosporinases, but they are capable of hydrolyzing all β-lactams to some extent (Hanson [18]).They have been named with inconsistency, according to the resistance phenotype to cephamycins (CMY), cefoxitin (FOX), and moxalactam (MOX) or latamoxef (LAT), to the type of β-lactamase, such as AmpC type (ACT) or Ambler class C (ACC), and to the site of discovery, such as the MiriamHospital in Providence, R.I. (MIR-1) or DhahranHospital in Saudi Arabia (DHA). BIL-1 was even named after the patient (Bilal) who provided the original sample (Philippon & al.[19]).Enterobacteriaphenotypically characterized as putative AmpC producers were evaluated by ampC multiplex PCR for the presence of plasmid mediated ampC genes. Two Salmonella sp. strains harbored the ACC type gene, one the CIT type, while one E. coli was positive for both act-1 and mir-1 and another E. coli strain harbored ampC genes of both FOX and EBC type (Fig. 2).

Plasmid-mediated ampC genes are derived from the chromosomal ampC genes of several members of the family Enterobacteriaceae, including E.cloacae, C. freundii, Morganella (M.) morganii, and H. alvei. The majority of plasmid-mediated ampC genes are found in nosocomial isolates of E. coli and K. pneumoniae(Barnaud & al.[20]).However they can sometimes be detected in other genera of the Enterobacteriaceae family. Plasmid-mediated AmpC-type enzymes are less common than the extended-spectrum β-lactamases (ESBLs), as a mechanism for resistance to ceftazidime and to other oxyimino-β-lactams in K. pneumoniae and E. coli; such enzymes are important to be recognized since they provide an even broader spectrum of resistance(Alvarez & al. [21]).

The act-1 and mir-1 genes are members of the EBC cluster. The act-1 is the first plasmid-mediated AmpC-type β-lactamase derived from Enterobacter which has been completely sequenced, while mir-1 gene product confers resistance to penicillins and broad-spectrum cephalosporins, including cefoxitin and ceftibuten, but not to cefepime, cefpirome, meropenem, or imipenem (Bradford & al. [22]; Papanicolaou & al. [23]).

Fig. 2 PCR products of ampC type genes (ACC; MIR-1 and ACT-1 (EBC cluster), FOXLanes: 1-Ladder 100bp; 2-K. oxytoca 3249; 3-C. farneri 322; 4-K. ornithynolitica 323; 5-S. odorifera 329; 6-S. pullorum 2128; 7-E. coli 2448; 8-E. coli 2429; 9-E. coli 2403; 10-E. coli 2471; 11-Salmonella sp. 2524; 12-E. coli 44.

The strains showing resistance to tetracycline were tested by PCR in order to detect the genes encoding this resistance – tetA, tetB, tetC and tetD. 55.5% of the tetracycline resistant strains harbored tetB gene, 33.3% were positive for tetA gene and 18.5% harbored both tetA and tetB genes (Fig. 3). None of the enterobacterial strains were positive for tetC or tetD genes. For a part of the tetracycline resistant strains the genetic support could not be established with the primers used in this study.