Journal of Medicine and Life Volume 7, Special Issue 2 , 2014 [620419]

Journal of Medicine and Life Volume 7, Special Issue 2 , 2014
80 Multiresistance to antibiotics of Sa lmonella enterica serovar Typhimurium
strains producing extended spect rum beta-lactamases (ESBLs)

Coculescu BI*, Palade AM**, Purcarea VL***
*“Titu Maiorescu” University, Faculty of Medicine, Bucharest
**”Cantacuzino” National Institute for Research and Development in Microbiology and Immunology, Bucharest
***“Carol Davila” University of Medicine and Pharmacy, Faculty of Medicine, Bucharest

Correspondence to : Coculescu Bogdan-Ioan, MD, PhD
“Titu Maiorescu” University, Bucharest, Romania, 22 Dambovnicului Street, code 040441, Bucharest Phone: 021 316 1646, E-mail: [anonimizat]

Abstract The purpose of the study is to give the rising incidence of Sa lmonellosis and the continuous decr ease of sensitivity of Salmone lla to
a range of antibiotics, the prime importance of the choice of opt imal anti-infective chemotherapy in order to prevent the selec tion of
multidrug-resistant strains (MDR). We have studied 54 strains of Salmonella sent for further investigation to “Cantacuzino” NIR DMI
Bucharest. The statistical analysis was based on the interp retation pathogen susceptibility and analyzed according to CLSI
recommendations 2009, which revealed that 8 (14,81%) identif ied as S. enterica serovar Typhimurium group, presented a
phenomenon of MDR. S. Typhimurium strains producing ESBL s howed resistance mechanisms associated with beta-lactam
resistance to aminoglycosides, quinolones, sulfonamides and tetracyclines.

Keywords : Salmonella enterica serovar Typhimurium, ESBL (extended spectrum beta-lactamase), multidrug-resistant strains (MDR)

Introduction
Gastrointestinal infections are caused by gram-negative amounts, ESBL-producing special problems in medical
practice, by decreased sensitivity to antibiotics, due to the ac quisition of various ways of achieving strength, including the
production of ESBL [ 1]. Given that intestinal infections themselves are a public health problem, etiological involvement of
microorganisms resistant to antibiotics increases their severity. A continuous monitorization of the emergence and spread of strains producing extended-spectrum beta-lactamases (ESBLs) is one of the major objectives of health programs that address infectious disease control.
Materials and methods
We have studied 54 strains of Salmonella sent for further investigation to “Cantacuzino” NIRDMI Bucharest. Antibiotic sensitivity was tested by disc diffusion antibiogram method (Kirby-Bauer), by plating each inoculum on Mueller-Hinton medium according to CLSI recommendations (Clinical and Laboratory Standards Institute) by using microtablets supplied by Oxoid Ltd. (Basingstoke, UK) or Mast Diagno stics (Bootle, UK) by using an inoculum of 0,5 McFarland
turbidity. E. coli ATCC 25922 and E. coli ATCC 35218 (for combinations of β-lactam beta-lactamase inhibitor) were used
as control strains of pathogen susceptibility performed (ref. no. 0335P, MicroBioLogics) [ 2]. The following antibiotics
were tested: Ampicillin (10 mg), Amoxicillin (10 mg), Am oxicillin/Clavulanic acid (20/10 mg), Cefoxitin (30 mg),
Cefotaxime (30 mg), Ceftazidime (30 mg), Imipenem (10 mg), Nalidixic acid (30 mg), Ciprofloxacin (5 mg), Gentamicin (10 mg), Kanamycin (30 mg), Streptomycin (10 mg), Sulfonamide/Sulfadiazine (300 mg), Trimethoprim (5 mg), Cotrimoxazole (Trimethoprim 1,25/Sulfamethoxazole 23,75 mg), Tetracycline (30 mg), Chloramphenicol (30 mg) [ 3,4].
Phenotype test for the confirmation of beta-l actam production – DDST ( double disk synergy test) [5]
The identification by producing extended-spectrum beta -lactamase (ESBL) was performed by using the double
disc. The phenotypic confirmation of S. serovar Typhimurium strains producing suspected ESBLs was achieved by a simultaneous testing by Kirby-Bauer disc diffusion method, the synergy between ce ftazidime discs, disc cefotaxime and
amoxicillin with clavulanic acid. Oxoid discs containing a combination amoxicillin/clavulanic acid (20 μg/10 mg),
ceftazidime (30 mg) and cefotaxime (30 mg) were used, placed at a distance of 2 cm (measured between centers disks) on the Mueller-Hinton medium. Plates were incubated for 18-20 hours at 37°C.

Journal of Medicine and Life Volume 7, Special Issue 2 , 2014
81 Results and Discussion
Prevalence of Salmonella strains studied is the Enteritis serotypes (51,85%) and Typhimurium (27,78%) – see
Table 1 .

Table 1. Prevalence of enteropathogenic strains of Salmonella serovar group after characterization

Species Group Serovar No strains Source of isolation (%)

S. enterica
D1
Enteritidis
28 21 ADD
7 FBD
51,85
S. enterica
B
Typhimurium
15 12 ADD
3 FBD
27,78

S. enterica
B
Agona
7 7 ADD
(isolated)
12,96

S. enterica
B
Saintpaul
4 3 ADD (isolated)
1 FBD
7,41
ADD = Acute Diarrheal Disease
FBD = Foodborne Disease

The statistical analysis was based on the interpretation pathogen susceptibility and analyzed according to CLSI,
revealing that of the 54 strains of Salmonella tested ant ibiotically, 8 (14,81%) identified as S. enterica serovar
Typhimurium group, presenting the phenomenon of multiresista nce to antibiotics (MDR), which meant being resistant to
beta-lactams, aminoglycosides, quinolones, tetracycline and sulfonamides. High rates of resistance (>70%) were recorded to ampicillin, cefota xime, ceftazidime, gentamicin, kanamycin and streptomycin, sulfonamide, trimethoprim,
respectively nalidixic acid. The highest level of resistance to tetracycline was obtained (87,5%). Instead, the patient
showed 100% sensitivity to cefoxitin, imipenem, and ciproflo xacin. Only 25% of the strains were sensitive to semi-
synthetic penicillins and cephalosporins (3
rd generation). In addition, a number of 7 strains showed resistance to
chloramphenicol (87,5%). The same eight strains were resistant to amoxicillin + clavulanic acid, showing on one hand,
the substrate specificity of beta-lactamases and, on the other hand, the possible adaptation of S. Typhimurium in
clavulanic acid (a beta-lactamase in hibitor which remains active, the ESBLs, therefore will enhance the action of
cephalosporins). According to the method of double diffusion synergy test, antimicrobial test results showed that the 8 strains tested produced beta-lactamase. Strains belonging to this phenotype (ESBLs) were characterized by cross-
resistance to most beta-lactam tested. All the 8 strain s showed ESBLs phenotype associated with phenotypes of
resistance to aminoglycosides, co trimoxazole and tetracyc line, and of these, sev en showed ESBLs phenotype
associated with chloramphenicol resistance phenotype.
This indicative study on the characterization of antibi otic resistance by phenotyp ic methods strains of S.
Typhimurium identified as producing beta-lactamases, showed that all the isolates were resistant to amoxicillin/clavulanic
acid and ceftazidime. No strain sensitive to these antibiotics was identified. Carbapenems (imipenem) are beta-lactam
antibiotics that were susceptible to a ll strains of S. Typhimurium producing ESBL s (100% of cases). Si milarly, there was
no resistant strain of isolated cefamicine (cefoxitim). The most active quinolone antibiotic ciprofloxacin proved that all the
8 strains of S. Typhimurium tested, showed sensitivity. All these antimicrobials may be used as antibiotics of choice in
the gastrointestinal infections caused by Salmonella serovar Typhimurium.
In contrast, a high percentage of isolates showed resistance to tetracycline (87,5%), ampicillin, cefotaxime,
gentamicin, kanamycin, streptomycin, sulfonamide, trimetho prim or nalidixic acid (the same percentage, 75%). In
addition, the study phenotypes of acquired resistance to anti-infective chemotherapies revealed that the 100% strains (8 of 8 selected) of S. Typhimurium showed ESBL-producing mu ltidrug-resistant phenomenon at medication (MDR) as
diffusion antibiogram.

Conclusions
Multiresistance to antibiotics of strains producing ESBLs was defined by the presence of concomitant
resistance to aminoglycosides, fluoroquinolones and sulfamethoxazole-trimethoprim. S. Typhimurium strains producing
ESBL showed resistance mechanisms associated with beta- lactam resistance to amin oglycosides, quinolones,
sulfonamides and tetracyclines.
In this context, the increasing prevalence of Salmonella enterica serovar Typhimurium strains resistant to
antibiotics and mainly those resistant betalactams, are worrying for the treatment of human salmonellosis.

Journal of Medicine and Life Volume 7, Special Issue 2 , 2014
82 References
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