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Revista argentina de microbiología

versión impresa ISSN 0325-7541versión On-line ISSN 1851-7617

Rev. argent. microbiol. v.40 n.4 Ciudad Autónoma de Buenos Aires oct./dic. 2008


Tetracycline and oxytetracycline resistance determinants detected in Bacillus cereus strains isolated from honey samples

A. C. López, R. V. M. De Ortúzar, A. M. Alippi *

Unidad de Bacteriología, Centro de Investigaciones de Fitopatología, Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, cc 31, calle 60 y 119, 1900 La Plata, Argentina.
*Correspondence. E-mail:


The aim of this study was to investigate the presence of tetracycline and oxytetracycline resistance determinants in Bacillus cereus strains isolated from honey samples. Of a total of 77 isolates analyzed, 30 (39%) exhibited resistance to tetracyclines according to the results of a disk diffusion method. Resistant strains (n=30) were screened by PCR for the presence of the resistant determinants tetK, tetL, tetM, tetO, tetW, otrA and otrB and their MIC values for tetracycline, oxytetracycline and minocycline were assessed. According to the PCR results, 23 isolates (77%) presented at least one tetracycline or oxytetracycline resistance determinant. The tetK genotype was present in 10 isolates while the tetL, tetM, and otrA genotypes were present in 3, 2, and 5 isolates, respectively. In addition, 2 isolates of the tetK plus tetM genotype, 1 of the tetK plus tetL genotype, and 1 of the tetK plus otrA genotype were found. All isolates were tetW, tetO and otrB negatives. On the other hand, 7 isolates (23%) showed a tetracycline-resistant and/or minocyclineresistant phenotype (MIC) but did not carry any of the tet or otr determinants investigated in this study. This research has shown that B. cereus isolates from honey samples contain a variety of tetracycline and oxytetracycline resistance genes, including the tetK and tetL determinants which encode for efflux proteins, and tetM and otrA, which encode for ribosomal protection proteins. These findings indicate that strains isolated from honeys could represent a reservoir for tetracycline resistance genes. To our knowledge, this is the first report of tetracycline-resistant and oxytetracyclineresistant B. cereus strains carrying the tetK determinant, and also the first report of oxytetracycline-resistant and tetracycline- resistant Bacillus species carrying the otrA determinant.

Key words: Bacillus cereus; Honey; Tetracycline resistance determinants; tetK; tetM; tetL; otrA.


Detección de determinantes de resistencia a tetraciclina y oxitetraciclina en cepas de Bacillus cereus aisladas de muestras de miel. El objetivo del presente estudio ha sido investigar la presencia de diversos determinantes de resistencia a tetraciclina y oxitetraciclina en las poblaciones de Bacillus cereus presentes en la miel. De un total de 77 aislamientos evaluados, 30 (39%) resultaron resistentes a tetraciclina y/o minociclina de acuerdo con los resultados de las pruebas de difusión en disco. Dentro del grupo que presentó un fenotipo resistente, se investigó la presencia de los determinantes tetK, tetL, tetM, tetO, tetW, otrA y otrB por PCR y se determinaron los valores de CIM para tetraciclina, oxitetraciclina y minociclina. De acuerdo con los resultados obtenidos por PCR, 23 aislamientos (77%) presentaron al menos un determinante de resistencia a tetraciclina o a oxitetraciclina; el genotipo tetK se encontró en 10 de esos aislamientos, mientras que los genotipos tetL, tetM y otrA se hallaron en 3, 2 y 5 aislamientos, respectivamente. Ningún aislamiento presentó los genotipos tetW, tetO ni otrB. Adicionalmente, se encontraron los genotipos tetK plus tetM (2 aislamientos); tetK plus tetL (1 aislamiento) y tetK plus otrA (1 aislamiento). Por otra parte, 7 cepas (23%) resultaron resistentes a tetraciclina, oxitetraciclina y/o minociclina por CIM, pero no presentaban ninguno de los determinantes tet u otr estudiados. Estos resultados indican la existencia de un alto porcentaje de cepas de B. cereus aisladas de miel con genes de resistencia a tetraciclina y oxitetraciclina, incluyendo los determinantes tetK, tetL, tetM y otrA. Este estudio constituye el primer registro de la presencia del determinante tetK de resistencia a tetraciclina en B. cereus, como así también la presencia del determinante otrA dentro del género Bacillus.

Palabras clave: Bacillus cereus; Miel; Determinantes de resistencia a tetraciclina; tetK; tetM; tetL; otrA.


Tetracycline (TC) is a broad-spectrum antibiotic used in the treatment of bacterial infections in humans, animals, and insects. Therefore, bacteria from different ecosystems
are exposed to this antibiotic.
There are three main mechanisms of resistance to tetracycline: energy-dependent efflux, protection of the bacterial ribosome, and enzymatic inactivation of the tetracycline molecule (17). The two first mechanisms are widely distributed among gram-negative and gram-positive bacteria. Resistance to TC is primarily due to the acquisition of tet determinants frequently associated with mobile elements (17, 18). Up to now, 39 different tet and otr genes have been reported (2, 8, 18, 22).
Oxytetracycline (OTC) is commonly used in apiculture for the prevention and control of American (AFB) and European Foulbrood (EFB) of honey bees, the two major bacterial diseases affecting the larval and pupal stages of honey bees (Apis mellifera L.) (19). Recently, oxytetracycline-resistant (OTCr) Paenibacillus larvae isolates, the cause of AFB, have been detected and this phenotype correlated with the presence of two different plasmids carrying the tetL gene (12) and the tetK gene (5).
Aerobic spore-forming bacteria of the genera Paenibacillus and Bacillus are commonly found in honey (6, 10, 20). Qualitative examination of honeys revealed that the most prevalent species of Bacillus are Bacillus cereus, B. megaterium, B. pumilus, and B. coagulans (6, 20). B. cereus is an ubiquitous species commonly found in diverse habitats including several types of food. It has been involved in food poisoning cases due to the production of diverse toxins (1, 9). Tetracycline resistant (TCr) strains of the B. cereus group (B. cereus, B. thuringiensis, B. anthracis) isolated from soil and animal manure samples were found to carry the tetL gene, while other isolates carried both the tetM and tetL genes (3). These genes can be mobilized in the presence of either conjugative plasmids or transposons (3).
Although the genotypic diversity of B. cereus recovered from honey has been reported (10), there is no information regarding the existence of B. cereus TCr strains isolated from honeys or other apiarian sources. The aim of this study was to investigate both phenotypic and genotypic features of TC and OTC resistance in B. cereus strains isolated from honey samples.


Bacterial strains
A total of 77 B. cereus isolated from honey samples were used in this work. Sixty six isolates were recovered from Argentinean honeys randomly collected from apiaries located in different geographical areas (6, 10). In addition, isolates obtained from commercial honeys from Mexico (n= 1), Brazil (n = 2), Italy (n= 2), USA (n= 6) and Chile (n= 1) and a reference strain (ATCC 11778) were also studied and are listed in Table 1. All isolates were frozen-stored at - 80 °C in tryptic-soy broth plus 20% glycerol (v/v) until used.

Table 1.
Geographical origin and designation of bacterial strains used in this study and susceptibility test results (disk diffusion) to tetracycline (TC) and minocycline (MN).

Table 1. Continued

Antimicrobial susceptibility tests for tetracyclines
Resistance to tetracyclines was preliminary determined by the double disk test method described by Trzcinski and co-workers (21), using a 30 μg tetracycline disk, a 30 μg minocycline disk and a 5 μg tetracycline disk in a straight line, 9 mm apart with the minocycline disk in the center. Isolates were considered susceptible when showing an inhibition zone around the 30 μg TC disk and/or the 30 μg MN disk of ≥ 19 mm in diameter, intermediate for those showing 15 to 18 mm and resistant when the inhibition zone was ≤ 14 mm (13). When using 5 μg TC disks, the breakpoint values for susceptible, intermediate and resistant strains were ≥ 20 mm, 15 to 18 mm, and ≤ 14 mm respectively (5).
For those isolates showing diminished inhibition zones to tetracycline, and/or minocycline, the MICs of tetracycline (TC), oxytetracycline (OTC), and minocycline (MN) were determined by the agar dilution method according to the CLSI guidelines (14). Isolates were considered susceptible when their MICs were ≤ 4 μg/ml, intermediate for MICs of 8 μg/ml, or resistant when their MICs were ≥ 16 μg/ml (14). For quality control in susceptibility testing, Escherichia coli ATCC 25922 and B. cereus ATCC 11778 were used (13, 14).

PCR analysis of tetracycline resistance genes
PCR analysis was performed only on those strains that showed resistant or intermediate values according to the double disk diffusion technique, and also on both ATCC reference strains.
Total DNA was extracted by a rapid procedure as described by Alippi and Aguilar (4). The presence of the tetracycline resistance determinants tetK, tetL and tetM was investigated by PCRs using specific primers according to Ng and co-workers (15). The presence of tetO and tetW tetracycline resistance determinants was examined according to Aminov et al (7). In addition, the presence of oxytetracycline resistance determinants otrA and otrB was evaluated according to Nikolakopoulou et al. (16).


Of a total of 77 isolates analyzed, 30 (39%) exhibited resistance to tetracycline and/or minocycline according to the results of a double disk diffusion method (Table 1). TC, OTC and MN MIC values for the resistant isolates (n=30) and the ATCC reference strains (n=2) were assessed and are listed in Table 2.

Table 2.
Susceptibility test results (MICs) to tetracycline (TC), minocycline (MN), and oxytetracycline (OTC) (in mg/l), and resistance determinants for selected strains of B. cereus isolated from honeys.

Of the 30 isolates that exhibited a TCr, OTCr, and/or MNr phenotype according to their MICs, 23 (77%) presented at least one tetracycline or oxytetracycline resistance determinant (Table 2). According to the PCR results, 10 B. cereus isolates were classified as tetK genotype, 3 as tetL, 3 as tetM and 5 as otrA (Figure 1). In addition, the tetK plus tetM genotype was found in 2 isolates (Bc22 and Bc 121); the tetK plus otrA genotype was found in 1 isolate (Bc14) and the tetK plus tetL genotype was found in 1 isolate (Bc 55). All isolates were tetW, tetO and otrB negative (Table 2).

Figure 1. Agarose gel electrophoresis of PCR products amplified from tetracycline-resistant strains of Bacillus cereus isolates from honey samples. Lanes: M: molecular weight marker (100 bp ladder Promega), 1. Bc50 strain showing the 267 bp amplicon compatible with the tetL genotype, 2. Bc 108 strain showing the 778 bp amplicon corresponding to the otrA genotype, 3. Bc93 strain showing the 406 bp amplicon corresponding to the tetM genotype, 4. Bc 123 strain showing the 169 bp amplicon corresponding to the tetK genotype, and M: Molecular weight marker.

On the other hand, 7 isolates (23%) were TCr, OTCr and/or MNr but did not carry any tet or otr genes studied here (Table 2), which suggests that they carry other determinants. A similar situation was reported by Agersø et al (3) when working with isolates belonging to the B. cereus group, where 81 out of 88 TCr isolates did not yield any positive amplicons for any of the tet genes examined. Other authors have also reported that not all gram-positive bacteria showing a TCr phenotype have been shown to carry any of the already known tet genes (17).
All the isolates of the tetK, tetL, or tetM genotypes and the combinations of tetK/tetM, tetK/tetL and tetK/otrA were from Argentina, while the genotype otrA was found in isolates from both Argentina and the USA. The highest MICs for TC and OTC were observed in isolates of the tetK and/or otrA genotypes (Table 2).
All the strains that exhibited genotypes tetK and/or tetL were resistant or intermediate to TC and OTC but susceptible to MN, and those with genotype tetM were resistant or intermediate to TC, OTC and MN, as previously reported for the genus Staphylococcus (17, 21). By using the double disk test, the tetK and/or tetL genotypes and also the tetM genotype might be predicted for the B. cereus isolates in the same manner as that reported for S. aureus (21).
The MIC and zone size values obtained for reference strain E. coli ATCC 25922 were within the range of acceptance for quality control (13, 14). B. cereus ATCC 11778 resulted susceptible to TC, OTC and MN and, as expected, did not produce any positive amplicons for any of the tet or otr genes tested here.
To our knowledge, this is the first report of TCr and OTCr B. cereus strains carrying tetK and/or otrA determinants. It is also the first report of B. cereus isolates from honeys carrying tetL or tetM determinants, although previously found in the B. cereus group (3). In addition, this is also the first register of a Bacillus species carrying the otrA determinant, only previously reported in streptomycete populations from environmental samples (16).
It is interesting to point out that 4 out of 6 isolates from the USA exhibited the otr(A) determinant while only 2 out of 23 Argentinean isolates from Laprida honeys, in Buenos Aires province, contained this determinant. OTC has been extensively used in many countries, including in the main honey producing areas of the USA and Argentina, to control bacterial diseases affecting honey bees. TCr and OTCr P. larvae isolates carrying the tetK (6) or tetM (11, 12) determinants in mobile elements have been recently found, which suggests that TC and OTC resistance could be transferred between gram-positive bacteria from the same ecological niche.
In conclusion, this study has shown that B. cereus isolates from honey samples contain a variety of TC and OTC resistant genes including the tetK and tetL determinants that encode efflux proteins, and tetM, and otrA that encode ribosomal protection proteins. This may indicate that strains isolated from honeys have access to and/or are a reservoir of resistance genes. Further studies will be needed to determine the genetic supports for these resistance genes in order to understand how they disseminate in gram-positive spore-forming bacteria from apiarian sources.

Acknowledgements. This research was supported by CIC (Comisión de Investigaciones Científicas de la Prov. de Bs. As., Argentina) and ANPCyT Argentina (Grant No. BID 1728 OC-AR PICT 2411). AMA is a Career Investigator of CIC and ACL is a recipient of a scholarship from CONICET, respectively.


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Recibido: 18/03/08
Aceptado: 30/09/08.

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