Nosocomial or intrahospital infections (IN or IIH) have been defined as those infections that are acquired within a hospital and whose manifestation, depending on the incubation period of the infection, can occur 48-72 hours later, or even once given discharge the patient, that is, they are not present or being incubated at the time of admission^[1,2]. They are preferentially caused by bacteria, being the most isolated from intensive human units Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Acinetobacter baumanni^[3,4].

In the case of veterinary medicine, there are few studies carried out in veterinary hospitals where the most prevalent nosocomial agents are isolated and identified. In Chile, some bacteria causing infections in operative wounds have been identified in a hospital of small animals, with Staphylococcus intermedius being the most recurrent agent in both dogs and cats, followed by Actinomyces pyogenes, Micrococcus spp., and Pseudomonas aeruginosa^[5]. We have also identified potentially nosocomial environmental bacteria such as Enterococcus faecium, Enterobacter cloacae, Escherichia coli and Pseudomonas aeruginosa, which presented a high percentage of resistance to 2 or more antimicrobials^[6].

To define if a bacterium is resistant to an antimicrobial, it is considered the minimum inhibitory concentration (MIC); when the concentration that the antimicrobial reaches in the tissue does not exceed the MIC, the bacteria have all the possibilities of surviving and it can be indicated that they are resistant^[7,8]. This resistance can be a natural property (intrinsic) or the result of a mutation or acquisition of genetic material in the form of plasmids or transposons, through different transfer processes, which is facilitated by the selection pressure generated when using antibacterials^[9-11].

Currently, one of the groups of antibacterials that have high levels of bacterial resistance is that of tetracyclines^[12-14]. The main resistance mechanisms described to counteract the effect of these antibacterials are: active expulsion pumps specific for tetracyclines and ribosomal protection proteins, with the enzymatic inactivation of the drug being of less importance^[15]. The first mechanism has been described mostly among Gram-negative bacteria, while the second among Gram-positive.

At least, 40 genetic determinants of tetracycline resistance (tet genes) and 3 determinants of resistance to oxytetracycline (otr genes) have been characterized^[16]. Most of these genes, including tet(A) and tet(B), encode a cytoplasmic membrane efflux (Tet) protein, which exchanges a proton for a cation-tetracycline complex against the concentration gradient^[17]. It is described that these two genes are widely disseminated in nature among Gram-negative bacteria, because they occur mainly in mobile genetic elements^[18].

Some studies have evaluated the prevalence of these genes in nosocomial bacteria and it is recognized that the tetgene (B) is the most prevalent in strains of Acinectobacerbaumanni^[18-20] while tet(A) and tet(B) present a high frequency in strains of Escherichia coli^[21]. To achieve this detection, the Polymerase Chain Reaction (PCR) has been used^[22]. This technique is based on the exponential amplification of a sequence of interest, in a sensitive and specific way. To perform it, DNA is required, two synthetic oligonucleotides (primers) that give specificity to the technique, the DNA polymerase originally from the bacteria Thermus aquaticus, the four deoxynucleotides and a thermal cycler, which allows to vary the temperature according to the stage in development, needing denaturation and extension, temperatures higher than alignment^[23,24]. The PCR technique requires positive controls, which are a fundamental element in the interpretation of the results. Currently, in the Microbiology laboratory of the Faculty of Veterinary and Animal Sciences of the University of Chile, there are no native strains that certify the presence of resistance genes that can be used as positive controls, so they must be obtained from phenotypically resistant strains. Due to this deficiency, this study aims to generate native positive controls useful in the detection of the tet(A) and tet(B) genes, complementing the investigations in which positive control strains for the blaTEM^[25] and mecA genes have been obtained, involved in β-lactam resistance (unpublished data).

These controls will allow confirming the presence or absence of these genes in bacteria isolated from hospital premises, being very useful as a predictive measure for greater control of bacterial resistance, and in the development of a surveillance system that two guide the rotation of antimicrobials to be used, according to the epidemiological situation of each hospital facility^[26-28].

Materials and Methods

This research was carried out in the Laboratory of Veterinary Bacteriology of the Department of Animal Preventive Medicine in the Faculty of Veterinary and Animal Sciences of the University of Chile.

Two bacterial strains were used: Pseudomonas aeruginosa and Pantoeaagglomerans, isolated and identified in a previous study^[29] that showed phenotypic resistance to tetracyclines according to the determination of antimicrobial susceptibility using the Kirby Bauer method and which, when analyzed previously by PCR, amplified bands of size compatible with what has been described for the tet (A) and tet (B) genes^[30].

The extraction of the bacterial DNA was carried out using a commercial kit (Genomic DNA Purification kit, Fermentas^®), following the manufacturer’s instructions. For its amplification, a commercial kit (2X PCR Master Mix Fermentas^®) was used, which contains the thermo stable polymerase, the deoxynucleotide triphosphates (dNTPs), the reaction buffer and MgCl₂.

The amplification conditions of the genes were the same for both tet genes. Briefly, the samples were subjected to 30 cycles, with denaturation at 94ºC for one minute, alignment at 55ºC for one minute, extension at 72ºC for one minute and finally a final elongation at 72ºC for five minutes. The used primers are 5’-GTAATTCTGAGCACTGTCGC-3' and 5'-CTGCCTGGACAACATTGCTT-3' for tet (A) gene and 5'-TTGGTTAGGGGCAAGTTTTG-3' and 5'-GTAATGGGCCAATAACACCG-3' for tet (B) gene and the expected band sizes for tet(A) and for tet(B) were 950 bp and 650 bp respectively. The visualization of the PCR products was done by electrophoresis in 2% agarose gel (Winkler^®) in Tris acetate EDTA (TAE) buffer (Fermentas^®). The PCR product was mixed with a commercial loading product (6X Mass Ruler Loading Dye Solution, Fermentas^®) and the electrophoresis was carried out at 90 V for 90 minutes. As a molecular size marker, a standard was used that contains DNA fragments between 100 and 1000 bp (DNA ladder, Fermentas^®). After electrophoresis, the gel was immersed in ethidium bromide (0.5 μg/mL, Fermelo^®) and the bands were visualized in a transilluminator of ultraviolet light (Transiluminator UVP^®) and photographed with a digital camera.

The biosafety measures included the use of sterile material, the use of long-sleeved apron and gloves. The use of a transilluminator of UV light and ethidium bromide, contemplated the use of an acrylic plate and glasses with filter, as well as the incineration of the gels.

The sequencing of the resulting DNA fragments was carried out by the Genytec Company (Genetics and Technology Ltd.). Subsequently, these sequences were aligned using the Clustal Ω online program (free access), obtaining a consensus sequence for each gene and each strain, comparing them with some described in the GenBank^® and finally determining the nucleotide identity percentage (NIP) for the genes of interest^[31]. The criterion for classifying the tet genes was based on that previously used, considering at least one NIP ≥ 80% to be classified within any of the genetic determinants already described^[32].

Results

Detection and sequencing of tet(A) and tet(B) genes in bacteria described as nosocomial.

In figure 1, the bands obtained when performing PCR are visualized: 300 bp bands for the gene tet(A) and 650 bp for the tet (B) genes.

Figure 1: PCR detection of tet(A) and tet(B) genes in two bacterial.

Determination of nucleotidic identity percentage (NIP) with respect to GenBank^®.

Once all these fragments were sequenced, a consensus sequence was obtained for each bacterial strain and tet gene studied (Table 1).

Table 1: Consensus sequences for BOR1 and BOR2 (tet(B)) and BOR3 and BOR4 (tet(A)) in two bacterial strains

Discussion

Nowadays, it is necessary to have tools to guide the control of bacterial resistance in hospitals. One of these tools could be based on the detection of resistance genes, which would allow a better choice of antimicrobials to be used, complementing the information obtained in the antibiogram.

For this molecular detection, an alternative is to use the PCR technique. An important risk in its realization is the contamination with exogenous DNA, which may come from mainly from previous reactions or using contaminated reagents. To avoid this, it is recommended to respect the work rules, use reagents of certified quality, disposable material and have a physical space separate from areas where other activities are carried out^[33,34].

The described methodology allowed obtaining amplicons of sizes close to 650 bp for the tet(B) gene and 300 bp for the tet(A) gene from samples from P. agglomerans and P. aeruginosa, two environmental bacterial strains described as potentially nosocomial. Obtaining amplicons of 650 bp was a first sign of the presence of the tet(B) gene. However, the presence of 300 bp amplicons with respect to the tet(A) gene generated an unresolved question, because these same strains were previously analyzed by PCR, amplifying 950 bp bands. The nucleotide sequences obtained allowed us to demonstrate the presence of the tet(B) gene and definitively rule out the alternative that the 300 bp fragment is part of the tet(A) gene, because despite reaching a relatively high PIN value (83%) in P. aeruginosa, its alignment with the other sequences described was not homogeneous, a situation that is even more evident in the strain of P. agglomerans. This result would not correspond to a failure in the extraction of bacterial DNA, which is corroborated by the presence of amplicons of 650 bp; nor would it be attributable to the primers, who were prepared again to avoid confusion; The reaction mixture was the same, therefore it does not represent an error either and finally, the program introduced in the thermo cycler is the same for both PCR protocols. Therefore, for the detection of this gene soon, it is advisable to use alternative primers, either previously used^[35] or designed by computer programs (in silico) using as reference sequences already described and published in gene banks.

When obtaining the nucleotide sequences and performing the alignment through the Clustal Ω program, the obtaining of two positive control strains for tet(B): P. agglomerans and P.aeruginosa was corroborated, since the NIP value reached (≥ 93%) allows classify them in this genetic determinant, surpassing the NIP value of 80%, recommended in the case of tet genes. However, the question remains about the presence of the tet(A) gene in the analyzed samples.

In relation to this, when incorporating the 300 bp sequence in the database of the free access on-line program called BLAST^[36], the sequence BOR4 (P.aeruginosa) shows a 93% nucleotide identity with a segment of the complete genome of P.aeruginosa, which was expected. However, by incorporating the sequence BOR3 (P. agglomerans), the program finds no identity in its vast nucleotide collection, suggesting perhaps non-specific amplification. For this reason, it would be interesting to reiterate the use of the in silico designed starters proposed in this study, to corroborate the obtaining of fragments compatible with the expected size and verify their identity through sequencing, evaluating their possible usefulness as positive controls.

Finally, the verification of the presence of the tet(B) gene in P.agglomerans and P.aeruginosa is -accordingly- a finding because this gene has not been previously described in any of these strains.

Conclusion

The methodology described allowed to obtain two native control strains: P. agglomerans and P.aeruginosa, to continue with the study of the tet(B) gene, one of the 40 genes involved in tetracycline resistance and constitutes a first step towards the study of the relationships between antimicrobial susceptibility determined by the Kirby Bauer method and the effective presence of a gene involved in resistance.

References

1. Boehme, A. K., Kumar, A.D., Dorsey, A.M., et al. Infections present on admission compared with hospital-acquired infections in acute ischemic stroke patients. (2013) J stroke Cerebrovasc Dis 22(8): 582-589.

   Pubmed| Crossref | Others

2. Prestinaci, F., Pezzotti, P., Pantosti, A. Antimicrobial resistance: a global multifaceted phenomenon. (2015) Pathog Glob Health 109(7): 309-18.

   Pubmed| Crossref | Others

3. Olaechea, P., Insausti, J., Blanco, A., et al. Epidemiología eimpacto de las infecciones nosocomiales. (2010) Med Intensiva 34(4): 256-267.

   Pubmed| Crossref | Others

4. Viderman, D., Khamzina, Y., Kaligozhin, Z., et al. An observational case study of hospital associated infections in a critical care unit in Astana, Kazakhstan. (2018) Antimicrob Resistance Infect Control 7: 57.

   Pubmed| Crossref | Others

5. Sanz, L., Junco, C. Identificación de la etiología de las infecciones bacterianasde las heridas operatorias. (2009) Hosp Vet 1: 21-32.

   Pubmed| Crossref | Others

6. Witt, N., Rodger, G., Vandesompele, J., et al. An assessment of air as a source of DNA contamination encountered when performing PCR. (2009) J Biomol Tech 20(5): 236-240.

   Pubmed| Crossref | Others

7. Errecalde, J. (2004). Uso de antimicrobianos en animales de consumo: incidencia del desarrollo de resistencias en salud pública. FAO. Roma, Italia. 61 p.

   Pubmed| Crossref | Others

8. Sandegren, L. Selection of antibiotic resistance at very low antibiotic concentrations. (2014) Ups J Med Sci 119(2): 103-107.

   Pubmed| Crossref | Others

9. Davies, J. Inactivation of antibiotics and dissemination of resistance genes. (1994) Science 264(5157): 375-382.

   Pubmed| Crossref | Others

10. Cabrera, C., Gómez, F., Zúñiga, A. La resistencia de bacterias antibióticos, antisépticos y desinfectantes una manifestación de los mecanismos de supervivencia y adaptación. (2007) Colomb Med 38: 149-158.

    Pubmed| Crossref | Others

11. Martinez, J.L., Baquero, F. Emergence and spread of antibiotic resistance: setting a parameter space. (2014) Ups J Med Sci 119(2): 68-77.

    Pubmed| Crossref | Others

12. Chopra, I., Roberts, M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. (2001) Microbiol Mol Biol Rev 65(2): 232-260.

    Pubmed| Crossref | Others

13. Fair, R.J., Tor, Y. Antibiotics and bacterial resistance in the 21st century. (2014) Perspect Medicin Chem 6: 25-64.

    Pubmed| Crossref | Others

14. Grossman, T.H. Tetracycline Antibiotics and Resistance. (2016) Cold Spring Harb Perspect Med. 6(4): 25387

    Pubmed| Crossref | Others

15. Trieber, C.A., Taylor, D.E. Mutations in the 16S rRNA genes of Helicobacter pylori mediate resistance to tetracycline. J Bacteriol 184(8): 2131-2140.

    Pubmed| Crossref | Others

16. Roberts, M. Tet mechanisms of resistance. (2010)

    Pubmed| Crossref | Others

17. Davies, J., Weeb, V. Antibiotic resistance of bacteria.In: Shaechter, M. (2004).The desk encyclopedia of microbiology. Elsevier. California, USA. 25-46.

    Pubmed| Crossref | Others

18. Martí, S., Fernández-Cuenca, F., Pascual, A., et al. Prevalencia de los genes tetAy tetB como mecanismode resistencia a tetraciclina y minociclina en aislamientos clínicos de Acinetobacter baumannii. (2006). Enferm Infecc Microbiol Clin 24(2): 77-80.

    Pubmed| Crossref | Others

19. Falagas, M.E., Vardakas, K.Z., Kapaskelis, A., et al. Tetracyclines for multidrug-resistant Acinetobacter baumannii infections. (2015) Int. J. Antimicrob. Agents 45(5): 455-460.

    Pubmed| Crossref | Others

20. Almasaudi, S.B. Acinetobacter spp. as nosocomial pathogens: Epidemiology and resistance features. (2016) Saudi J Biol Sci 25(3): 586-596.

    Pubmed| Crossref | Others

21. Tuckman, M., Petersen, P., Howe, A., et al. Occurrence of tetracycline resistance genes among Escherichia coli isolates from the phase 3 clinical trials for tigecycline. (2007) Antimicrob Agents Chemother 51(9): 3205-3211.

    Pubmed| Crossref | Others

22. Fluit, A.C., Visser, M., Schmitz, F.J. Molecular detection of antimicrobial resistance. (2001) Clin Microbiol Rev 14(4): 836-871.

    Pubmed| Crossref | Others

23. Mullis, K., Faloona, F. Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. (1987) Methods Enzymol 155: 335-350.

    Pubmed| Crossref | Others

24. Saiki, R., Gelfand, D., Stoffel, S., et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. (1988) Science 239(4839): 487-491.

    Pubmed| Crossref | Others

25. Yáñez, D., Jara, M.A., Navarro, C. Detection of BlaTEM resistance gene in bacteria described as nosocomial. (2018) J of Science Frontier Research 18(7): 21-28

    Pubmed| Crossref | Others

26. Oteo, J., Campos, J. Valor de los sistemas de vigilancia de resistencia a antibióticos. (2003) Enferm Infecc Clin 21(3): 123-125.

    Pubmed| Crossref | Others

27. Ogeer-Gyles, J., Mathews, K., Boerlin, P., et al. Nosocomial infections and antimicrobial resistance in critical care medicine. (2006) J Vet Emerg Crit Care 16(1): 1-18.

    Pubmed| Crossref | Others

28. Allcock, S., Young, E.H., Holmes, M., et al. Antimicrobial resistance in human populations: challenges and opportunities. (2017) Glob Health Epidemiol Genom 2: 4.

    Pubmed| Crossref | Others

29. Jara, MA., Navarro, C., Avendaño, P. et al. dentificación y estudio de susceptibilidad antimicrobiana de bacterias potencialmente responsables de infecciones nosocomiales en hospitales veterinarios de la Universidad de Chile. Av Cs Vet 24: 11-17.

    Pubmed| Crossref | Others

30. Oyarce, D. Detección de cuatro genes de resistencia a tetraciclinas en bacterias nosocomiales Gram-negativas, aisladas en recintos hospitalarios veterinarios. (2011) Memoria Título Médico Veterinario. Santiago, Chile. U. Chile, Fac Ciencias Veterinarias y Pecuarias. 21 .

    Pubmed| Crossref | Others

31. Thompson, J., Higginss, D., Gibson, T. Clustal W: Improving the sensitivity progressive multiple sequence alignments through sequence weighting position specific gap penalties and weight matrix choice. (1994) Nucl Acids Res 22(22): 4673-4680.

    Pubmed| Crossref | Others

32. Levy, S., McMurry, L., Barbosa, T., et al. Nomenclature for new tetracycline resistance determinants. (1999) Antimicrob Agents Chemother 43(6): 1523- 1524.

    Pubmed| Crossref | Others

33. Wolcott, M. Advances in nucleic acid-based detection methods. (1992) Clin Microbiol Rev 5(4): 370 -386.

    Pubmed| Crossref | Others

34. Corless, C.E., Guiver, M., Borrow, R., et al. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. (2000) J Clin Microbiol 38(5): 1747-1752.

    Pubmed| Crossref | Others

35. Guillaume, G., Verbrugge, D., Chasseur-Libotte, M. PCR typing of tetracycline resistance determinants (Tet A-E) in Salmonella enterica serotype Hadar and in the microbial community of activated sludges from hospital and urban wastewater treatment facilities in Belgium. (2000) FEMS Microbiol Ecol 32(1): 77-85.

    Pubmed| Crossref | Others

36. Zhang, Z., Schwartz, S., Wagner, L., et al. A greedy algorithm for aligning DNA sequences. (2000) J Comput Biol. 7(1-2): 203-214.

    Pubmed| Crossref | Others

37. WHO (world Health Organization) Epidemiología de las infecciones. (2003)

    Pubmed| Crossref | Others