Stress tolerant virulent strains of Cronobacter sakazakii from food

BACKGROUND: Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh. RESULT: Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress. CONCLUSION: Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Resistance to tetracycline and β-lactams and distribution of resistance markers in enteric microorganisms and pseudomonads isolated from the oral cavity

This study evaluated the occurrence of enteric bacteria and pseudomonads resistant to tetracycline and β-lactams in the oral cavity of patients exhibiting gingivitis (n=89), periodontitis (n=79), periodontally healthy (n=50) and wearing complete dentures (n=41). Microbial identification and presence of resistance markers associated with the production of β-lactamases and tetracycline resistance were performed by using biochemical tests and PCR. Susceptibility tests were carried out in 201 isolates of enteric cocci and rods. Resistance to ampicillin, amoxicillin/clavulanic acid, imipenem, meropenem and tetracycline was detected in 57.4 percent, 34.6 percent, 2.4 percent, 1.9 percent and 36.5 percent of the isolates, respectively. β-lactamase production was observed in 41.2 percent of tested microorganisms, while the most commonly found β-lactamase genetic determinant was gene blaTEM. Tetracycline resistance was disseminated and a wide scope of tet genes were detected in all studied microbial genus.

Tetracycline and oxytetracycline resistance determinants detected in Bacillus cereus strains isolated from honey samples / Detección de determinantes de resistencia a tetraciclina y oxitetraciclina en cepas de Bacillus cereus aisladas de muestras de miel

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.

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.

The involvement of tetA and tetE tetracycline resistance genes in plasmid and chromosomal resistance of Aeromonas in Brazilian strains

This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5 percent of the samples were positive for tetA, and also 37.5 percent were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.

Mini-Mu insertions in the tetracycline resistence determinant from proteus mirabilis

The inducible tetracycline resistance determinant isolated from Proteus mirabilis cloned into the plasmid pACYC177 was mutagenized by insertion of a mini-Mu-lac phage in order to define the regions in the cloned sequences encoding the structural and regulatory proteins. Three different types of mutants were obtained: one lost the resistance phenotype and became Lac+; another expressed the resistance at lowerlevels and constitutively; the third was still dependent on induction but showed a lower minimal inhibitory concentration. The mutant phenotypes and the locations of the insertions indicate that the determinant is composed of a repressor gene and a structural gene which are not transcribed divergently as are other known tetracycline determinants isolated from Gram-negative bacteria.

Plasmid mediated multidrug resistance in Salmonella typhi.

Between September 1989 and February 1991, strains of S. typhi showing multiple drug resistance were isolated from blood cultures of patients with typhoid fever. A total of 283 isolates were obtained over a period of 18 months. Forty four (11%) of these isolates were resistant to chloramphenicol, ampicillin, co-trimoxazole and tetracycline, the first three being drugs currently used in treating typhoid fever. Forty of the 44 multi-resistant strains tested showed transfer of resistance 'en block' to recipient Escherichia coli K 12 (J62-2). All 44 multi-resistant strains were sensitive to ciprofloxacin and ofloxacin. Since the resistance is plasmid-mediated, the problem is likely to get aggravated.