Tolerance response of multidrug-resistant Salmonella enterica strains to habituation to Origanum vulgare L. essential oil.

Multidrug-resistant Salmonella enterica isolates from human outbreaks or from poultry origin were investigated for their ability to develop direct-tolerance or cross-tolerance to sodium chloride, potassium chloride, lactic acid, acetic acid, and ciprofloxacin after habituation in subinhibitory amounts ( of the minimum inhibitory concentration - (MIC) and of the minimum inhibitory concentration - MIC) of Origanum vulgare L. essential oil (OVEO) at different time intervals. The habituation of S. enterica to OVEO did not induce direct-tolerance or cross-tolerance in the tested strains, as assessed by the modulation of MIC values. However, cells habituated to OVEO maintained or increased susceptibility to the tested antimicrobials agents, with up to fourfold double dilution decrease from previously determined MIC values. This study reports for the first time the non-inductive effect of OVEO on the acquisition of direct-tolerance or cross-tolerance in multidrug-resistant S. enterica strains to antimicrobial agents that are largely used in food preservation, as well as to CIP, the therapeutic drug of salmonellosis.

Commercially laid eggs vs. discarded hatching eggs: contamination by Salmonella spp.

Salmonella enterica is frequently associated with outbreaks of human salmonellosis, and products of avian origin, such as eggs and chicken meat, are the main vehicles of its transmission. The present study describes the occurrence of different serovars of Salmonella enterica and phagotypes of S. enterica serovar Enteritidis in eggs destined for human consumption. Four thousand eggs obtained from commercial egg laying farms and one thousand discarded hatching eggs from broiler farms, which were acquired at farmers' markets and informal shops, were analyzed. Salmonella spp. was isolated from 52.0% of the discarded hatching eggs, in which the predominant serovar was Enteritidis (84.6%), and the predominant Salmonella Enteritidis phagotype (PT) was PT7 (26.9%). Salmonella spp. was not isolated from eggs obtained from commercial egg laying farms. The antimicrobial resistance profile showed that 23.1% (n = 6) of the SE strains were resistant to nalidixic acid. The results suggest that the consumption of discarded hatching eggs represents an important source of Salmonella transmission to humans.

Plasmid-mediated quinolone resistance (PMQR) and mutations in the topoisomerase genes of Salmonella enterica strains from Brazil.

The objective of this study was to identify mutations in the Quinolone Resistance Determining sources Regions (QRDR) of the gyrA, gyrB, parC, and parE genes and to determine if any of the qnr variants or the aac(6')-Ib-cr variant were present in strains of Salmonella spp. isolated in Brazil. A total of 126 Salmonella spp. strains from epidemic (n = 114) and poultry (n = 12) origin were evaluated. One hundred and twelve strains (88.8%) were resistant to nalidixic acid (NAL) and 29 (23.01%) showed a reduced susceptibility to ciprofloxacin (Cip). The mutations identified were substitutions limited to the QRDR of the gyrA gene in the codons for Serine 83, Aspartate 87 and Alanine 131. The sensitivity to NAL seems to be a good phenotypic indication of distinguishing mutated and non-mutated strains in the QRDR, however the double mutation in gyrA did not cause resistance to ciprofloxacin. The qnrA1 and qnrB19 genes were detected, respectively, in one epidemic strain of S. Enteritidis and one strain of S. Corvallis of poultry origin. Despite previous detection of qnr genes in Brazil, this is the first report of qnr gene detection in Salmonella, and also the first detection of qnrB19 gene in this country. The results alert for the continuous monitoring of quinolone resistance determinants in order to minimize the emergence and selection of Salmonella spp. strains showing reduced susceptibility or resistance to quinolones.

Expression of the marA, soxS, acrB and ramA genes related to the AcrAB/TolC efflux pump in Salmonella entérica strains with and without quinolone resistance-determining regions gyrA gene mutations

Several studies have been conducted in recent years to elucidate the structure, function and significance of AcrB, MarA, SoxS and RamA in Salmonella enterica. In this study, the relative quantification of acrB, soxS, marA and ramA genes expression was evaluated in 14 strains of S. enterica, with or without accompanying mutations in the quinolone resistance-determining regions of the gyrA gene, that were exposed to ciprofloxacin during the exponential growth phase. The presence of ciprofloxacin during the log phase of bacterial growth activated the genes marA, soxS, ramA and acrB in all S. enterica strains analyzed in this study. The highest expression levels for acrB were observed in strains with gyrA mutation, and marA showed the highest expression in the strains without mutation. Considering only the strains with ciprofloxacin minimum inhibitory concentration values < 0.125 [1]g/mL (sensitive to ciprofloxacin), the most expressed gene in the strains both with and without mutations was acrB. In the strains with ciprofloxacin minimum inhibitory concentration values > 0.125 [1]g/mL (low susceptibility), with and without mutations in gyrA, the most expressed gene was marA. In this study, we observed that strains resistant to nalidixic acid may express genes associated with the efflux pump and the expression of the AcrAB-TolC pump genes seems to occur independently of mutations in gyrA.

Expression of the marA, soxS, acrB and ramA genes related to the AcrAB/TolC efflux pump in Salmonella enterica strains with and without quinolone resistance-determining regions gyrA gene mutations.

Several studies have been conducted in recent years to elucidate the structure, function and significance of AcrB, MarA, SoxS and RamA in Salmonella enterica. In this study, the relative quantification of acrB, soxS, marA and ramA genes expression was evaluated in 14 strains of S. enterica, with or without accompanying mutations in the quinolone resistance-determining regions of the gyrA gene, that were exposed to ciprofloxacin during the exponential growth phase. The presence of ciprofloxacin during the log phase of bacterial growth activated the genes marA, soxS, ramA and acrB in all S. enterica strains analyzed in this study. The highest expression levels for acrB were observed in strains with gyrA mutation, and marA showed the highest expression in the strains without mutation. Considering only the strains with ciprofloxacin minimum inhibitory concentration values<0.125 µg/mL (sensitive to ciprofloxacin), the most expressed gene in the strains both with and without mutations was acrB. In the strains with ciprofloxacin minimum inhibitory concentration values ≥ 0.125 µg/mL (low susceptibility), with and without mutations in gyrA, the most expressed gene was marA. In this study, we observed that strains resistant to nalidixic acid may express genes associated with the efflux pump and the expression of the AcrAB-TolC pump genes seems to occur independently of mutations in gyrA.

Commercially laid eggs vs. discarded hatching eggs: contamination by Salmonella spp

Salmonella enterica is frequently associated with outbreaks of human salmonellosis, and products of avian origin, such as eggs and chicken meat, are the main vehicles of its transmission. The present study describes the occurrence of different serovars of Salmonella enterica and phagotypes of S. enterica serovar Enteritidis in eggs destined for human consumption. Four thousand eggs obtained from commercial egg laying farms and one thousand discarded hatching eggs from broiler farms, which were acquired at farmers' markets and informal shops, were analyzed. Salmonella spp. was isolated from 52.0% of the discarded hatching eggs, in which the predominant serovar was Enteritidis (84.6%), and the predominant Salmonella Enteritidis phagotype (PT) was PT7 (26.9%). Salmonella spp. was not isolated from eggs obtained from commercial egg laying farms. The antimicrobial resistance profile showed that 23.1% (n = 6) of the SE strains were resistant to nalidixic acid. The results suggest that the consumption of discarded hatching eggs represents an important source of Salmonella transmission to humans.

Plasmid-mediated quinolone resistance (PMQR) and mutations in the topoisomerase genes of Salmonella enterica strains from Brazil

The objective of this study was to identify mutations in the Quinolone Resistance Determining sources Regions (QRDR) of the gyrA, gyrB, parC, and parE genes and to determine if any of the qnr variants or the aac(6')-Ib-cr variant were present in strains of Salmonella spp. isolated in Brazil. A total of 126 Salmonella spp. strains from epidemic (n = 114) and poultry (n = 12) origin were evaluated. One hundred and twelve strains (88.8%) were resistant to nalidixic acid (NAL) and 29 (23.01%) showed a reduced susceptibility to ciprofloxacin (Cip). The mutations identified were substitutions limited to the QRDR of the gyrA gene in the codons for Serine 83, Aspartate 87 and Alanine 131. The sensitivity to NAL seems to be a good phenotypic indication of distinguishing mutated and nonmutated strains in the QRDR, however the double mutation in gyrA did not cause resistance to ciprofloxacin. The qnrA1 and qnrB19 genes were detected, respectively, in one epidemic strain of S. Enteritidis and one strain of S. Corvallis of poultry origin. Despite previous detection of qnr genes in Brazil, this is the first report of qnr gene detection in Salmonella, and also the first detection of qnrB19 gene in this country. The results alert for the continuous monitoring of quinolone resistance determinants in order to minimize the emergence and selection of Salmonella spp. strains showing reduced susceptibility or resistance to quinolones.

Plasmid-mediated quinolone resistance by genes qnrA1 and qnrB19 in Salmonella strains isolated in Brazil.

Considering the importance of the mechanisms involved in quinolone resistance, this study evaluate the presence of PMQR in 126 epidemic and not epidemic strains of Salmonella spp. It was noted that presence of PMQR, by itself, did not generate resistance to ciprofloxacin; but detection of qnr genes in Salmonella spp. and the identification of the qnrB19 variant in a strain of poultry origin alert for the indiscriminate use of quinolones in poultry production, that can result in a pressure for mutant selection of resistant strains with a clinical limitation use of FQs in the near future. And last but not least, is the need to continued study of resistance mechanisms and to monitor the microbial resistance profile of epidemiological strains.

Molecular characterization and resistance profile of Salmonella Enteritidis PT4 and PT9 strains isolated in Brazil.

A total of 41 Salmonella Enteritidis strains, including phago-types (PTs) PT4 and PT9, were characterized by antimicrobial resistance profiles and PFGE. Of these strains, 34 were isolated from patients and foods, and 7 were of poultry origin. All strains were susceptible to ampicillin, chloramphenicol, cefotaxime, ciprofloxacin and trimethoprim/sulfamethoxazole, and 41.5 % (n = 17) were resistant to nalidixic acid. PFGE analysis using XbaI and SpeI restriction enzymes resulted in X1S1 as the prevalent pattern, which was present in 48.8 % (n = 20) of epidemic strains and in one strain isolated from discarded hatching eggs. Distinct patterns were found for the other strains isolated from poultry (X3S1, X8S8, X11S12, X11S13, X16S1 and X13S15). The S. Enteritidis PT9 strains associated with outbreaks of salmonellosis were highly similar (≥0.90), suggesting clonality. The PFGE genotypes were related to the PTs, and it was possible to differentiate strains isolated from patients with salmonellosis from other strains of non-epidemic origin. The PFGE results suggested that the S. Enteritidis strains of poultry origin were a possible source of human salmonellosis during the study period.

Ciprofloxacin susceptibility reduction of salmonella strains isolated from outbreaks

The antimicrobial susceptibility of 212 Salmonella strains isolated from patients and foods was evaluated and 45 percent were found to be resistant to nalidixic acid. Nalidixic acid resistant strains showed a higher minimal inhibitory concentration for ciprofloxacin than sensitive strains. During the study an increase of strains with reduced susceptibility to ciprofloxacin was also observed.

Ciprofloxacin susceptibility reduction of Salmonella strains isolated from outbreaks.

The antimicrobial susceptibility of 212 Salmonella strains isolated from patients and foods was evaluated and 45% were found to be resistant to nalidixic acid. Nalidixic acid resistant strains showed a higher minimal inhibitory concentration for ciprofloxacin than sensitive strains. During the study an increase of strains with reduced susceptibility to ciprofloxacin was also observed.

A robotic DNA purification protocol and real-time PCR for the detection of Campylobacter jejuni in foods.

Primers designed to amplify a Campylobacter jejuni cadF gene sequence were used in an SYBR Green I real-time PCR assay as an alternative to conventional bacteriological methods for the rapid detection of C. jejuni in foods. Twenty-five grams of chicken skin (breast and thigh) was contaminated by adding approximately 1, 10, or 50 CFU of C. jejuni ATCC 35560. Twenty-five grams of pork and 25-ml aliquots of milk were also inoculated with 1 and 10 CFU of the pathogen. The samples were incubated in Bolton broth for different periods at 37 and 42 degrees C under microaerophilic conditions. Using a commercial robotic DNA purification system, DNA was extracted and purified from 1-ml aliquots of the enrichment cultures before and after centrifugation of the 250-ml enrichment broth at 15,900 x g for 10 min at 4 degrees C. The DNA was used as the template in a real-time PCR assay. C. jejuni was detected after 12 h of enrichment from samples inoculated with about 50 CFU/25-g sample. After centrifugation, an enrichment step of 8 h was sufficient to allow detection of pathogen in samples inoculated with 10 CFU/25 g. However, 24 h of enrichment was necessary to detect pathogen in samples inoculated with approximately 1 CFU/25 g. The real-time PCR protocol developed in this study significantly reduced the detection time of C. jejuni in foods.

Detection of Campylobacter jejuni in naturally contaminated chicken skin by melting peak analysis of amplicons in real-time PCR.

Contamination of poultry by Campylobacter spp. is a significant source of human diarrheal diseases. Traditional methods currently used to detect Campylobacter in foods are time-consuming and labor-intensive. In this study, primers designed for the Campylobacter jejuni cadF gene sequence were used in a SYBR Green I real-time PCR assay as an alternative to a conventional bacteriological method for the rapid detection of C. jejuni from poultry. Twelve portions of chicken purchased from two local grocery stores and 39 portions obtained from a commercial processing plant were examined. Samples of the skin were enriched in Bolton broth at 37 degrees C for 3 h and then at 42 degrees C for 9, 21, or 45 h under microaerobic conditions. DNA was extracted from 1-ml aliquots of the enrichment cultures using 1% Triton X-100. The DNA was used as the template in a real-time polymerase chain reaction (PCR) assay. After 24 h of enrichment, C. jejuni was isolated from 13 samples and all of the positive cultures were also detected by the real-time PCR procedure. C. jejuni was detected by both methods from samples artificially contaminated with 1 or 10 CFU of C. jejuni per 10 g, after 24 h of enrichment. The real-time PCR method was found to be sensitive and specific. It significantly reduced the time required for the detection of C. jejuni in poultry following enrichment of samples.