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Chlortetracycline and florfenicol induce expression of genes associated with pathogenicity in multidrug-resistant Salmonella enterica serovar Typhimurium.

ABSTRACT: Background:Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Typhimurium) is a serious public health threat as infections caused by these strains are more difficult and expensive to treat. Livestock serve as a reservoir for MDR Salmonella, and the antibiotics chlortetracycline and florfenicol are frequently administrated to food-producing animals to treat and prevent various diseases. Therefore, we evaluated the response of MDR S. Typhimurium after exposure to these two antibiotics. Results:We exposed four MDR S. Typhimurium isolates to sub-inhibitory concentrations of chlortetracycline (16 and 32 µg/ml) or florfenicol (16 µg/ml) for 30 min during early-log phase. Differentially expressed genes following antibiotic treatment were identified using RNA-seq, and genes associated with attachment and those located within the Salmonella pathogenicity islands were significantly up-regulated following exposure to either antibiotic. The effect of antibiotic exposure on cellular invasion and motility was also assessed. Swimming and swarming motility were decreased due to antibiotic exposure. However, we observed chlortetracycline enhanced cellular invasion in two strains and florfenicol enhanced invasion in a third isolate. Conclusions:Chlortetracycline and florfenicol exposure during early-log growth altered the expression of nearly half of the genes in the S. Typhimurium genome, including a large number of genes associated with virulence and pathogenesis; this transcriptional alteration was not due to the SOS response. The results suggest that exposure to either of these two antibiotics may lead to the expression of virulence genes that are typically only transcribed in vivo, as well as only during late-log or stationary phase in vitro.

SUBMITTER: Holman DB

PROVIDER: S-EPMC5836442 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Chlortetracycline and florfenicol induce expression of genes associated with pathogenicity in multidrug-resistant Salmonella enterica serovar Typhimurium.

Holman Devin B DB Bearson Shawn M D SMD Bearson Bradley L BL Brunelle Brian W BW

Gut pathogens 20180305

<h4>Background</h4>Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Typhimurium) is a serious public health threat as infections caused by these strains are more difficult and expensive to treat. Livestock serve as a reservoir for MDR Salmonella, and the antibiotics chlortetracycline and florfenicol are frequently administrated to food-producing animals to treat and prevent various diseases. Therefore, we evaluated the response of MDR S. Typhimuri ...[more]

PMID: 29515658

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Project description:As part of a longitudinal study of antimicrobial resistance among salmonellae isolated from swine, we studied 484 Salmonella enterica subsp. enterica serovar Typhimurium (including serovar Typhimurium var. Copenhagen) isolates. We found two common pentaresistant phenotypes. The first was resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (the AmCmStSuTe phenotype; 36.2% of all isolates), mainly of the definitive type 104 (DT104) phage type (180 of 187 isolates). The second was resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline (the AmKmStSuTe phenotype; 44.6% of all isolates), most commonly of the DT193 phage type (77 of 165 isolates), which represents an unusual resistance pattern for DT193 isolates. We analyzed 64 representative isolates by amplified fragment length polymorphism (AFLP) analysis, which revealed DNA fingerprint similarities that correlated with both resistance patterns and phage types. To investigate the genetic basis for resistance among DT193 isolates, we characterized three AmKmStSuTe pentaresistant strains and one hexaresistant strain, which also expressed resistance to gentamicin (Gm phenotype), all of which had similar DNA fingerprints and all of which were collected during the same sampling. We found that the genes encoding the pentaresistance pattern were different from those from isolates of the DT104 phage type. We also found that all strains encoded all of their resistance genes on plasmids, unlike the chromosomally encoded genes of DT104 isolates, which could be transferred to Escherichia coli via conjugation, but that the plasmid compositions varied among the isolates. Two strains (strains UT08 and UT12) had a single, identical plasmid carrying bla(TEM) (which encodes ampicillin resistance), aphA1-Iab (which encodes kanamycin resistance), strA and strB (which encode streptomycin resistance), class B tetA (which encodes tetracycline resistance), and an unidentified sulfamethoxazole resistance allele. The third pentaresistant strain (strain UT20) was capable of transferring by conjugation two distinct resistance patterns, AmKmStSuTe and KmStSuTe, but the genes were carried on plasmids with slightly different restriction patterns (differing by a single band of 15 kb). The hexaresistant strain (strain UT30) had the same plasmid as strains UT08 and UT12, but it also carried a second plasmid that conferred the AmKmStSuGm phenotype. The second plasmid harbored the gentamicin resistance methylase (grm), which has not previously been reported in food-borne pathogenic bacteria. It also carried the sul1 gene for sulfamethoxazole resistance and a 1-kb class I integron bearing aadA for streptomycin resistance. We also characterized isolates of the DT104 phage type. We found a number of isolates that expressed resistance only to streptomycin and sulfamethoxazole (the StSu phenotype; 8.3% of serovar Typhimurium var. Copenhagen strains) but that had AFLP DNA fingerprints similar or identical to those of strains with genes encoding the typical AmCmStSuTe pentaresistance phenotype of DT104. These atypical StSu DT104 isolates were predominantly cultured from environmental samples and were found to carry only one class I integron of 1.0 kb, in contrast to the typical two integrons (InC and InD) of 1.0 and 1.2 kb, respectively, of the pentaresistant DT104 isolates. Our findings show the widespread existence of multidrug-resistant Salmonella strains and the diversity of multidrug resistance among epidemiologically related strains. The presence of resistance genes on conjugative plasmids and duplicate genes on multiple plasmids could have implications for the spread of resistance factors and for the stability of multidrug resistance among Salmonella serovar Typhimurium isolates.

Project description:Salmonella spp. are estimated to cause 1.2 million cases of human foodborne illness each year in the United States, and pigs can often be asymptomatically colonized with Salmonella spp. (>50% of farms). Recent reports state that 18.3% of Salmonella enterica serovar Typhimurium isolates are resistant to ≥3 antimicrobial classes, and multidrug-resistant (MDR) strains are associated with an increased hospitalization rate and other complications. Chlortetracycline is commonly used in swine production to prevent/treat various diseases; therefore, chlortetracycline treatment of pigs unknowingly colonized with MDR Salmonella may have collateral effects on Salmonella spp. (and other gut bacteria). In this study, we determined the effect of in-feed chlortetracycline (400 g/ton) on shedding and colonization of pigs challenged with the MDR S Typhimurium strain DT104 (n = 11/group). We also assessed the impact on the fecal microbiota over the 12-day experimental period and on the ileum, cecum, and tonsil microbiota at 7 days postinoculation (dpi). In MDR S Typhimurium-inoculated pigs, chlortetracycline administration significantly increased fecal shedding at 2 dpi (+1.4 log10 CFU/g; P < 0.001) and enhanced tonsil colonization (+3.1 log10 CFU/g; P < 0.001). There were few major alterations detected in the gut or tonsillar microbiota of pigs treated with MDR S Typhimurium and/or chlortetracycline. The tonsillar transcriptome was largely unaffected despite increased colonization by MDR S Typhimurium following inoculation of the chlortetracycline-treated pigs. These results highlight the idea that chlortetracycline administration can enhance shedding and colonization of MDR S Typhimurium in pigs, which could increase the risk of environmental dissemination of MDR Salmonella strains.IMPORTANCESalmonella spp. are an important cause of foodborne illness in North America, and pork products are associated with sporadic cases and outbreaks of human salmonellosis. Isolates of Salmonella may be resistant to multiple antibiotics, and infections with multidrug-resistant (MDR) Salmonella spp. are more difficult to treat, leading to increased hospitalization rates. Swine operations commonly use antimicrobials, such as chlortetracycline, to prevent/treat infections, which may have collateral effects on pig microbial populations. Recently, we demonstrated that chlortetracycline induces the expression of genes associated with pathogenesis and invasion in MDR Salmonella enterica serovar Typhimurium in vitro In our current study, we show increased tonsillar colonization and fecal shedding of the MDR S Typhimurium strain DT104 from pigs administered chlortetracycline. Therefore, pigs unknowingly colonized with multidrug-resistant Salmonella spp. and receiving chlortetracycline for an unrelated infection may be at a greater risk for disseminating MDR Salmonella spp. to other pigs and to humans through environmental or pork product contamination.

Project description:Motile bacteria employ one or more methods for movement, including darting, gliding, sliding, swarming, swimming, and twitching. Multidrug-resistant (MDR) Salmonella carries acquired genes that provide resistance to specific antibiotics, and the goal of our study was to determine how antibiotics influence swimming and swarming in such resistant Salmonella isolates. Differences in motility were examined for six MDR Salmonella enterica serovar Typhimurium isolates grown on swimming and swarming media containing subinhibitory concentrations of chloramphenicol, kanamycin, streptomycin, or tetracycline. Chloramphenicol and tetracycline reduced both swimming and swarming, though the effect was more pronounced for swimming than for swarming at the same antibiotic and concentration. Swimming was limited by kanamycin and streptomycin, but these antibiotics had much less influence on decreasing swarming. Interestingly, kanamycin significantly increased swarming in one of the isolates. Removal of the aphA1 kanamycin resistance gene and complementation with either the aphA1 or aphA2 kanamycin resistance gene revealed that aphA1, along with an unidentified Salmonella genetic factor, was required for the kanamycin-enhanced swarming phenotype. Screening of 25 additional kanamycin-resistant isolates identified two that also had significantly increased swarming motility in the presence of kanamycin. This study demonstrated that many variables influence how antibiotics impact swimming and swarming motility in MDR S. Typhimurium, including antibiotic type, antibiotic concentration, antibiotic resistance gene, and isolate-specific factors. Identifying these isolate-specific factors and how they interact will be important to better understand how antibiotics influence MDR Salmonella motility. IMPORTANCESalmonella is one of the most common causes of bacterial foodborne infections in the United States, and the Centers for Disease Control consider multidrug-resistant (MDR) Salmonella a "Serious Threat Level pathogen." Because MDR Salmonella can lead to more severe disease in patients than that caused by antibiotic-sensitive strains, it is important to identify the role that antibiotics may play in enhancing Salmonella virulence. The current study examined several MDR Salmonella isolates and determined the effect that various antibiotics had on Salmonella motility, an important virulence-associated factor. While most antibiotics had a neutral or negative effect on motility, we found that kanamycin actually enhanced MDR Salmonella swarming in some isolates. Subsequent experiments showed this phenotype as being dependent on a combination of several different genetic factors. Understanding the influence that antibiotics have on MDR Salmonella motility is critical to the proper selection and prudent use of antibiotics for efficacious treatment while minimizing potential collateral consequences.

Project description:BACKGROUND: Multidrug-resistant (MDR) Salmonella isolates are associated with increased morbidity compared to antibiotic-sensitive strains and are an important health and safety concern in both humans and animals. Salmonella enterica serovar Typhimurium is a prevalent cause of foodborne disease, and a considerable number of S. Typhimurium isolates from humans and livestock are resistant to three or more antibiotics. The majority of these MDR S. Typhimurium isolates are resistant to tetracycline, a commonly used and clinically and agriculturally relevant antibiotic. Because exposure of drug-resistant bacteria to antibiotics can affect cellular processes associated with virulence, such as invasion, we investigated the effect tetracycline had on the invasiveness of tetracycline-resistant MDR S. Typhimurium isolates. RESULTS: The isolates selected and tested were from two common definitive phage types of S. Typhimurium, DT104 and DT193, and were resistant to tetracycline and at least three other antibiotics. Although Salmonella invasiveness is temporally regulated and normally occurs during late-log growth phase, tetracycline exposure induced the full invasive phenotype in a cell culture assay during early-log growth in several DT193 isolates. No changes in invasiveness due to tetracycline exposure occurred in the DT104 isolates during early-log growth or in any of the isolates during late-log growth. Real-time PCR was used to test expression of the virulence genes hilA, prgH, and invF, and these genes were significantly up-regulated during early-log growth in most isolates due to tetracycline exposure; however, increased virulence gene expression did not always correspond with increased invasion, and therefore was not an accurate indicator of elevated invasiveness. This is the first report to assess DT193 isolates, as well as the early-log growth phase, in response to tetracycline exposure, and it was the combination of both parameters that was necessary to observe the induced invasion phenotype. CONCLUSIONS: In this report, we demonstrate that the invasiveness of MDR S. Typhimurium can be modulated in the presence of tetracycline, and this effect is dependent on growth phase, antibiotic concentration, and strain background. Identifying the conditions necessary to establish an invasive phenotype is important to elucidate the underlying factors associated with increased virulence of MDR Salmonella.

Dataset Information

Chlortetracycline and florfenicol induce expression of genes associated with pathogenicity in multidrug-resistant Salmonella enterica serovar Typhimurium.

Publications

Chlortetracycline and florfenicol induce expression of genes associated with pathogenicity in multidrug-resistant <i>Salmonella enterica</i> serovar Typhimurium.

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