Three-Year Trend in Antimicrobial Resistance and Genotypes among Salmonella in Swine and Humans

The aim of this study was to determine antimicrobial resistance among Salmonella isolated from swine and humans in North Carolina, compare genotypes among isolates from humans originated from pig-producing areas and characterize important genes. Resistance to 9 and 11 of the 12 antimicrobial agents tested was detected among isolates from swine and humans respectively. Frequency of resistance to tetracycline and b-lactams was significantly higher among isolates from swine than humans (p \u3c0.05). Two common multi-drug resistance (MDR) patterns were found among isolates from apparently healthy swine: AmKmStSuTe and AmCmStSuTe. However, the former MDR pattern was rare among clinical isolates. Genotyping revealed that two predominant genotypes, one composed of clinical isolates and the other non-clinical were noticed. Further characterization using Salmonella plasmid virulence; spvA gene also revealed that this gene is absent among the most common MDR pattern, AmKmStSuTe, in swine

Antimicrobial resistance among canine enterococci in the northeastern United States, 2007–2020

IntroductionAntimicrobial resistance (AMR) is a growing and complex One Health concern worldwide, threatening the practice of human and veterinary medicine. Although dogs are a potential reservoir of multidrug-resistant bacteria, there are very few surveillance studies on AMR from the canine population in the United States. Here, we assessed the antimicrobial susceptibility patterns, identified temporal resistance and minimum inhibitory concentration trends, and described associations between resistance phenotypes among canine clinical enterococci in the northeastern United States.MethodsThrough a large-scale retrospective study design, we collected species identification, minimum inhibitory concentration, and clinical data from 3,659 canine enterococci isolated at the Cornell University Animal Health Diagnostic Center between 2007 and 2020. We used the Mann-Kendall test, Sen’s slope, multivariable logistic regression, and survival analysis models to detect the presence of a significant trend in resistance over the study period.ResultsEnterococcus faecalis was the most prevalent species (67.1% of isolates), followed by Enterococcus faecium (20.4%). We found high levels of AMR among enterococci to almost all the tested antimicrobials, particularly E. faecium. The lowest percentage of resistance was to vancomycin and chloramphenicol. Multidrug resistance was common (80% of E. faecium and 33% of E. faecalis) and 31 isolates were extensively drug resistant. Multidrug resistance among E. faecium increased over time, but not in E. faecalis. Resistance to penicillins, enrofloxacin, and rifampin increased during the study period, but resistance to tetracyclines is on a downward trajectory compared to AMR data from the last decade. Emerging vancomycin-resistant E. faecalis (0.3%) and E. faecium (0.8%) infections in the canine population are of great concern to both human and animal health. One E. faecium isolate with acquired vancomycin resistance was identified in 2017 and four vancomycin-resistant enterococci isolates were identified in 2020.ConclusionThere is a crucial need to make rational prescribing decisions on the prudent use of antimicrobials and improve the quality of care for patients, especially when empirical antimicrobial treatment for enterococcal infection is common

Antimicrobial resistance, diversity and class-1 integrons among Salmonella serovars isolated from swine

The aim of this study was to determine frequency of antimicrobial resistance, identify and characterize multi-drug resistant Salmonella, determine genetic diversity and characterize class-! integrons. The frequency of resistance wasTe (85%), Am (47%), Pi (32%), Ax (23%), Cm (2 1 %), Gm (2%), Ce (2%) and TSx (I%). Two pentaresistant MDR phenotypes were predominant: AmCmStSuTe (36.2%) and AmKmStSuTe (44.6%). DNA fingerprinting revealed eight clusters of typhimurium and Copenhagen with less than 95% of fragment length identity. DT l 04 with StSu R-type, which carried only one class-1 integron of l .Ok b size, were also found from environmental samples. In addition, we identified class 1 integrons among uncommon serovars including Muenchen, Muenster, Worthington, and Bere. The genetic diversity and p resence of class-1 integrons among diverse serovars further imply the potentia l hazard and spread of multiresistant strains in Salmonella

Design and synthesis of boronic-acid-labeled thymidine triphosphate for incorporation into DNA

The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection

The Flagellar Regulator fliT Represses Salmonella Pathogenicity Island 1 through flhDC and fliZ

Salmonella pathogenicity island 1 (SPI1), comprising a type III section system that translocates effector proteins into host cells, is essential for the enteric pathogen Salmonella to penetrate the intestinal epithelium and subsequently to cause disease. Using random transposon mutagenesis, we found that a Tn10 disruption in the flagellar fliDST operon induced SPI1 expression when the strain was grown under conditions designed to repress SPI1, by mimicking the environment of the large intestine through the use of the intestinal fatty acid butyrate. Our genetic studies showed that only fliT within this operon was required for this effect, and that exogenous over-expression of fliT alone significantly reduced the expression of SPI1 genes, including the invasion regulator hilA and the sipBCDA operon, encoding type III section system effector proteins, and Salmonella invasion of cultured epithelial cells. fliT has been known to inhibit the flagellar machinery through repression of the flagellar master regulator flhDC. We found that the repressive effect of fliT on invasion genes was completely abolished in the absence of flhDC or fliZ, the latter previously shown to induce SPI1, indicating that this regulatory pathway is required for invasion control by fliT. Although this flhDC-fliZ pathway was necessary for fliT to negatively control invasion genes, fliZ was not essential for the repressive effect of fliT on motility, placing fliT high in the regulatory cascade for both invasion and motility

BarA-UvrY Two-Component System Regulates Virulence of Uropathogenic E. coli CFT073

Uropathogenic Escherichia coli (UPEC), a member of extraintestinal pathogenic E. coli, cause ∼80% of community-acquired urinary tract infections (UTI) in humans. UPEC initiates its colonization in epithelial cells lining the urinary tract with a complicated life cycle, replicating and persisting in intracellular and extracellular niches. Consequently, UPEC causes cystitis and more severe form of pyelonephritis. To further understand the virulence characteristics of UPEC, we investigated the roles of BarA-UvrY two-component system (TCS) in regulating UPEC virulence. Our results showed that mutation of BarA-UvrY TCS significantly decreased the virulence of UPEC CFT073, as assessed by mouse urinary tract infection, chicken embryo killing assay, and cytotoxicity assay on human kidney and uroepithelial cell lines. Furthermore, mutation of either barA or uvrY gene reduced the production of hemolysin, lipopolysaccharide (LPS), proinflammatory cytokines (TNF-α and IL-6) and chemokine (IL-8). The virulence phenotype was restored similar to that of wild-type by complementation of either barA or uvrY gene in trans. In addition, we discussed a possible link between the BarA-UvrY TCS and CsrA in positively and negatively controlling virulence in UPEC. Overall, this study provides the evidences for BarA-UvrY TCS regulates the virulence of UPEC CFT073 and may point to mechanisms by which virulence regulations are observed in different ways may control the long-term survival of UPEC in the urinary tract

SdiA, an N-Acylhomoserine Lactone Receptor, Becomes Active during the Transit of Salmonella enterica through the Gastrointestinal Tract of Turtles

encode a LuxR-type AHL receptor, SdiA, they cannot synthesize AHLs. In vitro, it is known that SdiA can detect AHLs produced by other bacterial species..We conclude that the normal gastrointestinal microbiota of most animal species do not produce AHLs of the correct type, in an appropriate location, or in sufficient quantities to activate SdiA. However, the results obtained with turtles represent the first demonstration of SdiA activity in animals

Molecular Characterization of Multidrug-Resistant Salmonella enterica subsp. enterica Serovar Typhimurium Isolates from Swine

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

Identification of CsrC and Characterization of Its Role in Epithelial Cell Invasion in Salmonella enterica Serovar Typhimurium

The csr regulatory system of Salmonella regulates the expression of the genes of Salmonella pathogenicity island 1 (SPI1) required for the invasion of epithelial cells. This system consists of the posttranscriptional regulator CsrA and an untranslated regulatory RNA, CsrB, that opposes the action of CsrA. Here we identify and characterize the role of a second regulatory RNA, CsrC, whose ortholog was discovered previously in Escherichia coli. We show that a mutant of csrC has only mild defects in invasion and the expression of SPI1 genes, as does a mutant of csrB, but that a double csrB csrC mutant is markedly deficient in these properties, suggesting that the two regulatory RNAs play redundant roles in the control of invasion. We further show that CsrC, like CsrB, is controlled by the BarA/SirA two-component regulator but that a csrB csrC mutant exhibits a loss of invasion equivalent to that of a barA or sirA mutant, indicating that much of the effect of BarA/SirA on invasion functions through its control of CsrB and CsrC. In addition to their control by BarA/SirA, each regulatory RNA is also controlled by other components of the csr system. The loss of csrB was found to increase the level of CsrC by sevenfold, while the loss of csrC increased CsrB by nearly twofold. Similarly, the overexpression of csrA increased CsrC by nearly 11-fold and CsrB by 3-fold and also significantly increased the stability of both RNAs