Critical Slowing Down at the Abrupt Mott Transition: When the First-Order Phase Transition Becomes Zeroth Order and Looks Like Second Order.

We report that the thermally induced Mott transition in vanadium sesquioxide shows critical slowing down and enhanced variance ("critical opalescence") of the order parameter fluctuations measured through low-frequency resistance-noise spectroscopy. Coupled with the observed increase of the phase-ordering time, these features suggest that the strong abrupt transition is controlled by a critical-like singularity in the hysteretic metastable phase. The singularity is identified with the spinodal point and is a likely consequence of the strain-induced long-range interaction.

Evaluation of the Genetics and Functionality of Plasmids in Incompatibility Group I1-Positive Salmonella enterica.

Salmonella is a predominant foodborne pathogen in the United States and other countries. Mobile genetic elements such as plasmids allow Salmonella to adapt to external stress factors such as nutrient deprivation and host factors. Incompatibility group I1 (IncI1) plasmid-carrying Salmonella enterica strains were examined to determine the presence of plasmid-associated genes and their influence on phenotypic characteristics. The objective of this study was to understand the genetic determinants on IncI1 plasmids and their impact on antimicrobial susceptibility, competitive growth inhibition of Escherichia coli, and plasmid transfer. Primers were designed for genes that play a role in virulence, antimicrobial resistance, and plasmid transfer based on previously sequenced IncI1 plasmids. Polymerase chain reaction assays were conducted on 92 incompatibility group I1 (IncI1)-positive S. enterica strains. Phenotypic characterization included conjugation assays, antimicrobial susceptibility testing, and bacteriocin production based on the inhibition of growth of colicin-negative E. coli J53. The antimicrobial resistance genes aadA1, tetA, sul1, and blaCMY were detected in 88%, 87%, 80%, and 48% of the strains, respectively. Over half of the strains were resistant or intermediately resistant to streptomycin (85%), sulfonamides (76%), tetracycline (74%), and ampicillin (68%) and 57% of the strains inhibited growth of E. coli J53 strain. Among putative virulence genes, colicin-associated colI and cib were detected in 23% and 35% of strains and imm and ccdA were present in 58% and 54% of strains, respectively. Approximately 61% of strains contained plasmids that conjugally transferred antimicrobial resistance, including 83% where the recipient received IncI1 plasmids. Most of the strains carried an assortment of transfer associated (pil and tra) genes with between 63% and 99% of strains being positive for individual genes. Taken together the study affirms that IncI1 plasmids likely play roles in the dissemination of antimicrobial resistance and virulence-associated factors among enteric organisms.

Molecular Characterization of Salmonella enterica Serovars Isolated from a Turkey Production Facility in the Absence of Selective Antimicrobial Pressure.

This study evaluated antimicrobial resistance and virulence factors in Salmonella enterica isolated from a turkey flock in which the birds were raised in an environment where antimicrobials were not administered to the birds, either through feed or water. Salmonella was isolated from turkeys and various environmental samples in the facility using conventional microbiological procedures. Isolates were serotyped and analyzed phenotypically by antimicrobial resistance profiling and genotypically by pulsed-field gel electrophoresis (PFGE) fingerprinting, integron analysis, plasmid profiling, replicon-based incompatibility (Inc) group typing, and virulence gene profiling. Ninety-five S. enterica isolates were isolated from cecal contents (n = 29), feed (n = 22), leftover feed (n = 13), litter (n = 12), drinkers (n = 10), environment (n = 8), and an insect. The following serotypes were identified: Montevideo (24%), Anatum (22%), Agona (17%), Kentucky and Worthington (12%), Senftenberg (11%), and rough phenotypes (3%). The majority of isolates (61/95; 64%) were susceptible to 12 antimicrobials tested; however, despite the absence of antimicrobials in the facility, approximately 36% of the isolates were resistant to two to five antimicrobials. Class 1 integrons were detected in 8% of the isolates. The integron sequence analysis revealed dihydrofolate reductase (dhfr) and aminoglycoside adenylyl transferase (aadA2) genes, which encode trimethoprim and streptomycin resistance, respectively. Furthermore, 71% of the isolates had at least one plasmid. There were five plasmid replicon types identified among the isolates, including IncI1, IncHI2, IncFIIA, IncB/O, and IncP, with variable prevalence among the serotypes. All 95 isolates tested polymerase chain reaction-positive for 19 virulence genes and negative for virD4 and virB4. The virulence gene profiles were similar within the isolates from the same serotype. Within particular serotypes, PFGE patterns revealed 100% similarity, even when the bacterial strains were isolated from different sources, indicating cross-colonization of sources within the turkey facility. On this antibiotic-free turkey farm, turkeys and feed appeared to be the major reservoirs of multidrug-resistant Salmonella, which harbored multiple virulence genes.

Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids.

Clostridium perfringens is an important pathogen, causing food poisoning and other mild to severe infections in humans and animals. Some strains of C. perfringens contain conjugative plasmids, which may carry antimicrobial resistance and toxin genes. We studied genomic and plasmid diversity of 145 C. perfringens type A strains isolated from soils, foods, chickens, clinical samples, and domestic animals (porcine, bovine and canine), from different geographic areas in the United States between 1994 and 2006, using multiple-locus variable-number tandem repeat analysis (MLVA) and/or pulsed-field gel electrophoresis (PFGE). MLVA detected the genetic diversity in a majority of the isolates. PFGE, using SmaI and KspI, confirmed the MLVA results but also detected differences among the strains that could not be differentiated by MLVA. All of the PFGE profiles of the strains were different, except for a few of the epidemiologically related strains, which were identical. The PFGE profiles of strains isolated from the same domestic animal species were clustered more closely with each other than with other strains. However, a variety of C. perfringens strains with distinct genetic backgrounds were found among the clinical isolates. Variation was also observed in the size and number of plasmids in the strains. Primers for the internal fragment of a conjugative tcpH gene of C. perfringens plasmid pCPF4969 amplified identical size fragments from a majority of strains tested; and this gene hybridized to the various-sized plasmids of these strains. The sequences of the PCR-amplified tcpH genes from 12 strains showed diversity among the tcpH genes. Regardless of the sources of the isolates, the genetic diversity of C. perfringens extended to the plasmids carrying conjugative genes.

Effect of loop composition on the stability and folding kinetics of RNA hairpins with large loops.

RNA hairpins are ubiquitous structural elements in biological RNAs, where they have the potential to regulate RNA folding and interactions with other molecules. There are established methods for predicting the thermodynamic stability of an RNA hairpin, but there are still relatively few detailed examinations of the kinetics of folding. Nonetheless, several recent studies indicate that hairpin folding does not proceed via a simple two-state model. Here, we monitor fluorescence from hairpins constructed as molecular beacons in ensemble, fluorescence correlation spectroscopy, and stopped-flow experiments to describe the folding of RNA hairpins with long (15 nucleotide) loops. Our results show that folding of these hairpins occurs through more than two states and that the mechanism of folding includes a fast intermediate phase observed on the tens of microseconds time scale and a slow phase, attributed to formation of the native folded hairpin loop and stem, observed on the milliseconds time scale. The composition of the RNA loop determines the time scale of intermediate and native folded states. Hairpins with a polyuracil loop sequence exhibit slower relaxation of the intermediate state and faster relaxation of the native folded state when compared to that of hairpins with cytosine or adenine in the loop. We hypothesize this composition dependence could be attributed to nucleobase stacking in cytosine and adenine containing regions of the loop, which would be absent in hairpins containing polyuracil loops. Such base stacking could destabilize the intermediate folds, thereby speeding the relaxation of the intermediate relative to similar sized hairpins with no base stacking in the loop. Likewise, the lower intermediate stability could prolong the relaxation of the native folded state.

Genotypic analyses of IncHI2 plasmids from enteric bacteria.

Incompatibility (Inc) HI2 plasmids are large (typically > 200 kb), transmissible plasmids that encode antimicrobial resistance (AMR), heavy metal resistance (HMR) and disinfectants/biocide resistance (DBR). To better understand the distribution and diversity of resistance-encoding genes among IncHI2 plasmids, computational approaches were used to evaluate resistance and transfer-associated genes among the plasmids. Complete IncHI2 plasmid (N = 667) sequences were extracted from GenBank and analyzed using AMRFinderPlus, IntegronFinder and Plasmid Transfer Factor database. The most common IncHI2-carrying genera included Enterobacter (N = 209), Escherichia (N = 208), and Salmonella (N = 204). Resistance genes distribution was diverse, with plasmids from Escherichia and Salmonella showing general similarity in comparison to Enterobacter and other taxa, which grouped together. Plasmids from Enterobacter and other taxa had a higher prevalence of multiple mercury resistance genes and arsenic resistance gene, arsC, compared to Escherichia and Salmonella. For sulfonamide resistance, sul1 was more common among Enterobacter and other taxa, compared to sul2 and sul3 for Escherichia and Salmonella. Similar gene diversity trends were also observed for tetracyclines, quinolones, ß-lactams, and colistin. Over 99% of plasmids carried at least 25 IncHI2-associated conjugal transfer genes. These findings highlight the diversity and dissemination potential for resistance across different enteric bacteria and value of computational-based approaches for the resistance-gene assessment.

Data mining tools for Salmonella characterization: application to gel-based fingerprinting analysis.

BACKGROUND: Pulsed field gel electrophoresis (PFGE) is currently the most widely and routinely used method by the Centers for Disease Control and Prevention (CDC) and state health labs in the United States for Salmonella surveillance and outbreak tracking. Major drawbacks of commercially available PFGE analysis programs have been their difficulty in dealing with large datasets and the limited availability of analysis tools. There exists a need to develop new analytical tools for PFGE data mining in order to make full use of valuable data in large surveillance databases. RESULTS: In this study, a software package was developed consisting of five types of bioinformatics approaches exploring and implementing for the analysis and visualization of PFGE fingerprinting. The approaches include PFGE band standardization, Salmonella serotype prediction, hierarchical cluster analysis, distance matrix analysis and two-way hierarchical cluster analysis. PFGE band standardization makes it possible for cross-group large dataset analysis. The Salmonella serotype prediction approach allows users to predict serotypes of Salmonella isolates based on their PFGE patterns. The hierarchical cluster analysis approach could be used to clarify subtypes and phylogenetic relationships among groups of PFGE patterns. The distance matrix and two-way hierarchical cluster analysis tools allow users to directly visualize the similarities/dissimilarities of any two individual patterns and the inter- and intra-serotype relationships of two or more serotypes, and provide a summary of the overall relationships between user-selected serotypes as well as the distinguishable band markers of these serotypes. The functionalities of these tools were illustrated on PFGE fingerprinting data from PulseNet of CDC. CONCLUSIONS: The bioinformatics approaches included in the software package developed in this study were integrated with the PFGE database to enhance the data mining of PFGE fingerprints. Fast and accurate prediction makes it possible to elucidate Salmonella serotype information before conventional serological methods are pursued. The development of bioinformatics tools to distinguish the PFGE markers and serotype specific patterns will enhance PFGE data retrieval, interpretation and serotype identification and will likely accelerate source tracking to identify the Salmonella isolates implicated in foodborne diseases.

Chromosomal and plasmid-mediated fluoroquinolone resistance mechanisms among broad-spectrum-cephalosporin-resistant Escherichia coli isolates recovered from companion animals in the USA.

OBJECTIVES: To determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and investigate mutations in gyrase and topoisomerase genes that may contribute to increased fluoroquinolone resistance in canine and feline Escherichia coli isolates in the USA that displayed reduced susceptibility to extended-spectrum cephalosporins. This study was undertaken because previous epidemiological studies identified a potential correlation between extended-spectrum cephalosporins and fluoroquinolone resistance. METHODS: Isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime were screened by PCR for the presence of PMQR determinants and gyrase and topoisomerase genes were sequenced. Isolates were further characterized by conjugation and phylogenetic analyses. RESULTS: PMQR determinants aac(6')-Ib-cr, qnrS and qepA were identified in 30, 23 and 5 isolates, respectively. Multiple mutations were identified in the quinolone resistance-determining region, including the novel substitutions of Glu-84 → Ala and Leu-88 → Gln in ParC and Arg-432 → Ser and Glu-460 → Val in ParE. The isolate that exhibited the highest level of enrofloxacin resistance (MIC > 256 mg/L) had a double mutation in gyrA (Ser-83 → Leu and Asp-87 → Asn) and a triple mutation in parC (Ser-80 → Ile, Glu-84 → Gly and a novel mutation, Leu-88 → Gln). The presence of PMQR genes increased the ciprofloxacin MIC values 4-fold to 8-fold in transconjugants relative to the recipient strain. Approximately 39% of the isolates belonged to phylogenetic group D and 30% to group B2, which typically contain an increased number of virulence determinants compared with other groups. CONCLUSIONS: Novel mutations in topoisomerase genes and PMQR determinants aac(6')-Ib-cr, qnrS and qepA genes were detected among extended-spectrum ß-lactamase-producing E. coli in the USA.

Longitudinal study of distributions of similar antimicrobial-resistant Salmonella serovars in pigs and their environment in two distinct swine production systems.

The aim of this longitudinal study was to determine and compare the prevalences and genotypic profiles of antimicrobial-resistant (AR) Salmonella isolates from pigs reared in antimicrobial-free (ABF) and conventional production systems at farm, at slaughter, and in their environment. We collected 2,889 pig fecal and 2,122 environmental (feed, water, soil, lagoon, truck, and floor swabs) samples from 10 conventional and eight ABF longitudinal cohorts at different stages of production (farrowing, nursery, finishing) and slaughter (postevisceration, postchill, and mesenteric lymph nodes [MLN]). In addition, we collected 1,363 carcass swabs and 205 lairage and truck samples at slaughter. A total of 1,090 Salmonella isolates were recovered from the samples; these were isolated with a significantly higher prevalence in conventionally reared pigs (4.0%; n = 66) and their environment (11.7%; n = 156) than in ABF pigs (0.2%; n = 2) and their environment (0.6%; n = 5) (P < 0.001). Salmonella was isolated from all stages at slaughter, including the postchill step, in the two production systems. Salmonella prevalence was significantly higher in MLN extracted from conventional carcasses than those extracted from ABF carcasses (P < 0.001). We identified a total of 24 different serotypes, with Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Anatum, Salmonella enterica serovar Infantis, and Salmonella enterica serovar Derby being predominant. The highest frequencies of antimicrobial resistance (AR) were exhibited to tetracycline (71%), sulfisoxazole (42%), and streptomycin (17%). Multidrug resistance (resistance to ≥ 3 antimicrobials; MDR) was detected in 27% (n = 254) of the Salmonella isolates from the conventional system. Our study reports a low prevalence of Salmonella in both production systems in pigs on farms, while a higher prevalence was detected among the carcasses at slaughter. The dynamics of Salmonella prevalence in pigs and carcasses were reciprocated in the farm and slaughter environment, clearly indicating an exchange of this pathogen between the pigs and their surroundings. Furthermore, the phenotypic and genotypic fingerprint profile results underscore the potential role played by environmental factors in dissemination of AR Salmonella to pigs.

Massively parallel sequencing of enriched target amplicons for high-resolution genotyping of Salmonella serovars.

With next generation sequencing (NGS) technology, it is now possible to carry out in-depth, large-scale sequencing projects, such as whole genome sequencing, in a fast and inexpensive manner. However, often it is more practical and convenient to sequence and analyze multiple, smaller regions of the bacterial genome to gain valuable information about an organism. One such application is genotyping of bacterial strains by multilocus sequence typing (MLST) that involves PCR and sequencing analysis of typically 7 housekeeping genes. Recently, we described a novel MLST method, called MLST-seq that combines a PCR-based target enrichment method and NGS technology to simultaneously analyze numerous target gene sequences, thereby improving the resolution and high-throughput capacity of current MLST approaches. However, the performance of the MLST-seq method was hampered from a substantial bias in target enrichment step. In this study, we used an improved target enrichment method using hairpin selectors to amplify 21 target genes simultaneously from each of 41 Salmonella strains. The resulting amplicons tagged with strain-specific barcodes were pooled and sequenced en masse by 454 pyrosequencing. Analysis of sequence data from 38 Salmonella strains using combinations of 3, 7 and 14 target genes resulted in 23, 32 and 37 distinct allelic profiles, respectively. These results demonstrated that MLST-seq with an increased number of target genes is an efficient way to improve discrimination among closely-related strains of Salmonella. With the rapidly increasing sequencing capacity of NGS technologies combined with further improvements in target capturing methods, MLST-seq could become a promising approach to perform high-resolution strain typing of a large collection of Salmonella, and likely other genera in a labor- and cost-efficient manner in the future.

Evaluation of virulence and antimicrobial resistance in Salmonella enterica serovar Enteritidis isolates from humans and chicken- and egg-associated sources.

Salmonella enterica serovar Enteritidis is a leading cause of salmonellosis throughout the world and is most commonly associated with the consumption of contaminated poultry and egg products. Salmonella Enteritidis has enhanced ability to colonize and persist in extraintestinal sites within chickens. In this study, 54 Salmonella Enteritidis isolates from human patients (n=28), retail chicken (n=9), broiler farms (n=9), and egg production facilities (n=8) were characterized by antimicrobial susceptibility testing, plasmid analysis, genetic relatedness using XbaI and AvrII pulsed-field gel electrophoresis (PFGE), and the presence of putative virulence genes. Nine isolates were evaluated for their abilities to invade and survive in intestinal epithelial and macrophage cell lines. Overall, 56% (n=30) of isolates were resistant to at least one antimicrobial agent tested, yet no isolates showed resistance to more than three antimicrobials. All isolates carried a common ∼55-kb plasmid, with some strains containing additional plasmids ranging from 3 to 50 kb. PFGE analysis revealed five XbaI and AvrII clusters. There were significant overlaps in the PFGE patterns of the isolates from human, chicken, and egg houses. All isolates tested PCR positive for iacP, purR, ttrB, spi4H, rmbA, sopE, invA, sopB, spvB, pagC, msgA, spaN, orgA, tolC, and sifA, and negative for iss, virB4, and sipB. Of the isolates selected for virulence testing, those containing the iron acquisition genes, iutA, sitA, and iucA, and ∼50-kb plasmids demonstrated among the highest levels of macrophage and epithelial cell invasion, which may indicate their importance in pathogenesis.

Dry eye symptoms and digital eyestrain - Emerging epidemics among university students due to online curriculum amid the COVID-19 pandemic. A cross-sectional study.

Purpose: Increased screen time has been a major concern among the students who have adopted the online curriculum amid the pandemic. The study was conducted to shed light on the changing trends of symptoms pertaining to dry eye disease and digital eyestrain due to the online curriculum and its negative implications on the ocular health of students. Methods: A cross-sectional study was conducted among students of Manipal Academy of Higher Education who are currently following the E-learning curriculum amid the COVID-19 pandemic the participants were surveyed using a pre-validated structured questionnaire. Results: The mean age of study participants was 23.33 ± 4.604 years. In total, 97.9% (321/352) of the respondents experienced at least three symptoms associated with the usage of digital devices. 88.1% of the participants were exposed to an average screen time of more than 4 hours per day. An increased number of hours of digital device usage was found to be associated with higher total symptom scores (P = 0.04). The total symptom scores were found to be considerably higher for those with continuous contact with the screen (P = 0.02). Headache (69.9%, n = 246) is the most commonly reported symptom followed by neck pain (65.3%, n = 230), tearing (44.6%, n = 157), eye pain (40.9%, n = 144), and burning sensation (40.1%, n = 141). Conclusion: This study highlights a tremendous increase in the prevalence of symptoms related to dry eye and digital eyestrain among students attending online classes during the COVID-19 pandemic. Eye care professionals need to be aware of this emerging public health threat and the appropriate measures for its prevention.

Millisecond time-scale folding and unfolding of DNA hairpins using rapid-mixing stopped-flow kinetics.

We report stopped-flow kinetics experiments to study the folding and unfolding of 5 base-pair stem and 21 nucleotide polythymidine loop DNA hairpins over various concentrations of NaCl. The reactions occurred on a time scale of milliseconds, considerably longer than the microsecond time scale suggested by previous kinetics studies of similar-sized hairpins. In comparison to a recent fluorescence correlation spectroscopy study (J. Am. Chem. Soc. 2006, 128, 1240-1249), we suggest the microsecond time-scale reactions are due to intermediate states and the millisecond time-scale reactions reported here are due to the formation of the fully folded DNA hairpin. These results support our view that DNA hairpin folding occurs via a minimum three-state mechanism.

Effect of heterogeneous substrate adhesivity of follower cells on speed and tension profile of leader cells in primary keratocyte collective cell migration.

In single keratocyte motility, membrane tension is reported to be high at cell-fronts and believed to establish front coherence. To understand role of membrane mechanics in collective cell migration, we study membrane height fluctuations in cell sheets from fish scales using interference reflection microscopy (IRM). We report the monolayer to have cells lacking substrate adhesion and show that such 'non-sticky' cells can form bridges between leader cells and far-away follower cells. Do such interactions alter motility and membrane mechanics in such leaders? We find non-significant, but reduced speed for leaders with 'non-sticky' followers in comparison to other leaders. Cells show high phenotypic variability in their membrane fluctuation tension profiles. On average, this tension is found to be lower at cell fronts than the mid-section. However, leaders with non-sticky followers are more prone to display higher tension at their front and have a negative correlation between cell speed and front-mid tension difference. Thus, we conclude that intracellular tension gradients are heterogeneous in cell sheets and substrate adhesivity of followers can control the coupling of the gradient to cell speed.

Detection of SARS-CoV-2 in conjunctival secretion and tears in patients with COVID-19 in a tertiary care centre, South India. .

Aims and objectives: Purpose of this study is to detect the presence of SAR-CoV-2 viral RNA in conjunctival secretions of COVID-19 patients and to compare the RT-PCR positivity rate for SARS-CoV-2 in conjunctival and nasopharyngeal swab. Materials and method: Eighty hospitalised COVID-19 patients whose nasopharyngeal swab tested positive for SARS-CoV-2 by RT-PCR were included in the study. Conjunctival swab was collected from eyes of these patients and sent for detection of SARS-CoV-2 by RT-PCR method. Results: Among the eighty patients, 51 (63.7%) were males and 29 (36.3%) were females. The mean age of the patients was 55.93 ± 16.59. Six patients had ocular manifestations. Eleven (13.75%) patients tested positive on conjunctival swab for SARS-CoV-2 viral RNA and only one of them had ocular manifestations out of the eleven. Conclusion: In our study the presence of SARS-CoV-2 in conjunctival secretions of COVID-19 patients was detected and this was not dependent on presence of ocular manifestations or duration of disease. Though the conjunctival positivity is lower compared to the nasopharyngeal swab sampling, ocular surface and secretions can be a potential route of viral transmission.

Microbial Genetics and Clonal Dissemination of Salmonella enterica Serotype Javiana Isolated from Human Populations in Arkansas, USA

Salmonella is estimated to cause over a million infections and ~400 deaths annually in the U.S. Salmonella enterica serotype Javiana strains (n = 409) that predominantly originated from the State of Arkansas over a six-year period (2003 to 2008) were studied. This period coincided with a rapid rise in the incidence of S. Javiana infections in the U.S. Children under the age of 10 displayed the highest prevalence of S. Javiana infections, regardless of sex or year of detection. Antimicrobial susceptibility to 15 different antimicrobials was assessed and 92% (n = 375) were resistant to at least one of the antimicrobials. Approximately 89% of the isolates were resistant to sulfisoxazole alone and 3% (n = 11) were resistant to different antimicrobials, including gentamicin, ciprofloxacin or ceftiofur. The pulsed-field gel electrophoresis (PFGE) analyses assessed the genotypic diversity and distribution of S. Javiana strains using XbaI restriction. Nine major clusters were identified and isolates from each group were digested with the restriction enzyme AvrII. Isolates with identical profiles of XbaI and AvrII were found to be disseminated in human populations. These distinct "types" of S. Javiana were persistent in human populations for multiple years. A subset of isolates (n = 19) with unique resistance phenotypes underwent plasmid and incompatibility (Inc) type analyses and the isolates resistant to more than one antimicrobial harbored multiple plasmids (<3 to 165 kb). Furthermore, these strains possessed 14 virulence genes, including pagC, cdtB, and iroN. The whole genome sequences (WGS) of 18 isolates that mostly originated from Arkansas from 2003 to 2011 were compared with isolates collected from different areas in the U.S. in 1999, indicating the perseverance of S. Javiana in disseminating antimicrobial resistance and virulence genes.

Molecular characterization of resistance to extended-spectrum cephalosporins in clinical Escherichia coli isolates from companion animals in the United States.

Resistance to extended-spectrum cephalosporins (ESC) among members of the family Enterobacteriaceae occurs worldwide; however, little is known about ESC resistance in Escherichia coli strains from companion animals. Clinical isolates of E. coli were collected from veterinary diagnostic laboratories throughout the United States from 2008 to 2009. E. coli isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime (MIC ≥ 16 µg/ml) and extended-spectrum-ß-lactamase (ESBL) phenotypes were analyzed. PCR and sequencing were used to detect mutations in ESBL-encoding genes and the regulatory region of the chromosomal gene ampC. Conjugation experiments and plasmid identification were conducted to examine the transferability of resistance to ESCs. All isolates carried the bla(CTX-M-1)-group ß-lactamase genes in addition to one or more of the following ß-lactamase genes: bla(TEM), bla(SHV-3), bla(CMY-2), bla(CTX-M-14-like), and bla(OXA-1.) Different bla(TEM) sequence variants were detected in some isolates (n = 40). Three isolates harbored a bla(TEM-181) gene with a novel mutation resulting in an Ala184Val substitution. Approximately 78% of the isolates had mutations in promoter/attenuator regions of the chromosomal gene ampC, one of which was a novel insertion of adenine between bases -28 and -29. Plasmids ranging in size from 11 to 233 kbp were detected in the isolates, with a common plasmid size of 93 kbp identified in 60% of isolates. Plasmid-mediated transfer of ß-lactamase genes increased the MICs (≥ 16-fold) of ESCs for transconjugants. Replicon typing among isolates revealed the predominance of IncI and IncFIA plasmids, followed by IncFIB plasmids. This study shows the emergence of conjugative plasmid-borne ESBLs among E. coli strains from companion animals in the United States, which may compromise the effective therapeutic use of ESCs in veterinary medicine.

Comparison of Salmonella enterica serovar Heidelberg isolates from human patients with those from animal and food sources.

Seventy-eight Salmonella enterica serovar Heidelberg isolates from humans were tested for antimicrobial susceptibility, resistance genes, and plasmids and genotyped by pulsed-field gel electrophoresis (PFGE). Most (88%) contained plasmids, and 47% were resistant to antimicrobials. The overall results were compared to those of previous S. Heidelberg studies of food- and animal-related sources, and multiple similarities were observed.

Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production.

Fresh and processed poultry have been frequently implicated in cases of human salmonellosis. Furthermore, increased consumption of meat and poultry has increased the potential for exposure to Salmonella enterica. While advances have been made in reducing the prevalence and frequency of Salmonella contamination in processed poultry, there is mounting pressure on commercial growers to prevent and/or eliminate these human pathogens in preharvest production facilities. Several factors contribute to Salmonella colonization in commercial poultry, including the serovar and the infectious dose. In the early 1900s, Salmonella enterica serovars Pullorum and Gallinarum caused widespread diseases in poultry, but vaccination and other voluntary programs helped eradicate pullorum disease and fowl typhoid from commercial flocks. However, the niche created by the eradication of these serovars was likely filled by S. Enteritidis, which proliferated in the bird populations. While this pathogen remains a significant problem in commercial egg and poultry production, its prevalence among poultry has been declining since the 1990s. Coinciding with the decrease of S. Enteritidis, S. Heidelberg and S. Kentucky have emerged as the predominant serovars in commercial broilers. In this review, we have highlighted bacterial genetic and host-related factors that may contribute to such shifts in Salmonella populations in commercial poultry and intervention strategies that could limit their colonization.

Pragmatic Strategy for Fecal Specimen Storage and the Corresponding Test Methods for Clostridioides difficile Diagnosis.

The quality of fecal specimens is one of the factors responsible for successful Clostridioides difficile infection (CDI) diagnosis. The quality depends largely on the storage conditions, including the temperature and time period. In this study, we organized the outputs of previous studies, filled experimental gaps in the knowledge of storage conditions, and introduced a pragmatic strategy for fecal storage for CDI diagnosis. A 5-step pathway was adopted to develop the fecal specimen storage strategy as follows: step 1, bibliomic analysis; step 2, experimental gap-filling; step 3, comparative evaluation; step 4, strategy development; step 5, internal review. Step 1 identified eight articles providing experimental information on the effects of fecal specimen storage conditions on the effectiveness of C. difficile detection methods. Step 2 provided additional quantitative data on C. difficile vegetative and spore cell viability and DNA stability. All previous and current results were compared (step 3). In step 4, fir general and nine special strategies were developed, followed by an internal review of the overall approaches (step 5). It is recommended to separate fecal samples into aliquots before testing and storing them. It is particularly recommended that fecal specimen samples be stored for CDI diagnosis at 4 °C for up to 60 days for all test methods.