Freshwater environment as a reservoir of extended-spectrum ß-lactamase-producing Enterobacteriaceae.

Surface water receives large quantities of wastes from human and animal sources, thus providing an ideal setting for the accumulation, development, and dissemination of antibiotic resistant bacteria, including extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. The rapid spread of ESBL-producing Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae, is a growing threat to public health, and there have been increasing reports on the prevalence and abundance of ESBL-producing Enterobacteriaceae in aquatic environments all over the globe. The objective of this review is to understand the extent of ESBL-producing Enterobacteriaceae contamination in aquatic environments and to enhance our knowledge on the role of the freshwater environment as a reservoir and transmission routes for these bacteria. In this review, we present the prevalence and distribution of ESBL-producing Enterobacteriaceae and their ESBL genes in the freshwater environment, potential sources of these bacteria in the aquatic environment, as well as their potential drivers in the environment, including anthropogenic and environmental factors.

Polymerase chain reaction for the in vitro detection of the pESI plasmid associated with the globally circulating Salmonella Infantis outbreak strain.

A globally circulating strain of Salmonella enterica serotype Infantis containing the pESI plasmid has increased in prevalence in poultry meat samples and cases of human infections. In this study, a polymerase chain reaction (PCR) protocol was designed to detect the pESI plasmid and confirm the Infantis serotype of Salmonella isolates. Primers were tested bioinformatically to predict specificity, sensitivity, and precision. A total of 54 isolates of Salmonella serotypes Infantis, Senftenberg, and Alachua were tested, with and without the pESI plasmid carriage. Isolates of 31 additional serotypes were also screened to confirm specificity to Infantis. Specificity, sensitivity, and precision of each primer were >0.95. All isolates tested produced the expected band sizes. This PCR protocol provides a rapid and clear result for the detection of the pESI plasmid and serotype Infantis and will allow for the in vitro detection for epidemiological studies where whole-genome sequencing is not available.

Analysis of Salmonella enterica Isolated from a Mixed-Use Watershed in Georgia, USA: Antimicrobial Resistance, Serotype Diversity, and Genetic Relatedness to Human Isolates.

As the cases of Salmonella enterica infections associated with contaminated water are increasing, this study was conducted to address the role of surface water as a reservoir of S. enterica serotypes. We sampled rivers and streams (n = 688) over a 3-year period (2015 to 2017) in a mixed-use watershed in Georgia, USA, and 70.2% of the total stream samples tested positive for Salmonella. A total of 1,190 isolates were recovered and characterized by serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). A wide range of serotypes was identified, including those commonly associated with humans and animals, with S. enterica serotype Muenchen being predominant (22.7%) and each serotype exhibiting a high degree of strain diversity by PFGE. About half (46.1%) of the isolates had PFGE patterns indistinguishable from those of human clinical isolates in the CDC PulseNet database. A total of 52 isolates (4.4%) were resistant to antimicrobials, out of which 43 isolates were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). These 52 resistant Salmonella isolates were screened for the presence of antimicrobial resistance genes, plasmid replicons, and class 1 integrons, out of which four representative MDR isolates were selected for whole-genome sequencing analysis. The results showed that 28 MDR isolates resistant to 10 antimicrobials had blacmy-2 on an A/C plasmid. Persistent contamination of surface water with a high diversity of Salmonella strains, some of which are drug resistant and genetically indistinguishable from human isolates, supports a role of environmental surface water as a reservoir for and transmission route of this pathogen. IMPORTANCE Salmonella has been traditionally considered a foodborne pathogen, as it is one of the most common etiologies of foodborne illnesses worldwide; however, recent Salmonella outbreaks attributed to fresh produce and water suggest a potential environmental source of Salmonella that causes some human illnesses. Here, we investigated the prevalence, diversity, and antimicrobial resistance of Salmonella isolated from a mixed-use watershed in Georgia, USA, in order to enhance the overall understanding of waterborne Salmonella. The persistence and widespread distribution of Salmonella in surface water confirm environmental sources of the pathogen. A high proportion of waterborne Salmonella with clinically significant serotypes and genetic similarity to strains of human origin supports the role of environmental water as a significant reservoir of Salmonella and indicates a potential waterborne transmission of Salmonella to humans. The presence of antimicrobial-resistant and MDR Salmonella demonstrates additional risks associated with exposure to contaminated environmental water.

Coproduction of Tet(X7) Conferring High-Level Tigecycline Resistance, Fosfomycin FosA4, and Colistin Mcr-1.1 in Escherichia coli Strains from Chickens in Egypt.

The plasmid-mediated tet(X7) conferring high-level tigecycline resistance was identified in five mcr-1.1-positive Escherichia coli strains (ST10 [n = 3] and ST155 [n = 2]) isolated from chickens in Egypt. Two fosfomycin-resistant fosA4-carrying IncFII plasmids (∼79 kb in size) were detected. Transposase ISCR3 (IS91 family) is syntenic with tet(X7) in all isolates, suggesting its role in the mobilization of tet(X7). To our knowledge, this is the first global report of ST4-IncHI2 plasmids cocarrying tet(X7) and mcr-1.1 from chickens.

Serotyping of sub-Saharan Africa Salmonella strains isolated from poultry feces using multiplex PCR and whole genome sequencing.

BACKGROUND: Salmonella enterica remains a leading cause of food-borne diseases worldwide. Serotype information is important in food safety and public health activities to reduce the burden of salmonellosis. In the current study, two methods were used to determine serotypes of 111 strains of Salmonella isolated from poultry feces in Burkina Faso. First, Salmonella Multiplex Assay for Rapid Typing (SMART) Polymerase Chain Reaction (PCR) was used to determine the serovars of the S. enterica isolates. Second, serovar prediction based on whole genome sequencing (WGS) data was performed using SeqSero 2.0. RESULTS: Among the 111 Salmonella isolates, serotypes for 17 (15.31%) isolates were identified based on comparison to a panel of representative SMART codes previously determined for the 50 most common serovars in the United States. Forty-four (44) new SMART codes were developed for common and uncommon serotypes. A total of 105 (94.59%) isolates were serotyped using SeqSero 2.0 for serovar prediction based on WGS data. CONCLUSION: We determined that SeqSero 2.0 was more comprehensive for identifying Salmonella serotypes from Burkina Faso than SMART PCR.

Genome Analysis of Multidrug-Resistant Escherichia coli Isolated from Poultry in Nigeria.

Escherichia coli is one of the most common commensal bacteria of the gastrointestinal tract of humans and warm-blooded animals. Contaminated poultry can lead to disease outbreaks in consumers causing massive economic losses in the poultry industry. Additionally, commensal E. coli can harbor antibiotic resistance genes that can be transferred to other bacteria, including pathogens, in a colonized human host. In a previous study on antimicrobial resistance of E. coli from food animals from Nigeria, multidrug-resistant E. coli were detected. Three of those isolates were selected for further study using whole-genome sequencing due to the extensive drug resistance exhibited. All of the isolates carried the extended-spectrum ß-lactamase (ESBL) genes, blaCTX-M15 and blaTEM-1, whereas one isolate harbored an additional ESBL, blaOXA-1. All of the tetracycline-resistant isolates carried tet(A). The genes aac3-IIa and aacA4, conferring resistance to aminoglycosides, were identified in an E. coli isolate resistant to gentamicin and tobramycin. In two E. coli isolates, dfrA14, qnrS1, and sulII, were detected conferring resistance to trimethoprim, fluoroquinolones, and sulfonamides, respectively. The third isolate carried dfrA17, no fluoroquinolone resistance gene, an additional sulI gene, and a chloramphenicol resistance gene, catB3. Mutations in candidate genes conferring resistance to fosfomycin and fluoroquinolones were also detected. Several efflux systems were detected in all the E. coli isolates and virulence-associated genes related to serum resistance, motility, and adhesion. E. coli and non-E. coli origin prophages were also identified in the isolates. The results underline the higher resolution power of whole-genome sequencing for investigation of antimicrobial resistance, virulence, and phage in E. coli.

Detection and Molecular Characterization of Staphylococci from Eggs of Household Chickens.

Eggs are a healthy and nutritious food source, but may be contaminated by bacteria. Previous studies have reported the presence of staphylococci in eggs of farmed chickens, but no study has evaluated the staphylococcal population of eggs from household chickens. In this study, staphylococci from eggs (n = 275) of household chickens collected from November 2016 to March 2017 from different villages of Khyber Pakhtunkhwa province, Pakistan, were characterized. Seven species of staphylococci were identified from 65 eggs, including the predominant species, Staphylococcus xylosus (49/275; 17.8%). S. xylosus isolates (n = 73) were tested for antimicrobial susceptibility, presence of resistance genes, genetic relatedness, and inhibitory activity against other bacteria. The majority of isolates were resistant to oxacillin (83.6%) and tetracycline (24.7%), but also exhibited resistance to daptomycin and linezolid (5.5% each). Of the 10 resistance genes tested, isolates were only positive for mecA (35.6%; 26/73), mecC/C1 (2.7%; 2/73), and tet(K) (14/73; 19%). Using pulsed-field gel electrophoresis (PFGE), nine clusters had identical PFGE patterns. Isolates produced inhibitory activity against a broad spectrum of bacteria; 20.5%, 19.2%, 17.8%, and 16.4% of S. xylosus were able to inhibit growth of Salmonella enterica serotype Typhi, methicillin-susceptible Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus, respectively. This study demonstrated the presence of genetically related antimicrobial-resistant S. xylosus from eggs from household chickens. Like table eggs, eggs of household chickens also contain staphylococci that may be resistant to antimicrobials used to treat human infections. These data will allow comparison between staphylococci from eggs from different sources and may indicate the relative safety of eggs from household chickens. Further study of these egg types and their microbial composition is warranted.

Detection and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus from Table Eggs in Haripur, Pakistan.

Table eggs are nutritionally important food consumed globally. Despite being protected inside the hard shell and a semipermeable membrane, the egg contents may be contaminated with microbes and thus become a possible carrier of infectious agents to humans. A number of medically significant bacterial species such as Salmonella enterica, Listeria monocytogenes, and Yersinia enterocolitica have already been reported from table eggs. More important is the presence of antimicrobial-resistant bacterial strains in this food source. The present study was aimed at detection and characterization of Staphylococcus aureus from table eggs collected from different retail shops in Haripur city of Pakistan. Staphylococci were isolated from 300 eggs collected from December 2015 to May 2016. S. aureus isolates were tested for antimicrobial susceptibility using broth microdilution and characterized using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and spa typing. The presence of Panton-Valentine leukocidin and antimicrobial resistance genes were detected using PCR. Staphylococci were isolated from 21.3% (64/300) of the table eggs tested. Of those, 59% (38/64) were identified as S. aureus, of which 33 (86.8%) were positive for mecA (MRSA, methicillin-resistant S. aureus). All MRSA were multidrug resistant (resistant to two or more antimicrobial classes), contained aac-aph (encoding aminoglycosides), and were pvl+. Using MLST, spa typing, and SCCmec typing, three genotypic patterns were assigned: ST8-t8645-MRSA-IV, associated with USA300; and ST772-t657-MRSA-IV and ST772-t8645-MRSA-IV, both characteristic of the Bengal Bay community-associated MRSA clone. Molecular typing by PFGE revealed that the bacterial population was highly homogenous with only two patterns observed. This study is the first report of detection of human-associated pvl+ MRSA from table eggs. The genetic similarities of MRSA present in the eggs to that of humans may suggest human to poultry transmission of MRSA via contamination.

Antibiotic Resistance Patterns of Major Zoonotic Pathogens from All-Natural, Antibiotic-Free, Pasture-Raised Broiler Flocks in the Southeastern United States.

The use of antibiotics in agroecosystems has been implicated in the rise in antibiotic resistance (AR), which can affect environmental, animal, and human health. To determine the environmental impact of antibiotic use in agroecosystems, appropriate background levels of AR in agricultural environments in the absence of antibiotic application must be determined. Therefore, to determine background levels of AR in broiler production, four target microbes (, , , and ) were isolated from 15 all-natural, antibiotic-free, pasture-raised broiler flocks from six farms within the southeastern United States. The AR profiles of these isolates were characterized using the CDC National Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS), and these resistance patterns were compared across target microbes and farms and throughout the life cycle of the flocks along the farm-to-fork continuum. Antibiotic resistances were most prevalent in and and least prevalent in . Although and were isolated from the same farms and characterized using the same NARMS plates, they exhibited distinct AR profiles, with demonstrating clear farm-specific resistance patterns. Multidrug resistance rates (three or more antibiotics), in order of prevalence, were (63.9%), (36.0%), (12.7%), and (1.4%). The results of this study demonstrate the variability in background AR among major food safety-related microbes, even when isolated from similar production and processing samples from the same farms, and indicate the need for the proper design of future broiler production studies to account for this highly dynamic background AR.

Genome analysis of Salmonella enterica serovar enteritis strains isolated from poultry and humans in Burkina Faso.

Whole-genome sequencing (WGS) was used to characterize four Salmonella enterica Enteritidis isolates from poultry (n=2) and human (n=2) from Ouagadougou, Burkina Faso. Antimicrobial resistance genes, chromosomal mutations, and mobile genetic elements were identified by analysis of WGS data using sequence homology.

Prevalence and characterization of methicillin-resistant Staphylococcus aureus isolates from retail meat and humans in Georgia.

There is increasing interest in the presence of Staphylococcus aureus, specifically methicillin-resistant S. aureus (MRSA), on retail meat products. In this study, staphylococci were isolated from retail pork and retail beef in Georgia, and MRSA from the products was compared to human MRSA from the same geographic area using broth microdilution antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 45% (45/100) of pork products and 63% (63/100) of beef products; mecA was detected in S. aureus from both pork (3/100; 3%) and beef (4/100; 4%). Fifty percent (50/100) of human S. aureus also contained mecA. Multidrug resistance was detected among MRSA from all sources. All MRSA (n = 57) was SCCmec type IV, and nine different spa types were present among the isolates (t002, t008, t012, t024, t179, t337, t548, t681, and t1062). Four sequence types (ST5, ST8, ST9, and ST30) were detected using MLST; the majority of MRSA isolates belonged to ST8, followed by ST5. One retail beef MRSA isolate belonged to ST8, while the remaining three were ST5. In retail pork MRSA, ST5, ST9, and ST30 were observed. The majority of human MRSA isolates belonged to ST8. Thirty-seven MRSA isolates, one of which was a retail beef MRSA isolate, were pvl(+). Using PFGE, MLST, and spa typing, three retail beef MRSA isolates were found to be identical in PFGE pattern, ST, and spa type to two human clonal MRSA isolates (USA100 and USA300). One additional retail beef MRSA isolate had a PFGE pattern similar to that of a human MRSA isolate, whereas none of the retail pork MRSA isolates had PFGE patterns similar to those of human MRSA isolates. These data suggest that the retail beef samples were contaminated by a human source, possibly during processing of the meat, and may present a source of MRSA for consumers and others who handle raw meat.

Clonally related methicillin-resistant Staphylococcus aureus isolated from short-finned pilot whales (Globicephala macrorhynchus), human volunteers, and a bayfront cetacean rehabilitation facility.

In May of 2011, a live mass stranding of 26 short-finned pilot whales (Globicephala macrorhynchus) occurred in the lower Florida Keys. Five surviving whales were transferred from the original stranding site to a nearby marine mammal rehabilitation facility where they were constantly attended to by a team of volunteers. Bacteria cultured during the routine clinical care of the whales and necropsy of a deceased whale included methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). In order to investigate potential sources or reservoirs of MSSA and MRSA, samples were obtained from human volunteers, whales, seawater, and sand from multiple sites at the facility, nearby recreational beaches, and a canal. Samples were collected on 3 days. The second collection day was 2 weeks after the first, and the third collection day was 2 months after the last animal was removed from the facility. MRSA and MSSA were isolated on each day from the facility when animals and volunteers were present. MSSA was found at an adjacent beach on all three collection days. Isolates were characterized by utilizing a combination of quantitative real-time PCR to determine the presence of mecA and genes associated with virulence, staphylococcal protein A typing, staphylococcal cassette chromosome mec typing, multilocus sequence typing, and pulsed field gel electrophoresis (PFGE). Using these methods, clonally related MRSA were isolated from multiple environmental locations as well as from humans and animals. Non-identical but genetically similar MSSA and MRSA were also identified from distinct sources within this sample pool. PFGE indicated that the majority of MRSA isolates were clonally related to the prototype human strain USA300. These studies support the notion that S. aureus may be shed into an environment by humans or pilot whales and subsequently colonize or infect exposed new hosts.

Human-associated methicillin-resistant Staphylococcus aureus from a subtropical recreational marine beach.

Reports of Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) detected in marine environments have occurred since the early 1990 s. This investigation sought to isolate and characterize S. aureus from marine waters and sand at a subtropical recreational beach, with and without bathers present, in order to investigate possible sources and to identify the risks to bathers of exposure to these organisms. During 40 days over 17 months, 1,001 water and 36 intertidal sand samples were collected by either bathers or investigators at a subtropical recreational beach. Methicillin-sensitive S. aureus (MSSA) and MRSA were isolated and identified using selective growth media and an organism-specific molecular marker. Antimicrobial susceptibility, staphylococcal cassette chromosome mec (SCCmec) type, pulsed-field gel electrophoresis (PFGE) pattern, multi-locus sequence type (MLST), and staphylococcal protein A (spa) type were characterized for all MRSA. S. aureus was isolated from 248 (37 %) bather nearby water samples at a concentration range of <2-780 colony forming units per ml, 102 (31 %) ambient water samples at a concentration range of <2-260 colony forming units per ml, and 9 (25 %) sand samples. Within the sand environment, S. aureus was isolated more often from above the intertidal zone than from intermittently wet or inundated sand. A total of 1334 MSSA were isolated from 37 sampling days and 22 MRSA were isolated from ten sampling days. Seventeen of the 22 MRSA were identified by PFGE as the community-associated MRSA USA300. MRSA isolates were all SCCmec type IVa, encompassed five spa types (t008, t064, t622, t688, and t723), two MLST types (ST8 and ST5), and 21 of 22 isolates carried the genes for Panton-Valentine leukocidin. There was a correlation (r = 0.45; p = 0.05) between the daily average number of bathers and S. aureus in the water; however, no association between exposure to S. aureus in these waters and reported illness was found. This report supports the concept that humans are a potential direct source for S. aureus in marine waters.

Epidemiology of Staphylococcus aureus Non-Susceptible to Vancomycin in South Asia.

Staphylococcus aureus is one of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens among which multidrug resistance has emerged. Resistance to methicillin has resulted in clinicians using the antibiotic of last resort, vancomycin, to treat infections caused by methicillin-resistant S. aureus (MRSA). However, excessive use and misuse of vancomycin are major causes of resistance among S. aureus strains. South Asia encompasses ~25% of the world's population, and countries in South Asia are often characterized as low- and middle-income with poor healthcare infrastructure that may contribute to the emergence of antibiotic resistance. Here, we briefly highlight the mechanism of vancomycin resistance, its emergence in S. aureus, and the molecular epidemiology of non-susceptible S. aureus to vancomycin in the South Asian region.

Use of automated capillary immunoassays for quantification of antibodies in chicken sera against recombinant Salmonella enterica serotype Heidelberg proteins.

The classic immunoblot technique is an important tool for identification and characterization of target proteins. However, a standard protocol for this classic immunoblot assay involves many steps that may cause experimental variations in each step and make quantification of antibodies in sera difficult. A capillary electrophoresis-based immunoblot system was developed to reduce potential problems in variations during the experimental process, enable protein identification in an automatic manner and quantitate various isotypes of antibodies in sera. In the present study, we used this system to examine the purity of the recombinant proteins and measure amounts of various isotypes of immunoglobins in chicken sera after immunization with two recombinant Salmonella FliD and FimA proteins. A single band of each protein was detected in the gel like images by this system after purification by nickel-chelated affinity chromatography. A good linear range of the protein concentrations was also obtained for each recombinant protein. This automated capillary immunoblot system was successfully used for detection and quantification of various immunoglobin isotypes against two recombinant Salmonella proteins from the immunized chicken sera, but not the un-immunized chicken sera. The chicken immunoglobin G (IgG) antibody response to the FliD protein from the immunized group was 1110- and 51,400-fold higher than that from the un-immunized chickens both two- and three-weeks post-vaccination, respectively. It was also observed that IgM antibody against the FliD protein from the immunized chickens was 1030-fold higher than that from the un-immunized chickens two weeks post-vaccination, but the IgM response declined to 120-fold between two groups from two weeks to three weeks after immunization. The IgM antibody response to the FimA protein from the immunized group was 1.84- and 1.12-fold higher than that from the un-immunized group, respectively, both two- and three-weeks post-vaccination, while the IgG antibody response from the immunized group was 8.07- and 27.6-fold higher than that from the un-immunized group, respectively, during the same period. These results suggest that this capillary-based immunoblot assay can be an alternative method for analyses and quantitation of chicken humoral immune response before and after immunization with any antigens and/or for investigation in Salmonella outbreaks.

ß-Lactamase Producing Escherichia coli Encoding blaCTX-M and blaCMY Genes in Chicken Carcasses from Egypt.

Escherichia coli with multidrug resistance and ß-lactamase genes may constitute a great public health hazard due to the potential for their transmission to humans through the food chain. This study determined the prevalence, antibiotic resistance profiles, phylogroups, and ß-lactamase genes of E. coli isolates from chicken carcasses marketed in Mansoura, Egypt. Interestingly, E. coli was detected in 98% (98/100) of the chicken carcasses examined, which seemed among the highest contamination rates by E. coli worldwide. From the 425 genetically verified uidA gene-positive E. coli, 85 isolates were further studied for antimicrobial resistance profiles, phylogroups, and ß-lactamase genes. Interestingly, 89.41% of E. coli (76/85) strains tested against 24 different antibiotics were multidrug-resistant. Of the examined 85 E. coli isolates, 22 (25.88%) isolates harbored blaCTX-M and were resistant to ampicillin, cefazoline, and ceftriaxone, while three of them were resistant to ceftazidime besides. Nine (10.59%) E. coli strains harbored AmpC- ß-lactamase blaCMY and were resistant to ampicillin. One isolate co-carried blaCMY and blaCTX-M genes, though it was negative for the blaTEM gene. Of the 35 isolates that harbored either extended-spectrum ß-lactamase (ESBL) and/or AmpC ß-lactamase genes, six strains (17.14%) were assigned to pathogenic phylogroup F and one to phylogroup E, whereas 28 (80%) isolates belonged to commensal phylogenetic groups.

Whole-genome sequencing of Listeria innocua recovered from retail milk and dairy products in Egypt.

The similarity of the Listeria innocua genome with Listeria monocytogenes and their presence in the same niche may facilitate gene transfer between them. A better understanding of the mechanisms responsible for bacterial virulence requires an in-depth knowledge of the genetic characteristics of these bacteria. In this context, draft whole genome sequences were completed on five L. innocua isolated from milk and dairy products in Egypt. The assembled sequences were screened for antimicrobial resistance and virulence genes, plasmid replicons and multilocus sequence types (MLST); phylogenetic analysis of the sequenced isolates was also performed. The sequencing results revealed the presence of only one antimicrobial resistance gene, fosX, in the L. innocua isolates. However, the five isolates carried 13 virulence genes involved in adhesion, invasion, surface protein anchoring, peptidoglycan degradation, intracellular survival, and heat stress; all five lacked the Listeria Pathogenicity Island 1 (LIPI-1) genes. MLST assigned these five isolates into the same sequence type (ST), ST-1085; however, single nucleotide polymorphism (SNP)-based phylogenetic analysis revealed 422-1,091 SNP differences between our isolates and global lineages of L. innocua. The five isolates possessed an ATP-dependent protease (clpL) gene, which mediates heat resistance, on a rep25 type plasmids. Blast analysis of clpL-carrying plasmid contigs showed approximately 99% sequence similarity to the corresponding parts of plasmids of L. monocytogenes strains 2015TE24968 and N1-011A previously isolated from Italy and the United States, respectively. Although this plasmid has been linked to L. monocytogenes that was responsible for a serious outbreak, this is the first report of L. innocua containing clpL-carrying plasmids. Various genetic mechanisms of virulence transfer among Listeria species and other genera could raise the possibility of the evolution of virulent strains of L. innocua. Such strains could challenge processing and preservation protocols and pose health risks from dairy products. Ongoing genomic research is necessary to identify these alarming genetic changes and develop preventive and control measures.

Tulathromycin metaphylaxis increases nasopharyngeal isolation of multidrug resistant Mannheimia haemolytica in stocker heifers.

Bovine respiratory disease (BRD) is a leading cause of disease in feedlot and stocker calves with Mannheimia haemolytica (MH) as one of the most common etiologies. One of the most effective means of controlling BRD is through metaphylaxis, which involves administering antimicrobials to all animals at high risk of developing BRD. However, increasing prevalence of multidrug resistant (MDR) MH may reduce efficacy of metaphylaxis due to decreased susceptibility to drugs used for metaphylaxis. Primarily, this study aimed to determine the effect of tulathromycin metaphylaxis and subsequent BRD treatment on antimicrobial resistance (AMR) in MH isolated from stocker calves. Secondary objectives included evaluating the effect of metaphylaxis and treatment for BRD on animal health and comparing the genetic relationship of MH isolated. Crossbred beef heifers (n = 331, mean weight = 232, SD = 17.8 kg) at high risk for BRD were randomly assigned to receive tulathromycin metaphylaxis (META, n = 167) or not (NO META, n = 164). Nasopharyngeal swabs were collected for MH isolation, antimicrobial susceptibility testing and whole genome sequencing at arrival and 3 (WK3) and 10 (WK10) weeks later. Mixed-effects logistic regression was used to identify risk factors for isolation of MH and MDR MH (resistant to ≥3 antimicrobial drug classes) at 3 and 10 weeks, BRD morbidity, and crude mortality. Animals in the META group had higher odds of isolation of MDR MH at 3 weeks [OR (95% CI) = 13.08 (5-30.9), p < 0.0001] and 10 weeks [OR (95% CI) = 5.92 (1.34-26.14), p = 0.019] after arrival. There was no difference in risk of isolation of any MH (resistant or susceptible) between META and NO META groups at all timepoints. Animals in the NO META group had 3 times higher odds of being treated for BRD [WK3: OR (95% CI) = 3.07 (1.70-5.52), p = 0.0002; WK10: OR (95% CI) = 2.76 (1.59-4.80), p = 0.0002]. Antimicrobial resistance genes found within isolates were associated with integrative conjugative element (ICE) genes. Tulathromycin metaphylaxis increased risk of isolation of MDR MH and in this population, the increase in MDR MH appeared to be associated with ICE containing antimicrobial resistance genes for multiple antimicrobial classes. This may have important implications for future efficacy of antimicrobials for control and treatment of BRD.