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.