Lari M. Hiott

Prevalence and antimicrobial resistance of enterococci isolated from retail fruits, vegetables, and meats

Although enterococci are considered opportunistic pathogens, they can be reservoirs of antimicrobial resistance. Antimicrobial resistance is increasingly important because of foodborne illnesses from meat and infections from produce. From 2000 through 2001, food items (vegetables, fruits, and meats) were obtained from grocery store chains in northern Georgia and cultured for the presence of enterococci; 47.7% (189 of 396) of these samples were positive for enterococci. For the fruits and vegetables, enterococci were cultured most often from tomatoes (9 of 27 samples, 33%) and radishes (10 of 11 samples, 91%), respectively. Among the meat items tested, enterococci were isolated from 95% (21 of 22) of the chicken samples, 73% (16 of 22) of the beef samples, 95% (20 of 21) of the turkey samples, and 68% (15 of 22) of the pork samples. The predominant species identified was Enterococcus faecalis (n = 80) from meat and Enterococcus casseliflavus (n = 66) from fruits and vegetables. Although high numbers of isolates were resistant to lincomycin (176 of 185 isolates, 95.1%) and bacitracin (150 of 185 isolates, 81.1%), very few isolates were resistant to salinomycin (2 isolates, 1.1%), penicillin (3 isolates, 1.6%), or nitrofurantoin (9 isolates, 4.9%). None of the isolates were resistant to linezolid or vancomycin. These data suggest that foods commonly purchased from grocery stores are a source of enterococci; however, overall resistance to antimicrobials is relatively low.

Antimicrobial Resistance in Enterococcus spp. Isolated from Environmental Samples in an Area of Intensive Poultry Production

Enterococcus spp. from two poultry farms and proximate surface and ground water sites in an area of intensive poultry production were tested for resistance to 16 clinical antibiotics. Resistance patterns were compared to assess trends and possible correlations for specific antimicrobials and levels of resistance. Enterococci were detected at all 12 surface water sites and three of 28 ground water sites. Resistance to lincomycin, tetracycline, penicillin and ciprofloxacin in poultry litter isolates was high (80.3%, 65.3%, 61.1% and 49.6%, respectively). Resistance in the surface water to the same antibiotics was 87.1%, 24.1%, 7.6% and 12.9%, respectively. Overall, 86% of litter isolates, 58% of surface water isolates and 100% of ground water isolates were resistant to more than one antibiotic. Fifty-four different resistance patterns were recognised in isolates obtained from litter and environmental samples and several E. faecium and E. faecalis isolates from litter and environment samples shared the same resistance pattern. Multiple antibiotic resistant (MAR) indices calculated to assess health risks due to the presence of resistant enterococci suggested an increased presence of antibiotics in surface water, likely from poultry sources as no other wastewater contributions in the area were documented.

Effect of media, temperature and culture conditions on the species population and antibiotic resistance of enterococci from broiler chickens*

Aims:  The effect of media type, incubation temperature and enrichment period on the species population and antibiotic susceptibility of enterococci from poultry carcass rinsates was determined.

Antimicrobial resistance, virulence determinants and genetic profiles of clinical and nonclinical Enterococcus cecorum from poultry

Enterococcus cecorum has been implicated as a possible cause of disease in poultry. However, the characteristics that contribute to pathogenesis of Ent. cecorum in poultry have not been defined. In this study, Ent. cecorum from carcass rinsates (n = 75) and diseased broilers and broiler breeders (n = 30) were compared based upon antimicrobial resistance phenotype, the presence of virulence determinants and genetic relatedness using pulsed‐field gel electrophoresis (PFGE). Of the 16 antimicrobials tested, Ent. cecorum from carcass rinsates and clinical cases were resistant to ten and six of the antimicrobials, respectively. The majority of Ent. cecorum from carcass rinsates was resistant to lincomycin (54/75; 72%) and tetracycline (46/75; 61·3%) while the highest level of resistance among clinical Ent. cecorum was to tetracycline (22/30; 73·3%) and erythromycin (11/30; 36·7%). Multidrug resistance (resistance to ≥2 antimicrobials) was identified in Ent. cecorum from carcass rinsates (53/75; 70·7%) and diseased poultry (18/30; 60%). Of the virulence determinants tested, efaAfm was present in almost all of the isolates (104/105; 99%). Using PFGE, the majority of clinical isolates clustered together; however, a few clinical isolates grouped with Ent. cecorum from carcass rinsates. These data suggest that distinguishing the two groups of isolates is difficult based upon the characterization criteria used.

Prevalence and multidrug resistance of Escherichia coli from community-acquired infections in Lagos, Nigeria

INTRODUCTION The emergence of multidrug resistance (MDR; resistance to ≥ 2 more antimicrobials) in Escherichia coli is of concern due to complications encountered in treatment. METHODOLOGY In this study, prevalence, antimicrobial resistance, and genetic characteristics of MDR community isolates of E. coli from Lagos, Nigeria were determined. Urine and stool samples were obtained from outpatients attending Lagos State hospitals and from animal handlers in abattoirs, poultries, and open markets, from December 2012 to July 2013. RESULTS Approximately 50% of urine (200/394) and 88% of stool samples (120/136) were positive for E. coli. Based upon β-lactamase production, a subset of those isolates was selected for further study. Of the 22 antimicrobials tested, E. coli exhibited resistance to all antimicrobials except amikacin and piperacillin/tazobactam. The highest levels of resistance were to tetracycline (182/247; 73.7%), trimethoprim/sulfamethoxazole (152/247; 61.5%), and ampicillin (147/247; 59.1%). Resistance to the cephalosporins ranged from 1.6%-15% including the third- and fourth-generation cephalosporins, cefpodoxime (20/247; 8.1%) and cefepime (4/247; 1.6%), respectively. MDR was observed in 69.6% (172/247) of the isolates. Forty-eight E. coli resistant to at least five antimicrobials were selected for further analysis using pulsed-field gel electrophoresis; seven distinct clusters were observed among the diverse patterns. Of the 48 MDR E. coli, 30 different sequence types (ST) were detected using multilocus sequence typing, including four ST131. CONCLUSIONS This study demonstrated circulating MDR E. coli in the Nigerian community. Monitoring of antimicrobial resistance in developing countries is necessary to optimize empiric treatment and the prudent use of antimicrobials.

Diversity of Plasmids and Antimicrobial Resistance Genes in Multidrug‐Resistant Escherichia coli Isolated from Healthy Companion Animals

The presence and transfer of antimicrobial resistance genes from commensal bacteria in companion animals to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. The purpose of this study was to determine antimicrobial resistance gene content and the presence of genetic elements in antimicrobial resistant Escherichia coli from healthy companion animals. In our previous study, from May to August, 2007, healthy companion animals (155 dogs and 121 cats) from three veterinary clinics in the Athens, GA, USA area were sampled and multidrug‐resistant E. coli (n = 36; MDR, resistance to ≥2 antimicrobial classes) were obtained. Of the 25 different plasmid replicon types tested by PCR, at least one plasmid replicon type was detected in 94% (34/36) of the MDR E. coli; four isolates contained as many as five different plasmid replicons. Nine replicon types (FIA, FIB, FII, I2, A/C, U, P, I1 and HI2) were identified with FIB, FII, I2 as the most common pattern. The presence of class I integrons (intI) was detected in 61% (22/36) of the isolates with eight isolates containing aminoglycoside‐ and/or trimethoprim‐resistance genes in the variable cassette region of intI. Microarray analysis of a subset of the MDR E. coli (n = 9) identified the presence of genes conferring resistance to aminoglycosides (aac, aad, aph and strA/B), β‐lactams (ampC, cmy, tem and vim), chloramphenicol (cat), sulfonamides (sulI and sulII), tetracycline [tet(A), tet(B), tet(C), tet(D) and regulator, tetR] and trimethoprim (dfrA). Antimicrobial resistance to eight antimicrobials (ampicillin, cefoxitin, ceftiofur, amoxicillin/clavulanic acid, streptomycin, gentamicin, sulfisoxazole and trimethoprim‐sulfamethoxazole) and five plasmid replicons (FIA, FIB, FII, I1 and I2) were transferred via conjugation. The presence of antimicrobial resistance genes, intI and transferable plasmid replicons indicate that E. coli from companion animals may play an important role in the dissemination of antimicrobial resistance, particularly to human hosts during contact.

A Comparison of BOX-PCR and Pulsed-Field Gel Electrophoresis to Determine Genetic Relatedness of Enterococci from Different Environments

Genetic relatedness of enterococci from poultry litter to enterococci from nearby surface water and groundwater in the Lower Fraser Valley regions of British Columbia, Canada was determined. A new automated BOX-PCR and Pulsed-Field Gel Electrophoresis (PFGE) were used to subtype enterococcal isolates from broiler and layer litter and surface and groundwater. All surface water samples (n = 12) were positive for enterococci, as were 11% (3/28) of groundwater samples. Enterococcus faecium (n = 90) was isolated from all sources, while Enterococcus faecalis (n = 59) was isolated from all sources except layer litter. The majority of E. faecalis originated from broiler litter (28/59; 47.5%) while the majority of E. faecium were isolated from layer litter (29/90; 32.2%). E. faecalis grouped primarily by source using BOX-PCR. Isolates from water samples were dispersed more frequently among PFGE groups containing isolates from poultry litter. E. faecium strains were genetically diverse as overall clustering was independent of source by both molecular methods. Subgroups of E. faecium isolates based upon source (layer litter) were present in BOX-PCR groups. Three individual E. faecalis groups and two individual E. faecium groups were 100% similar using BOX-PCR; only one instance of 100% similarity among isolates using PFGE was observed. Although enterococci from litter and water sources were grouped together using BOX-PCR and PFGE, isolates originating from water could not be definitively identified as originating from poultry litter. Automation of BOX-PCR amplicon separation and visualization increased the reproducibility and standardization of subtyping using this procedure.

Application of Multiplex PCR, Pulsed-Field Gel Electrophoresis (PFGE), and BOX-PCR for Molecular Analysis of Enterococci

Although it has been recovered from vegetation, soil, water, and food, Enterococcus is a ubiquitous Gram-positive bacterium found primarily in the intestine of nearly all animals (Giraffa 2002;Muller et al. 2001;Niemi et al. 1993;Svec and Sedlacek 1999). Different strains of enterococci populate the digestive tracts of humans and animals, making them a good indicator of water contamination (Svec and Sedlacek 1999). They are the second most studied group of bacteria in the field of microbial source tracking (following Escherichia coli) due to their connection to humans and animals as well as their recent significance as a clinical pathogen (Layton et al. 2010;Scott et al. 2005). The enterococci have been implicated in a number of clinical diseases including endocarditis, bacteremia, and urinary tract infections, most often in hospital settings (Huycke et al. 1998;Jett et al. 1994). They are a leading cause of nosocomial infections (hospital acquired infections), accounting for approximately 12% in the U.S. yearly; the majority of infections are caused by Enterococcus faecalis and E. faecium (Huycke et al. 1998). While their role as an opportunistic nosocomial pathogen has been well documented, their ability to cause foodborne illnesses still remains largely unknown. Their role in food processing can be desirable in some cases and unwanted in others. For example, they may be considered as beneficial because they harbor specific biochemical traits that are essential in manufacturing fermented milk products such as cheeses, but their presence can also indicate spoilage for fermented meats or unsanitary conditions in other food industries (Foulquie Moreno et al. 2006;Giraffa 2002). The production of biogenic amines in fermented foods by enterococci is also thought to result in food intoxication characterized by symptoms such as vomiting and headaches (Gardin et al. 2001;Giraffa 2002;Tham et al. 1990). In addition, because enterococci can potentially harbor antimicrobial resistance genes and genes which may

Multidrug resistant Mannheimia haemolytica isolated from high-risk beef stocker cattle after antimicrobial metaphylaxis and treatment for bovine respiratory disease

Antimicrobial resistance (AMR) in bacterial respiratory pathogens in high-risk stocker cattle has been poorly characterized. The objective of this study was to describe the prevalence of multidrug resistant (MDR; resistance to > 3 antimicrobial classes) respiratory pathogens in 50 conventionally managed stocker cattle over 21 days after arrival. Cattle received tildipirosin metaphylaxis on day 0 and were eligible to receive up to 3 additional antimicrobials for bovine respiratory disease (BRD): florfenicol, ceftiofur and enrofloxacin. Nasopharyngeal swabs were collected on days 0, 7, 14, and 21 for bacterial culture and antimicrobial susceptibility testing using disc diffusion and broth microdilution. Mannheimia haemolytica was isolated from 5 of 48, 27 of 50, 44 of 50, and 40 of 50 cattle on days 0, 7, 14, and 21, respectively. One of 5, 27 of 27, 43 of 44, and 40 of 40 M. haemolytica were MDR on days 0, 7, 14, and 21, respectively. Pasteurella multocida was isolated from 6 of 48 cattle on day 0 and none were MDR; no other pathogens were isolated. Twenty-four cattle required at least one BRD treatment; M. haemolytica was isolated before treatment from 13 of 24 cattle; all were MDR. One hundred-eighteen M. haemolytica isolates were subjected to pulsed-field gel electrophoresis (PFGE); multiple genotypes were identified. Whole genome sequencing of 33 isolates revealed 14 known AMR genes. Multidrug resistant M. haemolytica can be highly prevalent and genetically diverse in stocker cattle; additional research is necessary to determine factors that influence prevalence and the impact on cattle health.

Prevalence and characterization of Escherichia coli isolated from the Upper Oconee Watershed in Northeast Georgia

Surface waters are important sources of water for drinking, industrial, agricultural, and recreational uses; hence, contamination of water by fecal, pathogenic, or antimicrobial resistant (AR) bacteria is a major environmental and public health concern. However, very little data is available on prevalence of these bacteria in surface water throughout a watershed. This study aimed to characterize Escherichia coli present in the Upper Oconee Watershed, a mixed-use watershed in Athens, GA, USA for potential pathogenicity and AR. E. coli were enumerated by colony counts, cultured by enrichment and direct plating, and characterized by phylo-groups, diarrheagenic pathotypes, and antimicrobial susceptibility. From the analysis, 99.3% (455/458) of the total samples were positive for E. coli resulting in 496 isolates. E. coli counts were as high as 1.2×104 CFU/100 ml, which is above the United States Environmental Protection Agency (U.S. EPA) threshold for recreational water (235 CFU/100 ml based on a one-time measurement). Phylo-groups B2 (31.7%; 157/496) and B1 (30.8%; 153/496) were the most prevalent among the isolates. Enteropathogenic E. coli (EPEC) (19/496) and Shiga toxin-producing E. coli (STEC) (1/496) were the only diarrheagenic pathotypes detected. AR was observed in 6.9% (34/496) of the isolates, 15 of which were multidrug resistant (MDR; resistance to two or more classes of antimicrobials). Tetracycline resistance was most often detected (76.5%; 26/34), followed by ampicillin (32.4%; 11/34), streptomycin (23.5%; 8/34), sulfisoxazole (23.5%; 8/34), and nalidixic acid (14.7%; 5/34). Results from this study showed that E. coli is prevalent in high levels in the Upper Oconee Watershed, suggesting possible widespread fecal contamination. The presence of pathogenic, AR E. coli in the watershed indicates that environmental water can serve as a reservoir of resistant bacteria that may be transferred to humans through drinking and recreational activities.