Distribution and Transfer of Plasmid Replicon Families among Multidrug-Resistant Enterococcus faecalis and Enterococcus faecium from Poultry.

The presence and transfer of plasmids from commensal bacteria to more pathogenic bacteria may contribute to the dissemination of antimicrobial resistance. However, the prevalence of plasmids from commensal bacteria, such as the enterococci, in food animals remains largely unknown. In this study, the diversity and prevalence of plasmid families from multidrug-resistant (MDR; resistance to three or more antimicrobials) enterococci from poultry carcasses were determined. Plasmid-positive MDR enterococci were also tested for the ability to transfer plasmids to other enterococci using conjugation. MDR Enterococcus faecalis (n = 98) and Enterococcus faecium (n = 696) that were isolated from poultry carcass rinsates between 2004 and 2011 were tested for the presence of 21 plasmid replicon (rep) families using multiplex PCR. Approximately 48% of E. faecalis (47/98) and 16% of E. faecium (110/696) were positive for at least one rep-family. Fourteen rep-families were detected overall, and ten rep-families were shared between E. faecalis and E. faecium. The rep7 and rep17 families were unique to E. faecalis, while the rep5 and rep8 families were unique to E. faecium. The rep9 family was predominant in both E. faecalis and E. faecium for all the years tested. The greatest number of rep-families detected was in 2005 (n = 10), and the least was in 2009 (n = 1). Eight rep-families were transferred from E. faecalis donors to the E. faecalis JH2-2 recipient using conjugation. Results from this study showed that E. faecalis and E. faecium from poultry carcasses contain numerous and diverse rep-families that are capable of conjugal transfer.

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.

Genetic Diversity of Staphylococcus aureus Strains from a Tertiary Care Hospital in Rawalpindi, Pakistan.

Staphylococcus aureus is an important healthcare-associated bacterium that causes a multitude of infections in humans such as superficial skin and soft tissue infections, necrotizing pneumonia, foodborne illnesses and postsurgical infections. Treatment of S. aureus infections has become more complicated due to the emergence of Methicillin-Resistant Staphylococcus aureus (MRSA), some of which are multidrug resistant. The present study aimed to characterize S. aureus isolates from a tertiary care hospital in the Rawalpindi district of Pakistan. Staphylococci were isolated from 300 clinical samples collected from January 2018 to January 2019 and S. aureus isolates were tested for antimicrobial susceptibility and analyzed using Pulsed-Field Gel Electrophoresis (PFGE), Multi-Locus Sequence Typing (MLST), staphylococcal cassette chromosome mec (SCCmec) and spa typing. Approximately 25.3% (76/300) of the clinical samples were positive for S. aureus; of those, 88.2% (67/76) were mecA+ (MRSA). In addition to the ß-lactam antibiotics, high levels of resistance were also found to the fluoroquinolones (ciprofloxacin, gatifloxacin and levofloxacin (73.7% each)). Of the 23 different spa types identified, the majority of isolates belonged to spa type t632 and t657 (9/66; 13.6% each spa type). ST772-t657 (Bengal Bay clone) was the most commonly identified clone in this study although other clones circulating around different regions of the world were also found indicating the diversity in MRSA isolates from this area of Pakistan. This study emphasizes the need to monitor MRSA in the clinical setting for improved infection control and treatment options.

Emergence of Multidrug-Resistant Escherichia coli Producing CTX-M, MCR-1, and FosA in Retail Food From Egypt.

In this study, multidrug-resistant (MDR) Escherichia coli isolates from retail food and humans assigned into similar Multilocus Sequence Types (MLST) were analyzed using whole genome sequencing (WGS). In silico analysis of assembled sequences revealed the existence of multiple resistance genes among the examined E. coli isolates. Of the six CTX-M-producing isolates from retail food, blaCTX-M-14 was the prevalent variant identified (83.3%, 5/6). Two plasmid-mediated fosfomycin resistance genes, fosA3, and fosA4, were detected from retail food isolates (one each from chicken and beef), where fosA4 was identified in the chicken isolate 82CH that also carried the colistin resistance gene mcr-1. The blaCTX-M-14 and fosA genes in retail food isolates were located adjacent to insertion sequences ISEcp1 and IS26, respectively. Sequence analysis of the reconstructed mcr-1 plasmid (p82CH) showed 96-97% identity to mcr-1-carrying IncI2 plasmids previously identified in human and food E. coli isolates from Egypt. Hierarchical clustering of core genome MLST (HierCC) revealed clustering of chicken isolate 82CH, co-harboring mcr-1 and fosA4 genes, with a chicken E. coli isolate from China at the HC200 level (≤200 core genome allelic differences). As E. coli co-harboring mcr-1 and fosA4 genes has only been recently reported, this study shows rapid spread of this genotype that shares similar genetic structures with regional and international E. coli lineages originating from both humans and food animals. Adopting WGS-based surveillance system is warranted to facilitate monitoring the international spread of MDR pathogens.

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.

Evaluation of a new chromogenic agar for the detection of environmental Enterococcus.

CHROMagar Enterococcus (CHR), a new chromogenic medium not yet available for commercial purchase, was evaluated for the isolation of Enterococcus from environmental water samples. Its performance was evaluated in comparison to commercially available media, Enterococcosel agar and m-Enterococcus agar. Three consecutive tests were conducted with each test being performed with a newer batch of the CHR medium with improved media composition per batch. The recovery rate, positive predictive value, and sensitivity of the CHR medium improved with the subsequent re-formulation of the media components from 93.9%, 63%, and 92.6%, respectively, with the first batch of CHR, to 96.2%, 97.4%, and 95.7%, respectively, with the newest batch of CHR. The results showed that the newer batches of CHR performed better than the previous versions and are comparable to the other two commercial media tested. The CHR medium has been developed to decrease the turnaround time to approximately 18 h and be read more easily due to bigger colony morphology. The superior growth of colonies on CHR compared with other media in a shorter period of time can aid in the early detection of enterococci and may offer a user-friendly alternative to other media for the isolation of enterococci.

Staphylococci in poultry intestines: a comparison between farmed and household chickens.

Both pathogenic as well as nonpathogenic species of staphylococci have been reported in poultry, but these studies have not compared staphylococcal flora of both farmed and household broiler chickens. Staphylococci from farmed (n = 51) and household chicken intestines (n = 43) were isolated and tested for resistance to antimicrobials, presence of resistance genes, and inhibitory activity against other bacteria; correlation of resistance phenotype and genotype was also evaluated. At least 12 staphylococcal species were identified; Staphylococcus carnosus subspecies carnosus was the predominant species from both sources. Most farmed chicken staphylococci were resistant to tigecycline (38/51; 74.8%) while the highest level of resistance among the household chicken staphylococci was to clindamycin (31/43; 72.1%). The mecA gene was only detected in staphylococci from household chickens, whereas ermC and tetK or tetM were found in staphylococci from both groups of birds. Multidrug resistance (resistance ≥ 2 antimicrobial classes) was observed in 88% of resistant staphylococci ranging from 2 to 8 classes and up to 10 antimicrobials. Isolates produced inhibitory activity against 7 clinical bacterial strains primarily Enterococcus faecalis (25/88; 28.4%) and Escherichia coli (22/88; 25%). This study demonstrated that the staphylococcal population among farmed and household chickens varies by species and resistance to antimicrobials. These results may reflect the influence of the environment or habitat of each bird type on the intestinal microflora. As resistance in the staphylococci to antimicrobials used to treat human infections was detected, further study is warranted to determine strategies to prevent transfer of these resistant populations to humans via contamination of the poultry meat.

Antimicrobial Resistance, Genetic Diversity and Multilocus Sequence Typing of Escherichia coli from Humans, Retail Chicken and Ground Beef in Egypt.

Contamination of retail foods with foodborne pathogens, particularly the antimicrobial resistant ones, poses a persistent threat to human health. There is a dearth of information about the overlapping Escherichia coli (E. coli) lineages circulating among retail foods and humans in Egypt. This study aimed to determine the clonal diversity of 120 E. coli isolates from diarrheic patients (n = 32), retail chicken carcasses (n = 61) and ground beef (n = 27) from Mansoura, Egypt using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Simpson's index of diversity was calculated to compare the results of both typing methods. Antimicrobial resistance phenotypes, genotypes and phylogrouping of the isolates were also determined. Higher frequencies of antimicrobial resistance were found among chicken isolates compared to beef and human isolates; regardless of isolate source, the predominant antimicrobial resistances were found against ampicillin (87/120, 72.5%), tetracycline and sulfisoxazole (82/120, 68.3%, each), and streptomycin (79/120, 65.8%). None of the isolates displayed resistance to meropenem. The prevalent genes detected were tetA (64.2%), blaTEM (62.5%), sul1 (56.7%), floR (53.3%), sul2 (50%), strB (48.3%) and strA (47.5%) corresponding with resistance phenotypes. Alarmingly, blaCTX was detected in 63.9% (39/61) of chicken isolates. The majority of E. coli isolates from humans (90.6%), beef (81.5%) and chicken (70.5%) belonged to commensal phylogroups (A, B1, C). Using PFGE analysis, 16 out of 24 clusters (66.7%) contained isolates from different sources at a similarity level ≥75%. MLST results assigned E. coli isolates into 25, 19 and 13 sequence types (STs) from chicken, human and beef isolates, respectively. Six shared STs were identified including ST1011, ST156, ST48, ST224 (chicken and beef), ST10 (human and chicken) and ST226 (human and beef). Simpson's index of diversity was higher for MLST (0.98) than PFGE (0.94). In conclusion, the existence of common genetic determinants among isolates from retail foods and humans in Egypt as well as the circulation of shared STs indicates a possible epidemiological link with potential zoonotic hazards.

Comparison of Antimicrobial Resistance and Pan-Genome of Clinical and Non-Clinical Enterococcus cecorum from Poultry Using Whole-Genome Sequencing.

Enterococcus cecorum is an emerging avian pathogen, particularly in chickens, but can be found in both diseased (clinical) and healthy (non-clinical) poultry. To better define differences between E. cecorum from the two groups, whole-genome sequencing (WGS) was used to identify and compare antimicrobial resistance genes as well as the pan-genome among the isolates. Eighteen strains selected from our previous study were subjected to WGS using Illumina MiSeq and comparatively analyzed. Assembled contigs were analyzed for resistance genes using ARG-ANNOT. Resistance to erythromycin was mediated by ermB, ermG, and mefA, in clinical isolates and ermB and mefA, in non-clinical isolates. Lincomycin resistance genes were identified as linB, lnuB, lnuC, and lnuD with lnuD found only in non-clinical E. cecorum; however, lnuB and linB were found in only one clinical isolate. For both groups of isolates, kanamycin resistance was mediated by aph3-III, while tetracycline resistance was conferred by tetM, tetO, and tetL. No mutations or known resistance genes were found for isolates resistant to either linezolid or chloramphenicol, suggesting possible new mechanisms of resistance to these drugs. A comparison of WGS results confirmed that non-clinical isolates contained more resistance genes than clinical isolates. The pan-genome of clinical and non-clinical isolates resulted in 3651 and 4950 gene families, respectively, whereas the core gene sets were comprised of 1559 and 1534 gene families in clinical and non-clinical isolates, respectively. Unique genes were found more frequently in non-clinical isolates than clinical. Phylogenetic analysis of the isolates and all the available complete and draft genomes showed no correlation between healthy and diseased poultry. Additional genomic comparison is required to elucidate genetic factors in E. cecorum that contribute to disease in poultry.

Draft Genomic Sequences of Three Escherichia coli Sequence Type 131 Isolates (H45, H43ii, and H43iii) from Patients in Lagos, Nigeria.

Escherichia coli sequence type 131 (ST131) has recently emerged as a leading multidrug-resistant pathogen that causes urinary tract and bloodstream infections in humans. Here, we report the draft genomic sequences of three E. coli ST131 isolates, H45, H43ii, and H43iii, from urine samples of patients in Lagos, Nigeria.

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.

Circulation of emerging NDM-5-producing Escherichia coli among humans and dogs in Egypt.

The emergence of NDM-producing Escherichia coli has considerably threatened human and animal health worldwide. This study describes for the first time in Egypt, the draft genome sequences of emerging NDM-5-producing E. coli from humans and dogs, and investigates genetic relatedness between isolates from both sources. Two E. coli from human urine and seven from environmental clinical samples of dogs exhibited resistance to carbapenems and harbouring blaNDM were subjected to Illumina Miseq whole-genome sequencing (WGS). Assembly and analysis of the reads were performed to identify resistance genes, multilocus sequence types (MLST), plasmid replicon types (Inc) and insertion sequences (IS) of the blaNDM region; core genome MLST (cgMLST) analysis was also performed. Two different NDM alleles were identified; blaNDM-5 in E. coli HR119 from the urine of a healthy person and environmental samples of dogs, and blaNDM-1 in E. coli HR135 from a human patient's urine. Multiple mobilizable resistance genes to different antimicrobial classes were identified except the colistin resistance gene, mcr. E. coli isolates from humans and dogs were assigned to different sequence types (STs). Using cgMLST, dog isolates clustered together with only 1-2 allellic differences; however, human E. coli showed 1,978 different allelles compared with dog isolates. Plasmidfinder results indicated the presence of an IncX3 replicon in blaNDM-5 -producing E. coli; however, blaNDM-1 was linked to IncCoIKP3. Notably, the NDM region (3 Kb) in all isolates from humans and dogs was highly similar with variable flanking sequences that represented different IS elements. This study reports the first emergence of NDM-5-producing E. coli from dogs in Egypt that shared some genetic features with human isolates and could be considered potential public health threats.

Genetic Characterization of Antimicrobial-Resistant Escherichia coli Isolated from a Mixed-Use Watershed in Northeast Georgia, USA.

In order to determine the role of surface water in the development and spread of antibiotic-resistant (AR) bacteria, water samples were collected quarterly from 2015 to 2016 from a mixed-use watershed in Georgia. In our previous study, 496 Escherichia coli were isolated from surface water, out of which, 34 isolates were resistant to antimicrobials. For the current study, these 34 AR E. coli were characterized using pulsed-field gel electrophoresis, AR gene detection, plasmid replicon typing, class I integron detection, and multi-locus sequence typing. Genes were identified as conferring resistance to azithromycin (mph(A)); ß-lactams (blaCMY, blaCTX, blaTEM); chloramphenicol (floR); streptomycin (strA, strB); sulfisoxazole (sul1, sul2); tetracycline (tetA, tetB, tetC); and trimethoprim/sulfamethoxazole (dhfr5, dhfr12). Five ciprofloxacin- and/or nalidixic-resistant isolates contained point mutations in gyrA and/or parC. Most of the isolates (n = 28) carried plasmids and three were positive for class I integrons. Twenty-nine sequence types (ST) were detected, including three epidemic urinary-tract-infection-associated ST131 isolates. One of the ST131 E. coli isolates exhibited an extended-spectrum ß-lactamase (ESBL) phenotype and carried blaCTX-M-15 and blaTEM-1. To our knowledge, this is the first study on the emergence of an ESBL-producing E. coli ST131 from environmental water in the USA, which poses a potential risk to human health through the recreational, agricultural, or municipal use of this natural resource. This study identified E. coli with AR mechanisms to commonly used antimicrobials and carrying mobile genetic elements, which could transfer AR genes to other bacteria in the aquatic environment.

Plasmid Replicons and ß-Lactamase-Encoding Genes of Multidrug-Resistant Escherichia coli Isolated from Humans and Food Animals in Lagos, Southwest Nigeria.

As resistance to the ß-lactam class of antibiotics has become a worldwide problem, multidrug-resistant (MDR) human (n = 243) and food animal (n = 211) isolates from Lagos, Nigeria were further tested to characterize ß-lactamase-encoding genes and plasmid replicons. Four ß-lactamase-encoding genes (blaCMY, blaCTX-M, blaOXA, and blaTEM) were detected using PCR-based replicon typing, 13 and 17 different replicons were identified using a subset of MDR E. coli from humans (n = 48) and animals (n = 96), respectively. Replicon types FIB and X2 were detected in equal numbers (2/48; 4.2% each) from human isolates, while type Y (16/96; 16.7%) was the most common type from animals. Only two replicon types, FIB and Y, were detected in both groups; all other types were confined to one group or the other, but not both. Using conjugation, replicon type Y, present in three donors, transferred in all three instances, whereas FIA transferred in 75% (3/4) of the matings. This study showed that ß-lactamase genes were prevalent in MDR E. coli from both humans and animals in Nigeria and also contained diverse plasmid replicons. As the replicon-associated genes were mobile, they are likely to continue disseminating among E. coli and facilitating transfer of associated ß-lactamase genes in this region.

Antimicrobial Resistance Genes, Cassettes, and Plasmids Present in Salmonella enterica Associated With United States Food Animals.

The ability of antimicrobial resistance (AR) to transfer, on mobile genetic elements (MGEs) between bacteria, can cause the rapid establishment of multidrug resistance (MDR) in bacteria from animals, thus creating a foodborne risk to human health. To investigate MDR and its association with plasmids in Salmonella enterica, whole genome sequence (WGS) analysis was performed on 193 S. enterica isolated from sources associated with United States food animals between 1998 and 2011; 119 were resistant to at least one antibiotic tested. Isolates represented 86 serotypes and variants, as well as diverse phenotypic resistance profiles. A total of 923 AR genes and 212 plasmids were identified among the 193 strains. Every isolate contained at least one AR gene. At least one plasmid was detected in 157 isolates. Genes were identified for resistance to aminoglycosides (n = 472), ß-lactams (n = 84), tetracyclines (n = 171), sulfonamides (n = 91), phenicols (n = 42), trimethoprim (n = 8), macrolides (n = 5), fosfomycin (n = 48), and rifampicin (n = 2). Plasmid replicon types detected in the isolates were A/C (n = 32), ColE (n = 76), F (n = 43), HI1 (n = 4), HI2 (n = 20), I1 (n = 62), N (n = 4), Q (n = 7), and X (n = 35). Phenotypic resistance correlated with the AR genes identified in 95.4% of cases. Most AR genes were located on plasmids, with many plasmids harboring multiple AR genes. Six antibiotic resistance cassette structures (ARCs) and one pseudo-cassette were identified. ARCs contained between one and five resistance genes (ARC1: sul2, strAB, tetAR; ARC2: aac3-iid; ARC3: aph, sph; ARC4: cmy-2; ARC5: floR; ARC6: tetB; pseudo-ARC: aadA, aac3-VIa, sul1). These ARCs were present in multiple isolates and on plasmids of multiple replicon types. To determine the current distribution and frequency of these ARCs, the public NCBI database was analyzed, including WGS data on isolates collected by the USDA Food Safety and Inspection Service (FSIS) from 2014 to 2018. ARC1, ARC4, and ARC5 were significantly associated with cattle isolates, while ARC6 was significantly associated with chicken isolates. This study revealed that a diverse group of plasmids, carrying AR genes, are responsible for the phenotypic resistance seen in Salmonella isolated from United States food animals. It was also determined that many plasmids carry similar ARCs.

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.

Draft genome sequence of a human-associated streptogramin-resistant Staphylococcus aureus.

OBJECTIVES: Staphylococcus aureus is one of the leading causes of nosocomial and community-acquired infections. Treatment of these infections with macrolide-lincosamide-streptogramin (MLS) antibiotics has led to resistance to these antibiotics via various mechanisms. S. aureus strain CIP108540, isolated from a human in France, has been previously shown to exhibit resistance to the streptogramins quinupristin and dalfopristin; the presence of streptogramin resistance genes was verified by PCR. However, the extent of MLS resistance genes in this strain is unknown. This study analysed the genome sequence of S. aureus CIP108540 to assess genes associated with antimicrobial resistance, including to streptogramins. METHODS: Genomic DNA of S. aureus CIP108540 was sequenced using Illumina MiSeq. The generated sequencing reads were de novo assembled using A5-miseq. RESULTS: The draft genome size was 3014273bp with a GC content of 32.72%. There were 3063 predicted coding sequences with 59 tRNAs. Several antimicrobial resistance genes were identified conferring resistance to various antibiotics. CONCLUSION: The draft genome sequence of S. aureus CIP108540 released here will provide valuable information for a better understanding of its genetic makeup and resistome.

An assay for determining the susceptibility of Salmonella isolates to commercial and household biocides.

Poultry and meat products contaminated with Salmonella enterica are a major cause of foodborne illness in the United States. The food industries use a wide variety of antimicrobial interventions to reduce bacterial contamination. However, little is known about Salmonella susceptibility to these compounds and some studies have shown a concerning link between biocide resistance and antibiotic resistance. To investigate this, a 96 well panel of 17 common household and commercially used biocides was designed to determine the minimum inhibitory concentrations (MIC) of these compounds for Salmonella. The panel contained two-fold serial dilutions of chemicals including Dodecyltrimethylammonium chloride (DC), Benzalkonium chloride (BKC), Cetylpyridinium chloride (CPC), Hexadecyltrimethylammonium bromide (HB), Hexadecyltrimethylammonium chloride (HC), Acetic acid (AA), Lactic acid (LA), Citric acid (CA), Peroxyacetic acid (PXA), Acidified sodium chlorite (ASC), Sodium hypochlorite (SHB), 1,3 dibromo, 5,5 dimethylhydantoin (DBH), Chlorhexidine (CHX), Sodium metasilicate (SM), Trisodium phosphate (TSP), Arsenite (ARI), and Arsenate (ARA). The assay was used to test the susceptibility of 88 multidrug resistant (MDR) Salmonella isolates from animal sources. Bacteria are defined as multidrug resistant (MDR) if it exhibited non-susceptibility to at least one agent in three or more antimicrobial categories. The concentration of biocide at which ≥50% of the isolates could not grow was designated as the minimum inhibitory concentration or MIC50 and was used as the breakpoint in this study. The MIC50 (µg ml-1) for the tested MDR Salmonella was 256 for DC, 40 for BKC, 80 for CPC. HB and HC, 1,640 for AA, 5664 for LA, 3,156 for CA, 880 for PXA, 320 for ASC, 3.0 for CHX, 1,248 for DBH, 3,152 (6%) for SHB, 60,320 for SM, 37,712 for TSP, 56 for ARI and 832 for ARA. A few isolates were not susceptible at the MIC50 breakpoint to some chemicals indicating possible resistance. Isolates with MICs of two 2-fold dilutions above the MIC50 were considered resistant. Biocides for which resistant isolates were detected included CPC (n = 1 isolate), HB (1), CA (18), ASC (7), CHX (22), ARA (16), and ARI (4). There was no correlation detected between the biocide susceptibility of Salmonella isolates and antibiotic resistance. This assay can determine the MICs of bacteria to 17 biocides in a single test and will be useful in evaluating the efficacy of biocides and to detect the development of resistance to them.

Contribution of Healthy Chickens to Antimicrobial-Resistant Escherichia coli Associated with Human Extraintestinal Infections in Egypt.

BACKGROUND: Chickens are considered potential reservoirs for human extraintestinal infections with pathogenic Escherichia coli. However, information about genetic relatedness between E. coli from healthy chickens and human patients is still limited. METHODS AND RESULTS: In this study, clinical samples from patients with extraintestinal infections and healthy broiler chickens were collected from geographically related locations in Egypt during the 2nd half of 2015. The recovered isolates were tested for susceptibility against ß-lactam antimicrobials and screened for the presence of extended-spectrum ß-lactamases (ESBLs) and virulence genes; clonal and phylotypes were also determined. Forty-eight percent (48/100) and 31.3% (50/160) of human and chicken samples were positive for E. coli, respectively. Although only 4% (2/50) of the chicken isolates were resistant to the tested ß-lactams, over 58% of human E. coli isolates (28/48) exhibited resistance to cefotaxime. For ß-lactamases, 52.1%, 33.3%, 20.8%, and 6.25% of human E. coli were positive for blaCTX-M, blaTEM, blaOXA, and blaCMY, while blaTEM, blaOXA, and blaCMY were found in 32%, 4%, and 34% of chicken isolates, respectively. Low frequencies of virulence genes within human and chicken E. coli isolates were detected by PCR. The majority of E. coli isolates harboring ß-lactam resistance genes from human and chicken sources belonged to phylogroup C and B1, respectively. Using pulsed-field gel electrophoresis (PFGE), some E. coli grouped based upon source; however, most clusters contained isolates from both humans and chickens. CONCLUSIONS: The above findings suggest that although no single clone appeared to be circulating among E. coli isolates from human and chicken, some shared characteristics exist among isolates from both sources. Increased study will aid to track the dissemination of ß-lactam-resistant E. coli from healthy chickens to humans for implementation of effective intervention strategies.

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.