Associations between Isolation Source, Clonal Composition, and Antibiotic Resistance Genes in Escherichia coli Collected in Washington State, USA.

Antimicrobial resistance (AMR) is a global health problem stemming from the use of antibiotics in humans, animals, and the environment. This study used whole-genome sequencing (WGS) of E. coli to explore patterns of AMR across sectors in Washington State, USA (WA). The WGS data from 1449 E. coli isolates were evaluated for isolation source (humans, animals, food, or the environment) and the presence of antibiotic resistance genes (ARGs). We performed sequence typing using PubMLST and used ResFinder to identify ARGs. We categorized isolates as being pan-susceptible, resistant, or multidrug-resistant (MDR), defined as carrying resistance genes for at least three or more antimicrobial drug classes. In total, 60% of isolates were pan-susceptible, while 18% were resistant, and 22% exhibited MDR. The proportion of resistant isolates varied significantly according to the source of the isolates (p < 0.001). The greatest resistance was detected in isolates from humans and then animals, while environmental isolates showed the least resistance. This study demonstrates the feasibility of comparing AMR across various sectors in Washington using WGS and a One Health approach. Such analysis can complement other efforts for AMR surveillance and potentially lead to targeted interventions and monitoring activities to reduce the overall burden of AMR.

Study of heavy metal resistance genes in Escherichia coli isolates from a marine ecosystem with a history of environmental pollution (arsenic, cadmium, copper, and mercury).

We analyzed whole genome sequences of 308 Escherichia coli isolates from a marine ecosystem to determine the prevalence and relationships of heavy metal resistance genes (HMRGs) and antibiotic resistance genes (ARGs), as well as the presence of plasmid sequences. We screened all genomes for presence of 18 functional HMRGs conferring resistance to arsenic, cadmium, copper, or cadmium/mercury. In subset analyses, we examined geographic variations of HMRG carriage patterns in 224 isolates from water sources, and sought genetic linkages between HMRGs and ARGs in 25 genomes of isolates resistant to antibiotics. We found high carriage rates of HMRGs in all genomes, with 100% carrying at least one copy of 11 out of 18 HMRGs. A total of 173 (56%) of the isolates carried both HMRGs and plasmid sequences. In the 25 genomes of antibiotic-resistant isolates, 80% (n = 20) carried HMRGs, ARGs, and plasmid sequences, while 40% (n = 10) had linked HMRGs and ARGs on their assembled genomes. We found no evidence of geographic variation in HMRG frequency, nor any association between locational proximity to Superfund sites and co-carriage of HMRGs and ARGs. Our study findings indicate that HMRGs are common among E. coli in marine ecosystems, suggesting widespread heavy metal presence in water sources of a region with history of environmental pollution. Further research is needed to determine the role HMRGs play in driving antimicrobial resistance in human pathogens through genetic linkage and the value their detection in environmental bacterial genomes may offer as an indicator of environmental heavy metal pollution.

Differences in gut metagenomes between dairy workers and community controls: a cross-sectional study.

Background: As a nexus of routine antibiotic use and zoonotic pathogen presence, the livestock farming environment is a potential hotspot for the emergence of zoonotic diseases and antibiotic resistant bacteria. Livestock can further facilitate disease transmission by serving as intermediary hosts for pathogens as they undergo evolution prior to a spillover event. In light of this, we are interested in characterizing the microbiome and resistome of dairy workers, whose exposure to the livestock farming environment places them at risk for facilitating community transmission of antibiotic resistant genes and emerging zoonotic diseases. Results: Using shotgun sequencing, we investigated differences in the taxonomy, diversity and gene presence of the human gut microbiome of 10 dairy farm workers and 6 community controls, supplementing these samples with additional publicly available gut metagenomes. We observed greater abundance of tetracycline resistance genes and prevalence of cephamycin resistance genes in dairy workers' metagenomes, and lower average gene diversity. We also found evidence of commensal organism association with plasmid-mediated tetracycline resistance genes in both dairy workers and community controls (including Faecalibacterium prausnitzii, Ligilactobacillus animalis, and Simiaoa sunii). However, we did not find significant differences in the prevalence of resistance genes or virulence factors overall, nor differences in the taxonomic composition of dairy worker and community control metagenomes. Conclusions: This study presents the first metagenomics analysis of United States dairy workers, providing insights into potential risks of exposure to antibiotics and pathogens in animal farming environments. Previous metagenomic studies of livestock workers in China and Europe have reported increased abundance and carriage of antibiotic resistance genes in livestock workers. While our investigation found no strong evidence for differences in the abundance or carriage of antibiotic resistance genes and virulence factors between dairy worker and community control gut metagenomes, we did observe patterns in the abundance of tetracycline resistance genes and the prevalence of cephamycin resistance genes that is consistent with previous work.

Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton-Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Application of Nucleotide-Based Kinetic Modeling Approaches to Predict Antibiotic Resistance Gene Degradation during UV- and Chlorine-Based Wastewater Disinfection Processes: From Bench- to Full-Scale.

This study investigated antibiotic resistance gene (ARG) degradation kinetics in wastewaters during bench- and full-scale treatment with UV light and chlorine─with the latter maintained as free available chlorine (FAC) in low-ammonia wastewater and converted into monochloramine (NH2Cl) in high-ammonia wastewater. Twenty-three 142-1509 bp segments (i.e., amplicons) of seven ARGs (blt, mecA, vanA, tet(A), ampC, blaNDM, blaKPC) and the 16S rRNA gene from antibiotic resistant bacteria (ARB) strains Bacillus subtilis, Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were monitored as disinfection targets by qPCR. Rate constants for ARG and 16S rRNA gene amplicon degradation by UV, FAC, and NH2Cl were measured in phosphate buffer and used to expand and validate several recently developed approaches to predict DNA segment degradation rate constants based solely on their nucleotide contents, which were then applied to model ARG degradation during bench-scale treatment in buffer and wastewater matrixes. Kinetics of extracellular and intracellular ARG degradation by UV and FAC were well predicted up to ∼1-2-log10 elimination, although with decreasing accuracy at higher levels for intracellular genes, while NH2Cl yielded minimal degradation under all conditions (agreeing with predictions). ARB inactivation kinetics varied substantially across strains, with intracellular gene degradation lagging cell inactivation in each case. ARG degradation levels observed during full-scale disinfection at two wastewater treatment facilities were consistent with bench-scale measurements and predictions, where UV provided ∼1-log10 ARG degradation, and chlorination of high-ammonia wastewater (dominated by NH2Cl) yielded minimal ARG degradation.

Investigation of methicillin-resistant Staphylococcus aureus, methicillin-susceptible Staphylococcus aureus, and Staphylococcus argenteus from wild long-tailed macaques (Macaca fascicularis) at Kosumpee Forest Park, Maha Sarakham, Thailand.

Background and Aim: In the past, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infections in both humans and animals has increased across Thailand. Staphylococcus argenteus has been associated with infections among humans, exotic pets, and livestock. Both species have been identified in non-human primate species from geographically diverse locations but not from non-human primates in Thailand. This study aimed to determine the presence of MRSA/methicillin-susceptible S. aureus (MSSA) and S. argenteus isolates collected from buccal swab samples in Macaca fascicularis at Kosumpee Forest Park (KFP), Maha Sarakham, Northeast Thailand. Materials and Methods: Aseptic buccal swab samples were collected from 30 free-ranging macaques in November 2018. All isolates were tested using multiple biochemical tests and S. aureus latex slide agglutination test. Presumptive S. aureus isolates were tested for the presence of the mecA gene using polymerase chain reaction (PCR) assays. The isolates were phenotypically determined to be resistant to a ß-lactam antibiotic using the disk diffusion method with a 30 mg cefoxitin disk. The isolates were analyzed by PCR for the non-ribosomal peptide synthetase (NRPS) gene to distinguish S. argenteus from S. aureus. Results: Fifteen macaques (50%) were colonized with S. aureus and 21 isolates were characterized. Three of the macaques carried both the MRSA and MSSA isolate. One animal carried both MRSA and S. argenteus isolate, and one animal carried only S. argenteus. The NRPS gene analysis confirmed that 2 isolates (9.52%) were S. argenteus and 19 isolates (90.48%) were S. aureus [five MSSA and 14 MRSA]. Conclusion: This study is the first to identify MRSA/MSSA and S. argenteus in wild free-ranging M. fascicularis from Thailand at the KFP in Maha Sarakham. This study is also the first report on the occurrence of S. argenteus carriage in M. fascicularis from Thailand.

Surveillance for Antibiotic-Resistant E. coli in the Salish Sea Ecosystem.

E. coli was isolated from the Salish Sea (Puget Sound) ecosystem, including samples of marine and fresh water, and wildlife dependent on this environment. E. coli isolates were assessed for phenotypic and genotypic resistance to antibiotics. A total of 305 E. coli isolates was characterized from samples collected from: marine water obtained in four quadrants of the Salish Sea; select locations near beaches; fresh water from streams near marine beaches; and fecal samples from harbor porpoises (Phocoena phocoena), harbor seals (Phoca vitulina), river otters (Lontra canadensis), and English sole (Parophrys vetulus). Isolates were evaluated using antimicrobial susceptibility typing, whole-genome sequencing, fumC, and multilocus sequence typing. Resistance and virulence genes were identified from sequence data. Of the 305 isolates from Salish Sea samples, 20 (6.6%) of the E. coli were intermediate, and 31 (10.2%) were resistant to ≥1 class of antibiotics, with 26.9% of nonsusceptible (resistant and intermediate resistant) E. coli isolates from marine mammals and 70% from river otters. The proportion of nonsusceptible isolates from animals was significantly higher than samples taken from marine water (p < 0.0001). A total of 196 unique STs was identified including 37 extraintestinal pathogenic E. coli (ExPEC)-associated STs [ST10, ST38, ST58, ST69, ST73, ST117, ST131, and ST405]. The study suggests that animals may be potential sentinels for antibiotic-resistant and ExPEC E. coli in the Salish Sea ecosystem.

Genetic Characterization of the Tetracycline-Resistance Gene tet(X) Carried by Two Epilithonimonas Strains Isolated from Farmed Diseased Rainbow Trout, Oncorhynchus mykiss in Chile.

The main objective of this study was to characterize the tet(X) genes, which encode a monooxygenase that catalyzes the degradation of tetracycline antibiotics, carried by the resistant strains FP105 and FP233-J200, using whole-genome sequencing analysis. The isolates were recovered from fin lesion and kidney samples of diseased rainbow trout Oncorhynchus mykiss, during two Flavobacteriosis outbreaks occurring in freshwater farms located in Southern Chile. The strains were identified as Epilithonimonas spp. by using biochemical tests and by genome comparison analysis using the PATRIC bioinformatics platform and exhibited a minimum inhibitory concentration (MIC) of oxytetracycline of 128 µg/mL. The tet(X) genes were located on small contigs of the FP105 and FP233-J200 genomes. The sequences obtained for the tet(X) genes and their genetic environment were compared with the genomes available in the GenBank database of strains of the Chryseobacterium clade belonging to the Flavobacterium family, isolated from fish and carrying the tet(X) gene. The Tet(X) proteins synthesized by the Chilean Epilithonimonas strains showed a high amino acid similarity (range from 84% to 100%), with the available sequences found in strains belonging to the genus Chryseobacterium and Flavobacterium isolated from fish. An identical neighborhood of tet(X) genes from both Chilean strains was observed. The genetic environment of tet(X) observed in the two strains of Epilithonimonas studied was characterized by the upstream location of a sequence encoding a hypothetical protein and a downstream located alpha/beta hydrolase-encoding gene, similar to the observed in some of the tet(X) genes carried by Chryseobacterium and Flavobacterium strains isolated from fish, but the produced proteins exhibited a low amino acid identity (25-27%) when compared to these synthesized by the Chilean strains. This study reports for the first time the carriage of the tet(X) gene by the Epilithonimonas genus and their detection in fish pathogenic bacteria isolated from farmed salmonids in Chile, thus limiting the use of therapies based on oxytetracycline, the antimicrobial most widely used in Chilean freshwater salmonid farming. This results suggest that pathogenic strains of the Chryseobacterium clade occurring in Chilean salmonid farms may serve as important reservoirs of tet(X) genes.

Environmental Surveillance and Characterization of Antibiotic Resistant Staphylococcus aureus at Coastal Beaches and Rivers on the Island of Hawai'i.

Staphylococcus aureus are human facultative pathogenic bacteria and can be found as contaminants in the environment. The aim of our study was to determine whether methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolated from coastal beach and river waters, anchialine pools, sand, and wastewater on the island of Hawai'i, Hawai'i, are a potential health risk. Samples were collected from three regions on Hawai'i Island from July to December 2020 during the COVID-19 pandemic and were characterized using whole-genome sequencing (WGS). From WGS data, multilocus sequence typing (MLST), SCCmec type, antimicrobial resistance genes, virulence factors, and plasmids were identified. Of the 361 samples, 98.1% were positive for Staphylococcus spp. and 7.2% were S. aureus positive (n = 26); nine MRSA and 27 MSSA strains were characterized; multiple isolates were chosen from the same sample in two sand and seven coastal beach water samples. The nine MRSA isolates were multi-drug resistant (6-9 genes) sequence type (ST) 8, clonal complex (CC) 8, SCCmec type IVa (USA300 clone), and were clonally related (0-16 SNP differences), and carried 16-19 virulence factors. The 27 MSSA isolates were grouped into eight CCs and 12 STs. Seventy-eight percent of the MSSA isolates carried 1-5 different antibiotic resistance genes and carried 5-19 virulence factors. We found S. aureus in coastal beach and river waters, anchialine pools, and sand at locations with limited human activity on the island of Hawai'i. This may be a public health hazard.

Methicillin-Resistant and Methicillin-Susceptible Staphylococcus from Vervet Monkeys (Chlorocebus sabaeus) in Saint Kitts.

Antimicrobial resistance has been described in all ecosystems, including wildlife. Here we investigated the presence of methicillin-resistant and susceptible staphylococci in both colony-born and wild vervet monkeys (Chlorocebus sabaeus). Through selective isolation, PCR, MALDI-TOF, and whole-genome sequencing, methicillin-resistant and susceptible Staphylococcus spp. isolated from vervet monkeys were characterized. We obtained putatively methicillin-resistant staphylococci from 29 of the 34 nasal samples collected. Strains were identified by MALDI-TOF analysis. Staphylococcus cohnii (n = 15) was the most commonly isolated species, while nine other species were isolated one or two times. PCR analysis indicated that eight [28%] strains were mecA positive. The whole-genome sequencing [WGS] included eight methicillin-resistant strains (S. epidermidis (n = 2), S. cohnii (n = 3), S. arlettae (n = 2) and S. hominis (n = 1)), nine additional S. cohnii strains and two strains that could not be identified by MALDI-TOF, but genetically characterized as one S. cohnii and one S. warneri. Different resistance genes carried by different mobile genetic elements, mainly blaZ (n = 10) and tet(K) (n = 5) were found, while msr(A), cat, fosB, dfrG, erm(C), mph(C) and str were identified in one to three strains. Phylogenetic analysis of the S. cohnii strains based on SNPs indicated four clusters associated with colony born or wild. In addition, one singleton S. cohnii isolated did not form a separate group and clustered within other S. cohnii strains submitted to the NCBI. In this study, we demonstrated the presence of AMR and mobile genetic elements to both colony-born and wild vervet monkeys. We also identified a previously undescribed prevalence of S. cohnii in the nasal flora of these monkeys, which merits further investigation.

Staphylococcus aureus and Methicillin Resistant S. aureus in Nepalese Primates: Resistance to Antimicrobials, Virulence, and Genetic Lineages.

Staphylococcus aureus is a ubiquitous pathogen and colonizer in humans and animals. There are few studies on the molecular epidemiology of S. aureus in wild monkeys and apes. S. aureus carriage in rhesus macaques (Macaca mulatta) and Assam macaques (Macaca assamensis) is a species that has not previously been sampled and lives in remote environments with limited human contact. Forty Staphylococcus aureus isolates including 33 methicillin-susceptible S. aureus (MSSA) and seven methicillin-resistant S. aureus (MRSA) were characterized. Thirty-four isolates were from rhesus macaques and six isolates (five MSSA, one MRSA) were from Assam macaques. Isolates were characterized using StaphyType DNA microarrays. Five of the MRSA including one from Assam macaque were CC22 MRSA-IV (PVL+/tst+), which is a strain previously identified in Nepalese rhesus. One MRSA each were CC6 MRSA-IV and CC772 MRSA-V (PVL+). One MSSA each belonged to CC15, CC96, and CC2990. Six MRSA isolates carried the blaZ, while ten known CC isolates (seven MRSA, three MSSA) carried a variety of genes including aacA-aphD, aphA3, erm(C), mph(C), dfrA, msrA, and/or sat genes. The other 30 MSSA isolates belonged to 17 novel clonal complexes, carried no antibiotic resistance genes, lacked Panton-Valentine Leukocidin (PVL), and most examined exotoxin genes. Four clonal complexes carried egc enterotoxin genes, and four harbored edinB, which is an exfoliative toxin homologue.

ResFinder 4.0 for predictions of phenotypes from genotypes.

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

MRSA Strains in Nepalese Rhesus Macaques (Macaca mulatta) and Their Environment.

This study looked at 227 saliva samples from Rhesus macaques (Macaca mulatta) and 218 samples from the surrounding environments. From these samples, MRSA isolates were collected from Rhesus saliva samples (n = 13) and environmental samples (n = 19) near temple areas in Kathmandu, Nepal. For comparison, selected MRSA isolates (n = 5) were obtained from patients with wound infections from a Kathmandu hospital. All isolates were characterized using Abbott StaphyType® DNA microarrays. Eighteen isolates (62%) from monkeys (n = 4; 31%) and environmental samples (n = 14; 74%), were CC22-MRSA-IV. Most (n = 16) of them carried both, the PVL locus and toxic shock toxin gene (tst1), an unusual combination which is the same as in previously characterized strain from Nepalese macaques and pigs. The five human isolates also belonged to that strain type. Eight monkey MRSA isolates were CC361-MRSA-IV. One MRSA from a monkey and one from an environmental sample, were CC88-MRSA-V. Other environmental MRSA included one each, CC121-MRSA-VT, and CC772 -MRSA-V. Two were CC779-MRSA-VT, potentially a novel clone. All MRSA carried the blaZ gene. The aacA-aphD, dfrA, and erm (C) genes were very common in isolates from all sources. One macaque MRSA carried the resistance genes aphA3 and sat, neither previously identified in primate MRSA isolates. This current study suggests that humans could be a potential source of the MRSA in the macaques/environment and transmission may be linked to humans feeding the primates and/or living in close proximity to each other.

Whole-genome analysis of extraintestinal pathogenic Escherichia coli (ExPEC) MDR ST73 and ST127 isolated from endangered southern resident killer whales (Orcinus orca).

BACKGROUND: Limited studies have investigated the microbial diversity of wild marine mammals. OBJECTIVES: This study characterized Escherichia coli isolates collected from fresh faecal samples of endangered southern resident killer whales (Orcinus orca) located by detection dogs. METHODS: WGS of each strain was done to determine ST (using MLST), clonotype (C:H), antimicrobial resistance and virulence profile. Conjugation experiments were done to determine the mobility of the tet(B) tetracycline resistance gene. RESULTS: All isolates belonged to extraintestinal pathogenic E. coli (ExPEC) clonal lineages ST73 (8/9) and ST127 (1/9), often associated with human community-acquired urinary tract disease. Clonotyping using fumC and fimH alleles showed divergence in clonal lineages, with ST73 isolates belonging to the C24:H10 clade and the ST127 isolate belonging to C14:H2. The eight ST73 isolates carried multiple acquired antibiotic resistance genes, including aadA1, sul1 and tet(B), encoding aminoglycoside, sulphonamide and tetracycline resistance, respectively. Conjugative transfer of the resistance gene tet(B) was observed for three of the eight isolates. ST127 did not carry any of these acquired resistance genes. Virulence-associated genes identified included those encoding adhesins (iha, papC, sfaS), toxins (sat, vat, pic, hlyA, cnf1), siderophores (iutA, fyuA, iroN, ireA), serum survival/protectins (iss, ompT), capsule (kpsM) and pathogenicity island marker (malX). CONCLUSIONS: Orca whales can carry antibiotic-resistant potentially pathogenic strains of E. coli. Possible sources include contamination of the whale's environment and/or food. It is unknown whether these isolates cause disease in southern resident killer whales, which could contribute to the ongoing decline of this critically endangered population.

Molecular Analysis of Two Different MRSA Clones ST188 and ST3268 From Primates (Macaca spp.) in a United States Primate Center.

Methicillin-resistant Staphylococcus aureus (MRSA) were identified in macaques, their environmental facility, and nasal cultures of personnel from the Washington National Primate Research Center [WaNPRC] and included MRSA ST188 SCCmec IV and MRSA ST3268 SCCmec V. The aim of the current study was to determine the carriage of virulence genes, antibiotic resistance genes, and other characteristics of the primate MRSA isolates to determine if there were any obvious differences that would account for differences in transmission within the WaNPRC facility. In total, 1,199 samples from primates were tested for the presence of MRSA resulting in 158 MRSA-positive samples. Fifteen ST188 isolates (all from Macaca nemestrina) and nine ST3268 (four from Macaca mulatta, two from Macaca fascicularis, three from M. nemestrina), were selected for further characterization. All but one of the 15 ST188 isolates had spa type t189 and the remaining one had spa type t3887. These isolates were resistant to ß-lactams [blaZ, mecA], macrolides/lincosamides [erm(B)], aminoglycosides [aacA-aphD], and fluoroquinolones. Five isolates were additionally resistant to tetracyclines [tet(K)] and had elevated MICs for benzalkonium chloride [qacC]. In comparison, the nine ST3268 isolates had the related spa types t15469 (n = 5) and t13638 (n = 4). All nine ST3268 isolates were resistant to ß-lactams [blaZ, mecA], and tetracyclines [tet(K)]. Some isolates were additionally resistant to aminoglycosides [aacA-aphD], fluoroquinolones and/or showed elevated MICs for benzalkonium chloride [qacC]. In contrast to the ST188 isolates, the ST3268 isolates had the enterotoxin gene cluster egc [seg, sei, selm, seln, selo, selu] and enterotoxin genes sec and sel. The two clones have differences regarding their spa types, virulence and antibiotic resistance genes as well as ST and SCCmec types. However, the data presented does not provide insight into why ST188 spreads easily while ST3268 did not spread within the WaNPRC in-house primates.

Distribution of Staphylococcus species in dairy cows, workers and shared farm environments.

Dairy farming involves frequent contact among animals, workers and farm environments. To explore the Staphylococcus spp. diversity that occurs on dairy farms, a pilot study sampled dairy workers, cows and the farm environments from five farms, two organic and three conventional farms, in Washington State. Samples were taken from the nares and hands of consenting workers (n = 24), udders and nares of selected cows (n = 25) and representative environmental surfaces (n = 96) from each farm. To increase diversity of the Staphylococcus spp. characterized, five distinct colonies were selected from each sample for identification with 16S analysis. A total of 198 staphylococci were characterized representing 19 different Staphylococcus spp. The diversity of species ranged from 9-15 Staphylococcus spp./farm with no difference between conventional and organic farms. S. haemolyticus [n = 60 isolates] was the most common species and was isolated from all farms and from cows, humans and environmental samples. Whole genome sequencing of selected S. haemolyticus found no genetically related isolates among human, animal and environmental samples within the same farm. S. epidermidis, S. saprophyticus, S. sciuri and S. xylosus were also found in ≥1 farms from human, animal and environmental samples.

The human clone ST22 SCCmec IV methicillin-resistant Staphylococcus aureus isolated from swine herds and wild primates in Nepal: is man the common source?

Swine nasal samples [n = 282] were collected from 12 randomly selected farms around Kathmandu, Nepal, from healthy animals. In addition, wild monkey (Macaca mulatta) saliva samples [n = 59] were collected near temples areas in Kathmandu using a non-invasive sampling technique. All samples were processed for MRSA using standardized selective media and conventional biochemical tests. MRSA verification was done and isolates characterized by SCCmec, multilocus sequence typing, whole genome sequencing [WGS] and antibiotic susceptibilities. Six (2.1%) swine MRSA were isolated from five of the different swine herds tested, five were ST22 type IV and one ST88 type V. Four (6.8%) macaques MRSA were isolated, with three ST22 SCCmec type IV and one ST239 type III. WGS sequencing showed that the eight ciprofloxacin resistant ST22 isolates carried gyrA mutation [S84L]. Six isolates carried the erm(C) genes, five isolates carried aacC-aphD genes and four isolates carried blaZ genes. The swine linezolid resistant ST22 did not carry any known acquired linezolid resistance genes but had a mutation in ribosomal protein L22 [A29V] and an insertion in L4 [68KG69], both previously associated with linezolid resistance. Multiple virulence factors were also identified. This is the first time MRSA ST22 SCCmec IV has been isolated from livestock or primates.

Mechanisms of Bacterial Resistance to Antimicrobial Agents.

During the past decades resistance to virtually all antimicrobial agents has been observed in bacteria of animal origin. This chapter describes in detail the mechanisms so far encountered for the various classes of antimicrobial agents. The main mechanisms include enzymatic inactivation by either disintegration or chemical modification of antimicrobial agents, reduced intracellular accumulation by either decreased influx or increased efflux of antimicrobial agents, and modifications at the cellular target sites (i.e., mutational changes, chemical modification, protection, or even replacement of the target sites). Often several mechanisms interact to enhance bacterial resistance to antimicrobial agents. This is a completely revised version of the corresponding chapter in the book Antimicrobial Resistance in Bacteria of Animal Origin published in 2006. New sections have been added for oxazolidinones, polypeptides, mupirocin, ansamycins, fosfomycin, fusidic acid, and streptomycins, and the chapters for the remaining classes of antimicrobial agents have been completely updated to cover the advances in knowledge gained since 2006.

Assessment of Environmental Contamination with Pathogenic Bacteria at a Hospital Laundry Facility.

Little is known about exposure to pathogenic bacteria among industrial laundry workers who work with soiled clinical linen. To study worker exposures, an assessment of surface contamination was performed at an industrial laundry facility serving hospitals in Seattle, WA, USA. Surface swab samples (n = 240) from the environment were collected during four site visits at 3-month intervals. These samples were cultured for Clostridium difficile, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE). Voluntary participation of 23 employees consisted of nasal swabs for detection of MRSA, observations during work, and questionnaires. Contamination with all three pathogens was observed in both dirty (laundry handling prior to washing) and clean areas (subsequent to washing). The dirty area had higher odds of overall contamination (≥1 pathogen) than the clean area (odds ratio, OR = 18.0, 95% confidence interval 8.9-36.5, P < 0.001). The odds of contamination were high for each individual pathogen: C. difficile, OR = 15.5; MRSA, OR = 14.8; and VRE, OR = 12.6 (each, P < 0.001). The highest odds of finding surface contamination occurred in the primary and secondary sort areas where soiled linens were manually sorted by employees (OR = 63.0, P < 0.001). The study substantiates that the laundry facility environment can become contaminated by soiled linens. Workers who handle soiled linen may have a higher risk of exposure to C. difficile, MRSA, and VRE than those who handle clean linens. Improved protocols for prevention and reduction of environmental contamination were implemented because of this study.

Clostridium difficile environmental contamination within a clinical laundry facility in the USA.

Clostridium difficile is both a hospital and community-acquired pathogen. The current study determined if C. difficile could be cultured from clinical laundry facility surfaces. A total of 240 surface samples were collected from dirty areas (n = 120), which handle soiled clinical linens, and from clean areas (n = 120), which process and fold the clean linens, within the University of Washington Consolidated Laundry facility in 2015. Sampling was done four times over the course of 1 year. The dirty area was significantly more contaminated than the clean area (21% vs 2%, P < 0.001). Clostridium difficile isolates were genetically characterized using multilocus sequence typing and PCR for the detection of genes encoding toxin A and toxin B. The MLST types 1, 2, 3, 15, 26, 34, 35, 39, 42, 43, 44, 53, 63 and 284 were identified and have previously been found in both clinical and community settings. Toxin positive isolates were identified in both the dirty (n = 16/25) and clean areas (n = 2/2). Seasonal variation was observed with 40% of the 27 isolates cultured in April 2015. The study suggests that soiled clinical linens may be a source of C. difficile surface contamination.