Ronald R. Cutler

Could Public Restrooms Be an Environment for Bacterial Resistomes

Antibiotic resistance in bacteria remains a major problem and environments that help to maintain such resistance, represent a significant problem to infection control in the community. Restrooms have always been regarded as potential sources of infectious diseases and we suggest they have the potential to sustain bacterial “resistomes”. Recent studies have demonstrated the wide range of different bacterial phyla that can be found in non-healthcare restrooms. In our study we focused on the Staphylococci. These species are often skin contaminants on man and have been reported as common restroom isolates in recent molecular studies. We collected samples from 18 toilets sited in 4 different public buildings. Using MALDI-TOF-MS and other techniques, we identified a wide range of antibiotic resistant Staphylococci and other bacteria from our samples. We identified 19 different Staphylococcal species within our isolates and 37.8% of the isolates were drug resistant. We also identified different Staphylococcal species with the same antibiograms inhabiting the same restrooms. Bacterial “resistomes” are communities of bacteria often localised in specific areas and within these environments drug resistance determinants may be freely transferred. Our study shows that non-healthcare restrooms are a source of antibiotic resistant bacteria where a collection of antibiotic resistance genes in pathogenic and non-pathogenic bacteria could form a resistome containing a “nexus of genetic diversity”

Candidatus Rickettsia hoogstraalii in Ethiopian Argas persicus ticks.

Ethiopian soft ticks Argas persicus, hard ticks including both Amblyomma variegatum and Rhipicephalus (Boophilus) spp., and fleas were collected from livestock, traditional human dwellings, and cracks and crevices of trees. They were assessed in pools for the presence of Rickettsia using PCR-based methods. The extracted tick DNA was subjected to molecular screening for Rickettsia, which revealed 50.5% of the pooled samples to be positive for Rickettsia spp. These were then subjected to multi-gene analysis using both outer surface proteins and housekeeping genes with proven discriminatory potential. Sequencing of the citrate synthase and outer membrane genes clearly led to the identification of three distinct rickettsial species, Candidatus Rickettsia hoogstraalii in Argas persicus ticks; R. africae in hard tick pools, and R. felis in fleas. Furthermore, we demonstrated the presence of the plasmid-borne small heat-shock protein gene hsp2 in DNA from A. persicus ticks suggesting that Candidatus R. hoogstraalii carried by these ticks possess a plasmid. Unlike chromosomal gene sequences, the hsp2 gene failed to cluster with Candidatus R. hoogstraalii, instead falling into an isolated separate clade, suggesting a different origin for the plasmid.

Antibiotic resistance and mecA characterization of coagulase-negative staphylococci isolated from three hotels in London, UK.

Antibiotic resistance in bacteria isolated from non-healthcare environments, is a potential problem to public health. In our survey a total of 71 coagulase negative staphylococci (CNS) belonging to 11 different species were isolated from three large hotels in London, UK. The most prevalent species was Staphylococcus haemolyticus, with S. hominis, S. warneri, S. cohnii, and Staphylococcus epidermidis commonly detected. Antimicrobial susceptibilities and carriage of the mecA gene were determined for all of these isolates. Most (85.9%) staphylococci were resistant to multiple antibiotics with all displaying increased susceptibility toward penicillin, fusidic acid, erythromycin, and cefepime. Twenty-one (29.5%) of the isolates were mecA positive, however MIC values to oxacillin, normally associated with the carriage of mecA, varied widely in this group (from 0.06 to 256 mg/L). Fifteen of the twenty-one mecA positive isolates carried SCCmec of these seven were type V, one type I, one type II, and one type IV. Additionally, five of these 15 isolates carried a previously unreported type, 1A, which involves an association between class A mec complex and ccr type 1. The remaining six of the 21 isolates were non-typeable and carried a combination of class A mec complex and ccrC. In addition to this, we also report on new MLST types which were assigned for five S. epidermidis isolates. Four out of these five isolates had MICs between 0.06 and 256 mg/L to oxacillin and would be regarded as clinically susceptible but one isolate had a high oxacillin MIC of 256 mg/L. We demonstrated widespread multiple drug resistance among different staphylococcal species isolated from non-healthcare environments highlighting the potential for these species to act as a reservoir for methicillin and other forms of drug resistance.

The prevalence, antibiotic resistance and mecA characterization of coagulase negative staphylococci recovered from non-healthcare settings in London, UK

BackgroundCoagulase negative staphylococci (CoNS) are important reservoirs of antibiotic resistance genes and associated mobile genetic elements and are believed to contribute to the emergence of successful methicillin resistant Staphylococcus aureus (MRSA) clones. Although, these bacteria have been linked to various ecological niches, little is known about the dissemination and genetic diversity of antibiotic resistant CoNS in general public settings.MethodsFour hundred seventy-nine samples were collected from different non-healthcare/general public settings in various locations (n = 355) and from the hands of volunteers (n = 124) in London UK between April 2013 and Nov 2014.ResultsSix hundred forty-three staphylococcal isolates belonging to 19 staphylococcal species were identified. Five hundred seventy-two (94%) isolates were resistant to at least one antibiotic, and only 34 isolates were fully susceptible. Sixty-eight (11%) mecA positive staphylococcal isolates were determined in this study. SCCmec types were fully determined for forty-six isolates. Thirteen staphylococci (19%) carried SCCmec V, followed by 8 isolates carrying SCCmec type I (2%), 5 SCCmec type IV (7%), 4 SCCmec type II (6%), 1 SCCmec type III (2%), 1 SCCmec type VI (2%), and 1 SCCmec type VIII (2%). In addition, three isolates harboured a new SCCmec type 1A, which carried combination of class A mec complex and ccr type 1.MLST typing revealed that all S. epidermidis strains possess new MLST types and were assigned the following new sequence types: ST599, ST600, ST600, ST600, ST601, ST602, ST602, ST603, ST604, ST605, ST606, ST607 and ST608.ConclusionsThe prevalence of antibiotic resistant staphylococci in general public settings demonstrates that antibiotics in the natural environments contribute to the selection of antibiotic resistant microorganisms. The finding of various SCCmec types in non-healthcare associated environments indicates the complexity of SCCmec. We also report on new MLST types that were assigned for all S. epidermidis isolates, which demonstrates the genetic variability of these isolates.

Whole Genome Sequence and Comparative Genomics Analysis of Multi-drug Resistant Environmental Staphylococcus epidermidis ST59

Staphylococcus epidermidis is a major opportunistic pathogen primarily recovered from device-associated healthcare associated infections (DA-HAIs). Although S. epidermidis and other coagulase-negative staphylococci (CoNS) are less virulent than Staphylococcus aureus, these bacteria are an important reservoir of antimicrobial resistance genes and resistance-associated mobile genetic elements that can be transferred between staphylococcal species. We report a whole genome sequence of a multidrug resistant S. epidermidis (strain G6_2) representing multilocus sequence type (ST) 59 and isolated from an environmental sampling of a hotel room in London, UK. The genome of S. epidermidis G6_2 comprises of a 2408357 bp chromosome and six plasmids, with an average G+C content of 32%. The strain displayed a multi-drug resistance phenotype which was associated with carriage of 7 antibiotic resistance genes (blaZ, mecA, msrA, mphC, fosB, aacA-aphD, tetK) as well as resistance-conferring mutations in fusA and ileS. Antibiotic resistance genes were located on plasmids and chromosome. Comparative genomic analysis revealed that antibiotic resistance gene composition found in G6_2 was partly preserved across the ST59 lineage.

Detection of diverse genotypes of Methicillin-resistant Staphylococcus aureus from hospital personnel and the environment in Armenia

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a public health concern internationally. Studies examining a range of cohorts have been reported from various regions of the world, but little is known about the molecular epidemiology of MRSA in Armenia.MethodsBetween May and September 2013, twenty isolates of methicillin-resistant Staphylococcus aureus (MRSA; mecA positive) were recovered from hospital personnel (n = 10; 9 females, 1 male) and environmental sites (n = 10) in the maternity ward of one of the teaching hospitals in Armenia.ResultsMulti-locus sequence type clonal complex (MLST-CC) assignments inferred from spa typing data revealed the majority belonged to 3 pandemic lineages of MRSA including: t008-CC8-SCCmecV (n = 10; 7 from personnel); t021-CC30-SCCmecIV (n = 5; all environmental); and t1523-CC45 (n = 2; 1 from personnel), one harboured SCCmecV the other was SCCmec non-typable. The remainder identified as belonging to genotype t364-CC182, both of which harboured a novel SCCmec cassette with kdp, rif5, ccrB2 and ccrC detected by PCR (both from personnel); and t325-CC88-SCCmecIV (n = 1; environmental). All MRSA were negative for the Panton-Valentine Leukocidin (PVL) locus and three CC8 strains were positive for the arginine catabolic element (ACME).ConclusionsIn this small study, we report for the first time of the occurrence of diverse MRSA genotypes belonging to both pandemic and more sporadic international clones in Armenia harbouring the smaller SCCmec types and/or ACME, both of which have been associated with strain fitness. Further surveillance is warranted to better understand the prevalence, clinical and molecular epidemiology of MRSA throughout Armenia.

Fibroblast growth stimulation, DPPH antioxidant assay and antimicrobial activities of Funtumia elastica (Preuss) Stapf (Apocynaceae) leaf extracts.

Aims: To investigate the scientific basis for the wound-healing properties of Funtumia elastica (Apocynaceae) leaf extracts using relevant in vitro fibroblast growth stimulation, antimicrobial and DPPH-antioxidant assays. Place and Duration of Study: School of Health, Sports and Bioscience (Bioscience Laboratories), University of East London in the United Kingdom, between July 2007 and May 2010. Methodology: Methanolic extract of the leaves, and petroleum ether, ethyl acetate, n- butanol and aqueous fractions partitioned thereof were tested for antimicrobial activities against common wound pathogens (such as Staphylococcus spp, Pseudomonas aeroginosa and Escherichia coli). The Broth dilution method was used to determine the minimum inhibitory concentrations (MIC) of the extracts and fractions. The antioxidant activities were also determined using a 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay; whilst the ability to stimulate fibroblast growth was investigated using the MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Results: The n-butanol fraction exhibited the greatest overall activities. It stimulated the growth of fibroblast cells by 28%, and showed MIC range of 0.13 - 1.0 mg/mL against the Staphylococci species, P. aeruginosa, Bacillus subtilis and E. coli. The non-polar petroleum ether fraction exhibited MICs greater than 2.0 mg/mL against all the organisms. All the fractions exhibited antioxidant activities greater than or comparable to that of ascorbic acid. Conclusion: Collectively, the antioxidant activity, fibroblast growth stimulation and the antimicrobial activities demonstrated by F. elastica leaf extracts provide some evidence to support the use of the plant to manage wounds in African folklore medicine.