Global transmission of extended-spectrum cephalosporin resistance in Escherichia coli driven by epidemic plasmids.

BACKGROUND: Extended-spectrum cephalosporins (ESCs) are third and fourth generation cephalosporin antimicrobials used in humans and animals to treat infections due to multidrug-resistant (MDR) bacteria. Resistance to ESCs (ESC-R) in Enterobacterales is predominantly due to the production of extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (AmpCs). The dynamics of ESBLs and AmpCs are changing across countries and host species, the result of global transmission of ESC-R genes. Plasmids are known to play a key role in this dissemination, but the relative importance of different types of plasmids is not fully understood. METHODS: In this study, Escherichia coli with the major ESC-R genes blaCTX-M-1, blaCTX-M-15, blaCTX-M-14 (ESBLs) and blaCMY-2 (AmpC), were selected from diverse host species and other sources across Canada, France and Germany, collected between 2003 and 2017. To examine in detail the vehicles of transmission of the ESC-R genes, long- and short-read sequences were generated to obtain complete contiguous chromosome and plasmid sequences (n = 192 ESC-R E. coli). The types, gene composition and genetic relatedness of these plasmids were investigated, along with association with isolate year, source and geographical origin, and put in context with publicly available plasmid sequences. FINDINGS: We identified five epidemic resistance plasmid subtypes with distinct genetic properties that are associated with the global dissemination of ESC-R genes across multiple E. coli lineages and host species. The IncI1 pST3 blaCTX-M-1 plasmid subtype was found in more diverse sources than the other main plasmid subtypes, whereas IncI1 pST12 blaCMY-2 was more frequent in Canadian and German human and chicken isolates. Clonal expansion also contributed to the dissemination of the IncI1 pST12 blaCMY-2 plasmid in ST131 and ST117 E. coli harbouring this plasmid. The IncI1 pST2 blaCMY-2 subtype was predominant in isolates from humans in France, while the IncF F31:A4:B1 blaCTX-M-15 and F2:A-:B- blaCTX-M-14 plasmid subtypes were frequent in human and cattle isolates across multiple countries. Beyond their epidemic nature with respect to ESC-R genes, in our collection almost all IncI1 pST3 blaCTX-M-1 and IncF F31:A4:B1 blaCTX-M-15 epidemic plasmids also carried multiple antimicrobial resistance (AMR) genes conferring resistance to other antimicrobial classes. Finally, we found genetic signatures in the regions surrounding specific ESC-R genes, identifying the predominant mechanisms of ESC-R gene movement, and using publicly available databases, we identified these epidemic plasmids from widespread bacterial species, host species, countries and continents. INTERPRETATION: We provide evidence that epidemic resistance plasmid subtypes contribute to the global dissemination of ESC-R genes, and in addition, some of these epidemic plasmids confer resistance to multiple other antimicrobial classes. The success of these plasmids suggests that they may have a fitness advantage over other plasmid types and subtypes. Identification and understanding of the vehicles of AMR transmission are crucial to develop and target strategies and interventions to reduce the spread of AMR. FUNDING: This project was supported by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR), through the Medical Research Council (MRC, MR/R000948/1), the Canadian Institutes of Health Research (CFC-150770), and the Genomics Research and Development Initiative (Government of Canada), the German Federal Ministry of Education and Research (BMBF) grant no. 01KI1709, the French Agency for food environmental and occupational health & safety (Anses), and the French National Reference Center (CNR) for antimicrobial resistance. Support was also provided by the Biotechnology and Biological Sciences Research Council (BBSRC) through the BBSRC Institute Strategic Programme Microbes in the Food ChainBB/R012504/1 and its constituent project BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain).

Hospital-acquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia (VABP) in Canada: treatment update and the role of new IV antimicrobials.

INTRODUCTION: Hospital-acquired bacterial pneumonia (HABP) and ventilator-associated bacterial pneumonia (VABP) continue to be common infections causing significant morbidity and mortality worldwide. The timely initiation of empiric antimicrobial therapy is essential. In this paper, we provide a focused expert opinion on the current and potential empiric antimicrobial treatment options in HABP and VABP in Canada influenced by antimicrobial resistance impacting the use of older agents as well as available new intravenous (IV) antimicrobials. AREAS COVERED: The authors discuss treatment options for HABP and VABP in Canada. In addition, we focus on the potential role of new IV antimicrobials recently introduced to Canada. A literature search of HABP and VABP treatments was performed via PubMed (up to March 2023), using the following key words: monotherapy, combination therapy, aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, penicillins as well as amoxicillin/clavulanate, ceftobiprole, ceftolozane/tazobactam, dalbavancin, and fosfomycin. EXPERT OPINION: Empiric antimicrobial treatment for HABP and VABP in Canada continues to focus on both the severity of illness and the presence/absence of patient risk factors for antimicrobial resistance. The role of new IV antimicrobials in the empiric treatment for HABP and VABP depends on their antimicrobial activity and published data on efficacy and safety and influenced by Health Canada-approved indications.

The role of Fecal Microbiota Transplantation (FMT) in treating patients with multiple sclerosis.

INTRODUCTION: The associations between multiple sclerosis (MS) and altered intestinal microbiomes have clinicians considering the use of fecal microbiota transplantation (FMT). Animal data suggests that administering FMT from people with MS into healthy mice results in a microbiome with decreased abundance of Sutterella, reduced anti-inflammatory signals, increase in inflammation and experimental autoimmune encephalomyelitis (EAE). Animal studies that administered FMT (from normal healthy donors) into mice resulted in slowing down EAE development relieving symptoms, improving BBB integrity and restoration of microbiota diversity. Human studies indicated clinical benefits of FMT (from healthy donors) in people with MS including: improved intestinal motility and motor ability which lasted at least for the duration of the studies, ranging from 2 to 15 years. AREAS COVERED: The authors discuss the efficacy and safety of FMT in treatment of experimental MS in animals and humans with MS. A literature search was performed via PubMed (up to July 2023), using the key words: multiple sclerosis, fecal microbiota transplantation, microbiome. EXPERT OPINION: Limited associative data do not provide an understanding of role of FMT in the treatment for MS. Until appropriately designed randomized comparative trials which are underway, are completed, we cannot recommend routine use of FMT in people with MS.

Part 2: mucormycosis: focus on therapy.

INTRODUCTION: Mucormycosis (MCR), a rare but life-threatening infection, occurs primarily in immunocompromised hosts. Mortality rates with invasive MCR are high (>30-50%), up to 90% with disseminated disease, but lower (10-30%) with localized cutaneous disease. Due to the rarity of MCR, randomized, controlled therapeutic trials are lacking. Lipid formulations of amphotericin B (LFAB) are the mainstay of therapy, but oral triazoles (posaconazole and isavuconazole) may be effective as step-down therapy or in MCR cases refractory to or intolerant of LFAB. Early surgical debridement or excision plays important adjunctive roles in localized invasive disease. Control of hyperglycemia in diabetic patients, correction of neutropenia, and reduction of immunosuppressive therapy are critical for optimal survival. AREAS COVERED: The authors discuss various therapeutic options for mucormycosis. A literature search of mucormycosis therapies was performed via PubMed (up to December 2022), using the following keywords: invasive fungal infections; mold; mucormycosis; Mucorales; amphotericin B; isavuconazole; and posaconazole. EXPERT OPINION: Randomized, controlled therapeutic trials are lacking. Lipid formulations of amphotericin B (LFAB) are the mainstay of therapy, but oral triazoles (posaconazole and isavuconazole) may be effective as step-down therapy, in MCR cases refractory to or intolerant of LFAB. We encourage early surgical debridement or excision as adjunctive measures.

Part 1: Mucormycosis: prevalence, risk factors, clinical features, and diagnosis.

INTRODUCTION: Mucormycosis (MCR) is caused by filamentous molds within the Class Zygomycetes and Order Mucorales. Infections can result from inhalation of spores into the nares, oropharynx, or lungs, ingestion of contaminated food or water, or inoculation into disrupted skin or wounds. In developed countries, MCR occurs primarily in severely immunocompromised hosts. In contrast, in developing/low income countries, most cases of MCR occur in persons with poorly controlled diabetes mellitus and some cases in immunocompetent subjects following trauma. Mucormycosis exhibits a propensity to invade blood vessels, leading to thrombosis and infarction of tissue. Mortality rates associated with invasive MCR are high and can exceed 90% with disseminated disease. Mucormycosis can be classified as one of six forms: (1) rhino-orbital-cerebral mucormycosis (ROCM); (2) pulmonary; (3) cutaneous; (4) gastrointestinal or renal (5); disseminated; or (6) uncommon (focal) sites. AREAS COVERED: The authors discuss the prevalence, risk factors, and clinical features of mucormycosis. A literature search of mucormycosis was performed via PubMed (up to November 2022), using the key words: invasivefungal infections; mold; mucormycosis;Mucorales; Zyzomyces; Zygomycosis; Rhizopus, diagnosis. EXPERT OPINION: Mucormycosis occurs primarily in severely immunocompromised hosts. Mucormycosis can progress rapidly, and delay in initiating treatment by even a few days worsens outcomes.

Antimicrobial susceptibility testing of invasive isolates of Streptococcus pneumoniae from Canadian patients: the SAVE study, 2011-2020.

OBJECTIVES: To assess the antimicrobial susceptibility of 14 138 invasive Streptococcus pneumoniae isolates collected in Canada from 2011 to 2020. METHODS: Antimicrobial susceptibility testing was performed using the CLSI M07 broth microdilution reference method. MICs were interpreted using 2022 CLSI M100 breakpoints. RESULTS: In 2020, 90.1% and 98.6% of invasive pneumococci were penicillin-susceptible when MICs were interpreted using CLSI meningitis or oral and non-meningitis breakpoints, respectively; 96.9% (meningitis breakpoint) and 99.5% (non-meningitis breakpoint) of isolates were ceftriaxone-susceptible, and 99.9% were levofloxacin-susceptible. Numerically small, non-temporal, but statistically significant differences (P < 0.05) in the annual percentage of isolates susceptible to four of the 13 agents tested was observed across the 10-year study: chloramphenicol (4.4% difference), trimethoprim-sulfamethoxazole (3.9%), penicillin (non-meningitis breakpoint, 2.7%) and ceftriaxone (meningitis breakpoint, 2.7%; non-meningitis breakpoint, 1.2%). During the same period, annual differences in percent susceptible values for penicillin (meningitis and oral breakpoints) and all other agents did not achieve statistical significance. The percentage of isolates with an MDR phenotype (resistance to ≥3 antimicrobial classes) in 2011 and 2020 (8.5% and 9.4%) was not significantly different (P = 0.109), although there was a significant interim decrease observed between 2011 and 2015 (P < 0.001) followed by a significant increase between 2016 and 2020 (P < 0.001). Statistically significant associations were observed between resistance rates to most antimicrobial agents included in the MDR analysis (penicillin, clarithromycin, clindamycin, doxycycline, trimethoprim/sulfamethoxazole and chloramphenicol) and patient age, specimen source, geographic location in Canada or concurrent resistance to penicillin or clarithromycin, but not biological sex of patients. Given the large isolate collection studied, statistical significance did not necessarily imply clinical or public health significance in some analyses. CONCLUSIONS: Invasive pneumococcal isolates collected in Canada from 2011 to 2020 generally exhibited consistent in vitro susceptibility to commonly tested antimicrobial agents.

Streptococcus pneumoniae serotyping and antimicrobial susceptibility: assessment for vaccine efficacy in Canada after the introduction of PCV13.

BACKGROUND: Streptococcus pneumoniae continues to be an important bacterial pathogen associated with invasive (e.g. bacteraemia, meningitis) and non-invasive (e.g. community-acquired respiratory tract) infections worldwide. Surveillance studies conducted nationally and globally assist in determining trends over geographical areas and allow comparisons between countries. OBJECTIVES: To characterize invasive isolates of S. pneumoniae in terms of their serotype, antimicrobial resistance, genotype and virulence and to use the serotype data to determine the level of coverage by different generations of pneumococcal vaccines. METHODS: SAVE (Streptococcus pneumoniae Serotyping and Antimicrobial Susceptibility: Assessment for Vaccine Efficacy in Canada) is an ongoing, annual, national collaborative study between the Canadian Antimicrobial Resistance Alliance (CARE) and the National Microbiology Laboratory, focused on characterizing invasive isolates of S. pneumoniae obtained across Canada. Clinical isolates from normally sterile sites were forwarded by participating hospital public health laboratories to the Public Health Agency of Canada-National Microbiology Laboratory and CARE for centralized phenotypic and genotypic investigation. RESULTS: The four articles in this Supplement provide a comprehensive examination of the changing patterns of antimicrobial resistance and MDR, serotype distribution, genotypic relatedness and virulence of invasive S. pneumoniae obtained across Canada over a 10 year period (2011-2020). CONCLUSIONS: The data highlight the evolution of S. pneumoniae under pressure by vaccination and antimicrobial usage, as well as vaccine coverage, allowing both clinicians and researchers nationally and globally to view the current status of invasive pneumococcal infections in Canada.

Analysis of MDR in the predominant Streptococcus pneumoniae serotypes in Canada: the SAVE study, 2011-2020.

OBJECTIVES: To investigate the levels of MDR in the predominant serotypes of invasive Streptococcus pneumoniae isolated in Canada over a 10 year period. METHODS: All isolates were serotyped and had antimicrobial susceptibility testing performed, in accordance with CLSI guidelines (M07-11 Ed., 2018). Complete susceptibility profiles were available for 13 712 isolates. MDR was defined as resistance to three or more classes of antimicrobial agents (penicillin MIC ≥2 mg/L defined as resistant). Serotypes were determined by Quellung reaction. RESULTS: In total, 14 138 invasive isolates of S. pneumoniae were tested in the SAVE study (S. pneumoniae Serotyping and Antimicrobial Susceptibility: Assessment for Vaccine Efficacy in Canada), a collaboration between the Canadian Antimicrobial Resistance Alliance and Public Health Agency of Canada-National Microbiology Laboratory. The rate of MDR S. pneumoniae in SAVE was 6.6% (902/13 712). Annual rates of MDR S. pneumoniae decreased between 2011 and 2015 (8.5% to 5.7%) and increased between 2016 and 2020 (3.9% to 9.4%). Serotypes 19A and 15A were the most common serotypes demonstrating MDR (25.4% and 23.5% of the MDR isolates, respectively); however, the serotype diversity index increased from 0.7 in 2011 to 0.9 in 2020 with a statistically significant linear increasing trend (P < 0.001). In 2020, MDR isolates were frequently serotypes 4 and 12F in addition to serotypes 15A and 19A. In 2020, 27.3%, 45.5%, 50.5%, 65.7% and 68.7% of invasive MDR S. pneumoniae were serotypes included in the PCV10, PCV13, PCV15, PCV20 and PPSV23 vaccines, respectively. CONCLUSIONS: Although current vaccine coverage of MDR S. pneumoniae in Canada is high, the increasing diversity of serotypes observed among the MDR isolates highlights the ability of S. pneumoniae to rapidly evolve.

Genomic investigation of the most common Streptococcus pneumoniae serotypes causing invasive infections in Canada: the SAVE study, 2011-2020.

OBJECTIVES: To investigate the lineages and genomic antimicrobial resistance (AMR) determinants of the 10 most common pneumococcal serotypes identified in Canada during the five most recent years of the SAVE study, in the context of the 10-year post-PCV13 period in Canada. METHODS: The 10 most common invasive Streptococcus pneumoniae serotypes collected by the SAVE study from 2016 to 2020 were 3, 22F, 9N, 8, 4, 12F, 19A, 33F, 23A and 15A. A random sample comprising ∼5% of each of these serotypes collected during each year of the full SAVE study (2011-2020) were selected for whole-genome sequencing (WGS) using the Illumina NextSeq platform. Phylogenomic analysis was performed using the SNVPhyl pipeline. WGS data were used to identify virulence genes of interest, sequence types, global pneumococcal sequence clusters (GPSC) and AMR determinants. RESULTS: Of the 10 serotypes analysed in this study, six increased significantly in prevalence from 2011 to 2020: 3, 4, 8, 9N, 23A and 33F (P ≤ 0.0201). Serotypes 12F and 15A remained stable in prevalence over time, while serotype 19A decreased in prevalence (P < 0.0001). The investigated serotypes represented four of the most prevalent international lineages causing non-vaccine serotype pneumococcal disease in the PCV13 era: GPSC3 (serotypes 8/33F), GPSC19 (22F), GPSC5 (23A) and GPSC26 (12F). Of these lineages, GPSC5 isolates were found to consistently possess the most AMR determinants. Commonly collected vaccine serotypes 3 and 4 were associated with GPSC12 and GPSC27, respectively. However, a more recently collected lineage of serotype 4 (GPSC192) was highly clonal and possessed AMR determinants. CONCLUSIONS: Continued genomic surveillance of S. pneumoniae in Canada is essential to monitor for the appearance of new and evolving lineages, including antimicrobial-resistant GPSC5 and GPSC162.

Comparison of PCV10, PCV13, PCV15, PCV20 and PPSV23 vaccine coverage of invasive Streptococcus pneumoniae isolate serotypes in Canada: the SAVE study, 2011-20.

BACKGROUND: As pneumococci evolve under vaccine, antimicrobial and other selective pressures, it is important to track isolates covered by established (PCV10, PCV13 and PPSV23) and new (PCV15 and PCV20) vaccine formulations. OBJECTIVES: To compare invasive pneumococcal disease (IPD) isolates from serotypes covered by PCV10, PCV13, PCV15, PCV20 and PPSV23, collected in Canada from 2011 to 2020, by demographic category and antimicrobial resistance phenotype. METHODS: IPD isolates from the SAVE study were initially collected by members of the Canadian Public Health Laboratory Network (CPHLN) as part of a collaboration between the Canadian Antimicrobial Resistance Alliance (CARA) and the Public Health Agency of Canada (PHAC). Serotypes were determined by quellung reaction, and antimicrobial susceptibility testing was performed using the CLSI broth microdilution method. RESULTS: A total of 14 138 invasive isolates were collected from 2011 to 2020, with 30.7% of isolates covered by the PCV13 vaccine, 43.6% of isolates covered by the PCV15 vaccine (including 12.9% non-PCV13 serotypes 22F and 33F), and 62.6% of isolates covered by the PCV20 vaccine (including 19.0% non-PCV15 serotypes 8, 10A, 11A, 12F and 15B/C). Non-PCV20 serotypes 2, 9N, 17F and 20, but not 6A (present in PPSV23) represented 8.8% of all IPD isolates. Higher-valency vaccine formulations covered significantly more isolates by age, sex, region and resistance phenotype including MDR isolates. Coverage of XDR isolates did not significantly differ between vaccine formulations. CONCLUSIONS: When compared with PCV13 and PCV15, PCV20 covered significantly more IPD isolates stratified by patient age, region, sex, individual antimicrobial resistance phenotypes and MDR phenotype.

Escalation of antimicrobial resistance among MRSA part 2: focus on infections and treatment.

INTRODUCTION: MRSA is associated with causing a variety of infections including skin and skin structure infections, catheter and device-related (e.g. central venous catheter, prosthetic heart valve) infections, infectious endocarditis, blood stream infections, bone, and joint infections (e.g. osteomyelitis, prosthetic joint, surgical site), central nervous system infections (e.g. meningitis, brain/spinal cord abscess, ventriculitis, hydrocephalus), respiratory tract infections (e.g. hospital-acquired pneumonia, ventilator-associated pneumonia), urinary tract infections, and gastrointestinal infections. The emergence and spread of multidrug resistant (MDR) MRSA clones has limited therapeutic options. Older agents such as vancomycin, linezolid and daptomycin and a variety of newer MRSA antimicrobials and combination therapy are available to treat serious MRSA infections. AREAS COVERED: The authors discuss infections caused by MRSA as well as common older and newer antimicrobials and combination therapy for MRSA infections. A literature search of MRSA was performed via PubMed (up to September 2022), using the keywords: antimicrobial resistance; ß-lactams; multidrug resistance, Staphylococcus aureus, vancomycin; glycolipopeptides. EXPERT OPINION: Innovation, discovery, and development of new and novel classes of antimicrobial agents are critical to expand effective therapeutic options. The authors encourage the judicious use of antimicrobials in accordance with antimicrobial stewardship programs along with infection-control measures to minimize the spread of MRSA.

Escalation of antimicrobial resistance among MRSA part 1: focus on global spread.

INTRODUCTION: Staphylococcus aureus produce numerous virulence factors that influence tissue invasion, cytotoxicity, membrane damage, and intracellular persistence allowing them to be very common human pathogens. S. aureus isolates exhibit considerable diversity though specific genotypes have been associated with antimicrobial resistance (AMR) and toxin gene profiles. MRSA is an important pathogen causing both community-acquired (CA) and healthcare-acquired (HCA) infections. Importantly, over the past several decades, both HCA-MRSA and CA-MRSA have spread all over the globe. Even more concerning is that CA-MRSA clones have disseminated into hospitals and HCA-MRSA have entered the community. Factors that enhance spread of MRSA include: poor antimicrobial stewardship and inadequate infection control. The emergence and spread of multidrug resistant (MDR) MRSA has limited therapeutic options. AREAS COVERED: The authors discuss the escalation of MRSA, both HCA-MRSA and CA-MRSA across the globe. A literature search of MRSA was performed via PubMed (up to September 2022), using the key words: antimicrobial resistance; ß-lactams; community-associated MRSA; epidemiology; infection; multidrug resistance; Staphylococcus aureus. EXPERT OPINION: Over the past several decades, MRSA has spread all over the globe. We encourage the judicious use of antimicrobials in accordance with antimicrobial stewardship programs along with infection control measures to minimize the spread of MRSA.

Antimicrobial resistance patterns of bacterial pathogens recovered from the urine of patients at Canadian hospitals from 2009 to 2020.

Objectives: To investigate in vitro susceptibility patterns of bacterial pathogens recovered from the urine of outpatients (isolates from outpatient clinics or emergency departments) and hospital inpatients across Canada from 2009 to 2020 as part of the CANWARD study. Methods: Canadian hospital microbiology laboratories submitted bacterial pathogens cultured from urine to the CANWARD study coordinating laboratory on an annual basis (January 2009 to December 2020). Antimicrobial susceptibility testing was performed by CLSI broth microdilution, with MICs interpreted by current CLSI breakpoints. Results: In total, 4644 urinary pathogens were included in this study. Escherichia coli was recovered most frequently (53.3% of all isolates), followed by Enterococcus faecalis, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. Together, these six species accounted for 84.2% of study isolates. Nitrofurantoin demonstrated excellent in vitro activity versus E. coli, with 97.6% of outpatient and 96.1% of inpatient isolates remaining susceptible. In contrast, E. coli susceptibility rates were lower for ciprofloxacin (outpatient 79.5%, inpatient 65.9%) and trimethoprim/sulfamethoxazole (outpatient 75.2%, inpatient 73.5%). The percentage of E. coli isolates that were phenotypically positive for ESBL production significantly increased from 4.2% (2009-11) to 11.3% (2018-20). A similar although less pronounced temporal trend was observed with ESBL-producing K. pneumoniae. Conclusions: E. coli was the pathogen most frequently recovered from the urine of Canadian patients, and the proportion of isolates that were ESBL producers increased over time. Susceptibility data presented here suggest that ciprofloxacin and trimethoprim/sulfamethoxazole may be suboptimal for the empirical treatment of complicated urinary infections.

Dynamics of extended-spectrum cephalosporin resistance genes in Escherichia coli from Europe and North America.

Extended-spectrum cephalosporins (ESCs) are critically important antimicrobial agents for human and veterinary medicine. ESC resistance (ESC-R) genes have spread worldwide through plasmids and clonal expansion, yet the distribution and dynamics of ESC-R genes in different ecological compartments are poorly understood. Here we use whole genome sequence data of Enterobacterales isolates of human and animal origin from Europe and North America and identify contrasting temporal dynamics. AmpC ß-lactamases were initially more dominant in North America in humans and farm animals, only later emerging in Europe. In contrast, specific extended-spectrum ß-lactamases (ESBLs) were initially common in animals from Europe and later emerged in North America. This study identifies differences in the relative importance of plasmids and clonal expansion across different compartments for the spread of different ESC-R genes. Understanding the mechanisms of transmission will be critical in the design of interventions to reduce the spread of antimicrobial resistance.

A One Health Genomic Investigation of Gentamicin Resistance in Escherichia coli from Human and Chicken Sources in Canada, 2014 to 2017.

We investigated whether gentamicin resistance (Genr) in Escherichia coli isolates from human infections was related to Genr E. coli in chicken and whether resistance may be due to coselection from use of lincomycin-spectinomycin in chickens on farms. Whole-genome sequencing was performed on 483 Genr E. coli isolates isolated between 2014 and 2017. These included 205 human-source isolates collected by the Canadian Ward (CANWARD) program and 278 chicken-source isolates: 167 from live/recently slaughtered chickens (animals) and 111 from retail chicken meat collected by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). The predominant Genr gene was different in human and chicken sources; however, both sources carried aac(3)-IId, aac(3)-VIa, and aac(3)-IVa. Forty-one percent of human clinical isolates of Genr E. coli contained a blaCTX-M extended-spectrum beta-lactamase (ESBL) gene (84/205), and 53% of these were sequence type 131 (ST131). Phylogenomic analysis revealed a high diversity of Genr isolates; however, there were three small clusters of closely related isolates from human and chicken sources. Genr and spectinomycin resistance (Specr) genes were colocated in 148/167 (89%) chicken animal isolates, 94/111 (85%) chicken retail meat isolates, and 137/205 (67%) human-source isolates. Long-read sequencing of 23 isolates showed linkage of the Genr and Specr genes on the same plasmid in 14/15 (93%) isolates from chicken(s) and 6/8 (75%) isolates from humans. The use of lincomycin-spectinomycin on farms may be coselecting for gentamicin-resistant plasmids in E. coli in broiler chickens; however, Genr isolates and plasmids were mostly different in chickens and humans.

In vitro activity of sulopenem against 1880 bacterial pathogens isolated from Canadian patients with urinary tract infections (CANWARD, 2014-21).

INTRODUCTION: There are limited oral antimicrobial options for the treatment of urinary infections caused by ESBL-producing and MDR Enterobacterales. Sulopenem is an investigational thiopenem antimicrobial that is being developed as both an oral and IV formulation. The purpose of this study was to evaluate the in vitro activity of sulopenem versus bacterial pathogens recovered from the urine of patients admitted to or assessed at hospitals across Canada (CANWARD). MATERIALS AND METHODS: The in vitro activity of sulopenem and clinically relevant comparators was determined for 1880 Gram-negative and Gram-positive urinary isolates obtained as part of the CANWARD study (2014 to 2021) using the CLSI broth microdilution method. RESULTS: Sulopenem demonstrated excellent in vitro activity versus members of the Enterobacterales, with MIC90 values ranging from 0.06 to 0.5 mg/L for all species tested. Over 90% of ESBL-producing, AmpC-producing and MDR (not susceptible to ≥1 antimicrobial from ≥3 classes) Escherichia coli were inhibited by ≤0.25 mg/L of sulopenem. Sulopenem had an identical MIC90 to meropenem for ESBL-producing and MDR E. coli. The MIC90 of sulopenem and meropenem versus MSSA was 0.25 mg/L. Sulopenem was not active in vitro versus Pseudomonas aeruginosa (similar to ertapenem), and it demonstrated poor activity versus Enterococcus faecalis (similar to meropenem). CONCLUSIONS: Sulopenem demonstrated excellent in vitro activity versus bacterial pathogens recovered from the urine of Canadian patients. These data suggest that sulopenem may have a role in the treatment of urinary infections caused by antimicrobial-resistant Enterobacterales, but additional clinical studies are required.

In vitro activity of fosfomycin against bacterial pathogens isolated from urine specimens in Canada from 2007 to 2020: CANWARD surveillance study.

BACKGROUND: Multiple susceptible breakpoints are published to interpret fosfomycin MICs: ≤64 mg/L for Escherichia coli and Enterococcus faecalis grown from urine (CLSI M100); ≤32 mg/L for Enterobacterales and staphylococci when parenteral fosfomycin is prescribed (EUCAST); and ≤8 mg/L for uncomplicated urinary tract infection with E. coli when oral fosfomycin is used (EUCAST). Clinical laboratories are frequently requested to test fosfomycin against antimicrobial-resistant urinary isolates not included in standard documents. METHODS: The in vitro activity of fosfomycin was determined using the CLSI agar dilution method for a 2007-20 collection of clinically significant Gram-negative (3656 Enterobacterales; 140 Pseudomonas aeruginosa) and Gram-positive (346 E. faecalis; 94 Staphylococcus aureus) urinary isolates from the CANWARD surveillance study. Comparator agents were tested using CLSI broth microdilution. RESULTS: Using the CLSI MIC breakpoint (≤64 mg/L), 99.2% of E. coli (n = 2871; MIC90, 4 mg/L), including 96.7% of ESBL-positive isolates, were fosfomycin susceptible. Similarly, 95.8% of E. coli, including 95.2% of ESBL-positive isolates, were fosfomycin susceptible at ≤8 mg/L (EUCAST oral susceptible MIC breakpoint). All other species of Enterobacterales (except Citrobacter freundii) and P. aeruginosa had higher fosfomycin MICs (MIC90s, 64 to >512 mg/L) than E. coli. Using published breakpoints, 88.4% of E. faecalis (MIC ≤64 mg/L) and 97.9% of S. aureus (MIC ≤32 mg/L) isolates were fosfomycin susceptible. CONCLUSIONS: Fosfomycin demonstrated in vitro activity against frequently encountered Gram-positive and Gram-negative urinary pathogens; however, the extrapolation of current CLSI and EUCAST MIC breakpoints to pathogens not specified by standard methods requires further study and is currently not recommended.

Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps.

Bacterial resistance to the antiseptic chlorhexidine (CHX), is a growing problem, recently shown to be caused by deleterious mutations to the phospholipid transport system component (mlaA) as well as efflux pump overexpression. Comparisons of CHX resistance mechanisms, such as porin deletions (ompCF), and over-expressed efflux pumps (acrB, qacE, aceI), are lacking and may be distinguishable using antiseptic rapid fluorescent dye testing assays. Using E. coli K-12 CHX adapted isolates (CHXR1), gene deletion mutants, and over-expressed transformants the phenotypes of these CHX resistance genes were compared using antimicrobial susceptibility tests (AST), rapid fluorescent propidium iodide dye-based membrane integrity assays (RFDMIA), and scanning electron microscopy (SEM). AST findings showed CHXR1, ΔacrB, ΔompCF, and transformants pCA24N-aceI and pCA24N-mlaA conferred greater (two to fourfold) MIC changes when compared to matched controls. Examination of these mutants/transformants using CHX RFDMIA showed that porin dual-deletions (ΔompCF) and mlaA alterations (ΔmlaA; pCA24N-mlaA, CHXR1) were distinguishable from controls. Results for over-expressed (pMS119EH-aceI) and deleted (ΔacrB) efflux pump RFDMIA could not be distinguished with propidium iodide, only with ethidium bromide, suggesting propidium iodide is better suited for detecting porin and mlaA associated CHX resistance mechanisms. SEM of CHXR1 and unadapted E. coli cells exposed to increasing CHX concentrations revealed that CHX does not visibly damage cell envelope integrity at any tested concentration but did identify elongated CHXR1 cells. ΔmlaA confers similar levels of CHX resistance as efflux overexpression and porin deletions, however, only outer membrane-altering porin and mlaA deletions can be reliably distinguished using RFDMIA.

In Vitro Activity of Cefiderocol against Extensively Drug-Resistant Pseudomonas aeruginosa: CANWARD, 2007 to 2019.

Cefiderocol was evaluated by broth microdilution versus 1,050 highly antimicrobial-resistant Pseudomonas aeruginosa clinical isolates from the CANWARD study (2007 to 2019). Overall, 98.3% of isolates remained cefiderocol susceptible (MIC, ≤4 µg/mL), including 97.4% of extensively drug-resistant (XDR) (n = 235) and 97.9% of multidrug-resistant (MDR) (n = 771) isolates. Most isolates testing not susceptible to ceftolozane-tazobactam, ceftazidime-avibactam, and imipenem-relebactam remained susceptible to cefiderocol. In vitro data suggest that cefiderocol may be a treatment option for infections caused by MDR and XDR P. aeruginosa. IMPORTANCE After testing cefiderocol against a large collection of clinical isolates of highly antimicrobial-resistant Pseudomonas aeruginosa, we report that cefiderocol is active versus 97.4% of extensively drug-resistant (XDR) and 97.9% of multidrug-resistant (MDR) (n = 771) isolates. These data show that cefiderocol may be a treatment option for infections caused by MDR and XDR P. aeruginosa.

A Clostridioides difficile surveillance study of Canadian retail meat samples from 2016-2018.

In this study, we isolated and molecularly characterized 10 (1.6%) C. difficile isolates from 644 commercially available raw meat samples. Molecular typing by PFGE and ribotyping revealed NAP and ribotypes commonly associated with human clinical cases, suggesting retail meat could be a possible source of transmission warranting further investigation.