Emergence of KPC-31, a KPC-3 Variant Associated with Ceftazidime-Avibactam Resistance, in an Extensively Drug-Resistant ST235 Pseudomonas aeruginosa Clinical Isolate.

A ceftazidime-avibactam-resistant KPC-producing Pseudomonas aeruginosa strain was isolated in Argentina from a tracheal aspirate. The patient was treated with ceftazidime-avibactam in combination with other agents for 130 days. Whole-genome sequencing of P. aeruginosa identified a D179Y substitution in the Ω loop of KPC-3, corresponding to KPC-31, integrated at the chromosome. The strain belonged to the sequence type 235/O11 (ST235/O11) high-risk clone. Evaluation of carbapenemase detection assays most used by clinical laboratories failed to identify the isolate as a KPC producer.

Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates From Peru, 2018 and 2019.

BACKGROUND: Currently, in Latin America, including Peru, the treatment of gonorrhea is still empiric and information regarding antimicrobial resistance is scarce in some countries because of the limited resources, which can contribute to the rising rates of reported multidrug-resistant gonococcal strains. In that context, it is mandatory to continuously monitor and report antimicrobial resistance in N. gonorrhoeae to update treatment recommendations. METHODS: This descriptive study analyzed genital and anal samples from symptomatic patients who attended 15 sexually transmitted infections health facilities from 8 different regions in Peru during the years 2018 to 2019 within the framework of Sentinel Surveillance. After establishing the presumptive diagnosis, the isolates were sent to the Laboratory of Sexually Transmitted Bacteria of the National Institute of Health of Peru in Lima where the species were confirmed (N = 165) and susceptibility profiles were determined. RESULTS: Among the 165 isolates, 95.2% corresponded to male patients, between 18 and 22 years of age (40.6%), half reported having a sexual partner and being heterosexual. Clinically, 89.7% manifested the presence of urethral exudate. Microbiology showed 95.2% of the isolates resistant to ciprofloxacin and 9.1% non-susceptible to azithromycin. Reduced susceptibility to ceftriaxone and cefixime was observed in 1.2% and 3.6% of the isolates respectively. All strains tested were susceptible to spectinomycin. CONCLUSIONS: This study demonstrated that in Peru, fluoroquinolones should not be recommended or used in N. gonorrhoeae infections due to the high percentage of resistant strains. In addition, nationwide access to gonococcal resistance testing, molecular diagnostics and antimicrobial stewardship should be implemented to control the spread of gonococcal antimicrobial resistance.

Genomic Epidemiology of Carbapenemase-Producing Enterobacterales at a Hospital System in Toronto, Ontario, Canada, 2007 to 2018.

At a hospital system (H1) in Ontario, Canada, we investigated whether whole-genome sequencing (WGS) altered initial epidemiological interpretation of carbapenemase-producing Enterobacterales (CPE) transmission. We included patients with CPE colonization/infection identified by population-based surveillance from October 2007 to August 2018 who received health care at H1 in the year before/after CPE detection. H1 reported epidemiological transmission clusters. We combined single nucleotide variant (SNV) analysis, plasmid characterization, and epidemiological data. Eighty-five patients were included. H1 identified 7 epidemiological transmission clusters, namely, A to G, involving 24/85 (28%) patients. SNV analysis confirmed transmission clusters C, D, and G and identified two additional cases belonging to cluster A. One was a travel-related case that was the likely index case (0 to 6 SNVs from other isolates); this case stayed on the same unit as the initially presumed index case 4 months prior to detection of the initially presumed index case on another unit. The second additional case occupied a room previously occupied by 5 cluster A cases. Plasmid sequence analysis excluded a case from cluster A and identified clusters E and F as possibly two parts of a single cluster. SNV analysis also identified a case without direct epidemiologic links that was 18 to 21 SNVs away from cluster B, suggesting possible undetected interhospital transmission. SNV and plasmid sequence analysis identified cases belonging to transmission clusters that conventional epidemiology missed and excluded other cases. Implementation of routine WGS to complement epidemiological transmission investigations has the potential to improve prevention and control of CPE in hospitals.

Using Genetic Distance from Archived Samples for the Prediction of Antibiotic Resistance in Escherichia coli.

The rising rates of antibiotic resistance increasingly compromise empirical treatment. Knowing the antibiotic susceptibility of a pathogen's close genetic relative(s) may improve empirical antibiotic selection. Using genomic and phenotypic data for Escherichia coli isolates from three separate clinically derived databases, we evaluated multiple genomic methods and statistical models for predicting antibiotic susceptibility, focusing on potentially rapidly available information, such as lineage or genetic distance from archived isolates. We applied these methods to derive and validate the prediction of antibiotic susceptibility to common antibiotics. We evaluated 968 separate episodes of suspected and confirmed infection with Escherichia coli from three geographically and temporally separated databases in Ontario, Canada, from 2010 to 2018. Across all approaches, model performance (area under the curve [AUC]) ranges for predicting antibiotic susceptibility were the greatest for ciprofloxacin (AUC, 0.76 to 0.97) and the lowest for trimethoprim-sulfamethoxazole (AUC, 0.51 to 0.80). When a model predicted that an isolate was susceptible, the resulting (posttest) probabilities of susceptibility were sufficient to warrant empirical therapy for most antibiotics (mean, 92%). An approach combining multiple models could permit the use of narrower-spectrum oral agents in 2 out of every 3 patients while maintaining high treatment adequacy (∼90%). Methods based on genetic relatedness to archived samples of E. coli could be used to predict antibiotic resistance and improve antibiotic selection.

Comparing Patient Risk Factor-, Sequence Type-, and Resistance Locus Identification-Based Approaches for Predicting Antibiotic Resistance in Escherichia coli Bloodstream Infections.

Rapid diagnostic tests for antibiotic resistance that identify the presence or absence of antibiotic resistance genes/loci are increasingly being developed. However, these approaches usually neglect other sources of predictive information which could be identified over shorter time periods, including patient epidemiologic risk factors for antibiotic resistance and markers of lineage. Using a data set of 414 Escherichia coli isolates recovered from separate episodes of bacteremia at a single academic institution in Toronto, Ontario, Canada, between 2010 and 2015, we compared the potential predictive ability of three approaches (epidemiologic risk factor-, pathogen sequence type [ST]-, and resistance gene identification-based approaches) for classifying phenotypic resistance to three antibiotics representing classes of broad-spectrum antimicrobial therapy (ceftriaxone [a 3rd-generation cephalosporin], ciprofloxacin [a fluoroquinolone], and gentamicin [an aminoglycoside]). We used logistic regression models to generate model receiver operating characteristic (ROC) curves. Predictive discrimination was measured using apparent and corrected (bootstrapped) areas under the curves (AUCs). Epidemiologic risk factor-based models based on two simple risk factors (prior antibiotic exposure and recent prior susceptibility of Gram-negative bacteria) provided a modest predictive discrimination, with AUCs ranging from 0.65 to 0.74. Sequence type-based models demonstrated strong discrimination (AUCs, 0.83 to 0.94) across all three antibiotic classes. The addition of epidemiologic risk factors to sequence type significantly improved the ability to predict resistance for all antibiotics (P < 0.05). Resistance gene identification-based approaches provided the highest degree of discrimination (AUCs, 0.88 to 0.99), with no statistically significant benefit being achieved by adding the patient epidemiologic predictors. In summary, sequence type or other lineage-based approaches could produce an excellent discrimination of antibiotic resistance and may be improved by incorporating readily available patient epidemiologic predictors but are less discriminatory than identification of the presence of known resistance loci.

Genome-based epidemiology and antimicrobial resistance determinants of Neisseria gonorrhoeae isolates with decreased susceptibility and resistance to extended-spectrum cephalosporins in Argentina in 2011-16.

OBJECTIVES: Our aim was to describe the molecular epidemiology and antimicrobial resistance determinants of isolates of Neisseria gonorrhoeae with decreased susceptibility and resistance to extended-spectrum cephalosporins (ESCs) in Argentina in 2011-16. METHODS: Gonococcal isolates (n=158) with decreased susceptibility and resistance to ESCs collected in 2011-16 across Argentina were subjected to WGS and antimicrobial susceptibility testing for six antimicrobials. RESULTS: In total, 50% of the isolates were resistant to cefixime, 1.9% were resistant to ceftriaxone, 37.3% were resistant to azithromycin and 63.9% of the isolates showed an MDR phenotype. Resistance and decreased susceptibility to ESCs was mainly associated with isolates possessing the mosaic penA-34.001, in combination with an mtrR promoter A deletion, and PorB1b amino acid substitutions G120K/A121N. Phylogenetic analysis revealed two main clades of circulating strains, which were associated with the N. gonorrhoeae multiantigen sequence typing (NG-MAST) ST1407 and closely related STs, and characterized by a high prevalence rate, wide geographical distribution and temporal persistence. CONCLUSIONS: N. gonorrhoeae isolates with decreased susceptibility and resistance to ESCs in Argentina have emerged and rapidly spread mainly due to two clonal expansions after importation of one or two strains, which are associated with the international MDR NG-MAST ST1407 clone. The identification of the geographical dissemination and characteristics of these predominant clones may help to focus action plans and public health policies to control the spread of ESC resistance in Argentina. Dual antimicrobial therapy (ceftriaxone plus azithromycin) for gonorrhoea needs to be considered in Argentina.

Use of Whole Genome Sequencing for the Molecular Comparison of Neisseria gonorrhoeae Isolates With Decreased Susceptibility to Extended Spectrum Cephalosporins From 2 Geographically Different Regions in America.

BACKGROUND: Neisseria gonorrhoeae isolates with reduced susceptibility or resistance to the recommended first-line antimicrobial therapy have been described in several countries. The purpose of this study was to use genome analyses to compare the molecular characteristics of N. gonorrhoeae isolates with decreased susceptibility to extended-spectrum cephalosporin from Ontario, Canada, and Argentina. METHODS: A total of 128 N. gonorrhoeae isolates, collected in 2015, were included. The susceptibility to penicillin G, tetracycline, ciprofloxacin, cefixime, ceftriaxone, and azithromycin was determined using the agar dilution method. Isolates were subjected to whole genome sequencing, and an in silico analysis was performed to identify antimicrobial resistance determinants and for genotyping. RESULTS: Decreased susceptibility to extended-spectrum cephalosporin was mainly associated with penA mosaic allele 34.001, together with an mtrR promoter A deletion and porB1b alterations G120K/A121N. N. gonorrhoeae multiantigen sequence typing ST1407 or closely related genotypes were identified circulating in both regions. CONCLUSIONS: An international multi-drug resistant clone of N. gonorrhoeae was associated with decreased susceptibility to extended-spectrum cephalosporin (ESC) in 2 different regions in America. Evidence of clonal dissemination of the organism in some regions suggests that the strength of surveillance programs and establishment of collaborative projects are essential.

[Latin American consensus to define, categorize, and report multidrug-resistant, extensively drug-resistant, or pandrug-resistant pathogensConsenso latino-americano para definição, categorização e notificação de patógenos multirresistentes, com resistência ampliada ou panresistentes]. / Consenso latinoamericano para definir, categorizar y notificar patógenos multirresistentes, con resistencia extendida o panresistentes.

This document presents a Latin American consensus to standardize definitions of different levels of antimicrobial resistance in bacteria of public health importance. Inclusion and exclusion criteria are described for antibiotics to include (availability, relevance, and existence of cut-off values) and for methodologies to use. Three gram-negative microorganisms with a great impact in the hospital environment (Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp.) were selected as a pilot proposal. The lack of cut-off values for certain antibiotics (e.g., tigecycline, fosfomycin, and colistin), crucial in treating infections caused by multi-drug resistant or extensively drug-resistant pathogens, led to the need to discuss and agree on provisional cut-off values for monitoring resistance to these drugs. The work team also addressed and reached consensus on easier-to-use alternative susceptibility tests, other than methods approved by international guidelines, for routine testing in clinical bacteriology laboratories. The main benefit of this document is to provide Latin American laboratories with a standardized and consensual framework for the identification and constant and unified surveillance of resistant microorganisms. The recommendations included in this document are the result of consensus among representatives of the national reference laboratories in the countries belonging to the Latin American Surveillance Network of Antimicrobial Resistance, coordinated by the Pan American Health Organization.

É apresentado um consenso latino-americano para padronizar a definição dos graus de resistência antimicrobiana em bactérias de importância em saúde pública. São descritos os critérios de inclusão e exclusão para os antibióticos a serem incluídos (disponibilidade, relevância e pontos de corte de sensibilidade) e metodologias a serem usadas. Como proposta-piloto, foram selecionados três microrganismos Gram-negativos de grande impacto no ambiente hospitalar (Klebsiella pneumoniae, Pseudomonas aeruginosa e Acinetobacter spp.). Diante da falta de pontos de corte para alguns antibióticos (como tigeciclina, fosfomicina e colistina), essenciais para o tratamento de infecções causadas por patógenos com multirresistência ou resistência ampliada, foram debatidos e aprovados pela maioria pontos de corte provisórios para a vigilância da resistência a estes fármacos. Também foi discutido e aprovado o uso de testes de suscetibilidade alternativos aos métodos aprovados pelas diretrizes internacionais, mais simples de serem realizados como testes de rotina nos laboratórios de bacteriologia clínica. A principal contribuição deste documento é oferecer aos laboratórios latino-americanos um sistema padronizado e consensual para a identificação de microrganismos resistentes e a vigilância contínua e uniforme destes patógenos. As recomendações aqui contidas foram feitas por consenso por representantes dos laboratórios nacionais de referência dos países que integram a Rede Latino-Americana de Vigilância da Resistência Antimicrobiana, coordenada pela Organização Pan-Americana da Saúde (OPAS).

Emergence of Carbapenemase-Producing Enterobacteriaceae, South-Central Ontario, Canada1.

We analyzed population-based surveillance data from the Toronto Invasive Bacterial Diseases Network to describe carbapenemase-producing Enterobacteriaceae (CPE) infections during 2007-2015 in south-central Ontario, Canada. We reviewed patients' medical records and travel histories, analyzed microbiologic and clinical characteristics of CPE infections, and calculated incidence. Among 291 cases identified, New Delhi metallo-ß-lactamase was the predominant carbapenemase (51%). The proportion of CPE-positive patients with prior admission to a hospital in Canada who had not received healthcare abroad or traveled to high-risk areas was 13% for patients with oxacillinase-48, 24% for patients with New Delhi metallo-ß-lactamase, 55% for patients with Klebsiella pneumoniae carbapenemase, and 67% for patients with Verona integron-encoded metallo-ß-lactamase. Incidence of CPE infection increased, reaching 0.33 cases/100,000 population in 2015. For a substantial proportion of patients, no healthcare abroad or high-risk travel could be established, suggesting CPE acquisition in Canada. Policy and practice changes are needed to mitigate nosocomial CPE transmission in hospitals in Canada.

Characterization of OXA-48-like carbapenemase producers in Canada, 2011-14.

Objectives: Since the first identification of the OXA-48 carbapenemase in 2001, Enterobacteriaceae harbouring OXA-48-like enzymes have been reported globally. Here, we applied WGS to characterize the molecular epidemiology of these bacterial isolates. Methods: Enterobacteriaceae non-susceptible to carbapenems isolated from patients between 2011 and 2014 were voluntarily submitted to the Canadian National Microbiology Laboratory where they were screened for carbapenemase genes. WGS was conducted on OXA-48-like producers using the Illumina MiSeq platform. WGS data were used for single nucleotide variant (SNV) analysis, MLST analysis, detection of resistance genes and partial plasmid characterization. Susceptibilities were determined using Vitek2 and Etest. Patient data provided from sites were reviewed. Results: Sixty-seven non-duplicated cases were identified among Escherichia coli (n = 21) and Klebsiella pneumoniae (n = 46). Recent international travel was observed in 40.4% of cases. OXA-181 (52.2%) and OXA-48 (31.3%) were the most common variants, one E. coli OXA-48 producer was found to harbour the acquired colistin resistance gene mcr-1. The dominant STs were ST38 and ST410 in E. coli and ST14 in K. pneumoniae. Three common plasmid types were observed among isolates: IncL/M associated with OXA-48 producers, and ColKP3 and IncX3 associated with OXA-181/232 producers. Conclusions: Enterobacteriaceae with OXA-48-like carbapenemases are emerging in Canada. This study highlights the complexity of OXA-48-types identified in Canada owing to travel and the successful clones and plasmids harbouring the OXA-48-like enzyme.

qnrE1, a Member of a New Family of Plasmid-Located Quinolone Resistance Genes, Originated from the Chromosome of Enterobacter Species.

qnrE1, found in a clinical Klebsiella pneumoniae isolate, was undetectable by PCR assays used for the six qnr families. qnrE1 was located on a conjugative plasmid (ca. 185 kb) and differed from qnrB alleles by 25%. Phylogenetic reconstructions of qnr genes and proteins and analysis of the qnrE1 surroundings showed that this gene belongs to a new qnr family and was likely mobilized by ISEcp1 from the chromosome of Enterobacter spp. to plasmids of K. pneumoniae.

Lateral dissemination and inter-patient transmission of blaKPC-3: role of a conjugative plasmid in spreading carbapenem resistance.

OBJECTIVES: The objective of this study was to describe the nosocomial spread of carbapenemase-producing enterobacteria and characterize a plasmid involved in KPC dissemination. METHODS: Two Klebsiella pneumoniae, one Escherichia coli and one Citrobacter freundii isolated from two patients were studied. Susceptibility profiles were obtained using Etest. Carbapenemase activity was detected using the Carba NP test. ß-Lactamase gene content was screened by PCR and sequencing. K. pneumoniae isolates were genotyped by MLST and PFGE. KPC plasmid sizes were estimated by S1-DNA digestion and PFGE-Southern blot. Plasmids were sequenced using Illumina's technology and Sanger sequencing. RESULTS: Two patients sharing a room on a surgical unit were positive for carbapenemase-producing K. pneumoniae. One patient was also colonized with carbapenemase-producing C. freundii and E. coli. Neither patient had known risk factors for carbapenemase acquisition, although one patient had recent surgery at another Toronto hospital; the other patient's husband had surgery in New York City 3 years prior to her presentation. An extensive investigation was conducted at both hospitals, but no additional cases were identified. blaKPC-3 was detected in all clinical isolates. Variable carbapenem resistance levels were observed. Both K. pneumoniae belonged to the same clone by PFGE and MLST (ST277). pKPC-SMH (∼ 53 kb) was identified in all the clinical isolates, showing identity only with structurally similar IncN plasmids. CONCLUSIONS: We describe intra- and inter-patient dissemination of blaKPC. The involvement of a clone related to the successful K. pneumoniae ST258 and the blaKPC-3 gene detected in an active Tn4401 transposon carried on a conjugative broad-host-range plasmid increased the potential for this horizontal transmission.

Population structure and antimicrobial resistance of invasive serotype IV group B Streptococcus, Toronto, Ontario, Canada.

We recently showed that 37/600 (6.2%) invasive infections with group B Streptococcus (GBS) in Toronto, Ontario, Canada, were caused by serotype IV strains. We report a relatively high level of genetic diversity in 37 invasive strains of this emerging GBS serotype. Multilocus sequence typing identified 6 sequence types (STs) that belonged to 3 clonal complexes. Most isolates were ST-459 (19/37, 51%) and ST-452 (11/37, 30%), but we also identified ST-291, ST-3, ST-196, and a novel ST-682. We detected further diversity by performing whole-genome single-nucleotide polymorphism analysis and found evidence of recombination events contributing to variation in some serotype IV GBS strains. We also evaluated antimicrobial drug resistance and found that ST-459 strains were resistant to clindamycin and erythromycin, whereas strains of other STs were, for the most part, susceptible to these antimicrobial drugs.

Characterization of multiple NDM-1-producing Enterobacteriaceae isolates from the same patient.

A male patient was admitted to a community hospital in Ontario, Canada, with an infected sacral ulcer after returning from India, where he was hospitalized. Carbapenem-resistant Escherichia coli (isolated from blood cultures), Enterobacter cloacae, and Providencia stuartii (from urine samples), all positive for bla(NDM-1), were recovered. Comparative NDM-1 plasmid analysis suggests both lateral plasmid transfer and independent acquisition of the bla(NDM-1) gene in these clinical isolates.

What Is the Appropriate Meropenem MIC for Screening of Carbapenemase-Producing Enterobacteriaceae in Low-Prevalence Settings?

Infection with carbapenemase-producing Enterobacteriaceae (CPE) has been shown to cause significant illness among hospitalized patients. Given the paucity of treatment options, there is a critical need to stop the spread of CPE. However, screening for the presence of CPE in laboratory settings has been challenging. In order to assess the effectiveness of current CPE detection guidelines, we analyzed the meropenem MIC distribution for a large set of clinical Enterobacteriaceae isolates. A total of 1,022 isolates submitted to the Public Health Ontario Laboratories (PHOL) from January 2011 to March 2014 were examined. Only isolates displaying a meropenem or ertapenem MIC of ≥ 0.25 or ≥ 1 µg/ml, respectively, were included. Carbapenemase-positive isolates were identified by multiplex PCR. We identified 189 isolates positive for carbapenemases, which primarily comprised NDM, KPC, and OXA-48-like carbapenemases, and these isolates were largely Klebsiella spp., Escherichia coli, and Enterobacter spp. Interestingly, 14 to 20% of these isolates displayed meropenem MICs within the susceptible range on the basis of CLSI and EUCAST breakpoint interpretive criteria. While the majority of meropenem-susceptible CPE isolates were observed to be E. coli, meropenem susceptibility was not exclusive to any one species/genus or carbapenemase type. Application of CLSI screening recommendations captured only 86% of carbapenemase-producing isolates, whereas application of EUCAST recommendations detected 98.4% of CPE isolates. In a region with a low carbapenemase prevalence, meropenem-based screening approaches require a cutoff MIC near the epidemiological wild-type threshold in order to achieve nearly optimal CPE identification.

Simplified Protocol for Carba NP Test for Enhanced Detection of Carbapenemase Producers Directly from Bacterial Cultures.

We compared carbapenemase detection among 266 Gram-negative bacilli (161 carbapenemase producers) using the Carba NP tests issued by the CLSI (CNPt-CLSI) and a novel protocol (CNPt-direct) designed for carbapenemase detection direct from bacterial cultures (instead of bacterial extracts required by the CLSI tests). The specificities were comparable (100%), but the CNPt-direct was more sensitive (98% versus 84%). The CNPt-direct was easier to perform due to the direct use of colonies and offered a more robust detection of carbapenemase producers.

Characterization of an Enterococcus gallinarum Isolate Carrying a Dual vanA and vanB Cassette.

The ability of vancomycin resistance determinants to be horizontally transferred within enterococci species is a concern. Identification and characterization of vancomycin-resistant enterococci (VRE) in a clinical isolate have a significant impact on infection control practices. In this study, we describe a clinical isolate of Enterococcus gallinarum exhibiting high-level resistance to vancomycin and teicoplanin. The genetic characterization of this isolate showed the presence of vanA and vanB genes in addition to the naturally carried vanC gene. vanA was identified on pA6981, a 35,608-bp circular plasmid with significant homology to plasmid pS177. The vanB operon was integrated into the bacterial chromosome and showed a high level of homology to previously reported Tn1549 and Tn5382. To the best of our knowledge, this is the first report of E. gallinarum carrying both vanA and vanB operons, indicating the importance of identifying the vancomycin resistance mechanism in non-E. faecium and non-E. faecalis enterococcal species.

Emergence of Serotype IV Group B Streptococcus Adult Invasive Disease in Manitoba and Saskatchewan, Canada, Is Driven by Clonal Sequence Type 459 Strains.

Serotype IV group B Streptococcus (GBS) is emerging in Canada and the United States with rates as high as 5% of the total burden of adult invasive GBS disease. To understand this emergence, we studied the population structure and assessed the antimicrobial susceptibility of serotype IV isolates causing adult invasive infection in Manitoba and Saskatchewan, Canada, between 2010 and 2014. Whole-genome sequencing was used to determine multilocus sequence typing information and identify genes encoding antimicrobial resistance in 85 invasive serotype IV GBS strains. Antimicrobial susceptibility testing was performed by standard methods. Strain divergence was assessed using genome-wide single-nucleotide polymorphism analysis. Serotype IV strains were responsible for 16.9% of adult invasive GBS infections in Manitoba and Saskatchewan during the period. The majority of serotype IV isolates (89%) were clonally related, tetracycline-, erythromycin-, and clindamycin-resistant sequence type 459 (ST459) strains that possessed genes tetM and ermTR. Genome comparisons between ST459 and serotype V ST1 GBS identified several areas of recombination in an overall similar genomic background. Serotype IV ST459 GBS strains are expanding and causing a substantial percentage of adult invasive GBS disease. This emergence may be linked to the acquisition of resistance to tetracycline, macrolides, and lincosamides.

Azithromycin resistance is coevolving with reduced susceptibility to cephalosporins in Neisseria gonorrhoeae in Ontario, Canada.

Azithromycin (AZM) is routinely recommended as a component of dual therapy for gonorrhea in combination with third-generation cephalosporins (3GC). In this study, we examined the prevalence of AZM-resistant (AZM(r)) Neisseria gonorrhoeae from July 2010 to February 2013, assessed the rate of concurrent cephalosporin resistance under the current treatment recommendations, and analyzed the clonal distribution of AZM(r) isolates in Ontario, Canada. Nineteen AZM(r) clinical isolates (one per patient; MIC, ≥2 µg/ml) were included in the study. Susceptibility profiles of these isolates to 11 antibiotics, molecular typing, characterization of macrolide resistance mechanisms, and penicillin-binding protein 2 (PBP2) patterns were determined for all the isolates. Two groups were defined based on AZM(r) level; group A isolates displayed high-level resistance (MIC, ≥2,048 µg/ml) due to mutations (A2143G) in the four copies of the 23S rRNA rrl gene, and group B isolates had moderate resistance to AZM (MICs, 2 to 8 µg/ml, C2599T mutation in the rrl gene), with a subgroup belonging to sequence type 3158 (ST3158) (n = 8), which also showed reduced susceptibility to 3GC (MICs, 0.12 to 0.25 µg/ml, PBP2 pattern XXXIV). This AZM(r) phenotype was not observed in previous provincial surveillance in 2008 (the ST3158 clone was found, with AZM MICs of 0.25 to 0.5 µg/ml associated with mtrR mutations). We hypothesized that the AZM mutant prevention concentration (MPC) in the ST3158 subpopulation we found in 2008 was higher than the MPC in wild-type isolates (AZM MIC, ≤0.031 µg/ml), increasing the chances of additional selection of AZM(r) mutations. Full AZM resistance is now emerging in this clone together with reduced susceptibility to 3GC, threatening the future efficacy of these antibiotics as therapeutic options for treatment of gonorrhea.

Antimicrobial activity of solithromycin against clinical isolates of Legionella pneumophila serogroup 1.

The activity of solithromycin was evaluated against clinical Legionella pneumophila serogroup 1 (Lp1) isolates (n = 196) collected in Ontario, Canada, from 1980 to 2011. Its in vitro activity was compared to that of azithromycin (AZM) using the broth microdilution method. Solithromycin had a MIC50 of ≤0.015 µg/ml and a MIC90 of 0.031 µg/ml, making its activity at least 8-fold to 32-fold higher than that of AZM (MIC50 and MIC90, 0.125 µg/ml and 1 µg/ml, respectively). Ninety-nine percent of the isolates had MICs for solithromycin ranging from ≤0.015 µg/ml to 0.031 µg/ml, whereas 83.6% of the isolates showed MICs for AZM ranging from 0.062 µg/ml to 0.25 µg/ml. Interestingly, 96.7% (30 out of 31 clinical isolates) identified with higher AZM MICs (0.5 µg/ml to 2 µg/ml) belonged to the clinically prevalent sequence type 1. To investigate the intracellular activity of solithromycin, in vitro invasion assays were also performed against a subset of representative Lp1 isolates internalized within human lung epithelial cells. Solithromycin and AZM both inhibited growth of all intracellular Lp1 isolates at 1× or 8× MICs, displaying bacteriostatic effects, as would be expected with protein synthesis inhibitor rather than bactericidal activity. Solithromycin demonstrated the highest in vitro and intracellular potency against all Lp1 isolates compared to AZM. Given the rapid spread of resistance mechanisms among respiratory pathogens and the reported treatment failures in legionellosis, the development of this new fluoroketolide, already in phase 3 oral clinical studies, constitutes a promising alternative option for the treatment of legionellosis.