Sensitive and Specific Loop-Mediated Isothermal Amplification Assays for Detection of Salmonella, CTX-M-1 Group Genes, mph(A), and ermB in Stool and Blood Samples Based on Orange to Green Visible Dye.

Salmonella is a globally prevalent foodborne bacterium, and ceftriaxone and azithromycin have been regarded as drugs of choice for treating Salmonella infections, particularly in children. With the growing incidence of ceftriaxone and azithromycin resistance in Salmonella, there is an urgent requirement for a rapid and dependable gene testing approach to enhance the efficacy of treating Salmonella infections. Utilizing the orange to green visible dye approach, this study developed loop-mediated isothermal amplification (LAMP) assays for the sensitive and specific detection of Salmonella, ceftriaxone and azithromycin resistance genes (including CTX-M-1 group, mph(A), and ermB genes) in stool and blood samples. The specificity and sensitivity of primers during the LAMP assays for detection of Salmonella, CTX-M-1 group, mph(A), and ermB genes were determined in this study. The detection threshold for Salmonella was found to be 1.5 × 103 colony-forming units (CFU)/mL, while it was 1.5 × 102 CFU/mL for CTX-M-1 group genes (including blaCTX-M-3, blaCTX-M-15, and blaCTX-M-55), 1.5 × 102 CFU/mL for mph(A), and 1.5 × 102 CFU/mL for ermB, showing 10-103-fold, 103-fold, and 105-fold increased sensitivity compared with the polymerase chain reaction assay, respectively. Results indicated that the LAMP primers designed for Salmonella, CTX-M-1 group, mph(A), and ermB genes possess high specificity (100%) and sensitivity (over 94%). This novel approach advocates its application in detecting Salmonella, CTX-M-1 group, mph(A), and ermB genes.

Resistance and genomic characterization of a plasmid pkh2101 harbouring erm(B) isolated from emerging fish pathogen Lactococcus garvieae serotype II in Japan.

The emergence of antibiotic-resistant pathogenic strains of Lactococcus garvieae serotype II isolated from fish in Japan has become a growing concern in recent years. The data on drug susceptibility and its associated resistance mechanism are limited. Therefore, the present study was conducted to determine the minimum inhibitory concentrations (MICs) of chemotherapeutic agents against 98 pathogenic strains of emerging Lactococcus garvieae serotype II isolated from fish from six different prefectures in Japan from 2018 to 2021. The tested strains were resistant to erythromycin, lincomycin and tiamulin. PCR amplification revealed the presence of erm(B) in all erythromycin-resistant strains, while a conjugation experiment confirmed that these strains carried erm(B) that could be transferred to recipient Enterococcus faecalis OG1RF with frequencies from 10-4 to 10-6 per donor cells. Nucleotide sequencing of the representative isolated plasmid pkh2101 from an erythromycin-resistant strain showed that it was a 26,850 bp molecule with an average GC content of 33.49%, comprising 31 CDSs, 13 of which remained without any functional annotation. Comparative genomic analysis suggested that pkh2101 shared the highest similarity (97.57% identity) with the plasmid pAMbeta1, which was previously isolated clinically from Enterococcus faecalis DS-5. This study provides potential evidence that the plasmid harbouring erm(B) could be a source of antibiotic resistance transmission in emerging L. garvieae infection in aquaculture.

Development of Two Loop-Mediated Isothermal Amplification Assays for Rapid Detection of ermB and mefA Genes in Streptococcus suis.

Streptococcus suis is an important zoonotic pathogen that poses a serious threat to the pig industry and human health. The massive use of macrolides has led to the emergence of resistance in S. suis, and S. suis is suspected to be a reservoir of antimicrobial resistance genes. The mechanism to macrolide resistance in S. suis is mainly due to ermB and mefA. In this study, loop-mediated isothermal amplification (LAMP) methods were developed to detect ermB and mefA genes in S. suis through turbidimetry detection. The sensitivity and specificity of the LAMP reactions were determined. All results of LAMP and polymerase chain reaction (PCR) assay were compared to determine whether LAMP method was accurate and reliable. The results showed that all 100 nonstreptococcus clinical isolates tested negative, indicating the high specificity of LAMP assays. The detection limit of LAMP assay was 1 fg per reaction, and 102-104-fold lower than those of conventional PCR methods. Evaluation of the performance of the LAMP assay in S. suis clinical strains revealed a good consistency between LAMP and PCR assays. In conclusion, LAMP assays are specific, sensitive, and rapid methods to detect ermB and mefA in S. suis.

Genomic Insights of First ermB-Positive ST338-SCCmecVT/CC59 Taiwan Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus in Poland.

We report the first Polish representative of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), lukS/F-PV-positive, encoding the ermB gene, as a genetic determinant of constitutive resistance to macrolides, lincosamides, and streptogramin B antibiotics, cMLS-B. This is the first detection of the CA-MRSA strain responsible for nosocomial infection in the Warsaw Clinical Hospital. Resistance to ß-lactams associates with a composite genetic element, SCCmec cassette type VT (5C2&5). We assigned the strain to sequence type ST338 (single-locus variant of ST59), clonal complex CC59, spa-type t437, and agr-type I. Genomic-based comparison was designated SO574/12 as an international Taiwan clone, which has been so far described mainly in the Asia-Pacific region. The ermB gene locates on the chromosome within the 14,690 bp mobile element structure, i.e., the MESPM1-like structure, which also encodes aminoglycoside- and streptothricin-resistance genes. The MESPM1-like structure is a composite transposon containing Tn551, flanked by direct repeats of IS1216V insertion sequences, which probably originates from Enterococcus. The ermB is preceded by the 273 bp regulatory region that contains the regulatory 84 bp ermBL ORF, encoding the 27 amino acid leader peptides. The latest research suggests that a new leader peptide, ermBL2, also exists in the ermB regulatory region. Therefore, the detailed function of ermBL2 requires further investigations.

Molecular basis of macrolide-lincosamide-streptogramin (MLS) resistance in Finegoldia magna clinical isolates.

Of 69 clinical isolates of Finegoldia magna tested, 36% presented high-level MICs of erythromycin (>256 µg/ml), harboring erm(A) (n = 20) or erm(B) (n=5). Of nine isolates exhibiting an inducible resistance phenotype to macrolides-lincosamides-streptogramins B, four (44%) were susceptible with a potential risk of treatment failure due to emergence of resistant mutants.

Emerging erm(B)-Mediated Macrolide Resistance Associated with Novel Multidrug Resistance Genomic Islands in Campylobacter.

The rapid dissemination of the macrolide resistance gene erm(B) will likely compromise the efficacy of macrolides as the treatment of choice for campylobacteriosis. More importantly, erm(B) is always associated with several multidrug resistance genomic islands (MDRGIs), which confer resistance to multiple other antimicrobials. Continuous monitoring of the emergence of erm(B) and analysis of its associated genetic environments are crucial for our understanding of macrolide resistance in Campylobacter In this study, 290 Campylobacter isolates (216 Campylobacter coli isolates and 74 Campylobacter jejuni isolates) were obtained from 1,039 fecal samples collected in 2016 from pigs and chickens from three regions of China (344 samples from Guangdong, 335 samples from Shanghai, and 360 samples from Shandong). Overall, 74 isolates (72 C. coli isolates and 2 C. jejuni isolates) were PCR positive for erm(B). Combined with data from previous years, we observed a trend of increasing prevalence of erm(B) in C. coli Pulsed-field gel electrophoresis analyses suggested that both clonal expansion and horizontal transmission were involved in the dissemination of erm(B) in C. coli, and three novel types of erm(B)-associated MDRGIs were identified among the isolates. Furthermore, 2 erm(B)-harboring C. jejuni isolates also contained an aminoglycoside resistance genomic island and a multidrug-resistance-enhancing efflux pump, encoded by RE-cmeABC Antimicrobial susceptibility testing showed that most of the isolates were resistant to all clinically important antimicrobial agents used for the treatment of campylobacteriosis. These findings suggest that the increasing prevalence of erm(B)-associated MDRGIs might further limit treatment options for campylobacteriosis.

Short-term and long-term impacts of Helicobacter pylori eradication with reverse hybrid therapy on the gut microbiota.

BACKGROUND AND AIMS: Anti-Helicobacter pylori therapy may lead to the growth of pathogenic or antibiotic-resistant bacteria in the gut. The study aimed to investigate the short-term and long-term impacts of H. pylori eradication with reverse hybrid therapy on the components and macrolide resistance of the gut microbiota. METHODS: Helicobacter pylori-related gastritis patients were administered a 14-day reverse hybrid therapy. Fecal samples were collected before treatment and at the end of week 2, week 8, and week 48. The V3-V4 region of the bacterial 16S rRNA gene in fecal specimens was amplified by polymerase chain reaction and sequenced on Illumina MiSeq platform. Additionally, amplification of erm(B) gene (encoding erythromycin resistance methylase) was performed. RESULTS: Reverse hybrid therapy resulted in decreased relative abundances of Firmicutes (from 62.0% to 30.7%; P < 0.001) and Actinobacteria (from 3.4% to 0.6%; 0.032) at the end of therapy. In contrast, the relative abundance of Proteobacteria increased from 10.2% to 49.1% (0.002). These microbiota alterations did not persist but returned to the initial levels at week 8 and week 48. The amount of erm(B) gene in fecal specimens was comparable with the pretreatment level at week 2 but increased at week 8 (0.025) and then returned to the pretreatment level by week 48. CONCLUSIONS: Helicobacter pylori eradication with reverse hybrid therapy can lead to short-term gut dysbiosis. The amount of erm(B) gene in the stool increased transiently after treatment and returned to the pretreatment level at 1-year post-treatment.

Lack of Evidence for erm(B) Infiltration Into Erythromycin-Resistant Campylobacter coli and Campylobacter jejuni from Commercial Turkey Production in Eastern North Carolina: A Major Turkey-Growing Region in the United States.

In Campylobacter spp., resistance to erythromycin and other macrolides has typically implicated ribosomal mutations, especially substitutions in the 23S rRNA genes. However, in 2014, the macrolide resistance gene erm(B) was reported for the first time in Campylobacter and shown to be harbored by a multidrug resistance island in the chromosome of the swine-derived strain Campylobacter coli ZC113. erm(B)-positive C. coli and Campylobacter jejuni strains from the food supply have been mostly reported from China. However, erm(B)-positive C. coli isolates were also detected recently in fecal samples from turkeys in Spain. To determine whether erm(B) may be harbored by erythromycin-resistant Campylobacter from commercial turkey production in eastern North Carolina, a major turkey-growing region in the United States, we investigated a panel of 178 erythromycin-resistant isolates (174 C. coli, 4 C. jejuni) using PCR with erm(B)-specific primers. None of the isolates were PCR-positive for erm(B) and sequence analysis of a subset of these erythromycin-resistant isolates revealed that all harbored A2075G substitutions in the 23S rRNA genes. Data fail to provide evidence for infiltration of erm(B) into erythromycin-resistant Campylobacter from commercial turkey production in this region and suggest the need for continuing surveillance.

The Current State of Macrolide Resistance in Campylobacter spp.: Trends and Impacts of Resistance Mechanisms.

Campylobacter spp., especially Campylobacter jejuni and C. coli, are leading bacterial foodborne pathogens worldwide. In the United States, an estimated 0.8 million cases of campylobacteriosis occur annually, mostly involving C. jejuni Campylobacteriosis is generally self-limiting, but in severe cases, treatment with antibiotics may be mandated. The increasing incidence of fluoroquinolone resistance in Campylobacter has rendered macrolides such as erythromycin and azithromycin the drugs of choice for human campylobacteriosis. The prevalence of macrolide resistance in C. jejuni remains low, but macrolide resistance can be common in C. coli Substitutions in the 23S rRNA gene, specifically A2075G, and less frequently A2074C/G, remain the most common mechanism for high-level resistance to macrolides. In C. jejuni, resistance mediated by such substitutions is accompanied by a reduced ability to colonize chickens and other fitness costs, potentially contributing to the low incidence of macrolide resistance. Interestingly, similar fitness impacts have not been noted in C. coli Also noteworthy is a novel mechanism first reported in 2014 for a C. coli isolate from China and mediated by erm(B) harbored on multidrug resistance genomic islands. The incidence of erm(B) appears to reflect clonal expansion of certain strains, and whole-genome sequencing has been critical to the elucidation of erm(B)-associated macrolide resistance in Campylobacter spp. With the exception of one report from Spain, erm(B)-mediated macrolide resistance has been restricted to Campylobacter spp., mostly C. coli, of animal and human origin from China. If erm(B)-mediated macrolide resistance does not confer fitness costs in C. jejuni, the range of this gene may expand in C. jejuni, threatening to compromise treatment effectiveness for severe campylobacteriosis cases.

Molecular subtyping and erythromycin resistance of Campylobacter in China.

AIMS: To investigate the erythromycin resistance patterns and mechanism for Campylobacter isolates in China. METHODS AND RESULTS: The minimum inhibitory concentrations of erythromycin on 858 Chinese Campylobacter isolates were analysed. PCR and DNA sequencing were used to identify mutations in the 23S rRNA and the presence of the ermB gene in the 158 erythromycin resistance isolates (18·4%). About 83% (131/158) had A2075G mutation in their 23S rRNA; no A2074C/G mutants were found. The ermB gene was identified in 30 Campylobacter coli isolates (19%). Four types of multidrug-resistant gene islands (MDRGIs) were found. Fifty-three types were identified by multilocus sequence typing among the resistant isolates. All isolates of STs 6322 and 1145 had the ermB gene. CONCLUSIONS: The erythromycin resistance rate of Camp. coli (58·56%) was much higher than Campylobacter jejuni (0·67%). The insertion sites between cadF and CCO1582 and between nfsB and cinA on the chromosome might be hot spots for MDRGI transformation. SIGNIFICANCE AND IMPACT OF THE STUDY: Point mutation in domain V of the 23S rRNA and the ermB gene accounted for 100% of the erythromycin resistance of Campylobacter in China.

Improving the reproducibility of the NAP1/B1/027 epidemic strain R20291 in the hamster model of infection.

Comparative analysis of the Clostridium difficile BI/NAP1/027 strain R20291 and ClosTron-derived ermB mutants in the hamster infection model are compromised by the clindamycin susceptibility of the parent. Mutants can appear more virulent. We have rectified this anomaly by genome engineering. The variant created (CRG20291) represents an ideal control strain for virulence assays of ClosTron mutants.

Inducible Expression of both ermB and ermT Conferred High Macrolide Resistance in Streptococcus gallolyticus subsp. pasteurianus Isolates in China.

Streptococcus gallolyticus subsp. pasteurianus is an under-recognized pathogen and zoonotic agent causing opportunistic infections in humans. Despite increasing recognition of this subspecies as a cause for human infectious diseases, limited information is known about its antibiotic resistance mechanism. In this study, we aim to identify the molecular mechanism underlying the high macrolide resistance of six S. gallolyticus subsp. pasteurianus isolates from dead ducklings collected in several natural outbreaks in China during 2010-2013. All isolates exhibited multi-drug resistance including high macrolide resistance (MIC ≥ 1024 mg/L for erythromycin, and 512 mg/L for clarithromycin). Efflux-encoding mefA and mefE genes were not detectable in these isolates. The presence of 23S rRNA mutations in specific isolates did not significantly change macrolide MICs. No nucleotide substitutions were found in genes encoding ribosomal proteins L4 or L22. The ermB and ermT genes were found in the genomes of all isolates. These two genes were acquired independently in one highly virulent isolate AL101002, and clustered with Tn916 and IS1216, respectively. The expression of both ermB and ermT in all isolates was erythromycin inducible and yielded comparable macrolide MICs in all six isolates. Taken together, inducible expression of both ermB and ermT conferred high macrolide resistance in these S. gallolyticus subsp. pasterianus isolates. Our findings reveal new macrolide resistance features in S. gallolyticus subsp. pasteurianus by both ermB and ermT.

What causes decreased erythromycin resistance in Streptococcus pyogenes? Dynamics of four clones in a southern European region from 2005 to 2012.

OBJECTIVES: To survey antibiotic resistance among Streptococcus pyogenes isolates collected from 2005 to 2012, to characterize those showing erythromycin resistance and to analyse the association of certain emm types with erythromycin resistance or susceptibility. METHODS: Resistance determinants or mutations conferring erythromycin, clindamycin, tetracycline and fluoroquinolone resistance were analysed. All erythromycin-resistant isolates and a sample of erythromycin-susceptible isolates were emm typed. Multilocus sequence typing was performed for representative emm types. RESULTS: Antimicrobial susceptibility was studied for 12 346 S. pyogenes isolates. Erythromycin, clindamycin and tetracycline resistance showed a decreasing trend. In 2012, 2.8% of isolates were erythromycin resistant versus 7.5% in 2005 and 11.7% in 2006. Although 21 clones were involved, 4 clones accounted for almost 90% of erythromycin-resistant isolates. The emm12/ST36 clone, carrying the mef(A) gene, was the predominant (41.1%) erythromycin-resistant clone, with an incidence peak in 2008, followed by a gradual decline. The M phenotype predominated each year except for 2005, when two of the main erythromycin-resistant clones (emm11/ST403 and emm28/ST52) harboured an erm(B) gene. Erythromycin resistance was significantly higher in adults than in children. Skin isolates showed the highest erythromycin resistance rate; among these, perianal isolates frequently belonged to the emm28/ST52 clone. The emm type was not a predictor of erythromycin resistance; however, most emm11 and emm12 were erythromycin-resistant isolates. Macrolide consumption was similar throughout the study period. Only two isolates with a high level of levofloxacin resistance were detected. CONCLUSIONS: Resistance was mainly related to the circulation of emm12/ST36, emm11/ST403, emm28/ST52 and emm4/ST39 clones, all of which declined throughout the study period.

Temporal and spatial changes in the abundance of antibiotic resistance gene markers in a wastewater treatment plant.

In this study, we investigated the temporal and spatial quantitative changes in the concentration of antibiotic resistance gene (ARG) markers in a municipal wastewater treatment plant (WWTP). Four ARGs conferring resistance to different classes of antibiotics (ermB, sul1, tet[W], and blaCTXM) and a gene used as a proxy for ARG pollution (intl1) were quantified in two separate sampling campaigns covering two and half years of operation of the WWTP. First, a systematic monthly monitoring of multiple points in the inlet and the outlet revealed an absolute decrease in the concentration of all analyzed ARGs. However, the relative abundance of sul1 and intl1 genes relative to the total bacterial load (estimated using the universal marker 16S rDNA) increased in the outlet samples as compared to the inlet. To pinpoint the exact stage of removal and/or enrichment within the WWTP, a second sampling including the stages of the biological treatment was performed bimonthly. This revealed a distinct enrichment of sul1 and intl1 genes during the biological treatment phase. Moreover, the temporal and spatial variations in ARG abundance patterns within the WWTP underscored the complexity of the dynamics associated with the removal of ARGs during wastewater treatment. Understanding these dynamics is pivotal for developing efficient strategies to mitigate the dissemination of ARGs in aquatic environments. PRACTITIONER POINTS: Regular monitoring of ARG markers in WWTPs is essential to assess temporal and spatial changes, aiding in the development of effective mitigation strategies. Understanding the dynamics of ARG abundance during biological treatment is crucial for optimizing processes and minimizing dissemination in aquatic environments. Increased relative abundance of certain ARGs highlights potential enrichment during wastewater treatment, necessitating targeted interventions. Systematic monitoring of multiple points within WWTPs can provide valuable insights into the efficacy of treatment processes in reducing ARG levels over time. The complexity of ARG abundance patterns underscores the need to develop holistic approaches to tackle antibiotic resistance in wastewater systems.

Characterization of azithromycin-resistant Shigella flexneri serotype 2a isolates using whole genome sequencing in Ontario from 2016 to 2018.

Azithromycin-resistant shigellosis is increasing globally. This retrospective analysis of Shigella flexneri serotype 2a isolates from 2016 to 2018 in Ontario found nearly half were azithromycin (47.7%, 72/151) and ciprofloxacin (50.7%, 77/152) resistant. Moreover, 34.7% (25/72) of azithromycin-resistant isolates were also ciprofloxacin-resistant. Four isolates were ceftriaxone-resistant, although all azithromycin-resistant isolates were ceftriaxone-susceptible. Overall, 83.6% (127/152) of all S. flexneri 2a isolates were recovered from males and 97.2% (70/72) of the azithromycin-resistant cases were males. Among the azithromycin-resistant cases, some (8/72) reported international travel. Phylogenetic analysis of azithromycin-resistant isolates revealed two large male-dominated clusters, and one cluster may have been due to importation of resistant strain. Comparison of plasmids isolated from the clusters in Ontario revealed the presence of incFII plasmid with high percentage of similarity to plasmids present in global outbreaks affecting mostly males including men who have sex with men (MSM). These two large azithromycin-resistant clusters are suggestive of an outbreak among MSM, though disease exposure or sexual orientation of patients was unknown. The presence of plasmid-borne azithromycin resistance in ciprofloxacin-resistant isolates is a public health concern. Antimicrobial surveillance is important for patient management, understanding the spread of novel resistance types in local communities which sometimes is introduced by travel. We found ongoing multidrug-resistant outbreaks spanning multiple years affecting males. Reduction of future outbreaks in high-risk communities like MSM requires consorted information flow between laboratory, public health, and physicians. We impart genomic and antimicrobial characteristics of multidrug S. flexneri 2a which may serve as reference by clinicians and public health.IMPORTANCEOral ciprofloxacin and azithromycin are generally considered as the first-line therapy of shigellosis. Here, we report the emergence and transmission of azithromycin and ciprofloxacin-resistant S. flexneri serotype 2a among male adults in Ontario during 2016-2018. The percentage of azithromycin and ciprofloxacin resistance among S. flexneri 2a is higher compared to previous reports from Canada and United States. Here, we show the genetic basis of the antimicrobial resistance among these unique groups of S. flexneri 2a isolates. We describe a domestically acquired azithromycin-resistant and ciprofloxacin-resistant S. flexneri 2a lineage in Ontario. Combining whole-genome sequencing (WGS) data with travel-associated data helped in understanding dissemination and transmission. We employed WGS, which not only helped us in understanding the genetic-relationship between isolates but also mine information regarding plasmids. In the future, linking WGS, travel-related data, and clinical data can provide enhanced contact tracing and improve public-health management.

Machine learning-based classification reveals distinct clusters of non-coding genomic allelic variations associated with Erm-mediated antibiotic resistance.

The erythromycin resistance RNA methyltransferase (erm) confers cross-resistance to all therapeutically important macrolides, lincosamides, and streptogramins (MLS phenotype). The expression of erm is often induced by the macrolide-mediated ribosome stalling in the upstream co-transcribed leader sequence, thereby triggering a conformational switch of the intergenic RNA hairpins to allow the translational initiation of erm. We investigated the evolutionary emergence of the upstream erm regulatory elements and the impact of allelic variation on erm expression and the MLS phenotype. Through systematic profiling of the upstream regulatory sequences across all known erm operons, we observed that specific erm subfamilies, such as ermB and ermC, have independently evolved distinct configurations of small upstream ORFs and palindromic repeats. A population-wide genomic analysis of the upstream ermB regions revealed substantial non-random allelic variation at numerous positions. Utilizing machine learning-based classification coupled with RNA structure modeling, we found that many alleles cooperatively influence the stability of alternative RNA hairpin structures formed by the palindromic repeats, which, in turn, affects the inducibility of ermB expression and MLS phenotypes. Subsequent experimental validation of 11 randomly selected variants demonstrated an impressive 91% accuracy in predicting MLS phenotypes. Furthermore, we uncovered a mixed distribution of MLS-sensitive and MLS-resistant ermB loci within the evolutionary tree, indicating repeated and independent evolution of MLS resistance. Taken together, this study not only elucidates the evolutionary processes driving the emergence and development of MLS resistance but also highlights the potential of using non-coding genomic allele data to predict antibiotic resistance phenotypes. IMPORTANCE: Antibiotic resistance (AR) poses a global health threat as the efficacy of available antibiotics has rapidly eroded due to the widespread transmission of AR genes. Using Erm-dependent MLS resistance as a model, this study highlights the significance of non-coding genomic allelic variations. Through a comprehensive analysis of upstream regulatory elements within the erm family, we elucidated the evolutionary emergence and development of AR mechanisms. Leveraging population-wide machine learning (ML)-based genomic analysis, we transformed substantial non-random allelic variations into discernible clusters of elements, enabling precise prediction of MLS phenotypes from non-coding regions. These findings offer deeper insight into AR evolution and demonstrate the potential of harnessing non-coding genomic allele data for accurately predicting AR phenotypes.

Loss of erm(B)-Mediated rRNA Dimethylation and Restoration of Erythromycin Susceptibility in Erythromycin-Resistant Enterococci Following Induced Linezolid Resistance.

Linezolid has been reported to restore erythromycin susceptibility in erythromycin-resistant Staphylococcus aureus. This phenomenon has not been reported in enterococci and the mechanisms involved therein are still unknown. The purpose of this study was to investigate the mechanisms involved and the effect of combining linezolid with erythromycin on erythromycin-resistant enterococci. Checkerboard techniques were used to determine drug interactions, and 12 of 14 isolates showed a synergistic effect between erythromycin and linezolid (fractional inhibitory concentration <0.5). We observed that the erm(B) gene, which encodes a dimethyltransferase responsible for erythromycin resistance, was expressed from transposon Tn1545 in the tested erythromycin-resistant enterococci. After exposure to linezolid, erm(B)-mediated rRNA dimethylation at A2071 could not be detected, and the erm(B) gene was lost following acquisition of erythromycin susceptibility. Thus, in conclusion, linezolid combined with erythromycin exerts a synergistic effect against erythromycin-resistant enterococci. Linezolid treatment suppressed erm(B)-mediated rRNA dimethylation at A2071, which could lead to loss of the erm(B) gene.

Erythromycin resistance of clinical Campylobacter jejuni and Campylobacter coli in Shanghai, China.

Campylobacter species are zoonotic pathogens, as well as the prevalent cause of foodborne bacterial gastroenteritis. The spread of antimicrobial-resistant strains poses a serious threat to global public health and attracts attention worldwide, but information about clinical Campylobacter is relatively limited compared to isolates from food and animals. The current study illustrated the prevalence and antimicrobial resistance profiles of Campylobacter jejuni and Campylobacter coli isolates collected from a consecutive surveillance program between 2012 and 2019 in Shanghai, China, using antimicrobial susceptibility testing and whole-genome sequencing. Among the 891 Campylobacter strains (761 C. jejuni and 130 C. coli) isolates collected, high portions above 90% of resistance to ciprofloxacin, nalidixic acid, and tetracycline were observed for both C. jejuni and C. coli. The most common MDR profiles represented by C. jejuni and C. coli were combination of ciprofloxacin, tetracycline, florfenicol and nalidixic acid (5.39%), and azithromycin, ciprofloxacin, erythromycin, gentamicin, tetracycline, clindamycin, nalidixic acid (28.46%), respectively. The erythromycin resistance of C. coli (59.23%) is higher than C. jejuni (2.50%). A total of 76 erythromycin resistant isolates (16 C. jejuni and 60 C. coli) were sequenced using Illumina platform for determining the genotypes, antimicrobial resistance patterns and phylogeny analysis. Multilocus sequence typing (MLST) analysis showed a high genetic diversity with 47 sequence types (STs), including 4 novel alleles and 12 new STs. The most abundant clonal complexes (CCs) were CC-403 (31.25%) and CC-828 (88.33%) for C. jejuni and C. coli, respectively. Among the 76 erythromycin-resistant isolates, mutation A2075G in 23S rRNA and erm(B) gene were detected in 53.95 and 39.47%, respectively. The erm(B) gene was identified exclusively in 30 C. coli isolates. All these erm(B) positive isolates were multi-drug resistant. Furthermore, comparison of the erm(B)-carrying isolates of multiple sources worldwide demonstrated the possibility of zoonotic transmission of erm(B) in Campylobacter. These findings highlight the importance of continuous surveillance of erythromycin resistance dissemination in Campylobacter which may compromise the effectiveness of antimicrobial therapy.

Clostridioides difficile infections were predominantly driven by fluoroquinolone-resistant Clostridioides difficile ribotypes 176 and 001 in Slovakia in 2018-2019.

AIM: To investigate the epidemiology of Clostridioides difficile infection (CDI) in Slovakian hospitals after the emergence of ribotype 176 (027-like) in 2016. METHODS: Between 2018 and 2019, European Centre for Disease Control and Prevention CDI surveillance protocol v2.3 was applied to 14 hospitals, with additional data collected on recent antimicrobial use and the characterization of C. difficile isolates. RESULTS: The mean hospital incidence of CDI was 4.1 cases per 10,000 patient bed-days. One hundred and five (27.6%) in-hospital deaths were reported among the 381 cases. Antimicrobial treatment within the previous 4 weeks was recorded in 90.5% (333/368) of cases. Ribotype (RT)176 was detected in 50% (n=185/370, 14 hospitals) and RT001 was detected in 34.6% (n=128/370,13/14 hospitals) of cases with RT data. Overall, 86% (n=318/370) of isolates were resistant to moxifloxacin by Thr82Ile in GyrA (99.7%). Multi-locus variable tandem repeat analysis showed clonal relatedness of predominant RTs within and between hospitals. Seven of 14 sequenced RT176 isolates and five of 13 RT001 isolates showed between zero and three allelic differences by whole-genome multi-locus sequence typing. The majority of sequenced isolates (24/27) carried the erm(B) gene and 16/27 also carried the aac(6')-aph(2'') gene with the corresponding antimicrobial susceptibility phenotypes. Nine RT176 strains carried the cfr(E)gene and one RT001 strain carried the cfr(C) gene, but without linezolid resistance. CONCLUSIONS: The newly-predominant RT176 and endemic RT001 are driving the epidemiology of CDI in Slovakia. In addition to fluoroquinolones, the use of macrolide-lincosamide-streptogramin B antibiotics can represent another driving force for the spread of these epidemic lineages. In C. difficile, linezolid resistance should be confirmed phenotypically in strains with detected cfr gene(s).