Rapid fluoroquinolone resistance detection in Pseudomonas aeruginosa using mismatch amplification mutation assay-based real-time PCR

Antimicrobial resistance (AMR) is an ever-increasing global health concern. Here, we developed two SYBR Green-based mismatch amplification mutation assays (SYBR-MAMAs) targeting GyrA T83I, D87N, D87Y, and D87H, the predominant causes of fluoroquinolone AMR in Pseudomonas aeruginosa. SYBR-MAMAs were tested on 85 isolates, 45 with intermediate/full ciprofloxacin resistance. Assays identified the correct phenotype in 84% intermediate/full resistant and 100% sensitive strains. Clinical implementation of our rapid SYBR-MAMAs will permit timely treatment alterations to improve patient outcomes.

Antibiotic resistance among invasive Neisseria meningitidis isolates in England, Wales and Northern Ireland (2010/11 to 2018/19)

Invasive meningococcal disease (IMD), caused by Neisseria meningitidis, can have a fatality rate as high as 10%, even with appropriate treatment. In the UK, penicillin is administered to patients in primary care whilst third generation cephalosporins, cefotaxime and ceftriaxone, are administered in secondary care. The first-choice antibiotic for chemoprophylaxis of close contacts is ciprofloxacin, followed by rifampicin. Immunocompromised individuals are often recommended antibiotic chemoprophylaxis and vaccination due to a greater risk of IMD. Resistance to antibiotics among meningococci is relatively rare, however reduced susceptibility and resistance to penicillin are increasing globally. Resistance to third generation cephalosporins is seldom reported, however reduced susceptibility to both cefotaxime and ceftriaxone has been observed. Rifampicin resistance has been reported among meningococci, mainly following prophylaxis, and ciprofloxacin resistance, whilst uncommon, has also been reported across the globe. The Public Health England Meningococcal Reference Unit receives and characterises the majority of isolates from IMD cases in England, Wales and Northern Ireland. This study assessed the distribution of antibiotic resistance to penicillin, rifampicin, ciprofloxacin and cefotaxime among IMD isolates received at the MRU from 2010/11 to 2018/19 (n = 4,122). Out of the 4,122 IMD isolates, 113 were penicillin-resistant, five were ciprofloxacin-resistant, two were rifampicin-resistant, and one was cefotaxime-resistant. Penicillin resistance was due to altered penA alleles whilst rifampicin and ciprofloxacin resistance was due to altered rpoB and gyrA alleles, respectively. Cefotaxime resistance was observed in one isolate which had an altered penA allele containing additional mutations to those harboured by the penicillin-resistant isolates. This study identified several isolates with resistance to antibiotics used for current treatment and prophylaxis of IMD and highlights the need for continued surveillance of resistance among meningococci to ensure continued effective use.

Antibiotic resistance profile of E. coli isolates in 17 municipal wastewater utilities across Oregon

Wastewater treatment utilities are considered one of the main sources and reservoirs of antimicrobial resistance. The objective of this study was to determine the diversity and prevalence of antibiotic-resistant Escherichia coli in wastewater treatment systems across the state of Oregon. Influent, secondary effluent, final effluent, and biosolids were collected from 17 wastewater treatment utilities across Oregon during the winter and summer seasons of 2019 and 2020 (n = 246). E. coli strains were recovered from samples by culturing on mTEC, followed by confirmation with MacConkey with MUG agar plates. Antibiotic susceptibility of 1143 E. coli isolates against 8 antibiotics were determined, and resistance profiles and indices were analyzed between utilities, seasons, and flows. Antibiotic resistance phenotypes were detected in 31.6% of the collected E. coli isolates. Among those antibiotic-resistant E. coli isolates, multi-drug resistance (i.e., resistance to three or more classes of antibiotics) was harbored by 27.7% with some strains showing resistance to up to six classes of antibiotics. The most prevalent resistance was to ampicillin (n = 207) and the most common combinations of multi-drug resistance included simultaneous resistances to ampicillin, streptomycin, and tetracycline (n = 49), followed by ampicillin, streptomycin, and sulfamethoxazole/trimethoprim (n = 46). Significant correlations were observed between resistance to sulfamethoxazole/trimethoprim and resistances to ampicillin, ciprofloxacin, and tetracycline (p < 0.001). A small percentage (1.1%) of the E. coli isolates displayed extended-spectrum beta lactamase (ESBL) activity and a single isolate carried resistance to imipenem. Compared to wastewater influent, ciprofloxacin resistance was significantly more prevalent in biosolids (p <0.05) and tetracycline resistance was significantly lower in effluent (p <0.05). Seasonal impact on antibiotic-resistant E. coli in wastewater influent was observed through significantly higher multiple antibiotic resistance (MAR) index, ampicillin resistance prevalence, and ciprofloxacin resistance prevalence in summer compared to winter (p < 0.05). This state-wide study confirms the widespread distribution of antibiotic-resistant, multi-drug resistant, and extended-spectrum beta lactamase-producing E. coli in wastewater systems across different flows and seasonal variations, making them the recipients, reservoirs, and sources of antimicrobial resistance.

Australian Gonococcal Surveillance Programme Annual Report, 2020

The Australian Gonococcal Surveillance Programme (AGSP), established in 1981, has continuously monitored antimicrobial resistance in clinical isolates of Neisseria gonorrhoeae for more than 40 years. In 2020, a total of 7,222 clinical isolates of gonococci from patients in the public and private sectors, in all jurisdictions, were tested for in vitro antimicrobial susceptibility by standardised methods. Current treatment recommendations for gonorrhoea, for the majority of Australia, continues to be dual therapy with ceftriaxone and azithromycin. In 2020, decreased susceptibility (DS) to ceftriaxone (minimum inhibitory concentration [MIC] value ≥ 0.06 mg/L) was found nationally in 0.9% of isolates. There was one isolate, reported from Victoria in 2020, that was resistant to ceftriaxone (MIC value ≥ 0.25 mg/L). Resistance to azithromycin (MIC value ≥ 1.0 mg/L) was found nationally in 3.9% of N. gonorrhoeae isolates, continuing a downward trend observed and reported since 2017. Isolates with high-level resistance to azithromycin (MIC value ≥ 256 mg/L) are identified sporadically in Australia; in 2020, there was one such isolate reported in Queensland. In 2020, penicillin resistance was found in 27% of gonococcal isolates nationally, and ciprofloxacin resistance in 36%; however, there is considerable variation by jurisdiction. In some remote settings, penicillin resistance remains low, and this drug continues to be recommended as part of an empiric therapy strategy. In 2020, in remote Northern Territory, no penicillin resistance was reported, and in remote Western Australia 5/116 of gonococcal isolates (4.3%) were penicillin resistant. There was one ciprofloxacin-resistant isolate reported from remote Northern Territory, and ciprofloxacin resistance rates remain comparatively low in remote Western Australia (4/116; 3.4%).

A Novel Non-Coding RNA CsiR Regulates the Ciprofloxacin Resistance in Proteus vulgaris by Interacting with emrB mRNA

Bacterial non-coding RNAs (ncRNAs) play important regulatory roles in various physiological metabolic pathways. In this study, a novel ncRNA CsiR (ciprofloxacin stress-induced ncRNA) involved in the regulation of ciprofloxacin resistance in the foodborne multidrug-resistant Proteus vulgaris (P. vulgaris) strain P3M was identified. The survival rate of the CsiR-deficient strain was higher than that of the wild-type strain P3M under the ciprofloxacin treatment condition, indicating that CsiR played a negative regulatory role, and its target gene emrB was identified through further target prediction, quantitative real-time PCR (qRT-PCR), and microscale thermophoresis (MST). Further studies showed that the interaction between CsiR and emrB mRNA affected the stability of the latter at the post-transcriptional level to a large degree, and ultimately affected the ciprofloxacin resistance of P3M. Notably, the base-pairing sites between CsiR and emrB mRNAs were highly conserved in other sequenced P. vulgaris strains, suggesting that this regulatory mechanism may be ubiquitous in this species. To the best of our knowledge, this is the first identification of a novel ncRNA involved in the regulation of ciprofloxacin resistance in P. vulgaris species, which lays a solid foundation for comprehensively expounding the antibiotic resistance mechanism of P. vulgaris.

Changes of antibiotic resistance over time among Escherichia coli peritonitis in Southern China

Escherichia coli ( E. coli) is the main cause of Gram-negative bacterial peritonitis among peritoneal dialysis patients. According to the 2016 update of the International Society for Peritoneal Dialysis Peritonitis Recommendations, drug susceptibilities of specific organisms should be regularly monitored. The aim of this study was to examine the evolution of antimicrobial resistance of E. coli peritonitis from 2006 to 2018. Two hundred and fifty-three episodes of E. coli peritonitis were enrolled in our study, corresponding to a rate of 0.024 episodes per patient-year. According to drug sensitivity test results, isolates were most sensitive to carbapenems, followed by cefmetazole, piperacillin/tazobactam, cefotetan and amikacin, with an overall rate of more than 90% in both cohorts. Cefazolin and ciprofloxacin resistance increased significantly from 2006–2011 to 2012–2018. Conversely, cefepime and ceftazidime resistance decreased significantly. The extended-spectrum β-lactamase (ESBL) rate fluctuated from 34.7% in 2006–2011 to 46.8% in 2012–2018. Compared with the ESBL-negative strains, ESBL-producing E. coli were more likely be resistant to ampicillin, ampicillin/sulbactam, cephalosporins, quinolones, aminoglycosides, furadantin and sulfamethoxazole and accounted for over 50% of the drug resistance. In the correlation analysis, E. coli displayed significantly increased resistance to cefazolin and ciprofloxacin, a finding correlated with ESBL production ( r = 0.883 and 0.276 respectively, p < 0.001 and p = 0.003). In conclusion, the rate of E. coli peritonitis declined stably in recent years, but the resistance to antimicrobial was high.

Genetic interplay between type II topoisomerase enzymes and chromosomal ccdAB toxin-antitoxin in E. coli

Fluoroquinolones are one of the most widely used class of antibiotics. They target two type II topoisomerase enzymes: gyrase and topoisomerase IV. Resistance to these drugs, which is largely caused by mutations in their target enzymes, is on the rise and becoming a serious public health risk. In this work, we analyze the sequences of 352 extraintestinal E. coli clinical isolates to gain insights into the selective pressures shaping the type II topoisomerase mutation landscape in E. coli. We identify both Quinolone Resistance-Determining Region (QRDR) and non-QRDR mutations, outline their mutation trajectories, and show that they are likely driven by different selective pressures. We confirm that ciprofloxacin resistance is specifically and strongly associated with QRDR mutations. By contrast, non-QRDR mutations are associated with the presence of the chromosomal version of ccdAB, a toxin-antitoxin operon, where the toxin CcdB is known to target gyrase. We also find that ccdAB and the evolution of QRDR mutation trajectories are partially incompatible. Finally, we identify partial deletions in CcdB and additional mutations that likely facilitate the compatibility between the presence of the ccdAB operon and QRDR mutations. These "permissive" mutations are all found in ParC (a topoisomerase IV subunit). This, and the fact that CcdB-selected mutations frequently map to topoisomerase IV, strongly suggests that this enzyme (in addition to gyrase) is likely a target for the toxin CcdB in E. coli, although an indirect effect on global supercoiling cannot be excluded. This work opens the door for the use of the presence of ccdB and of the proposed permissive mutations in the genome as genetic markers to assess the risk of quinolone resistance evolution and implies that certain strains may be genetically more refractory to evolving quinolone resistance through mutations in target enzymes.

Quinolone Resistance of Actinobacillus pleuropneumoniae Revealed through Genome and Transcriptome Analyses

Actinobacillus pleuropneumoniae is a pathogen that infects pigs and poses a serious threat to the pig industry. The emergence of quinolone-resistant strains of A.pleuropneumoniae further limits the choice of treatment. However, the mechanisms behind quinolone resistance in A.pleuropneumoniae remain unclear. The genomes of a ciprofloxacin-resistant strain, A. pleuropneumoniae SC1810 and its isogenic drug-sensitive counterpart were sequenced and analyzed using various bioinformatics tools, revealing 559 differentially expressed genes. The biological membrane, plasmid-mediated quinolone resistance genes and quinolone resistance-determining region were detected. Upregulated expression of efflux pump genes led to ciprofloxacin resistance. The expression of two porins, OmpP2B and LamB, was significantly downregulated in the mutant. Three nonsynonymous mutations in the mutant strain disrupted the water–metal ion bridge, subsequently reducing the affinity of the quinolone–enzyme complex for metal ions and leading to cross-resistance to multiple quinolones. The mechanism of quinolone resistance in A. pleuropneumoniae may involve inhibition of expression of the outer membrane protein genes ompP2B and lamB to decrease drug influx, overexpression of AcrB in the efflux pump to enhance its drug-pumping ability, and mutation in the quinolone resistance-determining region to weaken the binding of the remaining drugs. These findings will provide new potential targets for treatment.

Enhanced Biosynthesis of Fatty Acids Is Associated with the Acquisition of Ciprofloxacin Resistance in Edwardsiella tarda

Edwardsiella tarda is the causative agent of edwardsiellosis, which imposes huge challenges on clinics and aquaculture. Due to the overuse of antibiotics, the emergence and spread of antibiotic-resistant E. tarda threaten human health and animal farming.