X-AMR, a pop-up journal

In 2018, the European Centre for Disease Prevention and Control reported the first cases of extensively drug-resistant Neisseria gonorrhoeae infections in Europe. Seeking new options for antimicrobial therapy we investigated the susceptibility of N. gonorrhoeae to nitroxoline (NIT) and mecillinam (MCM), both of which are currently only indicated to treat uncomplicated urinary tract infections. Clinical N. gonorrhoeae isolates with non-susceptibility to penicillin from two German medical centres were included (n =27). Most isolates were also non-susceptible to a range of other anti-gonococcal antimicrobials (cefotaxime, ciprofloxacin, azithromycin, tetracycline). All isolates were further characterized by multi-locus sequence typing. MICs of penicillin and cefotaxime were determined by agar gradient diffusion. Production of penicillinase was tested by cefinase disk test. Susceptibility of MCM was investigated by agar dilution, NIT by agar dilution and disk diffusion. Penicillin MICs ranged from 0.125 to 64 mg l−1 and MICs of cefotaxime ranged from < 0.016 to 1 mg l−1 . Five isolates were penicillinase-producers. MICs of MCM ranged from 16 to > 128 mg l−1 whereas MICs of NIT ranged from 0.125 to 2 mg l−1 . NIT disk diffusion (median zone diameter 32 mm) correlated well with results from agar dilution. We demonstrated excellent in vitro activity of NIT against clinical N. gonorrhoeae isolates with non-susceptibility to standard anti-gonococcal antibiotics. MCM activity was unsatisfactory. Correlation of agar dilution and disk diffusion in NIT susceptibility testing is an important aspect with potential clinical implications.

Biofilms may enhance the tolerance of bacterial pathogens to disinfectants, biocides and other stressors by restricting the penetration of antimicrobials into the matrix-enclosed cell aggregates, which contributes to the recalcitrance of biofilm-associated infections. In this work, we performed real-time monitoring of the penetration of nisin into the interior of Staphylococcus aureus biofilms under continuous flow and compared the efficacy of this lantibiotic against planktonic and sessile cells of S. aureus . Biofilms were grown in Center for Disease Control (CDC) reactors and the spatial and temporal effects of nisin action on S. aureus cells were monitored by real-time confocal microscopy. Under continuous flow, nisin caused loss of membrane integrity of sessile cells and reached the bottom of the biofilms within ~20 min of exposure. Viability analysis using propidium iodide staining indicated that nisin was bactericidal against S. aureus biofilm cells. Time-kill assays showed that S. aureus viability reduced 6.71 and 1.64 log c.f.u. ml-1 for homogenized planktonic cells in exponential and stationary phase, respectively. For the homogenized and intact S. aureus CDC biofilms, mean viability decreased 1.25 and 0.50 log c.f.u. ml-1, respectively. Our results demonstrate the kinetics of biofilm killing by nisin under continuous-flow conditions, and shows that alterations in the physiology of S. aureus cells contribute to variations in sensitivity to the lantibiotic. The approach developed here could be useful to evaluate the antibiofilm efficacy of other bacteriocins either independently or in combination with other antimicrobials.

Purpose. Multi-drug resistant (MDR) Acinetobacter baumannii has introduced a worldwide health crisis. The purposes of this study were to characterize the clonal relatedness among MDR clinical strains and to introduce a new two-locus typing method confirmed by multi-locus sequence typing (MLST).

Methodology. In this study, we determined antimicrobial resistance, detected genes associated with carbapenem resistance and characterized clonal relatedness among 99 clinical isolates extracted from 82 hospitalized inpatients in a university hospital.

Results. Of the 99 A. baumannii isolates, 92.9% (92/99) were resistant to imipenem and 97.9% (97/99) had an MDR profile. We found that the high prevalence of blaVIM [94.9% (94/99)] and blaOXA-23-like [93.93% (93/99)] is the main mechanism of carbapenem resistance. This study proposes a new two-locus typing (blaOXA-51-like and ampC) method for the rapid identification of clonal complexes (CCs). The results of this method and confirmation by MLST show that clinical isolates carry blaOXA-68 as well as ampC-10 or ampC-20 genes belonging to CC10 (ST10); blaOXA-66 and ampC-2 belonging to CC2 (ST2); and blaOXA-71 and ampC-3 belonging to CC3 (ST3). One isolate had blaOXA-90 with an undetermined allele number of ampC belonging to ST513.

Conclusion. The high prevalence of MDR strains and the circulation of four limited clones, including ST10 (45/99), ST2 (41/99), ST3 (12/99) and ST513 (1/99), in the clinical setting highlights the importance of a rigorous infection control programme. The two-locus typing method has more discrimination than the application of each method separately and it could be applied for the rapid determination of the CC without performing MLST.

Introduction. The last few years have seen the emergence of multi-drug resistant (MDR) Gram-negative infections, which are associated with high morbidity and mortality. The indiscriminate use of colistin has led to the development of resistance, which can be diagnosed effectively by broth microdilution. Studies from India are limited, and this study was conducted in order to determine the prevalence and risk factors associated with colistin resistance.

Methods. Urine samples from patients admitted with urinary tract infection (UTI), growing MDR Escherichia coli and Klebsiella pneumoniae , were tested for the minimum inhibitory concentration (MIC) of colistin by broth microdilution. Isolates with an MIC >2 µg ml−1 (resistant) were subjected to polymerase chain reaction (PCR) for the mcr1, mcr2 and mgrB genes. A case–control study with 21 cases (resistant) and 42 matched controls (sensitive) was designed to evaluate risk factors and outcomes (recurrent UTI, readmission and hospital stay >2 weeks).

Results. Two hundred and fifty MDR isolates ( E. coli =142/2319 and K.pneumoniae=108/775) from 216 patients were selected from the 25 046 isolates screened. Twenty-five isolates (20 K.pneumoniae and 5 E. coli ) were resistant to colistin, with a prevalence of 3.52  % in E. coli and 18.5  % in K. pneumoniae among the MDR isolates. PCR for the mcr1 and mcr2 genes was negative. Multivariate regression showed that multiple episodes of hospitalization, hospital stay >2 weeks, exposure to >three antibiotic classes and abnormality/surgery of the lower urinary tract were the significant risk factors for colistin resistance. Previous use of colistin and colistin resistance had a significant effect on all outcomes.

Conclusions. K. pneumoniae show six times higher prevalence of colistin resistance than E. coli , and the emergence of resistant organisms has led to an increase in morbidity in infected patients.

Purpose. The purpose of the present study was to determine the relatedness of Staphylococcus aureus strains successively isolated over a 7-day period from a single bacteraemic patient undergoing antibiotic treatment with vancomycin.

Methods. The S. aureus strains had been isolated and sequenced previously. Antibiotic susceptibility testing, population analysis profiling, and lysostaphin sensitivity and phagocytic killing assays were used to characterize these clonal isolates.

Results. The seven isolates (MEH1–MEH7) were determined to belong to a common multilocus sequence type (MLST) and spa type. Within the third and fifth day of vancomycin treatment, mutations were observed in the vraS and rpsU genes, respectively. Population analysis profiles revealed that the initial isolate (MEH1) was vancomycin-susceptible S. aureus (VSSA), while those isolated on day 7 were mostly heteroresistant vancomycin-intermediate S. aureus (hVISA). Supporting these findings, MEH7 was also observed to be slower in growth, to have an increase in cell wall width and to have reduced sensitivity to lysostaphin, all characteristics of VISA and hVISA strains. In addition, MEH7, although phagocytosed at numbers comparable to the initial isolate, MEH1, survived in higher numbers in RAW 264.7 macrophages. Macrophages infected with MEH7 also released more TNF-α and IFN-1β.

Conclusion. We report an increasing resistance to vancomycin coupled with daptomycin that occurred within approximately 3 days of receiving vancomycin and steadily increased until the infection was cleared with an alternative antibiotic therapy. This study reiterates the need for rapid, efficient and accurate detection of hVISA and VISA infections, especially in high-bacterial load, metastatic infections like bacteraemia.

Antibiotic resistance reservoirs within food-producing animals are thought to be a risk to animal and human health. This study describes the minimum natural resistome of pig faeces as the bacteria are under no direct antibiotic selective pressure. The faecal resistome of 257 different genes comprised 56 core and 201 accessory resistance genes. The genes present at the highest relative abundances across all samples were tetW, tetQ, tet44, tet37, tet40, mefA, aadE, ant(9)−1, ermB and cfxA2. This study characterized the baseline resistome, the microbiome composition and the metabolic components described by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in healthy pig faeces, without antibiotic selective pressures. The microbiome hierarchical analysis resulted in a cluster tree with a highly similar pattern to that of the accessory resistome cluster tree. Functional capacity profiling identified genes associated with horizontal gene transfer. We identified a statistically significant positive correlation between the total antibiotic resistome and suggested indicator genes, which agree with using these genes as indicators of the total resistomes. The correlation between total resistome and total microbiome in this study was positive and statistically significant. Therefore, the microbiome composition influenced the resistome composition. This study identified a core and accessory resistome present in a cohort of healthy pigs, in the same conditions without antibiotics. It highlights the presence of antibiotic resistance in the absence of antibiotic selective pressure and the variability between animals even under the same housing, food and living conditions. Antibiotic resistance will remain in the healthy pig gut even when antibiotics are not used. Therefore, the risk of antibiotic resistance transfer from animal faeces to human pathogens or the environment will remain in the absence of antibiotics.

Purpose. Comparative genomic analysis of strains may help us to better understand the wide diversity of their genetic profiles. The aim of this study was to analyse the genomic features of the resistome and virulome of Brazilian first methicillin-resistant Staphylococcus aureus (MRSA) isolates and their relationship to other Brazilian and international MRSA strains.

Methodology. The whole genomes of three MRSA strains previously isolated in Vitória da Conquista were sequenced, assembled, annotated and compared with other MRSA genomes. A phylogenetic tree was constructed and the pan-genome and accessory and core genomes were constructed. The resistomes and virulomes of all strains were identified.

Results/Key findings. Phylogenetic analysis of all 49 strains indicated different clones showing high similarity. The pan-genome of the analysed strains consisted of 4484 genes, with 31 % comprising the gene portion of the core genome, 47 % comprising the accessory genome and 22 % being singletons. Most strains showed at least one gene related to virulence factors associated with immune system evasion, followed by enterotoxins. The strains showed multiresistance, with the most recurrent genes conferring resistance to beta-lactams, fluoroquinolones, aminoglycosides and macrolides.

Conclusions. Our comparative genomic analysis showed that there is no pattern of virulence gene distribution among the clones analysed in the different regions. The Brazilian strains showed similarity with clones from several continents.

Biofilm model systems are used to study biofilm growth and predict the effects of anti-biofilm interventions within the human oral cavity. Many in vitro biofilm model systems use a confocal laser scanning microscope (CLSM) in conjunction with image analysis tools to study biofilms. The aim of this study was to evaluate an in-house developed image analysis software program that we call BAIT (Biofilm Architecture Inference Tool) to quantify the architecture of oral multi-species biofilms following anti-biofilm interventions using a microfluidic biofilm system. Differences in architecture were compared between untreated biofilms and those treated with water (negative control), sodium gluconate (‘placebo’) or stannous fluoride (SnF2). The microfluidic system was inoculated with pooled human saliva and biofilms were developed over 22 h in filter-sterilized 25 % pooled human saliva. During this period, biofilms were treated with water, sodium gluconate, or SnF2 (1000, 3439 or 10 000 p.p.m. Sn2+) 8 and 18 h post-inoculation. After 22 h of growth, biofilms were stained with LIVE/DEAD stain, and imaged by CLSM. BAIT was used to calculate biofilm biovolume, total number of objects, surface area, fluffiness, connectivity, convex hull porosity and viability. Image analysis showed oral biofilm architecture was significantly altered by 3439 and 10 000 p.p.m. Sn2+ treatment regimens, resulting in decreased biovolume, surface area, number of objects and connectivity, while fluffiness increased (P<0.01). In conclusion, BAIT was shown to be able to measure the changes in biofilm architecture and detects possible antimicrobial and anti-biofilm effects of candidate agents.

P urpose . Antimicrobial resistance (AMR) is of increasing global concern, threatening to undermine recent progress in reducing child and neonatal mortality. Repurposing older antimicrobials is a prominent strategy to combat multidrug-resistant sepsis. A potential agent is fosfomycin, however, there is scarce data regarding its in vitro activity and pharmacokinetics in the paediatric population.

M ethodology . We analysed a contemporary, systematically collected archive of community-acquired (CA) and hospital-acquired (HA) paediatric Gram-negative bacteraemia isolates for their susceptibility to fosfomcyin. MICs were determined using agar serial dilution methods and validated by disk diffusion testing where breakpoints are available. Disk diffusion antimicrobial susceptibility testing was also conducted for current empirical therapies (ampicillin, gentamicin, ceftriaxone) and amikacin (proposed in the literature as a new combination empirical therapeutic option).

R esults . Fosfomycin was highly active against invasive Gram-negative isolates, including 90  % (202/224) of Enterobacteriaceae and 96  % (22/23) of Pseudomonas spp. Fosfomycin showed high sensitivity against both CA isolates (94 %, 142/151) and HA isolates (81 %, 78/96; P =0.0015). CA isolates were significantly more likely to be susceptible to fosfomycin than the current first-line empirical therapy (96  % vs 59  %, P <0.0001). Extended spectrum β-lactamases (ESBL) production was detected in 34  % (85/247) of isolates with no significant difference in fosfomycin susceptibility between ESBL-positive or -negative isolates [73/85 (86  %) vs 147/162 (91  %) respectively, P =0.245]. All isolates were susceptible to a fosfomycin-amikacin combination.

C onclusion . Gram-negative paediatric bacteraemia isolates are highly susceptible to fosfomycin, which could be combined with aminoglycosides as a new, carbapenem-sparing regimen to achieve excellent coverage to treat antimicrobial-resistant neonatal and paediatric sepsis.

Bacitracin is a cell wall targeting antimicrobial with clinical and agricultural applications. With the growing mismatch between antimicrobial resistance and development, it is essential we understand the molecular mechanisms of resistance in order to prioritize and generate new effective antimicrobials. BcrR is a unique membrane-bound one-component system that regulates high-level bacitracin resistance in Enterococcus faecalis . In the presence of bacitracin, BcrR activates transcription of the bcrABD operon conferring resistance through a putative ATP-binding cassette (ABC) transporter (BcrAB). BcrR has three putative functional domains, an N-terminal helix–turn–helix DNA-binding domain, an intermediate oligomerization domain and a C-terminal transmembrane domain. However, the molecular mechanisms of signal transduction remain unknown. Random mutagenesis of bcrR was performed to generate loss- and gain-of-function mutants using transcriptional reporters fused to the target promoter P _bcrA . Fifteen unique mutants were isolated across all three proposed functional domains, comprising 14 loss-of-function and one gain-of-function mutant. The gain-of-function variant (G64D) mapped to the putative dimerization domain of BcrR, and functional analyses indicated that the G64D mutant constitutively expresses the P _bcrA-luxABCDE reporter. DNA-binding and membrane insertion were not affected in the five mutants chosen for further characterization. Homology modelling revealed putative roles for two key residues (R11 and S33) in BcrR activation. Here we present a new model of BcrR activation and signal transduction, providing valuable insight into the functional characterization of membrane-bound one-component systems and how they can coordinate critical bacterial responses, such as antimicrobial resistance.