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.

Rapid changes in the number and flow cytometric behaviour of cells of E. coli taken from a stationary phase and inoculated into rich medium.

Cells of E. coli were grown in LB medium, taken from a stationary phase of 2–4 h, and re-inoculated into fresh media at a concentration (105 ml−1 or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energization (using a carbocyanine dye) and DNA distribution. It transpired that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4 h, the true lag phase could be less than 15–20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of re-inoculation, providing the possibility of a very rapid antibiotic susceptibility test on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential urinary tract infection (UTI) samples taken from a doctor’s surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.

Purpose. Non-albicans Candida species have emerged as fungal pathogens that cause invasive infections, with many of these species displaying resistance to commonly used antifungal agents. This study was confined to studying the characteristics of clinical isolates of the C. rugosa complex and C. pararugosa species.

Methodology. Seven isolates of the C. rugosa complex and one isolate of C. pararugosa were obtained from two tertiary referral hospitals in Malaysia. Their antifungal susceptibilities, biofilm, proteinase, phospholipase, esterase and haemolysin activities were characterized. Biofilms were quantified using crystal violet (CV) and tetrazolium (XTT) reduction assays at 1.5, 6, 18, 24, 48 and 72 h.

Results/Key findings. The E-test antifungal tests showed that both species have elevated MICs compared to C. albicans and C. tropicalis. The highest biomass was observed in one of the C. rugosa isolates (0.237), followed by C. pararugosa (0.206) at 18 h of incubation. However, the highest bioactivity was observed in the C. rugosa ATCC 10571 strain at 24 h (0.075), followed by C. pararugosa at 48 h (0.048) and the same C. rugosa strain at 24 h (0.046), with P<0.05. All isolates exhibited high proteinase activity (+++) whereas six isolates showed very strong esterase activity (++++). All the isolates were alpha haemolytic producers. None of the isolates exhibited phospholipase activity.

Conclusion. Elevated MICs were shown for the C. rugosa complex and C. pararugosa for commonly used antifungal drugs. Further studies to identify virulence genes involved in the pathogenesis and genes that confer reduced drug susceptibility in these species are proposed.

Acinetobacter baumannii causes severe, fulminant, community-acquired pneumonia (CAP) in tropical and subtropical regions. We compared the population structure, virulence and antimicrobial resistance determinants of northern Australian community-onset A. baumannii strains with local and global strains. We performed whole-genome sequencing on 55 clinical and five throat colonization A. baumannii isolates collected in northern Australia between 1994 and 2016. Clinical isolates included CAP (n=41), healthcare-associated pneumonia (n=7) and nosocomial bloodstream (n=7) isolates. We also included 93 publicly available international A. baumannii genome sequences in the analyses. Patients with A. baumannii CAP were almost all critically unwell; 82 % required intensive care unit admission and 18 % died during their inpatient stay. Whole-genome phylogenetic analysis demonstrated that community-onset strains were not phylogenetically distinct from nosocomial strains. Some non-multidrug-resistant local strains were closely related to multidrug-resistant strains from geographically distant locations. Pasteur sequence type (ST)10 was the dominant ST and accounted for 31/60 (52 %) northern Australian strains; the remainder belonged to a diverse range of STs. The most recent common ancestor for ST10 was estimated to have occurred in 1738 (95 % highest posterior density, 1626–1826), with evidence of multiple introduction events between Australia and Southeast Asia between then and the present day. Virulence genes associated with biofilm formation and the type 6 secretion system (T6SS) were absent in many strains, and were not associated with in-hospital mortality. All strains were susceptible to gentamicin and meropenem; none carried an AbaR resistance island. Our results suggest that international dissemination of A. baumannii is occurring in the community on a contemporary timescale. Genes associated with biofilm formation and the T6SS may not be required for survival in community niches. The relative contributions of host and bacterial factors to the clinical severity of community-onset A. baumannii infection require further investigation.

Knowledge about biofilm-associated antibiotic tolerance mechanisms is warranted in order to develop effective treatments against biofilm infections. We performed a screen of a Streptococcus mutans transposon mutant library for mutants with reduced biofilm-associated antimicrobial tolerance, and found that the spxA1 gene plays a role in tolerance towards gentamicin and other antibiotics such as vancomycin and linezolid. SpxA1 is a regulator of genes involved in the oxidative stress response in S. mutans . The oxidative stress response genes gor and ahpC were found to be up-regulated upon antibiotic treatment of S. mutans wild-type biofilms, but not spxA1 mutant biofilms. The gor gene product catalyses the formation of glutathione which functions as an important antioxidant during oxidative stress, and accordingly biofilm-associated antibiotic tolerance of the spxA1 mutant could be restored by exogenous addition of glutathione. Our results indicate that the oxidative stress response plays a role in biofilm-associated antibiotic tolerance of S. mutans , and add to the on-going debate on the role of reactive oxygen species in antibiotic mediated killing of bacteria.

Non-typhoidal Salmonella is a leading cause of outbreak and sporadic-associated foodborne illnesses in the United States. These infections have been associated with a range of foods, including retail meats. Traditionally, pulsed-field gel electrophoresis (PFGE) and antibiotic susceptibility testing (AST) have been used to facilitate public health investigations of Salmonella infections. However, whole-genome sequencing (WGS) has emerged as an alternative tool that can be routinely implemented. To assess its potential in enhancing integrated surveillance in Pennsylvania, USA, WGS was used to directly compare the genetic characteristics of 7 retail meat and 43 clinical historic Salmonella isolates, subdivided into 3 subsets based on PFGE and AST results, to retrospectively resolve their genetic relatedness and identify antimicrobial resistance (AMR) determinants. Single nucleotide polymorphism (SNP) analyses revealed that the retail meat isolates within S. Heidelberg, S. Typhimurium var. O5- subset 1 and S. Typhimurium var. O5- subset 2 were separated from each primary PFGE pattern-matched clinical isolate by 6–12, 41–96 and 21–81 SNPs, respectively. Fifteen resistance genes were identified across all isolates, including fosA7, a gene only recently found in a limited number of Salmonella and a ≥95 % phenotype to genotype correlation was observed for all tested antimicrobials. Moreover, AMR was primarily plasmid-mediated in S. Heidelberg and S. Typhimurium var. O5- subset 2, whereas AMR was chromosomally carried in S. Typhimurium var. O5- subset 1. Similar plasmids were identified in both the retail meat and clinical isolates. Collectively, these data highlight the utility of WGS in retrospective analyses and enhancing integrated surveillance for Salmonella from multiple sources.

Purpose . To highlight the clinical significance of carbapenem-resistant Klebsiella pneumoniae (CRKP) rectal colonization by examining the risk factors for CRKP rectal colonization and subsequent bloodstream infection (BSI) in critically ill patients.

Methodology . Prospective study of CRKP rectal colonization in an intensive care unit (ICU) during a 39-month period. CRKP strains isolated from both the blood cultures and corresponding rectal specimens (n=96) of patients were screened by PCR for the presence of antibiotic resistance-associated genes. Molecular analyses were conducted to investigate the clonal relatedness of CRKP strains from the rectal and blood specimens.

Results . Among the 498 patients, 226 were rectally colonized by CRKP, 48 of whom developed a CRKP BSI. The median time from hospital admission to the detection of CRKP rectal colonization was 8 days, while the median time from colonization to BSI was 4 days. The duration of ICU stay, patient/nurse ratio and prior use of antianaerobic antimicrobials were associated with CRKP rectal colonization. No specific factor was associated with BSIs in the colonized patients. The bla _KPC-2 gene was detected in all 96 strains, which were all classified as sequence type ST-258. Representative pairs (n=48) of CRKP strains colonizing and infecting the same patient shared the same pulsotype.

Conclusion . Our results indicate that hospitalized patients become infected with their colonizing strains, supporting the strong association between colonization and BSI. Limiting antianaerobic antimicrobial administration, reducing the duration of ICU stay and maintaining a low patient/nurse ratio are possible strategies to restrict rectal CRKP colonization in ICUs.

The modified carbapenem inactivation method (mCIM) is a simple phenotypic screening method for detecting carbapenemase production by Enterobacteriaceae and Pseudomonas aeruginosa . We recently developed another modified carbapenem inactivation method (CIMTris), in which carbapenemase is extracted from bacteria with Tris-HCl buffer, to detect carbapenemase production by Acinetobacter and Pseudomonas species. This study describes an improved carbapenem inactivation method, CIMTrisII, for detecting carbapenemase production by Gram-negative pathogens, including Enterobacteriaceae , Acinetobacter and Pseudomonas species. CIMTrisII was different from CIMTris in the concentration of Meropenem disks (5-µg MEM disks vs. 10-µg MEM disks), the inoculum volume of the bacteria (a 5-µl loopful vs. a 10 µl loopful) and the incubation time (1 vs. 2 h). CIMTrisII showed an overall sensitivity of 99.3 % and an overall specificity of 95.0 % for tested isolates. In comparison, CIMTris showed a sensitivity of 96.1 % and a specificity of 96.3 %, and mCIM showed a sensitivity of 67.1 % and a specificity of 100 %. CIMTrisII is thus deemed useful for detecting carbapenemase production by Gram-negative pathogens.

A number of national and international organizations are advocating more intensive screening for Chlamydia trachomatis and Neisseria gonorrhoeae in high-prevalence populations as a way to reduce the prevalence of these infections. In this article, we review the available evidence and conclude that there is a paucity of evidence to support this approach. We further hypothesize that increasing screening intensity in high-prevalence populations will result in a considerable risk for the emergence of antimicrobial resistance in Neisseria gonorrhoeae and other pathobionts.

Resistance to carbapenem and aminoglycoside antibiotics is a critical problem in Acinetobacter baumannii , particularly when genes conferring resistance are acquired by multiply or extensively resistant members of successful globally distributed clonal complexes, such as global clone 1 (GC1) . Here, we investigate the evolution of an expanding clade of lineage 1 of the GC1 complex via repeated acquisition of carbapenem- and aminoglycoside-resistance genes. Lineage 1 arose in the late 1970s and the Tn6168/OCL3 clade arose in the late 1990s from an ancestor that had already acquired resistance to third-generation cephalosporins and fluoroquinolones. Between 2000 and 2002, two distinct subclades have emerged, and they are distinguishable via the presence of an integrated phage genome in subclade 1 and AbaR4 (carrying the oxa23 carbapenem-resistance gene in Tn2006) at a specific chromosomal location in subclade 2. Part or all of the original resistance gene cluster in the chromosomally located AbaR3 has been lost from some isolates, but plasmids carrying alternate resistance genes have been gained. In one group in subclade 2, the chromosomally located AbGRI3, carrying the armA aminoglycoside-resistance gene, has been acquired from a GC2 isolate and incorporated via homologous recombination. ISAba1 entered the common ancestor of this clade as part of the cephalosporin-resistance transposon Tn6168 and has dispersed differently in each subclade. Members of subclade 1 share an ISAba1 in one specific position in the chromosome and in subclade 2 two different ISAba1 locations are shared. Further shared ISAba1 locations distinguish further divisions, potentially providing simple markers for epidemiological studies.

Helicobacter cinaedi is an emerging pathogen causing bacteraemia and cellulitis. Nosocomial transmission of this microbe has been described, but detailed molecular-epidemiological analyses have not been performed. Here, we describe the results of a multi-step genome-wide phylogenetic analysis of a suspected intra-hospital outbreak of H. cinaedi that occurred in a hospital in Japan. The outbreak was recognized by the infectious control team (ICT) of the hospital as a sudden increase in H. cinaedi bacteraemia. ICT defined this outbreak case based on 16S rRNA sequence data and epidemiological information, but were unable to determine the source and route of the infections. We therefore re-investigated this case using whole-genome sequencing (WGS). We first performed a species-wide analysis using publicly available genome sequences to understand the level of genomic diversity of this under-studied species. The clusters identified were then separately analysed using the genome sequence of a representative strain in each cluster as a reference. These analyses provided a high-level phylogenetic resolution of each cluster, identified a confident set of outbreak isolates, and discriminated them from other closely related but distinct clones, which were locally circulating and invaded the hospital during the same period. By considering the epidemiological data, possible strain transmission chains were inferred, which highlighted the role of asymptomatic carriers or environmental contamination. The emergence of a subclone with increased resistance to fluoroquinolones in the outbreak was also recognized. Our results demonstrate the impact of the use of a closely related genome as a reference to maximize the power of WGS.

In this study, we characterized 7C, a spontaneous mutant selected from tigecycline-susceptible Mycobacterium abscessus ATCC 19977. Whole-genome sequencing (WGS) was used to identify possible resistance determinants in this mutant. Compared to the wild-type, 7C demonstrated resistance to tigecycline as well as cross-resistance to imipenem, and had a slightly retarded growth rate. WGS and subsequent biological verifications showed that these phenotypes were caused by a point mutation in MAB_3542c, which encodes an RshA-like protein. In Mycobacterium tuberculosis, RshA is an anti-sigma factor that negatively regulates the heat/oxidative stress response mechanisms. The MAB_3542c mutation may represent a novel determinant of tigecycline resistance. We hypothesize that this mutation may dysregulate the stress-response pathways which have been shown to be linked to antibiotic resistance in previous studies.

We have identified a Salmonella enterica serotype 4,[5],12:i:- containing a mcr-3.2 in a patient who travelled to Thailand 1 month prior to the identification of it in Canada. The isolate was multidrug resistant, but remained susceptible to the carbapenems, amikacin and piperacillin/tazobactam. The mcr-3.2 was carried on a 261 Kb variant of the IncHI2 pWJ1. This report provides further evidence of the emergence of a ST34 colistin-resistant clone.

Purpose. The emergence of carbapenem resistance in non-baumannii Acinetobacter has increased in clinical settings worldwide. We investigated the prevalence and mechanisms of carbapenem resistance in A. pittii and A. nosocomialis Thai isolates.

Methodology. Acinetobacter calcoaceticus–Acinetobacter baumannii (Acb) complex isolates were identified by gyrB mulitplex PCR. Carbapenem susceptibilities were studied by the agar dilution method and carbapenemase genes were detected by multiplex PCR. Reductions of the outer membrane proteins (OMPs) were evaluated by SDS-PAGE. Overexpressions of efflux pumps were detected by using efflux pump inhibitors and RT-PCR.

Results. Of the 346 Acb isolates, 22 and 19 were A. pittii and A. nosocomialis, respectively. The carbapenem resistance rates were 22.7 % in A. pittii and 26.3 % in A. nosocomialis. Three carbapenem-resistant A. pittii carried bla _OXA-23. One carbapenem-resistant A. pittii harboured bla _OXA-58, while another isolate co-harboured bla _OXA-58 and bla _IMP-14a. bla _OXA-58 was also found in three carbapenem-susceptible A. pittii. Five carbapenem-resistant A. nosocomialis carried bla _OXA-23. Eighteen A. pittii isolates carried bla _OXA-213-like. Reduced OMPs were found in carbapenem-resistant and -susceptible A. pittii carrying bla _OXA-58, but were not detected in carbapenem-resistant A. nosocomialis isolates. Overexpression of adeE was found in carbapenem-resistant A. pittii. No efflux pump genes were present in carbapenem-resistant A. nosocomialis.

Conclusion. The major mechanisms of carbapenem resistance in A. pittii and A. nosocomialis were the production of OXA-23 and OXA-58. Overexpression of adeE played a role in carbapenem resistance in A. pittii. Since bla _OXA-58 was found in carbapenem-susceptible A. pittii, using carbapenems in the treatment of A. pittii infection should be considered.

Much of the worldwide dissemination of antibiotic resistance has been driven by resistance gene associations with mobile genetic elements (MGEs), such as plasmids and transposons. Although increasing, our understanding of resistance spread remains relatively limited, as methods for tracking mobile resistance genes through multiple species, strains and plasmids are lacking. We have developed a bioinformatic pipeline for tracking variation within, and mobility of, specific transposable elements (TEs), such as transposons carrying antibiotic-resistance genes. TETyper takes short-read whole-genome sequencing data as input and identifies single-nucleotide mutations and deletions within the TE of interest, to enable tracking of specific sequence variants, as well as the surrounding genetic context(s), to enable identification of transposition events. A major advantage of TETyper over previous methods is that it does not require a genome reference. To investigate global dissemination of Klebsiella pneumoniae carbapenemase (KPC) and its associated transposon Tn4401, we applied TETyper to a collection of over 3000 publicly available Illumina datasets containing bla _KPC. This revealed surprising diversity, with over 200 distinct flanking genetic contexts for Tn4401, indicating high levels of transposition. Integration of sample metadata revealed insights into associations between geographic locations, host species, Tn4401 sequence variants and flanking genetic contexts. To demonstrate the ability of TETyper to cope with high-copy-number TEs and to track specific short-term evolutionary changes, we also applied it to the insertion sequence IS26 within a defined K. pneumoniae outbreak. TETyper is implemented in python and is freely available at https://github.com/aesheppard/TETyper.

The spread of antibiotic resistance within and between different bacterial populations is a major health problem on a global scale. The identification of genetic transformation in genomic data from Neisseria meningitidis, the meningococcus (Mc), and other bacteria is problematic, since similar or even identical alleles may be involved. A particular challenge in naturally transformable bacteria generally is to distinguish between common ancestry and true recombined sites in sampled genome sequences. Furthermore, the identification of recombination following experimental transformation of homologous alleles requires identifiable differences between donor and recipient, which in itself influences the propensity for homologous recombination (HR). This study identifies the distribution of HR events following intraspecies and interspecies Mc transformations of rpoB alleles encoding rifampicin resistance by whole-genome DNA sequencing and single nucleotide variant analysis. The HR events analysed were confined to the genomic region surrounding the single nucleotide genetic marker used for selection. An exponential length distribution of these recombined events was found, ranging from a few nucleotides to about 72 kb stretches. The lengths of imported sequences were on average found to be longer following experimental transformation of the recipient with genomic DNA from an intraspecies versus an interspecies donor (P<0.001). The recombination events were generally observed to be mosaic, with donor sequences interspersed with recipient sequence. Here, we present four models to explain these observations, by fragmentation of the transformed DNA, by interruptions of the recombination mechanism, by secondary recombination of endogenous self-DNA, or by repair/replication mechanisms.