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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.

A total of 153 non-repeat Acinetobacter spp. clinical isolates obtained in 2015 from Hospital Sultanah Nur Zahirah (HSNZ) in Terengganu, Malaysia, were characterized. Identification of the isolates at species level was performed by ribosomal DNA restriction analysis (ARDRA) followed by sequencing of the rpoB gene. The majority of the isolates (n=128; 83.7 %) were A. baumannii while the rest were identified as A. nosocomialis (n=16), A. calcoaceticus (n=5), A. soli (n=2), A. berezeniae (n=1) and A. variabilis (n=1). Multidrug resistance (MDR) was most prevalent in A. baumannnii (66.4 %) whereas only one non-baumannii isolate (A. nosocomialis) was MDR. The bla _OXA-23 gene was the predominant acquired carbapenemase gene (56.2 %) and was significantly associated (P<0.001) with carbapenem resistance. However, no significant association was found for carbapenem resistance and isolates that contained the ISAba1-bla _OXA-51 configuration.

Carbapenem resistance is a rapidly growing threat to our ability to treat refractory bacterial infections. To understand how carbapenem resistance is mobilized and spread between pathogens, it is important to study the genetic context of the underlying resistance mechanisms. In this study, the resistomes of six clinical carbapenem-resistant isolates of five different species – Acinetobacter baumannii, Escherichia coli, two Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa – were characterized using whole genome sequencing. All Enterobacteriaceae isolates and the A. baumannii isolate had acquired a large number of antimicrobial resistance genes (7–18 different genes per isolate), including the following encoding carbapenemases: bla _KPC-2, bla _OXA-48, bla _OXA-72, bla _NDM-1, bla _NDM-7 and bla _VIM-1. In addition, a novel version of bla _SHV was discovered. Four new resistance plasmids were identified and their fully assembled sequences were verified using optical DNA mapping. Most of the resistance genes were co-localized on these and other plasmids, suggesting a risk for co-selection. In contrast, five out of six carbapenemase genes were present on plasmids with no or few other resistance genes. The expected level of resistance – based on acquired resistance determinants – was concordant with measured levels in most cases. There were, however, several important discrepancies for four of the six isolates concerning multiple classes of antibiotics. In conclusion, our results further elucidate the diversity of carbapenemases, their mechanisms of horizontal transfer and possible patterns of co-selection. The study also emphasizes the difficulty of using whole genome sequencing for antimicrobial susceptibility testing of pathogens with complex genotypes.

The current paradigm for hospital outbreak detection and investigation is based on methodology first developed over 150 years ago. Daily surveillance to detect patients positive for pathogens of particular importance for nosocomial infection is supported by epidemiological investigation to determine their relationship in time and place, and to identify any other factor that could link them. The antibiotic resistance pattern is commonly used as a surrogate for bacterial relatedness, although this lacks sensitivity and specificity. Typing may be used to define bacterial relatedness, although routine methods lack sufficient discriminatory power to distinguish relatedness beyond the level of bacterial clones. Ultimately, the identification of an outbreak remains a predominately subjective process reliant on the intuition of experienced infection control professionals. Here, we propose a redesign of hospital outbreak detection and investigation in which bacterial species associated with nosocomial transmission and infection undergo routine prospective whole-genome sequencing. Further investigation is based on the probability that isolates are associated with an outbreak, which is based on the degree of genetic relatedness between isolates. Evidence is provided that supports this model based on studies of MRSA (methicillin-resistant Staphylococcus aureus), together with the benefits of a ‘Sequence First’ approach. The feasibility of implementation is discussed, together with residual barriers that need to be overcome prior to implementation.

Purpose. Antimicrobial-resistant bacterial infections in low- and middle-income countries (LMICs) are a well-established global health issue. We aimed to assess the prevalence of and epidemiological factors associated with the carriage of ciprofloxacin- and ceftriaxone-resistant Escherichia coli and associated resistance genes in a cohort of 498 healthy children residing in urban Vietnam.

Methodology. We cultured rectal swabs onto MacConkey agar supplemented with resistant concentrations of ciprofloxacin and ceftriaxone. Additionally, we screened meta-E. coli populations by conventional PCR to detect plasmid-mediated quinolone resistance (PMQR)- and extended-spectrum β-lactamase (ESBL)-encoding genes. We measured the associations between phenotypic/genotypic resistance and demographic characteristics using logistic regression.

Results/Key findings. Ciprofloxacin- and ceftriaxone-resistant E. coli were cultured from the faecal samples of 67.7 % (337/498) and 80.3 % (400/498) of children, respectively. The prevalence of any associated resistance marker in the individual samples was 86.7 % (432/498) for PMQR genes and 90.6 % (451/498) for β-lactamase genes. Overweight children were significantly more likely to carry qnr genes than children with lower weight-for-height z-scores [odds ratios (OR): 1.24; 95 % confidence interval (CI): 10.5–1.48 for each unit increase in weight for height; P=0.01]. Additionally, younger children were significantly more likely to carry ESBL CTX-M genes than older children (OR: 0.97, 95 % CI: 0.94–0.99 for each additional year, P=0.01).

Conclusion. The carriage of genotypic and phenotypic antimicrobial resistance is highly prevalent among E. coli in healthy children in the community in Vietnam. Future investigations on the carriage of antimicrobial resistant organisms in LMICs should focus on the progression of carriage from birth and structure of the microbiome in obesity.

Purpose. Nosocomial infections caused by multidrug resistant Acinetobacter baumannii have emerged as a serious problem in healthcare settings worldwide.

Methodology. A total of 100 A. baumannii clinical isolates from immunocompromised patients hospitalized in ICUs in Iran were investigated for antimicrobial susceptibility and the presence of carbapenemase and antiseptic resistance genes.

Results. All isolates were resistant to one or more antibiotics, with the most frequent resistance found against ciprofloxacin and imipenem (100 %) and piperacillin (99 %). The MICs of biocides were determined by the agar dilution method. No apparent resistance to biocides was seen among the 100 A. baumannii isolates. All isolates were effectively inhibited by the user’s defined concentrations of cetrimide, benzalkonium chloride and glutardaldehyde. The intrinsic β-lactamase gene, bla _OXA-51-like, was detected in all A. baumannii isolates. Coexistence of bla _OXA-51 andbla _OXA-23 was encountered in 89 % of isolates. However, genes bla _OXA-58, bla _SIM and bla _IMP were not detected in any isolates. While A. baumannii isolates were sensitive to biocides, they carried qac genes with the qacEΔ1 gene being the most common, at a frequency of 91 %.

Conclusion. Our study revealed the high frequency of multidrug- and carbapenem-resistant isolates of A. baumannii in ICU patients, with a high prevalence of the genes bla _OXA-23 and bla _OXA-51. However, no apparent biocide resistance was seen in A. baumannii isolates. It appears that appropriate surveillance and control measures are essential to prevent the emergence and transmission of MDR A. baumannii in Iran.