Aminoglycoside resistance genes in early members of the Acinetobacter baumannii ST78A (SMAL, Italian clone) reside in an IS26-bounded island in the chromosome.

BACKGROUND: The Acinetobacter baumannii isolate called SMAL, previously used to determine the structures of capsular polysaccharide and lipooligosaccharide, was recovered in Pavia, Italy in 2002 among the collection of aminoglycoside-resistant isolates designated as SMAL type. This type was later called the Italian clone, then ST78. ST78 isolates are now widely distributed. OBJECTIVES: To establish the resistance gene complement and the location and structure of acquired resistance regions in early members of the Italian/ST78 clone. METHODS: The draft genome of SMAL2002 was assembled from Illumina MiSeq reads. Contigs containing resistance genes were joined and located in the chromosome using PCR with custom primers. The resistance profile was determined using disc diffusion. RESULTS: SMAL2002 is an ST78A isolate and includes three aminoglycoside resistance genes, aadB (gentamicin, kanamycin, tobramycin) aphA1 (kanamycin, neomycin) and aac(6')-Ian (amikacin, kanamycin, tobramycin). The aadB gene cassette is incorporated at a secondary site in a relative of the aphA1-containing, IS26-bounded pseudo-compound transposon, PTn6020. The aac(6')-Ian gene is in an adjacent IS26-bounded structure that includes sul2 (sulphonamide) and floR (florfenicol) resistance genes. The two pseudo-compound transposons overlap and are in the chromosomal hutU gene flanked by an 8 bp target site duplication. Although aac(6')-Ian was not noticed previously, the same genes and structures were found in several available draft genomes of early ST78A isolates. CONCLUSIONS: This study highlights the importance of correlating resistance profiles with resistance gene content. The location of acquired resistance genes in the SMAL2002 chromosome represents the original location in the ST78A lineage of ST78.

Identification of further variation at the lipooligosaccharide outer core locus in Acinetobacter baumannii genomes and extension of the OCL reference sequence database for Kaptive.

The outer core locus (OCL) that includes genes for the synthesis of the variable outer core region of the lipooligosaccharide (LOS) is one of the key epidemiological markers used for tracing the spread of Acinetobacter baumannii, a bacterial pathogen of global concern. In this study, we screened 12 476 publicly available A. baumannii genome assemblies for novel OCL sequences, detecting six new OCL types that were designated OCL17-OCL22. These were compiled with previously characterized OCL sequences to create an updated version of the A. baumannii OCL reference database, providing a total of 22 OCL reference sequences for use with the bioinformatics tool Kaptive. Use of this database against the 12 476 downloaded assemblies found OCL1 to be the most common locus, present in 73.6 % of sequenced genomes assigned by Kaptive with a match confidence score of good or above. OCL1 was most common amongst isolates belonging to sequence types (STs) ST1, ST2, ST3 and ST78, all of which are over-represented clonal lineages. The highest level of diversity in OCL types was found in ST2, with eight different OCLs identified. The updated OCL reference database is available for download from GitHub (https://github.com/klebgenomics/Kaptive; under version v. 2.0.5), and has been integrated for use on Kaptive-Web (https://kaptive-web.erc.monash.edu/) and PathogenWatch (https://pathogen.watch/), enhancing current methods for A. baumannii strain identification, classification and surveillance.

An update to the database for Acinetobacter baumannii capsular polysaccharide locus typing extends the extensive and diverse repertoire of genes found at and outside the K locus.

Several novel non-antibiotic therapeutics for the critical priority bacterial pathogen, Acinetobacter baumannii, rely on specificity to the cell-surface capsular polysaccharide (CPS). Hence, prediction of CPS type deduced from genes in whole genome sequence data underpins the development and application of these therapies. In this study, we provide a comprehensive update to the A. baumannii K locus reference sequence database for CPS typing (available in Kaptive v. 2.0.1) to include 145 new KL, providing a total of 237 KL reference sequences. The database was also reconfigured for compatibility with the updated Kaptive v. 2.0.0 code that enables prediction of 'K type' from special logic parameters defined by detected combinations of KL and additional genes outside the K locus. Validation of the database against 8994 publicly available A. baumannii genome assemblies from NCBI databases identified the specific KL in 73.45 % of genomes with perfect, very high or high confidence. Poor sequence quality or the presence of insertion sequences were the main reasons for lower confidence levels. Overall, 17 KL were overrepresented in available genomes, with KL2 the most common followed by the related KL3 and KL22. Substantial variation in gene content of the central portion of the K locus, that usually includes genes specific to the CPS type, included 34 distinct groups of genes for synthesis of various complex sugars and >400 genes for forming linkages between sugars or adding non-sugar substituents. A repertoire of 681 gene types were found across the 237 KL, with 88.4 % found in <5 % of KL.

Achieving Antimicrobial Stewardship on the Global Scale: Challenges and Opportunities.

Antimicrobial resistance (AMR) has been clearly identified as a major global health challenge. It is a leading cause of human deaths and also has a toll on animals, plants, and the environment. Despite the considerable socio-economic impacts, the level of awareness of the problem remains woefully inadequate, and antimicrobials are not generally recognized as a global common good, one that everyone has a role and responsibility to conserve. It is imperative for antimicrobial stewardship to be more widely implemented to achieve better control of the AMR phenomenon. The Food and Agriculture Organization (FAO) of the United Nations plays an important role in promoting and facilitating antimicrobial stewardship. The specific needs to be addressed and barriers to be overcome, in particular, in low- and middle-income countries in order to implement antimicrobial stewardship practices in agrifood systems are being identified. As a global community, it is essential that we now move beyond discussing the AMR problem and focus on implementing solutions. Thus, FAO provides multi-pronged support for nations to improve antimicrobial stewardship through programs to strengthen governance, increase awareness, develop and enhance AMR surveillance, and implement best practices related to antimicrobial resistance in agrifood systems. For example, FAO is developing a platform to collect data on AMR in animals and antimicrobial use (AMU) in plants (InFARM), working on a campaign to reduce the need to use antimicrobials, studying the use of alternatives to the use of antimicrobials (especially those used for growth promotion) and actively promoting the implementation of the Codex Alimentarius AMR standards. Together, these will contribute to the control of AMR and also bring us closer to the achievement of multiple sustainable development goals.

The K139 capsular polysaccharide produced by Acinetobacter baumannii MAR17-1041 belongs to a group of related structures including K14, K37 and K116.

Capsular polysaccharide (CPS) is a key target for bacteriophage and vaccine therapies currently being developed for treatment of infections caused by the extensively antibiotic resistant bacterial species, Acinetobacter baumannii. Identification of new CPS structures and the genetics that drive their synthesis underpins tailored treatment strategies. A novel CPS biosynthesis gene cluster, designated KL139, was identified in the whole genome sequence of a multiply antibiotic resistant clinical isolate, A. baumannii MAR-17-1041, recovered in Russia in 2017. CPS material extracted from A. baumannii MAR-17-1041 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy, and the structure was found to include a branched pentasaccharide repeating unit containing neutral carbohydrates. This structure closely resembles the topology of the A. baumannii K14 CPS but differs in the presence of d-Glcp in place of a d-Galp sugar in the repeat-unit main chain. The difference was attributed to a change in the sequence for two glycosyltransferases. These two proteins are also encoded by the A. baumannii KL37 gene cluster, and a multiple sequence alignment of KL139 with KL14 and KL37 revealed a hybrid relationship. The global distribution of KL139 was also assessed by probing 9065 A. baumannii genomes available in the NCBI non-redundant and WGS databases for the KL139 gene cluster. KL139 was found in 16 genomes from four different countries. Eleven of these isolates belong to the multidrug resistant global lineage, ST25.

Identification of Acinetobacter baumannii loci for capsular polysaccharide (KL) and lipooligosaccharide outer core (OCL) synthesis in genome assemblies using curated reference databases compatible with Kaptive.

Multiply antibiotic-resistant Acinetobacter baumannii infections are a global public health concern and accurate tracking of the spread of specific lineages is needed. Variation in the composition and structure of capsular polysaccharide (CPS), a critical determinant of virulence and phage susceptibility, makes it an attractive epidemiological marker. The outer core (OC) of lipooligosaccharide also exhibits variation. To take better advantage of the untapped information available in whole genome sequences, we have created a curated reference database of 92 publicly available gene clusters at the locus encoding proteins responsible for biosynthesis and export of CPS (K locus), and a second database for 12 gene clusters at the locus for outer core biosynthesis (OC locus). Each entry has been assigned a unique KL or OCL number, and is fully annotated using a simple, transparent and standardized nomenclature. These databases are compatible with Kaptive, a tool for in silico typing of bacterial surface polysaccharide loci, and their utility was validated using (a) >630 assembled A. baumannii draft genomes for which the KL and OCL regions had been previously typed manually, and (b) 3386 A. baumannii genome assemblies downloaded from NCBI. Among the previously typed genomes, Kaptive was able to confidently assign KL and OCL types with 100 % accuracy. Among the genomes retrieved from NCBI, Kaptive detected known KL and OCL in 87 and 90 % of genomes, respectively, indicating that the majority of common KL and OCL types are captured within the databases; 13 of the 92 KL in the database were not detected in any publicly available whole genome assembly. The failure to assign a KL or OCL type may indicate incomplete or poor-quality genomes. However, further novel variants may remain to be documented. Combining outputs with multilocus sequence typing (Institut Pasteur scheme) revealed multiple KL and OCL types in collections of a single sequence type (ST) representing each of the two predominant globally distributed clones, ST1 of GC1 and ST2 of GC2, and in collections of other clones comprising >20 isolates each (ST10, ST25, and ST140), indicating extensive within-clone replacement of these loci. The databases are available at https://github.com/katholt/Kaptive and will be updated as further locus types become available.

Acinetobacter baumannii K116 capsular polysaccharide structure is a hybrid of the K14 and revised K37 structures.

The genome of Acinetobacter baumannii clinical isolate, MAR-303, recovered in Russia was sequenced and found to contain a novel gene cluster at the A. baumannii K locus for capsule biosynthesis. The gene cluster, designated KL116, included four genes for glycosyltransferases (Gtrs) and a gene for a Wzy polymerase responsible for joining oligosaccharide K units into the capsular polysaccharide (CPS). The arrangement of KL116 was a hybrid of previously described A. baumannii gene clusters, with two gtr genes and the wzy gene shared by KL37 and the two other gtr genes found in KL14. The structure of the K116 CPS was established by sugar analysis and Smith degradation, along with one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS is composed of branched pentasaccharide K units containing only neutral sugars, with three monosaccharides in the main chain and a disaccharide side chain. The K116 unit shares internal sugar linkages with the K14 and K37 units, corresponding to the presence of shared gtr genes in the gene clusters. However, the specific linkage formed by Wzy was discrepant between K116 and the previously reported K37 CPS produced by A. baumannii isolate NIPH146. The K37 structure was therefore revised in this study, and the corrected Wzy linkage found to be identical to the Wzy linkage in K116. The KL116, KL14 and KL37 gene clusters were found in genomes of a variety of A. baumannii strain backgrounds, indicating their global distribution.

Should Human Rights and Autonomy be The Primary Determinants for the Disclosure of a Decision to Withhold Futile Resuscitation?

Do not attempt cardiopulmonary resuscitation decisions (DNACPR) are considered good medical practice for those dying at the end of natural life. They avoid intrusive and inappropriate intervention. Historically, informing patients of these decisions was discretionary to avoid undue distress. Recent legal rulings have altered clinical guidance: disclosure is now all but obligatory. The basis for these legal judgments was respect for the patient's autonomy as an expression of their human rights. Through critical analysis, this paper explores other bioethical considerations and the potential harms if they are ignored. Arguably, disclosure of DNACPR status on its own will do little to improve patient experience. A focus on good communication with those identified as approaching end-of-life will facilitate personalized care. Discussions around DNACPR may still occur, but only if likely to be beneficial and at a patient-appropriate pace (not dictated by the need to activate the decision).

Interventions to reduce non-typhoidal Salmonella in pigs during transport to slaughter and lairage: Systematic review, meta-analysis, and research synthesis based infection models in support of assessment of effectiveness.

A systematic review of the effectiveness of interventions to reduce Salmonella prevalence or concentration in pork was undertaken. A broad search was conducted in two electronic databases. Each citation was appraised using screening tools designed and tested a priori. Level 1 relevance screening excluded irrelevant citations; level 2 confirmed relevance and categorized. Data were then extracted, and intervention categories were descriptively summarized. Meta-analysis was performed to provide a summary estimate of treatment effect where two or more studies investigated the same intervention in comparable populations. The Grading of Recommendation, Assessment, Development and Evaluation (GRADE) approach was used to assess the confidence in the estimated summary measures of intervention effect for each data subgroup. Data were also extracted from the control groups of 25 challenge trials captured by the review, to fit logistic regression models of Salmonella infection in pigs, using odds of infection as the outcome measure. The only intervention captured by the review which was significantly associated with reduced risk of Salmonella in field settings, was elimination of lairage, which is not currently feasible commercially. The logistic regression model for fecal Salmonella shedding in pigs with a random intercept for trial yielded the following predictors significantly associated with increased odds of infection: oral challenge route relative to intra-nasal, log increase in challenge dose, and elapsed time post-challenge. Univariable exact logistic regression modeling lymph node contamination post-challenge yielded the following predictors significantly associated with increased odds of Salmonella infection: younger animals relative to older ones; intra-nasal challenge route relative to oral route; and animals sampled within the first 7days post-challenge relative to those sampled at 14 or 21days. We hypothesize that the presence of absence of one or more of these predictors across studies could help to explain the inconsistent and/or non-significant findings reported for some interventions applied at lairage.

Rapid systematic review and meta-analysis of the evidence for effectiveness of primary production interventions to control Salmonella in beef and pork.

Non-typhoidal Salmonella spp. (hereafter referred to as Salmonella) on beef and pork is an important cause of foodborne illness and death globally. A systematic review of the effectiveness of interventions to reduce Salmonella prevalence or concentration in beef and pork was undertaken. A broad search was conducted in Scopus and CAB abstracts. Each citation was appraised using screening tools tested a priori. Level 1 relevance screening excluded irrelevant citations; level 2 confirmed relevance and categorized studies. Data were then extracted, and intervention categories were descriptively summarized. Meta-analysis was performed to provide a summary estimate of treatment effect where two or more studies investigated the same intervention in comparable populations. The Grading of Recommendation, Assessment, Development and Evaluation (GRADE) approach was used to assess the confidence in the estimated measures of intervention effect for data subgroups.

The Public Repository of Xenografts Enables Discovery and Randomized Phase II-like Trials in Mice.

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.

A Rapid Systematic Review and Meta-Analysis of the Efficacy of Slaughter and Processing Interventions to Control Non-Typhoidal Salmonella in Beef and Pork.

Pork is one of the major food sources of human salmonellosis worldwide, while beef products have been implicated in numerous foodborne outbreaks. As a result, effective interventions to reduce Salmonella contamination during beef and pork processing are of interest to both regulators and industry. We conducted a rapid systematic review and meta-analysis of literature investigating the efficacy of slaughter and processing interventions to control Salmonella in beef and pork. Review steps included: a comprehensive search strategy; relevance screening of abstracts; relevance confirmation of articles; data extraction; risk-of-bias assessment; meta-analysis (where appropriate); and a weight-of-evidence assessment. A total of 191 relevant experimental studies were identified. Two controlled trials indicated that hot water and steam treatments are effective at reducing the prevalence of Salmonella on beef carcasses (relative risk [RR] = 0.11, 95% confidence interval [CI]: 0.02, 0.58), while four trials found that pre-chill organic acid washes are effective at reducing Salmonella on pork carcasses (RR = 0.32, 95% CI: 0.13, 0.78), with high confidence in the estimates of effect. Four quasi-experimental studies found that post-exsanguination chemical washes were effective to reduce the prevalence of Salmonella on cattle hides, with low confidence in the specific estimate of effect; moderate confidence was found for the effect estimates of scalding (RR = 0.20, 95% CI: 0.14, 0.29) and singeing (RR = 0.34, 95% CI: 0.22, 0.52) of pork carcasses. The overall evidence supported enhanced reductions of Salmonella through a multiple-hurdle approach. In conclusion, various slaughter and processing interventions can contribute to reducing Salmonella on beef and pork carcasses, depending on the context of application; an appropriate combination should be selected, validated, and verified by establishment operators within their local conditions.

Application of a Rapid Knowledge Synthesis and Transfer Approach To Assess the Microbial Safety of Low-Moisture Foods.

Low-moisture foods (LMF) are increasingly implicated in outbreaks of foodborne illness, resulting in a significant public health burden. To inform the development of a new Codex Alimentarius code of hygienic practice for LMF, we applied a rapid knowledge synthesis and transfer approach to review global research on the burden of illness, prevalence, and interventions to control nine selected microbial hazards in eight categories of LMF. Knowledge synthesis methods included an integrated scoping review (search strategy, relevance screening and confirmation, and evidence mapping), systematic review (detailed data extraction), and meta-analysis of prevalence data. Knowledge transfer of the results was achieved through multiple reporting formats, including evidence summary cards. We identified 214 unique outbreaks and 204 prevalence and 126 intervention studies. Cereals and grains (n = 142) and Salmonella (n = 278) were the most commonly investigated LMF and microbial hazard categories, respectively. Salmonella was implicated in the most outbreaks (n = 96, 45%), several of which were large and widespread, resulting in the most hospitalizations (n = 895, 89%) and deaths (n = 14, 74%). Salmonella had a consistently low prevalence across all LMF categories (0 to 3%), but the prevalence of other hazards (e.g., Bacillus cereus) was highly variable. A variety of interventions were investigated in small challenge trials. Key knowledge gaps included underreporting of LMF outbreaks, limited reporting of microbial levels in prevalence studies, and a lack of intervention efficacy research under commercial conditions. Summary cards were a useful knowledge transfer format to inform complementary risk ranking activities. This review builds upon previous work in this area by synthesizing a broad range of evidence using a structured, transparent, and integrated approach to provide timely evidence informed inputs into international guidelines.

Powdered infant formula as a source of Salmonella infection in infants.

Powdered infant formula is not sterile and may be intrinsically contaminated with pathogens, such as Salmonella enterica, that can cause serious illness in infants. In recent years, at least 6 outbreaks of Salmonella infection in infants that have been linked to the consumption of powdered infant formula have been reported. Many of these outbreaks were identified because the Salmonella strains were unique in some way (e.g., a rare serotype) and a well-established Salmonella surveillance network, supported by laboratories capable of serotyping isolates, was in place. Another common feature of the outbreaks was the low level of salmonellae detected in the implicated formula (salmonellae may be missed in routine testing). These outbreaks likely represent only a small proportion of the actual number of Salmonella infections in infants that have been linked to powdered infant formula. Managing this problem requires a multidimensional approach in which manufacturers, regulators, and caregivers to infants can all play a role.

Microbiological risk assessment in developing countries.

Microbiological risk assessment (MRA) has been evolving at the national and international levels as a systematic and objective approach for evaluating information pertaining to microbiological hazards in foods and the risks they pose. This process has been catalyzed by international food trade requirements to base sanitary measures on sound scientific evidence and appropriate risk assessments. All countries, including developing countries, need to understand and use MRA. MRA is resource intensive, as has been demonstrated by some of the the assessments undertaken in industrialized countries. However, when used in the appropriate circumstances MRA offers many benefits. The process of undertaking MRA improves the understanding of key issues, enables an objective evaluation of risk management options, and provides a scientific justification for actions. Although the gap between developing countries and some industrialized countries is quite extensive with regard to MRA, many developing countries recognize the need to at least understand and move toward using MRA. This process requires development of infrastructure and enhancement of scientific and technical expertise while making optimal use of limited resources. International organizations, such as the Food and Agriculture Organization of the United Nations, are in a position to provide countries with guidance, training, information resources, and technical assistance to develop and/or strengthen food safety infrastructure. Enhanced cooperation and collaboration at all levels are needed for such efforts to be successful and to ensure that MRA, as a food safety tool, is available to all countries.