A Diverse Panel of Clinical Acinetobacter baumannii for Research and Development.

Over the past two decades, Acinetobacter baumannii has emerged as a leading cause of nosocomial infections worldwide. Of particular concern are panresistant strains, leading the World Health Organization (WHO) to designate carbapenem-resistant A. baumannii as a priority 1 (critical) pathogen for research and development of new antibiotics. A key component in supporting this effort is accessibility to diverse and clinically relevant strains for testing. Here, we describe a panel of 100 diverse A. baumannii strains for use in this endeavor. Whole-genome sequencing was performed on 3,505 A. baumannii isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. Isolates were cultured from clinical samples at health care facilities around the world between 2001 and 2017. Core-genome multilocus sequence typing and high-resolution single nucleotide polymorphism (SNP)-based phylogenetic analyses were used to select a final panel of 100 strains that captured the genetic diversity of the collection. Comprehensive antibiotic susceptibility testing was also performed on all 100 isolates using 14 clinically relevant antibiotics. The final 100-strain diversity panel contained representative strains from 70 different traditional Pasteur scheme multilocus sequence types, including major epidemic clones. This diversity was also reflected in antibiotic susceptibility and antimicrobial resistance (AMR) gene content, with phenotypes ranging from pansensitive to panresistant, and over 100 distinct AMR gene alleles identified from 32 gene families. This panel provides the most diverse and comprehensive set of A. baumannii strains for use in developing solutions for combating antibiotic resistance. The panel and all available metadata, including genome sequences, will be available to industry and academic institutions and federal and other laboratories free of charge.

A novel plasmid-encoded mcr-4.3 gene in a colistin-resistant Acinetobacter baumannii clinical strain.

OBJECTIVES: To identify the molecular mechanism of colistin resistance in an MDR Acinetobacter baumannii clinical strain isolated in 2008 from a meningitis case in Brazil. METHODS: Long- and short-read WGS was used to identify colistin resistance genes in A. baumannii strain 597A with a colistin MIC of 64 mg/L. MS was used to analyse lipid A content. mcr was cloned into pET-26b (+) and transformed into Escherichia coli BL21(λDE3)pLysS for analysis. RESULTS: A novel plasmid (pAb-MCR4.3) harbouring mcr-4.3 within a Tn3-like transposon was identified. The A. baumannii 597A lipid A MS spectra showed a main molecular ion peak at m/z=2034, which indicated the addition of phosphoethanolamine to the lipid A structure. E. coli BL21 transformed with pET-26b-mcr-4.3 gained colistin resistance with a colistin MIC of 8 mg/L. CONCLUSIONS: Colistin resistance in A. baumannii 597A was correlated with the presence of a novel plasmid-encoded mcr-4.3 gene.

Chromosomally Encoded mcr-5 in Colistin-Nonsusceptible Pseudomonas aeruginosa.

Whole-genome sequencing (WGS) of historical Pseudomonas aeruginosa clinical isolates identified a chromosomal copy of mcr-5 within a Tn3-like transposon in P. aeruginosa MRSN 12280. The isolate was nonsusceptible to colistin by broth microdilution, and genome analysis revealed no mutations known to confer colistin resistance. To the best of our knowledge, this is the first report of mcr in colistin-nonsusceptible P. aeruginosa.

An intervention to control an ICU outbreak of carbapenem-resistant Acinetobacter baumannii: long-term impact for the ICU and hospital.

BACKGROUND: Following a fatal intensive care unit (ICU) outbreak of carbapenem-resistant Acinetobacter baumanii (CRAB) in 2015, an aggressive infection control intervention was instituted. We outline the intervention and long-term changes in the incidence and prevalence of CRAB. METHODS: The infection control intervention included unit closure (3 days), environmental cleaning, hand hygiene interventions, and environmental culturing. CRAB acquisition and prevalence and colistin use were compared for the 1 year before and 2 years after the intervention. RESULTS: Following the intervention, ICU CRAB acquisition decreased significantly from 54.6 (preintervention) to 1.9 (year 1) to 5.6 cases (year 2)/1000 admissions (p < 0.01 for comparisons with preintervention period.). Unexpectedly, ICU CRAB admission prevalence also decreased from 56.5 to 5.8 to 13 cases/1000 admissions (p < 0.001) despite the infection control intervention's being directed at the ICU alone. In parallel, hospital CRAB prevalence decreased from 4.4 to 2.4 to 2.5 cases/1000 admissions (p < 0.001), possibly as a result of decreased discharge of CRAB carriers from the ICU to the wards (58.5 to 1.9 to 7.4 cases/1000 admissions; p < 0.001). ICU colistin consumption decreased from 200 to 132 to 75 defined daily dose (DDD)/1000 patient-days (p < 0.05). Hospital colistin consumption decreased from 21.2 to 19.4 to 14.1 DDD/1000 patient-days (p < 0.05). CONCLUSIONS: The ICU infection control intervention was highly effective, long-lasting, and associated with a decrease in last-line antibiotic use. The intervention was associated with the unexpected finding that hospital CRAB prevalence also decreased.

Analysis of Serial Isolates of mcr-1-Positive Escherichia coli Reveals a Highly Active ISApl1 Transposon.

The emergence of a transferable colistin resistance gene (mcr-1) is of global concern. The insertion sequence ISApl1 is a key component in the mobilization of this gene, but its role remains poorly understood. Six Escherichia coli isolates were cultured from the same patient over the course of 1 month in Germany and the United States after a brief hospitalization in Bahrain for an unconnected illness. Four carried mcr-1 as determined by real-time PCR, but two were negative. Two additional mcr-1-negative E. coli isolates were collected during follow-up surveillance 9 months later. All isolates were analyzed by whole-genome sequencing (WGS). WGS revealed that the six initial isolates were composed of two distinct strains: an initial ST-617 E. coli strain harboring mcr-1 and a second, unrelated, mcr-1-negative ST-32 E. coli strain that emerged 2 weeks after hospitalization. Follow-up swabs taken 9 months later were negative for the ST-617 strain, but the mcr-1-negative ST-32 strain was still present. mcr-1 was associated with a single copy of ISApl1, located on a 64.5-kb IncI2 plasmid that shared >95% homology with other mcr-1 IncI2 plasmids. ISApl1 copy numbers ranged from 2 for the first isolate to 6 for the final isolate, but ISApl1 movement was independent of mcr-1 Some movement was accompanied by gene disruption, including the loss of genes encoding proteins involved in stress responses, arginine catabolism, and l-arabinose utilization. These data represent the first comprehensive analysis of ISApl1 movement in serial clinical isolates and reveal that, under certain conditions, ISApl1 is a highly active IS element whose movement may be detrimental to the host cell.

Microbiota organization is a distinct feature of proximal colorectal cancers.

Environmental factors clearly affect colorectal cancer (CRC) incidence, but the mechanisms through which these factors function are unknown. One prime candidate is an altered colonic microbiota. Here we show that the mucosal microbiota organization is a critical factor associated with a subset of CRC. We identified invasive polymicrobial bacterial biofilms (bacterial aggregates), structures previously associated with nonmalignant intestinal pathology, nearly universally (89%) on right-sided tumors (13 of 15 CRCs, 4 of 4 adenomas) but on only 12% of left-sided tumors (2 of 15 CRCs, 0 of 2 adenomas). Surprisingly, patients with biofilm-positive tumors, whether cancers or adenomas, all had biofilms on their tumor-free mucosa far distant from their tumors. Bacterial biofilms were associated with diminished colonic epithelial cell E-cadherin and enhanced epithelial cell IL-6 and Stat3 activation, as well as increased crypt epithelial cell proliferation in normal colon mucosa. High-throughput sequencing revealed no consistent bacterial genus associated with tumors, regardless of biofilm status. However, principal coordinates analysis revealed that biofilm communities on paired normal mucosa, distant from the tumor itself, cluster with tumor microbiomes as opposed to biofilm-negative normal mucosa bacterial communities also from the tumor host. Colon mucosal biofilm detection may predict increased risk for development of sporadic CRC.

A Model for Transposition of the Colistin Resistance Gene mcr-1 by ISApl1.

Analysis of mcr-1-containing sequences identified a common ∼2,607-bp DNA segment that in many cases is flanked on one or both ends by ISApl1 We present evidence that mcr-1 is mobilized by an ISApl1 composite transposon which has, in some cases, subsequently lost one or both copies of ISApl1 We also show that mcr-1 can be mobilized in some circumstances by a single upstream copy of ISApl1 in conjunction with the remnants of a downstream ISApl1.

Fatal outbreak of an emerging clone of extensively drug-resistant Acinetobacter baumannii with enhanced virulence.

BACKGROUND: Severe Acinetobacter baumannii infections in immunocompetent patients are uncommon, and the virulence mechanisms of this organism are not fully understood. METHODS: Following an outbreak of fatal A. baumannii infections in a cohort of relatively immunocompetent patients (low comorbidity and illness severity scores), isolates were investigated with comparative genomics and in animal models. RESULTS: Two unrelated A. baumannii clades were associated with the outbreak. The clone associated with the majority of patient deaths, clade B, is evolutionarily distinct from the 3 international clonal complexes, belongs to multilocus sequence type (MLST) 10, and is most closely related to strains isolated from the Czech Republic, California, and Germany in 1994, 1997, and 2003, respectively. In 2 different murine models, clade B isolates were more virulent than comparator strains, including the highly virulent reference strain AB5075. The most virulent clade B derivative, MRSN 16897, was isolated from the patient with the lowest combined comorbidity/illness severity score. Clade B isolates possess a unique combination of putative virulence genes involved in iron metabolism, protein secretion, and glycosylation, which was leveraged to develop a rapid and specific clinical assay to detect this clade that cannot be distinguished by MLST. CONCLUSIONS: Clade B warrants continued surveillance and investigation.

War wound treatment complications due to transfer of an IncN plasmid harboring bla(OXA-181) from Morganella morganii to CTX-M-27-producing sequence type 131 Escherichia coli.

A 22-year-old male developed a recurrent sacral abscess associated with embedded shrapnel following a blast injury. Cultures grew extended-spectrum ß-lactamase (ESBL)-producing, carbapenem-susceptible Escherichia coli. Ertapenem was administered, but the infection recurred after each course of antibiotics. Initial surgical interventions were unsuccessful, and subsequent cultures yielded E. coli and Morganella morganii, both nonsusceptible to carbapenems. The isolates were Carba NP test negative, gave ambiguous results with the modified Hodge test, and amplified the bla(OXA48)-like gene by real-time PCR. All E. coli isolates were sequence type 131 (ST131), carried nine resistance genes (including bla(CTX-M-27)) on an IncF plasmid, and were identical by genome sequencing, except for 150 kb of plasmid DNA in carbapenem-nonsusceptible isolates only. Sixty kilobases of this was shared by M. morganii and represented an IncN plasmid harboring bla(OXA-181). In M. morganii, the gene was flanked by IS3000 and ISKpn19, but in all but one of the E. coli isolates containing bla(OXA-181), a second copy of ISKpn19 had inserted adjacent to IS3000. To the best of our knowledge, this is the first report of bla(OXA-181) in the virulent ST131 clonal group and carried by the promiscuous IncN family of plasmids. The tendency of M. morganii to have high MICs of imipenem, a bla(OXA-181) substrate profile that includes penicillins but not extended-spectrum cephalosporins, and weak carbapenemase activity almost resulted in the presence of bla(OXA-181) being overlooked. We highlight the importance of surveillance for carbapenem resistance in all species, even those with intrinsic resistances, and the value of advanced molecular techniques in detecting subtle genetic changes.

IS5 element integration, a novel mechanism for rapid in vivo emergence of tigecycline nonsusceptibility in Klebsiella pneumoniae.

Tigecycline nonsusceptibility is concerning because tigecycline is increasingly relied upon to treat carbapenem- or colistin-resistant organisms. In Enterobacteriaceae, tigecycline nonsusceptibility is mediated by the AcrAB-TolC efflux pump, among others, and pump activity is often a downstream effect of mutations in their transcriptional regulators, cognate repressor genes, or noncoding regions, as demonstrated in Enterobacteriaceae and Acinetobacter isolates. Here, we report the emergence of tigecycline nonsusceptibility in a longitudinal series of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Klebsiella pneumoniae isolates collected during tigecycline therapy and the elucidation of its resistance mechanisms. Clinical isolates were recovered prior to and during tigecycline therapy of a 2.5-month-old Honduran neonate. Antimicrobial susceptibility tests to tigecycline determined that the MIC increased from 1 to 4 µg/ml prior to the completion of tigecycline therapy. Unlike other studies, we did not find increased expression of ramA, ramR, oqxA, acrB, marA, or rarA genes by reverse transcription-quantitative PCR (qRT-PCR). Whole-genome sequencing revealed an IS5 insertion element in nonsusceptible isolates 85 bp upstream of a putative efflux pump operon, here named kpgABC, previously unknown to be involved in resistance. Introduction of the kpgABC genes in a non-kpgABC background increased the MIC of tigecycline 4-fold and is independent of a functional AcrAB-TolC pump. This is the first report to propose a function for kpgABC and identify an insertion element whose presence correlated with the in vivo development of tigecycline nonsusceptibility in K. pneumoniae.

Emergence of colistin-resistance in extremely drug-resistant Acinetobacter baumannii containing a novel pmrCAB operon during colistin therapy of wound infections.

BACKGROUND: Colistin resistance is of concern since it is increasingly needed to treat infections caused by bacteria resistant to all other antibiotics and has been associated with poorer outcomes. Longitudinal data from in vivo series are sparse. METHODS: Under a quality-improvement directive to intensify infection-control measures, extremely drug-resistant (XDR) bacteria undergo phenotypic and molecular analysis. RESULTS: Twenty-eight XDR Acinetobacter baumannii isolates were longitudinally recovered during colistin therapy. Fourteen were susceptible to colistin, and 14 were resistant to colistin. Acquisition of colistin resistance did not alter resistance to other antibiotics. Isolates had low minimum inhibitory concentrations of an investigational aminoglycoside, belonged to multi-locus sequence type 94, were indistinguishable by pulsed-field gel electrophoresis and optical mapping, and harbored a novel pmrC1A1B allele. Colistin resistance was associated with point mutations in the pmrA1 and/or pmrB genes. Additional pmrC homologs, designated eptA-1 and eptA-2, were at distant locations from the operon. Compared with colistin-susceptible isolates, colistin-resistant isolates displayed significantly enhanced expression of pmrC1A1B, eptA-1, and eptA-2; lower growth rates; and lowered fitness. Phylogenetic analysis suggested that colistin resistance emerged from a single progenitor colistin-susceptible isolate. CONCLUSIONS: We provide insights into the in vivo evolution of colistin resistance in a series of XDR A. baumannii isolates recovered during therapy of infections and emphasize the importance of antibiotic stewardship and surveillance.

Novel 16S rRNA methyltransferase RmtH produced by Klebsiella pneumoniae associated with war-related trauma.

Klebsiella pneumoniae strain MRSN2404 was isolated from the chronic wound of a soldier who had been wounded in Iraq in 2006. The strain displayed very high MICs of all aminoglycosides, including arbekacin. A gene encoding a novel 16S rRNA methyltransferase, now designated RmtH, was identified. RmtH had 64% identity with RmtB1 and RmtB2. rmtH was bracketed by two copies of ISCR2, which may have played a role in its mobilization.

Bacterial peritonitis due to Acinetobacter baumannii sequence type 25 with plasmid-borne new delhi metallo-ß-lactamase in Honduras.

A carbapenem-resistant Acinetobacter baumannii strain was isolated from the peritoneal fluid of a patient with complicated intra-abdominal infection and evaluated at the Multidrug-resistant Organism Repository and Surveillance Network by whole-genome sequencing and real-time PCR. The isolate was sequence type 25 and susceptible to colistin and minocycline, with low MICs of tigecycline. blaNDM-1 was located on a plasmid with >99% homology to pNDM-BJ02. The isolate carried numerous other antibiotic resistance genes, including the 16S methylase gene, armA.

Rapid and simultaneous detection of blaKPC and blaNDM by use of multiplex real-time PCR.

The increasing incidence of carbapenem nonsusceptibility among clinically important species is of global concern. Identification of the molecular mechanisms underlying carbapenem nonsusceptibility is critical for epidemiological investigations. In this report, we describe a real-time PCR-based assay capable of simultaneously detecting blaKPC and blaNDM, two of the most important carbapenemases, directly from culture in less than 90 min. The assay was validated with blaKPC- and blaNDM-carrying clinical isolates and demonstrated 100% concordance with the Carba NP test.

Detection of New Delhi metallo-ß-lactamase (encoded by blaNDM-1) in Acinetobacter schindleri during routine surveillance.

A carbapenem-resistant Alcaligenes faecalis strain was isolated from a surveillance swab of a service member injured in Afghanistan. The isolate was positive for bla(NDM) by real-time PCR. Species identification was reevaluated on three identification systems but was inconclusive. Genome sequencing indicated that the closest relative was Acinetobacter schindleri and that bla(NDM-1) was carried on a plasmid that shared >99% identity with one identified in an Acinetobacter lwoffii isolate. The isolate also carried a novel chromosomally encoded class D oxacillinase.

Investigating the genome diversity of B. cereus and evolutionary aspects of B. anthracis emergence.

Here we report the use of a multi-genome DNA microarray to investigate the genome diversity of Bacillus cereus group members and elucidate the events associated with the emergence of Bacillus anthracis the causative agent of anthrax-a lethal zoonotic disease. We initially performed directed genome sequencing of seven diverse B. cereus strains to identify novel sequences encoded in those genomes. The novel genes identified, combined with those publicly available, allowed the design of a "species" DNA microarray. Comparative genomic hybridization analyses of 41 strains indicate that substantial heterogeneity exists with respect to the genes comprising functional role categories. While the acquisition of the plasmid-encoded pathogenicity island (pXO1) and capsule genes (pXO2) represents a crucial landmark dictating the emergence of B. anthracis, the evolution of this species and its close relatives was associated with an overall shift in the fraction of genes devoted to energy metabolism, cellular processes, transport, as well as virulence.

Design of a Bacteriophage Cocktail Active against Shigella Species and Testing of Its Therapeutic Potential in Galleria mellonella.

Shigellosis is a leading global cause of diarrheal disease and travelers' diarrhea now being complicated by the dissemination of antibiotic resistance, necessitating the development of alternative antibacterials such as therapeutic bacteriophages (phages). Phages with lytic activity against Shigella strains were isolated from sewage. The genomes of 32 phages were sequenced, and based on genomic comparisons belong to seven taxonomic genera: Teetrevirus, Teseptimavirus, Kayfunavirus, Tequatrovirus, Mooglevirus, Mosigvirus and Hanrivervirus. Phage host ranges were determined with a diverse panel of 95 clinical isolates of Shigella from Southeast Asia and other geographic regions, representing different species and serotypes. Three-phage mixtures were designed, with one possessing lytic activity against 89% of the strain panel. This cocktail exhibited lytic activity against 100% of S. sonnei isolates, 97.2% of S. flexneri (multiple serotypes) and 100% of S. dysenteriae serotypes 1 and 2. Another 3-phage cocktail composed of two myophages and one podophage showed both a broad host range and the ability to completely sterilize liquid culture of a model virulent strain S. flexneri 2457T. In a Galleria mellonella model of lethal infection with S. flexneri 2457T, this 3-phage cocktail provided a significant increase in survival.

IS26-mediated plasmid reshuffling results in convergence of toxin-antitoxin systems but loss of resistance genes in XDR Klebsiella pneumoniae from a chronic infection.

Carbapenem-resistant Enterobacterales pose an urgent threat to human health worldwide. Klebsiella pneumoniae sequence type (ST) 14, initially identified in the Middle East and South-Asia and co-harbouring the carbapenemase genes bla OXA-232 and bla NDM-1, is now emerging globally. One such strain was detected in the USA in 2013 from a patient initially treated in India that also carried armA, a 16S rRNA methyltransferase that confers resistance to all clinically relevant aminoglycosides. Genetic and phenotypic changes were observed in 14 serial isolates collected from this chronically infected patient. The index isolate carried five plasmids, including an IncFIB-IncHI1B (harbouring armA and bla NDM-1), an IncFIA (bla CTX-M-15) and a ColE-like (bla OXA-232), and was extensively resistant to antibiotics. Four years later, a subsequent isolate had accumulated 34 variants, including a loss-of-function mutation in romA, resulting in tigecycline non-susceptibility. Importantly, this isolate now only carried two plasmids, including a large mosaic molecule made of fragments, all harbouring distinct toxin-antitoxin systems, from three of the canonical plasmids. Of the original acquired antibiotic resistance genes, this isolate only retained bla CTX-M-15, and as a result susceptibility to the carbapenems and amikacin was restored. Long-read sequencing of a subset of five representative isolates, collected between 2013 and 2017, allowed for the elucidation of the complex plasmid patterns and revealed the role of IS26-mediated plasmid reshuffling in the evolution of this clone. Such investigations of the mechanisms underlying plasmid stability, together with global and local surveillance programmes, are key to a better understanding of plasmid host range and dissemination.