BKC-2, a New BKC Variant Detected in MCR-9.1-Producing Enterobacter hormaechei subsp. xiangfangensis.

We characterized a multidrug-resistant (MDR) Enterobacter spp. isolate highlighting the genetic aspects of the antimicrobial resistance genes. An Enterobacter spp. isolate (Ec61) was recovered in 2014 from a transtracheal aspirate sample from a patient admitted to a Brazilian tertiary hospital and submitted to further microbiological and genomic characterization. Ec61 was identified as Enterobacter hormaechei subsp. xiangfangensis strain ST451, showing an MDR profile and the presence of genes codifying the new ß-lactamase variants BKC-2 and ACT-84 and the mobile colistin resistance gene mcr-9.1.

Human carriage of cefotaxime-resistant Escherichia coli in North-East India: an analysis of STs and associated resistance mechanisms.

OBJECTIVES: To determine the prevalence of Escherichia coli STs and associated resistance mechanisms carried by the community in North-East India. METHODS: E. coli (108) were isolated from sewage collected from 19 sites across the city of Silchar by plating on MacConkey agar with/without selection (50 mg/L cefotaxime). Species identification was confirmed by MALDI-TOF MS for 82 isolates. Common resistance mechanisms were determined by WGS of pooled E. coli isolates. PFGE combined with specific probes determined the presence of common resistance mechanisms in all isolates. Phylotypes, multilocus STs, core-genome multilocus STs, resistance genes and virulence genes were determined by in silico analysis of 38 genomes. RESULTS AND CONCLUSIONS: Analysis of isolates collected without selection (n=33) indicated that cefotaxime resistance in E. coli was 42% (14/33) and estimated meropenem resistance at 9%. The remaining 58% (19/33) were additionally susceptible to ampicillin, trimethoprim, ciprofloxacin and aminoglycosides. The most common ST among the cefotaxime-resistant E. coli was ST167 (29%), followed by ST410 (17%) and ST648 (10%). E. coli ST131 was absent from the collection. Sixty-three isolates were resistant to cefotaxime and harboured blaCTX-M-15 [54% (34/63)] or blaCMY-42 [46% (29/63)], of which 10% (6/63) harboured both genes. Carbapenem resistance was due to blaNDM-5, found in 10/63 cefotaxime-resistant isolates, and/or blaOXA-181, found in 4/63 isolates. NDM-5 was encoded by IncX3 and/or IncFII plasmids and CMY-42 was mostly encoded by IncI plasmids. NDM-5 appears to have replaced NDM-1 in this region and CMY-42 appears to be in the process of replacing CTX-M-15.

OXA-1 ß-lactamase and non-susceptibility to penicillin/ß-lactamase inhibitor combinations among ESBL-producing Escherichia coli.

Background: ESBL-producing Escherichia coli have expanded globally since the turn of the century and present a major public health issue. Their in vitro susceptibility to penicillin/inhibitor combinations is variable, and clinical use of these combinations against ESBL producers remains controversial. We hypothesized that this variability related to co-production of OXA-1 penicillinase. Methods: During a national study we collected 293 ESBL-producing E. coli from bacteraemias, determined MICs by BSAC agar dilution, and undertook genomic sequencing with Illumina methodology. Results: The collection was dominated by ST131 (n = 188 isolates, 64.2%) and blaCTX-M-15 (present in 229 isolates, 78.2%); over half the isolates (159/293, 54.3%) were ST131 with blaCTX-M-15. blaOXA-1 was found in 149 ESBL producers (50.9%) and blaTEM-1/191 in 137 (46.8%). Irrespective of whether all isolates were considered, or ST131 alone, there were strong associations (P < 0.001) between co-carriage of blaOXA-1 and reduced susceptibility to penicillin/inhibitor combinations, whereas there was no significant association with co-carriage of blaTEM-1/191. For piperacillin/tazobactam the modal MIC rose from 2 mg/L in the absence of blaOXA-1 to 8 or 16 mg/L in its presence; for co-amoxiclav the shift was smaller, from 4 or 8 to 16 mg/L, but crossed the breakpoint. blaOXA-1 was strongly associated with co-carriage also of aac(6')-Ib-cr, which compromises amikacin and tobramycin. Conclusions: Co-carriage of OXA-1, a penicillinase with weak affinity for inhibitors, is a major correlate of resistance to piperacillin/tazobactam and co-amoxiclav in E. coli and is commonly associated with co-carriage of aac(6')-Ib-cr, which narrows aminoglycoside options.

Analysis of Escherichia coli STs and resistance mechanisms in sewage from Islamabad, Pakistan indicates a difference in E. coli carriage types between South Asia and Europe.

Objectives: To discover the Escherichia coli STs and associated resistance mechanisms in the community in Islamabad, Pakistan by analysis of E. coli isolates in sewage. Methods: One hundred and ten E. coli were isolated from sewage across the city of Islamabad without antibiotic bias and confirmed as E. coli by MALDI-TOF MS. Isolates were characterized by fumC/fimH (CH) typing and core-genome MLST. Resistance mechanisms, virulence genes, phylotypes and plasmid incompatibility types were determined in a subset of isolates by in silico analysis. The genomic position of blaCTX-M-15 was determined using S1-PFGE, probing and Nanopore MinION sequencing. Results and conclusions: The most prevalent STs were ST394, ST10 and ST648, accounting for 39% of all isolates collected and were found at many sites across Islamabad. Carbapenemase genes were absent and only a single isolate of ST131 was found. The most prevalent resistance mechanisms were qnrS1 and blaCTX-M-15, with blaCTX-M-15 penetrating many STs and found in 31% of all collected isolates. However, the majority of the successful STs were blaCTX-M-15 negative indicating that resistance is not the main driver of prevalence. Twenty-three percent of blaCTX-M-15 genes were chromosomally encoded and large ISEcp1-mediated insertions included qnrS1 and several plasmid genes. In all chromosomally encoded isolates no plasmid copies of blaCTX-M-15 were found. The most prevalent ST (ST394) contained many enteroaggregative E. coli virulence genes and the fimH30 variant allele previously linked to the success of ST131.

Unconventional Human T Cells Accumulate at the Site of Infection in Response to Microbial Ligands and Induce Local Tissue Remodeling.

The antimicrobial responsiveness and function of unconventional human T cells are poorly understood, with only limited access to relevant specimens from sites of infection. Peritonitis is a common and serious complication in individuals with end-stage kidney disease receiving peritoneal dialysis. By analyzing local and systemic immune responses in peritoneal dialysis patients presenting with acute bacterial peritonitis and monitoring individuals before and during defined infectious episodes, our data show that Vγ9/Vδ2(+) γδ T cells and mucosal-associated invariant T cells accumulate at the site of infection with organisms producing (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and vitamin B2, respectively. Such unconventional human T cells are major producers of IFN-γ and TNF-α in response to these ligands that are shared by many microbial pathogens and affect the cells lining the peritoneal cavity by triggering local inflammation and inducing tissue remodeling with consequences for peritoneal membrane integrity. Our data uncover a crucial role for Vγ9/Vδ2 T cells and mucosal-associated invariant T cells in bacterial infection and suggest that they represent a useful predictive marker for important clinical outcomes, which may inform future stratification and patient management. These findings are likely to be applicable to other acute infections where local activation of unconventional T cells contributes to the antimicrobial inflammatory response.

World Health Organization Ranking of Antimicrobials According to Their Importance in Human Medicine: A Critical Step for Developing Risk Management Strategies to Control Antimicrobial Resistance From Food Animal Production.

Antimicrobial use in food animals selects for antimicrobial resistance in bacteria, which can spread to people. Reducing use of antimicrobials-particularly those deemed to be critically important for human medicine-in food production animals continues to be an important step for preserving the benefits of these antimicrobials for people. The World Health Organization ranking of antimicrobials according to their relative importance in human medicine was recently updated. Antimicrobials considered the highest priority among the critically important antimicrobials were quinolones, third- and fourth-generation cephalosporins, macrolides and ketolides, and glycopeptides. The updated ranking allows stakeholders in the agriculture sector and regulatory agencies to focus risk management efforts on drugs used in food animals that are the most important to human medicine. In particular, the current large-scale use of fluoroquinolones, macrolides, and third-generation cephalosporins and any potential use of glycopeptides and carbapenems need to be addressed urgently.

Extensively drug-resistant New Delhi metallo-ß-lactamase-encoding bacteria in the environment, Dhaka, Bangladesh, 2012.

Carriage of the New Delhi metallo-ß-lactamase variant 1 (NDM-1) enables drug resistance to move between communities and hospitals. In Bangladesh, we found the blaNDM-1 gene in 62% of environmental waters and in fermentative and nonfermentative gram-negative bacteria. Escherichia coli sequence type (ST) 101 was most commonly found, reflecting a common global relationship between ST101 and NDM-1.

Characterization of plasmids in extensively drug-resistant acinetobacter strains isolated in India and Pakistan.

The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae.

Plasmid carriage of bla NDM-1 in clinical Acinetobacter baumannii isolates from India.

NDM-1 probably emerged in Acinetobacter species prior to its dissemination among Enterobacteriaceae, and NDM-1-like enzymes are increasingly reported in Acinetobacter species. Here, we report on the genetic context of blaNDM-1 in the earliest known NDM-1-producing organisms, clinical isolates of Acinetobacter from India in 2005. These strains harbor blaNDM-1 plasmids of different sizes. The gene is associated with the remnants of the Tn125 transposon normally associated with blaNDM-1 in Acinetobacter spp. The transposon has been disrupted by the IS26 insertion and subsequent movement events.

Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.

While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models.

Safety and Quality Within the Healthcare Supply Chain: A Great Unknown.

Quality and safety in healthcare have emerged as key factors impacting on both clinical effectiveness and clinical outcomes. While improving the healthcare supply chain has been extensively researched, the impact of the healthcare supply chain on clinical safety has received little attention in the literature, largely due to the complexity of such studies and the involvement of multiple stakeholders. This research proposes an evaluation model using key performance measurements for an electronic procurement system that enables digital transformation of the healthcare supply chain. The model will be tested before and after the introduction of an electronic procurement system in the healthcare supply chain for small and medium sized healthcare providers to provide evidence of both the usefulness of the model itself within industry and to further contribute to the knowledge base. Future use of the model may provide benchmarking and important data and insights to enable enhanced clinical safety in the healthcare supply chain.

Genetic characterization and emergence of the metallo-ß-lactamase GIM-1 in Pseudomonas spp. and Enterobacteriaceae during a long-term outbreak.

Since the first isolation in 2002, the metallo-ß-lactamase GIM-1 has not been detected outside Germany. The data presented here, for 50 clinical blaGIM-1-positive isolates, including Pseudomonas spp. and Enterobacteriaceae (Enterobacter cloacae, Klebsiella oxytoca, Serratia marcescens, Escherichia coli, and Citrobacter freundii), collected between 2007 and 2012 at the original site in an ongoing outbreak, demonstrate a diverse genetic background and dissemination of the gene conferring resistance to enteric bacteria.

Identical miniature inverted repeat transposable elements flank class 1 integrons in clinical isolates of Acinetobacter spp.

Miniature inverted repeat transposable elements (MITEs) have been identified flanking class 1 integrons. We have identified and characterized a 439-bp MITE-like structure in seven Acinetobacter species isolates from Portugal and Brazil. The complete sequence similarity of the elements and flanking regions suggests that MITEs may act as mobilizable vectors for the dissemination of integrons.

Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection.

Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis--characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity--show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early infection and suggest novel diagnostic and therapeutic approaches.

Whole genome-based genetic insights of blaNDM producing clinical E. coli isolates in hospital settings of Pakistan.

Carbapenem resistance among Enterobacterales has become a global health concern. Clinical Escherichia coli isolates producing the metallo ß-lactamase NDM have been isolated from two hospitals in Faisalabad, Pakistan. These E. coli strains were characterized by MALDI-TOF, PCR, antimicrobial susceptibility testing, XbaI and S1 nuclease pulsed-field gel electrophoresis (PFGE), conjugation assay, DNA hybridization, whole genome sequencing, bioinformatic analysis, and Galleria mellonella experiments. Thirty-four blaNDM producing E. coli strains were identified among 52 nonduplicate carbapenem-resistant strains. More than 90% of the isolates were found to be multidrug resistant by antimicrobial susceptibility testing. S1 PFGE confirmed the presence of blaNDM gene on plasmids ranging from 40 kbps to 250 kbps, and conjugation assays demonstrated transfer frequencies of blaNDM harboring plasmids ranging from 1.59 × 10-1 to 6.46 × 10-8 per donor. Whole genome sequencing analysis revealed blaNDM-5 as the prominent NDM subtype with the highest prevalence of blaOXA-1, blaCTX-M-15, aadA2, aac(6')-Ib-cr, and tet(A) associated resistant determinants. E. coli sequence types: ST405, ST361, and ST167 were prominent, and plasmid Inc types: FII, FIA, FIB, FIC, X3, R, and Y, were observed among all isolates. The genetic environment of blaNDM region on IncF plasmids included partial ISAba125, the bleomycin ble gene, and a class I integron. The virulence genes terC, traT, gad, fyuA, irp2, capU, and sitA were frequently observed, and G. mellonella experiments showed that virulence correlated with the number of virulence determinants. A strong infection control management in the hospital is necessary to check the emergence of carbapenem resistance in Gram-negative bacteria.IMPORTANCEWe describe a detailed analysis of highly resistant clinical E. coli isolates from two tertiary care centers in Pakistan including carbapenem resistance as well as common co-resistance mechanisms. South Asia has a huge problem with highly resistant E. coli. However, we find that though these isolates are very difficult to treat they are of low virulence. Thus the Western world has an increasing problem with virulent E. coli that are mostly of low antibiotic resistance, whereas, South Asia has an increasing problem with highly resistant E. coli that are of low virulence potential. These observations allow us to start to devise methodologies to limit both virulence and resistance and combat problems in developing nations as well as the Western world.

Genetic and biochemical characterization of a novel metallo-ß-lactamase, TMB-1, from an Achromobacter xylosoxidans strain isolated in Tripoli, Libya.

An Achromobacter xylosoxidans strain from the Tripoli central hospital produced a unique metallo-ß-lactamase, designated TMB-1, which is related to DIM-1 (62%) and GIM-1 (51%). bla(TMB-1) was embedded in a class 1 integron and located on the chromosome. The TMB-1 ß-lactamase has lower k(cat) values than both DIM-1 and GIM-1 with cephalosporins and carbapenems. The K(m) values were more similar to those of GIM-1 than those of DIM-1, with the overall k(cat)/K(m) values being lower than those for GIM-1 and DIM-1.

Genetic and biochemical characterization of an acquired subgroup B3 metallo-ß-lactamase gene, blaAIM-1, and its unique genetic context in Pseudomonas aeruginosa from Australia.

Three clinical Pseudomonas aeruginosa isolates (WCH2677, WCH2813, and WCH2837) isolated from the Women's and Children's Hospital, Adelaide, Australia, produced a metallo-ß-lactamase (MBL)-positive Etest result. All isolates were PCR negative for known MBL genes. A gene bank was created, and an MBL gene, designated bla(AIM-1), was cloned and fully characterized. The encoded enzyme, AIM-1, is a group B3 MBL that has the highest level of identity to THIN-B and L1. It is chromosomal and flanked by two copies (one intact and one truncated) of an ISCR element, ISCR15. Southern hybridization studies indicated the movement of both ISCR15 and bla(AIM-1) within the three different clinical isolates. AIM-1 hydrolyzes most ß-lactams, with the exception of aztreonam and, to a lesser extent, ceftazidime; however, it possesses significantly higher k(cat) values for cefepime and carbapenems than most other MBLs. AIM-1 was the first mobile group B3 enzyme detected and signals further problems for already beleaguered antimicrobial regimes to treat serious P. aeruginosa and other Gram-negative infections.

Diverse sequence types of Klebsiella pneumoniae contribute to the dissemination of blaNDM-1 in India, Sweden, and the United Kingdom.

Clinical isolates of Klebsiella pneumoniae producing NDM-1 carbapenemase from India (n = 22), the United Kingdom (n = 13), and Sweden (n = 4) were subjected to multilocus sequence typing (MLST), automated repetitive sequence-based PCR (rep-PCR), serotyping, virulence gene screening, and plasmid replicon typing. The most frequently detected MLST sequence types (STs) were ST14 (n = 13; all serotype K2), ST11, ST149, ST231, and ST147. The correlation between MLST and automated rep-PCR was excellent. IncA/C was the most frequently detected plasmid replicon type (n = 14). ST14, ST11, and other successful clones may be important for the dissemination of bla(NDM-1).

The emergence of pan-resistant Gram-negative pathogens merits a rapid global political response.

Recent media coverage of New Delhi metallo-ß-lactamase (NDM-1) put antibiotic resistance back on the political map if only for the wrong reasons, mainly the reaction to the naming of NDM-1 and the incorrect assumption that medical tourism was being deliberately targeted. However, work on NDM-1 has most certainly highlighted the rapid dissemination of new antibiotic resistance mechanisms via economic globalization. The example of NDM-1 has also magnified the desperate need for a publicly funded global antibiotic surveillance system rather than just national or regional systems. Furthermore, there is a pressing need to establish a global task force to enforce international transparency and accountability on antibiotic stewardship and the implementation of measures to curb antibiotic resistance. An international antibiotic stewardship index should be established that is related to each country's gross domestic product (GDP) and assesses how much of their GDP is committed to publically funded health initiatives aimed at controlling antibiotic resistance.

Plasmid typing and genetic context of AmpC ß-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes: findings from a Spanish hospital 1999-2007.

OBJECTIVES: To gain insights into ampC transmission between bacterial strains. METHODS: We examined the genetic context of 117 acquired ampC genes from 27119 Enterobacteriaceae collected between 1999 and 2007. Plasmid analysis was carried out by PCR-based replicon or relaxase typing, S1-PFGE and Southern hybridization. I-CeuI/PFGE was used for isolates not characterized by plasmid analysis. PCR reactions were used to map the genetic organization of the ampC genes. RESULTS: Among the isolates studied, 81.2% of ampC genes were located on plasmids of known Inc/MOB groups, 7.7% were chromosomally located and 11.1% were not determined. A/C, I1 and K were the most commonly found replicons in plasmids carrying bla(CMY-2), while L/M replicons were associated with bla(DHA-1). bla(ACC-1) was linked to I1 and MOB(F11) plasmids; bla(CMY-27) was associated with IncF and MOB(P12) plasmids; the plasmid carrying bla(CMY-25) could not be typed, and bla(CMY-40) was chromosomally located. All 87 isolates carrying bla(CMY-2), bla(CMY-4), bla(CMY-25), bla(CMY-27), bla(CMY-40) or bla(ACC-1) displayed the transposon-like structures ISEcp1/ΔISEcp1-bla(CMY)-blc-sugE or ΔISEcp1-bla(ACC-1)-gdha. The most prevalent structure in bla(DHA-1) (93.3% of cases) was identical to that described in the Klebsiella pneumoniae pTN60013 plasmid. Remarkably, in three isolates containing chromosomal bla(CMY-2), this gene was mobilized by conjugation. CONCLUSIONS: Although plasmids are the main cause of the rapid dissemination of ampC genes among bacteria, we need to be aware that other mobile genetic elements such as integrative and conjugative elements (ICEs) can be involved in the mobilization of these genes.