Population-based surveillance of Enterobacter cloacae complex causing blood stream infections in a centralized Canadian region.

Active population-based surveillance determined clinical factors, susceptibility patterns, incidence rates (IR), and genomics among Enterobacter cloacae complex (n = 154) causing blood stream infections in a centralized Canadian region (2015-2017). The annual population IR was 1.2/100,000 (95% CI 0.9-16) in 2015, 1.4/100,000 (95% CI 1.1-1.9) in 2016, and 1.5/100,000 (95% CI 1.2-2.0) in 2017, affecting mainly elderly males with underlying comorbid conditions in the hospital setting. E. cloacae complex was dominated by polyclonal subspecies (i.e., E. hormaechei subsp. steigerwaltii, subsp. hoffmanni and subsp. xiangfangesis). Antimicrobial resistant determinants were rare. This study provided novel information about Enterobacter genomics in a well-defined human population.

Investigation of possible transmission of a susceptible microorganism through a contaminated duodenoscope; a case report.

BACKGROUND: Despite compliance to extensive reprocessing protocols, duodenoscopes have been linked to outbreaks of susceptible and multi-drug resistant organisms (MDRO) due to persistent duodenoscope contamination. Duodenoscope-associated infections (DAIs) based on transmission of susceptible microorganisms are likely to be underreported due to detection bias. CASE PRESENTATION: We describe the retrospective detection of a DAI case caused by a susceptible microorganism which at the time of clinical infection was not recognized as such. During 2017 and 2018, duodenoscopes were cultured on a daily basis due to research activities. While analyzing this data, it was found that a duodenoscope had been contaminated with Enterobacter cloacae complex over a period of 3 months. We checked whether patients treated with this duodenoscope had developed infections and found one patient with an E. cloacae cholangitis 3 months after the ERCP (Endoscopic retrograde cholangiopancreaticography) procedure. The isolates on the duodenoscope and in the patients' blood culture were indistinguishable by amplified fragment length polymorphism (AFLP). By classical multi-locus sequence typing (MLST), both strains were of the same (but novel) sequence type. Application of whole genome MLST showed 93 (out of 3757) allelic differences. CONCLUSION: This case report describes a plausible link between a contaminated duodenoscope and a patient infection with E. cloacae. Transmission of susceptible E. cloacae was highly suspected from AFLP and MLST results; by WGS, 93 allelic differences were found which proves closely related strains. This report shows that DAIs by susceptible microorganisms can be easily missed and therefore its true prevalence remains underscored.

Genome-wide Analysis of Four Enterobacter cloacae complex type strains: Insights into Virulence and Niche Adaptation.

Enterobacter cloacae complex (Ecc) species are widely distributed opportunistic pathogens mainly associated with humans and plants. In this study, the genomes of clinical isolates including E. hormaechei, E. kobei, and E. ludwigii and non-clinical isolate including E. nimipressuralis were analysed in combination with the genome of E. asburiae by using the reference strain E. cloacae subsp. cloacae ATCC 13047; the Ecc strains were tested on artificial sputum media (ASM), which mimics the host, to evaluate T6SS genes as a case study. All five Ecc strains were sequenced in our lab. Comparative genome analysis of the Ecc strains revealed that genes associated with the survival of Ecc strains, including genes of metal-requiring proteins, defence-associated genes and genes associated with general physiology, were highly conserved in the genomes. However, the genes involved in virulence and drug resistance, specifically those involved in bacterial secretion, host determination and colonization of different strains, were present in different genomic regions. For example, T6SS accessory and core components, T4SS, and multidrug resistance genes/efflux system genes seemed vital for the survival of Ecc strains in various environmental niches, such as humans and plants. Moreover, the ASM host-mimicking growth medium revealed significantly high expression of T6SS genes, including PrpC, which is a regulatory gene of the T6SS, in all tested Ecc strains compared to the control medium. The variations in T6SS gene expression in ASM vs. control showed that the ASM system represents a simple, reproducible and economical alternative to animal models for studies such as those aimed at understanding the divergence of Ecc populations. In summary, genome sequencing of clinical and environmental Ecc genomes will assist in understanding the epidemiology of Ecc strains, including the isolation, virulence characteristics, prevention and treatment of infectious disease caused by these broad-host-range niche-associated species.

PCR Detection of Oxacillinases in Bacteria.

Oxacillinases (OXA) have been mostly described in Enterobacteriaceae, Acinetobacter, and Pseudomonas species. Recent years have witnessed an increased prevalence of intrinsic and/or acquired ß-lactamase-producing Acinetobacter in food-producing animals. This study was conducted to assess the prevalence of OXA among selected bacterial species and to characterize these enzymes by in silico analysis. Screening of OXA was performed by PCR amplification using specific pairs of oligonucleotides. Overall, 40 pairs of primers were designed, of which 6 were experimentally tested in vitro. Among 49 bacterial isolates examined, the presence of blaOXA-1-like genes was confirmed in 20 cases (41%; 19 times in Klebsiella pneumoniae and once in Enterobacter cloacae). No OXA were found in animal isolates. The study results confirmed the specificity of the designed oligonucleotide pairs. Furthermore, the designed primers were found to possess the ability to specifically detect 90.2% of all OXA. These facts suggest that the in silico and in vitro tested primers could be used for single or multiplex PCR to screen for the presence of OXA in various bacteria, as well as to monitor their spread. At the same time, the presence of conserved characteristic amino acids and motifs was confirmed by in silico analysis of sequences of representative members of OXA.

The whole-genome sequence analysis of Enterobacter cloacae strain Ghats1: insights into endophytic lifestyle-associated genomic adaptations.

Enterobacter cloacae is normally considered to be an opportunistic human pathogen. Here, we report on the whole-genome sequence of an endophytic E. cloacae, strain "Ghats1", isolated from leaves of the medicinal plant Coscinium fenestratum Gaertn. Functional analysis of the Ghats1 genome revealed an enrichment for genes involved in the uptake and exchange of nutrients, for chemotaxis and for plant colonization. Unexpectedly though, there were no ORFs belonging to the "virulence factors and antibiotic resistance". Moreover, the presence of hydrolytic enzymes and motility functions reveals the characteristics of an endophyte lifestyle of a bacterium that can colonize and adapt to plant environment. These results provide a better understanding of an endophytic lifestyle through plant-microbe interaction, which can be further exploited as a biocontrol agent.

Epidemiology of Enterobacter cloacae strains producing a carbapenemase or metallo-beta-lactamase in Vietnamese clinical settings in 2014-2017.

Introduction. Little is known about the epidemiology of Enterobacter cloacae strains producing a carbapenemase or metallo-beta-lactamase in Vietnamese hospitals.Aim. This study analysed E. cloacae strains resistant to imipenem or meropenem that had been isolated from patients admitted to one of the largest hospitals in Vietnam in 2014-2017.Methodology. Eighteen Vietnamese (VN) strains were subjected to whole-genome sequencing and their sequences compared with those of 17 E. cloacae strains carrying a carbapenemase or metallo-beta-lactamase in the database (db strains).Results. Although the distribution of virulence factors did not differ significantly between VN and db strains, all 18 VN isolates harboured blaNDM-1, phylogenetic analysis revealed a high clonality of the VN strains. Bayesian phylogenetic analysis suggested that the VN strains speciated relatively recently.Conclusions. Several prevalent clones of carbapenem-resistant E. cloacae have circulated within Vietnamese hospitals. Adequate measures are needed to prevent their further spread.

Molecular characterization of metallo-ß-lactamase- producing carbapenem-resistant Enterobacter cloacae complex isolated in Heilongjiang Province of China.

BACKGROUND: Enterobacter cloacae complex (ECC) is one of the most common extended-spectrum ß-lactamase and carbapenemase-producing pathogen that threatens millions of the elderly and vulnerable sick persons. The objective of this study was to perform the molecular characteristics of the carbapenem-resistant E. cloacae complex (CREC) emerged in Heilongjiang Province of China. METHODS: Six CREC strains were isolated from the patients with infectious diseases. The identities of ECC isolates were confirmed by sequencing the polymerase chain reaction (PCR) products of 16S rRNA gene. The characterization of the CREC isolates were analyzed by sequencing PCR products of the carbapenemase, ampC and fluoroquinolone resistance genes and performing multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole genome sequencing. RESULTS: All 6 isolates harbored multiple resistance genes. Of them, 5 carried metallo-ß-lactamases and one was blaKPC-2-positive. The levofloxacin and ciprofloxacin-resistant strains had substitutions of gyrA83, gyrA87, and parC80 in the quinolone-resistance determining regions. The MLST analyses revealed that 6 isolates belonged to five sequence types (ST520, ST528, ST1119, ST1120, and ST93) while the PFGE patterns of the isolates fallen into four clusters. The strain ST1120 was found to carry two separated plasmids that encode blaNDM-1 and blaIMP-4. CONCLUSIONS: Our study, for the first time, identified a CREC strain that co-produces blaNDM-1 and blaIMP-4 in the Northeast China. Our finding emphasizes an urgent need for more intensive surveillance and precaution measures to prevent the CERC spread.

Characterization of NDM-1- and CMH-3-producing Enterobacter cloacae complex ST932 in a patient transferred from Ukraine to Spain.

INTRODUCTION: To characterize a carbapenem-resistant Enterobacter cloacae complex isolate recovered from a patient from Ukraine. METHODS: The isolate was sent to a regional reference laboratory for molecular characterization by whole genome sequencing. Susceptibility assays, carbapenemase identification, imipenem hydrolysis and clonality were performed. RESULTS: The isolate showed resistance or reduced susceptibility to all ß-lactam agents tested. Genome analysis led to the identification of an NDM-1-producing E. cloacae complex strain that was assigned to a new multilocus sequence type, ST932. The blaNDM-1 enzyme was located in a conjugative IncX3 plasmid of ca. 50kb. In addition, blaCMH-3, a recently described AmpC ß-lactamase sequence, which has not previously been reported in Europe, was also detected and its genetic environment was studied. CONCLUSION: To our knowledge, this is the first reported case in Europe of an E. cloacae complex strain that produces both blaNDM-1 and blaCMH-3.

Species-specific transcriptional profiles of the gut and gut microbiome of Ceratitis quilicii and Ceratitis rosa sensu stricto.

The fruit fly species, Ceratitis rosa sensu stricto and Ceratitis quilicii, are sibling species restricted to the lowland and highland regions, respectively. Until recently, these sibling species were considered as allopatric populations of C. rosa with distinct bionomics. We used deep Next Generation Sequencing (NGS) technology on intact guts of individuals from the two sibling species to compare their transcriptional profiles and simultaneously understand gut microbiome and host molecular processes and identify distinguishing genetic differences between the two species. Since the genomes of both species had not been published previously, the transcriptomes were assembled de novo into transcripts. Microbe-specific transcript orthologs were separated from the assembly by filtering searches of the transcripts against microbe databases using OrthoMCL. We then used differential expression analysis of host-specific transcripts (i.e. those remaining after the microbe-specific transcripts had been removed) and microbe-specific transcripts from the two-sibling species to identify defining species-specific transcripts that were present in only one fruit fly species or the other, but not in both. In C. quilicii females, bacterial transcripts of Pectobacterium spp., Enterobacterium buttiauxella, Enterobacter cloacae and Klebsiella variicola were upregulated compared to the C. rosa s.s. females. Comparison of expression levels of the host transcripts revealed a heavier investment by C. quilicii (compared with C. rosa s.s.) in: immunity; energy production; cell proliferation; insecticide resistance; reproduction and proliferation; and redox reactions that are usually associated with responses to stress and degradation of fruit metabolites.

Colistin-Resistant mcr-Positive Enterobacteriaceae in Fresh Vegetables, an Increasing Infectious Threat in China.

The presence of mobilized colistin resistance (mcr) genes is a global concern. However, data concerning mcr in fresh vegetables, a reservoir for antibiotic resistance genes, are still rare. In this study, mcr genes were analysed in 528 vegetable samples from 53 supermarkets or farmer's markets in 23 cities of 9 provinces in China, and the mcr-positive Enterobacteriaceae were characterized. Nineteen (3.6%) samples carried one or more mcr-positive isolates, and the highest three detection rates were found in carrot, pak choi and green pepper. Twenty-four mcr-1-positive isolates (23 Escherichia coli and one Enterobacter cloacae) were obtained, and E. coli isolates showed high genetic diversity. Different multilocus sequence type (MLST) isolates were also observed within the same sample. All 24 isolates showed multidrug resistance, and 14 carried blaCTX-M genes. Most isolates harbored similarly conjugative IncX4-type (∼33 kb) or IncI2-type (∼60 kb) mcr-1-bearing plasmids. The sequenced prevalent IncX4 plasmid and IncI2 plasmid from tomato were similar to the relevant plasmids from animals and clinical isolates in various countries. mcr-1-bearing IncHI2/ST3 plasmid highly similar to that carrying 14 resistance genes from E. coli of chicken was also observed. In conclusion, a high prevalence of mcr-1 in fresh vegetables was found in China, and the dissemination of mcr-1 was mediated by similar IncX4 or IncI2 plasmids. The plasmids from vegetables showed high similarity to plasmids from clinical isolates, indicating MCR-1-producers in ready-to-eat vegetables may pose a huge threat to public health and measures need to be taken to ensure food safety.

High distribution of 16S rRNA methylase genes rmtB and armA among Enterobacter cloacae strains isolated from an Ahvaz teaching hospital, Iran.

The emergence of 16S rRNA methylase genes encoded on plasmids confers high-level aminoglycoside resistance (HLAR). This study aimed to investigate the prevalence of 16S rRNA methylases among Enterobacter cloacae strains isolated from an Ahvaz teaching hospital, Iran. A total of 68 E. cloacae clinical strains were collected between November 2017 and September 2018. The MICs of aminoglycosides were assessed using the agar dilution method. The presence of 16S rRNA methylase genes, including armA, rmtA to rmtH, and nmpA was evaluated by PCR. The transferability of 16S rRNA methylase-harboring plasmids was evaluated by conjugation assay. The genetic diversity of all isolates was evaluated by ERIC-PCR. The armA and rmtB genes were the only 16S rRNA methylase genes detected in this study (29 out of 68 isolates; 42.64%). The transferability by conjugation was observed in 23 rmtB or/and armA positive donors. HLAR phenotype was in 33 of 68 strains. Ten clonal types were obtained by ERIC-PCR and significant associations (p < 0.05) were between the clone types and aminoglycoside susceptibility, as well as with profile of the 16S rRNA methylase genes. In conclusion, both horizontal transfer and clonal spread are responsible for dissemination of the rmtB and armA genes among E. cloacae strains.

A cluster of carbapenemase-producing Enterobacter cloacae complex ST171 at a tertiary care center demonstrating an ongoing regional threat.

BACKGROUND: In Minnesota and North Dakota, a clonal strain of blaKPC-3-producing Enterobacter cloacae complex has been reported with increasing frequency. METHODS: Between July 2015 and February 2016, 13 carbapenem-resistant E. cloacae complex isolates were identified at our institution. Five blaKPC-positive isolates were identified by polymerase chain reaction and underwent pulsed-field gel electrophoresis and whole genome sequencing. Medical records of these patients were reviewed. RESULTS: All 5 case-isolates belonged to sequence type 171 and were blaKPC-3-positive. Three pulsed-field gel electrophoresis patterns with >90% similarity were identified in the 5 case-isolates. We identified overlaps in time and location between case patients. Plasmid types and resistance genes were nearly identical between the isolates. Whole genome sequencing showed isolates A, B, and D to be closely related with <10 core single-nucleotide polymorphisms differences. Isolates C and E were also closely related to each other, but more distantly to A, B, and D; all belonged to the clonal lineage of the major circulating E. cloacae complex strain in Minnesota and North Dakota. Despite having overlapping hospital stays, isolates for patients C and D were not identical. CONCLUSIONS: Isolates A and D were nearly identical, indicating possible transmission during hospitalization. Transmission of the other isolates may have occurred elsewhere. This report highlights the importance of using both epidemiologic and molecular data to track the spread of carbapenemase-producers.

Nosocomial outbreak of extended-spectrum ß-lactamase-producing Enterobacter cloacae among cardiothoracic surgical patients: causes and consequences.

BACKGROUND: Enterobacteriaceae are recognized as leading pathogens of healthcare-associated infections. AIM: To report the investigation of a nosocomial outbreak of extended-spectrum ß-lactamase-producing Enterobacter cloacae affecting cardiothoracic surgery patients in a Belgian academic hospital. METHODS: Cases were defined based on epidemiological and microbiological investigations, including molecular typing using repetitive element-based polymerase chain reaction and multi-locus sequence typing. Case-control studies followed by field evaluations allowed the identification of a possible reservoir, and the retrospective assessment of human and financial consequences. FINDINGS: Over a three-month period, 42 patients were infected or colonized by CTX-M-15-producing E. cloacae strains that belonged to the same clonal lineage. Acquisition mainly occurred in the intensive care unit (N = 23) and in the cardiothoracic surgery ward (N = 16). All but one patient had, prior to acquisition, undergone a cardiothoracic surgical procedure, monitored by the same transoesophageal echocardiography (TOE) probe in the operating room. Despite negative microbiological culture results, the exclusion of the suspected probe resulted in rapid termination of the outbreak. Overall, the outbreak was associated with a high mortality rate among infected patients (40%) as well as significant costs (€266,550). CONCLUSION: The outbreak was indirectly shown to be associated with the contamination of a manually disinfected TOE probe used per-operatively during cardiothoracic surgery procedures, because withdrawal of the putative device led to rapid termination of the outbreak.

Genomic features of a multidrug-resistant Enterobacter cloacae ST279 producing CTX-M-15 and AAC(6')-Ib-cr isolated from fatal infectious stomatitis in a crossed pit viper (Bothrops alternatus).

OBJECTIVES: The widespread dissemination of extended-spectrum ß-lactamase-producing Enterobacteriaceae has become a major issue in veterinary medicine. However, until now, there has been no report of bacteria with such a phenotype in infected snakes. The aim of this study was to report the first draft genome sequence of an Enterobacter cloacae isolate (SERP1) recovered from a snake with infectious stomatitis. METHODS: The whole genome of E. cloacae strain SERP1 was sequenced on an Illumina NextSeq platform and was de novo assembled using CLC NGS Cell v.10. Data analysis was performed using online tools from the Center of Genomic Epidemiology. RESULTS: The genome size was calculated at 4966856bp, containing a total of 4796 protein-coding sequences. The strain was assigned to sequence type 279 (ST279) and, besides the clinically relevant blaCTX-M-15 and aac(6')-Ib-cr genes, it also presented resistance genes to ß-lactams, aminoglycosides, phenicols, sulphonamides, tetracyclines, trimethoprim, quinolones and fosfomycin. CONCLUSION: These data offer novel information regarding multidrug-resistant E. cloacae dissemination in wild animals and might contribute to further comparative genomic analysis.

Phenotypic and Genotypic Characterization of Carbapenem-resistant Enterobacteriaceae: Data From a Longitudinal Large-scale CRE Study in China (2012-2016).

Background: Carbapenem-resistant Enterobacteriaceae (CRE) strains are a major threat to global health. The development of effective control measures requires more detailed phenotypic and genotypic characterization of CRE. Methods: CRE isolates were collected from 65 hospitals in 25 provinces across China between January 1, 2012, and December 31, 2016. The isolates were characterized by antimicrobial susceptibility testing and multilocus sequence typing. Genes encoding carbapenemases, mobilized colistin resistance (mcr-1), and ß-lactamases were detected by polymerase chain reaction and DNA sequencing. Results: A total of 1801 independent CRE isolates (1201 Klebsiella pneumoniae, 282 Escherichia coli, and 179 Enterobacter cloacae) were collected during the study period. Overall, 96.9%, 89.7%, 54.5%, 49.9%, and 40% of CRE strains were susceptible to colistin, tigecycline, amikacin, minocycline, and fosfomycin, respectively. Notably, 1091/1201 (91%) K. pneumoniae, 225/282 (80%) E. coli, and 129/179 (72%) E. cloacae harbored carbapenemase gene. K. pneumoniae carbapenemase (KPC) was predominant in K. pneumoniae (77%), whereas New Delhi metallo-ß-lactamase (NDM) was predominant in E. coli (75%) and E. cloacae (53%). The mcr-1 gene was detected in 13 NDM-carrying E. coli isolates (4.6%). Sequence type (ST)11 and ST167 were predominant among the 100 K. pneumoniae and 47 E. coli STs, respectively. KPC-ST11, which accounted for 64% of K. pneumoniae isolates, had higher levels of resistance than non-ST11 strains to aztreonam, fosfomycin, and amikacin (P < .001). The proportions of KPC and NDM enzymes in CRE increased from 2012 to 2016 (54%-59% and 12%-28%, respectively). Conclusions: The number of CRE strains harboring carbapenemase is increasing. KPC-ST11 K. pneumoniae, the predominant strain, shows a reduced susceptibility to most available antibiotics.

Enterobacter cloacae Complex Sequence Type 171 Isolates Expressing KPC-4 Carbapenemase Recovered from Canine Patients in Ohio.

Companion animals are likely relevant in the transmission of antimicrobial-resistant bacteria. Enterobacter xiangfangensis sequence type 171 (ST171), a clone that has been implicated in clusters of infections in humans, was isolated from two dogs with clinical disease in Ohio. The canine isolates contained IncHI2 plasmids encoding blaKPC-4 Whole-genome sequencing was used to put the canine isolates in phylogenetic context with available human ST171 sequences, as well as to characterize their blaKPC-4 plasmids.

Effect of increasing meropenem MIC on the killing activity of meropenem in combination with amikacin or polymyxin B against MBL- and KPC-producing Enterobacter cloacae.

Carbapenem resistant Enterobacteriaceae (CRE) are a growing threat worldwide. Infections caused by these organisms have exhibited high rates of mortality (50%) for which there is no standard of care and a dearth of clinical trials. Most in vitro data on CRE focus on Klebsiella pneumoniae, but it is known that effective therapy may depend on species or even strain. To address this, meropenem, amikacin, and polymyxin B alone and in combination were evaluated by time kill against four carbapenem-producing Enterobacter cloacae clinical isolates representing a range of meropenem nonsusceptibility (2-32 mg/L) and resistance mechanisms (KPC 2 and/or VIM 1). As meropenem minimum inhibitory concentration (MIC) increased, bactericidal activity and synergy were maintained for 48 hours in isolates exposed to meropenem and amikacin, but synergy and bactericidal activity were not maintained in all isolates exposed to meropenem and polymyxin B.

Detection of virulence and ß-lactamase encoding genes in Enterobacter aerogenes and Enterobacter cloacae clinical isolates from Brazil.

Enterobacter cloacae and E. aerogenes have been increasingly reported as important opportunistic pathogens. In this study, a high prevalence of multi-drug resistant isolates from Brazil, harboring several ß-lactamase encoding genes was found. Several virulence genes were observed in E. aerogenes, contrasting with the E. cloacae isolates which presented none.

Detoxification of Jatropha curcas seed cake by a new soil-borne Enterobacter cloacae strain.

Jatropha curcas seed cake is a by-product generated after oil extraction from J. curcas seeds. Although the protein content is high, the cake contains phorbol esters and antinutritional factors such as phytates, trypsin inhibitors, lectins and tannins. Therefore, it cannot be directly used in food or feed. In this study, the toxic compounds and antinutrients present in J. curcas seed cake were detoxified by fermentation with Enterobacter Z11, a soil-borne isolate. Solid-state fermentation was undertaken under optimized conditions: deoiled cake, 5·0 g; initial moisture content, 50%; temperature, 30°C; and inoculum, 2 × 106 cells per gram of cake. Postfermentation, bacterial growth, pH and the amount of antinutrients were studied. Fermentation reduced the content of phorbol esters, phytates, lectins, tannins and trypsin inhibitors by 51·6, 82·6, 88·9, 37·8 and 90·5%, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The strain of Enterobacter cloacae Z11 was originally isolated from the soil. To the best of our knowledge, E. cloacae has never been used to remove toxins and antinutritional factors in Jatropha curcas seed cake (JSC). Under the optimized condition, fermentation with the Enterobacter strain decreased the phorbol esters content in JSC by 51·6%, and phytates, tannins, lectins and trypsin inhibitors contents by 83, 38, 89 and 90%, respectively. This study provided a new method with potential to render the seed cake suitable for use in feed. Further study is needed to focus on remaining toxicity and nutritional value post-treatment.