High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran.

OBJECTIVES: In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. RESULTS: In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX-M-15 gene (66.1%) followed by blaCTX-M (45.8%), blaCTX-M-14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Antimicrobial Resistance Genes, Virulence Genes, and Associated Mobile Genetic Elements of Eight Multidrug-Resistant Enterobacterales Isolated from Hospital-Acquired Urinary Tract Infections in Sri Lanka.

The study describes the first isolation of multidrug-resistant (MDR) Klebsiella pneumoniae ST16, Escherichia coli ST131 (Esc), and Enterobacter hormaechei subsp. steigerwaltii ST93 (Enterobacter cloacae complex [ECC]) in Sri Lanka. Eight MDR strains of uropathogenic Enterobacterales isolated from hospital acquired urinary tract infections (UTIs) were analyzed using genomic sequencing and comparative genomics. Isolates carried multiple carbapenemase, AmpC, and ESBL (extended-spectrum ß-lactamase) genes. ECC manifested both blaNDM-4 and blaOXA-181. The K. pneumoniae strains harbored fimbrial genes that facilitate pathogenesis of UTI. Several extraintestinal pathogenic E. coli associated virulence genes were identified in Esc. The efflux pump gene, acrA, and the T6SS gene cluster were detected in ECC. Many antimicrobial resistance (AMR) and virulence genes were identified associated with mobile genetic elements. ISEcp1 flanked upstream of blaCTX-M-15. The carbapenemase genes were carried on ColKP3 plasmids and were associated with ISEcp1. In Esc, the AMR gene blaTEM-1B and virulence gene traT were found on an IncF plasmid replicon. In K. pneumoniae the AMR genes sul1 and tetB present on IncR plasmid replicons and were associated with the insertion sequence IS6100. In Kp5, blaLAP-2 and qnrS1 coexisted and were flanked by ISEcl. AMR gene clusters, conferring resistance to multiple antimicrobial classes, flanked by mobile elements were identified in seven isolates.

A Novel Lipid-Based MALDI-TOF Assay for the Rapid Detection of Colistin-Resistant Enterobacter Species.

Enterobacter species are classified as high-priority pathogens due to high prevalence of multidrug resistance from persistent antibiotic use. For Enterobacter infections caused by multidrug-resistant isolates, colistin (polymyxin E), a last-resort antibiotic, is a potential treatment option. Treatment with colistin has been shown to lead to emergence of polymyxin resistance. The primary mechanism for colistin resistance is modification of terminal phosphate moieties of lipid A, leading to decreased membrane electronegativity and reducing colistin binding affinity. Detection of these modifications, including the addition of phosphoethanolamine and 4-amino-4-deoxy-l-arabinose (Ara4N), can be used for prediction of colistin resistance using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The objective of this study was to identify lipid A markers for colistin resistance in Enterobacter species and Klebsiella aerogenes (formerly Enterobacter aerogenes). Using a collection of Enterobacter and Klebsiella aerogenes clinical isolates, broth MICs for colistin were determined initially. Subsequently, killing assays were carried out to determine how the concentration of colistin at which there is approximately 50% survival (kill50) equates to their MICs. Finally, lipid A analysis was conducted via MALDI-TOF MS using the novel rapid extraction method, termed fast lipid analysis technique (FLAT), to correlate MIC and killing efficacy with predictive lipid A modifications. Sensitivity and specificity of the MS assay compared to MIC interpretation were 100% and 53.4%, respectively. A receiver operator characteristic (ROC) demonstrated that MS was highly correlated with killing, with area under the curve of 0.97. This analysis demonstrated the potential utility of MALDI-TOF MS as a rapid diagnostic platform of colistin resistance in Enterobacter species. IMPORTANCE In this study, we develop a novel method for identifying colistin resistance in Enterobacter species and Klebsiella aerogenes without performing antimicrobial susceptibility testing. Typically, susceptibility testing requires an additional 24 to 48 h, while the MS assay described in this study allows for resistant identifications in under 1 h after initial culture. Identification using MALDI-TOF MS would save time and prevent inappropriate use of colistin. MALDI-TOF MS is an easy-to-use, readily available, robust diagnostic tool in clinical laboratories. Furthermore, this study highlights limitations of polymyxin susceptibility testing. Use of a killing assay best captures how colistin treats infection and is shown to be highly correlated with our MS assay; thus, the MS assay in this study effectively predicts how colistin would treat a patient's infection. Use of MALDI-TOF MS for accurate and early identification of antimicrobial resistance can improve antimicrobial stewardship and patient outcomes.

Human Amnion-Derived Mesenchymal Stromal Cells: A New Potential Treatment for Carbapenem-Resistant Enterobacterales in Decompensated Cirrhosis.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) is a severe and often fatal infection in patients with decompensated cirrhosis and ascites. The only cure for SBP is antibiotic therapy, but the emerging problem of bacterial resistance requires novel therapeutic strategies. Human amniotic mesenchymal stromal cells (hA-MSCs) possess immunomodulatory and anti-inflammatory properties that can be harnessed as a therapy in such a context. METHODS: An in vitro applications of hA-MSCs in ascitic fluid (AF) of cirrhotic patients, subsequently infected with carbapenem-resistant Enterobacterales, was performed. We evaluated the effects of hA-MSCs on bacterial load, innate immunity factors, and macrophage phenotypic expression. RESULTS: hA-MSCs added to AF significantly reduce the proliferation of both bacterial strains at 24 h and diversely affect M1 and M2 polarization, C3a complement protein, and ficolin 3 concentrations during the course of infection, in a bacterial strain-dependent fashion. CONCLUSION: This study shows the potential usefulness of hA-MSC in treating ascites infected with carbapenem-resistant bacteria and lays the foundation to further investigate antibacterial and anti-inflammatory roles of hA-MSC in in vivo models.

Analysis of an IncR Plasmid Carrying blaNDM-1 Linked to an Azithromycin Resistance Region in Enterobacter hormaechei Involved in an Outbreak in Quebec.

In the context of a recent rise in prevalence of NDM-encoding carbapenemase-producing Enterobacterales (CPE) in the province of QC, Canada, the genetic environment of blaNDM-1 was investigated. Three NDM-producing clinical isolates of Enterobacter hormaechei recovered from hospitalized patients involved in a putative outbreak were further characterized by whole-genome sequencing (WGS). Two isolates were confirmed by pulsed-field gel electrophoresis and WGS to be closely related. In addition to a ∼128 kb IncFII conjugative multidrug-resistance (MDR) plasmid, these isolates possessed a ∼45 kb mobilizable IncR MDR plasmid containing 2 MDR regions: a complex class 1 integron harboring blaNDM-1 and 7 other AMR genes, and the IS26-mph(A)-mrx-mphR(A)-IS6100 azithromycin resistance unit. The predicted antimicrobial resistance (AMR) genes correlated with the antimicrobial susceptibility testing results. The multidrug-resistant phenotype in addition to the presence of two important mobile genetic elements, suggest a potent role as a reservoir of antibiotic resistance for such a small IncR plasmid. IMPORTANCE Analyzing the genetic environment of clinically relevant MDR genes can provide information on the way in which such genes are maintained and disseminated. Understanding this phenomenon is of interest for clinicians as it can also provide insight on where these genes might have been sourced, possibly supporting outbreak investigations.

A hospital-wide outbreak of IMI-17-producing Enterobacter ludwigii in an Israeli hospital.

ABSRACT: BACKGROUND: To describe the course and intervention of an hospital-wide IMI-Producing Enterobacter ludwigii outbreak. METHODS: This was an outbreak interventional study, done at a tertiary care center in Tel-Aviv, Israel. Data was collected on the course of the outbreak and the demographic and clinical characteristics of all patients involved in the outbreak. The intervention measures included patients' cohorting, contact isolation precautions, environmental cleaning and screening of contacts. The molecular features and phylogeny of outbreak-related isolates were studied by whole-genome based analysis. RESULTS: The outbreak included 34 patients that were colonized by IMI-Producing E. ludwigii and were identified in 24 wards throughout the hospital. Colonization was identified in the first 72 h of admission in 13/34 patients (38.2%). Most patients (91.2%) were admitted from home and had relatively low level of comorbidities. The majority of them (88%) had no recent use of invasive catheters and none had previous carriage of other multi-drug resistant bacteria. All available isolates harbored the blaIMI-17 allele and belonged to Sequence-Type 385. With the exception of two isolates, all isolates were closely related with less than a 20-SNP difference between them. CONCLUSIONS: This outbreak had most likely originated in the community and subsequently disseminated inside our institution. More studies are required in order to elucidate the epidemiology of IMI-Producing E. ludwigii and the possible role of environmental sources in its dissemination.

Has resistance to chlorhexidine increased among clinically-relevant bacteria? A systematic review of time course and subpopulation data.

Chlorhexidine (CHX) was introduced for use as an antimicrobial more than 70 years ago. CHX has been and continues to be used broadly for disinfecting surfaces in medical and food service facilities as well as directly on skin of humans and animals. Considering its widespread use over many decades, questions of resistance to CHX have been raised. Additionally, questions of possible coincident resistance to the biocide and resistance to clinically relevant antibiotics have also been raised. A number of important questions remain, including is there consistent evidence of resistance, what is the degree of resistance, especially among clinically isolated microbial strains, and what is the degree of resistance compared to the typical concentrations of the biocide used? Data for microbial species isolated over the last 70+ years were compiled to construct as complete a picture as practical regarding possible resistance, especially among species in which resistance to commonly used antibiotics has been noted to be increasing. This is a compilation and analysis of individual MIC values for CHX reported in the literature, not a compilation of the conclusions individual authors reached. The data were analyzed using straight-forward and robust statistical procedures to detect changes in susceptibility to CHX over time, i.e. linear regression. Linear regression was supplemented with the use of nonlinear least squares regression analysis to detect the presence of population parameters associated with subpopulations of microbial strains which exhibit increased resistance to CHX. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii were all found to have an increased resistance to CHX over time with the most profound change detected in A. baumannii. Additionally, subpopulations with log-normal distributions were found consistent with the presence of a baseline subpopulation of susceptible strains and a subpopulation with increased resistance to CHX. However, the CHX-resistant subpopulations did not correlate exactly with antibiotic resistance, so details of the relationship remain to be addressed. Increased resistance over time was not detected for Escherichia coli, Enterobacter faecalis, Staphylococcus aureus, or Candida albicans, although a subpopulation with greater than baseline resistance to CHX was detected among strains of E. faecalis and C. albicans. A difference in susceptibility to CHX was also detected between methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) S. aureus strains. The levels of resistance to CHX detected were all markedly lower than concentrations routinely used in medical and food service applications. Reaching conclusions regarding the relationship between antibiotic and CHX resistance was complicated by the limited overlap between tests of CHX and antibiotic resistance for several species. The results compiled here may serve as a foundation for monitoring changes in resistance to CHX and possible relationships between the use of CHX and resistance to antibiotics commonly used in clinical medicine.

Presence of ß-Lactamase-producing Enterobacterales and Salmonella Isolates in Marine Mammals.

Marine mammals have been described as sentinels of the health of marine ecosystems. Therefore, the aim of this study was to investigate (i) the presence of extended-spectrum ß-lactamase (ESBL)- and AmpC-producing Enterobacterales, which comprise several bacterial families important to the healthcare sector, as well as (ii) the presence of Salmonella in these coastal animals. The antimicrobial resistance pheno- and genotypes, as well as biocide susceptibility of Enterobacterales isolated from stranded marine mammals, were determined prior to their rehabilitation. All E. coli isolates (n = 27) were screened for virulence genes via DNA-based microarray, and twelve selected E. coli isolates were analyzed by whole-genome sequencing. Seventy-one percent of the Enterobacterales isolates exhibited a multidrug-resistant (MDR) pheno- and genotype. The gene blaCMY (n = 51) was the predominant ß-lactamase gene. In addition, blaTEM-1 (n = 38), blaSHV-33 (n = 8), blaCTX-M-15 (n = 7), blaOXA-1 (n = 7), blaSHV-11 (n = 3), and blaDHA-1 (n = 2) were detected. The most prevalent non-ß-lactamase genes were sul2 (n = 38), strA (n = 34), strB (n = 34), and tet(A) (n = 34). Escherichia coli isolates belonging to the pandemic sequence types (STs) ST38, ST167, and ST648 were identified. Among Salmonella isolates (n = 18), S. Havana was the most prevalent serotype. The present study revealed a high prevalence of MDR bacteria and the presence of pandemic high-risk clones, both of which are indicators of anthropogenic antimicrobial pollution, in marine mammals.

Evaluation of the MICRONAUT MIC-strip colistin assay for colistin susceptibility testing of carbapenem-resistant Acinetobacter baumannii and Enterobacterales.

We sought to evaluate the MICRONAUT MIC-Strip colistin (MMS) assay, a commercial broth microdilution (BMD) panel, using 273 carbapenem-resistant Acinetobacter baumannii and Enterobacterales isolates. BMD was used as gold standard. MMS had 98.5% sensitivity and 99.5% specificity. All 37 isolates with MICs close to the breakpoint exhibited complete agreement.

Effects of vancomycin-induced gut microbiome alteration on the pharmacodynamics of metformin in healthy male subjects.

Metformin is a major treatment for type 2 diabetes. This study was conducted to investigate the impact of gut microbiome dysbiosis on the pharmacokinetics and antihyperglycemic effects of metformin. Healthy adult males aged 19-45 years with no defecation abnormalities were recruited for this 4-period clinical study: baseline; post-metformin (i.e., multiple oral doses of 1000 mg metformin on days 1-4); post-vancomycin (i.e., multiple oral doses of 500 mg vancomycin on days 11-17 inducing gut microbiome changes); and post-metformin + vancomycin (i.e., multiple oral doses of 1000 mg metformin on days 16-19). In each period, serum glucose and insulin concentrations following an oral glucose tolerance test, fecal samples for gut microbiome composition, and safety data were obtained. Following metformin dosing, plasma and urine samples for pharmacokinetics were collected. Nine subjects completed the study. The pharmacokinetics of metformin remained unchanged, and the antihyperglycemic effect was significantly decreased after vancomycin administration (p value = 0.039), demonstrating the weak relationship between the pharmacokinetics and pharmacodynamics of metformin. Relative abundances of some genus were changed after vancomycin administration, and tended to correlate with the antihyperglycemic effects of metformin (p value = 0.062 for Erysipelatoclostridium; p value = 0.039 for Enterobacter; and p value = 0.086 for Faecalibacterium). Adverse events occurred in all subjects and were resolved without sequelae. In conclusion, a decrease in the antihyperglycemic effect of metformin was observed after concomitant administration with vancomycin, without changes in metformin pharmacokinetics. The antihyperglycemic effect was tended to correlate with the relative abundance of several genus, suggesting that the effect of metformin is partly attributable to the gut microbiome (ClinicalTrials.gov, NCT03809260).

Risk factor analysis for piperacillin-tazobactam-resistant Enterobacter spp. bacteremia at a tertiary hospital.

This study aimed to analyze the risk factors for piperacillin-tazobactam (TZP1)-resistant Enterobacter spp. bacteremia. The medical records of 111 patients with Enterobacter spp. bacteremia divided into a TZP-susceptible group (minimum inhibitory concentrations [MICs2] ≤16 µg/mL) and TZP-resistant group (MICs >16 µg/mL) were retrospectively reviewed. The male-to-female ratio, age, underlying disease, and infection site did not differ between the 2 groups. Multivariate analysis revealed that the independent predictor associated with TZP-resistant Enterobacter spp. bacteremia was the previous usage of third-generation cephalosporins (P = 0.036). In conclusion, TZP administration in cases of suspected Enterobacter spp. bacteremia previously treated with third-generation cephalosporin should be cautiously considered.

Institutional outbreak involving multiple clades of IMP-producing Enterobacter cloacae complex sequence type 78 at a cancer center in Tokyo, Japan.

BACKGROUND: Information about the clinical and microbiological characteristics of IMP-producing Enterobacterales has been limited. Here, we describe an institutional outbreak of IMP-producing Enterobacter cloacae complex (ECC) involving multiple clades of ECC sequence type (ST) 78 strains. METHODS: Antimicrobial susceptibility testing, whole-genome sequencing, and conjugation experiments of 18 IMP-producing ECC strains isolated during four-year study period were performed. Species and subspecies were determined by average nucleotide identity analysis and clonal relatedness of the isolates was analyzed with multilocus sequence typing and core-genome single nucleotide polymorphism (SNP) analysis. Relevant clinical information was extracted from medical records. RESULTS: Fourteen of 18 IMP-producing ECC isolates were determined as Enterobacter hormaechei ST78. Sixteen isolates, including 13 isolates belonging to ST78, carried blaIMP-1 in In316-like class 1 integron and also carried IncHI2 plasmids. Conjugation experiments were successful for 12 isolates carrying blaIMP-1 on IncHI2 plasmids and for an isolate carrying blaIMP-11 on an IncL/M plasmid. Although isolation of ST78 strains was clustered in a 14-months period suggesting nosocomial transmission, these strains were subdivided into three clades by SNP analysis: clade A (n = 10), clade B (n = 1), clade C (n = 3). A part of clonal relatedness was unexpected by the epidemiological information at the time of isolation of the strains. Most of the IMP-producing ECC strains were susceptible to non-ß-lactam antibiotics and had relatively low minimum inhibitory concentrations to carbapenems (≤4 µg/mL). Five of six infections caused by IMP-producing ECC were treated successfully. CONCLUSIONS: Whole-genome sequencing analysis revealed the outbreak was caused by three different clades of ST78 strains, where patients had favorable treatment outcome of the infections compared with that caused by Enterobacterales producing other carbapenemases, possibly due to their non-multidrug-resistant phenotype.

Co-infections of two carbapenemase-producing Enterobacter hormaechei clinical strains isolated from the same diabetes individual in China.

Introduction. Since mcr-1 was first reported in China, there have been ten variants of MCR appearing nationwide so far. Multidrug-resistant Enterobacteriaceae bacteria carrying both NDM and MCR have become a serious threat to global public health.Hypothesis/Gap Statement. The genetic structure of mcr-9 needs to be better understood in order to better prevent and control the transmission of drug-resistant genes.Aims. The aim of this study was to characterize the presence of two Enterobacter hormaechei isolates, which carries bla NDM-5 CME2 and the coexistence of mcr-9 and bla NDM-1 strain CMD2, which were isolated from a patient with diabetes in Sichuan, China.Methodology. The microbroth dilution method was used for antibiotic susceptibility. Conjugation experiment was used to investigate the transferability of bla NDM-1, bla NDM-5 and mcr-9. Whole-genome sequencing was performed on Illumina HiSeq platform. The ability of biofilm formation was detected by crystal-violet staining, the virulence of the bacteria was measured by Galleria mellonella killing assay.Results. bla NDM-5 carrier CME2 and CMD2 with bla NDM-1 and mcr-9 were resistant to carbapenems, ß-lactam, aminoglycoside, quinolone and tetracycline, while CMD2 was also resistant to colistin. Conjugation assay and plasmid replicon typing further demonstrated that both bla NDM-1 and bla NDM-5 were respectively present on the self-transferrable IncX3 plasmid, mcr-9 was located on the self-transferrable IncHI2 plasmid. Through the analysis of mcr-9 gene context, the structure was DUF4942-rcnR-rcnA-copS-IS903-mcr-9-wbuC-qseC-qseB-IS1R-ΔsilR-IS903, bla NDM-1 context was IS3000-ΔISAba125-IS5-bla NDM-1-ble-trpF-groS-groL-insE-ΔIS26 structure, bla NDM-5 structure was IS3000-bla NDM-5-ble-trpF-dsbC-ΔIS26-umuD-ISKox3-tnpR-parA. Biofilm formation of CME2 was stronger than CMD2. There was no significant difference in virulence between the two strains.Conclusion. This study reveals two multiple drug-resistant E. hormaechei isolates from diabetes patient samples. E. hormaechei carrying two NDM-resistant genes is already a serious threat, where MCR is an important cause of treatment failure in bacterial infections. This study is a reminder not only to prevent infection in patients with diabetes, but also to constantly monitor the epidemic and spread of the drug-resistant gene.

Carbapenem and Colistin Resistance in Enterobacter: Determinants and Clones.

Enterobacter is a globally important pathogen. Here we clarify its taxonomy and review recent developments in its resistance to carbapenem and colistin, illustrating that Enterobacter has a large arsenal of mechanisms to grow under antimicrobial pressure. Further studies are required to decipher colistin heteroresistance and understand why certain Enterobacter lineages have emerged clinically.

Functional Differences between E. coli and ESKAPE Pathogen GroES/GroEL.

As the GroES/GroEL chaperonin system is the only bacterial chaperone that is essential under all conditions, we have been interested in the development of GroES/GroEL inhibitors as potential antibiotics. Using Escherichia coli GroES/GroEL as a surrogate, we have discovered several classes of GroES/GroEL inhibitors that show potent antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it remains unknown if E. coli GroES/GroEL is functionally identical to other GroES/GroEL chaperonins and hence if our inhibitors will function against other chaperonins. Herein we report our initial efforts to characterize the GroES/GroEL chaperonins from clinically significant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). We used complementation experiments in GroES/GroEL-deficient and -null E. coli strains to report on exogenous ESKAPE chaperone function. In GroES/GroEL-deficient (but not knocked-out) E. coli, we found that only a subset of the ESKAPE GroES/GroEL chaperone systems could complement to produce a viable organism. Surprisingly, GroES/GroEL chaperone systems from two of the ESKAPE pathogens were found to complement in E. coli, but only in the strict absence of either E. coli GroEL (P. aeruginosa) or both E. coli GroES and GroEL (E. faecium). In addition, GroES/GroEL from S. aureus was unable to complement E. coli GroES/GroEL under all conditions. The resulting viable strains, in which E. coligroESL was replaced with ESKAPE groESL, demonstrated similar growth kinetics to wild-type E. coli, but displayed an elongated phenotype (potentially indicating compromised GroEL function) at some temperatures. These results suggest functional differences between GroES/GroEL chaperonins despite high conservation of amino acid identity.IMPORTANCE The GroES/GroEL chaperonin from E. coli has long served as the model system for other chaperonins. This assumption seemed valid because of the high conservation between the chaperonins. It was, therefore, shocking to discover ESKAPE pathogen GroES/GroEL formed mixed-complex chaperonins in the presence of E. coli GroES/GroEL, leading to loss of organism viability in some cases. Complete replacement of E. coligroESL with ESKAPE groESL restored organism viability, but produced an elongated phenotype, suggesting differences in chaperonin function, including client specificity and/or refolding cycle rates. These data offer important mechanistic insight into these remarkable machines, and the new strains developed allow for the synthesis of homogeneous chaperonins for biochemical studies and to further our efforts to develop chaperonin-targeted antibiotics.

Complete genome sequence of plant growth-promoting and heavy metal-tolerant Enterobacter tabaci 4M9 (CCB-MBL 5004).

Enterobacter tabaci 4M9 (CCB-MBL 5004) was reported to have plant growth-promoting and heavy metal tolerance traits. It was able to tolerate more than 300 mg/L Cd, 600 mg/L As, and 500 mg/L Pb and still maintained the ability to produce plant growth-promoting substances under metal stress conditions. To explore the genetic basis of these beneficial traits, the complete genome sequencing of 4M9 was carried out using Pacific Bioscience (PacBio) sequencing technology. The complete genome consisted of one chromosome of 4,654,430 bp with a GC content of 54.6% and one plasmid of 51,135 bp with a GC content of 49.4%. Genome annotation revealed several genes involved in plant growth-promoting traits, including the production of siderophore, indole acetic acid, and 1-aminocyclopropane-1-carboxylate deaminase; solubilization of phosphate and potassium; and nitrogen metabolism. Similarly, genes involved in heavy metals (As, Co, Zn, Cu, Mn, Se, Cd, and Fe) tolerance were detected. These support its potential as a heavy metal-tolerant plant growth-promoting bacterium and a good genetic resource that can be employed to improve phytoremediation efficiency of heavy metal-contaminated soil via biotechnological techniques. This, to the best of our knowledge, is the first report on the complete genome sequence of heavy metal-tolerant plant growth-promoting E. tabaci.

Genomic analysis of VIM-2-producing Enterobacter hormaechei subsp. steigerwaltii.

Carbapenemase-producing Enterobacterales (CPE) is a major public-health concern. Here we describe the occurrence of blaVIM-2 in three isolates of Enterobacter hormaechei subsp. steigerwaltii. The blaVIM-2 gene was part of a class II transposon Tn1332 and was embedded in a remnant of a class 1 integron. Tn1332 was carried by a large, conjugative, non-typeable plasmid. The three isolates belonged to sequence type 90 (ST90). Two isolates (90H2 and 90H3) were highly related [<10 single nucleotide polymorphisms (SNPs)], whereas isolate 104D2 exhibited more than 50 SNPs and Tn1332 was inserted in a different place in the plasmid. Another IncHI-type plasmid carrying the extended-spectrum ß-lactamase (ESBL) gene blaCTX-M-15 was identified in 90H2 and 90H3. Among the three isolates, isolate 104D2 was negative for detection of carbapenemase activity using the biochemical Carba NP test, despite the presence of Tn1332 on the same plasmid. Mutants of 104D2 with higher minimum inhibitory concentrations (MICs) for carbapenems were obtained and one mutant (m104D2) was analysed. In contrast to 104D2, mutant m104D2 gave a positive Carba NP test. The mutant possessed two copies of Tn1332 per cell and a nonsense mutation in WecA, an enzyme involved in enterobacterial common antigen and peptidoglycan intermediate biosynthesis. This study describes the first occurrence of Tn1332 in Enterobacterales and the phenotypic diversity of VIM-2-producing E. hormaechei.

Clay-assisted protection of Enterobacter sp. from Pb (II) stress.

Clay minerals can adsorb both microorganisms and heavy metals. In this study, typical soil bacterium, Enterobacter sp. was applied to investigate the potential protection of the bacterial cells from Pb2+ stress by clay minerals. The sorption by two representative types of montmorillonite (Mt) were contrasted, i.e., Mts/Mtw with strong/weak CEC. There was no significant difference between the two clay minerals regarding their adsorption of Pb2+ cations in water (i.e., ~55 mg L-1). However, the sorption of bacterial cells on the two clay minerals showed evident contrasts, which resulted in the different capacity of Pb sorption. Mts with high CEC preferentially adsorbed abundant bacterial cells (rather than Pb2+) on its surface. The residual Pb2+ concentration in solution actually raised by 7.5% after the addition of Enterobacter sp. In addition, both the Pb-contaminated cells and "healthy" cells (with low Pb contamination) could be adsorbed onto Mt surface, whereas the latter dominated the adsorbents on Mts. This was due to that the Mts with high CEC could provide more exchangeable cations, building more cation bridging ligands between the microbial cells (whatever the types of cells) and clay surface. Furthermore, the adsorbed "healthy" bacterial cells might escape from clay surface via "self-liberating" mechanism, i.e., increasing electrostatic repulsion between the bacteria and clay during microbial decomposition of the medium. This study hence elucidated the protection of microorganisms from Pb2+ stress by Mt.

Importance of Reviewing Antibiotic Courses by 48 Hours: Risk Factors for Third-Generation Cephalosporin Resistance Among AmpC Harboring Organisms in Urine and Respiratory Cultures.

BACKGROUND: Citrobacter, Enterobacter, Morganella, and Serratia (AmpC organisms) species can exhibit third-generation cephalosporin (TGC) resistance after TGC exposure. We aimed to assess if institutional TGC utilization correlated with institutional AmpC organism susceptibility and if prior TGC exposure ≤48 hours were associated with TGC resistance in the first culture of a future infection episode caused by an AmpC organism. METHODS: A 5-year retrospective cohort study was performed, including AmpC organisms isolated from pediatric urinary and respiratory tract cultures at an institution with TGC courses reviewed by the antimicrobial stewardship program at 48 hours. Correlations were assessed by Pearson's correlation. Multivariable logistic regression identified factors independently associated with TGC resistance in a subcohort of infection episodes. RESULTS: Among 654 cultures, AmpC organism TGC susceptibility increased from 74% in 2013 to 89.3% in 2017, and this correlated with a 26.1% decrease in TGC utilization (R = -0.906; P = 0.034). Among 275 AmpC organism infections, 21.1% were resistant. Resistance occurred in 13.6%, 17.4%, and 56.5% of infections with no exposure, ≤48 hours, and >48 hours of TGC exposure in the past 30 days, respectively. TGC exposure ≤48 hours was not associated with resistance (odds ratio [OR], 1.26; 95% confidence interval [CI], 0.32-4.94; P = 0.74), whereas, TGC exposure >48 hours was (OR, 8.7; 95% CI, 3.67-20.6; P < 0.001). Infections in 2017 were less likely to be resistant (OR, 0.25; 95% CI, 0.08-0.8; P = 0.019). CONCLUSIONS: Decreased TGC utilization, likely related to antimicrobial stewardship, correlated with increased AmpC organism susceptibility. Limiting TGC exposure to ≤48 hours when possible may reduce AmpC organism resistance in future infections.

Emergence and Persistence over Time of Carbapenemase-Producing Enterobacter Isolates in a Spanish University Hospital in Madrid, Spain (2005-2018).

Carbapenemase production is constantly increasing among different Enterobacterales species. We analyzed the microbiological characteristics and population structure of all carbapenemase-producing Enterobacter spp. (CP-Ent) isolates recovered at the Ramón y Cajal Hospital between 2005 and 2018. Overall, 178 CP-Ent isolates (60.7% colonization, 39.3% clinical) were recovered from 165 hospitalized patients (165/176, 93.7%; medical [102/165], surgical [34/165], and intensive care unit [29/165] areas), emergency unit (4/176, 2.3%), and ambulatory patients (7/176, 4.0%). In addition, three CP-Ent were found in environmental sources. Clinical samples were mainly urine (37.1%). The most frequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-identified species were Enterobacter cloacae (n = 85) and Enterobacter asburiae (n = 49). hsp60 gene sequencing showed a higher species diversity than MALDI-TOF: 70 Enterobacter hormaechei-clusters III, VI, VIII; 69 Enterobacter roggenkampii-IV; 15 Enterobacter kobei-II; 9 E. asburiae-I; 3 Enterobacter ludwigii-V; and 1 E. cloacae subsp. dissolvens-XII. Nine Klebsiella aerogenes were also identified. Overall, a high clonal diversity (Simpson Diversity Index >0.90) was found among CP-Ent-clusters. Environmental isolates were clonally related to clinical ones. Amikacin and tigecycline showed the highest susceptibility (>93%). VIM-1 (n = 133/181, 73.5%) and OXA-48 (n = 34/181, 18.8%) carbapenemases were predominant, followed by KPC-2 (n = 9/181, 5.0%), KPC-3 (n = 2/181, 1.1%), VIM-2 (n = 1/181, 0.6%), and two coproducers (VIM-1+KPC-2 and VIM-1+KPC-3). Extended-spectrum beta-lactamase (ESBL) coproduction (14.4%) emerged in 2012, mainly associated with blaSHV-12 (p < 0.001), E. roggenkampii (p < 0.001), and colonization (p = 0.03). VIM-1- and OXA-48-CP-Ent fecal carriers increased in our hospital, particularly between 2011 and 2018 (p < 0.001). Moreover, KPC and OXA-48 producers emerged in 2010 and 2012, respectively. They superimposed over VIM producers, which were persistently recovered since first detection in 2005. These results depict increased complexity over time of CP-Ent.