NagZ modulates the virulence of E. cloacae by acting through the gene of unknown function, ECL_03795.

ß-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout significantly eradicated AmpC-dependent ß-lactam resistance in Enterobacter cloacae. However, NagZ's role in the virulence of E. cloacae remains unclear. Our study, incorporating data on mouse and Galleria mellonella larval mortality rates, inflammation markers, and histopathological examinations, revealed a substantial reduction in the virulence of E. cloacae following NagZ knockout. Transcriptome sequencing uncovered differential gene expression between NagZ knockout and wild-type strains, particularly in nucleotide metabolism pathways. Further investigation demonstrated that NagZ deletion led to a significant increase in cyclic diguanosine monophosphate (c-di-GMP) levels. Additionally, transcriptome sequencing and RT-qPCR confirmed significant differences in the expression of ECL_03795, a gene with an unknown function but speculated to be involved in c-di-GMP metabolism due to its EAL domain known for phosphodiesterase activity. Interestingly, in ECL_03795 knockout strains, a notable reduction in the virulence was observed, and virulence was rescued upon complementation with ECL_03795. Consequently, our study suggests that NagZ's function on virulence is partially mediated through the ECL_03795→c-di-GMP pathway, providing insight into the development of novel therapies and strongly supporting the interest in creating highly efficient NagZ inhibitors.

Enterobacter cloacae aggravates metabolic disease by inducing inflammation and lipid accumulation.

It is well known that gut microbiota imbalance can promote the development of metabolic disease. Enterobacter cloacae (E. cloacae) is a kind of opportunistic pathogen in the intestine. Therefore, we hypothesized that E. cloacae accelerated the development of metabolic disease. To answer this question, we used E. cloacae to induce disease in guinea pigs. We used H&E staining to detect the pathological changes of liver and aorta and used Oil Red O staining to evaluate the lipid accumulation in the liver. And that we used a kit to detect AST content and used Western blot to detect protein levels in the liver. We found that E. cloacae could induce liver pathological changes and lipid accumulation as well as aortic wall pathological changes in guinea pigs. And E. cloacae increased the liver index to 5.94% and the serum AST level to 41.93 U/L. Importantly, E. cloacae activated liver high mobility group protein (HMGB1)/toll-like receptor 4 (TLR4)/myeloiddifferentiationfactor88 (MYD88)/nuclear factor-kappa B (NF-κB) signal and sterol regulatory element-binding protein 1c (SREBP-1c) and inhibited AMP-activated protein kinase (AMPK). We conclude that E. cloacae promote nonalcoholic fatty liver disease (NAFLD) by inducing inflammation and lipid accumulation, and E. cloacae also promote atherosclerosis. These findings are important for study on the pathogenesis and drug screening of NAFLD and atherosclerosis.

Distribution of Antimicrobial Resistance and Virulence Genes within the Prophage-Associated Regions in Nosocomial Pathogens.

Prophages are often involved in host survival strategies and contribute toward increasing the genetic diversity of the host genome. Prophages also drive horizontal propagation of various genes as vehicles. However, there are few retrospective studies contributing to the propagation of antimicrobial resistance (AMR) and virulence factor (VF) genes by prophage. We extracted the complete genome sequences of seven pathogens, including ESKAPE bacteria and Escherichia coli from a public database, and examined the distribution of both the AMR and VF genes in prophage-like regions. We found that the ratios of AMR and VF genes greatly varied among the seven species. More than 70% of Enterobacter cloacae strains had VF genes, but only 1.2% of Klebsiella pneumoniae strains had VF genes from prophages. AMR and VF genes are unlikely to exist together in the same prophage region except in E. coli and Staphylococcus aureus, and the distribution patterns of prophage types containing AMR genes are distinct from those of VF gene-carrying prophage types. AMR genes in the prophage were located near transposase and/or integrase. The prophage containing class 1 integrase possessed a significantly greater number of AMR genes than did prophages with no class 1 integrase. The results of this study present a comprehensive picture of AMR and VF genes present within, or close to, prophage-like elements and different prophage patterns between AMR- or VF-encoding prophage-like elements. IMPORTANCE Although we believe phages play an important role in horizontal gene transfer in exchanging genetic material, we do not know the distribution of the antimicrobial resistance (AMR) and/or virulence factor (VF) genes in prophages. We collected different prophage elements from the complete genome sequences of seven species-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Escherichia coli-and characterized the distribution of antimicrobial resistance and virulence genes located in the prophage region. While virulence genes in prophage were species specific, antimicrobial resistance genes in prophages were highly conserved in various species. An integron structure was detected within specific prophage regions such as P1-like prophage element. Maximum of 10 antimicrobial resistance genes were found in a single prophage region, suggesting that prophages act as a reservoir for antimicrobial resistance genes. The results of this study show the different characteristic structures between AMR- or VF-encoding prophages.

Liquid Extraction Surface Analysis Mass Spectrometry of ESKAPE Pathogens.

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae) represent clinically important bacterial species that are responsible for most hospital-acquired drug-resistant infections; hence, the need for rapid identification is of high importance. Previous work has demonstrated the suitability of liquid extraction surface analysis mass spectrometry (LESA MS) for the direct analysis of colonies of two of the ESKAPE pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) growing on agar. Here, we apply LESA MS to the remaining four ESKAPE species (E. faecium E745, K. pneumoniae KP257, A. baumannii AYE, and E. cloacae S11) as well as E. faecalis V583 (a close relative of E. faecium) and a clinical isolate of A. baumannii AC02 using an optimized solvent sampling system. In each case, top-down LESA MS/MS was employed for protein identification. In total, 24 proteins were identified from 37 MS/MS spectra by searching against protein databases for the individual species. The MS/MS spectra for the identified proteins were subsequently searched against multiple databases from multiple species in an automated data analysis workflow with a view to determining the accuracy of identification of unknowns. Out of 24 proteins, 19 were correctly assigned at the protein and species level, corresponding to an identification success rate of 79%.

Drug resistance of pathogens causing nosocomial infection in orthopedics from 2012 to 2017: a 6-year retrospective study.

BACKGROUND: Hospital-acquired infections (HAIs) are an emerging global problem that increases in-hospital mortality, length of stay, and cost. We performed a 6-year retrospective study to provide valuable insight into appropriate antibiotic use in HAI cases. We also aimed to understand how hospitals could reduce pathogen drug resistance in a population that overuses antibiotics. METHODS: All data (2012-2017) were obtained from the hospital information warehouse and clinical microbiology laboratory. RESULTS: We isolated 1392 pathogen strains from patients admitted to the orthopedics department during 2012-2017. Escherichia coli (14.7%, 204/1392), Enterobacter cloacae (13.9%, 193/1392), and Staphylococcus aureus (11.3%, 157/1392) were the most common pathogens causing nosocomial infections. The dominant Gram-negative bacterium was E. coli, with high resistance to ampicillin, levofloxacin, cotrimoxazole, gentamicin, and ciprofloxacin, in that order. E. coli was least resistant to amikacin, cefoperazone-sulbactam. The most dominant Gram-positive bacterium was S. aureus, highly resistant to penicillin and ampicillin, but not resistant to fluoroquinolones and cotrimoxazole. Analysis of risk factors related to multidrug-resistant bacteria showed that patients with open fractures (Gustillo III B and IIIC) were significantly more susceptible to methicillin-resistant S. aureus infections (p < 0.05). Additionally, extended-spectrum ß-lactamase-producing E. coli infections occurred significantly more often in patients with degenerative diseases (p < 0.05). Elderly patients tended to be more susceptible to multidrug-resistant bacterial infections, but this outcome was not statistically significant. CONCLUSIONS: Antimicrobial resistance is a serious problem in orthopedics. To effectively control antimicrobial resistance among pathogens, we advocate extensive and dynamic monitoring of MDR bacteria, coupled with careful use of antibiotics.

Enterobacter cloacae colonisation and infection in a neonatal intensive care unit: retrospective investigation of preventive measures implemented after a multiclonal outbreak.

BACKGROUND: Enterobacter cloacae species is responsible for nosocomial outbreaks in vulnerable patients in neonatal intensive care units (NICU). The environment can constitute the reservoir and source of infection in NICUs. Herein we report the impact of preventive measures implemented after an Enterobacter cloacae outbreak inside a NICU. METHODS: This retrospective study was conducted in one level 3 NICU in Lyon, France, over a 6 year-period (2012-2018). After an outbreak of Enterobacter cloacae infections in hospitalized neonates in 2013, several measures were implemented including intensive biocleaning and education of medical staff. Clinical and microbiological characteristics of infected patients and evolution of colonization/infection with Enterobacter spp. in this NICU were retrieved. Moreover, whole genome sequencing was performed on 6 outbreak strains. RESULTS: Enterobacter spp. was isolated in 469 patients and 30 patients developed an infection including 2 meningitis and 12 fatal cases. Preventive measures and education of medical staff were not associated with a significant decrease in patient colonisation but led to a persistent decreased use of cephalosporin in the NICU. Infection strains were genetically diverse, supporting the hypothesis of multiple hygiene defects rather than the diffusion of a single clone. CONCLUSIONS: Grouped cases of infections inside one setting are not necessarily related to a single-clone outbreak and could reveal other environmental and organisational problematics. The fight against implementation and transmission of Enterobacter spp. in NICUs remains a major challenge.

Elucidating the pathogenic potential of Enterobacter cloacae SBP-8 using Caenorhabditis elegans as a model host.

Enterobacter cloacae, an opportunistic nosocomial pathogen, is reported to possess different virulence factors that could potentially influence its pathogenesis. Generally, the E. cloacae infections are of endogenous origin occurring in immunocompromised patients. The mechanisms of pathogenicity remain elusive, possibly due to the absence of established model hosts. Thus, we explored the utility of Caenorhabditis elegans as a model host to test the pathogenicity of E. cloacae SBP-8, a soil isolate. E. cloacae SBP-8 progressively colonized the intestine of C. elegans. It induced cell death (as assessed through DNA damage), reproductive defect and reduction of lifespan, comparable to a clinical isolate, E. cloacae (MTCC 509). Observation with Nomarski microscopy revealed significant anterior pharyngeal distention, and altered egg arrangement with internal egg hatching in 70% infected worms. The internal egg hatching was observed as early as 48 h post infection. E. cloacae SBP-8 infection reduced the brood size by 16%. A 2',7'-dichlorodihydrofluorescein diacetate staining confirmed the 10-fold induction of reactive oxygen species implicating either mitochondrial damage or septic shock in infected worms. Expression analysis through RT-PCR indicated stimulation of immune response by E. cloacae SBP-8 in worms by upregulating tol-1, a Toll-like receptor, within 6 h of exposure. During the initial phase of infection (up to 24 h) the nematodes exhibited protective immune response by upregulating antimicrobial peptide genes, lys-1, clec-60, clec-85, and clec-87. However, these genes were downregulated at later hours (48 h), indicating the nematodes surrendered to the infection. A similar trend was observed for reproductive genes (lin-29 and let-23), suggesting a struggle to maintain functional reproduction by the nematodes. These results clearly demonstrate the pathogenic potential of E. cloacae SBP-8 and suggest the suitability of C. elegans as a model organism to study its pathogenesis. This is the first study indicating that E. cloacae infections could potentially originate from an exogenic source (here soil).

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.

Cooccurrence of NDM-1, ESBL, RmtC, AAC(6')-Ib, and QnrB in Clonally Related Klebsiella pneumoniae Isolates Together with Coexistence of CMY-4 and AAC(6')-Ib in Enterobacter cloacae Isolates from Saudi Arabia.

The aim of this study was to investigate the mechanisms responsible for resistance to antimicrobials in a collection of enterobacterial isolates recovered from two hospitals in Saudi Arabia. A total of six strains isolated from different patients showing high resistance to carbapenems was recovered in 2015 from two different hospitals, with four being Klebsiella pneumoniae and two Enterobacter cloacae. All isolates except one K. pneumoniae were resistant to tigecycline, but only one K. pneumoniae was resistant to colistin. All produced a carbapenemase according to the Carba NP test, and all were positive for the EDTA-disk synergy test for detection of MBL. Using PCR followed by sequencing, the four K. pneumoniae isolates produced the carbapenemase NDM-1, while the two E. cloacae isolates produced the carbapenemase VIM-1. Genotyping analysis by Multilocus Sequence Typing (MLST) showed that three out of the four K. pneumoniae isolates were clonally related. They had been recovered from the same hospital and belonged to Sequence Type (ST) ST152. In contrast, the fourth K. pneumoniae isolate belonged to ST572. Noticeably, the NDM-1-producing K. pneumoniae additionally produced an extended-spectrum ß-lactamase (ESBL) of the CTX-M type, together with OXA-1 and TEM-1. Surprisingly, the three clonally related isolates produced different CTX-M variants, namely, CTX-M-3, CTX-M-57, and CTX-M-82, and coproduced QnrB, which confers quinolone resistance, and the 16S rRNA methylase RmtC, which confers high resistance to all aminoglycosides. The AAC(6')-Ib acetyltransferase was detected in both K. pneumoniae and E. cloacae. Mating-out assays using Escherichia coli as recipient were successful for all isolates. The bla NDM-1 gene was always identified on a 70-kb plasmid, whereas the bla VIM-1 gene was located on either a 60-kb or a 150-kb plasmid the two E. cloacae isolates, respectively. To the best of our knowledge, this is the first report of the coexistence of an MBL (NDM-1), an ESBL (CTX-M), a 16S rRNA methylase (RmtC), an acetyltransferase (AAC[6']-Ib), and a quinolone resistance enzyme (QnrB) in K. pneumoniae isolates recovered from different patients during an outbreak in a Saudi Arabian hospital.

Induction of plasmid-mediated AmpC ß-lactamase DHA-1 by piperacillin/tazobactam and other ß-lactams in Enterobacteriaceae.

Chromosomal AmpC ß-lactamase induction by several types of ß-lactams has been reported, but not enough data are available on DHA-1 ß-lactamase, a plasmid-mediated AmpC ß-lactamase. Therefore, we evaluated the DHA-1 ß-lactamase induction by various antibiotics including piperacillin/tazobactam (PIP/TZB) in this study. Six strains (Enterobacter cloacae 2 strains, Citrobacter freundii 1 strain, Serratia marcescens 2 strain, and Morganella morganii 1 strain) possessing chromosomal inducible AmpC ß-lactamase were used as controls. Four strains (Escherichia coli 2 strains, Klebsiella pneumoniae 1 strain, and C. koseri 1 strain) possessing DHA-1 ß-lactamase were used. The ß-lactamase activities were determined by a spectrophotometer using nitrocefin. ß-lactamase induction by PIP, PIP/TZB was not observed in any strains and ß-lactamase induction by third- and fourth-generation cephems was not observed in most strains. The induction ratios of the chromosomal AmpC ß-lactamase in the reference group by PIP/TZB were <1.51, and those of the DHA-1 ß-lactamase were <1.36, except for K. pneumoniae Rkp2004 (2.22). The ß-lactamase induction by first- and second-generation cephems, flomoxef, and carbapenem differed in each strain. Cefmetazole (CMZ) strongly induced ß-lactamase. This study demonstrated that the induction of DHA-1 ß-lactamase was similar to that of chromosomal AmpC using various Enterobacteriaceae, although the induction of ß-lactamase in both groups by PIP/TZB was low. We also reported that the induction of PIP/TZB, a ß-lactamase inhibitor combination antibiotic, against various AmpC-producing Enterobacteriaceae, including DHA-1 producers, was low.

Whole Genome Sequencing of Extended Spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae Isolated from Hospitalized Patients in KwaZulu-Natal, South Africa.

Extended spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae remain a critical clinical concern worldwide. The aim of this study was to characterize ESBL-producing K. pneumoniae detected within and between two hospitals in uMgungundlovu district, South Africa, using whole genome sequencing (WGS). An observational period prevalence study on antibiotic-resistant ESKAPE (i.e. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) bacteria was carried out in hospitalized patients during a two-month period in 2017. Rectal swabs and clinical specimens were collected from patients hospitalized and were screened for ESBL-producing, Gram-negative ESKAPE bacteria using cefotaxime-containing MacConkey agar and ESBL combination disk tests. Nine confirmed ESBL-K. pneumoniae isolated from six patients and two hospitals were whole genome sequenced using an Illumina MiSeq platform. Genome sequences were screened for presence of integrons, insertion sequences, plasmid replicons, CRISPR regions, resistance genes and virulence genes using different software tools. Of the 159 resistant Gram-negative isolates collected, 31 (19.50%) were ESBL-producers, of which, nine (29.03%) were ESBL-K. pneumoniae. The nine K. pneumoniae isolates harboured several ß-lactamase genes, including blaCTX-M-15, blaTEM-1b, blaSHV-1, blaOXA-1 concomitantly with many other resistance genes e.g. acc(6')-lb-cr, aadAI6, oqxA and oqxB that confer resistance to aminoglycosides and/or fluoroquinolones, respectively. Three replicon plasmid types were detected in both clinical and carriage isolates, namely ColRNAI, IncFIB(K), IncF(II). Sequence type ST152 was confirmed in two patients (one carriage isolate detected on admission and one isolate implicated in infection) in one hospital. In contrast, ST983 was confirmed in a clinical and a carriage isolate of two patients in two different hospitals. Our data indicate introduction of ESBL-producing K. pneumoniae isolates into hospitals from the community. We also found evidence of nosocomial transmission within a hospital and transmission between different hospitals. The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-associated cas3 genes were further detected in two of the nine ESBL-KP isolates. This study showed that both district and tertiary hospital in uMgungundlovu District were reservoirs for several resistance determinants and highlighted the necessity to efficiently and routinely screen patients, particularly those receiving extensive antibiotic treatment and long-term hospitalization stay. It also reinforced the importance of infection, prevention and control measures to reduce the dissemination of antibiotic resistance within the hospital referral system in this district.

The therapeutic potential of bacteriophages targeting gram-negative bacteria using Galleria mellonella infection model.

BACKGROUND: Phage therapy is the therapeutic use of bacteriophages to treat highly drug resistant bacterial infections. The current surge in bacteriophage therapy is motivated mainly because of the emergence of antibiotic-resistant bacteria in clinics. This study evaluated the therapeutic potential of three bacteriophages isolated against Escherichia coli ec311, Klebsiella pneumoniae kp235 and Enterobacter cloacae el140 strains using Galleria mellonella. The in vitro activity of three different phages belonging to Podoviridae and Myoviridae families was studied by the double agar overlay method against multi-drug resistant strains. Larval survivability studies were performed to evaluate the potential of phages against infection using G. mellonella. RESULTS: All the three phages were found to have potential to infect the host bacterial strains. For in vivo studies it was observed that E. coli and E. cloacae infected larvae, should be treated with three phage doses (20 µL, 104 PFU/mL) at 6 h interval to achieve 100% survival rate. But in the case of K. pneumoniae, a single phage dose treatment showed promising outcome. When mixed bacterial infections (all three bacterial cultures at 108 CFU/mL) were tested, minimum of four doses of phage cocktail (three phages) at 6 h interval was necessary to recover the larvae. All the results were confirmed by enumerating bacteria from the larvae. CONCLUSION: Our data shows that although in vitro studies showed high infectivity of phages, for in vivo models multiple phage doses were required for effective treatment.

Carbapenemase-producing Enterobacteriaceae in Mexico: report of seven non-clonal cases in a pediatric hospital.

BACKGROUND: Carbapenemases-producing Enterobacteriaceae (CPE) are a worldwide public health emergency. In Mexico, reports of CPE are limited, particularly in the pediatric population. Here, we describe the clinical, epidemiological, and molecular characteristics of seven consecutive cases in a third-level pediatric hospital in Mexico City over a four-month period during 2016. RESULTS: The Enterobacteriaceae identified were three Escherichia coli strains (producing OXA-232, NDM-1 and KPC-2), two Klebsiella pneumoniae strains (producing KPC-2 and NDM-1), one Klebsiella oxytoca strain producing OXA-48 and one Enterobacter cloacae strain producing NDM-1. The majority of patients had underlying disesases, three were immunocompromised, and three had infections involved the skin and soft tissues. Half patients died as a result of CPE infection. CONCLUSIONS: This study represents the first report of E. coli ST131-O25b clone producing NDM-1 in Latin America. In addition, this study is the first finding of K. oxytoca producing OXA-48 and E. coli producing OXA-232 in Mexican pediatric patients.

Factors associated to prevalence and treatment of carbapenem-resistant Enterobacteriaceae infections: a seven years retrospective study in three tertiary care hospitals.

BACKGROUND: The increasing incidence of carbapenem-resistant Enterobacteriaceae (CRE), has resulted in a difficult problem in the current clinical anti-infective treatment. We performed a retrospective analysis of prevalence and treatment for CRE infections patients. METHODS: This study was conducted in three tertiary care hospitals from January 1, 2010 to December 30, 2016. Baseline data, treatment, and outcomes were collected in patients with ventilator-associated bacterial pneumonia (VABP), bacteremia, complicated urinary tract infection (cUTI)/acute pyelonephritis (AP), hospital-acquired bacterial pneumonia (HABP), superficial wound infection (SWI), biliary tract infection (BTI), deep wound infection (DWI) and sterile body fluids infection (SBFI) due to CRE. RESULTS: One hundred twenty-four cases of CRE infection were identified: 31 VABP, 22 bacteremia, 18 cUTI/AP, 16 HABP, 16 SWI, 9 BTI, 7 DWI and 5 SBFI. The patient population had significant immunocompromised (33 of 124, 26.6%) and severe sepsis (43 of 124, 34.7%). The most common CRE pathogens were Klebsiella pneumoniae (84 of 124, 67.7%) and Enterobacter cloacae (24 of 124, 19.4%). And the production of IMP-type carbapenemase was the main antibiotic resistance mechanism. The majority of patients to take monotherapy for empiric therapy and dual therapy for direct treatment. Outcomes were universally poor (28-day mortality was 22.6%, 28 of 124) across all sites of infection. CONCLUSIONS: We identified a large number of cases of CRE infection in 7 years from different parts, most of these pathogens have been confirmed to produce IMP-type carbapenemases. The retrospective analysis of cases of such bacterial infections will help to control future infections of these pathogens. Despite the high mortality rate, we still found that the selection of quinolone antibiotics can be effective in the treatment of CRE producing IMP type enzymes.

Antimicrobial susceptibility and molecular epidemiology of clinical Enterobacter cloacae bloodstream isolates in Shanghai, China.

BACKGROUND: Enterobacter cloacae is a major nosocomial pathogen causing bloodstream infections. We retrospectively conducted a study to assess antimicrobial susceptibility and phylogenetic relationships of E. cloacae bloodstream isolates in two tertiary university-affiliated hospitals in Shanghai, in order to facilitate managements of E. cloacae bloodstream infections and highlight some unknowns for future prevention. METHODS: Fifty-three non-duplicate E. cloacae bloodstream isolates were consecutively collected from 2013 to 2016. Antimicrobial susceptibility was determined by disk diffusion. PCR was performed to detect extended-spectrum ß-lactamase (ESBL), carbapenemase and colistin resistance (MCR-1) gene. Plasmid-mediated AmpC ß-lactamase (pAmpC) genes were detected using a multiplex PCR assay targeting MIR/ACT gene (closely related to chromosomal EBC family gene) and other plasmid-mediated genes, including DHA, MOX, CMY, ACC, and FOX. eBURST was applied to analyze multi-locus sequence typing (MLST). RESULTS: The rates of resistance to all tested antibiotics were <40%. Among 53 E. cloacae isolates, 8(15.1%) were ESBL producers, 3(5.7%) were carbapenemase producers and 18(34.0%) were pAmpC producers. ESBL producers bear significantly higher resistance to cefotaxime (100.0%), ceftazidime (100.0%), aztreonam (100.0%), piperacillin (87.5%), tetracycline (75.0%), and trimethoprim-sulfamethoxazole (62.5%) than non-producers (p<0.05). PAmpC- and non-producers both presented low resistance rates (<40%) to all antibiotics (p>0.05). SHV (6/8, 75.0%) and MIR/ACT (15/18, 83.3%) predominated in ESBL and pAmpC producers respectively. Moreover, 2 isolates co-carried TEM-1, SHV-12, IMP-26 and DHA-1. MLST analysis distinguished the 53 isolates into 51 STs and only ST414 and ST520 were assigned two isolates of each (2/53). CONCLUSION: The antimicrobial resistance rates were low among 53 E. cloacae bloodstream isolates in the two hospitals. Multiclonality disclosed no evidence on spread of these isolates in Shanghai. The simultaneous presence of ESBL, carbapenemase and pAmpC detected in 2 isolates was firstly reported in Shanghai, which necessitated active ongoing surveillances and consistent prevention and control of E. cloacae.

Actividad sinérgica y eficacia clínica de la asociación fosfomicina-ciprofloxacino en el tratamiento de una infección de malla quirúrgica con absceso de partes blandas por Enterobacter cloacae productor de carbapenemasa / Synergistic activity and clinical efficacy of fosfomycin and ciprofloxacin combination treatment for soft tissue infection caused by carbapenemase-producing Enterobacter cloacae

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A Prolonged Outbreak of KPC-3-Producing Enterobacter cloacae and Klebsiella pneumoniae Driven by Multiple Mechanisms of Resistance Transmission at a Large Academic Burn Center.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter cloacae has been recently recognized in the United States. Whole-genome sequencing (WGS) has become a useful tool for analysis of outbreaks and for determining transmission networks of multidrug-resistant organisms in health care settings, including carbapenem-resistant Enterobacteriaceae (CRE). We experienced a prolonged outbreak of CRE E. cloacae and K. pneumoniae over a 3-year period at a large academic burn center despite rigorous infection control measures. To understand the molecular mechanisms that sustained this outbreak, we investigated the CRE outbreak isolates by using WGS. Twenty-two clinical isolates of CRE, including E. cloacae (n = 15) and K. pneumoniae (n = 7), were sequenced and analyzed genetically. WGS revealed that this outbreak, which seemed epidemiologically unlinked, was in fact genetically linked over a prolonged period. Multiple mechanisms were found to account for the ongoing outbreak of KPC-3-producing E. cloacae and K. pneumoniae This outbreak was primarily maintained by a clonal expansion of E. cloacae sequence type 114 (ST114) with distribution of multiple resistance determinants. Plasmid and transposon analyses suggested that the majority of blaKPC-3 was transmitted via an identical Tn4401b element on part of a common plasmid. WGS analysis demonstrated complex transmission dynamics within the burn center at levels of the strain and/or plasmid in association with a transposon, highlighting the versatility of KPC-producing Enterobacteriaceae in their ability to utilize multiple modes to resistance gene propagation.

High third-generation cephalosporin resistant Enterobacteriaceae prevalence rate among neonatal infections in Dakar, Senegal.

BACKGROUND: Neonatal infection constitutes one of Senegal's most important public health problems, with a mortality rate of 41 deaths per 1,000 live births. METHODS: Between January 2007 and March 2008, 242 neonates with suspected infection were recruited at three neonatal intensive care units in three major tertiary care centers in Dakar, the capital of Senegal. Neonatal infections were confirmed by positive bacterial blood or cerebrospinal fluid culture. The microbiological pattern of neonatal infections and the antibiotic susceptibility of the isolates were characterized. In addition, the genetic basis for antibiotic resistance and the genetic background of third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae were studied. RESULTS: A bacteriological infection was confirmed in 36.4 % (88/242) of neonates: 22.7 % (30/132) during the early-onset and 52.7 % (58/110) during the late-onset periods (p > 0.20). Group B streptococci accounted for 6.8 % of the 88 collected bacterial isolates, while most of them were Enterobacteriaceae (n = 69, 78.4 %). Of these, 55/69 (79.7 %) were 3GC-R. The bla CTX-M-15 allele, the bla SHV and the bla TEM were highly prevalent (63.5, 65.4 and 53.8 %, respectively), usually associated with qnr genes (65.4 %). Clonally related strains of 3GC-R Klebsiella pneumoniae and 3GC-R Enterobacter cloacae, the two most commonly recovered 3GC-R Enterobacteriaceae (48/55), were detected at the three hospitals, underlining the role of cross-transmission in their spread. The overall case fatality rate was 18.6 %. CONCLUSIONS: Measures should be taken to prevent nosocomial infections and the selection of resistant bacteria.

Brote de Enterobacter cloacae complex multirresistente productor de CTX-M-9 en una unidad de cuidados intensivos / Outbreak of multidrug-resistant CTX-M-9-producing Enterobacter cloacae complex in an intensive care unit

INTRODUCCIÓN: Descripción clínica y epidemiológica de un brote en una unidad de cuidados intensivos (UCI) causado por Enterobacter cloacae complex multirresistente productor de una beta-lactamasa de espectro extendido (BLEE) tipo CTX-M-9. MÉTODOS: Se realizó un estudio retrospectivo de las características clínicas y epidemiológicas del brote causado por E. cloacae complex. La identificación y estudio de sensibilidad de las cepas fueron realizados mediante el sistema semiautomático BD Phoenix™, y la caracterización de la BLEE, por PCR y secuenciación. La tipificación molecular se realizó mediante electroforesis en gel de campo pulsado (PFGE). RESULTADOS: Durante febrero de 2014, 6 pacientes (50% mujeres; media de edad: 61,5 años; rango de edad: 44-76 años) ingresados en la UCI del Complejo Hospitalario de Pontevedra (CHOP) presentaron aislamientos de E. cloacae complex resistente a cefalosporinas de amplio espectro. Tres pacientes desarrollaron infección; uno presentó bacteriemia primaria y shock séptico, y 2 neumonía asociada a ventilación mecánica. En los 3 casos restantes los aislamientos de E. cloacae complex se consideraron colonización. El análisis fenotípico y genotípico reveló que todos los aislados presentaban el mismo perfil por PFGE y que portaban la misma BLEE del tipo CTX-M-9. El brote se controló mediante la mejora de las medidas universales y el aislamiento de contacto de los pacientes infectados y/o colonizados. CONCLUSIÓN: Se describe desde un punto de vista clínico y epidemiológico un brote de E. cloacae complex portador de CTX-M-9 en una UCI

INTRODUCTION: Clinical and epidemiological description of an outbreak in an intensive care unit (ICU) caused by a strain of multidrug-resistant Enterobacter cloacae complex carrying a CTX-M-9-type extended-spectrum beta-lactamase (ESBL). METHODS: A retrospective study of the clinical and epidemiological features of the outbreak caused by E. cloacaecomplex was performed. Identifying and studying the sensitivity of the strains were performed using the semi-automated system BD Phoenix™, and the characterisation of ESBL using PCR and sequencing. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE). RESULTS: During February 2014, 6 patients (50% women; mean age: 61.5 years; age range: 44-76 years) admitted to the ICU of the Hospital of Pontevedra (CHOP) presented resistant E. cloacae complex isolates to extended-spectrum cephalosporins. Three patients developed infection; one had primary bacteraemia and septic shock, and 2 with ventilator-associated pneumonia. In the remaining three cases E. cloacae complex isolates were considered as colonisation. Phenotypic and genotypic analysis revealed that all isolates had the same PFGE profile and carried the same CTX-M-9 ESBL. The outbreak was controlled by improving universal precautions and contact isolation of patients infected and/or colonized. CONCLUSION: The clinical and epidemiological features of an outbreak in an ICU caused by E. cloacae complex carrying CTX-M-9 are described