Sequencing of five poultry strains elucidates phylogenetic relationships and divergence in virulence genes in Morganella morganii.
ABSTRACT: BACKGROUND:M. morganii is a bacterium frequently associated with urinary infections in humans. While many human strains are sequenced, only the genomes of few poultry strains are available. Here, we performed a detailed characterization of five highly resistant Morganella morganii strains isolated in association with Escherichia coli from diseased domestic Austrian poultry flocks, namely geese, turkeys and chicken layers. Additionally, we sequenced the genomes of these strains by NGS and analyzed phylogenetic clustering, resistance and virulence genes in the context of host-specificity. RESULTS:Two strains were identified to be Extended Spectrum Beta Lactamase (ESBL) and one as AmpC beta-lactamases (AMP-C) phenotype, while two were ESBL negative. By integrating the genome sequences of these five poultry strains with all the available M. morganii genomes, we constructed a phylogenetic tree that clearly separates the Morganella genus into two clusters (M1 and M2), which approximately reflect the proposed subspecies classification (morganii and sibonii). Additionally, we found no association between phylogenetic structure and host, suggesting interspecies transmission. All five poultry strains contained genes for resistance to aminocoumarins, beta-lactams, colistin, elfamycins, fluoroquinolones, phenicol, rifampin and tetracycline. A comparative genomics analysis of virulence genes showed acquisition of novel virulence genes involved in secretion system and adherence in cluster M2. We showed that some of these genes were acquired by horizontal gene transfer from closely related Morganellaceae species and propose that novel virulence genes could be responsible for expansion of tissue tropism in M. morganii. Finally, we detected variability in copy number and high sequence divergence in toxin genes and provided evidence for positive selection in insecticidal toxins genes, likely reflecting host-related adaptations. CONCLUSIONS:In summary, this study describes i) the first isolation and characterization of M. morganii from goose and turkey, ii) a large-scale genetic analysis of M. morganii and an attempt to generate a global picture of the M. morganii intraspecific phylogenetic structure.
Project description:A serious concern is arising on the coexistence of extended-spectrum beta-lactamase (ESBL) and plasmid mediated quinolone resistance (PMQR) producing bacteria in animal husbandry, which could be transferred to humans, especially in strains that may not be routinely screened for resistance. This study therefore tested the prevalence of ESBL and PMQR genes in selected bacteria isolated from poultry faeces. Faecal droppings of birds were collected from 11 farms in five states in South Western Nigeria. Bacteria were isolated from the samples on cefotaxime supplemented plates and identified with MALDI-TOF. The MIC was determined using VITEK system and resistance genes were detected with PCR. A total of 350 strains were isolated from different samples and selected strains were identified as 23 Klebsiella pneumonia, 12 Morganella morganii, seven Leclercia adecarboxylata and one Citrobacter freundii. All the species were resistant to gentamycin, trimethoprim/sulphamethaxole, tobramycin, piperacillin, cefotaxime and aztreonam (except Morganella morganii strains which were mostly susceptible to aztreonam). All the tested strains were susceptible to imipenem, meropenem and amikacin. All Leclercia adecarboxylata strains were resistant to ceftazidime, cefepime and fosfomycin while all Morganella morganii strains were resistant to fosfomycin, moxifloxacin and ciprofloxacin. All tested species were generally sensitive to ciprofloxacin except Morganella morganii strains which were resistant to ciprofloxacin. The resistance to ciprofloxacin, ceftazidime, cefepime, tigercylin, colistin and fosfomycin were 65%, 40%, 23%,, 7%, 33%, 48% respectively while the prevalence of SHV, TEM and CTX genes were 42%, 63%, 35% respectively. 9.3% of the isolates had the three ESBL genes, 2.33% had qnrA gene, 4.65% had qnr B gene while none had qnrS gene. The most prevalent PMQR gene is Oqxb (25.58%) while 6.98% had the qep gene. Klebsiella pneumoniae generally had both ESBL and PMQR genes. The high prevalence of extended spectrum beta-lactamase genes in the studied strains calls for caution in the use of beta lactam antibiotics in poultry feeds. This is the first report of the occurrence of extended spectrum beta-lactamase and plasmid mediated quinolone resistance genes in Morganella morganii and Leclercia adecarboxylata strains isolated from poultry faeces.
Project description:Salmonella genomic island 1 (SGI1) is a multidrug resistance integrative mobilizable element that harbors a great diversity of antimicrobial resistance gene clusters described in numerous Salmonella enterica serovars and also in Proteus mirabilis. A serious threat to public health was revealed in the recent description in P. mirabilis of a SGI1-derivative multidrug resistance island named PGI1 (Proteus genomic island 1) carrying extended-spectrum-?-lactamase (ESBL) and metallo-?-lactamase resistance genes, blaVEB-6 and blaNDM-1, respectively. Here, we report the first description of Salmonella genomic island 1 (SGI1) in a multidrug-resistant clinical Morganella morganii subsp. morganii strain isolated from a patient in France in 2013. Complete-genome sequencing of the strain revealed SGI1 variant SGI1-L carrying resistance genes dfrA15, floR, tetA(G), blaPSE-1 (now referred to as blaCARB-2), and sul1, conferring resistance to trimethoprim, phenicols, tetracyclines, amoxicillin, and sulfonamides, respectively. The SGI1-L variant was integrated into the usual chromosome-specific integration site at the 3' end of the trmE gene. Beyond Salmonella enterica and Proteus mirabilis, the SGI1 integrative mobilizable element may thus also disseminate its multidrug resistance phenotype in another genus belonging to the Proteae tribe of the family Enterobacteriaceae. IMPORTANCE Since its initial identification in epidemic multidrug-resistant Salmonella enterica serovar Typhimurium DT104 strains, several SGI1 variants, SGI1 lineages, and SGI1-related elements (SGI2, PGI1, and AGI1) have been described in many bacterial genera (Salmonella, Proteus, Morganella, Vibrio, Shewanella, etc.). They constitute a family of multidrug resistance site-specific integrative elements acquired by horizontal gene transfer, SGI1 being the best-characterized element. The horizontal transfer of SGI1/PGI1 elements into other genera is of public health concern, notably with regard to the spread of critically important resistance genes such as ESBL and carbapenemase genes. The identification of SGI1 in Morganella morganii raises the issue of (i) the potential for SGI1 to emerge in other human pathogens and (ii) its bacterial host range. Further surveillance and research are needed to understand the epidemiology, the spread, and the importance of the members of this SGI1 family of integrative elements in contributing to antibiotic resistance development.
Project description:To investigate the molecular characteristics of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae collected during a cross-sectional study examining the prevalence and risk factors for faecal carriage of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in humans living in areas with high or low broiler density.ESC-resistant Enterobacteriaceae were identified by combination disc-diffusion test. ESBL/AmpC/carbapenemase genes were analysed using PCR and sequencing. For E. coli, phylogenetic groups and MLST were determined. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid multilocus sequence typing.175 ESC-resistant Enterobacteriaceae were cultured from 165/1,033 individuals. The isolates were Escherichia coli(n=65), Citrobacter freundii (n=52), Enterobacter cloacae (n=38), Morganella morganii (n=5), Enterobacter aerogenes (n=4), Klebsiella pneumoniae (n=3), Hafnia alvei (n=2), Shigella spp. (n=2), Citrobacter amalonaticus (n=1), Escherichia hermannii (n=1), Kluyvera cryocrescens (n=1), and Pantoea agglomerans (n=1). The following ESBL genes were recovered in 55 isolates originating from 49 of 1,033 (4.7 %) persons: blaCTX-M-1 (n=17), blaCTX-M-15 (n=16), blaCTX-M-14 (n=9), blaCTX-M-2 (n=3), blaCTX-M-3 (n=2), blaCTX-M-24 (n=2), blaCTX-M-27 (n=1), blaCTX-M-32 (n=1), blaSHV-12 (n=2), blaSHV-65 (n=1) and blaTEM-52 (n=1). Plasmidic AmpC (pAmpC) genes were discovered in 6 out of 1,033 (0.6 %) persons. One person carried two different E. coli isolates, one with blaCTX-M-1 and the other with blaCMY-2 and therefore the prevalence of persons carrying Enterobacteriaceae harboring ESBL and/or pAmpC genes was 5.2 %. In eight E. coli isolates the AmpC phenotype was caused by mutations in the AmpC promoter region. No carbapenemase genes were identified. A large variety of E. coli genotypes was found, ST131 and ST10 being most common.ESBL/pAmpC genes resembled those from patients in Dutch hospitals, indicating that healthy humans form a reservoir for transmission of these determinants to vulnerable people. The role of poultry in the transmission to humans in the community remains to be elucidated.
Project description:In 1999, 39 of 2,599 isolates of the family Enterobacteriaceae (1.5%) collected by eight private laboratories in the Aquitaine region in France produced an extended-spectrum beta-lactamase (ESBL). Among these were 19 Enterobacter aerogenes isolates; 8 Klebsiella pneumoniae isolates; 6 Escherichia coli isolates; 3 Proteus mirabilis isolates; and 1 isolate each of Serratia marcescens, Morganella morganii, and Providencia stuartii. ESBL producers were isolated from 38 patients, including 33 residents of 11 clinics or nursing homes and 5 ambulatory patients. Seven different ESBLs were characterized. These mainly consisted of TEM-24 (25 isolates) and TEM-21 (9 isolates), but TEM-15 (2 isolates) and TEM-3, TEM-19, SHV-4, and CTX-M-1 (1 isolate each) were also characterized. Seven strains showed the coexistence of different TEM- and/or SHV-encoding genes, including a new SHV-1 variant, SHV-44, defined by the substitution R205L previously reported for SHV-3 in association with S238G. The epidemiology of the ESBL producers was investigated by random amplification of polymorphic DNA, typing by enterobacterial repetitive intergenic consensus PCR, analysis of resistance cotransferred with the ESBL, and analysis of the restriction profiles of the ESBL-encoding plasmids. Of the TEM-24-expressing strains, 18 were E. aerogenes isolates, including 9 from the same clinic, that were representatives of the epidemic clone disseminating in France. Of the TEM-21-producing strains that belonged to different species of the family Enterobacteriaceae (E. coli, K. pneumoniae, and P. mirabilis), 8 were isolated in the same nursing home. Outbreaks due to strain and/or plasmid dissemination in these clinic and nursing home were demonstrated. The presence of ESBL producers in five ambulatory patients probably resulted from nosocomial acquisition. Our data highlight the serious need to monitor patients for ESBL-producing Enterobacteriaceae in general practice.
Project description:Morganella morganii is an opportunistic bacterial pathogen shown to cause a wide range of clinical and community-acquired infections. This study was aimed at sequencing and comparing the genomes of three M. morganii strains isolated from the urine samples of patients with community-acquired urinary tract infections. Draft genome sequencing was conducted using the Illumina HiSeq platform. The genomes of MM 1, MM 4, and MM 190 strains have a size of 3.82-3.97 Mb and a GC content of 50.9-51%. Protein-coding sequences (CDS) represent 96.1% of the genomes, RNAs are encoded by 2.7% of genes and pseudogenes account for 1.2% of the genomes. The pan-genome containes 4,038 CDS, of which 3,279 represent core genes. Six to ten prophages and 21-33 genomic islands were identified in the genomes of MM 1, MM 4, and MM 190. More than 30 genes encode capsular biosynthesis proteins, an average of 60 genes encode motility and chemotaxis proteins, and about 70 genes are associated with fimbrial biogenesis and adhesion. We determined that all strains contained urease gene cluster ureABCEFGD and had a urease activity. Both MM 4 and MM 190 strains are capable of hemolysis and their activity correlates well with a cytotoxicity level on T-24 bladder carcinoma cells. These activities were associated with expression of RTX toxin gene hlyA, which was introduced into the genomes by a phage similar to Salmonella phage 118970_sal4.
Project description:Introduction: The increasing incidence of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in sub-Saharan Africa is of serious concern. Studies from countries with a highly industrialized poultry industry suggest the poultry production-food-consumer chain as a potential transmission route. In Africa, integrated studies at this human-animal interface are still missing. Aim: To determine the molecular epidemiology of ESBL-producing E. coli from the intestinal tract of humans and poultry in rural Ghana. Methods: During a 6-month period, fecal samples from all children admitted to the Agogo Hospital (Ghana) and broilers at eight poultry farms located within the hospital catchment area were collected. After screening on selective ESBL agar, whole genome sequencing (WGS) was performed on all ESBL isolates. The genomes were analyzed using multilocus sequence typing (MLST), ESBL genotyping and genome-based phylogenetic analyses. Results: Of 140 broilers and 54 children, 41 (29%) and 33 (61%) harbored ESBL E. coli, respectively, with prevalences on farms ranging between 0 and 85%. No predominant sequence type (ST) was detected among humans. ST10 was most prevalent among broilers (n = 31, 69%). The ESBL gene bla CTX-M-15 was predominant among broilers (n = 43, 96%) and humans (n = 32, 97%). Whole-genome-based phylogenetic analysis revealed three very closely related broiler/human isolate clusters (10% of ESBL isolates) with chromosomal and plasmid-mediated ESBL genes. Conclusion: The findings demonstrate a high frequency of intestinal ESBL-producing E. coli in rural Ghana. Considering that animal and human samples are independent specimens from the same geographic location, the number of closely related ESBL isolates circulating across these two reservoirs is substantial. Hence, poultry farms or meat products might be an important source for ESBL-producing bacteria in rural Ghana leading to difficult-to-treat infections in humans.
Project description:BACKGROUND: The opportunistic enterobacterium, Morganella morganii, which can cause bacteraemia, is the ninth most prevalent cause of clinical infections in patients at Changhua Christian Hospital, Taiwan. The KT strain of M. morganii was isolated during postoperative care of a cancer patient with a gallbladder stone who developed sepsis caused by bacteraemia. M. morganii is sometimes encountered in nosocomial settings and has been causally linked to catheter-associated bacteriuria, complex infections of the urinary and/or hepatobiliary tracts, wound infection, and septicaemia. M. morganii infection is associated with a high mortality rate, although most patients respond well to appropriate antibiotic therapy. To obtain insights into the genome biology of M. morganii and the mechanisms underlying its pathogenicity, we used Illumina technology to sequence the genome of the KT strain and compared its sequence with the genome sequences of related bacteria. RESULTS: The 3,826,919-bp sequence contained in 58 contigs has a GC content of 51.15% and includes 3,565 protein-coding sequences, 72 tRNA genes, and 10 rRNA genes. The pathogenicity-related genes encode determinants of drug resistance, fimbrial adhesins, an IgA protease, haemolysins, ureases, and insecticidal and apoptotic toxins as well as proteins found in flagellae, the iron acquisition system, a type-3 secretion system (T3SS), and several two-component systems. Comparison with 14 genome sequences from other members of Enterobacteriaceae revealed different degrees of similarity to several systems found in M. morganii. The most striking similarities were found in the IS4 family of transposases, insecticidal toxins, T3SS components, and proteins required for ethanolamine use (eut operon) and cobalamin (vitamin B12) biosynthesis. The eut operon and the gene cluster for cobalamin biosynthesis are not present in the other Proteeae genomes analysed. Moreover, organisation of the 19 genes of the eut operon differs from that found in the other non-Proteeae enterobacterial genomes. CONCLUSIONS: This is the first genome sequence of M. morganii, which is a clinically relevant pathogen. Comparative genome analysis revealed several pathogenicity-related genes and novel genes not found in the genomes of other members of Proteeae. Thus, the genome sequence of M. morganii provides important information concerning virulence and determinants of fitness in this pathogen.
Project description:To ascertain whether on animal farms there reside extended-spectrum beta-lactamase (ESBL) and plasmidic class C beta-lactamase-producing Escherichia coli isolates potentially pathogenic for humans, phylogenetic analyses, pulsed-field gel electrophoresis (PFGE) typing, serotyping, and virulence genotyping were performed for 86 isolates from poultry (57 isolates) and pig (29 isolates) farms. E. coli isolates from poultry farms carried genes encoding enzymes of the CTX-M-9 group as well as CMY-2, whereas those from pig farms mainly carried genes encoding CTX-M-1 enzymes. Poultry and pig isolates differed significantly in their phylogenetic group assignments, with phylogroup A predominating in pig isolates and phylogroup D predominating in avian isolates. Among the 86 farm isolates, 23 (26.7%) carried two or more virulence genes typical of extraintestinal pathogenic E. coli (ExPEC). Of these, 20 were isolated from poultry farms and only 3 from pig farms. Ten of the 23 isolates belonged to the classic human ExPEC serotypes O2:H6, O2:HNM, O2:H7, O15:H1, and O25:H4. Despite the high diversity of serotypes and pulsotypes detected among the 86 farm isolates, 13 PFGE clusters were identified. Four of these clusters contained isolates with two or more virulence genes, and two clusters exhibited the classic human ExPEC serotypes O2:HNM (ST10) and O2:H6 (ST115). Although O2:HNM and O2:H6 isolates of human and animal origins differed with respect to their virulence genes and PFGE pulsotypes, the O2:HNM isolates from pigs showed the same sequence type (ST10) as those from humans. The single avian O15:H1 isolate was compared with human clinical isolates of this serotype. Although all were found to belong to phylogroup D and shared the same virulence gene profile, they differed in their sequence types (ST362-avian and ST393-human) and PFGE pulsotypes. Noteworthy was the detection, for the first time, in poultry farms of the clonal groups O25b:H4-ST131-B2, producing CTX-M-9, and O25a-ST648-D, producing CTX-M-32. The virulence genes and PFGE profiles of these two groups were very similar to those of clinical human isolates. While further studies are required to determine the true zoonotic potential of these clonal groups, our results emphasize the zoonotic risk posed especially by poultry farms, but also by pig farms, as reservoirs of ESBL- and CMY-2-encoding E. coli.
Project description:To evaluate in vitro susceptibilities of aerobic and facultative Gram-negative bacterial (GNB) isolates from intra-abdominal infections (IAIs) to 12 selected antimicrobials in Chinese hospitals from 2012 to 2014.Hospital acquired (HA) and community acquired (CA) IAIs were collected from 21 centers in 16 Chinese cities. Extended spectrum beta-lactamase (ESBL) status and antimicrobial susceptibilities were determined at a central laboratory using CLSI broth microdilution and interpretive standards.From all isolated strains the Enterobacteriaceae (81.1%) Escherichia coli accounted for 45.4% and Klebsiella pneumoniae for 20.1%, followed by Enterobacter cloacae (5.2%), Proteus mirabilis (2.1%), Citrobacter freundii (1.8%), Enterobacter aerogenes (1.8%), Klebsiella oxytoca (1.4%), Morganella morganii (1.2%), Serratia marcescens (0.7%), Citrobacter koseri (0.3%), Proteus vulgaris (0.3%) and others (1.0%). Non- Enterobacteriaceae (18.9%) included Pseudomonas aeruginosa (9.8%), Acinetobacter baumannii (6.7%), Stenotrophomonas maltophilia (0.9%), Aeromonas hydrophila (0.4%) and others (1.1%). ESBL-screen positive Escherichia coli isolates (ESBL+) showed a decreasing trend from 67.5% in 2012 to 58.9% in 2014 of all Escherichia coli isolates and the percentage of ESBL+ Klebsiella pneumoniae isolates also decreased from 2012 through 2014 (40.4% to 26.6%), which was due to reduced percentages of ESBL+ isolates in HA IAIs for both bacteria. The overall susceptibilities of all 5160 IAI isolates were 87.53% to amikacin (AMK), 78.12% to piperacillin-tazobactam (TZP) 81.41% to imipenem (IMP) and 73.12% to ertapenem (ETP). The susceptibility of ESBL-screen positive Escherichia coli strains was 96.77%-98.8% to IPM, 91.26%-93.16% to ETP, 89.48%-92.75% to AMK and 84.86%-89.34% to TZP, while ESBL-screen positive Klebsiella pneumoniae strains were 70.56%-80.15% susceptible to ETP, 80.0%-87.5% to IPM, 83.82%-87.06% to AMK and 63.53%-68.38% to TZP within the three year study. Susceptibilities to all cephalosporins and fluoroquinolones were less than 50% beside 66.5% and 56.07% to cefoxitin (FOX) for ESBL+ Escherichia coli and Klebsiella pneumoniae strains respectively.The total ESBL+ rates decreased in Escherichia coli and Klebsiella pneumoniae IAI isolates due to fewer prevalence in HA infections. IPM, ETP and AMK were the most effective antimicrobials against ESBL+ Escherichia coli and Klebsiella pneumoniae IAI isolates in 2012-2014 and a change of fluoroquinolone regimens for Chinese IAIs is recommended.
Project description:The worldwide dissemination of extended-spectrum beta-lactamase producing Enterobacteriaceae, (ESBL-E) and their subset producing carbapenemases (CPE), is alarming. Limited data on the prevalence of such strains in infections from patients from Sub-Saharan Africa are currently available. We determined, here, the prevalence of ESBL-E/CPE in bacteriemic patients in two teaching hospitals from Bamako (Mali), which are at the top of the health care pyramid in the country. During one year, all Enterobacteriaceae isolated from bloodstream infections (E-BSI), were collected from patients hospitalized at the Point G University Teaching Hospital and the pediatric units of Gabriel Touré University Teaching Hospital. Antibiotic susceptibility testing, enzyme characterization and strain relatedness were determined. A total of 77 patients had an E-BSI and as many as 48 (62.3%) were infected with an ESBL-E. ESBL-E BSI were associated with a previous hospitalization (OR 3.97 95% IC [1.32; 13.21]) and were more frequent in hospital-acquired episodes (OR 3.66 95% IC [1.07; 13.38]). Among the 82 isolated Enterobacteriaceae, 58.5% were ESBL-E (20/31 Escherichia coli, 20/26 Klebsiella pneumoniae and 8/15 Enterobacter cloacae). The remaining (5 Salmonella Enteritidis, 3 Morganella morganii 1 Proteus mirabilis and 1 Leclercia adecarboxylata) were ESBL negative. CTX-M-1 group enzymes were highly prevalent (89.6%) among ESBLs; the remaining ones being SHV. One E. coli produced an OXA-181 carbapenemase, which is the first CPE described in Mali. The analysis of ESBL-E relatedness suggested a high rate of cross transmission between patients. In conclusion, even if CPE are still rare for the moment, the high rate of ESBL-BSI and frequent cross transmission probably impose a high medical and economic burden to Malian hospitals.