Spread of carbapenemase-producing Morganella spp from 2013 to 2021: a comparative genomic study.

BACKGROUND: Morganella spp are opportunistic pathogens involved in various infections. Intrinsic resistance to multiple antibiotics (including colistin) combined with the emergence of carbapenemase producers reduces the number of active antimicrobials. The aim of this study was to characterise genetic features related to the spread of carbapenem-resistant Morganella spp. METHODS: This comparative genomic study included extensively drug-resistant Morganella spp isolates collected between Jan 1, 2013, and March 1, 2021, by the French National Reference Center (NRC; n=68) and European antimicrobial resistance reference centres in seven European countries (n=104), as well as one isolate from Canada, two reference strains from the Pasteur Institute collection (Paris, France), and two colistin-susceptible isolates from Bicêtre Hospital (Kremlin-Bicêtre, France). The isolates were characterised by whole-genome sequencing, antimicrobial susceptibility testing, and biochemical tests. Complete genomes from GenBank (n=103) were also included for genomic analysis, including phylogeny and determination of core genomes and resistomes. Genetic distance between different species or subspecies was performed using average nucleotide identity (ANI). Intrinsic resistance mechanisms to polymyxins were investigated by combining genetic analysis with mass spectrometry on lipid A. FINDINGS: Distance analysis by ANI of 275 isolates identified three groups: Morganella psychrotolerans, Morganella morganii subspecies sibonii, and M morganii subspecies morganii, and a core genome maximum likelihood phylogenetic tree showed that the M morganii isolates can be separated into four subpopulations. On the basis of these findings and of phenotypic divergences between isolates, we propose a modified taxonomy for the Morganella genus including four species, Morganella psychrotolerans, Morganella sibonii, Morganella morganii, and a new species represented by a unique environmental isolate. We propose that M morganii include two subspecies: M morganii subspecies morganii (the most prevalent) and M morganii subspecies intermedius. This modified taxonomy was supported by a difference in intrinsic resistance to tetracycline and conservation of metabolic pathways such as trehalose assimilation, both only present in M sibonii. Carbapenemase producers were mostly identified among five high-risk clones of M morganii subspecies morganii. The most prevalent carbapenemase corresponded to NDM-1, followed by KPC-2, and OXA-48. A cefepime-zidebactam combination was the most potent antimicrobial against the 172 extensively drug-resistant Morganella spp isolates in our collection from different European countries, which includes metallo-ß-lactamase producers. Lipid A analysis showed that the intrinsic resistance to colistin was associated with the presence of L-ARA4N on lipid A. INTERPRETATION: This global characterisation of, to our knowledge, the widest collection of extensively drug-resistant Morganella spp highlights the need to clarify the taxonomy and decipher intrinsic resistance mechanisms, and paves the way for further genomic comparisons. FUNDING: None.

A Genomic and Bioinformatics View of the Classification and Evolution of Morganella Species and Their Chromosomal Accessory Genetic Elements Harboring Antimicrobial Resistance Genes.

In this study, draft-genome sequencing was conducted for 60 Chinese Morganella isolates, and furthermore, 12 of them were fully sequenced. Then, a total of 166 global sequenced Morganella isolates, including the above 60, were collected to perform average nucleotide identity-based genomic classification and core single nucleotide polymorphism-based phylogenomic analysis. A genome sequence-based species classification scheme for Morganella was established, and accordingly, the two conventional Morganella species were redefined as two complexes and further divided into four and two genospecies, respectively. At least 88 acquired antimicrobial resistance genes (ARGs) were disseminated in these 166 isolates and were prevalent mostly in the isolates from hospital settings. IS26/IS15DI, IS10 and IS1R, and Tn3-, Tn21-, and Tn7-subfamily unit transposons were frequently presented in these 166 isolates. Furthermore, a detailed sequence comparison was applied to 18 Morganella chromosomal accessory genetic elements (AGEs) from the fully sequenced 12 isolates, together with 5 prototype AGEs from GenBank. These 23 AGEs were divided into eight different groups belonging to composite/unit transposons, transposable prophages, integrative and mobilizable elements, and integrative and conjugative elements, and they harbored at least 52 ARGs involved in resistance to 15 categories of antimicrobials. Eleven of these 23 AGEs acquired large accessory modules, which exhibited complex mosaic structures and contained many antimicrobial resistance loci and associated ARGs. Integration of ARG-containing AGEs into Morganella chromosomes would contribute to the accumulation and dissemination of ARGs in Morganella and enhance the adaption and survival of Morganella under complex and diverse antimicrobial selection pressures. IMPORTANCE This study presents a comprehensive genomic epidemiology analysis on global sequenced Morganella isolates. First, a genome sequence-based species classification scheme for Morganella is established with a higher resolution and accuracy than those of the conventional scheme. Second, the prevalence of accessory genetic elements (AGEs) and associated antimicrobial resistance genes (ARGs) among Morganella isolates is disclosed based on genome sequences. Finally, a detailed sequence comparison of eight groups of 23 AGEs (including 19 Morganella chromosomal AGEs) reveals that Morganella chromosomes have evolved to acquire diverse AGEs harboring different profiles of ARGs and that some of these AGEs harbor large accessory modules that exhibit complex mosaic structures and contain a large number of ARGs. Data presented here provide a deeper understanding of the classification and evolution of Morganella species and also those of ARG-containing AGEs in Morganella at the genomic scale.

Histamine Production Behaviors of a Psychrotolerant Histamine-Producer, Morganella psychrotolerans, in Various Environmental Conditions.

Histamine food poisoning is a major safety concern related to seafood consumption worldwide. Morganella psychrotolerans is a novel psychrotolerant histamine-producer. In this study, the histamine production behaviors of M. psychrotolerans and two other major histamine-producers, mesophilic Morganella morganii and psychrotrophic Photobacterium phosphoreum, were compared in seafood products, and histamine accumulation by M. psychrotolerans was characterized at various pH and temperature levels in culture broth. The growth of M. psychrotolerans and P. phosphoreum increased similarly at 4 °C in canned tuna, but M. psychrotolerans produced much higher levels of histamine than P. phosphoreum. Histamine accumulation by M. psychrotolerans was induced at lower environmental pH condition at 4 and 20 °C. The optimal temperature and pH for producing histamine by crude histidine decarboxylase of M. psychrotolerans were 30 °C and pH 7, respectively. The activity of the crude HDC extracted from M. psychrotolerans cells at 10 °C retained 45% of the activity at 30 °C. Histidine decarboxylase gene expression of M. psychrotolerans was induced by low pH conditions. These results suggest that M. psychrotolerans are also a very important histamine-producer leading to histamine poisoning associated with seafood below the refrigeration temperature.

Proteae: a reservoir of class 2 integrons?

OBJECTIVES: Our aim was to confirm with a large panel of clinical isolates that class 2 integrons are highly prevalent in Proteae and to analyse their genetic characteristics. METHODS: Proteae (Proteus spp., Morganella spp. and Providencia spp.) isolates were collected from clinical samples during 2013 at Limoges University Hospital, France. The presence of class 1, 2 and 3 integrons was investigated by quantitative PCR. The presence of a stop codon in the intI2 gene was determined by Sanger sequencing. The gene cassette arrays of class 2 integrons were determined by PCR-RFLP and Sanger sequencing or next-generation sequencing when needed. RESULTS: Of the 327 Proteae collected, 103 (31.5%) harboured a class 2 integron and 45 (13.8%) a class 1 integron. No class 3 integrons were detected. One functional IntI2 integrase was detected in a Morganella morganii isolate. Six different gene cassette arrays were detected. Four had already been described in the literature: dfrA1-sat2-aadA1 (72 isolates), dfrA1-catB2-sat2-aadA1 (17), sat2-aadA1 (6) and lnu(F), dfrA1, aadA1 (1). We identified two new gene cassette arrays: (i) a new variant of the dfrA1 gene cassette (one isolate; the one with the functional IntI2); and (ii) the array dfrA1-gcu115-sat2 harbouring the new gcu115 gene cassette with two ORFs encoding proteins of unknown functions (five isolates). CONCLUSIONS: We showed a high frequency of class 2 integrons, as well as a diversity of gene cassette arrays, among Proteae. This work highlights that the Proteae tribe plays an important role as a reservoir of class 2 integrons.

Development of a real-time PCR method coupled with a selective pre-enrichment step for quantification of Morganella morganii and Morganella psychrotolerans in fish products.

Histamine fish poisoning is common and due to toxic concentrations of histamine often produced by Gram-negative bacteria in fin-fish products with a high content of the free amino acid histidine. The genus Morganella includes two species previously reported to cause incidents of histamine fish poisoning. Morganella morganii and Morganella psychrotolerans are both strong producer of histamine. However, little is known about the occurrence and critical stages for fish contamination with these bacteria. To elucidate contamination routes of Morganella, specific real-time quantitative PCR (RTi qPCR) methods for quantification of M. morganii and M. psychrotolerans have been developed. Selective primers amplified a 110 bp region of the vasD gene for M. psychrotolerans and a 171 bp region of the galactokinase gene for M. morganii. These primer-sets showed high specificity as demonstrated by using purified DNA from 23 other histamine producing bacteria and 26 isolates with no or limited histamine production. The efficiency of the qPCR reactions on artificially contaminated fish samples were 100.8% and 96.3% respectively. The limit of quantification (LOQ) without enrichment was 4 log CFU/g. A quantitative enrichment step with a selective medium was included and improved the sensitivity of the methods to a LOQ of below 50 CFU/g in seafood. RTi qPCR methods with or without enrichment were evaluated for enumeration of Morganella species in naturally contaminated fresh fish and lightly preserved seafood from Denmark. These new methods will contribute to a better understanding of the occurrence and histamine production by Morganella species in fish products, information that is essential to reduce the unacceptably high frequency of histamine fish poisoning.

Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury.

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.

Morganella psychrotolerans sp. nov., a histamine-producing bacterium isolated from various seafoods.

Mesophilic Morganella morganii (n=6) and psychrotolerant M. morganii-like isolates from various seafoods (n=13), as well as clinical M. morganii isolates (n=3), were characterized by using a polyphasic approach including multi-locus sequencing. Based on the phylogenetic analysis, the 22 strains were divided into two distinct groups comprising mesophilic and psychrotolerant isolates, respectively. This classification was supported by DNA-DNA hybridization studies, whereby a psychrotolerant isolate (strain U2/3(T)) showed 41.0 and 17.8 % relatedness to the type strains of the mesophilic species Morganella morganii subsp. morganii (strain LMG 7874(T)) and Morganella morganii subsp. sibonii (strain DSM 14850(T)), respectively. Analysis of the 16S rRNA gene sequences showed a similarity of 98.6 % between mesophilic and psychrotolerant isolates. However, fragments of seven protein-encoding housekeeping genes (atpD, dnaN, gyrB, hdc, infB, rpoB and tuf) all showed less than 90.9 % sequence similarity between the two groups. The psychrotolerant isolates grew at 0-2 degrees C and also differed from the mesophilic M. morganii isolates with respect to growth at 37 degrees C and in 8.5 % (w/v) NaCl and fermentation of d-galactose. The psychrotolerant strains appear to represent a novel species, for which the name Morganella psychrotolerans sp. nov. is proposed. The type strain is U2/3(T) (=LMG 23374(T)=DSM 17886(T)).

Classification, identification, and clinical significance of Proteus, Providencia, and Morganella.

This review presents the current taxonomy of the genera Proteus, Providencia, and Morganella, along with the current methods for the identification of each species within the three genera, incorporating both conventional biochemical and commercial methods. While all of these organisms are ubiquitous in the environment, individual case reports and nosocomial outbreak reports that demonstrate their ability to cause major infectious disease problems are presented. Lastly, anticipated antimicrobial susceptibility patterns are reviewed. Many of these organisms are easily controlled, but the advent of newer and more powerful antimicrobial agents has led to some problems of which laboratorians need to be aware.