Characterization of KPC-Encoding Plasmids from Enterobacteriaceae Isolated in a Czech Hospital.

Ten Enterobacteriaceae isolates collected in a Czech hospital carried blaKPC-positive plasmids of different sizes (∼30, ∼45, and ∼80 kb). Sequencing revealed three types of plasmids (A to C) with the Tn4401a transposon. Type A plasmids comprised an IncR backbone and a KPC-2-encoding multidrug resistance (MDR) region. Type B plasmids were derivatives of type A plasmids carrying an IncN3-like segment, while type C plasmids were IncP6 plasmids sharing the same KPC-2-encoding MDR region with type A and B plasmids.

Investigation of acid phosphatase variants for the synthesis of phosphate monoesters.

The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000). Biotechnol. Bioeng. 2017;114: 2187-2195. © 2017 Wiley Periodicals, Inc.

War wound treatment complications due to transfer of an IncN plasmid harboring bla(OXA-181) from Morganella morganii to CTX-M-27-producing sequence type 131 Escherichia coli.

A 22-year-old male developed a recurrent sacral abscess associated with embedded shrapnel following a blast injury. Cultures grew extended-spectrum ß-lactamase (ESBL)-producing, carbapenem-susceptible Escherichia coli. Ertapenem was administered, but the infection recurred after each course of antibiotics. Initial surgical interventions were unsuccessful, and subsequent cultures yielded E. coli and Morganella morganii, both nonsusceptible to carbapenems. The isolates were Carba NP test negative, gave ambiguous results with the modified Hodge test, and amplified the bla(OXA48)-like gene by real-time PCR. All E. coli isolates were sequence type 131 (ST131), carried nine resistance genes (including bla(CTX-M-27)) on an IncF plasmid, and were identical by genome sequencing, except for 150 kb of plasmid DNA in carbapenem-nonsusceptible isolates only. Sixty kilobases of this was shared by M. morganii and represented an IncN plasmid harboring bla(OXA-181). In M. morganii, the gene was flanked by IS3000 and ISKpn19, but in all but one of the E. coli isolates containing bla(OXA-181), a second copy of ISKpn19 had inserted adjacent to IS3000. To the best of our knowledge, this is the first report of bla(OXA-181) in the virulent ST131 clonal group and carried by the promiscuous IncN family of plasmids. The tendency of M. morganii to have high MICs of imipenem, a bla(OXA-181) substrate profile that includes penicillins but not extended-spectrum cephalosporins, and weak carbapenemase activity almost resulted in the presence of bla(OXA-181) being overlooked. We highlight the importance of surveillance for carbapenem resistance in all species, even those with intrinsic resistances, and the value of advanced molecular techniques in detecting subtle genetic changes.

Type II and type IV topoisomerase mutations in clinical isolates of Morganella morganii harbouring the qnrD gene.

INTRODUCTION: The aim of this study was to show the emergence of the qnrD gene among fluoroquinolone-resistant Morganella morganii isolate. The occurrence of mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC,parE) genes was also investigated in this strain. METHODOLOGY: 95 clinical Enterobacteria were screened for harbouring the qnrD gene. The clinical isolate of M. morganii was recovered from urine from a patient hospitalized in the urology unit at Fattouma Bourguiba Hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method. Quinolone susceptibility was studied with microbroth dilution technique. The investigations of plasmid mediated quinolone resistance (PMQR) and topoisomerases mutations were performed by polymerase chain reaction and nucleotide sequencing. RESULTS: This isolate showed high level of resistance to quinolones. The MIC with microbroth dilution technique was 512 µg/ml for norfloxacin, 256 µg/ml for ofloxacin and ciprofloxacin and 64µg/ml for levofloxacin.This strain was found to harbour the quinolone resistance determinant qnrD. In addition, this strain harboured two new gyrB mutations (S463A, S464Y) and one parC mutation (S80I). CONCLUSIONS: This is the first report in Tunisia of qnrD determinant and tow new gyrB muations in M. morganii. The nosocomial infection due to this proteeae invites further study of its epidemiologic evolution.

Sequencing, characterization, and gene expression analysis of the histidine decarboxylase gene cluster of Morganella morganii.

The histidine decarboxylase gene cluster of Morganella morganii DSM30146(T) was sequenced, and four open reading frames, named hdcT1, hdc, hdcT2, and hisRS were identified. Two putative histidine/histamine antiporters (hdcT1 and hdcT2) were located upstream and downstream the hdc gene, codifying a pyridoxal-P dependent histidine decarboxylase, and followed by hisRS gene encoding a histidyl-tRNA synthetase. This organization was comparable with the gene cluster of other known Gram negative bacteria, particularly with that of Klebsiella oxytoca. Recombinant Escherichia coli strains harboring plasmids carrying the M. morganii hdc gene were shown to overproduce histidine decarboxylase, after IPTG induction at 37 °C for 4 h. Quantitative RT-PCR experiments revealed the hdc and hisRS genes were highly induced under acidic and histidine-rich conditions. This work represents the first description and identification of the hdc-related genes in M. morganii. Results support the hypothesis that the histidine decarboxylation reaction in this prolific histamine producing species may play a role in acid survival. The knowledge of the role and the regulation of genes involved in histidine decarboxylation should improve the design of rational strategies to avoid toxic histamine production in foods.

Histamine fish poisoning in Australia, 2001 to 2013.

We report on human illness due to histamine fish poisoning outbreaks in Australia from 2001 to 2013. Histamine fish poisoning results from the ingestion of histamine contained within the flesh of certain fish species that naturally contain histidine, which has been converted to histamine by spoilage bacteria following poor handling or temperature control after harvesting. While symptoms vary, allergic symptoms such as facial flushing, headaches and rashes are frequently reported. Using the OzFoodNet outbreak register, published case reports and surveillance reports, we found data on 57 outbreaks of histamine fish poisoning, which affected 187 people, of whom 14% were hospitalised. There were no deaths reported. Outbreaks were generally small in size, with a median of 2 cases per outbreak (range 1 to 22 people), with 88% of outbreaks comprising less than 5 people. Tuna (in the family Scombridae) was the most frequently reported food vehicle, while 18 outbreaks involved non-scombridae fish. Median incubation periods among the outbreaks were short; being less than 1 hour for 22 outbreaks. The most frequently reported symptoms were diarrhoea and rash. Symptoms of facial/body flushing were reported for at least one case in 19 outbreaks and tingling, burning or swelling of the skin, especially around the lips for at least 1 case in 13 outbreaks. In 3 outbreaks, one or more cases were reported to have had respiratory distress or difficulty breathing. While the condition is often mild, improved recognition and appropriate treatment is important, as it will reduce the possibility of any severe health effects resulting from this condition. Key features of histamine fish poisoning outbreaks are the high attack rate, rapid onset, the typical symptoms and their short duration.

[New metalloendopeptidase of Morganella morganii ZM].

Proteolytic activity which is inhibited in the presence of o-phenanthroline was found in M. morganii ZM. Intracellular proteases of M. morganii ZM unlimited split musculoskeletal actin in contrast to grimelysin. Several proteolitic proteins of M. morganii ZM cells were identified by zymography with gelatin. Metalloproteinase of M. morganii ZM cell lysate was purified by hydrophobic chromatography fractionation. The molecular weight of the protein was 35 kDa.

Identification of bla KPC-2 on different plasmids of three Morganella morganii isolates.

Three Morganella morganii strains resistant to carbapenems were recovered from the surgical intensive care unit (SICU) in our hospital. Carbapenemases and extended-spectrum ß-lactamases (ESBLs) were respectively detected by the modified Hodge test and the modified Clinical and Laboratory Standards Institute (CLSI) ESBL confirmatory test in all isolates. Amplification of whole-cell and plasmid DNAs extracted from isolates with primers specific for the bla (KPC) produced an amplicon confirmed to be bla (KPC-2) by sequence analysis. Pulsed-field gel electrophoresis (PFGE) typing revealed that three isolates belonged to two closely related types. Plasmids electrophoresis and restriction analysis revealed that the bla (KPC-2) was located on different plasmids. The transfer of carbapenem resistance from the three original isolates to Escherichia coli EC600 was successful by conjugation. An examination of the outer membrane proteins showed a lack of a 38-kDa outer membrane protein (OMP) compared with M. morganii susceptible to carbapenems. The production of KPC-2 and ESBLs, combined with OMP deficiency, resulted in high-level carbapenem resistance in the M. morganii strains. The genetic environment around bla (KPC-2) analysis revealed that this ß-lactamase was located on the same mobile genetic elements which could transfer between different plasmids.

New Delhi metallo-ß-lactamase-1: local acquisition in Ontario, Canada, and challenges in detection.

New Delhi metallo-ß-lactamase-1 (NDM-1) is a recently identified metallo-ß-lactamase that confers resistance to carbapenems and all other ß-lactam antibiotics, with the exception of aztreonam. NDM-1 is also associated with resistance to many other classes of antibiotics. The enzyme was first identified in organisms isolated from a patient in Sweden who had previously received medical treatment in India, but it is now recognized as endemic throughout India and Pakistan and has spread worldwide. The gene encoding NDM-1 has been found predominantly in Escherichia coli and Klebsiella pneumoniae. We describe the isolation NDM-1-producing organisms from two patients in Toronto, Ontario. To the best of our knowledge, this is the first report of an organism producing NDM-1 that was locally acquired in Canada. We also discuss the evidence that NDM-1 can affect bacterial species other than E. coli and K. pneumoniae, the limited options for treatment and the difficulty laboratories face in detecting organisms that produce NDM-1.

Molecular Analysis of blaKPC-2-Harboring Plasmids: Tn4401a Interplasmid Transposition and Tn4401a-Carrying ColRNAI Plasmid Mobilization from Klebsiella pneumoniae to Citrobacter europaeus and Morganella morganii in a Single Patient.

The spread of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales is a public health concern. KPC-encoding blaKPC is predominantly spread by strains of a particular phylogenetic lineage, clonal group 258, but can also be spread by horizontal transfer of blaKPC-carrying plasmids. Here, we report the transfer of a blaKPC-2-harboring plasmid via mobilization from K. pneumoniae to Citrobacter freundii complex and Morganella morganii strains in a single patient. We performed draft whole-genome sequencing to analyze 20 carbapenemase-producing Enterobacterales strains (15 of K. pneumoniae, two of C. freundii complex, and three of M. morganii) and all K. pneumoniae strains using MiSeq and/or MinION isolated from a patient who was hospitalized in New York and Montreal before returning to Japan. All strains harbored blaKPC-2-containing Tn4401a. The 15 K. pneumoniae strains each belonged to sequence type 258 and harbored a Tn4401a-carrying multireplicon-type plasmid, IncN and IncR (IncN+R). Three of these K. pneumoniae strains also possessed a Tn4401a-carrying ColRNAI plasmid, suggesting that Tn4401a underwent interplasmid transposition. Of these three ColRNAI plasmids, two and one were identical to plasmids harbored by two Citrobacter europaeus and three M. morganii strains, respectively. The Tn4401a-carrying ColRNAI plasmids were each 23,753 bp long and incapable of conjugal transfer via their own genes alone, but they mobilized during the conjugal transfer of Tn4401a-carrying IncN+R plasmids in K. pneumoniae. Interplasmid transposition of Tn4401a from an IncN+R plasmid to a ColRNAI plasmid in K. pneumoniae and mobilization of Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of blaKPC-2 in C. europaeus and M. morganii. IMPORTANCE Plasmid transfer plays an important role in the interspecies spread of carbapenemase genes, including the Klebsiella pneumoniae carbapenemase (KPC)-coding gene, blaKPC. We conducted whole-genome sequencing (WGS) analysis and transmission experiments to analyze blaKPC-2-carrying mobile genetic elements (MGEs) between the blaKPC-2-harboring K. pneumoniae, Citrobacter europaeus, and Morganella morganii strains isolated from a single patient. blaKPC-2 was contained within an MGE, Tn4401a. WGS of blaKPC-2-carrying K. pneumoniae, C. europaeus, and M. morganii strains isolated from one patient revealed that Tn4401a-carrying ColRNAI plasmids were generated by plasmid-to-plasmid transfer of Tn4401a from a multireplicon-type IncN and IncR (IncN+R) plasmid in K. pneumoniae strains. Tn4401a-carrying ColRNAI plasmids were incapable of conjugal transfer in C. europaeus and M. morganii but mobilized from K. pneumoniae to a recipient Escherichia coli strain during the conjugal transfer of Tn4401a-carrying IncN+R plasmid. Therefore, Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of blaKPC-2 in C. europaeus and M. morganii.

Purification and Biochemical Characterization of a Tyrosine Phenol-lyase from Morganella morganii.

Tyrosine phenol-lyase (TPL) is a valuable and cost-effective biocatalyst for the biosynthesis of L-tyrosine and its derivatives, which are valuable intermediates in the pharmaceutical industry. A TPL from Morganella morganii (Mm-TPL) was overexpressed in Escherichia coli and characterized. Mm-TPL was determined as a homotetramer with molecular weight of 52 kDa per subunit. Its optimal temperature and pH for ß-elimination of L-tyrosine were 45 °C and pH 8.5, respectively. Mm-TPL manifested strict substrate specificity for the reverse reaction of ß-elimination and ortho- and meta-substituted phenols with small steric size were preferred substrates. The enzyme showed excellent catalytic performance for synthesis of L-tyrosine, 3-fluoro-L-tyrosine, and L-DOPA with a yield of 98.1%, 95.1%, and 87.2%, respectively. Furthermore, the fed-batch bioprocess displayed space-time yields of 9.6 g L-1 h-1 for L-tyrosine and 4.2 g L-1 h-1 for 3-fluoro-L-tyrosine with a yield of 67.4 g L-1 and 29.5 g L-1, respectively. These results demonstrated the great potential of Mm-TPL for industrial application.

Highly multidrug-resistant Morganella morganii clinical isolates from Nepal co-producing NDM-type metallo-ß-lactamases and the 16S rRNA methylase ArmA.

Morganella morganii can harbour extended-spectrum ß-lactamases and carbapenemases, resulting in increased resistance to multiple antibiotics and a high mortality rate. This study describes the emergence of highly multidrug-resistant clinical isolates of M. morganii from Nepal co-producing NDM-type metallo-ß-lactamases, including NDM-1 and NDM-5, and the 16S rRNA methylase ArmA. This is the first report of M. morganii clinical isolates from Nepal co-producing NDM-1/-5 and ArmA. It is important to establish infection control systems and effective treatments against multidrug-resistant M. morganii.

Characterization of a second ornithine decarboxylase isolated from Morganella morganii.

The genes involved in the putrescine formation by Morganella morganii were investigated because putrescine is an indicator of food process deterioration. We report here on the existence of a new gene for ornithine decarboxylase (ODC) in M. morganii. The sequenced 5,311-bp DNA region showed the presence of four complete and one partial open reading frame. Putative functions have been assigned to several gene products by sequence comparison with the proteins included in the databases. The third open reading frame (speC) encoded a 722-amino acid protein showing 70.9% identity to the M. morganii ODC previously characterized (SpeF). The speC gene has been expressed in Escherichia coli, resulting in ODC activity. The presence of a functional promoter (PspeC) located upstream of speC has been demonstrated. Quantitative real-time reverse transcription PCR assay was used to quantify expression of both M. morganii ODC-encoding genes, speC and speF, under different growth conditions. This assay allows us to identify SpeF as the inducible M. morganii ODC, since it was highly expressed in the presence of ornithine.

Failure of a multiplex polymerase chain reaction assay to detect IMP-27 in a clinical isolate of Morganella morganii.

A case of an IMP-27-positive Morganella morganii isolate is reported, where the carbapenemase enzyme was demonstrated by whole genome sequencing. Carbapenemase detection using a multiplex PCR assay was negative due to mutations in the primer binding site. This case serves to illustrate the limitations of multiplex PCR for carbapenemase detection.

Early onset Morganella morganii sepsis in a newborn infant with emergence of cephalosporin resistance caused by depression of AMPC beta-lactamase production..

A preterm infant with early onset Morganella morganii sepsis was treated with cefotaxime and gentamicin after confirmation of antimicrobial susceptibility. The infant developed persistent ventriculitis caused by the emergence of a cefotaxime-resistant Morganella variant with derepression of its AmpC beta-lactamase. When choosing antibiotic therapy, the risk of development of resistance to cephalosporins should be considered in infections caused by M. morganii and other Gram-negative organisms with inducible AmpC beta-lactamases.

Detection of bacterial strains producing sulbactam- or tazobactam-sensitive beta-lactamases by the use of disks containing the inhibitors alone instead of combining them with antibiotics.

The author demonstrated that disks containing beta-lactamase inhibitors sulbactam or tazobactam combined with ampicillin (SAM) and piperacillin (TZP) are not suitable for performing the double-disk synergy test (DDST) with the aim of determining the sensitivity of beta-lactamases to these inhibitors. The presence of antibiotics (especially of piperacillin) is so disturbing that the results of testing are not specific. In contrast, the use of disks containing sulbactam or tazobactam alone yields very specific results. The author suggested to the firms producing sensi-disks that they make these commercially available to laboratory workers.

MmoSTI restriction endonuclease, isolated from Morganella morganii infecting a tropical moth, Actias selene, cleaving 5'-|CCNGG-3' sequences.

A type II restriction endonuclease, MmoSTI, from the pathogenic bacterium Morganella morganii infecting a tropical moth, Actias selene, has been detected and biochemically characterized, as a potential etiological differentiation factor. The described REase recognizes interrupted palindromes, i.e., 5'-CCNGG-3' sequences and cleaves DNA leaving 5-nucleotide (nt) long, single-stranded (ss), 5'-cohesive ends, which was determined by three complementary methods: (i) cleavage of custom and standard DNA substrates, (ii) run-off sequencing of cleavage products, and (iii) shotgun cloning and sequencing of bacteriophage lambda (λ) DNA digested with MmoSTI. MmoSTI, the first 5'-CCNGG-3' REase characterized from M. morganii, is a neoschizomer of ScrFI, which cleaves DNA leaving 1-nt long, ss, 5'-cohesive ends. It is a high-frequency cutter and can be isolated from easily cultured bacteria, thus it can potentially serve as a tool for DNA manipulations.

Molecular investigation of extended-spectrum beta-lactamase genes and potential drug resistance in clinical isolates of Morganella morganii.

BACKGROUND: Resistance to beta-lactam antibiotics has become more common in Morganella morganii, which can cause of outbreaks of bacteremia and septicemia in postoperative patients. OBJECTIVE: Investigate drug susceptibility of M morganii, identify the gene responsible for extended-spectrum beta-lactamase (ESBL) production and explore treatment options. DESIGN: Descriptive study. SETTING: Hospitals in An Najaf, Iraq. METHODS: M morganii isolates were identified based on morphology, biochemical tests and VITEK® 2 compact system using (GN-ID) card. M morganii isolates were subjected to antibiotic resistance tests using the minimum inhibitory concentration (MIC) technique and an antibiogram was produced. Molecular studies were conducted using the polymerase chain reaction technique. MAIN OUTCOME MEASURE(S): Minimum inhibitory concentration. RESULTS: From 395 gram-negative bacteria, only 17 isolates M morganii grew on MacConkey agar. M morganii isolates strongly resistant to several antibiotics were considered multidrug resistant. All M morganii isolates were ESBL producers. Four genes (CTX-M, SHV, TEM and OXA) encoding the b-lactamase enzyme were detected. Meropenem and imipenem were highly active against the M morganii isolates. CONCLUSIONS: All isolates showed resistance to most common antibiotics, which limits options for treatment. This study provided useful information for selecting antibiotics to precisely target infections caused by M morganii. LIMITATIONS: Limited to antibiotic susceptibility and genotype.

Inhibition of Morganella morganii Histidine Decarboxylase Activity and Histamine Accumulation in Mackerel Muscle Derived from Filipendula ulumaria Extracts.

Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganii histidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria (rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganii HDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 µM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmaria exhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.

Emergence of New Sequence Type OXA-48 Carbapenemase-Producing Enterobacteriaceae in Kuwait.

The aim of this study was to investigate the infections due to OXA-48 carbapenemase-producing bacteria in tertiary hospitals in Kuwait (September 2011 to April 2013) and to determine the sequence types (STs) of the corresponding isolates. Eleven OXA-48 carbapenemase-producing Enterobacteriaceae isolates were recovered from patients treated in nine different hospitals in Kuwait. Susceptibility testing to eighteen antibiotics was done using the E-test. PCR assays were performed for the detection of genes encoding extended-spectrum ß-lactamases (ESBLs) (blaCTX-M, blaSHV, and blaTEM) and carbapenemases (blaOXA-48, blaVIM, blaNDM, blaIMP, blaGIM, and blaKPC). STs were determined by Multilocus Sequence Typing. Seven Klebsiella pneumoniae, two Escherichia coli, one Enterobacter cloacae, and one Morganella morganii harbored the blaOXA-48 gene. The K. pneumoniae and E. coli belonged to seven and two different STs, respectively, which were not related to those reported from this region. The majority of the isolates carried either blaCTX-M or blaSHV genes and showed a multidrug-resistant phenotype, including resistance to tigecycline. Multidrug-resistant Enterobacteriaceae harboring the blaOXA-48 gene are emerging in Kuwait with different STs compared to those identified in other countries of the region. Detection of OXA-48-producing Enterobacteriaceae in Kuwait is important to prevent its rapid spread.