Metallo-ß-lactamases and a tug-of-war for the available zinc at the host-pathogen interface.

Metallo-ß-lactamases (MBLs) are zinc-dependent hydrolases that inactivate virtually all ß-lactam antibiotics. The expression of MBLs by Gram-negative bacteria severely limits the therapeutic options to treat infections. MBLs bind the essential metal ions in the bacterial periplasm, and their activity is challenged upon the zinc starvation conditions elicited by the native immune response. Metal depletion compromises both the enzyme activity and stability in the periplasm, impacting on the resistance profile in vivo. Thus, novel inhibitory approaches involve the use of chelating agents or metal-based drugs that displace the native metal ion. However, newer MBL variants incorporate mutations that improve their metal binding abilities or stabilize the metal-depleted form, revealing that metal starvation is a driving force acting on MBL evolution. Future challenges require addressing the gap between in cell and in vitro studies, dissecting the mechanism for MBL metalation and determining the metal content in situ.

Specific Protein-Membrane Interactions Promote Packaging of Metallo-ß-Lactamases into Outer Membrane Vesicles.

Outer membrane vesicles (OMVs) act as carriers of bacterial products such as plasmids and resistance determinants, including metallo-ß-lactamases. The lipidated, membrane-anchored metallo-ß-lactamase NDM-1 can be detected in Gram-negative OMVs. The soluble domain of NDM-1 also forms electrostatic interactions with the membrane. Here, we show that these interactions promote its packaging into OMVs produced by Escherichia coli. We report that favorable electrostatic protein-membrane interactions are also at work in the soluble enzyme IMP-1 while being absent in VIM-2. These interactions correlate with an enhanced incorporation of IMP-1 compared to VIM-2 into OMVs. Disruption of these interactions in NDM-1 and IMP-1 impairs their inclusion into vesicles, confirming their role in defining the protein cargo in OMVs. These results also indicate that packaging of metallo-ß-lactamases into vesicles in their active form is a common phenomenon that involves cargo selection based on specific molecular interactions.

Molecular Evolution of Transition Metal Bioavailability at the Host-Pathogen Interface.

The molecular evolution of the adaptive response at the host-pathogen interface has been frequently referred to as an 'arms race' between the host and bacterial pathogens. The innate immune system employs multiple strategies to starve microbes of metals. Pathogens, in turn, develop successful strategies to maintain access to bioavailable metal ions under conditions of extreme restriction of transition metals, or nutritional immunity. However, the processes by which evolution repurposes or re-engineers host and pathogen proteins to perform or refine new functions have been explored only recently. Here we review the molecular evolution of several human metalloproteins charged with restricting bacterial access to transition metals. These include the transition metal-chelating S100 proteins, natural resistance-associated macrophage protein-1 (NRAMP-1), transferrin, lactoferrin, and heme-binding proteins. We examine their coevolution with bacterial transition metal acquisition systems, involving siderophores and membrane-spanning metal importers, and the biological specificity of allosteric transcriptional regulatory proteins tasked with maintaining bacterial metallostasis. We also discuss the evolution of metallo-ß-lactamases; this illustrates how rapid antibiotic-mediated evolution of a zinc metalloenzyme obligatorily occurs in the context of host-imposed nutritional immunity.

Extensively drug-resistant IMP-16-producing Pseudomonas monteilii isolated from cerebrospinal fluid.

IMP-1-producing Pseudomonas aeruginosa was first reported in Japan and since then, bacteria with this metallo-ß-lactamase have been detected worldwide. Pseudomonas monteilii (part of P. putida group) were considered an environmental pathogen with low virulence potential; however, multidrug-resistant and carbapenem-resistant P. monteilii have emerged. The present study reports the draft sequence of an extensively drug-resistant IMP-16-producing P. monteilii 597/14 isolated from cerebrospinal fluid in 2014. The sequencing data revealed blaIMP-16 as a gene cassette on class 1 integron, In1738 characterized in this study. Furthermore, the resistome of Pm597/14 consisted of 7 resistance genes (aadA1b, strA, strB, aacA4, blaIMP-16, blaOXA-2, sul1) and diverse virulence determinants involved in the adherence, LPS, antiphagocytosis, iron uptake and mercuric resistance. Although different virulence determinants were found in this study, using Galleria mellonella infection model, Pm597/14 did not kill any larvae between 7 days post-infection. P. monteilii isolates have been reported from clinical and environmental sources, carrying different MBL genes showing its potential role as their reservoir.

Caracterización de carbapenemasas en enterobacterias de muestras de pacientes que acudieron al Hospital General San Juan de Dios de la ciudad de Guatemala durante 2014 y 2015 / Characterization of carbapenemases in Enterobacteriaceae of samples of patients who attended the San Juan de Dios General Hospital in Guatemala City during 2014 and 2015

En salud pública a nivel mundial, la producción de carbapenemasas es actualmente el mayor problema de resistencia antimicrobiana. El objetivo de este estudio fue caracterizar las carbapenemasas en enterobacterias en pacientes que acudieron al Hospital General San Juan de Dios de la ciudad de Guatemala y determinar servicios hospitalarios y tipos de muestras más frecuentes. Se usaron datos de 2014 y 2015 del área de bacteriología del hospital; se realizó una revisión sistemática, selección, ordenamiento y cálculo de frecuencias y porcentajes. En 2014, 165/165 (100 %) de las carbapenemasas fueron de tipo metalo-ß-lactamasas (MBL); en 2015, 90/118 (76 %) MBL y 28/118 (24 %) Klebsiella pneumoniae carbapenemasa (KPC). Klebsiella pneumoniae fue la enterobacteria productora de carbapenemasas (CPE) aislada con más frecuencia, 134/165 (81 %) en 2014 y 82/118 (69 %) en 2015. En 2014 la unidad de cuidados intensivos de neonatos obtuvo el mayor porcentaje de aislamientos de CPE, 30/165 (18 %); en 2015, medicina de hombres fue el servicio con el mayor porcentaje de CPE, 13/118 (11 %). El tipo de muestra más frecuente en 2014 fue sangre, 67/165 (41 %); en el 2015 fue orina, 31/118 (26 %). Los resultados evidencian la persistencia de carbapenemasas tipo MBL y la aparición de nuevos tipos, específicamente carbapenemasas tipo KPC, que destacan la necesidad de actuar urgentemente ante el riesgo que suponen para la salud de la población.

In public health worldwide, carbapenemase production is currently the biggest problem of antimicrobial resistance. The objective of this study was to characterize carbapenemases in Enterobacteriaceae of patients who attended the San Juan de Dios General Hospital in Guatemala City and to determine hospital services and types of samples more frequent. Data from 2014 and 2015 of the bacteriology department of the hospital were used; a systematic review, selection, ordering and calculation of frequencies and percentages was conducted. In 2014, 165/165 (100 %) of the carbapenemases were metallo-ß-lactamases (MBL); in 2015, 90/118 (76 %) MBL and 28/118 (24 %) Klebsiella pneumoniae carbapenemase (KPC). Klebsiella pneumoniae was the carbapenemases-producing Enterobacteriaceae (CPE) most frequently isolated, 134/165 (81 %) in 2014 and 82/118 (69 %) in 2015. In 2014 the neonatal intensive care unit obtained the highest percentage in CPE, 30/165 (18 %); in 2015, men's medicine was the service with the highest percentage of carbapenemases, 11/138 (11 %). The most frequent type of sample in 2014 was blood, 67/165 (41 %). In 2015 it was urine, 31/118 (26 %). The results obtained highlight the persistence of MBL-type carbapenemases and the appearance of new types of carbapenemases, specifically KPC. These results underline the need to act urgently in Guatemala in the face of the problems that carbapenemases-producing Enterobacteriaceae pose for the health of the population.

Metallo-ß-Lactamase Inhibitors Inspired on Snapshots from the Catalytic Mechanism.

ß-Lactam antibiotics are the most widely prescribed antibacterial drugs due to their low toxicity and broad spectrum. Their action is counteracted by different resistance mechanisms developed by bacteria. Among them, the most common strategy is the expression of ß-lactamases, enzymes that hydrolyze the amide bond present in all ß-lactam compounds. There are several inhibitors against serine-ß-lactamases (SBLs). Metallo-ß-lactamases (MBLs) are Zn(II)-dependent enzymes able to hydrolyze most ß-lactam antibiotics, and no clinically useful inhibitors against them have yet been approved. Despite their large structural diversity, MBLs have a common catalytic mechanism with similar reaction species. Here, we describe a number of MBL inhibitors that mimic different species formed during the hydrolysis process: substrate, transition state, intermediate, or product. Recent advances in the development of boron-based and thiol-based inhibitors are discussed in the light of the mechanism of MBLs. We also discuss the use of chelators as a possible strategy, since Zn(II) ions are essential for substrate binding and catalysis.

The Resistome of Low-Impacted Marine Environments Is Composed by Distant Metallo-ß-Lactamases Homologs.

The worldwide dispersion and sudden emergence of new antibiotic resistance genes (ARGs) determined the need in uncovering which environment participate most as their source and reservoir. ARGs closely related to those currently found in human pathogens occur in the resistome of anthropogenic impacted environments. However, the role of pristine environment as the origin and source of ARGs remains underexplored and controversy, particularly, the marine environments represented by the oceans. Here, due to the ocean nature, we hypothesized that the resistome of this pristine/low-impacted marine environment is represented by distant ARG homologs. To test this hypothesis we performed an in silico analysis on the Global Ocean Sampling (GOS) metagenomic project dataset focusing on the metallo-ß-lactamases (MßLs) as the ARG model. MßLs have been a challenge to public health, since they hydrolyze the carbapenems, one of the last therapeutic choice in clinics. Using Hidden Markov Model (HMM) profiles, we were successful in identifying a high diversity of distant MßL homologs, related to the B1, B2, and B3 subclasses. The majority of them were distributed across the Atlantic, Indian, and Pacific Oceans being related to the chromosomally encoded MßL GOB present in Elizabethkingia genus. It was observed only a reduced number of metagenomic sequence homologs related to the acquired MßL enzymes (VIM, SPM-1, and AIM-1) that currently have impact in clinics. Therefore, low antibiotic impacted marine environment, as the ocean, are unlikely the source of ARGs that have been causing enormous threat to the public health.

Global Molecular Epidemiology of IMP-Producing Enterobacteriaceae.

International data on the molecular epidemiology of Enterobacteriaceae with IMP carbapenemases are lacking. We performed short-read (Illumina) whole-genome sequencing on a global collection of 38 IMP-producing clinical Enterobacteriaceae (2008 to 2014). IMP-producing Enterobacteriaceae (7 varieties within 11 class 1 integrons) were mainly present in the South Pacific and Asia. Specific blaIMP-containing integrons (In809 with blaIMP-4, In722 with blaIMP-6, and In687 with blaIMP-14) were circulating among different bacteria in countries such as Australia, Japan, and Thailand. In1312 with blaIMP-1 was present in Klebsiella pneumoniae from Japan and Citrobacter freundii from Brazil. Klebsiella pneumoniae (n = 22) was the most common species; clonal complex 14 (CC14) from Philippines and Japan was the most common clone and contained In1310 with blaIMP-26 and In1321 with blaIMP-6 The Enterobacter cloacae complex (n = 9) consisted of Enterobacter hormaechei and E. cloacae cluster III. CC78 (from Taiwan) containing In73 with blaIMP-8 was the most common clone among the E. cloacae complex. This study highlights the importance of surveillance programs using the latest molecular techniques for providing insight into the characteristics and global distribution of Enterobacteriaceae with blaIMP genes.

Carbapenem-resistant Pseudomonas aeruginosa: association with virulence genes and biofilm formation.

Pseudomonas aeruginosa is an opportunistic pathogen that causes frequently nosocomial infections, currently becoming more difficult to treat due to the various resistance mechanisms and different virulence factors. The purpose of this study was to determine the risk factors independently associated with the development of bacteremia by carbapenem-resistant P. aeruginosa, the frequency of virulence genes in metallo-ß-lactamases producers and to evaluate their ability to produce biofilm. We conducted a case-control study in the Uberlândia Federal University - Hospital Clinic, Brazil. Polymerase Chain Reaction was performed for metallo-ß-lactamases and virulence genes. Adhesion and biofilm assays were done by quantitative tests. Among the 157 strains analyzed, 73.9% were multidrug-resistant, 43.9% were resistant to carbapenems, 16.1% were phenotypically positive for metallo-ß-lactamases, and of these, 10.7% were positive for blaSPM gene and 5.3% positive for blaVIM. The multivariable analysis showed that mechanical ventilation, enteral/nasogastric tubes, primary bacteremia with unknown focus, and inappropriate therapy were independent risk factors associated with bacteremia. All tested strains were characterized as strongly biofilm producers. A higher mortality was found among patients with bacteremia by carbapenem-resistant P. aeruginosa strains, associated independently with extrinsic risk factors, however it was not evident the association with the presence of virulence and metallo-ß-lactamases genes.

High prevalence of blaVIM-1 gene in bacteria from Brazilian soil.

This study investigated bacteria from soil samples to (i) determine the main bacterial genera and species having resistance to carbapenem and other ß-lactams and (ii) establish if the mechanism of resistance was due to the production of metallo-ß-lactamases. The isolates were characterized by PCR for metallo-ß-lactamases and integrons, by antimicrobial susceptibility testing, and by sequencing. The antimicrobial profile of 40 imipenem-resistant Gram-positive soil isolates from all Brazilian regions demonstrated that 31 (77.5%) of them were multidrug resistant. Among the 40 isolates, 19 presented the blaVIM gene and class 1 integrons by PCR. Six of the 19 isolates were identified as Paenibacillus sp., 12 as Bacillus sp., and just 1 was classified as Staphylococcus sp., by sequencing of the 16S rRNA gene. These results suggest that bacteria from soil can act as a source of blaVIM-1 genes, representing a threat to public health.

Evolution of Metallo-ß-lactamases: Trends Revealed by Natural Diversity and in vitro Evolution.

The production of ß-lactamase enzymes is one of the most distributed resistance mechanisms towards ß-lactam antibiotics. Metallo-ß-lactamases constitute a worrisome group of these kinds of enzymes, since they present a broad spectrum profile, being able to hydrolyze not only penicillins, but also the latest generation of cephalosporins and carbapenems, which constitute at present the last resource antibiotics. The VIM, IMP, and NDM enzymes comprise the main groups of clinically relevant metallo-ß-lactamases. Here we present an update of the features of the natural variants that have emerged and of the ones that have been engineered in the laboratory, in an effort to find sequence and structural determinants of substrate preferences. This knowledge is of upmost importance in novel drug design efforts. We also discuss the advances in knowledge achieved by means of in vitro directed evolution experiments, and the potential of this approach to predict natural evolution of metallo-ß-lactamases.

Detection of carbapenemase activity directly from blood culture vials using MALDI-TOF MS: a quick answer for the right decision.

OBJECTIVES: Recently, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was successfully applied for the detection of carbapenemase activity directly from Gram-negative colonies. Based on this principle, we evaluated the performance of MALDI-TOF MS for rapid detection of carbapenemase activity directly from positive blood culture vials. METHODS: A total of 100 blood culture vials were randomly selected. MALDI-TOF MS carbapenemase assay results were confirmed by the detection of carbapenemase-encoding genes. RESULTS: A total of 110 bacterial isolates were recovered. The MALDI-TOF MS carbapenemase assay identified 21 of 29 (72.4%) of the carbapenemase-producing isolates directly from the blood culture vials, especially those encoding KPC-2 (100%) and SPM-1 (100%), after a 4 h incubation period. Although the majority of OXA-23-producing Acinetobacter baumannii isolates were not identified on day 1, all isolates were identified as carbapenemase producers directly from the colony on the next day. CONCLUSIONS: The MALDI-TOF MS carbapenemase assay is a feasible and rapid test to identify carbapenemase activity directly from blood culture vials. It may contribute to faster readjustment of empirical antimicrobial therapy and implementation of infection control measures.

Isolation of the first IMP-4 metallo-ß-lactamase producing Klebsiella pneumoniae in Tianjin, China.

This study shows for the first time the mechanism of carbapenem resistance of a Klebsiella pneumoniae clinical isolate TJ8 recovered from Tianjin Medical University General Hospital ,China. The modified Hodge test and EDTA synergy test were performed for the screening of carbapenemases and metallo-ß-lactamases (MBLs), respectively. Polymerase chain reactions and DNA sequencing confirmed that the strain carried IMP-4 metallo-ß-lactamase (MBL) , SHV-11 and TEM-1 ß-lactamase. Class I integron was positive and gave a 3.0-kb PCR amplicon .IMP-4 was located in Class I integron 5'CS. The gene determinants were organized in the order of bla IMP-4-orfII-orfIII.In all, the results show that IMP-4 MBL production caused the TJ8 resistance to carbapenems.

Actividad in vitro de piperacilina-tazobactam en combinación con aminoglucósidos y fluoroquinolonas en Pseudomonas aeruginosa productoras de metalo-β-lactamasas / In vitro piperacillin-tazobactam activity in combination with aminoglycosides and fluoroquinolones in Pseudomonas aeruginosa metallo-β-lactamases producers

Pseudomonas aeruginosa es un bacilo gramnegativo, capaz de adquirir genes que codifican la producción de metalo-β-lactamasas (MBL) haciéndose multirresistente, lo cual dificulta el tratamiento de las infecciones causadas por este microorganismo. Se evaluó la actividad in vitro de piperacilina-tazobactam, en combinación con aminoglucósidos y fluoroquinolonas, en 8 aislamientos de P. aeruginosa productoras de MBL, provenientes de cuatro hospitales del oriente y sur de Venezuela mediante el método epsilométrico. Las combinaciones de antimicrobianos que presentaron mayor efecto sinérgico fueron piperacilina-tazobactam/gentamicina y piperacilina-tazobactam/ciprofloxacina en 5/8 aislamientos. La combinación de piperacilina-tazobactam/amikacina presentó sinergia en 4 de los casos y adición en los otros 4, manifestando el menor índice de concentración inhibitoria fraccionada promedio. No hubo antagonismo en ninguna combinación de antimicrobianos. Los resultados de este estudio sugieren que piperacilina-tazobactam en combinación con gentamicina, amikacina o ciprofloxacina podrían ser utilizadas como alternativas terapéuticas en las infecciones por P. aeruginosa multirresistentes productoras de MBL en los hospitales del estado Bolívar, mientras que en los hospitales del oriente de Venezuela se sugiere la combinación piperacilina-tazobactam/amikacina.

Pseudomonas aeruginosa is a gram negative bacillus able to acquire genes which codify for production of metallo-β-lactamases (MBL) becoming multiresistant, which difficults the treatment of infections produced by this microorganism. The in vitro activity of piperacilline-tazobactam, in combination with aminoglycosides and fluoroquinonolones was evaluated in 8 P. aeruginosa MBL producers obtained at four hospitals of the west and south areas of Venezuela through an epsilometric method. The antimicrobial combinations which showed the greatest synergic effect were piperacillin-tazobactam/gentamicin and piperacillin-tazobactam/amikacin in 5/8 isolates. The piperacilline-tazobactam/amikacin combination showed synergy in 4 cases and addition in the other 4, with the lowest index of mean fractionated inhibitory concentration. There was no antagonism in any of the antimicrobial combinations. The results of this study suggest that piperacilline-tazobactam in combination with gentamicin, amikacin or ciprofloxacin, could be used as therapeutic alternatives in multiresistant P. aeruginosa MBL producers at all the hospitals of Bolivar State, while in hospitals located in the west of Venezuela, the piperacilline-tazobactan/amikacin combination is suggested.

Detection of extended spectrum beta lactamases, ampc beta lactamases and metallobetalactamases in clinical isolates of ceftazidime resistant Pseudomonas Aeruginosa.

We studied the prevalence of ceftazidime resistance in Pseudomonas aeruginosa and the rates of extended-spectrum ß-lactamase (ESBL), AmpC ß-lactamase (AmpC) and metallo-ß-lactamase (MBL) production among the ceftazidime resistant Pseudomonas aeruginosa. A very high rate of MBL production was observed, which suggested it to be an important contributing factor for ceftazidime resistance among Pseudomonas aeruginosa.

Detección del gen blaVIM-2 en cepas de Pseudomonas aeruginosa productoras de metalo ß-lactamasa aisladas en una unidad de cuidados intensivos en Ciudad Bolívar, Venezuela / blaVIM-2 gene detection in metallo-ß-lactamase-producing Pseudomonas aeruginosa strains isolated in an Intensive Care Unit in Ciudad Bolívar, Venezuela

Ten Pseudomonas aeruginosa strains with resistance to broad-spectrum cephalosporin and carbapenems were studied to determine the presence of genes that mediate the productionof metallo-p-lactamases. These strains were isolated from patients with nosocomial infection at the Intensive Care Unit of the Complejo Hospitalario "Ruiz y Paéz" of Ciudad Bolívar, Bolívar State, Venezuela, from 2003 to 2006. In all isolates a metallo-enzyme activity was detected by using the double disk synergism test. PCR amplification of genes encoding the families IMP, VIM and SPM metallo-ß-lactamases showed the presence of a blaVIM gene in all strains studied. DNA sequencing revealed that all isolates showed the presence of blaVIM-2These results suggest that it is necessary to keep these strains under epidemiologic surveillance, establish laboratory strategies for opportune detection and the imple-mentation of new policies to ensure the appropriate use of antibiotics in this institution.

Se estudiaron 10 cepas de Pseudomonas aeruginosa con resistencia a cefalosporinas y carbapenémicos con el objeto de determinar la presencia de genes que median la producción de metalo ß-lactamasas. Estas cepas se aislaron de pacientes con infección nosocomial hospitalizados en la Unidad de Cuidados Intensivos (UCI) del Complejo Hospitalario "Ruiz y Paéz" de Ciudad Bolívar, estado Bolívar, Venezuela, durante el período 2003 - 2006. En todas las cepas se detectó la actividad de una metalo-enzima, mediante la prueba del sinergismo del doble disco. La amplificación por la reacción de polimerasa en cadena de los genes que codifican para las metalo ß-lactamasas de las familias IMP, VIM y SPM, y su posterior secuenciación, permitió confirmar la presencia de metalo p-lactamasas de tipo VIM-2. Estos resultados sugieren que es necesario mantener bajo vigilancia epidemiológica a estas cepas, establecer estrategias de laboratorio para su detección oportuna e implementar nuevas políticas que aseguren el uso adecuado de los antibacterianos en esta institución.

Spread of Metallo-ß-lactamases: screening reveals the presence of aBLA SPM-1 gene in hospital sewage in southern Brazil.

Of 396 Pseudomonas aeruginosa strains isolated from hospital sewage, the blaSPM-1 gene was confirmed in nine. This is the first report of environmental P. aeruginosa strains carrying the blaSPM-1 gene in Brazil. The carbapenem resistance, already disseminated among clinical isolates, has been detected among environmental isolates.